Vascular Smooth Muscle Cell Contraction Research Articles

Arginase II activity regulates cytosolic Ca2+ level in a p32-dependent manner that contributes to Ca2+-dependent vasoconstriction in native low-density lipoprotein-stimulated vascular smooth muscle cells

Although arginase II (ArgII) is abundant in mitochondria, Ca2+-accumulating organelles, the relationship between ArgII activity and Ca2+ translocation into mitochondria and the regulation of cytosolic Ca2+ signaling are completely unknown. We investigated the effects of ArgII activity on mitochondrial Ca2+ uptake through mitochondrial p32 protein (p32m) and on CaMKII-dependent vascular smooth muscle cell (VSMC) contraction. Native low-density lipoprotein stimulation induced an increase in [Ca2+]m as measured by CoCl2-quenched calcein-AM fluorescence, which was prevented by Arg inhibition in hAoSMCs and reduced in mAoSMCs from ArgII−/− mice. Conversely, [Ca2+]c analyzed with Fluo-4 AM was increased by Arg inhibition and ArgII gene knockout. The increased [Ca2+]c resulted in CaMKII and MLC 20 phosphorylation, which was associated with enhanced vasoconstriction activity to phenylephrine (PE) in the vascular tension assay. Cy5-tagged siRNA against mitochondrial p32 mRNA (sip32m) abolished mitochondrial Ca2+ uptake and induced activation of CaMKII. Spermine, a polyamine, induced mitochondrial Ca2+ uptake and dephosphorylation of CaMKII and was completely inhibited by sip32m incubation. In mAoSMCs from ApoE-null mice fed a high-cholesterol diet (ApoE−/− +HCD), Arg activity was increased, and spermine concentration was higher than that of wild-type mice. Furthermore, [Ca2+]m and p32m levels were elevated, and CaMKII phosphorylation was reduced in mAoSMCs from ApoE−/− +HCD. In vascular tension experiments, an attenuated response to vasoconstrictors in de-endothelialized aorta from ApoE−/− +HCD was recovered by incubation of sip32m. ArgII activity-dependent production of spermine augments Ca2+ transition from the cytosol to the mitochondria in a p32m-dependent manner and regulates CaMKII-dependent constriction in VSMCs.

Update on Clinical Trials of Losartan With and Without β-Blockers to Block Aneurysm Growth in Patients With Marfan Syndrome

ImportanceThoracic aortic aneurysms leading to acute aortic dissections are a major cause of morbidity and mortality despite significant advances in surgical treatment, which remains the main intervention to prevent type A dissections. In the past 2 decades progress has been made toward a better understanding of molecular mechanisms that lead to aneurysm formation and dissections of the thoracic aorta. This focused review emphasizes the results of clinical trials using β-blocker, losartan potassium, and irbesartan in patients with Marfan syndrome and comments briefly on mechanisms of aortic remodeling, including fibrosis and transforming growth factor β signaling.ObservationThe major risk factors for the disease are increased hemodynamic forces, typically owing to poorly controlled hypertension, and heritable genetic variants. The altered genes predisposing to thoracic aortic disease have been shown or are predicted to decrease vascular smooth muscle cell contraction, decrease transforming growth factor β signaling, or alter the extracellular matrix. Preclinical models of Marfan syndrome showed promising results for losartan as a potential therapy to attenuate aortic dilation in mice. However, several clinical trials did not conclusively confirm that losartan attenuated aortic aneurysm expansion better than β-blockers. Most importantly, clinical trials assessing whether losartan therapy not only reduces aortic growth but also improves adverse aortic outcomes, including dissection, need for surgery, and death, have not been conducted. The largest trial to date to our knowledge, the Pediatric Heart Network trial, sponsored by the National Heart, Lung, and Blood Institute, showed a nonsignificant increase in adverse aortic outcomes, with almost a doubling of adverse events in patients randomized to losartan treatment compared with β-blockers, suggesting that this study was underpowered to assess adverse aortic outcomes. On the other hand, the evidence for β-blocker therapy to reduce morbidity and mortality in Marfan syndrome is limited to a single small, prospective randomized and nonblinded clinical trial.Conclusions and RelevanceTaken together, these data emphasize the need for clinical trials adequately powered to assess both aortic aneurysm growth and adverse aortic outcomes to identify effective medical therapies for Marfan syndrome and other aortopathies.

MiR-204 regulates type 1 IP3R to control vascular smooth muscle cell contractility and blood pressure

MiR-204 is expressed in vascular smooth muscle cells (VSMC). However, its role in VSMC contraction is not known. We determined if miR-204 controls VSMC contractility and blood pressure through regulation of sarcoplasmic reticulum (SR) calcium (Ca2+) release. Systolic blood pressure (SBP) and vasoreactivity to VSMC contractile agonists (phenylephrine (PE), thromboxane analogue (U46619), endothelin-1 (ET-1), angiotensin-II (Ang II) and norepinephrine (NE) were compared in aortas and mesenteric resistance arteries (MRA) from miR-204−/− mice and wildtype mice (WT). There was no difference in basal systolic blood pressure (SBP) between the two genotypes; however, hypertensive response to Ang II was significantly greater in miR-204−/− mice compared to WT mice. Aortas and MRA of miR-204−/− mice had heightened contractility to all VSMC agonists. In silico algorithms predicted the type 1 Inositol 1, 4, 5-trisphosphate receptor (IP3R1) as a target of miR-204. Aortas and MRA of miR-204−/− mice had higher expression of IP3R1 compared to WT mice. Difference in agonist-induced vasoconstriction between miR-204−/− and WT mice was abolished with pharmacologic inhibition of IP3R1. Furthermore, Ang II-induced aortic IP3R1 was greater in miR-204−/− mice compared to WT mice. In addition, difference in aortic vasoconstriction to VSMC agonists between miR-204−/− and WT mice persisted after Ang II infusion. Inhibition of miR-204 in VSMC in vitro increased IP3R1, and boosted SR Ca2+ release in response to PE, while overexpression of miR-204 downregulated IP3R1. Finally, a sequence-specific nucleotide blocker that targets the miR-204-IP3R1 interaction rescued miR-204-induced downregulation of IP3R1. We conclude that miR-204 controls VSMC contractility and blood pressure through IP3R1-dependent regulation of SR calcium release.

α1D-adrenoceptor involves the relaxation effect of farrerol in rat aortic vascular smooth muscle cells

The aim of this study was to investigate the relaxation effect of farrerol on rat aortic vascular smooth muscle cells (VSMCs) and its underlying mechanism. VSMCs were cultured primarily and were used to examine the relaxation effect of farrerol. Cells surface and length were measured by dynamic observation, or by rhodamine-phalloidin labeling and hematoxylin-eosin staining. Cells contractive activity were tested using collagen gel contraction assay. The [Ca2+]in was measured with molecular probe fluo-4-AM. The mRNA and protein expression of regulatory proteins for contraction were measured. In addition, rat aortic VSMCs were transfected with lentivirus-mediated α1D-adrenoceptor gene-shRNA, then the effect of farrerol were detected by the above experimental methods. The results revealed that 10 μΜ AngⅡ promoted cell contraction, increased [Ca2+]in and enhanced collagen contraction in rat aortic VSMCs. 10 μΜ AngⅡ not only increased expression of myosin light chain kinase (MLCK) and smooth muscle protein 22α (SM22α), but also increased phosphorylation level of myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1). The above effects induced by AngⅡ could be significantly inhibited by farrerol in a concentration dependent manner. When the cells were transfected with lentivirus mediated α1D-adrenoceptor gene-shRNA, the effects of farrerol on changes induced by AngⅡ in rat aortic VSMCs were markedly reversed. In conclusion, farrerol could produce relaxtion effect in rat aortic VSMCs precontracted by 10 μΜ AngⅡ, which was involved in downregulation expression of MLCK and SM22α, and inhibition phosphorylation level of MYPT1 and MLC via activating α1D-adrenoceptor gene.

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

Over the past few decades, isometric contraction studies of isolated thoracic aorta segments have significantly contributed to our overall understanding of the active, contractile properties of aortic vascular smooth muscle cells (VSMCs) and their cross‐talk with endothelial cells. However, the physiological role of VSMC contraction or relaxation in the healthy aorta and its contribution to the pulse‐smoothening capacity of the aorta is currently unclear. Therefore, we investigated the acute effects of VSMC contraction and relaxation on the isobaric biomechanical properties of healthy mouse aorta. An in‐house developed set‐up was used to measure isobaric stiffness parameters of periodically stretched (10 Hz) aortic segments at an extended pressure range, while pharmacologically modulating VSMC tone and endothelial cell function. We found that the effects of α1‐adrenergic stimulation with phenylephrine on the pressure‐stiffness relationship varied in sensitivity, magnitude and direction, with the basal, unstimulated NO production by the endothelium playing a pivotal role. We also investigated how arterial disease affected this system by using the angiotensin‐II‐treated mouse. Our results show that isobaric stiffness was increased and that the aortic segments demonstrated a reduced capacity for modulating the pressure‐stiffness relationship. This suggests that not only increased isobaric stiffness at normal pressure, but also a reduced capacity of the VSMCs to limit the pressure‐associated increase in aortic stiffness, may contribute to the pathogenesis of this mouse model. Overall, this study provides more insight in how aortic VSMC tone affects the pressure‐dependency of aortic biomechanics at different physiological and pathological conditions.

The place of ARBs in heart failure therapy: is aldosterone suppression the key?

Since the launch of the first orally available angiotensin II (AngII) type 1 receptor (AT1R) blocker (ARB) losartan (Cozaar) in the late 1990s, the class of ARBs (or ‘sartans’, short for Angiotensin-RecepTor-ANtagonistS) quickly expanded to include candesartan, eprosartan, irbesartan, valsartan, telmisartan, and olmesartan. All ARBs have high affinity for the AT1 receptor, expressed in various tissues, including smooth muscle cells, heart, kidney, and brain. Since activation of AT1R, the target of these drugs, leads, among other effects, to vascular smooth muscle cell growth, proliferation and contraction, activation of fibroblasts, cardiac hypertrophy, aldosterone secretion from the adrenal cortex, thirst-fluid intake (hypervolemia), etc., the ARBs are nowadays one of the most useful cardiovascular drug classes used in clinical practice. However, significant differences in their pharmacological and clinical properties exist that may favor use of particular agents over others within the class, and, in fact, two of these drugs, candesartan and valsartan, continuously appear to distinguish themselves from the rest of the ‘pack’ in recent clinical trials. The reason(s) for the potential superiority of these two agents within the ARB class are currently unclear but under intense investigation. The present short review gives an overview of the clinical properties of the ARBs currently approved by the United States Food and Drug Administration, with a particular focus on candesartan and valsartan and the areas where these two drugs seem to have a therapeutic edge. In the second part of our review, we outline recent data from our laboratory (mainly) on the molecular effects of the ARB drugs on aldosterone production and on circulating aldosterone levels, which may underlie (at least in part) the apparent clinical superiority of candesartan (and valsartan) over most other ARBs currently in clinical use.

A Calcium Mediated Mechanism Coordinating Vascular Smooth Muscle Cell Adhesion During KCl Activation.

Efficient mechanotransduction in vascular smooth muscle cells (VSMCs) is intimately coupled to physical coupling of the cell to extracellular matrix proteins (ECM) by integrins. Integrin adhesion receptors are essential for normal vascular function and defective integrin signaling is associated with cardiovascular disease. However, less is known about the mechanism of integrin activation in VSMCs in relation to vasoregulation. Our laboratory previously demonstrated that the vasoconstrictor Angiotensin II increases VSMC stiffness in concert with enhanced adhesion to fibronectin (FN), indicating an important role for adhesion in contraction. However, the mechanism of this coordination remains to be clarified. In this study, intracellular Ca2+ ([Ca2+]i) was hypothesized to link integrin activation through inside-out signaling pathways leading to enhanced adhesion in response to AII. By using atomic force microscopy (AFM) with an anti-α5 antibody coated AFM probe, we confirmed that cell stiffness was increased by AII, while we observed no change in adhesion to an α5 integrin antibody. This indicated that increases in cell adhesion to FN induced by AII were occurring through an integrin activation process, as increased membrane integrin expression/receptor density would have been accompanied by increased adhesion to the anti-α5 antibody. Further studies were performed using either KCl or BAPTA-AM to modulate the level of [Ca2+]i. After KCl, VSMCs showed a rapid transient increase in cell stiffness as well as cell adhesion to FN, and these two events were synchronized with superimposed transient increases in the level of [Ca2+]i, which was measured using the Ca2+ indicator, fluo-4. These relationships were unaffected in VSMCs pretreated with the myosin light chain kinase inhibitor, ML-7. In contrast, unstimulated VSMCs incubated with an intracellular calcium chelator, BAPTA-AM, showed reduced cell adhesion to FN as well the expected decrease in [Ca2+]i. These data suggest that in VSMCs, integrin activation is linked to signaling events tied to levels of [Ca2+]i while being less dependent on events at the level of contractile protein activation. These findings provide additional evidence to support a role for adhesion in VSMC contraction and suggest that following cell contractile activation, that adhesion may be regulated in tandem with the contractile event.

Vascular smooth muscle cell contractile function and mechanotransduction

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the arterial wall and normally adopt a quiescent, contractile phenotype to regulate vascular tone. In the arterial wall, VSMCs are exposed to multiple mechanical cues, including stretch and matrix stiffness, which regulate VSMC contraction. However, during ageing and in vascular disease, such as atherosclerosis, hypertension and vascular calcification, the arterial wall stiffens and VSMC contraction contributes to this process. VSMCs display remarkable plasticity and changes in their mechanical environment promote VSMCs to adopt a proliferative, synthetic phenotype. VSMC phenotypic modulation is associated with altered expression of contractile proteins that generate actomyosin-based force. However, our understanding of precise mechanisms whereby altered mechanical landscape and mechanotransduction influence VSMC contraction remains limited. In this review, we discuss the present literature describing how VSMCs sense and respond to changes in their mechanical environment and how these changes influence VSMC contraction.

LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Regulates Smooth Muscle Contractility by Modulating Ca2+ Signaling and Expression of Cytoskeleton-Related Proteins.

Objective- Mutations affecting contractile-related proteins in the ECM (extracellular matrix), microfibrils, or vascular smooth muscle cells can predispose the aorta to aneurysms. We reported previously that the LRP1 (low-density lipoprotein receptor-related protein 1) maintains vessel wall integrity, and smLRP1-/- mice exhibited aortic dilatation. The current study focused on defining the mechanisms by which LRP1 regulates vessel wall function and integrity. Approach and Results- Isometric contraction assays demonstrated that vasoreactivity of LRP1-deficient aortic rings was significantly attenuated when stimulated with vasoconstrictors, including phenylephrine, thromboxane receptor agonist U-46619, increased potassium, and L-type Ca2+ channel ligand FPL-64176. Quantitative proteomics revealed proteins involved in actin polymerization and contraction were significantly downregulated in aortas of smLRP1-/- mice. However, studies with calyculin A indicated that although aortic muscle from smLRP1-/- mice can contract in response to calyculin A, a role for LRP1 in regulating the contractile machinery is not revealed. Furthermore, intracellular calcium imaging experiments identified defects in calcium release in response to a RyR (ryanodine receptor) agonist in smLRP1-/- aortic rings and cultured vascular smooth muscle cells. Conclusions- These results identify a critical role for LRP1 in modulating vascular smooth muscle cell contraction by regulating calcium signaling events that potentially protect against aneurysm development.

Contribution of L-type Ca2+ channel-sarcoplasmic reticulum coupling to depolarization-induced arterial contraction in spontaneously hypertensive rats

Evidence has shown that vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs) are depolarized and that the expression of L-type Ca2+ channels (LTCCs) and the sarcoplasmic reticulum (SR) Ca2+ buffering system are upregulated. Arterial rings exposed to high K+ solutions develop a contraction with two components, namely, an initial or phasic component and a sustained or tonic component. Because LTCCs and SR have different functions in the phasic and tonic components of depolarization-induced contraction, this study investigated the role of LTCC-SR coupling in depolarized arterial rings of SHRs. In the absence of extracellular Ca2+, high external K+ or LTCC agonists elicited a transitory contraction, which was sensitive to nifedipine and was potentiated in SHRs. In the presence of extracellular Ca2+, cyclopiazonic acid (CPA), an SR Ca2+-ATPase (SERCA) inhibitor, evoked a transient contraction that was significantly increased in SHRs. Although the phasic and tonic components were markedly increased in depolarized arterial rings of SHRs, they showed different voltage-dependence and sensitivity to SERCA inhibition. The tonic component was more sensitive to moderate depolarizations, and CPA selectively reduced the tonic component to the level observed in WKY rats. These results suggested that LTCC-SR coupling is potentiated in the sustained contraction of hypertensive VSMCs.

Inositol 1,4,5-Trisphosphate Receptors in Hypertension.

Chronic hypertension remains a major cause of global mortality and morbidity. It is a complex disease that is the clinical manifestation of multiple genetic, environmental, nutritional, hormonal, and aging-related disorders. Evidence supports a role for vascular aging in the development of hypertension involving an impairment in endothelial function together with an alteration in vascular smooth muscle cells (VSMCs) calcium homeostasis leading to increased myogenic tone. Changes in free intracellular calcium levels ([Ca2+]i) are mediated either by the influx of Ca2+ from the extracellular space or release of Ca2+ from intracellular stores, mainly the sarcoplasmic reticulum (SR). The influx of extracellular Ca2+ occurs primarily through voltage-gated Ca2+ channels (VGCCs), store-operated Ca2+ channels (SOC), and Ca2+ release-activated channels (CRAC), whereas SR-Ca2+ release occurs through inositol trisphosphate receptor (IP3R) and ryanodine receptors (RyRs). IP3R-mediated SR-Ca2+ release, in the form of Ca2+ waves, not only contributes to VSMC contraction and regulates VGCC function but is also intimately involved in structural remodeling of resistance arteries in hypertension. This involves a phenotypic switch of VSMCs as well as an alteration of cytoplasmic Ca2+ signaling machinery, a phenomena tightly related to the aging process. Several lines of evidence implicate changes in expression/function levels of IP3R isoforms in the development of hypertension, VSMC phenotypic switch, and vascular aging. The present review discusses the current knowledge of these mechanisms in an integrative approach and further suggests potential new targets for hypertension management and treatment.

Purinergic receptor stimulation induces calcium oscillations and smooth muscle contraction in small pulmonary veins.

We investigated the excitation-contraction coupling mechanisms in small pulmonary veins (SPVs) in rat precision-cut lung slices. We found that SPVs contract strongly and reversibly in response to extracellular ATP and other vasoconstrictors, including angiotensin-II and endothelin-1. ATP-induced vasoconstriction in SPVs was associated with the stimulation of purinergic P2Y2 receptors in vascular smooth muscle cell, activation of phospholipase C-β and the generation of intracellular Ca2+ oscillations mediated by cyclic Ca2+ release events via the inositol 1,4,5-trisphosphate receptor. Active constriction of SPVs may play an important role in the development of pulmonary hypertension and pulmonary oedema. The small pulmonary veins (SPVs) may play a role in the development of pulmonary hypertension and pulmonary oedema via active changes in SPV diameter, mediated by vascular smooth muscle cell (VSMC) contraction. However, the excitation-contraction coupling mechanisms during vasoconstrictor stimulation remain poorly understood in these veins. We used rat precision-cut lung slices and phase-contrast and confocal microscopy to investigate dynamic changes in SPV cross-sectional luminal area and intracellular Ca2+ signalling in their VSMCs. We found that the SPV (∼150μm in diameter) contract strongly in response to extracellular ATP and other vasoconstrictors, including angiotensin-II and endothelin-1. ATP-induced SPV contraction was fast, concentration-dependent, completely reversible upon ATP washout, and inhibited by purinergic receptor antagonists suramin and AR-C118925 but not by MRS2179. Immunofluorescence showed purinergic P2Y2 receptors expressed in SPV VSMCs. ATP-induced SPV contraction was inhibited by phospholipase Cβ inhibitor U73122 and accompanied by intracellular Ca2+ oscillations in the VSMCs. These Ca2+ oscillations and SPV contraction were inhibited by the inositol 1,4,5-trisphosphate receptor inhibitor 2-APB but not by ryanodine. The results of the present study suggest that ATP-induced vasoconstriction in SPVs is associated with the activation of purinergic P2Y2 receptors in VSMCs and the generation of Ca2+ oscillations.

The T2238C Human Atrial Natriuretic Peptide Molecular Variant and the Risk of Cardiovascular Diseases.

Atrial natriuretic peptide (ANP) is a cardiac hormone which plays important functions to maintain cardio-renal homeostasis. The peptide structure is highly conserved among species. However, a few gene variants are known to fall within the human ANP gene. The variant rs5065 (T2238C) exerts the most substantial effects. The T to C transition at the 2238 position of the gene (13–23% allele frequency in the general population) leads to the production of a 30-, instead of 28-, amino-acid-long α-carboxy-terminal peptide. In vitro, CC2238/αANP increases the levels of reactive oxygen species and causes endothelial damage, vascular smooth muscle cells contraction, and increased platelet aggregation. These effects are achieved through the deregulated activation of type C natriuretic peptide receptor, the consequent inhibition of adenylate cyclase activity, and the activation of Giα proteins. In vivo, endothelial dysfunction and increased platelet aggregation are present in human subjects carrying the C2238/αANP allele variant. Several studies documented an increased risk of stroke and of myocardial infarction in C2238/αANP carriers. Recently, an incomplete response to antiplatelet therapy in ischemic heart disease patients carrying the C2238/αANP variant and undergoing percutaneous coronary revascularization has been reported. In summary, the overall evidence supports the concept that T2238C/ANP is a cardiovascular genetic risk factor that needs to be taken into account in daily clinical practice.

Aberrant endoplasmic reticulum stress mediates coronary artery spasm through regulating MLCK/MLC2 pathway

Coronary artery spasm (CAS) is a pathophysiological phenomenon that may cause myocardial infarction and lead to circulatory collapse and death. Aberrant endoplasmic reticulum (ER) stress causes accumulation of misfolding proteins and has been reported to be involved in a variety of vascular diseases. The present study investigated the role of ER stress in the development of CAS and explored the possible molecular mechanisms. Initially, it was found that ER stress markers were elevated in response to drug-induced vascular smooth muscle cells (VSMCs) contraction. Pharmacologic activation of ER stress using Tunicamycin (Tm) persistently induced CAS and significantly promoted Pituitrin-induced CAS in mice as well as in a collagen gel contraction assay. On the contrary, pharmacologic inhibition of ER stress using 4-phenylacetic acid (4-PBA) completely blunted Pituitrin-induced CAS development in mice. Moreover, during the drug-induced VSMCs contraction, expression of ER stress markers were increased in parallel to those of myosin light chain kinase (MLCK) and phosphor-MLC2 (p-MLC2, at Ser19). After inhibiting MLCK activity by using its specific inhibitor ML-7, the ER stress activator Tm failed to activate the MLCK/MLC2 pathway and could neither trigger CAS in mice nor induce VSMCs contraction in vitro. Our results suggested that aberrant ER stress mediated CAS via regulating the MLCK/MLC2 pathway. ER stress activators might be more robust than the common drugs (Pituitrin or acetylcholine) as to induce vasocontraction and thus may serve as potential therapeutics against chronic bleeding, while its inhibitor might be useful for treatment of severe CAS caused by other medication.

Quercetin-Induced AMP-Activated Protein Kinase Activation Attenuates Vasoconstriction Through LKB1-AMPK Signaling Pathway.

The vascular tone plays an important role in blood pressure and flow. It is influenced by the contraction of vascular smooth muscle cells (VSMCs), which in turn is regulated by the balance between the myosin light chain kinase (MLCK) and the phosphorylated myosin light chain (p-MLC). Quercetin is a common flavonoid which is found in many fruits and red wine. Although quercetin has been widely reported to be involved in cell proliferation, migration, and apoptosis in VSMCs, it has not yet been demonstrated whether quercetin is related to vasocontraction, a function regulated by the AMP-activated protein kinase (AMPK) signaling pathway. Accordingly, the aim of this study is to investigate the molecular mechanism through which the quercetin-activated LKB1-AMPK signaling pathway regulates the contraction of VSMCs. In cultured VSMCs, quercetin activated AMPK in a dose- and time-dependent manner. Quercetin inhibited the phenylephrine (PE)-induced expression of MLCK and p-MLC through the LKB1-AMPK signaling pathway and decreased the mRNA level of MLCK. Adenovirus-AMPK DN α1 and AMPK DN α2-transduced VSMCs displayed higher p-MLC expression. Moreover, quercetin inhibited the PE-mediated contraction in rat aorta. These data suggest that the quercetin-activated LKB1-AMPK signaling pathway regulates VSMC contraction by inhibiting MLCK and p-MLC; hence, it may be a therapeutic intervention for the treatment of cardiovascular disorders such as atherosclerosis and hypertension.

Loss of SPRR3 in ApoE-/- mice leads to atheroma vulnerability through Akt dependent and independent effects in VSMCs.

Vascular smooth muscle cells (VSMCs) represent important modulators of plaque stability in advanced lesions. We previously reported that loss of small proline-rich repeat protein 3 (Sprr3), leads to VSMC apoptosis in a PI3K/Akt-dependent manner and accelerates lesion progression. Here, we investigated the role of Sprr3 in modulating plaque stability in hyperlipidemic ApoE-/- mice. We show that loss of Sprr3 increased necrotic core size and reduced cap collagen content of atheromas in brachiocephalic arteries with evidence of plaque rupture and development of intraluminal thrombi. Moreover, Sprr3-/-ApoE-/- mice developed advanced coronary artery lesions accompanied by intraplaque hemorrhage and left ventricle microinfarcts. SPRR3 is known to reduce VSMC survival in lesions by promoting their apoptosis. In addition, we demonstrated that Sprr3-/- VSMCs displayed reduced expression of procollagen in a PI3K/Akt dependent manner. SPRR3 loss also increased MMP gelatinase activity in lesions, and increased MMP2 expression, migration and contraction of VSMCs independently of PI3K/Akt. Consequently, Sprr3 represents the first described VSMC modulator of each of the critical features of cap stability, including VSMC numbers, collagen type I synthesis, and protease activity through Akt dependent and independent pathways.

A Novel Regulatory Mechanism of Smooth Muscle α-Actin Expression by NRG-1/circACTA2/miR-548f-5p Axis.

Neuregulin-1 (NRG-1) includes an extracellular epidermal growth factor-like domain and an intracellular domain (NRG-1-ICD). In response to transforming growth factor-β1, its cleavage by proteolytic enzymes releases a bioactive fragment, which suppresses the vascular smooth muscle cell (VSMC) proliferation by activating ErbB (erythroblastic leukemia viral oncogene homolog) receptor. However, NRG-1-ICD function in VSMCs remains unknown. Here, we characterize the function of NRG-1-ICD and underlying mechanisms in VSMCs. Immunofluorescence staining, Western blotting, and quantitative real-time polymerase chain reaction showed that NRG-1 was expressed in rat, mouse, and human VSMCs and was upregulated and cleaved in response to transforming growth factor-β1. In the cytoplasm of HASMCs (human aortic smooth muscle cells), the NRG-1-ICD participated in filamentous actin formation by interacting with α-SMA (smooth muscle α-actin). In the nucleus, the Nrg-1-ICD induced circular ACTA2 (alpha-actin-2; circACTA2) formation by recruitment of the zinc-finger transcription factor IKZF1 (IKAROS family zinc finger 1) to the first intron of α-SMA gene. We further confirmed that circACTA2, acting as a sponge binding microRNA (miR)-548f-5p, interacted with miR-548f-5p targeting 3' untranslated region of α-SMA mRNA, which in turn relieves miR-548f-5p repression of the α-SMA expression and thus upregulates α-SMA expression, thereby facilitating stress fiber formation and cell contraction in HASMCs. Accordingly, in vivo studies demonstrated that the localization of the interaction of circACTA2 with miR-548f-5p is significantly decreased in human intimal hyperplastic arteries compared with normal arteries, implicating that dysregulation of circACTA2 and miR-548f-5p expression is involved in intimal hyperplasia. These results suggest that circACTA2 mediates NRG-1-ICD regulation of α-SMA expression in HASMCs via the NRG-1-ICD/circACTA2/miR-548f-5p axis. Our data provide a molecular basis for fine-tuning α-SMA expression and VSMC contraction by transcription factor, circular RNA, and microRNA.

KCNQ1 variants associate with hypertension in type 2 diabetes and affect smooth muscle contractility in vitro.

KCNQ1 encodes a potassium voltage-gated channel and represents a susceptibility locus for type 2 diabetes mellitus (T2DM). Here, we explored the association between KCNQ1 polymorphisms and hypertension risk in individuals with T2DM, as well as the role of KCNQ1 in vascular smooth muscle cell contraction in vitro. To investigate the relationship between KCNQ1 and the risk of developing hypertension in patients with T2DM, we divided the T2DM cohort into hypertension (n = 452) and non-hypertension (n = 541) groups. The Mann-Whitney U test, chi-square test, and multivariate regression analyses were used to assess the clinical characteristics and genotypic frequencies. In vitro studies utilized the rat aortic smooth muscle A10 cell line. Patients in the hypertension group were significantly older at the time of enrollment and had higher levels of body mass index, waist-to-hip ratio, and triglyceride than those in the non-hypertension group. The KCNQ1 rs3864884 and rs12576239 genetic variants were associated with hypertension in T2DM. KCNQ1 expression was lower in the individuals with the CC versus the CT and TT genotypes. Smooth muscle cell contractility was inhibited by treatment with a KCNQ1 inhibitor. These results suggest that KCNQ1 might be associated with hypertension in individuals with T2DM.

Purinergic signalling in mesenteric vascular smooth muscle in a mice model of essential hypertension

In vascular smooth muscle cells (VSMCs) non‐selective cation channels such as TRPC3 and TRPC6 have been proposed as the molecular constituents of receptor‐(ROCs) and stretch‐(SOCs) operated channels linking DAG signaling cascade pathway with increased vascular tone. The membrane depolarization caused by TRPCs can increase Ca2+ entry leading to augmented [Ca2+]I and VSMC contraction. Therefore, by influencing membrane potential, TRPC channels could play an important role controlling the increased vascular tone present in several pathologies, such as essential hypertension. We have previously described an increased functional expression of TRPC3 channels in mesenteric VSMCs from hypertensive mice that can contribute to increased vascular tone by favouring depolarization (Alvarez‐Miguel et al., J Physiol, in press). As several mechanosensitive G‐protein coupled receptors (GPCR) are linked to the DAG‐dependent activation of TRPC3/6 channels, we explore here the contribution of this signalling pathway to the increased vascular tone in hypertension.Using a mouse model of essential hypertension (BPH mice) and its normotensive control (BPN mice) we explored changes in the molecular components of the mechanotransduction pathway in hypertension. We analized the expression of mechanosensitive ion channels and some VSMCs sensor elements, including GPCRs, channels and transporters with qPCR. Their functional contribution was studied in isolated VSMCs (using electrophysiology) and in intact mesenteric arteries with pressure myography.Real‐time PCR showed an increased expression of some of the elements of the purinergic signalling pathway, including purinergic receptors P2Y2 and P2Y6, TRPC3 channels and the Ca2+‐activated Cl− channel Clca1. Pressure myography of mesenteric arteries after endothelium removal showed a vasoconstriction response to ATP, UTP and UDP. ATP‐induced vasoconstriction was not different between BPN and BPH arteries. The EC50 of the effect was around 7–10 μM in both preparations, and it could be blocked by α‐β‐methilene‐ATP. UTP‐induced vasoconstriction was not affected by α‐β‐methilene‐ATP and was significantly larger in BPH arteries: 50 μM UTP‐elicited vasoconstriction represented 40% of the maximal response to ATP in BPN arteries and 150% in BPH arteries. Our data indicate that mesenteric vascular reactivity to ATP is not altered in our hypertension model, but there is an increased contribution of the purinergic GPCR signalling pathway to the increased vascular tone. Together with the observed upregulation of P2YR receptors in BPH VSMCs, the augmented functional expression of TRPC3 channels could participate in the increased vasoconstriction observed in response to uridine nucleotides in BPH mesenteric arterieSupport or Funding InformationSupported by grants ISCIII‐FEDER RD12/0042/0006, (Heracles Program), BFU2013‐45867‐R (MINECO) and by a Junta de Castilla y Leon fellowship to IAM

23 NADPH OXIDASE 5 (NOX5), cholesterol-rich microdomains and ANG II signallingsignaling in human primary vascular smooth muscle cells

Background and hypothesis Nox5 has been identified in human vessels. However the trafficking, regulation and function of vascular Nox5 is unclear. We questioned the function of Nox5 and other Nox isoforms in signalling associated with vascular smooth muscle cell contraction, growth and cytoskeletal organisation and assessed whether lipid rafts/caveolae play a role in Nox5 regulation. Methods Human primary vascular smooth muscle cells (VSMCs) isolated from small arteries were studied. Nox isoforms were sequentially downregulated by siRNA. siRNA to p22phox reduced activity of Nox1,2 and 4, since these isoforms are p22phox-dependent. Nox5 was targeted by Nox5 siRNA. Signalling pathways associated with contraction (MLC, MYPT1), cell growth (PCNA, p53) and cytoskeletal organisation (Ezrin-Radixin-Moiesin) were assessed by immunostudies. Cells were stimulated with Ang II in the absence and presence of disrupters and enhancers of cholesterol-rich microdomains. Nox5 interaction with lipid rafts/caveolae was examined by immunohistochemistry in intact vessels and by sucrose gradient cell fractionation in cultured cells. Results Nox5 inhibition by siRNA was associated with reduced Ang II-induced phosphorylation of MLC20 (by 0.74-fold, 5 mins stimulation) and MYPT1 (by 0.5-fold, 5 mins stimulation). p22phox siRNA did not significantly alter activation of MLC20 or MYPT1. siRNA to p22phox and Nox5 decreased Ang II-induced increase in PCNA expression (by 0.18-fold and 0.3-fold in Ang II 2 hour and 8 hour stimulation respectively, p Conclusions These results indicate that in human VSMCs Ang II stimulates contractile pathways through Nox5-dependent pathways whereas signalling associated with VSMC growth and cytoskeletal organisation involve multiple Nox isoforms. Nox5 co-localises in lipid rafts/caveolae, which may play a role in Nox5 trafficking. Our data identify novel redox signalling processes through cholesterol-rich microdomains and highlight a potentially important function of Nox5 in vascular contraction.