Angiotensin II-induced Smooth Muscle Cell Research Articles

Dexmedetomidine protects cells from Angiotensin II-induced smooth muscle cell phenotype switch and endothelial cell dysfunction

ABSTRACT Abdominal aortic aneurysm (AAA) is a vascular disorder greatly threatening life of the elderly population. Dexmedetomidine (DEX), an α2-adrenergic receptor agonist, has been shown to suppress AAA development. Nevertheless, the signaling pathways that might be mediated by DEX in AAA has not been clarified. Vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) were treated with Angiotensin II (Ang II) to mimic AAA in vitro. BrdU, wound healing, and Transwell assays were utilized for measuring VSMC proliferation and migration. Western blotting was used for evaluating protein levels of contractile VSMC markers, collagens and matrix metalloproteinases (MMPs) in VSMCs as well as apoptosis- and HMGB1/TLR4/NF-κB signaling-related markers in ECs. Cell adhesion molecule expression and monocyte-endothelial adhesion were assessed by immunofluorescence staining and adhesion assays. Flow cytometry was implemented for analyzing EC apoptosis. Hematoxylin-eosin staining and ELISA were used to detect the effect of DEX in vivo. In this study, DEX inhibited Ang II-evoked VSMC phenotype switch and extracellular matrix degradation. DEX suppressed the inflammatory response and apoptosis of ECs induced by Ang II. DEX inhibited HMGB1/TLR4/NF-κB signaling pathway in Ang II-treated ECs. DEX attenuated Ang II-induced AAA and inflammation in mice. Overall, DEX ameliorates Ang II-induced VSMC phenotype switch, and inactivates HMGB1/TLR4/NF-κB signaling pathway to alleviate Ang II-induced EC dysfunction.

KLF4 prevented angiotensin II-induced smooth muscle cell senescence by enhancing autophagic activity.

Vascular aging is an important risk factor for various cardiovascular diseases. Transcription factor krüppel-like factor 4 (KLF4) could regulate the phenotypic transformation of the vascular smooth muscle cell (VSMC) in the pathogenesis of aortic diseases. The present study aimed to explore the role and mechanism of KLF4 in angiotensin II (Ang II)-induced VSMC senescence. The VSMC senescence mouse model was induced by sustained release of Ang II (1.0μg/kg/min) for 4 weeks. The premature senescent VSMCs were induced by Ang II (0.1μmol/L) for 72 h. Cellular senescence was measured by senescence-associated β-galactosidase (SA-β-gal) activity and p53/p16 expression. The autophagic activity was evaluated by autophagic flux and autophagic marker expression. The expression of KLF4 was extremely increased in abdominal aorta tissues after 1-week Ang II stimulation (p < .01) but began to decrease in later periods. Decreased expression of KLF4 was also detected in premature senescent VSMCs. Overexpression of KLF4 could enhance the antisenescence ability of VSMCs. Significantly decreased amounts of SA-β-gal-positive cells and lower p53/p16 expression were detected in KLF4-overexpressing VSMCs (p < .01). Next, telomerase reverse transcriptase (TERT) was identified as a direct downstream target of KLF4 in VSMCs. Overexpression of KLF4 in VSMCs prevented the decreased expression of TERT under Ang II stimulation condition, which could in turn, contribute to the enhanced autophagic activity, and ultimately to the improved antisenescence ability of VSMCs. Our results demonstrated that overexpression of KLF4 prevented Ang II-induced VSMC senescence by promoting TERT-mediated autophagy. These findings provided novel potential targets for the prevention and therapy of vascular aging.

Sestrin2 increases in aortas and plasma from aortic dissection patients and alleviates angiotensin II-induced smooth muscle cell apoptosis via the Nrf2 pathway

BackgroundPrevious studies have demonstrated that oxidative stress is closely related to aortic dissection (AD). Sestrin2 (Sesn2) is an important antioxidant protein, and this study aimed to investigate whether Sesn2 participates in AD and the possible mechanisms. MethodsSesn2 expression was detected in aortas collected from AD patients and normal donors. In addition, blood samples were collected from AD patients and non-AD (NAD) patients, and the plasma Sesn2 levels were measured. Furthermore, the effects of Sesn2 on angiotensin (Ang) II-induced smooth muscle cell (SMC) apoptosis were investigated in vitro. ResultsCompared with the aortas from normal donors, aortas from AD patients had significantly increased Sesn2. Sesn2 was mainly secreted by macrophages, and low levels were secreted by CD4+ T lymphocytes, but not SMCs. Plasma Sesn2 levels were also increased in AD patients compared with NAD patients. Sesn2 levels were negatively corrected with superoxide dismutase (SOD) levels but positively corrected with malondialdehyde (MDA) levels in AD patients. In co-cultures of macrophages and SMCs, Sesn2 overexpression in macrophages significantly reduced Ang II-induced SMC apoptosis, and this effect could be reversed by Nrf2 silencing. ConclusionsSesn2 is increased in both aortas and plasma from AD patients. Sesn2 may alleviate Ang II-induced SMC apoptosis and participate in AD via the Nrf2 pathway. Sesn2 may be a new target in the treatment and prevention of AD.

Reactive oxygen species derived from NADPH oxidase 1 and mitochondria mediate angiotensin II-induced smooth muscle cell senescence

Cellular senescence has emerged as an important player in both physiology and pathology. Excessive reactive oxygen species (ROS) is known to mediate cellular senescence. NADPH oxidases are major sources for ROS production in the vascular wall; the roles of different NADPH oxidase isoforms in cellular senescence remain unclear, however. We investigated the roles of two NADPH oxidase isoforms in mitochondrial dysfunction during angiotensin II (Ang II)-induced cellular senescence of human aortic vascular smooth muscle cells (VSMCs). Ang II (10−7M) stimulated ROS generation, exhibiting early increases between 30 and 60min and sustained increases between 24h and 72h, and induced VSMCs senescence after 48h or 72h treatment as assessed with senescence-associated β-galactosidase activity and the expression of two cell cycle inhibitors, p21 and p16. ROS scavengers and membrane-permeable catalase (catalase-PEG) reduced Ang II-stimulated cellular senescence. Furthermore, small interfering RNA (siRNA) of NADPH oxidase catalytic subunit Nox1, but not that of another isoform Nox4, inhibited Ang II-induced cellular senescence. Nox1 siRNA inhibited both early and sustained ROS increases induced by Ang II. In addition, a mitochondrial-specific antioxidant, mitoQ10, effectively inhibited Ang II-induced ROS increases and cellular senescence. Ang II decreased ATP synthesis and induced mitochondrial membrane depolarization, which were attenuated by pre-treating cells with Nox1 siRNA, mitoQ10 or catalase-PEG. The effect of Ang II on the mitochondrial regulator peroxisome-proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its downstream genes was examined. Ang II stimulated S570 phosphorylation of PGC-1α with concomitant decreases in catalase and uncoupling protein-2 (UCP-2) levels between 12h and 72h, which were inhibited by Nox1 siRNA. Knockdown of both catalase and UCP-2 mimicked Ang II-induced VSMC senescence. These results suggested that Ang II-stimulated Nox1 activation mediates mitochondrial dysfunction, probably by decreasing PGC-1α activity and increasing mitochondrial oxidative stress, and leads to cellular senescence of VSMCs.

Angiotensin II-induced smooth muscle cell migration is mediated by LDL receptor-related protein 1 via regulation of matrix metalloproteinase 2 expression

Angiotensin II (Ang II), one of the main vasoactive hormones of the renin-angiotensin system, contributes to the development and progression of atherosclerosis by inducing vascular smooth muscle cells (VSMCs) migration. Although previous studies have shown that Ang II upregulates low density lipoprotein receptor-related protein 1 (LRP1) expression in VSMCs and increases VSMCs migration, the role of LRP1 in Ang II-induced VSMCs migration remains unclear. Here, we reveal a novel mechanism by which LRP1 induces the expression of matrix metalloproteinase 2 (MMP2) and thereby promotes the migration of rat aortic SMCs (RAoSMCs). Knockdown of LRP1 expression greatly decreased RAoSMCs migration, which was rescued by forced expression of a functional LRP1 minireceptor, suggesting that LRP1 is a key regulator of Ang II-induced RAoSMCs migration. Inhibition of ligand binding to LRP1 by the specific antagonist receptor-associated protein (RAP) also led to reduced RAoSMCs migration. Because MMPs play critical roles in RAoSMCs migration, we examined the expression of several MMPs and found that the expression of functional MMP2 was selectively increased by Ang II treatment and decreased in LRP1-knockdown RAoSMCs. More interestingly, reduced MMP2 expression in LRP1-knockdown cells was completely rescued by exogenous expression of mLRP4, suggesting that MMP2 is a downstream regulator of LRP1 in Ang II-induced RAoSMCs migration. Together, our data strongly suggest that LRP1 promotes the migration of RAoSMCs by regulating the expression and function of MMP2.

Pigment Epithelium-Derived Factor (PEDF) Inhibits Angiotensin IIInduced Smooth Muscle Cell Proliferation Through Its Anti-Oxidative Properties

Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis, suggesting that loss of this factor may be involved in angiogenic eye disease. Here, we show that PEDF inhibits angiotensin II-induced smooth muscle cell proliferation through its anti-oxidative properties. Our present study suggests that PEDF may play a protective role against atherosclerosis.

Decreased blood pressure in NOX1-deficient mice

To understand the role of the superoxide-generating NADPH oxidase NOX1 in the vascular system, we have generated NOX1-deficient mice. NOX1-deficient mice had a moderately decreased basal blood pressure. In response to angiotensin II they showed an almost complete loss of the sustained blood pressure response, while the initial increase was conserved. NOX1-deficient mice showed a marked reduction in aortic media hypertrophy. Angiotensin II-induced smooth muscle cell proliferation was conserved, but there was a marked decrease in extracellular matrix accumulation. Our results establish a role for NOX1 in blood pressure regulation and vascular angiotensin II response.

Doxazosin blocks the angiotensin II-induced smooth muscle cell DNA synthesis in the media, but not in the neointima of the rat carotid artery after balloon injury

Doxazosin blocks the angiotensin II-induced smooth muscle cell DNA synthesis in the media, but not in the neointima of the rat carotid artery after balloon injury

Doxazosin blocks the angiotensin II-induced smooth muscle cell DNA synthesis in the media, but not in the neointima of the rat carotid artery after balloon injury

Infusion of angiotensin II (AngII) during the third and fourth week after balloon injury of the left common carotid artery of the rat induces smooth muscle cell (SMC) DNA synthesis. In this study we wanted to investigate whether alpha 1-adrenoreceptors are involved in AngII-induced SMC DNA synthesis in the neointima. Adult male Wistar Kyoto rats were subcutaneously infused for 2 weeks with AngII and the alpha 1-adrenoreceptor antagonist doxazosin during the 3rd and the 4th week after balloon injury of the left common carotid artery. Control groups received AngII, 0.9% NaCl, AngII + 50% dimethylsulfoxide (DMSO, the solvent of doxazosin), doxazosin or 50% dimethylsulfoxide. Each rat received 5-bromo-2'-deoxyuridine in a separate osmotic minipump to label DNA-synthesizing SMC. Systolic blood pressures were measured in all groups. Angiotensin II caused an increase in systolic blood pressure, whereas addition of doxazosin did not affect the increase in SBP caused by AngII. In the media of the non-injured carotid artery, AngII increased SMC DNA synthesis, as the BrdUrd labeling fraction increased from 0.2 +/- 0.1% (mean +/- s.e.m.) in the NaCl group towards 3.4 +/- 0.6% in the AngII group. Coinfusion with doxazosin reduced the AngII-induced increase in BrdUrd labeling fraction from 3.2 +/- 0.8% in the AngII + DMSO group towards 0.6 +/- 0.2% in the AngII+doxazosin group. A similar effect of doxazosin was found in the media of the injured left carotid artery, in which coinfusion with doxazosin also reduced the BrdUrd labeling fraction from 2.6 +/- 0.8% in the AngII+DMSO group towards 0.3 +/- 0.1% in the AngII+doxazosin group. In the neointima of the injured left carotid artery, AngII increased the BrdUrd labeling fraction from 11.7 +/- 1.6% in the NaCl group towards 28.0 +/- 3.4% in the AngII group. Coinfusion with doxazosin did not influence the AngII-induced SMC DNA synthesis, since the BrdUrd labeling fraction in the neointima of the AngII+doxazosin group was 22.5 +/- 2.9%, whereas the neointimal BrdUrd labeling fraction in the AngII+DMSO group was 22.9 +/- 2.3%. Little effect was found on the medial cross-sectional area. The neointimal cross-sectional area was increased as a result of infusion of AngII (0.12 +/- 0.01 mm2 vs. 0.18 +/- 0.01 mm2), and coinfusion of doxazosin did not reduce the AngII-induced increase in neointimal cross-sectional area (0.18 +/- 0.03 mm2). These data suggest that alpha 1-adrenoreceptors are not involved in AngII-induced neointimal SMC DNA synthesis and cross-sectional area, but only play a role in the media of the carotid artery.

Effect of SR 47436, a novel angiotensin II AT 1 receptor antagonist, on human vascular smooth muscle cells in vitro

Proliferation of smooth muscle cells within the intima plays a key role in vascular occlusive disorders such as atherosclerosis and restenosis following balloon angioplasty. Among the factors that may be important in the development of vascular lesions, several authors have reported that the local angiotensin system participates in modulating the proliferation of smooth muscle cells after arterial injury. This study was therefore designed to characterize the antagonistic properties and to investigate the antiproliferative effect of a newly developed non-peptide angiotensin II AT 1 receptor antagonist, SR 47436. This compound is a potent and competitive antagonist of the binding of [ 125I]angiotensin II to its receptor on cultured human aortic smooth muscle cells, exhibiting an IC 50 value of 1.7 ± 0.6 nM. SR 47436 was 10-fold more potent than DuP 753 (Losartan) (IC 50 = 20.8 ± 3.7 nM). In these same cells, SR 47436 potently inhibited the angiotensin II-induced [Ca 2+] i increase (IC 50 = 0.53 ± 0.13 vs. 7.4 ± 1.3 nM for DuP 753). Angiotensin II is a potent mitogen for human aortic smooth muscle cells in culture, exhibiting a maximum proliferative response at 1 μM. SR 47436 and Losartan prevented angiotensin II-induced proliferation of these cells in a dose-dependent manner (IC 50 = 0.32 ± 0.09 and 0.71 ± 0.8 μM, respectively). SR 47436 displayed a marked in vitro inhibition of serum-induced smooth muscle cell proliferation (IC 50 = 5.5 ± 0.8 μM). A selective AT 2 receptor antagonist, PD 123177 did not affect angiotensin II-induced responses in these cells. This in vitro study shows that SR 47436 is a potent inhibitor of angiotensin II-induced smooth muscle cell growth and thus may have beneficial effects in the development and regression of vascular hypertrophy, which is associated with the development of atherosclerosis, restenosis and hypertension.

Identification of three types of PDGF-A chain gene transcripts in rabbit vascular smooth muscle and their regulated expression during development and by angiotensin II

PDGF-like peptides secreted from smooth muscles have been suggested to be responsible for the smooth muscle growth. In order to elucidate the nature of PDGF-like molecules expressed in vascular smooth muscles, we have isolated and characterized cDNA clones for PDGF-A chain from a rabbit embryonic aorta cDNA library. One of the cDNA clones was found to encode a novel PDGF-A chain, named PDGF-A3 in this report. PDGF-A3 arises from a single PDGF-A chain gene by alternative RNA splicing and differs from the sequences of previously reported endothelial- or the glioma-type transcripts by a 110 bp insertion. Expression of PDGF-A3 mRNA was selectively induced by Angiotensin II in the smooth muscle cell in vitro. Total PDGF-A mRNA is most enriched in embryonic aortas, but its expression is down-regulated with vascular development. PDGF-A mRNA is markedly increased in primary-cultured smooth muscle cells during the log-phase growth. Our results suggest that autocrine production of PDGF-A chains from the smooth muscle cell may play a role in early vascular development and in Angiotensin II-induced smooth muscle cell proliferation.

Angiotensin II-stimulated protein synthesis in cultured vascular smooth muscle cells.

To investigate the role of vasoconstrictor hormones in vascular smooth muscle cell growth we have studied the effects of the potent vasoconstrictor angiotensin II on cell growth in a cultured rat aortic cell model. Angiotensin II was not mitogenic for these cells, as assessed by determining cell number, nor was it synergistic in this regard with 10% calf serum. However, 24-hour exposure to 100 nM angiotensin II caused an 80% increase in protein synthesis (compared with 0.4% increase with serum control) as measured by tritiated leucine incorporation. This was a "hypertrophic" response as indicated by a 30% increase in protein content and a 45% increase in cell volume. Angiotensin II-induced smooth muscle cell hypertrophy was maximal at 100 nM, had an ED50 of 1 nM, and was inhibited by the competitive antagonist [Sar1, Ile8]angiotensin II. The increase in protein synthesis required continuous presence of angiotensin II for 6 hours and required messenger RNA (mRNA) synthesis as suggested by complete inhibition after exposure to actinomycin D. Angiotensin II-stimulated protein synthesis was dependent on a rise in intracellular Ca2+ concentration evidenced by a 70% decrease in tritiated leucine incorporation after chelation of Ca2+ with 25 microM quin 2-AM. This treatment did not alter protein synthesis induced by 10% calf serum. Decreasing extracellular Na+ to prevent Na+/H+ exchange and intracellular alkalinization did not inhibit the angiotensin II response but decreased the 10% calf serum-stimulated protein synthesis by 35%. Downregulation of protein kinase C by 24-hour treatment with phorbol 12,13-dibutyrate did not inhibit angiotensin II-induced protein synthesis, while phorbol 12-myristate 13-acetate-stimulated protein synthesis was abolished. These findings suggest that angiotensin II-induced hypertrophy, acting via a Ca2+ mechanism, may play an important role in abnormal vascular smooth muscle cell growth in certain forms of hypertension.