Production and Release of Inactive Renin by Human Vascular Smooth Muscle Cells

Human arterial smooth muscle cells were obtained from surgically excised tissues and cultured by the explant method. The cultured cells had both active and inactive forms of an angiotensin I forming enzyme. About a five-fold increase in the activity was obtained by trypsin treatment. This renin-like enzyme was also found in abundance in the culture media, mostly in an inactive form. Most of the enzyme activity, either before or after the activation, was suppressed by an antibody specific to human renin. The inactive enzyme was activated to some extent also by acidification and by cold exposure. The molecular weight of the inactive enzyme was estimated to be approximately 49,000 by gel filtration. These results suggest that human vascular smooth muscle cells can produce renin and release an inactive form of renin, and can be a potential source of plasma inactive renin under certain conditions such as the anephric state.     

Phosphatidylethanol in high density lipoproteins increases the vascular endothelial growth factor in smooth muscle cells

To study whether qualitative changes in high density lipoprotein (HDL) phospholipids mediate part of the advantageous effects of ethanol on atherosclerosis, we investigated whether HDL associated phosphatidylethanol (PEth) affects the secretion of vascular endothelial growth factor (VEGF) from cultured human smooth muscle cells. Serum-starved human umbilical vein HUVS-112D smooth muscle cells were incubated in the presence of PEth–HDL, HDL, or buffer. The phosphorylation of protein kinase C (PKC) and mitogen activated protein kinase (p44/42 MAPK) was determined by specific antibodies against phosphorylated and total proteins. VEGF concentrations were measured from cell culture medium of the cells. PEth increased the secretion of VEGF into the culture medium of HUVS cells. PEth–HDL increased the PKC phosphorylation by 2.1-fold and p44/42 MAPK phosphorylation by 3.3-fold compared with HDL, indicating that PEth-containing HDL particles influence vascular smooth muscle cells by PKC and p44/42 MAPK signalling. This may mediate the effects of ethanol on vascular wall by increasing the VEGF secretion from smooth muscle cells. The secreted VEGF may inhibit the formation of neointima and in doing so helps prevent atherosclerosis.     

Platelet-derived growth factor suppresses and fibroblast growth factor enhances cytokine-induced production of nitric oxide by cultured smooth muscle cells. Effects on cell proliferation.

Stimulation of thymidine incorporation by basic fibroblast growth factor or epidermal growth factor treatment of cultured quiescent smooth muscle cells (rat and human) was attenuated by the concomitant treatment with interleukin-1 beta in the presence of indomethacin. Platelet-derived growth factor-AB and -BB-induced thymidine incorporation was not inhibited by the presence of the cytokine under similar experimental conditions. Elevation of nitrite levels in the conditioned medium of cultures exposed to interleukin-1 beta correlated with the inhibition of thymidine incorporation. Platelet-derived growth factor-AB and -BB inhibited the production of nitric oxide (measured as nitrite levels in conditioned medium) by cells treated simultaneously with interleukin-1 beta and growth factor. However, platelet-derived growth factor-AA neither affected nitrite production nor thymidine incorporation by smooth muscle cells. Levels of cytokine-stimulated nitrite production by smooth muscle cells were increased synergistically by the presence of fibroblast growth factors or epidermal growth factor. The inhibition of thymidine incorporation and concomitant elevation of nitrite production was abolished in the presence of nitro-L-arginine. Cultures maintained in the presence of low levels of the cytokine for 9 days were growth-inhibited, and this was reversed when culture medium was supplemented with nitro-L-arginine. The treatment of smooth muscle cells, which were grown in coculture inserts with the cytokine to induce nitric oxide production, before their combination with other quiescent layers of cells resulted in the inhibition of thymidine incorporation by this second layer of cells regardless of the growth factor used for stimulation. Nitric oxide may act as an endogenous inhibitor of smooth muscle cell proliferation in the vessel wall, and impairment of its production may be one action of potent vascular mitogens such as platelet-derived growth factor.     

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   

Insulin stimulates endogenous angiotensin II production via a mitogen-activated protein kinase pathway in vascular smooth muscle cells

OBJECTIVE: The present study was designed to determine the effects of insulin on cytosolic angiotensin II production and proliferation in cultured rat vascular smooth muscle cells. DESIGN AND METHODS: Vascular smooth muscle cells were incubated with insulin for 48 h. Cytosolic angiotensin I and II were determined by radioimmunoassays of purified cell homogenates. Angiotensin II was also detected by immunohistochemistry of intact cells. Cell proliferation was determined by pulse labeling with radiolabeled thymidine. Angiotensinogen mRNA expression was determined by slot-blot analysis. RESULTS: Insulin significantly increased cytosolic angiotensin II concentration in vascular smooth muscle cells. Lisinopril, omapatrilat and irbesartan inhibited this increase of angiotensin II, but had no effect on angiotensin I levels. Immunohistochemical staining confirmed the presence of angiotensin II in control and insulin-treated vascular smooth muscle cells. Insulin increased cell proliferation, and addition of lisinopril, omapatrilat or irbesartan inhibited this effect. Insulin also increased expression of angiotensinogen mRNA in cultured vascular smooth muscle cells, but PD98059, a mitogen-activated protein kinase inhibitor, prevented the rise in angiotensinogen expression. CONCLUSION: These results support the concept that insulin stimulates angiotensin II production in cultured vascular smooth muscle cells through a mitogen-activated, protein kinase-dependent pathway that might be a factor in the progression of atherosclerosis. Agents that block the renin-angiotensin system have direct protective effects, reducing vascular angiotensin II and growth of vascular smooth muscle cells and are thus of cardiovascular benefit.     

Human vascular smooth muscle cells both express and respond to heparin-binding growth factor I (endothelial cell growth factor).

The control of vascular endothelial and smooth muscle cell proliferation is important in such processes as tumor angiogenesis, wound healing, and the pathogenesis of atherosclerosis. Class I heparin-binding growth factor (HBGF-I) is a potent mitogen and chemoattractant for human endothelial cells in vitro and will induce angiogenesis in vivo. RNA gel blot hybridization experiments demonstrate that cultured human vascular smooth muscle cells, but not human umbilical vein endothelial cells, express HBGF-I mRNA. Smooth muscle cells also synthesize an HBGF-I-like polypeptide since (i) extract prepared from smooth muscle cells will compete with 125I-labeled HBGF-I for binding to the HBGF-I cell surface receptor, and (ii) the competing ligand is eluted from heparin-Sepharose affinity resin at a NaCl concentration similar to that required by purified bovine brain HBGF-I and stimulates endothelial cell proliferation in vitro. Furthermore, like endothelial cells, smooth muscle cells possess cell-surface-associated HBGF-I receptors and respond to HBGF-I as a mitogen. These results indicate the potential for an additional autocrine component of vascular smooth muscle cell growth control and establish a vessel wall source of HBGF-I for endothelial cell division in vivo.     

血小板衍生生长因子—BB对血管内皮细胞、平滑肌细胞及成纤维细胞增殖的影响

目的 观察血小板衍生生长因子—BB对培养的人血管内皮细胞、兔平滑肌细胞和人成纤维细胞增殖的影响。方法 采用培养的人脐静脉血管内皮细胞、兔动脉血管平滑肌细胞和人血管成纤维细胞，应用^3H—TdR掺入方法，观察血小板衍生生长因子—BB对三种细胞DNA合成的影响。结果 血小板衍生生长因子—BB可促进处于静止状态的三种细胞DNA的合成，并呈现出明显的浓度依赖关系，在30ng／ml的浓度时成纤维细胞DNA的合成达到高峰，在40ng／ml的浓度时内皮细胞、平滑肌细胞DNA的合成达到高峰。成纤维细胞、平滑肌细胞分别在PDCF-BB作用24h和36h年到DNA合成的高峰，内皮细胞在48h时DNA合成量最高。结论 血小板衍生生长因子—BB可明显促进培养的人脐静脉血管内皮细胞、兔动脉血管平滑肌细胞和人血管成纤维细胞的增殖。     

氯化锂通过上调焦磷酸水平抑制血管平滑肌细胞钙化

目的探讨氯化锂对血管平滑肌细胞钙化的影响及其可能的作用机制。方法体外培养人主动脉血管平滑肌细胞,用钙化培养基(无机磷浓度为3 mmol/L)建立平滑肌细胞钙化模型。药物处理组用氯化锂(10 mmol/L)预处理细胞4 h后加入3 mmol/L的无机磷,继续培养细胞数天后用茜素红染色检测细胞钙盐沉积水平,同时应用焦磷酸偶联酶反应底物烟酰胺腺嘌呤二核苷酸(NADH)消耗法,酶标仪(OD340)检测细胞外焦磷酸的分泌;实时荧光定量聚合酶链式反应(real-time PCR)和免疫印迹法(Western blot)检测细胞中ankh基因的表达;采用慢病毒感染人主动脉平滑肌细胞的方法,建立对照细胞组和ankh敲低的实验细胞组,并观察其对细胞钙化的影响。结果高磷细胞组钙化检测值为(65.00±2.11)ng/g,较对照细胞组(12.39±0.38)ng/g钙化水平明显升高(P<0.01)。氯化锂预处理细胞的钙化检测值为(24.92±1.87)ng/g,与高磷对照组(60.94±4.51)ng/g比较能显著抑制高磷诱导的血管平滑肌细胞钙化(P<0.01);氯化锂预处理明显上调细胞外液中焦磷酸的含量,基础条件下预处理组为(51.70±7.26)×10^-3 mmol/g,对照组为(28.71±2.55)×10^-3mmol/g(P<0.01);高磷刺激下预处理组为(34.35±4.27)×10^-3mmol/g,对照组为(20.89±4.93)×10^-3mmol/g(P<0.05),同时氯化锂预处理显著升高细胞ankh表达水平(P<0.01);而敲低ankh使无机磷诱导的血管平滑肌细胞钙化程度显著加重,敲低ankh细胞钙化检测值为(71.73±2.45)ng/g,对照组为(56.19±3.59)ng/g(P<0.01)。结论氯化锂通过上调平滑肌ANKH的表达,升高细胞外液中的焦磷酸水平,抑制高磷诱导下血管平滑肌细胞的钙化。     

Short- and long-term interactions of endothelium and vascular smooth muscle in coculture: effects on cyclic GMP production

In intact blood vessels, many vasodilators act by stimulating the release from endothelium of factor(s) that relax vascular smooth muscle and stimulate increases in cGMP. To investigate how endothelium regulates cGMP production in vascular smooth muscle, bovine aortic endothelial cells and rat aortic smooth muscle cells were cultured both separately and together in cocultures for 48 hr. Nitroprusside (1 mM) increased intracellular cGMP concentration 30-fold in smooth muscle cells (from a basal level of 103 +/- 54 fmol/mg of cell protein to 2920 +/- 1800 fmol/mg) but only 2-fold in endothelial cells (from 41 +/- 7 fmol/mg to 93 +/- 23 fmol/mg). When endothelial and smooth muscle cells were cocultured as a mixed cell population (1:1 cell ratio), both basal and nitroprusside-stimulated cGMP levels were significantly increased (550 +/- 250 and 13,240 +/- 9950 fmol/mg of total cell protein, respectively). The calcium ionophore A23187 (10 microM) caused no increase in cGMP concentration in either cell type cultured alone but produced a 6-fold increase in cocultures. Neither aspirin nor 5,8,11,14-icosatetraynoic acid influenced these results. No changes in cAMP levels were detected. Using cocultures in which one cell type was grown on microcarrier beads, we have shown that cGMP increased only in vascular smooth muscle cells and was not dependent upon the formation of junctions between endothelium and smooth muscle cells. In long-term (48-hr) mixed-cell cocultures, but not in short-term microcarrier cocultures, amplification of the nitroprusside-induced increase in cGMP was observed. These results show that responses associated with endothelium-dependent relaxation can be reconstituted in cultured endothelial and vascular smooth muscle cells and that endothelium generates a humoral factor(s) that stimulates accumulation of smooth muscle cGMP and has a longer-term effect that amplifies guanylate cyclase stimulation by nitroprusside, a drug acting directly upon smooth muscle to stimulate formation of the cyclic nucleotide. Cultured cells provide a valuable model system for the study of endothelium-vascular smooth muscle interactions.     

Oxysterol-induced apoptosis of vascular smooth muscle cells is reduced by HMG-CoA reductase inhibitor,pravastatin

We investigated the mechanism by which 7-ketocholesterol damages vascular smooth muscle cells and the protective effect of the hydroxymethyl glutary CoA reductase inhibitor, pravastatin on it. When 7-ketocholesterol (50 micromol/L) was added to cultured human vascular smooth muscle cells, the extent of cell detachment increased and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling was positive. DNA extracted from the smooth muscle cells exposed to 7-ketocholesterol showed a ladder pattern on agarose electrophoresis. The fragmented DNA also increased in smooth muscle cells incubated with 7-ketocholesterol dose-dependently. In the presence of pravastatin, the cell detachment induced by 7-ketocholesterol was inhibited and the amount of fragmented DNA decreased significantly. These effects of pravastatin were inhibited by mevalonate. The results suggest that 7-ketocholesterol-induced apoptosis of vascular smooth muscle cells is inhibited by pravastatin, and mevalonate acts as a trigger of the apoptosis.     

miR-155通过调控CaSR表达影响VSMC生长的机制研究

目的 miR-155可以通过干扰血管平滑肌细胞(VMSC)生物活性来抑制动脉粥样硬化斑块的形成,但具体机制不十分清楚.本研究主要观察miR-155对VSMC中钙敏感受体(CaSR)表达的影响,同时探讨CaSR在miR-155调控VSMC生物活性过程中的作用.方法 培养VSMC,将miR-155 mimic、Ad-CaS...     

Carvedilol, a cardiovascular drug, prevents vascular smooth muscle cell proliferation, migration, and neointimal formation following vascular injury.

Carvedilol is a cardiovascular drug currently used for the treatment of hypertension. Clinical studies have recently demonstrated efficacy in angina and congestive heart failure. Recently, carvedilol has been shown to attenuate oxygen free radical-initiated lipid peroxidation and to inhibit vascular smooth muscle mitogenesis induced by a wide variety of growth factors. These findings are of interest since smooth muscle proliferation and abnormal lipid metabolism are proposed to play an important role in the pathogenesis of atherosclerotic plaque formation and in development of stenotic lesions following vascular injury by balloon angioplasty and coronary artery bypass grafting. On the basis of these observations, the antiproliferative actions of carvedilol have been explored in detail. In human cultured pulmonary artery vascular smooth muscle cells, carvedilol (0.1-10 microM) produced a concentration-dependent inhibition of the mitogenesis stimulated by platelet-derived growth factor, epidermal growth factor, thrombin, and serum, with IC50 values ranging from 0.3 to 2.0 microM. Carvedilol also produced a concentration-dependent inhibition of vascular smooth muscle cell migration induced by platelet-derived growth factor, with an IC50 value of 3 microM. The extensive neointimal formation that occurs following balloon angioplasty of rat carotid arteries was markedly attenuated by carvedilol (1 mg/kg, i.p.; twice daily starting 3 days before angioplasty and continuing until 14 days after angioplasty). Quantitative image analysis demonstrated that carvedilol reduced the neointimal growth following angioplasty by 84% without altering either medial or adventitial cross-sectional areas. These observations indicate that carvedilol may also be effective in the treatment of pathological disorders principally associated with abnormal vascular smooth muscle growth, such as atherosclerosis and acute vascular wall injury induced by angioplasty or coronary artery bypass grafting.     

体外转染野生型p16基因对兔血管平滑肌细胞生长的抑制作用

目的 :研究体外转染外源性野生型 p16基因对兔血管平滑肌细胞生长的影响。　　方法 :构建野生型 p16基因的重组逆转录病毒载体 ,并体外转染兔血管平滑肌细胞 ,应用 Northern blot及 Westernblot检测外源 p16基因的表达 ,并用四唑盐 ( MTT)法及流式细胞仪检测其对细胞生长的抑制作用。　　结果 :逆转录病毒载体可有效地将外源性 p16基因导入平滑肌细胞 ,表达 P16蛋白 ,并显著抑制血管平滑肌细胞的生长使细胞滞留于 G1 期。　　结论 :体外转染外源性野生型 p16基因可有效抑制血管平滑肌细胞的生长。     

有机阴离子转运蛋白3在人血管平滑肌细胞的表达

目的检测有机阴离子转运蛋白3（OAT3）及其mRNA在人血管平滑肌细胞的表达,并观察尿酸刺激对其表达的影响。方法分离培养人脐动脉血管平滑肌细胞,随机分为正常对照组及尿酸处理组,提取细胞总RNA,RT-PCR方法扩增特异性OAT3 cDNA片段,制备探针,用Northern blot方法检测各组OAT3 mRNA的表达。提取细胞膜蛋白,用Westernblot方法检测各组OAT3的蛋白表达。结果在正常对照组人血管平滑肌细胞可检测到OAT3mRNA和蛋白的表达。与对照组相比,尿酸处理组血管平滑肌细胞OAT3的表达在mRNA及蛋白水平均显著上调（P<0.05,n=3）。结论人血管平滑肌细胞表达OAT3,尿酸处理可以使其表达上调。提示OAT3可能部分介导血管平滑肌细胞对尿酸的摄取过程。     

Growth factors and extracellular signal-regulated kinase in vascular smooth muscle cells of normotensive and spontaneously hypertensive rats

OBJECTIVE: Transforming growth factor-beta1 (TGF-beta1) stimulates vascular smooth muscle cell growth in spontaneously hypertensive rats (SHR), but inhibits cell growth in normotensive Wistar- Kyoto (WKY) rats. The present study was undertaken to test the hypothesis that TGF-beta1 might differentially modulate the activities of mitogen-activated protein (MAP) kinase family members (ERK, JNK and p38) in vascular smooth muscle cells of SHR and WKY rats. METHODS: MAP kinase activity was measured from cultured vascular smooth muscle cells in response to TGF-1 by specific substrate phosphorylation of myelin basic protein, GST-c-Jun and GST-ATF2. RESULTS: Exposure of cultured vascular smooth muscle cells from SHR or WKY rats to TGF-beta1 resulted in a marked increase in the activity of ERK, but not of JNK or p38. The increase of ERK activity stimulated by TGF-beta1 appeared similar in time course and extent in both WKY and SHR cells, with increased activity peaking at 15 min of incubation. Epidermal growth factor (EGF) also stimulated the activity of ERK, in both WKY and SHR cells, but nor of JNK or p38, with stimulation of ERK activity by EGF occurring more rapidly in SHR cells than in those from WKY rats. Co-incubation of SHR cells with TGF-beta1 and EGF showed additive effect on ERK activity. CONCLUSIONS: The results provide the first evidence that TGF-beta1 activates ERK in vascular smooth muscle cells of both normotensive and hypertensive rats. The matching response of ERK activation to TGF-1 in SHR cells suggests that the MAP kinase-signaling pathway remains largely unchanged in the regulation of vascular smooth muscle growth by TGF-1 in spontaneously hypertensive rats.     

Low density lipoproteins in human plasma make vascular smooth muscle cells resistant to growth inhibition by heparin

OBJECTIVE: Vascular smooth muscle cell hyperplasia plays a role in atherosclerosis and restenosis. While heparin has shown promise as an inhibitor of smooth muscle cell proliferation in vitro and in some animal models, it has failed to reduce restenosis in clinical trials. We have previously shown that human smooth muscle cells grown in the presence of human serum are heparin resistant, whereas in the presence of bovine serum they are heparin sensitive. In this report, we demonstrate that the heparin resistance factor is present in human plasma as well as serum, and characterise it further. METHODS: Human vascular smooth muscle cells were cultured as explants from the media of redundant adult internal mammary or umbilical cord arteries. They were tested for sensitivity to heparin at 100 microg/ml in the growth medium, in the presence of foetal bovine serum, human-plasma-derived serum, human whole blood serum, or fractions derived from these. RESULTS: In the presence of foetal bovine serum, heparin inhibited cell proliferation, while human-plasma-derived or whole blood sera conveyed heparin resistance. This activity was contained within the fraction of plasma/serum which bound to heparin Sepharose, and the sub-fraction which was retained by a membrane filter of molecular weight cut off of 100000. All the heparin resistance in this latter fraction was supplied by lipoproteins. LDL prepared directly from human plasma conveyed similar heparin resistance to the lipoproteins from the above sub-fraction. CONCLUSION: LDL in human plasma/serum conveys resistance to the anti-proliferative effects of heparin upon vascular smooth muscle cells. This activity may interfere with potential therapeutic effects of heparin as an anti-restenosis agent.     

Effect of glycated collagen on proliferation of human smooth muscle cells in vitro

Summary While non-enzymatic glycation of long-lived tissue proteins such as collagen has been implicated in chronic complications of diabetes mellitus, its role in the aetiology of diabetic macroangiopathy has not been elucidated. To test the hypothesis that glycation of collagen abolishes the inhibitory effect of native collagen on the proliferation of human smooth muscle cells, we obtained smooth muscle cells from human gastric arteries and cultured them on dishes coated with glycated or non-glycated collagen. The proliferation of human smooth muscle cells in the presence of 10 % fetal calf serum or platelet derived growth factor-BB (10 ng/ml) was inhibited by type 1 collagen coated on the dishes. Glycation of collagen with glucose 6-phosphate for 7 days abolished the growth-inhibitory effect of native collagen. Succinylation of collagen, which like glycation blocked the lysyl residues in collagen, also abolished the growth-inhibitory effect. Adhesion of human smooth muscle cells to collagen-coated dishes was not affected by glycation of collagen. Addition of glycated albumin to the medium did not affect the growth of human smooth muscle cells on plastic dishes. The inhibition of human smooth muscle cell proliferation by collagen was not reversed by the glycation of collagen in the presence of aminoguanidine. Results suggest that early glycation abolishes the inhibitory effect of collagen on human smooth muscle cell proliferation and may thus participate in the progression of macroangiopathy in diabetes. [Diabetologia (1996) 39: 800–806] Received: 22 August 1995 and in revised form: 19 February 1996