Arthur J. A. Leloup

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. # Novelty and Significance {#article-title-28}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.

Endothelial Senescence Contributes to Heart Failure With Preserved Ejection Fraction in an Aging Mouse Model.

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.

Elastic and Muscular Arteries Differ in Structure, Basal NO Production and Voltage-Gated Ca2+-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 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.

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.

Continuous administration of the mTORC1 inhibitor everolimus induces tolerance and decreases autophagy in mice.

Everolimus is an allosteric inhibitor of the mechanistic target of rapamycin complex 1 (mTORC1) widely known for its potent autophagy stimulating properties. Because everolimus shows poor solubility and stability in aqueous solutions, long‐term in vivo administration in preclinical models is challenging. The aim of the present study was to evaluate the effects of short‐term and long‐term everolimus administration on mTORC1 inhibition and autophagy induction in mice.

Basal activity of voltage-gated Ca2+ channels controls the IP3-mediated contraction by α1-adrenoceptor stimulation of mouse aorta segments

α1-Adrenoceptor stimulation of mouse aorta causes intracellular Ca(2+) release from sarcoplasmic reticulum Ca(2+) stores via stimulation of inositoltriphosphate (IP3) receptors. It is hypothesized that this Ca(2+) release from the contractile and IP3-sensitive Ca(2+) store is under the continuous dynamic control of time-independent basal Ca(2+) influx via L-type voltage-gated Ca(2+) channels (LCC) residing in their window voltage range. Mouse aortic segments were α1-adrenoceptor stimulated with phenylephrine in the absence of external Ca(2+) (0Ca) to measure phasic isometric contractions. They gradually decreased with time in 0Ca, were inhibited with 2-aminoethoxydiphenyl borate, and declined with previous membrane potential hyperpolarization (levcromakalim) or with previous inhibition of LCC (diltiazem). Former basal stimulation of LCC with depolarization (15 mM K(+)) or with BAY K8644 increased the subsequent phasic contractions by phenylephrine in 0Ca. Although exogenous NO (diethylamine NONOate) reduced the phasic contractions by phenylephrine, stimulation of endothelial cells with acetylcholine in 0Ca failed to attenuate these phasic contractions. Finally, inhibition of the basal release of NO with N(Ω)-nitro-L-arginine methyl ester also attenuated the phasic contractions by phenylephrine. Results indicated that α1-adrenoceptor stimulation with phenylephrine causes phasic contractions, which are controlled by basal LCC and endothelial NO synthase activity. Endothelial NO release by acetylcholine was absent in 0Ca. Given the growing interest in the active regulation of arterial compliance, the dependence of contractile SR Ca(2+) store-refilling in basal conditions on the activity of LCC and basal eNOS may contribute to a more thorough understanding of physiological mechanisms leading to arterial stiffness.

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

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.

Isometric Stretch Alters Vascular Reactivity of Mouse Aortic Segments

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 Laplaces 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.

Longitudinal micro-ultrasound assessment of the ob/ob mouse model: evaluation of cardiovascular, renal and hepatic parameters

Background/Objectives:Obesity is reaching epidemic proportions and is associated with increased risk factors for cardiovascular disease. Leptin-deficient mice (ob/ob) are widely employed to investigate obesity. Aim of this study was to provide a micro-ultrasound (μUS) longitudinal evaluation of the ob/ob mouse model in terms of cardiovascular effects, consequences for renal microcirculation and liver fat accumulation.Subjects/Methods:Sixteen wild-type (wt) and eleven ob/ob male mice were studied at 8 (T0) and 25 (T1) weeks of age with a μUS system (Vevo2100) and B-mode and Doppler images were acquired. Cardiac output (CO), ejection fraction (EF), stroke volume (SV), fractional shortening (FS) and E/A ratio were measured from cardiac images. Mean diameter (Dmabd, Dmcar), relative distension (relDabd and relDcar) and pulse wave velocity (PWVabd and PWVcar) were obtained for both abdominal aorta and common carotid. As regards renal microcirculation, renal resistivity and pulsatility index (RI and PI) were assessed. The ratio between grey levels related to liver and kidney (Steato-Score) was used as index of hepatic steatosis.Results:At T0, ob/ob mice showed reduced SV, EF, CO and relDabd values and increased LVmass, PWVabd, RI, PI and Steato-score measurements. The same comparison repeated at T1 highlighted similar results for SV, EF, CO, RI, PI and Steato-Score; furthermore, obese mice showed reduced Dmabd and Dmcar measurements in comparison with lean controls. The longitudinal analysis showed an increase in LVmass and Dmabd and a reduction of FS, EF, CO, relDabd and relDcar for wt animals, while no differences were found for the ob/ob group.Conclusions:ob/ob mice presented a premature cardiac dysfunction without a further age-related deterioration and a reduction in the abdominal aorta and carotid artery mean diameter in adult age. The proposed analysis can represent a valid approach for longitudinal studies aimed at testing new therapeutic strategies or for characterizing other mouse models.

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.