Localized cysteine sulfenic acid formation by vascular endothelial growth factor: role in endothelial cell migration and angiogenesis

Abstract

Reactive oxygen species (ROS) are important mediators for VEGF receptor 2 (VEGFR2) signalling involved in angiogenesis. The initial product of Cys oxidation, cysteine sulfenic acid (Cys-OH), is a key intermediate in redox signal transduction; however, its role in VEGF signalling is unknown. We have previously demonstrated IQGAP1 as a VEGFR2 binding scaffold protein involved in ROS-dependent EC migration and post-ischemic angiogenesis. Using a biotin-labelled Cys-OH trapping reagent, we show that VEGF increases protein-Cys-OH formation at the lamellipodial leading edge where it co-localizes with NADPH oxidase and IQGAP1 in migrating ECs, which is prevented by IQGAP1 siRNA or trapping of Cys-OH with dimedone. VEGF increases IQGAP1-Cys-OH formation, which is prevented by N-acetyl cysteine or dimedone, which inhibits VEGF-induced EC migration and capillary network formation. In vivo, hindlimb ischemia in mice increases Cys-OH formation in small vessels and IQGAP1 in ischemic tissues. In summary, VEGF stimulates localized formation of Cys-OH-IQGAP1 at the leading edge, thereby promoting directional EC migration, which may contribute to post-natal angiogenesis in vivo. Thus, targeting Cys-oxidized proteins at specific compartments may be the potential therapeutic strategy for various angiogenesis-dependent diseases.     

Tetraspanin CD63 Promotes Vascular Endothelial Growth Factor Receptor 2-β1 Integrin Complex Formation,Thereby Regulating Activation and Downstream Signaling in Endothelial Cells in Vitro and in Vivo

CD63 is a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) implicated in the regulation of membrane protein trafficking, leukocyte recruitment, and adhesion processes. We have investigated the involvement of CD63 in endothelial cell (EC) signaling downstream of β1 integrin and VEGF. We report that silencing of CD63 in primary ECs arrested capillary sprouting and tube formation in vitro because of impaired adhesion and migration of ECs. Mechanistically, CD63 associated with both β1 integrin and the main VEGF receptor on ECs, VEGFR2. Our data suggest that CD63 serves to bridge between β1 integrin and VEGFR2 because CD63 silencing disrupted VEGFR2-β1 integrin complex formation identified using proximity ligation assays. Signaling downstream of β1 integrin and VEGFR2 was attenuated in CD63-silenced cells, although their cell surface expression levels remained unaffected. CD63 was furthermore required for efficient internalization of VEGFR2 in response to VEGF. Importantly, systemic delivery of VEGF failed to potently induce VEGFR2 phosphorylation and downstream signaling in CD63-deficient mouse lungs. Taken together, our findings demonstrate a previously unrecognized role for CD63 in coordinated integrin and receptor tyrosine kinase signaling in vitro and in vivo.     

Essential Role for Endocytosis in the Growth Factor-stimulated Activation of ERK1/2 in Endothelial Cells

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to VEGF receptor 2 (VEGFR2) on endothelial cells (ECs). Downstream activation of the extracellular related kinases 1/2 (ERK1/2) is important for angiogenesis to proceed. Receptor internalization has been implicated in VEGFR2 signaling, but its role in the activation of ERK1/2 is unclear. To explore this question we utilized pitstop and dynasore, two small molecule inhibitors of endocytosis. First, we confirmed that both inhibitors block the internalization of VEGFR2 in ECs. We then stimulated ECs with VEGF in the presence and absence of the inhibitors and examined VEGFR2 signaling to ERK1/2. Activation of VEGFR2 and C-Raf still occurred in the presence of the inhibitors, whereas the activation of MEK1/2 and ERK1/2 was abrogated. Therefore, although internalization is not required for activation of either VEGFR2 or C-Raf in ECs stimulated with VEGF, internalization is necessary to activate the more distal kinases in the cascade. Importantly, inhibition of internalization also prevented activation of ERK1/2 when ECs were stimulated with other pro-angiogenic growth factors, namely fibroblast growth factor 2 and hepatocyte growth factor. In contrast, the same inhibitors did not block ERK1/2 activation in fibroblasts or cancer cells stimulated with growth factors. Finally, we show that these small molecule inhibitors of endocytosis block angiogenesis in vitro and in vivo. Therefore, receptor internalization may be a generic requirement for pro-angiogenic growth factors to activate ERK1/2 signaling in human ECs, and targeting receptor trafficking may present a therapeutic opportunity to block tumor angiogenesis.     

Adipose stromal cell and sarpogrelate orchestrate the recovery of inflammation‐induced angiogenesis in aged hindlimb ischemic mice

Aging population displays a much higher risk of peripheral arterial disease (PAD) possibly due to the higher susceptibility, poor prognosis, and fewer therapeutic options. This study was designed to examine the impact of combined multipotent adipose‐derived stromal cells (mADSCs) and sarpogrelate treatment on aging hindlimb ischemia and the mechanism of action involved. mADSCs (1.0 × 10⁷) constitutively expressing enhanced green fluorescent protein (eGFP) or firefly luciferase (Fluc) reporter were engrafted into the hindlimb of aged Vegfr2‐luc transgenic or FVB/N mice subjected to unilateral femoral artery occlusion, followed by a further administration of sarpogrelate. Multimodality molecular imaging was employed to noninvasively evaluate mADSCs' survival and therapeutic efficacy against aging hindlimb ischemia. Aged Tg(Vegfr2‐luc) mice exhibited decreased inflammatory response, and downregulation of vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor‐2 (VEGFR2) compared with young ones following hindlimb ischemia induction, resulting in angiogenesis insufficiency and decompensation for ischemia recovery. Engrafted mADSCs augmented inflammation‐induced angiogenesis to yield pro‐angiogenic/anti‐apoptotic effects partly via the VEGF/VEGFR2/mTOR/STAT3 pathway. Nonetheless, mADSCs displayed limited survival and efficacy following transplantation. Sarpogrelate treatment with mADSCs further upregulated mammalian target of rapamycin (mTOR)/STAT3 signal and modulated pro‐/anti‐inflammatory markers including IL‐1β/TNF‐α/IFN‐γ and IL‐6/IL‐10, which ultimately facilitated mADSCs' survival and therapeutic benefit in vivo. Sarpogrelate prevented mADSCs from hypoxia/reoxygenation‐induced cell death via a mTOR/STAT3‐dependent pathway in vitro. This study demonstrated a role of in vivo kinetics of VEGFR2 as a biomarker to evaluate cell‐derived therapeutic angiogenesis in aging. mADSCs and sarpogrelate synergistically restored impaired angiogenesis and inflammation modulatory capacity in aged hindlimb ischemic mice, indicating its therapeutic promise for PAD in the elderly.     

<Emphasis Type="Italic">In vivo</Emphasis> monitoring of fetoplacental Vegfr2 gene activity in a murine pregnancy model using a Vegfr2-luc reporter gene and bioluminescent imaging

Background  

Vascular endothelial growth factor receptor-2 (VEGFR2) plays a pivotal role in angiogenesis by eliciting vascular endothelial cell growth when bound to VEGF, a powerful pro-angiogenic ligand. While Vegf and Vegfr2 are expressed throughout gestation, the latter third of gestation in mice is characterized by a marked increase in fetoplacental angiogenesis. Thus, the objective of this study was to determine the feasibility of monitoring fetoplacental Vegfr2 gene activity non-invasively using a Vegfr2-luc reporter transgenic mouse and bioluminescent imaging.     

Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cells

p66Shc, a longevity adaptor protein, is demonstrated as a key regulator of reactive oxygen species (ROS) metabolism involved in aging and cardiovascular diseases. Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that ROS derived from Rac1-dependent NADPH oxidase are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. However, a role of p66Shc in VEGF signaling and physiological responses in ECs is unknown. Here we show that VEGF promotes p66Shc phosphorylation at Ser36 through the JNK/ERK or PKC pathway as well as Rac1 binding to a nonphosphorylated form of p66Shc in ECs. Depletion of endogenous p66Shc with short interfering RNA inhibits VEGF-induced Rac1 activity and ROS production. Fractionation of caveolin-enriched lipid raft demonstrates that p66Shc plays a critical role in VEGFR2 phosphorylation in caveolae/lipid rafts as well as downstream p38MAP kinase activation. This in turn stimulates VEGF-induced EC migration, proliferation, and capillary-like tube formation. These studies uncover a novel role of p66Shc as a positive regulator for ROS-dependent VEGFR2 signaling linked to angiogenesis in ECs and suggest p66Shc as a potential therapeutic target for various angiogenesis-dependent diseases.     

Anti-Cancer Activity of an Osthole Derivative,NBM-T-BMX-OS01: Targeting Vascular Endothelial Growth Factor Receptor Signaling and Angiogenesis

Angiogenesis occurs during tissue growth, development and wound healing. It is also required for tumor progression and represents a rational target for therapeutic intervention. NBM-T-BMX-OS01 (BMX), derived from the semisynthesis of osthole, an active ingredient isolated from Chinese herb Cnidium monnieri (L.) Cuss., was recently shown to enhance learning and memory in rats. In this study, we characterized the anti-angiogenic activities of NBM-T-BMX-OS01 (BMX) in an effort to develop novel inhibitors to suppress angiogenesis and tumor growth. BMX inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and endothelial tube formation in human umbilical endothelial cells (HUVECs). BMX also attenuated VEGF-induced microvessel sprouting from aortic rings ex vivo and reduced HCT116 colorectal cancer cells-induced angiogenesis in vivo. Moreover, BMX inhibited the phosphorylation of VEGFR2, FAK, Akt and ERK in HUVECs exposed to VEGF. BMX was also shown to inhibit HCT116 cell proliferation and to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. Taken together, this study provides evidence that BMX modulates vascular endothelial cell remodeling and leads to the inhibition of tumor angiogenesis. These results also support the role of BMX as a potential drug candidate and warrant the clinical development in the treatment of cancer.     

Localized cysteine sulfenic acid formation by vascular endothelial growth factor: role in endothelial cell migration and angiogenesis

Reactive oxygen species (ROS) are important mediators for VEGF receptor 2 (VEGFR2) signalling involved in angiogenesis. The initial product of Cys oxidation, cysteine sulfenic acid (Cys-OH), is a key intermediate in redox signal transduction; however, its role in VEGF signalling is unknown. We have previously demonstrated IQGAP1 as a VEGFR2 binding scaffold protein involved in ROS-dependent EC migration and post-ischemic angiogenesis. Using a biotin-labelled Cys-OH trapping reagent, we show that VEGF increases protein-Cys-OH formation at the lamellipodial leading edge where it co-localizes with NADPH oxidase and IQGAP1 in migrating ECs, which is prevented by IQGAP1 siRNA or trapping of Cys-OH with dimedone. VEGF increases IQGAP1-Cys-OH formation, which is prevented by N-acetyl cysteine or dimedone, which inhibits VEGF-induced EC migration and capillary network formation. In vivo, hindlimb ischemia in mice increases Cys-OH formation in small vessels and IQGAP1 in ischemic tissues. In summary, VEGF stimulates localized formation of Cys-OH-IQGAP1 at the leading edge, thereby promoting directional EC migration, which may contribute to post-natal angiogenesis in vivo. Thus, targeting Cys-oxidized proteins at specific compartments may be the potential therapeutic strategy for various angiogenesis-dependent diseases.     

MICAL2 is expressed in cancer associated neo-angiogenic capillary endothelia and it is required for endothelial cell viability,motility and VEGF response

The capacity of inducing angiogenesis is a recognized hallmark of cancer cells. The cancer microenvironment, characterized by hypoxia and inflammatory signals, promotes proliferation, migration and activation of quiescent endothelial cells (EC) from surrounding vascular network. Current anti-angiogenic drugs present side effects, temporary efficacy, and issues of primary resistance, thereby calling for the identification of new therapeutic targets.MICALs are a unique family of redox enzymes that destabilize F-actin in cytoskeletal dynamics. MICAL2 mediates Semaphorin3A-NRP2 response to VEGFR1 in rat ECs. MICAL2 also enters the p130Cas interactome in response to VEGF in HUVEC. Previously, we showed that MICAL2 is overexpressed in metastatic cancer. A small-molecule inhibitor of MICAL2 exists (CCG-1423).Here we report that 1) MICAL2 is expressed in neo-angiogenic ECs in human solid tumors (kidney and breast carcinoma, glioblastoma and cardiac myxoma, n = 67, were analyzed with immunohistochemistry) and in animal models of ischemia/inflammation neo-angiogenesis, but not in normal capillary bed; 2) MICAL2 protein pharmacological inhibition (CCG-1423) or gene KD reduce EC viability and functional performance; 3) MICAL2 KD disables ECs response to VEGF in vitro. Whole-genome gene expression profiling reveals MICAL2 involvement in angiogenesis and vascular development pathways.Based on these results, we propose that MICAL2 expression in ECs participates to inflammation-induced neo-angiogenesis and that MICAL2 inhibition should be tested in cancer- and noncancer-associated neo-angiogenesis, where chronic inflammation represents a relevant pathophysiological mechanism.     

Heterotypic contact reveals a COX-2-mediated suppression of osteoblast differentiation by endothelial cells: A negative modulatory role for prostanoids in VEGF-mediated cell: cell communication?

In bone, angiogenesis must be initiated appropriately, but limited once remodelling or repair is complete. Our recent findings have supported a role for prostaglandins (PG), known modulators of osteoblast (OB) and endothelial cell (EC) behaviour, in facilitating VEGF-mediated paracrine communication from OBs to ‘remotely located’ ECs, but the mechanism(s) regulating OB:EC crosstalk when these cells are closely opposed are undefined. In this study we have examined: (i) the effects of exogenous PGE₂ on VEGF-driven events in ECs, and (ii) the role of endogenous COX-2-derived prostanoids in mediating communication between intimately opposed OBs and ECs in direct contact. Exposure of ECs to PGE₂ increased ERK1/2 phosphorylation, COX-2 induction, 6-keto-PGF_1α release and EC proliferation. In contrast, PGE₂ attenuated VEGF₁₆₅-induced VEGFR2/Flk1 phosphorylation, ERK1/2 activation and proliferation of ECs, suggesting that exogenous PGE₂ restricts the actions of VEGF. However, the COX-2-selective inhibitor, NS398, also attenuated VEGF-induced proliferation, implying a distinct role for endogenous COX-2 activity in regulating EC behaviour. To examine the effect of OB:EC proximity and the role of COX-2 products further, we used a confrontational co-culture model. These studies showed that COX-2 blockade with NS398 enhanced EC-dependent increases in OB differentiation, that this effect was reversed by exogenous PGH₂ (immediate COX-2 product), and that exogenous VEGF did not influence EC-dependent OB differentiation under these conditions. Our findings indicate that locally produced prostanoids may serve distinct roles depending on OB:EC proximity and negatively modulate VEGF-mediated changes in EC behaviour when these cells are closely opposed to control angiogenesis during bone (re)modelling.     

Molecular MRI assessment of vascular endothelial growth factor receptor‐2 in rat C6 gliomas

Angiogenesis is essential to tumour progression and a precise evaluation of angiogenesis is important for tumour early diagnosis and treatment. The quantitative and dynamic in vivo assessment of tumour angiogenesis can be achieved by molecular magnetic resonance imaging (mMRI). Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are the main regulatory systems in angiogenesis and have been used as hot targets for radionuclide‐based molecular imaging. However, little research has been accomplished in targeting VEGF/VEGFRs by mMRI. In our study, we aimed to assess the expression of VEGFR2 in C6 gliomas by using a specific molecular probe with mMRI. The differential uptake of the probe conjugated to anti‐VEGFR2 monoclonal antibody, shown by varied increases in T₁ signal intensity during a 2 hr period, demonstrated the heterogeneous expression of VEGFR2 in different tumour regions. Microscopic fluorescence imaging, obtained for the biotin group in the probe with streptavidin‐Cy3, along with staining for cellular VEGFR2 levels, laminin and CD45, confirmed the differential distribution of the probe which targeted VEGFR2 on endothelial cells. The angiogenesis process was also assessed using magnetic resonance angiography, which quantified tumour blood volume and provided a macroscopic view and a dynamic change of the correlation between tumour vasculature and VEGFR2 expression. Together these results suggest mMRI can be very useful in assessing and characterizing the expression of specific angiogenic markers in vivo and help evaluate angiogenesis associated with tumour progression.     

Vascular endothelial growth factor receptor-2 expression is down-regulated by 17beta-estradiol in MCF-7 breast cancer cells by estrogen receptor alpha/Sp proteins

17beta-Estradiol (E2) induces and represses gene expression in breast cancer cells; however, the mechanisms of gene repression are not well understood. In this study, we show that E2 decreases vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels in MCF-7 cells, and this gene was used as a model for investigating pathways associated with E2-dependent gene repression. Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich motifs at -58 and -44 are critical for the E2-dependent decreased response in MCF-7 cells. Mutation or deletion of these GC-rich elements results in loss of hormone responsiveness and shows that the -60 to -37 region of the VEGFR2 promoter is critical for both basal and hormone-dependent decreased VEGFR2 expression in MCF-7 cells. Western blot, immunofluorescent staining, RNA interference, and EMSAs support a role for Sp proteins in hormone-dependent down-regulation of VEGFR2 in MCF-7 cells, primarily through estrogen receptor (ER)alpha/Sp1 and ERalpha/Sp3 interactions with the VEGFR2 promoter. Using chromatin immuno-precipitation and transient transfection/RNA interference assays we show that the ERalpha/Sp protein-promoter interactions are accompanied by recruitment of the co-repressors SMRT (silencing mediator of retinoid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) to the promoter and that SMRT and NCoR knockdown reverse E2-mediated down-regulation of VEGFR2 expression in MCF-7 cells. This study illustrates that both SMRT and NCoR are involved in E2-dependent repression of VEGFR2 in MCF-7 cells.     

Laminin promotes vascular network formation in 3D in vitro collagen scaffolds by regulating VEGF uptake

Angiogenesis is an essential neovascularisation process, which if recapitulated in 3D in vitro, will provide better understanding of endothelial cell (EC) behaviour. Various cell types and growth factors are involved, with vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 key components. We were able to control the aggregation pattern of ECs in 3D collagen hydrogels, by varying the matrix composition and/or having a source of cells signalling angiogenic proteins. These aggregation patterns reflect the different developmental pathways that ECs take to form different sized tubular structures. Cultures with added laminin and thus increased expression of α6 integrin showed a significant increase (p<0.05) in VEGFR2 positive ECs and increased VEGF uptake. This resulted in the end-to-end network aggregation of ECs. In cultures without laminin and therefore low α6 integrin expression, VEGFR2 levels and VEGF uptake were significantly lower (p<0.05). These ECs formed contiguous sheets, analogous to the ‘wrapping’ pathway in development. We have identified a key linkage between integrin expression on ECs and their uptake of VEGF, regulated by VEGFR2, resulting in different aggregation patterns in 3D.     

The Bartonella henselae VirB/Bep system interferes with vascular endothelial growth factor (VEGF) signalling in human vascular endothelial cells

The vasculotropic pathogen Bartonella henselae (Bh) intimately interacts with human endothelial cells (ECs) and subverts multiple cellular functions. Here we report that Bh specifically interferes with vascular endothelial growth factor (VEGF) signalling in ECs. Bh infection abrogated VEGF-induced proliferation and wound closure of EC monolayers as well as the capillary-like sprouting of EC spheroids. On the molecular level, Bh infection did not alter VEGF receptor 2 (VEGFR2) expression or cell surface localization, but impeded VEGF-stimulated phosphorylation of VEGFR2 at tyrosine(1175) . Consistently, we observed that Bh infection diminished downstream events of the tyrosine(1175) -dependent VEGFR2-signalling pathway leading to EC proliferation, i.e. phospholipase-Cγ activation, cytosolic calcium fluxes and mitogen-activated protein kinase ERK1/2 phosphorylation. Pervanadate treatment neutralized the inhibitory activity of Bh on VEGF signalling, suggesting that Bh infection may activate a phosphatase that alleviates VEGFR2 phosphorylation. Inhibition of VEGFR2 signalling by Bh infection was strictly dependent on a functional VirB type IV secretion system and thereby translocated Bep effector proteins. The data presented in this study underscore the role of the VirB/Bep system as important factor controlling EC proliferation in response to Bh infection; not only as previously reported by counter-acting an intrinsic bacterial mitogenic stimulus, but also by restricting the exogenous angiogenic stimulation by Bh-induced VEGF.     

Retinal pigment epithelium-derived transforming growth factor-β2 inhibits the angiogenic response of endothelial cells by decreasing vascular endothelial growth factor receptor-2 expression

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that is known to modulate various aspects of endothelial cell (EC) biology. Retinal pigment epithelium (RPE) is important for regulating angiogenesis of choriocapillaris and one of the main cell sources of TGF-β secretion, particularly TGF-β2. However, it is largely unclear whether and how TGF-β2 affects angiogenic responses of ECs. In the current study, we demonstrated that TGF-β2 reduces vascular endothelial growth factor receptor-2 (VEGFR-2) expression in ECs and thereby inhibits vascular endothelial growth factor (VEGF) signaling and VEGF-induced angiogenic responses such as EC migration and tube formation. We also demonstrated that the reduction of VEGFR-2 expression by TGF-β2 is due to the suppression of JNK signaling. In coculture of RPE cells and ECs, RPE cells decreased VEGFR-2 levels in ECs and EC migration. In addition, we showed that TGF-β2 derived from RPE cells is involved in the reduction of VEGFR-2 expression and inhibition of EC migration. These results suggest that TGF-β2 plays an important role in inhibiting the angiogenic responses of ECs during the interaction between RPE cells and ECs and that angiogenic responses of ECs may be amplified by a decrease in TGF-β2 expression in RPE cells under pathologic conditions.     

Extracellular SOD-Derived H2O2 Promotes VEGF Signaling in Caveolae/Lipid Rafts and Post-Ischemic Angiogenesis in Mice

Reactive oxygen species (ROS), in particular, H₂O₂, is essential for full activation of VEGF receptor2 (VEGFR2) signaling involved in endothelial cell (EC) proliferation and migration. Extracellular superoxide dismutase (ecSOD) is a major secreted extracellular enzyme that catalyzes the dismutation of superoxide to H₂O₂, and anchors to EC surface through heparin-binding domain (HBD). Mice lacking ecSOD show impaired postnatal angiogenesis. However, it is unknown whether ecSOD-derived H₂O₂ regulates VEGF signaling. Here we show that gene transfer of ecSOD, but not ecSOD lacking HBD (ecSOD-ΔHBD), increases H₂O₂ levels in adductor muscle of mice, and promotes angiogenesis after hindlimb ischemia. Mice lacking ecSOD show reduction of H₂O₂ in non-ischemic and ischemic limbs. In vitro, overexpression of ecSOD, but not ecSOD-ΔHBD, in cultured medium in ECs enhances VEGF-induced tyrosine phosphorylation of VEGFR2 (VEGFR2-pY), which is prevented by short-term pretreatment with catalase that scavenges extracellular H₂O₂. Either exogenous H₂O₂ (<500 µM), which is diffusible, or nitric oxide donor has no effect on VEGF-induced VEGFR2-pY. These suggest that ecSOD binding to ECs via HBD is required for localized generation of extracellular H₂O₂ to regulate VEGFR2-pY. Mechanistically, VEGF-induced VEGFR2-pY in caveolae/lipid rafts, but non-lipid rafts, is enhanced by ecSOD, which localizes at lipid rafts via HBD. One of the targets of ROS is protein tyrosine phosphatases (PTPs). ecSOD induces oxidation and inactivation of both PTP1B and DEP1, which negatively regulates VEGFR2-pY, in caveolae/lipid rafts, but not non-lipid rafts. Disruption of caveolae/lipid rafts, or PTPs inhibitor orthovanadate, or siRNAs for PTP1B and DEP1 enhances VEGF-induced VEGFR2-pY, which prevents ecSOD-induced effect. Functionally, ecSOD promotes VEGF-stimulated EC migration and proliferation. In summary, extracellular H₂O₂ generated by ecSOD localized at caveolae/lipid rafts via HBD promotes VEGFR2 signaling via oxidative inactivation of PTPs in these microdomains. Thus, ecSOD is a potential therapeutic target for angiogenesis-dependent cardiovascular diseases.