Vascular Smooth Muscle Cell Contraction Research Articles

Generation and proliferation assessment of HEK293T cells stably expressing Kv1.5 channel with and without regulatory subunits β1.1 or β1.2

Potassium channels, including voltage-gated potassium (Kv) channels, play an important role in regulation of vascular smooth muscle cell (VSMC) contraction and proliferation. Our previous studies indicate that KV1.5 is a major type of shaker-family KV1 channels expressed in human coronary and adipose arterioles. The function of KV1.5 channels is impaired in disease, contributing to reduced KV1.5-dependent VSMC relaxation and leading to vascular dysfunction in subjects with coronary artery disease (CAD). However, it remains largely unknown whether KV1.5, alone and with its regulatory subunits (β1.1 and β1.2), also regulates VSMC proliferation in CAD. This study was designed to examine if expression of KV1.5 with and without β1.1 or β1.2) modulates cell proliferation in cultured HEK293T cells. We created a Kv1.5 cell line with lentiviral transduction at a multiplicity of infection (MOI) of 20. We used this cell line to introduce β1.1 subunit with the same protocol (MOI=20). However, when we attempted to introduce β1.2 subunit to Kv1.5 cell line, transduction efficiency was only 40% prompting us to repeat the experiment with MOI of 40 to obtain transduction efficiency of over 90%. These results suggest that the β1.2 subunit may be responsible for inhibiting cell proliferation. To test our hypothesis, we took images of transduced cultured cells every 24 hours from 24 to 96 hours and analyzed them with ImageJ software. Each cell was outlined to indicate the surface area taken up by the cells as compared to total surface area of the image for estimation of the overall cell density/confluency. ImageJ analysis revealed that the lowest density was observed in Kv1.5+ β1.2 cell line (25±3% at 48 hours, n=15 images from 6 independent experiments P<0.001) as compared with densities of Kv1.5 (72±3%, n=18); Kv1.5+ β1.1 (75±1%, n=15); and GFP (65±2%, n=9). Kv1.5+ β1.2 consistently exhibited the lowest confluency at all time points (24, 72, and 96 hours). Next, we performed cell counting with a trypan blue solution using a hemocytometer to assess the cell density of the entire sample at 48 hours. After we converted the number of cells into percentages relative to GFP control, our data indicated that Kv1.5 was at 97±1%, n=6; Kv1.5+ β1.1 at 96±1%, n=6; and Kv1.5+ β1.2 was at 41±0.5%, n=6 (P<0.001). In conclusion, our results indicate that, expression of Kv1.5 channel + β1.2 regulatory subunit, but not KV1.5 alone or with β1.1 subunit, inhibits cell proliferation in heterologous expression cell systems. Future studies will identify the role and mechanism of KV1.5 and its β1.2 subunit in VSMC proliferation in CAD or other vascular diseases.

MicroRNA-212-5p and its target PAFAH1B2 suppress vascular proliferation and contraction via the downregulation of RhoA.

Vascular remodeling and contraction contribute to the development of hypertension. We investigated the role of miR-212-5p and its downstream target in vascular smooth muscle cell (VSMC) proliferation, migration, and contraction. MicroRNA microarray and PCR analyses showed that miR-212-5p expression was increased with angiotensin II treatment in vivo and in vitro. Moreover, miR-212-5p mimic treatment attenuated and miR-212-5p inhibitor treatment increased VSMC proliferation and migration. Additionally, miR-212-5p mimic treatment suppressed VSMC contraction and related gene expression [Ras homolog gene family member A (RhoA) and Rho-associated protein kinase 2], while miR-212-5p inhibitor treatment exerted opposite effects. Bioinformatics analysis revealed that platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) is a target of miR-212-5p. miR-212-5p mimic treatment significantly reduced and miR-212-5p inhibitor treatment increased PAFAH1B2 expression. Furthermore, PAFAH1B2 expression was decreased in angiotensin II-treated aortic tissues and VSMCs. PAFAH1B2 was ubiquitously expressed in most adult rat tissues. In the vasculature, PAFAH1B2 was only distributed in the cytoplasm. PAFAH1B2 overexpression decreased A10 cell proliferation, while PAFAH1B2 knockdown increased A10 cell proliferation and cyclin D1 mRNA levels. PAFAH1B2 knockdown stimulated VSMC contraction and RhoA expression. These results suggest that miR-212-5p and PAFAH1B2 are novel negative regulators of VSMC proliferation, migration, and contraction in hypertension.

Synthetic VSMCs Induce BBB Disruption Mediated by MYPT1 in Ischemic Stroke

Vascular smooth muscle cells (VSMCs) have been widely recognized as key players in regulating blood-brain barrier (BBB) function, their roles are unclear in ischemic stroke. Myosin phosphatase target subunit 1 (MYPT1) is essential for VSMCs contraction and contributes to maintaining healthy vasculature. We generated VSMC-specific MYPT1 knockout mice (MYPT1SMKO mice) and cultured VSMCs infected with Lv-shMYPT1 to explore BBB integrity, brain infarction and neurological deficits after ischemic stroke. MYPT1 deficiency induced phenotypic switching of synthetic VSMCs, which aggravated BBB disruption. Proteomic analysis identified evolutionarily conserved signaling intermediates in Toll pathways (ECSIT) as a downstream molecule that promotes phenotypic switching of VSMCs and contributed to IL-6 expression. Knocking down ECSIT rescued the phenotypic switching of VSMCs and BBB disruption in ischemic stroke. Additionally, inhibition of IL-6 decreased BBB permeability. These findings reveal that MYPT1 deficiency activated phenotypic switching of VSMCs and induced BBB disruption after ischemic stroke through ECSIT-IL-6 signaling.

Physical interaction of tropomyosin 3 and STIM1 regulates vascular smooth muscle contractility and contributes to hypertension

ScopeTropomyosin (TPM), an actin-binding protein widely expressed across different cell types, is primarily involved in cellular contractile processes. We investigated whether TPM3 physically and functionally interacts with stromal interaction molecule 1 (STIM1) to contribute to vascular smooth muscle cell (VSMC) contraction, store-operated calcium entry (SOCE), and high-salt intake–induced hypertension in rats. Methods and resultsAnalysis of a rat RNA-seq data set of 80 samples showed that the STIM1 and Tpm3 transcriptome expression pattern is highly correlated, and co-immunoprecipitation results indicated that TPM3 and STIM1 proteins physically interacted in rat VSMCs. Immunohistochemical data displayed obvious co-localization of TPM3 and STIM1 in rat VSMCs. Knockdown of TPM3 or STIM1 in VSMCs with specific small interfering RNA significantly suppressed contractions in tension measurement assays and decreased SOCE in calcium assays. Rats fed a high-salt diet for 4 weeks had significantly higher systolic blood pressure than controls, with significantly increased contractility and markedly increased TPM3 and STIM1 expression levels in the mesenteric resistance artery (shown by tension measurements and immunoblotting, respectively). Additionally, high salt environment in vitro induced significant enhancement of TPM3 and STIM1 expression levels in VSMCs. ConclusionsWe showed for the first time that TPM3 and STIM1 physically and functionally interact to contribute to VSMC contraction, SOCE, and high-salt intake–induced hypertension. Our findings provide mechanistic insights and offer a potential therapeutic target for high-salt intake–induced hypertension.

The Calcilytic Drug Calhex-231 Ameliorates Vascular Hyporesponsiveness in Traumatic Hemorrhagic Shock by Inhibiting Oxidative Stress and miR-208a-Mediated Mitochondrial Fission

Background The calcium-sensing receptor (CaSR) plays a fundamental role in extracellular calcium homeostasis in humans. Surprisingly, CaSR is also expressed in nonhomeostatic tissues and is involved in regulating diverse cellular functions. The objective of this study was to determine if Calhex-231 (Cal), a negative modulator of CaSR, may be beneficial in the treatment of traumatic hemorrhagic shock (THS) by improving cardiovascular function and investigated the mechanisms. Methods Rats that had been subjected to THS and hypoxia-treated vascular smooth muscle cells (VSMCs) were used in this study. The effects of Cal on cardiovascular function, animal survival, hemodynamics, and vital organ function in THS rats and the relationship to oxidative stress, mitochondrial fusion-fission, and microRNA (miR-208a) were investigated. Results Cal significantly improved hemodynamics, elevated blood pressure, increased vital organ blood perfusion and local oxygen supply, and markedly improved the survival outcomes of THS rats. Furthermore, Cal significantly improved vascular reactivity after THS in vivo and in vitro. Cal also restored the THS-induced decrease in myosin light chain (MLC) phosphorylation (the key element for VSMC contraction). Inhibition of MLC phosphorylation antagonized the Cal-induced restoration of vascular reactivity following THS. Cal suppressed oxidative stress in THS rats and hypoxic-VSMCs. Meanwhile, THS induced expression of mitochondrial fission proteins Drp1 and Fis1 and decreased expression of mitochondrial fusion protein Mfn1 in vascular tissues. Cal reduced expression of Drp1 and Fis1. In hypoxic-VSMCs, Cal inhibited mitochondrial fragmentation and preserved mitochondrial morphology. In addition, miR-208a mimic decreased Fis1 expression, and miR-208a inhibitor prevented Cal-induced Fis1 downregulation in hypoxic-VSMCs. Conclusion Calhex-231 exhibits outstanding potential for effective therapy of traumatic hemorrhagic shock, and the beneficial effects result from its protection of vascular function via inhibition of oxidative stress and miR-208a-mediated mitochondrial fission.

Matrix Metalloproteinases and Hypertension-Mediated Organ Damage: Current Insights.

Matrix metalloproteinases (MMPs) are important extracellular enzymes involved in many physiological and pathological processes. Changes in the activity and concentration of specific MMPs, as well as the unbalance with their inhibitors (tissue inhibitors of metalloproteinases – TIMPs), have been described as a part of the pathogenic cascade promoted by arterial hypertension. MMPs are able to degrade various protein substrates in the extracellular matrix, to influence endothelial cells function, vascular smooth muscle cells migration, proliferation and contraction, and to stimulate cardiomyocytes changes. All these processes can be activated by chronically elevated blood pressure values. Animal and human studies demonstrated the key function of MMPs in the pathogenesis of hypertension-mediated vascular, cardiac, and renal damage, besides age and blood pressure values. Thus, the role of MMPs as biomarkers of hypertension-mediated organ damage and potential pharmacological treatment targets to prevent further cardiovascular and renal complications in hypertensive population is increasingly supported. In this review, we aimed to describe the main scientific evidence about the behavior of MMPs in the development of vascular, cardiac, and renal damage in hypertensive patients.

Variants of Focal Adhesion Scaffold Genes Cause Thoracic Aortic Aneurysm.

Thoracic aortic aneurysm (TAA) leads to substantial mortality worldwide. Familial and syndromic TAAs are highly correlated with genetics. However, the incidence of sporadic isolated TAA (iTAA) is much higher, and the genetic contribution is not yet clear. Here, we examined the genetic characteristics of sporadic iTAA. We performed a genetic screen of 551 sporadic iTAA cases and 1071 controls via whole-exome sequencing. The prevalence of pathogenic mutations in known causal genes was 5.08% in the iTAA cohort. We selected 100 novel candidate genes using a strict strategy, and the suspected functional variants of these genes were significantly enriched in cases compared with controls and carried by 60.43% of patients. We found more severe phenotypes and a lower proportion of hypertension in cases with pathogenic mutations or suspected functional variants. Among the candidate genes, Testin (TES), which encodes a focal adhesion scaffold protein, was identified as a potential TAA causal gene, accounting for 4 patients with 2 missense variants in the LIM1 domain (c.751T>C encoding p.Y251H; c.838T>C encoding p.Y280H) and highly expressed in the aorta. The 2 variants led to a decrease in TES expression. The thoracic aorta was spontaneously dilated in the TesY249H knock-in and Tes-/- mice. Mechanistically, the p.Y249H variant or knockdown of TES led to the repression of vascular smooth muscle cell contraction genes and disturbed the vascular smooth muscle cell contractile phenotype. Interestingly, suspected functional variants of other focal adhesion scaffold genes, including TLN1 (Talin-1) and ZYX (zyxin), were also significantly enriched in patients with iTAA; moreover, their knockdown resulted in decreased contractility of vascular smooth muscle cells. For the first time, this study revealed the genetic landscape across iTAA and showed that the focal adhesion scaffold genes are critical in the pathogenesis of iTAA.

Effects of Extracellular Matrix Softening on Vascular Smooth Muscle Cell Dysfunction.

Vascular smooth muscle cells (VSMCs) shift from a physiological contractile phenotype to an adverse proliferative or synthetic state, which is a major event leading to aortic disease. VSMCs are exposed to multiple mechanical signals from their microenvironment including vascular extracellular matrix (ECM) stiffness and stretch which regulate VSMC contraction. How ECM stiffness regulates the function and phenotype of VSMCs is not well understood. In this study, we introduce in vitro and in vivo models to evaluate the impact of ECM stiffnesses on VSMC function. Through unbiased transcriptome sequencing analysis, we detected upregulation of synthetic phenotype-related genes including osteopontin, matrix metalloproteinases, and inflammatory cytokines in VSMCs cultured using soft matrix hydrogels in vitro, suggesting VSMC dedifferentiation toward a synthetic phenotype upon ECM softening. For the in vivo model, the lysyl oxidase inhibitor β-aminopropionitrile monofumarate (BAPN) was administrated to disrupt the cross-linking of collagen to induce ECM softening. Consistently, decreased ECM stiffnesses promoted VSMC phenotypic switching to a synthetic phenotype as evidenced by upregulation of synthetic phenotype-related genes in the aortas of mice following BAPN treatment. Finally, BAPN-treated mice showed severe expansion and developed aortic dissection. Our study reveals the pivotal role of ECM softening in regulating the VSMC phenotype switch and provides a potential target for treating VSMC dysfunction and aortic dissection disease.

Increase in Endothelin-1 Expression in Umbilical Cord Arteries in Preeclampsia

Objective: Endothelin1 (ET1) is 21- amino acid vasoconstrictor peptide secreted by endotheliumwhich has an important role in the pathoohysiology of preeclampsia (PE). The objective of this study was to evaluate the binding sites and quantitative changes in ET1 in umbilical cord vessels of PE patients. Methods: This study recruited 40 pregnant women between 20-40 years old at 3rd trimester. All cases selected for this study underwent an elective cesarean section, grouped into 2 groups; PE group of 20 pregnant women (at 3rd trimester) who proved to have pregnancy induced hypertension and proteinuria. The control group was of 20 healthy pregnant females at the same average of gestational age and with the same exclusion criteria and no PE, underwent elective caesarean section. Umbilical cord tissues were taken from the maternal side, fixed with formalin, paraffin, embedded sections of umbilical cord were treated with Endothelin1 antibody. The immunoreactivity of ET1 was assessed using Aperio image scope software. Statistical analysis was done using SPSS program. Results: The results demonstrated a significant increase (P = 0.001) of ET1 expression in cord vessels of PE group with respect to control group (mean 28.5±1.7, 2.6±0.4 respectively). Conclusion: It is concluded that ET1 is markedly increase in PE and may be the cause behind promoted vascular smooth muscle cell contraction and blood pressure elevation in PE.

Smooth Muscle Cell – Extracellular Matrix Alterations in Genetic Vasculopathies

Pathogenic variants in 11 genes predispose individuals to aneurysms and dissections of the thoracic aorta and in some cases, vascular complications throughout the arterial tree, termed heritable thoracic aortic disease (HTAD) (Renard M et al. JACC, 2018;72:605–615). These genes are involved in vascular smooth muscle cell (SMC) contraction and metabolism, extracellular matrix (ECM) integrity and transforming growth factor β (TGF β) signaling. A unifying hypothesis is that the mutated gene products contribute to altered mechano‐transduction leading to vasculopathy (Humphrey JD et al. Circ Res 2015;116:1448–1461; Pinard A, Jones GT, Milewicz DM. Circ Res 2019;124:588–606). The objective of this study was to employ light (LM) and electron (EM) microscopy to evaluate the thoracic aortic and other arterial tissue from individuals harboring pathogenic variants in the 11 established predisposition genes who underwent surgical aneurysm or dissection repair. These individuals (n) harbored pathogenic variants in the following genes: FBN1 (13), ACTA2 (6), TGFBR1 (7), TGFBR2 (3), TGFβ2 (3), SMAD3 (3), LOX (1), MYLK (1), PRKG1 (1), and COL3A1 (4); total 42. Arterial specimens were evaluated using LM consensus criteria (Halushka MK et al. Cardiovasc Pathol 2016;25:247–257). EM was performed on selected cases (Davis EC. Lab Invest 1993;68:89–99). By LM, specimens were graded (0 – 3+) for mucoid extracellular matrix accumulation (MEMA), elastic fiber fragmentation and/or loss (EFFL), SMC nuclear loss/laminar medial collapse (SMCNL/LMC), and overall medial degeneration (A 1–2, B 3–4, C 5–6, D 7–9); intimal lesions (0–3+) and medial dissection (+/−) also were noted. The 42 specimens included 17 with medial dissection and 25 non‐dissected aneurysms. By LM, cases showed a spectrum of extent and severity of medial degenerative changes: A mild 8 cases, B moderate 18 cases, C moderately severe 14 cases, and D severe 2 cases. By EM, COL3A1 specimens showed reduction of collagen fibrils and shape variations of residual collagen fibrils; elastin fiber connections to SMCs were intact. In contrast, specimens with mutations in the TGF β pathway exhibited multiple foci with deficiency of elastic fibers and loss of elastic fiber connections to SMC but had normal collagen ultrastructure. These results support the hypothesis that the pathogenesis of genetic vasculopathies involves structural defects in the interaction of ECM components and SMCs, including impairment of the elastin‐contractile unit directly or through loss of structural support provided by collagen.Support or Funding InformationLocal funds

Reactive oxygen species in cardiovascular health and disease: special references to nitric oxide, hydrogen peroxide, and Rho-kinase.

The interaction between endothelial cells and vascular smooth muscle cells (VSMC) plays an important role in regulating cardiovascular homeostasis. Endothelial cells synthesize and release endothelium-derived relaxing factors (EDRFs), including vasodilator prostaglandins, nitric oxide (NO), and endothelium-dependent hyperpolarization (EDH) factors. Importantly, the contribution of EDRFs to endothelium-dependent vasodilatation markedly varies in a vessel size-dependent manner; NO mainly mediates vasodilatation of relatively large vessels, while EDH factors in small resistance vessels. We have previously identified that endothelium-derived hydrogen peroxide (H2O2) is an EDH factor especially in microcirculation. Several lines of evidence indicate the importance of the physiological balance between NO and H2O2/EDH factor. Rho-kinase was identified as the effectors of the small GTP-binding protein, RhoA. Both endothelial NO production and NO-mediated signaling in VSMC are targets and effectors of the RhoA/Rho-kinase pathway. In endothelial cells, the RhoA/Rho-kinase pathway negatively regulates NO production. On the contrary, the pathway enhances VSMC contraction with resultant occurrence of coronary artery spasm and promotes the development of oxidative stress and vascular remodeling. In this review, I will briefly summarize the current knowledge on the regulatory roles of endothelium-derived relaxing factors, with special references to NO and H2O2/EDH factor, in relation to Rho-kinase, in cardiovascular health and disease.

Activation of AMPK/proteasome/MLCK degradation signaling axis by telmisartan inhibits VSMC contractility and vessel contraction

Telmisartan, an angiotensin II type 1 receptor blocker (ARB), is widely used to treat hypertension. Dysfunction of vascular smooth muscle cells (VSMCs) is well-established to contribute to the pathogenesis of various vascular diseases. A growing body of evidence indicates that increased VSMC contractility plays a primary role in the development of pathological artery spasms. Nevertheless, effect of telmisartan on VSMC contractility, and its mechanism of action remain unknown. Here, we investigated the mechanism by which telmisartan inhibits VSMC contractility and vessel contraction in rat VSMCs and endothelium-deprived aortas. Telmisartan inhibited phenylephrine-induced vessel contraction in endothelium-deprived aortas, and decreased myosin light chain kinase (MLCK) levels (without altering corresponding mRNA levels) and myosin light chain (MLC) phosphorylation at Ser19 (p-MLC-Ser19) in VSMCs. MG-132 but not doxycycline significantly restored telmisartan-inhibited MLCK expression and p-MLC-Ser19. Telmisartan induced AMP-activated protein kinase (AMPK) phosphorylation at Thr172 (p-AMPK-Thr172), and compound C or ectopic expression of the dominant negative (dn)-AMPKα1 gene significantly reversed telmisartan-inhibited MLCK expression and p-MLC-Ser19. Of the ARBs tested (including losartan and fimasartan), only telmisartan increased p-AMPK-Thr172, and inhibited MLCK expression and p-MLC-Ser19. GW9662 had no effects on telmisartan-induced changes. Similar to the in vitro results, telmisartan enhanced p-AMPK-Thr172, and inhibited MLCK expression and p-MLC-Ser19 in endothelium-deprived aortas. Furthermore, the telmisartan-inhibited vessel contraction in the aortas was significantly reversed by MG-132 or compound C. In conclusion, we demonstrated that telmisartan inhibits VSMC contractility and vessel contraction by activating AMPK/proteasome/MLCK degradation signaling axis. These results suggest that telmisartan can be used to treat pathological vasospasms.

MicroRNA-29a Involvement in Phenotypic Transformation of Venous Smooth Muscle Cells Via Ten-Eleven Translocation Methylcytosinedioxygenase 1 in Response to Mechanical Cyclic Stretch.

Mechanical stimuli play an important role in vein graft restenosis and the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) are pathological processes contributing to this disorder. Here, based on previous high-throughput sequencing data from vein grafts, miR-29a-3p and its target, the role of Ten-eleven translocation methylcytosinedioxygenase 1 (TET1) in phenotypic transformation of VSMCs induced by mechanical stretch was investigated. Vein grafts were generated by using the "cuff" technique in rats. Deep transcriptome sequencing revealed that the expression of TET1 was significantly decreased, a process confirmed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. MicroRNA-seq showed that miR-29a-3p was significantly up-regulated, targeting TET1 as predicted by Targetscan. Bioinformatics analysis indicated that the co-expressed genes with TET1 might modulate VSMC contraction. Venous VSMCs exposed to 10%-1.25 Hz cyclic stretch by using the Flexcell system were used to simulate arterial mechanical conditions in vitro. RT-qPCR revealed that mechanical stretch increased the expression of miR-29a-3p at 3 h. Western blot analysis showed that TET1 was significantly decreased, switching contractile VSMCs to cells with a synthetic phenotype. miR-29a-3p mimics (MI) and inhibitor (IN) transfection confirmed the negative impact of miR-29a-3p on TET1. Taken together, results from this investigation demonstrate that mechanical stretch modulates venous VSMC phenotypic transformation via the mediation of the miR-29a-3p/TET1 signaling pathway. miR-29a-3p may have potential clinical implications in the pathogenesis of remodeling of vein graft restenosis.

Activation of Cav1.2 and BKCa is involved in the downregulation of caffeine-induced contraction in mice mesenteric arteries

AimsCaffeine is a methylxanthine with multiple actions in vascular smooth muscle cells (VSMCs), including the increase in the intracellular Ca2+ (iCa2+) concentration by the activation of ryanodine receptors (RyRs). The present study aimed at investigating the participation of Ca2+-influx through different Ca2+-channels on the transient contraction (TC) induced by caffeine in mice mesenteric arteries. Main methodsSecond-order of mesenteric arteries was isolated from male Swiss mice. Vessels without functional endothelium were stimulated with caffeine (10 mM). The caffeine-induced TC was evaluated after the incubation of artery rings for 30 min with the following drugs: nifedipine (10 μM), a Cav1.2 blocker; 2-aminoethoxydiphenyl borate (2-APB; 10 μM) and ruthenium red (RuR; 10 μM), transient receptor potential (TRPs) channels blockers; capsazepine (10 μM) and HC067047 (10 μM), TRPV1 and TRPV4 antagonists, respectively; paxilline (1 μM), a selective BKCa blocker; and SKF-96365 (30 μM), an Orai blocker. Ca2+-fluorescence measurements were also performed on the investigated arteries. Key findingsThe TC induced by caffeine was partially dependent on Ca2+-influx. However, the blockage of Cav1.2 increased the TC while reduced the iCa2+ signal. Similar results were observed after the blockage of TRPs or BKCa. Therefore, caffeine promoted Ca2+-influx via TRPs and Cav1.2, and hyperpolarization through the activation of BKCa, inducing negative feedback of TC. SignificanceOur results indicate an alternative mechanism for the control of VSMCs contraction in resistance arteries. The evidence of the negative feedback of contraction via TRP-Cav1.2-BKCa provides a new perspective for understanding the mechanism involved in the vascular responses triggered by caffeine.

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.