Francis J. Miller

Superoxide Production in Vascular Smooth Muscle Contributes to Oxidative Stress and Impaired Relaxation in Atherosclerosis

The endothelium is a source of reactive oxygen species in short-term models of hypercholesterolemia and atherosclerosis. We examined a chronic model of atherosclerosis for increased vascular production of superoxide (O2-.) and determined whether endothelial overexpression of superoxide dismutase (SOD) would improve endothelium-dependent relaxation. Superoxide generation was 3 times higher in isolated aortas from Watanabe heritable hyperlipidemic (WHHL) rabbits (2 to 4 years old) compared with aortas from New Zealand White (NZ) rabbits (43+/-10 versus 14+/-2 relative light units x min(-1) x mm(-2), n=9, P<0.05). After in vitro transduction with adenovirus containing the gene for CuZn-SOD (AdCMVCuZn-SOD) or extracellular SOD (AdCMVEC-SOD), endothelial O2-. levels in WHHL aortas were significantly reduced. Gene transfer of SOD to WHHL aortas, however, failed to improve the impaired relaxation to acetylcholine or calcium ionophore. By use of the oxidative fluorescent dye hydroethidine, an in situ assay indicated markedly increased generation of O2-. throughout the wall of WHHL aorta, especially within layers of smooth muscle. This finding was confirmed by demonstrating increased O2-. levels in smooth muscle cells cultured from WHHL aorta. We conclude that elevated O2-. levels in atherosclerotic vessels are not confined to the endothelium but occur throughout the vascular wall, including smooth muscle cells. Reduction in endothelial O2-. levels is not sufficient to improve endothelium-dependent relaxation. Generation of reactive oxygen species within the media may contribute to vasomotor dysfunction in atherosclerosis.

Reactive oxygen species: from health to disease

Upon reaction with electrons, oxygen is transformed into reactive oxygen species (ROS). It has long been known that ROS can destroy bacteria and destroy human cells, but research in recent decades has highlighted new roles for ROS in health and disease. Indeed, while prolonged exposure to high ROS concentrations may lead to non-specific damage to proteins, lipids, and nucleic acids, low to intermediate ROS concentrations exert their effects rather through regulation of cell signalling cascades. Biological specificity is achieved through the amount, duration, and localisation of ROS production. ROS have crucial roles in normal physiological processes, such as through redox regulation of protein phosphorylation, ion channels, and transcription factors. ROS are also required for biosynthetic processes, including thyroid hormone production and crosslinking of extracellular matrix. There are multiple sources of ROS, including NADPH oxidase enzymes; similarly, there are a large number of ROS-degrading systems. ROS-related disease can be either due to a lack of ROS (e.g., chronic granulomatous disease, certain autoimmune disorders) or a surplus of ROS (e.g., cardiovascular and neurodegenerative diseases). For diseases caused by a surplus of ROS, antioxidant supplementation has proven largely ineffective in clinical studies, most probably because their action is too late, too little, and too non-specific. Specific inhibition of ROS-producing enzymes is an approach more promising of clinical efficacy.

Overexpression of Human Catalase Inhibits Proliferation and Promotes Apoptosis in Vascular Smooth Muscle Cells

The role of reactive oxygen species, such as superoxide anions (O(2). (-)) and hydrogen peroxide (H(2)O(2)), in modulating vascular smooth muscle cell proliferation and viability is controversial. To investigate the role of endogenously produced H(2)O(2), rat aortic smooth muscle cells were infected with adenoviral vectors containing cDNA for human catalase (AdCat) or a control gene, beta-galactosidase (AdLacZ). Infection with AdCat resulted in dose-dependent increases in intracellular catalase protein, which was predominantly localized to peroxisomes. After infection with 100 multiplicity of infection (MOI) of AdCat, cellular catalase activity was increased by 50- to 100-fold, and intracellular H(2)O(2) concentration was reduced, as compared with control. Infection with AdCat reduced [(3)H]thymidine uptake, an index of DNA synthesis, in cells maintained in medium supplemented with 2% serum (0.37+/-0.09 disintegrations per minute per cell [AdLacZ] versus 0.22+/-0.08 disintegrations per minute per cell [AdCat], P<0.05). Five days after infection with 100 MOI of AdCat, cell numbers were reduced as compared with noninfected or AdLacZ-infected cells (157 780+/-8413 [AdCat], P<0.05 versus 233 700+/-3032 [noninfected] or 222 410+/-5332 [AdLacZ]). Furthermore, the number of apoptotic cells was increased 5-fold after infection with 100 MOI of AdCat as compared with control. Infection with AdCat resulted in induction of cyclooxygenase (COX)-2, and treatment with a COX-2 inhibitor overcame the AdCat-induced reduction in cell numbers. These findings indicate that overexpression of catalase inhibited smooth muscle proliferation while increasing the rate of apoptosis, possibly through a COX-2-dependent mechanism. Our results suggest that endogenously produced H(2)O(2) importantly modulates survival and proliferation of vascular smooth muscle cells.

Oxidative Stress in Human Abdominal Aortic Aneurysms. A Potential Mediator of Aneurysmal Remodeling

Abdominal aortic aneurysm (AAA) is an inflammatory disorder characterized by localized connective tissue degradation and smooth muscle cell (SMC) apoptosis, leading to aortic dilatation and rupture. Reactive oxygen species are abundantly produced during inflammatory processes and can stimulate connective tissue–degrading proteases and apoptosis of SMCs. We hypothesized that reactive oxygen species are locally increased in AAA and lead to enhanced oxidative stress. In aortas from patients undergoing surgical repair, superoxide levels (measured by lucigenin-enhanced chemiluminescence) were 2.5-fold higher in the AAA segments compared with the adjacent nonaneurysmal aortic (NA) segments (6638±2164 versus 2675±1027 relative light units for 5 minutes per millimeter squared, respectively; n=7). Formation of thiobarbituric acid–reactive substances and conjugated dienes, 2 indices of lipid peroxidation, were increased 3-fold in AAA compared with NA segments. Immunostaining for nitrotyrosine was significantly greater in AAA tissue. Dihydroethidium staining indicated that increased superoxide in AAA segments was localized to infiltrating inflammatory cells and to SMCs. Expression of the NADPH oxidase subunits p47phox and p22phox and NAD(P)H oxidase activity were increased in AAA segments compared with NA segments. Thus, oxidative stress is markedly increased in AAA, in part through the activation of NAD(P)H oxidase, and may contribute to the disease pathogenesis.

Cytokine Activation of Nuclear Factor κB in Vascular Smooth Muscle Cells Requires Signaling Endosomes Containing Nox1 and ClC-3

Reactive oxygen species (ROS) are mediators of intracellular signals for a myriad of normal and pathologic cellular events, including differentiation, hypertrophy, proliferation, and apoptosis. NADPH oxidases are important sources of ROS that are present in diverse tissues throughout the body and activate many redox-sensitive signal transduction and gene expression pathways. To avoid toxicity and provide specificity of signaling, ROS production and metabolism necessitate tight regulation that likely includes subcellular compartmentalization. However, the constituent elements of NADPH oxidase-dependent cell signaling are not known. To address this issue, we examined cytokine generation of ROS and subsequent activation of the transcription factor nuclear factor &kgr;B in vascular smooth muscle cells (SMCs). Tumor necrosis factor-&agr; and interleukin (IL)-1&bgr; stimulation of SMCs resulted in diphenylene iodonium-sensitive ROS production within intracellular vesicles. Nox1 and p22phox, integral membrane subunits of NADPH oxidase, coimmunoprecipitated with early endosomal markers in SMCs. ClC-3, an anion transporter that is primarily found in intracellular vesicles, also colocalized with Nox1 in early endosomes and was necessary for tumor necrosis factor-&agr; and interleukin-1&bgr; generation of ROS. Cytokine activation of nuclear factor &kgr;B in SMCs required both Nox1 and ClC-3. We conclude that in response to tumor necrosis factor-&agr; and interleukin-1&bgr;, NADPH oxidase generates ROS within early endosomes and that Nox1 cannot produce sufficient ROS for cell signaling in the absence of ClC-3. These data best support a model whereby ClC-3 is required for charge neutralization of the electron flow generated by Nox1 across the membrane of signaling endosomes.

Intracellular Protein Aggregation Is a Proximal Trigger of Cardiomyocyte Autophagy

Background— Recent reports demonstrate that multiple forms of cardiovascular stress, including pressure overload, chronic ischemia, and infarction-reperfusion injury, provoke an increase in autophagic activity in cardiomyocytes. However, nothing is known regarding molecular events that stimulate autophagic activity in stressed myocardium. Because autophagy is a highly conserved process through which damaged proteins and organelles can be degraded, we hypothesized that stress-induced protein aggregation is a proximal trigger of cardiomyocyte autophagy. Methods and Results— Here, we report that pressure overload promotes accumulation of ubiquitinated protein aggregates in the left ventricle, development of aggresome-like structures, and a corresponding induction of autophagy. To test for causal links, we induced protein accumulation in cultured cardiomyocytes by inhibiting proteasome activity, finding that aggregation of polyubiquitinated proteins was sufficient to induce cardiomyocyte autophagy. Furthermore, attenuation of autophagic activity dramatically enhanced both aggresome size and abundance, consistent with a role for autophagic activity in protein aggregate clearance. Conclusions— We conclude that protein aggregation is a proximal trigger of cardiomyocyte autophagy and that autophagic activity functions to attenuate aggregate/aggresome formation in heart. Findings reported here are the first to demonstrate that protein aggregation occurs in response to hemodynamic stress, situating pressure-overload heart disease in the category of proteinopathies.

Deletion of p47phox Attenuates Angiotensin II–Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E–Deficient Mice

Background— Angiotensin II (Ang II) contributes to vascular pathology in part by stimulating NADPH oxidase activity, leading to increased formation of superoxide (O2−). We reported that O2− levels, NADPH oxidase activity, and expression of the p47phox subunit of NADPH oxidase are increased in human abdominal aortic aneurysms (AAAs). Here, we tested the hypothesis that deletion of p47phox will attenuate oxidative stress and AAA formation in Ang II–infused apoE−/− mice. Methods and Results— Male apoE−/− and apoE−/−p47phox−/− mice received saline or Ang II (1000 ng · kg−1 · min−1) infusion for 28 days, after which abdominal aortic weight and maximal diameter were determined. Aortic tissues and blood were examined for parameters of aneurysmal disease and oxidative stress. Ang II infusion induced AAAs in 90% of apoE−/− versus 16% of apo−/−p47phox−/− mice (P<0.05). Abdominal aortic weight (14.1±3.2 versus 35.6±9.0 mg), maximal aortic diameter (1.5±0.2 versus 2.4±0.4 mm), aortic NADPH oxidase activity, and parameters of oxidative stress were reduced in apoE−/−p47phox−/− mice compared with apoE−/− mice (P<0.05). In addition, aortic macrophage infiltration and matrix metalloproteinase-2 activity were reduced in apoE−/−p47phox−/− mice compared with apoE−/− mice. Deletion of p47phox attenuated the pressor response to Ang II; however, coinfusion of phenylephrine with Ang II, which restored the Ang II pressor response, did not alter the protective effects of p47phox deletion on AAA formation. Conclusions— Deletion of p47phox attenuates Ang II–induced AAA formation in apoE−/− mice, suggesting that NADPH oxidase plays a critical role in AAA formation in this model.

Vitamin E Inhibits Abdominal Aortic Aneurysm Formation in Angiotensin II–Infused Apolipoprotein E–Deficient Mice

Background—Abdominal aortic aneurysms (AAAs) in humans are associated with locally increased oxidative stress and activity of NADPH oxidase. We investigated the hypothesis that vitamin E, an antioxidant with documented efficacy in mice, can attenuate AAA formation during angiotensin II (Ang II) infusion in apolipoprotein E–deficient mice. Methods and Results—Six-month-old male apolipoprotein E–deficient mice were infused with Ang II at 1000 ng/kg per minute for 4 weeks via osmotic minipumps while consuming either a regular diet or a diet enriched with vitamin E (2 IU/g of diet). After 4 weeks, abdominal aortic weight and maximal diameter were determined, and aortic tissues were sectioned and examined using biochemical and histological techniques. Vitamin E attenuated formation of AAA, decreasing maximal aortic diameter by 24% and abdominal aortic weight by 34% (P<0.05, respectively). Importantly, animals treated with vitamin E showed a 44% reduction in the combined end point of fatal+nonfatal aortic rupture (P<0.05). Vitamin E also decreased aortic 8-isoprostane content (a marker of oxidative stress) and reduced both aortic macrophage infiltration and osteopontin expression (P<0.05, respectively). Vitamin E treatment had no significant effect on the extent of aortic root atherosclerosis, activation of matrix metalloproteinases 2 or 9, serum lipid profile, or systolic blood pressure. Conclusions—Vitamin E ameliorates AAAs and reduces the combined end point of fatal+nonfatal aortic rupture in this animal model. These findings are consistent with the concept that oxidative stress plays a pivotal role in Ang II–driven AAA formation in hyperlipidemic mice.

Endothelial Dysfunction in Chronic Inflammatory Diseases

Chronic inflammatory diseases are associated with accelerated atherosclerosis and increased risk of cardiovascular diseases (CVD). As the pathogenesis of atherosclerosis is increasingly recognized as an inflammatory process, similarities between atherosclerosis and systemic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel diseases, lupus, psoriasis, spondyloarthritis and others have become a topic of interest. Endothelial dysfunction represents a key step in the initiation and maintenance of atherosclerosis and may serve as a marker for future risk of cardiovascular events. Patients with chronic inflammatory diseases manifest endothelial dysfunction, often early in the course of the disease. Therefore, mechanisms linking systemic inflammatory diseases and atherosclerosis may be best understood at the level of the endothelium. Multiple factors, including circulating inflammatory cytokines, TNF-α (tumor necrosis factor-α), reactive oxygen species, oxidized LDL (low density lipoprotein), autoantibodies and traditional risk factors directly and indirectly activate endothelial cells, leading to impaired vascular relaxation, increased leukocyte adhesion, increased endothelial permeability and generation of a pro-thrombotic state. Pharmacologic agents directed against TNF-α-mediated inflammation may decrease the risk of endothelial dysfunction and cardiovascular disease in these patients. Understanding the precise mechanisms driving endothelial dysfunction in patients with systemic inflammatory diseases may help elucidate the pathogenesis of atherosclerosis in the general population.

Gene Transfer of Endothelial Nitric Oxide Synthase Improves Relaxation of Carotid Arteries From Diabetic Rabbits

BACKGROUND Diabetes mellitus is associated with impairment of NO-mediated vascular relaxation. The purpose of this study was to determine whether adenovirus-mediated gene transfer of endothelial NO synthase (eNOS) or Cu/Zn superoxide dismutase (SOD1) improves responsiveness to acetylcholine in alloxan-induced diabetic rabbits. METHODS AND RESULTS After 8 weeks, plasma glucose was greater in diabetic rabbits (418+/-35 mg/dL) (mean+/-SEM) than in normal rabbits (105+/-4 mg/dL). Carotid arteries were removed and cut into ring segments. Arteries were incubated for 2 hours with adenoviral vectors driven by a CMV promoter expressing beta-galactosidase (beta-gal), eNOS, SOD1, or vehicle. After incubation with virus, arteries were incubated for an additional 24 hours to allow transgene expression. Vascular reactivity was examined by recording isometric tension. After precontraction with phenylephrine, responses to the endothelium-independent vasodilator sodium nitroprusside were similar in diabetic and normal arteries. Endothelium-dependent relaxation to acetylcholine (3x10(-6) mol/L) was significantly less in arteries from diabetic animals (68+/-5%) than in normal vessels (90+/-3%). Adenoviral transfection of arteries with eNOS improved relaxation in response to acetylcholine in diabetic (EC(50) eNOS=0.64+/-0.12x10(-7) mol/L versus vehicle =1. 70+/-0.43x10(-7) mol/L) but not normal arteries. Vasorelaxation in response to acetylcholine was inhibited by N(omega)-nitro-L-arginine (100 micromol/L) in all groups. Responses to acetylcholine were unchanged after gene transfection of SOD1 or beta-gal in arteries from diabetic or normal rabbits. CONCLUSIONS Adenovirus-mediated gene transfer of eNOS, but not SOD, improves impaired NO-mediated relaxation in vessels from diabetic rabbits.