Cardiovascular protection by DPP-4 inhibitors in preclinical studies: an updated review of molecular mechanisms

Dipeptidyl peptidase 4 (DPP4) inhibitors are a class of antidiabetic medications that cause glucose-dependent increase in incretins in diabetic patients. One of the two incretins, glucagon-like peptide-1 (GLP-1), beside its insulinotropic activity, has been studied for extra pancreatic effects. Most of DPP4 inhibitors (DPP4i) have been investigated in in vivo and in vitro models of diabetic and nondiabetic cardiovascular diseases including heart failure, hypertension, myocardial ischemia or infarction, atherosclerosis, and stroke. Results of preclinical studies proved prominent therapeutic potential of DPP4i in cardiovascular diseases, regardless the presence of diabetes. This review aims to present an updated summary of the cardiovascular protective and therapeutic effects of DPP4 inhibitors through the past 5 years focusing on the molecular mechanisms beneath these effects. Additionally, based on the results summary presented here, future studies may be conducted to elucidate or illustrate some of these findings which can add clinical benefits towards management of diabetic cardiovascular complications.


Introduction
Type 2 diabetes mellitus (DM) is associated with various cardiovascular (CV) complications including hypertension, ischemic heart disease, heart failure, and atherosclerosis. Diabetic patients tend to develop cardiovascular disease at a younger age and at higher incidence than non-diabetic patients (Chen et al. 2016;Leon and Maddox 2015). In addition to the deleterious effects of hyperglycemia, studies proved that diabetic patients exhibit an impaired response to intestinal hormones called incretins compared to individuals with normal blood glucose level. This sheds lights on a new family of antidiabetic medications, the dipeptidyl peptidase 4 inhibitors (DPP4i) or gliptins, which target incretin hormones. Several DPP4i are commercially available including alogliptin, linagliptin, saxagliptin, sitagliptin, anagliptin, vildagliptin, teneligliptin, and omarigliptin, in addition to several others which are under development.
Incretins are hormones released by the intestinal mucosa to stimulate insulin secretion following oral nutrient intake. Humans express two types of incretins: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). GLP-1 is secreted by the neuroendocrine L cells in the ileum, while GIP is secreted by K cells in the duodenum and jejunum. Both hormones promote insulin release from pancreatic β-cells by activation of intracellular cAMP. They also stimulate β-cell proliferation and inhibit apoptosis, which increases β-cell mass (Drucker 2006). In addition, GLP-1, but not GIP, inhibits gastric emptying, food intake, and pancreatic release of glucagon, the hormone which stimulates hepatic glucose production.
GLP-1 has extra pancreatic effects and its receptor is expressed on many sites including the brain, intestine, lung, adipose tissue, myocardium, and vascular smooth muscle cells (Bullock et al. 1996). On the other hand, GIP has no major role in the cardiovascular system (Nauck and Meier 2018).  is the active form that is degraded to an inactive form, GLP-1 (9-36), within 1 to 2 min by the enzyme DPP4 (Mafong and Henry 2009). DPP-4 is a widely expressed enzyme that exists in 2 forms, an extracellular protein that, when activated, initiates intracellular signal transduction pathways independent of its enzymatic activity, as well as a circulating soluble form which is enzymatically active (Drucker 2006). Current developments in incretin research have demonstrated a significant role of incretins in cardiovascular pathology. This comprises 2 approaches. Firstly, the role of GLP-1 which is mediated through receptor-dependent and receptor-independent mechanisms. GLP-1 signaling seems to play a role in cardiac development, because mice with a targeted gene deletion of the GLP-1 receptors showed enlarged hearts (Gros et al. 2003). Diebold et al. (2018) demonstrated a significant role of GLP-1 secretion for left ventricular contractility during myocardial infarction. Mechanistically, it was shown that DPP-4 inhibition increased AMP-activated protein kinase (AMPK) activity and stimulated the mitochondrial respiratory capacity of non-infarcted myocardial tissues.
Secondly, it is now known that DPP-4 also cleaves several other peptides, some of which have direct actions on the cardiac and vascular cells. Hence, DPP4 inhibition provides favorable cardiovascular outcomes independent of GLP-1. DPP-4 is responsible for degradation of B-type natriuretic peptide (BNP, Brandt et al. 2006), stromal cell-derived factor 1α (SDF-1α, Zaruba et al. 2009), and substance P (Wang et al. 1991). These findings suggest DPP4i as potential cardioprotective agents in diabetic and non-diabetic patients specially when considering that DPP4i have no risk of hyperglycemia or weight gain.
This review presents an updated overview on the cardiovascular protective effects of DPP4i in experimental preclinical studies during the past 5 years. In addition, this review describes and evaluates the underlying molecular mechanisms for these effects.

Literature search
To gather the most recent experimental studies on DPP4i in animal models of cardiovascular disease, a literature search was performed in "PubMed" and "Google Scholar" search engines up to 20 February 2022. The keywords "DPP4 inhibitors or gliptins or linagliptin or vildagliptin or saxagliptin or sitagliptin or alogliptin or anagliptin or teneligliptin or omarigliptin," were matched with "cardiovascular or myocardial ischemia reperfusion or myocardial infarction or heart failure or atherosclerosis or stroke," and with "experimental animals." We included studies published in trustful scientific journals starting from 2017 and thereafter. To ensure a comprehensive search, we also included some related articles from references. Articles were carefully read, and data related to disease models, DPP4i dose, treatment duration, and main findings as well as the underlying molecular mechanisms were summarized in a tabular form.

Results
Results of our search of DPP4i cardiovascular effects are summarized in a tabular form. Results are classified based on the CV disease then the type of gliptin (Table 1).

Discussion
This review covers the recent research of the cardiovascular (CV) protective potential of DPP4 inhibitors (DPP4i) during the past 5 years. It is believed that the protective effects of DPP4i are maintained through 2 approaches, (I) Glucagon-like peptide-1 (GLP-1)-mediated mechanisms and (II) conservation of some peptides that are physiologically degraded by the DPP4 enzyme. Researchers reported that DPP4 is responsible for degradation of B-type natriuretic peptide (BNP), stromal cell-derived factor 1α (SDF-1α), and substance P. Gliptins effectively mitigate the deleterious effects of postprandial hyperglycemia on oxidative stress, inflammation, and CV remodeling. However, in models of CV disease in non-diabetic animals, gliptins show protective effects through various pathways that will be discussed here.
GLP-1 deficiency is a consequence rather than a cause of diabetes as GLP-1 secretion is decreased in hyperglycemia. Also, DPP4 activity is increased in diabetes, both type 1 and 2, and is negatively correlated with adiponectin levels (Vollmer et al. 2009). The effect of DPP4 antagonism was studied in models of myocardial ischemia/reperfusion (I/R) and infarction in normoglycemic and hyperglycemic animal models of CV disease by genetic deletion of DPP4 or by using DPP4i. Both approaches resulted in improved cardiac function and hemodynamics (Sauvé et al. 2010). These promising finding may be attributed to (I) increased GLP-1 level which exerts its effects through receptor-dependent as well as receptor-independent mechanisms. (II) DPP4 inhibition preserves some peptides that have advantages on CV function in various disease conditions (see Fig. 1).

I-DPP4 inhibition increases GLP-1
It is well known that hyperglycemia underlines diabetic cardiovascular complications through various pathways.
Glucotoxicity increases reactive oxygen species (ROS) production through many mechanisms including polyol and hexosamine pathways, increased advanced glycation end products (AGEs) production, activation of protein kinase C (PKC) and poly(ADP-ribose) polymerase-1 (PARP-1) enzyme (Mapanga and Essop 2016). Increased ROS production triggers a closed cycle of myocardial and vascular inflammation and oxidative stress leading to apoptosis, endothelial dysfunction, and cardiomyopathies. In healthy individuals, GLP-1 is secreted by the neuroendocrine L cells in the ileum in response to food ingestion and stimulates pancreatic beta cells to release insulin. Postprandial insulin augmentation helps regulate blood glucose with minimal hypoglycemic effect. As GLP-1 is impaired in diabetes, inhibiting its degradation helps decreasing blood glucose in diabetic patients and consequently accentuate the rush through these oxidative and nonoxidative glucotoxicity pathways. In addition, enhanced insulin secretion improves cardiac function and increase inotropy, regulate blood flow to myocardial tissue, and exerts anti-inflammatory, antioxidant, and antiapoptotic effects (Ng et al. 2012).
Beside the glycemic effect of GLP-1, it exerts direct cardiovascular protection. Since the discovery of GLP-1 receptor expression in the cardiac muscle, cardioprotective role of GLP-1 was extensively studied and was proved. Infusions of GLP-1 in animal models and human subjects with heart failure have demonstrated significant improvement in cardiac parameters (Ban et al., 2008). In patients with type 2 diabetes (T2DM), GLP-1 infusion significantly improved endothelial function, irrespective of changes in insulin sensitivity (Bullock et al. 1996). Moreover, infusion of GLP-1 in patients with T2DM and established coronary artery disease significantly improved endothelial dysfunction as measured by flow-mediated vasodilation (Nyström et al. 2004). Observations suggest that part of the cardioprotective and vasodilatory effects of GLP-1 on myocardial metabolism is direct, insulin-independent, and GLP-1-receptor-independent (Ban et al. 2008). A study by Gros et al. (2003) on GLP-1 receptor knock out mice (GLP-1R − / − ) showed that lack of GLP-1R is associated with decreased resting heart rate and increased left ventricular end diastolic (LVED) pressure and LV thickness compared with CD-1 wild-type controls. In addition, GLP-1R deletion resulted in impaired LV contractility and diastolic function after insulin or epinephrine administration. In another way, a study on a mouse model of I/R found that GLP-1(9-36), the primary metabolite of GLP-1, has nearly identical results, suggesting the presence of an alternative signaling mechanism for GLP-1 and its metabolite independent of its known receptor.

II-DPP4 inhibition preserves physiologically active cardioprotective peptides
Beside GLP-1, other peptides are substrates of DPP4 enzyme including atrial natriuretic peptide (ANP), brain  natriuretic peptide (BNP), stromal cell-derived factor-1α (SDF-1α), and substance P. ANP is synthesized in the atria while BNP is produced by heart ventricles. They act locally and systemically to exert several biological functions including diuresis, natriuresis, and vasodilatation as well as inhibition of renin and aldosterone secretion (Nishikimi et al. 2006). Physiological levels of ANP and BNP are low but they increase as a compensatory mechanism in heart failure. The active form, BNP(1-32), is degraded by DPP4 by removing the two N-terminal amino acids (serine and proline) to produce BNP(3-32), which has reduced biological activity. Elevated levels of NPs were proved in hyperglycemia and decrease by improved glycemic control (Dal et al. 2014). However, in cardiac pathology, it seems that DPP4 is implicated in high levels of NPs as it was found that genetic deletion of DPP4 improved the elevated levels of ANP and BNP in rats subjected to myocardial ischemia/reperfusion (Ku et al. 2011). However, a recent meta-analysis of clinical studies on diabetic patients treated with DPP4i reported no significant effect of DPP4 on NP levels (Mu et al. 2022) and this finding needs further explanation. SDF-1α is a chemokine that promotes cardiac homing of endothelial progenitor cells, to stimulate angiogenesis, which consequently improves myocardial perfusion. SDF-1α is a substrate of DPP4 enzyme and DPP4 inhibition preserves SDF-1α actions and promotes cardiac recovery after I/R (Pala and Rotella 2013), acute myocardial infarction (Li et al. 2019), or stroke (Chiazza et al. 2018). Another DPP4 substrate is substance P which has role in regulating heart rate and blood pressure. Substance P showed protective effects in some animal models of heart disease through inhibiting apoptosis, myocardial cell injury (Chen et al. 2022), and fibrosis (Widiapradja et al. 2021). On the contrary, in vitro studies reported that DPP4 inactivates fibrin by cleavage of fibrin a-chain leading to inhibition of fibrin polymerization and clot formation (Mentlein and Heymann 1982). This points to possible thrombolytic effect of DPP4 enzyme.

Molecular pathways behind DPP4i effects on CV diseases
Diabetes is linked to a variety of cardiovascular diseases that lower the life quality of diabetic patients. Diabetic patients have many fold increase in the risk of atherosclerosis, myocardial ischemia, myocardial infarction, and heart failure. A common theme shared among these pathologies is massive ROS production that affects glucose metabolism and increase fatty acid oxidation. Additionally, ROS activates proinflammatory mediators, NRLP3 inflammasomes, and proatherogenic transcription factors. They also reduce mediators of tissue repair such as Nrf-2, sirtuin, and AMPK. Moreover, ROS stimulates mitochondrial fission leading to reduced efficiency of the mitochondrial electron transport chain and ATP synthesis, hence, myocardial ischemia and endothelial dysfunction. DPP4 mediates ROS production through several mechanisms of which glucotoxicity is major (see Fig. 1).

DPP4i inhibit oxidative stress via controlling glucotoxicity and lipotoxicity
Studies showed that many gliptins significantly decreased ROS, RNS, DNA fragmentation, AGEs, and Nox4 and increased antioxidants in most of animal models of CV disease. In addition, a recent study by Wang et al. (2021) on liver inflammation in diabetic mice found direct ROS scavenging activity of sitagliptin (Wang et al. 2021). DPP4i work through several mechanisms that can improve myocardial perfusion. DPP4i preserve endothelial function by increasing eNOS phosphorylation and decreasing Ang II-mediated Nox-4 production. They decrease ischemia-induced damage by minimizing oxidative stress. They also increase the level of intracellular cAMP and activate cAMP-dependent protein kinase (PKA) and SDF-1α. Besides, they enhance eNOS activity with subsequent augmentation of endothelialdependent vasodilatation and myocardial perfusion. Incretins might target postprandial lipid metabolism and thereby favorably influence several endothelial and cardiovascular functions. DPP4 release strongly correlates with adipocyte size and is considered risk factor for obesity (Pala and Rotella 2013). Several studies covered by this review found decreased serum TG and total cholesterol and LDL in models of obesity or insulin resistance. DPP4i improve insulin sensitivity which is mediated partially by Sirt-1 and Sirt-6 beside other mechanisms that collectively decrease oxLDL and saturated fatty acids.

DPP4i improve CV inflammation
Preclinical studies show that DPP4i reduce myocardial inflammation via inhibition of cytokine release, monocyte activation, and chemotaxis. It is known that DPP4 significantly activate MAPK and NF-κB signaling pathway leading to vascular aging and dysfunction. Recent studies summarized in the "Results" section show that DPP4i control the release of many proinflammatory mediators such as NFkB, TNF-α, ILs, COX, MAPK, TLR4, CCl2, MCP-1, and MMPs. Inhibition of MMP activity maintains cellular architecture and prevents remodeling and fibrosis. DPP4 at least indirectly is implicated in endothelial and vascular smooth muscle cells structural remodeling and aging through inflammation and oxidative stress. In addition, ROS increases mitochondrial stress leading to energy shortage and subsequent CV senescence. The recent studies on animal models of myocardial infarction, I/R, and diabetes or obesity-induced cardiac remodeling reported that DPP4i significantly reduced hypertrophy, left ventricular interstitial, and periarterial fibrosis as evidenced by decreased expression of TGF-β, collagen, and components of cAMP/ PKA/RhoA/ROCK2 pathway. Decreased fibrotic lesions in myocardial tissue will improve heart conductance and contractility.

DPP4i inhibit cell death
DPP4i decrease overall cell death as evidenced by decreasing infarct size, serum cardiac enzymes (CK, LDH), and troponin T. In addition, they decrease proapoptotic markers MMP-2, HSP-70, and caspase-3 but increased antiapoptotic marker Bcl-2. In another way, DPP4i promote tissue repair mechanisms such as Sirt1,6 (vildagliptin, saxagliptin and sitagliptin) and Nrf-2 (saxagliptin). Also, DPP4 inhibition by sitagliptin or vildagliptin modulated the disturbed autophagy responses in CV disease (LC3II and P65 levels) and vildagliptin enhanced mitochondrial fusion (increased mfn-2 level). These findings are partly resulted from decreased oxidative stress and inflammation due to improved hyperglycemia but may also be attributed to direct action of GLP-1 on its receptor in the CVS.

DPP4i improve CV hemodynamics
Diabetic cardiomyopathies affect almost all parameters of CV hemodynamics with negative impact on both conductance and contractility. Diabetes in animal and human increases peripheral resistance due to endothelial dysfunction and atherosclerosis. Long-term increase in blood pressure attenuates cardiac output specially in the presence of other dependent or independent by CV pathologies. Results of our search in models of diabetes-, obesity-, or druginduced cardiomyopathy showed that treatment with gliptins resulted in increased cardiac output and left ventricular ejection fraction, but reduced systolic, diastolic, mean arterial, and left ventricular end diastolic pressures. These changes help preserve cardiac function. The mechanism of gliptinmediated decrease in BP may be attributed in part to reduced plasma renin concentration and cardiac angiotensin II (Ang II) contents as well as increasing ACE2 and Angiotensin 1-7 (see Fig. 2). It can be also due to less atherosclerotic lesions in large arteries due to controlled lipotoxicity and CV lipid accumulation which is evident in many studies. Taken together, the studies presented in this review clearly found that gliptins improved systolic and diastolic left ventricular dysfunction in many CV disease models and so improved overall cardiac performance.

Conclusions and prospective
Uncontrolled diabetes is associated with CV complications and predispose the patient to end-organ damage such as stroke and heart failure. Since their introduction into drug market, DPP4i have gained attention due to their effective glucose regulation and due to their beneficial CV effects. Studies on DPP4i proved that these effects are partly due to control of hyperglycemia and are also due to direct effect on the CV system via receptor-dependent and possibly receptor-independent effects too. DPP4i modulate not only the level of GLP-1, but also the concentration of other peptides that might exert vasoactive, and CV protective effects such as BNP, SDF-1α, and substance P. In this review, we summarized the result of most recent preclinical studies on CV protective effects of gliptins during the past 5 years. DPP4i control hyperglycemia, decreasing oxidative stress and inflammation, leading to less mitochondrial stress and cell death. They also enhance tissue repair and preserve endothelial function leading to improved myocardial perfusion. Moreover, DPP4i can significantly decrease cardiac Ang II but increase Ang1-7 which can also improve cardiac perfusion. These consistent findings in various CV diseases suggest promising cardioprotective potential of DPP4i, especially when considering their ability to improve glucose control without affecting body weight or causing hypoglycemia. However, further investigations on their mechanism and long-term safety data are required before recommending gliptins as CV-protecting agents in diabetes.
Author contribution Esraa M. Zakaria raised the idea and contributed to all steps of manuscript construction and revision. Walaa M.
Tawfeek participated in constructing the manuscript. Mohammed Y. Hassaballah contributed to data collection, extraction, and manuscript revision. Mohammed Hassan created the figure. All authors approved the submitted version.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Competing interests
The authors declare no competing interests.
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