Endothelial dysfunction aggravates arterial media calcification in warfarin administered rats.

Arterial media calcification is an active cell process. This encompasses osteochondrogenic transdifferentiation of vascular smooth muscle cells followed by the deposition of calcium-phosphate crystals. Increasing evidence suggests a significant role for endothelial cells (ECs) in the development of arterial media calcification. This manuscript explores a role for endothelial dysfunction in the disease progression of arterial media calcification. Male rats were randomly assigned to four different groups. The first group received standard chow. The second group was given L-NAME (≈50 mg kg-1 · d-1 ), to induce endothelial dysfunction, in addition to standard chow. The third group and fourth group received a warfarin-supplemented diet to induce mild calcification and the latter group was co-administered L-NAME. Prior to sacrifice, non-invasive measurement of aortic distensibility was performed. Animals were sacrificed after 6 weeks. Arterial media calcification was quantified by measuring aortic calcium and visualized on paraffin-embedded slices by the Von Kossa method. Arterial stiffness and aortic reactivity was assessed on isolated carotid segments using specialized organ chamber setups. Warfarin administration induced mineralization. Simultaneous administration of warfarin and L-NAME aggravated the arterial media calcification process. Through organ chamber experiments an increased vessel tonus was found, which could be linked to reduced basal NO availability, in arteries of warfarin-treated animals. Furthermore, increased calcification because of L-NAME administration was related to a further compromised endothelial function (next to deteriorated basal NO release also deteriorated stimulated NO release). Our findings suggest early EC changes to impact the disease progression of arterial media calcification.

Prdm16 Supports Arterial Flow Recovery by Maintaining Endothelial Function.

[Figure: see text].

Altered stress hormone levels affect in vivo vascular function in the hAPP23+/- overexpressing mouse model of Alzheimer's disease.

Alzheimer's disease (AD) has long been considered a brain-specific dementia syndrome. However, in recent decades, the occurrence of cardiovascular (CV) disease in the progression of AD has been confirmed by increasing epidemiological evidence. In this study, we conducted an in-depth cardiovascular characterization of a humanized amyloid precursor protein (APP) overexpressing mouse model (hAPP23+/-), which overexpresses the Swedish mutation (KM670/671NL). At the age of 6 mo, hAPP23+/- mice had a lower survival, lower body weight, and increased corticosterone and VMA levels compared with C57BL/6 littermates. Systolic blood pressure was increased in hAPP23+/- animals compared with C57BL/6 littermates, but diastolic blood pressure was not statistically different. Pulse pressure remained unchanged but abdominal and carotid pulse-wave velocity (aPWV and cPWV) were increased in hAPP23+/- compared with C57BL/6 mice. Echocardiography showed no differences in systolic or diastolic cardiac function. Ex vivo evaluation of vascular function showed decreased adreno receptor dependent vasoconstriction of hAPP23+/- aortic segments, although the isobaric biomechanics of the aortic wall were similar to C57BL/6 aortic segments. In conclusion, hAPP23+/- mice exhibited high serum corticosterone levels, elevated systolic blood pressure, and increased arterial stiffness in vivo. However, ex vivo aortic stiffness of hAPP23+/- aortic segments was not changed and vascular reactivity to α1-adrenoceptor stimulation was attenuated. These findings highlight the need for more frequent assessment of circulating stress hormone levels and PWV measurements in daily clinical practice for people at risk of AD.NEW & NOTEWORTHY We showed that male amyloid precursor protein (APP) transgenic mice have higher circulating stress hormone levels. As a result, higher systolic blood pressure and pulse-wave velocity were measured in vivo in addition to a smaller α-adrenergic receptor-dependent contraction upon ex vivo stimulation with phenylephrine. Our findings highlight the need for more frequent assessment of circulating stress hormone levels and PWV measurements in daily clinical practice for people at risk of Alzheimer's disease.

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.

Elastin fragmentation in atherosclerotic mice leads to intraplaque neovascularization, plaque rupture, myocardial infarction, stroke, and sudden death.

AIMS: There is a need for animal models of plaque rupture. We previously reported that elastin fragmentation, due to a mutation (C1039G(+/-)) in the fibrillin-1 (Fbn1) gene, promotes atherogenesis and a highly unstable plaque phenotype in apolipoprotein E deficient (ApoE(-/-)) mice on a Western-type diet (WD). Here, we investigated whether plaque rupture occurred in ApoE(-/-)Fbn1(C1039G+/-) mice and was associated with myocardial infarction, stroke, and sudden death. METHODS AND RESULTS: Female ApoE(-/-)Fbn1(C1039G+/-) and ApoE(-/-) mice were fed a WD for up to 35 weeks. Compared to ApoE(-/-) mice, plaques of ApoE(-/-)Fbn1(C1039G+/-) mice showed a threefold increase in necrotic core size, augmented T-cell infiltration, a decreased collagen I content (70 ± 10%), extensive neovascularization, intraplaque haemorrhage, and a significant increase in matrix metalloproteinase-2, -9, -12, and -13 expression or activity. Plaque rupture was observed in 70% of ascending aortas and in 50% of brachiocephalic arteries of ApoE(-/-)Fbn1(C1039G+/-) mice. In ApoE(-/-) mice, plaque rupture was not seen in ascending aortas and only in 10% of brachiocephalic arteries. Seventy percent of ApoE(-/-)Fbn1(C1039G+/-) mice died suddenly, whereas all ApoE(-/-) mice survived. ApoE(-/-)Fbn1(C1039G+/-) mice showed coronary plaques and myocardial infarction (75% of mice). Furthermore, they displayed head tilt, disorientation, and motor disturbances (66% of cases), disturbed cerebral blood flow (73% of cases; MR angiograms) and brain hypoxia (64% of cases), indicative of stroke. CONCLUSIONS: Elastin fragmentation plays a key role in plaque destabilization and rupture. ApoE(-/-)Fbn1(C1039G+/-) mice represent a unique model of acute plaque rupture with human-like complications.

Defective autophagy in vascular smooth muscle cells alters contractility and Ca²âº homeostasis in mice.

Autophagy is an evolutionary preserved process that prevents the accumulation of unwanted cytosolic material through the formation of autophagosomes. Although autophagy has been extensively studied to understand its function in normal physiology, the role of vascular smooth muscle (SM) cell (VSMC) autophagy in Ca(2+) mobilization and contraction remains poorly understood. Recent evidence shows that autophagy is involved in controlling contractile function and Ca(2+) homeostasis in certain cell types. Therefore, autophagy might also regulate contractile capacity and Ca(2+)-mobilizing pathways in VSMCs. Contractility (organ chambers) and Ca(2+) homeostasis (myograph) were investigated in aortic segments of 3.5-mo-old mice containing a SM cell-specific deletion of autophagy-related 7 (Atg7; Atg7(fl/fl) SM22α-Cre(+) mice) and in segments of corresponding control mice (Atg7(+/+) SM22α-Cre(+)). Our results indicate that voltage-gated Ca(2+) channels (VGCCs) of Atg7(fl/fl) SM22α-Cre(+) VSMCs were more sensitive to depolarization, independent of changes in resting membrane potential. Contractions elicited with K(+) (50 mM) or the VGCC agonist BAY K8644 (100 nM) were significantly higher due to increased VGCC expression and activity. Interestingly, the sarcoplasmic reticulum of Atg7(fl/fl) SM22α-Cre(+) VSMCs was enlarged, which, combined with increased sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 expression and higher store-operated Ca(2+) entry, promoted inositol 1,4,5-trisphosphate-mediated contractions of Atg7(fl/fl) SM22α-Cre(+) segments and maximized the Ca(2+) storing capacity of the sarcoplasmic reticulum. Moreover, decreased plasma membrane Ca(2+)-ATPase expression in Atg7(fl/fl) SM22α-Cre(+) VSMCs hampered Ca(2+) extrusion to the extracellular environment. Overall, our study indicates that defective autophagy in VSMCs leads to an imbalance between Ca(2+) release/influx and Ca(2+) reuptake/extrusion, resulting in higher basal Ca(2+) concentrations and significant effects on vascular reactivity.

Contribution of α-adrenoceptor stimulation by phenylephrine to basal nitric oxide production in the isolated mouse aorta.

In the mouse aorta, contractions evoked by the α(1)-adrenoceptor agonist phenylephrine are strongly suppressed by the continuous production of nitric oxide (NO). We investigated whether phenylephrine itself stimulated NO production by activating endothelial α(2)-adrenoceptors. On a prostaglandin F(2α) contraction, the α(2)-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) induced 29.3 ± 7.4% relaxation, which was inhibited by 0.1 µM 2-[(4,5-Dihydro-1H-imidazol-2-yl)methyl]-2,3-dihydro-1-methyl-1H-isoindole (BRL44408) with a pKB' corresponding to α(2)-antagonism. In the presence of NO synthase blockers, UK14304 elicited small contractions above 1 µM that were inhibited by 0.1 µM prazosin, but not influenced by 0.1 µM rauwolscine. At 3 µM or higher concentrations, phenylephrine caused only modest relaxation (up to 7.4 ± 2.3%) of segments constricted with prostaglandin F(2α) in the presence of prazosin, which was abolished with 0.1 µM BRL44408. Furthermore, BRL44408 did not increase contractions induced with 1 µM phenylephrine. These results confirm that α(1)- but not α(2)-adrenoceptors are expressed on aortic smooth muscle cells, whereas endothelial cells only express α(2)-adrenoceptors. Moreover, phenylephrine exerted a very modest relaxing effect through nonspecific stimulation of α(2)-adrenoceptors, but only at concentrations higher than 1 µM. It is concluded that the high basal output of NO in the isolated mouse aorta is not due to stimulation of α-adrenoceptors.

Increasing pulse pressure ex vivo, mimicking acute physical exercise, induces smooth muscle cell-mediated de-stiffening of murine aortic segments.

The mechanisms by which physical activity affects cardiovascular function and physiology are complex and multifactorial. In the present study, cardiac output during rest or acute physical activity was simulated in isolated aortic segments of healthy C57BL/6J wild-type mice. This was performed using the Rodent Oscillatory Tension Set-up to study Arterial Compliance (ROTSAC) by applying cyclic stretch of different amplitude, duration and frequency in well-controlled and manageable experimental conditions. Our data show that vascular smooth muscle cells (VSMCs) of the aorta have the intrinsic ability to "de-stiffen" or "relax" after periods of high cyclic stretch and to "re-stiffen" slowly thereafter upon return to normal distension pressures. Thereby, certain conditions have to be fulfilled: 1) VSMC contraction and repetitive stretching (loading/unloading cycles) are a prerequisite to induce post-exercise de-stiffening; 2) one bout of high cyclic stretch is enough to induce de- and re-stiffening. Aortic de-stiffening was highly dependent on cyclic stretch amplitude and on the manner and timing of contraction with probable involvement of focal adhesion phosphorylation/activation. Results of this study may have implications for the therapeutic potential of regular and acute physical activity and its role in the prevention and/or treatment of cardiovascular disease.

Progression of the prothrombotic state in aging Bmal1-deficient mice.

OBJECTIVE: The goal of this study was to examine the functional relationship between aging endothelium and thrombogenicity in a mouse model of premature aging. METHODS AND RESULTS: Coagulation tests and factors, blood cell counts, aorta endothelial function, aorta gene expression, and FeCl(3)-induced thrombosis in mesenteric blood vessels were analyzed in 10- to 30-week-old brain and muscle ARNT-like protein-1 (Bmal1)-deficient (knockout [KO]) mice and wild-type littermates. Ten-week-old KO mice manifested shortened prothrombin times (9.7 versus 11.3 seconds in wild-type) and elevated plasma fibrinogen (264 versus 172 mg/dL). At 30 weeks, factor VII (198% versus 149%), and platelet counts (2049 versus 1354 K/µL) were increased in KO mice. Gene deficiency reduced the vasoactive nitric oxide production at 10 and 30 weeks and tended to reduce and increase the protein expression of thrombomodulin and von Willebrand factor, respectively, with aging. Shortened venular and arteriolar occlusion times on FeCl(3)-induced injury in 10-week-old KO mice confirmed higher thrombogenicity, culminating in priapism, observed in 60% of 25- to 30-week-old KO males. CONCLUSION: Endothelial dysfunction and a hypercoagulable state cause early arterial and venous thrombogenicity in Bmal1 KO mice. With aging, progressive endothelial dysfunction, rising platelet counts, and high factor VII further enhance thrombogenicity, provoking priapism.

Contribution of transient and sustained calcium influx, and sensitization to depolarization-induced contractions of the intact mouse aorta.

BACKGROUND: Electrophysiological studies of L-type Ca2+ channels in isolated vascular smooth muscle cells revealed that depolarization of these cells evoked a transient and a time-independent Ca2+ current. The sustained, non-inactivating current occurred at voltages where voltage-dependent activation and inactivation overlapped (voltage window) and its contribution to basal tone or active tension in larger multicellular blood vessel preparations is unknown at present. This study investigated whether window Ca2+ influx affects isometric contraction of multicellular C57Bl6 mouse aortic segments. RESULTS: Intracellular Ca2+ (Cai2+, Fura-2), membrane potential and isometric force were measured in aortic segments, which were clamped at fixed membrane potentials by increasing extracellular K+ concentrations. K+ above 20 mM evoked biphasic contractions, which were not affected by inhibition of IP3- or Ca2+ induced Ca2+ release with 2-aminoethoxydiphenyl borate or ryanodine, respectively, ruling out the contribution of intracellular Ca2+ release. The fast force component paralleled Cai2+ increase, but the slow contraction coincided with Cai2+ decrease. In the absence of extracellular Ca2+, basal tension and Cai2+ declined, and depolarization failed to evoke Cai2+ signals or contraction. Subsequent re-introduction of external Ca2+ elicited only slow contractions, which were now matched by Cai2+ increase. After Cai2+ attained steady-state, isometric force kept increasing due to Ca2+- sensitization of the contractile elements. The slow force responses displayed a bell-shaped voltage-dependence, were suppressed by hyperpolarization with levcromakalim, and enhanced by an agonist of L-type Ca2+ channels (BAY K8644). CONCLUSION: The isometric response of mouse aortic segments to depolarization consists of a fast, transient contraction paralleled by a transient Ca2+ influx via Ca2+ channels which completely inactivate. Ca2+ channels, which did not completely inactivate during the depolarization, initiated a second, sustained phase of contraction, which was matched by a sustained non-inactivating window Ca2+ influx. Together with sensitization, this window L-type Ca2+ influx is a major determinant of basal and active tension of mouse aortic smooth muscle.

Basal Vascular Smooth Muscle Cell Tone in eNOS Knockout Mice Can Be Reversed by Cyclic Stretch and Is Independent of Age.

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.

Disparate biomechanical properties of the aorta in non-aneurysmal and aneurysmal mice treated with angiotensin II.

In vivo angiotensin II (AngII)-treatment is a widely used experimental model to induce cardiovascular disease and results in a high likelihood of abdominal aorta aneurysm (AAA) formation. This involves progressive and irreversible focal dilation of the abdominal aorta and induces adverse aortic connective tissue remodeling contributing to aortic wall stiffening through inflammation, elastin degradation, and collagen restructuring. Hence, the present study aimed to investigate how AAA formation in AngII-treated mice affects aortic function and biomechanics. To this end, C57Bl/6J mice were treated with AngII (1000 ng/[kg.min]) or PBS infusion for 28 days. Peripheral blood pressure, echocardiography, and aortic pulse wave velocity were measured in vivo. Thoracic aorta rings were studied ex vivo in organ chambers, while aortic vascular smooth muscle cell (VSMC) phenotype was investigated histologically. We confirmed peripheral hypertension, cardiac hypertrophy, aortic stiffening, and increased VSMC proliferation and migration after AngII-treatment. Abdominal aorta aneurysm formation was observed in 8/13 AngII-treated mice. Ex vivo thoracic aortic rings of both aneurysmal and non-aneurysmal AngII-treated mice showed high isobaric aortic stiffness, endothelial dysfunction, heightened α1 -adrenergic contractility, and altered VSMC contractile calcium signaling. However, aortic biomechanics were differently affected, with heightened α1 -adrenoreceptor mediated aortic stiffening in non-aneurysmal mice, whereas contraction-dependent stiffening was impaired in aneurysmal mice. In conclusion, although aneurysmal and non-aneurysmal 4-week AngII-treated mice displayed similar changes in aortic physiology, aortic biomechanics were dissimilarly affected.

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.

Aortic Stiffness in L-NAME Treated C57Bl/6 Mice Displays a Shift From Early Endothelial Dysfunction to Late-Term Vascular Smooth Muscle Cell Dysfunction.

Introduction and Aims: Endothelial dysfunction is recognized as a cardiovascular aging hallmark. Administration of nitric oxide synthase blocker N-Ω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) constitutes a well-known small animal model of cardiovascular aging. Despite extensive phenotypic characterization, the exact aortic function changes in L-NAME treated mice are largely unknown. Therefore, this study presents a longitudinal characterization of the aortic reactivity and biomechanical alterations in L-NAME treated C57Bl/6 mice. Methods and Results: Male C57Bl/6 mice were treated with L-NAME (0.5 mg/ml drinking water) for 1, 2, 4, 8, or 16 weeks. Peripheral blood pressure measurement (tail-cuff) and transthoracic echocardiograms were recorded, showing progressive hypertension after 4 weeks of treatment and progressive cardiac hypertrophy after 8-16 weeks of treatment. Aortic stiffness was measured in vivo as aortic pulse wave velocity (aPWV, ultrasound) and ex vivo as Peterson modulus (Ep). Aortic reactivity and biomechanics were investigated ex vivo in thoracic aortic rings, mounted isometrically or dynamically-stretched in organ bath set-ups. Aortic stiffening was heightened in L-NAME treated mice after all treatment durations, thereby preceding the development of hypertension and cardiac aging. L-NAME treatment doubled the rate of arterial stiffening compared to control mice, and displayed an attenuation of the elevated aortic stiffness at high distending pressure, possibly due to late-term reduction of medial collagen types I, III, and IV content. Remarkably, endothelial dysfunction, measured by acetylcholine concentration-response stimulation in precontracted aortic rings, was only observed after short-term (1-4 weeks) treatment, followed by restoration of endothelial function which coincided with increased phosphorylation of endothelial nitric oxide synthase (S1177). In the late-disease phase (8-16 weeks), vascular smooth muscle cell (VSMC) dysfunction developed, including increased contribution of voltage-dependent calcium channels (assessed by inhibition with diltiazem), basal VSMC cytoplasmic calcium loading (assessed by removal of extracellular calcium), and heightened intracellular contractile calcium handling (assessed by measurement of sarcoplasmic reticulum-mediated transient contractions). Conclusion: Arterial stiffness precedes peripheral hypertension and cardiac hypertrophy in chronic L-NAME treated male C57Bl/6 mice. The underlying aortic disease mechanisms underwent a distinct shift from early endothelial dysfunction to late-term VSMC dysfunction, with continued disease progression.

Progressive aortic stiffness in aging C57Bl/6 mice displays altered contractile behaviour and extracellular matrix changes.

Aortic stiffness is a hallmark of cardiovascular disease, but its pathophysiology remains incompletely understood. This study presents an in-dept characterization of aortic aging in male C57Bl/6 mice (2-24 months). Cardiovascular measurements include echocardiography, blood pressure measurement, and ex vivo organ chamber experiments. In vivo and ex vivo aortic stiffness increases with age, and precede the development of cardiac hypertrophy and peripheral blood pressure alterations. Contraction-independent stiffening (due to extracellular matrix changes) is pressure-dependent. Contraction-dependent aortic stiffening develops through heightened α1-adrenergic contractility, aberrant voltage-gated calcium channel function, and altered vascular smooth muscle cell calcium handling. Endothelial dysfunction is limited to a modest decrease in sensitivity to acetylcholine-induced relaxation with age. Our findings demonstrate that progressive arterial stiffening in C57Bl/6 mice precedes associated cardiovascular disease. Aortic aging is due to changes in extracellular matrix and vascular smooth muscle cell signalling, and not to altered endothelial function.

Transcription profiles of aortic smooth muscle cells from atherosclerosis-prone and -resistant regions in young apolipoprotein E-deficient mice before plaque development.

BACKGROUND/AIMS: Site-specific atherosclerosis is generally attributed to differential gene expression in endothelial cells. We investigated whether the transcriptome of smooth muscle cells is different between atherosclerosis-prone and atherosclerosis-resistant regions in apolipoprotein E-deficient (apoE-/-) mice before plaque development, and in C57Bl/6 mice. METHODS: De-endothelialized aortas (both strains: 3 males, 3 females, age 4 months) were divided into atherosclerosis-prone (AA: ascending aorta, aortic arch and proximal 2 mm of thoracic aorta) and -resistant (CTA: central thoracic aorta, i.e. 6 mm distal from the proximal 2 mm) regions. The transcriptome of these two regions was compared using whole-genome mouse microarrays. RESULTS: Microarray analysis revealed differential expression (>2-fold difference) of 70 and 244 genes in C57Bl/6 and apoE-/- mice. This was confirmed for 6 genes using the real-time quantitative polymerase chain reaction. Up- or downregulation in the AA was observed for 33 and 37 genes in C57Bl/6, and for 186 and 58 genes in apoE-/- mice, respectively. The 201 genes that showed exclusively differential expression in apoE-/- mice were related to atherosclerotic processes, such as cell adhesion, proliferation, differentiation, motility, cell death, lipid metabolism and immune responses. CONCLUSION: Our findings indicate that smooth muscle cells display an altered transcriptome at atherosclerosis-prone locations before actual lesion development.

Effects of nebivolol on vascular endothelial and myocardial function in diabetes mellitus.

BACKGROUND: Nebivolol is a ß1-adrenergic receptor (ß1-AR) antagonist, inducing endothelial nitric oxide (NO) release, most likely due to ß3-AR agonism. Nebivolol is vasculoprotective and cardioprotective in the setting of hypertension. In this study, we investigated the effects of nebivolol, compared with those of bisoprolol, on vascular and myocardial function in diabetic mice. METHODS: Diabetic (Lepr) and nondiabetic mice (Lepr) were treated with vehicle, nebivolol, or bisoprolol for 16 weeks. Endothelium-dependent and endothelial-independent relaxations were studied in isolated aortic segments. Myocardial twitch performance was studied in isolated right ventricular papillary muscles. RESULTS: In aortic segments of diabetic mice, endothelium-dependent relaxations were significantly shifted to the right. This shift was not prevented by chronic nebivolol or bisoprolol treatment. Papillary muscle twitches of diabetic mice displayed a significant delay in the onset of relaxation, and an increased time from peak active force to 50% relaxation, leading to prolonged twitch activity, without changes in twitch amplitude. These changes were not prevented in nebivolol- and bisoprolol-treated diabetic mice. Surprisingly, in nebivolol-treated mice, regardless of diabetic status, twitch duration was further increased. Applying nebivolol to papillary muscles in the organ bath reproduced the changes induced by chronic treatment in vivo; this was not the case for bisoprolol. Nebivolol-induced changes were blunted by a ß3-AR antagonist and by NO synthase (NOS) inhibition. CONCLUSIONS: Diabetes-induced changes in vascular and myocardial function were not prevented by treatment with nebivolol or bisoprolol. Strikingly, myocardial effects of nebivolol differed from those of bisoprolol, by inducing a ß3-AR and NOS-dependent prolongation of twitch activity.