Due to its viscoelastic properties, the aorta aids in dampening blood pressure pulsatility. At the level of resistance-arteries, the pulsatile flow will be transformed into a continuous flow to allow for optimal perfusion of end organs such as the kidneys and the brain. In this study, we investigated the ex vivo viscoelastic properties of different regions of the aorta of healthy C57Bl6/J adult mice as well as the interplay between (altered) cyclic stretch and viscoelasticity. We demonstrated that the viscoelastic parameters increase along the distal aorta and that the effect of altered cyclic stretch is region dependent. Increased cyclic stretch, either by increased pulse pressure or pulse frequency, resulted in decreased aortic viscoelasticity. Furthermore, we identified that the vascular smooth muscle cell (VSMC) is an important modulator of viscoelasticity, as we have shown that VSMC contraction increases viscoelastic parameters by, in part, increasing elastin fiber tortuosity. Interestingly, an acute increase in stretch amplitude reverted the changes in viscoelastic properties induced by VSMC contraction, such as a decreasing contraction-induced elastin fiber tortuosity. Finally, the effects of altered cyclic stretch and VSMC contraction on viscoelasticity were more pronounced in the abdominal infrarenal aorta, compared to both the thoracic ascending and descending aorta, and were attributed to the activity and stability of VSMC focal adhesion. Our results indicate that cyclic stretch is a modulator of aortic viscoelasticity, acting on VSMC focal adhesion. Conditions of (acute) changes in cyclic stretch amplitude and/or frequency, such as physical exercise or hypertension, can alter the viscoelastic properties of the aorta.

Measuring arterial stiffness has recently gained a lot of interest because it is a strong predictor for cardiovascular events and all-cause mortality. However, assessing blood vessel stiffness is not easy and the in vivo measurements currently used provide only limited information. Ex vivo experiments allow for a more thorough investigation of (altered) arterial biomechanical properties. Such experiments can be performed either statically or dynamically, where the latter better corresponds to physiological conditions. In a dynamic setup, arterial segments oscillate between two predefined forces, mimicking the diastolic and systolic pressures from an in vivo setting. Consequently, these oscillations result in a pulsatile load (i.e., the pulse pressure). The importance of pulse pressure on the ex vivo measurement of arterial stiffness is not completely understood. Here, we demonstrate that pulsatile load modulates the overall stiffness of the aortic tissue in an ex vivo setup. More specifically, increasing pulsatile load softens the aortic tissue. Moreover, vascular smooth muscle cell (VSMC) function was affected by pulse pressure. VSMC contraction and basal tonus showed a dependence on the amplitude of the applied pulse pressure. In addition, two distinct regions of the aorta, namely the thoracic descending aorta (TDA) and the abdominal infrarenal aorta (AIA), responded differently to changes in pulse pressure. Our data indicate that pulse pressure alters ex vivo measurements of arterial stiffness and should be considered as an important variable in future experiments. More research should be conducted in order to determine which biomechanical properties are affected due to changes in pulse pressure. The elucidation of the underlying pulse pressure-sensitive properties would improve our understanding of blood vessel biomechanics and could potentially yield new therapeutic insights.

Autophagy is an important cellular survival process that enables degradation and recycling of defective organelles and proteins to maintain cellular homeostasis. Hence, defective autophagy plays a role in many age-associated diseases, such as atherosclerosis, arterial stiffening and hypertension. Recently, we showed in mice that autophagy in vascular smooth muscle cells (VSMCs) of large elastic arteries such as the aorta is important for Ca2+ mobilization and vascular reactivity. Whether autophagy plays a role in the smaller muscular arteries, such as the femoral artery, and thereby contributes to for example, blood pressure regulation is currently unknown. Therefore, we determined vascular reactivity of femoral artery segments of mice containing a VSMC specific deletion of the essential autophagy gene Atg7 (Atg7F/F SM22α-Cre+) and compared them to femoral artery segments of corresponding control mice (Atg7+/+ SM22α-Cre+). Our results indicate that similar to the aorta, femoral artery segments showed enhanced contractility. Specifically, femoral artery segments of Atg7F/F SM22α-Cre+ mice showed an increase in phasic phenylephrine (PE) induced contractions, together with an enhanced sensitivity to depolarization induced contractions. In addition, and importantly, VSMC sensitivity to exogenous nitric oxide (NO) was significantly increased in femoral artery segments of Atg7F/F SM22α-Cre+ mice. Notwithstanding the fact that small artery contractility is a significant pathophysiological determinant for the development of hypertension, 7 days of treatment with angiotensin II (AngII), which increased systolic blood pressure in control mice, was ineffective in Atg7F/F SM22α-Cre+ mice. It is likely that this was due to the increased sensitivity of VSMCs to NO in the femoral artery, although changes in the heart upon AngII treatment were also present, which could also be (partially) accountable for the lack of an AngII-induced rise in blood pressure in Atg7F/F SM22α-Cre+ mice. Overall, our study indicates that apart from previously shown effects on large elastic arteries, VSMC autophagy also plays a pivotal role in the regulation of the contractile and relaxing properties of the smaller muscular arteries. This may suggest a role for autophagy in vascular pathologies, such as hypertension and arterial stiffness.

Introduction and Aims: Endothelial nitric oxide synthase (eNOS) knockout mice develop pronounced cardiovascular disease. In the present study, we describe the alterations in aortic physiology and biomechanics of eNOS knockout and C57Bl/6 control mice at 2-12 months of age, including a thorough physiological investigation of age and cyclic stretch-dependent VSMC contractility and aortic stiffness. Methods and Results: Peripheral blood pressure and aortic pulse wave velocity were measured in vivo, and aortic biomechanical studies and isometric contractions were investigated ex vivo. Age-dependent progression of aortic stiffness, peripheral hypertension, and aortic contractility in eNOS knockout mice was absent, attenuated, or similar to C57Bl/6 control mice. Voltage-gated calcium channel (VGCC)-dependent calcium influx inversely affected isometric contraction and aortic stiffening by α1-adrenergic stimulation in eNOS knockout mice. Baseline aortic stiffness was selectively reduced in eNOS knockout mice after ex vivo cyclic stretch exposure in an amplitude-dependent manner, which prompted us to investigate cyclic stretch dependent regulation of aortic contractility and stiffness. Aortic stiffness, both in baseline conditions and after activation of vascular smooth muscle cell (VSMC) contraction, was reduced with increasing cyclic stretch amplitude. This cyclic stretch dependency was attenuated with age, although aged eNOS knockout mice displayed better preservation of cyclic stretch-dependency compared to C57Bl/6 control mice. Store operated calcium entry-medicated aortic stiffening as induced by inhibiting sarcoplasmic reticulum calcium ATPase pumps with 10 µM CPA was most pronounced in the aorta of aged mice and at low cyclic stretch amplitude, but independent of eNOS. Basal aortic tonus and VSMC depolarization were highly dependent on eNOS, and were most pronounced at low cyclic stretch, with attenuation at increasing cyclic stretch amplitude. Conclusion: eNOS knockout mice display attenuated progression of arterial disease as compared to C57Bl/6 control mice. Basal VSMC tone in eNOS knockout mice could be reduced by ex vivo exposure to cyclic stretch through stretch-dependent regulation of cytosolic calcium. Both baseline and active aortic stiffness were highly dependent on cyclic stretch regulation, which was more pronounced in young versus aged mice. Other mediators of VSMC contraction and calcium handling were dependent on cyclic stretch mechanotransduction, but independent of eNOS.

In the last decades, the search for mechanisms underlying progressive arterial stiffening and for interventions to avoid or reverse this process has gained much attention. In general, arterial stiffening displays regional variation and is, for example, during aging more prominent in elastic than in muscular arteries. We hypothesize that besides passive also active regulators of arterial compliance [i.e., endothelial and vascular smooth muscle cell (VSMC) function] differ between these arteries. Hence, it is conceivable that these vessel types will display different time frames of stiffening. To investigate this hypothesis segments of muscular arteries such as femoral and mesenteric arteries and elastic arteries such as the aorta and carotid artery were isolated from female C57Bl6 mice (5-6 months of age, n = 8). Both microscopy and passive stretching of the segments in a myograph confirmed that passive mechanical properties (elastin, collagen) of elastic and muscular arteries were significantly different. Endothelial function, more specifically basal nitric oxide (NO) efficacy, and VSMC function, more specifically L-type voltage-gated Ca(2+) channel (VGCC)-mediated contractions, were determined by α1-adrenoceptor stimulation with phenylephrine (PE) and by gradual depolarization with elevated extracellular K(+) in the absence and presence of eNOS inhibition with L-NAME. PE-mediated isometric contractions significantly increased after inhibition of NO release with L-NAME in elastic, but not in muscular vessel segments. This high basal eNOS activity in elastic vessels was also responsible for shifts of K(+) concentration-contraction curves to higher external K(+). VGCC-mediated contractions were similarly affected by depolarization with elevated K(+) in muscular artery segments or in elastic artery segments in the absence of basal NO. However, K(+)-induced contractions were inhibited by the VGCC blocker diltiazem with significantly higher sensitivity in the muscular arteries, suggestive of different populations of VGCC isoforms in both vessel types. The results from the present study demonstrate that, besides passive arterial wall components, also active functional components contribute to the heterogeneity of arterial compliance along the vascular tree. This crucially facilitates the search for (patho) physiological mechanisms and potential therapeutic targets to treat or reverse large artery stiffening as occurring in aging-induced arterial stiffening.