To cite this article: Madamanchi NR, Hakim ZS, Runge MS. Oxidative stress in atherogenesis and arterial thrombosis: the disconnect between cellular studies and clinical outcomes. J Thromb Haemost 2005; 3: 254-67.Summary. Atherosclerosis is a multifactorial disease for which the molecular etiology of many of the risk factors is still unknown. As no single genetic marker or test accurately predicts cardiovascular death, phenotyping for markers of inflammation may identify the individuals at risk for vascular diseases. Reactive oxygen species (ROS) are key mediators of signaling pathways that underlie vascular inflammation in atherogenesis, starting from the initiation of fatty streak development through lesion progression to ultimate plaque rupture. Various animal models of atherosclerosis support the notion that ROS released from NAD(P)H oxidases, xanthine oxidase, lipoxygenases, and enhanced ROS production from dysfunctional mitochondrial respiratory chain indeed have a causatory role in atherosclerosis and other vascular diseases. Human investigations also support the oxidative stress hypothesis of atherogenesis. This is further supported by the observed impairment of vascular function and enhanced atherogenesis in animal models that have deficiencies in antioxidant enzymes. The importance of oxidative stress in atherosclerosis is further emphasized because of its role as a unifying mechanism across many vascular diseases. The main contraindicator for the role oxidative stress plays in atherosclerosis is the lack of effectiveness of antioxidants in reducing primary endpoints of cardiovascular death and morbidity. However, this lack of effectiveness by itself does not negate the existence or causatory role of oxidative stress in vascular disease. Lack of proven markers of oxidative stress, which could help to identify a subset of population that can benefit from antioxidant supplementation, and the complexity and subcellular localization of redox reactions, are among the factors responsible for the mixed outcomes in the use of antioxidants for the prevention of cardiovascular diseases. To better understand the role of oxidative stress in vascular diseases, future studies should be aimed at using advances in mouse and human genetics to define oxidative stress phenotypes and link phenotype with genotype.

Objective-We previously showed that NAD(P)H oxidase deficiency significantly reduces atherosclerosis in apoE Ϫ/Ϫ mice. The present study was designed to determine the relative contribution of monocyte/macrophage versus vascular wall cell NAD(P)H oxidase to atherogenesis in this model. Methods and Results-Cell-specific NAD(P)H oxidase inhibition was achieved via allogenic, sex-mismatched bone marrow transplantation. Aortic atherosclerosis and superoxide production in apoE Ϫ/Ϫ mice (Control) with functional NAD(P)H oxidase in both monocytes/macrophages and vascular wall cells was compared with that in apoE Ϫ/Ϫ mice with nonfunctional monocyte/macrophage NAD(P)H oxidase (BMO) or nonfunctional vessel wall NAD(P)H oxidase (VWO). A significant decrease in superoxide production and atherosclerotic lesions was observed in BMO and VWO mice compared with control mice. Interestingly, BMO mice had significantly lower plasma oxidized LDL levels compared with control and VWO mice, whereas aortic sections of VWO mice showed decreased expression of cellular adhesion molecules compared with control and BMO mice. NAD(P)H oxidase deficiency also attenuated neointimal hyperplasia and mitogenic protein activation in apoE Ϫ/Ϫ mice after arterial injury. Conclusions-We conclude that (1) both monocyte/macrophages and vessel wall cells play critical roles in atherogenesis;(2) decrease in atherosclerosis results from attenuated superoxide generation in monocyte/macrophages or vessel wall cells; and (3) Key Words: Adhesion molecules Ⅲ signal transduction Ⅲ oxidized lipids Ⅲ reactive oxygen species Ⅲ thrombin R eactive oxygen species (ROS) derived from NAD(P)H oxidase have been strongly associated with experimental hypertension, 1 cardiac hypertrophy, 2 thrombosis, 3 restenosis, and atherosclerosis. 4 In humans, higher expression of NAD(P)H oxidase subunit proteins is associated with increased superoxide (O 2 ⅐Ϫ ) production and severity of atherosclerosis. 5 NAD(P)H oxidase is also an important source of increased O 2 ⅐Ϫ production in human diabetes mellitus, a risk factor for atherosclerosis. 6 All vascular wall cells-endothelial cells, smooth muscle cells (SMCs), and fibroblasts-as well as monocytes/macrophages contain NAD(P)H oxidases which are activated under pathophysiological conditions. The resultant ROS induce redox-sensitive signaling pathways that contribute to atherogenesis. 7 The phagocytic NAD(P)H oxidase contains the membranebound subunits gp91phox (Nox2) and p22phox, the catalytic site of the oxidase and the cytosolic components p47phox, p67phox, and G-protein Rac1 or Rac2. 8 Vascular NAD(P)H oxidases are similar in structure to phagocytic NAD(P)H oxidase, but have a distinct molecular composition. Endothelial cells and adventitial fibroblasts possess all the components of the phagocytic oxidase, whereas SMCs predominantly express homologues of gp91phox, Nox1 and Nox4. Mouse SMCs also express a p67phox homologue, Noxa1. 9 The activation of vascular NAD(P)H oxidases is constitutive as well as inducible in a manner similar ...

Myocyte apoptosis is central to myocardial dysfunction following ischemia/reperfusion (I/R) and during the transition from hypertrophy to heart failure. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase regulates adhesion-dependent survival signals and unopposed FAK activation has been linked to tumor development. We previously showed that conditional myocyte-specific deletion of FAK (MFKO) in the adult heart did not affect basal cardiomyocyte survival or cardiac function but led to dilated cardiomyopathy and heart failure following pressure overload. In the present study, we sought to determine if FAK functions to limit stress-induced cardiomyocyte apoptosis. We reasoned that (I/R), which stimulates robust apoptotic cell death, might uncover an important cardioprotective function for FAK. We found that depletion of FAK markedly exacerbates hypoxia/re-oxygenation-induced cardiomyocyte cell death in vitro. Moreover, deletion of FAK in the adult myocardium resulted in significant increases in I/R-induced infarct size and cardiomyocyte apoptosis with a concomitant reduction in left ventricular function. Finally, our results suggest that NF-κB signaling may play a key role in modulating FAK-dependent cardioprotection, since FAK inactivation blunted activation of the NF-κB survival signaling pathway and reduced levels of the NF-κB target genes, Bcl2 and Bcl-xl. Since the toggling between pro-survival and pro-apoptotic signals remains central to preventing irreversible damage to the heart, we conclude that targeted FAK activation may be beneficial for protecting stress-dependent cardiac remodeling.

Objective-Reactive oxygen species (ROS) integrate cellular signaling pathways involved in aortic smooth muscle cell (SMC) proliferation and migration associated with atherosclerosis. However, the effect of subcellular localization of ROS on SMC mitogenic signaling is not yet fully understood. Methods and Results-We used superoxide dismutase (SOD)-deficient mouse aortic SMCs to address the role of subcellular ROS localization on SMC phenotype and mitogenic signaling. Compared with wild-type, a 54% decrease in total SOD activity (Ϸ50% decrease in SOD1 protein levels) and a 42% reduction in SOD2 activity (Ϸ50% decrease in SOD2 protein levels) were observed in SOD1 ϩ/Ϫ and SOD2 ϩ/Ϫ SMCs, respectively. Consistent with this, basal and thrombin-induced superoxide levels increased in these SMCs. SOD1ϩ/Ϫ and SOD2 ϩ/Ϫ SMCs exhibit increased basal proliferation and enhanced [3 H]-thymidine and [ 3 H]-leucine incorporation in basal and thrombin-stimulated conditions. Our results indicate preferential activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinases in SOD1 ϩ/Ϫ and janus kinase/signal transducer and activator of transcriptase (JAK/STAT) pathway in SOD2 ϩ/Ϫ SMCs. Pharmacological inhibitors of ERK1/2 p38 and JAK2 confirm the SOD genotypedependent SMC proliferation. Conclusions-Our results suggest that SOD1 and SOD2 regulate SMC quiescence by suppressing divergent mitogenic signaling pathways, and dysregulation of these enzymes under pathophysiological conditions may lead to SMC hyperplasia and hypertrophy. Key Words: ROS Ⅲ SMC Ⅲ thrombin Ⅲ SOD Ⅲ cell signaling S mooth muscle cells (SMCs) are characterized by marked plasticity in their proliferative potential and differentiation status. As the primary constituents of the arterial media, they provide mechanical support for blood vessels and participate in regulation of vasomotor tone. In addition, SMCs contribute to neointimal formation after vascular injury and during atherosclerotic lesion progression in humans. In advanced atherosclerotic lesions, SMCs are prone to apoptosis and release proteolytic enzymes such as matrix metalloproteinases that may contribute to plaque instability. The remarkable diversity of SMC phenotype and function is determined in large part through regulatory cues in the extracellular microenvironment and interactions among intracellular signaling pathways. In addition, SMC phenotypes are modified over time by the accumulation of injuries to cellular macromolecules. See page 887Reactive oxygen species (ROS) are key components for integration of SMC signaling events, whereas at the same time, they are major contributors to the degradation of cellular function through their interactions with proteins and structural components of the cell. Exogenous ROS are potent stimuli for activation of SMC signaling and mitogenesis, 1 although in high (but physiological) concentrations, they may induce DNA damage and mitochondrial dysfunction. 2 SMCs have several intrinsic mechanisms for generating ROS, the majo...

Abstract-To characterize novel signaling pathways that underlie NAD(P)H oxidase-mediated signaling in atherosclerosis, we first examined differences in thrombin-induced gene expression between wild-type and p47phox Ϫ/Ϫ (NAD[P]H oxidase-deficient) VSMC. Of the 9000 genes analyzed by cDNA microarray method at the G 1 /S transition point, 76 genes were similarly and significantly modulated in both the cell types, whereas another 22 genes that encompass various functional groups were regulated in NAD(P)H oxidase-dependent manner. Among these 22 genes, thrombininduced NAD(P)H oxidase-mediated regulation of Klf15, Igbp1, Ak4, Adamts5, Ech1, Serp1, Sec61a2, Aox1, Aoh1, Fxyd5, Rai14, and Serpinh1 was shown for the first time in VSMC. The role of NAD(P)H oxidase in the regulation of a subset of these genes (CD44, BMP4, Id1, and Id3) was confirmed using modulators of reactive oxygen species (ROS) generation, a ROS scavenger and in gain-of-function experiments. We then characterized regulation of these genes in restenosis and atherosclerosis. In both apoE Ϫ/Ϫ mice and in a mouse vascular injury model, these genes are regulated in NAD(P)H oxidase-dependent manner during vascular lesion formation. Based on these findings, we propose that NAD(P)H oxidase-dependent gene expression in general, and the CD44 and BMP4-Id signaling pathway in particular, is important in restenosis and atherosclerosis. Key Words: vascular smooth muscle cells Ⅲ reactive oxygen species Ⅲ microarray Ⅲ redox regulation T he development of molecular therapies for atherosclerosis has lagged behind other diseases, perhaps because of the complexity of redundant signaling pathways that govern vascular cell function. Although the precise role of reactive oxygen species (ROS) in vascular smooth muscle cell (VSMC) biology remains controversial, the importance of ROS generation in signaling is not. In VSMC, several signal transduction pathways induced by growth factors and cytokines are mediated by ROS generated by the activation of membrane-bound NAD(P)H oxidase. 1 The VSMC NAD(P)H oxidase includes membrane-bound components: Nox1/4 and p22phox; and cytosolic components: Rac1 and p47phox. 2 A third cytosolic component, p67phox, found in other vascular cells, is not present in VSMC. 3 Recently, p67phox and also p47phox homologs have been reported in VSMC. 2 The key role of p47phox in NAD(P)H oxidase activation in response to agonist stimulation and subsequent increase in cell proliferation was demonstrated using wild-type and p47phox Ϫ/Ϫ VSMC. 4 With few exceptions, 5 predominant data support strong association of ROS with atherosclerosis. ApoE Ϫ/Ϫ / p47phoxϪ/Ϫ mice have lower levels of aortic ROS production and less atherosclerosis than apoE Ϫ/Ϫ mice. 4 Other mouse models with altered levels of manganese superoxide dismutase (SOD2) 6 and p66Shc 7 have produced a consistent theme that increased levels of vascular ROS promote, whereas decreased levels reduce atherogenesis. ROS may enhance atherosclerosis by several mechanisms. Increased ROS production enhances...