VEGF-Induced Endothelial Podosomes via ROCK2-Dependent Thrombomodulin Expression Initiate Sprouting Angiogenesis.

[Figure: see text].

CM082, a novel VEGF receptor tyrosine kinase inhibitor, can inhibit angiogenesis in vitro and in vivo.

The goal of this study was to evaluate the effects of CM082, a novel vascular endothelial growth factor (VEGF) receptor-2 tyrosine kinase inhibitor, on human umbilical vein endothelial cells (HUVECs), and oxygen-induced retinopathy (OIR) mice. HUVECs were stimulated with rHuVEGF165 and then treated with CM082 to assess the antiangiogenic effects of CM082; subsequently, proliferation, wound-healing migration, Transwell invasion, tube formation assays, and Western blotting were performed in vitro. Retinal neovascularization tufts, avascular area, and TUNEL assays were estimated for OIR mice after intraperitoneal injection with CM082. CM082 significantly inhibited proliferation, migration, invasion, and tube formation induced by stimulation of HUVECs with rHuVEGF165; this inhibitory effect was mediated by blocking VEGFR2 activation. CM082 significantly inhibited retinal neovascularization and avascular area and did not increase apoptosis in the retina of OIR mice. The findings demonstrated that CM082 exhibits highly antiangiogenic effects in HUVECs and OIR mice. Thus, it may serve as an alternative treatment for neovascular eye disease in the future.

A Novel Small Peptide H-KI20 Inhibits Retinal Neovascularization Through the JNK/ATF2 Signaling Pathway.

Purpose: Abundant evidence has shown benefits of antivascular endothelial growth factor (anti-VEGF) therapies in neovascular eye diseases. However, the high cost, side effects, and inconvenience of frequent injections demand alternative novel drug candidates. This study aimed to analyze antiangiogenic effects of peptide H-KI20 and illustrated signaling mechanisms. Methods: Live cell culture and tracing, wound healing assay, and tube formation were performed in human retinal microvascular endothelial cells (HRECs). The chick embryo chorioallantoic membrane and mouse oxygen-induced ischemic retinopathy model were applied to examine the effects of H-KI20 in vivo. The intracellular signaling pathways were examined. Molecular docking and surface plasmon resonance assay were used to validate the direct interaction of H-KI20 and c-Jun N-terminal kinase 2 (JNK2). Results: H-KI20 had high penetration ability in vitro and in vivo. It inhibited motility, migration, and tube formation of HRECs, without cytotoxicity, and inhibited angiogenesis in vivo. Furthermore, H-KI20 treatment reduced the phosphorylation level of activating transcription factor 2 (ATF2) stimulated by VEGF via downregulating p-JNK. H-KI20 bound to JNK2 directly with a dissociation constant value of 83.68 µM. The knockdown of ATF2 attenuated VEGF-induced tube formation and decreased the movement speed of HRECs. Conclusions: H-KI20 inhibited angiogenesis both in vitro and in vivo. The ratios of p-ATF2/ATF2 and p-JNK/JNK stimulated by VEGF were decreased by H-KI20, and H-KI20 targeted JNK2 directly. In addition, the pivotal role of ATF2 in VEGF-induced retinal neovascularization was elucidated for the first time. Taken together, H-KI20 displays potential for pathological retinal angiogenesis as a sustained and low-toxic peptide.

Therapeutic Potential of Tpl2 (Tumor Progression Locus 2) Inhibition on Diabetic Vasculopathy Through the Blockage of the Inflammasome Complex.

OBJECTIVE: Diabetic retinopathy, one of retinal vasculopathy, is characterized by retinal inflammation, vascular leakage, blood-retinal barrier breakdown, and neovascularization. However, the molecular mechanisms that contribute to diabetic retinopathy progression remain unclear. Approach and Results: Tpl2 (tumor progression locus 2) is a protein kinase implicated in inflammation and pathological vascular angiogenesis. Nε-carboxymethyllysine (CML) and inflammatory cytokines levels in human sera and in several diabetic murine models were detected by ELISA, whereas liquid chromatography-tandem mass spectrometry analysis was used for whole eye tissues. The CML and p-Tpl2 expressions on the human retinal pigment epithelium (RPE) cells were determined by immunofluorescence. Intravitreal injection of pharmacological inhibitor or NA (neutralizing antibody) was used in a diabetic rat model. Retinal leukostasis, optical coherence tomography, and H&E staining were used to observe pathological features. Sera of diabetic retinopathy patients had significantly increased CML levels that positively correlated with diabetic retinopathy severity and foveal thickness. CML and p-Tpl2 expressions also significantly increased in the RPE of both T1DM and T2DM diabetes animal models. Mechanistic studies on RPE revealed that CML-induced Tpl2 activation and NADPH oxidase, and inflammasome complex activation were all effectively attenuated by Tpl2 inhibition. Tpl2 inhibition by NA also effectively reduced inflammatory/angiogenic factors, retinal leukostasis in streptozotocin-induced diabetic rats, and RPE secretion of inflammatory cytokines. The attenuated release of angiogenic factors led to inhibited vascular abnormalities in the diabetic animal model. CONCLUSIONS: The inhibition of Tpl2 can block the inflammasome signaling pathway in RPE and has potential clinical and therapeutic implications in diabetes-associated retinal microvascular dysfunction.

Endothelium-specific deletion of Nox4 delays retinal vascular development and mitigates pathological angiogenesis.

NADPH oxidase 4 (Nox4) is a major isoform of NADPH oxidases playing an important role in many biological processes. Previously we have shown that Nox4 is highly expressed in retinal blood vessels and is upregulated in oxygen-induced retinopathy (OIR). However, the exact role of endothelial Nox4 in retinal angiogenesis remains elusive. Herein, using endothelial cell (EC)-specific Nox4 knockout (Nox4EC-KO) mice, we investigated the impact of endothelial Nox4 deletion on retinal vascular development and pathological angiogenesis during OIR. Our results show that deletion of Nox4 in ECs led to retarded retinal vasculature development with fewer, blunted-end tip cells and sparser, dysmorphic filopodia at vascular front, and reduced density of vascular network in superficial, deep, and intermediate layers in postnatal day 7 (P7), P12, and P17 retinas, respectively. In OIR, loss of endothelial Nox4 had no effect on hyperoxia-induced retinal vaso-obliteration at P9 but significantly reduced aberrant retinal neovascularization at P17 and decreased the deep layer capillary density at P25. Ex vivo study confirmed that lack of Nox4 in ECs impaired vascular sprouting. Mechanistically, loss of Nox4 significantly reduced expression of VEGF, p-VEGFR2, integrin αV, angiopoietin-2, and p-ERK1/2, attenuating EC migration and proliferation. Taken together, our results indicate that endothelial Nox4 is important for retinal vascular development and contributes to pathological angiogenesis, likely through regulation of VEGF/VEGFR2 and angiopoietin-2/integrin αV/ERK pathways. In addition, our study suggests that endothelial Nox4 appears to be essential for intraretinal revascularization after hypoxia. These findings call for caution on targeting endothelial Nox4 in ischemic/hypoxic retinal diseases.

p38 promoted retinal micro-angiogenesis through up-regulated RUNX1 expression in diabetic retinopathy.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and is characterized by visible microvascular alterations including retinal ischemia-reperfusion injury, inflammation, abnormal permeability, neovascularization and macular edema. Despite the available treatments, some patients present late in the course of the disease when treatment is more difficult. Hence, it is crucial that the new targets are found and utilized in the clinical therapy of DR. In the present study, we constructed a DR animal model and a model in HRMECs to investigate the relationship between p38 and RUNX1 in retinal micro-angiogenesis in diabetic retinopathy. We found that p38 could promote retinal micro-angiogenesis by up-regulating RUNX1 expression in diabetic retinopathy. This suggested that the p38/ RUNX1 pathway could become a new retinal micro-angiogenesis target in DR treatment.

Association analysis of genetic polymorphisms and expression levels of selected genes involved in extracellular matrix turnover and angiogenesis with the risk of age-related macular degeneration.

BACKGROUND: Age-related macular degeneration is a progressive eye disease affecting the macula and causing acute visual loss particularly in elder people. The aim of the study was an attempt to discern an influence of expression levels and functional genetic polymorphisms of selected genes related to the extracellular matrix turnover or neovascularization on age-related macular degeneration occurrence and progression. METHODS: We conducted a case-control study of 200 polish patients with recognized age-related macular degeneration (dry and wet) and compared the results with those obtained from matched 100 healthy control subjects. TaqMan Genotyping Assays were employed to examine the following single nucleotide polymorphisms: matrix metalloproteinase (MMP)-2 -735C/T, MMP-7 -181A/G, MMP-9 -1702T/A, and -1562C/T; tissue inhibitors of metalloproteinase (TIMP)-2 -418G/C; vascular endothelial growth factor (VEGF) +405 G/C and +936 C/T, VEGFR-2 +1719 T/A and -271 G/A. Real-time polymerase chain reaction was assessed to determine the mRNA quantity. Serum levels of proteins were measured using enzyme-linked immunosorbent assay. RESULTS: The single nucleotide polymorphism genotyping showed that TT genotype for MMP-9 -1702T/A and CC genotype for VEGF +936C/T increase markedly the risk of age-related macular degeneration but do not influence on its progression. Additionally, the possible protective effect of CC genetic variant in MMP-9 -1562C/T polymorphism against progression of age-related macular degeneration was observed. We also found significant differences in systemic expression levels of MMP-2, -7, -9, TIMP-2, vascular endothelial growth factor, VEGFR-2, and pigment epithelium-derived factor between studied group. The research demonstrated evident differences in serum levels of MMP-2, -7, -9, TIMP-2, vascular endothelial growth factor, and pigment epithelium-derived factor between wet and dry age-related macular degeneration patients. CONCLUSIONS: We can conclude that disturbances in angiogenic homeostasis and processes of extracellular matrix turnover occurring in age-related macular degeneration-affected ocular tissues may be reflected in changes in systemic expression levels of the investigated genes.

Chebulagic acid Chebulinic acid and Gallic acid, the active principles of Triphala, inhibit TNFα induced pro-angiogenic and pro-inflammatory activities in retinal capillary endothelial cells by inhibiting p38, ERK and NFkB phosphorylation.

Tumor necrosis factor-α (TNFα) a pleiotropic cytokine induces pro-inflammatory and pro-angiogenic changes in conditions such as diabetic retinopathy (DR) and neovascular age related macular degeneration (NV-AMD). Hence, inhibition of TNFα mediated changes can benefit the management of DR and NV-AMD. Triphala, an ayurvedic herbal preparation is known to have immunomodulatry functions. In this study we evaluated the alcoholic extract of triphala (AlE) and its compounds Chebulagic acid (CA), Chebulinic acid (CI) and Gallic acid (GA) for their anti-TNFα activity. TNFα induced pro-inflammatory and pro-angiogenic changes in the retinal-choroid microvascular endothelial cells (RF/6A). Treatment with CA/CI/GA and the whole Triphala extract showed characteristic inhibition of MMP-9, cell proliferation/migration and tube formation as well the expression of IL-6, IL-8 and MCP-1 without affecting cell viability. This was mediated by inhibition of p38, ERK and NFκB phosphorylation. Ex vivo angiogenesis assay using chick chorioallantoic membrane (CAM) model also showed that TNFα-induced angiogenesis and it was inhibited by AlE and its active principles. Further, in silico studies revealed that CA, CI and GA are capable of binding the TNFα-receptor-1 to mediate anti-TNFα activity. This study explains the immunomodulatory function of Triphala, evaluated in the context of retinal and choroid vasculopathies in vitro and ex vivo; which showed that CA, CI and GA can be a potential pharmacological agents in the management of DR and NV-AMD.

Sphingosine Kinase 2 Modulates Retinal Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy.

Purpose: Neovascularization is a major cause of blindness in various ocular diseases. Bioactive sphingosine 1-phosphate (S1P), synthesized by two sphingosine kinases (Sphk1, Sphk2), emerged as a key player in a multitude of cellular processes, including cell survival, proliferation, inflammation, migration, and angiogenesis. We investigated the role of Sphk2, S1P, and S1P receptors (S1PR) during retinal neovascularization using the oxygen-induced retinopathy mouse model (OIR). Methods: Sphk2 overexpressing (tgSphk2) and Sphk2 knockout (Sphk2-/-) mice were used in the OIR model, exposed to 75% O2 over 5 days from postnatal day (P)7 to 12 to initiate vessel regression. After returning to room air, these mice developed a marked neovascularization. Retinae recovered from untreated and treated eyes at P7, P12, P14, and P17 were used for lectin-stained retinal whole mounts, mass spectrometry, and quantitative real-time PCR. Results: tgSphk2 mice showed higher retinal S1P concentrations, accelerated retinal angiogenesis, and increased neovascularization. Expression of S1PR, vascular endothelial growth factor α (VEGFα), and angiopoietin 1 and 2 was differentially regulated during the course of OIR in the different genotypes. Sphk2-/- displayed a markedly reduced retinal angiogenesis and neovascularization as well as decreased VEGFα and angiopoietin expression. Conclusions: Using genetic models of Sphk2 overexpression or deletion we demonstrate a strong impact of Sphk2/S1P on retinal vasculopathy and expression of vascular growth factors like VEGF and angiopoietin in the retina. Consequently, Sphk2, S1P, and S1PR may offer attractive novel therapeutic targets for ischemic retinopathies.

Thioredoxin-interacting protein deficiency ameliorates diabetic retinal angiogenesis.

Diabetic retinopathy is the leading cause of blindness among working-aged adults around the world. Hyperglycemia and intraocular vascular endothelial growth factor (VEGF) over-accumulation are essential for the progression of diabetic retinopathy, which eventually results in proliferative diabetic retinopathy, characterized by pathologic angiogenesis and impaired vision. Thioredoxin-interacting protein (TXNIP) was highly induced in retinal endothelial cells under diabetic conditions. However, the role of TXNIP in diabetes-associated retinal angiogenesis remains elusive. Here, we investigated whether the absence of TXNIP alters diabetes-associated retinal angiogenesis. Exposure of human retinal microvascular endothelial cells (HRMECs) to moderately high glucose (MHG) promoted cell migration and tube formation, but not proliferation. Knockdown of TXNIP suppressed moderately high glucose (MHG)-induced reactive oxygen species (ROS) generation, migration, tube formation and activation of Akt/mTOR pathway in HRMECs. Moreover, gene silencing of TXNIP inhibited VEGF-induced angiogenic response by blocking VEGFR2 and downstream signal pathway Akt/mTOR activation in HRMECs. Furthermore, TXNIP knockout inhibited VEGF or VEGF and MHG-induced retinal angiogenesis ex vivo compared with wild-type mice. In conclusion, our study demonstrated that TXNIP deficiency inhibited VEGF or/and MHG-induced angiogenic response in HRMECs and mice retinas and suggested TXNIP may be a potential therapy target for treating proliferative diabetic retinopathy.

Cytochrome P450 Oxidase 2C Inhibition Adds to ω-3 Long-Chain Polyunsaturated Fatty Acids Protection Against Retinal and Choroidal Neovascularization.

OBJECTIVE: Pathological ocular neovascularization is a major cause of blindness. Increased dietary intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFA) reduces retinal neovascularization and choroidal neovascularization (CNV), but ω-3 LCPUFA metabolites of a major metabolizing pathway, cytochrome P450 oxidase (CYP) 2C, promote ocular pathological angiogenesis. We hypothesized that inhibition of CYP2C activity will add to the protective effects of ω-3 LCPUFA on neovascular eye diseases. APPROACH AND RESULTS: The mouse models of oxygen-induced retinopathy and laser-induced CNV were used to investigate pathological angiogenesis in the retina and choroid, respectively. The plasma levels of ω-3 LCPUFA metabolites of CYP2C were determined by mass spectroscopy. Aortic ring and choroidal explant sprouting assays were used to investigate the effects of CYP2C inhibition and ω-3 LCPUFA-derived CYP2C metabolic products on angiogenesis ex vivo. We found that inhibition of CYP2C activity by montelukast added to the protective effects of ω-3 LCPUFA on retinal neovascularization and CNV by 30% and 20%, respectively. In CYP2C8-overexpressing mice fed a ω-3 LCPUFA diet, montelukast suppressed retinal neovascularization and CNV by 36% and 39% and reduced the plasma levels of CYP2C8 products. Soluble epoxide hydrolase inhibition, which blocks breakdown and inactivation of CYP2C ω-3 LCPUFA-derived active metabolites, increased oxygen-induced retinopathy and CNV in vivo. Exposure to selected ω-3 LCPUFA metabolites of CYP2C significantly reversed the suppression of both angiogenesis ex vivo and endothelial cell functions in vitro by the CYP2C inhibitor montelukast. CONCLUSIONS: Inhibition of CYP2C activity adds to the protective effects of ω-3 LCPUFA on pathological retinal neovascularization and CNV.

The Cytosolic NADPH Oxidase Subunit NoxO1 Promotes an Endothelial Stalk Cell Phenotype.

OBJECTIVE: Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases contribute to angiogenesis and vascular repair. NADPH oxidase organizer 1 (NoxO1) is a cytosolic protein facilitating assembly of constitutively active NADPH oxidases. We speculate that NoxO1 also contributes to basal reactive oxygen species formation in the vascular system and thus modulates angiogenesis. APPROACH AND RESULTS: A NoxO1 knockout mouse was generated, and angiogenesis was studied in cultured cells and in vivo. Angiogenesis of the developing retina and after femoral artery ligation was increased in NoxO1(-/-) when compared with wild-type animals. Spheroid outgrowth assays revealed greater angiogenic capacity of NoxO1(-/-) lung endothelial cells (LECs) and a more tip-cell-like phenotype than wild-type LECs. Usually signaling by the Notch pathway switches endothelial cells from a tip into a stalk cell phenotype. NoxO1(-/-) LECs exhibited attenuated Notch signaling as a consequence of an attenuated release of the Notch intracellular domain on ligand stimulation. This release is mediated by proteolytic cleavage involving the α-secretase ADAM17. For maximal activity, ADAM17 has to be oxidized, and overexpression of NoxO1 promoted this mode of activation. Moreover, the activity of ADAM17 was reduced in NoxO1(-/-) LECs when compared with wild-type LECs. CONCLUSIONS: NoxO1 stimulates α-secretase activity probably through reactive oxygen species-mediated oxidation. Deletion of NoxO1 attenuates Notch signaling and thereby promotes a tip-cell phenotype that results in increased angiogenesis.

LRP1 Regulates Retinal Angiogenesis by Inhibiting PARP-1 Activity and Endothelial Cell Proliferation.

OBJECTIVE: We recently demonstrated that low-density lipoprotein receptor-related protein 1 (LRP1) is required for cardiovascular development in zebrafish. However, what role LRP1 plays in angiogenesis remains to be determined. To better understand the role of LRP1 in endothelial cell function, we investigated how LRP1 regulates mouse retinal angiogenesis. APPROACH AND RESULTS: Depletion of LRP1 in endothelial cells results in increased retinal neovascularization in a mouse model of oxygen-induced retinopathy. Specifically, retinas in mice lacking endothelial LRP1 have more branching points and angiogenic sprouts at the leading edge of the newly formed vasculature. Increased endothelial proliferation as detected by Ki67 staining was observed in LRP1-deleted retinal endothelium in response to hypoxia. Using an array of biochemical and cell biology approaches, we demonstrate that poly(ADP-ribose) polymerase-1 (PARP-1) directly interacts with LRP1 in human retinal microvascular endothelial cells. This interaction between LRP1 and PARP-1 decreases under hypoxic condition. Moreover, LRP1 knockdown results in increased PARP-1 activity and subsequent phosphorylation of both retinoblastoma protein and cyclin-dependent kinase 2, which function to promote cell cycle progression and angiogenesis. CONCLUSIONS: Together, these data reveal a pivotal role for LRP1 in endothelial cell proliferation and retinal neovascularization induced by hypoxia. In addition, we demonstrate for the first time the interaction between LRP1 and PARP-1 and the LRP1-dependent regulation of PARP-1-signaling pathways. These data bring forth the possibility of novel therapeutic approaches for pathological angiogenesis.

[Research advance of retinal pathological angiogenesis related to ATM protein kinase].

Ataxia-telangiectasia mutated (ATM) gene could cause ataxia telangiectasia which is an autosomal recessive disease. The ATM protein kinase encoded by the ATM gene mainly distributed in nucleus as a master regulator of the DNA damage response and apoptosis via cell signaling pathways. The ATM kinase plays a key role in the pathogenesis of cellular senescence and tumor genesis. Recently, some studies have indicated that ATM protein kinase is involved in pathological neovascularization, suggesting that it could be a novel potential therapeutic target in diseases associated with pathological angiogenesis.

Characterization of oxygen-induced retinopathy in mice carrying an inactivating point mutation in the catalytic site of ADAM15.

PURPOSE: Retinal neovascularization is found in diseases such as macular degeneration, diabetic retinopathy, or retinopathy of prematurity and is usually caused by alterations in oxygen supply. We have previously described that mice lacking the membrane-anchored metalloproteinase ADAM15 (a Disintegrin and Metalloprotease 15) have decreased pathological neovascularization of the retina in the oxygen-induced retinopathy (OIR) model. The main purpose of the present study was to determine the contribution of the catalytic activity of ADAM15 to OIR. METHODS: To address this question, we generated knock-in mice carrying an inactivating Glutamate to Alanine (E>A) point mutation in the catalytic site of ADAM15 (Adam15E>A mice) and subjected these animals to the OIR model and a heterotopic tumor model. Moreover, we used cell-based assays to determine whether ADAM15 can process cell surface receptors involved in angiogenesis. RESULTS: We found that pathological neovascularization in the OIR model in Adam15E>A mice was comparable to that observed in wild type mice, but tumor implantation by heterotopically injected melanoma cells was reduced. In cell-based assays, overexpressed ADAM15 could process the FGFR2iiib, but was unable to process several receptors with roles in angiogenesis. CONCLUSIONS: Collectively, these results suggest that the catalytic activity of ADAM15 is not crucial for its function in promoting pathological neovascularization in the mouse OIR model, most likely because of the very limited substrate repertoire of ADAM15. Instead, other noncatalytic functions of ADAM15 must be important for its role in the OIR model.

Nucleoside diphosphate kinase B regulates angiogenesis through modulation of vascular endothelial growth factor receptor type 2 and endothelial adherens junction proteins.

OBJECTIVE: Nucleoside diphosphate kinase B (NDPKB) participates in the activation of heterotrimeric and monomeric G proteins, which are pivotal mediators in angiogenic signaling. The role of NDPKB in angiogenesis has to date not been defined. Therefore, we analyzed the contribution of NDPKB to angiogenesis and its underlying mechanisms in well-characterized in vivo and in vitro models. APPROACH AND RESULTS: Zebrafish embryos were depleted of NDPKB by morpholino-mediated knockdown. These larvae displayed severe malformations specifically in vessels formed by angiogenesis. NDPKB-deficient (NDPKB(-/-)) mice were subjected to oxygen-induced retinopathy. In this model, the number of preretinal neovascularizations in NDPKB(-/-) mice was strongly reduced in comparison with wild-type littermates. In accordance, a delayed blood flow recovery was detected in the NDPKB(-/-) mice after hindlimb ligation. In in vitro studies, a small interfering RNA-mediated knockdown of NDPKB was performed in human umbilical endothelial cells. NDPKB depletion impaired vascular endothelial growth factor (VEGF)-induced sprouting and hampered the VEGF-induced spatial redistributions of the VEGF receptor type 2 and VE-cadherin at the plasma membrane. Concomitantly, NDPKB depletion increased the permeability of the human umbilical endothelial cell monolayer. CONCLUSIONS: This is the first report to show that NDPKB is required for VEGF-induced angiogenesis and contributes to the correct localization of VEGF receptor type 2 and VE-cadherin at the endothelial adherens junctions. Therefore, our data identify NDPKB as a novel molecular target to modulate VEGF-dependent angiogenesis.

Endothelial deletion of phospholipase D2 reduces hypoxic response and pathological angiogenesis.

OBJECTIVE: Aberrant regulation of the proliferation, survival, and migration of endothelial cells (ECs) is closely related to the abnormal angiogenesis that occurs in hypoxia-induced pathological situations, such as cancer and vascular retinopathy. Hypoxic conditions and the subsequent upregulation of hypoxia-inducible factor-1α and target genes are important for the angiogenic functions of ECs. Phospholipase D2 (PLD2) is a crucial signaling mediator that stimulates the production of the second messenger phosphatidic acid. PLD2 is involved in various cellular functions; however, its specific roles in ECs under hypoxia and in vivo angiogenesis remain unclear. In the present study, we investigated the potential roles of PLD2 in ECs under hypoxia and in hypoxia-induced pathological angiogenesis in vivo. APPROACH AND RESULTS: Pld2 knockout ECs exhibited decreased hypoxia-induced cellular responses in survival, migration, and thus vessel sprouting. Analysis of hypoxia-induced gene expression revealed that PLD2 deficiency disrupted the upregulation of hypoxia-inducible factor-1α target genes, including VEGF, PFKFB3, HMOX-1, and NTRK2. Consistent with this, PLD2 contributed to hypoxia-induced hypoxia-inducible factor-1α expression at the translational level. The roles of PLD2 in hypoxia-induced in vivo pathological angiogenesis were assessed using oxygen-induced retinopathy and tumor implantation models in endothelial-specific Pld2 knockout mice. Pld2 endothelial-specific knockout retinae showed decreased neovascular tuft formation, despite a larger avascular region. Tumor growth and tumor blood vessel formation were also reduced in Pld2 endothelial-specific knockout mice. CONCLUSIONS: Our findings demonstrate a novel role for endothelial PLD2 in the survival and migration of ECs under hypoxia via the expression of hypoxia-inducible factor-1α and in pathological retinal angiogenesis and tumor angiogenesis in vivo.

Involvement of Nox2 NADPH oxidase in retinal neovascularization.

PURPOSE: The proliferation of new blood vessels in the retina is a leading cause of vision impairment. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) is involved in cell signaling for ischemia-induced angiogenesis, but its role in retinal neovascularization is unclear. We have analyzed the dependence of retinal neovascularization on the Nox2 isoform in oxygen-induced retinopathy (OIR) in mice. METHODS: Neonatal C57BL/6 mice aged 7 days (P7) were placed in a hyperoxic chamber (75% O2) for 5 days, followed by 5 days of exposure to room air. Eyes were harvested on P8 and P17 for the quantification of retinal vaso-obliteration and neovascularization, respectively. The retinal expression of Nox2 and VEGF-A were measured by RT-PCR, while superoxide generation was detected by in situ dihydroethidium (DHE) staining of fresh frozen sections. RESULTS: In wild type (WT) mice, OIR was characterized by central retinal vaso-obliteration at P8 and neovascularization at P17, which was associated with increases in Nox2 and VEGF-A gene expression, superoxide generation, and accumulation of Iba-1 positive cells in the inner retina. In contrast, Nox2 knockout mice exhibited markedly less retinal neovascularization and VEGF-A mRNA expression at P17, despite showing comparable vaso-obliteration at P8. These changes were accompanied by reductions in DHE fluorescence and Iba-1-positive cell accumulation in the hypoxic retina. CONCLUSIONS: The Nox2-generated reactive oxygen species (ROS) facilitate the retinal expression of VEGF-A and neovascularization in this mouse model of OIR. Therapies targeting Nox2 could be of value to reduce aberrant retinal neovascularization in retinopathy of prematurity, diabetes, and other disease processes driven by VEGF.