NADPH oxidase is involved in prostaglandin F2alpha-induced hypertrophy of vascular smooth muscle cells: induction of NOX1 by PGF2alpha

Prostaglandin (PG) F(2alpha), one of the primary prostanoids generated in vascular tissue, is known to cause hypertrophy in vascular smooth muscle cells. To clarify the molecular mechanisms underlying PGF(2alpha)-induced hypertrophy, the involvement of reactive oxygen species was examined in a rat vascular smooth muscle cell line, A7r5. PGF(2alpha) and (+)-fluprostenol, a selective agonist of the PGF receptor, significantly increased intracellular O(2)(-) in A7r5. The PGF(2alpha)-induced O(2)(-) increase was suppressed by diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase that has been reported to be the major source of O(2)(-) in vascular cells. The augmented synthesis of the protein induced by PGF(2alpha) or (+)-fluprostenol was suppressed in the presence of DPI. In PGF(2alpha) or (+)-fluprostenol-treated cells, a dose-dependent increase in the expression of NOX1, a homolog of the catalytic subunit of the phagocyte NADPH oxidase gp91(phox), was demonstrated by Northern blot analysis. Finally, depletion of NOX1 mRNA in the cells transfected with ribozymes targeted for three independent cleavage sites on the mRNA sequence significantly reduced the PGF(2alpha)-induced increase in protein synthesis. Taken together, these results suggest that hypertrophy of vascular smooth muscle cells caused by PGF(2alpha) is mediated by NOX1 induction and the resultant overproduction of O(2)(-) by NADPH oxidase.     

NADPH氧化酶活性不影响主动脉平滑肌细胞负荷胆固醇

NADPH氧化酶产生的活性氧促进血管平滑肌细胞的增殖和迁移,与动脉粥样硬化的发生密切相关.为了观察NADPH氧化酶的亚基p47phox对血管平滑肌细胞胆固醇代谢的影响,把p47phox基因敲除小鼠的主动脉血管平滑肌细胞与10 mg/L水溶性胆固醇共孵育72 h,然后用0.3 mg/L凝血酶处理10 min,采用免疫组织化学和油红O染色、实时定量逆转录PCR、免疫蛋白印迹、细胞内胆固醇测定等方法,观察细胞内胆固醇的改变,与平滑肌细胞、巨噬细胞、炎症反应细胞内胆固醇代谢相关蛋白的表达.结果显示,与未孵育的对照组相比,水溶性胆固醇孵育过的主动脉血管平滑肌细胞内胆固醇明显增加,差别有显著性意义:细胞内中性脂滴明显增加;α-肌动蛋白的表达下降,半乳糖凝集素3表达升高,单核细胞趋化蛋白1及血管细胞黏附分子1的表达不变;ATP结合盒转运体A1、酰基辅酶A:胆固醇酰基转移酶1及脂肪分化相关蛋白的表达增加.但是,与野生型血管平滑肌细胞相比,敲除p47phox基因并不能使所测定的指标发生变化.结果提示,负荷胆固醇后,p47phox依赖的NADPH氧化酶并不能改变血管平滑肌细胞向泡沫细胞的转变.单纯敲除p47phox基因不能改变细胞内胆固醇代谢的状态.     

Tyrosine kinase-mediated activation of NADPH oxidase enhances proliferative capacity of diabetic vascular smooth muscle cells

To investigate a potential molecular basis for a link between diabetes and atherosclerosis, experiments were performed to determine the role of NADPH oxidase in the enhanced proliferative capacity of vascular smooth muscle cells (VSMC) from OLETF rat, an animal model of type 2 diabetes. An enhanced proliferative response to 10% fetal bovine serum with an increased cell cycle progression from G1 to S phase as well as an augmented superoxide generation with an increased NADPH oxidase activity were observed in diabetic versus control VSMC. Both the enhanced proliferation and superoxide generation in diabetic VSMC were significantly attenuated not only by diphenyleneiodonium (10 microM) and apocynin (100 microM), NADPH oxidase inhibitors but also by protein tyrosine kinase inhibitors such as genistein (100 microM) and AG 112 (100 microM). Furthermore, the enhanced NADPH oxidase activity in diabetic VSMC was significantly attenuated by genistein and AG112, but not by daidzein (100 microM), a genistein analogue devoid of protein tyrosine kinase inhibitory properties. Based on these results, it is suggested that the enhanced proliferative capacity of diabetic VSMC is closely related to the activation of NADPH oxidase that is induced through activation of protein tyrosine kinase.     

Regulation of smooth muscle by inducible nitric oxide synthase and NADPH oxidase in vascular proliferative diseases

Inflammation plays a critical role in promoting smooth muscle migration and proliferation during vascular diseases such as postangioplasty restenosis and atherosclerosis. Another common feature of many vascular diseases is the contribution of reactive oxygen (ROS) and reactive nitrogen (RNS) species to vascular injury. Primary sources of ROS and RNS in smooth muscle are several isoforms of NADPH oxidase (Nox) and the cytokine-regulated inducible nitric oxide (NO) synthase (iNOS). One important example of the interaction between NO and ROS is the reaction of NO with superoxide to yield peroxynitrite, which may contribute to the pathogenesis of hypertension. In this review, we discuss the literature that supports an alternate possibility: Nox-derived ROS modulate NO bioavailability by altering the expression of iNOS. We highlight data showing coexpression of iNOS and Nox in vascular smooth muscle demonstrating the functional consequences of iNOS and Nox during vascular injury. We describe the relevant literature demonstrating that the mitogen-activated protein kinases are important modulators of proinflammatory cytokine-dependent expression of iNOS. A central hypothesis discussed is that ROS-dependent regulation of the serine/threonine kinase protein kinase Cdelta is essential to understanding how Nox may regulate signaling pathways leading to iNOS expression. Overall, the integration of nonphagocytic NADPH oxidase with cytokine signaling in general and in vascular smooth muscle in particular is poorly understood and merits further investigation.     

The emerging role of ROS-generating NADPH oxidase NOX4 in DNA-damage responses

The human genome is continuously exposed to such potentially deleterious agents as the highly reactive molecules known as reactive oxygen species (ROS). ROS include superoxide anions (O(2)(-)) and hydrogen peroxide (H(2)O(2)). Over the last decade, the ROS-generating NADPH oxidases (NOXs) have been recognized as one of the main sources of ROS production in numerous human cell types. In addition to regulating normal physiological redox-dependent processes, the NOXs are involved in cellular oxidative stress. In contrast to the other NOXs, the NADPH oxidase NOX4 exists in the immediate environment of the nucleus. There is accumulating evidence for the involvement of NOX4-derived ROS in genomic instability as well as in cancer and other inflammation-related diseases. We recently showed that NOX4 plays a critical role in oncogenic Ras-induced DNA damage. Here we reflect upon the growing awareness of NOX4, review its role in inducing genomic instability, and call attention to its possible role in nuclear redox-sensitive mechanisms underlying DNA-damage signaling and repair.     

Effects of cholesterol oxidase on cultured vascular smooth muscle cells

Summary Cholesterol oxidase (3-hydroxy-steroid oxidase) catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. The purpose of the present study was to investigate its effects on cultured vascular smooth muscle cells. Cultured rabbit aortic smooth muscle cells were morphologically altered after exposure to cholesterol oxidase in the presence of culture medium containing 10% fetal calf serum. If fetal calf serum was absent, cells were unaffected by the treatment. The extent of morphological change of the smooth muscle cells was dependent upon the time of exposure to the enzyme and the concentration of cholesterol oxidase employed. After moderate treatment with cholesterol oxidase, cells excluded trypan blue. Further, a specific mitochondrial marker DASPMI (dimethyl aminostyryl-methyl-pyridiniumiodine) which was used as a fluorescent index of cell viability, revealed that cell viability was unchanged after moderate cholesterol oxidase treatment. Nile red, a hydrophobic probe which selectively stains intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with cholesterol oxidase. Cellular nile red fluorescence intensity increased linearly with the time and concentration of cholesterol oxidase treatment. These results demonstrate that cholesterol oxidase alters lipid deposition in the cell and changes cell morphology. The primary site of action of cholesterol oxidase appears to be independent of the cell membrane itself and instead is dependent upon the lipid content in the surrounding culture media. These changes occur prior to the cytotoxic effects of extensive oxidation. Because oxidized cholesterol may play an important role in the pathogenesis of atherosclerosis, our results have implications for intracellular accumulation of lipids in smooth muscle cells during the atherosclerotic lesion.     

Ca2+ regulation of vascular smooth muscle

Regulation of intracellular free Ca2+ concentrations in vascular smooth muscle is accomplished mainly by Ca2+ channels and ATP-dependent Ca2+ pumps in the plasmalemma and sarcoplasmic reticulum (SR). Ca2+ entry through the plasmalemma is apparently mediated by four different pathways: leak; receptor-operated Ca2+ channels; potential sensitive Ca2+ channels; and stretch-activated channels. The agonist releasable intracellular Ca2+ store appears to be identical with the SR. Evidence for the involvement of Ca2+-induced Ca2+ release and inositol-1,4,5-trisphosphate in the release of SR Ca2+ is discussed. Smooth muscle contractions induced by certain agonists may be further enhanced by inhibition of Ca2+ uptake by the SR and of active Ca2+ extrusion across the plasmalemma. At the moment it is not clear from a consideration of the Ca2+ regulatory mechanisms present in vascular smooth muscle how dietary Ca2+ affects vascular tone. The increased Ca2+ permeation through smooth muscle cell membranes of resistance arteries taken from spontaneously hypertensive rats may be relevant to this problem.     

Role of NADPH oxidase in cytomegalovirus-induced proliferation of human coronary artery smooth muscle cells

A number of infectious agents have been implicated in the development of vascular diseases such as atherosclerosis and posttransplantation arterial restenosis. Cytomegalovirus (CMV) has been reported to cause obliteration of coronary arteries by a progressive vasculopathy that involves proliferation of medial smooth muscle cells (SMC). In this study, we report that CMV enhances the serum-induced proliferation of human coronary SMC through activation of a superoxide-generating NADPH oxidase. Exposure of SMC to CMV for 2 h was associated with an 80% increase in NADPH oxidase. This increase in oxidase activity was associated with a two-fold increase in serum-induced DNA synthesis (5-bromo-2'-deoxyuridine incorporation) and significant interleukin-8 (IL-8) production by SMC. Diphenylene iodonium, an inhibitor of NADPH oxidase, significantly inhibited CMV-induced IL-8 production and promotion of serum-induced DNA synthesis. Similar effects were seen following pretreatment of SMC with N-acetyl cysteine, a potent antioxidant, suggesting that oxidative stress following CMV exposure might be responsible for triggering the proliferation of SMC. From this study, we conclude that CMV-mediated promotion of SMC growth is redox sensitive and may be mediated by NADPH oxidase.     

Reciprocal regulation of syndecan-2 and Notch signaling in vascular smooth muscle cells

Vascular cell interactions mediated through cell surface receptors play a critical role in the assembly and maintenance of blood vessels. These signaling interactions transmit important information that alters cell function through changes in protein dynamics and gene expression. Here, we identify syndecan-2 (SDC2) as a gene whose expression is induced in smooth muscle cells upon physical contact with endothelial cells. Syndecan-2 is a heparan sulfate proteoglycan that is known to be important for developmental processes, including angiogenesis. Our results show that endothelial cells induce mRNA expression of syndecan-2 in smooth muscle cells by activating Notch receptor signaling. Both NOTCH2 and NOTCH3 contribute to the increased expression of syndecan-2 and are themselves sufficient to promote its expression independent of endothelial cells. Syndecan family members serve as coreceptors for signaling molecules, and interestingly, our data show that syndecan-2 regulates Notch signaling and physically interacts with NOTCH3. Notch activity is attenuated in smooth muscle cells made deficient in syndecan-2, and this specifically prevents expression of the differentiation marker smooth muscle α-actin. These results show a novel mechanism in which Notch receptors control their own activity by inducing the expression of syndecan-2, which then acts to propagate Notch signaling by direct receptor interaction.     

Ca2+ regulation in vascular smooth muscle.

Regulation of aorta smooth muscle contraction by Ca ion requires the collaboration of the 80,000 dalton factor and tropomyosin. A method for preparing pure actin from aorta smooth muscle is described.     

Reduced expression of the NADPH oxidase NOX4 is a hallmark of adipocyte differentiation

Adipocyte differentiation is a complex process regulated among other factors by insulin and the production of reactive oxygen species (ROS). NOX4 is a ROS generating NADPH oxidase enzyme mediating insulin's action in 3T3L1 adipocytes. In the present paper we show that NOX4 is expressed at high levels both in white and brown preadipocytes and that differentiation into adipocytes results in a decrease in their NOX4 mRNA content. These in vitro results were confirmed in vivo by demonstrating that in intact adipose tissue the majority of NOX4 expressing cells are localized within the preadipocyte containing stromal/vascular fraction, rather than in the portion consisting of mature adipocytes. In line with these observations, quantification of NOX4 mRNA in fat derived from different rodent models of insulin resistance indicated that alteration in NOX4 expression reflects changes in the ratio of adipocyte/interstitial fractions. In conclusion, we reveal that decreased NOX4 mRNA content is a hallmark of adipocyte differentiation and that NOX4 expression measured in whole adipose tissue is not an unequivocal indicator of intact or impaired insulin action.