Background— Because of global aging, the prevalence of heart failure with preserved ejection fraction (HFpEF) continues to rise. Although HFpEF pathophysiology remains incompletely understood, endothelial inflammation is stated to play a central role. Cellular senescence is a process of cellular growth arrest linked with aging and inflammation. We used mice with accelerated aging to investigate the role of cellular senescence in HFpEF development. Methods and Results— Senescence-accelerated mice (SAM, n=18) and control mice with normal senescence (n=15) were fed normal chow or a high-fat, high-salt diet (WD). Vascular and cardiac function was assessed at 8, 16, and 24 weeks of age. At 24 weeks, both SAM on WD (SAM-WD) and SAM on regular diet displayed endothelial dysfunction, as evidenced by impaired acetylcholine-induced relaxation of aortic segments and reduced basal nitric oxide. At week 24, SAM-WD had developed HFpEF, characterized by diastolic dysfunction, left ventricular hypertrophy, left atrial dilatation, and interstitial fibrosis. Also, exercise capacity was reduced and lung weight increased. Cardiovascular inflammation and senescence were assessed by immunohistochemical and immunofluorescence staining of hearts and aortas. SAM-WD showed increased endothelial inflammation (intercellular adhesion molecule 1 expression) and increased endothelial senescence (acetyl-p53/CD31 costaining). The latter correlated with diastolic function and intercellular adhesion molecule 1 expression. Conclusions— SAM develop endothelial dysfunction. Adding a high-salt, high-fat diet accelerates endothelial senescence and instigates endothelial inflammation. This coincides with hemodynamic and structural changes typical of HFpEF. Targeting endothelial senescence could be a new therapeutic avenue in HFpEF.

L-type Ca2+ channel (VGCC) mediated Ca2+ influx in vascular smooth muscle cells (VSMC) contributes to the functional properties of large arteries in arterial stiffening and central blood pressure regulation. How this influx relates to steady-state contractions elicited by α1-adrenoreceptor stimulation and how it is modulated by small variations in resting membrane potential (Vm) of VSMC is not clear yet. Here, we show that α1-adrenoreceptor stimulation of aortic segments of C57Bl6 mice with phenylephrine (PE) causes phasic and tonic contractions. By studying the relationship between Ca2+ mobilisation and isometric tension, it was found that the phasic contraction was due to intracellular Ca2+ release and the tonic contraction determined by Ca2+ influx. The latter component involves both Ca2+ influx via VGCC and via non-selective cation channels (NSCC). Influx via VGCC occurs only within the window voltage range of the channel. Modulation of this window Ca2+ influx by small variations of the VSMC Vm causes substantial effects on the contractile performance of aortic segments. The relative contribution of VGCC and NSCC to the contraction by α1-adrenoceptor stimulation could be manipulated by increasing intracellular Ca2+ release from non-contractile sarcoplasmic reticulum Ca2+ stores. Results of this study point to a complex interactions between α1-adrenoceptor-mediated VSMC contractile performance and Ca2+ release form contractile or non-contractile Ca2+ stores with concomitant Ca2+ influx. Given the importance of VGCC and their blockers in arterial stiffening and hypertension, they further point toward an additional role of NSCC (and NSCC blockers) herein.

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 Ca2+ 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.

Over the past few decades, isometric contraction studies of isolated thoracic aorta segments have significantly contributed to our overall understanding of the active, contractile properties of aortic vascular smooth muscle cells ( VSMC s) and their cross‐talk with endothelial cells. However, the physiological role of VSMC contraction or relaxation in the healthy aorta and its contribution to the pulse‐smoothening capacity of the aorta is currently unclear. Therefore, we investigated the acute effects of VSMC contraction and relaxation on the isobaric biomechanical properties of healthy mouse aorta. An in‐house developed set‐up was used to measure isobaric stiffness parameters of periodically stretched (10 Hz) aortic segments at an extended pressure range, while pharmacologically modulating VSMC tone and endothelial cell function. We found that the effects of α1‐adrenergic stimulation with phenylephrine on the pressure‐stiffness relationship varied in sensitivity, magnitude and direction, with the basal, unstimulated NO production by the endothelium playing a pivotal role. We also investigated how arterial disease affected this system by using the angiotensin‐ II ‐treated mouse. Our results show that isobaric stiffness was increased and that the aortic segments demonstrated a reduced capacity for modulating the pressure‐stiffness relationship. This suggests that not only increased isobaric stiffness at normal pressure, but also a reduced capacity of the VSMC s to limit the pressure‐associated increase in aortic stiffness, may contribute to the pathogenesis of this mouse model. Overall, this study provides more insight in how aortic VSMC tone affects the pressure‐dependency of aortic biomechanics at different physiological and pathological conditions.

Cyclic stretch is a major contributor to vascular function. However, isolated mouse aortas are frequently studied at low stretch frequency or even in isometric conditions. Pacing experiments in rodents and humans show that arterial compliance is stretch frequency dependent. The Rodent Oscillatory Tension Set-up to study Arterial Compliance is an in-house developed organ bath set-up that clamps aortic segments to imposed preloads at physiological rates up to 600 beats min . The technique enables us to derive pressure-diameter loops and assess biomechanical properties of the segment. To validate the applicability of this set-up we aimed to confirm the effects of distension pressure and vascular smooth muscle tone on arterial stiffness. At physiological stretch frequency (10 Hz), the Peterson modulus (E ; 293 (10) mmHg) for wild-type mouse aorta increased 22% upon a rise in pressure from 80-120 mmHg to 100-140 mmHg, while, at normal pressure, E increased 80% upon maximal contraction of the vascular smooth muscle cells. We further validated the method using a mouse model with a mutation in the fibrillin-1 gene and an endothelial nitric oxide synthase knock-out model. Both models are known to have increased arterial stiffness, and this was confirmed using the set-up. To our knowledge, this is the first set-up that facilitates the study of biomechanical properties of mouse aortic segments at physiological stretch frequency and pressure. We believe that this set-up can contribute to a better understanding of how cyclic stretch frequency, amplitude and active vessel wall components influence arterial stiffening.

Arterial hypertension (AHT) affects the voltage dependency of L-type Ca(2+) channels in cardiomyocytes. We analyzed the effect of angiotensin II (AngII)-induced AHT on L-type Ca(2+) channel-mediated isometric contractions in conduit arteries. AHT was induced in C57Bl6 mice with AngII-filled osmotic mini-pumps (4 weeks). Normotensive mice treated with saline-filled osmotic mini-pumps were used for comparison. Voltage-dependent contractions mediated by L-type Ca(2+) channels were studied in vaso-reactive studies in vitro in isolated aortic and femoral arteries by using extracellular K(+) concentration-response (KDR) experiments. In aortic segments, AngII-induced AHT significantly sensitized isometric contractions induced by elevated extracellular K(+) and depolarization. This sensitization was partly prevented by normalizing blood pressure with hydralazine, suggesting that it was caused by AHT rather than by direct AngII effects on aortic smooth muscle cells. The EC50 for extracellular K(+) obtained in vitro correlated significantly with the rise in arterial blood pressure induced by AngII in vivo. The AHT-induced sensitization persisted when aortic segments were exposed to levcromakalim or to inhibitors of basal nitric oxide release. Consistent with these observations, AngII-treatment also sensitized the vaso-relaxing effects of the L-type Ca(2+) channel blocker diltiazem during K(+)-induced contractions. Unlike aorta, AngII-treatment desensitized the isometric contractions to depolarization in femoral arteries pointing to vascular bed specific responses of arteries to hypertension. AHT affects the voltage-dependent L-type Ca(2+) channel-mediated contraction of conduit arteries. This effect may contribute to the decreased vascular compliance in AHT and explain the efficacy of Ca(2+) channel blockers to reduce vascular stiffness and central blood pressure in AHT.

Most vaso-reactive studies in mouse aortic segments are performed in isometric conditions and at an optimal preload, which is the preload corresponding to a maximal contraction by non-receptor or receptor-mediated stimulation. In general, this optimal preload ranges from about 1.2 to 8.0 mN/mm, which according to Laplace's law roughly correlates with transmural pressures of 10–65 mmHg. For physiologic transmural pressures around 100 mmHg, preloads of 15.0 mN/mm should be implemented. The present study aimed to compare vascular reactivity of 2 mm mouse (C57Bl6) aortic segments preloaded at optimal (8.0 mN/mm) vs. (patho) physiological (10.0–32.5 mN/mm) preload. Voltage-dependent contractions of aortic segments, induced by increasing extracellular K+, and contractions by α1-adrenergic stimulation with phenylephrine (PE) were studied at these preloads in the absence and presence of L-NAME to inhibit basal release of NO from endothelial cells (EC). In the absence of basal NO release and with higher than optimal preload, contractions evoked by depolarization or PE were attenuated, whereas in the presence of basal release of NO PE-, but not depolarization-induced contractions were preload-independent. Phasic contractions by PE, as measured in the absence of external Ca2+, were decreased at higher than optimal preload suggestive for a lower contractile SR Ca2+ content at physiological preload. Further, in the presence of external Ca2+, contractions by Ca2+ influx via voltage-dependent Ca2+ channels were preload-independent, whereas non-selective cation channel-mediated contractions were increased. The latter contractions were very sensitive to the basal release of NO, which itself seemed to be preload-independent. Relaxation by endogenous NO (acetylcholine) of aortic segments pre-contracted with PE was preload-independent, whereas relaxation by exogenous NO (diethylamine NONOate) displayed higher sensitivity at high preload. Results indicated that stretching aortic segments to higher than optimal preload depolarizes the SMC and causes Ca2+ unloading of the contractile SR, making them extremely sensitive to small changes in the basal release of NO from EC as can occur in hypertension or arterial stiffening.

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