Losartan Inhibits Vascular Smooth Muscle Cell Proliferation through Activation of AMP-Activated Protein Kinase

Losartan is a selective angiotensin II (Ang II) type 1 (AT₁) receptor antagonist which inhibits vascular smooth muscle cells (VSMCs) contraction and proliferation. We hypothesized that losartan may prevent cell proliferation by activating AMP-activated protein kinase (AMPK) in VSMCs. VSMCs were treated with various concentrations of losartan. AMPK activation was measured by Western blot analysis and cell proliferation was measured by MTT assay and flowcytometry. Losartan dose- and time-dependently increased the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase (ACC) in VSMCs. Losartan also significantly decreased the Ang II- or 15% FBS-induced VSMC proliferation by inhibiting the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. Compound C, a specific inhibitor of AMPK, or AMPK siRNA blocked the losartan-induced inhibition of cell proliferation and the G₀/G₁ cell cycle arrest. These data suggest that losartan-induced AMPK activation might attenuate Ang II-induced VSMC proliferation through the inhibition of cell cycle progression.     

Heme Oxygenase-1 Induced by Aprotinin Inhibits Vascular Smooth Muscle Cell Proliferation Through Cell Cycle Arrest in Hypertensive Rats

Spontaneous hypertensive rats (SHR) are an established model of genetic hypertension. Vascular smooth muscle cells (VSMC) from SHR proliferate faster than those of control rats (Wistar-Kyoto rats; WKY). We tested the hypothesis that induction of heme oxygenase (HO)-1 induced by aprotinin inhibits VSMC proliferation through cell cycle arrest in hypertensive rats. Aprotinin treatment inhibited VSMC proliferation in SHR more than in normotensive rats. These inhibitory effects were associated with cell cycle arrest in the G1 phase. Tin protoporphyrin IX (SnPPIX) reversed the anti-proliferative effect of aprotinin in VSMC from SHR. The level of cyclin D was higher in VSMC of SHR than those of WKY. Aprotinin treatment downregulated the cell cycle regulator, cyclin D, but upregulated the cyclin-dependent kinase inhibitor, p21, in VSMC of SHR. Aprotinin induced HO-1 in VSMC of SHR, but not in those of control rats. Furthermore, aprotinin-induced HO-1 inhibited VSMC proliferation of SHR. Consistently, VSMC proliferation in SHR was significantly inhibited by transfection with the HO-1 gene. These results indicate that induction of HO-1 by aprotinin inhibits VSMC proliferation through cell cycle arrest in hypertensive rats.     

Aprotinin Inhibits Vascular Smooth Muscle Cell Inflammation and Proliferation via Induction of HO-1

Aprotinin is used clinically in cardiopulmonary bypass surgery to reduce transfusion requirements and the inflammatory response. The mechanism of action for the anti-inflammatory effects of aprotinin is still unclear. We examined our hypothesis whether inhibitory effects of aprotinin on cytokine-induced inducible nitric oxide synthase (iNOS) expression (IL-1β plus TNF-α), reactive oxygen species (ROS) generation, and vascular smooth muscle cell (VSMC) proliferation were due to HO-1 induction in rat VSMCs. Aprotinin induced HO-1 protein expression in a dose-dependent manner, which was potentiated during inflammatory condition. Aprotinin reduced cytokine mixture (CM)-induced iNOS expression in a dose dependent manner. Furthermore, aprotinin reduced CM-induced ROS generation, cell proliferation, and phosphorylation of JNK but not of P38 and ERK1/2 kinases. Aprotinin effects were reversed by pre-treatment with the HO-1 inhibitor, tin protoporphyrin IX (SnPPIX). HO-1 is therefore closely involved in inflammatory-stimulated VSMC proliferation through the regulation of ROS generation and JNK phosphorylation. Our results suggest a new molecular basis for aprotinin anti-inflammatory properties.     

Reactive Oxygen Species Mediate ET-1-Induced Activation of ERK1/2 Signaling in Cultured Feline Esophageal Smooth Muscle Cells

Reactive oxygen species (ROS) have been shown to play a critical role in propagating the signals of several growth factors, peptide hormones, and cytokines, such as epidermal growth factor, insulin, and interleukin-1. We investigated a possible role for ROS generation in mediating the action of ET-1 on activation of ERK1/2 in cultured feline esophageal smooth muscle cells (ESMC). Confluent layers of ESMC were stimulated by 10nM ET-1; activation of ERK was examined by western blot analysis with phospho-specific antibodies of ERKs. ET-1 induced ERK1/2 phosphorylation in a dose- and time- dependent manner. ERK1/2 activation by ET-1 reached the maximal levels at 5min showing slight activation up to 20min, and then slowly declined. It was confirmed that the activation of ERK1/2 was reduced by MEK inhibitor PD98059. We observed the dose-dependent inhibitory effect of diphenyleneiodonium (DPI), an inhibitor of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase on the ET-1-enhanced ERK1/2 phosphorylation in ESMC. Pretreatment of ESMC with N-acetylcysteine, a ROS scavenger, also attenuated the ET-1-induced ERK1/2 activation. In addition, DPI significantly inhibited the ET-1- induced ROS production when ROS was measured as a function of DCF fluorescence. The results suggest that ROS might be critical mediators of the ET-1-induced ERK1/2 signaling events in ESMC.     

Olibanum Extract Inhibits Vascular Smooth Muscle Cell Migration and Proliferation in Response to Platelet-Derived Growth Factor

Olibanum (Boswellia serrata) has been shown to have anti-inflammatory, anti-arthritic and anti-cancer effects. This study determined the role of a water extract of olibanum in platelet-derived growth factor (PDGF)-stimulated proliferation and migration of rat aortic smooth muscle cells (RASMCs). PDGF-BB induced the migration and proliferation of RASMCs that were inhibited by olibanum extract in a dose-dependent manner. The PDGF-BB-increased phosphorylation of p38 mitogen-activated protein kinase (MAPK); the heat shock protein (Hsp) 27 was significantly inhibited by the olibanum extract. The effects of PDGF-BB-induced extracellular signal-regulated kinase1/2 was not altered by the olibanum extract. Treatment with olibanum extract inhibited PDGF-BB-stimulated sprout out growth of aortic rings. These results suggest that the water extract of olibanum inhibits PDGF-BB-stimulated migration and proliferation in RASMCs as well as sprout out growth, which may be mediated by the inhibition of the p38 MAPK and Hsp27 pathways.     

首乌复方冲剂对ox-LDL引起的血管平滑肌细胞增殖的影响

采用氧化型低密度脂蛋白(ox-LDL)造成小牛主动脉平滑肌细胞(VSMC)增殖,观察首乌复方冲剂对ox-LDL诱导VSMC增殖的影响。将培养的VSMC分为对照组、ox-LDL组和用药组(ox-LDL 不同剂量首乌复方冲剂),应用硫代巴比妥酸比色法测定VSMC培养液中丙二醛(MDA)的水平。用MTT法测定VSMC数量和应用流式细胞仪测定VSMC周期。结果显示首乌复方冲剂能明显降低细胞培养液中MDA的水平,MTT检测显示首乌复方冲剂抑制ox-LDL诱导的VSMC增殖,流式细胞仪检测表明,用药组(S G2M)期降低。首乌复方冲剂能抑制ox-LDL刺激的VSMC的增殖,并具有抗脂质过氧化作用。     

Quercetin Glucuronide Inhibits Cell Migration and Proliferation by Platelet-Derived Growth Factor in Vascular Smooth Muscle Cells

Many epidemiologic studies have reported that dietary flavonoids provide protection against cardiovascular disease. Quercetin, a member of the bioflavonoids family, has been proposed to have anti-inflammatory, anti-atherogenic, and anti-hypertensive properties leading to the beneficial effects against cardiovascular diseases. Recent studies demonstrated that orally administered quercetin appeared in plasma as glucuronide-conjugated forms in rats and humans. Therefore, we examined the effect of chemically synthesized quercetin glucuronide on platelet-derived growth factor (PDGF)-induced cell migration and kinase activation in cultured rat aortic smooth muscle cells (RASMCs). PDGF-induced RASMC migration was inhibited by quercetin 3-O-β-D-glucuronide (Q3GA). Q3GA also attenuated PDGF-induced cell proliferation in RASMCs. PDGF activated extracellular-signal regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, and Akt in RASMCs. PDGF-induced JNK and Akt activations were suppressed by Q3GA, whereas ERK1/2 and p38 MAP kinase activations were not affected. We also confirmed that PDGF-induced JNK and Akt activations were inhibited by antioxidants, N-acetylcysteine and diphenyleneiodonium chloride, in RASMCs. These findings suggest Q3GA would be an active metabolite of quercetin in plasma and may possess preventing effects for cardiovascular diseases relevant to vascular smooth muscle cell disorders.     

吡格列酮和罗格列酮对脂多糖诱导大鼠血管平滑肌细胞增殖的影响

目的:观察胰岛素增敏剂——噻唑烷二酮类(TZD)药物吡格列酮和罗格列酮对脂多糖(LPS)诱导的血管平滑肌细胞(VSMCs)异常增殖的影响。方法:组织块贴壁法培养大鼠胸主动脉血管平滑肌细胞;LPS(0.1,1,10mg·L-1)干预血管平滑肌细胞不同时间(0,12,24,48,72h)后,分别用吡格列酮和罗格列酮与10mg·L-1LPS共同孵育72h,四甲基偶氮咗盐微量酶比色(MTT)法检测血管平滑肌细胞增殖。结果:10mg·L-1LPS孵育72h导致血管平滑肌细胞增殖程度最明显(P<0.01);吡格列酮和罗格列酮可明显抑制LPS诱导的血管平滑肌细胞增殖。结论:噻唑烷二酮类代表药吡格列酮和罗格列酮均对LPS诱导的血管平滑肌细胞异常增殖具有抑制作用,该类药物可能在治疗心血管疾病方面有较好的应用前景。     

离体和在体机械损伤致血管平滑肌增殖模型的建立

目的建立离体和在体机械损伤致血管平滑肌增殖模型。方法原代培养大鼠胸主动脉血管平滑肌细胞(VSMCs),体外建立机械划伤致VSMCs增殖模型,采用5-溴脱氧尿嘧啶(5-BrUd)掺入法,流式细胞术检测细胞增殖情况;整体动物上,采用新西兰兔左侧颈总动脉球囊损伤,术后7d取颈总动脉段,常规病理切片,HE染色,观测血管内膜厚度、内膜面积、内膜厚度/中膜厚度、内膜面积/中膜面积。结果机械划痕损伤12 h后划伤处两侧的细胞开始有部分增殖和迁移,24 h后细胞增殖较为明显。流式细胞术检测机械损伤后细胞增殖增多(P<0.05)。整体动物上,与假手术组比较,模型组术后7 d血管内膜厚度、内膜面积、内膜厚度/中膜厚度、内膜面积/中膜面积显著增加(P<0.01)。结论机械损伤可以导致离体和在体血管平滑肌细胞增殖。     

(‐)‐Epigallocatechin Gallate Inhibits Endothelin‐1‐Induced C‐Reactive Protein Production in Vascular Smooth Muscle Cells

Abstract: Numerous studies have shown that C‐reactive protein (CRP), a pro‐inflammation cytokine, makes a direct contribution to atherosclerosis, and that (–)‐epigallocatechin gallate (EGCG) is able to exert an anti‐atherosclerotic effect by anti‐oxidative and anti‐inflammatory activities. Based on our previous study, the effect of EGCG on endothelin‐1 (ET‐1)‐induced CRP production in rat vascular smooth muscle cells (VSMCs) and the possible mechanism were observed. The in vitro experiments showed that EGCG concentration‐dependently inhibited ET‐1‐stimulated expression of CRP both in protein and mRNA levels in VSMCs as determined by the immunocytochemical staining, the enzyme‐linked immunosorbent assay and the real‐time quantitative polymerase chain reaction (RT‐qPCR). The in vivo investigation with the double‐labelled immunofluorescence staining and RT‐qPCR displayed that EGCG also prevented ET‐1‐induced CRP expression in protein and mRNA levels in the aortic VSMCs of rats receiving the subchronic infusion of ET‐1. In addition, EGCG suppressed reactive oxygen species (ROS) generation evoked by ET‐1 in VSMCs as observed by the fluorescence probe. These demonstrate that EGCG may inhibit ET‐1‐stimulated generation of CRP in VSMCs so to relieve the inflammatory response and oxidative stress via blocking ROS signal, which provides new evidence for an anti‐atherosclerotic effect of EGCG.     

Synergistic Efficacy of Concurrent Treatment with Cilostazol and Probucol on the Suppression of Reactive Oxygen Species and Inflammatory Markers in Cultured Human Coronary Artery Endothelial Cells

In the present study, we aimed to identify the synergistic effects of concurrent treatment of low concentrations of cilostazol and probucol to inhibit the oxidative stress with suppression of inflammatory markers in the cultured human coronary artery endothelial cells (HCAECs). Combination of cilostazol (0.3~3 µM) with probucol (0.03~0.3 µM) significantly suppressed TNF-α-stimulated NAD(P)H-dependent superoxide, lipopolysaccharide (LPS)-induced intracellular reactive oxygen species (ROS) production and TNF-α release in comparison with probucol or cilostazol alone. The combination of cilostazol (0.3~3 µM) with probucol (0.1~0.3 µM) inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) more significantly than did the monotherapy with either probucol or cilostazol. In line with these results, combination therapy significantly suppressed monocyte adhesion to endothelial cells. Taken together, it is suggested that the synergistic effectiveness of the combination therapy with cilostazol and probucol may provide a beneficial therapeutic window in preventing atherosclerosis and protecting from cerebral ischemic injury.     

The Protective Effect of Heme Oxygenase-1 on Liver Injury Caused by DON-Induced Oxidative Stress and Cytotoxicity

Deoxynivalenol (DON) is a kind of Fusarium toxin that can cause a variety of toxic effects. Oxidative stress and DNA damage play a critical role in the toxicity of DON. However, previous studies focused more on acute toxicity in vivo/vitro models and lacked subchronic toxicity study in vivo. The potentially harmful effect of DON given at doses comparable to the daily human consumption in target organs, especially the liver, which is the main detoxification organ of DON, is also still not fully understood. Otherwise, Heme Oxygenase-1 (HO-1) has also reduced cell damage under the DON condition according to our previous study. Therefore, we used a rodent model that mimicked daily human exposure to DON and further explored its mechanism of toxic effects on liver tissue and Hepa 1–6 cell line. We also used adeno-associated virus (AAV)-modified HO-1 expressing by tail vein injection and constructed lentivirus-Hepa 1–6 cell line for mimicking HO-1 protective ability under the DON condition. The main results showed that both 30 d and 90 d exposures of DON could cause low-grade inflammatory infiltration around hepatic centrilobular veins. The reactive oxygen species (ROS) and 8-hydroxy-2 deoxyguanosine (8-OHdG) increased during DON exposure, indicating oxidation stress and DNA damage. Significantly, AAV-mediated liver-specific overexpression of HO-1 reduced DON-induced liver damage and indirectly protected the abilities of antioxidant enzyme/DNA damage repair system, while AAV-mediated silence of HO-1 produced the opposite effect. In addition, we found that overexpression of HO-1 could enhance autophagy and combined it with an antioxidant enzyme/DNA damage repair system to inhibit DON-induced hepatocyte damage. Altogether, these data suggest that HO-1 reduces the oxidative stress and DNA damage caused by DON sub-chronic exposure through maintaining DNA repair, antioxidant activity, as well as autophagy.     

白细胞介素1诱导牛脑微血管平滑肌细胞增殖及甘糖酯的拮抗作用

用牛脑微血管平滑肌细胞（ＢＣＭＳＭＣ）体外培养技术,研究白细胞介素１（ＩＬ－１）对ＢＣＭＳＭＣ增殖的影响。浓度为５０ｕ／ｍｌ的 ＩＬ－１与ＢＣＭＳＭＣ共同孵育７２ｈ,即可明显诱导ＢＣＭＳＭＣ增殖。甘糖酯在０．２、０．４、０．８、１．６ｍｇ／ｍｌ的浓度下可显著抑制ＩＬ－１诱导的ＢＣＭＳＭＣ增殖。提示甘糖酯对脑血管疾病的防治作用可能与抑制血管平滑肌细胞的异常增殖有关。     

Downregulation of Angiotensin II-Induced 12-Lipoxygenase Expression and Cell Proliferation in Vascular Smooth Muscle Cells from Spontaneously Hypertensive Rats by CCL5

Angiotensin II (Ang II) plays an important role in vascular hypertension. The role of the chemokine CCL5 on Ang II-induced activities in vascular smooth muscle cells (VSMCs) has not been studied. In this study, we elucidated the effect of CCL5 on Ang II-induced 12-lipoxygenase (LO) expression and cell proliferation in spontaneously hypertensive rats (SHR) VSMCs. CCL5 decreased Ang II-induced 12-LO mRNA expression and protein production, and it increased Ang II type 2 (AT₂) receptor expression in SHR VSMCs. The inhibitory effect of CCL5 on Ang II-induced 12-LO mRNA expression was mediated through the AT₂ receptor. Although treatment of CCL5 alone induced SHR VSMCs proliferation, CCL5 inhibited Ang II-induced VSMCs proliferation and PD123,319, an AT₂ receptor antagonist, blocked the inhibitory effect of CCL5 on Ang II-induced VSMCs proliferation. Phosphorylation of p38 was detected in VSMCs treated with Ang II or CCL5 alone. But, decrease of p38 phosphorylation was detected in VSMCs treated with Ang II and CCL5 simultaneously (Ang II/CCL5) and PD123,319 increased p38 phosphorylation in VSMCs treated with Ang II/CCL5. Therefore, these results suggest that the inhibitory effect of CCL5 on Ang II-induced VSMCs proliferation is mediated by the AT₂ receptor via p38 inactivation, and CCL5 may play a beneficial role in Ang II-induced vascular hypertension.     

不同浓度白头翁皂苷对口腔鳞癌细胞增殖、迁移和钙蛋白酶1、E cadherin、N cadherin蛋白的影响

摘 要 目的：探究白头翁皂苷对口腔鳞癌细胞增殖、迁移及钙蛋白酶1（calpain1）表达的影响。方法: 用0,1.0,2.0,4.0,8.0,12.0 mg·L-1白头翁皂苷处理人口腔鳞癌CAL27细胞。四甲基偶氮唑（MTT）法检测白头翁皂苷对CAL27细胞增殖的影响;细胞划痕实验与Transwell实验检测白头翁皂苷对CAL27细胞迁移与侵袭能力的影响;蛋白质印迹（Western blot）技术检测CAL27细胞calpain1、E cadherin、N cadherin蛋白的表达变化。结果:与空白对照组比较,白头翁皂苷对CAL27细胞的增殖抑制率显著增加（P<0.05）,且具有时间 剂量依赖关系。12.0 mg·L-1白头翁皂苷处理48 h后CAL27细胞细胞形态从长梭形间质样变成鹅卵石状上皮样。随着白头翁皂苷处理浓度的增加,CAL27细胞迁移与侵袭能力逐渐减弱（P<0.05）;CAL27细胞中calpain1、N cadherin蛋白表达水平逐渐降低（P<0.05）,E cadherin蛋白表达水平逐渐升高（P>0.05）。结论:白头翁皂苷可抑制口腔鳞癌CAL27细胞增殖、迁移与侵袭过程,其机制可能与calpain1、N cadherin表达下调、E cadherin表达上调有关。