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

β-Adrenergic Receptor-PI3K Signaling Crosstalk in Mouse Heart: Elucidation of Immediate Downstream Signaling Cascades

Sustained β-adrenergic receptors (βAR) activation leads to cardiac hypertrophy and prevents left ventricular (LV) atrophy during LV unloading. The immediate signaling pathways downstream from βAR stimulation, however, have not been well investigated. The current study was to examine the early cardiac signaling mechanism(s) following βAR stimulation. In adult C57BL/6 mice, acute βAR stimulation induced significant increases in PI3K activity and activation of Akt and ERK1/2 in the heart, but not in lungs or livers. In contrast, the same treatment did not elicit these changes in β1/β2AR double knockout mice. We further showed the specificity of β2AR in this crosstalk as treatment with formoterol, a β2AR-selective agonist, but not dobutamine, a predominantly β1AR agonist, activated cardiac Akt and ERK1/2. Acute βAR stimulation also significantly increased the phosphorylation of mTOR (the mammalian target of rapamycin), P70S6K, ribosomal protein S6, GSK-3α/β (glycogen synthase kinase-3α/β), and FOXO1/3a (the forkhead box family of transcription factors 1 and 3a). Moreover, acute βAR stimulation time-dependently decreased the mRNA levels of the muscle-specific E3 ligases atrogin-1 and muscle ring finger protein-1 (MuRF1) in mouse heart. Our results indicate that acute βAR stimulation in vivo affects multiple cardiac signaling cascades, including the PI3K signaling pathway, ERK1/2, atrogin-1 and MuRF1. These data 1) provide convincing evidence for the crosstalk between βAR and PI3K signaling pathways; 2) confirm the β2AR specificity in this crosstalk in vivo; and 3) identify novel signaling factors involved in cardiac hypertrophy and LV unloading. Understanding of the intricate interplay between β2AR activation and these signaling cascades should provide critical clues to the pathogenesis of cardiac hypertrophy and enable identification of targets for early clinical interaction of cardiac lesions

Embracing Diversity, Equity, and Inclusion in the Scientific Community-Viewpoints of the Diversity, Equity, and Inclusion Committee of the North American Vascular Biology Organization

Recent increased visibility on racial issues in the United States elicited public outcry and a collective call for action. The social justice movement has facilitated energetic discussions about race, sexual orientation, and various issues of diversity, equity, and inclusion. This article discusses issues faced by people of color that we as scientists can address, as well as challenges faced by women and internationally trained scientists in the scientific community that need immediate attention. Moreover, we highlight various ways to resolve such issues at both institutional and individual levels. Silence and incremental solutions are no longer acceptable to achieving lasting social justice and ensure prosperous societies that work for all

Direct Sensing of Endothelial Oxidants by Vascular Endothelial Growth Factor Receptor-2 and c-Src

BACKGROUND: ADPH oxidase-derived reactive oxygen species (ROS) play important roles in redox homeostasis and signal transduction in endothelial cells (ECs). We previously demonstrated that c-Src plays a key role in VEGF-induced, ROS-dependent selective activation of PI3K-Akt but not PLCγ-1-ERK1/2 signaling pathways. The aim of the present study was to understand how VEGFR-2-c-Src signaling axis 'senses' NADPH oxidase-derived ROS levels and couples VEGF activation of c-Src to the redox state of ECs. METHODOLOGY/PRINCIPAL FINDINGS: Using biotinylated probe that detects oxidation of cysteine thiol (cys-OH) in intracellular proteins, we demonstrate that VEGF induced oxidative modification in c-Src and VEGFR-2, and that reduction in ROS levels using siRNA against p47(phox) subunit of Rac1-dependent NADPH oxidase inhibited this phenomenon. Co-immunoprecipitation studies using human coronary artery ECs (HCAEC) showed that VEGF-induced ROS-dependent interaction between VEGFR-2 and c-Src correlated with their thiol oxidation status. Immunofluorescence studies using antibodies against internalized VEGFR-2 and c-Src demonstrated that VEGF-induced subcellular co-localization of these tyrosine kinases were also dependent on NADPH oxidsase-derived ROS. CONCLUSION/SIGNIFICANCE: These results demonstrate that VEGF induces cysteine oxidation in VEGFR-2 and c-Src in an NADPH oxidase-derived ROS-dependent manner, suggesting that VEGFR-2 and c-Src can 'sense' redox levels in ECs. The data also suggest that thiol oxidation status of VEGFR-2 and c-Src correlates with their ability to physically interact with each other and c-Src activation. Taken together, these findings suggest that prior to activating downstream c-Src-PI3K-Akt signaling pathway, VEGFR-2-c-Src axis requires an NADPH oxidase-derived ROS threshold in ECs

Mechanosignaling in the vasculature: emerging concepts in sensing, transduction and physiological responses

Cells are constantly exposed to mechanical forces that play a role in modulating cellular structure and function. The cardiovascular system experiences physical forces in the form of shear stress and stretch associated with blood flow and contraction, respectively. These forces are sensed by endothelial cells and cardiomyocytes and lead to responses that control vascular and cardiac homeostasis. This was highlighted at the Pan American Physiological Society meeting at Iguassu Falls, Brazil, in a symposium titled “Mechanosignaling in the Vasculature.” This symposium presented recent research that showed the existence of a vital link between mechanosensing and downstream redox sensitive signaling cascades. This link helps to transduce and transmit the physical force into an observable physiological response. The speakers showcased how mechanosensors such as ion channels, membrane receptor kinases, adhesion molecules, and other cellular components transduce the force via redox signals (such as reactive oxygen species and nitric oxide) to receptors (transcription factors, growth factors, etc.). Receptor activated pathways then lead to cellular responses including cellular proliferation, contraction, and remodeling. These responses have major relevance to the physiology and pathophysiology of various cardiovascular diseases. Thus an understanding of the complex series of events, from the initial sensing through the final response, is essential for progress in this field. Overall, this symposium addressed some important emerging concepts in the field of mechanosignaling and the eventual pathophysiological responses.Centro de Investigaciones Cardiovasculare

MicroRNA regulation of endothelial homeostasis and commitment—implications for vascular regeneration strategies using stem cell therapies

Human embryonic (hESC) and induced pluripotent (hiPSC) stem cells have broad therapeutic potential in the treatment of a range of diseases, including those of the vascular system. Both hESCs and hiPSCs have the capacity for indefinite self-renewal, in addition to their ability to differentiate into any adult cell type. These cells could provide a potentially unlimited source of cells for transplantation and, therefore, provide novel treatments, e.g. in the production of endothelial cells for vascular regeneration. MicroRNAs are short, noncoding RNAs that act posttranscriptionally to control gene expression and thereby exert influence over a wide range of cellular processes, including maintenance of pluripotency and differentiation. Expression patterns of these small RNAs are tissue specific, and changes in microRNA levels have often been associated with disease states in humans, including vascular pathologies. Here, we review the roles of microRNAs in endothelial cell function and vascular disease, as well as their role in the differentiation of pluripotent stem cells to the vascular endothelial lineage. Furthermore, we discuss the therapeutic potential of stem cells and how knowledge and manipulation of microRNAs in stem cells may enhance their capacity for vascular regeneration