Aleksandr E. Vendrov

Oxidative Stress and Vascular Disease

Growing evidence indicates that chronic and acute overproduction of reactive oxygen species (ROS) under pathophysiologic conditions is integral in the development of cardiovascular diseases (CVD). These ROS can be released from nicotinamide adenine dinucleotide (phosphate) oxidase, xanthine oxidase, lipoxygenase, mitochondria, or the uncoupling of nitric oxide synthase in vascular cells. ROS mediate various signaling pathways that underlie vascular inflammation in atherogenesis: from the initiation of fatty streak development through lesion progress to ultimate plaque rupture. Various animal models of oxidative stress support the notion that ROS have a causal role in atherosclerosis and other cardiovascular diseases. Human investigations also support the oxidative stress hypothesis of atherosclerosis. Oxidative stress is the unifying mechanism for many CVD risk factors, which additionally supports its central role in CVD. Despite the demonstrated role of antioxidants in cellular and animal studies, the ineffectiveness of antioxidants in reducing cardiovascular death and morbidity in clinical trials has led many investigators to question the importance of oxidative stress in human atherosclerosis. Others have argued that the prime factor for the mixed outcomes from using antioxidants to prevent CVD may be the lack of specific and sensitive biomarkers by which to assess the oxidative stress phenotypes underlying CVD. A better understanding of the complexity of cellular redox reactions, development of a new class of antioxidants targeted to specific subcellular locales, and the phenotype-genotype linkage analysis for oxidative stress will likely be avenues for future research in this area as we move toward the broader use of pharmacological and regenerative therapies in the treatment and prevention of CVD.

Atherosclerosis Is Attenuated by Limiting Superoxide Generation in Both Macrophages and Vessel Wall Cells

Objective—We previously showed that NAD(P)H oxidase deficiency significantly reduces atherosclerosis in apoE−/− 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−/− mice (Control) with functional NAD(P)H oxidase in both monocytes/macrophages and vascular wall cells was compared with that in apoE−/− 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−/− 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) superoxide generation may impact atherosclerosis, in part, by activating smooth muscle cell mitogenic signaling pathways.

ECC-1 Cells: A Well-Differentiated Steroid-Responsive Endometrial Cell Line with Characteristics of Luminal Epithelium

Abstract Endometrial cancer cell lines have provided a valuable model to study endometrial epithelial cells in vitro. Since the first development of HEC1B over 35 yr ago, many different cell lines have been isolated and described. One valuable cell line that maintains hormone responsiveness and unique stability over time is the ECC-1 cell line, developed originally by the late P.G. Satyaswaroop. In this study, we investigated some of the properties of these cells and present their salient characteristics. Like Ishikawa cells, ECC-1 cells maintain both estrogen receptors (ESR1 [ER alpha] and ESR2 [ER beta]), progesterone receptors (PR A and B; PGRs), and androgen receptors (ARs), along with the p160 steroid receptor coactivators NCOA1 (formerly SRC1), NCOA2 (formerly TIF2), and NCOA3 (formerly AIB1). The karyotype of these cells is abnormal, with multiple structural rearrangements in all cells analyzed. Unlike Ishikawa cells that express glandular epithelial antigens, ECC-1 cells maintain a luminal phenotype, with expression of KRT13 (cytokeratin 13) and KRT18 (cytokeratin 18). Apparent differences in the regulation of ESR2 also were evident in ECC-1 cells compared to Ishikawa cells. Like other endometrial cell lines, ECC-1 cells express the steroid receptor coactivators and exhibit epidermal growth factor-stimulated expression of known luminal proteins thought to be involved in implantation, including the hyaluronate receptor CD44 and SPP1 (formerly osteopontin) and CD55 (decay-accelerating factor). These characteristics appear to be stable and persistent over multiple cell passages, making this well-differentiated cell line an excellent choice to study endocrine and paracrine regulation of endometrial epithelium in vitro.

Nox activator 1: A potential target for modulation of vascular reactive oxygen species in atherosclerotic arteries

Background— Despite a concerted effort by many laboratories, the critical subunits that participate in vascular smooth muscle cell (VSMC) NADPH oxidase function have yet to be elucidated. Given the potential therapeutic importance of cell-specific inhibition of NADPH oxidase, we investigated the role of Nox activator 1 (NoxA1), a homolog of p67phox, in VSMC NADPH oxidase function and atherosclerosis. Methods and Results— The presence of NoxA1 in mouse aortic VSMCs was confirmed by reverse-transcription polymerase chain reaction and sequencing. NoxA1/p47phox interaction after thrombin treatment was observed by immunoprecipitation/Western analysis of lysates from p47phox−/− VSMCs transfected with adenoviral HA-NoxA1 and Myc-p47phox. Infection with adenoviral NoxA1 significantly enhanced thrombin-induced reactive oxygen species generation in wild-type but not in p47phox−/− and Nox1−/− VSMCs. Thrombin-induced reactive oxygen species production and VSMC proliferation were significantly reduced after downregulation of NoxA1 with shRNA. Infection with NoxA1 shRNA but not scrambled shRNA significantly decreased thrombin-induced activation of the redox-sensitive protein kinases (Janus kinase 2, Akt, and p38 mitogen-activated protein kinase) in VSMCs. Adenovirus-mediated overexpression of NoxA1 in guidewire-injured mouse carotid arteries significantly increased superoxide production in medial VSMCs and enhanced neointimal hyperplasia. NoxA1 expression was significantly increased in aortas and atherosclerotic lesions of ApoE−/− mice compared with age-matched wild-type mice. Furthermore, in contrast to p67phox, immunoreactive NoxA1 is present in intimal and medial SMCs of human early carotid atherosclerotic lesions. Conclusions— NoxA1 is the functional homolog of p67phox in VSMCs that regulates redox signaling and VSMC phenotype. These findings support the potential for modulation of NoxA1 expression as a viable approach for the treatment of vascular diseases.

Mitochondrial Oxidative Stress in Aortic Stiffening With Age: The Role of Smooth Muscle Cell Function

Objective—Age-related aortic stiffness is an independent risk factor for cardiovascular diseases. Although oxidative stress is implicated in aortic stiffness, the underlying molecular mechanisms remain unelucidated. Here, we examined the source of oxidative stress in aging and its effect on smooth muscle cell (SMC) function and aortic compliance using mutant mouse models. Methods and Results—Pulse wave velocity, determined using Doppler, increased with age in superoxide dismutase 2 (SOD2)+/− but not in wild-type, p47phox−/− and SOD1+/− mice. Echocardiography showed impaired cardiac function in these mice. Increased collagen I expression, impaired elastic lamellae integrity, and increased medial SMC apoptosis were observed in the aortic wall of aged SOD2+/− versus wild-type (16-month-old) mice. Aortic SMCs from aged SOD2+/− mice showed increased collagen I and decreased elastin expression, increased matrix metalloproteinase-2 expression and activity, and increased sensitivity to staurosporine-induced apoptosis versus aged wild-type and young (4-month-old) SOD2+/− mice. Smooth muscle &agr;-actin levels were increased with age in SOD2+/− versus wild-type SMCs. Aged SOD2+/− SMCs had attenuated insulin-like growth factor-1-induced Akt and Forkhead box O3a phosphorylation and prolonged tumor necrosis factor-&agr;–induced Jun N-terminal kinase 1 activation. Aged SOD2+/− SMCs had increased mitochondrial superoxide but decreased hydrogen peroxide levels. Finally, dominant-negative Forkhead box O3a overexpression attenuated staurosporine-induced apoptosis in aged SOD2+/− SMCs. Conclusion—Mitochondrial oxidative stress over a lifetime causes aortic stiffening, in part by inducing vascular wall remodeling, intrinsic changes in SMC stiffness, and aortic SMC apoptosis.

NADPH oxidases regulate CD44 and hyaluronic acid expression in thrombin-treated vascular smooth muscle cells and in atherosclerosis

The intracellular signaling events by which NADPH oxidase-generated reactive oxygen species (ROS) modulate vascular smooth muscle cell (VSMC) function and atherogenesis are yet to be entirely elucidated. We previously demonstrated that NADPH oxidase deficiency decreased atherosclerosis in apoE−/− mice and identified adhesion protein CD44 as an important ROS-sensitive gene expressed in VSMC and atherosclerotic lesions. Here, we examined the molecular mechanisms by which NADPH oxidase-generated ROS regulate the expression of CD44 and its principal ligand, hyaluronan (HA), and how CD44-HA interaction affects VSMC proliferation and migration and inflammatory gene expression in apoE−/− mice aortas. Thrombin-induced CD44 expression is mediated by transcription factor AP-1 in a NADPH oxidase-dependent manner. NADPH oxidase-mediated ROS generation enhanced thrombin-induced HA synthesis, and hyaluronan synthase 2 expression in VSMC. Hyaluronidase, which generates low molecular weight HA (LMW-HA), is induced in VSMC in a NADPH oxidase-dependent manner and LMW-HA stimulated ROS generation and cell proliferation in wild-type but not p47phox−/− VSMC, effects that were enhanced by thrombin pretreatment. Haptotactic VSMC migration toward HA was increased by thrombin in a CD44-dependent manner. HA expression in atherosclerotic lesions and plasma-soluble CD44 and HA levels were higher in apoE−/− compared with apoE−/−/p47phox−/− mice. HA-regulated pro-inflammatory gene expression was higher in apoE−/− than apoE−/−/p47phox−/− mouse aortas. GKT136901, a specific inhibitor of Nox1- and Nox4-containing NADPH oxidase activity, attenuated ROS generation and atherosclerosis and decreased CD44 and HA expression in atherosclerotic lesions. Together, these data suggest that increased CD44 and HA expression and CD44-HA-dependent gene regulation may play a role in atherosclerosis stimulated by NADPH oxidase activation.

Thrombin and NAD(P)H Oxidase–Mediated Regulation of CD44 and BMP4-Id Pathway in VSMC, Restenosis, and Atherosclerosis

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−/− (NAD[P]H oxidase–deficient) VSMC. Of the 9000 genes analyzed by cDNA microarray method at the G1/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, thrombin-induced 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−/− 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.

NOX4 NADPH Oxidase-Dependent Mitochondrial Oxidative Stress in Aging-Associated Cardiovascular Disease

AIMS Increased oxidative stress and vascular inflammation are implicated in increased cardiovascular disease (CVD) incidence with age. We and others demonstrated that NOX1/2 NADPH oxidase inhibition, by genetic deletion of p47phox, in Apoe(-/-) mice decreases vascular reactive oxygen species (ROS) generation and atherosclerosis in young age. The present study examined whether NOX1/2 NADPH oxidases are also pivotal to aging-associated CVD. RESULTS Both aged (16 months) Apoe(-/-) and Apoe(-/-)/p47phox(-/-) mice had increased atherosclerotic lesion area, aortic stiffness, and systolic dysfunction compared with young (4 months) cohorts. Cellular and mitochondrial ROS (mtROS) levels were significantly higher in aortic wall and vascular smooth muscle cells (VSMCs) from aged wild-type and p47phox(-/-) mice. VSMCs from aged mice had increased mitochondrial protein oxidation and dysfunction and increased vascular cell adhesion molecule 1 expression, which was abrogated with (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) treatment. NOX4 expression was increased in the vasculature and mitochondria of aged mice and its suppression with shRNA in VSMCs from aged mice decreased mtROS levels and improved function. Increased mtROS levels were associated with enhanced mitochondrial NOX4 expression in aortic VSMCs from aged subjects, and NOX4 expression levels in arterial wall correlated with age and atherosclerotic severity. Aged Apoe(-/-) mice treated with MitoTEMPO and 2-(2-chlorophenyl)-4-methyl-5-(pyridin-2-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione had decreased vascular ROS levels and atherosclerosis and preserved vascular and cardiac function. INNOVATION AND CONCLUSION These data suggest that NOX4, but not NOX1/2, and mitochondrial oxidative stress are mediators of CVD in aging under hyperlipidemic conditions. Regulating NOX4 activity/expression and using mitochondrial antioxidants are potential approaches to reducing aging-associated CVD.

The Role of Vascular Endothelial Growth Factor-Induced Activation of NADPH Oxidase in Choroidal Endothelial Cells and Choroidal Neovascularization

Rac1, a subunit of NADPH oxidase, plays an important role in directed endothelial cell motility. We reported previously that Rac1 activation was necessary for choroidal endothelial cell migration across the retinal pigment epithelium, a critical step in the development of vision-threatening neovascular age-related macular degeneration. Here we explored the roles of Rac1 and NADPH oxidase activation in response to vascular endothelial growth factor treatment in vitro and in a model of laser-induced choroidal neovascularization. We found that vascular endothelial growth factor induced the activation of Rac1 and of NADPH oxidase in cultured human choroidal endothelial cells. Further, vascular endothelial growth factor led to heightened generation of reactive oxygen species from cultured human choroidal endothelial cells, which was prevented by the NADPH oxidase inhibitors, apocynin and diphenyleneiodonium, or the antioxidant, N-acetyl-L-cysteine. In a model of laser-induced injury, inhibition of NADPH oxidase with apocynin significantly reduced reactive oxygen species levels as measured by dihydroethidium fluorescence and the volume of laser-induced choroidal neovascularization. Mice lacking functional p47phox, a subunit of NADPH oxidase, had reduced dihydroethidium fluorescence and choroidal neovascularization compared with wild-type controls. Taken together, these results indicate that vascular endothelial growth factor activates Rac1 upstream from NADPH oxidase in human choroidal endothelial cells and increases generation of reactive oxygen species, contributing to choroidal neovascularization. These steps may contributed to the pathology of neovascular age-related macular degeneration.

Identification of a Protective Role for Protein Phosphatase 1cγ1 against Oxidative Stress-induced Vascular Smooth Muscle Cell Apoptosis

The development of therapeutic strategies to inhibit reactive oxygen species (ROS)-mediated damage in blood vessels has been limited by a lack of specific targets for intervention. Targeting ROS-mediated events in the vessel wall is of interest, because ROS play important roles throughout atherogenesis. In early atherosclerosis, ROS stimulate vascular smooth muscle cell (VSMC) growth, whereas in late stages of lesion development, ROS induce VSMC apoptosis, causing atherosclerotic plaque instability. To identify putative protective genes against oxidative stress, mouse aortic VSMC were infected with a retroviral human heart cDNA expression library, and apoptosis was induced in virus-infected cells by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) treatment. A total of 17 different, complete cDNAs were identified from the DMNQ-resistant VSMC clones by PCR amplification and sequencing. The cDNA encoding PP1cγ1 (catalytic subunit of protein phosphatase 1) was present in several independent DMNQ-resistant VSMC clones. DMNQ increased mitochondrial ROS production, caspase-3/7 activity, DNA fragmentation, and decreased mitochondrial transmembrane potential in VSMC while decreasing PP1cγ1 activity and expression. Depletion of PP1cγ1 expression by short hairpin RNA significantly enhanced basal as well as DMNQ-induced VSMC apoptosis. PP1cγ1 overexpression abrogated DMNQ-induced JNK1 activity, p53 Ser15 phosphorylation, and Bax expression and protected VSMC against DMNQ-induced apoptosis. In addition, PP1cγ1 overexpression attenuated DMNQ-induced caspase-3/7 activation and DNA fragmentation. Inhibition of p53 protein expression using small interfering RNA abrogated DMNQ-induced Bax expression and significantly attenuated VSMC apoptosis. Together, these data indicate that PP1cγ1 overexpression promotes VSMC survival by interfering with JNK1 and p53 phosphorylation cascades involved in apoptosis.