Calcium In Vascular Smooth Muscle Research Articles

Mechanistic study on vasodilatory and antihypertensive effects of hesperetin: ex vivo and in vivo approaches.

Hesperetin is one of the prominent flavonoids found in citrus fruit. Several research studies have reported that hesperetin can promote vasodilation in vascular tissue by increasing the level of nitric oxide and cyclic nucleotides. However, these may not be the only pathway for hesperetin to exert its vasodilatory effect. In addition to vasodilation, hesperetin has been found to carry an antihypertensive effect through intraperitoneal injection, although no study has comprehensively investigated the antihypertensive effect of hesperetin through oral administration. Therefore, this study aimed to determine the possible mechanism pathways involved in hesperetin-induced vasodilation and investigated its antihypertensive effects on hypertensive rats' model via oral administration. The ex vivo experimental findings showed that the NO/sGC/cGMP signalling pathway was involved in hesperetin-mediated vasodilation. Moreover, hesperetin activated the AC/cAMP/PKA pathway through PGI2 and activated the β2-adrenergic receptor. Hesperetin can act as a voltage-gated potassium channel (KV) and ATP-sensitive potassium channel (KATP) opener. The intracellular calcium in vascular smooth muscle was reduced by hesperetin through blocking the voltage-operated calcium channels (VOCC) and inositol triphosphate receptor (IP3R). In the in vivo assessment, hesperetin shows a significant decrease in Spontaneously Hypertensive rats' blood pressure following 21 days of oral treatment. The sub-chronic toxicity assessment demonstrated that hesperetin exhibited no deleterious effects on the body weights, clinical biochemistry and haematological profile of Sprague-Dawley rats. This study implies that hesperetin holds promise as a potential medication for hypertension treatment, devoid of undesirable side effects.

Cdon suppresses vascular smooth muscle calcification via repression of the Wnt/Runx2 Axis

Osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) is a risk factor associated with vascular diseases. Wnt signaling is one of the major mechanisms implicated in the osteogenic conversion of VSMCs. Since Cdon has a negative effect on Wnt signaling in distinct cellular processes, we sought to investigate the role of Cdon in vascular calcification. The expression of Cdon was significantly downregulated in VSMCs of the aortas of patients with atherosclerosis and aortic stenosis. Consistently, calcification models, including vitamin D3 (VD3)-injected mice and VSMCs cultured with calcifying media, exhibited reduced Cdon expression. Cdon ablation mice (cKO) exhibited exacerbated aortic stiffness and calcification in response to VD3 compared to the controls. Cdon depletion induced the osteogenic conversion of VSMCs accompanied by cellular senescence. The Cdon-deficient aortas showed a significant alteration in gene expression related to cell proliferation and differentiation together with Wnt signaling regulators. Consistently, Cdon depletion or overexpression in VSMCs elevated or attenuated Wnt-reporter activities, respectively. The deletion mutant of the second immunoglobulin domain (Ig2) in the Cdon ectodomain failed to suppress Wnt signaling and osteogenic conversion of VSMCs. Furthermore, treatment with purified recombinant proteins of the entire ectodomain or Ig2 domain of Cdon displayed suppressive effects on Wnt signaling and VSMC calcification. Our results demonstrate a protective role of Cdon in VSMC calcification by suppressing Wnt signaling. The Ig2 domain of Cdon has the potential as a therapeutic tool to prevent vascular calcification.

Activating BK channels ameliorates vascular smooth muscle calcification through Akt signaling.

Vascular calcification (VC) is characterized by pathological depositions of calcium and phosphate in the arteries and veins via an active cell-regulated process, in which vascular smooth muscle cells (VSMCs) transform into osteoblast/chondrocyte-like cells as in bone formation. VC is associated with significant morbidity and mortality in chronic kidney disease (CKD) and cardiovascular disease, but the underlying mechanisms remain unclear. In this study we investigated the role of large-conductance calcium-activated potassium (BK) channels in 3 experimental VC models. VC was induced in vascular smooth muscle cells (VSMCs) by β-glycerophosphate (β-GP), or in rats by subtotal nephrectomy, or in mice by high-dosage vitamin D3. We showed that the expression of BK channels in the artery of CKD rats with VC and in β-GP-treated VSMCs was significantly decreased, which was functionally confirmed by patch-clamp recording. In β-GP-treated VSMCs, BK channel opener NS1619 (20 μM) significantly alleviated VC by decreasing calcium content and alkaline phosphatase activity. Furthermore, NS1619 decreased mRNA expression of ostoegenic genes OCN and OPN, as well as Runx2 (a key transcription factor involved in preosteoblast to osteoblast differentiation), and increased the expression of α-SMA protein, whereas BK channel inhibitor paxilline (10 μM) caused the opposite effects. In primary cultured VSMCs from BK-/- mice, BK deficiency aggravated calcification as did BK channel inhibitor in normal VSMCs. Moreover, calcification was more severe in thoracic aorta rings of BK-/- mice than in those of wild-type littermates. Administration of BK channel activator BMS191011 (10 mg· kg-1 ·d-1) in high-dosage vitamin D3-treated mice significantly ameliorated calcification. Finally, co-treatment with Akt inhibitor MK2206 (1 μM) or FoxO1 inhibitor AS1842856 (3 μM) in calcified VSMCs abrogated the effects of BK channel opener NS1619. Taken together, activation of BK channels ameliorates VC via Akt/FoxO1 signaling pathways. Strategies to activate BK channels and/or enhance BK channel expression may offer therapeutic avenues to control VC.

The Novel gem-Dihydroperoxide 12AC3O Suppresses High Phosphate-Induced Calcification via Antioxidant Effects in p53LMAco1 Smooth Muscle Cells.

The excessive intake of phosphate (Pi), or chronic kidney disease (CKD), can cause hyperphosphatemia and eventually lead to ectopic calcification, resulting in cerebrovascular diseases. It has been reported that reactive oxygen species (ROS), induced by high concentrations of Pi loading, play a key role in vascular calcification. Therefore, ROS suppression may be a useful treatment strategy for vascular calcification. 12AC3O is a newly synthesized gem-dihydroperoxide (DHP) that has potent antioxidant effects. In the present study, we investigated whether 12AC3O inhibited vascular calcification via its antioxidative capacity. To examine whether 12AC3O prevents vascular calcification under high Pi conditions, we performed Alizarin red and von Kossa staining, using the mouse aortic smooth muscle cell line p53LMAco1. Additionally, the effect of 12AC3O against oxidative stress, induced by high concentrations of Pi loading, was investigated using redox- sensitive dyes. Further, the direct trapping effect of 12AC3O on reactive oxygen species (ROS) was investigated by ESR analysis. Although high concentrations of Pi loading exacerbated vascular smooth muscle calcification, calcium deposition was suppressed by the treatment of both antioxidants and 12AC3O, suggesting that the suppression of ROS may be a candidate therapeutic approach for treating vascular calcification induced by high concentrations of Pi loading. Importantly, 12AC3O also attenuated oxidative stress. Furthermore, 12AC3O directly trapped superoxide anion and hydroxyl radical. These results suggest that ROS are closely involved in high concentrations of Pi-induced vascular calcification and that 12AC3O inhibits vascular calcification by directly trapping ROS.

Enhanced Vascular Smooth Muscle Calcium Sensitivity and Loss of Endothelial Vasodilator Influence Contribute to Myogenic Tone Development in Rat Radial Uterine Arteries during Gestation

Uterine artery myogenic tone (MT) develops during pregnancy in hemochorial placentates such as rats and humans. The physiological reason for its appearance is not clear, and we reasoned that it may be a late pregnancy (LP) event in preparation for controlling hemorrhage during parturition. We also hypothesized that gestational increases in RhoA-induced vascular smooth muscle (VSM) calcium sensitivity are contributory and occur under the tonic influence of nitric oxide (NO). Second-order pre-placental radial arteries from early-pregnant (day 12, n = 5), mid-pregnant (day 16, n = 5) and LP (day 20, n = 20) rats were used in combination with arteriography, VSM calcium measurements, pharmacological RHO/Rho-associated protein kinase (ROCK) and nitric oxide synthase (NOS) inhibition, and Western blotting. A subgroup of LP animals (LP + LN; n = 5) treated with L-NAME from gestational days 10 to 20 were used to determine the effects of NOS inhibition on MT and RhoA expression. MT was evident throughout pregnancy, but its expression in pressurized vessels was masked by endothelial NO-induced vasodilation during early gestation. RhoA protein expression was upregulated in LP and attenuated by in vivo NOS inhibition (as was MT). In vitro RHO/ROCK inhibition decreased MT in a concentration-dependent manner without reducing VSM calcium. In summary, pressure-dependent uterine artery tone increases with gestational age due to a combination of RhoA-mediated increases in VSM calcium sensitivity and a loss of endothelial NO influence.

Exercise, but not statins improve endothelial function in obese rats

IntroductionStatins and exercise training (ET) improve endothelial function. The mechanisms by which statins in combination with ET alter vascular function are largely unknown. We investigated how statins influence the effects of ET on vascular function in obese rats.MethodsEight‐week‐old male Wistar rats (n = 46) received a high‐fat diet for 20 weeks. The rats were randomized after into four groups after eight weeks: sedentary (SED; n =11), exercise (EX; n=11), statin (STAT; n=13) and exercise with statin (EX/STAT; n=11). Simvastatin (10mg/d/kg) was administered in drinking water. The rats exercised for 12 weeks, 5 days/week for 1 h/day at 18 m/min. Endothelium‐dependent (acetylcholine [Ach]) and – independent (sodium‐nitroprusside [SNP]) vascular function was assessed in the abdominal aorta using in vitro myography. Gene expression and protein abundance of nitric‐oxide soluble guanylate cyclase (NO‐sGC) signaling was assessed using q‐PCR and Western blot.ResultsStatin treatment significantly reduced cholesterol levels and did not influence exercise duration or intensity. ET independent of statins improved endothelium dependent vasodilation in EX and EX/STAT compared to SED and STAT, respectively. In untreated rats ET did not alter SNP vasodilation. However, in statin treated animals, ET improved SNP vasodilation. ET and statins increased NO protein abundance by 40% and 80%, respectively, compared to SED. However, in treated rats ET reduced NO to 40% above SED. Protein phosphatase 1 regulatory subunit 14A (CPI17), an inhibitor protein of smooth muscle myosin phosphatase showed a significant statin and exercise interaction at gene expression level (p < .0001). In untreated rats, ET upregulated CPI17 gene expression by 50% (p < .05). In treated rats a downregulation of 35% (p < .05) was observed. The results were similar on the protein level. The myosin phosphatase target subunit 1 (MYPT1) which drives the cGMP independent smooth muscle myosin phosphatase calcium desensitization was increased due to statin treatment (40%) and not affected by ET. Statin reduced sGCα protein content by more than 80%. Protein kinase cGMP‐dependent 1 (PRKG1), which phosphorylates multiple targets implicated in modulating cellular calcium was significantly increased (50%; p < .05) in non‐treated but decreased in treated rats. Protein abundance of the sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA), which drives the uptake of calcium into the sarcoplasmic reticulum was reduced by statins (50%) and not rescued by ET.ConclusionET independent of statin treatment improved endothelium‐dependent vasodilation. Interestingly, in untreated animals these improvements were most likely driven by NO, while in animals receiving statins, ET altered vascular smooth muscle calcium handling and desensitization. Overall our results suggests that individuals receiving statins should exercise to maintain vascular health.Support or Funding InformationDZHK (German Centre for Cardiovascular Research)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuropeptide-Y causes coronary microvascular constriction and is associated with reduced ejection fraction following ST-elevation myocardial infarction.

AimsThe co-transmitter neuropeptide-Y (NPY) is released during high sympathetic drive, including ST-elevation myocardial infarction (STEMI), and can be a potent vasoconstrictor. We hypothesized that myocardial NPY levels correlate with reperfusion and subsequent recovery following primary percutaneous coronary intervention (PPCI), and sought to determine if and how NPY constricts the coronary microvasculature.Methods and resultsPeripheral venous NPY levels were significantly higher in patients with STEMI (n = 45) compared to acute coronary syndromes/stable angina ( n = 48) or with normal coronary arteries (NC, n = 16). Overall coronary sinus (CS) and peripheral venous NPY levels were significantly positively correlated (r = 0.79). STEMI patients with the highest CS NPY levels had significantly lower coronary flow reserve, and higher index of microvascular resistance measured with a coronary flow wire. After 2 days they also had significantly higher levels of myocardial oedema and microvascular obstruction on cardiac magnetic resonance imaging, and significantly lower ejection fractions and ventricular dilatation 6 months later. NPY (100–250 nM) caused significant vasoconstriction of rat microvascular coronary arteries via increasing vascular smooth muscle calcium waves, and also significantly increased coronary vascular resistance and infarct size in Langendorff hearts. These effects were blocked by the Y1 receptor antagonist BIBO3304 (1 μM). Immunohistochemistry of the human coronary microvasculature demonstrated the presence of vascular smooth muscle Y1 receptors.ConclusionHigh CS NPY levels immediately after reperfusion correlate with microvascular dysfunction, greater myocardial injury, and reduced ejection fraction 6 months after STEMI. NPY constricts the coronary microcirculation via the Y1 receptor, and antagonists may be a useful PPCI adjunct therapy.

Abstract 032: Reconstitution of Autophagy Ameliorates Endothelium-Dependent Relaxation, Vascular Smooth Muscle Calcium Sensitization, & Arterial Stiffening in Spontaneously Hypertensive Rats

Insufficient autophagy has been proposed as a mechanism of cellular aging, as this leads to the accumulation of dysfunctional macromolecules and organelles. Premature vascular aging occurs in hypertension. In fact, many factors that contribute to the deterioration of vascular function as we age are accelerated in clinical and experimental hypertension. Previously, we have reported decreased autophagic activity in arteries from spontaneously hypertensive rats (SHR); however, the effects of restoring autophagic activity on blood pressure and vascular function are currently unknown. We hypothesized that reconstitution of arterial autophagy in SHR would decrease blood pressure and improve endothelium-dependent relaxation. We treated 13-18 week old Wistar and SHR with autophagy activator trehalose (2% in drinking water) for 28 days. Blood pressure was measured by telemetry and vascular function was performed on isolated mesenteric resistance arteries using wire and pressure myographs. Treatment with trehalose had no effect on mean arterial pressure in SHR (p>0.05). Trehalose treatment in SHR improved the relaxation to acetylcholine (ACh) [%Relaxation (ACh 1 μM), vehicle: 59.6±8.4 vs. trehalose: 82.4±4.2; p<0.05], and this was not different from arteries incubated with cyclooxygenase inhibitor indomethacin and reactive oxygen species scavenger tempol [%Relaxation (ACh 1 μM), trehalose: 82.4±4.2 vs. vehicle+indomethacin: 92.7±3.9 and vehicle+tempol: 89.3±5.4; both p>0.05]. Trehalose treatment in SHR also prevented the relaxation to Rho kinase inhibition [%Relaxation (Y-27632 0.3 μM), vehicle: 30.4±4.3 vs. trehalose: 8.4±3.5; p<0.05]. Finally, trehalose treatment in SHR decreased arterial stiffness as indicated by the slope of the stress-strain curve [β (stress-strain), vehicle: 8.3±0.6 vs. trehalose: 5.5±0.2; p<0.05]. Overall these data indicate that reconstitution of arterial autophagy in SHR improves endothelial and vascular smooth muscle function, which could synergize to prevent arterial stiffening. As a result, restoration of arterial autophagic activity could be a novel therapeutic for premature vascular aging in hypertension, a recently adopted clinical guideline for cardiovascular disease prevention.

Adenine-induced chronic renal failure in rats decreases aortic relaxation rate and alters expression of proteins involved in vascular smooth muscle calcium handling.

Rats with adenine-induced chronic renal failure (A-CRF) develop a reduced rate of relaxation of the thoracic aorta. The aim of this study was to elucidate the mechanisms underlying this abnormality. Male Sprague Dawley rats received either chow containing adenine or were pair-fed with normal chow (controls). After 8-14weeks, arterial function was analysed exvivo using wire myography and the expression of proteins involved in vascular smooth muscle excitation-contraction coupling in the thoracic aorta was analysed. The rate of relaxation following washout of KCl was reduced in A-CRF rats vs. controls in the thoracic aorta (P<0.01), abdominal aorta (P<0.05), and common carotid artery (P<0.05), but not in the common femoral artery. Relaxation rates of thoracic aortas increased (P<0.01), but were not normalized, in response to washout of KCl with Ca2+ -free buffer. Microarray and qRT-PCR analyses of genes involved in excitation-contraction coupling identified 10 genes, which showed significantly altered expression in A-CRF thoracic aortas. At the protein level, the α2 subunit of the Na,K-ATPase (P<0.001) and SERCA2 (P<0.05) was significantly downregulated, whereas stromal interaction molecule 1 and calsequestrin-1 and calsequestrin-2 were significantly upregulated (P<0.05). Rats with A-CRF show a marked alteration in relaxation of larger conduit arteries localized proximal to the common femoral artery. This abnormality may be caused by reduced cytosolic Ca2+ clearance in vascular smooth muscle cells secondary to dysregulation of proteins crucially involved in this process.

Abstract 617: Wnt-Containing Microvesicles Are Upregulated in Vascular Smooth Muscle Cell Cultures and Plasma From SM22-Cre;LRP6(fl/fl);LDLR-/- Mice

When fed a high fat diet (HFD) typical of westernized societies, male LDL receptor -deficient (LDLR-/-) mice develop obesity and insulin-resistant diabetes, with concomitant arteriosclerotic calcification and fibrosis. We recently demonstrated that noncanonical Wnt signals and ligands (including Wnt4, Wnt7b, and Wnt10a) were upregulated with LRP6 deficiency in aortic vascular smooth muscle (VSM) of LDLR-/- mice, with concomitant worsening of vascular disease. Because Wnt ligands are hydrophobic due to fatty acid acylation, we examined whether lipidaceous microvesicles (MVs) containing Wnt ligands were elaborated by VSM, and whether LRP6 impacted plasma MV numbers and contents. Microvesicles were isolated using both polymer based- and differential ultracentrifugation - based purification methods, and Malvern NS300 NanoSight nanoparticle tracking analysis implemented to quantify vesicle concentrations, focusing upon the 50-200 nm size range of exosomes and mineralizing vesicles. As compared to Cre-negative control cultures, SM22-Cre;LRP6(fl/fl);LDLR-/- VSM elaborated 2.9-fold fold higher numbers of MVs over a 2 day period (143 +/-27 million/ml +/- vs. 49+/- 5 million/ml; p = 0.01). ML141 -- a cdc42/rac1 antagonist that reduces VSM calcification and noncanonical Wnt signals activated with LRP6 deficiency — reduces MV numbers in VSM cultures (42+/- 10 million / ml with 10 uM ML141 vs.120 +/- 19 million/ ml vehicle; p&lt; 0.001). Consistent with primary VSM results, a 2-fold increase in the circulating plasma MV concentration was observed in aging SM22-Cre;LRP6(fl/fl);LDLR-/- mice vs. LRP6(fl/fl);LDLR-/- sibling controls (4.7+/- 1.1 billion/ml vs. 2.3+/-0.3 billion/ml, p = 0.07, n = 7-8 per genotype). Western blot analysis of extracts prepared from plasma MVs confirmed the presence of Wnt5a/b, Wnt10a, Wnt4 in this circulating nanoparticle population, and that Wnt4 was significantly increased in the MV fraction with LRP6 deficiency (1.4-fold, p = 0.01). Thus, Wnt-containing microvesicles are elaborated by VSM in vitro and in vivo. Quantitative characterization of plasma MV protein and lipid composition may yield novel biomarkers of pro-sclerotic Wnt signaling in diabetic vascular disease.

MicroRNAs 29b, 133b, and 211 Regulate Vascular Smooth Muscle Calcification Mediated by High Phosphorus.

Vascular calcification is a frequent cause of morbidity and mortality in patients with CKD and the general population. The common association between vascular calcification and osteoporosis suggests a link between bone and vascular disorders. Because microRNAs (miRs) are involved in the transdifferentiation of vascular smooth muscle cells into osteoblast-like cells, we investigated whether miRs implicated in osteoblast differentiation and bone formation are involved in vascular calcification. Different levels of uremia, hyperphosphatemia, and aortic calcification were induced by feeding nephrectomized rats a normal or high-phosphorus diet for 12 or 20 weeks, at which times the levels of eight miRs (miR-29b, miR-125, miR-133b, miR-135, miR-141, miR-200a, miR-204, and miR-211) in the aorta were analyzed. Compared with controls and uremic rats fed a normal diet, uremic rats fed a high-phosphorous diet had lower levels of miR-133b and miR-211 and higher levels of miR-29b that correlated respectively with greater expression of osteogenic RUNX2 and with lower expression of several inhibitors of osteoblastic differentiation. Uremia per se mildly reduced miR-133b levels only. Similar results were obtained in two in vitro models of vascular calcification (uremic serum and high-calcium and -phosphorus medium), and experiments using antagomirs and mimics to modify miR-29b, miR-133b, and miR-211 expression levels in these models confirmed that these miRs regulate the calcification process. We conclude that miR-29b, miR-133b, and miR-211 have direct roles in the vascular smooth muscle calcification induced by high phosphorus and may be new therapeutic targets in the management of vascular calcification.

Resveratrol Attenuates Vasocontractile Responses to Phenylephrine and Potassium Chloride via a Nitric Oxide Synthase– and Endothelium–Independent Mechanism

This study examined the effect of acute pre‐incubation without or with resveratrol (RSV) at increasing in‐bath concentrations (0, 20, 50, or 100 µM) on the dose‐dependent vasocontractile response stimulated by either the α1‐adrenergic receptor agonist phenylephrine (PE; ‐9.0 to ‐5.0 LogM) or potassium chloride (KCl; 0 to 60 mM) in isometrically‐mounted common carotid artery rings isolated from 20‐30 week old Spontaneously Hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). In both SHR and WKY, the contractile dose‐response curve to PE was increasingly blunted by preincubation with 20, 50, and 100 µM RSV, as evidenced by a RSV concentration‐dependent rightward shift of EC50 and decrease in maximum amplitude and area under the curve (p&lt;0.05). The contractile dose‐response curve to KCl was similarly affected by preincubation with increasing concentrations of RSV (p&lt;0.05). Neither nitric oxide synthase (NOS) inhibition nor endothelium removal was found to affect the ability of 50 µM RSV to attenuate either PE‐ or KCl‐stimulated contraction in SHR or WKY. These data indicate that RSV can attenuate vasocontractile activity stimulated by either receptor‐mediated (PE) activation or voltage‐mediated (KCl) activation via a vascular wall cell‐signaling mechanism that is independent of NOS or the endothelium. This suggests that RSV may act to attenuate contraction in the vascular wall by directly affecting central pathways of contractile activation, such as vascular smooth muscle calcium handling and/or sensitivity.Funded by NSERC Canada

Increased pressure-induced tone in rat parenchymal arterioles vs. middle cerebral arteries: role of ion channels and calcium sensitivity.

Brain parenchymal arterioles (PAs) are high-resistance vessels that branch off pial arteries and perfuse the brain parenchyma. PAs are the target of cerebral small vessel disease and have been shown to have greater pressure-induced tone at lower pressures than pial arteries. We investigated mechanisms by which brain PAs have increased myogenic tone compared with middle cerebral arteries (MCAs), focusing on differences in vascular smooth muscle (VSM) calcium and ion channel function. The amount of myogenic tone and VSM calcium was measured using Fura 2 in isolated and pressurized PAs and MCAs. Increases in intraluminal pressure caused larger increases in tone and cytosolic calcium in PAs compared with MCAs. At 50 mmHg, myogenic tone was 37 ± 5% for PAs vs. 6.5 ± 4% for MCAs (P < 0.01), and VSM calcium was 200 ± 20 nmol/l in PAs vs. 104 ± 15 nmol/l in MCAs (P < 0.01). In vessels permeabilized with Staphylococcus aureus α-toxin, PAs were not more sensitive to calcium, suggesting calcium sensitization was not at the level of the contractile apparatus. PAs were 30-fold more sensitive to the voltage-dependent calcium channel (VDCC) inhibitor nifedipine than MCAs (EC50 for PAs was 3.5 ± 0.4 vs. 82.1 ± 2.1 nmol/l for MCAs;P < 0.01); however, electrophysiological properties of the VDCC were not different in VSM. PAs had little to no response to the calcium-activated potassium channel inhibitor iberiotoxin, whereas MCAs constricted ∼15%. Thus increased myogenic tone in PAs appears related to differences in ion channel activity that promotes VSM membrane depolarization but not to a direct sensitization of the contractile apparatus to calcium.

Attenuation of Chondrogenic Transformation in Vascular Smooth Muscle by Dietary Quercetin in the MGP-Deficient Mouse Model

RationaleCartilaginous metaplasia of vascular smooth muscle (VSM) is characteristic for arterial calcification in diabetes and uremia and in the background of genetic alterations in matrix Gla protein (MGP). A better understanding of the molecular details of this process is critical for the development of novel therapeutic approaches to VSM transformation and arterial calcification.ObjectiveThis study aimed to identify the effects of bioflavonoid quercetin on chondrogenic transformation and calcification of VSM in the MGP-null mouse model and upon TGF-β3 stimulation in vitro, and to characterize the associated alterations in cell signaling.Methods and ResultsMolecular analysis revealed activation of β-catenin signaling in cartilaginous metaplasia in Mgp-/- aortae in vivo and during chondrogenic transformation of VSMCs in vitro. Quercetin intercepted chondrogenic transformation of VSM and blocked activation of β-catenin both in vivo and in vitro. Although dietary quercetin drastically attenuated calcifying cartilaginous metaplasia in Mgp-/- animals, approximately one-half of total vascular calcium mineral remained as depositions along elastic lamellae.ConclusionQuercetin is potent in preventing VSM chondrogenic transformation caused by diverse stimuli. Combined with the demonstrated efficiency of dietary quercetin in preventing ectopic chondrogenesis in the MGP-null vasculature, these findings indicate a potentially broad therapeutic applicability of this safe for human consumption bioflavonoid in the therapy of cardiovascular conditions linked to cartilaginous metaplasia of VSM. Elastocalcinosis is a major component of MGP-null vascular disease and is controlled by a mechanism different from chondrogenic transformation of VSM and not sensitive to quercetin.

Upregulation of a Disintegrin and Metalloproteinase With Thrombospondin Motifs-7 by miR-29 Repression Mediates Vascular Smooth Muscle Calcification

Vascular calcification significantly increases cardiovascular morbidity and mortality. We recently reported that the deficiency of cartilage oligomeric matrix protein (COMP) leads to vascular mineralization. We characterized the COMP-degrading metalloproteinase, a disintegrin and metalloproteinase with thrombospondin motifs-7 (ADAMTS-7). Here, we tested whether ADAMTS-7 facilitates vascular calcification. ADAMTS-7 expression was markedly upregulated in calcifying rat vascular smooth muscle cells (VSMCs) in vitro, calcified arteries of rats with chronic renal failure in vivo, and radial arteries of uraemic patients. Silencing of ADAMTS-7 markedly reduced COMP degradation and ameliorated VSMC calcification, whereas ectopic expression of ADAMTS-7 greatly enhanced COMP degradation and exacerbated mineralization. The transcriptional activity of ADAMTS-7 promoter was not altered by high phosphate. We used bioinformatics and quantitative polymerase chain reaction analysis to demonstrate that high-phosphate upregulated ADAMTS-7 mRNA and protein via miR-29a/b repression, which directly targeted the 3' untranslated region of ADAMTS-7 in VSMCs. MicroRNA (MiR)-29a/b mimic markedly inhibited but miR-29a/b inhibitor greatly enhanced high-phosphate-induced ADAMTS-7 expression, COMP degradation, and subsequent VSMC calcification. ADAMTS-7 silencing significantly diminished miR-29a/b repression-exaggerated VSMC calcification. Our data reveal a novel mechanism by which ADAMTS-7 upregulation by miR-29a/b repression mediates vascular calcification, which may shed light on preventing cardiovascular morbidity and mortality.

Ethanol increases phosphate‐mediated mineralization and osteoblastic transformation of vascular smooth muscle cells

Vascular calcification is implicated in the pathogenesis of atherosclerosis, diabetes and chronic kidney disease. Human vascular smooth muscle cells (HSMCs) undergo mineralization in response to elevated levels of inorganic phosphate (Pi) in an active and well-regulated process. This process involves increased activity of alkaline phosphatase and increased expression of core binding factor α-1 (CBF-α1), a bone-specific transcription factor, with the subsequent induction of osteocalcin. It has been shown that heavy alcohol consumption is associated with greater calcification in coronary arteries. The goal of our study was to examine whether ethanol alters mineralization of HSMCs provoked by high Pi. Exposure of HSMCs to ethanol increased extracellular matrix calcification in a dose responsive manner, providing a significant additional calcium deposition at concentrations of ≥60 mmol/l. HSMC calcification was accompanied by further enhancement in alkaline phosphatase activity. Ethanol also provoked a significant increase in the synthesis of osteocalcin. Moreover, in cells challenged with ethanol the expression of CBF-α1, a transcription factor involved in the regulation of osteoblastic transformation of HSMCs, was elevated. The observed effects of ethanol were not due to alterations of phosphate uptake by HSMCs. We conclude that ethanol enhances Pi-mediated human vascular smooth muscle calcification and transition of these cells into osteoblast-like cells.

Impaired myogenic tone in mesenteric arteries from overweight rats

BackgroundRats fed high fat (HFD) or high sucrose (HSD) diets develop increased adiposity as well as impaired vasodilatory responsiveness stemming from oxidative stress. Moreover, HFD rats become hypertensive compared to either control (Chow) or HSD fed rats, suggesting elevated vascular tone. We hypothesized that rats with increased adiposity and oxidative stress demonstrate augmented pressure-induced vasoconstriction (i.e. myogenic tone) that could account for the hypertensive state.MethodsMale Sprague-Dawley rats were fed Chow, HFD or HSD for 6 weeks. The effects of oxidative stress and endogenous nitric oxide on myogenic responses were examined in small mesenteric arteries by exposing the arteries to incremental intraluminal pressure steps in the presence of antioxidants or an inhibitor of nitric oxide synthase, LNNA (100 μM).ResultsContrary to the hypothesis, rats fed either HSD or HFD had significantly impaired myogenic responses despite similar vascular morphology and passive diameter responses to increasing pressures. Vascular smooth muscle (VSM) calcium levels were normal in HFD arteries suggesting that diminished calcium sensitivity was responsible for the impaired myogenic response. In contrast, VSM calcium levels were reduced in HSD arteries but were increased with pre-exposure of arteries to the antioxidants tiron (10 mM) and catalase (1200 U/mL), also resulting in enhanced myogenic tone. These findings show that oxidative stress impairs myogenic tone in arteries from HSD rats by decreasing VSM calcium. Similarly, VSM calcium responses were increased in arteries from HFD rats following treatment with tiron and catalase, but this did not result in improved myogenic tone. Nitric oxide is involved in the impaired myogenic response in HFD, but not HSD, rats since inhibition with LNNA resulted in maximal myogenic responses at lower intraluminal pressures and VSM calcium levels, further implicating reduced calcium sensitivity in the impaired response.ConclusionThe impaired myogenic responses observed in isolated arteries from HSD and HFD rats are attributed to changes in VSM calcium signaling.

A Genome-Wide Association Study in Europeans and South Asians Identifies 5 New Loci for Coronary Artery Disease

### Study Hypothesis Recently, genome-wide association studies (GWAS) have identified several common variants associated with increased risk of coronary artery disease (CAD) and myocardial infarction (MI) by 10% to 30%. The authors state that these loci explain only a small proportion of the predicted genetic risk and that all of the current loci for CAD and MI by GWAS have been discovered in European populations. The authors hypothesized that discovery of new susceptibility loci of smaller effect sizes would be aided by conducting much larger studies in addition to an emphasis on early-onset CAD and clearly defined clinical end points. Therefore, they assembled the Coronary Artery Disease (C4D) Genetics Consortium.1 ### How Was the Hypothesis Tested? The authors performed a meta-analysis of 4 large GWAS of CAD, 2 of European ancestry (PROCARDIS and HPS) and 2 of South Asian ancestry (PROMIS and LOLLIPOP), with ≈575 000 genotyped single-nucleotide polymorphisms (SNPs) in a discovery data set comprising 15 420 individuals with CAD (cases) (8424 Europeans and 6996 South Asians) and 15 062 control subjects. They observed little evidence for ancestry-specific associations, supporting the use of combined analyses. Furthermore, the authors state that because all individuals were genotyped on the same platform (whole-genome Illumina BeadChips), a meta-analysis of actual genotypes rather than imputed data enabled analysis of low-frequency variants (1% to 5%), which have been excluded from GWAS either because of sample size or because imputation has been required to combine data from different genotyping platforms. After the meta-analysis, they selected 59 SNPs from 50 loci that showed potential new associations from the meta-analysis of the European and South Asian studies (41 SNPs; P <1.0×10−4), the European-only meta-analysis (8 SNPs; P <3.0×10−5), the South …

Cartilage Oligomeric Matrix Protein Inhibits Vascular Smooth Muscle Calcification by Interacting With Bone Morphogenetic Protein-2

Vascular calcification is a significant contributor to cardiovascular morbidity and mortality. We recently reported that cartilage oligomeric matrix protein (COMP) is pivotal for maintaining the homeostasis of vascular smooth muscle cells (VSMCs). Whether COMP affects the process of vascular calcification is unknown. We aimed to test whether COMP modulates vascular calcification. VSMC calcification in vitro was induced by calcifying media containing high inorganic phosphate or calcium. In vivo medial vessel calcification was induced in rats by 5/6 nephrectomy with a high-phosphate diet or by periadventitial application of CaCl(2) to the abdominal aorta. COMP protein level was markedly reduced in both calcified VSMCs and arteries. COMP deficiency remarkably exacerbated VSMC calcification, whereas ectopic expression of COMP greatly reduced calcification. Furthermore, COMP knockdown facilitated osteogenic markers expression by VSMCs even in the absence of calcifying media. By contrast, COMP overexpression significantly inhibited high phosphate- or high calcium-induced VSMC osteochondrogenic transition. Induction of osteogenic marker expression by COMP silencing was reversed by a soluble form of bone morphogenetic protein (BMP)-2 receptor IA, which suggests a BMP-2-dependent mechanism. Our data revealed that COMP bound directly to BMP-2 through the C terminus, inhibited BMP-2 receptor binding, and blocked BMP-2 osteogenic signaling, indicating COMP inhibits osteochondrogenic transition of VSMCs at least partially through inhibiting BMP-2. Our data strongly suggest that COMP is a novel inhibitor of vascular calcification. The imbalance between the effects of COMP and BMP-2 may provide new insights into the pathophysiology of vascular calcification.

Antioxidant and Vasodilatory Effects of Heme Oxygenase on Mesenteric Vasoreactivity Following Chronic Hypoxia

Chronic hypoxia (CH) results in impaired vasoconstriction associated with increased expression of heme oxygenase (HO). We hypothesized that enhanced HO activity minimizes reactive oxygen species (ROS) in arteries from CH rats, thereby normalizing endothelium-dependent vasodilation and concurrently produces carbon monoxide (CO), resulting in tonic vasodilation. ROS were quantified in mesenteric arteries from control and CH Sprague-Dawley rats. Reactivity to the endothelium-dependent vasodilator, acetylcholine (ACh), and the vasoconstrictor, phenylephrine (PE), were also assessed. Basal ROS levels did not differ between groups and were similarly increased by HO inhibition. In contrast, catalase inhibition increased ROS in CH rats only. Vasodilatory responses to ACh were not different between groups. Combined inhibition of catalase and HO impaired PE-induced vasoconstriction in both groups. CH-induced impairment of vasoconstriction was reversed by either catalase or HO inhibition supporting the protective roles of the HO and catalase pathways following CH. Increased vascular smooth muscle calcium was observed with inhibition in the CH group, suggesting that catalase and HO-derived CO elicit reduced calcium influx, leading to the impaired vasoconstriction. Our data suggest that although the HO pathway is an important antioxidant influence, impaired vasoconstriction following CH appears to be due to effects of ROS and HO-derived CO.