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Integrin regulation and signaling play a central role in the hemostasis process, particularly at the level of endothelial cells by regulating the contractility and barrier function of these cells and in platelets by controlling adhesion and aggregation at the site of cell injury. Reactive oxygen species (ROS) have emerged as an important mediator both transducing the signals associated with integrin activation and modulating integrin function. Ligation of integrins in endothelial cells and platelets induces activation of the Ras/mitogen-activated protein kinase, nuclear factor-kappaB, and phosphatidylinositol 3-kinase and Rho-GTPases pathways. Following vessel-wall injury and associated with activation and recruitment of platelets, there is a production of ROS concomitant with the stimulation of the blood coagulation. Moreover, ROS are capable of inducing conformational changes in integrins to change their binding affinity and function. This review will explore how ROS have emerged as an important modulator of integrins in coagulation through both outside-in (integrins stimulating ROS production to effect intracellular events) and inside-out signaling (intracellular ROS altering integrin function).

The catalytic subunit of the NADPH oxidase complex, Nox1 (homologue of gp91phox/Nox2), expressed mainly in intestinal epithelial and vascular smooth muscle cells, functions in innate immune defense and cell proliferation. The molecular mechanisms underlying these functions, however, are not completely understood. We measured Nox1-dependent O 2 2 production during cell spreading on Collagen IV (Coll IV) in colon carcinoma cell lines. Knocking down Nox1 by shRNA, we showed that Nox1-dependent O 2 2 production is activated during cell spreading after 4 hr of adhesion on Collagen IV. Nox1 activation during cell spreading relies on Rac1 activation and arachidonic metabolism. Our results showed that manoalide (a secreted phospholipase A2 inhibitor) and cinnamyl-3,4-dihydroxy-a-cyanocinnamate (a 12-lipoxygenase inhibitor) inhibit O 2 2 production, cell spreading and cell proliferation in these colonic epithelial cells. 12-Lipoxygenase inhibition of ROS production and cell spreading can be reversed by adding 12-HETE, a 12-lipoxygenase product, supporting the specific effect observed with cinnamyl-3,4-dihydroxy-a-cyanocinnamate. In contrast, Nox1 shRNA and DPI (NADPH oxidase inhibitor) weakly affect cell spreading while inhibiting O 2 2 production and cell proliferation. These results suggest that the 12-lipoxygenase pathway is upstream of Nox1 activation and controls cell spreading and proliferation, while Nox1 specifically affects cell proliferation. ' 2007 Wiley-Liss, Inc.Key words: NADPH oxidase; Nox1; colon; integrins; arachidonic acidThe phagocytic NADPH oxidase is composed of 5 subunits, including p22phox and gp91 phox that constitute the membranebound flavocytochrome b 558 , and p40 phox , p47phox and p67 phox that translocate from the cytoplasm to the membrane upon cell activation.1 In addition, the small Rac GTPase have emerged as important functional components that are indispensable for NADPH oxidase activity. Since the initial identification of Nox1 (gp91 phox homolog) in human Caco-2 colon carcinoma cells, the biological significance of superoxide production by NAPDH oxidase in nonphagocytic cells has garnered much interest.2 In contrast to gp91phox/Nox2, which catalyzes a massive and explosive production of superoxide involved in host defense, Nox1 controls a lower superoxide production that acts as mediator of cell signaling. 2Nox1 is expressed mainly in normal and cancerous intestinal epithelial cells and vascular smooth muscle cells. Although being the subject of intense investigation, the role and the endogenous regulation of Nox1 in colon epithelial cells is still unclear.3,4 In colon carcinoma cells, homologs of p47 phox and p67 phox , respectively called Noxo1 (for Nox organizer 1) and Noxa1 (for Nox activator 1), have recently been identified. 5,6 In contrast to p47phox , Noxo1 appears to be constitutively associated with the NADPH oxidase complex.Activation of NADPH oxidase is dependent upon lipid mediators, such as phosphatidic and arachidonic acids and phosphatidylinositol. Arachidonic acid (AA)...

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PurposeTherapeutic strategies that modulate ventricular remodeling can be useful after acute myocardial infarction (MI). In particular, statins may exert effects on molecular pathways involved in collagen metabolism. The aim of this study was to determine whether treatment with atorvastatin for 4 weeks would lead to changes in collagen metabolism and ventricular remodeling in a rat model of MI.MethodsMale Wistar rats were used in this study. MI was induced in rats by ligation of the left anterior descending coronary artery (LAD). Animals were randomized into three groups, according to treatment: sham surgery without LAD ligation (sham group, n = 14), LAD ligation followed by 10mg atorvastatin/kg/day for 4 weeks (atorvastatin group, n = 24), or LAD ligation followed by saline solution for 4 weeks (control group, n = 27). After 4 weeks, hemodynamic characteristics were obtained by a pressure-volume catheter. Hearts were removed, and the left ventricles were subjected to histologic analysis of the extents of fibrosis and collagen deposition, as well as the myocyte cross-sectional area. Expression levels of mediators involved in collagen metabolism and inflammation were also assessed.ResultsEnd-diastolic volume, fibrotic content, and myocyte cross-sectional area were significantly reduced in the atorvastatin compared to the control group. Atorvastatin modulated expression levels of proteins related to collagen metabolism, including MMP1, TIMP1, COL I, PCPE, and SPARC, in remote infarct regions. Atorvastatin had anti-inflammatory effects, as indicated by lower expression levels of TLR4, IL-1, and NF-kB p50.ConclusionTreatment with atorvastatin for 4 weeks was able to attenuate ventricular dysfunction, fibrosis, and left ventricular hypertrophy after MI in rats, perhaps in part through effects on collagen metabolism and inflammation. Atorvastatin may be useful for limiting ventricular remodeling after myocardial ischemic events.

BackgroundThe inflammatory response has been implicated in the pathogenesis of left ventricular (LV) remodeling after myocardial infarction (MI). An anthraquinone compound with anti-inflammatory properties, diacerein inhibits the synthesis and activity of pro-inflammatory cytokines, such as tumor necrosis factor and interleukins 1 and 6. The purpose of this study was to investigate the effects of diacerein on ventricular remodeling in vivo.Methods and ResultsLigation of the left anterior descending artery was used to induce MI in an experimental rat model. Rats were divided into two groups: a control group that received saline solution (n = 16) and a group that received diacerein (80 mg/kg) daily (n = 10). After 4 weeks, the LV volume, cellular signaling, caspase 3 activity, and nuclear factor kappa B (NF-κB) transcription were compared between the two groups. After 4 weeks, end-diastolic and end-systolic LV volumes were reduced in the treatment group compared to the control group (p < .01 and p < .01, respectively). Compared to control rats, diacerein-treated rats exhibited less fibrosis in the LV (14.65%± 7.27% vs. 22.57%± 8.94%; p < .01), lower levels of caspase-3 activity, and lower levels of NF-κB p65 transcription.ConclusionsTreatment with diacerein once a day for 4 weeks after MI improved ventricular remodeling by promoting lower end-systolic and end-diastolic LV volumes. Diacerein also reduced fibrosis in the LV. These effects might be associated with partial blockage of the NF-κB pathway.

A431 cells escape EGF-induced apoptosis by forming cell aggregates. We show that these clusters migrate and merge with neighboring ones, resulting in larger structures composed of a multilayer central (3D) population surrounded by a cell monolayer (2D). We found that after 48 hr of 10 nM EGF treatment, 3D structure formation correlates with ␣2␤1 integrin upregulation. Blockade of ␣2 integrin impairs 3D structure formation. We studied the involvement of reactive oxygen species (ROS) in this process. We show that A431 cells express the NADPH oxidase catalytic subunits Nox1. EGF-induced dose-dependant ROS production was inhibited by the NADPH oxidase inhibitor, diphenylene iodonium (DPI), in these cells while rotenone was ineffective. Inhibition of ROS level in A431 cells with DPI or ebselen (glutathione peroxydase mimic) as well as P38 MAP kinase inhibition by SB203580 decreases ␣2 integrin subunit expression and induces a shift to 3D versus 2D populations. Cell cycle analysis of 2D cells shows that DPI, ebselen and SB203580 decrease the number of cells in S/G2 phase without affecting the cell number in mitosis phase. On the contrary, for 3D cells, these treatments increased the proportion of cells in mitosis without modification of the cell number in S/G2 phase. For both populations, apoptosis was increased by DPI and ebselen. Resistance of cell aggregates by paclitaxel to cell death is usually described. We show that DPI abolishes paclitaxel resistance of 3D cell aggregates. We observed a greater than additive effect between paclitaxel and DPI resulting in an increased proportion of cells in S/G2 phase for 3D populations. These results suggested that the ROS-P38 MAP kinase-␣2␤1 integrin pathway was implicated in the A431 survival process by modulating the balance between 2D/3D cells. © 2004 Wiley-Liss, Inc. Key words: reactive oxygen species; ␣2␤1 integrin; P38 MAP kinase; EGF receptor; adenocarcinoma A431 cell; apoptosis resistance; paclitaxel Epidermal growth factor (EGF) stimulates growth of a great variety of normal and tumoral cells. 1 Nevertheless, a number of tumor cells that overexpress EGF receptors (EGFR) are growthinhibited upon EGF treatment. 2,3 This paradoxical phenomenon has been best characterized in human squamous carcinoma A431 cells, which express a high number of EGFR. 4 These cells have thus been shown to exhibit a biphasic response to EGF in that they are weakly stimulated by picomolar concentrations of EGF but exhibit marked inhibition of proliferation in the presence of nanomolar EGF. 3,5 The growth-inhibitory action of EGF can lead to cell cycle block due to elevation of cyclin-dependent kinase inhibitor p21/CIP1 protein level correlated to G1/S arrest 6 -8 and/or to apoptosis. 9 -11 In A431 cells and in MDA-MB-468, another human cancer cell line overexpressing EGFR, the p21/CIP1-dependent STAT signaling pathway was shown to be required for apoptosis. 12,13 Nevertheless, a proportion of A431 cells escape the EGF-induced apoptosis process and these survival cells were characteriz...

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