白介素-10抑制TNF-α诱导的血管平滑肌细胞增殖

研究观察了重组人白介素 10 (rhIL 10 )对肿瘤坏死因子 (TNF α)刺激的离体大鼠胸主动脉血管平滑肌细胞增殖、细胞周期及对p4 4 /p4 2丝裂素活化蛋白激酶的影响。实验培养大鼠主动脉血管平滑肌细胞 ,采用MTS/PES法确定血管平滑肌细胞 (vascularsmoothmusclecells,VSMCs)的增殖状态 ;应用流式细胞术测定细胞周期 ;利用p4 4 / 4 2磷酸化抗MAPK抗体的蛋白免疫印迹法测定MAPK蛋白表达。结果显示 :( 1)TNF α处理组与对照组相比 ,TNF α对VSMC增殖具有明显的刺激作用 (P <0 0 5 )。rhIL 10单独应用对VSMCs生长没有影响 (P >0 0 5 )。在TNF α刺激下 ,低至 10ng/ml的rhIL 10可抑制VSMCs的生长 (P <0 0 5 )。流式细胞术测定的结果显示 ,rhIL 10分别可使TNF α作用下的VSMC大部分处于G0 /G1期 ,与对照组相比有明显差异 (P <0 0 1)。 ( 2 )TNF α对p4 4 /p4 2MAPK蛋白表达有显著的增强作用 ,此作用可被rhIL 10抑制。结果提示 ,rhIL 10可抑制TNF α诱导的VSMC增殖及p4 4 /p4 2丝裂素活化蛋白激酶的表达     

几种扩血管多肽对bFGF促血管平滑肌细胞增殖作用的影响

目的和方法：研究肾上腺髓质素（Ａｄｍ）、降钙素基因相关肽（ＣＧＲＰ）及Ｃ－型心房利太（ＣＮＰ）对碱性成纤维细胞生长因子（ｂＦＧＦ）促血管平滑细胞（ＶＳＭＣ）增殖作用的影响及其机制。结果：孵育２４ｈ后,ｂＦＧＦ刺激ＶＳＭＣ增殖较对照组增加２．１倍（Ｐ〈０．０１）,细胞内蛋白磷酸化程度增加１．４倍（Ｐ〈０．０１）,ＰＫＣ及ＭＡＰＫ活性分别增加１．５和２．５倍（Ｐ〈０．０１０;Ａｄｍ．ＣＧＲＰｔ ＣＮＰ     

Role of caveolin-1 in p42/p44 MAP kinase activation and proliferation of human airway smooth muscle

Chronic airways diseases, including asthma, are associated with an increased airway smooth muscle (ASM) mass, which may contribute to chronic airway hyperresponsiveness. Increased muscle mass is due, in part, to increased ASM proliferation, although the precise molecular mechanisms for this response are not completely clear. Caveolae, which are abundant in smooth muscle cells, are membrane microdomains where receptors and signaling effectors can be sequestered. We hypothesized that caveolae and caveolin-1 play an important regulatory role in ASM proliferation. Therefore, we investigated their role in p42/p44 MAPK signaling and proliferation using human ASM cell lines. Disruption of caveolae using methyl-beta-cyclodextrin and small interfering (si)RNA-knockdown of caveolin-1 caused spontaneous p42/p44 MAPK activation; additionally, caveolin-1 siRNA induced ASM proliferation in mitogen deficient conditions, suggesting a key role for caveolae and caveolin-1 in maintaining quiescence. Moreover, caveolin-1 accumulates twofold in myocytes induced to a contractile phenotype compared with proliferating ASM cells. Caveolin-1 siRNA failed to increase PDGF-induced p42/p44 MAPK activation and cell proliferation, however, indicating that PDGF stimulation actively reversed the antimitogenic control by caveolin-1. Notably, the PDGF induced loss of antimitogenic control by caveolin-1 coincided with a marked increase in caveolin-1 phosphorylation. Furthermore, the strong association of PDGF receptor-beta with caveolin-1 that exists in quiescent cells was rapidly and markedly reduced with agonist addition. This suggests a dynamic relationship in which mitogen stimulation actively reverses caveolin-1 suppression of p42/p44 MAPK signal transduction. As such, caveolae and caveolin-1 coordinate PDGF receptor signaling, leading to myocyte proliferation, and inhibit constitutive activity of p42/p44 MAPK to sustain cell quiescence.     

Effects of TH-142177 on angiotensin II-induced proliferation, migration and intracellular signaling in vascular smooth muscle cells and on neointimal thickening after balloon injury.

We investigated the effects of TH-142177 (N-n-butyl-N-[2'-(1-H-tetrazole-5-yl) biphenyl-4-yl]-methyl-(N-carboxy methyl-benzylamino)-acetamide), a novel selective antagonist of angiotensin II type 1-receptor (AT1-R) on angiotensin II (AII)-induced proliferation and migration of vascular smooth muscle cells (VSMC), and on neointimal formation in the rat carotid artery after balloon injury, and on the intracellular signaling by the stimulation of AT1-R. High affinity AII receptor sites were detected in rat VSMC by the use of [125I]Sar1,Ile8-AII. TH-142177 and losartan competed with [125I]Sar1,Ile8-AII for the binding sites in VSMC in a monophasic manner, although PD123177, a selective antagonist of angiotensin II type 2-receptor (AT2-R), had little inhibitory effect, demonstrating the predominant existence of AT1-R in rat VSMC. TH-142177 prevented AII-induced DNA synthesis and migration, with a significant inhibition of 74 and 55%, respectively, at the concentration of 100 nM. AII-induced activation of p21ras, mitogen-activated protein kinase (p42MAPK and p44MAPK), and protein kinase C was significantly (50-78%) inhibited by TH-142177 (100 nM), suggesting that the activation of these enzymes is mediated through the stimulation of AT1-R. Balloon-injured left carotid arteries in rats showed extensive neointimal thickening, and TH-142177 (3 mg/kg) brought out a marked decrease in the neointimal thickening after balloon injury. In conclusion, TH-142177 inhibited AII-induced proliferation and migration of rat VSMC and neointimal formation in the carotid artery after balloon injury, and these effects may be related, in part, to the suppression of ras, p42MAPK and p44MAPK, and protein kinase C activities through the blockade of AT1-R. Thus, TH-142177 may have therapeutic potential for the treatment of vascular diseases such as atherosclerosis and restenosis.     

17beta -Estradiol modulates mechanical strain-induced MAPK activation in mesangial cells.

Gender is an important determinant of clinical outcome across a broad spectrum of kidney diseases, but the mechanism(s) responsible for the protective effect of female gender have not been fully elucidated. Remnant kidney glomerular injury is limited in female rats compared with male rats despite similar elevations in glomerular capillary pressure. In vitro, mechanical strain leads to the activation of p44/42 mitogen-activated kinase (p44/42 MAPK) and Jun N-terminal kinase/stress-activated protein kinase (SAPK) in glomerular mesangial cells (MC). Accordingly, we studied the effect of 17beta-estradiol on mechanical strain-induced signal transduction in MC. Exposure of MC to mechanical strain increased p44/42 MAPK activation (3-fold) and SAPK activation (2.5-fold), and kinase activation was inhibited by pretreatment with 17beta-estradiol (10(minus sign8) to 10(minus sign11) m) for 24 h in a dose-dependent manner. Mechanical strain-induced nuclear translocation of p44/42 MAPK and SAPK and nuclear protein binding to AP-1 were also attenuated by 17beta-estradiol. The inhibitory effects of 17beta-estradiol were not reproduced by the cell-impermeable estrogen, BSA/17beta-estradiol, nor did preincubation with 17beta-estradiol lead to actin cytoskeleton disassembly or impaired stress fiber formation. However, 17beta-estradiol did increase base-line levels of the dual specificity phosphatase MKP-1. The inhibitory effects of 17beta-estradiol on p44/42 MAPK activation and SAPK activation, translocation, and AP-1 binding were all abrogated by the estrogen receptor antagonist, ICI-182,780. We conclude that attenuation of mechanical strain-induced MAPK activation by 17beta-estradiol is dependent on intracellular estrogen receptor. The attenuation of stretch-induced kinase activation may be due, at least in part, to an effect of 17beta-estradiol on MKP-1 expression. Together, these findings add insight into the protective effect of gender on renal disease progression.     

一氧化氮在17-β雌二醇抑制血管平滑肌细胞增殖和原癌基因c -fos表达中的作用

实验利用大鼠血管平滑肌细胞（vascular smooth muscle cells,VSMC)作为模型,观察17-β雌二醇（E2）对VSMC增殖和原癌基因c-fos表达的影响,并探讨VSMC源性一氧化氮（NO）在基中的作用,检测指标包括NO释放的测定,细胞计数、^3H-Tdr掺入,噻唑蓝（MTT）测定和c-fosmRNA表达,结果显示,E2（10^-12-10^-8mol/L)呈浓度依赖性地促进VSMC中NO的释放;10^-8mol/LE2能明显抑制10%小牛血清（FCS）和10^-7mol/L内皮素-1（ET-1）诱导的细胞增殖和DNA合成,E2的抑制作用均可被雌激素受体（ER）拮抗剂tamoxifen(10^-7mol/L)和一氧化氮合酶抑制剂L-NAME（10^-6mol/L)明显减轻;E2（10^-8mol/L)可明显抑制10^-7mol/LET-1诱导的VSMCc-fos表达,这种抑制作用可被L-NAME（10^-6mol/L)明显减轻,这些结果提示E2能抑制VSMC增殖和原癌基因c-fos表达,这种促进VSMC的NO释放密切相关,而且该作用至少部分通过ER介导。     

Activation of mitogen-activated protein kinase by oxidized low-density lipoprotein in canine cultured vascular smooth muscle cells

Oxidized low-density lipoprotein (OX-LDL) contributes significantly to the development of atherosclerosis. However, the mechanisms of OX-LDL-induced vascular smooth muscle cell (VSMC) proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with a specific pattern of mitogen-activated protein kinase (MAPK) by [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in canine cultured VSMCs. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in VSMCs. Pretreatment of these cells with pertussis toxin (PTX) for 24 hours attenuated the OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating that these responses were mediated through a receptor coupled to a PTX-sensitive G protein. In cells pretreated with PMA for 24 h and with either the PKC inhibitor staurosporine or the tyrosine kinase inhibitor genistein for 1h, substantially reduced the [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to OX-LDL. Removal of Ca(2+) by addition of BAPTA/AM plus EGTA significantly inhibited OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating the requirement of Ca(2+) for these responses. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK). Furthermore, we also showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. Taken together, these results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G-protein-coupled receptor that involves the activation o Ras/Raf/MEK/MAPK pathway similar to those of PDGF-BB in canine cultured VSMCs.     

Bradykinin B<Subscript>2</Subscript> receptor-mediated proliferation via activation of the Ras/Raf/MEK/MAPK pathway in rat vascular smooth muscle cells

It has been suggested that bradykinin (BK) plays an important role in regulating neointimal formation after vascular injury. However, implication of BK in the growth of rat vascular smooth muscle cells (VSMCs) is controversial. Therefore, we examined the mitogenic effect of BK on VSMCs associated with activation of mitogen-activated protein kinase (MAPK). Both [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation were activated by BK in time- and concentration-dependent manners. Pretreatment of these cells with neither pertussis toxin nor cholera toxin attenuated the BK-induced responses. Pretreatment of VSMCs with Hoe 140 (a selective B(2) receptor antagonist), U73122 (an inhibitor of phospholipase C), and BAPTA/AM (an intracellular Ca(2+) chelator) inhibited both [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to BK. BK-induced [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation were inhibited by pretreatment of VSMCs with tyrosine kinase inhibitors (genistein and herbimycin A), protein kinase C (PKC) inhibitors (staurosporine, Go-6976, and Ro-318220), an MAPK kinase inhibitor (PD98059), and a p38 MAPK inhibitor (SB203580). Overexpression of the dominant negative mutants, H-Ras-15A and Raf-N4, suppressed p42/p44 MAPK activation induced by BK and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. From these results, we concluded that the mitogenic effect of BK is mediated through activation of the Ras/Raf/MEK/MAPK pathway similar to that of PDGF-BB. BK-mediated MAPK activation was modulated by Ca(2+), PKC, and tyrosine kinase all of which are associated with cell proliferation in rat cultured VSMCs.     

Amphiregulin is a potent mitogen for the vascular smooth muscle cell line,A7r5

The regulation of amphiregulin, an epidermal growth factor (EGF) family member, and its effect on vascular smooth muscle cells (VSMC) were examined. Amphiregulin mRNA was upregulated by amphiregulin itself as well as alpha-thrombin. Amphiregulin caused an approximate 3-fold increase in DNA synthesis. Its effect on growth was compared with those of other mitogens, and was found to be approximately 3.5-, 2.4-, and 1.0-fold greater than those of endothelin-I (ET-I), alpha-thrombin, and platelet-derived growth factor-AB (PDGF-AB), respectively. As evidenced by Western blot analysis, amphiregulin stimulated the phosphorylation of p42/p44-mitogen-activated protein kinase (MAPK), p38-MAPK, c-Jun NH2-terminal protein kinase (JNK), and Akt/protein kinase B (PKB), respectively. By statistical analysis, the amphiregulin-induced growth effect was significantly decreased by the MAP kinase/ extracellular regulated kinase kinase-1 (MEK-1) inhibitor PD98059, p38-MAPK inhibitor SB203580, and phosphatidylinositol 3-kinase (PI-3 kinase) inhibitor wortmannin, respectively, but was not decreased by JNK inhibitor SP600125. These results suggest that amphiregulin is the most potent mitogen of the mitogens tested, and its growth effect is mediated at least in part through the p42/p44-MAPK, p38-MAPK, and PI-3 kinase-Akt/PKB pathways in VSMC.     

Regional myocardial ischemia-induced activation of MAPKs is associated with subcellular redistribution of caveolin and cholesterol

Ischemia-reperfusion activates ERK and p38 MAPK in cardiac membranes, but the role of caveolae in MAPK signaling during this stress has not been studied. The purpose of this study was to determine the effect of in vivo myocardial ischemia-reperfusion on the level and distribution of caveolin-1 and -3 and cholesterol as well as MAPK activation in caveolin-enriched fractions. Adult male rats were subjected to in vivo regional myocardial ischemia induced by 25 min of coronary artery occlusion and 10 min (n = 5) or 2 h (n = 4) of reperfusion. Another group of rats served as appropriate nonischemic time controls (n = 4). A discontinuous sucrose density gradient was used to isolate caveolae/lipid rafts from ischemic and nonischemic heart tissue. Caveolin-1 and -3, as well as cholesterol, were enriched in the light fractions. A redistribution of caveolin-3 and a reduction in caveolin-1 and cholesterol levels in the light fractions occurred after 10 min of reperfusion. The ERKs were activated in ischemic zone light and heavy fractions by 10 min of reperfusion. p44 ERK was activated after 2 h of reperfusion only in the light fractions, whereas p42 ERK phosphorylation was increased in the light and heavy fractions. Although no p38 MAPK activation occurred after 10 min of reperfusion, 2 h of reperfusion caused significant activation of p38 MAPK in nonischemic zone light and heavy fractions. These results show the importance of caveolar membrane/lipid rafts in MAPK signaling and suggest that subcellular compartmentation of p44/p42 ERKs and p38 MAPK may play distinct roles in the response to myocardial ischemia-reperfusion.     

Caveolin-1, Id3a and two LIM protein genes are upregulated by estrogen in vascular smooth muscle cells

Estrogen has diverse effects on the vasculature, such as vasodilation, endothelial growth and inhibition of vascular smooth muscle cell (VSMC) proliferation and migration. However, little is known about the genes that are regulated by estrogen in the vascular wall. Wistar rats were ovariectomized or sham-operated (Sham group), and 2 weeks after the operation, were subjected to subcutaneous implantation of placebo pellets (OVX + V group) or estradiol pellets (OVX + E group). Endothelium-denuded aortic tissue was examined 2 weeks after implantation. By applying high-density oligonucleotide microarray analysis, the expression of approximately 7000 genes was analyzed. Among the genes with different expression levels between the OVX + E group and the OVX + V group, those that have been reported to be expressed in the vasculature or muscle tissue, were chosen. Finally, four genes, caveolin-1, two LIM proteins (enigma and SmLIM) and Id3a, were identified. Microarray as well as real-time polymerase chain reaction showed that the expression levels of these genes were significantly higher in the OVX + E group than in the OVX + V group. To clarify whether estrogen directly upregulates these genes in the vascular wall, Northern blot analysis was performed using cultured rat VSMC. Addition of 100 nmol/L estradiol for 24 hours increased the mRNA levels of all four genes. Although the precise mechanism remains unclear, regulation of these genes by estrogen might contribute to its effect on VSMC.     

Manumycin A, inhibitor of ras farnesyltransferase, inhibits proliferation and migration of rat vascular smooth muscle cells.

Restenosis after angioplasty is thought to be caused by proliferation and migration of vascular smooth muscle cells (VSMCs), and it is a most serious problem in medical treatment. A low dose (50 ng/ml) of manumycin A, an inhibitor of p21(ras) (ras) farnesylation, significantly inhibited proliferation of rat VSMCs stimulated by the platelet-derived growth factor (PDGF). The mitoinhibitory effect of manumycin A was dose- and time-dependent but was independent of cell density. Western blot analysis showed that manumycin A reduced the amount of functional ras localized at the cytoplasmic membrane and inhibited the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK). Manumycin A also inhibited VSMC migration and disorganized alpha actin fibers, as shown by immnofluorecence staining. These results indicate that the interruption of the ras/MAPK signal transduction pathway and the disorganization of alpha actin fibers are the main cause of manumycin A inhibition of VSMC proliferation and migration induced by PDGF.     

胰岛素增强糖基化白蛋白对大鼠血管平滑肌细胞的促增殖作用

在糖尿病性大血管病变的发病过程中,高血糖以及晚期糖基化终末产物（advanced glycation end products,AGEs）、脂质异常和高胰岛素血症的相互作用较其单独作用可能更重要。本研究采用糖基化白蛋白（glycated serum albumin,GSA）模拟AGEs,观察胰岛素和GSA对大鼠血管平滑肌细胞（vascular smooth muscle cells,VSMCs）的增殖是否存在协同作用,并初步探讨其作用机制。采用组织贴块法分离培养大鼠VSMCs。经过或不经过各种丝裂原激活蛋白激酶（mitogen-activated protein kinases．MAPKs）抑制剂和氧自由基清除剂N-acetylcysteine（NAC）处理后,加入不同浓度的胰岛素、GSA或GSA＋胰岛素,用MTT法和细胞计数法检测VSMCs的增殖。采用Western blot检测p38MAPK和C-Jun N-terminal kinase1／2（JNK1／2）的磷酸化。结果显示,GSA和胰岛素联合作用促进p38MAPK的磷酸化,而对JNK1／2的磷酸化无明显影响。GSA和胰岛素均可促进VSMCs增殖,而且两者具有协同作用。p38MAPK抑制剂SB203580和NAC可以抑制GSA和胰岛素联合作用引起的VSMCs增殖。以上结果提示,胰岛素和GSA对促进VSMCs增殖有协同作用,这可能是通过氧化应激敏感的p38MAPK通路实现的。胰岛素和AGEs的协同作用在糖尿病性动脉粥样硬化和再狭窄的发病过程中可能起重要作用。     

Characterization of phospholipase A1, A2, C activity in Ureaplasma urealyticum membranes

Neointimal thickening following catheter injury is characterized, in part, by growth factor-induced vascular smooth muscle cell (VSMC) proliferation. It was hypothesized that a reduction in serum insulin-like growth factor-1 (IGF-1), characteristic of chemically-induced diabetes, would result in decreased VSMC proliferation and attenuate neointimal thickening. It was found that alloxan-treated New Zealand White rabbits exhibit varying degrees of glycemia. Rabbits classified as diabetic (glucose = 400 mg/dL) had significantly decreased serum concentration of IGF-1 (87.4 ± 14 nmol/L vs. 170 ± 14 nmol/L) and significantly decreased intimal/medial (I/M) ratios 2, 4, and 8 weeks after aortic injury compared to euglycemic rabbits (13.7 ± 2, 21.1 + mn; 2, 32.4 ± 3 in euglycemics and 6.6 ± 1, 14 ± 2, 19 ± 5 in diabetics, respectively). The I/M for high hyperglycemic animals (glucose 286-399 mg/dL) was comparable to diabetic animals yet their serum IGF-1 levels were normal rather than depressed. Vascular IGF-1 content similarly increased upon injury in both diabetic and euglycemic animals. In diabetic animals, proliferating cell nuclear antigen (PCNA) immunostaining was present by day 1 peaked by day 5 and returned to control by day 14. In euglycemic animals, staining by day 1 continued to increase through day 14. A similar increase in mitogen-activated protein kinase (MAPK) activity occurred from day 1 through day 5 in both diabetic and euglycemic animals. This is the first demonstration of an association between MAPK activity and VSMC proliferation following vascular injury in diabetic animals as previously reported in euglycemic animals. In conclusion, this study provides evidence against a direct effect of IGF-1 in the reduction in neointimal thickening, VSMC proliferation, and MAPK activity upon catheter injury in chemically-induced diabetic rabbits.     

内源性CO对内皮素促大鼠主动脉平滑肌细胞增殖及MAPK活性的影响

血红素加氧酶（ｈｅｍｅ ｏｘｙｇｅｎａｓｅ,ＨＯ）是血红素分解代谢过程中的限速酶,它能使细胞内的血红素降解成胆绿素和一氧化碳（ｃａｒｂｏｎｍｏｎｏｘｉｄｅ,ＣＯ）,近来资料表明内源性一氧化碳对生理和病理状态下的血管张力有重要的调节作用,目前尚不不禁内源性ＨＯ／ＣＯ刘否参与平滑肌细胞增殖过程的调节,本实验在体内培养的大鼠主动脉平滑肌细胞模型上,用血色素加氧酶抑制剂卟啉锌－９（ｚｉｎｃ ｐｒｏｔｏｐｏ     

Inhibition of ras-mediated cell proliferation by benzyloxybenzaldehyde

Excessive proliferation of vascular smooth muscle cells (VSMC) in the intima is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis after balloon angioplasty. Therefore, control of VSMC growth may be a suitable therapeutic intervention in vascular proliferative disorders. In the present work, we have studied the 2-benzyloxybenzaldehyde (CCY1a)-mediated antiproliferative effect and its mechanisms of action. CCY1a inhibited serum-induced VSMC proliferation in a concentration-dependent manner, as demonstrated using [³H]thymidine incorporation and MTT assays; the IC₅₀ values were calculated to be 7.0 × 10^–6 and 1.2 × 10^–5 M, respectively. Furthermore, it also significantly suppressed serum-induced progression of the cell cycle, as shown by flow cytometric analysis. CCY1a as well as PD98059 almost completely abolished serum-induced activation of p42/44 mitogen-activated protein kinase (MAPK), the downstream effectors of c-fos and c-jun mRNA expression and activator protein-1 (AP-1) DNA binding activity, suggesting the central roles of these signaling cascades. Interestingly, CCY1a also effectively blocked serum-induced IB- phosphorylation, IB- degradation and nuclear factor-B (NF-B) binding activity. Based on these observations, we examined the effect of CCY1a on serum-mediated Ras activity, an upstream regulator of the above signaling events. The data demonstrated a marked inhibition of Ras activation by CCY1a. We conclude that CCY1a blocks cell proliferation via inhibition of the upstream effector of Ras and downstream events, including p42/44 MAPK activation and c-fos and c-jun mRNA expression, as well as NF-B and AP-1 DNA binding activities.     

Mechanisms of angiotensin II-induced expression of B2 kinin receptors

Although the primary roles of the kallikreinkinin system and the renin-angiotensin system are quite divergent, they are often intertwined under pathophysiological conditions. We examined the effect of ANG II on regulation of B(2) kinin receptors (B2KR) in vascular cells. Vascular smooth muscle cells (VSMC) were treated with ANG II in a concentration (10(-9)-10(-6) M)- and time (0-24 h)-dependent manner, and B2KR protein and mRNA levels were measured by Western blots and PCR, respectively. A threefold increase in B2KR protein levels was observed as early as 6 h, with a peak response at 10(-7) M. ANG II (10(-7) M) also increased B2KR mRNA levels twofold 4 h after stimulation. Actinomycin D suppressed the increase in B2KR mRNA and protein levels induced by ANG II. To elucidate the receptor subtype involved in mediating this regulation, VSMC were pretreated with losartan (AT(1) receptor antagonist) and/or PD-123319 (AT(2) receptor antagonist) at 10 microM for 30 min, followed by ANG II (10(-7) M) stimulation. Losartan completely blocked the ANG II-induced B2KR increase, whereas PD-123319 had no effect. In addition, expression of B2KR mRNA levels was decreased in AT(1A) receptor knockout mice. Finally, to determine whether ANG II stimulates B2KR expression via activation of the MAPK pathway, VSMC were pretreated with an inhibitor of p42/p44(mapk) (PD-98059) and/or an inhibitor of p38(mapk) (SB-202190), followed by ANG II (10(-7) M) for 24 h. Selective inhibition of the p42/p44(mapk) pathway significantly blocked the ANG II-induced increase in B2KR expression. These findings demonstrate that ANG II regulates expression of B2KR in VSMC and provide a rationale for studying the interaction between ANG II and bradykinin in the pathogenesis of vascular dysfunction.     

血管平滑肌细胞的体外氧化应激反应及雌激素的影响

目的探讨雌激素对VSMC氧化应激反应的影响及机制。方法第3-4代雌性大鼠血管平滑肌细胞(VSMC)在有或无10-8mol/L 17b-雌二醇(E2)存在下用不同浓度(0-250mmol/L)H2O2诱导氧化应激;MTT法,流式细胞术,Western blot分别检测VSMC活力,细胞周期,MAPK信号通路活性的变化。结果当浓度低于25mmol/L时,H2O2对VSMC活力无影响;当浓度为50-150mmol/L时,VSMC活力呈剂量依赖性下降,并伴随G0/G1期细胞升高;当浓度达到200mmol/L时,细胞活力下降至最低点并出现凋亡峰。10-8mol/L E2可显著抑制150mmol/L H2O2诱导的VSMCERK和p38的磷酸化、从而缓解VSMC G0/G1期阻滞,并降低高浓度H2O2诱导的VSMC凋亡。结论中等浓度的H2O2(50-150mmol/L)抑制VSMC增殖;高浓度H2O2(≥200mmol/L)不仅抑制VSMC生长、且导致部分VSMC凋亡;雌激素可通过抑制ERK和p38活性对氧化应激的VSMC起保护作用。     

重组人白介素-10抑制晚期糖基化终产物诱导的大鼠血管平滑肌细胞和血管新生内膜的增殖

研究观察了重组人白介素10(rhIL-l0)对晚期糖基化终产物(AGE)刺激下离体大鼠胸主动脉血管平滑肌细胞增殖及对SD大鼠血管损伤后新生内膜增殖的影响。体外培养大鼠主动脉血管平滑肌细胞,采用MTS／PES法确定血管平滑肌细胞的增殖状态;应用流式细胞术测定细胞周期;利用p44／42磷酸化抗MAPK抗体的蛋白免疫印迹法测定p44／42 MAPK磷酸化蛋白表达。利用大鼠颈动脉血管损伤模型,观察rhIL—10对新生内膜增殖的影响。结果显示：(1)AGE处理组与对照组相比,AGE对血管平滑肌细胞增殖具有明显的刺激作用(P＜0．05)。rhIL-l0单独应用对血管平滑肌细胞生长没有影响(P＞0．05)。在AGE刺激下,低至100ng／ml的rhIL-l0可抑制血管平滑肌细胞的生长(P＜0．05)。(2)流式细胞术测定的结果显示,rhIL—10可以使AGE作用下的VSMC大部分处于Go／G1期,与对照组相比有明显差异(P＜0．01)。(3)AGE对p44／p42 MAPK磷酸化蛋白表达有显著的增强作用,此作用可被rhIL—10抑制(P＜0．001)。(4)大鼠颈动脉损伤后,rhIL—10治疗组的动脉血管新生内膜/中层面积比低于对照组约45％(P＜0．01)。表明抗炎细胞因子rhIL—10可抑制AGE诱导的大鼠血管平滑肌细胞增殖和血管新生内膜的增殖。     

Bradykinin-induced p42/p44 MAPK phosphorylation and cell proliferation via Src, EGF receptors, and PI3-K/Akt in vascular smooth muscle cells

In our previous study, bradykinin (BK) exerts its mitogenic effect through Ras/Raf/MEK/MAPK pathway in vascular smooth muscle cells (VSMCs). In addition to this pathway, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3-K) have been implicated in linking a variety of G-protein coupled receptors to MAPK cascades. Here, we investigated whether these different mechanisms participating in BK-induced activation of p42/p44 MAPK and cell proliferation in VSMCs. We initially observed that BK- and EGF-dependent activation of Src, EGFR, Akt, and p42/p44 MAPK and [3H]thymidine incorporation were mediated by Src and EGFR, because the Src inhibitor PP1 and EGFR kinase inhibitor AG1478 abrogated BK- and EGF-dependent effects. Inhibition of PI3-K by LY294002 attenuated BK-induced Akt and p42/p44 MAPK phosphorylation and [3H]thymidine incorporation, but had no effect on EGFR phosphorylation, suggesting that EGFR may be an upstream component of PI3-K/Akt and MAPK in these responses. This hypothesis was supported by the tranfection with dominant negative plasmids of p85 and Akt which significantly attenuated BK-induced Akt and p42/p44 MAPK phosphorylation. Pretreatment with U0126 (a MEK1/2 inhibitor) attenuated the p42/p44 MAPK phosphorylation and [3H]thymidine incorporation stimulated by BK, but had no effect on Akt activation. Moreover, BK-induced transactivation of EGFR and cell proliferation was blocked by matrix metalloproteinase inhibitor GM6001. These results suggest that, in VSMCs, the mechanism of BK-stimulated activation of p42/p44 MAPK and cell proliferation was mediated, at least in part, through activation of Src family kinases, EGFR transactivation, and PI3-K/Akt.