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Wnt16 is expressed in bone and arteries, and maintains bone mass in mice and humans, but its role in cardiovascular physiology is unknown. We show that Wnt16 protein accumulates in murine and human vascular smooth muscle (VSM). WNT16 genotypes that convey risk for bone frailty also convey risk for cardiovascular events in the Dallas Heart Study. Murine Wnt16 deficiency, which causes postnatal bone loss, also reduced systolic blood pressure. Electron microscopy demonstrated abnormal VSM mitochondrial morphology in Wnt16-null mice, with reductions in mitochondrial respiration. Following angiotensin-II (AngII) infusion, thoracic ascending aorta (TAA) dilatation was greater in Wnt16 −/− vs Wnt16+/+ mice (LDLR −/− background). Acta2 (vascular smooth muscle alpha actin) deficiency has been shown to impair contractile phenotype and worsen TAA aneurysm with concomitant reductions in blood pressure. Wnt16 deficiency reduced expression of Acta2 , SM22 (transgelin), and other contractile genes, and reduced VSM contraction induced by TGFβ. Acta2 and SM22 proteins were reduced in Wnt16 −/− VSM as was Ankrd1, a prototypic contractile target of Yap1 and Taz activation via TEA domain (TEAD)-directed transcription. Wnt16 −/− VSM exhibited reduced nuclear Taz and Yap1 protein accumulation. SiRNA targeting Wnt16 or Taz, but not Yap1, phenocopied Wnt16 deficiency, and Taz siRNA inhibited contractile gene upregulation by Wnt16. Wnt16 incubation stimulated mitochondrial respiration and contraction (reversed by verteporfin, a Yap/Taz inhibitor). SiRNA targeting Taz inhibitors Ccm2 and Lats1/2 mimicked Wnt16 treatment. Wnt16 stimulated Taz binding to Acta2 chromatin and H3K4me3 methylation. TEAD cognates in the Acta2 promoter conveyed transcriptional responses to Wnt16 and Taz. Wnt16 regulates cardiovascular physiology and VSM contractile phenotype, mediated via Taz signaling. [ABSTRACT FROM AUTHOR]

The reaction field of abnormal vascular contraction induced by sphingosylphosphorylcholine (SPC) and the action point of SPC around the plasma membranes remain unknown. However, we found in a previous study that fisetin prevents SPC-induced vascular smooth muscle cells contraction, while the mechanism remains unknown. Therefore, in this study, we aimed to address the action point of SPC around the plasma membranes and the involvement of fisetin. We focused on microdomains and evaluated their markers flotillin-1 and caveolin-1 and the localization of SPC to investigate their action point. The results showed that microdomains of vascular smooth muscle cells were not involved in SPC-induced contraction. However, we found that after SPC had been affected on the plasma membrane, cells took up SPC via endocytosis. Moreover, SPC remained in the cells and did not undergo transcytosis, and SPC-induced contracting cells produced exosomes. These phenomena were similar to those observed in fisetin-treated cells. Thus, we speculated that, although not involved in the reaction field of SPC-induced contractions, the microdomain induced the endocytosis of SPCs, and fisetin prevented the contractions by directly targeting vascular smooth muscle cells. Notably, this preventive mechanism involves the cellular uptake of SPC via endocytosis. [ABSTRACT FROM AUTHOR]

Hypertension is a key risk factor for cardiovascular disease and it is a growing public health problem worldwide. The pathophysiological mechanisms of vascular smooth muscle (VSM) contraction contribute to the development of hypertension. Calcium (Ca2+)-dependent and -independent signaling mechanisms regulate the balance of the myosin light chain kinase and myosin light chain phosphatase to induce myosin phosphorylation, which activates VSM contraction to control blood pressure (BP). Here, we discuss the mechanism of the contractile machinery in VSM, especially RhoA/Rho kinase and PKC/CPI-17 of Ca2+ sensitization pathway in hypertension. The two signaling pathways affect BP in physiological and pathophysiological conditions and are highlighted in pulmonary, pregnancy, and salt-sensitive hypertension.

Vascular smooth muscle (VSM) plays an important role in the regulation of vascular function. Identifying the mechanisms of VSM contraction has been a major research goal in order to determine the causes of vascular dysfunction and exaggerated vasoconstriction in vascular disease. Major discoveries over several decades have helped to better understand the mechanisms of VSM contraction. Ca 2+ has been established as a major regulator of VSM contraction, and its sources, cytosolic levels, homeostatic mechanisms and subcellular distribution have been defined. Biochemical studies have also suggested that stimulation of Gq protein-coupled membrane receptors activates phospholipase C and promotes the hydrolysis of membrane phospholipids into inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 stimulates initial Ca 2+ release from the sarcoplasmic reticulum, and is buttressed by Ca 2+ influx through voltage-dependent, receptor-operated, transient receptor potential and store-operated channels. In order to prevent large increases in cytosolic Ca 2+ concentration ([Ca 2+ ] c ), Ca 2+ removal mechanisms promote Ca 2+ extrusion via the plasmalemmal Ca 2+ pump and Na + /Ca 2+ exchanger, and Ca 2+ uptake by the sarcoplasmic reticulum and mitochondria, and the coordinated activities of these Ca 2+ handling mechanisms help to create subplasmalemmal Ca 2+ domains. Threshold increases in [Ca 2+ ] c form a Ca 2+ -calmodulin complex, which activates myosin light chain (MLC) kinase, and causes MLC phosphorylation, actin–myosin interaction, and VSM contraction. Dissociations in the relationships between [Ca 2+ ] c , MLC phosphorylation, and force have suggested additional Ca 2+ sensitization mechanisms. DAG activates protein kinase C (PKC) isoforms, which directly or indirectly via mitogen-activated protein kinase phosphorylate the actin-binding proteins calponin and caldesmon and thereby enhance the myofilaments force sensitivity to Ca 2+ . PKC-mediated phosphorylation of PKC-potentiated phosphatase inhibitor protein-17 (CPI-17), and RhoA-mediated activation of Rho-kinase (ROCK) inhibit MLC phosphatase and in turn increase MLC phosphorylation and VSM contraction. Abnormalities in the Ca 2+ handling mechanisms and PKC and ROCK activity have been associated with vascular dysfunction in multiple vascular disorders. Modulators of [Ca 2+ ] c , PKC and ROCK activity could be useful in mitigating the increased vasoconstriction associated with vascular disease. [ABSTRACT FROM AUTHOR]

There is accumulating evidence that makes the link between the circadian variation in blood pressure and circadian variations in vascular contraction. The importance of vascular endothelium-derived redox-active and redox-derived species in the signalling pathways involved in controlling vascular smooth muscle contraction are well known, and when linked to the circadian variations in the processes involved in generating these species, suggests a cellular mechanism for the circadian variations in blood pressure that links directly to the peripheral circadian clock. Relaxation of vascular smooth muscle cells involves endothelial-derived relaxing factor (EDRF) which is nitric oxide (NO) produced by endothelial NO synthase (eNOS), and endothelial-derived hyperpolarising factor (EDHF) which includes hydrogen peroxide (H 2 O 2 ) produced by NADPH oxidase (Nox). Both of these enzymes appear to be under the direct control of the circadian clock mechanism in the endothelial cells, and disruption to the clock results in endothelial and vascular dysfunction. In this review, we focus on EDRF and EDHF and summarise the recent findings on the influence of the peripheral circadian clock mechanism on processes involved in generating the redox species involved and how this influences vascular contractility, which may account for some of the circadian variations in blood pressure and peripheral resistance. Moreover, the direct link between the peripheral circadian clock and redox-signalling pathways in the vasculature, has a bearing on vascular endothelial dysfunction in disease and aging, which are both known to lead to dysfunction of the circadian clock. [ABSTRACT FROM AUTHOR]

S-nitrosothiols derived from nitric oxide are known to regulate cell signaling through thiol modification. Since small G protein RhoA contains cysteine residues in the GTP-binding domain which is critical for its function, modification these thiols may alter RhoA activity and lead to changes in the downstream signaling such as myosin light chain phosphorylation. However, it is still unclear that if RhoA activity and its downstream signals might be modulated by S-nitrosothiols and if the two cysteine residues located in the GTP-binding domain are critical for the regulation. In this study we show that S-nitroso-L-cysteine (CSNO) blocked RhoA activation as determined by either GDP/GTP exchange, active RhoA binding to rhotekin or RhoA translocation. CSNO was shown to lead to RhoA nitrosylation and RhoA thiol oxidation status was found to be consistent with loss of its activity. Mutation of all 6 single cysteine residues to serine showed that purified recombinant C20S mutant and C26/20S mutant were resistant to CSNO, but interestingly, in the intact cells only the double C16/20S mutant was resistant to CSNO. Moreover, inhibition of RhoA activation led to Rho-kinase inhibition and inhibition of Rho pathway signaling by CSNO. In both smooth muscle cells and aortic tissue, the outcome was inhibition of agonist-stimulated MYPT1 phosphorylation and reduced levels of myosin light chain phosphorylation. These effects of CSNO on MYPT1 and myosin light chain phosphorylation appear to be cGMP-independent since they were unaffected by inhibition of guanylyl cyclase. In contrast to CSNO, spermine NONOate did not alter RhoA GDP/GTP exchange and the effects of this compound on myosin light chain phosphorylation were blocked by guanylyl cyclase inhibition. And importantly, in C16/20S overexpressed smooth muscle cells, MYPT1 phosphorylation was resistant to the inhibitory effect of CSNO. Together, these data suggest that S-nitrosothiols regulate myosin light chain phosphorylation by inhibiting RhoA/Rho-kinase signaling through modification of RhoA cysteine residues at 16 and 20 in its GTP-binding domain, which might be an important therapeutic target for diseases with imbalanced vascular resistance. [ABSTRACT FROM AUTHOR]

Hypertension is a major risk factor for many common chronic diseases, such as heart failure, myocardial infarction, stroke, vascular dementia, and chronic kidney disease. Pathophysiological mechanisms contributing to the development of hypertension include increased vascular resistance, determined in large part by reduced vascular diameter due to increased vascular contraction and arterial remodelling. These processes are regulated by complexinteracting systems such as the renin-angiotensin-aldosterone system, sympathetic nervous system, immune activation, and oxidative stress, which influence vascular smooth muscle function. Vascular smooth muscle cells are highly plastic and in pathological conditions undergo phenotypic changes from a contractile to a proliferative state. Vascular smooth muscle contraction is triggered by an increase in intracellular free calcium concentration ([Ca2+]i), promoting actin-myosin cross-bridge formation. Growing evidence indicates that contraction is also regulated by calcium-independent mechanisms involving RhoA-Rho kinase, protein Kinase C and mitogen-activated protein kinase signalling, reactive oxygen species, and reorganization of the actin cytoskeleton. Activation of immune/inflammatory pathways and non-coding RNAs are also emerging as important regulators of vascular function. Vascular smooth muscle cell [Ca2+]i not only determines the contractile state but also influences activity of many calciumdependent transcription factors and proteins thereby impacting the cellular phenotype and function. Perturbations in vascular smooth muscle cell signalling and altered function influence vascular reactivity and tone, important determinants of vascular resistance and blood pressure. Here, we discuss mechanisms regulating vascular reactivity and contraction in physiological and pathophysiological conditions and highlight some new advances in the field, focusing specifically on hypertension. [ABSTRACT FROM AUTHOR]

Introduction It is well known that moderate to high doses of ionizing radiation have a toxic effect on the organism. However, there are few experimental studies on the mechanisms of LDR ionizing radiation on nerve regeneration after peripheral nerve injury. Methods We established the rats’ peripheral nerve injury model via repaired Peripheral nerve injury nerve, vascular endothelial growth factor a and Growth associated protein-43 were detected from different treatment groups. We performed transcriptome sequencing focusing on investigating the differentially expressed genes and gene functions between the control group and 1 Gy group. Sequencing was done by using high-throughput RNA-sequencing (RNA-seq) technologies. Results The results showed the 1 Gy group to be the most effective promoting repair. RNA-sequencing identified 619 differently expressed genes between control and treated groups. A Gene Ontology analysis of the differentially expressed genes revealed enrichment in the functional pathways. Among them, candidate genes associated with nerve repair were identified. Discussion Pathways involved in cell-substrate adhesion, vascular smooth muscle contraction and cell adhesion molecule signaling may be involved in recovery from peripheral nerve injury. [ABSTRACT FROM AUTHOR]

Vascular smooth muscle contraction is suppressed by feedback dilation mediated by the endothelium. In skeletal muscle arterioles, this feedback can be activated by Ca2+ signals passing from smooth muscle through gap junctions to endothelial cells, which protrude through holes in the internal elastic lamina to make contact with vascular smooth muscle cells. Although hypothetically either Ca2+ or inositol trisphosphate (IP3) may provide the intercellular signal, it is generally thought that IP3 diffusion is responsible. We provide evidence that Ca2+ entry through L-type voltage-dependent Ca2+ channels (VDCCs) in vascular smooth muscle can pass to the endothelium through positions aligned with holes in the internal elastic lamina in amounts sufficient to activate endothelial cell Ca2+ signaling. In endothelial cells in which IP3 receptors (IP3Rs) were blocked, VDCC-driven Ca2+ events were transient and localized to the endothelium that protrudes through the internal elastic lamina to contact vascular smooth muscle cells. In endothelial cells in which IP3Rs were not blocked, VDCC-driven Ca2+ events in endothelial cells were amplified to form propagating waves. These waves activated voltage-insensitive, intermediate-conductance, Ca2+-activated K+ (IKCa) channels, thereby providing feedback that effectively suppressed vasoconstriction and enabled cycles of constriction and dilation called vasomotion. Thus, agonists that stimulate vascular smooth muscle depolarization provide Ca2+ to endothelial cells to activate a feedback circuit that protects tissue blood flow. [ABSTRACT FROM AUTHOR]

The preglomerular microcirculation of spontaneously hypertensive rats (SHR) is hypersensitive to angiotensin (ANG) II, and studies have shown that this is likely due to enhanced coincident signaling between G protein subunits αq (Gαq; released by ANG II) and βγ (Gβγ released by Gi-coupled receptors) to active phospholipase C (PLC). Here we investigated the molecular basis for the enhanced coincident signaling between Gβγ and Gαq in SHR preglomerular vascular smooth muscle cells (PGVSMCs). Because receptor for activated C kinase 1 (RACK1; a scaffolding protein) organizes interactions between Gβγ, Gαq, and PLC, we included RACK1 in this investigation. Cell fractionation studies demonstrated increased levels of membrane (but not cytosolic) Gγ, Gαq, PLCγ3, and RACK1 in SHR PGVSMCs compared with Wistar-Kyoto rat PGVSMCs. In SHR PGVSMCs, coimmunoprecipitation demonstrated RACK1 binding to Gγ and PLCγ3, but only at cell membranes. Pertussis toxin (which blocks Gβγ) and U73122 (which blocks PLC) reduced membrane RACK1; however, RACK1 knockdown (shRNA) did not affect membrane levels of Gγ, Gαq, or PLCγ3. In a novel gel contraction assay, RACK1 knockdown in SHR PGVSMCs attenuated contractions to ANG II and abrogated the ability of neuropeptide Y (which signals via Gβγ) to enhance ANG II-induced contractions. We conclude that in SHR PGVSMCs the enlarged pool of Gβγ and PLCγ3 recruits RACK1 to membranes and RACK1 then organizes signaling. Consequently, knockdown of RACK1 prevents coincident signaling between ANG II and the Gi pathway. This is the first study to implicate RACK1 in vascular smooth muscle cell contraction and suggests that RACK1 inhibitors could be effective cardiovascular drugs. [ABSTRACT FROM AUTHOR]

Key points Depletion of Ca2+ stores activates store-operated channels (SOCs), which mediate Ca2+ entry pathways that regulate cellular processes such as contraction, proliferation and gene expression., In vascular smooth muscle cells (VSMCs), stimulation of SOCs composed of canonical transient receptor potential channel 1 (TRPC1) proteins requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1/protein kinase C (PKC) activity. We studied the role of stromal interaction molecule 1 (STIM1) in coupling store depletion to this activation pathway using patch clamp recording, GFP-PLCδ1-PH imaging and co-localization techniques., Store-operated TRPC1 channel and PLCβ1 activities were inhibited by STIM1 short hairpin RNA (shRNA) and absent in TRPC1−/− cells, and store-operated PKC phosphorylation of TRPC1 was inhibited by STIM1 shRNA. Store depletion induced interactions between STIM1 and TRPC1, Gαq and PLCβ1, which required STIM1 and TRPC1. Similar effects were produced with noradrenaline., These findings identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, where store-operated STIM1-TRPC1 interactions stimulate Gαq/PLCβ1/PKC activity to induce channel gating., Abstract In vascular smooth muscle cells (VSMCs), stimulation of canonical transient receptor potential channel 1 (TRPC1) protein-based store-operated channels (SOCs) mediates Ca2+ entry pathways that regulate contractility, proliferation and migration. It is therefore important to understand how these channels are activated. Studies have shown that stimulation of TRPC1-based SOCs requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1 activities and protein kinase C (PKC) phosphorylation, although it is unclear how store depletion stimulates this gating pathway. The present study examines this issue by focusing on the role of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum Ca2+ sensor. Store-operated TRPC1 channel activity was inhibited by TRPC1 and STIM1 antibodies and STIM1 short hairpin RNA (shRNA) in wild-type VSMCs, and was absent in TRPC1−/− VSMCs. Store-operated PKC phosphorylation of TRPC1 was reduced by knockdown of STIM1. Moreover, store-operated PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH was reduced by STIM1 shRNA and absent in TRPC1−/− cells. Immunocytochemistry, co-immunoprecipitation and proximity ligation assays revealed that store depletion activated STIM1 translocation from within the cell to the plasma membrane (PM) where it formed STIM1-TRPC1 complexes, which then associated with Gαq and PLCβ1. Noradrenaline also evoked TRPC1 channel activity and associations between TRPC1, STIM1, Gαq and PLCβ1, which were inhibited by STIM1 knockdown. Effects of N-terminal and C-terminal STIM1 antibodies on TRPC1-based SOCs and STIM1 staining suggest that channel activation may involve insertion of STIM1 into the PM. The findings of the present study identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, in which store-operated STIM1-TRPC1 interactions stimulate PLCβ1 activity to induce PKC phosphorylation of TRPC1 and channel gating. [ABSTRACT FROM AUTHOR]

Ion channels underlie a wide variety of physiological processes that involve rapid changes in cell dynamics, such as cardiac and vascular smooth muscle contraction. Overexpression or dysfunction of these membrane proteins are the basis of many cardiovascular diseases that represent the leading cause of morbidity and mortality for human beings. In the last few years, flavonoids, widely distributed in the plant kingdom, have attracted the interest of many laboratories as an emerging class of fine ion, in particular Ca v , channels modulators. Pieces of in vitro evidence for direct as well as indirect effects exerted by various flavonoids on ion channel currents are now accumulating in the scientific literature. This activity may be responsible, at least in part, for the beneficial and protective effects of dietary flavonoids toward cardiovascular diseases highlighted in several epidemiological studies. Here we examine numerous studies aimed at analysing this feature of flavonoids, focusing on the mechanisms that promote their sometimes controversial activities at cardiovascular Ca v channels. New methodological approaches, such as molecular modelling and docking to Ca v 1.2 channel α 1c subunit, used to elucidate flavonoids intrinsic mechanism of action, are introduced. Moreover, flavonoid-membrane interaction, bioavailability, and antioxidant activity are taken into account and discussed. [ABSTRACT FROM AUTHOR]

Context:Calcium channel blockers may theoretically exhibit relaxing effects not only on vascular smooth muscle but also on airway smooth muscle. Objective:To investigate possible effects of nifedipine on respiratory mechanics in the rat. Methods:Respiratory system mechanical parameters were measured by the end-inflation occlusion method in the ratin vivobefore and after the intraperitoneal administration of nifedipine. Results:We found that nifedipine affects respiratory mechanics, inducing a reduction of airway resistance and of respiratory system elastance, probably because of a relaxing action on airway and parenchimal smooth muscle cells. Conclusion:Should these results be further confirmed by human investigations, a possible role of nifedipine in pharmacological respiratory system’s diseases treatment may be suggested. [ABSTRACT FROM PUBLISHER]

Chemerin is an adipokine associated with increased blood pressure, and may link obesity with hypertension. We tested the hypothesis that chemerin-induced contraction of the vasculature occurs via calcium flux in smooth muscle cells. Isometric contraction of rat aortic rings was performed in parallel with calcium kinetics of rat aortic smooth muscle cells to assess the possible signaling pathway. Chemerin-9 (nonapeptide of the chemerin S 157 isoform) caused a concentration-dependent contraction of isolated aorta (EC 50 100 nM) and elicited a concentration-dependent intracellular calcium response (EC 50 10 nM). Pertussis toxin (G i inhibitor), verapamil (L-type Ca 2 + channel inhibitor), PP1 (Src inhibitor), and Y27632 (Rho kinase inhibitor) reduced both calcium influx and isometric contraction to chemerin-9 but PD098059 (Erk MAPK inhibitor) and U73122 (PLC inhibitor) had little to no effect on either measure of chemerin signaling. Although our primary aim was to examine chemerin signaling, we also highlight differences in the mechanisms of chemerin-9 and recombinant chemerin S 157 . These data support a chemerin-induced contractile mechanism in vascular smooth muscle that functions through G i proteins to activate L-type Ca 2 + channels, Src, and Rho kinase. There is mounting evidence linking chemerin to hypertension and this mechanism brings us closer to targeting chemerin as a form of therapy. [ABSTRACT FROM AUTHOR]

Aim 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. Methods Male Sprague Dawley rats received either chow containing adenine or were pair-fed with normal chow (controls). After 8-14 weeks, arterial function was analysed ex vivo using wire myography and the expression of proteins involved in vascular smooth muscle excitation-contraction coupling in the thoracic aorta was analysed. Results 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). Conclusions 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 FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Phenotype switching of vascular smooth muscle cells (VSMC) from the contractile type to the synthetic type is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. Inward rectifier K + channel 2.1 (Kir2.1) has been identified in VSMC. However, whether it plays a functional role in regulating cellular transformation remains obscure. In this study, we evaluated the role of Kir2.1 on VSMC proliferation, migration, phenotype switching, and post-injury carotid neointimal formation. Kir2.1 knockdown significantly suppressed platelet-derived growth factor BB-stimulated rat vascular smooth muscle cells (rat-VSMC) proliferation and migration. Deficiency in Kir2.1 contributed to the restoration of smooth muscle α-actin, smooth muscle 22α, and calponin and to a reduction in osteopontin expression in rat-VSMC. Moreover, the in vivo study showed that rat-VSMC switched to proliferative phenotypes and that knockdown of Kir2.1 significantly inhibited neointimal formation after rat carotid injury. Kir2.1 may be a potential therapeutic target in the treatment of cardiovascular diseases, such as atherosclerosis and restenosis following percutaneous coronary intervention. [ABSTRACT FROM AUTHOR]

The perivascular adipose tissue (PVAT) releases a variety of factors that affect vascular function. PVAT in the thoracic aorta shares characteristics with the brown adipose tissue, including a large amount of mitochondria. PVAT-derived factors influence both endothelial and smooth muscle function via several signaling mechanisms including the release/generation of reactive nitrogen and oxygen species. Considering the importance of reactive oxygen species (ROS) on vascular function and that mitochondria are an important source of ROS, we hypothesized that mitochondria-derived ROS in the PVAT modulates vascular reactivity. Vascular reactivity to norephinephrine (NE) was evaluated in thoracic aortic rings, with or without endothelium and/or PVAT, from male Wistar rats. Mitochondrial uncoupling, as well as hydrogen peroxide (H 2 O 2 ) removal, increased the contraction in vessels surrounded by PVAT. PVAT stimulated with NE exhibited increased protein expression, determined by Western blot analysis, of manganese superoxide dismutase (Mn-SOD) and decreased protein expression of catalase. Ultimately, NE increased superoxide anion (O 2 − ) generation in PVAT via increases in intracellular calcium. These results clearly demonstrate that mitochondrial electron transport chain (mETC) in PVAT contributes to modulation of aortic muscle contraction by generating higher amounts of O 2 − that is, in turn, dismutated to hydrogen peroxide, which then acts as a pivotal signaling molecule regulating vascular smooth muscle contraction. [ABSTRACT FROM AUTHOR]

Epithelial–Mesenchymal Transformation (EMT) and the subsequent invasion of epicardial and endocardial cells during cardiac development is critical to the development of the coronary vessels and heart valves. The transformed cells give rise to cardiac fibroblasts and vascular smooth muscle cells or valvular interstitial cells, respectively. The Type III Transforming Growth Factor β (TGFβR3) receptor regulates EMT and cell invasion in both cell types, but the signaling mechanisms downstream of TGFβR3 are not well understood. Here we use epicardial and endocardial cells in in vitro cell invasion assays to identify common mechanisms downstream of TGFβR3 that regulate cell invasion. Inhibition of NF-κB activity blocked cell invasion in epicardial and endocardial cells. NF-κB signaling was found to be dysregulated in Tgfbr3 − / − epicardial cells which also show impaired cell invasion in response to ligand. TGFβR3-dependent cell invasion is also dependent upon Activin Receptor-Like Kinase (ALK) 2, ALK3, and ALK5 activity. A TGFβR3 mutant that contains a threonine to alanine substitution at residue 841 (TGFβR3-T841A) induces ligand-independent cell invasion in both epicardial and endocardial cells in vitro . These findings reveal a role for NF-κB signaling in the regulation of epicardial and endocardial cell invasion and identify a mutation in TGFβR3 which stimulates ligand-independent signaling. [ABSTRACT FROM AUTHOR]

EPH kinases and their ligands, ephrins (EFNs), have vital and diverse biological functions, although their function in blood pressure (BP) control has not been studied in detail. In the present study, we report that Efnb3 gene knockout (KO) led to increased BP in female but not male mice. Vascular smooth muscle cells (VSMCs) were target cells for EFNB3 function in BP regulation. The deletion of EFNB3 augmented contractility of VSMCs from female but not male KO mice, compared with their wild-type (WT) counterparts. Estrogen augmented VSMC contractility while testosterone reduced it in the absence of EFNB3, although these sex hormones had no effect on the contractility of VSMCs from WT mice. The effect of estrogen on KO VSMC contractility was via a nongenomic pathway involving GPER, while that of testosterone was likely via a genomic pathway, according to VSMC contractility assays and GPER knockdown assays. The sex hormone-dependent contraction phenotypes in KO VSMCs were reflected in BP in vivo. Ovariectomy rendered female KO mice normotensive. At the molecular level, EFNB3 KO in VSMCs resulted in reduced myosin light chain kinase phosphorylation, an event enhancing sensitivity to Ca2+ flux in VSMCs. Our investigation has revealed previously unknown EFNB3 functions in BP regulation and show that EFNB3 might be a hypertension risk gene in certain individuals. [ABSTRACT FROM AUTHOR]

Studies conducted in divers indicate that endothelium function is impaired following a dive even without decompression sickness (DCS). Our previous experiment conducted on rat isolated vessels showed no differences in endothelium- dependent vasodilation after a simulated dive even in the presence of DCS, while contractile response to phenylephrine was progressively impaired with increased decompression stress. This study aimed to further investigate the effect of DCS on vascular smooth muscle. Thirty-two male Sprague-Dawley rats were submitted to the same hyperbaric protocol and classified according to the severity of DCS: no-DCS (without clinical symptoms), mild-DCS, or severe-DCS (dead within 1 h). A control group remained at atmospheric pressure. Isometric tension was measured in rings of abdominal aorta and mesenteric arteries. Single dose contraction was assessed with KCl solution. Dose-response curves were obtained with phenylephrine and endothelin- 1. Phenylephrine-induced contraction was observed in the presence of antioxidant tempol. Additionally, plasma concentrations of angiotensin II, angiotensin-converting enzyme, and thiobarbituric acid reactive substances (TBARS) were assessed. Response to phenylephrine was impaired only among mild-DCS in both vessels. Doseresponse curves to endothelin-1 were impaired after mild-DCS in mesenteric and severe-DCS in aorta. KCl-induced contraction was affected after hyperbaric exposure regardless of DCS status in aorta only. These results confirm postdive vascular dysfunction is dependent on the type of vessel. It further evidenced that vascular dysfunction is triggered by DCS rather than by diving itself and suggest the influence of circulating factor/s. Diving-induced impairment of the L-type voltage-dependent Ca2+ channels and/or influence of reninangiotensin system is proposed. [ABSTRACT FROM AUTHOR]

Atherosclerosis is a chronic inflammatory disease associated with increased expression of adhesion molecules in vascular smooth muscle cells (VSMCs). Matrine is a main active ingredient of Sophora flavescens r oots, which a re used to treat inflammatory diseases. However, the effects of matrine on the expression of adhesion molecules in VSMCs have largely remained elusive. Therefore, the present study investigated the effects of matrine on the expression of adhesion molecules in tumor necrosis factor (TNF)-α-stimulated human aortic smooth muscle cells (HASMCs). The results showed that matrine inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in TNF-α-stimulated HASMCs. Matrine markedly inhibited the TNF-α-induced expression of nuclear factor (NF)-κB p65 and prevented the TNF-α-caused degradation of inhibitor of NF-κB; it also inhibited TNF-α-induced activation of mitogen-activated protein kinases (MAPKs). Furthermore, matrine inhibited the production of intracellular reactive oxygen species (ROS) in TNF-α-stimulated HASMCs. In conclusion, the results of the present study demonstrated that matrine inhibited the expression of VCAM-1 and ICAM-1 in TNF-α-stimulated HASMCs via the suppression of ROS production as well as NF-κB and MAPK pathway activation. Therefore, matrine may have a potential therapeutic use for preventing the advancement of atherosclerotic lesions. [ABSTRACT FROM AUTHOR]

Background and Purpose: We investigated the hypothesis that elevated glucose increases contractile responses in vascular smooth muscle and that this enhanced constriction occurs due to the glucose-induced PKC-dependent inhibition of voltage-gated potassium channels.Experimental Approach: Patch-clamp electrophysiology in rat isolated mesenteric arterial myocytes was performed to investigate the glucose-induced inhibition of voltage-gated potassium (Kv ) current. To determine the effects of glucose in whole vessel, wire myography was performed in rat mesenteric, porcine coronary and human internal mammary arteries.Key Results: Glucose-induced inhibition of Kv was PKC-dependent and could be pharmacologically dissected using PKC isoenzyme-specific inhibitors to reveal a PKCβ-dependent component of Kv inhibition dominating between 0 and 10 mM glucose with an additional PKCα-dependent component becoming evident at concentrations greater than 10 mM. These findings were supported using wire myography in all artery types used, where contractile responses to vessel depolarization and vasoconstrictors were enhanced by increasing bathing glucose concentration, again with evidence for distinct and complementary PKCα/PKCβ-mediated components.Conclusions and Implications: Our results provide compelling evidence that glucose-induced PKCα/PKCβ-mediated inhibition of Kv current in vascular smooth muscle causes an enhanced constrictor response. Inhibition of Kv current causes a significant depolarization of vascular myocytes leading to marked vasoconstriction. The PKC dependence of this enhanced constrictor response may present a potential therapeutic target for improving microvascular perfusion following percutaneous coronary intervention after myocardial infarction in hyperglycaemic patients. [ABSTRACT FROM AUTHOR]

Introduction: Smooth muscle cell contraction is an essential function of arteries and relies on the integrity of the actin-myosin apparatus. The tissue-specific α2-smooth muscle actin, encoded by ACTA2, is predominantly expressed in vascular smooth muscle cells. ACTA2 mutations predispose to development of aortic aneurysms and early onset coronary and cerebrovascular disease. Based on arteriographic findings, a distinct cerebrovascular disease has been proposed for ACTA2 heterozygous patients carrying the R179H mutation. Results: We present the first integrated analysis of a severely compromised patient with the R179H mutation and define the arterial pathology of ACTA2-related cerebrovascular disease. Histologically, striking morphological abnormalities were present in cerebral arteries of all sizes. Massive intimal smooth muscle cell proliferation, fragmentation of the elastic laminae and medial fibromuscular proliferation characterized large arteries whereas prominent vessel wall thickening, fibrosis and smooth muscle cell proliferation were unique changes in small arteries. The medial fibrosis and smooth muscle cell proliferation explain the characteristic radiologic appearance of “straight arteries” and suggest impaired function of mutant smooth muscle cells. Actin three-dimensional molecular modeling revealed critical positioning of R179 at the interface between the two strands of filamentous actin and destabilization of inter-strand bundling by the R179H mutation, explaining the severe associated phenotype. Conclusions: In conclusion, these characteristic clinical and pathologic findings confirm ACTA2-related cerebrovascular disease as a new cerebrovascular disorder for which new therapeutic strategies need to be designed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aim:Tanshinone II-A sodium sulfonate (DS-201), a water-soluble derivative of Tanshinone II-A, has been found to induce vascular relaxation and activate BKCa channels. The aim of this study was to explore the mechanisms underlying the action of DS-201 on BKCa channels.Methods:Human BKCa channels containing α subunit alone or α plus β1 subunits were expressed in HEK293 cells. BKCa currents were recorded from the cells using patch-clamp technique. The expression and trafficking of BKCa subunits in HEK293 cells or vascular smooth muscle cells (VSMCs) were detected by Western blotting, flow cytometry and confocal microscopy.Results:DS-201 (40-160 μmol/L) concentration-dependently increased the total open probability of BKCa channels in HEK293 cells, associated with enhancements of Ca2+ and voltage dependence as well as a delay in deactivation. Coexpression of β1 subunit did not affect the action of DS-201: the values of EC50 for BKCa channels containing α subunit alone and α plus β1 subunit were 66.6±1.5 and 62.0±1.1 μmol/L, respectively. In both HEK293 cells and VSMCs, DS-201 (80 μmol/L) markedly increased the expression of α subunit without affecting β1 subunit. In HEK293 cells, DS-201 enriched the membranous level of α subunit, likely by accelerating the trafficking and suppressing the internalization of α subunit. In both HEK293 cells and VSMCs, DS-201 (≥320 μmol/L) induced significant cytotoxicity.Conclusion:DS-201 selectively targets the pore-forming α subunit of human BKCa channels, thus enhancing the channel activities and increasing the subunit expression and trafficking, whereas the β1 subunit does not contribute to the action of DS-201. [ABSTRACT FROM AUTHOR]

Background and Purpose Calcium-activated chloride channels ( CaCCs) are key depolarizing mechanisms that have an important role in vascular smooth muscle contraction. Here, we investigated whether these channels are regulated by phosphatidylinositol (4,5) bisphosphate [ P(4,5) P2], a known regulator of various ion channels. Experimental Approach Calcium-activated Cl− currents ( IClCa) were recorded by patch clamp electrophysiology of rat isolated pulmonary artery smooth muscle cells. TMEM16A protein-phosphoinositide interaction was studied by co-immunoprecipitation and phosphoinositide binding arrays on protein lysates from whole pulmonary arteries and HEK293 cells overexpressing TMEM16A, the molecular correlate. Key Results PI(4,5) P2 and other phospholipids were shown to bind directly to TMEM16A isolated from whole pulmonary artery ( PA) and TMEM16A-eGFP expressed in HEK293 cells. Agents that reduced PI(4,5) P2 levels through different routes [PL C activation, PI4K inhibition, PI(4,5) P2 scavenging and absorption] all increased IClCa evoked by solutions containing clamped-free [ Ca2+], whereas enrichment of activating solutions with PI(4,5) P2 inhibited IClca in PA smooth muscle cells with approximately 50% reduction at 1 μM. Conclusions and Implications These data are the first to show a negative regulation of TMEM16A-encoded CaCCs by PI(4,5) P2 and propose that control of PI(4,5) P2 levels is a key determinant of arterial physiology. [ABSTRACT FROM AUTHOR]

The most prevalent cardiovascular diseases arise from alterations in vascular smooth muscle cell (VSMC) morphology and function. Tetraspanin CD9 has been previously implicated in regulating vascular pathologies; however, insight into how CD9 may regulate adverse VSMC phenotypes has not been provided. We utilized a human model of aortic smooth muscle cells to understand the consequences of CD9 deficiency on VSMC phenotypes. Upon knocking down CD9, the cells developed an abnormally small and rounded morphology. We determined that this morphological change was due to a lack of typical parallel actin arrangement. We also found similar total RhoA but decreased GTP-bound (active) RhoA levels in CD9 deficient cells. As a result, cells lacking a full complement of CD9 were less contractile than their control treated counterparts. Upon restoration of RhoA activity in the CD9 deficient cells, the phenotype was reversed and cell contraction was restored. Conversely, inhibition of RhoA activity in the control cells mimicked the CD9-deficient cell phenotype. Thus, alteration in CD9 expression was sufficient to profoundly disrupt cellular actin arrangement and endogenous cell contraction by interfering with RhoA signaling. This study provides insight into how CD9 may regulate previously described vascular smooth muscle cell pathophysiology. [ABSTRACT FROM AUTHOR]

Background: Phosphodiesterases (PDEs) cleave phosphodiester bonds in cyclic nucleotides and play diverse roles in cell biology. PDE5A is a cytoplasmic phosphodiesterase which specifically degrades cyclic guanosine monophosphate (cGMP), a cell signaling molecule that plays important roles in neuronal signaling and vascular smooth muscle contraction. Inhibition of PDE5A induces headache resembling migraine headaches. Aim: To test the hypothesis that Ser102 and Ser104 in PDE5A and/or their phosphoserine derivatives 1) regulate the intracellular localization and/or activity of PDE5A, and 2) modulate the interaction between PDE5A and pharmaceutical reagents in clinical or pre-clinical use for migraine headaches and other types of vascular dysfunction. Methods: Wild type PDE5A or PDE5A with substitution mutations (Ser102Ala, Ser104Ala or Ser102Ala/Ser104Ala) were overexpressed in SK-N-AS neuroblastoma cells as C-terminal fusions with green fluorescent protein. Transfected cells were treated with sildenafil, cilostazol, glyceryl trinitrate, calcitonin gene-related peptide (CGRP) or sumatriptan. PDE5A-GFP fusion proteins were localized in fixed cells by immunofluorescence and PDE activity was quantified in cell extracts by standard in vitro assay using [3H] cGMP. Results: The intracellular distribution of wild-type, single and double mutant PDE5A was similar and was not altered by exposure to sildenafil, cilostazol, glyceryl trinitrate, calcitonin gene-related peptide (CGRP) or sumatriptan. PDE5 activity was similar for wild type, Ser102Ala and Ser104Ala PDE5A, but activity of the Ser102Ala/Ser104Ala mutant was approximately two-fold higher than wild type. Double mutant Ser102Ala/Ser104Ala migrated as a single band on a native acrylamide gel, while wild-type and single mutant PDE5A migrated as a doublet. Interpretation: Ser102 and Ser104 may influence the conformational flexibility of PDE5A, which may in turn influence phosphorylation status, allosteric regulation by cGMP or other as yet unknown regulatory mechanisms for PDE5A. PDE5A activation could be important in reversal of migraine-like headache. [ABSTRACT FROM AUTHOR]

Cardiovascular homeostasis and health is maintained through the balanced interactions of cardiac generated blood flow and cross-talk between the cellular components that comprise blood vessels. Central to this cross-talk is endothelial generated nitric oxide (NO) that stimulates relaxation of the contractile vascular smooth muscle (VSMC) layer of blood vessels. In cardiovascular disease this balanced interaction is disrupted and NO signaling is lost. Work over the last several years indicates that regulation of NO is much more complex than previously believed. It is now apparent that the secreted protein thrombospondin-1 (TSP1), that is upregulated in cardiovascular disease and animal models of the same, on activating cell surface receptor CD47, redundantly inhibits NO production and NO signaling. This inhibitory event has implications for baseline and disease-related responses mediated by NO. Further work has identified that TSP1-CD47 signaling stimulates enzymatic reactive oxygen species (ROS) production to further limit blood flow and promote vascular disease. Herein consideration is given to the most recent discoveries in this regard which identify the TSP1-CD47 axis as a major proximate governor of cardiovascular health. [ABSTRACT FROM AUTHOR]

Following the dispersal out of Africa, where hominins evolved in warm environments for millions of years, our species has colonised different climate zones of the world, including high latitudes and cold environments. The extent to which human habitation in (sub-)Arctic regions has been enabled by cultural buffering, short-term acclimatization and genetic adaptations is not clearly understood. Present day indigenous populations of Siberia show a number of phenotypic features, such as increased basal metabolic rate, low serum lipid levels and increased blood pressure that have been attributed to adaptation to the extreme cold climate. In this study we introduce a dataset of 200 individuals from ten indigenous Siberian populations that were genotyped for 730,525 SNPs across the genome to identify genes and non-coding regions that have undergone unusually rapid allele frequency and long-range haplotype homozygosity change in the recent past. At least three distinct population clusters could be identified among the Siberians, each of which showed a number of unique signals of selection. A region on chromosome 11 (chr11:66–69 Mb) contained the largest amount of clustering of significant signals and also the strongest signals in all the different selection tests performed. We present a list of candidate cold adaption genes that showed significant signals of positive selection with our strongest signals associated with genes involved in energy regulation and metabolism (CPT1A, LRP5, THADA) and vascular smooth muscle contraction (PRKG1). By employing a new method that paints phased chromosome chunks by their ancestry we distinguish local Siberian-specific long-range haplotype signals from those introduced by admixture. [ABSTRACT FROM AUTHOR]

Vascular smooth muscle contraction and the myogenic response regulate blood flow in the resistance vascular and contribute to systemic blood pressure. Three pathways are currently known to contribute to the development of the myogenic response: (i) Ca2+-dependent phosphorylation of LC20; (ii) Ca2+ sensitization through inhibition of myosin phosphatase; and (iii) cortical actin polymerization. A number of regulatory smooth muscle proteins are integrated with these pathways to fine tune the response and facilitate adaptations to vascular (patho)physiologies. Of particular interest is the SMTN family of proteins, consisting of SMTN-A, SMTN-B, and the SMTN-like protein, SMTNL1. The SMTN-B and SMTNL1 proteins are both implicated in regulating smooth muscle contractility and contributing to vascular adaptations associated with hypertension, pregnancy, and exercise training. In the case of SMTNL1, the protein plays multiple roles in regulating contraction through functional interactions with contractile regulators as well as transcriptional control of the contractile phenotype and Ca2+-sensitizing capacity. For the first time, preliminary results suggest SMTNL1 is involved in the myogenic response of the cerebral resistance vasculature. In this regard, global SMTNL1 deletion is associated with greater myogenic reactivity of cerebral arterioles, although the precise mechanism accounting for this finding remains to be defined. [ABSTRACT FROM AUTHOR]

The contractility status of trabecular meshwork (TM) cells influences aqueous humor outflow resistance and intraocular pressure. Using human TM cells as a model, the goal of the present study was to examine concentration–response relationships of two prototypical molecules, nitric oxide (NO) and endothelin-1 (ET-1), known to differentially influence vascular smooth muscle contractility. Efficacy of ET-1, two NO donors (DETA-NO and SNP) and a cGMP analog (8-Br-cGMP) were assessed using two complementary methods: functionally in a gel contraction assay and biochemically using a myosin light chain phosphorylation assay. The NO donors DETA-NO and SNP dose dependently relaxed cultured human TM cells (EC50 for DETA-NO = 6.0 ± 2.4 μM, SNP = 12.6 ± 8.8 μM), with maximum effects at 100 μM. Interestingly, at concentrations of NO donors above 100 μM, the relaxing effect was lost. Relaxation caused by DETA-NO (100 μM) was dose dependently blocked by the soluble guanylate cyclase specific inhibitor ODQ (IC50 = 460 ± 190 nM). In contrast to the NO donors, treatment of cells with the cGMP analog, 8-Br-cGMP produced the largest relaxation (109.4%) that persisted at high concentrations (EC50 = 110 ± 40 μM). ET-1 caused a dose-dependent contraction of human TM cells (EC50 = 1.5 ± 0.5 pM), with maximum effect at 100 pM (56.1%) and this contraction was reversed by DETA-NO (100 μM). Consistent with functional data, phosphorylation status of myosin light chain was dose dependently reduced with DETA-NO, and increased with ET-1. Together, data show that TM cells rapidly change their contractility status over a wide dynamic range, well suited for the regulation of outflow resistance and intraocular pressure. [ABSTRACT FROM AUTHOR]

Background--Phenotypic modulation or switching of vascular smooth muscle cells from a contractile/quiescent to a proliferative/synthetic phenotype plays a key role in vascular proliferative disorders such as atherosclerosis and restenosis. Although several calcium handling proteins that control differentiation of smooth muscle cells have been identified, the role of protein phosphatase inhibitor 1 (I-1) in the acquisition or maintenance of the contractile phenotype modulation remains unknown. Methods and Results--In human coronary arteries, I-1 and sarco/endoplasmic reticulum Ca2+-ATPase expression is specific to contractile vascular smooth muscle cells. In synthetic cultured human coronary artery smooth muscle cells, protein phosphatase inhibitor 1 (I-1 target) is highly expressed, leading to a decrease in phospholamban phosphorylation, sarco/endoplasmic reticulum Ca2+-ATPase, and cAMP-responsive element binding activity. I-1 knockout mice lack phospholamban phosphorylation and exhibit vascular smooth muscle cell arrest in the synthetic state with excessive neointimal proliferation after carotid injury, as well as significant modifications of contractile properties and relaxant response to acetylcholine of femoral artery in vivo. Constitutively active I-1 gene transfer decreased neointimal formation in an angioplasty rat model by preventing vascular smooth muscle cell contractile to synthetic phenotype change. Conclusions--I-1 and sarco/endoplasmic reticulum Ca2+-ATPase synergistically induce the vascular smooth muscle cell contractile phenotype. Gene transfer of constitutively active I-1 is a promising therapeutic strategy for preventing vascular proliferative disorders. [ABSTRACT FROM AUTHOR]

Pre-existing diabetes increases the risk of maternal and fetal complications during pregnancy, which may be due to underlying maternal vascular dysfunction and impaired blood supply to the uteroplacental unit. Endothelial dysfunction and reduced vascular smooth muscle responsiveness to nitric oxide (NO) are common vascular impairments in type 2 diabetes (T2D). We hypothesized that uterine arteries from diabetic rats would have reduced vascular smooth muscle sensitivity to NO compared with nondiabetic rats due to impairment in the NO/soluble guanylate cyclase (sGC)/cGMP signaling pathway. Uterine arteries from pregnant Goto-Kakizaki (GK; model of T2D) and Wistar (nondiabetic) rats were studied in a wire myograph. GK nonpregnant uterine arteries had reduced responses to ACh and sodium nitroprusside (SNP) but increased responses to propylamine propylamine NONOate and greater sensitivity to sildenafil compared with Wistar nonpregnant arteries. In late pregnancy, Wistar rats had reduced uterine vascular smooth muscle responsiveness to SNP, but GK rats failed to show this adaptation and had reduced expression of sGC compared with the nonpregnant state. GK rats had a smaller litter size (13.9 ± 0.48 vs. 9.8 ± 0.75; P < 0.05) and a greater number of resorptions compared with Wistar controls (0.8 ± 0.76% vs. 19.9 ± 6.06%; P < 0.05). These results suggest that uterine arteries from rats with T2D show reduced sensitivity of uterine vascular smooth muscle sGC to NO. During pregnancy, the GK uterine vascular smooth muscle fails to show relaxation responses similar to those of arteries from nondiabetic rats. [ABSTRACT FROM AUTHOR]

Objective: The sphingosylphosphorylcholine-Rho-kinase pathway plays an important role in Ca2+ sensitization of vascular smooth muscle contraction. Eicosapentaenoic acid (EPA) inhibits sphingosylphosphorylcholine -Rho-kinase−activated Ca2+-sensitization in vitro and in subarachnoid hemorrhage (SAH) models in vivo and has also been shown to inhibit the occurrence of cerebral vasospasm (CIV) after the onset of SAH in a prospective, nonrandomized study. The current prospective, multicenter, randomized study was performed to confirm the preventive effects of EPA on CIV in patients with SAH. Methods: The trial population comprised 162 patients who underwent surgical clipping within 72 hours of the onset of SAH. Of these patients, 81 received 2700 mg/day EPA from the day after surgery until day 30 (EPA group), and 81 did not receive EPA (control group). The primary end point was the occurrence of symptomatic vasospasm (SV) or cerebral infarction caused by CIV. Results: The occurrences of SV (15% vs. 30%; P = 0.022) and CIV (7% vs. 21%; P = 0.012) were lower in the EPA group. Multivariate analysis revealed an adjusted odds ratio of 0.39 (95% confidence interval, 0.17–0.89; P = 0.028) for SV inhibition by EPA and 0.27 (95% confidence interval, 0.09–0.72; P = 0.012) for CIV inhibition. Conclusions: These results indicate that oral EPA reduces the frequency of SV and CIV after the onset of aneurysmal SAH. [ABSTRACT FROM AUTHOR]

The second messenger cAMP plays a central role in mediating vascular smooth muscle relaxation in response to vasoactive transmitters and in strengthening endothelial cell-cell junctions that regulate the movement of solutes, cells and macromolecules between the blood and the surrounding tissue. The vasculature expresses three cAMP effector proteins: PKA (protein kinase A), CNG (cyclic-nucleotide-gated) ion channels, and the most recently discovered Epacs (exchange proteins directly activated by cAMP). Epacs are a family of GEFs (guanine-nucleotide-exchange factors) for the small Ras-related GTPases Rap1 and Rap2, and are being increasingly implicated as important mediators of cAMP signalling, both in their own right and in parallel with the prototypical cAMP target PKA. In the present paper, we review what is currently known about the role of Epac within blood vessels, particularly with regard to the regulation of vascular tone, endothelial barrier function and inflammation. [ABSTRACT FROM AUTHOR]