Angiogenesis, a new vessel formation from the pre-existing ones, is essential for embryonic development, wound repair and treatment of ischemic heart and limb diseases. However, dysregulated angiogenesis contributes to various pathologies such as diabetic retinopathy, atherosclerosis and cancer. Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) as well as mitochondria play an important role in promoting the angiogenic switch from quiescent endothelial cells (ECs). However, how highly diffusible ROS produced from different sources and location can communicate with each other to regulate angiogenesis remains unclear. To detect a localized ROS signal in distinct subcellular compartments in real time in situ, compartment-specific genetically encoded redox-sensitive fluorescence biosensors have been developed. Recently, the intercellular communication, “cross-talk”, between ROS derived from NOX and mitochondria, termed “ROS-induced ROS release”, has been proposed as a mechanism for ROS amplification at distinct subcellular compartments, which are essential for activation of redox signaling. This “ROS-induced ROS release” may represent a feed-forward mechanism of localized ROS production to maintain sustained signaling, which can be targeted under pathological conditions with oxidative stress or enhanced to promote therapeutic angiogenesis. In this review, we summarize the recent knowledge regarding the role of the cross-talk between NOX and mitochondria organizing the sustained ROS signaling involved in VEGF signaling, neovascularization and tissue repair.

Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) and mitochondria play a critical role in growth factor-induced switch from a quiescent to an angiogenic phenotype in endothelial cells (ECs). However, how highly diffusible ROS produced from different sources can coordinate to stimulate VEGF signaling and drive the angiogenic process remains unknown. Using the cytosol- and mitochondria-targeted redox-sensitive RoGFP biosensors with real-time imaging, here we show that VEGF stimulation in human ECs rapidly increases cytosolic RoGFP oxidation within 1 min, followed by mitochondrial RoGFP oxidation within 5 min, which continues at least for 60 min. Silencing of Nox4 or Nox2 or overexpression of mitochondria-targeted catalase significantly inhibits VEGF-induced tyrosine phosphorylation of VEGF receptor type 2 (VEGFR2-pY), EC migration and proliferation at the similar extent. Exogenous hydrogen peroxide (H₂O₂) or overexpression of Nox4, which produces H₂O₂, increases mitochondrial ROS (mtROS), which is prevented by Nox2 siRNA, suggesting that Nox2 senses Nox4-derived H₂O₂ to promote mtROS production. Mechanistically, H₂O₂ increases S36 phosphorylation of p66Shc, a key mtROS regulator, which is inhibited by siNox2, but not by siNox4. Moreover, Nox2 or Nox4 knockdown or overexpression of S36 phosphorylation-defective mutant p66Shc(S36A) inhibits VEGF-induced mtROS, VEGFR2-pY, EC migration, and proliferation. In summary, Nox4-derived H₂O₂ in part activates Nox2 to increase mtROS via pSer36-p66Shc, thereby enhancing VEGFR2 signaling and angiogenesis in ECs. This may represent a novel feed-forward mechanism of ROS-induced ROS release orchestrated by the Nox4/Nox2/pSer36-p66Shc/mtROS axis, which drives sustained activation of angiogenesis signaling program.

Mitochondrial dynamics are tightly controlled by fusion and fission, and their dysregulation and excess reactive oxygen species (ROS) contribute to endothelial cell (EC) dysfunction. How redox signals regulate coupling between mitochondrial dynamics and endothelial (dys)function remains unknown. Here, we identify protein disulfide isomerase A1 (PDIA1) as a thiol reductase for the mitochondrial fission protein Drp1. A biotin-labeled Cys-OH trapping probe and rescue experiments reveal that PDIA1 depletion in ECs induces sulfenylation of Drp1 at Cys644, promoting mitochondrial fragmentation and ROS elevation without inducing ER stress, which drives EC senescence. Mechanistically, PDIA1 associates with Drp1 to reduce its redox status and activity. Defective wound healing and angiogenesis in diabetic or PDIA1+/- mice are restored by EC-targeted PDIA1 or the Cys oxidation-defective mutant Drp1. Thus, this study uncovers a molecular link between PDIA1 and Drp1 oxidoreduction, which maintains normal mitochondrial dynamics and limits endothelial senescence with potential translational implications for vascular diseases associated with diabetes or aging.

Reactive oxygen species (ROS; e.g ., superoxide [O ₂ ^•− ] and hydrogen peroxide [H ₂ O ₂ ]) and reactive nitrogen species (RNS; e.g ., nitric oxide [NO ^• ]) at the physiological level function as signaling molecules that mediate many biological responses, including cell proliferation, migration, differentiation, and gene expression. By contrast, excess ROS/RNS, a consequence of dysregulated redox homeostasis, is a hallmark of cardiovascular disease. Accumulating evidence suggests that both ROS and RNS regulate various metabolic pathways and enzymes. Recent studies indicate that cells have mechanisms that fine-tune ROS/RNS levels by tight regulation of metabolic pathways, such as glycolysis and oxidative phosphorylation. The ROS/RNS-mediated inhibition of glycolytic pathways promotes metabolic reprogramming away from glycolytic flux toward the oxidative pentose phosphate pathway to generate nicotinamide adenine dinucleotide phosphate (NADPH) for antioxidant defense. This review summarizes our current knowledge of the mechanisms by which ROS/RNS regulate metabolic enzymes and cellular metabolism and how cellular metabolism influences redox homeostasis and the pathogenesis of disease. A full understanding of these mechanisms will be important for the development of new therapeutic strategies to treat diseases associated with dysregulated redox homeostasis and metabolism. Antioxid. Redox Signal . 34, 1319–1354.

VEGFR2 (KDR/Flk1) signaling in endothelial cells (ECs) plays a central role in angiogenesis. The P-type ATPase transporter ATP7A regulates copper homeostasis, and its role in VEGFR2 signaling and angiogenesis is entirely unknown. Here, we describe the unexpected crosstalk between the Copper transporter ATP7A, autophagy, and VEGFR2 degradation. The functional significance of this Copper transporter was demonstrated by the finding that inducible EC-specific ATP7A deficient mice or ATP7A-dysfunctional ATP7Amut mice showed impaired post-ischemic neovascularization. In ECs, loss of ATP7A inhibited VEGF-induced VEGFR2 signaling and angiogenic responses, in part by promoting ligand-induced VEGFR2 protein degradation. Mechanistically, VEGF stimulated ATP7A translocation from the trans-Golgi network to the plasma membrane where it bound to VEGFR2, which prevented autophagy-mediated lysosomal VEGFR2 degradation by inhibiting autophagic cargo/adapter p62/SQSTM1 binding to ubiquitinated VEGFR2. Enhanced autophagy flux due to ATP7A dysfunction in vivo was confirmed by autophagy reporter CAG-ATP7Amut -RFP-EGFP-LC3 transgenic mice. In summary, our study uncovers a novel function of ATP7A to limit autophagy-mediated degradation of VEGFR2, thereby promoting VEGFR2 signaling and angiogenesis, which restores perfusion recovery and neovascularization. Thus, endothelial ATP7A is identified as a potential therapeutic target for treatment of ischemic cardiovascular diseases.