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.

Role of prostaglandin D2 receptors in the pathogenesis of abdominal aortic aneurysm formation.

Prostaglandin D2 (PGD2) released from immune cells or other cell types activates its receptors, D prostanoid receptor (DP)1 and 2 (DP1 and DP2), to promote inflammatory responses in allergic and lung diseases. Prostaglandin-mediated inflammation may also contribute to vascular diseases such as abdominal aortic aneurysm (AAA). However, the role of DP receptors in the pathogenesis of AAA has not been systematically investigated. In the present study, DP1-deficient mice and pharmacological inhibitors of either DP1 or DP2 were tested in two distinct mouse models of AAA formation: angiotensin II (AngII) infusion and calcium chloride (CaCl2) application. DP1-deficient mice [both heterozygous (DP1+/-) and homozygous (DP1-/-)] were protected against CaCl2-induced AAA formation, in conjunction with decreased matrix metallopeptidase (MMP) activity and adventitial inflammatory cell infiltration. In the AngII infusion model, DP1+/- mice, but not DP1-/- mice, exhibited reduced AAA formation. Interestingly, compensatory up-regulation of the DP2 receptor was detected in DP1-/- mice in response to AngII infusion, suggesting a potential role for DP2 receptors in AAA. Treatment with selective antagonists of DP1 (laropiprant) or DP2 (fevipiprant) protected against AAA formation, in conjunction with reduced elastin degradation and aortic inflammatory responses. In conclusion, PGD2 signaling contributes to AAA formation in mice, suggesting that antagonists of DP receptors, which have been extensively tested in allergic and lung diseases, may be promising candidates to ameliorate AAA.

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.

Oxidant Signaling Mediated by Nox2 in Neutrophils Promotes Regenerative Myelopoiesis and Tissue Recovery following Ischemic Damage.

Ischemic tissue damage activates hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM)-generating myeloid cells, and persistent HSPC activity may drive chronic inflammation and impair tissue recovery. Although increased reactive oxygen species in the BM regulate HSPC functions, their roles in myelopoiesis of activated HSPCs and subsequent tissue recovery during ischemic damage are not well understood. In this paper, we report that deletion of Nox2 NADPH oxidase in mice results in persistent elevations in BM HSPC activity and levels of inflammatory monocytes/macrophages in BM and ischemic tissue in a model of hindlimb ischemia. Ischemic tissue damage induces oxidants in BM such as elevations of hydrogen peroxide and oxidized phospholipids, which activate redox-sensitive Lyn kinase in a Nox2-dependent manner. Moreover, during tissue recovery after ischemic injury, this Nox2-ROS-Lyn kinase axis is induced by Nox2 in neutrophils that home to the BM, which inhibits HSPC activity and inflammatory monocyte generation and promotes tissue regeneration after ischemic damage. Thus, oxidant signaling in the BM mediated by Nox2 in neutrophils regulates myelopoiesis of HSPCs to promote regeneration of damaged tissue.

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 transporters and copper chaperones: roles in cardiovascular physiology and disease.

Copper (Cu) is an essential micronutrient but excess Cu is potentially toxic. Its important propensity to cycle between two oxidation states accounts for its frequent presence as a cofactor in many physiological processes through Cu-containing enzymes, including mitochondrial energy production (via cytochrome c-oxidase), protection against oxidative stress (via superoxide dismutase), and extracellular matrix stability (via lysyl oxidase). Since free Cu is potentially toxic, the bioavailability of intracellular Cu is tightly controlled by Cu transporters and Cu chaperones. Recent evidence reveals that these Cu transport systems play an essential role in the physiological responses of cardiovascular cells, including cell growth, migration, angiogenesis and wound repair. In response to growth factors, cytokines, and hypoxia, their expression, subcellular localization, and function are tightly regulated. Cu transport systems and their regulators have also been linked to various cardiovascular pathophysiologies such as hypertension, inflammation, atherosclerosis, diabetes, cardiac hypertrophy, and cardiomyopathy. A greater appreciation of the central importance of Cu transporters and Cu chaperones in cell signaling and gene expression in cardiovascular biology offers the possibility of identifying new therapeutic targets for cardiovascular disease.

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.

ROS-induced ROS release orchestrated by Nox4, Nox2, and mitochondria in VEGF signaling and angiogenesis.

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.

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.

Low-dose 6-bromoindirubin-3'-oxime induces partial dedifferentiation of endothelial cells to promote increased neovascularization.

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-3ß, induced the stabilization of ß-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 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 vascular endothelial growth factor (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(+) cell recruitment around the neovessels. Thus, these results demonstrate 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 ß-catenin and the NANOG signaling pathway.

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.

A novel regulator of angiogenesis in endothelial cells: 5-hydroxytriptamine 4 receptor.

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.

NADPH oxidase 2 regulates bone marrow microenvironment following hindlimb ischemia: role in reparative mobilization of progenitor cells.

Bone marrow (BM) microenvironment, which is regulated by hypoxia and proteolytic enzymes, is crucial for stem/progenitor cell function and mobilization involved in postnatal neovascularization. We demonstrated that NADPH oxidase 2 (Nox2)-derived reactive oxygen species (ROS) are involved in postischemic mobilization of BM cells and revascularization. However, role of Nox2 in regulating BM microenvironment in response to ischemic injury remains unknown. Here, we show that hindlimb ischemia of mice increases ROS production in both the endosteal and central region of BM tissue in situ, which is almost completely abolished in Nox2 knockout (KO) mice. This Nox2-dependent ROS production is mainly derived from Gr-1(+) myeloid cells in BM. In vivo injection of hypoxyprobe reveals that endosteum at the BM is hypoxic with high expression of hypoxia-inducible factor-1α in basal state. Following hindlimb ischemia, hypoxic areas and HIF-1α expression are expanded throughout the BM, which is inhibited in Nox2 KO mice. This ischemia-induced alteration of Nox2-dependent BM microenvironment is associated with an increase in vascular endothelial growth factor expression and Akt phosphorylation in BM tissue, thereby promoting Lin(-) progenitor cell survival and expansion, leading to their mobilization from BM. Furthermore, hindlimb ischemia increases proteolytic enzymes membrane type 1-matrix metalloproteinase (MMP) expression and MMP-9 activity in BM, which is inhibited in Nox2 KO mice. In summary, Nox2-dependent increase in ROS plays a critical role in regulating hypoxia expansion and proteolytic activities in BM microenvironment in response to tissue ischemia. This in turn promotes progenitor cell expansion and reparative mobilization from BM, leading to postischemic neovascularization and tissue repair.

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.