Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis.

VEGFR2 signaling in endothelial cells (ECs) is regulated by reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria, which plays an important role in postnatal angiogenesis. However, it remains unclear how highly diffusible ROS signal enhances VEGFR2 signaling and reparative angiogenesis. Protein disulfide isomerase A1 (PDIA1) functions as an oxidoreductase depending on the redox environment. We hypothesized that PDIA1 functions as a redox sensor to enhance angiogenesis. Here we showed that PDIA1 co-immunoprecipitated with VEGFR2 or colocalized with either VEGFR2 or an early endosome marker Rab5 at the perinuclear region upon stimulation of human ECs with VEGF. PDIA1 silencing significantly reduced VEGF-induced EC migration, proliferation and spheroid sprouting via inhibiting VEGFR2 signaling. Mechanistically, VEGF stimulation rapidly increased Cys-OH formation of PDIA1 via the NOX4-mitochondrial ROS axis. Overexpression of "redox-dead" mutant PDIA1 with replacement of the active four Cys residues with Ser significantly inhibited VEGF-induced PDIA1-CysOH formation and angiogenic responses via reducing VEGFR2 phosphorylation. Pdia1+/- mice showed impaired angiogenesis in developmental retina and Matrigel plug models as well as ex vivo aortic ring sprouting model. Study using hindlimb ischemia model revealed that PDIA1 expression was markedly increased in angiogenic ECs of ischemic muscles, and that ischemia-induced limb perfusion recovery and neovascularization were impaired in EC-specific Pdia1 conditional knockout mice. These results suggest that PDIA1 can sense VEGF-induced H2O2 signal via CysOH formation to promote VEGFR2 signaling and angiogenesis in ECs, thereby enhancing postnatal angiogenesis. The oxidized PDIA1 is a potential therapeutic target for treatment of ischemic vascular diseases.

Exercise improves angiogenic function of circulating exosomes in type 2 diabetes: Role of exosomal SOD3.

Exosomes, key mediators of cell-cell communication, derived from type 2 diabetes mellitus (T2DM) exhibit detrimental effects. Exercise improves endothelial function in part via the secretion of exosomes into circulation. Extracellular superoxide dismutase (SOD3) is a major secretory copper (Cu) antioxidant enzyme that catalyzes the dismutation of O2•- to H2 O2 whose activity requires the Cu transporter ATP7A. However, the role of SOD3 in exercise-induced angiogenic effects of circulating plasma exosomes on endothelial cells (ECs) in T2DM remains unknown. Here, we show that both SOD3 and ATP7A proteins were present in plasma exosomes in mice, which was significantly increased after two weeks of volunteer wheel exercise. A single bout of exercise in humans also showed a significant increase in SOD3 and ATP7A protein expression in plasma exosomes. Plasma exosomes from T2DM mice significantly reduced angiogenic responses in human ECs or mouse skin wound healing models, which was associated with a decrease in ATP7A, but not SOD3 expression in exosomes. Exercise training in T2DM mice restored the angiogenic effects of T2DM exosomes in ECs by increasing ATP7A in exosomes, which was not observed in exercised T2DM/SOD3-/- mice. Furthermore, exosomes overexpressing SOD3 significantly enhanced angiogenesis in ECs by increasing local H2 O2  levels in a heparin-binding domain-dependent manner as well as restored defective wound healing and angiogenesis in T2DM or SOD3-/- mice. In conclusion, exercise improves the angiogenic potential of circulating exosomes in T2DM in a SOD3-dependent manner. Exosomal SOD3 may provide an exercise mimetic therapy that supports neovascularization and wound repair in cardiometabolic disease.

Caveolin-1 stabilizes ATP7A, a copper transporter for extracellular SOD, in vascular tissue to maintain endothelial function.

Caveolin-1 (Cav-1) is a scaffolding protein and a major component of caveolae/lipid rafts. Previous reports have shown that endothelial dysfunction in Cav-1-deficient (Cav-1-/-) mice is mediated by elevated oxidative stress through endothelial nitric oxide synthase (eNOS) uncoupling and increased NADPH oxidase. Oxidant stress is the net balance of oxidant generation and scavenging, and the role of Cav-1 as a regulator of antioxidant enzymes in vascular tissue is poorly understood. Extracellular SOD (SOD3) is a copper (Cu)-containing enzyme that is secreted from vascular smooth muscle cells/fibroblasts and subsequently binds to the endothelial cells surface, where it scavenges extracellular [Formula: see text] and preserves endothelial function. SOD3 activity is dependent on Cu, supplied by the Cu transporter ATP7A, but whether Cav-1 regulates the ATP7A-SOD3 axis and its role in oxidative stress-mediated vascular dysfunction has not been studied. Here we show that the activity of SOD3, but not SOD1, was significantly decreased in Cav-1-/- vessels, which was rescued by re-expression of Cav-1 or Cu supplementation. Loss of Cav-1 reduced ATP7A protein, but not mRNA, and this was mediated by ubiquitination of ATP7A and proteasomal degradation. ATP7A bound to Cav-1 and was colocalized with SOD3 in caveolae/lipid rafts or perinucleus in vascular tissues or cells. Impaired endothelium-dependent vasorelaxation in Cav-1-/- mice was rescued by gene transfer of SOD3 or by ATP7A-overexpressing transgenic mice. These data reveal an unexpected role of Cav-1 in stabilizing ATP7A protein expression by preventing its ubiquitination and proteasomal degradation, thereby increasing SOD3 activity, which in turn protects against vascular oxidative stress-mediated endothelial dysfunction.

Copper Transporter ATP7A (Copper-Transporting P-Type ATPase/Menkes ATPase) Limits Vascular Inflammation and Aortic Aneurysm Development: Role of MicroRNA-125b.

OBJECTIVE: Copper (Cu) is essential micronutrient, and its dysregulation is implicated in aortic aneurysm (AA) development. The Cu exporter ATP7A (copper-transporting P-type ATPase/Menkes ATPase) delivers Cu via the Cu chaperone Atox1 (antioxidant 1) to secretory Cu enzymes, such as lysyl oxidase, and excludes excess Cu. Lysyl oxidase is shown to protect against AA formation. However, the role and mechanism of ATP7A in AA pathogenesis remain unknown. Approach and Results: Here, we show that Cu chelator markedly inhibited Ang II (angiotensin II)-induced abdominal AA (AAA) in which ATP7A expression was markedly downregulated. Transgenic ATP7A overexpression prevented Ang II-induced AAA formation. Conversely, Cu transport dysfunctional ATP7Amut/+/ApoE-/- mice exhibited robust AAA formation and dissection, excess aortic Cu accumulation as assessed by X-ray fluorescence microscopy, and reduced lysyl oxidase activity. In contrast, AAA formation was not observed in Atox1-/-/ApoE-/- mice, suggesting that decreased lysyl oxidase activity, which depends on both ATP7A and Atox1, was not sufficient to develop AAA. Bone marrow transplantation suggested importance of ATP7A in vascular cells, not bone marrow cells, in AAA development. MicroRNA (miR) array identified miR-125b as a highly upregulated miR in AAA from ATP7Amut/+/ApoE-/- mice. Furthermore, miR-125b target genes (histone methyltransferase Suv39h1 and the NF-κB negative regulator TNFAIP3 [tumor necrosis factor alpha induced protein 3]) were downregulated, which resulted in increased proinflammatory cytokine expression, aortic macrophage recruitment, MMP (matrix metalloproteinase)-2/9 activity, elastin fragmentation, and vascular smooth muscle cell loss in ATP7Amut/+/ApoE-/- mice and reversed by locked nucleic acid-anti-miR-125b infusion. CONCLUSIONS: ATP7A downregulation/dysfunction promotes AAA formation via upregulating miR-125b, which augments proinflammatory signaling in a Cu-dependent manner. Thus, ATP7A is a potential therapeutic target for inflammatory vascular disease.

Novel interaction of antioxidant-1 with TRAF4: role in inflammatory responses in endothelial cells.

NADPH oxidase (NOX)-derived reactive oxygen species (ROS) and copper (Cu), an essential micronutrient, have been implicated in vascular inflammatory diseases. We reported that in proinflammatory cytokine TNF-α-stimulated endothelial cells (ECs), cytosolic Cu chaperone antioxidant-1 (Atox1) functions as a Cu-dependent transcription factor for the NOX organizer p47phox, thereby increasing ROS-dependent inflammatory gene expression. However, the role and mechanism of Atox1 nuclear translocation in inflamed ECs remain unclear. Using enface staining and nuclear fractionation, here we show that Atox1 was localized in the nucleus in inflamed aortas from ApoE-/- mice with angiotensin II infusion on a high-fat diet, while it was found in cytosol in those from control mice. In cultured human ECs, TNF-α stimulation promoted Atox1 nuclear translocation within 15 min, which was associated with Atox1 binding to TNF-α receptor-associated factor 4 (TRAF4) in a Cu-dependent manner. TRAF4 depletion by siRNA significantly inhibited Atox1 nuclear translocation, p47phox expression, and ROS production as well as its downstream VCAM1/ICAM1 expression and monocyte adhesion to inflamed ECs, which were rescued by overexpression of nuclear targeted Atox1. Furthermore, Atox1 colocalized with TRAF4 at the nucleus in TNF-α-stimulated inflamed ECs and vessels. In summary, Cu-dependent Atox1 binding to TRAF4 plays an important role in Atox1 nuclear translocation and ROS-dependent inflammatory responses in TNF-α-stimulated ECs. Thus the Atox1-TRAF4 axis is a novel therapeutic target for vascular inflammatory disease such as atherosclerosis.

Akt2 (Protein Kinase B Beta) Stabilizes ATP7A, a Copper Transporter for Extracellular Superoxide Dismutase, in Vascular Smooth Muscle: Novel Mechanism to Limit Endothelial Dysfunction in Type 2 Diabetes Mellitus.

OBJECTIVE: Copper transporter ATP7A (copper-transporting/ATPase) is required for full activation of SOD3 (extracellular superoxide dismutase), which is secreted from vascular smooth muscle cells (VSMCs) and anchors to endothelial cell surface to preserve endothelial function by scavenging extracellular superoxide. We reported that ATP7A protein expression and SOD3 activity are decreased in insulin-deficient type 1 diabetes mellitus vessels, thereby, inducing superoxide-mediated endothelial dysfunction, which are rescued by insulin treatment. However, it is unknown regarding the mechanism by which insulin increases ATP7A expression in VSMCs and whether ATP7A downregulation is observed in T2DM (type2 diabetes mellitus) mice and human in which insulin-Akt (protein kinase B) pathway is selectively impaired. APPROACH AND RESULTS: Here we show that ATP7A protein is markedly downregulated in vessels isolated from T2DM patients, as well as those from high-fat diet-induced or db/db T2DM mice. Akt2 (protein kinase B beta) activated by insulin promotes ATP7A stabilization via preventing ubiquitination/degradation as well as translocation to plasma membrane in VSMCs, which contributes to activation of SOD3 that protects against T2DM-induced endothelial dysfunction. Downregulation of ATP7A in T2DM vessels is restored by constitutive active Akt or PTP1B-/- (protein-tyrosine phosphatase 1B-deficient) T2DM mice, which enhance insulin-Akt signaling. Immunoprecipitation, in vitro kinase assay, and mass spectrometry analysis reveal that insulin stimulates Akt2 binding to ATP7A to induce phosphorylation at Ser1424/1463/1466. Furthermore, SOD3 activity is reduced in Akt2-/- vessels or VSMCs, which is rescued by ATP7A overexpression. CONCLUSION: Akt2 plays a critical role in ATP7A protein stabilization and translocation to plasma membrane in VSMCs, which contributes to full activation of vascular SOD3 that protects against endothelial dysfunction in T2DM.

Copper transporter ATP7A interacts with IQGAP1, a Rac1 binding scaffolding protein: role in PDGF-induced VSMC migration and vascular remodeling.

Vascular smooth muscle cell (VSMC) migration contributes to neointimal formation after vascular injury. We previously demonstrated that copper (Cu) transporter ATP7A is involved in platelet-derived growth factor (PDGF)-induced VSMC migration in a Cu- and Rac1-dependent manner. The underlying mechanism is still unknown. Here we show that ATP7A interacts with IQGAP1, a Rac1 and receptor tyrosine kinase binding scaffolding proteins, which mediates PDGF-induced VSMC migration and vascular remodeling. In cultured rat aortic SMCs, PDGF stimulation rapidly promoted ATP7A association with IQGAP1 and Rac1 and their translocation to the lipid rafts and leading edge. Cotransfection assay revealed that ATP7A directly bound to NH2-terminal domain of IQGAP1. Functionally, either ATP7A or IQGAP1 depletion using siRNA significantly inhibited PDGF-induced VSMC migration without additive effects, suggesting that IQGAP1 and ATP7A are in the same axis to promote migration. Furthermore, IQGAP1 siRNA blocked PDGF-induced ATP7A association with Rac1 as well as its translocation to leading edge, while PDGF-induced IQGAP1 translocation was not affected by ATP7A siRNA or Cu chelator. Overexpression of mutant IQGAP1 lacking a Rac1 binding site prevented PDGF-induced translocation of Rac1, but not ATP7A, to the leading edge, thereby inhibiting lamellipodia formation and VSMC migration. In vivo, ATP7A colocalized with IQGAP1 at neointimal VSMCs in a mice wire injury model, while neointimal formation and extracellular matrix deposition induced by vascular injury were inhibited in ATP7A mutant mice with reduced Cu transporter function. In summary, IQGAP1 functions as ATP7A and Rac1 binding scaffolding protein to organize PDGF-dependent ATP7A translocation to the lamellipodial leading edge, thereby promoting VSMC migration and vascular remodeling.

Short-term regular aerobic exercise reduces oxidative stress produced by acute in the adipose microvasculature.

High blood pressure has been shown to elicit impaired dilation in the vasculature. The purpose of this investigation was to elucidate the mechanisms through which high pressure may elicit vascular dysfunction and determine the mechanisms through which regular aerobic exercise protects arteries against high pressure. Male C57BL/6J mice were subjected to 2 wk of voluntary running (~6 km/day) for comparison with sedentary controls. Hindlimb adipose resistance arteries were dissected from mice for measurements of flow-induced dilation (FID; with or without high intraluminal pressure exposure) or protein expression of NADPH oxidase II (NOX II) and superoxide dismutase (SOD). Microvascular endothelial cells were subjected to high physiological laminar shear stress (20 dyn/cm2) or static condition and treated with ANG II + pharmacological inhibitors. Cells were analyzed for the detection of ROS or collected for Western blot determination of NOX II and SOD. Resistance arteries from exercised mice demonstrated preserved FID after high pressure exposure, whereas FID was impaired in control mouse arteries. Inhibition of ANG II or NOX II restored impaired FID in control mouse arteries. High pressure increased superoxide levels in control mouse arteries but not in exercise mouse arteries, which exhibited greater ability to convert superoxide to H2O2 Arteries from exercised mice exhibited less NOX II protein expression, more SOD isoform expression, and less sensitivity to ANG II. Endothelial cells subjected to laminar shear stress exhibited less NOX II subunit expression. In conclusion, aerobic exercise prevents high pressure-induced vascular dysfunction through an improved redox environment in the adipose microvasculature.NEW & NOTEWORTHY We describe potential mechanisms contributing to aerobic exercise-conferred protection against high intravascular pressure. Subcutaneous adipose microvessels from exercise mice express less NADPH oxidase (NOX) II and more superoxide dismutase (SOD) and demonstrate less sensitivity to ANG II. In microvascular endothelial cells, shear stress reduced NOX II but did not influence SOD expression.

Novel role of reactive oxygen species-activated Trp melastatin channel-2 in mediating angiogenesis and postischemic neovascularization.

OBJECTIVE: Transient receptor potential melastatin-2 (TRPM2) channel is a nonselective cation channel that mediates influx of Ca(2+) and Na(+) with relative permeability of PCa:PNa ≈0.6 in response to cellular oxidative stress. As angiogenesis and ischemic neovascularization are both significantly dependent on oxidant signaling, here we investigated the possible role of vascular endothelial growth factor (VEGF)-induced reactive oxygen species production in activating TRPM2-dependent Ca(2+) signaling and in the mechanism of angiogenesis and ischemic neovascularization. APPROACH AND RESULTS: We observed that VEGF stimulation rapidly induced the association of TRPM2 and cellular Src kinase with vascular endothelial-cadherin forming a signalplex at vascular endothelial-cadherin junctions in endothelial cells. Using endothelial cells isolated from TRPM2(-/-) mice or after small interfering RNA depletion of TRPM2, we demonstrated that TRPM2-activated Ca(2+) signaling was required for cellular Src kinase-induced phosphorylation of vascular endothelial-cadherin at Y658 and Y731, the crucial sites involved in vascular endothelial-cadherin internalization in response to VEGF. VEGF-induced reactive oxygen species generation activated TRPM2-induced Ca(2+) entry, whereas the reactive oxygen species-insensitive TRPM2 mutant (C1008→A) showed impaired Ca(2+) entry. Endothelial cells depleted of TRPM2 also displayed significantly perturbed migratory phenotype and impaired activation of cellular Src in response to VEGF. TRPM2(-/-) mice reconstituted with wild-type myeloid cells demonstrated aberrant angiogenesis and neovascularization in the hindlimb ischemia model as compared with wild-type mice. CONCLUSIONS: VEGF-induced angiogenesis and postischemic neovascularization in mice required reactive oxygen species generation in endothelial cells and resultant TRPM2 activation. Thus, our findings provide novel insight into the role of TRPM2 in mechanism of angiogenesis and ischemic neovascularization.

Novel role of copper transport protein antioxidant-1 in neointimal formation after vascular injury.

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. CONCLUSIONS: 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.

Endothelial Drp1 Couples VEGF-induced Redox Signaling with Glycolysis Through Cysteine Oxidation to Drive Angiogenesis.

Angiogenesis plays a vital role for postnatal development and tissue repair following ischemia. Reactive oxygen species (ROS) generated by NADPH oxidases (NOXes) and mitochondria act as signaling molecules that promote angiogenesis in endothelial cells (ECs) which mainly relies on aerobic glycolysis for ATP production. However, the connections linking redox signaling with glycolysis are not well understood. The GTPase Drp1 is a member of the dynamin superfamily that moves from cytosol to mitochondria through posttranslational modifications to induce mitochondrial fission. The role of Drp1 in ROS-dependent VEGF signaling and angiogenesis in ECs has not been previously described. Here, we identify an unexpected function of endothelial Drp1 as a redox sensor, transmitting VEGF-induced H 2 O 2 signals to enhance glycolysis and angiogenesis. Loss of Drp1 expression in ECs inhibited VEGF-induced angiogenic responses. Mechanistically, VEGF rapidly induced the NOX4-dependent sulfenylation (CysOH) of Drp1 on Cys 644 , promoting disulfide bond formation with the metabolic kinase AMPK and subsequent sulfenylation of AMPK at Cys 299 / 304 via the mitochondrial fission-mitoROS axis. This cysteine oxidation of AMPK, in turn, enhanced glycolysis and angiogenesis. In vivo , mice with EC-specific Drp1 deficiency or CRISPR/Cas9-engineered "redox-dead" (Cys to Ala) Drp1 knock-in mutations exhibited impaired retinal angiogenesis and post-ischemic neovascularization. Our findings uncover a novel role for endothelial Drp1 in linking VEGF-induced mitochondrial redox signaling to glycolysis through a cysteine oxidation-mediated Drp1-AMPK redox relay, driving both developmental and reparative angiogenesis.

IQGAP1 links PDGF receptor-ß signal to focal adhesions involved in vascular smooth muscle cell migration: role in neointimal formation after vascular injury.

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 receptor 2 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-ß (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/FA signaling protein complex. Functionally, IQGAP1 siRNA inhibits PDGF-induced FA 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 FA 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.

Pentose Pathway Activation Is Superior to Increased Glycolysis for Therapeutic Angiogenesis in Peripheral Arterial Disease.

Background In endothelial cells (ECs), glycolysis, regulated by PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase, isoform-3), is the major metabolic pathway for ATP generation. In preclinical peripheral artery disease models, VEGF165a (vascular endothelial growth factor165a) and microRNA-93 both promote angiogenesis. Methods and Results Mice following hind-limb ischemia (HLI) and ECs with, and without, hypoxia and serum starvation were examined with, and without, microRNA-93 and VEGF165a. Post-HLI perfusion recovery was monitored. EC metabolism was studied using seahorse assay, and the expression and activity of major metabolism genes were assessed. Reactive oxygen species levels and EC permeability were evaluated. C57Bl/6J mice generated a robust angiogenic response to HLI, with ECs from ischemic versus nonischemic muscle demonstrating no increase in glycolysis. Balb/CJ mice generated a poor angiogenic response post-HLI; ischemic versus nonischemic ECs demonstrated significant increase in glycolysis. MicroRNA-93-treated Balb/CJ mice post-HLI showed better perfusion recovery, with ischemic versus nonischemic ECs showing no increase in glycolysis. VEGF165a-treated Balb/CJ mice post-HLI showed no improvement in perfusion recovery with ischemic versus nonischemic ECs showing significant increase in glycolysis. ECs under hypoxia and serum starvation upregulated PFKFB3. In ECs under hypoxia and serum starvation, VEGF165a versus control significantly upregulated PFKFB3 and glycolysis, whereas miR-93 versus control demonstrated no increase in PFKFB3 or glycolysis. MicroRNA-93 versus VEGF165a upregulated glucose-6-phosphate dehydrogenase expression and activity, activating the pentose phosphate pathway. MicroRNA-93 versus control increased reduced nicotinamide adenine dinucleotide phosphate and virtually eliminated the increase in reactive oxygen species. In ECs under hypoxia and serum starvation, VEGF165a significantly increased and miR-93 decreased EC permeability. Conclusions In peripheral artery disease, activation of the pentose phosphate pathway to promote angiogenesis may offer potential therapeutic advantages.

Myeloid Drp1 Deficiency Limits Revascularization in Ischemic Muscles via Inflammatory Macrophage Polarization and Metabolic Reprograming.

In the preclinical model of peripheral arterial disease (PAD), M2-like anti-inflammatory macrophage polarization and angiogenesis are required for revascularization. The regulation of cell metabolism and inflammation in macrophages is tightly linked to mitochondrial dynamics. Drp1, a mitochondrial fission protein, has shown context-dependent macrophage phenotypes with both pro- and anti-inflammatory characteristics. However, the role of macrophage Drp1 in reparative neovascularization remains unexplored. Here we show that Drp1 expression was significantly increased in F4/80+ macrophages within ischemic muscle at day 3 following hindlimb ischemia (HLI), an animal model of PAD. Myeloid-specific Drp1 -/- mice exhibited reduced limb perfusion recovery, angiogenesis and muscle regeneration after HLI. These effects were concomitant with enhancement of pro-inflammatory M1-like macrophages, p-NFkB, and TNFα levels, while showing reduction in anti-inflammatory M2-like macrophages and p-AMPK in ischemic muscle of myeloid Drp1 -/- mice. In vitro, Drp1 -/- macrophages under hypoxia serum starvation (HSS), an in vitro PAD model, demonstrated enhanced glycolysis via reducing p-AMPK as well as mitochondrial dysfunction and excessive mitochondrial ROS, resulting in increased M1-gene and reduced M2-gene expression. Conditioned media from HSS-treated Drp1 -/- macrophages exhibited increased secretion of pro-inflammatory cytokines and suppressed angiogenic responses in cultured endothelial cells. Thus, Drp1 deficiency in macrophages under ischemia drives inflammatory metabolic reprogramming and macrophage polarization, thereby limiting revascularization in experimental PAD.

Unexpected role of the copper transporter ATP7A in PDGF-induced vascular smooth muscle cell migration.

RATIONALE: Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. OBJECTIVE: To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. METHODS AND RESULTS: Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. CONCLUSIONS: These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.

Whole-Transcriptome Sequencing Analyses of Nuclear Antixoxidant-1 in Endothelial Cells: Role in Inflammation and Atherosclerosis.

Inflammation, oxidative stress, and copper (Cu) play an important role in cardiovascular disease, including atherosclerosis. We previously reported that cytosolic Cu chaperone antioxidant-1 (Atox1) translocates to the nucleus in response to inflammatory cytokines or exogenous Cu and that Atox1 is localized at the nucleus in the endothelium of inflamed atherosclerotic aorta. However, the roles of nuclear Atox1 and their function are poorly understood. Here we showed that Atox1 deficiency in ApoE-/- mice with a Western diet exhibited a significant reduction of atherosclerotic lesion formation. In vitro, adenovirus-mediated overexpression of nuclear-targeted Atox1 (Ad-Atox1-NLS) in cultured human endothelial cells (ECs) increased monocyte adhesion and reactive oxygen species (ROS) production compared to control cells (Ad-null). To address the underlying mechanisms, we performed genome-wide mapping of Atox1-regulated targets in ECs, using an unbiased systemic approach integrating sequencing data. Combination of ChIP-Seq and RNA-Seq analyses in ECs transfected with Ad-Atox1-NLS or Ad-null identified 1387 differentially expressed genes (DEG). Motif enrichment assay and KEGG pathway enrichment analysis revealed that 248 differentially expressed genes, including inflammatory and angiogenic genes, were regulated by Atox1-NLS, which was then confirmed by real-time qPCR. Among these genes, functional analysis of inflammatory responses identified CD137, CSF1, and IL5RA as new nuclear Atox1-targeted inflammatory genes, while CD137 is also a key regulator of Atox1-NLS-induced ROS production. These findings uncover new nuclear Atox1 downstream targets involved in inflammation and ROS production and provide insights into the nuclear Atox1 as a potential therapeutic target for the treatment of inflammatory diseases such as atherosclerosis.

Cysteine oxidation of copper transporter CTR1 drives VEGFR2 signalling and angiogenesis.

Vascular endothelial growth factor receptor type 2 (VEGFR2, also known as KDR and FLK1) signalling in endothelial cells (ECs) is essential for developmental and reparative angiogenesis. Reactive oxygen species and copper (Cu) are also involved in these processes. However, their inter-relationship is poorly understood. Evidence of the role of the endothelial Cu importer CTR1 (also known as SLC31A1) in VEGFR2 signalling and angiogenesis in vivo is lacking. Here, we show that CTR1 functions as a redox sensor to promote angiogenesis in ECs. CTR1-depleted ECs showed reduced VEGF-induced VEGFR2 signalling and angiogenic responses. Mechanistically, CTR1 was rapidly sulfenylated at Cys189 at its cytosolic C terminus after stimulation with VEGF, which induced CTR1-VEGFR2 disulfide bond formation and their co-internalization to early endosomes, driving sustained VEGFR2 signalling. In vivo, EC-specific Ctr1-deficient mice or CRISPR-Cas9-generated redox-dead Ctr1(C187A)-knockin mutant mice had impaired developmental and reparative angiogenesis. Thus, oxidation of CTR1 at Cys189 promotes VEGFR2 internalization and signalling to enhance angiogenesis. Our study uncovers an important mechanism for sensing reactive oxygen species through CTR1 to drive neovascularization.

The P-type ATPase transporter ATP7A promotes angiogenesis by limiting autophagic degradation of VEGFR2.

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.

Modification of Cardiac Progenitor Cell-Derived Exosomes by miR-322 Provides Protection against Myocardial Infarction through Nox2-Dependent Angiogenesis.

Myocardial infarction (MI) is the primary cause of cardiovascular mortality, and therapeutic strategies to prevent or mitigate the consequences of MI are a high priority. Cardiac progenitor cells (CPCs) have been used to treat cardiac injury post-MI, and despite poor engraftment, they have been shown to inhibit apoptosis and to promote angiogenesis through poorly understood paracrine effects. We previously reported that the direct injection of exosomes derived from CPCs (CPCexo) into mouse hearts provides protection against apoptosis in a model of acute ischemia/reperfusion injury. Moreover, we and others have reported that reactive oxygen species (ROS) derived from NADPH oxidase (NOX) can enhance angiogenesis in endothelial cells (ECs). Here we examined whether bioengineered CPCexo transfected with a pro-angiogenic miR-322 (CPCexo-322) can improve therapeutic efficacy in a mouse model of MI as compared to CPCexo. Systemic administration of CPCexo-322 in mice after ischemic injury provided greater protection post-MI than control CPCexo, in part, through enhanced angiogenesis in the border zones of infarcted hearts. Mechanistically, the treatment of cultured human ECs with CPCexo-322 resulted in a greater angiogenic response, as determined by increased EC migration and capillary tube formation via increased Nox2-derived ROS. Our study reveals that the engineering of CPCexo via microRNA (miR) programing can enhance angiogenesis, and this may be an effective therapeutic strategy for the treatment of ischemic cardiovascular diseases.

Redox Regulation of Mitochondrial Fission Protein Drp1 by Protein Disulfide Isomerase Limits Endothelial Senescence.

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.