Cardiovascular risk is assessed for the prediction and appropriate management of patients using collections of identified risk markers obtained from clinical questionnaire information, concentrations of certain blood molecules (e.g., N-terminal proB-type natriuretic peptide fragment and soluble receptors of tumor-necrosis factor-α and interleukin-2 [IL-2]), imaging data using various modalities, and electrocardiographic variables, in addition to traditional risk factors. These risk markers and factors can be combined to form standard cardiovascular risk scores (e.g., the Framingham risk score). These risk markers and factors can also be ranked according to their efficiency in predicting each cardiovascular outcome.

The term biomarker has been defined as an indicator of physiological or pathophysiological processes and hence used for diagnostic, prognostic,Footnote 1 and/or therapeutic purposes. Biomarker is defined by the National Institutes of Health as an entity “that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention.” In a pathophysiological context, the useless prefix bio is omitted.

Markers of redox stress encompass molecules modified by interactions with reactive oxygen species (ROS) (i.e., proteins, lipids, carbohydrates, and DNA) and molecules of the antioxidant defense [90]. Examples of circulating markers that have a value in addition to traditional cardiovascular risk factors include glycated hemoglobin (HbA1c) for glycemic control in diabetes, high-sensitivity troponin-I (hsTnnI) and C-reactive protein (hsCRP) for cardiovascular risk prediction, and N-terminal proB-type natriuretic peptide for heart failure (HF).

A risk marker is not necessarily involved in the cause or set of causes of a cardiovascular disease (CVD), like a risk factor.

Markers of pathological conditions serve to:

  1. 1.

    Detect and sort individuals at risk for CVDs

  2. 2.

    Monitor disease progression

  3. 3.

    Select therapy and assess treatment efficacy

Hence, features that determine the clinical utility of a marker include (1) the ease and cost of measurement, (2) its sensitivity and specificity, and (3) evidence for guiding management and improving patient outcome.

A competing risk or cause precludes the occurrence of another risk or cause. For example, an adverse event is a competing cause with respect to death attributable to any cardiovascular event.

The health of individuals can be defined, at least partly, by low levels of adverse markers of CVD. Cardiovascular health also relies on sets of risk factors that determine long-term survival and quality of life (Vol. 7, Chap. 2. Context of Cardiac Diseases). Clinical markers and factors, in particular, those related to inflammation, adverse remodeling, and thrombosis, assess the risk for CVD, which is refined using biological constants and imaging data.

The risk factors comprise behavioral habits, such as smoking, physical inactivity, eating pattern (food quantity, meal composition and frequency, and use of processed, canned, and frozen food), which have an impact on the body weight, along with blood concentrations of cholesterol and glucose, and secondarily on arterial pressure [224].

In addition to clinical markers that are used for early detection, prevention of acute cardiovascular events targets modifiable risk factors, especially changes in habits (e.g., smoking cessation and supervised exercise) and therapy.

Most CVD is indeed related to risk factors that can be controlled or treated, such as hypertension (systolic and diastolic arterial pressure greater than 18.6 kPa [SBP> 140 mmHg] and 12.0 kPa [DBP> 90 mmHg], respectively), dyslipoproteinemia, obesity, tobacco smoking, physical inactivity, and diabetes. The leading CVD risk factors comprise hypertension (∼13% deaths), followed by tobacco use (∼9%), hyperglycemia (∼6%), lack of physical activity (∼6%), and obesity (∼5%) [30].

Cardiovascular risk factors are similar for men and women.

  • The total risk of a person can be estimated by summing the risk imparted by each of the major risk factors.

  • The absolute risk is defined as the probability of developing CVD over a given time period.

  • The relative risk is the ratio of the absolute risk of a given patient to that of a low-risk group, that is:

    • Systolic and diastolic blood pressure lower than 130 and 80 mmHg

    • Total cholesterol concentration ranging from 160 to 199 mg/dl

    • Low-density lipoprotein (LDL)CS ranging from 100 to 129 mg/dlFootnote 2 and a 1-mmol reduction in LDLCS in middle-aged individuals for 5 years being estimated to yield a 20% decrease in CVD risk [121]

    • High-density lipoproteins (HDLs)CS greater than or equal to 45 mg/dl for men or 55 mg/dl for women

    • Nonsmoker

    • Absence of diabetes mellitus [226]

The major independent cardiovascular risk factors include cigarette smoking, physical inactivity, aging, hypertension, obesity, diabetes, and dyslipoproteinemia with hypercholesterolemia and elevated LDLCS concentration but decreased HDLCS concentration [226].

Behavioral risk factors are shared by atherosclerosis complications (myocardial infarction [MI] and stroke) and diabetes, cancer, and respiratory disease [30].

Hypertension is a primary risk factor for stroke, especially long-term large variations of both systolic and diastolic arterial pressure observed in a series of medical examinations (either measured or recorded by ambulatory blood pressure monitoring), such as early morning (arousal) arterial pressure surges [227]. Large variations in arterial pressure can result from abnormal baroreceptor functioning, excessive sympathetic activity, elevated sensitivity to the renin–angiotensin axis, transient high-salt diet, acute fluctuations in body weight, sleep disorders, and ingestion of nonsteroidal anti-inflammatory drugs [228]. In addition, excessive arterial pressure reductions during treatment (SBP< 16.0 kPa [< 120 mmHg]; DBP< 9.3 kPa [< 70 mmHg) can increase the cardiovascular event rate.

Coronary arterial calcium score is the most important predictor of adverse coronary events, whereas fasting glycemia and carotid ultrasonographic measures are salient predictors of stroke [229].

Predisposing risk factors, such as a family history of premature coronary artery disease, ethnic characteristics, and psychosocial factors (hostility, depression, and social isolation), worsen the independent risk factors.

Conditional risk factors encompass hypertriglyceridemia; a high proportion of small, dense LDLs; elevated plasmatic concentrations of homocysteine and lipoprotein-A; and the presence of prothrombotic factors (e.g., fibrinogen) and inflammatory markers (e.g., C-reactive protein [CRP]).

Risk factors can be also categorized as modifiable and nonmodifiable. Modifiable risk factors include behavioral, metabolic, and other controllable risk factors.

  • Behavioral risk factors include:

    1. 1.

      Tobacco smoking

    2. 2.

      Physical inactivity

    3. 3.

      Unhealthy hypercaloric diet rich in lipids and salt

    4. 4.

      Abuse of illicit substances (e.g., amphetamines, cocaine, ecstasy, and heroin), which is associated with an increased risk of ischemic and hemorrhagic strokes

    5. 5.

      Harmful use of alcohol

  • Metabolic risk factors comprise:

    1. 1.


    2. 2.

      Hyperglycemia and diabetes

    3. 3.

      Hyperlipidemia and hypercholesterolemia

    4. 4.


  • Other modifiable risk factors consist of:

    1. 1.

      Poverty and low educational status

    2. 2.

      Psychological factors (e.g., stressful life and depression)

    3. 3.


    4. 4.

      Air pollution, particularly in populated agglomerations, which, in general, do not meet the ambient air quality standards

Some major CVD risk factors cannot be controlled:

  • Aging, with augmented frequency of CVD

  • Gender, a man being at a greater risk for CVD than a premenopausal woman

  • Family history, that is, inherited genetic predisposition

  • EthnicityFootnote 3

Additional risk factors encompass inflammatory disorders, infection, pollution, and cardiac atrial disorders, independently of atrial fibrillation [230]. Almost all patients with atherosclerosis have lesions in many arterial territories and risk factors. Major risk factors for limb and visceral ischemia (i.e., kidney, bowel, liver, or spleen), which are usually late-stage manifestations of atherosclerosis, include hypertension, smoking, and diabetes [231].

Because atherosclerosis is involved in both abdominal aortic aneurysms (AAAs) and coronary stenoses, these two types of vascular lesions share primary risk factors (i.e., age, smoking, high LDLCS or total cholesterol concentration, and male sex). On the other hand, the role of hypertension in AAA genesis is less well established than in chronic obstructive airways disease (CoAD), and White race is more relevant to AAA than to CoAD [232].

In a sample of 4232 men and women aged 60 years, the plasmatic concentration of PCSK9Footnote 4 is associated with the future risk for CVD (e.g., myocardial infarction and ischemic stroke), even after adjustments for established CVD risk factors (LDLCS, HDLCS, LPA, TGs, hypertension, diabetes, smoking, overweight, obesity, and physical inactivity), and statin use [234].

The American Heart Association cardiovascular health score is inversely related to CVD. According to the Framingham Study (mean age 58 years, 55% women), circulation markers of subclinical CVD comprise [235]:

  1. 1.

    Structural and functional criteria:

    • Reduced ankle–brachial index (ABI)

    • Carotid intima–media thickness and possible stenosis

    • Left ventricular hypertrophy

    • Left ventricular systolic dysfunction

    • Microalbuminuria

  2. 2.

    In addition to lifestyle:

  3. 3.

    In addition to mechanical parameter values:

    • Blood pressure

  4. 4.

    Blood concentrations of agents related to lipid and glucose metabolism, inflammation, and thrombosis (Tables 2.1, 2.2, 2.3, and 2.4):

    Table 2.1 Examples of risk factors related to lipid metabolism (Source: [238]; lpPLA2 lipoprotein-associated phospholipase-A2, PCSK proprotein convertase subtilisin/kexin, S PLA2 secretory PLA2)
    Table 2.2 Examples of risk markers related to inflammation and thrombosis (Source: [238])
    Table 2.3 Examples of risk markers related to injury, healing, and fibrosis (Part 1; Source: [238]; DAMP damage-associated molecular pattern, GDF growth differentiation factor, IL interleukin, MMP matrix metallopeptidase)
    Table 2.4 Examples of risk markers related to injury, healing, and fibrosis (Part 2; Source: [238]; MR proAdm midregional fragment of adrenomedullin precursor, MR proANP midregional fragment of type-A natriuretic peptide [ANP] precursor, NT proBNP inactive N-terminal fragment of BNP precursor, TGF transforming growth factor)
    • Cholesterol, in particular, LDP–cholesterol (LDLCS) and its regulator proprotein convertase subtilisin/kexin PCSK9

    • Glucose

    • Natriuretic peptides (Table 2.5) in addition to N-terminal fragments of A- (atrial; ANP) and B-type (brain; BNP) natriuretic peptide precursors (NTproANP and NTproBNP)Footnote 7

      Table 2.5 Several cardiokines can serve as CVD markers (Source: [71])
    • Plasminogen activator inhibitor-1

    • Aldosterone

    • C-reactive protein (or pentraxin-1)

    • DDimerFootnote 8

    • Fibrinogen

    • Homocysteine

    • TroponinsFootnote 9

    • Growth differentiation factor (GDF15)

Hence, an overlap exists between risk factors and markers.

Lipoproteins (i.e., water-soluble protein–lipid complexes) mediate the transport of cholesterol and lipids in blood. Linkage of cholesterol to lipoproteins (HDLs and LDLs) enables its back and forth motion in blood between the liver and other organs. Relatively high concentration of cholesterol tethered to ApoB-containing (ApoB+ ) LDLs is an atherosclerosis risk marker. Cholesterol ester links to ApoA1+ HDLs. Therefore, concentrations of low- (LDLCS) and HDL –cholesterol (HDLCS) and TG (or triacylglycerol [TAG]) are measured to identify patients with a high risk for CVD.

The measure non-HDLCS combines the cholesterol content of LDLs, lipoprotein-A (LPA), an LDL-like particle linked to ApoA, and TG-rich lipoproteins (remnants, i.e., intermediate-density lipoproteins (IDLs), VLDLs, and chylomicron remnants), TGs being mainly carried by remnants. In treated patients, non-HDLCS level is more closely associated with coronary atherosclerosis progression than LDLCS concentration [239].

Values of certain markers differ significantly according to gender, such as markers of lipid metabolism, adiposity, inflammation, endothelial and renal dysfunction, and cardiac stress [240].

  • Women have higher concentrations of HDL and HDLCS, leptin,Ddimer, homoarginine, and NTproBNP.

  • Women have lower concentrations of LDLCS, adiponectin, the chemokine CCL2, troponin-T (TNNT), cystatin-C, symmetrical and asymmetrical dimethylarginine, lipoprotein-associated phospholipase-A2, and soluble endothelial cell adhesion molecules.

The benefit of drugs administered in addition to a recommended healthy lifestyle generally exceeds the therapeutic risk. However, cholesterol-lowering therapy in patients with LDLCS concentrations below a certain threshold is unfounded. A treatment threshold is aimed at avoiding treating patients at a low risk for atherosclerosis-associated complications. The LDLCS concentration is monitored to adjust therapy according to its effects.

Measurements of alternative cardiovascular markers (e.g., coronary artery calcium) in addition to concentrations of LDLCS and high-sensitivity C-reactive protein (hsCRP), PWV index (e.g., ABI), etc., used in therapy decisions (e.g., statins), provide additional information when a treatment decision is uncertain.Footnote 10 These markers are aimed at identifying individuals a priori defined in the lower risk population who nevertheless can experience an adverse event (MI and stroke) if left untreated, as, in fact, they can belong to a higher risk population when these alternative markers are incorporated in scoring.

Dying parenchymal cells and leukocytes release small DNA fragments into the bloodstream. Blood concentrations of fragmented DNA increase in CVD, among other maladies. Because each cell type has a unique DNA methylation pattern, a source of plasmatic DNA can be identified using cell-specific DNA methylation markers [241].

Single-stranded DNA aptamers are oligonucleotides that contain approximately 50 bases. They can bind proteins with high specificity and affinity and can be used to identify early CVD markers.

Dickkopf-related protein Dkk4, a Wnt inhibitor, and cryptic, a transforming growth factor (TGF) inhibitor, are relevant targets in the context of myocardial injury [242].

Dietary habits are modifiable risk factors. Intestinal microbiota and bacterial metabolites are connected to dietary composition. Gut flora-derived metabolite trimethylamine Noxide (TMAO) is a small organic compound generated from betaine, carnitine, and choline [243].Footnote 11 It accumulates in fishes, especially elasmobranchs,Footnote 12 and protects against the protein-destabilizing effectFootnote 13 and ligand-binding inhibition of urea at high concentrations in addition to adverse effects of temperature, salinity, and hydrostatic pressure. Its blood concentration, which depends on diet composition in addition to the activity of gut flora and liver FMO, increases after ingesting trimethylammonium-containing nutrients (Lcarnitine and phosphatidylcholine-rich food), such as red meat, eggs, and fish [244]. Trimethylamine oxide is a risk factor for major adverse cardio- and cerebrovascular events (ACaVCeVE), especially in patients with chronic kidney disease. Plasmatic TMAO concentration correlates positively with ACaVCeVE and mortality [244]. Trimethylamine oxide, which impairs cholesterol and bile acid metabolism and the activity of sterol transporters in the liver and intestine, activates inflammation, and markedly reduces reverse cholesterol transport, may favor the formation of macrophage scavenger receptors and foam cells in the arterial wall [243, 244]. In mice, the inhibition of hepatic FMO3, which is controlled by the bile acid-activated farnesoid X receptor (FXR), and microbial TMAO production counters atherogenesis.

Current clinical risk stratification methods (e.g., Framingham score) do not suffice to predict CVD. Many events happen in patients identified to be at a low or moderate risk; a large fraction of patients at risk are identified by a major event. Therefore, better risk stratification tools are needed.

2.1 Environmental Stressors

Environmental stressors, such as air pollution and noise, are cardiovascular risk factors. In particular, Asian windblown sand dust (Asian dust [kosa]) originating in the deserts of China and East Asia that mixes with air pollutants during atmospheric transport and contains microbes correlates not only with respiratory diseases but also with acute MI at least in men older than 75 years, living in southwestern Japan and having hypertension, diabetes mellitus, and, above all, chronic kidney disease [245].

2.1.1 Air Pollution

Air pollution is a major environmental risk factor. Air pollutants include particulate matter, gaseous pollutants, and volatile organic compounds (VOCs).

Particulate matter is classified into three categories according to the size of airborne microparticles:

  1. 1.

    Coarse (2.5≤aerodynamic caliber < 10 μm [10−5 m]; PM10)

  2. 2.

    Fine (0.1≤aerodynamic caliber < 2.5 μm; PM2.5)

  3. 3.

    Ultrafine particles (aerodynamic caliber < 0.1 μm [10−7 m]; PM0.1), also called nanoparticles

Particles are represented by concentrations of PM2.5 and PM10 particles, the latter comprising ultrafine, fine, and coarse particles.Footnote 14 Nanoparticles suspended in air (nanoaerosol; size \({\mathcal O}\)[10−9 m]) contribute to indoor air pollution exposure.

Among PM, inhalable particles of aerodynamic caliber less than 10 μm can travel deep into the bronchial tree inside lungs. Air pollution facilitates and exacerbates respiratory infections.

In fact, environmental toxins cause numerous acute and chronic illnesses, particularly cardiovascular maladies, especially in individuals with diabetes during episodes of high air pollution exposure [247]. Exposure to air polluted with fine PM (PM2.5) augments the risk for cardiovascular morbidity and mortality via endothelial dysfunction and systemic inflammation. Air pollution exacerbates existing maladies and can be involved in the development of CVDs (atherosclerosis, cardiac arrhythmias, HF, stroke, and venous thromboembolism) [248]. In mice, concentrated ambient PM2.5 potentiates atherosclerotic plaque vulnerability.

In Canada, Europe, and the USA, a 10 μg/m3 increase in PM10 is associated with a 0.2–0.6% increase in all-cause mortality [248]. In Europe, a 10 μg/m3 elevation of NO2 causes a 0.4% augmentation in the cardiovascular mortality rate. In China, outdoor air pollution ranks fourth among preventable causes of diseases, current PM2.5 concentrations (61 μg/m3) having increased from those during the Beijing Olympic games (55 μg/m3), which were already much higher than World Health Organization standard (10 μg/m3) [249].

According to the World Health Organization, outdoor air pollution in both cities and rural areas in 2012 is estimated to cause 3.7 million premature deaths worldwide per year, 88% of those premature deaths occurring in low- and middle-income countries [250]. Reducing outdoor emissions from household coal and biomass energy systems, agricultural waste incineration, forest fires, and certain agroforestry activities (e.g., charcoal production) may reduce rural air pollution sources in developing regions.

According to the Global Burden of Disease study in 2010, air pollution may be responsible for about 6% of all-cause and all-age deaths, in addition to development and exacerbation of respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease, and pulmonary cancers) [248].

Air pollution ranks as the ninth contributor among modifiable disease risk factors, that is, above low physical activity, high-sodium diet, high cholesterol, and drug use [248].

Acute exposure to environmental toxicants can lead to angina, arrhythmia, MI, and HF. Repeated exposure to air pollution can cause vascular inflammation and redox stress, thereby promoting atherosclerotic plaque expansion and rupture [246]. Long-term exposure increases the risk for CoAD-caused death.

Inhalation of combustion-derived nanoparticles that incorporate soluble reactive organic compounds, polycyclic aromatic hydrocarbons, and oxidized transition metals causes pulmonary inflammation with secondary or direct cardiovascular effects after passage from airways and pulmonary alveoli into the blood circulation.

Combustion-derived nanoparticulate matter induces cardiovascular effects via classical and alternative pathways (Table 2.6).

Table 2.6 Direct and indirect vascular effects of combustion-derived nanoparticulate matter (Source: [246])
  • The classical pathway is defined by indirect effects that result from pulmonary inflammation caused by inhaled particulate matter. Lung inflammation provokes heightened plasma concentrations of cytokines, such as IL1β and IL6 and colony-stimulating factor CSF2, in addition to the release of bone marrow-derived neutrophils and monocytes into blood circulation [246].

  • The alternative pathway is related to the passage from the lung into the blood circulation of inhaled fine PM and nanoparticles, directly across the alveolocapillary membrane or after ingestion by alveolar macrophages. Particles can cross the lung–blood barrier according to particle size, charge, and chemical composition. Once in the blood, particles can interact with the vascular endothelium and provoke endothelium dysfunction, redox stress, and inflammation.

Short-duration exposure (hours to weeks) to particulate matter PM2.5 can provoke the occurrence of an event (fatal or not) related to a cardiovascular malady [247].

In young, healthy, nonsmoking adults, episodic exposures to elevated PM2.5 levels raise the endotheliocyte (EC) apoptosis rate as well as densities of circulating monocytes and T lymphocytes (but not B lymphocytes) and concentrations of Anx5+ Itgα2b− pecam1+ endothelial microvesicles containing arterial-, venous-, and lung-specific markers (but not selectin+ microvesicles)Footnote 15 and cause an antiangiogenic profile [251]. These microparticles carry metallopeptidases, which can damage endothelium, erode eventual atherosclerotic plaques, and cleave soluble adhesion molecules. Elevated blood concentrations of endothelium-derived microvesicles shed from activated and apoptotic cells demonstrate endothelial damage, even in young, healthy nonsmokers.

On the one hand, blood concentrations of proangiogenic factors (EGF, PDGF, VEGF, soluble TNFSF5, CCL5, and CXCL1) decrease. Exposure to PM2.5 provokes loss of trophic and angiogenic factors, thereby increasing the apoptosis rate of ECs and endothelial injury [251]. On the other hand, blood concentrations of antiangiogenic factors (TNFSF1 and CXCL10) and other proinflammatory cytokines (IL1β and IL6) and chemokines (CCL2 to CCL4 and CXCL8), in addition to soluble endothelial adhesion molecules (icam1S and vcam1S) rise. Proinflammatory cytokines and chemokines (e.g., TNFSF1, IL6, and CXCL8) can be released by alveolar macrophages or bronchial epitheliocytes exposed to airborne particles. Both TNFSF1 and CXCL10 are secreted by activated T lymphocytes, monocytes, and ECs. The mild inflammatory state is characterized by augmented densities of circulating CD4+ CD8+ T lymphocytes, CD14+ monocytes, and Fcγ R3+ natural killer cells, but not CD19+ cells. Moreover, upon PM2.5 exposure, platelet–monocyte aggregates increase, establishing a procoagulation state [251]. Systemic inflammation due to PM2.5 inhalation can thus favor athero- and thrombogenesis.

The PM2.5-induced pattern of released cytokines, chemokines, and endothelial microparticles differs from that resulting from inhalation of other toxins, such as tobacco smoke. Although smoking is also associated with EC apoptosis and a decrease in plasmatic concentrations of EGF, TNFSF5, and CXCL1, it is generally characterized by elevated blood concentrations of VEGF, owing to the stimulatory effect of nicotine and hypoxia induced by CO in cigarette smoke and not by augmented plasmatic concentrations of TNFSF1 and CCL2 [251].

In long-term PM2.5 exposure, an approximately 10% increase in all-cause mortality is observed per 10-μg/m3 average elevation. Longer-term exposure (years) reduces life expectancy (months or a few years). Long-term exposure to air pollution correlates with cardiovascular mortality. Air pollution is linked to cardiovascular morbidity via redox stress, inflammation, endothelial dysfunction, platelet hyperreactivity, altered fibrinolysis, and hence thrombogenicity and arrhythmogenesis.

Exposure to ultrafine carbon particles alters cardiac frequency and its variability (i.e., perturbation of the autonomic function in addition to that caused by diabetes) in type 2 diabetes mellitus (T2DM) patients [252]. These disturbances persist many hours after the end of exposure.

Recently emitted ultrafine particles and aged fine PM are both associated with changes in cardiac function in T2DM patients and individuals with impaired glucose tolerance in urban zones [253].

Environmental risk factors include indoor and outdoor air pollution. Most of the pollutant exposure commonly occurs indoors (i.e., at home, school, working place, and other community sites), as, along with indoor pollutants, outdoor air pollutants infiltrate buildings.

In addition to outdoor air pollution, indoor smoke resulting from cooking and home heating with biomass fuels and coal is a health risk for about 3 billion people [250].

Air pollution in accommodation, particularly due to the use of solid fuels, was a leading risk factor of altered health in sub-Saharan Africa and southern Asia in 2010 [254]. Mitigation of air pollution can reduce the burden of CVDs [250].

Approximately 80% of outdoor air pollution-related premature deaths are caused by cardiac ischemia and stroke, 14% by chronic obstructive pulmonary disease or acute lower respiratory infections, and 6% by pulmonary cancer.Footnote 16

Temporal variation of daily average air pollution concentrations is more related to weather (i.e., wind direction and speed and atmospheric stability) that affects pollutant dispersion than the intensity of pollutant sources [248]. In addition, temperature and sunlight affect chemical reaction rates (e.g., ozone formation rate). Airborne Pollutants

Air components encompass airborne particulate matter (or toxic aerosol; i.e., particles from multiple sources and various sizes and compositions; Vol. 6, Chap. 4. Physiology of Ventilation) and gaseous pollutants (ozone [O3], nitrogen dioxide [NO2], carbon monoxide [CO], sulfur dioxide [SO2], and volatile organic compounds [e.g., benzene]).

Environmental toxins exist in air, water, and in the food supply. They include volatile organic compounds, polychlorinated biphenyls, phthalates, pesticides, dioxins, asbestos, heavy metals (aluminum, arsenic, cadmium, lead, and mercury), chlorine, and mycotoxins, among others. Although many phytochemicals are normally toxins at high doses, their consumption at relatively low doses activates an adaptive cellular stress response that protects cells.

Among pollutants, transition metals (e.g., copper, iron, nickel, vanadium, and zinc), organic compounds, semiquinones, and endotoxin (e.g., lipopolysaccharide) are relevant to cardiovascular pathological conditions, as these PM-related components are able to prime redox stress and inflammation. Certain features of ultrafine particles (e.g., high surface area, particle number, metal and organic carbon content) may augment cardiovascular risk after short-term exposure [247].

Semivolatile organic compounds found in both the gas and particle phases constitute with volatile organic compounds (VOCs) a large category of pollutants (e.g., benzene, (1,3)-butadiene, toluene, xylene, and polycyclic aromatic hydrocarbons) [247]. They emanate from both combustion and fugitive emissions and with secondary formation. Volatile organic compounds can be converted into less or more toxic substances. They can be oxidized in the atmosphere, becoming semivolatile organic compounds, and then contribute to the composition of PM2.5.

Concentrations of PM10 and PM2.5 are typically measured in mass per volume of air (μg/m3), whereas ultrafine particles are often measured by their number per cubic centimeter (Table 2.7) [247].

Table 2.7 Typical concentration peaks of various types of ambient air pollutants (Source: [247] ppb parts per billion, ppm parts per million)

Aerosol, a suspension of liquid or solid particles of organic and inorganic substances in air, is thus constituted of particles that have various sizes, shapes, chemical and physical properties, and chemical components. Major components include sulfate, nitrate, ammonia, sodium chloride, black carbon, mineral dust, and water.

Environmental tobacco smoke, organic compounds (polycyclic aromatic hydrocarbons, volatile organic substances, formaldehyde, naphthalene), carbon monoxide, and benzo[a]pyrene have a detrimental effect on health [248].

However, PM affects health more than any other pollutant [250]. In addition, it plays a major role in global climate change, which alters ecosystems, along with food production and the water supply.

Analysis of mixtures of pollutants emitted by given sources (e.g., traffic-related pollution, power plants, steel mills, and wood smoke) can be more pertinent to investigations of health outcomes and to the development of effective air quality strategies [247].

Primary pollutants, such as soot particles and oxides of nitrogen and sulfur, are emitted from the combustion of fossil fuels. Major sources of nitrogen dioxide are motorized vehicles, industrial sources, and residential heating.

Secondary pollutants are formed in the atmosphere from other components. In particular, ozone is formed from photochemical reactions of nitrogen oxides and volatile organic components.

Both acute and chronic exposure to high levels of air pollution disturb the endothelial function [256]. Expression of redox stress markers (e.g., NOS2 and nitrotyrosine) increases after exposure to diesel gas exhaust.

Oxidized lipids can mediate adverse vascular effects of air pollutants, especially particulate matter PM2.5. In mice, PM2.5 increases 7-ketocholesterol, an oxidated form of cholesterol and a major component of oxysterols, in plasma IDLs and LDLs and in aortic atherosclerotic plaques concomitantly with increased ScaRb3 expression in plaque macrophages [256]. 7-Ketocholesterol can be continuously formed on the surface of cholesterol-loaded lipoproteins.

In addition, air pollution favors uptake of 7-ketocholesterol through ScaRb3 by macrophages. Exposure to engine emissions increases not only the scavenger receptor ScaRb3 but also ScaRe1, thereby dysregulating the metabolism of oxidized lipids [256]. On the other hand, 7-ketocholesterol can be exported from cells to lipoproteins. For example, ABCg1 carries 7-ketocholesterol and 7β-hydroxycholesterol to HDLs, hence protecting cells. Once they are subjected to 7-ketocholesterol, cholesterol ingress increases in ECs and cholesterol egress decreases in macrophages [256].

Chronic exposure to PM2.5 increases the generation of derivatives of oxidized phospholipids. In airways, derivatives (oxPAPC) of 1-palmitoyl 2-arachidonyl sn-glycero 3-phosphorylcholine (PAPC), a dominant phospholipid of surfactant, comprise 1-palmitoyl 2-(5-oxovaleroyl) sn-glycero 3-phosphocholine and 1-palmitoyl 2-glutaroyl sn-glycero 3-phosphocholine). These derivatives have elevated concentrations in circulating lipoproteins (LDLs and HDLs). The generation of derivatives of oxidized phospholipids, such as oxPAPCs, stimulates NFκB and then expression of proinflammatory genes (e.g., the chemokine CCL2) via toll-like receptor TLR4 and NADPH oxidase (TLR4–NOx2 pathway), hence mobilizing bone marrow-derived mononuclear cells [256]. Determinants of Air Pollution Exposure

Pollutant emission or formation rate, atmospheric lifetime, weather pattern, and diurnal and seasonal cycles in solar radiation and temperature impact on pollutant concentrations.

The gaseous pollutants nitrogen oxides and carbon monoxide, in addition to the particle constituents elemental carbon and volatile organic compounds, are emitted during combustion, and their concentrations peak during rush hour [247]. Photochemical oxidants (e.g., ozone and secondary PM2.5 and VOCs) peak in the afternoon.

Meteorological conditions affect pollutant concentrations. A stationary atmosphere, that is, a period of low horizontal and vertical mixing in the lower atmosphere, gradually accumulates regionally multiple pollutant types.

The microenvironment and individual activities (e.g., trafficking duration, indoor sources, tobacco smoke and occupational exposure, and degree of indoor penetration of background pollution) cause between-subject variability of pollutant exposure. Aerosol Classification

Aerosol classifiers select a monodisperse aerosol. Aerosol particles can be classified according to electrostatic forces and drag using a differential mobility analyzer. Charged particles move in an electric field with a velocity that depends on the particle size and number of elementary charges on the particle. However, particles can have the same electrical mobility, but different numbers of charges and sizes.

Aerosol particle mass analyzer that comprises two coaxial electrodes rotating at an equal angular velocity categorizes aerosol particles according to their mass-to-charge ratio using centrifugal and electrostatic forces applied on charged particles in opposite directions. Particles with mass-to-charge ratios greater and smaller than that set by the force balance adhere to the outer and inner cylinder, respectively. In fact, the Couette centrifugal particle mass analyzer, in which the inner cylinder rotates slightly faster than the outer cylinder, has better performance [257].

In addition, equivalent aerodynamic diameter can be derived from particle relaxation time.Footnote 17 The aerodynamic aerosol classifier uses a centrifugal force and sheath flow between two concentric cylinders rotating at the same angular velocity to produce a monodisperse aerosol [258]. Different particle sizes are distinguished by changing the rotational speed. Particles with short relaxation times exit the classifier through a gap in the outer cylinder with the exhaust flow, those with long relaxation times impact and adhere to the outer cylinder.

Coarse particles result mainly from the resuspension of soil and road dust by wind or moving vehicles along with construction work and industrial emissions. Fine particles are engendered by combustion. They are composed of carbon, transition metals, and complex organic molecules, in addition to sulfate and nitrate formed in the atmosphere from volatile organic compounds, SO2, and NO2. They can travel large distances (> 100 km). Ultrafine particles are generated by motorized vehicles.

Air pollution-linked mortality is due to exposure to suspended PM of 10 μm or less in diameter (PM10), which contributes to cardiovascular (Vol 7, Chap. 2. Context of Cardiac Diseases) and respiratory diseases and cancers [250]. Small particulate pollution has an impact on health, even at very low concentrations. No health-damage threshold has been identified. However, guideline values are proposed (PM2.5: annual mean 10 μg/m3 and 24-h mean 25 μg/m3; PM10: annual mean 20 μg/m3 and 24-h mean 50 μg/m3). Air Filtration

The effects of PM can be attenuated by technological advancements and regulations. For example, enhanced urban public transport associated with proper traveling of cyclists and pedestrians can lower urban air pollution, encourage physical activity, lessen traffic injuries, and reduce the mobility cost [254]. Improved home insulation in addition to ameliorated heating and cooking systems and indoor ventilation can reduce vulnerability to diseases.

Air filtration is the most effective method for air sampling and cleaning. Nanofiber filtration has a good filtration efficiency and low resistance to air flow. However, nanoparticles with their given physical properties (surface area, shape, density, diffusivity, rigidity, electric charge, and aerodynamics) behave differently than microparticles. Yet, conventional filters are replaced by nanofibrous filters. The interfacial behavior between nanoparticles and nanofibers (caliber 10–100 nm) remains to be investigated.

Factors that influence the efficiency of a fiber filter in capturing particles include:

  • Particle characteristics (size, physical state, chemical composition, density, charge distribution, and concentration)

  • Filter characteristics (material type, fiber caliber, filter thickness, porosity, packing fiber density, filter fiber orientation, and electrical property)

  • Collection (mechanical and electrostatic) mechanisms

  • Operating conditions (temperature, filtrated air flow rate [face velocity], pressure, and viscosity)

  • Thermal rebound associated with the Brownian motion

Nanoparticles (size≤1 nm) may have a sufficiently high impact velocity to rebound from the surface upon collision (thermal rebound) [259]. Rebound of nanoparticles depends on the relative humidity of the air. However, the influence of thermal rebound on the collection efficiency may be low, as the filter is a fibrous medium consisting of entangled fibers; hence, the probability of rebound throughout the finite filter thickness is low.

Particle removal relies on four particle collection mechanisms.

  • Inertial impaction occurs when the particle near a filter fiber changes its direction of travel and collides with the filter fiber.

  • Interception appears when a particle is carried by air that flows very close to a filter fiber.

  • Diffusion due to Brownian motion causes the particle to impact the filter fiber.

  • Electrostatic attraction between particles and filter fibers is caused by Coulombic and dielectrophoretic attraction forces. In a heterogeneous electric field, a dielectric particle becomes electrically polarized due to charge separation that creates a dipole and bears a net dielectrophoretic force. The magnitude of this force, which can be exerted on neutral particles, depends on the medium and particle properties (shape, size, and electrical features) and on the frequency of the electric field. If a suspended particle has a polarizability higher than the medium, the dielectrophoretic force attracts the particle toward regions of higher electric field (positive dielectrophoretic force) and conversely (negative dielectrophoretic force).

The combined effect of these mechanisms governs the penetration of particles through the filter. Interception and inertial impaction are dominant collection processes for microparticles and diffusion for nanoparticles. At low air flow rates (face velocities) and hence high residence times, diffusion and electrostatic forces can contribute to the capture efficiency. For an order of magnitude of the particle size ranging from 10 to 100 nm and face velocity from 10 to 100 mm/s, the filtration efficiency augments at a low face velocity, whatever the particles’ shape [260].

Humidity influences the filtration performance, at least for a particle size greater than 100 nm. Using mechanical filters, elevated relative humidity raises capillary force and filtration [260]. On the other hand, using charged filters, the filtration decreases as the humidity increases, as water molecules can remove charges from filter fibers.

Two major classes of filters comprise fibrous and membrane filters. The important filter characteristics are efficiency (capacity to arrest the passage of and hold airborne particles), dust holding capacity, face velocity, and pressure drop. The quality factor (QF) evaluates the filter performance:

$$\displaystyle \begin{aligned} \mathrm{QF} = \dfrac{\ln(1/P)}{\varDelta p}, \end{aligned} $$

where P is the penetration and Δp the pressure drop across the filter. The latter increases for a given face velocity when the filter becomes dirty.

In conventional aerosol filtration theory, diffusion is supposed to be the dominant transport means of submicron particles, and impaction of these aerosol particles is followed by adhesion. The classical filtration theory also assumes that nanoparticlesFootnote 18 adhere when they strike a filter with a Brownian motion. However, nanoparticles (size< 10 nm) may have a sufficiently high impact velocity to rebound from the surface upon collision (thermal rebound) [259]. Rebound of nanoparticles depends on the relative humidity of the air. Membrane-coated filters, that is, filters coated with a polytetrafluoroethylene membrane, can be considered as two combined layers, a fibrous and membrane layer. They may control nanoparticle spreading.

Investigations quantify atmospheric pollution, measure immersed particle properties, particle size distributions, and particle filtration efficiency; and determine nanoparticle toxicology and aerosol pulmonary dose.

The relative particle velocity with respect to the air flow velocity is the migration velocity. An external force, such as gravity and electrostatic force, is required to separate particles and select a given particle size. Impactors endowed with a nozzle and an impaction plate or a receiving tube enable particle classification according to inertia. Large particles with high inertia tend to cross gas flow streamlines, whereas small particles with low inertia remain in the gas flow. Biological Mechanisms

Inhalation of PM can operate via [247] (1) the release of proinflammatory mediators and cells (e.g., cytokines along with activated immunocytes and platelets), vasculoactive molecules (e.g., angiotensin, endothelin, and histamine), or microparticles from pulmonary cells, (2) perturbation of the autonomic nervous system via stimulation of lung receptors and irritant and afferent nerves, and (3) passage of ultrafine particles or particle constituents (e.g., organic compounds and metals) into the blood.

In addition to the role of copollutants, pollutant features (size, charge, solubility, surface chemistry [e.g., the ROS-producing potential of metals, organic compounds, and semiquinones], and aggregation) are involved in their clearance (or retention rate) and interaction with pulmonary epitheliocytes and immunocytes, and within cells with organelles (e.g., mitochondrion) or molecules (e.g., cytochrome P450 and NADPH oxidase), and hence in pulmonary inflammation and redox stress [247].

Blood concentrations of stress hormones (corticotropin-releasing and adrenocorticotropic hormone, cortisol, cortisone, and catecholamines, adrenaline and noradrenaline), metabolites (amino acids, glucose, fatty acids, and other lipid types), and markers of redox stress and inflammation were investigated in response to PM exposure in 55 healthy Shanghai college students. Exposure to PM2.5 increases significantly concentrations of stress hormones and markers of insulin resistance, along with blood pressure, and provokes redox stress and inflammation [261]. Exposure to PM2.5 can thus activate the hypothalamus–pituitary–adrenal and sympathetic–adrenal axes, contributing to adverse metabolic and cardiovascular effects. The hypothalamus releases corticotropin-releasing hormone, which stimulates the anterior pituitary gland, which secretes adrenocorticotropic hormone (ACTH) into the blood, reaching the adrenal cortex, where it stimulates the synthesis and release of glucocorticoids, mainly cortisol and cortisone. The synthesis and secretion of catecholamines in the adrenal medulla is regulated by preganglionic sympathetic neurons. Glucocorticoids can also modulate catecholamine synthesis via phenylethanolamine methyltransferase. Conversely, adrenaline can stimulate ACTH secretion from the pituitary gland. Glucocorticoids provoke vasoconstriction. In addition, they increase metabolic rate and energy expenditure, glycemia, as well as lipolysis, lipid oxidation, and proteolysis, and they favor insulin resistance. Catecholamines augment glycolysis and lipolysis, cause vasoconstriction, and raise cardiac output and formation of proinflammatory cytokines. Exposure to PM2.5 may also stimulate the secretion in the pineal gland via β-adrenoceptors of melatonin, a circadian cycle regulator, which has an anti-inflammatory effect and is a ROS scavenger. In addition, greater levels of lysolipids (e.g., lysophosphatidylcholine and lysophosphatidylethanolamine) accompany higher PM exposure. Indoor air purification for 48 h not only reduces personal exposure to fine PM but also yields cardiopulmonary benefits following consecutive exposures [261]. Cardiovascular Events

Some individuals can be more susceptible to air pollution than the general population at a given pollutant concentration, especially the elderly, those who are obese, and patients with diabetes, chronic lung disease, coronary atherosclerosis, or HF, in addition to humans with low socioeconomic status. On the other hand, vulnerability refers to subjects at greater risk, as they are exposed more frequently to high pollutant concentrations.

Infraclinical changes can occur in individuals in response to PM2.5 exposure. Short-term exposure to PM2.5 (hours to weeks) can contribute to arrhythmias, myocardial ischemia, and strokes. Long-term PM2.5 exposure increases the risk for cardiovascular mortality.

Inflammation with elevated circulating markers (CRP, fibrinogen, TNFSF1, IL1β, IL6, and soluble adhesion molecules [e.g., icam1]) can be detected after PM inhalation [247]. Pollutant exposure raises plasma concentrations of CRP and fibrinogen that alter expression by leukocytes of adhesion molecules. Artery wall inflammation destabilizes atheromatous plaques.

Moreover, pollutant nanoparticles that alter the features of arterial and venous ECs and enhance platelet activation and aggregation may act as foci for thrombus formation [246]. Exposure to PM10 can be associated with elevated platelet aggregation and concentrations of fibrinogen, von Willebrand factor, and plasminogen activator inhibitor PAI1 [247]. In addition, release of tissue plasminogen activator that participates in fibrinolysis is reduced after diesel exhaust inhalation.

Furthermore, prolonged exposure to PM generates vasoconstriction, hence favoring hypertension. In particular, exposure to black carbon, a component of fine PM derived from vehicular pollution, is associated with hypertension. In older adults, short- to moderate-term ambient black carbon concentrations are correlated with blood mitochondrial abundance, a compensatory response that attenuates the cardiac effects of pollution [262]. A 2-h exposure to diesel exhaust alters the pulmonary vasomotor tone, thereby increasing pulmonary vascular resistance, under stress (but not rest) conditions (i.e., at high cardiac output) [263]. On the other hand, diesel exhaust exposure does not affect concentration of plasmatic endothelin-1 or exhaled nitric oxide (NO). Air pollution can thus potentiate acquired pulmonary hypertension.

Redox stress is linked to inflammation, as it is often induced by and elicits inflammation. It can remain confined to the cell level and hence not be directly observable by circulating markers. However, some studies show that ambient PM can increase the plasmatic concentration of homocysteine, a mediator of redox stress [247].

Lead can be absorbed via the respiratory and gastrointestinal tracts (inhalation of lead-containing dust and gas exhaust) and through the skin. Ninety-nine per cent of blood lead content is bound to RBCs. Bones serve as the main lead repository in the body. The kidney is a principal organ of lead excretion.

Chronic lead exposure causes hypertension and cardiovascular disease. It favors redox stress. It activates NFκB, promoting inflammation [264]. In addition, it downregulates expression of soluble guanylate cyclase β subunit and limits NO availability, hence reducing endothelium-dependent vasorelaxation; augments activity of adrenoceptors, protein kinase-C, and plasma angiotensin convertase, as well as endothelin production; upregulates PGhS2 expression, raising concentration of vasoconstrictory thromboxane and lowering that of vasodilatory prostacyclin; attenuates atrial natriuretic peptide production; and disturbs calcium signaling in vSMCs, thereby inducing vasoconstriction [264]. Moreover, lead induces endothelial injury, impedes endothelial repair and angiogenesis, diminishes endotheliocyte growth, but stimulates vSMC proliferation. It also reduces production of proteoglycans and tissue plasminogen activator, and raises that of plasminogen activator inhibitor (PAI1).

In two large European cohorts (144,082 adult participants), greater road traffic noise exposure is associated with a higher level of high-sensitivity CRP, TGs, and HDL. Greater air pollution exposure (PM10 and NO2) is linked to higher TG and CRP concentrations [265]. Moreover, exposure to road traffic noise and air pollution raises fasting glycemia. Heart Ischemia and Stroke

Ischemic cardiac events are related to long-term exposure to elevated PM2.5 levels [247]. The risk for an adverse post-MI outcome (death, subsequent infarction, or congestive HF) rises with greater exposure to PM10. Fatal coronary events can be statistically correlated with PM2.5 exposure.

Nonfatal and fatal cerebrovascular diseases increase with prolonged exposure to PM2.5, with a small statistically significant correlation [247]. Cardiac Arrhythmias and Heart Failure

Cardiac arrhythmias can appear in the context of exposure to fine PM and related pollutants. Death due to arrhythmias and HF is associated with prolonged exposure to PM2.5 [247]. Hospitalization for heart failure can be linked to short-term changes in PM exposure. Arterial and Venous Diseases

A nearly statistically significant positive association can be found between daily changes in PM pollution and hospitalizations for peripheral vascular diseases [247]. The risk for deep vein thrombosis augments with long-term PM10 exposure.

2.1.2 Noise

Whereas certain types of sounds and music have beneficial effects on cardiovascular physiology, tempo, melodic structure, and periods of silence being crucial factors that have an impact on blood flow parameters and baroreflex, chronic low-intensity noise disturbs sleep and activity. Disturbed sleep and repetitive exposure to noise are risk factors [266]. Noise engendered by road, railway, and air traffic augments the incidence of hypertension and atherosclerosis complications (MI and stroke). Night-time noise alters endothelial function and sleep quality and tends to increase arterial pressure [267]. Vitamin C significantly improves flow-mediated dilation, suggesting the occurrence of redox stress upon noise exposure.

Noise does indeed cause redox stress and endothelial dysfunction. Although noise increases NOS3 production, it decreases NO concentrations due to NOS3 uncoupling. It raises not only vascular ROS production but also inflammation marker levels along with circulating concentrations of stress hormones (adrenaline and cortisol). Monocytes, macrophages, natural killer cells, and neutrophils infiltrate vascular walls. Reactive oxygen species are linked to various pathways (PI3K–PKB, FoxO, TGFβ1, NFκB, and ET1). Noise increases vascular expression not only of endothelin-1 but also of NOx2 mainly via the Agt2–AT1 axis [267].

Moreover, noise activates the sympathetic nervous system. In mice exposed to intermittent noise mimicking aircraft noise delivered in short episodes (peak sound A-weighting intensity 85 dBA; mean sound intensity 72 dBA) for 4 days at the most increases systolic blood pressure, cardiac frequency, and plasmatic concentrations of IL-6, noradrenaline, and angiotensin-2 (Agt2) [267].Footnote 19

2.2 Transcripts

Precision medicine relies on identification of additional risk markers linked to pathogenic genetic variations (Chap. 7). Many gene variants affect blood pressure and lipid concentrations.

Transcription, the first stage of gene expression, consists of translating information from a DNA segment, that is, a protein-coding gene (among> 22,000 protein-coding genes), to a pre-messenger RNA molecule, which is spliced and exported into the cytosol. Translation converts information contained in a mature mRNA into a polypeptide that engenders a protein properly folded and located within the cell.

Ribonucleic acids are classified into protein-coding messenger RNAs (mRNAs) and nonprotein-coding RNAs (ncRNAs). In addition, the superclass of RNAs include many nucleotide-derived coenzymes, self-processing ribozymes, metabolite-binding riboswitches, and even ribosomes [268]. Many of the most common signaling molecules are also formed from RNA nucleotides or their precursors. The most widespread second messengers include the cyclic ribonucleotides cAMP and cGMP, in addition to pyrimidine nucleotides such as cytidine and uridine triphosphate. Other signaling effectors comprise cyclic adenosine diphosphate–ribose and nicotinic acid adenine dinucleotide phosphate, in addition to the dimeric signaling mediator diadenosine tetraphosphate (AP4A).

Messenger RNA elements, riboswitches, bind to small molecules to regulate gene expression. Among numerous riboswitch classes, many regulate gene transcription in response to the selective binding of coenzymes and signaling mediators derived from RNA monomers or their precursors.

Nonprotein-coding RNAs (ncRNAs) comprise functional RNAs that are not translated into proteins. They include spliceosomal (small nuclear) RNAs, transfer (tRNAs), ribosomal (rRNAs), and other types of small (sncRNAs; < 200 nucleotides [nts]) and long ncRNAs (lncRNAs; > 200 nts).

Aminoacyl–tRNA synthetases (aaRSs) act during the first stage of ribosome-dependent protein synthesis. These enzymes attach the appropriate amino acid (aminoacylation) onto cognate tRNA isoacceptors (AA–tRNAAA; AA: a given type of amino acid). They are categorized into two groups according to their chemical properties, catalytic domain architecture, and consensus sequencesFootnote 20 in addition to subgroups based on their mechanistic properties, anticodon-binding domain characteristics, and structural motif organization [269].

Mammalian aaRSs differ from those of prokaryotes and lower eukaryotes, as they associate as multienzyme complexes. Nine aaRSs (ArgRS, GlnRS, IleRS, LeuRS, LysRS, and MetRS, along with aspartyl– [AspRS or DaRS] and glutamyl–prolyl–tRNA synthetases [Glu–ProRS or EPRS]) and three auxiliary aminoacyl–tRNA synthetase-interacting multifunctional proteins (AIMP1–AIMP3) assemble into a multi-aaRS complex that operates in alternating activities of its members [269]. Under some circumstances, one of the aaRSs or AIMPs is released to trigger cell signaling (Table 2.8).Footnote 21

Table 2.8 Activities of free aminoacyl–tRNA synthetases (aaRSs) and aaRS-interacting multifunctional proteins (AIMPs) released from the human aaRS–AIMP complex (Source: [269])

In addition to their canonical role in translation, aaRSs have auxiliary functions in nutritional stress response, apoptosis, and cell survival along with the regulation of gene transcription and transcript translation via linkage to transcription factors or tRNA-like molecule, RNA splicing,Footnote 22 inflammation via recruitment of immunocytes, and angiogenesis, as some aaRSs and/or their secreted cleavage products contribute to controlling EC survival, proliferation, and migration via activation of ERK1, ERK2, P38MAPK, and PKB (Table 2.9) [271].

Table 2.9 Aminoacyl–tRNA synthetases (aaRSs) and aaRS-associated anti- and pro-angiogenic and angiostatic proteins (Source: [271])

Loss-of-function mutations in TARS and IARS genes that encode threonyl– and isoleucyl–tRNA synthetases (premature stop codon and point mutation, respectively) upregulate synthesis of proteins implicated in the unfolded protein response (UPR; e.g., ATF4, ATF6, and XBP1)Footnote 23 in addition to vascular endothelial growth factor (VEGFa) via UPR-stimulated activating transcription factor ATF4 and increase branching angiogenesis [273]. The ATF4–VEGF pathway does indeed prime angiogenesis,Footnote 24 whereas secreted TaRS signaling supports angiogenesis independently of its synthetase activity. In fact, many aaRSs can promote angiogenesis upon nutrient depletion, which launches UPR and VEGF production, or via extracellular mechanisms [272].

In murine embryonic ECs, formation of JmjD8, which belongs to the JmjC categoryFootnote 25 and promotes TNFSF1-primed NFκB signaling, is upregulated during their differentiation. It regulates both endothelial sprouting and metabolism, as it interacts with pyruvate kinase-M2 [274].Footnote 26

Small nonprotein-coding RNAs include microRNAs (miRs), P-element-induced wimpy testis-interacting-associated RNAs (piRNAs), and endogenous small interfering RNAs (endo-siRNAs).

Long nonprotein-coding RNAs operate as signals,Footnote 27 decoys,Footnote 28 guides,Footnote 29 and scaffolds.Footnote 30 They control the chromatin structure and hence can repress gene transcription. They also participate in post-transcriptional regulation, in particular in cell metabolism and differentiation [275].

The nucleoplasm and cytoplasm contain numerous small ribonucleoprotein complexes, the small nuclear (snRNPs) and cytoplasmic RNPs (scRNPs), respectively. They are composed of at least one protein and a small stable RNA (< ∼300 nt). SnRNPs and scRNPs regulate nuclear and cytosolic RNA processing (i.e., pre-mRNA splicing and mRNA translation). Hence, other subsets of ncRNAs include (1) Components of the Ro (Ro60) ribonucleoprotein, YRNAs,Footnote 31 (2) small cytoplasmic RNAs (scRNAs or7SLRNAs), and (3) constituents of large cytoplasmic ribonucleoproteins, the vaults.

YRNAs are small ncRNAs (∼100 nt) transcribed by RNA polymerase-3 that form stem–loop structures. Ro small cytoplasmic ribonucleoproteins are a subgroup of La ribonucleoproteins.Footnote 32 The Ro RNPs are stable and cytoplasmic, whereas the majority of the La RNPs turn over rapidly and reside in the nucleus [277]. These components of Ro RNPs are bound to 60-kDa Ro protein, which can be detected in the blood of patients with autoimmune diseases, such as systemic lupus erythematosus (or simply lupus)Footnote 33 and Sjögren’s syndrome.Footnote 34 Binding to Ro60 is a prerequisite for the export of YRNAs from the nucleus to the cytoplasm [276].

Ubiquitous small cytoplasmic RNA component 7SL of the signal recognition particle (SRP) serves as a scaffold for the six SRP proteins (SRP9, SRP14, SRP19, SRP54, SRP68, and SRP72) [278]. It links to the ribosome and assists translocation of nascent presecretory proteins to the endoplasmic reticulumFootnote 35 and then to secretory pathway, sorting them for membrane insertion or secretion from the cell.

Vaults participate in nucleocytoplasmic transfer. They include the major Footnote 36 and two minor vault proteins, PARP4Footnote 37 and TEP1,Footnote 38 in addition to various small untranslated RNAs (i.e., vault RNA components vaultrc1–vaultrc3 [vtRNA1-1–vtRNA1-3] and vtRNA2-1 [or pre-miR886]).Footnote 39

However, within the transcriptome, bifunctional RNAs have both coding and noncoding functions and can serve as both messenger and nonprotein-coding RNAs [279], although bifunctional RNAs comprise RNAs that have two different ncRNA functions.Footnote 40 They include [282]:

  1. 1.

    mRNAs (e.g., TNFRSF17as, Dmpk, H2B, Mdfic,Footnote 41 fortilin,Footnote 42 Sra) either with an intrinsic noncoding function (base pairing, scaffolding, and quenching) or with a nonprotein-coding isoform due to alternative splicing or allele-specific transcripts with variants that destroy coding potential

  2. 2.

    lncRNAs (e.g., ac074183.4, cgat1, ctd2651B20.6, dancr, gas5, malat1, snhg8, rp5-875o13.1, rp11-21n3.1, rp11-220i1.1, rp11-354p17.9, and rpph1) containing a small open reading frame that are strongly associated with ribosomes and thus encoding small peptides (< 100 amino acids)

Nonprotein-coding RNAs can be identified within exosomes, which are intercellular communication shuttles formed from multivesicular bodies and then secreted. They contain miRs, rRNAs, tRNAs, lncRNAs, piRNAs, and small nuclear (snRNAs) and nucleolar RNAs (snoRNAs). Among them, miRs and lncRNAs, which regulate cell signaling, have an altered expression in pathogenesis and hence are additional markers.

2.2.1 MicroRNAs

Aberrantly regulated nonprotein-coding RNAs, such as short microRNAs and long nonprotein-coding RNAs that are released into the bloodstream, where their concentrations differ thus from those in healthy subjects, contribute to cardiovascular pathophysiology.

MicroRNAs are linked to CVD risk factors, such as hyperlipidemia, hypertension, obesity, diabetes, lack of physical activity, and smoking. They participate in CVD, especially atherosclerosis and aneurysms.

MicroRNAs (content 18–22 nucleotides; Vol. 11, Chap. 3. Small Nonprotein-Coding RNAs—MicroRNAs) are transcribed from genomic mir loci by RNA polymerase-2 assisted by epigenetic regulators, enhancers, and transcription factors into transcripts, the primary microRNAs, which are capped, polyadenylated, and spliced. These mir transcripts contain either a single miR or a miR cluster. The primary mir transcript is processed by the nuclear microprocessor complex that contains the ribonuclease-3 drosha and the double-stranded RNA-binding protein DGCR8 (or pasha).Footnote 43 The precursor microRNA is then exported into the cytoplasm using exportin-5 in conjunction with Ran. The cytosolic ribonuclease-3 dicer generates the mature miR–miR duplex. Then, dicer, in cooperation with the RNA-induced silencing complex (RISC)-loading complex subunit transactivation-responsive RNA-binding protein TARBP2, recruits argonaute-2 to the bound miR. Argonaute proteins, which unwind the double strand and incorporate one of the two miR strands into the RISC, lead to mRNA translation repression or degradation.Footnote 44 Therefore, argonaute proteins are key elements of gene silencing and microRNAs regulate amounts of proteins produced from target mRNAs.

For example, miR504, which resides in an intron of the Fgf13 gene, targets p53 mRNA [285]. The FGF subtype, FGF13, does not function like a regular growth factor. In the nucleolus, it represses transcription of ribosomal RNA and hence protein synthesis. On the other hand, P53 prevents transcription of the Fgf13 locus and thus synthesis of both FGF13 and miR504 (negative feedback loop).

MiR33 targets autophagy regulators and effectors in macrophages, diminishing lipid droplet catabolism and free cholesterol generation and subsequent efflux [286]. Concentration of oxysterol-binding protein-like protein OSBPL6 and hence cholesterol transfer is also regulated by microRNAs. MiR548p reduces ApoB secretion and lipid synthesis via HMGCoAR and long-chain fatty acylCoA synthase ACSL4 [286].

Some microRNAs, such as miR1, miR126, miR138, miR143, and miR145, participate in cardiogenesis via angiogenesis, establishment of cardiac cell polarity, development of the cardiac conduction system, cardiac patterning, or smooth myocyte differentiation. Often, these miRs regulate the core cardiac transcriptional network [287].

Two miRs are highly expressed in vascular smooth myocytes and ECs, miR221 and miR222, which preclude EC migration and proliferation and hence angiogenesis, but favor vSMC proliferation and migration and counter apoptosis [288]. MiR221 and miR222 reduce NOS3 expression and can cause endothelial dysfunction.

In patients, circulating miR15b-5p allows the differentiation of individuals with well and poorly developed collateral vessels, as miR15b-5p produced in vascular ECs regulates angiogenesis [286]. Proangiogenic miR126a is implicated in lymphangiogenesis via VEGFR3 signaling. Suppression of angiopoietin-2 formation by miR150 enables repair of vascular injury.

  • MiR182-3p modulates a vascular smooth myocyte phenotype.

  • MiR22 plays a role in smooth myocyte differentiation.

  • MiR155 supports the migration of smooth myocyte progenitors via CCL2.

  • MiR590-3p increases vSMC contractility and represents a risk factor for hypertension in the presence of genetic defects [286].

MiR125b is associated with vascular calcification [286]. MiR146a targets the transcript encoding polo-like kinase PLK2 to induce bone marrow cell apoptosis and senescence [286].

Redox stress due to excessive ROS production and insufficient scavenging is linked to aging, hypertension, hypercholesterolemia, diabetic vasculopathy, atherosclerosis, and ischemia. Reactive oxygen species increase expression of MIR200 family members (miR141, miR200a–miR200c, and miR429) [289].Footnote 45 Endothelial Mechanical Stress-Responsive MicroRNAs

The vascular endothelium forms a barrier that regulates its permeability. Moreover, this mechanosensor controls leukocyte adhesion, blood coagulation, the vasomotor tone, and vascular cell proliferation, especially that of smooth myocytes, in addition to angiogenesis.

MicroRNAs and histone deacetylases (HDACs) control gene expression and hence endothelial function. MiR10a pertains to the endothelial mechanical stress-responsive microRNAs.Footnote 46 It targets Gata6, which elicits vcam1 synthesis (an anti-inflammatory effect). Mechano-miR10a expression is regulated by KLF2 via RARα–RARE binding, which heterodimerizes with RXRα to bind RARE in the enhancer region of target genes [294].Footnote 47 According to the local flow pattern, HDAC3, HDAC5, and HDAC7, which link to myocyte enhancer factor MEF2, thereby hampering production of anti-inflammatory KLF2, or NR2b1, operate as mechanosensitive repressors and enhancers, respectively.

Vascular ECs in strongly curved segments with low wall shear stress (WSS) induce a quiescent-to-activated phenotypic transition of adjacent smooth myocytes and hyperplasia. Endothelial microRNAs, such as miR126-3p and miR200a-3p, are released in low WSS regions via activation of the SNAREs, vesicle-associated membrane protein VAMP3Footnote 48 and synaptosomal-associated protein SnAP23 [295]. These miRs then favor hyperplasia of recipient vSMCs. Regulators of Vascular Smooth Myocyte Phenotype

Vascular smooth myocytes (vSMCs) are not irreversibly differentiated, because they can modify their phenotype according to environmental conditions, whereas fully differentiated smooth myocytes (SMCs) are refractive to PDGFbb stimulation.

In large and medium-sized blood vessels, they constitute the main cellular population of the media. They are organized in a variable number of monolayers of circumferentially oriented vSMCs.

The arterial media contains quiescent and contractile vSMCS surrounded by their own basement membrane, immersed in an extracellular matrix, and a small quantity of macrophages and fibroblasts.

Heparan sulfate proteoglycans (HSPGs) are major components of the vSMC glycocalyx. The glycocalyx contains heparan and chondroitin sulfate proteoglycans and hyaluronic acid in addition to glycoproteins with terminal sialic acids.

The basement membrane surrounding vSMCs contains predominantly collagen-4, laminin, and HSPGs, such as perlecan and syndecan. HSPGs are low-affinity receptors that sequester growth factors such as FGF2, which can modify vSMC phenotype, hence priming proliferation.

Proteoglycans are proteins bearing glycosaminoglycans (GAGs). Glycosaminoglycans are long linear (unbranched) polysaccharide chains, which are also termed mucopolysaccharides. They encompass chondroitin (ChS), dermatan (DeS), heparan (HeS), and keratan sulfate (KeS), as well as heparin and hyaluronan, DeS being stereoisomeric variants of ChS. ChS and DeS interact with various types of extracellular molecules, such as growth factors, cytokines, chemokines, and morphogens, and are therefore involved in cell adhesion, proliferation, and migration.

The medial interstitial matrix contains collagen-1 and -3, various types of glycoproteins (e.g., fibronectin, tenascin, thrombospondin, and vitronectin), and chondroitin sulfate proteoglycans such as versican [296].

Under normal conditions, vascular SMC differentiation is controlled by messengers such as TGFβ; plasma membrane components, such as adhesion molecules; matrix constituents, such as collagen and elastin; mechanical forces; neuronal signals; and intercellular interactions.

On the other hand, dedifferentiated vSMCs synthesize matrix metallopeptidases (MMPs), tissue inhibitors of metallopeptidases (TIMPs), and matrix components.

Lysosomal heparin lyases (or heparinase)Footnote 49 released by macrophages remove HS chains and favor vSMC proliferation.

Heparanase is an endoglycosidase that cleaves HS from the cell surfaces and matrix constituents, hence facilitating the release of exosomes and subsequent intercellular communication in addition to remodeling the extracellular matrix.

The dedifferentiated vSMC can activate programmed cell death pathways and elaborate proinflammatory cytokines [298]. Migration, proliferation, and death of vSMCs are events regulated by HSPGs and hence cell surface (extracellular) HS sulfataseheparan sulfate sulfatase-1 [299]Footnote 50

Furthermore, activated vSMCs can be involved in efferocytosis and phagocytosis, ossification, reverse cholesterol transport, and foam cell formation.

Phenotype switch and abnormal vSMC proliferation and migration constitute a major process in the genesis of arterial wall diseases, especially neointima formation, a hallmark of atherosclerosis and post-stenting restenosis.

Once they are exposed to stressors, vSMCs change their phenotype, that is, they dedifferentiate from their quiescent contractile phenotype to a state characterized by abnormal proliferation, migration, matrix synthesis, and inflammation. This dedifferentiation contributes to maladaptive remodeling of the vascular wall (e.g., atherogenesis, post-angioplasty intimal hyperplasia, aneurysmal degeneration, and medial calcinosis).

Cell motility relies on cycles of cell protrusion and retraction and of formation and disruption of cell–matrix adhesion sites, on polarized vesicular transfer and exocytosis, and, at the nanoscale, extracellular signal-regulated kinase (ERK). The MAPK module component ERK and its effector kinase, p90 ribosomal S6 kinase (RSK), regulate cell migration mainly via phosphorylation of various substrates [300].

The Ras–Raf–MAP2K–ERK–RSK pathway is activated by liganded receptor protein Tyr kinases (i.e., growth factors) and stimulated integrins. Activated ERK translocates into the nucleus, where it phosphorylates some transcription factors, thereby regulating gene expression. It also phosphorylates (activates) various types of kinases (RSKs, MSKs, and MNKs). Cytosolic MKnKs regulate mRNA translation, as they phosphorylate eukaryotic translation initiation factor eIF4e. Nuclear MSKs also regulate gene transcription, as they phosphorylate transcription factors, high-mobility group nucleosome-binding domain-containing protein HMGN1, and histone H3 [300]. Nuclear and cytosolic RSKs phosphorylate various transcription factors in the nucleus and the 40S ribosomal subunit protein S6 and eIF4b in the cytoplasm.

Therefore, ERK regulates cell motility via genomic and nongenomic effects. It engenders motile mesenchymal cells from polarized epitheliocytes. Epithelial–mesenchymal transition is triggered by various transcription factors (e.g., Snai1 and Snai2, Twist1, and ZEB1 and ZEB2) [300]. Both ERK and RSK phosphorylate regulators of cell protrusion and retraction, cell–matrix adhesion, and exocytosis (Table 2.10).

Table 2.10 Components of the cell motility machinery phosphorylated by ERK or RSK (Source: [300]; AbI Abelson kinase interactor, CKI cyclin-dependent kinase inhibitor, EPH erythropoietin-producing hepatocyte receptor protein Tyr kinase, Exo exocyst subunit, FAK focal adhesion kinase, MLCK myosin light chain kinase, SH3P SH3 domain-containing protein, SorbS3 sorbin and SH3 domain-containing protein-3 [vinexin], vasp vasodilator-stimulated phosphoprotein, WAVe WASP family verprolin homolog)

Cell movement is induced by activation of the small GTPase Ras, which in turn stimulates both PI3K and ERK axes. The PI3K pathway stimulates actin polymerization and the formation of a protrusion (a lamellipodium) at the leading edge of the moving cell via activation of the small GTPase Rac1 and protein kinase PKB.

The ERK kinase phosphorylates MLC kinase, cortactin, WASP family protein member WASF2 (wave2), and FAK, among other substrates, thereby promoting cell migration.Footnote 51

In addition, ERK phosphorylates (activates) MLCK, which phosphorylates myosin light chain and activates myosin-2 [300]. Its effector RSK phosphorylates (inactivates) SH3P2,Footnote 52 an inhibitor of the nanomotor myosin-1E,Footnote 53 disrupting SH3P2–Myo1e tethering and enabling Myo1e to move to the lamellipodium tip, where it facilitates cell motion [301].

Among the four isozymes (RSK1–RSK4), RSK1 impedes actin elongation, as it phosphorylates (inactivates) VASP [300]. On the other hand, VASP is also phosphorylated by PKA and then re-localizes to lamellipodia.

Ribosomal s6 kinase (RSK) phosphorylates filamin-A, a dimeric actin crosslinker, at the same site phosphorylated by PAK, and, among plastins (Pls1–Pls3), Pls2 that then form actin bundles.

Focal cell–matrix adhesions are mediated by heterodimeric transmembrane integrins, which interact with matrix components (e.g., collagen, fibronectin, and vitronectin) and cytosolic constituents, transducing bidirectional signals between the extra- and intracellular media (outside–in and inside–out signaling).

Focal adhesion kinase (FAK) and SRC family kinases (SFKs) participate in proteic complex formation at the cytoplasmic tail of integrins. Autophosphorylated FAK recruits various SFKs, which further phosphorylate FAK and process FAK-associated proteins such as the docker paxillin, thereby accelerating formation of signaling complexes at focal adhesion sites. Extracellular-signal-regulated kinase (ERK) phosphorylates both FAK and paxillin in focal adhesion sites, increasing FAK–Pax binding and eliciting cell adhesions and protrusions [300].

Sorbin and SH3 domain-containing protein Sorbs3 (or vinexin) are other regulators of cell spreading and growth factor signaling at focal adhesion sites [300]. Among several alternative forms (Sorbs3α–Sorbs3γ), Sorbs3β is phosphorylated by ERK at the leading edge of moving cells, decreasing cell spreading and motility.

Calpain-2 cleaves proteins involved in cell–matrix adhesion and cell protrusion, such as FAK, SFKs, cortactin, integrins, and paxillin [300]. ERK phosphorylates (activates) calpain-2. Calpain-2 can complex with ERK and FAK at focal adhesion sites. Calpain-2 also cleaves linkers of focal adhesions to the cytoskeleton, such as spectrin and talin, thereby eliciting the turnover of adhesion complexes.

In the retracting tail of migrating cells, RhoA, RhoB, and RhoC are implicated in the detachment of cell–matrix adhesions and myosin-2-mediated cell retraction. These small Rho GTPases activate rock that phosphorylates (activates) LIMK, which phosphorylates (inhibits) cofilin, precluding further actin polymerization. Rock also phosphorylates (inhibits) myosin phosphatase, supporting myosin-based contractile force.

Extracellular-signal-regulated kinase (ERK) phosphorylates (activates) RhoGEF2 in focal adhesions in response to stretch. On the other hand, ERK phosphorylates (activates) RhoGAP7 in focal adhesions, thereby enabling cycles of Rho deactivation–reactivation. Therefore, ERK functions as a spatiotemporal regulator of cell retraction. In addition, ERK phosphorylates RhoA, priming its ubiquitination and subsequent proteasomal degradation [300]. Furthermore, RSK phosphorylates cyclin-dependent kinase inhibitor CKI1b, which is then sequestered in the cytoplasm, where it binds to Rho, hampering its activity.

Polarized endo- and exocytosis during cell migration is defined by internalization of membrane proteins, such as integrins and RTKs, at the cell trailing edge to be recycled to the leading edge and displacement of secretory vesicles carrying newly synthesized proteins from the Golgi body to the leading edge. These processes are controlled by the small GTPases of the ARF, Rab, Ral, and Rho superfamilies. The octameric proteic complex exocyst tethers secretory vesicles to the plasma membrane. ERK phosphorylates the exocyst component Exo70, promoting its binding to other exocyst components and secretion of matrix metallopeptidases [300]. Transcription Factors

Phenotypic switching of vSMCs involves both activators of SMC dedifferentiation and repressors of SMC differentiation, such as ETS-like factor ELk1, once it is phosphorylated by ERK, E26 transformation-specific sequence ETS1, forkhead box-containing FoxO4, four-and-a-half LIM-only FHL2 hairy and enhancer of split-related HRT2, and Krüppel-like transcription factor KLF4, specificity protein SP1, and yin yang transcription factor YY1, which act in a coordinated manner [302].

Serum response factor and its cofactor promote differentiation of vSMCs. Myocardin (Myocd) is the most powerful serum response factor (SRF) cofactor from embryogenesis to adulthood, as myocardin stimulates SRF-initiated gene transcription. The SRF–Myocd couple activates smooth and cardiac muscle gene promoters. Both SRF and Myocd undergo post-translational modification (e.g., phosphorylation), which affects SRF–Myocd activity in SMC differentiation.

The SRF homodimer connects to the CC(A/T)6GG (CArG) DNA consensus sequence, which is found in promoters and introns of SMC-specific genes. Transforming growth factor-β provokes SRF expression and stimulates SMC differentiation.

Myocardin selectively induces transcription of most of the CArG-containing genes, especially SMC marker genes, such as those encoding smooth muscle α-actin (actα2) and myosin heavy chain (MyH11), transgelin, calponin,Footnote 54 and telokin, but not the CArG-containing early response gene FOS [302]. Myocardin mediates inducible expression of CArG-containing SMC marker genes in response to Agt2, TGFβ, PDGFbb, and the CaV1–RhoA axis.

On the other hand, the osteogenic Runt-related transcription factor Runx2 is a SRF corepressor, as it tethers to SRF and hinders SRF–Myocd complex formation.

Olfactomedin-2 (Olfm2)Footnote 55 regulates TGFβ-induced SMC differentiation from mesenchymal stem cells, Runx2 being undetectable [307]. Olfm2 promotes the dissociation of SRF from HRT2, a transcriptional repressor for SMC genes.

On the other hand, in a different cellular context, Olfm2 also participates in mature vSMC phenotype switching, Runx2 being substantially present in mature SMCs. It promotes their proliferation and migration, hence favoring vascular wall remodeling [307]. Although TGFβ triggers formation of Olfm2 and SMC markers in mesenchymal progenitor cells, it does not significantly induce Olfm2 expression in mature rat aortic SMCs, whereas PDGFbb primes synthesis of Runx2 and Olfm2 in SMCs [307]. Olfm2 facilitates interaction of SRF with Runx2, thereby hampering SRF–Myocd interaction.

The gene promoters contain not only a distal CArG element but also a G/C-rich repressor element located proximal to the CArG element. The G/C element binds to specificity proteins SP1 and SP3, members of the KLF family, and early growth response transcription factor EGR1, which are normally absent in differentiated SMCs, but rapidly induced upon vascular injury [302]. For example, PDGFbb counters SRF attachment to the CArG–G/C–CArG motif within the MYH11 and TAGLN promoters of genes encoding smooth muscle myosin heavy chain and transgelin, respectively.

FHL2 interacts with SRF and binds to the promoter of SMC-specific CArG-containing genes in response to RhoA [302]. It precludes SRF-mediated transcription of SMC-specific genes, as it competes with MRTFa for SRF binding.

HRT2 prevents myocardin-dependent transactivation of the MYH11 and TAGLN gene promoters [302]. In addition, it tethers to SRF and hampers SRF binding to CArG box motifs.

Yin yang-YY1 represses SRF binding and Fos activity, hence skeletal and cardiac muscle α-actin formation. However, its overexpression can transactivate the TAGLN promoter in cultured rat aortic SMCs [302]. Krüppel-Like Factors

Krüppel-like factor KLF4 was identified as a binding factor to the G/C-rich TGFB promoter and also connects to the MYH11 and TAGLN promoters. It suppresses expression of myocardin and activation by myocardin of SMC marker genes; competes for binding of SRF to CArG regions, hence reducing SRF binding to CArG-containing SMC marker genes; and provokes hypoacetylation of histone H4 at CArG regions (transcriptional silencing) [302].

KLF4 along with platelet-derived growth factor (PDGFbb), notch, and TGF-β affects the vSMC differentiation status [298]. Moreover, contractile and synthetic vSMC phenotypes are controlled by numerous microRNAs. MicroRNAs are indeed major regulators in vSMC proliferation and migration.

Among 20 members of the KLF family, anti-inflammatory KLF2, KLF4, proproliferative KLF5, and KLF6 play important roles in the vasculature. KLF1 is essential for erythrocyte maturation.

In the vascular wall, KLF2 is exclusively synthesized in ECs, its synthesis being inhibited by inflammatory IL-1β. It induces NOS3 expression; but impedes cytokine-primed formation of adhesion molecules (e.g., E-selectin and vcam1) [308].

KLF4 operates in endothelial inflammation, macrophage gene expression, and vSMC phenotype [309]. In ECs, KLF4, the production of which is induced by proinflammatory agents, induces expression of multiple anti-inflammatory and anti-thrombotic factors (e.g., NOS3 and thrombomodulin) [310].Footnote 56

KLF4 prevents Jun expression and hence intimal hyperplasia. It can also block the PDGFbb–PDGFR–ERK axis and vSMC proliferation primed by PDGFbb. Furthermore, it induces the expression of P53 and subsequently that of CKI1a and represses that of ID3 and retinoic acid receptor RARα (NR1b1), which suppresses CKI1a synthesis. It also promotes the production of several cell cycle inhibitors (CKI1b, DM2, and RB) and represses that of cell cycle stimulators (cyclin-D1 and ornithine decarboxylase) [309].

On the other hand, KLF4 can be a potent transcriptional repressor of SMC differentiation markers (ACTA2, CNN1, TAGLN, and MYH11 gene encoding smooth muscle α-actin, calponin-1, smooth muscle myosin heavy chain, and transgelin, respectively), functioning as an effector of PDGFbb, which induces phosphorylation of ELk1 via the MAP2K1/2–ERK1/2 axis, re-localizing ELk1 to the nucleus, where it competes with myocardin, thereby blocking SRF–myocardin interaction [309]. In the context of gene repression, KLF4 recruits HDAC2, which deacetylates histone H4, hence causing chromatin compaction. It also downregulates the formation of paired-related homeobox-derived protein PrRx1 and MRTFs.

However, acetylated KLF4 can also promote synthesis of smooth muscle actin-α2, myosin heavy chain-11, and transgelin [309]. In the context of gene activation, KLF4 recruits KAT3b to gene promoters. KAT3b acetylates KLF4 in addition to histones. Moreover, KLF4 phosphorylation enhances its transactivation capacity, hence facilitating vSMC differentiation.

Therefore, KLF4 activity depends on post-translational modifications (acetylation, phosphorylation, and sumoylation) induced by various messengers and effectors (PDGFR, Tβ R, and RAR).

In addition, KLF4 supports the synergistic action of ECs and mural macrophages, which originate from circulating and then resident monocytes. It transactivates liver X receptor (NR1h2/3) and cholesterol 25-hydroxylase, which forms 25-hydroxycholesterol, thereby reducing inflammasome activity in ECs, promoting M1-to-M2 phenotypic transition in macrophages, and protecting against atherosclerosis susceptibility [311].

Cyclosporine-A (CSa), a PP3 inhibitor, suppresses vSMC proliferation. In vSMC, it upregulates the expression of P53 and KLF4 [312]. KLF4 hinders vSMC proliferation, as it elicits CDKN1A gene transcription. It facilitates migration, as it also promotes Mmp3 gene transcription. In addition, it supports the formation of MMP9 and collagen-8. It represses transcription of MYOCD and vSMC differentiation marker genes (ACTA2, SMTN, and TAGLN). In addition, CSa acutely and transiently represses MyH11 production.

KLF5 activates the PDGFA genes, thereby favoring in-stent neointima formation in rabbits [302]. In response to vascular injury, KLF5 is induced in proliferating vSMCs; it initiates the production of EGR1, PDGFa, PDGFb, PAI1 (serpin-E1), NOS2, and VEGFRs [309]. KLF5 can reverse KLF4-induced suppression of SMC marker gene expression. It can also induce the expression of NMHCB and PDGFA genes, which encode nonmuscle myosin heavy chain NMHCb (myosin-10) and PDGFa genes in vSMCs.

Upon vascular injury, in ECs, KLF6 triggers synthesis of TGFβ and its receptors and urokinase plasminogen activator [309]. Growth Factors and Autacoids

Under normal conditions, vSMCs, endowed with their contractile apparatus, are maintained in a quiescent and contractile state. On the other hand, vSMCs can switch their phenotype from a quiescent to a synthetic state associated with formation of a new extracellular matrix and the capacity of proliferation and migration from the media to the intima.

The pathogenic phenotype can be countered by HSPGs, NO, prostaglandins, and TGFβ.

The vSMC phenotype change is triggered by various growth factors, cytokines, and other stimulators released upon vascular injury, such as epidermal (EGF), fibroblast (particularly FGF1 and FGF2), and IGF1, PDGFbb, vascular endothelial growth factor (VEGF), angiotensin-2, endothelin-1, thrombin, tumor-necrosis factor-α (TNFSF1), and IL-1 [313]. Among these factors, PDGFs and FGFs are the most potent for initiating the phenotypic switch and for controlling vSMC proliferation and migration [316]. Platelet-Derived Growth Factors

Three dimeric PDGF isoforms (PDGFaa, PDGFab, and PDGFbb) tether to two reeceptor protomers (PDGFRα–PDGFRβ). PDGFs stimulate proliferation and migration of SMCs in addition to fibroblasts and gliocytes via PDGFRβ. Among PDGFs, PDGFab and PDGFbb support vSMC migration, whereas PDGFaa inhibits migration induced by PDGFab and PDGFbb via heparin-binding fibronectin [314]. Cell adhesion to fibronectin (but not to collagen-4) suppresses vSMC migration. Vascular SMCs and platelets produce PDGFbb in response to acute vascular injury.

However, administration of an antibody that neutralizes activity of PDGFaa, PDGFbb, and PDGFab reduces proliferation and migration of medial SMCs in a rat carotid artery balloon injury model [302]. Whereas inhibition of PDGFRβ prevents SMC accumulation after vascular injury, that of PDGFRα does not impede SMC accumulation. Vascular SMC fate is thus mainly affected by PDGFbb, which signals via PDGFRβ, rather than PDGFRα.

Platelet-derived growth factor (PDGFbb) functions not only as a growth stimulator but also as a repressor of SMC protein production at both the transcriptional and post-transcriptional levels linked to degradation of SMC marker transcripts and proteins. In addition, PDGFbb cooperates with IL1β to repress vSMC differentiation via the PI3K–PKB–S6K pathway.

The principal plasma membrane-associated HSPGs include transmembrane syndecans and membrane-anchored glypican using a glycosylphosphatidylinositol chain. PDGFaa increases syndecan-1 production and causes HSPG accumulation on vSMCs more efficiently than does PDGFbb, whereas both PDGF isoforms decrease formation of perlecan, another HSPG. PDGFaa thus influences the interaction of SMCs with heparin-binding matrix proteins, thereby suppressing vSMC migration. Fibroblast Growth Factors

The FGF family comprises 22 members that act via their receptors (FGFR1–FGFR4), triggering assembly of a ternary complex with the adaptors FRS2 and GRB2 and the GEF, SOS, which activates the Ras–MAPK signaling cascade.

Among FGFs, FGF12 expression is elevated in contractile vSMCs but downregulated in synthetic vSMCs. It induces and maintains the quiescent vSMC phenotype [315]. Moreover, it prevents cell proliferation via P53. It upregulates synthesis of myocardin and serum response factor via P38MAPK. Synthesis of FGF12 is inhibited by PDGFbb [315]. Transforming Growth Factor

TGFβ mediates vSMC phenotype switch from the synthetic to the contractile state. It connects to and activates Tβ R2, which then associates with and activates Tβ R1, which phosphorylates SMAD2 and SMAD3, eventually priming production of contractile proteins in pericytes and smooth myocytes [316].

On the other hand, TGFβ which signals via SMAD3 stimulates rather than inhibits vSMC proliferation via phosphorylation and nuclear export of CKI1b [317]. The TGFβ–SMAD3 axis also launches the synthesis of fibronectin and collagen-1 in vSMCs.

The inhibitor of both vSMC proliferation and migration and the stimulator of vSMC proliferation according to the circumstances, TGFβ, signal via various pathways. It does indeed operate via ERK, JNK, and P38MAPK along with PI3K and most commonly SMADs (receptor-activated SMAD2 and SMAD3, common SMAD4, and inhibitory SMAD6 and SMAD7) [317]. Activated TGFβ receptor phosphorylates (activates) SMAD3, which then complexes with SMAD4. The SMAD3–SMAD4 complex translocates to the nucleus, where it connects to SMAD-binding elements and regulates transcription of target genes. Inhibitory SMADs antagonize TGFβ signaling, as they either prevent activation of receptor-activated SMADs or prime Tβ R degradation.

The repressor of TGFβ signaling, SKi, precludes vSMC proliferation stimulated by TGFβ in addition to vSMC autophagy induced by oxLDLs and PDGF via hampering of PKB phosphorylation, thereby downregulating production of proliferating cell nuclear antigen (PCNA) [318]. Autophagy inducers PDGF and oxLDLs are also involved in vSMC phenotype transition and aberrant proliferation. Decayed production of SKi is thus implicated in intimal hyperplasia. MiR21 impedes SKi synthesis, thereby suppressing P38MAPK–CKI1a/CKI1b signaling and favoring vSMC proliferation [319].

Upon endothelium and internal elastic lamina injury (after angioplasty, near anastomoses of vascular grafts, and in ruptured atherosclerotic lesions), vSMCs can be exposed to blood flow and hence subjected to shear and stretch engendered by the hemodynamic stress field. They align in the streamwise direction. These mechanical stresses enhance NO production but impede vSMC migration and proliferation predominantly via TGFβ1 in an autocrine manner [320]. After exposure of vSMCs to flow, synthesis of TGFβ1 and tissue-type plasminogen activator, which converts plasminogen to plasmin, an activator of latent inactive TGFβ1 form, rises [321]. In addition, hemodynamic stress can prevent vSMC migration, as it lowers PDGFRβ and MMP2 formation [322]. Hemodynamic stress suppresses SMC migration, as it increases NO production and impedes MMP2 activity [323]. Vascular ECs and SMCs directly exposed to hemodynamic stress (shear) detected by mechanosensors (glycocalyx, GPCRs, adhesion molecules, and ion channels) modify their NO production rate. Semaphorins

Semaphorin-3E is a regulator of cell migration and proliferation; its expression progressively decays during intimal hyperplasia. Sema3e prevents vSMC migration and proliferation via its receptor plexin-D1 and inactivation of the Rap1–PKB axis [324]. On the other hand, after vascular injury, the mutual PDGFbb–sema3e inhibition favors neointimal formation. On the one hand, PDGFbb downregulates sema3e formation in vSMCs. On the other, sema3e precludes synthesis of the PDGFb protomer in ECs via EGR1, PDGFb being the most potent mitogen for vSMCs. In addition, P53 induces sema3e expression, hence suppressing vSMC proliferation and intimal hyperplasia after vascular injury [324]. Heparin

Heparin is a GAG that prevents cell migration and proliferation, particularly vSMC proliferation via inhibition of PKCα, suppressing phosphorylation of myristoylated alanine-rich PKC substrate, and promotes the contractile phenotype [325]. Heparin impedes the transcription of several genes associated with cell cycle progression (e.g., MyB, MyC, and Fos), the mitochondrial adenine nucleotide translocator (or ATP–ADP carrier) SLC25a5, and histone H3. Prostaglandins

The balance between thromboxane and prostacyclin is altered in cardiovascular disease. Prostacyclin counters vSMC dedifferentiation using the PtgIR–Gs–AC–cAMP–PKA axis [326]. Prostacyclin is a vasodilator and a potent inhibitor of platelet activation and vSMC proliferation. This main product of prostaglandin-H synthase PGhS2 (or cyclooxygenase COx2) is derived from arachidonic acid in vascular endotheliocytes. It acts via its cognate PtgIR (IP) receptor. On the other hand, thromboxane TxA2 is a potent activator of platelets and a vasoconstrictor. It is produced by PGhS1 (COx1) from arachidonic acid and released by activated platelets. Loss of prostacyclin and NO causes EC dysfunction and SMC dedifferentiation. Lactate

Lactate concentration that increases upon ischemia can shift vSMC behavior to the synthetic phenotype [327]. Vascular smooth myocytes cultured in lactate-enriched medium have a synthetic phenotype and a larger proliferation and migration rate with respect to those grown in glucose-free and standard media. These changes are accompanied by an increased expression of monocarboxylic acid transporters. In pigs, concentrations of lactate, lactate dehydrogenase, and monocarboxylic acid transporter in addition to vSMC proliferation rate are greater in ischemic regions than in other sites. Intracellular Regulators

Protein kinase-B is activated by hormones such as insulin, growth factors, and cytokines. It regulates cell metabolism, survival, differentiation, proliferation, and migration. Members of the PKB family (PKB1–PKB3) play distinct roles. Multiple factors can explain the distinct role of PKB isoforms, such as cell type, subcellular localization, expression level of the PKB subtypes in addition to their substrates and regulators, and interference with other signaling cascades. For example, PKB1 promotes and PKB2 inhibits migration of mouse embryonic fibroblasts using distinct facets of the Rac–PAK pathway [328].

In vECs, PKB1, the major isoform, is involved in angiogenesis and in inflammation. In vSMCs, the structurally similar (but not identical) PKB1 and PKB2, which are formed at similar concentrations, exert opposing effects on the cell phenotype, although they share many substrates. PKB1 promotes vSMC proliferation and migration and hence the dedifferentiated phenotype, PKB2, supports the differentiated phenotype [328]. In addition, PKB1 can play an antiapoptotic role. Moreover, PKB1 can inhibit PKB2.

Rapamycin, a TORC1 inhibitor stored in a drug-eluting stent, is aimed at avoiding intimal hyperplasia, as it prevents vSMC proliferation and migration. TORC1 represses insulin and IGF1 action and hence PI3K signaling, as it phosphorylates (inhibits) insulin receptor substrate IRS1. Its inhibition thus activates PKB. The latter phosphorylates (inactivates) transcription factors of the FOXO family (FoxO1, FoxO3a, and FoxO4), leading to their nuclear exclusion and cytosolic sequestration by 14-3-3 proteins. Rapamycin activates the PKB2 isoform.

Rapamycin launches vSMC differentiation via myocardin and PKB2 and subsequent nuclear exclusion of forkhead box-derived transcription factor FoxO4 (but neither FoxO1 nor FoxO3), thereby preventing its binding to the MYOCD promoter and subsequent MYOCD gene transcription [328]. FoxO4 interacts with both myocardin and SRF and thus hinders myocardin action. In addition, FoxO4 supports MMP9 formation, thereby assisting vSMC migration. On the other hand, myocardin promotes synthesis of smooth muscle contractile proteins. In addition, the transcription factor GATA6 is another PKB2 substrate that promotes SMC differentiation [328]. MicroRNAs

Let7g, miR21, miR29, miR126, miR132, miR145, miR146a, miR155, miR221, and miR222 are implicated in vSMC dysfunction associated with aberrant vSMC proliferation and migration and adverse intimal remodeling in atherosclerosis and restenosis. Vascular injury downregulates formation of miR143 and miR145, which cause vSMC differentiation and repress vSMC proliferation, but upregulates miR21, miR146a, and miR222, eliciting synthetic vSMC phenotype. Other important miRs include antiproliferative miR1, miR133, and miR195 and proproliferative miR21, miR221, miR222, and miR146a.

Let7g controls autophagy and apoptosis of vSMCs exposed to oxidized LDLs [329]. Oxidized LDLs (10–40 μg/ml) elicit autophagy via beclin-1 (Atg6), MAP1LC3 (Atg8), and Atg5 in addition to apoptosis via BCL2 and BCL2L1 on the one hand and BAX and caspase-3 on the other. Let7g impedes autophagy, as lipoxygenase LOx1 expression and intracellular ROS generation decline. In airway SMCs, Let7g also hampers synthesis of LOx1 and Oct1.

Myocardin is a cardiac and smooth myocyte-specific transcription cofactor that promotes their differentiation, but impedes proliferation, partly via HDAC4 and HAND2. Myocardin induces expression of miR1 in CMCs and in SMCs [330]. Transcription of the MIR1 gene depends on SRF in the myocardium and transcription factors of the MyoD family in skeletal muscles. Spindle-shaped SMCs possess higher amounts of both myocardin and miR1 than epithelioid SMCs. MicroRNA-1 targets PIM1 and prevents vSMC proliferation.

MiR21 favors vSMC proliferation and intimal hyperplasia after vascular injury via downregulation of the expression of proapoptotic proteins BCL2 and PTen [298]. It also prevents SKi action, hence priming vSMC proliferation and migration induced by oxLDLs [319]. It also contributes to TGFβ-regulated endothelial-to-mesenchymal transition. Differentiation of pulmonary SMCs induced by BMPs is mediated by miR21, partly via suppression of programmed cell death protein PdCD4 [331]. On the other hand, differentiation of aortic SMCs can reduce miR21 expression.

Like miR21, miR31 regulates aberrant vSMC proliferation [332]. In proliferative vSMCs, proproliferative miR31 expression is upregulated by ERK. It then targets large tumor suppressor homolog LATS2.

Formation of miR24 and miR29a is primed by myocardin, the former being implicated in atherosclerosis retardation and the latter in matrix remodeling and fibrosis [298].

MiR26a hampers vSMC differentiation, thereby favoring their proliferation and migration via the TGFβ pathway, as it hinders production of SMAD1 and SMAD4 [298].

MiR29 expression is lost in calcified vSMCs in vitro and in calcified rat arteries in vivo. MiR29 targets ADAMTS7, which represses vSMC calcification [298].

MicroRNAs contribute to sexual differentiation. Female-specific miR29b targets DNA methyltransferases DNMT3a and DNMT3b [333]. Oxidized LDL raises aortic SMC migration, as it upregulates synthesis of MMP2 and MMP9 via diminished DNA methylation by DNMT3b [334]. Upon oxLDL exposure, miR29b targets DNMT3b and promotes migration of arterial SMCs.

Apoptosis of vSMCs favored by redox stress, partly via JNK, intervenes in vascular remodeling and atherosclerotic plaque instability. JNK is a substrate of miR92a in macrophages. In quiescent vSMCs, miR92a is produced at a low level. Its synthesis is hampered by H2O2 [335]. Conversely, miR92a represses H2O2-primed vSMC apoptosis, as it inhibits MAP2K4–JNK1 signaling.

MicroRNA-124 expression is downregulated in proliferative vascular diseases. MiR124 targets specificity protein SP1 and attenuates PDGFbb-induced vSMC proliferation and differentiation [336].

Histone H3 Lys9 methyltransferase KMT1a and associated repressive epigenetic mark H3K9me3 prevents sustained inflammatory gene expression (heterochromatic silencing and euchromatic transcriptional repression). MiR125b targets KMT1a, thereby favoring production of inflammatory cytokines and chemokines, such as IL6 and CCL2 [337]. MiR125b promotes the proinflammatory response in diabetic vSMCs that possess a preactivated phenotype with augmented inflammatory gene expression. Hence, miR125b favors diabetic vSMC phenotype. Expression of miR125b and MIR200 family members is upregulated in vSMCs of T2DM db/db mice with respect to control db/+ mice.

MicroRNA-126, one of the most abundant endothelial miRs, the transcription of which is regulated by ETS1 (a substrate of miR155, miR221, and miR222), ETS2, and flow-dependent KLF2, attenuates the rate of EC proliferation and supports VEGF-primed apoptosis decay and migration [331]. Thus, endothelial miR126 modulates angiogenesis. Activated and apoptotic ECs release miR126 in microvesicles and transfer it to adjoining cells, regulating SMC turnover. Argonaute-2, a component of the miR-induced silencing complex, is transmitted with miR126 via extracellular vesicles (EVs) and facilitates miR126 transfer from ECs to SMCs. Endothelial miR126 represses transcripts encoding FoxO3, BCL2, and IRS1 [338].

Angiotensin-2 induces miR132–miR212 cluster synthesis in vSMCs and increases inflammatory gene transcription [339]. MiR132 targets the PTEN transcript. The miR132–miR212 cluster mediates Agt2-induced CCL2 production in vSMCs.

MiR133 is synthesized in vSMCs, whereas miR1 concentration is negligible. It represses vSMC phenotype switch. The miR133a1–miR1-2, miR133a2–miR1-1, and miR206–miR133b clusters are bicistronic, the latter being produced in skeletal muscles, but not in the heart. MiR133 production is regulated by ERK1 and ERK2 [340]. MiR133 suppresses formation of the transcription factor SP1, which induces synthesis of cyclin-dependent kinase inhibitor CKI1b and subsequently cell cycle arrest.

In diabetes, miR138 also regulates vSMC proliferation and migration [286].

Cardiovascular-specific miR143 and miR145 encoded by a bicistronic gene cluster abound in vSMCs. Transcription of the miR143–miR145 cluster is primed by SRF and its coactivators, myocardin and myocardin-related transcription factors. They are atheroprotective and prevent intimal hyperplasia induced by vascular injury, which launches vSMC dedifferentiation and cytoskeletal remodeling. MiR143 and miR145 are also transferred from ECs to vSMCs by extracellular vesicles; KLF2 increases their amount in ECs and triggers their release via microvesicles [331]. They play a crucial role in cooperation with other miRNAs, such as miR1 and miR24, in the differentiation and proliferation of vSMCs. MiR1, the formation of which is triggered by myocardin in vSMCs, precludes cell proliferation but inhibits myocardin-induced vSMC contractility. The Jag1–notch axis can also induce transcription of the miR143–miR145 cluster. MiR143 and miR145 repress Krüppel-like factors KLF4 and KLF5 that inhibit myocardin, thereby supporting vSMC differentiation and repressing their proliferation. TGFβ and BMP4 are potent inducers of contractile vSMC phenotype; they prevent vSMC proliferation and migration. KLF4 hampers activation of the CArG box by SRF and its coactivators. TGFβ and BMP4 downregulate KLF4 synthesis via miR143 and miR145 [341]. In addition, miR143 also reduces the production of ETS-like factor ELk1, hence promoting synthetic phenotype. Furthermore, miR145 downregulates ACE. Angiotensin-2 is a vasoconstrictor that also exerts a proinflammatory effect via IL6 and CCL2 and elicits vSMC proliferation and hypertrophy, mostly via AT1, the MAPK module, NFκB, and CREB [339]. Conversely, miR143 and miR145 are repressed by ACE, KLF4, and KLF5, which then favor neointima formation [298]. In addition, miR145 targets slit-robo GTPase-activating protein SRGAP1 (RhoGAP13) and SRGAP2 (RhoGAP34), adducin-3, slingshot-2 phosphatase, and calmodulin kinase CamK2δ, which are involved in the regulation of SRF activity and actin dynamics [331]. Genetic deficiency in miR143 and miR145 in mice shifts the vSMC phenotype from a contractile to a synthetic state.

MiR146a targets Krüppel-like factor KLF4, thereby promoting vSMC proliferation and intimal hyperplasia [342]. KLF4 competes with KLF5 for MIR146A promoter binding; KLF4 and KLF5 exert opposing effects on the MIR146A promoter, KLF4 impeding MIR146A gene transcription. MiR146a and KLF4 form a feedback loop to regulate each other’s expression and vSMC proliferation.

MiR155 supports vSMC proliferation [343]. STK3 (MST2), a component of MAPK modules and the hippo pathway,Footnote 57 mediates miR155-primed inflammation and redox stress, which lead to vSMC proliferation and vascular remodeling. Inflammation and redox stress, which occur after vascular injury, are coordinated by the ERK1/2 pathway. STK3 interacts with cRaf, thereby competing with MAP2K and regulating ERK1/2 signaling. Downregulation of STK3 expression by proproliferative miR155 activates ERK1/2 signaling, as it facilitates the interaction between cRaf and MAP2K, and then boosts vSMC proliferation. In addition, miR155 supports migration of smooth myocyte progenitors via CCL2 [286].

MiR155, which is synthesized and secreted upon KLF5 stimulation in vSMCs, is a potent regulator of the endothelial barrier, as it regulates production of endothelial tight junction proteins [344]. Exosomes originating from vSMCs transfer miR155 from SMCs to ECs, which then alter tight junction renewal and hence the integrity of the endothelial barrier, increasing endothelial permeability. Moreover, miR155 in ECs impedes their proliferation and migration and hence re-endothelialization.

MiR182 expression is markedly downregulated during vSMC dedifferentiation [345]. MiR182 increases synthesis of SMC-specific contractile proteins and prevents SMC proliferation and migration in basal conditions and upon PDGFbb exposure. It targets the Fgf9 transcript. FGF9 is a potent mitogen secreted from bone marrow-derived cells that drives neovascular maturation via PDGFRβ, which resides particularly on vSMCs and pericytes, the production of which is upregulated by sonic hedgehog [346].

MiR182-3p counters vSMC proliferation and migration via ERK [347]. This microRNA targets myeloid-associated differentiation marker (MyADM), hence repressing intimal hyperplasia. The NOS inhibitor, asymmetrical dimethylarginine (ADMA), downregulates miR182-3p production, thereby supporting intimal hyperplasia.

The subunit of the AP1 transcription factor complex, JunB, promotes blood vessel development and maintenance but precludes cell proliferation and inflammation. Under hypoxia, JunB regulates angiogenesis, as it upregulates synthesis of VEGFa, core-binding factor CBFβ, which with Runx forms the heterodimeric core-binding transcription complex CBF, and MMP13, a CBF target. Components of the AP1 complex regulate expression of various microRNAs (miR21, miR101, miR144, miR155, and miR203) [348]. MiR182 is a JunB target implicated in lymphangiogenesis, at least in zebrafish [348]. MiR182 attenuates FoxO1 formation to avoid its overproduction, hence driving proper lymphangiogenesis.

MicroRNA-195 abounds in vSMCs [349]. It impedes vSMC proliferation, migration, and synthesis of IL1β and IL6 and the CXCL8 chemokine. It represses translation from Cdc42, CCND1, and Fgf1 transcripts.

MiR200 downregulates expression of the zinc finger E-box-binding homeobox-containing transcription factor ZEB1, hence raising production of proinflammatory PGhS2 (COx2) and CCL2 [298].

Estrogens such as estradiol (E2) protect vascular walls via miR203 production, which is upregulated in vSMCs via NR3a1 (ERα) [350]. The transcription factors ZEB1 and AP1 mediate E2-induced upregulation of miR203 transcription. MiR203 represses Abl1 and P63 production and vSMC (but not vEC) proliferation.

MiR221 and miR222 are expressed by a gene cluster of the X chromosome in vSMCs and ECs [331]. They favor neointima growth. Therefore, downregulation of miR143 and miR145 expression and upregulation of miR221 and miR222 after vascular injury support intimal hyperplasia. Proliferation of vSMCs results from the elimination of cyclin-dependent kinase inhibitors CKI1b and CKI1c by miR221 and miR222. On the other hand, they impede EC proliferation. Whereas miR221 and miR222 lower the vSMC apoptosis rate and support the latter’s migration, they hinder EC migration and favor apoptosis. Moreover, miR221 and miR222 prevent angiotensin-2-induced inflammation, as they suppress the formation of VEGFR1, vcam1, and CCL2, thereby reducing leukocyte recruitment and adhesion [331]. In addition, miR221 suppresses the synthesis of SCFR in SMCs, which initiates myocardin production.

Angiotensin-2 regulates the long nonprotein-coding RNA lncAng362, which is responsible for the production of two microRNAs implicated in vSMC proliferation, miR221 and miR222 [351]. In fact, angiotensin-2 controls the production of many lncRNAs, downregulating that of lncAng219 and lncAng249 and upregulating that of lncAng112, lncAng162, and lncAng362. Production of lncAng362, miR221, and miR222 is upregulated once vSMCs are exposed to Agt2.

Hyperglycemia contributes to abnormal vSMC activation. Hyperglycemia stimulates vSMC proliferation and migration via ERK and ROS. In addition, hyperglycemia hinders vSMC apoptosis, as it upregulates the expression of antiapoptotic proteins BCL2 and BCL2L1. Furthermore, hyperglycemia inactivates AMPK, which can improve endothelial function and suppress vSMC proliferation and migration. On the other hand, family with sequence similarity protein Fam3b (its secretory form named pancreatic-derived factor and abbreviated pander),Footnote 58 which is secreted from the endocrine pancreas, can provoke apoptosis of α and β cells. It is cosecreted with insulin from β cells by hyperglycemia [352]. It boosts vSMC proliferation and migration and precludes miR322-5p expression. Conversely, miR322-5p impedes Fam3b-primed vSMC proliferation and migration.

MiR424 (ortholog of rat miR322) concentration decays shortly after induction of vSMC proliferation and then rises. It precludes vSMC proliferation and migration without affecting apoptosis [353]. It targets cyclin-D1 and Ca2+-regulating proteins calumenin (directly) and stromal-interacting molecule STIM1 (indirectly), lowering store-operated Ca2+ entry. It is upregulated after vascular injury, thereby protecting against restenosis.

Cyclin-D1 transcript is a substrate of antiproliferative miR365 [354]. It lowers PDGFbb-primed vSMC proliferation, as it blocks G1–S transition. On the other hand, vSMC proliferation triggered by PDGFbb and angiotensin-2 downregulates miR365 expression.

Oxidized LDLs induce vSMC proliferation, downregulate the synthesis of miR490-3p and IGF1, but upregulate that of pappalysin-1 (or PAPPa)Footnote 59 and IGF2 in vSMCs [355]. Overproduction of PAPPa and the resulting activation of proinflammatory IGF in vSMCs may promote their proliferation, but IGF1 may have anti-inflammatory and antioxidant effects. MiR490-3p targets PAPPA, countering the effect of oxLDLs and suppressing the PAPPa metallopeptidase effect and hence IGFBP4 cleavage.

In diabetes mellitus, vascular smooth myocytes acquire a proatherogenic and proinflammatory phenotype. Diabetes mellitus is linked to elevated concentrations of glucose, advanced glycation end products, growth factors, and oxidized lipids that favor vSMC dysfunction. In vSMCs of T2DM mice, 135miRs are differentially expressed, among which several regulate vSMC function. MiR504, the production of which is upregulated, targets the adaptor GRB10 and transcription factor EGR2 [356]. It provokes a vSMC phenotype switch, as it downregulates expression of contractile genes and upregulates that of ERK1 and ERK2, thereby promoting vSMC proliferation and migration. High concentrations of glucose and palmitic acid increase miR504 formation and the expression of inflammatory genes.

The PDGFbb stimulant elicits vSMC proliferation and suppresses miR599 synthesis [357]. Conversely, miR599 represses vSMC proliferation and migration in addition to the synthesis of PCNA, an auxiliary protein of DNA polymerase-δ involved in the control of DNA replication, and of cellular proliferation marker protein MKi67, an antigen identified by monoclonal antibody Ki67, which is encoded by the MKI67 gene and linked to the perichromosomal layer during mitosis, acting as a surfactant [358]. In addition, miR599 suppresses production of collagen-1 and -5 and proteoglycan. In vSMCs, the TGFB2 transcript is a substrate of miR599 [357]. TGFβ2 can reverse miR599-primed prevention of vSMC proliferation along with collagen and proteoglycan synthesis. MiR590-3p increases vSMC contractility and represents a risk factor for hypertension in the presence of genetic defects [286].

miR638 is one of the most significantly downregulated in human aortic vSMCs during their proliferation and migration launched by PDGFbb [359]. Conversely, miR638 hinders vSMC proliferation, as it targets the nuclear receptor NR4a3 (NOR1), subsequently impeding cyclin-D1 production. Platelet MicroRNAs

Platelets, which contain mRNA and can thus synthesize new proteins (e.g., integrin-α3, or glycoprotein-3A, and fibrinogen receptor), are a major source of miRs, YRNAs, circular RNAs, and lncRNAs.

Newly formed platelets are larger, more reticulated, and more reactive than older platelets; they also contain a higher amount of RNA and can thus produce greater amounts of proteins, especially α granule-stored proteins. On the other hand, platelets may inherit their miR content during shedding from megakaryocytes.

Platelets release microvesicles and exosomes, especially during activation. Inappropriate activation of platelets initiates thrombosis. MicroRNAs can thus be used as markers of platelet activation [360].

Whereas the platelet miR expression pattern remains stable over the lifespan of the platelet in healthy individuals, it is markedly altered in diabetes, after myocardial infarction, and in cancer (Table 2.11) [360]. MiR release from platelets may result from shedding of microvesicles and synthesis of mature miRs from precursor miRs. On the other hand, activation of calpain that targets dicer lowers the concentrations of several platelet miRs.

Table 2.11 Platelet microRNA] (Source: [360]) Hepatic MicroRNAs Operating in Metabolism

Because the liver is a major organ in the regulation of lipid, cholesterol, and glucose metabolism,Footnote 60 hepatic microRNAs, such as miR24,Footnote 61 miR29,Footnote 62 miR30c,Footnote 63 miR33,Footnote 64 miR33,Footnote 65 miR122,Footnote 66 miR144,Footnote 67 miR148a,Footnote 68 and miR223,Footnote 69 packaged in microvesicles and proteic complexes,Footnote 70 affect hepatic metabolism and hence are linked to metabolic syndrome, non-alcoholic fatty liver disease, T2DM, and atherosclerosis; they can thus serve as markers [361].

Apolipoprotein-B is an essential scaffold in VLDL assembly. MiR548p interacts with the 3-untranslated region of the APOB mRNA and promotes its post-transcriptional degradation, thereby reducing ApoB synthesis and secretion from hepatocytes. Furthermore, miR548p significantly decreases lipoprotein production in addition to lipid synthesis in hepatocytes, as it lowers the activity of 3-hydroxy 3-methylglutarylCoA reductase and long-chain acylCoA synthetase ACSL4Footnote 71 involved in cholesterol and fatty acid synthesis, but does not affect fatty acid oxidation [366]. MicroRNAs and Mitochondrial Function and Dysfunction

MicroRNAs participate in the regulation of mitochondrial metabolism and ATP production via proteins implicated in substrate transport, glycolysis, fatty acid β-oxidation, ketone catabolism, and coupled processing by the tricarboxylic acid cycle (TCAC) and electron transport chain (ETC).

The metabolism evolves from fatty acid oxidation to glycolysis during hypoxia, especially in the context of hypertension-induced HF, as expression of fatty acid oxidation enzymes is downregulated and that of glycolytic enzymes is upregulated [367].

Mitochondria import various species of ncRNAs, such as tRNAs; 5S rRNA, which is the most abundant mitochondrial RNA type; miRs; and lncRNAs [368]. Polynucleotide phosphorylase of the mitochondrial intermembrane space that processes mitochondrial transcripts promotes the import of ncRNAs through the inner mitochondrial membrane into the mitochondrial matrix.

The term mitomicroRNAs refers to microRNAs located inside the mitochondrion, category 1 MtmiRs being encoded by the mitochondrial genome and category 2 MtmiRs being transcribed in the nucleus and imported to the mitochondrion (Table 2.12). For example, preLet7b, pre-miR302a, and their corresponding mature miRs lodge in the mitochondrion. Most category-2 MtmiRs originate from loci pertaining to mitochondrial gene clusters or close to mitochondrial genes. MitomicroRNAs may interact with transcripts encoded in the mitochondrion.

Table 2.12 MicroRNAs and mitochondrial function (Source: [368]; CcOx4 cytochrome-C oxidase subunit-4, CcOx10 cytochrome-C oxidase protoheme-9 farnesyltransferase, ISCu iron–sulfur cluster assembly scaffold)

Certain mitomicroRNAs can act as translational activators, as they promote translation of mitochondrial transcripts using a specific miR–mRNA base pairing [368]. Under certain conditions, once it is recruited to the 5-end of target mRNAs and/or internal ribosome entry sites, miR1 can activate translation in the cytosol. MiR1 recruits the ND1 and COX1 mRNAs to mitochondrial ribosomes.

In addition to MtmiRs, several cytosolic microRNAs modulate mitochondrial function. Brain-specific miR338 lowers the formation of the cytochrome-C oxidase subunit CcOx4 encoded in the nucleus [368]. MiR210, which is induced by HIF1α under hypoxia, can repress oxidative phosphorylation, as it targets transcripts encoding cytochrome-C oxidase assembly protein CcOx10 and iron–sulfur cluster scaffold (ISCu).

Several miRs participate in intrinsic apoptosis. In cardiac and skeletal myocytes, miR1, the formation of which is induced by hyperglycemia, diminishes mitochondrial membrane potential and provokes cytochrome-C release via IGF1 [368]. In the heart, miR378 downregulates caspase-3 expression and hence impedes apoptosis.

Certain miRs such as those of the MIR30 family, which abound in the heart, modulate mitochondrial dynamics. They regulate mitochondrial fission and fusion via the P53–DRP1 axis, as they decrease amounts of P53 and subsequently of DRP1, hence favoring fusion to support energy demand and raising the threshold of apoptotic activation in CMCs [368].

In HF, impaired fatty acid β-oxidation increases circulating free fatty acid (FFA) concentration, which is detected by the liver that uses FFAs as substrates for ketogenesis. Ketones enter the bloodstream, from which they are taken up by CMCs, the rate of oxidation matching the rate of delivery. Cardiac ketolysis carried out by 3-hydroxybutyrate dehydrogenase BDH1, succinylCoA:3-oxoacidCoA transferase (SCOT), and acetoacetyl-CoA thiolase produces acetyl-CoA [367].

In CMCs, some microRNAs (e.g., miR181c and miR378) translocate to mitochondria and affect expression of the mitochondrial transcripts (Table 2.13) [367].

Table 2.13 MicroRNAs affecting the mitochondrial metabolism, the expression of which is upregulated in heart failure (Source: [367]; Arl ADP ribosylation factor-like transcript, ETC electron transport chain, FABP fatty acid-binding protein, Hk hexokinase, Hsp heat shock protein, ISCu iron–sulfur cluster assembly scaffold homolog, MTATP6 mitochondrially encoded ATP synthase (ETCcomplex-V ) F0 subunit-6, MTCO1 mitochondrially encoded cytochrome-C oxidase subunit-1 [ETCcomplex-IV component], PDHB mitochondrial pyruvate dehydrogenase subunit-β, PFKM phosphofructokinase muscle isozyme, PGC PPARγ coactivator, PKM pyruvate kinase muscle isozyme, Ppar peroxisome proliferator-activated receptor, Vdac voltage-dependent anion channel)

Hypoxia and redox stress affect expression of microRNAs and hence mRNAs. Furthermore, these stressors contribute to oxidation of metabolic enzymes, altering their activity.

Formation of both NOx2 and NOx4 can be induced by HIF1α during hypoxia. In HF, upregulated expression of NOx2 can partly result from downregulated miR17. In addition, the baseline constitutive expression of NOx4 is augmented by hypercholesterolemia that downregulates miR25 production. On the other hand, increased miR25 expression in HF inhibits serca2a production. In HF, downregulated synthesis of serca2 and RyR2 is associated with an altered expression of CK [369].

In most aging-related diseases, mitochondrial ROS are produced by the ETC, which requires NADH and is hence countered by enzymes that need NADPH as cofactor. The nicotinamide nucleotide transhydrogenase (NNT) is an antioxidative enzyme that regenerates NADPH from NADH; it uses the proton gradient to generate NADPH from NADH and NADP+, hence linking mitochondrial respiration and detoxification by the thioredoxin–peroxiredoxin processor (∼80%) or glutathione peroxidase (∼20%) of H2O2, which is produced from mitochondrial superoxide radical by spontaneous dismutation and/or by manganese-containing superoxide dismutase SOD2 and by citric acid cycle enzymes, such as aconitase and α-ketoglutarate dehydrogenase [370].Footnote 72

The enzyme NNT promotes TRdx and TRdx reductase activity. Mitochondrial NADPH can also be regenerated by isocitrate dehydrogenase IDH2 and mitochondrial NADP-dependent malic enzymes (ME2 and ME3).

An adverse metabolic context reverses the NNT processing direction, consuming NADPH to support NADH and ATP production [371]. In HF, changes in the mitochondrial proteome lead to decreased expression of two NADPH-producing proteins, mitochondrial NNT and IDH2 [369]. The latter yields the highest contribution to overall NADPH regenerating capacity in cardiac mitochondria (∼70%; NNT: ∼22%; ME: ∼8%) [371].

Redox stress in HF is caused by increased expression of prooxidant aconitase, reduced formation of antioxidant superoxide dismutase, in addition to hypersensitivity of the ETC and ATP synthase to glutathionylation [367].

Apoptosis of cardiac cells is caused by upregulated synthesis of apoptotic proteins such as voltage-dependent anion channel, which acts in the mitochondrial permeability transition pore. The major outer mitochondrial membrane importer of ATP constituents and exporter of ATP, VDAC1, has downregulated expression in end-stage heart failure owing to upregulated miR29a [367].

Synthesis of adenine nucleotide transporter ANT1, the inner mitochondrial membrane transporter of ADP and ATP, is inhibited by miR424, which suppresses production of ARL2 [372]. ARL2 complexes with its partner ARL2BP, and, both being predominantly cytosolic, enter the mitochondrion and bind to ANT1 (but not to ANT2).

MiR141, a member of the MIR200 family, targets the inner mitochondrial membrane phosphate transporter, SLC25a3, which provides inorganic phosphate to the mitochondrial matrix for ATP production [288].

Mitochondria constitute the most redox-active cellular compartment; the mitochondrial matrix of CMCs has a highly reduced redox potential with respect to the cytosol [69, 373]. Glutathione peroxidase inactivates H2O2 using reduced glutathione. Glutathione is produced in the cytosol and accumulates in the mitochondrial matrix, where it is primarily found in the reduced state, mainly because of NADPH produced by the TCAC. Distinct glutathione nanodomains exist in the redoxomes of CMCs, that is, the cytosol, mitochondrion, and other redox-active organelles. Oxidants are also produced in nanodomains inside redoxomes.

Increased ROS generation upon cellular stress provokes a reductive stress in the cytosol and mitochondria and oxidative stress upon reoxygenation [69]. On the other hand, upon prolonged ROS exposure and chronic ischemia, the cytosol and mitochondria are more oxidized media. In addition, ROS scavenging by antioxidants influences redox status. Increased production of reductants such as accumulation of reduced glutathione and NADPH in addition to elevated activity of antioxidant enzymes can result from reductive countering of radicals upon oxidative stress.

Redox stress can involve NFE2L2, HSPs, ROS, and G6PD [69]. Antioxidant flux depends on mitochondrial substrate catabolism along with thioredoxin and glutathione.

Because they modulate mitochondrial metabolism, several miRNAs act as oncogenes or tumor suppressors [368]. Production of miR23a and miR23b, which control expression of mitochondrial glutaminase,Footnote 73 is downregulated upon oncogenic activation (e.g., MyC overexpression)Footnote 74 and accompanied by decreased ROS synthesis, as glutamine is a substrate for glutathione formation, and by increased ATP synthesis via the TCAC.

Expression of both miR23a and miR23b is induced in cardiac hypertrophy. In addition, miR15b, miR16,Footnote 75 miR195, and miR424, which preclude formation of ARL2 and lower ATP concentration, affect mitochondrial integrity in CMCs [288]. These members of the MIR15 family undergo upregulated expression in ischemia–reperfusion injury and cardiac hypertrophy.

Ischemia is characterized by hypoxia. The HIF pathway provokes a metabolic shift (Pasteur effect) from aerobic mitochondrial oxidative phosphorylation to anaerobic glycolysis, an event linked to repression of mitochondrial genesis and production of glycolytic enzymes (e.g., lactate dehydrogenase, LDHa, which converts pyruvate to lactate) and activation of pyruvate dehydrogenase kinase (PDHK, which decreases glucose utilization and increases fatty acid metabolism and intervenes in ROS generation).Footnote 76 During hypoxia, mitochondrial ROS formation rises and repression of the oxidative metabolism by HIF maintains redox homeostasis. During chronic hypoxia and hence ischemia, oxidative metabolism can be maintained, at least in fibroblasts under hypoxia, but generates toxic ROS levels [288].

miR210, which is regulated by NFκB and ELk1, is upregulated by hypoxia. HIF1α represses oxidative phosphorylation and activity of mitochondrial ETC via miR210 in many cell types [288].

MiR210 impedes synthesis of ISCu1 and ISCu2 (which participates in the assembly of iron–sulfur clusters present in several ETC and TCAC components), transferrin receptor, mitochondrial cytochrome-C oxidase assembly protein (protoheme-9 farnesyltransferase) CcOx10 (a component of ETCcomplex-I and -IV ), mitochondrial heme synthase (ferrochelatase [FeCh], the subunit of succinate dehydrogenase SDHd, which oxidizes succinate to fumarate), glycerol 3-phosphate dehydrogenase-1-like protein (GPD1L, which modulates the NAD+/ NADH ratio), and NADH dehydrogenase (ubiquinone)-1α (NADH–ubiquinone oxidoreductase) subunit NDUFα4 [288]. MiR210 may protect ECs and skeletal and cardiac myocytes against redox stress. However, miR210 can raise ROS production at least in some cell types and provokes cellular senescence. MicroRNAs as Risk Markers

MicroRNAs can be released into the extracellular space and blood circulation in vesicles or linked to proteic binding partners, which avoid miR destruction.

Certain microRNAs originating from the heart or other sources can be used as risk markers (Tables 2.14, 2.15, and 2.16) [238, 430]. Concentrations of the cardiac and muscular miRs (miR1, miR133, miR208, and miR499) are elevated in the bloodstream after MI.

Table 2.14 Examples of microRNAs used as risk markers (Source: [238]; BNP brain natriuretic peptide, vSMC vascular smooth myocyte)
Table 2.15 Cardiac microRNAs as markers of cardiovascular maladies (Source: [376])
Table 2.16 Noncardiac microRNAs as markers of cardiovascular maladies (Source: [376]) MicroRNAs Involved in Adverse Cardiac Remodeling

MicroRNAs participate in regulating transcript translation and degradation during cardiac genesis and remodeling (Table 2.17). MiR21, miR29a, miR129, miR210, miR211, miR320, and miR423, along with Let7c, are highly expressed in the fetal heart and in postnatal cardiac development [377]. Members of the MIR1 (miR1-1 and miR1-2) and MIR133 (miR133a1–miR133a2 and miR133b) and miR206 are muscle-specific microRNAs; miR1 and miR133 are also produced in adequate amounts during the early stage of cardiogenesis. MiR1 targets the transcription factor HAND2, which promotes CMC proliferation, in addition to the transcriptional repressor HDAC4. MiR133 acts on the nuclear factor NELFA.Footnote 77

Table 2.17 MicroRNAs in cardiac fate (Source: [377])

Loss of miR1 and miR133 leads to ventricular septal defects and cardiac chamber dilation. In ventricular septal defects, expression of miR181c rises. In the human heart, miR196a is associated with the HoxB8 (or Hox2D)–sonic hedgehog pathway implicated in cardiac septation, valve formation, and outflow tract morphogenesis. Tetralogy of Fallot, the most common congenital cyanotic disorder, is linked to an overexpression of miR421 in ventriculomyocytes, whereas miRNA940 is underexpressed [377]. In bicuspid aortic valve, a congenital valvulopathy, miR26a, miR30b, miR141, and miR195 concentrations are weak.

In adverse cardiac remodeling, that is, hypertension-induced hypertrophy, miR1 and miR133 expression is downregulated, whereas that of miR21, miR195, miR208, and miR214 is upregulated [377].

In ventricular arrhythmias, arrhythmogenic miR1 represses expression of the KCNJ2 and GJA1 genes that encode KIR2.1 channel and connexin-43 [377]. On the other hand, miR1 concentration is reduced in atrial fibrillation, which is linked to miR328.

Profibrotic miR33a is produced predominantly by cardiofibroblasts, which ensures necessary acute wound healing (cardiac reactive fibrosis) [378]. On the other hand, cardiac replacement fibrosis linked to adverse remodeling, which upregulates miR33a expression in the heart, is deleterious. In CFBmir33−∕− (i.e., only in cardiofibroblasts), cardiac fibrosis is attenuated in response to hypertension. Depletion of miR33a impedes PKB activation but favors ABCa1 expression. MiR33 can control cardiofibroblast proliferation, as it can target CDK6, CcnD1, and P53 in some cell types [364], but CDK4 is predominant over CDK6 in fibroblasts [378]. In fact, miR33a preserves the cholesterol content of membrane rafts, which enable PKB activation and cardiofibroblast proliferation.Footnote 78 On the other hand, upregulated synthesis of ABCa1, which permits efflux of intracellular free cholesterol to extracellular cholesterol acceptor ApoA1, depletes the cholesterol content of membrane rafts and alters the proliferative capacity of cardiofibroblasts.

Some microRNAs are associated with matrix degradation and pathological cardiac remodeling caused by prolonged overactivation of the renin–angiotensin–aldosterone axis and sympathetic nervous system in addition to hypertension, valve stenosis or regurgitation, myocarditis, familial and acquired cardiomyopathies, and MI (e.g., miR100 and miR101) [381]. MiR101 regulates Rab1a formation; miR100 and miR101 (along with miR15b, miR20a, miR92a, and miR132), prime apoptosis, as they target inhibitors of the intrinsic apoptosis pathway.

Many microRNAs favor or counter maladaptive cardiac hypertrophy (Tables 2.18 and 2.19) and fibrosis (Tables 2.20 and 2.21).

Table 2.18 Antihypertrophic microRNAs in the heart (Source: [381] AtG autophagy-related gene product, CamK calcium–calmodulin-dependent kinase, Ccn cyclin, FABP fatty acid-binding protein, Fbln fibulin, GATA GATA-binding protein, HAND heart and neural crest derivatives expressed protein, IGF insulin-like growth factor, KAT lysine (K) acetyltransferase, Ksr kinase suppressor of Ras, MAPK mitogen-activated protein kinase, NCX sodium–calcium exchanger, NFAT nuclear factor of activated T-cells, TnnI3K troponin-I type-3-interacting kinase, Twf actin-binding twinfilin homolog [PTK9], WHSC2 Wolf–Hirschhorn syndrome candidate protein-2, or negative elongation factor NeLFa, XBP1 X-box-binding protein)
Table 2.19 Prohypertrophic microRNAs in the heart (Source: [381] BECN1 autophagy-related beclin-1, CDKN1B transcript encoding cyclin-dependent kinase inhibitor-1B [CKI1b], DYRK dual-specificity Tyr (Y) phosphorylation-regulated kinase, FBxO32 F-box only protein-32, or atrogin-1, Fox forkhead box transcription factor, GRB growth factor receptor-bound protein, GSK glycogen synthase kinase, HDAC histone deacetylase, LPA 1, lysophosphatidic acid receptor-1, MAPK mitogen-activated protein kinase, MED mediator complex subunit, MSTN myostatin, PPARγ, peroxisome proliferator-activated receptor-γ, or nuclear receptor NR1c3, PDLIM5 PDZ and LIM domain-containing protein-5, or enigma homolog [enigma-like PDZ and LIM domain-containing protein EnH1], PTen phosphatase and tensin homolog, serca sarco(endo)plasmic reticulum Ca2+ ATPase, SIRT silent information regulator-2, SorbS sorbin and SH3 domain-containing protein, TP53INP1 tumor protein P53-inducible nuclear protein-1, TriM63 tripartite motif-containing protein-63, or muscle-specific RING finger-containing protein MuRF1)
Table 2.20 Antifibrotic microRNAs in the heart (Source: [381] Col collagen, CTGF connective tissue growth factor, Eln elastin, Fbn fibrillin, Fos FBJ murine osteosarcoma viral proto-oncogene homolog, IL interleukin, Snai1 zinc finger-containing snail homolog, TGF transforming growth factor)
Table 2.21 Profibrotic microRNAs in the heart (Source: [381] Apln apelin, PTen phosphatase and tensin homolog, Spry sprouty homolog)

Concentrations of circulating microRNAs depend on disease states and can thus serve as markers of CVD evolution. Microvesicles and HDLs can transport miRs in blood and deliver them to cells.

MiR208a and miR208b, which are encoded by introns of the MYH6 and MYH7 genes respectively, are also involved in the mutually incompatible regulation of MHC6 and MHC7 expression in adverse cardiac hypertrophy, downregulating MYH6 and upregulating MYH7 expression [383]. They regulate MYH7 expression via the mediator complex subunit Med13 and myostatin [384].

MicroRNA-146a concentration moderately increases in the left ventricular wall of patients with aortic stenosis. Overexpression of miR146a in CMCs provoked cardiac hypertrophy [385]. MiR146a reduces the formation of dihydrolipoyl succinyltransferase (DLST).Footnote 79 MiR181c can act in the mitochondrion of CMCs, where it targets MTCOI mRNA, which encodes cytochrome-C oxidase catalytic subunit-1 (CcOx1) and provokes remodeling of ETCcomplex-IV [386]. MicroRNAs in Obesity and Diabetes

Increased adiposity, obesity, and diabetes are linked to redox stress and inflammation. Mitochondrial ROS overproduction results from hyperglycemia in the diabetic microvasculature, whereas in the diabetic macrovascular and heart, it is the consequence of increased fatty acid oxidation and insulin resistance [288].

In obese subjects, redox stress in adipose tissue is associated with chronic low-grade inflammation. Three miRs target antioxidant, anti-inflammatory, and antiapoptotic heme oxygenase HOx1, miR155, miR183, and miR872 [288].

In adipose tissue of obese mice, miR221 and miR222 trigger hypoxia and the formation of miR27, which hampers production of PPARγ and C/EBPα [288].

In diabetes, concentrations of proinflammatory mediators, such as advanced glycation end products, PGhS2, cytokines IL6 and TNFSF1, and chemokines CCL2 and CX3CL1, rise, thereby favoring vSMC proliferation, redox stress, and vascular complications. In vSMCs of diabetic mice, expression of miR125b and MIR200 family members is upregulated, enhancing transcription of proinflammatory genes via an epigenetic process [288].

In diabetic subjects, miR34a and miR504 also play a role in vascular smooth myocyte dysfunction, whereas miR126 targets tissue factor and reduces thrombogenicity and miR191 impedes wound repair, as it hinders migration of fibroblasts and favors apoptosis of dermal fibroblasts [286].

The circular RNA Hipk3 (circHipk3) acts as a miR30a-3p sponge, thereby increasing VEGFc, Fzd4, and Wnt2 formation. Its production is upregulated in diabetic retinal ECs [387]. CircHIPK3 affects survival, proliferation, and migration, and hence the angiogenic capacity of retinal ECs. In vivo, circHIPK3 thus controls diabetic proliferative retinopathy; the silencing of circHIPK3 alleviates retinal vascular dysfunction. MicroRNAs Involved in Atherosclerosis

When atherosclerotic lesions become unstable, miR concentrations increase. MiR29 may contribute to inflammation and plaque destabilization [388]. Other microRNAs, such as miR92, miR126, miR145, and miR155, are implicated in the initiation of endothelial dysfunction and macrophage activity.

MiR155 has both anti- and proatherogenic effects according to the disease stage [286]. MiR150-5p, miR365a-3p, and miR1275 are linked to coronary atherosclerosis. The angiomicroRNAs miR378 and Let7b are upregulated in CD34+ + progenitor cells in response to MI.

In patients with atherosclerosis, especially with coronary stenoses and acute coronary syndrome (ACS),Footnote 80 miR133a, miR133b, miR208a, miR208b, and miR499, can serve as diagnostic and/or prognostic markers across different progression stages [389]. MiR1Footnote 81 and miR145bFootnote 82 are also ACS markers, with miR1 having a higher sensitivity for all types of MI and miR145 for ST segment elevation myocardial infarction (STEMI). In addition, miR1, miR21, miR122, miR134, miR14525, miR37026, and miR49931 correlate positively with CoAD severity. The greatest prognostic or diagnostic value is yielded by the miR132–miR150–miR186 combination. Both miR208a and miR208b, which are exclusively produced in CMCs, are the early detectable specific diagnostic MI markers with respect to cTnIn and cTnnT. MiR320a and miR49922 are also sensitive and specific MI diagnostic markers. For some investigators, only miR126,Footnote 83 miR223, and miR19736 provide an MI risk.

The identified miRs regulate endothelial function and angiogenesis (miR1 and miR133), vSMC differentiation (miR133 and miR145), communication between vEC and vSMC (miR145), apoptosis (miR1, miR133, and miR499), and CMC differentiation (miR1, miR133, miR145, miR208, and miR499), and repress cardiac hypertrophy (miR133). Their shared targets include mRNAs encoding CKI1a, ETS1,Footnote 84 Fscn1,Footnote 85 HCN4,Footnote 86 IGF1R,Footnote 87 LASP1,Footnote 88 PNP,Footnote 89 and Tagln2 [389].Footnote 90

Matrix metallopeptidases degrade collagens, elastin, proteoglycans, and glycoproteins, such as fibronectin and laminin. They are counteracted by TIMPs. In unstable atherosclerotic lesions, the myelopoietic growth factor CSF2,Footnote 91 which is involved in the development of mononuclear phagocytes only during inflammation, favors the TIMP3− phenotype of macrophages, which are more invasive and sensitive to apoptosis than TIMP3+ macrophages [391]. The density of TIMP3− macrophages is higher in unstable than in stable plaques. In macrophages, CSF2 reduces TIMP3 production via miR181b, thereby favoring plaque instability and aneurysm development [392]. Tissue inhibitors of metallopeptidases protect against the progression of atherosclerosis and AAAs.Footnote 92 In addition, miR181b, which is overexpressed in developed atherosclerotic plaques and AAAs, precludes elastin production by vSMCs. Moreover, miR181b is a potent regulator of NFκB signaling in the vascular endothelium, as it targets the KPNA4 (or IPOA3) gene, which encodes karyopherin-α4 (or importin-α3), required for the nuclear translocation of NFκB, thereby downregulating Kpnα4 and NFκB nuclear translocation in the vascular endothelium [391, 392].Footnote 93 MiR181b also favors the M2 phenotype in atherosclerotic lesions, as it also targets notch-1 [393], in addition to promoting development of NK cells from hematopoietic progenitor cells [394].

Some miRs (miR29b-3p, miR126-3p, miR145-5p, and miR155-5p) exhibit a transcoronary concentration gradient (TCG) within the coronary circulation between its entry (aortic orifices) and exit (venous coronary sinus; Table 2.22) [382]. They appear in the coronary circulation in the early stages of coronary atherosclerosis. They are related to vulnerable plaques observed by optical coherence tomography with the presence of thin-cap fibroatheroma [382]. On the other hand, miR29b-3p, miR126-3p, miR145-5p, and miR155-5p have negative TCGs in subjects without vulnerable plaques. Uptake of circulating atheroprotective miR126-3p and miR145-5p in the coronary vessels stabilizes atherosclerotic lesions. Platelets can contribute to transcoronary miR concentrations; miR223-3p, which is highly expressed in platelets, but weakly expressed in ECs, also has a negative TCG [382].

Table 2.22 MicroRNA (miR) transcoronary concentration gradients (TCGs) during the main evolution phases of coronary atherosclerosis (Source: [388])

Cardiomyocytes cultured under ischemic conditions release exosomes that promote angiogenesis via miR143 and miR222, the most abundant microRNAs in ischemic exosomes [395]. Intramyocardial delivery of ischemic exosomes improves neovascularization following MI. MicroRNAs Involved in Stroke

Two major categories of strokes include ischemic (∼87%) and hemorrhagic strokes, which are mainly caused by emboli and bleeding respectively. Ischemic stroke can result from carotid artery atherosclerosis, either from hypoperfusion due to high-grade stenosis or emboli upon plaque ulceration, even in relatively small lesions (∼20%) [396].

MicroRNAs can serve as markers to assess atherosclerotic plaque rupture and embolic stroke risk. MicroRNA expression is deregulated in human carotid plaques, causing lesion growth, instability, and rupture (Tables 2.23, 2.24, 2.25, and 2.26).

Table 2.23 MicroRNAs and atherogenesis (Part 1; Source: [396]; asterisk [miR ] microRNAs regulated by hemodynamic stress, + [−] miR protecting [amplifying] effect on atherosclerogenesis)
Table 2.24 MicroRNAs and atherogenesis (Part 2; Source: [396]; asterisk [miR ] microRNAs regulated by hemodynamic stress, + [−] miR’s protective [amplifying] effect on atherosclerogenesis)
Table 2.25 Targets and effects of main microRNA species involved in atherosclerosis (Part 1; Source: [396]; ABC ATP-binding cassette transporter, BCL B-cell lymphoma protein, DNMT DNA methyltransferase, FABP fatty acid-binding protein, LDLR low-density lipoprotein receptor, LPL lipoprotein lipase, MMP matrix metallopeptidase, Sema semaphorin, SIRT sirtuin, TGF transforming growth factor, TIMP tissue inhibitor of metallopeptidase)
Table 2.26 Targets and effects of main microRNA species involved in atherosclerosis (Part 2; Source: [396]; AT1R angiotensin-2 type-1 receptor, Col collagen, CKI cyclin-dependent kinase inhibitor, ELk ETS-like transcription factor, ETS E26 transformation-specific sequence transcription factor, IRAK interleukin-1 receptor-associated kinase, IRF interferon regulatory factor, KLF Krüppel-like factor, OSBPL oxysterol-binding protein-like protein, PTGS prostaglandin endoperoxide synthase [prostaglandin-G/H synthase and cyclooxygenase], ScaR scavenger receptor, SCFR stem cell factor receptor, TLR toll-like receptor, TOR target of rapamycin, TNFSF TNF superfamily member, TRAF TNFR-associated factor, vcam vascular cell adhesion molecule, VEGF vascular endothelial growth factor)

After a stroke, expression of Let7f, miR15b, miR126, miR142-3p, miR186, miR519e, miR768-5p, and miR1259 is downregulated [396]. Circulating concentrations of miR21 and miR221 are higher in patients with stroke and carotid atherosclerosis than in healthy controls. Plasmatic concentrations of miR106b-5p and miR4306 increase after stroke, whereas those of miR320d and miR320e decline. In peripheral blood mononuclear cells (lymphocytes and monocytes) of patients with stroke, intracellular concentrations of Let7i, miR19a, miR122, miR148a, miR320d, and miR4429 are lower and those of miR363 and miR487b are elevated. Blood concentrations of miR151a-3p and miR140-5p increase, whereas that of miR18b-5p decreases. Concentrations of miR100, miR125a, miR127, miR133a, miR133b, miR145, and miR221 are higher in symptomatic than in asymptomatic plaques examined after endarterectomy. MiR145 concentration is higher in carotid plaques from hypertensive patients; plasmatic miR92a concentration is higher in hypertensive patients. Prognosis is better in patients with a high miR210 level in peripheral blood leukocytes, especially if measurement is combined with those of fibrin degradation product and IL-6 [396]. An elevated miR17 concentration predicts early stroke recurrence. A high exosomal miR223 level in stroke patients is related to a poor outcome.

Acute ischemic stroke is associated in a small sample of patients with abnormal blood concentrations of miR125a-5p, miR125b-5p, and miR143-3p, concentrations of which increase significantly, platelets rather than cerebral cells being the major source of these microRNAs [397].

2.2.2 Circular RNAs

Circular RNAs (circRNAs) can serve as miR sponges, which sequester miRs, and nuclear transcriptional regulators. They can be generated from back-spliced exons or intron-derived RNAs.

Platelets and RBCs contain large amounts of ubiquitous cell type-unspecific circular RNAs. Circular RNAs commonly derive from exons of protein-coding genes via backsplicing [360]. On the other hand, some arise from intronic or intergenic regions via alternative splicing. They generally localize to the cytoplasm. RNA-binding proteins, such as quaking homolog and muscleblind-like protein, affect the amount of circRNA in cells, as the interaction of RNA-binding proteins and each RNA end facilitates the formation of a circle. Quaking elicits circRNA genesis.

In the heart, highly expressed circRNAs correspond to key cardiac genes such as desmin (DES), dystrophin, four and a half LIM domain-containing protein-2 (FHL2), myoglobin (MB), cardiac myosin heavy chain-7 (MYH7), cardiac myosin regulatory light chain-2 (MYL2), cardiac ryanodine receptor-2 (RYR2), Na+–Ca2+ exchanger NCX1 (SLC8A1), tropomyosin-1 (TPM1), titin (TTN), and cardiac type-2 troponin-T (TNNT2) [398]. The longest human transcript TTN generates up to 415 different exonic circRNA isoforms.

In vascular ECs, hypoxia upregulates many circRNAs, such as proangiogenic znf292,Footnote 94 caff1,Footnote 95 cdennd4c,Footnote 96 and slightly csrsf4,Footnote 97 but downregulates cthsd1 [399].Footnote 98 Cznf292 promotes angiogenesis via AP1, E2F1, HIF1, NFκB, SP1, STAT3, and STAT5.

In atherosclerotic plaques, the atheroprotective circANRIL regulates ribosome genesis in vSMCs and macrophages [399].

Atherosclerosis and post-stenting restenosis are characterized by the dedifferentiation of vascular smooth myocytes and intimal hyperplasia. Differentiated vSMCs synthesize SMC-specific contractile proteins and related molecules, such as myosin heavy chain MHC11, actin Actα2, transgelin, and calponin, under control of the transcription factor serum response factor and its cofactor myocardin, which maintain SMC differentiation via the transcription factors and regulators Nkx3.1 and Nkx3.2, GATA4 and GATA6, SMADs, and Prx1, and the cysteine-rich LIM-only proteins, CRP1 and CRP2, which are associated with SRF and GATA proteins [400]. PTen interacts with SRF in the nucleus to maintain the differentiated SMC phenotype. In addition, TGFβ, notch, RhoA, and P38MAPK promote SMC differentiation via transcriptional regulators that interact with SRF–myocardin. Furthermore, among epigenetic factors, histone modifications promote SMC contractile gene expression via SRF binding to genes, such as histone modifiers that acetylate (e.g., KAT3b) and methylate histones flanking CArG boxes in the MYH11 and ACTA2 genes. Short and long nonprotein-coding RNAs stimulate or preclude differentiated SMC phenotype. Components of the miR143–miR145 cluster target transcriptional repressors such as KLF4. On the other hand, miR21 and components of the miR221–miR222 cluster favor SMC dedifferentiation via growth repressors (e.g., PTen). Dedifferentiation of SMCs leads to a highly proliferative, migratory, and inflammatory phenotype characterized by the downregulated transcription of SMC-specific genes and upregulated production of inflammatory mediators and matrix components.

Transforming growth factor-β induces the formation and cleavage of transmembrane neuregulin-1.Footnote 99 The extracellular EGF-like domain-containing domain (Nrg1ECD) binds to nearby receptors such as receptor protein Tyr kinases of the EGFR family, thereby acting as an auto- and paracrine messenger, in particular, during cardiovasculogenesis. Neuregulin-1, which has 11 isoforms, binds to HER3 and HER4 [401]. Released soluble Nrg1 suppresses SMC proliferation via the HER receptor and then activates ERK1 and ERK2 [402].

On the other hand, in response to TGFβ1, the intracellular domain of neuregulin-1 (Nrg1ICD) translocates to the nucleus and interacts with the transcription factor Ikaros family zinc finger protein IkZF1 (ZnFn1a1 or simply Ikaros) [402].Footnote 100 In the nucleus, the Nrg1ICD–IkZF1 complex binds to the first intron of the ACTA2 gene and launches the formation of the circRNA, circActa2, thereby promoting Actα2 production, as it operates as a sponge for miR548f-5p repressing Actα2 synthesis. In human intimal hyperplasia, expression of circActa2 decays and miR548f-5p dissociates from circActa2. On the other hand, PDGFbb decreases NRg1 expression.

2.2.3 YRNAs

YRNAs are small RNA species, the size of which ranges from 112 (Y1RNA), 101 (Y3RNA), 98 (Y4RNA), to 84 nucleotides (Y5RNA). They originate from four genes (RNY1 and RNY3–RNY5) and about 900 pseudogenes [360]. Full-length YRNAs reside in the nucleus or cytoplasm. They are involved in the initiation of DNA replication and ribosomal RNA quality control. They are components of Ro ribonucleoproteins. Fragments of YRNAs generated during cell stress or apoptosis circulate in the bloodstream, 5 fragments being much more abundant than 3 fragments for most RNY gene products.

2.2.4 Long Nonprotein-Coding RNAs

In addition to proteins and microRNAs, long nonprotein-coding RNAs (lncRNAs; content> 200 nt), lack significant open reading frames and hence a protein-coding potential. Nevertheless, lncRNAs can act as cofactors that permit the interaction of a transcription factor with a gene promoter, thereby regulating gene transcription. In addition, they can interact with histone modifiers to repress or activate gene expression.

LncRNAs can be used as markers to identify and categorize individuals at risk for developing any type of cardiovascular pathological condition. They can hence support prevention, diagnosis, surveillance, and prognosis of cardiovascular maladies and help to adapt lifestyle and eventually optimize treatments.

Numerous genes encoding lncRNAs have been identified [376]. LncRNAs can be categorized into different groups of transcripts (Vol. 11, Chap. 2. Nonprotein-Coding Transcripts and Regulation of Protein-Coding Gene Transcription).

  1. 1.

    Sense lncRNAs overlap exons or introns of messenger RNAs.

  2. 2.

    Antisense lncRNAs originate from the opposite strand of protein-coding genes; their transcription start site localizes downstream from that of the coding gene; these RNAs transcribed in the antisense direction often display at least partial overlap with the coding sequence of the corresponding mRNA.

  3. 3.

    Long intergenic ncRNAs are encoded between coding genes and do not overlap with exons of other genes; they are transcribed independently.

  4. 4.

    Circular lncRNAs form closed loops derived from the splicing of protein-coding RNAs.

  5. 5.

    Enhancer RNAs are located to genomic loci that activate gene expression.

  6. 6.

    Bidirectional RNAs are transcribed from the same promoter as coding genes, but in the opposite direction.

LncRNA genesis encompasses chromatin remodeling, lncrna gene transcription, and generally, but not always, transcript 5-capping, polyadenylation, and alternative splicing [287].

Chromatin that packages DNA controls access to genetic information; it must be remodeled for gene transcription. Messenger RNAs, synthesized in the nucleus and packaged by RNA-binding proteins that form ribonucleoparticles, are exported to the cytoplasm.

Certain enzymes change states of DNA methylation and histone modifications, whereas ATP-dependent chromatin-remodeling complexes modify nucleosome composition or positioning.

LncRNAs contribute to the regulation of gene expression, as they are involved in epigenetic, transcriptional, or post-transcriptional regulation. They can partner with DNA- and histone-modifying enzymes, such as DNA methyltransferase DNMT1 and histone demethylase KDM1a, polycomb repressive complex and mixed lineage (myeloid–lymphoid) leukemia (MLL) factor Footnote 101 activatory complex, in addition to nucleosome remodelers such as the Swi/SNF-like BAF complex [404]. They can also serve as scaffolds.

Nuclear lncRNAs are involved in epigenetic and transcriptional regulation of neighboring (in cis) or distant genes (in trans). Cis-acting lncRNAs can regulate lncRNA transcription. Trans-acting lncRNAs operate as recruiters or decoys of chromatin modifiers and transcription factors to activate or silence gene transcription. They can interact with RNA-binding proteins, such as members of the polycomb and trithorax groups. LncRNAs that recruit the polycomb repressive complex, a chromatin remodeler, provoke gene silencing. Other lncRNAs sequester RNA-binding proteins and transcription factors. On the other hand, some lncRNAs enhance protein-coding gene transcription. Activating nonprotein-coding RNAs have enhancer-like activity; they activate neighboring genes using a mechanism involving DNA looping between the lncRNA and its target gene [287]. In addition, lncRNAs can bind to RNA polymerase-2, thereby regulating the transcriptional machinery.

Cytoplasmic lncRNAs control mRNA splicing, translation, stability, and degradation, as they bind to mRNAs or proteic components of ribonucleoproteic complexes. Additionally, LncRNAs can act as decoys or sponges for proteins or microRNAs, preventing their activity, and they can serve as templates for small peptide synthesis. Some lncRNAs undergo a posttranscriptional processing. The lncRNA neat1 men𝜖Footnote 102 and neat2 (malat1),Footnote 103 which mainly localize to the nucleus, are processed into menRNA and mascRNAFootnote 104 respectively, the latter lodging exclusively in the cytoplasm [406]. Both neat2 and its product mascRNA serve as immunoregulators.

Unlike microRNAs and YRNA fragments, which mainly lodge in the cytosol, lncRNAs are predominantly retained in the nucleus. Whereas microRNAs and YRNA fragments are secreted in microvesicles with cytoplasmic components, even in the absence of injury, lncRNAs are released upon cellular damage with nuclear material [360]. In general, because their longer sequence is more vulnerable to cleavage and less protected by RNA-binding proteins, lncRNAs are more susceptible to ribonuclease than small ncRNAs.

LncRNAs cross the plasma membrane, their egress being regulated by ceramide.Footnote 105 They can thus reside in body fluids (e.g., blood, urine, and breast milk) [376]. They are observed in exosomes, microvesicles, and apoptotic bodies and are linked to RNA-binding proteins, such as nucleolin, nucleophosmin NPm1, and argonaute protein Ago2, a factor of the RINC, LDLs, and HDLs. A substantial number of circulating ncRNAs are associated with ribonucleoproteins.

Chromatin remodeling determines, at least partly, transcriptome programming during cardiogenesis and its reprogramming during pathogenesis. Improper re-organization of epigenetic marks leads to aberrant gene expression and organ dysfunction. LncRNAs participate in epigenetic regulation; they are designated EpilncRNAs.

For example, HOX transcript antisense intergenic RNA (hotair) binds to PRC2 and promotes methylation of histone H3 at Lys27 trimethylation (H3K27me3) at the promoter region of target genes, causing gene silencing, in addition to the demethylase KDM1a that suppresses methylation of histone H3 at Lys4 (H3K4) carried out by the MLL complex [407].

Antisense long nonprotein-coding RNA in the CDKN2B gene locus in the chromosomal 9p21 region suppresses synthesis of the cell cycle regulator CKI2b. It is linked to AAA, myocardial infarction, and stroke [408].

Hypoxia upregulates some lncRNAs. Hypoxia-induced endoplasmic reticulum stress (ERS)-regulating lncRNA (hyperlnc)Footnote 106 enables expression of human pericyte markers, such as PDGFRβ, DES, α-smooth muscle actin, and neural and glial antigen NG2 [409]. In addition, hyperlnc supports the connection between pericytes and ECs, thereby reducing endothelial permeability. On the other hand, hyperlnc deficiency favors ERS, ERS-related transcription factors being activated, and reduces the viability and proliferation of pericytes and primes their dedifferentiation. Therefore, hyperlnc modulates pericyte differentiation, proliferation, and recruitment to ECs.

Pericytes stabilize the microvasculature (arterioles, venules, and lymphatics). They can be a source of regenerative cells that participate in the repair after injury and ischemia [408]. A subset of pericytes can serve as a reservoir of multipotential mesenchymal stem cells that may replace lost CMCs or other cardiovascular cell types.

Hyperlnc concentration is markedly reduced in the myocardium of failing hearts, which can be linked to abnormal microvasculature and fibrosis [409]. In addition, hyperlnc expression correlates significantly with pericyte markers in human lungs from patients with idiopathic pulmonary arterial hypertension.

Liver-expressed LXR-induced sequence lncRNA (lexis) regulates cholesterol efflux and synthesis.

The role played by 16401 lncRNAs was assessed in seven types of human cell lines (six transformed cell lines and one human-induced pluripotent stem cells) [410]. The function of most of the tested lncRNAs was specific to a given cell type and often limited to a single cell type. A total of 499 lncRNAs influence cell proliferation, exclusively for a single cell type, among which 372 and 299 have loci distal from a protein-coding gene or mapped enhancer, respectively [410].

Endotheliocytes express relatively high concentrations of the lncRNAs neat2,Footnote 107 tug1,Footnote 108 meg3,Footnote 109 linc00657, and linc00493 [411]. Production of the lncRNAs neat2, tug1, meg3, and linc00657 increases in ECs exposed to hypoxia, but not that of linc00493. Neat2 controls both epigenetic gene regulation and RNA splicing. It promotes proliferation of ECs, but prevents their migration. It supports expression of endothelial cyclins CcnA2, CcnB1, and CcnB2 and hampers that of CKI1a and CKI1b. It interacts with CBx4,Footnote 110 assisting recruitment of coactivators to the promoters of cell cycle control genes, which are linked to an increased number of transcriptionally active histone marks. Endothelial Long Nonprotein-Coding RNAs

Endotheliocytes respond to local and systemic stimuli and can change from quiescence to activation, then becoming a source of prooxidant, proinflammatory, vasoconstrictory, and clotting mediators. LncRNAs operate at the onset and after EC activation. The formation of 27 lncRNAs is markedly changed (two-fold), but among them, that of a single one, the PDZD2-206 transcript (ENST00000509256.1), which encodes PDZ domain-containing protein PDZD2,Footnote 111 is downregulated [412]. Many genes with a regulatory function are coexpressed with lncRNAs (e.g., CDK13, EIF3A, HNRNPH2, LHX2, PDSS2, SMEK2, RIF1, SPRY2, STAU2, Wdr20, and WDR42A [Dcaf8]).

The proangiogenic lncRNA linc00323-003Footnote 112 and mir503hgFootnote 113 are induced in ECs subjected to hypoxia [413]. However, linc00323-003 elicits tube formation, whereas mir503hg does not markedly affect tube formation.

The lncRNA meg3Footnote 114 represses the production of VEGFa and VEGFR1 [413]. Its expression increases in senescent ECs, precluding their angiogenic activity.

The lncRNA neat2Footnote 115 is proangiogenic [413]. It upregulates VEGF expression. In particular, it favors tumor angiogenesis, as it promotes FGF2 production in tumor-associated macrophages.

Formation of the lncRNA igf2asFootnote 116 increases in myocardial microvascular ECs of T2DM rats, impeding cell proliferation via downregulation of IGF2 and VEGF expression [413].

The lncRNA miatFootnote 117 is an angiogenic activator [413]. Hyperglycemia can launch miat formation in microvascular and retinal ECs.

The epigenetically controlled endothelial nuclear lncRNA mantis,Footnote 118 the gene of which lodges in the antisense strand of an intron of the gene encoding annexin-A4, is upregulated during angiogenesis; it maintains the endothelial angiogenic phenotype [414]. Its synthesis is prevented by the histone demethylase KDM5b. Its production is downregulated in patients with idiopathic pulmonary arterial hypertension. It favors not only angiogenic sprouting, but also alignment of ECs subjected to hemodynamic stress. It interacts with SMARCa4 (BRG1), the catalytic subunit of the Swi/SNF-related, matrix-associated, actin-dependent regulator of chromatin, thereby promoting transcription of the NR2F2, Smad6, and SOX18 genes.

In ECs, the lncRNA sencrFootnote 119 induces cell proliferation and migration in addition to differentiation (i.e., embryonic stem cell commitment into ECs) and hence angiogenesis [415]. It stimulates EC migration, as it upregulates proangiogenic chemokines CCL5 and CX3CL1. It also governs the differentiation status of vascular smooth myocytes. This EC- and vSMC-enriched lncRNA exists in a full-length (sencrV1) and alternatively spliced variant (sencrV2). Long Nonprotein-Coding RNAs and Mitochondrial Function

Mitochondria serve as controllers of the cellular redox status and as stress sensors via proteins encoded by mitochondrial and nuclear DNA and integrators of stressors.Footnote 120 These hubs also integrate cues from several metabolic pathways (ATP production and fatty acid and amino acid metabolism), modulating nuclear gene expressionFootnote 121 implicated in cell survival and growth [368].

Mitochondria and the nucleus mutually communicate to adapt their respective activities to the environment. The anterograde signaling, that is, the nuclear-to-mitochondrial control, modulates mitochondrial genesis and activity according to cellular needs. The nuclearly encoded mitochondrial proteome is regulated in a coordinated manner by transcription factors (e.g., NRF1–NRF2), among which are some nuclear receptors (e.g., ERRs and PPARs) and their cofactors (PGC1α–PGC1β). These effectors are regulated by sensors of changes in metabolic conditions.Footnote 122 Under nuclear stress (e.g., DNA damage), the anterograde signaling lowers mitochondrial metabolism.Footnote 123

On the other hand, mitochondria signal their functional state and can trigger a compensatory cellular response. The retrograde signaling launches a metabolic reprogramming, ultimately leading to cell adaptation or death. In addition, when mitochondria are severely damaged, the retrograde signaling engages cellular stress responses without nuclear involvement, such as the intrinsic apoptotic pathway [368].

Mitochondrial function is monitored via their products, which act as signaling effectors that modulate the activity of regulators (e.g., sirtuins, HIF1α, AMPK, TOR, and calmodulin).Footnote 124

Redox stress inactivates TOR, the master cytosolic modulator of protein synthesis. In addition, TOR controls mitochondrial genesis and activity; this hub integrates antero- and retrograde signaling [368].

Nonprotein-coding RNAs act as messengers between the nucleus and mitochondria, hence contributing to the synchronization of cellular processes. The term mitolncRNAs (MtlncRNAs) refers to lncRNAs lodging inside the mitochondrion, which are encoded either by the mitochondrial genome (category 1)Footnote 125 or in the nucleus and subsequently enter the mitochondrion (category 2; Table 2.27) [368].Footnote 126

Table 2.27 Long nonprotein-coding RNAs (lncRNAs) and mitochondrial function (Source: [368]; GAS growth arrest-specific noncoding, single-stranded RNA, lipcar long intergenic noncoding RNA predicting cardiac remodeling, sammson survival-associated mitochondrial melanoma-specific oncogenic long intergenic noncoding RNA, sncmtRNA sense nonprotein-coding mitochondrial RNA)

In addition to functioning as messengers, lncRNAs can also indirectly modulate mitochondrial function via regulation of metabolic components, such as AMPK, TOR, HIF1α, and sirtuins. The TOR kinase regulates synthesis of mitochondrial proteins encoded by the nuclear genes such as MtTFa, components of ETCcomplex-I and -V , and mitochondrial ribosomal proteins. The gas5 lncRNA promotes growth arrest upon TOR inhibition. The hif1as lncRNA is associated with angiogenesis. The hypoxia-induced lincrnap21 disrupts the connection between VHL and HIF1α, hence impeding HIF1α ubiquitination and proteasomal degradation. Long Nonprotein-Coding RNAs in Adverse Cardiac Remodeling

As CMCs are particularly enriched in mitochondria, mitochondrial lncRNAs are released during myocardial infarction [360].

Three lncRNAs, CoroMarker,Footnote 127 bat5,Footnote 128 and il21ras1,Footnote 129 are CoAD markers (Table 2.28) [420]. In a population of patients suffering from chest pain, CoroMarker correlates with genes involved in atherosclerosis, such as IL15RA and ADORA1, which encode IL15 receptor-α and adenosine receptor-A1, respectively. On the other hand, CoroMarker correlates negatively with genes implicated in apoptosis and redox stress, such as BAX and GSTT1, which encodes glutathione Stransferase GSTθ1. CoroMarker plays an important role in inflammation involved in atherogenesis, as it favors expression of the proinflammatory cytokines IL1β, IL6, and TNFSF1 [420].

Table 2.28 Long nonprotein-coding RNA (lncRNA) markers in CVD (Source: [376]; lipcar long intergenic ncRNA predicting cardiac remodeling, myheart myosin heavy-chain-associated RNA transcript, sencr smooth muscle and endothelial cell-enriched migration and differentiation-associated lncRNA, uca1 urothelial carcinoma-associated lncRNA-1)

LncRNAs include regulators of CMC differentiation, such as braveheart and fendrr,Footnote 130 and of cardiac remodeling, such as myheart or chast.Footnote 131

In hypertrophic cardiomyopathy, BRG1,Footnote 132 which interacts with multiple transcription regulators, in addition to several HDACs and polyADPribose polymerases, is activated. The epigenetic regulator BRG1 induces MYH7 gene transcription and represses that of the MYH6 gene, which encode fetal β- and adult α-myosin heavy chain, respectively.Footnote 133 Pathological cardiac stress activates the BRG1–HDAC2–HDAC9–PARP1 chromatin repressor complex to complex on MYH6 and MYH7 promoters for antithetical control, thereby precluding transcription of the lncRNA myheart (mhrt; myosin heavy chain [MYH7]-associated RNA transcript) in the heart, activating aberrant MYH7 gene expression and repressing MYH6 gene transcription, hence re-inducing fetal gene expression that is linked to maladaptive hypertrophy and that can evolve to HF.

The lncRNA cardiac hypertrophy-related factor (chrf) regulates MYH7 expression, as it acts as a sponge for antihypertrophic miR489 [384]. Whereas miR489 reduces adverse hypertrophy initiated by angiotensin-2 via MyD88, chrf downregulates miR489 expression [423].

Cardiac-specific myheart, which abounds in adult CMCs, protects the heart from adverse remodeling. Its concentration is elevated after myocardial infarction [376].

The most abundant isoform. Mhrt779, is a potent inhibitor of adverse cardiac hypertrophy. Alternative splicing of MYH7 antisense transcripts engenders a cluster of Mhrts (size 709–1147 nucleotides) [424]. The mhrt lncRNAs are linked to a transcriptional start site overlapping, but in the opposite direction, the MYH6 promoter, and the coding sequence extends into that of the MYH7 gene; hence, Mhrts correspond to both promoter-associated lncRNAs and antisense lncRNAs with respect to MYH6 and MYH7 respectively [384]. In addition, these alternatively spliced antisense transcripts are implicated not only in cis-regulation at the MYH6–MYH7 locus, but also in the trans-regulation of other cardiac genes.

The Mhrt splice variants abound in the adult myocardium (but in neither endo- nor the epicardium) [424]. Cellular stress activates the BRG1–HDAC–PARP chromatin repressor complex and prevents Mhrt transcription, enabling cardiomyopathy development. In a given cell, the BRG1 complex inhibits both MYH6 and Mhrt and reactivates MYH7 expression. On the other hand, the Mhrt spliced isoforms tether to and antagonize the chromatin remodeler and helicase BRG1, which can connect to mhrt and chromatinized (but not naked) DNA, preventing cardiac adverse hypertrophic gene reprogramming. Mhrt directly interacts with BRG1 helicase domain [424], as a decoy to regulate its chromatin remodeling activity, similar to braveheart, which operates as a decoy for the PRC2 complex [383].

Cardiac apoptosis-related lncRNA, which was identified in CMCs exposed to anoxia, is another sponge for miR539 that regulates PHB2 expression [425]. The prohibitin complex, with its two subunits (Phb1–Phb2), which localizes to the inner mitochondrial membrane, operates in mitochondrial genesis. In addition, Phb2 is an estrogen receptor-binding protein that represses gene transcription and a regulator of mitochondrial fission and fusion dynamics. Its production declines with the increased formation of miR539, causing mitochondrial fission and CMC apoptosis.

In addition to carl and chrf, hulcFootnote 134 lincMD1Footnote 135 LncRNA H19, which contributes to cardiofibroblast proliferation and fibrosis via repression of the DuSP5–ERK1/2 axis [428], and the circular RNAs cdr1asFootnote 136 also sequester miRs.

The long nonprotein-coding RNA cardiac hypertrophy-associated epigenetic regulator (chaer) acts at the onset of cardiac hypertrophy via a stress-induced transient interaction between chaer and the polycomb repressor complex PRC2, which depends on TOR [430]. Chaer recognizes the epigenetic modulator KTM6a, a PRC2 component, and sequesters PRC2, which forms repressive histone marks, thereby hampering histone H3 Lys27 methylation at the promoter regions of genes involved in cardiac hypertrophy and enabling prohypertrophic gene expression [407]. Interaction between chaer and PRC2 is transiently induced after hormone or stress stimulation via TORC1, and causes epigenetic reprogramming.Footnote 137 Inhibition of chaer production in the heart before, but not after, the onset of hypertension attenuates cardiac hypertrophy.

The cardiac hypertrophy-associated transcript chast is upregulated in maladaptive cardiac hypertrophy [432]. Chast impedes the autophagy regulator PlekHm1Footnote 138 impeding CMC autophagy.

The mitochondrial long intergenic ncRNA predicting cardiac remodeling lipcarin (i.e., lncRNA lipcar or uc022bqs.1) is downregulated in an early phase after MI but is upregulated during later stages [433]. It has a strong prognostic power for the future development of detrimental cardiac remodeling, HF after MI, and death.

Concentration of urothelial carcinoma-associated transcript-1 (uca1), an indicator of pulmonary cancer, declines shortly after MI and increases 3 days after cardiac injury. Concentration of miR1, which may regulate uca1 expression, follows an opposite time course [376].

Blood concentrations of uc004cov4 and uc022bqu1 are higher in hypertrophic obstructive cardiomyopathy than in hypertrophic non-obstructive cardiomyopathy and healthy subjects [376].

The long nonprotein-coding RNAs wisperFootnote 139 abounds in cardiofibroblasts and regulates their survival, proliferation, and migration in addition to extracellular matrix protein deposition and hence cardiac fibrosis after injury [434]. Repression of wisper attenuates fibrosis after MI. Wisper cooperates with T-cell intracellular antigen TIA1-related protein (TIAR), an RNA-binding protein involved in the regulation of alternative pre-mRNA splicing, to control the expression of a profibrotic form of procollagen lysine 2-oxoglutarate 5-dioxygenase PLOD2,Footnote 140 which is implicated in collagen crosslinking and stabilization of the matrix.

2.2.5 Ribosomal RNA

Ribosomal RNA participates in protein synthesis. It is also detected in the blood circulation. It is released upon tissue damage and cell death.

Extracellular ribosomal RNA may play several pathophysiological roles in atherosclerotic plaques, as it can increase vascular permeability, initiate blood coagulation and hence thrombus formation, recruit leukocytes and support a proinflammatory phenotype of monocytes and macrophages, and favor CMC death [376].

2.3 Clinical Types of Markers

Markers of diseases can be classified into three main groups:

  1. 1.

    Screening markers for individuals who have not yet developed any apparent disease (i.e., for early diagnosis and evolution prevention), as these markers modulate diet and activity in addition to potentially beneficial drug effects

  2. 2.

    Diagnostic markers in subjects in whom advanced-stage disease is already suspected

  3. 3.

    Prognostic markers in patients who have the disease

2.3.1 Screening

Potential screening markers comprise molecules associated with [435]:

  1. 1.

    Inflammation, such as CRP (or pentraxin-1; Sects. and, IL-6 (Sect., and secreted and lipoprotein-associated phospholipase-A2.

  2. 2.

    Hemostasis, such as fibrinogen and plasminogen-activator inhibitor-1 (or serpin-E1)

  3. 3.

    Neurohormonal control, such as renin and BNP

  4. 4.

    Insulin resistance, such as insulin and hemoglobin-A1c.Footnote 141

  5. 5.

    Endothelial dysfunction, such as homocysteine and albuminuria (urinary albumin).

  6. 6.

    Myocardial damage, such as cardiac FABP (HeFABP), which diffuses rapidly into the circulation after CMC injury, and growth differentiation factor GDF15, which also rises sharply after myocardial ischemia

Screening tests incorporate two features: sensitivity and specificity. Sensitivity and specificity refer to the proportion of diseased persons with a positive test and to the proportion of healthy individuals with a negative test, respectively.

Markers fulfill several criteria:

  • Marker expression in the vascular tissue correlates with blood concentration.

  • Marker blood level is related to structural and functional changes.

  • Its method of identification is easy, reproducible, and has a low cost.

  • The marker yields good sensitivity and specificity in detecting the pathological condition. Hemostasis Factors

Many components of hemostasis, such as fibrinogen, Ddimer, coagulation factor-VIII, plasmin–antiplasmin complexes, and von Willebrand factor, in addition to adamts13, correlate with an increased risk of coronary atherosclerosis complications, stroke, and mortality [436].

Von Willebrand factor (vWF) carries coagulation factor-VIII and promotes platelet adhesion and aggregation at sites of vascular injury. Its activity depends on the metallopeptidase adamts13, which cleaves large and highly procoagulant vWF multimers into smaller and less procoagulant forms. Deficiency in vWF quantity or activity causes bleeding in von Willebrand disease. Severe deficiency in adamts13 engenders the clotting thrombocytopenic purpura. Elevated plasmatic vWF and reduced adamts13 concentrations provoke a procoagulant state, and hence the combination of the two is associated with an increased risk of atherosclerosis complications [437]. In addition, both vWF and adamts13 are dysregulated in inflammation. Periodontitis

Periodontitis is a chronic inflammatory disease caused by bacterial infections that destroy the tissue between the tooth surface and gingiva and provoke the loss of connective tissue attachment and teeth in addition to erosion of alveolar bone, which defines mild and moderate (radiographic bone loss 21–44%) or severe periodontitis (< 66% remaining bone).

Periodontitis is more commonly observed in patients with atherosclerosis and its acute complications (MI and stroke) along with abdominal aortic aneurysm [438]. The risk for coronary atherosclerosis increases in these individuals.

2.3.2 Diagnosis

CRP, brain natriuretic peptide, urinary albumin, renin, and homocysteine are predictors of cardiovascular events with possible high death risk, but they moderately yield supplementary prediction in addition to known risk factors. In fact, markers often provide information that correlates with known data. Troponin, Myosin-Binding Protein-C, and CyR61

In emergency departments, triage of patients with acute chest pain relies on the measurements in the blood circulation of cardiac troponins cTnnI or cTnnT, as only a small proportion of patients with acute myocardial infarction (AMI) have ST segment elevation on ECG trace. After the onset of myocardial ischemia, CMC death can occur within 15 min and necrosis within 4–6 h [439].

The fundamental contractile unit of the CMC, sarcomere, comprises thick (myosin) and thin filaments (actin; Vol. 5, Chap. 5. Cardiomyocytes). The thin filament is a helix of two filamentous actins, the groove of which contains TPM, to which troponin is attached. The troponin heterotrimer controls the Ca2+-dependent interaction of actin and myosin:

  1. 1.

    Inhibitory cTnnI prevents actin–myosin cross-bridge formation in the absence of Ca2+ ions.

  2. 2.

    Tropomyosin-bound cTnnT links TPM on actin to the troponin complex.

  3. 3.

    Calcium-binding cTnnC regulates activation of actin.

Troponins are synthesized in fast- (ftTnns encoded by the TNNC2, TNNI2, and TNNT3 genes) and slow-twitch skeletal (stTnns encoded by the TNNC1, TNNI1, and TNNT1 genes) and cardiac myocytes (cTnns encoded by the TNNC1, TNNI3, and TNNT2 genes]). Hence, troponins TnnI and TnnT are selectively produced in the heart, but not TnnC, which is shared with the slow-twitch skeletal muscle. Both cTnnI and cTnnT are distinguished by regions of different amino acid sequences.

The TNNT2 mRNA generates multiple alternatively spliced transcripts encoding different proteins (cTnnT1–cTnnT4), cTnnT3 being the dominant isoform in the normal adult heart [439].

During embryo- and fetogenesis, stTnnI is expressed in the heart instead of cTnnI. Troponin fetal isoforms, the expression of which is downregulated postnatally, can be re-expressed postnatally under certain pathological circumstances such as chronic HF. The cTnnT2 subtype is expressed to a greater extent than cTnnT3 in failing hearts.

Cardiac troponins commonly serve as markers for acute myocardial necrosis. Both cTnnI and cTnnT are released in free forms and cleared by cardiac lymphatics. They are also liberated as noncovalent binary (cTnnI–cTnnT and predominantly cTnnI–cTnnC dimers) and ternary complexes (cTnnT–cTnnI–cTnnC trimer) [439]. Moreover, all troponin isoforms undergo redox modifications and circulate as oxidized and reduced forms. Troponin is also released from the sarcomere both as intact proteins and degradation products after proteolysis by calpain-1, caspase, or MMP2 [439]. These enzymes also reside in blood and the circulating cTnn complexes can also be degraded within blood.

However, cTnn concentrations are commonly elevated in acute, reversible or irreversible, myocardial injury and are thus unrelated to necrosis linked to ACS, in addition to chronic diseases (e.g., chronic HF [myocardial strain], diabetes mellitus, pulmonary arterial hypertension, stable coronary artery disease, and chronic kidney disease, especially end-stage renal disease), along with extreme exercise [439]. Cardiac troponins thus serve as organ-specific, disease-nonspecific markers.

In addition, cTnnI and cTnnT are released slowly. Cardiac myosin-binding protein-C (MyBPc3),Footnote 142 another marker of cardiac injury, is more abundant than cardiac troponins and is released more rapidly after MI. Its blood concentration rises and falls more rapidly than those of cTnnT and cTnnI [440].

Known diagnostic markers include cardiac troponin-I and -T for MI and B-type natriuretic peptide for HF.

In mouse HF and human cardiomyopathies, accumulation of branched-chain α-keto acids (BCKAs) and associated defective catabolism of branched-chain amino acids (BCAAs) are markers of metabolic reprogramming [441]. The transcription factor KLF15 regulates BCAA catabolism in the heart. Impaired BCAA catabolism causes redox stress in response to hypertension. Elevated BCKA concentrations provoke superoxide production in mitochondria.

Cardiac-specific troponins are characterized by better specificity and sensitivity than that of the cardiac marker CK-MB (which combines two isozymes SkMCK [muscle] and BrCK [brain]). Elevated troponin level is a CoAD diagnosis criterion that can require early revascularization. However, blood troponins are not necessarily augmented before 12 h after the onset of symptoms.Footnote 143

Concentration of MyBPC3 is significantly higher in patients with acute myocardial necrosis than those without AMI [440]. The discriminatory power of MyBPC3 (> 10 ng/l) for AMI is similar to that of high-sensitivity troponins hscTnnT and hscTnnI, but higher than that of hscTnnT in patients with chest pain for less than 3 h (i.e., in early attenders to emergency departments).

In coronary thrombi of ACS patients, the amount of cysteine-rich angiogenic inducer CYR61 transcriptsFootnote 144 is elevated with respect to that in lymphocytes and monocytes [442]. In a murine ischemia–reperfusion model, myocardial Cyr61 expression markedly rises. Concentration of CyR61 also significantly increases in STEMI with respect to non-STEMI, unstable angina, and stable CoAD, whether coronary thrombi are present or not. Therefore, soluble CyR61 can serve as a marker of ACS due to a thromboembolic event or acute myocardial injury. It can surpass the diagnostic potential of troponin [442]. Fatty Acid-Binding Proteins

Fatty acid-binding proteins (FABPs) are small cytosolic widespread proteins that are members of the ILBP setFootnote 145 (Sect. They reversibly bind to intracellular hydrophobic ligands, such as fatty acids and their acylCoA derivatives, with high affinity and broad specificity for transfer to various cellular compartments, (e.g., endoplasmic reticulum, nucleus, lipid droplets, peroxisomes, and mitochondria) for storage, oxidation, membrane synthesis, signaling, and activation of nuclear receptors, which are transcription factors, such as peroxisome proliferator-activated receptors (PPARs [NR1c1–NR1c3]) [445].

Synthesis of FABPs is mostly regulated at the transcriptional level. The human genome encodes at least nine FABPs (Table 2.29). These isoforms differ with regard to ligand affinity and specificity.

Table 2.29 Fatty acid-binding proteins (Source: [445])

Circulating FABPs are markers of injury in the liver (FABP1), intestine (FABP2), myocardium (FABP3), kidney (FABP4), and brain (FABP7) [167].

Cardiac FABP3, which can be detected 1 h after acute coronary occlusion, is a more reliable early index of cardiac damage than MB [446]. Moreover, it has a sensitivity superior to that of cardiac TNNT, but lacks specificity in detecting acute MI [447].

Plasmatic FABP4 concentration is correlated with atherosclerosis, insulin resistance, type-2 diabetes, obesity, hypertension, left ventricular hypertrophy, intimal hyperplasia, and endothelial and cardiac systolic and diastolic dysfunction [167]. FABP4 is synthesized in adipocytes, macrophages, and ECs of capillaries and veins, but not arteries. However, ectopic production in and secretion of FABP4 from coronary arterial ECs contribute to neointima formation and restenosis after stenting [448].

Plasmatic FABP5 concentration is also associated with metabolic syndrome, although the correlation was not as strong as that of FABP4 [167]. The metabolic syndrome results from interference between metabolic and inflammatory pathways, that is, metaflammation (Vol. 12, Chap. 3. Metabolic Syndrome). C-Reactive Protein (Pentraxin-1)

The circulating pentameric molecule CRPFootnote 146 (median concentration 0.8 mg/l in healthy young adults; half-life ∼19 h [450]), a prototypical acute phase reactant,Footnote 147 is the first identified pattern recognition receptor. It binds to phosphocholine on the surface of dead or dying cells and some types of bacteria, thereby activating the complement system via the C1q complex.Footnote 148

It is synthesized mainly in the liver at a low rate in normal conditions. CRP isoforms have distinct functions. Its production increases, and it is secreted following tissue injury and inflammation, especially under the control of IL-6 released by immunocytes [451]. Concentration of CRP hence rises in response to inflammation (> 500 mg/l [450]).

C-reactive protein (CRP) pertains to the pentraxin family with pentraxin-2Footnote 149 and pentraxin-3.Footnote 150 Short monomeric, insoluble, tissue-associated, proinflammatory, pentraxin-1 (Ptx1) dissociates from pentameric CRP.

Circulating pentameric CRP binds to activated platelets and microparticles enriched in lysophosphatidylcholine, which predominantly derive from activated cell membranes and carry microRNAs. On microparticles, the pentamer is converted into CRP monomers that reside on the microparticle surface [452].

In vitro, monomeric CRP provokes synthesis of chemokines in neutrophils and integrins in monocytes, enhances neutrophil adhesion to ECs, and delays neutrophil apoptosis. Monomeric Ptx1 enhances platelet adhesion to ECs and supports formation of neutrophil–platelet aggregates.

Pentraxin-1 can be detected in atherosclerotic plaques and infarcted myocardium, where it colocalizes with inflammatory leukocytes.

Plasmatic concentrations of CRP, nitrotyrosine, a molecular marker for NO-derived oxidants, and the CXCL8 chemokine (or IL-8) are predictors of acute cardiovascular events in patients with stable or unstable angina [453]. Plasmatic CRP concentration is a stronger predictor of cardiovascular events than that of LDLCS [451]. In women, 77% of cardiovascular events occur with LDLCS concentrations below 160 mg/dl (4.1 mmol/l) and 46% below 130 mg/dl (3.4 mmol/l) [454]. Plasmatic CRP concentration rises in patients at moderate (1–3 μ g/ml [or mg/l]) and high risk (> 3 μ g/ml) for future cardiovascular events [454].

Plasma concentration of pentraxin-3, which is highly expressed in the heart and mainly by dendrocytes, macrophages, and ECs in response to inflammatory stimuli, reaches a peak approximately 7 h after MI. It also lodges in atherosclerotic lesions and vasculitis in humans. It is induced by oxidized LDLs in smooth myocytes. This acute phase protein, but neither Ptx1 nor other cardiac markers (NTproBNP, TNNT, and CK), predicts 3-month mortality [455]. Other Inflammatory Indices

The neutrophil-to-lymphocyte ratio and monocyte-to-high-density lipoprotein ratio are used as indicators of inflammation and redox stress. These markers are related to adverse cardiovascular outcomes, especially after ACS treated by primary percutaneous coronary intervention (e.g., post-stenting restenosis and altered cardiac rhythm such as atrial fibrillation). They are also associated with a bad prognosis in cancers, which is also linked to systemic inflammation (throughout the body).

In HF, neurohumoral processes are aimed at preserving contractile performance to compensate for a defective myocardium. However, sympathetic nervous system hyperactivity is associated with a poor prognosis in HF patients.

Concentration of β ARs and GRK2 in circulating lymphocytes correlates with those in the myocardium, particularly in right atrial appendages of HF subjects [456]. Moreover, after left ventricular assist device implantation, partial restoration of myocardial function and, in CMCs, of β AR signaling and attenuated GRK2 activity is associated with diminished GRK2 activity in lymphocytes. Therefore, lymphocytic GRK2 concentration may complement the blood concentration of NTproBNP to assess HF severity. Furthermore, lymphocyte GRK2 expression correlates significantly with age and left ventricular ejection fraction [457]. The prognostic power of LCGRK2 concentration is similar to that of NTproBNP in HF patient surveillance, as LCGRK2 concentration independently predicts mortality.

Inhibitors of the adrenergic and angiotensin signaling are used in HF patients. β-Adrenoceptor antagonists (β-blockers) that prevent CMC apoptosis and normalize inotropy are used in congestive heart failure and hypertension [458]. Among them, carvedilol is a β-arrestin-based agonist of β-adrenoceptors that elicits GPCR phosphorylation by GRK, β-arrestin recruitment, β AR internalization, ERK activation, and EGF receptor transactivation. Pepducins are lipidated peptides derived from the GPCR intracellular loop that can stimulate or inhibit downstream signaling from their cognate receptors. β2ARPepducin operates in a similar manner to carvedilol, stabilizing the β2AR conformation used as a substrate for GRK-mediated phosphorylation and β-arrestin-1 binding, but induces CMC contraction, whereas carvedilol does not [458]. Lymphocytic GRK2 concentration is not linked to therapy efficiency and does not predict mortality [456].

Elevated plasmatic concentration of γ-glutamyl transferase (GGT)Footnote 151 correlates with hypertension, diabetes, and vascular diseases, in addition to liver damage. GGT is an indicator for an early diagnosis and prognosis in HF patients [459].

Cytokines released by inflammatory leukocytes cause vSMC apoptosis or transdifferentiation to an osteochondrogenic lineage, mineralizing atherosclerotic plaques. Arterial wall calcification (Vol. 10, Chap. 3. Adverse Wall Remodeling), which is linked to altered stress field and phosphate metabolism in addition to hyperglycemia, hyperlipidemia, inflammation, and redox stress, is a frequent and severe complication of atherosclerosis [460]. Calcium released in apoptotic bodies forms a nidus of microcalcification that can destabilize the lesion, but can also later fuse to form macrocalcification, which may protect against plaque rupture.

The cytokine TNFSF1 released primarily by macrophages not only primes apoptosis and hence the accumulation of apoptotic bodies but also activates osteogenic reprogramming in vSMCs via BMP2, Msx2, and Wnt [461].

Signaling by GTPases of the Rac category influences the synthesis of growth factors and cytokines. In macrophages, Rac2 promotes VEGFa formation and hence arteriogenesis in response to ischemia [461]. In addition, Rac2 precludes Rac1 activation. On the other hand, activated Rac1 supports NFκB signaling and ROS production via NOx and hence the action of NLRP3 inflammasome, which promotes IL1β maturation. Pattern recognition receptors and cholesterol crystals, which destabilize lysosomes, trigger the expression of IL1β via the Rac1–NFκB pathway.

Interleukin-1β released by SMC-derived plaque macrophages also favors vascular calcification and can thus serve as a marker of advanced atherosclerotic plaque calcification. On the other hand, Rac2 hinders Rac1-mediated IL1β production in bone marrow-derived macrophages [461]. In the late stages of atherosclerosis, the Rac2 concentration diminishes. Extracellular Vesicles

Information is exchanged between cells using (1) short-distance signaling via intercellular contact, (2) mid-range intercellular transfer of paracrine messengers (autacoids) and EVs (size 50 nm–2 μ m), and (3) remote transmission of hormones, growth factors, and cytokines. Intercellular communication is fostered by juxta-, para-, and endocrine messengers (i.e., autacoids and endocrine regulators), which are secreted either as isolated messengers or contained in EVs.

Once released, EVs travel to neighboring or relatively remote recipient cells, in which they can be incorporated via receptors or fusion of vesicle and target cell plasma membranes and subsequently enter the cytoplasm and even the nucleus.

Extracellular vesicles of multiple cellular origin mediate intercellular communication in physiological and pathological conditions. Circulating EVs yield information on the disease state.

Most cell types, in particular ECs, vascular smooth myocytes, CMCs, monocytes, macrophages, and platelets, release extracellular vesicles of different sizes, composition, and content, and hence subcellular origin. Extracellular vesicle composition and content are markers of cellular activation and injury.

Extracellular vesicles originate from diverse subcellular compartments. They contain cytoplasmic and plasmalemmal components of source cells. Membrane-encapsulated extracellular particles are formed in different pathways, carry diverse cargos to target cells, and have distinct functions. They are detected in all bodily fluids (e.g., cerebrospinal fluid, saliva, and urine), especially in the blood circulation.

A given cell type secretes various types of EVs characterized by their size, density, intracellular origin, and tetraspanin composition. In particular, cells release many types of nanovesicles with similar size, density, and surface markers.

Extracellular vesicles are secreted under basal conditions and upon stress exposure, particularly in inflammatory sites [462]. The origin, composition, and concentration of EVs can also be used as diagnostic and prognostic markers. Their composition, in particular the microRNA content, indeed signals cell activation and injury.

Extracellular vesicles are covered by a lipid bilayer, which contains transmembrane proteins. They serve as containers of numerous soluble proteins (e.g., growth factors, cytokines, chemokines, receptors, and cell adhesion molecules) in addition to genetic information (DNA, mRNA, premature and mature microRNAs, long nonprotein-coding RNAs, and other nucleic acid species) and free ribonucleoprotein complexes.

The vesicular content is transmitted to acceptors (e.g., ECs, monocytes, and platelets). The surface receptors of EVs allow their capture by neighboring recipient cells that can then incorporate proteic, lipidic, and RNA messengers. Extracellular vesicles that circulate in the extracellular space (including the blood) protect RNA from degradation by nucleases.

Plasmatic concentrations of EVs result from the balance between production and rapid clearance by Kupffer cells in the liverFootnote 152 and macrophages in the spleen and lung that renders the long-range transmission of information unlikely [462].

Red blood capsule-derived EVs undergo major uptake by the liver (∼45%), followed by bone (∼23%), skin (∼10%), muscle (∼6%), spleen (∼3%), kidney (∼3%), and lung (∼2%) [464].

The EV formation rate depends on the activation status of releasing cells. On the other hand, EVs are cleared using different mechanisms. Endothelial microvesicles are taken up by the endothelium owing to an annexin-5-phosphatidylserine receptor [464]. Platelet-derived microvesicles are taken up by ECs via EDIL3Footnote 153 in addition to macrophages via lactadherinFootnote 154 Microvesicle clearance also implicate their uptake from the circulation by Kupffer cells and splenocytes. Exosome turnover relies on uptake via tetraspanins, integrins, and lactadherin [464].

Extracellular vesicles can be categorized into large, mid-size, and small particles. Small EVs can be further subdivided into subcategories according to proteic markers (e.g., Tspan28–Tspan30).

Many subtypes of EVs are described in the literature, such as ectosomes, exosomes, microvesicles, microparticles, membrane particles, and apoptotic vesicles (Vol. 10, Chap. 1. Architecture and Structure of the Vasculature). Extracellular vesicles mainly comprise (Table 2.30):

Table 2.30 Characteristics of different types of extracellular vesicles, the size of vesicle subtype varying according to investigators (Source: [462])
  1. 1.

    Exosomes (size 30–150 nm) originating from the endosomal circuit that are released upon fusion of multivesicular bodies with the plasma membrane

  2. 2.

    Microvesicles (size 150 nm–1 μm) that are directly shed from the plasma membrane

  3. 3.

    Apoptotic bodies (size 1–5 μm) that are released as blebs of apoptotic cells

Exosomes are permanently exported from cells, but microvesicles are released in response to cell activation or injury [464].

Extracellular vesicles formed and released from the plasma membrane are often called microparticles, shed microvesicles, or ectosomes. Their sizes range from a few tens of nanometers to a few micrometers.

Intracellular vesicles generated in the endosomal and multivesicular compartment are secreted when they fuse with the plasma membrane. These endosome-derived vesicles released in the extracellular medium are named exosomes. Exosomes are defined by their size (< 150 nm) and content (i.e., endosomal proteins).

Extracellular vesicles, at least those from monocyte-derived dendrocytes, are categorized into large, medium-sized, and small vesicles [466].

  • Large EVs contain HSP90b1 (endoplasmin) and possibly other endoplasmic reticulum-associated proteins.

  • Large and medium-sized EVs possess actinin-4, mitofilin, and possibly other mitochondrial proteins, but not small EVs.

  • Small EVs are characterized by the presence of flotillins, annexin-5, -6, and -11, syntenin-1, endosomal sorting complexes required for transport (ESCRT1) complex subunits TSG101, ChMP4a, ChMP4b, and VPS4b, ADAM10, ACE, and EHD4. Among them, syntenin-1 and TSG101 are specific to exosomes.

Patients with risk factors, such as hypertension, metabolic syndrome, diabetes, and/or hypercholesterolemia, have elevated concentrations of circulating microvesicles derived from ECs and platelets [464].

In addition, microvesicular content changes. Diabetic patients have a reduced amount of endothelial miR26 and miR126-3p in circulating endothelial microvesicles [464]. In EVs from smokers, the quantity of platelet-derived miR223 declines, whereas amounts of miR29b and type-2 U6 small nuclear RNA (RNU6-2) increase with respect to controls. Familial hypercholesterolemia is associated with higher numbers of platelet-derived microvesicles in addition to tissue factor-rich microvesicles, which have a procoagulant activity. Patients with low miR26a and miR126 levels are at a high risk for CoAD.

In patients at a high CoAD risk, levels of circulating CD144+ endothelial microvesicles and/or CD31+ annexin-5+ microvesicles in addition to low concentrations of miR126-3p and miR199-5p predict future cardiovascular events [464]. In patients with pulmonary hypertension, the circulating level of SelE+ endothelial microvesicles is related to a bad mid-term prognosis. Microvesicular cystatin-C (Cst3), an inhibitor of cysteine peptidases, serpin-F2, and CD14, a coreceptor for TLR2–TLR6 heterodimer and bacterial lipopolysaccharide, are related to an elevated risk for future vascular events. Small Extracellular Vesicles

Small EVs cross 220-nm pore filters. They incorporate subpopulations of exosomes and other types of extracellular vesicles.

Flotillin-1, 70-kDa heat shock proteins (inducible HSPa1 and constitutive HSPa8), and MHC class-I and II proteins are present in all types of EVs, albeit to various extents [466].

Four subcategories of small extracellular vesicles (SECVs) can be defined according to the types of contained tetraspanins among the three species used as markers (Tspan28–Tspan30) [466].

  1. 1.

    SECVs enriched in Tspan28 to Tspan30 and endosomal markers are genuine exosomes.

  2. 2.

    SECVs devoid of Tspan28 and Tspan30, but enriched in Tspan29 are linked to the plasma membrane and have an early endocytic signature. Tspan29+ small EVs have transmembrane proteins, integrin-α2B, and MMP14.

  3. 3.

    SECVs devoid of Tspan28 to Tspan30 are not associated with the endosomal route.

  4. 4.

    SECVs enriched in matrix constituents (collagen, fibronectin, S100a18, and serpin-F1), complement (C3 and C4a), and serum-derived factors (prothrombin [coagulation factor FII] and albumin) are also not linked to the endosomal route.

Pulmonary arterial hypertension (PAHT) is characterized by hyperplasia and hypertrophy of vascular smooth myocytes and proliferation of ECs in small pulmonary arteries, increasing the mean pulmonary arterial pressure and hence right ventricle afterload. The severity of PAHT is associated with elevated amounts of circulating microvesicles released from stressed and apoptotic cells [467].

Circulating small vesicles from PAHT mouse models are heterogeneous in size (30–430 nm) and carry proteins characteristic of exosomes and microvesicles. Administration of lung- and plasma-derived vesicles from these mice to healthy mice increases right ventricular mass and pulmonary vascular wall thickness [468]. Lung-derived small vesicles (LDSVs), but not plasma-derived small vesicles (PDSVs), contain high amounts of endothelial mRNAs. In addition, small vesicles from monocrotaline-treated mice possess altered levels of microRNAs, such as miR145, miR328, and miR451, which are involved in pulmonary arterial hypertensive remodeling. Furthermore, the concentration of phosphodiesterase-5 is augmented in both LDSVs and PDSVs.

Extracellular vesicles from monocrotaline-treated mice induce differentiation of bone marrow cells into endothelial progenitor cells. When these cells are transplanted into healthy mice, they home to the lung, where they raise the muscularization of pulmonary arterial walls, thereby contributing to adverse pulmonary vascular remodeling; mobilization of bone marrow-derived progenitor cells and their recruitment to the pulmonary vasculature are also observed in experimental hypoxia-induced PAHT [467]. Extracellular vesicles from monocrotaline-treated mice upregulate the activity of growth factor implicated in PAHT genesis from bone marrow-derived progenitor cells. Exosomes

Exosomes are produced on the endosomal route from multivesicular bodies spontaneously or in response to external mechanical and chemical cues (e.g., shear stress, hypoxia, cytokines, and chemotherapeutic agents), whereas larger vesicles are expelled from the plasma membrane.

Exosomes contain membrane-anchored receptors, adhesion molecules, signaling mediators, and nucleic acids. They transfer their cargos to recipient cells, thereby affecting cell fate.

Exosomes influence immune response and are involved in pathogenesis and in tumor growth. Cancerous cells exploit exosomes to favor tumor growth and control premetastatic niche formation [469]. Exosomes can intervene in cancer growth, angiogenesis, metastasis, and immune surveillance.

Syntenin, a cytosolic adaptor that binds to the intracellular domain of syndecans, is implicated in the genesis of a subset of exosomes. Syntenin also binds to programmed cell death-6-interacting protein (PdCD6IP), which is involved in endocytosis, multivesicular body genesis, membrane repair, cytokinesis, apoptosis, and maintenance of tight junction integrity. PdCD6IP connects the syntenin–syndecan complex to the ESCRT machinery, thereby operating in endosomal membrane budding and scission and generating intraluminal vesicles that are released as exosomes when multivesicular endosomes fuse with the plasma membrane [469]. Regulators of these processes include, on the cytosolic side, the small GTPase ARF6 and its effector phospholipase-D2 and, on the luminal side, the endoglycosidase heparanase.

The cytoplasmic protein Tyr kinase Src, which controls cell growth, proliferation, adhesion, and motility, and signal transduction induced by growth factors and integrins, stimulates the secretion of exosomes loaded with syntenin and syndecans [469]. This regulator of exosomal communication phosphorylates syndecans and syntenin and stimulates syndecan internalization, endosomal genesis, and secretion of exosomes having promigratory activity on ECs. It operates in syndecan endocytosis and syntenin–syndecan endosomal budding upstream from the ArfGEF cytohesin-2, ARF6, and PLD2. Syntenin endosomal budding depends on interaction with phosphatidic acid, the PLD2 product [469].

Exosomes can cause pulmonary hypertension, but not microvesicles [470]. Hypoxic lung-derived exosomes from patients with idiopathic pulmonary artery hypertension contain increased concentrations of miR19b, miR20a, miR20b, and miR145. On the other hand, mesenchymal stem cell-derived exosomes have elevated concentrations of anti-inflammatory, antiproliferative, and antimigratory microRNAs (miR34a, miR122, miR124, and miR127),Footnote 155 thereby preventing vascular remodeling and the development of hypoxic pulmonary hypertension. However, nonmesenchymal bone marrow stem and progenitor cells have no effect.

Numerous microRNAs of mesenchymal stem cell-derived exosomes (e.g., miR101a, miR122, miR193, miR224, and miR302b) provoke apoptosis or suppress proliferation or migration of cancerous cells [470].

Adipose tissue macrophages (ATMφs) release exosomes containing microRNAs that modulate insulin sensitivity. MicroRNA-155 is differentially produced in ATMs in lean and obese rodents. Diet-induced obesity alters the amounts of various microRNAs in ATMφ-generated exosomes. In particular, the amount of miR155 rises. MicroRNA-155 diminishes glucose uptake and impedes insulin signaling [471].

In addition, macrophage-derived leukotriene-B4, which operates via its cognate receptor LTb4R,Footnote 156 participates in metabolic dysfunction; it can reduce insulin signaling in hepatocytes and myocytes [472]. It supports macrophage chemotaxis, elicits inflammatory phenotype in macrophages, promotes hepatic lipogenesis, decreases insulin-stimulated glucose uptake in myocytes, increases gluconeogenesis, and alters insulin-mediated suppression of glucose output in hepatocytes, as it reduces insulin-primed Gi- and JNK-dependent PKB phosphorylation and augments IRS1 and IRS2 phosphorylation [472].

Galectin-3 (GaL3), a β-galactoside-binding lectin on monocytes and macrophages, the concentration of which rises progressively during differentiation of monocytes into macrophages, is a macrophage-secreted molecule that can promote inflammation and inhibit insulin receptor. Galectin-3 serves as a chemoattractant for monocytes and macrophages. Intracellular GaL3 participates in cell migration and apoptosis. Extracellular GaL3 mediates cellular adhesion and cytokine production. Homocysteine, which is produced by methionine processing, stimulates activity of the GaL3 inducer, NFκB. Galectin-3 contributes to macrophage activation, especially in atherosclerotic plaques [473]. Hyperhomocysteinemia is observed in patients with unstable CoAD. In monocytes, it participates in the production of superoxide and cooperates with ROS in exosome formation. Galectin-3 is thus involved in inflammation, redox stress, and cardiac fibrosis.

ATMφ-derived TSG101+ ,Footnote 157 syntenin-1+ ,Footnote 158 TSpan29+ , and TSpan30+ exosomes are taken up by adipocytes, hepatocytes, and skeletal myocytes, the major insulin target cells [471]. In particular, neighboring adipocytes take up exosomes containing myeloid cell-specific microRNAs. MicroRNA-155 contributes to insulin resistance in obesity. MicroRNA-155 released by proinflammatory macrophages targets the ligand-activated transcription factor, nuclear receptor NR1c3 (PPARγ) [471]. However, other exosomal microRNAs contribute to the modulation of insulin sensitivity. ATMφ-derived exosomal miRs from obese mice impair cellular insulin sensitivity. On the other hand, ATMφ-derived exosomal miRs from lean mice attenuate obesity-induced insulin resistance. Lean mice receiving ATMφ-derived exosomes from obese mice develop glucose intolerance and insulin resistance. Conversely, in obese mice, injection of ATMφ-engendered exosomes from lean mice normalizes insulin signaling.

Exosomes mediate intercellular communication among tumoral cells and between them and their surrounding stroma. Sunitinib-resistant renal carcinoma cellsFootnote 159 transfer the long nonprotein-coding RNA activated in renal cell carcinoma with sunitinib resistance (lncarsr) to sensitive cells via exosomes. Lncarsr competitively connects to tumor-suppressive microRNAs miR34 and miR449 and raises amounts of receptor protein Tyr kinases Axl and hepatocyte growth factor receptor in recipient cells [475]. Lncarsr packaging into exosomes involved the ribonucleoprotein hnRNPa2b1. Inhibitors of both Axl and HGFR resensitize lncarsr+ cells to sunitinib. Microvesicles

Plasmatic concentration of endothelial microvesicles is a marker of endothelial dysfunction. Plasmatic concentration of platelet and leukocyte microparticles rises in HF and cardiac valvulopathies [462].

Phosphatidylserine+ microvesicles of different cellular origin bind to the platelet scavenger receptor ScaRb3 and increase ADP-dependent platelet activation, thereby facilitating thrombosis. On the other hand, phosphatidylserine of endothelial microvesicles can interact with endothelial phosphatidylserine receptor in an annexin-1-dependent manner to prevent apoptosis [462]. LactadherinFootnote 160 allows the clearance of circulating PSer+ microvesicles by macrophages in the spleen and liver.

Microvesicles contain MMP2, MMP3, MMP7, and MMP13, which can assist crossing of the extracellular matrix to reach target neighboring cells (mainly) or blood circulation [462].Footnote 161 Microvesicles derived from atherosclerotic plaques possess the adamalysin ADAM17. Microvesicles can also engender ROS production.

Exposure of ECs to microvesicles shed from senescent ECs or circulating endothelial microvesicles from ACS patients provokes premature aging, elevated β-galactosidase activity, redox stress, abnormal phosphorylation of MAPK and PKB, and upregulation of P53, CKI1a, and CKI2a [476]. Endotheliocyte senescence is associated with a progressive shedding of procoagulant microvesicles, augmented tissue factor activity, attenuated NOS3 activity and hence NO-mediated inhibition of platelet aggregation. These microvesicles are endowed with an angiotensin convertase activity and AT1 receptors. Inhibitors of AT1, MAPK, or PKB prevent microvesicle-primed endothelial senescence. Premature endothelial senescence and thrombogenicity result from angiotensin-2-induced redox-sensitive activation of the MAPK module and PKB. Genomic Loci and Variants

New markers can be found from reproducible variants in genes, RNAs, and metabolites associated with the investigated disease. Current technology allows the examination of hundreds to thousands of single-nucleotide polymorphisms to search for significant associations with any disease (Chap. 7).

For example, a locus on chromosome 9p21 that does not contain genes linked to established CoAD risk factors, but near genes encoding cyclin-dependent kinase inhibitors CKI2a and CKI2b implicated in cellular senescence and apoptosis, is associated with the early onset of myocardial infarction [435].

2.3.3 Prognosis

PrognosisFootnote 162 predicting the likely outcome of a disease, upon treatment or not. In addition, appropriate use of markers can optimize treatment [477]. Prognostic indicators and estimators can be used to assess the disease evolution. Renin

Reninemia (blood [plasmatic] concentration of renin) is inversely related to oral sodium intake. Reninemia can serve as a prognostic index for the cardiovascular event risk, independently of the blood pressure value [478]. IL1RL1 (ST2 Marker of Cardiac Stress)

Membrane-bound receptor mIL1RL1Footnote 163 also exists as a soluble truncated form (\({ }_{\mathrm {t}}^{\mathrm {S}}\)IL1RL1 or ST2), which is secreted into the bloodstream. The messenger RNA of IL33 is markedly upregulated in cultured CMCs exposed to hypertension. Binding of IL33 to IL1RL1 ensures cardioprotection. However, IL33 connects competitively to \({ }_{\mathrm {t}}^{\mathrm {S}}\)IL1RL1 and is then unavailable for its IL1RL1 receptor, hence functioning as a decoy receptor for the IL33 cytokine. The soluble truncated IL1RL1 can be used to estimate the severity of adverse cardiac remodeling; elevated blood concentration in \({ }_{\mathrm {t}}^{\mathrm {S}}\)IL1RL1 can then predict outcomes in HF patients [479, 480]. S100

The secreted calcium-binding protein S100a9Footnote 164 is a discriminator between stable and unstable CoAD. Other proteins can be targeted using nuclear magnetic resonance spectroscopy and tandem mass spectrometry coupled with chromatography, but generally their amounts are low, as they are within nanomolar (e.g., troponin), picomolar (e.g., insulin), or femtomolar (e.g., tumor-necrosis factor) ranges [435]. Quinones

Quinones are metabolic intermediates of several endogenous and dietary molecules that can form stable adducts with DNA and proteins, which can be toxic products. The most common quinones are derivatives of hormones and neurotransmitters, such as catecholamines (adrenaline, noradrenaline, and dopamine). Others are produced in the human organism from tea catechins and other vegetable-derived flavonoids (with anti-inflammatory, -oxidant, and -carcinogenic effects) in addition to environmental pollutants such as pesticides. Molecular sources share a catechol (1,2)-dihydroxy benzene group that is generally metabolized to a quinone moiety. Moreover, quinones can also be generated during redox stress, as they can be produced from catechol-containing molecules by reaction with free radicals and other reactive species.

Quinones are involved in the onset of several pathological conditions. Systematic analysis of the reactivity of quinone derivatives with amino acids and DNA bases based on computational chemistry techniques is aimed at addressing the formation and stability of quinone adducts to predict the most reactive quinones and their adducts with biological and pathophysiological relevance. Computational results can then be used to guide experiments to validate the identification of reliable markers in the early detection and prognosis evaluation of pathological conditions. Rock

Two isoforms of rock kinases (rock1–rock2) are effectors of small GTPases of the RHOA subfamily that have different functions in the vascular circuit. Hence, rock is an effector of the small RhoA, RhoB, and RhoC GTPases [481].

The RhoA–rock pathway is implicated in cell contraction (and hence vasospasm and systemic and pulmonary hypertension), motility, proliferation, and apoptosis.

Its major substrates include PP1r12a (Thr697, Ser854, and Thr855, attenuating MLCP activity), PP1r14a (at Thr38), MLC2, LIMKs (LIMK1–LIMK2, which phosphorylate [inhibit] cofilin, hence impeding actin depolymerization), the plasma membrane–actin filament crosslinkers ezrin, radixin, and moesin (phosphorylated in response to vasoconstrictors signaling via PKC and rock), and adducin. These substrates are also phosphorylated by MLCK, PKA, PKC, and PKG [482].

The kinase rock participates in organizing the actin cytoskeleton and controlling its dynamics and hence stress fiber formation, using the contraction-mediating rock–PP1r12a–MLC and actin-stabilizing rock–LIMK–cofilin pathways. It is thus involved in cell contraction, adhesion, migration, proliferation, and apoptosis, in addition to remodeling of the extracellular matrix. Its activity is modulated by interaction with lipid mediators (e.g., arachidonic acid and sphingosylphosphorylcholine), mechanical stress, autophosphorylation, and proteolytic cleavage of its inhibitory C-terminus.

Because rock stabilizes PTen, the RhoA–rock–PTen pathway antagonizes the PI3K–PKB pathway. Rock also phosphorylates IRS1 and IRS2, thereby improving or impairing insulin signaling according to the context.

Its ubiquitous isoforms, rock1 and rock2, are functionally redundant. They both homodimerize. The autoinhibitory domain obviates constitutive activation; activation by RhoGTP thus relies on derepression. Rock activity does not depend on autophosphorylation. Full catalytic rock2 activity relies on several phosphorylation sites (PLK1 targets Thr967, Ser1099, Ser1133, and Ser1374).

Cytosolic ubiquitous rock1 is linked to the plasma membrane, centrosome, cellular adhesion sites, and vesicles [481]. Cytosolic and nuclear rock2, which abounds in the brain, heart, muscle, lung, and placenta, localizes to the centrosome and colocalizes with actin and vimentin filaments, in addition to the intercalated and Z discs of striated myocytes. In the myocardium and skeletal muscles, a unique rock2 splice variant exists (SkMrock2) [481]. Both rock1 and rock2 are cleaved and activated by peptidases.

Only rock2 binds directly to and phosphorylates PP1r12a [482]. In addition, rock2 tethers to PIP3 and PI(4,5)P2, but not rock1 [481]. Rock1, but not rock2, phosphorylates RhoE, a rock-specific substrate [481].

In the vasculature, The RhoA–rock pathway is implicated in vasoconstriction, using the rock–PP1r12a–MLC2 pathway, and inflammatory cell recruitment, in addition to endothelial dysfunction and vascular and cardiac remodeling [482]. It is involved in vascular effects of redox stress (ROS production by NOxs) and various vasoconstrictors, in particular the renin–angiotensin–aldosterone axis, and hence hypertension.

The rock is also involved in transcriptional regulation, especially in SRF activity [482]. Myocardin and myocardin-related transcription factors MRTFa and MRTFb are SRF coactivators, the nuclear translocation of which depends on actin dynamics. It can thus regulate MRTFa-mediated myofibroblast activation in cardiac fibrosis.

In addition, rock mediates CMC hypertrophy caused by angiotensin-2, endothelin-1, and α-adrenoceptor [482]. In fact, rock1 and rock2 operates in cardiac fibrosis and hypertrophy, respectively [481].

The rock also favors inflammation, as it can induce expression of proinflammatory cytokines and adhesion molecules in endothelial and smooth muscle cells. It stimulates several molecules involved in inflammation, fibrosis, and thrombosis. Activity of the rock in circulating leukocytes serves as a marker for the assessment of disease severity and therapeutic response [483].

Myosin light chain (MLC) is phosphorylated by Ca2+–Cam-activated MLC kinase (MLCK) and is dephosphorylated by MLC phosphatase (MLCP),Footnote 165 hence causing vSMC contraction and relaxation, respectively. In vascular smooth myocytes, the RhoA–rock axis leads to MLC phosphorylation directly and via PP1r12a inhibition. The substrates of rock include not only MLC and MLCP, but also the members of the ERM (ezrin–radixin–moesin) family, adducin, PTen phosphatase, and LIM kinases [483].

The vascular effect of the rock is augmented by mechanical stress, hypoxia, growth factors (e.g., PDGF), and some vasoactive agents (e.g., angiotensin-2, thrombin, thromboxanes, extracellular nucleotides, and urotensin) [483]. The rock upregulates NADPH oxidase expression.

The rock participates in interactions between vascular endothelial and vSMCs. Endotheliocytes release vasodilators, such as NO and prostacyclin, and endothelium-derived hyperpolarizing factors such as hydrogen peroxide. Hydrogen peroxide transmitted through myoendothelial junctions rapidly cause vasodilation via PGhS (cyclooxygenase), cAMP, and the cGMP–PKG1α–KCa pathway (vSMC membrane hyperpolarization) [483].

In ECs, the RhoA–rock axis prevents nitric oxide synthesis. It also supports actin–myosin contractility, hence disrupting the endothelial barrier and raising endothelial permeability.

In addition, activated rock enhances transcriptional regulation of SRF, provoking stress fiber formation and vSMC contraction. It also mediates vSMC contraction elicited by vasoconstrictory GPCR ligands sent by ECs.

Cyclophilin-A mediates redox stress. Reactive oxygen species and the RhoA–rock axis promote cyclophilin-A secretion via VAMP2+ Footnote 166 vesicle formation and actin–myosin-2 cytoskeletal dynamics [483].

In ECs, released cyclophilin-A decreases NOS3 activity. In addition, it stimulates expression of E-selectin and vcam1 on vECs. It is also a chemoattractant for inflammatory leukocytes and activates MMPs.

In vascular smooth myocytes, extracellular cyclophilin-A activates ERK1, ERK2, PKB, and JaK, contributing to ROS production. Reactive oxygen species foster vSMC proliferation in an auto- and paracrine manner.

The rock is active not only in vascular cells, but also in leukocytes. It inhibits reverse cholesterol transport in macrophages, thereby promoting foam cell formation [484].

Extracellular cyclophilin-A also activates platelets via basigin and the PI3K–PKB axis, enhancing their adhesion and thrombus formation.

Platelets are not only involved in hemostasis but also in inflammation and immunity. Platelet activation results from signaling initiated and then amplified by plasmalemmal receptors and involves re-organization of the actin cytoskeleton for the secretion of granule content, adhesion, spreading, aggregation, and blood clot retraction, and hence monomeric GTPases of the RHO superfamily (RhoA, Rac1, and CDC42). RhoA is implicated in platelet contractility and thrombus stability, Rac1 in lamellipodium formation, and CDC42 in filopodium formation and granule secretion [484].

Platelet activators such as thrombin connect to their cognate GPCRs and launch RhoA–rock2 signaling. In addition, RhoA and Rock2 regulate platelet formation from megakaryocytes, albeit to a considerably lesser extent than Rac1 and CDC42 [484].

Rock2 operates in platelet activation and thrombosis. It promotes α2Bβ3-integrin binding to fibrinogen and expression of P-selectin, which elicits hetero- (platelet–leukocyte) and homotypic aggregates [485]. Rock2 does not influence expression of plasmalemmal receptors, but their activation and function, as binding of fibrinogen to α2Bβ3Itg relies on its structural changes to bind to fibrinogen. Rock2 is necessary for initial activation of α2Bβ3Itg, but not for the maintenance of of activation. α-Granules contain P-selectin. Upon platelet activation, P-selectin lodges on the plasma membrane. Rock2 is necessary for the initial release of granules at low thrombin concentration, but not for release maintenance at a higher thrombin concentration.

Rock2 influences blood clot formation, but not its growth. In platelets, rock2 may also regulate rock1 concentration. Rock2 deficiency does indeed mildly reduce rock1 concentration [485].

Coronary microvascular resistance is controlled by numerous myogenic, metabolic, neurohumoral, and endothelium-derived factors to adjust myocardial nutrient oxygen delivery to its metabolism. Microvascular dysfunction is generally characterized by the decreased production of vasodilators (NO, hydrogen peroxide, prostacyclin, and endothelium-derived hyperpolarizing factors) and increased formation of vasoconstrictors (endothelin, thromboxane-A2, and superoxide). Adverse remodeling and hypoperfusion perpetuate cardiac dysfunction. Activation of the RhoA–rock axis and redox stress in different cardiac cell types alters cardiac function either directly via hypertrophy and fibrosis or indirectly via hypoperfusion, that is, whether abnormal activation of the RhoA–rock axis and redox stress occur in cardiac myocytes and fibroblasts or in arteriolar endothelial and smooth muscle cells [486].

Hypertensive cardiac hypertrophy is associated with reduced coronary flow reserve. In hypertension, rock1 and rock2 cause NO deficiency, augment ET activity, and increase superoxide production, thereby provoking redox stress and contributing to coronary arteriolar dysfunction, that is, the inability to increase blood flow in response to metabolic stimulation. Myocardial and microvascular redox stress, elevated vasoconstriction, perivascular fibrosis, and CMC hypertrophy are attenuated by rock inhibition [487]. Expression of both rock isoforms is significantly upregulated in the coronary arterioles of hypertensive mice. In mice, deletion of rock1 reduces fibrogenesis, but not ventricular hypertrophy, although it improves cardiac contractility. On the other hand, rock2 deletion reduces cardiac cell apoptosis and ventricular fibrosis and hypertrophy. In addition, rock inhibition improves aortic endothelial and cardiac function in rat models of streptozotocin-induced diabetes [486].