KLF5与c-Jun相互作用促进Ang II诱导的血管平滑肌细胞增殖

KLF5（Krüppel-like factor 5）是KLF家族转录因子中的成员,参与多种与增 殖相关基因的转录调节.通过体外培养大鼠血管平滑肌细胞（vascular smooth muscle cells, VSMC）,以Ang Ⅱ为增殖诱导因素,研究KLF5介导VSMC增殖的机制. 研究发现,Ang Ⅱ可剂量依赖性诱导VSMC增殖,并伴有KLF5和c-Jun蛋白表达水平的 升高.为了证实KLF5参与Ang Ⅱ诱导的细胞增殖过程,用siRNA敲低内源性KLF5的表达后,发现细胞增殖活力受到明显的抑制;交互免疫沉淀和GST pull down分析结果显示,KLF5与c-Jun在体内和体外存在物理学上的相互作用,并且Ang Ⅱ可诱导二者之间的相互缔合.这些结果表明,KLF5通过与c-Jun相互作用进而介导Ang Ⅱ诱导的VSMC 增殖.     

Transforming Growth Factor β Inhibits Platelet Derived Growth Factor-Induced Vascular Smooth Muscle Cell Proliferation via Akt-Independent,Smad-Mediated Cyclin D1 Downregulation

In adult tissue, vascular smooth muscle cells (VSMCs) exist in a differentiated phenotype, which is defined by the expression of contractile proteins and lack of proliferation. After vascular injury, VSMC adopt a synthetic phenotype associated with proliferation, migration and matrix secretion. The transition between phenotypes is a consequence of the extracellular environment, and in particular, is regulated by agonists such as the pro-differentiating cytokine transforming growth factor β (TGFβ) and the pro-proliferative cytokine platelet derived growth factor (PDGF). In this study, we investigated the interplay between TGFβ and PDGF with respect to their ability to regulate VSMC proliferation. Stimulation of human aortic VSMC with TGFβ completely blocked proliferation induced by all isoforms of PDGF, as measured by DNA synthesis and total cell number. Mechanistically, PDGF-induced Cyclin D1 mRNA and protein expression was inhibited by TGFβ. TGFβ had no effect on PDGF activation of its receptor and ERK1/2, but inhibited Akt activation. However, constitutively active Akt did not reverse the inhibitory effect of TGFβ on Cyclin D1 expression even though inhibition of the proteasome blocked the effect of TGFβ. siRNA against Smad4 completely reversed the inhibitory effect of TGFβ on PDGF-induced Cyclin D1 expression and restored proliferation in response to PDGF. Moreover, siRNA against KLF5 prevented Cyclin D1 upregulation by PDGF and overexpression of KLF5 partially reversed TGFβ-induced inhibition of Cyclin D1 expression. Taken together, our results demonstrate that KLF5 is required for PDGF-induced Cyclin D1 expression, which is inhibited by TGFβ via a Smad dependent mechanism, resulting in arrest of VSMCs in the G1 phase of the cell cycle.     

Anti-proliferative effect of rosiglitazone on angiotensin II-induced vascular smooth muscle cell proliferation is mediated by the mTOR pathway

VSMC (vascular smooth muscle cell) proliferation contributes significantly to intimal thickening in atherosclerosis, restenosis and venous bypass graft diseases. Ang II (angiotensin II) has been implicated in VSMC proliferation though the activation of multiple growth-promoting signals. Although TZDs (thiazolidinediones) can inhibit VSMC proliferation and reduce Ang II-induced fibrosis, the mechanism underlying the inhibition of VSMC proliferation and fibrosis needs elucidation. We have used primary cultured rat aortic VSMCs and specific antibodies to investigate the inhibitory mechanism of rosiglitazone on Ang II-induced VSMC proliferation. Rosiglitazone treatment significantly inhibited Ang II-induced rat aortic VSMC proliferation in a dose-dependent manner. Western blot analysis showed that rosiglitazone significantly lowered phosphorylated ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt (also known as protein kinase B), mTOR (mammalian target of rapamycin), p70S6K (70 kDa S6 kinase) and 4EBP1 (eukaryotic initiation factor 4E-binding protein) levels in Ang II-treated VSMCs. In addition, PPAR-γ (peroxisome-proliferator-activated receptor γ) mRNA increased significantly and CTGF (connective tissue growth factor), Fn (fibronectin) and Col III (collagen III) levels decreased significantly. The results demonstrate that the rosiglitazone directly inhibits the pro-atherosclerotic effect of Ang II on rat aortic VSMCs. It also attenuates Ang II-induced ECM (extracellular matrix) molecules and CTGF production in rat aortic VSMCs, reducing fibrosis. Importantly, PPAR-γ activation mediates these effects, in part, through the mTOR-p70S6K and -4EBP1 system.     

Krüppel样因子5介导血管平滑肌细胞的增殖和迁移

Krüppel样因子5（krüppel-like factor 5,KLF5）是KLF家族中与胚胎发育、细胞增殖和肿瘤发生密切相关的转录调节因子。为观察KLF5在体外培养的大鼠血管平滑肌细胞（vascularsmooth muscle cells,VSMCs）增殖和迁移活性中的作用,通过构建KLF5腺病毒表达载体并感染细胞以过表达KLF5或用特异性siRNA敲低KLF5,用MTT、流式细胞术以及免疫细胞化学染色和伤口愈合实验检测其对VSMCs增殖和迁移活性的影响。结果发现,KLF5过表达可加速细胞由G0/G1期向S期转变,促进细胞增殖和迁移;反之,敲低KLF5后细胞增殖活性明显低于转染无关序列NS-siRNA对照组细胞,G0/G1期细胞数所占比例增多,S期细胞数所占比例减少,VSMCs迁移活性也明显降低。结果表明KLF5可参与介导VSMCs的增殖和迁移。     

Dual Regulation of Myocardin Expression by Tumor Necrosis Factor-α in Vascular Smooth Muscle Cells

De-differentiation of vascular smooth muscle cells (VSMCs) plays a critical role in the development of atherosclerosis, a chronic inflammatory disease involving various cytokines such as tumor necrosis factor-α (TNFα). Myocardin is a co-factor of serum response factor (SRF) and is considered to be the master regulator of VSMC differentiation. It binds to SRF and regulates the expression of contractile proteins in VSMCs. Myocardin is also known to inhibit VSMC proliferation by inhibiting the NF-κB pathway, whereas TNFα is known to activate the NF-κB pathway in VSMCs. NF-κB activation has also been shown to inhibit myocardin expression and smooth muscle contractile marker genes. However, it is not definitively known whether TNFα regulates the expression and activity of myocardin in VSMCs. The current study aimed to investigate the role of TNFα in regulating myocardin and VSMC function. Our studies showed that TNFα down-regulated myocardin expression and activity in cultured VSMCs by activating the NF-κB pathway, resulting in decreased VSMC contractility and increased VSMC proliferation. Surprisingly, we also found that TNFα prevented myocardin mRNA degradation, and resulted in a further significant increase in myocardin expression and activity in differentiated VSMCs. Both the NF-κB and p44/42 MAPK pathways were involved in TNFα regulation of myocardin, which further increased the contractility of VSMCs. These differential effects of TNFα on myocardin seemingly depended on whether VSMCs were in a differentiated or de-differentiated state. Taken together, our results demonstrate that TNFα differentially regulates myocardin expression and activity, which may play a key role in regulating VSMC functions.     

Simultaneous Increases in Proliferation and Apoptosis of Vascular Smooth Muscle Cells Accelerate Diabetic Mouse Venous Atherosclerosis

Aims

This study was designed to demonstrate simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) leading to accelerated vein graft remodeling and to explore the underlying mechanisms.

Methods

Vein grafts were performed in non-diabetic and diabetic mice. The cultured quiescent VSMCs were subjected to mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs). Harvested vein grafts and treated VSMCs were used to detect cell proliferation, apoptosis, mitogen-activated protein kinases (MAPKs) activation and SM-α-actin expression.

Results

Significantly thicker vessel walls and greater increases in proliferation and apoptosis were observed in diabetic vein grafts than those in non-diabetic. Both SS and AGEs were found to induce different activation of three members of MAPKs and simultaneous increases in proliferation and apoptosis of VSMCs, and combined treatment with both had a synergistic effect. VSMCs with strong SM-α-actin expression represented more activated JNKs or p38MAPK, and cell apoptosis, while the cells with weak SM-α-actin expression demonstrated preferential activation of ERKs and cell proliferation. In contrast, inhibition of MAPKs signals triggered significant decreases in VSMC proliferation, and apoptosis. Treatment of the cells with RNA interference of receptor of AGEs (RAGE) also resulted in significant decreases in both proliferation and apoptosis.

Conclusions

Increased pressure-induced SS triggers simultaneous increases in proliferation and apoptosis of VSMCs in the vein grafts leading to vein arterializations, which can be synergistically accelerated by high glucose-induced AGEs resulting in vein graft atherosclerosis. Either SS or AGEs and their combination induce simultaneous increases in proliferation and apoptosis of VSMCs via different activation of three members of MAPKs resulting from different VSMC subtypes classified by SM-α-actin expression levels.     

PGC-1α Is a Key Regulator of Glucose-Induced Proliferation and Migration in Vascular Smooth Muscle Cells

Background

Atherosclerosis is a complex pathological condition caused by a number of mechanisms including the accelerated proliferation of vascular smooth muscle cells (VSMCs). Diabetes is likely to be an important risk factor for atherosclerosis, as hyperglycemia induces vascular smooth muscle cell (VSMC) proliferation and migration and may thus contribute to the formation of atherosclerotic lesions. This study was performed to investigate whether PGC-1α, a PPARγ coactivator and metabolic master regulator, plays a role in regulating VSMC proliferation and migration induced by high glucose.

Methodology/Principal Findings

PGC-1α mRNA levels are decreased in blood vessel media of STZ-treated diabetic rats. In cultured rat VSMCs, high glucose dose-dependently inhibits PGC-1α mRNA expression. Overexpression of PGC-1α either by infection with adenovirus, or by stimulation with palmitic acid, significantly reduces high glucose-induced VSMC proliferation and migration. In contrast, suppression of PGC-1α by siRNA mimics the effects of glucose on VSMCs. Finally, mechanistic studies suggest that PGC-1α-mediated inhibition of VSMC proliferation and migration is regulated through preventing ERK1/2 phosphorylation.

Conclusions/Significance

These results indicate that PGC-1α is a key regulator of high glucose-induced proliferation and migration in VSMCs, and suggest that elevation of PGC-1α in VSMC could be a useful strategy in preventing the development of diabetic atherosclerosis.     

川芎嗪抑制血管紧张素Ⅱ诱导的平滑肌细胞NF-κB激活和骨形成蛋白-2表达降低

本文旨在观察血管紧张素Ⅱ（angiotensinⅡ,AngⅡ）对血管平滑肌细胞核转录因子-κB（nuclear factor-κB,NF-κB）的活性及骨形成蛋白-2（bone morphogenetic protein-2,BMP-2）表达的影响,以探讨AngⅡ参与动脉粥样硬化的机制,并探讨川芎嗪是否能抑制AngⅡ的促动脉粥样硬化作用。采用Western blot、免疫组化和原位杂交等方法分别检测AngⅡ刺激和川芎嗪干预后NF-κB活性、BMP-2蛋白和mRNA表达的变化。结果显示：（1）AngⅡ刺激激活NF-κB。AngⅡ刺激15min即有NF-κB p65核转移,30min达高峰（P〈0．01）,1h后减退。川芎嗪抑制AngⅡ诱导的NF-κB激活,与AngⅡ组比较,川芎嗪＋AngⅡ组NF-κB活性显著降低（P〈0．01）。（2）AngⅡ刺激6h时BMP-2表达增强（P〈0．05）,12h时减弱（P〈0．01）,24h时更弱（P〈0．01）。川芎嗪＋AngⅡ组中,川芎嗪干预6h时BMP-2表达亦增强,12与24h时保持正常水平。（3）川芎嗪对正常细胞的NF-κB活性和BMP-2表达无影响。以上结果表明,AngⅡ刺激后激活NF-κB并最终使生长抑制因子BMP-2表达下降,这可能是其参与动脉粥样硬化发生的机制之一。BMP-2一过性增高可能不依赖NF-κB通路的激活。川芎嗪可抑制AngⅡ诱导的NF-κB激活与BMP-2表达降低,提示它在抗动脉粥样硬化形成中起重要作用。     

Exendin-4 Prevents Vascular Smooth Muscle Cell Proliferation and Migration by Angiotensin II via the Inhibition of ERK1/2 and JNK Signaling Pathways

Angiotensin II (Ang II) is a main pathophysiological culprit peptide for hypertension and atherosclerosis by causing vascular smooth muscle cell (VSMC) proliferation and migration. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, is currently used for the treatment of type-2 diabetes, and is believed to have beneficial effects for cardiovascular diseases. However, the vascular protective mechanisms of GLP-1 receptor agonists remain largely unexplained. In the present study, we examined the effect of exendin-4 on Ang II-induced proliferation and migration of cultured rat aortic smooth muscle cells (RASMC). The major findings of the present study are as follows: (1) Ang II caused a phenotypic switch of RASMC from contractile type to synthetic proliferative type cells; (2) Ang II caused concentration-dependent RASMC proliferation, which was significantly inhibited by the pretreatment with exendin-4; (3) Ang II caused concentration-dependent RASMC migration, which was effectively inhibited by the pretreatment with exendin-4; (4) exendin-4 inhibited Ang II-induced phosphorylation of ERK1/2 and JNK in a pre-incubation time-dependent manner; and (5) U0126 (an ERK1/2 kinase inhibitor) and SP600125 (a JNK inhibitor) also inhibited both RASMC proliferation and migration induced by Ang II stimulation. These results suggest that exendin-4 prevented Ang II-induced VSMC proliferation and migration through the inhibition of ERK1/2 and JNK phosphorylation caused by Ang II stimulation. This indicates that GLP-1 receptor agonists should be considered for use in the treatment of cardiovascular diseases in addition to their current use in the treatment of diabetes mellitus.     

YS 49, 1-(alpha-naphtylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, regulates angiotensin II-stimulated ROS production, JNK phosphorylation and vascular smooth muscle cell proliferation via the induction of heme oxygenase-1

Overexpression of the gene for heme oxygenase (HO)-1 leads to a reduction in pressor responsiveness to angiotensin II (Ang II) in experimental animals. Using rat vascular smooth muscle cells (VSMCs), we tested whether YS 49 [1-(alpha-naphtylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] inhibits Ang II-stimulated proliferation of VSMCs via induction of HO-1. YS 49 induced HO-1 protein production in a dose-and time-dependent manner in VSMCs. Treatment with YS 49 significantly and dose-dependently inhibited Ang II-induced VSMC proliferation, ROS production, and phosphorylation of JNK, but not P38 MAP kinase or ERK1/2. The antiproliferation effect of YS 49 was reversed by pretreatment with the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX), or with hemoglobin, a carbon monoxide (CO) scavenger. Similarly, VSMC proliferation, ROS production and phosphorylation of JNK by Ang II were significantly inhibited in VSMCs transfected with the HO-1 gene. Thus, HO-1 and the HO-1 product CO play, at least in part, a crucial role in Ang II-stimulated VSMC proliferation through the regulation of ROS production and JNK phosphorylation. Therefore, YS 49 has potential as a therapeutic strategy for the pathogenesis of Ang II-related vascular diseases such as hypertension and atherosclerosis, via the induction of HO-1 gene activity.     

Dimethylarginine dimethylaminohydrolase-1 mediates inhibitory effect of interleukin-10 on angiotensin II-induced hypertensive effects in vascular smooth muscle cells of spontaneously hypertensive rats

In hypertension studies, anti-inflammatory cytokine interleukin-10 (IL-10) has been shown to prevent angiotensin II (Ang II)-induced vasoconstriction and regulate vascular function by down-regulating pro-inflammatory cytokine and superoxide production in vascular cells. However, little is known about the mechanism behind the down-regulatory effect of IL-10 on Ang II-induced hypertensive mediators. In this study, we demonstrated the effects of IL-10 on expression of dimethylarginine dimethylaminohydrolase (DDAH)-1, a regulator of NO bioavailability, as well as the down-regulatory mechanism of action of IL-10 in relation to Ang II-induced hypertensive mediator expression and cell proliferation in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). IL-10 increased DDAH-1 but not DDAH-2 expression and increased DDAH activity. Additionally, IL-10 attenuated Ang II-induced DDAH-1 inhibition in SHR VSMCs. Increased DDAH activity due to IL-10 was mediated mainly through Ang II subtype II receptor (AT₂ R) and AMP-activated protein kinase (AMPK) activation. DDAH-1 induced by IL-10 partially mediated the inhibitory action of IL-10 on Ang II-induced 12-lipoxygenase (LO) and endothelin (ET)-1 expression in SHR VSMCs. In addition, the inhibitory effect of IL-10 on proliferation of Ang II-induced VSMCs was mediated partially via DDAH-1 activity. These results suggest that DDAH-1 plays a potentially important role in the anti-hypertensive activity of IL-10 during Ang II-induced hypertension.