Prdm16 Supports Arterial Flow Recovery by Maintaining Endothelial Function.

[Figure: see text].

Neuregulin-1 compensates for endothelial nitric oxide synthase deficiency.

Endothelial cells (ECs) secrete different paracrine signals that modulate the function of adjacent cells; two examples of these paracrine signals are nitric oxide (NO) and neuregulin-1 (NRG1), a cardioprotective growth factor. Currently, it is undetermined whether one paracrine factor can compensate for the loss of another. Herein, we hypothesized that NRG1 can compensate for endothelial NO synthase (eNOS) deficiency. We characterized eNOS null and wild-type (WT) mice by cardiac ultrasound and histology and we determined circulating NRG1 levels. In a separate experiment, eight groups of mice were divided into four groups of eNOS null mice and WT mice; half of the mice received angiotensin II (ANG II) to induce a more severe phenotype. Mice were randomized to daily injections with NRG1 or vehicle for 28 days. eNOS deficiency increased NRG1 plasma levels, indicating that ECs increase their NRG1 expression when NO production is deleted. eNOS deficiency also increased blood pressure, lowered heart rate, induced cardiac fibrosis, and affected diastolic function. In eNOS null mice, ANG II administration not only increased cardiac fibrosis but also induced cardiac hypertrophy and renal fibrosis. NRG1 administration prevented cardiac and renal hypertrophy and fibrosis caused by ANG II infusion and eNOS deficiency. Moreover, Nrg1 expression in the myocardium is shown to be regulated by miR-134. This study indicates that administration of endothelium-derived NRG1 can compensate for eNOS deficiency in the heart and kidneys.NEW & NOTEWORTHY ECs compensate for eNOS deficiency by increasing the secretion of NRG1. NRG1 administration prevents cardiac and renal hypertrophy and fibrosis caused by ANG II infusion and eNOS deficiency. NRG1 expression is regulated by miR-134.

Defective autophagy in vascular smooth muscle cells increases passive stiffness of the mouse aortic vessel wall.

Aging and associated progressive arterial stiffening are both important predictors for the development of cardiovascular diseases. Recent evidence showed that autophagy, a catabolic cellular mechanism responsible for nutrient recycling, plays a major role in the physiology of vascular cells such as endothelial cells and vascular smooth muscle cells (VSMCs). Moreover, several autophagy inducing compounds are effective in treating arterial stiffness. Yet, a direct link between VSMC autophagy and arterial stiffness remains largely unidentified. Therefore, we investigated the effects of a VSMC-specific deletion of the essential autophagy-related gene Atg7 in young mice (3.5 months) (Atg7F/F SM22α-Cre+ mice) on the biomechanical properties of the aorta, using an in-house developed Rodent Oscillatory Tension Set-up to study Arterial Compliance (ROTSAC). Aortic segments of Atg7F/F SM22α-Cre+ mice displayed attenuated compliance and higher arterial stiffness, which was more evident at higher distention pressures. Passive aortic wall remodeling, rather than differences in VSMC tone, is responsible for these phenomena, since differences in compliance and stiffness between Atg7+/+ SM22α-Cre+ and Atg7F/F SM22α-Cre+ aortas were more pronounced when VSMCs were completely relaxed by the addition of exogenous nitric oxide. These observations are supported by histological data showing a 13% increase in medial wall thickness and a 14% decrease in elastin along with elevated elastin fragmentation. In addition, expression of the calcium-binding protein S100A4, which is linked to matrix remodeling, was elevated in aortic segments of Atg7F/F SM22α-Cre+ mice. Overall, these findings illustrate that autophagy exerts a crucial role in defining arterial wall compliance.

Ex vivo aortic stiffness in mice with different eNOS activity.

An important physiological role of the aorta is to convert the pulsatile blood flow that originates in the heart to a nearly continuous flow in the peripheral vessels. Previously, we demonstrated that basal, unstimulated nitric oxide (NO) production is more abundant in large as compared with muscular arteries and that it is an important regulator of arterial (aortic) stiffness. Hence, endothelial function and NO bioavailability are important determinants of aortic biomechanics, and mouse models with altered NO signaling might be of interest to investigate the (patho)physiological role of the NO signaling as a dynamic regulator of arterial stiffness. We aimed to characterize the ex vivo biomechanical properties of aortic segments from mice with no (eNOS-/-), normal [wild type (WT)], or high (eNOS-tg) endothelial NO synthase (eNOS) expression. Isobaric aortic diameter and compliance were lower in eNOS-/- mice and increased in eNOS-tg mice as compared with WT mice. Interestingly, these differences remained when NO levels were pharmacologically restored ex vivo, suggesting that they were not merely the result of a lack or excess of the vasodilator effects of NO. Analysis of basal vascular smooth muscle cell tone and the phasic as well as the tonic contraction in response to α1-adrenergic stimulation with phenylephrine revealed that the chronic lack of eNOS expression affected aortic reactivity similarly but with different magnitude as compared with acute eNOS blockade using Nω-nitro-l-arginine methyl ester in WT and eNOS-tg mice, suggesting that chronical distortion of NO signaling triggered several compensatory mechanisms that reflect the organism's attempt to restore the contractile imbalance and maintain optimal central hemodynamics.NEW & NOTEWORTHY Endothelial function and NO bioavailability are important determinants of aortic biomechanics and function. With a new technique we investigated the ex vivo aortic segment biomechanics of different mouse models with altered NO signaling. Our experiments clearly show that chronic distortion of NO signaling triggered several compensatory mechanisms that reflect the organism's attempt to maintain optimal central hemodynamics.

A novel set-up for the ex vivo analysis of mechanical properties of mouse aortic segments stretched at physiological pressure and frequency.

KEY POINTS: Cyclic stretch is known to alter intracellular pathways involved in vessel tone regulation. We developed a novel set-up that allows straightforward characterization of the biomechanical properties of the mouse aorta while stretched at a physiological heart rate (600 beats min-1 ). Active vessel tone was shown to have surprisingly large effects on isobaric stiffness. The effect of structural vessel wall alterations was confirmed using a genetic mouse model. This set-up will contribute to a better understanding of how active vessel wall components and mechanical stimuli such as stretch frequency and amplitude regulate aortic mechanics. ABSTRACT: 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-1 . 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 (EP ; 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, EP 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.

Effect of angiotensin II-induced arterial hypertension on the voltage-dependent contractions of mouse arteries.

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.

Mouse aortic biomechanics are affected by short-term defective autophagy in vascular smooth muscle cells.

The physiology of vascular smooth muscle (VSMC) cells is affected by autophagy, a catabolic cellular mechanism responsible for nutrient recycling. Autophagy-inducing compounds may reverse arterial stiffening, whereas congenital VSMC-specific autophagy deficiency promotes arterial stiffening. The elevated aortic stiffness in 3.5-month-old C57Bl/6 mice, in which the essential autophagy-related gene Atg7 was specifically deleted in the VSMCs (Atg7F/F SM22α-Cre+ mice) was mainly due to passive aortic wall remodeling. The present study investigated whether aortic stiffness was also modulated by a shorter duration of autophagy deficiency. Therefore, aortic segments of 2-month-old Atg7F/F SM22α-Cre+ mice were studied. Similarly to the older mice, autophagy deficiency in VSMCs promoted aortic stiffening by elastin degradation and elastin breaks, and increased the expression of the calcium binding protein S100A4 (+ 157%), the aortic wall thickness (+ 27%), the sensitivity of the VSMCs to depolarization and the contribution of VGCC mediated Ca2+ influx to α1 adrenergic contractions. Hence, all these phenomena occurred before the age of 2 months. When compared to autophagy deficiency in VSMCs at 3.5 months, shorter term autophagy deficiency led to higher segment diameter at 80 mmHg (+ 7% versus - 2%), normal baseline tonus (versus increased), unchanged IP3-mediated phasic contractions (versus enhanced), and enhanced endothelial cell function (versus normal). Overall, and because in vivo cardiac parameters or aortic pulse wave velocity were not affected, these observations indicate that congenital autophagy deficiency in VSMCs of Atg7F/F SM22α-Cre+ mice initiates compensatory mechanisms to maintain circulatory homeostasis.

Vascular smooth muscle cell contraction and relaxation in the isolated aorta: a critical regulator of large artery compliance.

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 (VSMCs) 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 VSMCs 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.

Short-Term Angiotensin II Treatment Affects Large Artery Biomechanics and Function in the Absence of Small Artery Alterations in Mice.

Induction of hypertension by angiotensin II (AngII) is a widely used experimental stimulus to study vascular aging in mice. It is associated with large artery stiffness, a hallmark of arterial aging and a root cause of increased cardiovascular risk. We reported earlier that long term (4 week) AngII treatment in mice altered the active, contractile properties of the arteries in a vascular bed-specific manner and that, in healthy mice aorta, active contractile properties of the aortic wall determine isobaric aortic stiffness. Given the huge physiological relevance of large artery stiffening, we aimed to characterize the early (1 week) changes in the active properties of the aorta of AngII-treated mice. We were not able to detect a significant effect of AngII treatment on anesthetized blood pressure or abdominal aorta pulse wave velocity. Ex vivo biomechanical and functional studies of the aorta revealed increased arterial stiffness and altered vascular smooth muscle cell (VSMC) and endothelial cell reactivity. Interestingly, the AngII-associated changes in the aorta could be largely attributed to alterations in basal VSMC tone and basal nitric oxide efficacy, indicating that, besides structural remodeling of the arterial wall, dysfunctional active components of the aorta play a crucial role in the pathophysiological mechanisms by which AngII treatment induces arterial stiffness.

Elastic and Muscular Arteries Differ in Structure, Basal NO Production and Voltage-Gated Ca(2+)-Channels.

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.

Dissecting out the complex Ca2+-mediated phenylephrine-induced contractions of mouse aortic segments.

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.

Applanation tonometry in mice: a novel noninvasive technique to assess pulse wave velocity and arterial stiffness.

Arterial stiffening is the root cause of a range of cardiovascular complications, including myocardial infarction, left ventricular hypertrophy, stroke, renal failure, dementia, and death, and a hallmark of the aging process. The most important in vivo parameter of arterial stiffness is pulse wave velocity (PWV). Clinically, PWV is determined noninvasively using applanation tonometry. Unlike the clinical value of arterial stiffness and PWV, techniques to determine PWV in mice are scarce. The only way to determine aortic PWV noninvasively in the mouse is by using ultrasound echo Doppler velocimetry. It is a fast, efficient, and accurate technique, but the required tools are expensive and technically complex. Here, we describe the development and validation of a novel technique to assess carotid-femoral PWV noninvasively in mice. This technique is based on applanation tonometry as used clinically. We were able to establish a reproducible reference value in wild-type mice (3.96±0.05 m/s) and to detect altered carotid-femoral PWV values in endothelial nitric oxide synthase knockout mice (4.66±0.05 m/s; P<0.001 compared with control), and in mice sedated with sodium pentobarbital (2.89±0.17 m/s; P<0.001 compared with control). Also, carotid-femoral PWV was pharmacologically modulated and measured in a longitudinal experiment with endothelial nitric oxide synthase knockout mice to demonstrate the applicability of this technique. In general, applanation tonometry can be used to measure carotid-femoral PWV noninvasively in mice. The experimental setup is simple, and the technical requirements are basic, making this technique readily implementable in any mouse model-based research facility interested in arterial stiffness.