Abstract
Arterial stiffness, a key indicator of vascular health, encompassing active (vascular tone) and passive (extracellular matrix) components. This study aims to address how these different components affect arterial stiffness along the aorta and the influence of aging. Aortic segments of 12 week and 24 month old (both n = 6) male C57BL/6J mice were mounted in a Rodent Oscillatory Set-up to study Arterial Compliance, in order to measure arterial stiffness and vascular reactivity. Regional variations in arterial stiffness were evident, with abdominal infrarenal aorta (AIA) exhibiting highest stiffness and smallest diameters. AIA displayed both the highest amount of collagen and collagen:elastin ratio. Regional ex vivo vascular reactivity revealed heightened AIA contractions and lowered NO availability. Aging is a significant factor contributing towards vessel remodelling and arterial stiffness. Aging increased arterial stiffness, aortic diameters, collagen content, and reduced VSMC contraction. The results of this study could identify specific regions or mechanisms to target in the development of innovative therapeutic interventions aimed at enhancing overall vascular health.
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Introduction
Vascular health is intricately linked to the ability of arteries to extend and recoil, playing a pivotal role in maintaining blood flow, regulating blood pressure and ensuring adequate oxygen and nutrient delivery to tissues1. Arterial stiffness, a recognized marker of vascular health, reflects the elasticity and compliance of arteries2. Mitchell et al.3 demonstrated that central arterial stiffness, as measured by pulse wave velocity, is a predictor of cardiovascular events and mortality independent of traditional risk factors. Subsequent studies, such as those by Sharif et al.4 and Vlachopoulos et al.5 expanded the field of arterial stiffness, highlighting the significance of arterial stiffness in predicting adverse cardiovascular outcomes. Arterial stiffness is multifaceted, comprised of both active and passive stiffness2,6. The active contribution to arterial stiffness or otherwise known vascular tone, is regulated by vascular smooth muscle cells (VSMCs) and endothelial cells (ECs)7. ECs, lining the inner surface of blood vessels, contribute towards vascular tone by releasing bioactive molecules that modulate vasoconstriction and vasodilation of VSMCs8. Nitric oxide (NO), a key endothelium-derived vasodilator, is crucial for maintaining proper vessel function9. EC dysfunction leads to impaired NO production, contributing towards augmented vasoconstriction, oxidative stress, and ultimately, cardiovascular diseases10. Passive stiffness encompasses extra cellular matrix (ECM) structural proteins2. Collagen and elastin are important structural proteins in ECM, contributing to mechanical properties of blood vessels. Collagen provides tensile strength, while elastin confers elasticity11. The balanced interaction between collagen and elastin is vital for maintaining arterial integrity, allowing blood vessels to withstand mechanical stress12. Arterial tissues from different anatomical regions on the aorta exhibit distinct mechanical properties and endothelial responses due to variations in structure, hemodynamics, and local microenvironments13. Additionally, aging is associated with increased arterial stiffness, caused by changes in active and passive arterial stiffness14.
Hence, and based on current literature, we hypothesised that tissue stiffness increases along the aortic tree and that aging augments the ex vivo mechanical stiffness of the aorta. Therefore, the current study aims to comprehensively characterise both active and passive components influencing regional arterial stiffness and its association with aging. Providing a comprehensive insight into cardiovascular physiology and pathology, offering valuable insights for the development of targeted therapeutic approaches.
Methods
Mice and tissue preparation
12 week old wild type (WT) C57BL/6J male mice (n = 6) were purchased from Charles River (Ecully, France), whereas 24 month old WT C57BL/6J male mice (n = 6) originated from internal breeding. All mice were housed in the University of Antwerp animal facility in standard cages with 12 h–12 h light–dark cycles, with free access to regular chow and tap water. The research adhered to the standards outlined in the ARRIVE guidelines, and all experimental procedures complied with Directive 2010/63/EU of The European Parliament and The Council regarding the protection of animals used for scientific purposes. Furthermore, per the Belgium Royal Decree of May 2013, harvesting of organs for ex vivo analysis is not considered an animal experiment and therefore is exempt from ethical approval. All methods were performed in accordance with these guidelines and regulations. The mice were humanely killed by perforating the diaphragm whilst under anaesthesia (sodium pentobarbital (Sanofi, Belgium), 200 mg/kg i.p.). The aorta was carefully dissected and stripped of any adherent tissue. Subsequently, the aorta was sectioned into 2 mm segments of different anatomical locations (Fig. 1). Experiments related to aging utilized thoracic descending aorta (TDA) segments for comparative purposes whilst thoracic ascending aorta (TAA), TDA and abdominal infrarenal aorta (AIA) were used in regional investigations.
Ex vivo stiffness
Ex vivo stiffness of aortic segments was determined via the Rodent Oscillatory Set-up to study Arterial Compliance (ROTSAC) as previously described by Leloup et al.6 (Fig. 2). In brief, 2 mm aortic segments (TAA, TDA, and AIA) were mounted between two parallel hooks in 10 mL organ baths. Segments were immersed in Krebs Ringer (KR) solution (37 °C, 95% O2/5% CO2, pH 7.4) containing (in mM): NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, CaEDTA 0.025 and glucose 11.1. Force and displacement of the upper hooks were controlled and assessed using a force length transducer. Segments were subjected to cyclic stretching, between alternating preloads, emulating “diastolic” and “systolic” transmural pressures at 10 Hz frequency mimicking the physiological heart rate in mice (600 beats per minute). Transmural pressure was determined using the Laplace relationship. Aortic extension was calibrated using an optical camera to obtain photographs at different stretches, providing calibration of the upper hook and allowing vessel diameters during systole and diastole to be calculated. Subsequently, the Peterson pressure-strain modulus of elasticity (Ep) was calculated at various pressure levels. The Ep was calculated as follows: Ep = D0. ΔP/ΔD, with ΔP = difference in pressure (kept constant at 40 mmHg), D0 = “diastolic” diameter and ΔD = the change in diameter between “diastolic” and “systolic” pressure. The protocol included the evaluation of arterial stiffness (Ep) at different pressures (i.e., ranging from 60–100 mmHg until 140–180 mmHg with 20 mmHg incremental intervals). The pressure-stiffness relationship was determined under a physiological (Krebs–Ringer solution) condition, a maximally-contracted state (PE 2 µM, reflecting active components), as well as in the absence of active stiffness (2 μM of 2-(N,N-diethylamino)-diazenolate-2-oxide sodium salt hydrate (DEANO), a NO donor to remove any contribution of VSMC contraction).
Evaluation of vascular reactivity
For assessment of vascular reactivity, the force–length transducer was switched (after completion of the stiffness protocol) to isometric modus and aortic segments were preloaded equivalent to 100 mmHg mean pressure. VSMC contraction was evaluated by adding cumulative concentrations of phenylephrine (PE; 1 nM–10 μM), an α1-adrenergic receptor agonist. Subsequently, EC-dependent relaxations were evaluated by adding cumulative concentrations of acetylcholine (ACh; 3 nM–10 μM), a muscarinic receptor agonist, or Adenosine-5′-triphosphate (ATP; 10 nM–100 µM), a purinergic receptor agonist. ACh and ATP engage different intracellular signalling pathways, providing complementary assessment of complex regulatory mechanisms involved in regional vascular reactivity. To negate the influence of NO, l-Nitro arginine methyl ester (l-NAME; 300 μM), a non-selective NO synthase inhibitor, was administered. Basal NO index was calculated by assessing the change in contractile response (Δ force) before and after the introduction of L-NAME and expressed as a percentage as previously reported15,16. DEANO (0.03 nM–10 μM), an exogenic nitric oxide donor was administered to investigate EC-independent VSMC relaxation.
Histology
Aortic segments (TAA, TDA and AIA) were fixed in 4% buffered formaldehyde solution (BDH Prolabo, VWR, Belgium) for 24 h, subsequently dehydrated in 60% isopropanol (BDH Prolabo, VWR, Belgium), followed by paraffin-embedding. Total collagen of the medial area was evaluated by a Sirius red staining (Abcam, ab150681). Elastin content was evaluated by staining with orcein (Abcam, ab245881). Elastin breaks were characterised by the degradation or breakdown of elastic fibres, which appear discontinuous, disrupted, or broken in aortic tissue cross-sections. Total collagen and elastin was expressed as percentage area positivity in the region of interest (i.e., the medial layer). The Olympus BX40 microscope and Universal Graph 6.1 software were used to capture mosaic images at 20× magnification, quantified using ImageJ software.
Chemical compounds
PE, L-NAME, ACh, ATP, and DEANO were obtained from Sigma-Aldrich (Overijse, Belgium).
Statistics
Statistical analyses were performed in GraphPad Prism 10.0.0. All data are presented as mean ± SEM. Dots represent n samples from the experiments. Regional disparities were evaluated through a one-way ANOVA, with a Sidak post hoc test to address multiple comparisons. To address regional distinctions across pressure gradients and concentration responses, a two-way ANOVA with repeated measures was conducted, alongside a Sidak post hoc test. The impact of aging was assessed using unpaired T-tests. Moreover, to examine age-related effects across pressure gradients and concentration responses, a two-way ANOVA with repeated measures was employed, accompanied by a Sidak post hoc test. Statistical tests are specified in the figure legends. Differences were considered significant when p < 0.05.
Results
The relationship between pressure and stiffness was investigated in aortic tissue from different anatomical locations (Fig. 2A) in three different conditions. Under physiological conditions, regional differences were observed in the pressure-stiffness relationship (p < 0.001) (Fig. 2B). Additionally, differences in arterial stiffness were reported regionally at basal (i.e. 80–120 mmHg) pressure (Fig. 2C). The AIA showed the highest overall arterial stiffness and smallest ex vivo diameter compared to TAA (p < 0.0001) and TDA (p < 0.0001), whereas the ex vivo diameter of the TDA was smaller (p < 0.0001) to TAA (Fig. 2D, Supplementary Fig. 1).
Investigating active and passive factors affecting arterial stiffness through analysis of pressure-stiffness curves, using PE (2 μM, active) and DEANO (2 μM, passive) respectively, unveiled subtle distinctions in curve morphology consistent regionally. The pressure-stiffness curves resulting from PE showed a slight shift upwards to the right, suggesting a trend towards higher stiffness at lower pressures and lower stiffness at higher pressures when compared to the pressure-stiffness curves from DEANO (Fig. 3A–C). Additionally, PE resulted in smaller diameters regionally compared to DEANO (Fig. 3D–F), revealing an increased buffering capacity and lowered recruitment of stiffer collagen fibres at higher pressure.
As differences in the mechanical properties between tissues from different regions of the aorta were apparent, alterations in passive load-bearing components were postulated. Characterization of ECM structural proteins followed to understand passive disparities in regional pressure-stiffness relationships. Sirius Red and Orcein stains were performed for collagen and elastin, respectively, to assess regional differences in the load-bearing components of the arterial wall (Fig. 4A). Whereas arterial tissues from the AIA had a significantly higher amount of collagen than the TAA (p < 0.01) and TDA (p < 0.05) (Fig. 4B), they had lower amounts of elastin compared to tissue from the TAA (p < 0.05) and TDA (p < 0.01) (Fig. 4C). Hence, tissue samples from AIA have higher (p < 0.05) collagen:elastin ratio (Fig. 4D).
Next, investigations into active disparities revealed region specific alterations in ex vivo vascular reactivity. AIA showed increased PE induced contractions after an incremental concentration response (p < 0.05) (Fig. 5A). However, contractions in the presence of an endothelial NO synthase inhibitor (L-NAME) revealed, AIA experiencing highest contractions and TDA lowest (Fig. 5B). Therefore, basal NO index, an indicator of EC functionality was reduced in AIA when compared to TAA (p < 0.01) and TDA (p < 0.05) (Fig. 5C). Variations in endothelial-dependent relaxations were evident under both ACh (p < 0.01) (Fig. 5D) and ATP (p < 0.05) (Fig. 5E), suggesting a lowered capacity of the ECs, present in the AIA to generate NO upon stimulation compared to TAA, with a similar trend observed in TDA. Administration of DEANO resulted in no differences in regional relaxations, revealing no regional alternations in ability of VSMCs to interact with NO (Fig. 5F).
In a second part, the effect of aging on the active and passive components of arterial stiffness were evaluated by assessing TDA segments of 24 month vs 12 week old mice. Overall, aging increased arterial stiffness ex vivo in a pressure dependent manner (Fig. 6A, (p < 0.0001)). The diameters of aged mice were also larger (p < 0.001) when compared to those of younger mice (Fig. 6B). This occurrence is indicative of the outward remodelling processes commonly associated with the aging vasculature. Sirius Red (total collagen) and Orcein (i.e. elastin breaks) stains (Fig. 7A,C) were performed to assess possible aged related ECM remodelling. Collagen content was found to be higher (p < 0.001) in aged mice when compared to young mice (Fig. 7B). Fragmentation of elastin fibres was not present in either young nor aged aortic tissue (Fig. 7D).
Investigating ex vivo vascular reactivity further revealed the effects of aging on active components of the arterial wall. A significant reduction (p < 0.01) in PE mediated VSMC contraction was observed in aged mice (Fig. 8A). Furthermore, maximum contraction (i.e., PE contraction in the presence of LNAME) was reduced in aortic samples from aged mice as well (Fig. 8B). Alternatively, no changes in basal NO index were observed between both groups (Fig. 8C). EC-dependent relaxation with ATP was increased in aortic samples from aged mice (p < 0.0001, Fig. 8E). However, no differences were observed in ACh-mediated vasorelaxation (Fig. 8D). Finally, administration of DEANO resulted in a left shift (p < 0.01) in aged mice, revealing an increased sensitivity of VSMCs to NO (Fig. 8F).
Discussion
The current study showcased regional differences in arterial stiffness and vascular reactivity, indicating that tissue stiffness is not uniform along the aorta. Furthermore, also VSMC contractility differed in different regions of the aorta, with heightened responses observed in more distal region. Furthermore, we demonstrated that aging increased arterial stiffness paralleled with increased collagen content and VSMC dysfunction.
Ex vivo measurements incorporated our in-house developed organ bath setup (ROTSAC) possessing the unique capability to study active and passive contributions of arterial stiffness under dynamic oscillations, yet independently from in vivo confounders6. A growing body of literature has sought to unravel the intricate relationship between arterial stiffness and vascular function at a regional level, however current data is fragmented with limited mechanistic insight. Nevertheless, advancements in imaging modalities, such as high-resolution ultrasound and magnetic resonance imaging, have allowed for a more detailed assessment of regional variations in arterial stiffness8. In a study by Bia et al.10 utilizing in vitro pressure diameter loops as well as mathematical modelling to evaluate regional differences in arterial elastic modulus, arterial stiffness was demonstrated to be increased in distal aortic regions compared to proximal aortic regions. Our ex vivo findings complement and align with previous research, such as work by Bia et al.17 and Wilkinson et al.18 who reported discrepancies in stiffness between proximal and distal aortic regions. Regional variability can be attributed to variations in composition of ECM and vascular tone. Indeed, the buffering of cardiac output mainly occurs in TAA and TDA and less in AIA as reflected in collagen and elastin percentages19, hence making the AIA less compliant and more stiff. On the other hand, in case of hypertension, the AIA would become more stiff compared to TDA20, as evident by AIA having highest overall vascular tone and lowest basal NO index. Further, a theoretical de-stiffening effect of anti-hypertensives at different regions is conceivable. However, passive stiffness (e.g. elastin breaks with aging) holds most importance with regional variations in arterial stiffness, with active contribution being notably smaller. Nonetheless, this active stiffness plays a crucial role in managing acute alterations in hemodynamics without directly inducing arterial remodelling, which is not needed to accommodate acute rises in cardiac output during exercise21,22. The current findings are further supported by AIA experiencing a significantly higher amount of collagen and collagen:elastin ratio compared with TAA and TDA. Collagen is fundamental in providing structural support and tensile strength, therefore segments with a more abundant collagen network as well as increased collagen:elastin ratios will result in elevated arterial stiffness and reduced compliance23,24. Differences in elastin content were also observed regionally, with TAA and TDA containing higher amounts of elastin compared to AIA. Elastin, imparts elasticity and recoil properties to arteries, enabling expansion in response to changes in blood flow and pressure23,25. Consequently, aortic segments situated in closer proximity to the heart must adapt to increased pressure and blood flow resulting in elevated elastin content26, supported by our current findings.
Differences in vascular reactivity were observed regionally, with basal NO index serving as a metric for EC function15. NO availability was evaluated by comparing VSMC contractions in the presence and absence of NO, revealing noteworthy distinctions among different regions. TAA observed highest levels of basal NO index accompanied with AIA lowest. Compromised basal NO production is linked to endothelial dysfunction, increased vascular tone, and impaired blood flow regulation9. To assess EC function regionally and through aging, relaxation induced by ACh and ATP were evaluated. The rationale for employing a dual-compound approach lies in distinct actions of ACh and ATP on ECs. ACh primarily stimulates NO through muscarinic signalling pathways, whereas ATP activates ECs through purinergic signally pathways27,28, offering valuable insight into signalling mechanisms that underlie vasodilation regionally and through aging. Interestingly, the AIA exhibited a significantly lower ability to produce NO when compared with TAA and TDA under both conditions. Most notable differences were present under EC dependant relaxations with ATP. Interestingly, Morato et al.29 found an elevated distribution of P2X receptors in small arteries, potentially concealing the vasodilatory impact of P2Y receptors. This observation may contribute to regional variations observed in our present study. Indicating possible variances in distribution of purinergic receptor subtypes across regions. No differences in endothelium-independent relaxation were observed with DEANO. Therefore, we can conclude no discernible regional variations in sensitivity of VSMC to NO were present within aortic segments of young mice.
In a subsequent sub-analysis, we examined the contributions of active and passive constituents of arterial stiffness in the context of aging. Specifically, we conducted comparisons between TDA segments of old (24-month) and young (12-week) mice. TDA segments were chosen due to practical considerations but are representative of the central aorta30. Selection of 24-month-old mice represented an advanced aging model, associated with increased PWV and outwards remodelling, known-features of aging31. It is well established that VSMCs exhibit a phenotype switch from a contractile to a synthetic state with aging, altering contractile properties of the aorta32,33. Additionally, ECs have a reduced ability to produce NO accompanied with increasing amounts of oxidative stress34,35. Concurrently, passive augmentation in collagen content and fragmentation of elastin are present, leading to heightened vessel stiffness and diminished compliance, thereby amplifying vulnerabilities to cardiovascular diseases36,37. As expected, aging was associated with increased arterial stiffness in a pressure dependent manner accompanied with significantly higher collagen content compared to younger counterparts, resulting in heightened arterial stiffness. However, no differences in elastin fragmentation were present. As suggested by Neutel et al.16 the TAA may be more appropriate for studying elastin breaks in aged animals than the TDA. Interestingly, most physiological changes in vascular reactivity with aging involved diminished VSMC function, whereas EC-dependent relaxations were mostly unaffected. The current findings contradict clinical literature where endothelial dysfunction is a hallmark of cardiovascular aging38,39. However, previous work by our lab16,31 has shown that the thoracic descending aorta of C57BL6/J mice at 24 months of age did not exhibit a sufficiently advanced state of aging to exhibit endothelial dysfunction. Nonetheless, VSMC dysfunction further exacerbates passive arterial stiffness as VSMC cannot buffer elevated arterial pressure resulting in recruitment of stiffer collagen fibres, subsequently increasing arterial stiffness40,41, emphasizing the synergistic interplay between active and passive components of arterial stiffness.
In the current study, we were primarily interested in studying the regional differences in mechanical properties of the aorta ex vivo, in that regard we focused exclusively on male mice. However, we acknowledge and recognise the potential limitations inherent in our study methodology. Concentrating solely on male mice could introduce a sex bias, as arterial responses and age-related changes in female mice might diverge from those observed in males, possibly neglecting sex-specific disparities in vascular physiology and pathology. Furthermore, the analysis of age-related changes was limited to the thoracic descending aorta, while similar assessments were conducted on the thoracic ascending aorta, thoracic descending aorta and abdominal infrarenal aorta in young mice. This discrepancy in regional analysis may overlook potential variations in aging effects across different regions of the aorta. The thoracic descending aorta represents the central aorta with a balanced collagen-to-elastin ratio and hence is interesting for studying the aging effect of central arteries. Nonetheless, for future studies, the incorporation of both male and female mice as well as regional differences with aging should be investigated.
To conclude, our current study provides comprehensive insight into both active and passive components influencing regional arterial stiffness and their association with aging. Moreover, the observed changes in vascular reactivity across various vascular regions and during aging provide important information for the creation of targeted treatment strategies. In particular, these strategies could focus on improving endothelial function or reducing arterial stiffness in specific areas of the aorta. This tailored approach, aligning interventions with regional vascular characteristics, has potential to optimise treatment efficacy. Additionally, regional differences in arterial stiffness and reactivity play a significant role in hemodynamics, influencing blood flow patterns and pressure distribution. A comprehensive understanding of these dynamics holds implications for managing conditions associated with hemodynamic alterations. The results of this study could identify specific regions or mechanisms that could be targeted in the development of innovative therapeutic interventions aimed at enhancing overall vascular health.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author (Callan Wesley) on reasonable request.
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Acknowledgements
The authors would like to thank Mandy Vermont, Hermine Fret and Abbi Van Tilborg for technical help.
Funding
C.D.W. is an Early Stage Researcher (ESR) in the INSPIRE project, which has received funding from the European Union’s Horizon 2020 Research and Innovation Program (H2020-MSCA-ITN program, Grant Agreement: No858070) and the Research Council of the University of Antwerp (BOF DOCPRO1 funding). C.H.G.N. is a predoctoral fellow of the Fund for Scientific Research (FWO)-Flanders.
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C.W. wrote the main manuscript text and prepared all figures. C.W. and C.N. executed all experiments. C.W., W.M. and P.J.G. were involved in the conceptualization, design and planning of all experiments. G.D.M, W.M. and P.J.G. provided interpretation of study results. All authors reviewed the manuscript.
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Wesley, C.D., Neutel, C.H.G., De Meyer, G.R.Y. et al. Unravelling the impact of active and passive contributors to arterial stiffness in male mice and their role in vascular aging. Sci Rep 14, 18337 (2024). https://doi.org/10.1038/s41598-024-68725-9
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DOI: https://doi.org/10.1038/s41598-024-68725-9
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