HMG-CoA reductase inhibitors reduce interleukin-6 synthesis in human vascular smooth muscle cells

Interleukin-6 (IL-6) is a key molecule in chronic inflammation and has been implicated in the progression of atherosclerosis. HMG-CoA reductase inhibitors (statins) may reduce the cardiovascular risk and vulnerability of atherosclerotic plaque through nonlipid as well as lipid-lowering mechanisms, but their anti-inflammatory effects on the vascular tissue have not been fully elucidated. We investigated the effects of fluvastatin on IL-6 synthesis in human vascular smooth muscle cells (VSMCs). Addition of fluvastatin decreased IL-6 synthesis in VSMCs in a time (0–24 hours)- and dose (10^–8–10^–5 mol/L)-dependent manner. Fluvastatin also decreased IL-6 mRNA expression in VSMCs. The effects of fluvastatin on IL-6 expression were completely reversed in the presence of mevalonate or geranylgeranyl-pyrophosphate, but not squalene. Inhibition of Rho by C3 exoenzyme or Rho kinase by Y-27632 significantly decreased IL-6 expression in VSMCs. In conclusion, fluvastatin decreases IL-6 synthesis in human VSMCs through inhibition of Rho pathway. These findings suggested that reduction of IL-6 expression by statins may partially explain their therapeutic effects in patients with coronary artery disease.     

Regulation of plasminogen activator inhibitor-1 secretion by urokinase and tissue plasminogen activator in rat epithelioid-type smooth muscle cells

Tissue plasminogen activator (tPA) and urokinase (uPA) are targets of plasminogen activator inhibitor-1 (PAI-1) inhibition. We have previously shown that both proteases can also induce PAI-1 secretion in rat smooth muscle cells (SMCs). We now report that both proteases appear to use very similar cellular mechanisms for signal transduction. They induced PAI-1 secretion using a pathway(s) involving protein kinase C (PKC). They also activated the Raf/Mek/mitogen-activated protein kinase (MAPK) pathway, which lies downstream of PKC activation. Activation of protein kinase A (PKA), however, lowered PAI-1 secretion induced by uPA and tPA, as a result of an inhibition of the PKC pathway and inhibition of Raf, Mek and MAPK phosphorylations. Src and syk family non-receptor tyrosine kinases (TK) were also involved in PAI-1 induction. The mechanisms of interaction of these tyrosine kinases with other pathways appeared to be quite different: src appeared to act within the PKC and PKA pathways, while syk operated independently of these pathways. Furthermore, whereas src inhibition resulted in inhibition of Raf/Mek/Erk phosphorylations, syk inhibition could only inhibit Mek and Erk phosphorylations but not the phosphorylation of Raf. These multiple pathways utilized by uPA and tPA to modulate PAI-1 secretion might be involved in determining the proteolytic or antiproteolytic potential of the SMCs under different pathophysiological conditions.     

Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells

We analyzed the secretory dynamics of tissue plasminogen activator (tPA) in EA.hy926 cells, an established vascular endothelial cell (VEC) line producing GFP-tagged tPA, using total internal reflection-fluorescence (TIR-F) microscopy. tPA-GFP was detected in small granules in EA.hy926 cells, the distribution of which was indistinguishable from intrinsically expressed tPA. Its secretory dynamics were unique, with prolonged (> 5 minutes) retention of the tPA-GFP on the cell surface, appearing as fluorescent spots in two-thirds of the exocytosis events. The rapid disappearance (mostly by 250 ms) of a domain-deletion mutant of tPA-GFP possessing only the signal peptide and catalytic domain indicates that the amino-terminal heavy chain of tPA-GFP is essential for binding to the membrane surface. The addition of PAI-1 dose-dependently facilitated the dissociation of membrane-retained tPA and increased the amounts of tPA-PAI-1 high-molecular-weight complexes in the medium. Accordingly, suppression of PAI-1 synthesis in EA.hy926 cells by siRNA prolonged the dissociation of tPA-GFP, whereas a catalytically inactive mutant of tPA-GFP not forming complexes with PAI-1 remained on the membrane even after PAI-1 treatment. Our results provide new insights into the relationship between exocytosed, membrane-retained tPA and PAI-1, which would modulate cell surface-associated fibrinolytic potential.     

吡格列酮对AGE-BSA诱导大鼠平滑肌细胞增殖和PAI-1 mRNA表达水平的影响

目的探讨吡格列酮（Pio）对糖基化修饰的牛血清白蛋白（AGE-BSA）诱导大鼠血管平滑肌细胞（VSMCs）增殖以及纤溶酶原激活物抑制剂1（PAI-1）mRNA表达的影响。方法体外制备的AGE-BSA以不同浓度和不同时间干预大鼠VSMCs,观察Pio对AGE-BSA作用的影响（1）MTT法观察VSMCs增殖;（2）半定量RT-PCR测定PAI-1 mRNA表达水平。结果（1）各浓度的AGE-BSA作用16h开始导致VSMCs增殖（P〈0.05）;（2）AGE-BSA导致PAI-1 mRNA表达水平升高,呈浓度、时间依赖性;（3）Pio可明显抑制AGE-BSA诱导的VSMCs增殖以及PAI-1 mRNA表达水平。结论Pio在2型糖尿病中可能通过抑制AGE-BSA诱导的VSMCs增殖和PAI-1 mRNA表达水平,在血管并发症方面起到有益作用。     

Regulation of plasminogen activator inhibitor-1 secretion by growth factors in smooth muscle cells.

Epithelioid-type vascular smooth muscle cells are metabolically active and secrete many proteases and protease inhibitors. We have previously cloned epithelioid-type smooth muscle cells from rat carotid arteries, and showed that polypeptide growth factors basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) could dose-dependently induce plasminogen activator inhibitor-1 (PAI-1) secretion from these cells. In the present study, we have used these cells to investigate the growth factor-induced signal transduction pathways leading to PAI-1 secretion. We report here that PAI-1 induction was dependent on protein kinase C (PKC) and tyrosine kinase but not on protein kinase A (PKA), ras and phosphoinositol-3-kinase inhibitor. Induction of PAI-1 by bFGF and PDGF was also accompanied by activation of a mitogen-activated protein kinase pathway involving Raf/Mek/Erk1/2, and the family non-receptor tyrosine kinases., another non-receptor tyrosine kinase, on the contrary, behaved differently from in that it was part of a pathway leading to PAI-1 induction by bFGF, but not when PDGF was used as the stimulating reagent. Activation of a PKA-dependent pathway(s) opposed PAI-1 induction. One mechanism for PKA activators to inhibit PAI-1 secretion was that they markedly inhibited the phosphorylations of Mek and mitogen-activated protein kinase that were up-regulated in the presence of bFGF and PDGF.     

Regulation of interleukin-8 expression by HMG-CoA reductase inhibitors in human vascular smooth muscle cells

Interleukin-8 (IL-8) is a potent chemotactic factor that has been implicated in atherogenesis. HMG-CoA reductase inhibitors (statins) may reduce the cardiovascular risk and vulnerability of atherosclerotic plaque through nonlipid mechanisms such as inhibition of cytokine expression. In this study, we investigated the effects of statins on IL-8 synthesis in human vascular smooth muscle cells (VSMCs). Addition of angiotensin II (Ang II) increased IL-8 production in VSMCs in a time (0-24 h)- and dose (10(-8)-10(-6) mol/l)-dependent manner with increased IL-8 mRNA accumulation. The Ang II type 1 receptor (AT1R) antagonist candesartan, but not the Ang II type 2 receptor (AT2R) antagonist PD123319, significantly blocked Ang II-induced IL-8 production. Addition of fluvastatin decreased the basal and Ang II-induced IL-8 production in VSMCs in a dose (10(-8)-10(-5) mol/l)-dependent manner with a decrease in IL-8 mRNA accumulation. The effect of fluvastatin on IL-8 production was completely reversed in the presence of mevalonate or geranylgeranyl-pyrophosphate, but not in the presence of squalene or farnesyl-pyrophosphate. Lipophilic cerivastatin also significantly decreased IL-8 production, while hydrophilic pravastatin showed no effect on IL-8 levels. In conclusion, we demonstrated for the first time that Ang II increased IL-8 production and fluvastatin decreased the basal and Ang II-induced IL-8 production in human VSMCs. These findings suggested that Ang II may exacerbate atherosclerosis through induction of IL-8 in VSMCs, while statins may exert therapeutic effects by modulating IL-8 synthesis in patients with atherosclerotic disease.     

Vitamin D analogs modulate the expression of plasminogen activator inhibitor-1, thrombospondin-1 and thrombomodulin in human aortic smooth muscle cells

BACKGROUND/AIMS: Plasminogen activator inhibitor-1 (PAI-1), thrombospondin-1 (THBS1) and thrombomodulin (TM) are involved in atherothrombosis. Vitamin D receptor agonists (VDRAs) provide survival/cardiovascular benefits for chronic kidney disease patients. METHODS: The effects of VDRAs on regulating PAI-1, THBS1 and TM in human aortic smooth muscle cells (SMC) and endothelial cells (EC) were studied. RESULTS: In SMC, paricalcitol and calcitriol downregulated the expression of PAI-1 mRNA and protein in a dose-dependent manner (EC(50) = 0.7 and 4.4 nM, respectively). Both drugs also downregulated THBS1 mRNA and protein (EC(50) = 1.6 and 3.9 nM, respectively). In contrast, paricalcitol and calcitriol upregulated TM mRNA and protein (EC(50) = 28.9 and 25.5 nM, respectively). EC did not express VDR, and VDRAs failed to induce CYP24A1, a VDR target gene. The effect of paricalcitol on THBS1 in SMC was blocked by cycloheximide, while its effect on TM and CYP24A1 was not affected, suggesting that the regulation of THBS1 by VDR may be mediated through intermediate factors, but that TM is likely a direct target of VDR. CONCLUSION: VDR may play a role in atherothrombosis via regulation of PAI-1, THBS1 and TM.     

Type 1 plasminogen activator inhibitor synthesis of endothelial cells is downregulated by smooth muscle cells

Plasminogen activator inhibitor type 1 (PAI-1), the physiologic inhibitor of both tissue-type plasminogen activator (tPA) and urokinase- type plasminogen activator (uPA), is a major biosynthetic product of endothelial cells in vitro; endothelial cells in vivo, in contrast, do not appear to produce significant amounts of PAI-1 as made evident by in situ-hybridization studies in normal mice. This suggests that the high rate of PAI-1 synthesis of endothelial cells in vitro might be a result of the culture conditions. When human umbilical vein endothelial cells (HUVEC) were grown on human amniotic membranes, resembling the natural growth support instead of coated plastic, their morphology was changed from the cobblestone-like appearance on plastic to an in vivo like flagstone pattern. However, this morphological change had no significant effect on the synthesis and secretion of PAI-1. When smooth muscle cell (SMC) conditioned media (CM) were added to HUVEC cultures, PAI-1 antigen secretion of HUVEC was reduced by 40% to 60% as measured by enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation experiments using 36S-methionine metabolically labeled HUVEC and Northern blot analysis of HUVEC PAI-1 mRNA indicate that this reduction was attributable to decreased PAI-1 synthesis and reduced steady-state levels of both the 3.2 kb and 2.2 kb form of PAI-1 mRNA. This effect was dose-dependent and observed under serum-containing as well as serum- free conditions, in the absence or presence of endothelial cell growth supplement (ECGS, 0 to 100 micrograms/mL) and attributable to a nondialyzable factor. Our data suggest that the high level of PAI-1 biosynthesis of endothelial cells in vitro may be attributable to the lack of a soluble factor produced by SMC, which controls and suppresses PAI-1 biosynthesis of endothelial cells in vivo.     

Effect of HMG-CoA reductase inhibitors on extracellular matrix expression in human vascular smooth muscle cells

Clinical studies have shown that treatment with 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors can stabilize atherosclerotic plaques and slow their progression. One determinant of plaque stability and size is the composition of the vascular extracellular matrix. The aim of this study was to evaluate the effects of different HMG-CoA reductase inhibitors on the expression of major components of the vascular extracellular matrix in smooth muscle cells. Cultured human vascular smooth muscle cells were incubated for 24–72 h with the HMG-CoA reductase inhibitors lovastatin (1–50 μmol/L), simvastatin (0.05–20 μmol/L), and pravastatin ( 1–100 μmol/L). RNA expression of the extracellular matrix proteins thrombospondin-1, fibronectin, collagen type I, and biglycan as well as expression of the cytokine TGF-β1 was determined by Northern blotting. Extracellular matrix protein secretion was visualized by immunofluorescence. In addition, cell proliferation and viability were measured using BrDU-ELISAs, MTT-tests, and direct cell counting. Expression of thrombospondin-1 was significantly decreased after 24 h incubations with lovastatin in concentrations as low as 1 μmol/L. Coincubation with the cholesterol precursor mevalonate completely reversed this effect. The downregulation of thrombospondin-1 expression occured in the same concentration range that also inhibited cell proliferation. In contrast, lovatatin did not affect expression of fibronectin, whereas collagen type I and biglycan expression decreased only after long incubations with high, toxic lovastatin concentrations. Simvastatin, but not the very hydrophilic compound pravastatin, had a similar effect on extracellular matrix expression as lovastatin. In summary, lovastatin and simvastatin predominantly decrease the expression of the glycoprotein thrombospondin-1, which is functionally associated with smooth muscle cell migration and proliferation. In contrast, expression of plaque-stabilizing extracellular proteins such as collagen type I and biglycan are much less affected.     

The HMG-CoA reductase inhibitor rosuvastatin inhibits plasminogen activator inhibitor-1 expression and secretion in human adipocytes

Human preadipocytes and adipocytes are known to produce the proatherogenic factor PAI-1 and proinflammatory cytokines, and obesity was found to be state of increased adipose production of these factors. In the present study, we investigated the effect of rosuvastatin on the regulation of PAI-1 gene expression in human adipocytes. Human preadipocytes, adipocytes in primary culture and the SGBS cell line were used as cell models. Cells were transfected using various constructs and promoter activity was measured as luciferase activity. PAI-1 expression was measured by quantitative RT-PCR and ELISA. Rosuvastatin inhibited PAI-1 mRNA expression and secretion of the protein in a concentration-dependent manner. This effect was reversed by isoprenoids. Addition of MEK-inhibitors and NFkappaB inhibitors also reduced PAI-1 expression and PAI-1 promoter luciferase activity. Further experiments revealed that rosuvastatin down-regulated the MEKK-1 mediated activation of the PAI-1 promoter. In conclusion our data suggest that rosuvastatin inhibits PAI-1 expression and release from human adipocytes via a MEKK-1-dependent but not a NFkappaB-dependent mechanism.     

Effect of valsartan on angiotensin II-induced plasminogen activator inhibitor-1 biosynthesis in arterial smooth muscle cells

Previous studies have shown that angiotensin II stimulates the synthesis of plasminogen activator inhibitor-1 in cultured vascular cells, which suggests that activation of the renin-angiotensin system may impair fibrinolysis. We have investigated the effects of angiotensin II and of valsartan, a recently developed angiotensin II antagonist that is highly specific and selective for the angiotensin II subtype 1 receptor, on plasminogen activator inhibitor-1 secretion by smooth muscle cells isolated from rat and human vessels. Angiotensin II induced a time- and concentration-dependent increase of plasminogen activator inhibitor activity in supernatants of rat aortic cells, which reached a plateau after 6 hours of incubation with 100 nmol/L angiotensin II (2.4+/-0.6-fold over control value; P:<0.001). The angiotensin II-induced plasminogen activator inhibitor activity was inhibited, in a concentration-dependent manner, by valsartan with an IC(50) value of 21 nmol/L. Valsartan fully prevented the angiotensin II-induced increase in plasminogen activator inhibitor-1 protein and mRNA. Furthermore, angiotensin II doubled the secretion of plasminogen activator inhibitor-1 by smooth muscle cells obtained from human umbilical and internal mammary arteries, and valsartan fully prevented it. Angiotensin II did not affect the secretion of tissue plasminogen activator antigen by any of the cell systems tested. Thus, valsartan effectively inhibits angiotensin II-induced plasminogen activator inhibitor-1 secretion without affecting that of tissue plasminogen activator in arterial rat and human smooth muscle cells.     

细胞因子诱导人胸膜细胞释放纤溶酶原激活物抑制剂-1的研究

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Thrombin regulation of mRNA levels of tissue plasminogen activator and plasminogen activator inhibitor-1 in cultured human umbilical vein endothelial cells

Cultured human umbilical vein endothelial cells release tissue plasminogen activator (t-PA) and type 1 plasminogen activator inhibitor (PAI-1) in response to alpha thrombin stimulation. In order to study the mechanisms of thrombin stimulation, we measured changes in levels of mRNA for t-PA and PAI-1 following exposure of endothelial cells to 3 U/mL alpha thrombin. Alpha thrombin causes a significant and time- dependent increase in the mRNA levels of both t-PA and PAI-1. Catalytically inactivated diisofluorophosphate (DIP) treated thrombin and alpha thrombin pretreated with hirudin do not alter t-PA and PAI-1 mRNA levels. We conclude that the increased secretion of t-PA and PAI-1 by human umbilical vein endothelial cells in response to alpha thrombin is mediated at least partially through an increase in mRNA levels. In addition, an active thrombin catalytic site is required for these increases in mRNA to occur.