Human Brain Vascular Smooth Muscle Cells Research Articles

Methamphetamine and Designer Stimulants Modulate Tonic Human Cerebrovascular Smooth Muscle Contractility: Relevance to Drug-Induced Neurovascular Stress.

To avoid criminal prosecution, clandestine chemists produce designer stimulants that mimic the pharmacological and psychoactive effects of conventional stimulants, such as methamphetamine. Following persistent or high-dose exposure, both acute vasoconstriction and loss of vascular homeostasis are reported dangers of conventional stimulants, and designer stimulants may pose even greater dangers. To compare the effects of a conventional stimulant and two designer stimulants on vascular contraction, this study examined the direct effects of 1,3-benzodioxolylbutanamine (BDB) and N-butylpentylone in comparison to methamphetamine on the function of human brain vascular smooth muscle cells (HBVSMCs). HBVSMCs suspended in collagen gels were exposed to varying concentrations of each drug, and the degree of constriction was assessed over one week. The MTT assay was used to measure the impact of the three drugs on the cellular metabolic activity as a marker of cellular toxicity. The highest concentration tested of either methamphetamine or N-butylpentylone produced a loss of HBVSMC contractility and impaired cellular metabolism. BDB showed a similar pattern of effects, but, uniquely, it also induced vasoconstrictive effects at substantially lower concentrations. Each drug produced direct effects on HBVSMC contraction that may be a mechanism by which the cardiovascular system is damaged following high-dose or persistent exposure, and this could be exacerbated by any sympathomimetic effects of these compounds in whole organisms. BDB appears to impact HBVSMC function in ways distinct from methamphetamine and N-butylpentylone, which may present unique dangers.

MiR-140 promotes the progression of intracranial aneurysms by targeting BCL2L2.

To investigate the role of miR-140/BCL2L2 axis on the formation of intracranial aneurysms. The expression of miR-140 in the serum of patients with intracranial aneurysms and healthy volunteers was detected. CCK-8 assay and Annexin V-FITC/PI double staining flow cytometry were used to evaluate the effect of miR-140 knockdown on the proliferation and apoptosis of human brain vascular smooth muscle cells (HBVSMCs). Meanwhile, the relationship between miR-140 and BCL2L2 was examined. MiR-140 was found to be upregulation in intracranial aneurysm patients. MiR-140 knock-out significantly inhibited the apoptosis of HBVSMCs and promoted cell proliferation. BCL2L2 was a direct target gene of miR-140 and suppressed its expression. Knockdown of miR-140 alleviates the development of intracranial aneurysms. MiR-140/BCL2L2 axis promotes the progression of intracranial aneurysms by regulating apoptosis of HBVSMCs. Therefore, miR-140 is a potential therapeutic target for intracranial aneurysms.

Circ_FOXO3 regulates KLF6 through sponge adsorption of miR-122-5p to repress H2O2-induced HBVSMC proliferation, thus promoting IA development in vitro model.

The phenotypic transformation of human brain vascular smooth muscle cells (HBVSMC) is widely involved in the appearance and progression of intracranial aneurysms (IA). Aneurysm (IA) Circular RNA circ_FOXO3 functions pivotally in vascular diseases and tumors, but its regulatory role as well as its molecular mechanism in IA is still uncertain. This research was to explore how circ_FOXO3 works and its mechanism in vitro model of HBVSMC IA induced by H2O2. Thirty-eight patients with IA and their normal tissues were clinically collected. Examination of endothelin-1, vascular hematoma factor, circ_FOXO3, microRNA (miR)-122-5p and KLF6 and the correlation of circ_FOXO3 with clinical case information were ensured. Establishment of an in vitro IA model was through HBVSMC induced by H2O2 and transfection with circ_FOXO3, miR-122-5p and KLF6 related plasmids was to figure out their roles in cell growth. The relationship among circ_FOXO3, miR-122-5p with KLF6 was detected. Up-regulated circ_FOXO3 and KLF6 and reduced miR-122-5p were in IA tissues; Circ_FOXO3 was associated with smoking history, Hunt-Hess grading and endothelial injury degree. Repressive circ_FOXO3 or KLF6 and strengthening miR-122-5p facilitated H2O2-induced proliferation and repressed HBVSMC apoptosis, while elevation of circ_FOXO3 or depressive miR-122-5p was opposite. circ_FOXO3 bound to miR-122-5p, whose target was KLF6, which participated in controlling IA by mediating the circ_FOXO3/miR-122-5p axis. In summary, the findings suggest that circ_FOXO3 suppresses H2O2-induced proliferation of HBVSMC but promotes apoptosis via modulation of miR-122-5p/KLF6 axis. Targeted therapy of circ_FOXO3/miR-122-5p/KLF6 axis is supposed to be a promising treatment approach for IA patients.

ATF4 promotes brain vascular smooth muscle cells proliferation, invasion and migration by targeting miR-552-SKI axis

BackgroundStudies have indicated vascular smooth muscle cells (VSMCs) played a crucial role in atherosclerosis and microRNAs (miRNAs) played key roles in biological functions of VSMCs. Whereas, the potential function and mechanism of miR-552 in VSMCs remains unclear. Our aim was to explore the role of miR-552 on VSMCs and underlying mechanism.Material/MethodsMTT assay and transwell assay were used to measure the proliferation, invasion, and migration of human brain VSMCs (HBVSMCs) and mice VSMCs (mVSMCs), respectively. Bioinformatics tools and luciferase assay were adopted to verify the association between miR-552 and SKI. Rescue experiments were employed to assess the interaction of miR-552 and SKI in modulating biological functions in HBVSMCs and mVSMCs. The expression level of transcription factors (TFs)was measured via qRT-PCR assay. The effect of ATF4 on miR-552 and SKI expression was tested by qRT-PCR or western blot assay. Finally, chromatin immunoprecipitation (ChIP) assay and JASPAR databases were used to analyze the regulatory linkage between ATF4 and miR-552.ResultsWe found that miR-552 was upregulated in HBVSMCs treated with PDGF-bb and miR-552 overexpression could promote proliferation, invasion, and migration of HBVSMCs and mVSMCs, whereas, miR-552 knockdown had the opposite impact. In addition, we also found that SKI was a direct target of miR-552, which reversed miR-552-mediated proliferation, invasion, and migration in HBVSMCs and mVSMCs. Furthermore, we also discovered that miR-552 overexpression promoted the effects of ATF4 elevation on proliferation, migration and invasion of HBVSMCs and mVSMCs, but, miR-552 decline had the opposite impact.ConclusionsATF4-miR-552-SKI axis played critical roles in the proliferation and migration of HBVSMCs and mVSMCs, which were closely involved in atherosclerosis (AS). Therefore, our findings might offer a novel therapeutic target for AS.

STAT3 inhibitor mitigates cerebral amyloid angiopathy and parenchymal amyloid plaques while improving cognitive functions and brain networks

Previous reports indicate a potential role for signal transducer and activator of transcription 3 (STAT3) in amyloid-β (Aβ) processing and neuritic plaque pathogenesis. In the present study, the impact of STAT3 inhibition on cognition, cerebrovascular function, amyloid pathology, oxidative stress, and neuroinflammation was studied using in vitro and in vivo models of Alzheimer’s disease (AD)-related pathology. For in vitro experiments, human brain vascular smooth muscle cells (HBVSMC) and human brain microvascular endothelial cells (HBMEC) were used, and these cultured cells were exposed to Aβ peptides followed by measurement of activated forms of STAT3 expression and reactive oxygen species (ROS) generation. Further, 6 months old 5XFAD/APOE4 (5XE4) mice and age-matched negative littermates were used for in vivo experiments. These mice were treated with STAT3 specific inhibitor, LLL-12 for 2 months followed by neurobehavioral and histopathological assessment. In vitro experiments showed exposure of cerebrovascular cells to Aβ peptides upregulated activated forms of STAT3 and produced STAT3-mediated vascular oxidative stress. 5XE4 mice treated with the STAT3-specific inhibitor (LLL-12) improved cognitive functions and functional connectivity and augmented cerebral blood flow. These functional improvements were associated with a reduction in neuritic plaques, cerebral amyloid angiopathy (CAA), oxidative stress, and neuroinflammation. Reduction in amyloid precursor protein (APP) processing and attenuation of oxidative modification of lipoprotein receptor related protein-1 (LRP-1) were identified as potential underlying mechanisms. These results demonstrate the broad impact of STAT3 on cognitive functions, parenchymal and vascular amyloid pathology and highlight the therapeutic potential of STAT3 specific inhibition for treatment of AD and CAA.

CircRNA DOCK1 Regulates miR-409-3p/MCL1 Axis to Modulate Proliferation and Apoptosis of Human Brain Vascular Smooth Muscle Cells.

BackgroundIntracranial aneurysm is an abnormal expansion in the intracranial arteries, which is associated with growth and apoptosis of vascular smooth muscle cells. Circular RNAs (circRNAs) have implicated in the progression of intracranial aneurysms. The purpose of this paper is to study the function and mechanism of circRNA dedicator of cytokinesis 1 (circ_DOCK1) in regulating proliferation and apoptosis of human brain vascular smooth muscle cells (HBVSMCs).MethodsHBVSMCs were exposed to hydrogen peroxide (H2O2). Cell proliferation and apoptosis were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and flow cytometry, respectively. Circ_DOCK1, microRNA (miR)-409-3p, and myeloid cell leukemia sequence 1 (MCL1) levels were examined by quantitative reverse transcription polymerase chain reaction or western blotting. The target association was assessed by dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation assays.ResultsExposure to H2O2 decreased proliferation and increased apoptosis of HBVSMCs. Circ_DOCK1 expression was reduced in H2O2-treated HBVSMCs. Circ_DOCK1 overexpression rescued H2O2-caused reduction of proliferation and PCNA expression and attenuated H2O2-induced apoptosis and expression of Bcl-2, Bax, and cleaved PARP. MiR-409-3p was targeted by circ_DOCK1 and upregulated in H2O2-treated HBVSMCs. MiR-409-3p upregulation mitigated the role of circ_DOCK1 in proliferation and apoptosis of H2O2-treated HBVSMCs. MCL1 was targeted via miR-409-3p and downregulated via H2O2 treatment. Circ_DOCK1 overexpression enhanced MCL1 expression via modulating miR-409-3p. MiR-409-3p knockdown weakened H2O2-induced proliferation reduction and apoptosis promotion via regulating MCL1.ConclusionCirc_DOCK1 overexpression mitigated H2O2-caused proliferation inhibition and apoptosis promotion in HBVSMCs by modulating miR-409-3p/MCL1 axis.

Amyloid Beta Peptides and Th1 Cytokines Modulate Human Brain Vascular Smooth Muscle Tonic Contractile Capacity In Vitro: Relevance to Alzheimer's Disease?

Alzheimer’s Disease (AD) is a neurodegenerative condition characterized both by the presence of tau protein neurofibrillary tangles and amyloid beta (Aβ) containing extracellular “plaques”. The cleavage of amyloid precursor protein (APP) yields several Aβ peptides. Although Aβ toxicity to neurons has been described extensively, its effects on other components of the neurovasculature such as vascular smooth muscle cells have been less well characterized. AD is now also recognized as a neurovascular disease characterized by cerebral microbleeds and disturbances in autoregulation. AD is also a neuroinflammatory condition in which several proinflammatory cytokines are elevated and may contribute to the intensification of AD severity. Cerebral autoregulation (the mechanism by which brain blood flow is maintained despite changes in perfusion pressure) is extremely tightly controlled in the brain and shows disturbances in AD. The failure of autoregulation in AD may make the brain susceptible to cerebral microbleeds through a reduced capacity to limit blood flow when pressure is increased. Conversely, reduced vasodilation during low flow might could also exacerbate tissue hypoxia. Currently, whether and how Aβ peptides and inflammatory cytokines depress brain smooth muscle cell tonic contraction is not known, but could reveal important targets in the preservation of autoregulation which is disturbed in AD. We used a collagen gel contractility assay to evaluate the influence of Aβ25-35, Aβ1-40 and Aβ1-42 peptides and inflammatory cytokines on the tonic contractility of human brain vascular smooth muscle cells (HBVSMC) as an in vitro model of cerebral autoregulation. We found that 5 and 10 μM Aβ1-42 significantly depressed HBVSM contractility, while Aβ1-40 5–20 μM had no effect on contractility. Conversely, Aβ25-35 (1–50 μM) increased contractility. Interestingly, the inflammatory cytokines TNF-α (20 ng/mL), IL-1β (20 ng/mL) and IFN-γ (1000 U/mL) also depressed HBVSM tonic contractility alone and in combination. These data suggest that both the inflammatory milieu in AD as well as the abundance of Aβ peptides may promote autoregulatory failure and increase brain susceptibility to dysregulated perfusion and microbleeds which are an important and devastating characteristic of AD.

Interleukin Enhancer Binding Factor 2 Regulates Cell Viability and Apoptosis of Human Brain Vascular Smooth Muscle Cells.

The proliferation and migration of vascular smooth muscle cells (VSMCs) are involved in the pathogenesis of intracranial aneurysm (IA) formation and rupture. Interleukin enhancer binding factor 2 (ILF2) is known as the nuclear factor of activated T cells and regulates cell growth. This study was aimed to explore the effects of ILF2 on IA progression. Human brain VSMCs (hBVSMCs) were transfected with pCDNA3.1(+), pCDNA3.1(+)-ILF2, siRNA-negative control, and siRNA-ILF2. The transfection efficiency was then evaluated by determining ILF2 expression. The cell viability and apoptosis were determined using Cell Counting Kit-8 and Annexin V-FITC cell apoptosis assay kit, respectively. Real-time quantification PCR (RT-qPCR) was applied to measure the expression levels of apoptosis-related and inflammation-related genes. Finally, western blot was used to detect the expression level of Fas cell surface death receptor 95 (CD95) and Caspase 8. Overexpression of ILF2 could significantly increase cell viability and decrease cell apoptosis (P < 0.05), while knock-down of ILF2 showed opposite trends for hBVSMCs on cell viability and apoptosis (P < 0.05). RT-qPCR results showed that ILF2 knock-down downregulated the expression levels of BCL2 apoptosis regulator (BCL2), transcriptional regulator Myc-like (c-Myc), and caspase 1 (ICE) whereas upregulated the expression levels of CD95, p21, p53, and interleukin-13 (IL-13). Additionally, the protein expression levels of CD95 and Caspase 8 were significantly decreased after ILF2 overexpression while were significantly increased after ILF2 knock-down (P < 0.05). ILF2 knock-down may inhibit cell viability and promote cell apoptosis of hBVSMCs by regulating the expression levels of apoptosis-related genes and suppressing inflammatory response.

Induction of SPARC on Oxidative Stress, Inflammatory Phenotype Transformation, and Apoptosis of Human Brain Smooth Muscle Cells Via TGF-β1-NOX4 Pathway.

Secreted protein acidic and rich in cysteine (SPARC) has a close association with inflammatory response and oxidative stress in tissues and is widely expressed in intracranial aneurysms (IAs), especially in smooth muscle cells. Therefore, it is inferred that SPARC might be involved in the formation and development of IAs through the inflammatory response pathway or oxidative stress pathway. The aim of this study is to investigate the pathological mechanism of SPARC in oxidative stress, inflammation, and apoptosis during the formation of IAs, as well as the involvement of TGF-β1 and NOX4 molecules. Human brain vascular smooth muscle cells (HBVSMCs) were selected as experimental objects. After the cells were stimulated by recombinant human SPARC protein in vitro, the ROS level in the cells was measured using an ID/ROS fluorescence analysis kit combined with fluorescence microscope and flow cytometry. The related protein expression in HBVSMCs was measured using western blotting. The mitochondrial membrane potential change was detected using a mitochondrial membrane potential kit and laser confocal microscope. The mechanism was explored by intervention with reactive oxygen scavengers N-acetylcysteine (NAC), TGF-β1 inhibitor (SD-208), and siRNA knockout. The results showed that SPARC upregulated the expression of NOX4 through the TGF-β1-dependent signaling pathway, leading to oxidative stress and pro-inflammatory matrix behavior and apoptosis in HBVSMCs. These findings demonstrated that SPARC may promote the progression of IAs.

Ozanimod Protects Against Hypoxia plus Glucose Deprivation‐Induced Morphological Changes and Improves Cell Health in Human Brain Vascular Smooth Muscle

Vascular smooth muscle cells regulate arterial contractile tone, blood pressure, and flow in relation to specific metabolic demands during normal and pathophysiological (i.e. hypoxic or ischemic) conditions. The plasticity of vascular smooth muscle cells allows for phenotypic transitions from contractile to synthetic‐like dedifferentiated states during pathophysiological conditions which then can lead to the development and progression of vascular inflammation and blood vessel wall remodeling. Our previous studies demonstrated that hypoxia plus glucose deprivation (HGD), an in vitro model of ischemia, alters human vascular cell morphology and increases cell death. We further demonstrated that the selective sphingosine‐1‐phosphate receptor 1 (S1PR1) ligand, ozanimod, attenuated these HGD‐mediated responses. In this study, we characterized HGD mediated phenotypic transitions and assessed expression of proteins involved in contraction (myosin light chain kinase, MLCK) and structure formation (alpha‐smooth muscle actin, vimentin) following HGD exposure. We further investigated the effect of ozanimod HGD‐mediated phenotypic transitions by assessing protein levels and localization LC3B as an indicator of autophagocytosis. Using immunocytochemistry and standard immunoblotting, we determined the localization and expression levels of α‐smooth muscle actin, MLCK, vimentin, and LC3B protein during normoxia (21% O2, control) or HGD (1%O2) for 9 h in male human brain vascular smooth muscle cells (HBVSMC) at passage 6–7. We observed that LC3B protein, a vacuole marker associated with autophagy, is ubiquitously expressed in the cytoplasm of HBVSMC during normoxia. During HGD exposure, LC3B protein levels were increased and immunohistochemical staining demonstrated LC3B localization in what appears to be autophagic vacuole formation. Both responses were attenuated by ozanimod. Although alpha‐smooth muscle actin in HBVSMC was effective in visualization of morphological changes from spindle (contractile) to rhomboid (synthetic) shape following HGD, basal protein expression levels were not altered. Similarly, MLCK, an enzyme responsible for contractile protein phosphorylation, was not altered following HGD. Levels of the synthetic phenotype marker, vimentin, was basally expressed in HBVSMC, however, levels were not increased following HGD. In conclusion, although vacuole formation is prominent, indicating poor health, HBVSMCs retained a contractile phenotype during ischemic‐like conditions. Attenuation of HGD‐mediated LC3B via ozanimod suggests a novel mechanism associated with the beneficial effect of this selective S1PR1 ligand during ischemic‐like exposure.Support or Funding InformationAmerican Heart Association 19AIREA34480018 (RJG).

SPARC induces phenotypic modulation of human brain vascular smooth muscle cells via AMPK/mTOR-mediated autophagy

Secreted protein acidic and rich in cysteine (SPARC) was widely expressed in VSMCs of human IAs and could reduce the capability of self-repair. This indicates that SPARC may play a role in the promotion of IAs formation and progression, but the mechanism remains unclear. In this study, we further investigated whether SPARC could induce phenotypic modulation of Human Brain Vascular Smooth Muscle Cells (HBVSMCs) and sought to elucidate the role of SPARC-mediated autophagy involved in it. The results demonstrated that SPARC inhibited the expression of contractile genes in HBVSMCs and induced a synthetic phenotype. More importantly, SPARC significantly up-regulated multiple proteins including autophagy marker microtubule-associated protein light chain 3-II (LC3-II), Beclin-1, and autophagy-related gene 5(ATG5). Furthermore, SPARC could promote p62 degradation. The autophagy inhibitor 3- methyladenine (3-MA) significantly blocked SPARC-induced phenotypic modulation of HBVSMCs. We further sought to elucidate the molecular mechanism involved in SPARC-induced autophagy, and found that SPARC could activate the AMPK/mTOR signaling pathway in HBVSMCs. AMPK could be pharmacologically inhibited by Compound C (CC), which significantly decreased the phosphorylation of AMPK into p-AMPK, increased the phosphorylation of mTOR into p-mTOR, and decreased LC3-II, Beclin-1 and ATG5 levels. This suggested that activated AMPK/ mTOR signaling is related to SPARC-mediated autophagy. These results indicated that SPARC plays a role in the phenotypic modulation of HBVSMCs through autophagy activation by AMPK/mTOR signaling pathway.

Inhibition of angiotensin II-induced cerebrovascular smooth muscle cell proliferation by LRRC8A downregulation through suppressing PI3K/AKT activation.

Cerebrovascular smooth muscle cell proliferation is the major contributor to cerebrovascular remodeling and stroke. Chloride channels have been suggested to play an important role in the regulation of smooth muscle cell proliferation. This study aims to investigate the effect of leucine-rich repeat-containing 8A (LRRC8A), an essential component of volume-sensitive chloride channels, on cerebrovascular smooth muscle cell proliferation. The data showed that LRRC8A expression was increased in mouse brain artery during angiotensin II (AngII)-induced cerebrovascular remodeling. Similarly, AngII also increased the expression of LRRC8A in human brain vascular smooth muscle cells (HBVSMCs). Knockdown of LRRC8A by siRNA significantly inhibited AngII-induced the proliferation, migration, and invasion in HBVSMCs. The inhibition of HBVSMCs proliferation by LRRC8A downregulation appeared to be involved in suppression of cell-cycle transition. AngII-induced the decrease in p21 and p27 expression and the increase in CDK4 and cyclin D1 expression were attenuated by LRRC8A downregulation. Moreover, inhibition of LRRC8A suppressed AngII-induced PI3K/AKT activation and reactive oxygen species generation, but had no effect on JNK, ERK, and p38 phosphorylation. In addition, activation of PI3K/AKT-signaling pathways with specific agonists significantly abolished the effect of LRRC8A deficiency on HBVSMC proliferation. This present study demonstrates that knockdown of LRRC8A ameliorates AngII-induced cerebrovascular smooth muscle cell proliferation via inhibiting PI3K/AKT pathway, suggesting that LRRC8A may be a novel molecular target in the treatment of vascular remodeling and stroke.

Icariin ameliorates angiotensin II-induced cerebrovascular remodeling by inhibiting Nox2-containing NADPH oxidase activation.

Cerebrovascular smooth muscle cells (SMCs) hyperplasia is an important contributor to cerebrovascular remodeling during hypertension. The aim of present study was to investigate the effects of Icariin on cerebrovascular SMCs proliferation and remodeling and the underlying mechanisms. The results revealed that Icariin administration attenuated the enhanced basilar artery constriction in angiotensin II (AngII)-induced hypertension rat model, as well as the inhibition of basilar artery diameter reduction in response to AngII and phenylephrine. In addition, histological analyses showed that Icariin also significantly ameliorated basilar artery remodeling in AngII hypertensive rats. In human brain vascular SMCs (HBVSMCs), AngII-induced cell proliferation, migration and invasion were markedly inhibited by Icariin treatment. Moreover, Icariin treatment largely limited AngII-induced the increase of reactive oxygen species (ROS) production in HBVSMCs, which was closely associated with cell proliferation. Analysis of the mechanisms showed that Icariin decreased ROS production via inhibiting NADPH oxidase activity but not mitochondria-derived ROS production. Further, Icariin promoted Nox2 degradation and consequently reduced its protein expression. In conclusion, these findings demonstrate that Icariin attenuates cerebrovascular SMCs hyperplasia and subsequent remodeling through inhibiting Nox2-containing NADPH oxidase activation, suggesting Icariin may be a potential therapeutic agent to prevent the onset and progression of stroke.

ClC-2 knockdown prevents cerebrovascular remodeling via inhibition of the Wnt/\u03b2-catenin signaling pathway

BackgroundMishandling of intracellular chloride (Cl−) concentration ([Cl−]i) in cerebrovascular smooth muscle cells is implicated in several pathological processes, including hyperplasia and remodeling. We investigated the effects of ClC-2-mediated Cl− efflux on the proliferation of human brain vascular smooth muscle cells (HBVSMCs) induced by angiotensin II (AngII).MethodsCell proliferation and motility were determined using the CCK-8, bromodeoxyuridine staining, wound healing and invasion assays. ClC-2, PCNA, Ki67, survivin and cyclin D1 expression, and β-catenin and GSK-3β phosphorylation were examined using western blotting. Histological analyses were performed using hematoxylin and eosin staining and α-SMA staining.ResultsOur results showed that AngII-induced HBVSMC proliferation was accompanied by a decrease in [Cl−]i and an increase in ClC-2 expression. Inhibition of ClC-2 by siRNA prevented AngII from inducing the efflux of Cl−. AngII-induced HBVSMC proliferation, migration and invasion were significantly attenuated by ClC-2 downregulation. The inhibitory effects of ClC-2 knockout on HBVSMC proliferation and motility were associated with inactivation of the Wnt/β-catenin signaling pathway, as evidenced by inhibition of β-catenin phosphorylation and nuclear translocation, and decrease of GSK-3β phosphorylation and survivin and cyclin D1 expression. Recombinant Wnt3a treatment markedly reversed the effect of ClC-2 knockdown on HBVSMC viability. An in vivo study revealed that knockdown of ClC-2 with shRNA adenovirus ameliorated basilar artery remodeling by inhibiting Wnt/β-catenin signaling in AngII-treated mice.ConclusionThis study demonstrates that blocking ClC-2-mediated Cl− efflux inhibits AngII-induced cerebrovascular smooth muscle cell proliferation and migration by inhibiting the Wnt/β-catenin pathway. Our data indicate that downregulation of ClC-2 may be a viable strategy in the prevention of hyperplasia and remodeling of cerebrovascular smooth muscle cells.

Salidroside attenuates hypoxia/reoxygenation-induced human brain vascular smooth muscle cell injury by activating the SIRT1/FOXO3α pathway.

It has been reported that salidroside (SAL), a natural dietary isothiocyanate, exhibits neuroprotective roles in cerebral ischemia-reperfusion injury. However, to the best of our knowledge, its underlying protective mechanism remains unknown. Sirtuin 1 (SIRT1) is a class III histone deacetylase involved in a variety of cellular functions. SIRT1 has been identified as a mediator of cerebral ischemia and may induce neuroprotection by activating various intracellular downstream targets, such as forkhead box protein O3α (FOXO3α). Therefore, the present study aimed to investigate whether SAL protects human brain vascular smooth muscle cells (HBVSMC) against hypoxia/reoxygenation (H/R) injury, which is a cell model of cerebral ischemia-reperfusion injury, through regulating the SIRT1-activited signaling pathway. The present study revealed that H/R treatment significantly reduced the expression of SIRT1 protein in HBVSMCs. Additionally, pretreatment with SAL reversed the H/R-induced decrease in cellular viability, increased caspase-3 activity, the appearance of apoptotic cells and the apoptosis rate in HBVSMCs. SAL attenuated the H/R-induced decrease in the expression of SIRT1 and phosphorylated FOXO3α protein in HBVSMCs, suggesting that the protective role of SAL in H/R injury occurs via the SIRT1/FOXO3α pathway. Furthermore, sirtinol, a SIRT1-specific inhibitor, suppressed the inhibitory effects of SAL on H/R-induced cytotoxicity and apoptosis as indicated by the downregulation of cell viability and upregulation of caspase-3 activity and apoptosis rate induced by sirtinol treatment in HBVSMCs. The reversal effects of SAL on H/R-induced alternation of B-cell lymphoma (Bcl-2) and Bcl-2 associated X protein expression were also attenuated by sirtinol. These results suggest that SAL exhibits neuroprotective effects against H/R injury by activating the SIRT1/FOXO3α pathway, which may become a novel potential therapeutic target for the treatment of cerebral ischemic disease.

Varicella zoster virus-infected cerebrovascular cells produce a proinflammatory environment.

Objective:To test whether varicella zoster virus (VZV) infection of human brain vascular cells and of lung fibroblasts directly increases proinflammatory cytokine levels, consistent with VZV as a causative agent in intracerebral VZV vasculopathy and giant-cell arteritis (GCA).Methods:Conditioned supernatant from mock- and VZV-infected human brain vascular adventitial fibroblasts (HBVAFs), human perineurial cells (HPNCs), human brain vascular smooth muscle cells (HBVSMCs), and human fetal lung fibroblasts (HFLs) were collected at 72 hours postinfection and analyzed for levels of 30 proinflammatory cytokines using the Meso Scale Discovery Multiplex ELISA platform.Results:Compared with mock infection, VZV infection led to significantly increased levels of the following: interleukin-8 (IL-8) in all cell lines examined; IL-6 in HBVAFs, HPNCs, and HFLs, with no change in HBVSMCs; and vascular endothelial growth factor A in HBVAFs, HBVSMCs, and HFLs, with a significant decrease in HPNCs. Other cytokines, including IL-2, IL-4, IL-15, IL-16, TGF-b, Eotaxin-1, Eotaxin-3, IP-10, MCP-1, and granulocyte macrophage colony-stimulating factor, were also significantly altered upon VZV infection in a cell type–specific manner.Conclusions:VZV infection of vascular cells can directly produce a proinflammatory environment that may potentially lead to prolonged arterial wall inflammation and vasculitis. The VZV-mediated increase in IL-8 and IL-6 is consistent with that seen in the CSF of patients with intracerebral VZV vasculopathy, and the VZV-mediated increase in IL-6 is consistent with the cytokine's elevated levels in temporal arteries and plasma of patients with GCA.

Nox4 contributes to the hypoxia-mediated regulation of actin cytoskeleton in cerebrovascular smooth muscle

Ischemia/reperfusion and the resulting oxidative/nitrative stress impair cerebral myogenic tone via actin depolymerization. While it is known that NADPH oxidase (Nox) family is a major source of vascular oxidative stress; the extent and mechanisms by which Nox activation contributes to actin depolymerization, and equally important, the relative role of Nox isoforms in this response is not clear. Aim: To determine the role of Nox4 in hypoxia-mediated actin depolymerization and myogenic-tone impairment in cerebral vascular smooth muscle. Main methods: Control and Nox4 deficient (siRNA knock-down) human brain vascular smooth muscle cells (HBVSMC) were exposed to 30-min hypoxia/45-min reoxygenation. Nox2, Nox4, inducible and neuronal nitric oxide synthase (iNOS and nNOS) and nitrotyrosine levels as well as F:G actin were determined. Myogenic-tone was measured using pressurized arteriography in middle cerebral artery isolated from rats subjected to sham, 30-min ischemia/45-min reperfusion or ex-vivo oxygen glucose deprivation in the presence and absence of Nox inhibitors. Results: Nox4 and iNOS expression were significantly upregulated following hypoxia or ischemia/reperfusion. Hypoxia augmented nitrotyrosine levels while reducing F actin. These effects were nullified by inhibiting nitration with epicatechin or pharmacological or molecular inhibition of Nox4. Ischemia/reperfusion impaired myogenic-tone, which was restored by the selective inhibition of Nox4. Conclusion: Nox4 activation in VSMCs contributes to actin depolymerization after hypoxia, which could be the underlying mechanism for myogenic-tone impairment following ischemia/reperfusion.

Angiotensin-(1–7) counteracts the effects of Ang II on vascular smooth muscle cells, vascular remodeling and hemorrhagic stroke: Role of the NFкB inflammatory pathway

Angiotensin (Ang)-(1–7) is a potential vasoprotective peptide. In the present study, we investigated its counteractive effects to Ang II on vascular smooth muscle cells (VSMCs) and intracerebral hemorrhagic stroke (ICH) through inflammatory mechanism. In in vitro experiments, human brain VSMCs (HBVSMCs) were treated with vehicle, Ang II, Ang II+Ang-(1–7), Ang II+A-779 or Ang II+Ang-(1–7)+A-779 (Mas receptor antagonist). HBVSMC proliferation, migration and apoptosis were determined by methyl thiazolyltetrazolium, wound healing assay and flow cytometry, respectively. In in vivo experiments, C57BL/6 mice were divided into vehicle, Ang II, Ang II+Ang-(1–7), Ang II+A-779 or Ang II+Ang-(1–7)+A-779 groups before they were subjected to collagenase-induced ICH or sham surgery. Hemorrhage volume and middle cerebral artery (MCA) remodeling were determined by histological analyses. Levels of NFκB, inhibitor of κBα (IκBα), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein 1 (MCP-1) and interleukin (IL-8) were measured by western blot or ELISA. We found that 1) Ang II increased HBVSMC migration, proliferation and apoptosis, and increased the blood pressure (BP), neurological deficit score, MCA remodeling and hemorrhage volume in ICH mice. 2) Ang-(1–7) counteracted these effects of Ang II, which was independent of BP, with the down-regulation of NFκB, up-regulation of IκBα, and decreased levels of TNF-α, MCP-1 and IL-8. 3) The beneficial effects of Ang-(1–7) could be abolished by A-779. In conclusion, Ang-(1–7) counteracts the effects of Ang II on ICH via modulating NFκB inflammation pathway in HBVSMCs and cerebral microvessels.

Upregulation of relaxin after experimental subarachnoid hemorrhage in rabbits.

Background. Although relaxin causes vasodilatation in systemic arteries, little is known about its role in cerebral arteries. We investigated the expression and role of relaxin in basilar arteries after subarachnoid hemorrhage (SAH) in rabbits. Methods. Microarray analysis with rabbit basilar artery RNA was performed. Messenger RNA expression of relaxin-1 and relaxin/insulin-like family peptide receptor 1 (RXFP1) was investigated with quantitative RT-PCR. RXFP1 expression in the basilar artery was investigated with immunohistochemistry. Relaxin concentrations in cerebrospinal fluid (CSF) and serum were investigated with an enzyme-linked immunosorbent assay. Using human brain vascular smooth muscle cells (HBVSMC) preincubated with relaxin, myosin light chain phosphorylation (MLC) was investigated with immunoblotting after endothelin-1 stimulation. Results. After SAH, RXFP1 mRNA and protein were significantly downregulated on day 3, whereas relaxin-1 mRNA was significantly upregulated on day 7. The relaxin concentration in CSF was significantly elevated on days 5 and 7. Pretreatment with relaxin reduced sustained MLC phosphorylation induced by endothelin-1 in HBVSMC. Conclusion. Upregulation of relaxin and downregulation of RXFP1 after SAH may participate in development of cerebral vasospasm. Downregulation of RXFP1 may induce a functional decrease in relaxin activity during vasospasm. Understanding the role of relaxin may provide further insight into the mechanisms of cerebral vasospasm.

Involvement of Organic Cation Transporter-3 and Plasma Membrane Monoamine Transporter in Serotonin Uptake in Human Brain Vascular Smooth Muscle Cells

The serotonin (5-HT) uptake system is supposed to play a crucial part in vascular functions by “fine-tuning” the local concentration of 5-HT in the vicinity of 5-HT2 receptors in vascular smooth muscle cells. In this study, the mechanism of 5-HT uptake in human brain vascular smooth muscle cells (HBVSMCs) was investigated. [3H]5-HT uptake in HBVSMCs was Na+-independent. Kinetic analyses of [3H]5-HT uptake in HBVSMCs revealed a Km of 50.36 ± 10.2 mM and a Vmax of 1033.61 ± 98.86 pmol/mg protein/min. The specific serotonin re-uptake transporter (SERT) inhibitor citalopram, the specific norepinephrine transporter (NET) inhibitor desipramine, and the dopamine transporter (DAT) inhibitor GBR12935 inhibited 5-HT uptake in HBVSMCs with IC50 values of 97.03 ± 40.10, 10.49 ± 5.98, and 2.80 ± 1.04 μM, respectively. These IC50 values were 100-fold higher than data reported by other authors, suggesting that those inhibitors were not blocking their corresponding transporters. Reverse transcription-polymerase chain reaction results demonstrated the presence of mRNA for organic cation transporter (OCT)-3 and plasma membrane monoamine transporter (PMAT), but the absence of OCT-1, OCT-2, SERT, NET, and DAT. siRNA knockdown of OCT-3 and PMAT specifically attenuated 5-HT uptake in HBVSMCs. It is concluded that 5-HT uptake in HBVSMCs was mediated predominantly by a low-affinity and Na+-independent mechanism. The most probable candidates are OCT-3 and PMAT, but not the SERT.