P2Y receptor regulation of PAI-1 expression in vascular smooth muscle cells

P2Y-type purine and pyrimidine nucleotide receptors play important roles in the regulation of vascular hemostasis. In this article, the regulation of plasminogen activator inhibitor-1 (PAI-1) expression in rat aortic smooth muscle cells (RASMCs) by adenine and uridine nucleotides was examined and compared. Northern analysis revealed that RASMCs express multiple P2Y receptor subtypes, including P2Y(1), P2Y(2), and P2Y(6). Treatment of RASMCs with UTP increased PAI-1 mRNA expression and extracellular PAI-1 protein levels by 21-fold (P<0.001) and 7-fold (P<0.001), respectively. The ED(50) for the effect of UTP on PAI-1 expression was approximately 1 micromol/L, and its maximal effect occurred at 3 hours. UDP stimulated a 5-fold increase (P<0.005) in PAI-1 expression. In contrast to these potent stimulatory effects of uridine nucleotides, ATP and 2-methylthioadenosine triphosphate (2-MeSATP) caused a small and transient increase in PAI-1 mRNA at 1 hour, followed by a rapid decrease to baseline levels. ADP produced only an inhibitory effect, reducing PAI-1 mRNA levels by 63% (P<0.05) at 3 hours. The relative nucleotide potency in stimulating PAI-1 expression is UTP>UDP>ATP=2-MeSATP, consistent with a predominant role of the P2Y(6) receptor. Further studies revealed that exposure of RASMCs to either ATP or ADP for 3 hours inhibited both UTP- and angiotensin II-stimulated PAI-1 expression by up to 90% (P<0.001). Thus, ATP induced a small and transient upregulation of PAI-1 that was followed by a strong inhibition of PAI-1 expression. These results show that extracellular adenine and uridine nucleotides exert potent and opposing effects on vascular PAI-1 expression.     

SSeCKS gene expression in vascular smooth muscle cells: regulation by angiotensin II and a potential role in the regulation of PAI-1 gene expression

Rat aortic smooth muscle cells (RASM) express the src suppressed C-kinase substrate (SSeCKS), which is thought to be an integral regulatory component of cytoskeletal dynamics and G-protein coupled-receptor signaling modules. The specific sub-classes of growth factor receptors that regulate the genomic changes in SSeCKS expression in smooth muscle cells have not been characterized. In this study we identify SSeCKS as an angiotensin type 1 (AT(1)) receptor-dependent target gene in RASM cells treated with angiotensin II (Ang II). SSeCKS mRNA levels increase up to three-fold relative to the control within 3.5 h of Ang II treatment and are followed by a slight decrease of mRNA relative to the control levels after 24 h of stimulation. SSeCKS gene expression and plasminogen activator inhibitor-1 (PAI-1) gene expression correlate in RASM cells treated with Ang II. By co-transfecting plasmids bearing recombinant-SSeCKS and a PAI-1-promoter/luciferase reporter into Cos-1 cells, we show that alternative forms of recombinant-SSeCKS protein differentially influence PAI-1 promoter activity. These data indicate a biochemical linkage between SSeCKS activity and one or more of the cytoplasmic signaling pathways that are involved in the control of PAI-1 promoter activity. Finally, we show that the alternative forms of recombinant-SSeCKS protein differentially influence cell-spreading when ectopically expressed in ras -transformed rat kidney (KNRK) fibroblasts. Taken together, our data suggest that SSeCKS interacts with intracellular signaling pathways that control cytoskeletal remodeling and extracellular matrix remodeling following Ang II stimulation of the RASM cell.     

全反式维甲酸对培养的大鼠动脉平滑肌细胞增殖的影响

目的 研究全反式维甲酸(ATRA)对培养的大鼠动脉平滑肌细胞(VSMCs)的影响。方法 血管平滑肌细胞采用组织贴块法培养。ATRA(2.5×10-6mol/L)分别作用于经PDGF—BB(10 ng/ml)和20％FCS刺激后的VSM—Cs,作用24h后分别行3H—TdR,3H—Leucine掺入实验测定。结果 ATRA明显抑制由PDGF—BB(10ng/ml)和20％FCS促进的VSMCs的DNA和蛋白质合成。结论ATRA具有抑制VSMCs增殖的作用。     

全反式维甲酸对培养大鼠主动脉平滑肌细胞Cyclin A表达水平及增生的影响

目的:探讨全反式维甲酸(ATRA)对培养的大鼠主动脉平滑肌细胞细胞周期蛋白A(Cyclin A)表达水平及增生的影响。 方法:血管平滑肌细胞采用组织贴块法培养。ATRA(2.5×10-6mol／L)作用于经20％FBS刺激后的VSMC,作用24h后分别行蛋白印迹检测Cyc-lin A蛋白表达和3H-TdR掺入实验测定。 结果:ATRA明显抑制由20％FBS促进的DNA合成[每孔每分钟脉冲数(3725.48±643.77)对(11567.09±1974.67)],同时降低Cyclin A蛋白的表达[平均光密度(122.50±9.59)对(135.92±11.95)]。 结论:ATRA抑制Cyclin A蛋白的表达是抑制VSMC增生的原因之一。ATRA可能成为预防和治疗动脉粥样硬化(AS)的有效药物。     

可罗卡林对血管平滑肌细胞增殖及c-myc基因表达的影响

目的：观察对大鼠主动脉平滑肌细胞起负调节作用的可罗卡林(cromakalim对同型半胱氨酸(hcy)刺激的血管平滑肌细胞(vascular smooth muscle cells，VSMC)增殖及c—myc基因表达的影响。方法：在建立hcy诱导的平滑肌细胞增殖模型后，应用流式细胞术观察VSMC增殖周期的变化；并用免疫细胞化学方法观察可罗卡林对VSMC增殖及c—myc基因蛋白表达的影响。结果：可罗卡林使VSMC处于G0／G1期的细胞数显著增多(P＜0．01)，S期G2 M期的细胞数显著减少(P＜0．01．)，能够抑制hcy诱导的VSMC增殖和c—myc基因蛋白表达的增加。结论：可罗卡林对hcy诱导的VSMC增殖有显著的抑制作用，其作用机制与抑制c—myc基因表达有关。     

Estradiol inhibits hyaluronic acid synthase 1 expression in human vascular smooth muscle cells

Epidemiological and clinical data suggest that estrogen retards the progression of atherosclerosis. This study aims to elucidate whether the phenotypic regulation of human vascular smooth muscle cells (VSMC) by estrogen may involve effects on the hyaluronan matrix. VSMC were synchronized by serum withdrawal and subsequently stimulated with 0.001, 0.01, 0.1 and 1 μM estradiol (E₂) in the presence or absence of platelet-derived growth factor BB (PDGF-BB) for 24 h. E₂ reduced mRNA-expression of hyaluronic acid synthase (HAS) 1 in the presence and absence of PDGF-BB. In contrast, HAS2- and HAS3-mRNA-expression were not affected. This E₂-mediated effect on HAS1 mRNA-expression was accompanied by reduced hyaluronan secretion and a shift of HA toward lower molecular weight as evidenced by molecular sieve chromatography. The downregulation of HAS1 was abrogated by the estrogen receptor (ER) α and β antagonist ICI182780 and could be mimicked by the ERα-agonist propyl-pyrazole triol (PPT). On the contrary, the ERβ-agonist diarylpropionitrile (DPN) had no effect on HAS1 mRNA-expression. To investigate whether the downregulation of HAS1 was causally involved in the phenotypic regulation of human VSMC by E₂, lentiviral overexpression of HAS1 was conducted. Overexpression of HAS1 abrogated the inhibition of sustained ERK1/2 phosphorylation and in turn inhibition of DNA-synthesis by E₂. For the first time this study provides strong evidence that HAS1-driven HA-synthesis is a target of E₂ in human VSMC and that E₂ mediates part of its anti-proliferative effects through an ERα-dependent inhibition of HA-synthesis.     

全反式维甲酸对培养大鼠主动脉平滑肌细胞Cyclin A表达水平及增生的影响

目的探讨全反式维甲酸(ATRA)对培养的大鼠主动脉平滑肌细胞细胞周期蛋白A(Cyclin A)表达水平及增生的影响.方法血管平滑肌细胞采用组织贴块法培养.ATRA(2.5×10-6mol/L)作用于经20%FBS刺激后的VSMC,作用24h后分别行蛋白印迹检测Cyclin A蛋白表达和3H-TdR掺入实验测定. 结果ATRA明显抑制由20%FBS促进的DNA合成[每孔每分钟脉冲数(3725.48±643.77)对(11567.09±1974.67)],同时降低Cyclin A蛋白的表达[平均光密度(122.50±9.59)对(135.92±11.95)]. 结论ATRA抑制Cyclin A蛋白的表达是抑制VSMC增生的原因之一.ATRA可能成为预防和治疗动脉粥样硬化(AS)的有效药物.     

BMP-2 gene expression and effects on human vascular smooth muscle cells

Bone morphogenetic proteins (BMPs) and their serine/threonine kinase receptors have been identified in atherosclerotic arteries and vascular smooth muscle cells, respectively. Thus, BMPs (the largest subfamily of the TGF-beta superfamily) have been implicated in the pathogenesis of atherosclerosis. However, the origins of BMP biosynthesis and the functional roles of BMP in blood vessels are unclear. The present study explored BMP-2 gene expression in various human blood vessels and vascular cell types. Functional in vitro studies were also performed to determine the effects of recombinant human BMP-2 on migration (transwell assay) and proliferation ([3H]-thymidine incorporation) of human aortic vascular smooth muscle cells (HASMC). RT-PCR experiments revealed BMP-2 gene expression in normal and atherosclerotic human arteries as well as cultured human aortic and coronary vascular smooth muscle cells, human umbilical vein endothelial cells (HUVECs) and human macrophages. In cellular migration studies, incubation with BMP-2 produced efficacious (相似文献   

粉防已碱对血管平滑肌细胞增殖及相关基因表达的影响

目的 :研究粉防已碱 (Tetrandrine,Tet)对内皮素诱导的大鼠血管平滑肌细胞 (VSMC)增殖及相关基因表达的影响。方法 :采用细胞计数及3H TDR掺入实验检测VSMC增殖状态 ;应用Northern杂交检测Tet对VSMC诱导型一氧化氮合酶 (iNOS)基因和ET诱导的c jun基因表达的影响。结果 :Tet可明显抑制VSMC的增殖 (P <0 0 5 ) ,抑制ET对VSMC的促丝裂作用 (P <0 0 5 ) ;抑制原癌基因c jun的表达 ,促进iNOS转录。结论 :Tet可能通过调节iNOS和细胞增殖相关基因的表达而抑制VSMC的增殖。     

Stimulatory effect of retinoic acid on GH gene expression: the interaction of retinoic acid and tri-iodothyronine in rat pituitary cells

We have previously demonstrated that retinoic acid (RA) as well as thyroid hormone stimulates GH gene expression. To clarify the relationship between the action of RA and thyroid hormone, pituitary-specific gene expression was investigated further in rat pituitary cells. Rat clonal pituitary cells, GH3, were treated with RA with or without tri-iodothyronine (T3) for up to 3 days. After treatment with 10-1000 nmol RA/l with or without 0.1-10 nmol T3/l, medium was collected for radioimmunoassay and cells were subjected to RNA extraction, and GH and prolactin gene expression was analysed using 32P-labelled rat GH and rat prolactin cDNA probes respectively. The data demonstrated the dose-responsive manner of the stimulatory effects of RA and T3 on GH secretion with T3-depleted media. The action of RA was additive to that of T3 for GH secretion when maximum effective doses of RA or T3 were used. Using dot blot and Northern gel analysis, it was shown that RA increased GH mRNA levels in T3-depleted media, and that this action of RA was additive to that of T3 on the induction of GH mRNA levels. In contrast, neither RA nor T3 stimulated the secretion of prolactin and prolactin mRNA levels in these cells. Our results indicate that RA stimulates GH mRNA increment and GH secretion in T3-depleted media, and that the stimulatory effect of RA is additive to the maximum effective dose of T3.     

Atorvastatin inhibits myocardin expression in vascular smooth muscle cells

Atorvastatin (ATV), an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, is widely prescribed as a lipid-lowering drug. It also inhibits the RhoA-Rho-associated kinase pathway in vascular smooth muscle (SM) cells and critically inhibits SM function. Myocardin is a coactivator of serum response factor, which upregulates SM contractile proteins. The RhoA-Rho-associated kinase pathway, which directly triggers SM contraction, also increases myocardin gene expression. Therefore, we investigated whether ATV inhibits myocardin gene expression in SM cells. In mice injected with ATV (IP 20 μg/g per day) for 5 days, myocardin gene expression was significantly downregulated in aortic and carotid arterial tissues with decreased expression of myocardin target genes SM α-actin and SM22. Correspondingly, the contractility of aortic rings in mice treated with ATV or the Rho-associated kinase inhibitor Y-27632 was reduced in response to treatment with either KCl or phenylephrine. In cultured mouse and human aortic SM cells, KCl treatment stimulated the expression of myocardin, SM α-actin, and SM22. These stimulatory effects were prevented by ATV treatment. ATV-induced inhibition of myocardin expression was prevented by pretreatment with either mevalonate or geranylgeranylpyrophosphate but not farnesylpyrophosphate. Treatment with Y-27632 mimicked ATV effects on the gene expression of myocardin, SM α-actin, and SM22, further suggesting a role for the RhoA-Rho-associated kinase pathway in ATV effects. Furthermore, ATV treatment inhibited RhoA membrane translocation and activation; these effects were prevented by pretreatment with mevalonate. We conclude that ATV inhibits myocardin gene expression in vivo and in vitro, suggesting a novel mechanism for ATV inhibition of vascular contraction.