Angiotensin II-induced Vascular Smooth Muscle Research Articles

Retracted: EZH2 Regulates ANXA6 Expression via H3K27me3 and Is Involved in Angiotensin II-Induced Vascular Smooth Muscle Cell Senescence.

[This retracts the article DOI: 10.1155/2022/4838760.].

Preparation and Characterization of Polysaccharides from Turpiniae Folium and Its Antioxidative, Anti-Inflammatory Activities and Antiproliferative Effect on VSMCs.

Turpiniae Folium, the dried leaves of Turpinia arguta Seem., is a kind of historic traditional Chinese medicine. Here, based on our previous study, we extracted the Turpiniae Folium polysaccharides (TFP) and isolated three polysaccharide fractions from TFP. Then, TFP and one of the major polysaccharide fractions (TFP-1a) were identified through HPLC, HPGPC, and ATR-FTIR. Furthermore, the evaluations of their antioxidative, anti-inflammatory activities and inhibitory effect on angiotensin II-induced vascular smooth muscle cells (VSCMs) proliferation in vitro were conducted. Both TFP and TFP-1a showed strong hydroxyl radical scavenging, DPPH radical scavenging, and Fe2+ chelating activities, and exerted strong anti-inflammatory activity. Moreover, TFP and TFP-1a also possessed a strong inhibitory effect on Ang II-induced VSCMs proliferation. On these premises, we inferred that TFP and TFP-1a could be potential and promising natural antioxidants, anti-inflammatory agents, and implicated to treat cardiovascular disease.

Quercetin inhibits angiotensin II-induced vascular smooth muscle cell proliferation and activation of JAK2/STAT3 pathway: A target based networking pharmacology approach.

The rapid growth of vascular smooth muscle cells (VSMCs) represents crucial pathological changes during the development of hypertensive vascular remodeling. Although quercetin exhibits significantly therapeutic effects on antihypertension, the systematic role of quercetin and its exact mode of action in relation to the VSMCs growth and its hypertension-related networking pharmacology is not well-documented. Therefore, the effect of quercetin was investigated using networking pharmacology followed by in vitro strategies to explore its efficacy against angiotensin II (Ang II)-induced cell proliferation. Putative genes of hypertension and quercetin were collected using database mining, and their correlation was investigated. Subsequently, a network of protein-protein interactions was constructed and gene ontology (GO) analysis was performed to identify the role of important genes (including CCND1) and key signaling pathways [including cell proliferation and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway]. We therefore further investigated the effects of quercetin in Ang II-stimulated VSMCs. This current research revealed that quercetin significantly reduced the cell confluency, cell number, and cell viability, as well as expression of proliferating cell nuclear antigen (PCNA) in Ang II-stimulated VSMCs. Mechanistic study by western blotting confirmed that quercetin treatment attenuated the activation of JAK2 and STAT3 by reducing its phosphorylation in Ang II stimulated VSMCs. Collectively, the current study revealed the inhibitory effects of quercetin on proliferation of Ang II stimulated VSMCs, by inhibiting the activation of JAK2/STAT3 signaling might be one of underlying mechanisms.

EZH2 Regulates ANXA6 Expression via H3K27me3 and Is Involved in Angiotensin II-Induced Vascular Smooth Muscle Cell Senescence.

Objectives Abdominal aortic aneurysm (AAA) has a high risk of rupture of the aorta and is one of the leading causes of death in older adults. This study is aimed at confirming the influence and mechanism of the abnormally expressed ANXA6 gene in AAA. Methods Clinical samples were collected for proteome sequencing to screen for differentially expressed proteins. An Ang II-induced vascular smooth muscle cell (VSMC) aging model as well as an AAA animal model was used. Using RT-qPCR to detect the mRNA levels of EZH2, ANXA6, IK-6, and IL-8 in cells and tissues were assessed. Western blotting and immunohistochemistry staining were used apply for the expression of associated proteins in cells and tissues. SA-β-gal staining, flow cytometry, and DHE staining were used to detect senescent cells and the level of ROS. The cell cycle was assessed by flow cytometry. Arterial pathology was observed by HE staining. The aging of VSMCs in arterial tissue was assessed by coimmunofluorescence for α-SMA and p53. Results There were 24 differentially expressed proteins in the AAA clinical samples, including 10 upregulated protein and 14 downregulated protein, and the differential expression of ANXA6 was associated with vascular disease. Our study found that ANXA6 was highly expressed and EZH2 was lowly expressed in an Ang II-induced VSMC aging model. Knockdown of ANXA6 or overexpression of EZH2 inhibited Ang II-induced ROS, inhibited cell senescence, decreased Ang II evoked G1 arrest, and increased cells in G2 phase, while overexpression of ANXA6 played the opposite role. Overexpression of EZH2 inhibited ANXA6 expression by increasing H3K27me3 modification at the ANXA6 promoter. Simultaneous overexpression of EZH2 and the protective effect of EZH2 on cell senescence were partially reversed by ANXA6. Similarly, ANXA6 was highly expressed and EZH2 was lowly expressed in an Ang II-induced AAA animal model. Knockdown of ANXA6 and overexpression of EZH2 alleviated Ang II-induced VSMC senescence and inhibited AAA progression, while simultaneous overexpression of EZH2 and ANXA6 partially reversed the protective effect of EZH2 on AAA. Conclusion EZH2 regulates the ANXA6 promoter H3K27me3 modification, inhibits ANXA6 expression, alleviates Ang II-induced VSMC senescence, and inhibits AAA progression.

Ginsenoside Rh1 Inhibits Angiotensin II-Induced Vascular Smooth Muscle Cell Migration and Proliferation through Suppression of the ROS-Mediated ERK1/2/p90RSK/KLF4 Signaling Pathway.

Vascular smooth muscle cell (VSMC) proliferation and migration play key roles in the progression of atherosclerosis and restenosis. A variety of ginsenosides exert various cardiovascular benefits. However, whether and how ginsenoside Rh1 (Rh1) inhibits VSMC dysfunction remain unclear. Here, we investigated the inhibitory effects of Rh1 on rat aortic smooth muscle cell (RASMC) migration and proliferation induced by angiotensin II (Ang II) and the underlying mechanisms. Cell proliferation and migration were evaluated using sulforhodamine B and wound-healing assay. The molecular mechanisms were investigated using Western blotting, quantitative reverse-transcription polymerase chain reaction analysis, immunofluorescence staining, and luciferase assay. Reactive oxygen species (ROS) production was measured using dihydroethidium and MitoSOX staining. We found that Rh1 dose-dependently suppressed Ang II-induced cell proliferation and migration. Concomitantly, Ang II increased protein levels of osteopontin, vimentin, MMP2, MMP9, PCNA, and cyclin D1, while these were reduced by Rh1 pretreatment. Notably, Ang II enhanced both the protein expression and promoter activity of KLF4, a key regulator of phenotypic switching, whereas pretreatment with Rh1 reversed these effects. Mechanistically, the effects of Rh1 on VSMC proliferation and migration were found to be associated with inhibition of ERK1/2/p90RSK signaling. Furthermore, the inhibitory effects of Rh1 were accompanied by inhibition of ROS production. In conclusion, Rh1 inhibited the Ang II-induced migration and proliferation of RASMCs by suppressing the ROS-mediated ERK1/2/p90RSK signaling pathway.

Chromodomain Helicase DNA Binding Protein 1-like, a negative regulator of Forkhead box O3a, promotes the proliferation and migration of Angiotensin II-induced vascular smooth muscle cells

ABSTRACT Essential hypertension (EH) represents a major risk factor for stroke, myocardial infarction, and heart failure. Dysregulated proliferation and migration of vascular smooth muscle cells (VSMCs) play an important role in pathogenesis of EH. This study aims to investigate the effect of Chromodomain Helicase DNA Binding Protein 1-Like (CHD1L) on Angiotensin II (AngII)-induced VSMCs injury and reveal the underlying mechanism. The expression of CHD1L in EH patients was determined by bioinformatics analysis, and then it was silenced in AngII-induced VSMCs to detect the changes in cellular functions including proliferation, migration, invasion and phenotypic switching via CCK-8, EDU staining, wound healing, transwell and Western blot assays, respectively. Inflammation and oxidative stress were also measured by detecting related markers via commercial kits. After confirming the binding sites between forkhead box O3A (FOXO3a) and CHD1L and their negative association by bioinformatics analysis, FOXO3a was further silenced, and the cellular functions were assessed again to reveal the underlying mechanism. Results showed that CHD1L was highly expressed in EH, and interference of CHD1L suppressed the proliferation, migration, invasion and phenotypic switching in VSMCs. Inflammation and oxidative stress were also restrained by CHD1L knockdown. After validating the negative role of FOXO3a in regulating CHD1L, it was found that FOXO3a abrogated the effect of CHD1L knockdown on the cellular functions of AngII-induced VSMCs. In conclusion, FOXO3a suppresses the proliferation and migration of AngII-induced VSMCs by down-regulating CHD1L.

PKCβ inhibitor blocked angiotensin II-induced vascular smooth muscle proliferation and oxidative stress

PKCβ inhibitor blocked angiotensin II-induced vascular smooth muscle proliferation and oxidative stress

Gly14]-Humanin inhibits an angiotensin II-induced vascular smooth muscle cell phenotypic switch via ameliorating intracellular oxidative stress.

Angiotensin II (AngII) is involved in the pathogenesis of hypertensive artery remodeling by inducing a phenotypic switch in vascular smooth muscle cells [Gly14]-Humanin (HNG), a humanin analogue, exerts potent cytoprotective effects both in vitro and in vivo. This study aimed to investigate the effects of HNG on an AngII-induced phenotypic switch in VSMCs and the potential mechanisms underlying these effects. The roles of [Gly14]-Humanin in AngII-stimulated VSMCs proliferation and migration was detected by CCK-8 assay, Cell cycle analysis, wound healing assay, trsnswell assay and western blot. The mechanism by which [Gly14]-Humanin regulates VSMC phenotypic switch was determined by intracellular oxidative stress detection, transcriptomic analysis and qRT-PCR. The results showed that HNG inhibited AngII-induced VSMC proliferation and migration and maintained a stable VSMC contractile phenotype. In addition, HNG reduced the level of AngII-induced oxidative stress in vascular smooth muscle cells. This process could be accomplished by inhibiting nicotinamide adenine dinucleotide phosphate oxidase activity. In conclusion, the results suggested that HNG ameliorated intracellular oxidative stress by inhibiting NAD(P)H oxidase activity, thereby suppressing the AngII-induced VSMC phenotype switch. Thus, HNG is a potential drug to ameliorate artery remodeling in hypertension.

Angptl2 gene knockdown is critical for abolishing angiotensin II-induced vascular smooth muscle cell proliferation and migration.

Angiopoietin-like 2 (Angptl2) is reported to be correlated with cardiovascular diseases, but its role in hypertension remains unclear. This study aimed to investigate the role and potential mechanism of Angptl2 in hypertension. Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were used to detect the expression of Angptl2. Angiotensin II (Ang II) stimulates vascular smooth muscle cells (VSMCs) to mimic hypertension in vitro. Cell proliferation, migration, and invasion abilities were determined using CCK-8, cell colony formation, wound healing, and transwell assays, respectively. The cell cycle distribution was detected by flow cytometry. The expression of Ki67 was determined by immunofluorescence, and protein expression was measured using western blotting. Angptl2 was found to be elevated in hypertensive rats in vivo and in VSMCs upon Ang II stimulation in vitro. Angptl2 knockdown suppressed cell proliferation, colony formation, cell migration, and invasion as well as the downregulation of Ki67. Additionally, Angptl2 knockdown hindered cell cycle progression and downregulated protein expression of CDK2/4 and cyclin D1, but upregulated p21 expression. Furthermore, Angptl2 knockdown inhibited activation of the NLRP3 inflammasome. Our findings suggest that Angptl2 knockdown suppresses VSMC proliferation, migration, and invasion induced by Ang II. Angptl2 may be a new target for vascular remodeling in hypertension.

The Protective Effects of Tripeptides VPP and IPP against Small Extracellular Vesicles from Angiotensin II-Induced Vascular Smooth Muscle Cells Mediating Endothelial Dysfunction in Human Umbilical Vein Endothelial Cells.

Endothelial dysfunction is a common disorder of vascular homeostasis in hypertension characterized by oxidative stress, malignant migration, inflammatory response, and active adhesion response of endothelial cells. The extracellular vesicles (EVs), a vital participant in vascular cell communication, have been considered responsible for vascular disease progression. However, the potential mechanism of antihypertensive peptides against the EVs-induced endothelial dysfunction is still unclear. In this study, we investigated whether the antihypertensive peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) ameliorate the effects of EVs from Ang II-induced vascular smooth muscles (VSMCs) on the endothelial dysfunction. The dihydroethidium staining, wound healing assay, 3D cell culture, and co-culture with U937 monocyte were used to investigate the oxidant/antioxidant balance, migration, tube formation, and cell adhesion in EV-induced human umbilical vein endothelial cells. VPP and IPP treatment reduced the level of reactive oxygen species and EV-induced expression of adhesion molecules and restored the ability of tube formation by upregulating endothelial nitric oxide synthase expression. VPP and IPP reduced the protein levels of IL-6 to 227.34 ± 10.56 and 273.84 ± 22.28 pg/mL, of IL-1β protein to 131.56 ± 23.18 and 221.14 ± 13.8 pg/mL, and of MCP-1 to 301.48 ± 19.75 and 428.68 ± 9.59 pg/mL. These results suggested that the VPP and IPP are potential agents that can improve the endothelial dysfunction caused by EVs from Ang II-induced VSMCs.

DNA N6-methyladenine modification in hypertension.

DNA methylation has a role in the pathogenesis of essential hypertension. DNA N6-methyladenine (6mA) modification as a novel adenine methylation exists in human tissues, but whether it plays a role in hypertension development remains unclear. Here, we reported that the global 6mA DNA level in leukocytes was significantly reduced in patients with hypertension and was reversed with successful treatment. Age, systolic blood pressure, and serum total cholesterol and high-density lipoprotein levels were associated with decreased leukocyte 6mA DNA level. Elevated ALKBH1 (AlkB homolog 1), a demethylase of 6mA, level mediated this dynamic change in 6mA level in leukocytes and vascular smooth muscle cells in hypertension mouse and rat models. Knockdown of ALKBH1 suppressed angiotensin II-induced vascular smooth muscle phenotype transformation, proliferation and migration. ALKBH1-6mA directly and negatively regulated hypoxia inducible factor 1 α (HIF1α), which responded to angiotensin II-induced vascular remodeling. Collectively, our results demonstrate a potential epigenetic role for ALKBH1-6mA regulation in hypertension development, diagnosis and treatment.

MicroRNA-27a regulates angiotensin II-induced vascular smooth muscle cell proliferation and migration by targeting α-smooth muscle-actin in vitro

Angiotensin II (Ang II) modulates VSMCs phenotypic switch that play a critical role in the cardiovascular diseases. MicroRNA-27a (miR-27a) has been proven to be involved in regulating vascular remodeling; however, the functional role of miR-27a in VSMCs in response to Ang II stimulation need to be elucidated. Cell proliferation and migration were measured by Cell counting kit-8 (CCK-8), BrdU incorporation and scratch wound assay in VSMCs transfected with miR-27a or its inhibitor. The target of miR-27a was confirmed using bioinformatics analysis and luciferase reporter assay. Ang II treatment time-dependently increased proliferation and migration of VSMCs accompanied with downregulation of α-smooth muscle-actin (α-SMA) and upregulation of miR-27a expression. Moreover, knockdown of miR-27a in VSMCs significantly attenuated Ang II-induced cell proliferation and migration, whereas this effect was aggravated by overexpression of miR-27a. A potential mechanistic analysis revealed that miR-27a directly targeted α-SMA, which mediated miR-27a-induced cell proliferation and migration. In conclusion, these results indicate that miR-27a acts as a novel regulator of Ang II-induced proliferation and migration by directly targeting α-SMA expression in VSMCs in vitro, and may be a potential therapeutic target for treating vascular diseases.

Klotho Inhibits Proliferation and Migration of Angiotensin II-Induced Vascular Smooth Muscle Cells (VSMCs) by Modulating NF-κB p65, Akt, and Extracellular Signal Regulated Kinase (ERK) Signaling Activities.

BackgroundIt has been proven that phenotype shifting, from the contractile phenotype to the synthetic phenotype, of vascular smooth muscle cells (VSMCs), plays an important role in vascular diseases such as atherosclerosis, restenosis, and hypertension. Recently, accumulating evidence suggests that Klotho is associated with many cardiovascular diseases or damage. Through the estimation of the proliferation and migration of Ang II-induced VSMCs and the related intracellular signal transduction pathways, we researched the effects of Klotho on phenotype modulation in this study.Material/MethodsA rat vascular smooth muscle cell line was grown in vitro with or without Ang II or Klotho, and cell proliferation and migration were evaluated.ResultsThe dose-dependent inhibition of Ang II-induced proliferation and migration by Klotho was shown in VSMCs. The phenotype modulation was inhibited by Klotho co-treatment; this co-treatment promoted the expression of contractile phenotype marker proteins, including SM22α, and also the proliferation phenotype marker protein PCNA compared with Ang II alone, which was suppressed, and activated VSMCs. Furthermore, by reducing the expression of G0/G1-specific regulatory proteins such as cyclin D1, cyclin-dependent kinase (CDK) 4, cyclin E, and CDK2, cell cycle arrest was induced by Klotho at G0/G1 phase. Although Ang II strongly stimulated NF-κB, p65, Akt, and ERK phosphorylation, these activation events were diminished by co-treatment with Ang II and Klotho.ConclusionsPhenotype modulation of Ang II-induced VSMCs and stimulation of the NF-κB, p65, Akt, and ERK signaling pathways were inhibited by Klotho, which suggests that Klotho may play an important role in the phenotype modulation of VSMCs.

Valsartan inhibits angiotensin II-induced vascular smooth muscle cell apoptosis

Objective To investigate the impacts of valsartan on cell apoptosis induced by angiotensin Ⅱ in vascular smooth muscle cells, and discuss whether the mechanism is relevant to AMP-Activated Protein Kinases. Methods Vascular smooth muscle cells (A7r5) were designated to 5 groups: ①control(DMSO) group, ②Angiotensin Ⅱ(Ang Ⅱ) 100 μmol/L group, ③Angiotensin Ⅱ 100 μmol/L+ valsartan 10 μmol/L group, ④Angiotensin Ⅱ 100 μmol/L+ valsartan 10 μmol/L+ compound C 1 μmol/L group, ⑤Angiotensin Ⅱ 100 μmol/L+ 5-Aminoimidazole-4earboxamide-ribo-nucle-oside(AICAR) 100 μmol/L group, after 24h incubation, the intracellular activity of Caspase 3 was measured by spectrophotometry, the cell apoptosis were enumerated by low cytometry, the intracellular AMP-Activated Protein Kinases (AMPK) phosphorylation and total expression quantity were examined by western blot, the intracellular reactive oxygen species (ROS) was measured by fluorescent probe DCFH-DA, the intracellular activity of total superoxide dismutase(SOD) was measured by WST-1 method, the intracellular activity of Malondialdehyde (MDA) was measured by TBA method. Two groups were compared by using Student t test. Differences among multiple groups were evaluated by ANOVA. Results Compared with control group, the cell apoptosis of Angiotensin Ⅱ group was increased[(45.46±15.40)% vs.(1.88±3.28)%, P=0.002], the synthesis of ROS was increased [(9.24±0.46)vs.(1.00±0.00), P<0.01], the activity of Caspase 3 was increased [(35.03±3.54)vs.(13.33±1.79), P<0.01], the activity of MDA was increased [(4.32±0.73)vs.(2.05±0.18), P<0.01)], the phosphorylation of AMPK was decreased, the activity of SOD was decreased [(90.29±14.73)vs.(136.02±18.82), P=0.001]; compared with Angiotensin Ⅱ group, the cell apoptosis of Angiotensin Ⅱ+ valsartan group and Angiotensin Ⅱ+ AICAR group were decreased[(24.91±8.46)% vs.(45.46±15.40)%, P=0.031]; [(27.90±4.39)% vs.(45.46±15.40)%, P=0.038], the synthesis of ROS was decreased[(2.37±0.05)vs.(9.24±0.46), P<0.01]; [(2.79±0.31)vs.(9.24±0.46), P<0.01], the activity of Caspase 3 was decreased [(18.08±2.69)vs.(35.03±3.54), P<0.01]; [(27.83±3.56)vs.(35.03±3.54), P=0.002], the activity of MDA were decreased [(3.25±0.55)vs.(4.32±0.73), P=0.017]; [(3.46±0.60)vs.(4.32±0.73), P=0.047], the phosphorylation of AMPK was increased, the activity of SOD was increased [(140.71±20.27)vs.(90.29±14.73), P<0.01]; [(116.73±17.96)vs.(90.29±14.73), P=0.029]; compared with Angiotensin Ⅱ+ valsarntan group, the cell apoptosis of Angiotensin Ⅱ+ valsartan+ compound C group was increased[(43.84±12.00)% vs.(24.91±8.46)%, P=0.043], the synthesis of ROS was increased [(4.64±0.15)vs.(2.37±0.05), P<0.01], the activity of Caspase 3 was increased[(25.64±3.52)vs. (18.08±2.69), P=0.011], the activity of MDA was increased[(5.12±0.92)vs.(3.25±0.55), P<0.01], the phosphorylation of AMPK was decreased, the activity of SOD was decreased [(99.48±16.59)vs.(90.29±14.73), P=0.002)]. Conclusions Valsartan could inhibit angiotensin Ⅱ-induced vascular smooth muscle cell apoptosis via activating AMPK, suppressing the synthesis of ROS and the activity of MDA, elevating the activity of SOD. Key words: Valsartan; Angiotensin Ⅱ; Vascular smooth muscle cell; Apoptosis; AMP-Activated Protein Kinases; Reactive oxygen species; Total superoxide dismutase; Malondialdehyde

Polyacetylenic Oleanane-Type Triterpene Saponins from the Roots of Panax japonicus.

Three new polyacetylenic oleanane-type triterpenoids, baisanqisaponins A-C (1-3), and one new oleanane-type triterpenoid, chikusetsusaponin-V ethyl ester (4), together with 19 known compounds (5-23), were isolated from the roots of Panax japonicus. The structures were elucidated on the basis of spectroscopic analyses and chemical methods. Compounds 1-3 feature a rare panaxytriol group containing a polyacetylene on the saponin skeleton. Neuroprotective activity was evaluated for compounds 1-17, and angiotensin II-induced vascular smooth muscle cell proliferation inhibition was tested for compounds 5-7 and 10-12.

Estrogenic compound attenuates angiotensin II-induced vascular smooth muscle cell proliferation through interaction between LKB1 and estrogen receptor α

The prevalence rate of cardiovascular disease is higher for males than females, and estradiol (E2) induces AMP-activated protein kinase (AMPK) activation, which is known to regulate proliferation of VSMC. We identified the estrogenic properties of nordihydroguaiaretic acid (NDGA, a lignan phytoestrogen) that inhibit VSMC proliferation and explored the underlying mechanisms. Both the phosphorylation and expression of LKB1 were increased by NDGA. In addition, NDGA significantly attenuated angiotensin II (Ang II)-induced VSMC proliferation. To elucidate the estrogenic effects, we confirmed that NDGA increased estrogen receptor α (ERα) expression, similar to treatment with E2 and estriol (E3). Furthermore, tamoxifen and ERα siRNA obstructed the effects of NDGA including ERα expression, AMPK phosphorylation and both LKB1 phosphorylation and expression. VSMC proliferation was restored by tamoxifen and ERα siRNA. LKB1 siRNA also reversed the NDGA-mediated inhibition of VSMC proliferation. The estrogenic activity of NDGA induced LKB1 translocation from nucleus to cytosol, and tamoxifen obstructed LKB1 translocation. The absence of LKB1 completely abolished the increase of ERα expression induced by NDGA. Taken together, the beneficial effects of estrogenic compound (E2 and NDGA) on inhibition of VSMC proliferation are mediated by interaction between LKB1 and ERα, suggesting a potential mechanism for females having less cardiovascular disease.

Activation of Nrf2 contributes to the protective effect of Exendin-4 against angiotensin II-induced vascular smooth muscle cell senescence.

Oxidative stress and impaired antioxidant defense are believed to be contributors to the cardiovascular aging process. The transcription factor nuclear factor-E2-related factor 2 (Nrf2) plays a key role in orchestrating cellular antioxidant defenses and maintaining redox homeostasis. Our previous study showed that Exendin-4, a glucagon-like peptide-1 analog, alleviates angiotensin II (ANG II)-induced vascular smooth muscle cell (VSMC) senescence by inhibiting Rac1 activation via cAMP/PKA (Zhao L, Li AQ, Zhou TF, Zhang MQ, Qin XM. Am J Physiol Cell Physiol 307: C1130-C1141, 2014). The objective of this study is to investigate if Nrf2 mediates the antisenescent effect of Exendin-4 in ANG II-induced VSMCs. Here we report that Exendin-4 triggered Nrf2 nuclear translocation, a downstream target of cAMP-responsive element-binding protein (CREB) and expressions of antioxidant genes heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO-1) in a dose- and time-dependent manner. In addition, knock-down of Nrf2 attenuated the inhibitory effects of Exendin-4 on ANG II-induced superoxidant generation and VSMC senescence. PKA/CREB pathway participated in the upregulations of HO-1 and NQO-1 induced by Exendin-4. Notably, our study revealed that Exendin-4 dose-dependently increased the acetylation of Nrf2 and the recruitment of transcriptional coactivator CREB binding protein (CBP) to Nrf2. The Exendin-4-induced Nrf2 transactivation was diminished in the presence of CBP small interfering RNA. Microscope imaging of Nrf2, as well as immunoblotting for Nrf2, showed that the Exendin-4-evoked Nrf2 acetylation favored its nuclear retention. Importantly, CBP silencing attenuated the suppressing effects of Exendin-4 on ANG II-induced VSMC senescence and superoxidant production. In conclusion, these results provide a mechanistic insight into how Nrf2 signaling mediates the antisenescent and antioxidative effects induced by Exendin-4 in VSMCs.

MicroRNA-761 inhibits Angiotensin II-induced vascular smooth muscle cell proliferation and migration by targeting mammalian target of rapamycin

Aberrant vascular smooth muscle cell (VSMC) proliferation and migration are a major pathological phenomenon in vascular disease characterized by intimal thickening. The important role of the mammalian target of rapamycin (mTOR) signaling in VSMC proliferation has been previously reported. Consequently, down-regulation of mTOR pathway may be an effective way of controlling excessive VSMC proliferation. Since microRNAs (miRNA) are newly emerging regulators of virtually all the biological processes including cellular proliferation, miRNAs targeting mTOR pathway may be utilized to suppress aberrant VSMC proliferation during pathologic conditions. Thus, in the present study, we screened miRNAs targeting mTOR, and we identified miR-761 as a new mTOR targeting miRNA. Luciferase assay using luciferase vector containing 3'UTR of mTOR indicated that miR-761 directly targets mTOR mRNA leading to suppression of mTOR protein expression. Our data also indicate that miR-761 expression decreases during angiotensin II (AngII)-induced proliferation of VSMCs, and exogenous miR-761 delivery effectively inhibit the AngII-induced VSMC proliferation. Additionally, the results of migration tests demonstrate that down-regulation of mTOR using exogenous miR-761 suppresses AngII-induced migration of VSMCs as well. Taken together, the present study provided evidence that miR-761 can be a potent anti-proliferative agent for vascular diseases such as atherosclerosis and restenosis, and warrants further studies to validate the effectiveness of miR-761 in vivo.

Adiponectin Attenuates Angiotensin II-Induced Vascular Smooth Muscle Cell Remodeling through Nitric Oxide and the RhoA/ROCK Pathway.

Introduction: Adiponectin (APN), an adipocytokine, exerts protective effects on cardiac remodeling, while angiotensin II (Ang II) induces hypertension and vascular remodeling. The potential protective role of APN on the vasculature during hypertension has not been fully elucidated yet. Here, we evaluate the molecular mechanisms of the protective role of APN in the physiological response of the vascular wall to Ang II.Methods and Results: Rat aortic tissues were used to investigate the effect of APN on Ang II-induced vascular remodeling and hypertrophy. We investigated whether nitric oxide (NO), the RhoA/ROCK pathway, actin cytoskeleton remodeling, and reactive oxygen species (ROS) mediate the anti-hypertrophic effect of APN. Ang II-induced protein synthesis was attenuated by pre-treatment with APN, NO donor S-nitroso-N-acetylpenicillamine (SNAP), or cGMP. The hypertrophic response to Ang II was associated with a significant increase in RhoA activation and vascular force production, which were prevented by APN and SNAP. NO was also associated with inhibition of Ang II-induced phosphorylation of cofilin. In addition, immunohistochemistry revealed that 24 h Ang II treatment increased the F- to G-actin ratio, an effect that was inhibited by SNAP. Ang II-induced ROS formation and upregulation of p22phox mRNA expression were inhibited by APN and NO. Both compounds failed to inhibit Nox1 and p47phox expression.Conclusion: Our results suggest that the anti-hypertrophic effects of APN are due, in part, to NO-dependent inhibition of the RhoA/ROCK pathway and ROS formation.

High sodium augments angiotensin II-induced vascular smooth muscle cell proliferation through the ERK 1/2-dependent pathway

Angiotensin II (Ang II)-induced vascular injury is exacerbated by high-salt diets. This study examined the effects of high-sodium level on Ang II-induced cell proliferation in rat vascular smooth muscle cells (VSMCs). The cells were cultured in a standard medium containing 137.5 mmol l(-1) of sodium. The high-sodium medium (140 mmol l(-1)) contained additional sodium chloride. Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation was determined by western blot analysis. Cell proliferation was evaluated by [(3)H]-thymidine incorporation. Ang II (100 nmol l(-1)) significantly increased ERK 1/2 phosphorylation and cell proliferation in the both medium containing standard sodium and high sodium. High-sodium level augmented Ang II-induced ERK 1/2 phosphorylation and cell proliferation compared with standard sodium. Pre-treatment with candesartan (1 μmol l(-1), Ang II type 1 receptor blocker) or PD98095 (10 μmol l(-1), ERK kinase iinhibitor) abolished the proliferative effect induced by high sodium/Ang II. Pre-treatment with 5-N,N-hexamethylene amiloride (30 μmol l(-1), Na(+)/H(+) exchanger type 1 (NHE-1) inhibitor), but not SN-6 (10 μmol l(-1), Na(+)/Ca(2+) exchanger inhibitor) or ouabain (1 mmol l(-1), Na(+)/K(+)-ATPase inhibitor) attenuated ERK 1/2 phosphorylation or cell proliferation. Osmotic pressure or chloride had no effect on Ang II-induced proliferative changes. High-sodium level did not affect Ang II receptor expression. Ang II increased intracellular pH via NHE-1 activation, and high-sodium level augmented the pH increase induced by Ang II. These data suggest that high-sodium level directly augments Ang II-induced VSMC proliferation through NHE-1- and ERK 1/2-dependent pathways and may offer new insights into the mechanisms of vascular remodeling by high-sodium/Ang II.