Superoxide Dismutases: Role in Redox Signaling, Vascular Function and Diseases

Excessive reactive oxygen species Revised abstract, especially superoxide anion (O2•−), play important roles in the pathogenesis of many cardiovascular diseases, including hypertension and atherosclerosis. Superoxide dismutases (SODs) are the major antioxidant defense systems against O2•−, which consist of three isoforms of SOD in mammals: the cytoplasmic Cu/ZnSOD (SOD1), the mitochondrial MnSOD (SOD2), and the extracellular Cu/ZnSOD (SOD3), all of which require catalytic metal (Cu or Mn) for their activation. Recent evidence suggests that in each subcellular location, SODs catalyze the conversion of O2•− H2O2, which may participate in cell signaling. In addition, SODs play a critical role in inhibiting oxidative inactivation of nitric oxide, thereby preventing peroxynitrite formation and endothelial and mitochondrial dysfunction. The importance of each SOD isoform is further illustrated by studies from the use of genetically altered mice and viral-mediated gene transfer. Given the essential role of SODs in cardiovascular disease, the concept of antioxidant therapies, that is, reinforcement of endogenous antioxidant defenses to more effectively protect against oxidative stress, is of substantial interest. However, the clinical evidence remains controversial. In this review, we will update the role of each SOD in vascular biologies, physiologies, and pathophysiologies such as atherosclerosis, hypertension, and angiogenesis. Because of the importance of metal cofactors in the activity of SODs, we will also discuss how each SOD obtains catalytic metal in the active sites. Finally, we will discuss the development of future SOD-dependent therapeutic strategies. Antioxid. Redox Signal. 15, 000–000

Hydrogen Peroxide Regulates Extracellular Superoxide Dismutase Activity and Expression in Neonatal Pulmonary Hypertension

We previously demonstrated that superoxide and H(2)O(2) promote pulmonary arterial vasoconstriction in a lamb model of persistent pulmonary hypertension of the newborn (PPHN). Because extracellular superoxide dismutase (ecSOD) augments vasodilation, we hypothesized that H(2)O(2)-mediated ecSOD inactivation contributes to pulmonary arterial vasoconstriction in PPHN lambs. ecSOD activity was decreased in pulmonary arterial smooth muscle cells (PASMCs) isolated from PPHN lambs relative to controls. Exposure to 95% O(2) to mimic hyperoxic ventilation reduced ecSOD activity in control PASMCs. In both cases, these events were associated with increased protein thiol oxidation, as detected by the redox sensor roGFP. Accordingly, exogenous H(2)O(2) decreased ecSOD activity in control PASMCs, and PEG-catalase restored ecSOD activity in PPHN PASMCs. In intact animal studies, ecSOD activity was decreased in fetal PPHN lambs, and in PPHN lambs ventilated with 100% O(2) relative to controls. In ventilated PPHN lambs, administration of a single dose of intratracheal PEG-catalase enhanced ecSOD activity, reduced superoxide levels, and improved oxygenation. We propose that H(2)O(2) generated by PPHN and hyperoxia inactivates ecSOD, and intratracheal catalase enhances enzyme function. The associated decrease in extracellular superoxide augments vasodilation, suggesting that H(2)O(2) scavengers may represent an effective therapy in the clinical management of PPHN

Critical Role of Endothelial Hydrogen Peroxide in Post-Ischemic Neovascularization

<div><p>Background</p><p>Reactive oxygen species (ROS) play an important role in angiogenesis in endothelial cells (ECs) <i>in vitro</i> and neovascularization <i>in vivo</i>. However, little is known about the role of endogenous vascular hydrogen peroxide (H₂O₂) in postnatal neovascularization.</p> <p>Methodology/Principal Findings</p><p>We used Tie2-driven endothelial specific catalase transgenic mice (Cat-Tg mice) and hindlimb ischemia model to address the role of endogenous H₂O₂ in ECs in post-ischemic neovascularization <i>in vivo</i>. Here we show that Cat-Tg mice exhibit significant reduction in intracellular H₂O₂ in ECs, blood flow recovery, capillary formation, collateral remodeling with larger extent of tissue damage after hindlimb ischemia, as compared to wild-type (WT) littermates. In the early stage of ischemia-induced angiogenesis, Cat-Tg mice show a morphologically disorganized microvasculature. Vascular sprouting and tube elongation are significantly impaired in isolated aorta from Cat-Tg mice. Furthermore, Cat-Tg mice show a decrease in myeloid cell recruitment after hindlimb ischemia. Mechanistically, Cat-Tg mice show significant decrease in eNOS phosphorylation at Ser1177 as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1) in ischemic muscles, which is required for inflammatory cell recruitment to the ischemic tissues. We also observed impaired endothelium-dependent relaxation in resistant vessels from Cat-Tg mice.</p> <p>Conclusions/Significance</p><p>Endogenous ECs-derived H₂O₂ plays a critical role in reparative neovascularization in response to ischemia by upregulating adhesion molecules and activating eNOS in ECs. Redox-regulation in ECs is a potential therapeutic strategy for angiogenesis-dependent cardiovascular diseases.</p> </div

Endothelial catalase overexpression blunts endothelium-dependent relaxation of resistant vessels.

<p><b>A</b>, the first branches mesenteric arteries were harvested from Wild-type (WT) and Tie2-driven catalase transgenic (Cat-Tg) mice and assessed for endothelium-dependent or – independent relaxation by acetylcholine or sodium nitroprusside, respectively (n = 4 per group and *p<0.05). Data shown are mean+SE. <b>B</b>, a proposed model for the role of endogenous H₂O₂ in endothelial cells during ischemia-induced neovascularization. Tissue ischemia induces endogenous reactive oxygen species production including hydrogen peroxide (H₂O₂) intracellularly and extracellularly for endothelial cells. Intracellular H₂O₂, which can be reduced by Tie2-driven catalase overexpression in this study, promote crucial neovascular signaling regulating endothelial sprouting and tube formation, endothelial nitric oxide synthase (eNOS) activation as well as the expression of vascular adhesion molecule (VCAM)-1 and monocyte chemoattractant protein (MCP)-1. Endothelial H₂O₂ could be involved in vascular progenitor mobilization. H₂O₂ is thought to be diffusible across cellular membrane (blue arrow). Myeloid recruitment, vascular endothelial growth factor (VEGF) and potential nitric oxide (NO) production are regulated by endogenous H₂O₂ in endothelial cells during neovascularization.</p

Endothelial catalase overexpression decreases the recruitment of F4/80+ myeloid cells to the ischemic tissue.

<p><b>A</b>, the ischemic area of gastrocnemius muscles from wild-type (WT) and Tie2-driven catalase transgenic (Cat-Tg) mice at day 7 was analyzed for myeloid cell recruitment with immunostaining for F4/80 (brown and arrows). The percentage of F4/80+ cell infiltrated area in the damaged region of gastrocnemius muscles is shown (n = 3 mice per group). <b>B</b>, adductor muscles in the upper limb were harvested at day 3 and analyzed for F4/80+ myeloid accumulation (brown and arrows) at the perivascular space of collateral arteries. Eosin staining was performed to show the structures. (n = 3 mice per group and *p<0.05). <b>C</b>, ischemic tibialis anterior muscles were harvested at day 3 and analyzed for mRNA expression of intercellular adhesion molecule 1 (<i>icam1</i>), vascular cell adhesion molecule 1 (<i>vcam1</i>) and monocyte chemotactic protein-1 (MCP-1 (<i>ccl2</i>)) by real-time polymerase chain reaction. Ribosomal 18S and <i>hprt</i> were used as internal controls. Relative expression for WT is shown (n = 3 mice per group). <b>D</b>, vascular endothelial growth factor (VEGF) expression was analyzed by Western blotting of protein lysate from ischemic tibialis anterior muscle at day 7. Alpha tubulin is shown as control. Densitometry analysis is shown (n = 3 mice per group). All data shown are mean+SE (*p<0.05).</p

Endothelial catalase overexpression impairs vessel sprouting and tube elongation in <i>ex vivo</i> aortic ring assay.

<p>Aortas were harvested from Wild-type (WT) and Tie2-driven catalase transgenic (Cat-Tg) mice and cultured in Matrigel for 7 days. Capillary sprouts and average tube length were measured in 5 rings from each aorta under microscopy (n = 3 mice per group and *p<0.05). Data shown are mean+SE.</p

Intracellular H₂O₂ in endothelial cells regulate endothelial nitric oxide synthase activation <i>in vivo</i>.

<p><b>A</b>, intracellular redox status was measured by 2′,7′-dichlorfluorescein-diacetate (DCF-DA) staining in gated CD31⁺/CD45⁻ population of collagenase-digested ischemic muscles at day 3. The dotted lines indicate the background signals without DCF-DA. <b>B</b>, ischemic muscles from Wild-type (WT) and Tie2-driven catalase transgenic (Cat-Tg) mice at day 3 were isolated and incubated. Their H₂O₂ production was measured by Amplex Ultra Red assay. <b>C</b>, harvested ischemic and non-ischemic muscles at day 3 were analyzed for protein expression of phosphorylated and total form of endothelial nitric oxide synthase (eNOS), Akt and ERK1/2 (as control) by Western analysis. Densitometry analysis in activation (phosphorylation) of each protein is shown. All data shown are mean+SE (n = 3 mice per group, *p<0.05, **p<0.01 and ***p<0.001).</p

Endothelial catalase overexpression impairs collateral remodeling and stabilization of vessels undergoing neovascularization.

<p><b>A</b>, collateral remodelling after hindlimb ischemia was analyzed in the same anatomical arteries localized at semimembranosus muscles in the upper limbs between wild-type (WT) and Tie2-driven catalase transgenic (Cat-Tg) mice. The luminal diameter and wall area are calculated from the measurements of luminal and perivascular tracing. Arrows indicate collateral wall. <b>B</b>, ischemic gastrocnemius muscles were analyzed for the morphology of vessels at day 3 with immunostaining for an endothelial-marker, CD31 (green). Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) (blue). Magnified images show that morphologically disorganized vessels with varying size, enlarged lumen and irregular shape are often seen in Cat-Tg mice. C, capillary densities in the same region as B were analyzed by CD31 staining at day 7. Bars indicate 20 μm in A, and 50 μm in B and C. All data shown are mean+SE (n = 3–4 mice per group, *p<0.05 and **p<0.01).</p

Tie2-driven catalase overexpression affects circulating progenitor cell level.

<p>The levels of white blood cells, monocytes and vascular progenitors (Sca1⁺/Flk1⁺ cells) were analyzed in the peripheral blood at indicated time points. Representative plots of vascular progenitors at day 2 are shown. All data shown are mean+SE (n = 4 mice per group and *p<0.05).</p

Nitroglycerin Tolerance in Caveolin-1 Deficient Mice

<div><p>Nitrate tolerance developed after persistent nitroglycerin (GTN) exposure limits its clinical utility. Previously, we have shown that the vasodilatory action of GTN is dependent on endothelial nitric oxide synthase (eNOS/NOS3) activity. Caveolin-1 (Cav-1) is known to interact with NOS3 on the cytoplasmic side of cholesterol-enriched plasma membrane microdomains (caveolae) and to inhibit NOS3 activity. Loss of Cav-1 expression results in NOS3 hyperactivation and uncoupling, converting NOS3 into a source of superoxide radicals, peroxynitrite, and oxidative stress. Therefore, we hypothesized that nitrate tolerance induced by persistent GTN treatment results from NOS3 dysfunction and vascular toxicity. Exposure to GTN for 48–72 h resulted in nitrosation and depletion (>50%) of Cav-1, NOS3 uncoupling as measured by an increase in peroxynitrite production (>100%), and endothelial toxicity in cultured cells. In the Cav-1 deficient mice, NOS3 dysfunction was accompanied by GTN tolerance (>50% dilation inhibition at low GTN concentrations). In conclusion, GTN tolerance results from Cav-1 modification and depletion by GTN that causes persistent NOS3 activation and uncoupling, preventing it from participating in GTN-medicated vasodilation.</p></div