[Show abstract][Hide abstract] ABSTRACT: Objective:
Comprehensive understanding of the mechanisms regulating angiogenesis might provide new strategies for angiogenic therapies for treating diverse physiological and pathological ischemic conditions. The ETS factor Ets variant 2 (ETV2; aka Ets-related protein 71) is essential for the formation of hematopoietic and vascular systems. Despite its indispensable function in vessel development, ETV2 role in adult angiogenesis has not yet been addressed. We have therefore investigated the role of ETV2 in vascular regeneration.
Approach and results:
We used endothelial Etv2 conditional knockout mice and ischemic injury models to assess the role of ETV2 in vascular regeneration. Although Etv2 expression was not detectable under steady-state conditions, its expression was readily observed in endothelial cells after injury. Mice lacking endothelial Etv2 displayed impaired neovascularization in response to eye injury, wounding, or hindlimb ischemic injury. Lentiviral Etv2 expression in ischemic hindlimbs led to improved recovery of blood flow with enhanced vessel formation. After injury, fetal liver kinase 1 (Flk1) expression and neovascularization were significantly upregulated by Etv2, whereas Flk1 expression and vascular endothelial growth factor response were significantly blunted in Etv2-deficient endothelial cells. Conversely, enforced Etv2 expression enhanced vascular endothelial growth factor-mediated endothelial sprouting from embryoid bodies. Lentiviral Flk1 expression rescued angiogenesis defects in endothelial Etv2 conditional knockout mice after hindlimb ischemic injury. Furthermore, Etv2(+/-); Flk1(+/-) double heterozygous mice displayed a more severe hindlimb ischemic injury response compared with Etv2(+/-) or Flk1(+/-) heterozygous mice, revealing an epistatic interaction between ETV2 and FLK1 in vascular regeneration.
Our study demonstrates a novel obligatory role for the ETV2 in postnatal vascular repair and regeneration.
[Show abstract][Hide abstract] ABSTRACT: Copper (Cu), an essential micronutrient, plays a fundamental role in inflammation and angiogenesis; however, its precise mechanism remains undefined. Here we uncover a novel role of Cu transport protein Antioxidant-1 (Atox1), which is originally appreciated as a Cu chaperone and recently discovered as a Cu-dependent transcription factor, in inflammatory neovascularization. Atox1 expression is upregulated in patients and mice with critical limb ischemia. Atox1-deficient mice show impaired limb perfusion recovery with reduced arteriogenesis, angiogenesis, and recruitment of inflammatory cells. In vivo intravital microscopy, bone marrow reconstitution, and Atox1 gene transfer in Atox1-/-mice show that Atox1 in endothelial cells (ECs) is essential for neovascularization and recruitment of inflammatory cells which release VEGF and TNFα. Mechanistically, Atox1-depleted ECs demonstrate that Cu chaperone function of Atox1 mediated through Cu transporter ATP7A is required for VEGF-induced angiogenesis via activation of Cu enzyme lysyl oxidase. Moreover, Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer p47phox, thereby increasing ROS-NFÎ° B-VCAM-1/ICAM-1 expression and monocyte adhesion in ECs inflamed with TNFα in an ATP7A-independent manner. These findings demonstrate a novel linkage between Atox1 and NADPH oxidase involved in inflammatory neovascularization and suggest Atox1 as a potential therapeutic target for treatment of ischemic disease.
[Show abstract][Hide abstract] ABSTRACT: Stem cells are defined as cells that have the capacity to self-renew and exhibit multipotency or pluripotency, whereas progenitor cells are committed to selected lineages but retain their self-renewal capacity. The stem or progenitor cell niche refers to the microenvironment of the regenerative cells in the bone marrow (BM) or other tissues such as the heart. It can regulate self-renewal, differentiation, migration and proliferation of regenerative stem/progenitor cells. The precise regulatory mechanisms by which the niche and the stem/progenitor cells interact are an active area of research. Reactive oxygen species (ROS) are one such niche regulatory mechanism. Quiescent stem cells in a hypoxic niche exhibit low ROS levels due to well-organized antioxidant defense systems which protect stem cells from extrinsic oxidative stress, whereas high levels of ROS promote the differentiation or migration of stem/progenitor cells. In pathophysiological conditions such as diabetes, BM niche dysfunction induced by oxidative stress contributes to reduction of the angiogenic and vasculogenic potential of BM-derived regenerative cells, thereby leading to a less efficient healing and revascularization. Cells have evolved mechanisms to fine-tune ROS levels by tightly regulated metabolic pathways such as glycolysis rather than oxidative phosphorylation to reduce oxidative stress. This FORUM will summarize the recent progress regarding the redox and metabolic regulation of hematopoietic and cardiac stem/progenitor cells as well as their niche interactions involved in tissue regeneration and repair under physiological and pathological conditions. Understanding such mechanisms will contribute to the development of novel therapeutic strategies to enhance regeneration and repair of diseased tissues.
[Show abstract][Hide abstract] ABSTRACT: Although trafficking of newly synthetized VEGFR2 to the plasma membrane is a key determinant of angiogenesis, the molecular mechanisms of Golgi to plasma membrane trafficking are unknown. Here we identified the key role of the kinesin family plus-end molecular motor KIF13B in delivering VEGFR2 cargo from Golgi to the endothelial cell surface. KIF13B was shown to interact directly with VEGFR2 on microtubules. We also observed that over-expression of the KIF13B binding domain interacting with VEGFR2 inhibited VEGF-induced capillary tube formation. KIF13B depletion prevented VEGF-mediated endothelial migration, capillary tube formation, and neo-vascularization in mice. Impairment in trafficking induced by knockdown of KIF13B shunted VEGFR2 towards the lysosomal degradation pathway. Thus, KIF13B is an essential molecular motor required for the trafficking of VEGFR2 from the Golgi and its delivery to the endothelial cell surface mediates angiogenesis.
[Show abstract][Hide abstract] ABSTRACT: Endothelial cell (EC) dedifferentiation in relation to neovascularization is a poorly understood process. In this report we addressed the role of Wnt signaling in the mechanisms of neovascularization in adult tissues. Here, we show that a low-dose of 6-bromoindirubin-3'-oxime (BIO), a competitive inhibitor of Glycogen Synthase Kinase (GSK)-3β induced the stabilization of β-catenin and its subsequent direct interaction with the transcription factor NANOG in the nucleus of ECs. This event induced loss of VE-cadherin from the adherens junctions, increased EC proliferation accompanied by asymmetric cell division (ACD), and formed cellular aggregates in a hanging drop assays indicating the acquisition of a dedifferentiated state. In a chromatin immunoprecipitation assay, nuclear NANOG protein bound to the NANOG- and VEGFR2-promoters in ECs, and the addition of BIO activated the NANOG-promoter-luciferase reporter system in a cell-based assay. Consequently, NANOG-knockdown decreased BIO-induced NOTCH-1 expression, thereby decreasing cell proliferation, ACD and neovascularization. In a Matrigel plug assay, BIO induced increased neovascularization, secondary to the presence of VEGF. Moreover, in a mouse model of hind limb ischemia, BIO augmented neovascularization that was coupled with increased expression of NOTCH-1 in ECs and increased smooth muscle α-actin (SMA)(+) cell recruitment around the neovessels. Thus, these results show the ability of a low-dose of BIO to augment neovascularization secondary to VEGF, a process that was accompanied by a partial dedifferentiation of ECs via β-catenin and the NANOG signaling pathway. Stem Cells 2014.
[Show abstract][Hide abstract] ABSTRACT: P-21 activated kinases, or PAKs, are serine-threonine kinases that serve a role in diverse biological functions and organ system diseases. Although PAK signaling has been the focus of many investigations, still our understanding of the role of PAK in inflammation is incomplete. This review consolidates what is known about PAK1 across several cell types, highlighting the role of PAK1 and PAK2 in inflammation in relation to NADPH oxidase activation. This review explores the physiological functions of PAK during inflammation, the role of PAK in several organ diseases with an emphasis on cardiovascular disease, and the PAK signaling pathway, including activators and targets of PAK. Also, we discuss PAK1 as a pharmacological anti-inflammatory target, explore the potentials and the limitations of the current pharmacological tools to regulate PAK1 activity during inflammation, and provide indications for future research. We conclude that a vast amount of evidence supports the idea that PAK is a central molecule in inflammatory signaling, thus making PAK1 itself a promising prospective pharmacological target.
[Show abstract][Hide abstract] ABSTRACT: Induced pluripotent stem (iPS) cells have emerged as a source of potentially unlimited supply of autologous endothelial cells (ECs) for vascularization. However, the regenerative function of these cells relative to adult ECs and ECs derived from embryonic stem (ES) cells is unknown. The objective was to define the differentiation characteristics and vascularization potential of Fetal liver kinase (Flk)1(+) and Vascular Endothelial (VE)-cadherin(+) ECs derived identically from mouse (m)ES and miPS cells.
Naive mES and miPS cells cultured in type IV collagen (IV Col) in defined media for 5 days induced the formation of adherent cell populations, which demonstrated similar expression of Flk1 and VE-cadherin and the emergence of EC progenies. FACS purification resulted in 100% Flk1(+) VE-cadherin(+) cells from both mES and miPS cells. Emergence of Flk1(+)VE-cadherin(+) cells entailed expression of the vascular developmental transcription factor Er71, which bound identically to Flk1, VE-cadherin, and CD31 promoters in both populations. Immunostaining with anti-VE-cadherin and anti-CD31 antibodies and microscopy demonstrated the endothelial nature of these cells. Each cell population (unlike mature ECs) organized into well-developed vascular structures in vitro and incorporated into CD31(+) neovessels in matrigel plugs implanted in nude mice in vivo.
Thus, iPS cell-derived Flk1(+)VE-cadherin(+) cells expressing the Er71 are as angiogenic as mES cell-derived cells and incorporate into CD31(+) neovessels. Their vessel forming capacity highlights the potential of autologous iPS cells-derived EC progeny for therapeutic angiogenesis.
PLoS ONE 12/2013; 8(12):e85549. DOI:10.1371/journal.pone.0085549 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Platelet-derived growth factor (PDGF) stimulates vascular smooth muscle cell (VSMC) migration and neointimal formation in response to injury. We previously identified IQ-domain GTPase-activating protein 1 (IQGAP1) as a novel VEGF receptor2 binding scaffold protein involved in endothelial migration. However, its role in VSMC migration and neointimal formation in vivo is unknown. Here we show that PDGF stimulation rapidly promotes IQGAP1 association with PDGF receptor β (PDGFR) as well as IQGAP1 tyrosine phosphorylation in cultured VSMC. Overexpression or knockdown of IQGAP1 enhances or inhibits PDGFR autophosphorylation (p-PDGFR), respectively. Immunofluorescence and cell fractionation analysis reveals that PDGF-induced p-PDGFR localized in focal adhesions (FAs), but not caveolae/lipid rafts, is inhibited by IQGAP1 knockdown with siRNA. PDGF stimulation promotes IQGAP1 association with PDGFR/FAs signaling proteins complex. Functionally, IQGAP1 siRNA inhibits PDGF-induced FAs formation as well as VSMC migration induced by PDGF. In vivo, IQGAP1 expression is markedly increased at neointimal VSMC in wire-injured femoral arteries. Mice lacking IQGAP1 exhibit impaired neointimal formation in response to vascular injury. In summary, IQGAP1, through interaction with PDGFR and FAs signaling proteins, promotes activation of PDGFR in FAs as well as FA formation, which may contribute to VSMC migration and neointimal formation after injury. Our findings provide insight into IQGAP1 as a potential therapeutic target for vascular migration-related diseases.
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress and endothelial dysfunction contribute to vascular complication in diabetes mellitus (DM). Extracellular superoxide dismutase (ecSOD, SOD3) is one of the key antioxidant enzymes which obtains copper via copper transporter ATP7A. SOD3 is secreted from vascular smooth muscles cells (VSMCs) and anchors at endothelial surface. Role of SOD3 and ATP7A in endothelial dysfunction in type 1 DM is entirely unknown. Here we show that the specific activity of SOD3, but not SOD1, is decreased, which is associated with increased O2(•-) production in aortas of streptozotocin-induced and genetically-induced Ins2(Akita) type 1 DM mice. Exogenous copper partially rescued SOD3 activity in isolated DM vessels. Functionally, acetylcholine-induced endothelium-dependent relaxation is impaired in DM mesenteric arteries, which is rescued by SOD mimetic tempol or gene transfer of SOD3. Mechanistically, ATP7A expression in DM vessels is dramatically decreased while other copper transport proteins are not altered. DM-induced endothelial dysfunction and decrease of SOD3 activity are rescued in transgenic mice overexpressing ATP7A. Furthermore, SOD3 deficient DM mice or ATP7A mutant DM mice augment endothelial dysfunction and vascular O2(•-) production vs. DM mice. These effects are in part due to hypoinsulinemia in type1-DM mice, since insulin treatment, but not high glucose, increases ATP7A expression in VSMCs and restores SOD3 activity in the organoid culture of DM vessels. In summary, decrease in ATP7A protein expression contributes to impaired SOD3 activity, resulting in O2(•-) overproduction and endothelial dysfunction in blood vessels of type1 DM. Thus, restoring copper transporter function is an essential therapeutic approach for oxidant stress-dependent vascular and metabolic diseases.
[Show abstract][Hide abstract] ABSTRACT: Protein disulfide isomerase (PDI) derived from intravascular cells is required for thrombus formation. However, it remains unclear whether platelet PDI contributes to the process. Using platelet-specific PDI-deficient mice, we demonstrate that PDI-null platelets have defects in aggregation and ATP secretion induced by thrombin, collagen, and ADP. Such defects were rescued by exogenously-added wild-type but not mutant PDI, indicating that the isomerase activity of platelet surface PDI is critical for the regulatory effect. PDI-deficient platelets expressed increased levels of intracellular ERp57 and ERp72. Platelet PDI regulated αIIbβ3 integrin activation but not P-selectin exposure, Ca(2+) mobilization, β3-talin interaction, and platelet spreading on immobilized fibrinogen. Inhibition of ERp57 further diminished αIIbβ3 integrin activation, aggregation and ATP secretion of activated PDI-deficient platelets, suggesting distinct roles of PDI and ERp57 in platelet functions. We found that platelet PDI is important for thrombus formation on collagen-coated surfaces under arteriolar shear. Intravital microscopy demonstrates that platelet PDI is important for platelet accumulation but not initial adhesion and fibrin generation following laser-induced arteriolar injury. Tail bleeding time and blood loss in platelet-specific PDI-deficient mice were not significantly increased. Our results provide important evidence that platelet PDI is essential for thrombus formation but not for hemostasis in mice.
[Show abstract][Hide abstract] ABSTRACT: Background
Reactive oxygen species (ROS) play an important role in angiogenesis in endothelial cells (ECs) in vitro and neovascularization in vivo. However, little is known about the role of endogenous vascular hydrogen peroxide (H2O2) in postnatal neovascularization.
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 in vivo. 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.
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.
PLoS ONE 03/2013; 8(3):e57618. DOI:10.1371/journal.pone.0057618 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective:
Vascular smooth muscle cell (VSMC) migration is critically important for neointimal formation after vascular injury and atherosclerosis lesion formation. Copper (Cu) chelator inhibits neointimal formation, and we previously demonstrated that Cu transport protein antioxidant-1 (Atox1) is involved in Cu-induced cell growth. However, role of Atox1 in VSMC migration and neointimal formation after vascular injury is unknown.
Approach and results:
Here, we show that Atox1 expression is upregulated in injured vessel, and it is colocalized with the Cu transporter ATP7A, one of the downstream targets of Atox1, mainly in neointimal VSMCs at day 14 after wire injury. Atox1(-/-) mice show inhibition of neointimal formation and extracellular matrix expansion, which is associated with a decreased VSMCs accumulation within neointima and lysyl oxidase activity. Mechanistically, in cultured VSMC, Atox1 depletion with siRNA inhibits platelet-derived growth factor-induced Cu-dependent VSMC migration by preventing translocation of ATP7A and small G protein Rac1 to the leading edge, as well as Cu- and Rac1-dependent lamellipodia formation. Furthermore, Atox1(-/-) mice show decreased perivascular macrophage infiltration in wire-injured vessels, as well as thioglycollate-induced peritoneal macrophage recruitment.
Atox1 is involved in neointimal formation after vascular injury through promoting VSMC migration and inflammatory cell recruitment in injured vessels. Thus, Atox1 is a potential therapeutic target for VSMC migration and inflammation-related vascular diseases.
[Show abstract][Hide abstract] ABSTRACT: PDGF stimulates vascular smooth muscle cell (VSMC) migration and neointimal formation in response to injury primary through the PDGF receptor β (PDGFR). However, underlying molecular mechanisms are poorly understood. We previously identified IQGAP1 as a novel VEGFR-2 and Rac1 binding scaffold protein expressed in endothelial cell. However, its function in VSMC is unknown. Here we show that PDGF stimulation rapidly promotes IQGAP1 association with PDGFR within 5 min (2.1-fold) in cultured VSMC. Functional significance of IQGAP1 binding to PDGFR is demonstrated by showing that overexpression of IQGAP1 using adenovirus enhances PDGF-induced PDGFR autophosphorylation (1.6-fold), while IQGAP1 knockdown by siRNA inhibits this response (69%). Immunofluorescence and subcellular fractionation analysis reveals that p-PDGFR is found at both focal adhesions (FAs) and caveolae/lipid rafts (C/LR), but p-PDGFR at FAs, but not at C/LR, is inhibited by IQGAP1 siRNA. PDGF stimulation promotes recruitment of Rac1 (1.9-fold) and FAs signaling proteins such as paxillin, vinculin, and FAK (1.7-, 2.7-, 1.6-fold) to the IQGAP1/PDGFR complex. Functionally, IQGAP1 siRNA inhibits PDGF-induced Rac1 translocation and FAs formation at the leading edge as well as VSMC migration induced by PDGF (70%) or wound scratch (73%). Overexpression of IQGAP1 mutant lacking GRD domain, which blocks PDGF-induced Rac1 translocation, also inhibits FAs formation without affecting PDGFR autophosphorylation, thereby inhibiting VSMC migration, suggesting that IQGAP1/Rac1-dependent FA formation is involved in VSMC migration. In vivo, IQGAP1 expression is markedly increased at neointimal VSMC after wire-induced femoral artery injury in wild type (WT) mice. Neointimal formation at day 21 after vascular injury is significantly inhibited in mice lacking IQGAP1 (I/M ratio: 57%) as compared to WT. In summary, IQGAP1, through interaction with PDGFR, Rac1, and components of FAs, promotes activation of PDGFR in FAs as well as Rac1 translocation and FA formation, which may contribute to neointimal formation after vascular injury. Our results will provide new insight into IQGAP1 as a potential therapeutic target for the crucially important events in VSMC migration-related vascular diseases.
[Show abstract][Hide abstract] ABSTRACT: Bone marrow (BM)-derived stem and progenitor cell functions including self-renewal, differentiation, survival, migration, proliferation and mobilization are regulated by unique cell-intrinsic signals and -extrinsic signals provided by their microenvironment, also termed the 'niche'. Reactive oxygen species (ROS), especially hydrogen peroxide (H(2)O(2)), play important roles in regulating stem and progenitor cell function in various physiologic and pathologic responses. The low level of H(2)O(2) in quiescent hematopoietic stem cells (HSCs) contributes to maintain their stemness, whereas a higher level of H(2)O(2) within HSCs or their niche promotes differentiation, proliferation, migration, and survival of HSCs or stem/progenitor cells. Major sources of ROS are NADPH oxidase and mitochondria. In response to ischemic injury, ROS derived from NADPH oxidase are increased in the BM microenvironment, which is required for hypoxia and HIF1α expression and expansion throughout the BM. This, in turn, promotes progenitor cell expansion and mobilization from BM, leading to reparative neovascularization and tissue repair. In pathophysiological states such as aging, atherosclerosis, heart failure, hypertension and diabetes, excess amounts of ROS create an inflammatory and oxidative microenvironment, which induces cell damage and apoptosis of stem and progenitor cells. Understanding the molecular mechanisms of how ROS regulate the functions of stem and progenitor cells and their niche in physiological and pathological conditions will lead to the development of novel therapeutic strategies.
Free Radical Biology and Medicine 10/2012; 54. DOI:10.1016/j.freeradbiomed.2012.10.532 · 5.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The 5-hydroxytryptamine type 4 receptor (5-HT(4)R) regulates many physiological processes, including learning and memory, cognition, and gastrointestinal motility. Little is known about its role in angiogenesis. Using mouse hindlimb ischemia model of angiogenesis, we observed a significant reduction of limb blood flow recovery 14 days after ischemia and a decrease in density of CD31-positive vessels in adductor muscles in 5-HT(4)R(-/-) mice compared to wild type littermates. Our in vitro data indicated that 5-HT(4)R endogenously expressed in endothelial cells (ECs) may promote angiogenesis. Inhibition of the receptor with 5-HT(4)R antagonist RS 39604 reduced EC capillary tube formation in the reconstituted basement membrane. Using Boyden chamber migration assay and wound healing "scratch" assay, we demonstrated that RS 39604 treatment significantly suppressed EC migration. Transendothelial resistance measurement and immunofluorescence analysis showed that a 5-HT(4)R agonist RS 67333 led to an increase in endothelial permeability, actin stress fiber and interendothelial gap formation. Importantly, we provided the evidence that 5-HT(4)R-regulated EC migration may be mediated by Gα13 and RhoA. Our results suggest a prominent role of 5-HT(4)R in promoting angiogenesis and identify 5-HT(4)R as a potential therapeutic target for modulating angiogenesis under pathological conditions.
[Show abstract][Hide abstract] ABSTRACT: Extracellular superoxide dismutase (SOD3) is a secretory copper enzyme involved in protecting angiotensin II (Ang II)-induced hypertension. We found previously that Ang II upregulates SOD3 expression and activity as a counterregulatory mechanism; however, underlying mechanisms are unclear. Antioxidant 1 (Atox1) is shown to act as a copper-dependent transcription factor, as well as a copper chaperone, for SOD3 in vitro, but its role in Ang II-induced hypertension in vivo is unknown. Here we show that Ang II infusion increases Atox1 expression, as well as SOD3 expression and activity, in aortas of wild-type mice, which are inhibited in mice lacking Atox1. Accordingly, Ang II increases vascular superoxide production, reduces endothelium-dependent vasodilation, and increases vasoconstriction in mesenteric arteries to a greater extent in Atox1(-/-) than in wild-type mice. This contributes to augmented hypertensive response to Ang II in Atox1(-/-) mice. In cultured vascular smooth muscle cells, Ang II promotes translocation of Atox1 to the nucleus, thereby increasing SOD3 transcription by binding to Atox1-responsive element in the SOD3 promoter. Furthermore, Ang II increases Atox1 binding to the copper exporter ATP7A, which obtains copper from Atox1, as well as translocation of ATP7A to plasma membranes, where it colocalizes with SOD3. As its consequence, Ang II decreases vascular copper levels, which is inhibited in Atox1(-/-) mice. In summary, Atox1 functions to prevent Ang II-induced endothelial dysfunction and hypercontraction in resistant vessels, as well as hypertension, in vivo by reducing extracellular superoxide levels via increasing vascular SOD3 expression and activity.