Karine Laude

Vascular Oxidant Stress Enhances Progression and Angiogenesis of Experimental Atheroma

Background—Although multiple pathological processes have been associated with oxidative stress, the causative relation between oxidative stress and arterial lesion progression remains unclear. Methods and Results—To test the effect of creating arterial wall oxidative stress, we compared progression of mouse carotid lesions induced by flow cessation in the wild-type (WT) versus transgenic mice (Tgp22vsmc), in which overexpression of p22phox, a critical component of NAD(P)H oxidase was targeted to smooth muscle cell (SMC). Compared with WT mice, arterial lesions grew significantly larger in Tgp22vsmc (P <0.001) and demonstrated elevated hydrogen peroxide (H2O2) and vascular endothelial growth factor (VEGF) levels at all time points examined (P <0.001, n=4 animals per time point), probably related to increased expression of hypoxia inducible factor (HIF)-1&agr; via SMC oxidative stress in the Tgp22vsmc arteries, both basally (203±12% versus WT, P <0.001, n=3) and after lesion formation. Interestingly, Tgp22vsmc lesions were complicated by extensive neointimal angiogenesis. In vitro experiments confirmed SMCs isolated from Tgp22vsmc to be the source for increased H2O2, VEGF, and HIF-1&agr; and their capacity to induce angiogenic cord-like structures when cocultured with endothelial cells. The antioxidant ebselen inhibited SMC activities in vitro and intralesion angiogenesis and lesion progression in vivo. Conclusions—We have demonstrated a novel pathway by which oxidative stress can trigger in vivo an angiogenic switch associated with experimental plaque progression and angiogenesis. This pathway may be related to human atheroma progression and destabilization through intraplaque hemorrhage.

Role of Extracellular Superoxide Dismutase in Hypertension

We previously found that angiotensin II–induced hypertension increases vascular extracellular superoxide dismutase (ecSOD), and proposed that this is a compensatory mechanism that blunts the hypertensive response and preserves endothelium-dependent vasodilatation. To test this hypothesis, we studied ecSOD-deficient mice. ecSOD−/− and C57Blk/6 mice had similar blood pressure at baseline; however, the hypertension caused by angiotensin II was greater in ecSOD−/− compared with wild-type mice (168 versus 147 mm Hg, respectively; P<0.01). In keeping with this, angiotensin II increased superoxide and reduced endothelium-dependent vasodilatation in small mesenteric arterioles to a greater extent in ecSOD−/− than in wild-type mice. In contrast to these findings in resistance vessels, angiotensin II paradoxically improved endothelium-dependent vasodilatation, reduced intracellular and extracellular superoxide, and increased NO production in aortas of ecSOD−/− mice. Whereas aortic expression of endothelial NO synthase, Cu/ZnSOD, and MnSOD were not altered in ecSOD−/− mice, the activity of Cu/ZnSOD was increased by 80% after angiotensin II infusion. This was associated with a concomitant increase in expression of the copper chaperone for Cu/ZnSOD in the aorta but not in the mesenteric arteries. Moreover, the angiotensin II–induced increase in aortic reduced nicotinamide-adenine dinucleotide phosphate oxidase activity was diminished in ecSOD−/− mice as compared with controls. Thus, during angiotensin II infusion, ecSOD reduces hypertension, minimizes vascular superoxide production, and preserves endothelial function in resistance arterioles. We also identified novel compensatory mechanisms involving upregulation of copper chaperone for Cu/ZnSOD, increased Cu/ZnSOD activity, and decreased reduced nicotinamide-adenine dinucleotide phosphate oxidase activity in larger vessels. These compensatory mechanisms preserve large vessel function when ecSOD is absent in hypertension.

Toll-Like Receptors 2-Deficient Mice Are Protected Against Postischemic Coronary Endothelial Dysfunction

Objectives—Toll-like receptors (TLR) 2 are expressed in cardiac and inflammatory cells, and regulate leukocyte function. Because leukocyte adhesion is a critical event in endothelial injury induced by ischemia/reperfusion (I/R), we assessed whether TLR2 were involved in I/R - induced coronary endothelial injury. Methods and Results—Ischemia - reperfusion markedly decreased NO-mediated coronary relaxations to acetylcholine assessed ex vivo. In contrast, in TLR2 deficient mice, I/R paradoxically improved the NO-mediated responses to acetylcholine. To precise the cellular compartment expressing TLR2 which is involved in endothelial injury, we developed bone-marrow chimeric mice by transplanting TLR2−/− bone marrow to WT mice or WT bone marrow to TLR2−/− mice and submitted them to I/R 5 weeks after transplant. Both chimeric mice displayed similar protection as TLR2−/− mice against I/R-induced endothelial dysfunction, suggesting a role of TLR2 expressed on both non-bone marrow cells (in our case presumably endothelial cells and/or cardiomyocytes) and cells of bone marrow origin (presumably neutrophils). TLR2 deficiency was also associated with a smaller infarct size, and reduced reperfusion-induced production of reactive oxygen species and leukocyte infiltration. Conclusions—TLR2 contribute to coronary endothelial dysfunction after I/R, possibly through stimulation of neutrophil- (and free radical-) mediated endothelial injury.

Targeted detoxification of selected reactive oxygen species in the vascular endothelium.

Oxidative stress underlies diverse vascular diseases, but its management remains elusive, in part because of our inability to selectively detoxify reactive oxygen species (ROS) in pathological sites and our limited understanding which species need to be eliminated. The antioxidant enzymes (AOEs) superoxide dismutase (SOD) and catalase (which decompose and H2O2, respectively), conjugated with an antibody to platelet-endothelial cell adhesion molecule-1 (PECAM-1), bind to endothelial cells and alleviate oxidative stress in cell culture models. Here, we studied the effects of these antioxidant conjugates in mouse models of vascular oxidative stress. Anti-PECAM/catalase and anti-PECAM/SOD conjugates, in contrast to control IgG/AOE conjugates, accumulated in the lungs and vascularized organs after intravenous injection in wild-type, but not PECAM KO mice. Anti-PECAM/catalase, but not anti-PECAM/SOD, protected mice from lung injury induced by H2O2 produced by glucose oxidase deposited in the pulmonary vasculature. Anti-PECAM/catalase also reduced alveolar edema and attenuated decline in arterial oxygen in mice that underwent unilateral lung ischemia/reperfusion, whereas anti-PECAM/SOD was not effective, implying the key role of H2O2 in tissue damage in this pathology. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase prevented oxidation of tetrahydrobiopterin and normalized vasoreactivity in the vessels of mice rendered hypertensive by pretreatment with angiotensin-II. This outcome agrees with reports implicating superoxide and peroxynitrite in altered endothelium-dependent vasodilatation in hypertension. Therefore, the use of endothelial cell-targeted antioxidants identifies the key specific species of ROS involved in various forms of vascular disease and holds promise for the mechanistically tailored treatment of these pathologies.