A requirement for the rac1 GTPase in the signal transduction pathway leading to cardiac myocyte hypertrophy.

We have used adenoviral-mediated gene transfer of a constitutively active (V12rac1) and dominant negative (N17rac1) isoform of rac1 to assess the role of this small GTPase in cardiac myocyte hypertrophy. Expression of V12rac1 in neonatal cardiac myocytes results in sarcomeric reorganization and an increase in cell size that is indistinguishable from ligand-stimulated hypertrophy. In addition, V12rac1 expression leads to an increase in atrial natriuretic peptide secretion. In contrast, expression of N17rac1, but not a truncated form of Raf-1, attenuated the morphological hypertrophy associated with phenylephrine stimulation. Consistent with the observed effects on morphology, expression of V12rac1 resulted in an increase in new protein synthesis, while N17rac1 expression inhibited phenylephrine-induced leucine incorporation. These results suggest rac1 is an essential element of the signaling pathway leading to cardiac myocyte hypertrophy.     

颈总动脉损伤大鼠成纤维细胞生长因子受体1、p21ras表达与辛伐他汀的抑制效应

背景：辛伐他汀可通过抑制血管平滑肌细胞增殖发挥抗血管狭窄作用,但其作用机制尚不完全清楚。目的：观察辛伐他汀对血管损伤后狭窄时成纤维细胞生长因子受体1、p21ras蛋白表达的影响。方法：建立SD大鼠颈总动脉损伤模型,随机分为假手术组、模型组、低剂量和高剂量辛伐他汀组,后2组分别于术前3d灌胃辛伐他汀5,10mg/（kg？d）直至术后14d。结果与结论：大鼠颈总动脉损伤后：①苏木精-伊红染色显示,血管中膜血管平滑肌增殖显著,排列紊乱,管腔严重狭窄;应用辛伐他汀后血管内膜相对光滑,中膜血管平滑肌细胞排列趋于规整,管腔狭窄程度有不同程度减轻。②Western blot检测显示,颈总动脉成纤维细胞生长因子受体1、p21ras表达明显升高（P〈0.05）,低剂量辛伐他汀干预后两种蛋白表达无明显变化,应用高剂量辛伐他汀干预后两种蛋白表达明显下降（P〈0.05）。表明辛伐他汀可能通过抑制成纤维细胞生长因子受体1、p21ras蛋白的表达抑制血管平滑肌细胞增殖,减轻血管损伤后狭窄。     

人类巨细胞病毒感染对血管平滑肌细胞脂代谢相关基因表达的影响

目的：探讨血管平滑肌细胞被感染人类巨细胞病毒后,其细胞内胆固醇脂代谢及相关基因表达的变化情况。方法通过分离原代脐动脉血管平滑肌细胞,以1个感染复数的人类巨细胞病毒攻击作为实验组,对照组则采用含3％小牛血清的DMEM/F12培养基模拟感染。检测平滑肌细胞内胆固醇含量在病毒攻击后的变化情况,以基因表达谱芯片技术检测基因表达情况。结果血管平滑肌细胞被感染人类巨细胞病毒后,细胞内相关基因表达发生显著变化,其中低密度脂蛋白受体相关蛋白10、11、12以及 HMG-CoA 合成酶,HMG-CoA 还原酶,B 型清道夫受体表达上调。载脂蛋白 A1、载脂蛋白 M 表达下调。结论人类巨细胞病毒感染血管平滑肌细胞后可能通过改变脂代谢途径的相关基因表达,增强细胞内胆固醇合成,导致胆固醇代谢失衡,从而参与动脉粥样硬化的发病过程。     

短期缺氧-复氧对大鼠血管平滑肌细胞myocardin基因表达的影响

目的 研究短期缺氧-复氧对大鼠血管平滑肌细胞myocardin基因表达的影响.方法 无菌条件下留取大鼠胸主动脉中膜,采用组织贴块法培养的第4～7代细胞,通过观察细胞在缺氧环境下形态学变化、死亡细胞计数等,判断缺氧刺激对体外培养平滑肌细胞活性的影响,并与正常培养箱内对照进行比较.提取细胞总RNA,用RT-PCR法,研究平滑肌细胞在缺氧及复氧后不同时间段myocardin mRNA的表达变化.结果培养细胞呈典型的"峰谷"样生长.自制缺氧装置24 h内氧分压明显降低,且pH值波动不明显.RT-PCR结果表明,与正常对照组相比,平滑肌细胞myocardin mRNA表达在缺氧12、24 h后逐渐降低[(0.871±0.053)与(0.573±0.074)、(1.329±0.042),P＜0.05].缺氧12 h降低最明显.且随着缺氧时间的延长,myocardin基因的表达水平逐渐回升,并增高;缺氧24 h再复氧6 h后,其表达水平逐渐回升,复氧12 h接近正常表达水平.结论 短期缺氧-复氧影响大鼠血管平滑肌细胞myocardin基因的表达.     

Beta2-adrenergic agonist protects human endothelial cells from hypoxia/reoxygenation injury in vitro

OBJECTIVE: Circulatory shock results in hypoxia/reoxygenation processes that lead to the release of reactive oxygen species, endothelial injury, and multiple organ failure. Previous data suggest that beta2-adrenergic agonists prevent endothelial dysfunction. The study aimed at determining whether the beta2-adrenergic agonist formoterol protects endothelial cells against hypoxia/reoxygenation injury in vitro. DESIGN: Prospective controlled trial. SETTING: University hospital research laboratory. SUBJECTS: Cultured human umbilical vein endothelial cells (HUVECs). INTERVENTIONS: Confluent HUVECs were sealed in a flow-through chamber mounted on an inverted microscope and perfused with a constant flow of Krebs medium. After 1 hr of equilibration, HUVECs underwent 2 hrs of hypoxia and 1 hr of reoxygenation. Cell death at the end of reoxygenation and reactive oxygen species formation were assessed with fluorescent probes propidium iodide and 2',7'-dichlorodihydrofluorescein diacetate, respectively. The effects of the beta2-adrenergic agonist formoterol, the beta2-adrenergic antagonist ICI 118,551 and the nitric oxide synthase inhibitor L-NNA were investigated. Statistical analysis was performed with analysis of variance followed by post hoc Fisher's test. MEASUREMENTS AND MAIN RESULTS: Hypoxia/reoxygenation increased cell death (hypoxia/reoxygenation 29 +/- 4% vs. control 1 +/- 5%, p < .05) and endothelial reactive oxygen species production (hypoxia/reoxygenation 126 +/- 4% vs. control 108 +/- 4%, p < .05). Formoterol reduced cell death in a concentration-dependent manner (EC95 = 10 mol/L) and reduced endothelial reactive oxygen species production (hypoxia/reoxygenation + formoterol EC95 109 +/- 4% vs. hypoxia/reoxygenation 126 +/- 4%, p < .05). When added to formoterol EC95, ICI 118,551 and L-NNA abolished the formoterol-induced cell protection and reduced reactive oxygen species production. CONCLUSIONS: These results indicate that formoterol reduces endothelial cell death and reactive oxygen species production in this in vitro hypoxia/reoxygenation model. These effects are beta2-adrenergic specific and are partially mediated by nitric oxide synthase.     

Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques.

We studied death of human vascular smooth muscle cells derived from coronary plaques and normal coronary arteries and aorta. Cells from normal arteries underwent death only upon removal of serum growth factors. In contrast, plaque-derived cells died even in high serum conditions, and death increased after serum withdrawal. Death was characteristically by apoptosis in both normal and plaque-derived cells, as determined by time-lapse videomicroscopy, electron microscopy, and DNA fragmentation patterns. IGF-1 and PDGF were identified as potent survival factors in serum, whereas EGF and basic fibroblast growth factor had little effect. Stable expression of bcl-2, a protooncogene that regulates apoptosis in other cell lines, protected smooth muscle cells from apoptosis, although there was no detectable difference in endogenous bcl-2 expression between cells from plaques or normal vessels. We conclude that apoptosis of human vascular smooth muscle cells is regulated by both specific gene products and local cytokines acting as survival factors. Apoptosis may therefore regulate cell mass in the normal arterial wall and the higher rates of apoptosis seen in plaque smooth muscle cells may ultimately contribute to plaque rupture and breakdown and thus to the clinical sequelae of atherosclerosis.     

Protamine and protamine-insulins exacerbate the vascular response to injury.

Endothelial cells and smooth muscle cells produce heparinlike compounds that are growth inhibitory for vascular smooth muscle cells, and it has been suggested that these compounds play a regulatory role that is perturbed with vascular injury. Indeed, exogenous heparin preparations effectively suppress smooth muscle cell proliferation following injury imposed on vascular endothelium. We now report that protamine, an agent that binds heparin and negates its anticoagulant properties, has potent stimulatory effects on vascular smooth muscle cell proliferation. The administration of protamine, alone or as part of commonly used insulin preparations, stimulated the proliferation of cultured smooth muscle cells, exacerbated vascular smooth muscle cell proliferative lesions in laboratory rats, and interfered with the growth-inhibitory effects of heparin in culture and in vivo. These results confirm the importance of endogenous heparinlike compounds in arterial homeostasis and may require reconsideration of protamine use following vascular reparative procedures and in diabetics.     

Synthesis and release of interleukin 1 by reoxygenated human mononuclear phagocytes.

To examine the possible involvement of cytokines in reperfusion injury, we have studied production of IL-1 by human vascular cells, including smooth muscle and mononuclear phagocytes. Exposure of cells to hypoxia (pO2 approximately 14 torr) followed by reoxygenation led to significant release of IL-1 only from the mononuclear phagocytes. Elaboration of IL-1 was dependent on the oxygen tension and duration of hypoxia (optimal at lower pO2s, approximately 14-20 torr, and after 9 h), as well as the time in reoxygenation (maximal IL-1 release at 6-9 h). Although a period of hypoxia was necessary for subsequent IL-1 production during reoxygenation of either peripheral blood monocytes or cultured monocyte-derived macrophages, no IL-1 release occurred during the hypoxic exposure. IL-1 released during reoxygenation was newly synthesized, and its production was triggered by the generation of oxygen free radicals, as it could be blocked by the addition of either allopurinol or free radical scavengers to cultures and could be stimulated in part by low concentrations of hydrogen peroxide or xanthine/xanthine oxidase. The potential pathophysiological effects of IL-1-containing supernatants from reoxygenated macrophages was shown by their induction of endothelial tissue factor and enhancement of endothelial adhesiveness for neutrophils, both of which could be blocked by anti-IL-1 antibody. The relevance of IL-1 to hypoxia/reoxygenation in vivo was suggested by the presence of circulating nanogram amounts of this cytokine in the plasma of mice during the reoxygenation period following a hypoxia.     

The discoidin domain receptor tyrosine kinase DDR1 in arterial wound repair

Collagens act as important signaling molecules regulating vascular smooth muscle cell responses during arterial wound repair. Discoidin domain receptors (DDRs) are a novel class of receptor tyrosine kinases that bind to several collagens and stimulate matrix metalloproteinase (MMP) production, but little is known about their expression and function in the vasculature. We posited a critical role for the DDRs controlling smooth muscle cell migration and proliferation and thus repair following arterial injury. Smooth muscle cells were isolated from the aortas of mice with a targeted deletion of the DDR1 gene (DDR1-null) and studied in culture using models that mimic critical steps in neointimal thickening. Our studies suggest that DDR1 plays an important role in regulating attachment to collagen, chemotaxis, proliferation, and MMP production in smooth muscle cells. Following mechanical injury to the carotid arteries, cross-sectional area of the neointima was significantly lower in DDR1-null mice than in wild-type mice. There was also a significant decrease in collagen deposition in the injured arteries of the DDR1-null mice. Our results support the hypothesis that DDR1 plays an important role as a collagen receptor, mediating intimal thickening after vascular injury.     

Role of CD44 in the reaction of vascular smooth muscle cells to arterial wall injury.

CD44, the principal receptor for hyaluronic acid, is a widely distributed cell surface proteoglycan involved in cellular activation, proliferation, and migration. These processes are also central to the vascular smooth muscle cell's response to arterial wall injury. We evaluated the expression of CD44 and its isoform, CD44-V6, on vascular smooth muscle cells in vitro and in vivo and assessed the role of CD44 in DNA synthesis. Cultured vascular smooth muscle cells expressed CD44 and CD44-V6 at levels equal to or higher than those of the beta 1 and beta 2 integrins. In a rat carotid artery balloon injury model, CD44 and CD44-V6 mRNAs were unregulated in vascular smooth muscle cells after injury, and CD44 protein expression was greatest at the luminal edge of the growing neointima. CD44-expressing smooth muscle cells proliferated actively, and hyaluronic acid expression increased after injury in a temporal pattern similar to that of CD44. Through binding to hyaluronic acid, CD44 augmented DNA synthesis in cultured human and rat smooth muscle cells by 48 +/- 7.8 and 100 +/- 12.5%, respectively, an effect inhibited by an anti-CD44 antibody that blocked hyaluronate binding. These observations support a role for CD44 in the reaction of vascular smooth muscle cells to arterial wall injury.     

Rac1 and superoxide are required for the expression of cell adhesion molecules induced by tumor necrosis factor-alpha in endothelial cells

Oxidative signals play an important role in the regulation of endothelial cell adhesion molecule expression. Small GTP-binding protein Rac1 is activated by various proinflammatory substances and regulates superoxide generation in endothelial cells. In the present study, we demonstrate that adenoviral-mediated expression of dominant negative N17Rac1 (Ad.N17Rac1) suppresses tumor necrosis factor-alpha (TNF-alpha)-induced vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin gene expression in a dose-dependent manner. Ad.N17Rac1 did not inhibit TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB) binding activity or inhibitor of NF-kappaB-alpha degradation. In contrast, Ad.N17Rac1 inhibited TNF-alpha-induced NF-kappaB-driven HIV(kappaB)(4)-CAT and p288VCAM-Luc promoter activity, suggesting that N17Rac1 inhibits TNF-alpha-induced VCAM-1, E-selectin, and ICAM-1 through suppressing NF-kappaB-mediated transactivation. In addition, expression of superoxide dismutase by adenovirus suppressed TNF-alpha-induced VCAM-1, E-selectin, and ICAM-1 mRNA accumulation. However, adenoviral-mediated expression of catalase only partially inhibited TNF-alpha-induced E-selectin gene expression and had no effect on VCAM-1 and ICAM-1 gene expression. These data suggest that Rac1 and superoxide play crucial roles in the regulation of expression of cell adhesion molecules in endothelial cells.     

硫氧还蛋白-1对代谢型谷氨酸受体1a毒性的抑制作用

目的 研究在HEK293细胞中过表达代谢型谷氨酸受体1a(Metabotropic gluatarnate receptor 1,mGluRla),引起激动剂非依赖型细胞死亡的分子机制和硫氧还蛋白-1(Trxl)在此过程中的作用.方法 采用噻唑蓝法、DCF法、免疫印迹法以及氧化还原蛋白印迹法分别检测了Trxl对细胞活力...     

Dominant negative c-jun gene transfer inhibits vascular smooth muscle cell proliferation and neointimal hyperplasia in rats

We previously reported that activator protein-1 (AP-1), containing c-Jun, is rapidly activated in balloon-injured artery. Therefore, we examined the role of c-Jun in vascular smooth muscle cell (SMC) proliferation, by using in vitro and in vivo gene transfer techniques. (1) Serum (2%) stimulation significantly increased AP-1 DNA binding activity in aortic SMCs, followed by the increase in both 3H-thymidine incorporation and cell number. Aortic SMCs were infected with recombinant adenovirus containing TAM67, a dominant negative c-Jun lacking transactivation domain of wild c-Jun (Ad-DN-c-Jun), to specifically inhibit AP-1. Ad-DN-c-Jun significantly inhibited serum-induced SMC proliferation, by inhibiting the entrance of SMC into S phase. (2) The effect of DN-c-Jun was examined on balloon injury-induced intimal hyperplasia in rats. Before balloon injury, DN-c-Jun was transfected into rat carotid artery using the hemagglutinating virus of Japan-liposome method. In vivo transfection of DN-c-Jun significantly inhibited vascular SMC proliferation in the intima and the media and subsequently prevented intimal thickening at 14 days after balloon injury. We obtained the first evidence that DN-c-Jun gene transfer prevented vascular SMC proliferation in vitro and in vivo, and c-Jun was involved in balloon injury-induced intimal hyperplasia. Thus, AP-1 seems to be the new therapeutic target for treatment of vascular diseases.     

Evidence for altered Na+/H+ antiport activity in cultured skeletal muscle cells and vascular smooth muscle cells from the spontaneously hypertensive rat.

1. Intracellular pH and Na+/H+ antiport activity were determined by a fluorimetric method in cultured skeletal muscle cells (myoblasts) and aortic vascular smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats. 2. The intracellular pH was significantly more alkaline at three different extracellular pH values in both myoblasts and vascular smooth muscle cells from the spontaneously hypertensive rats than in those from the normotensive control rats. 3. A kinetic analysis of the Na+/H+ antiport activity in these cells showed that the raised activity in the spontaneously hypertensive rats was due to an increased maximal transport capacity in vascular smooth muscle cells and to an increase in the affinity of the antiport for internal H+ in the myoblasts. 4. When the extracellular pH was reduced in the skeletal muscle cells of both types of rat, the intracellular pH fell. However, in vascular smooth muscle cells, a reduction in the extracellular pH was not associated with a fall in the intracellular pH. This resistance of the intracellular pH to changes in the extracellular pH differentiates vascular smooth muscle cells from other cells that have been studied in this way.     

Human salivary histatin 5 fungicidal action does not induce programmed cell death pathways in Candida albicans

Salivary histatins (Hsts) are potent candidacidal proteins that induce a nonlytic form of cell death in Candida albicans accompanied by loss of mean cell volume, cell cycle arrest, and elevation of intracellular levels of reactive oxygen species (ROS). Since these phenotypes are often markers of programmed cell death and apoptosis, we investigated whether other classical markers of apoptosis, including generation of intracellular ROS and protein carbonyl groups, chromosomal fragmentation (laddering), and cytochrome c release, are found in Hst 5-mediated cell death. Increased intracellular levels of ROS in C. albicans were detected in cells both following exogenous application of Hst 5 and following intracellular expression of Hst 5. However, Western blot analysis failed to detect specifically increased protein carbonylation in Hst 5-treated cells. There was no evidence of chromosomal laddering and no cytochrome c release was observed following treatment of C. albicans mitochondria with Hst 5. Superoxide dismutase enzymes of C. albicans and Saccharomyces cerevisiae provide essential protection against oxidative stress; therefore, we tested whether SOD mutants have increased susceptibility to Hst 5, as expected if ROS mediate fungicidal effects. Cell survival of S. cerevisiae SOD1/SOD2 mutants and C. albicans SOD1 mutants following Hst 5 treatment (31 micro M) was indistinguishable from the survival of wild-type cells treated with Hst 5. We conclude that ROS may not play a direct role in fungicidal activity and that Hst 5 does not initiate apoptosis or programmed cell death pathways.     

骨骼肌细胞缺氧复氧损伤的保护机制研究

目的探讨碱性成纤维细胞生长肽(bFGF)对新生大鼠骨骼肌细胞缺氧复氧(A/R)损伤的保护作用。方法应用培养的新生Wistar大鼠骨骼肌细胞制备A/R损伤模型,观察bFGF对A/R损伤后肌细胞存活率、胞浆乳酸脱氢酶(LDH)漏出的影响。并用免疫组化法检测bFGF对A/R损伤后肌细胞凋亡(apoptosis)相关基因蛋白bcl-2表达的影响。结果bFGF预处理呈浓度依赖性地提高了A/R后肌细胞存活率,减少了胞浆LDH的漏出,明显上调bcl-2的表达。结论bFGF对A/R损伤的骨骼肌细胞有明显保护作用。     

Enhanced pulmonary expression of endothelin-1 in an ovine model of smoke inhalation injury.

Recent studies suggest a role of endothelin-1 (ET-1) in mediating airway inflammation and lung injury. The aim of this study was to assess the immunohistochemical expression of ET-1 in the lung following smoke inhalation injury. ET-1 immunoreactivity was assessed in normal sheep (N = 4) and in sheep at 1 (N = 2), 6 (N = 3), 12 (N = 3), and 24 (N = 3) hours after inhalation injury. In normal animals, ET-1 expression was limited to the basal cell layer of the tracheal epithelium, main bronchi, and associated mucous glands. One hour after injury, ET-1 immunoreactivity was enhanced in upper airway epithelium and mucus glands with new expression in bronchioles. Airway smooth muscle, vascular tissue, and alveolar duct smooth muscle cells expressed moderate levels of ET-1 at 12 and 24 hours. ET-1 immunoreactivity was absent in areas of parenchymal edema and inflammation. The pattern of ET-1 expression following inhalation injury suggests that this peptide may contribute to the airway inflammation, mucus secretion, pulmonary hypertension, increased airway resistance, and decreased lung compliance, which are evident in our ovine model of inhalation injury.     

Pluripotent stem cell differentiation into vascular cells: a novel technology with promises for vascular re(generation)

Several types of stem and progenitor cells are currently under investigation for their potential to accomplish vascular regeneration. This review focuses on embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We will discuss the technologies allowing for their derivation, culture expansion and maintenance in a pluripotent status. Moreover, both ESCs and iPSCs can be differentiated in endothelial cells (ECs) and mural cell, including vascular smooth muscle cells (VSMCs). Here, we will describe the involvements of growth factors (vascular endothelial growth factors-VEGFs-, platet-derived growth factors-PDGFs-), Wnt and Notch signal pathways, reactive oxygen species (ROS), histone deacetylases (HDACs), and microRNAs (miRNAs) in vascular cell differentiation from pluripotent stem cells. We will additionally describe the therapeutic potential of stem cells for vascular medicine.     

基质细胞衍生因子1α及受体CXCR4对颈总动脉球囊损伤血管平滑肌细胞和骨髓基质细胞的影响

背景：在细胞因子的作用下,骨髓基质细胞可以向内皮祖细胞分化,并迁移至损伤部位增殖,参与血管新生内膜的修复。目的：观察基质细胞衍生因子1α及其受体CXCR4在大鼠颈总动脉球囊损伤后对血管平滑肌细胞和骨髓基质细胞的影响。方法：体外培养大鼠颈总动脉球囊损伤后各时间点（即刻、1,4,7,14及1个月）的血管平滑肌细胞和骨髓基质细胞,检测损伤后平滑肌细胞中基质细胞衍生因子1α及骨髓基质细胞中CXCR4的表达情况,并应用基质细胞衍生因子1αsiRNA转染损伤后血管平滑肌细胞,同时应用transwell小室观察基质细胞衍生因子1α和损伤后血管平滑肌细胞对骨髓基质细胞的趋化作用。结果与结论：大鼠颈总动脉球囊损伤后,平滑肌细胞中基质细胞衍生因子1αmRNA表达开始增加,至损伤第7天后逐渐减弱;血管损伤后4d起,骨髓基质细胞中CXCR4mRNA开始出现,并持续表达;transwell小室显示损伤后的骨髓基质细胞对基质细胞衍生因子1α的趋化性增强;CXCR4受体拮抗剂AMD3100可以明显减弱基质细胞衍生因子1α对骨髓基质细胞的趋化作用;损伤后的血管平滑肌细胞对骨髓基质细胞的趋化作用强于基质细胞衍生因子1α（P〈0.05）,应用AMD3100干预或细胞内转染基质细胞衍生因子1αsiRNA后,此趋化作用亦减弱（P〈0.05）。结果提示,基质细胞衍生因子1α/CXCR4轴在骨髓基质细胞向损伤血管迁移中起重要的作用。