Formation Of Neovascular Tufts Research Articles

The Anti-Diabetic Drug Metformin Suppresses Pathological Retinal Angiogenesis via Blocking the mTORC1 Signaling Pathway in Mice (Metformin Suppresses Pathological Angiogenesis)

Purpose Metformin, a biguanide antihyperglycemic drug, can exert various beneficial effects in addition to its glucose-lowering effect. The effects of metformin are mainly mediated by AMP-activated protein kinase (AMPK)-dependent pathway. AMPK activation interferes with the action of the mammalian target of rapamycin complex 1 (mTORC1), and blockade of mTORC1 pathway suppresses pathological retinal angiogenesis. Therefore, in this study, we examined the effects of metformin on pathological angiogenesis and mTORC1 activity in the retinas of mice with oxygen-induced retinopathy (OIR). Methods OIR was induced by exposing the mice to 80% oxygen from postnatal day (P) 7 to P10. The OIR mice were treated with metformin, rapamycin (an inhibitor of mTORC1), or the vehicle from P10 to P12 or P14. The formation of neovascular tufts, revascularization in the central avascular areas, expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) 2, and phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTORC1 activity, were evaluated at P10, P13, or P15. Results Neovascular tufts and vascular growth in the central avascular areas were observed in the retinas of P15 OIR mice. The formation of neovascular tufts, but not the revascularization in the central avascular areas, was attenuated by metformin administration from P10 to P14. Metformin had no significant inhibitory effect on the expression of VEGF and VEGFR2, but it reduced the pS6 immunoreactivity in vascular cells at the sites of angiogenesis. Rapamycin completely blocked the phosphorylation of ribosomal protein S6 and markedly reduced the formation of neovascular tufts. Conclusions These results suggest that metformin partially suppresses the formation of neovascular tufts on the retinal surface by blocking the mTORC1 signaling pathway. Metformin may exert beneficial effects against the progression of ocular diseases in which abnormal angiogenesis is associated with the pathogenesis.

Triciribine attenuates pathological neovascularization and vascular permeability in a mouse model of proliferative retinopathy

Proliferative retinopathies are the leading cause of irreversible blindness in all ages, and there is a critical need to identify novel therapies. We investigated the impact of triciribine (TCBN), a tricyclic nucleoside analog and a weak Akt inhibitor, on retinal neurovascular injury, vascular permeability, and inflammation in oxygen-induced retinopathy (OIR). Post-natal day 7 (P7) mouse pups were subjected to OIR, and treated (i.p.) with TCBN or vehicle from P14-P16 and compared with age-matched, normoxic, vehicle or TCBN-treated controls. P17 retinas were processed for flat mounts, immunostaining, Western blotting, and qRT-PCR studies. Fluorescein angiography, electroretinography, and spectral domain optical coherence tomography were performed on days P21, P26, and P30, respectively. TCBN treatment significantly reduced pathological neovascularization, vaso-obliteration, and inflammation marked by reduced TNFα, IL6, MCP-1, Iba1, and F4/80 (macrophage/microglia markers) expression compared to the vehicle-treated OIR mouse retinas. Pathological expression of VEGF (vascular endothelial growth factor), and claudin-5 compromised the blood-retinal barrier integrity in the OIR retinas correlating with increased vascular permeability and neovascular tuft formation, which were blunted by TCBN treatment. Of note, there were no changes in the retinal architecture or retinal cell function in response to TCBN in the normoxia or OIR mice. We conclude that TCBN protects against pathological neovascularization, restores blood-retinal barrier homeostasis, and reduces retinal inflammation without adversely affecting the retinal structure and neuronal function in a mouse model of OIR. Our data suggest that TCBN may provide a novel therapeutic option for proliferative retinopathy.

Therapeutic activation of endothelial sphingosine-1-phosphate receptor 1 by chaperone-bound S1P suppresses proliferative retinal neovascularization.

Sphingosine-1-phosphate (S1P), the circulating HDL-bound lipid mediator that acts via S1P receptors (S1PR), is required for normal vascular development. The role of this signaling axis in vascular retinopathies is unclear. Here, we show in a mouse model of oxygen-induced retinopathy (OIR) that endothelial overexpression of S1pr1 suppresses while endothelial knockout of S1pr1 worsens neovascular tuft formation. Furthermore, neovascular tufts are increased in Apom-/- mice which lack HDL-bound S1P while they are suppressed in ApomTG mice which have more circulating HDL-S1P. These results suggest that circulating HDL-S1P activation of endothelial S1PR1 suppresses neovascular pathology in OIR. Additionally, systemic administration of ApoM-Fc-bound S1P or a small-molecule Gi-biased S1PR1 agonist suppressed neovascular tuft formation. Circulating HDL-S1P activation of endothelial S1PR1 may be a key protective mechanism to guard against neovascular retinopathies that occur not only in premature infants but also in diabetic patients and aging people.

Normalization of Oxygen‐Induced Retinopathy in the Mouse Supplementation with Myo/Nog Cells

IntroductionRetinopathy of Prematurity (ROP) is the leading cause of blindness in children, affecting 50% of infants born prior to gestational week 32. ROP is the result of postpartum supplemental oxygen administered to compensate for underdeveloped lungs. However, hyperoxia inhibits proper development supplying the retina. When the infants are returned to ambient air, an imbalance is present between the oxygen concentration and metabolic demands of retinal neurons. This leads to a rapid induction of angiogenesis, leaky blood vessels, neovascular tuft formation, and cell death. Myo/Nog cells that express MyoD, Noggin, and brain‐specific angiogenesis inhibitor 1 (BAI1) exhibit neuroprotective properties in the eye and brain and are proposed to play a role in regulating angiogenesis in ROP.MethodsAn Oxygen‐induced retinopathy (OIR) protocol was implemented in neonatal C57BL/6J mice by exposing them on postnatal days 7‐12 (P7‐P12) to hyperoxia (75% oxygen) and then returning them to normal air (21% oxygen) from P12 until P21. On P15, groups receiving treatments were intravitreally injected with 1 µL of phosphate‐buffered saline (PBS) containing 2000 Myo/Nog cells isolated from the brain with the BAI1 antibody (BAI1+ group), 2000 unsorted brain cells (BAI1‐ group), or PBS alone (PBS group). Electroretinography (ERG) was conducted on P21 to assess retinal function. Eyes were extracted and either dissected for flat‐mount observation of vasculature and tufts, or processed for histology. The thickness of the retina and number of TUNEL+ apoptotic cells were quantified in tissue sections of eyes processed for histology. Adobe Photoshop (Adobe, San Jose, CA) was used to splice together 9 images that made up each whole retina from flat‐mounted eyes. The blood vessels were selected using the “Lasso tool” on photoshop, quantified by number of pixels, and then compared to the total number of pixels in the whole retina.ResultsOIR induced neovascular tuft formation. Exogenously added Myo/Nog cells normalized the retinopathy induced by the OIR model, reducing the number of tufts and improving the quality of the vasculature. The vasculature was most developed in retinas injected with brain derived Myo/Nog cells. ERG data shows a trend towards improvement of visual function with Myo/Nog cell injection.ConclusionMyo/Nog cell regulation of retinal vascularization may be related to the production of BAI1. Improved visual function seen with injection of Myo/Nog cells may be related to both a reduction of neovascular tufts and direct effects on retinal neurons. Therefore Myo/Nog cells have therapeutic potential in the prevention of ROP by normalizing the vasculature and preventing neuronal cell death.

Hyaloid Vasculature as a Major Source of STAT3+ (Signal Transducer and Activator of Transcription 3) Myeloid Cells for Pathogenic Retinal Neovascularization in Oxygen-Induced Retinopathy.

Myeloid cells are critically involved in inflammation-induced angiogenesis, although their pathogenic role in the ischemic retina remains controversial. We hypothesize that myeloid cells contribute to pathogenic neovascularization in retinopathy of prematurity through STAT3 (signal transducer and activator of transcription 3) activation. Approach and Results: Using the mouse model of oxygen-induced retinopathy, we show that myeloid cells (CD45+IsolectinB4 [IB4]+) and particularly M2-type macrophages (CD45+ Arg1+), comprise a major source of STAT3 activation (pSTAT3) in the immature ischemic retina. Most of the pSTAT3-expressing myeloid cells concentrated at the hyaloid vasculature and their numbers were strongly correlated with the severity of pathogenic neovascular tuft formation. Pharmacological inhibition of STAT3 reduced the load of IB4+ cells in the hyaloid vasculature and significantly reduced the formation of pathogenic neovascular tufts during oxygen-induced retinopathy, leading to improved long-term visual outcomes (ie, increased retinal thickness and scotopic b-wave electroretinogram responses). Genetic deletion of SOCS3 (suppressor of cytokine signaling 3), an endogenous inhibitor of STAT3, in myeloid cells, enhanced pathological and physiological neovascularization in oxygen-induced retinopathy, indicating that myeloid-STAT3 signaling is crucial for retinal angiogenesis. Circulating myeloid cells may migrate to the immature ischemic retina through the hyaloid vasculature and contribute to retinal neovascularization via activation of STAT3. Understanding how STAT3 modulates myeloid cells for vascular repair/pathology may provide novel therapeutic options in pathogenic angiogenesis.

CNTF Prevents Development of Outer Retinal Neovascularization Through Upregulation of CxCl10.

PurposeCiliary neurotrophic factor (CNTF) is a well-characterized neurotrophic factor currently in clinical trials for the treatment of macular telangiectasia type II. Our previous work showed that CNTF-induced STAT3 signaling is a potent inhibitor of pathologic preretinal neovascular tuft formation in the mouse model of oxygen-induced retinopathy. In this study, we investigated the effect of CNTF on outer retinal and choroidal angiogenesis and the mechanisms that underpin the observed decrease in outer retinal neovascularization following CNTF treatment.MethodsIn the Vldlr–/– and laser-CNV mouse models, mice received a one-time injection (on postnatal day [P] 12 in the Vldlr–/– model and 1 day after laser in the Choroidal Neovascularization (CNV) model) of recombinant CNTF or CxCl10, and the extent of neovascular lesions was assessed 6 days posttreatment. STAT3 downstream targets affected by CNTF treatment were identified using quantitative PCR analysis. A proteome array was used to compare media conditioned by CNTF-treated and control-treated primary Müller cells to screen for CNTF-induced changes in secreted angiogenic factors.ResultsIntravitreal treatment with recombinant CNTF led to significant reduction in neovascularization in the Vldlr–/– and laser-CNV mouse models. Treatment effect in the Vldlr–/– was long-lasting but time sensitive, requiring intravitreal treatment before P19. Mechanistic workup in vitro as well as in vivo confirmed significant activation of the STAT3-signaling pathway in Müller cells in response to CNTF treatment and upregulation of CxCl10. Intravitreal injections of recombinant CxCl10 significantly reduced outer retinal neovascularization in vivo in both the Vldlr–/– and laser-CNV mouse models.ConclusionsCNTF treatment indirectly affects outer retinal and choroidal neovascularization by inducing CxCl10 secretion from retinal Müller cells.

The investigation of endothelial nitric oxide synthase (eNOS) activation in retinopathy

AbstractPurposeTo elucidate the role of endothelial nitric oxide synthase (eNOS) activation in the induction of vascular endothelial growth factor (VEGF)‐induced permeability, the underlying mechanism and the contribution to exaggerated vascular leakage in eye disease.MethodsRetinal neovascularization and pathological edema in the superficial and deep retinal vascular plexa were investigated using the oxygen‐induced retinopathy (OIR) mouse model. eNOS activity was blocked either chemically by using the NO inhibitor or genetically by using mutant mice (Mut) in which serine 1176 in eNOS is replaced by alanine (Nos3S1176A/S1176A,therefore is unable to induce eNOS activity). Retina avascular area and proliferative vessel tuft formation were assessed at postnatal (P) day 17. Leakage was determined by injecting 25 nm fluorescent microspheres in the circulation followed by microscopy. Isolectin B4, eNOS, vascular endothelial cadherin (VEC), and phosphoY685‐VEC immunostaining was assessed by confocal imaging followed by image analysis using ImageJ. Students t‐test was used for statistical evaluation. p < 0.05 was regarded as statistically significant.ResultsAdministration of the NO inhibitor during P12‐P17 reduced overall pathological tuft area (p < 0.05), and decreased the area of individual tufts (p < 0.01) compared to control. Mut displayed reduced neovascular tuft formation (p < 0.01) and decreased tuft size compared to wildtype mice (WT) (p < 0.05). Mice unable to activate eNOS displayed a junctional phenotype. Firstly, phosphoY685‐VEC in tufts was decreased in Mut compared to WT (p < 0.05). Secondly, immunostaining for eNOS colocalized with VEC in vessels of WT but not in Mut (p < 0.05). Thirdly, vascular leakage was reduced in OIR‐challanged Mut (p < 0.01).ConclusionOur data establish that eNOS is critical for VEC‐regulated endothelial junction stability and vascular leakage. Thus, eNOS is an important target for the development of therapeutic agents to treat eye disease.

Kruppel-like factor 4 regulates developmental angiogenesis through disruption of the RBP-J-NICD-MAML complex in intron 3 of Dll4.

Angiogenesis is a multistep process that requires highly regulated endothelial cell (EC) behavior. The transcription factor Krüppel-like factor 4 (KLF4) is a critical regulator of several basic EC functions; we have recently shown that KLF4 disturbs pathological (tumor) angiogenesis by mediating the expression of members of VEGF and Notch signaling pathways. Notch signaling is central to orchestration of sprouting angiogenesis but little is known about the upstream regulation of Notch itself. To determine the role of KLF4 in normal (developmental) angiogenesis, we used a mouse retinal angiogenesis model. We found that endothelial-specific overexpression of KLF4 in transgenic mice (EC-K4 Tg) leads to increased vessel density, branching and number of tip cell filopodia as assessed on postnatal day 6 (P6). The hypertrophic vasculature seen with sustained KLF4 overexpression is not stable and undergoes prominent remodeling during P7-P12 resulting in a normal appearing retinal vasculature in adult EC-K4 Tg mice. We find that KLF4 inhibits Delta-like 4 (DLL4) expression in the angiogenic front during retinal vascular development. Furthermore, in an oxygen-induced retinopathy model, overexpression of KLF4 results in decreased vaso-obliteration and neovascular tuft formation that is similar to genetic or pharmacologic DLL4 inhibition. Mechanistically, we show that KLF4 disables the activity of the essential Notch transcriptional activator RBP-J by interfering with binding of co-activators NICD and MAML at intron 3 of the Notch ligand DLL4. In summary, our experimental results demonstrate a regulatory role of KLF4 in developmental angiogenesis through regulation of DLL4 transcription.

Novel Small Molecule JP-153 Targets the Src-FAK-Paxillin Signaling Complex to Inhibit VEGF-Induced Retinal Angiogenesis.

Targeting vascular endothelial growth factor (VEGF) is a common treatment strategy for neovascular eye disease, a major cause of vision loss in diabetic retinopathy and age-related macular degeneration. However, the decline in clinical efficacy over time in many patients suggests that monotherapy of anti-VEGF protein therapeutics may benefit from adjunctive treatments. Our previous work has shown that through decreased activation of the cytoskeletal protein paxillin, growth factor-induced ischemic retinopathy in the murine oxygen-induced retinopathy model could be inhibited. In this study, we demonstrated that VEGF-dependent activation of the Src/FAK/paxillin signalsome is required for human retinal endothelial cell migration and proliferation. Specifically, the disruption of focal adhesion kinase (FAK) and paxillin interactions using the small molecule JP-153 inhibited Src-dependent phosphorylation of paxillin (Y118) and downstream activation of Akt (S473), resulting in reduced migration and proliferation of retinal endothelial cells stimulated with VEGF. However, this effect did not prevent the initial activation of either Src or FAK. Furthermore, topical application of a JP-153-loaded microemulsion affected the hallmark features of pathologic retinal angiogenesis, reducing neovascular tuft formation and increased avascular area, in a dose-dependent manner. In conclusion, our results suggest that using small molecules to modulate the focal adhesion protein paxillin is an effective strategy for treating pathologic retinal neovascularization. To our knowledge, this is the first paradigm validating modulation of paxillin to inhibit angiogenesis. As such, we have identified and developed a novel class of small molecules aimed at targeting focal adhesion protein interactions that are essential for pathologic neovascularization in the eye.

Antiangiogenic Effectiveness of the Urokinase Receptor-Derived Peptide UPARANT in a Model of Oxygen-Induced Retinopathy.

Pharmacologic control of neovascularization is a promising approach for the treatment of retinal angiogenesis. UPARANT, an inhibitor of the urokinase-type plasminogen activator receptor (uPAR), inhibits VEGF-driven angiogenesis in vitro and in vivo. This study investigates for the first time the effectiveness of UPARANT in counteracting pathologic neovascularization in the retina. Murine retinal fragments and a mouse model of oxygen-induced retinopathy (OIR) were used. In mice with OIR, UPARANT-treated retinas were analyzed for avascular area and neovascular tuft formation. Levels of transcription and proangiogenic factors were determined. UPARANT effects on the blood-retinal barrier (BRB), visual function, retinal cytoarchitecture, and inflammatory markers were also assessed. Human umbilical vein endothelial cells (HUVECs) and chick embryo chorioallantoic membrane (CAM) in which angiogenesis was induced by the vitreous fluid from patients with proliferative diabetic retinopathy (PDR) were also used. UPARANT reduced VEGF-induced angiogenesis in retinal fragments. In mice with OIR, UPARANT decreased neovascular response, VEGF, and VEGF receptor-2 activity. Transcription factors regulating VEGF expression were also reduced. UPARANT restored BRB integrity, recovered visual loss, and reduced levels of inflammatory markers. Restored electroretinogram does not involve any rescue in the retinal cytoarchitecture. Finally, UPARANT blocked PDR vitreous fluid-induced angiogenesis in HUVEC and CAM assays. The finding that UPARANT is effective against neovascularization may help to establish uPAR as a target in the treatment of proliferative retinopathies. The potential application of UPARANT in retinal diseases is further supported by UPARANT capacity to counteract the angiogenic activity of PDR vitreous fluid.

Aquaporin 4 is required to induce retinal angiogenesis in a mouse model of oxygen‐induced retinopathy

AbstractPurpose Aquaporin 4 (AQP4) is the most abundant water channel in the retina and participates in the formation of blood‐retinal barrier (BRB). In several retinal pathologies accompanied by BRB dysfunction the expression of AQP4 is altered. In the present study, we investigated the effects of AQP4 deletion in the vascular retinal response to hypoxia.Methods We used wild type (WT) and AQP4 knockout (KO) mice. The retinal angiogenic response to hypoxia was studied in a mouse model of oxygen‐induced retinopathy (OIR) using real time RT‐PCR, Western blot, ELISA and immunohistochemistry.Results In WT mice, retinal levels of AQP4 were increased in response to hypoxia. In OIR mice, the BRB dysfunction was more pronounced in KO than in WT mice. BRB tight junction proteins, occludin, ZO‐1 and JAM‐C, were analyzed and significant alteration was found for JAM‐C in KO mice, indicating a role for this protein in the observed dysfunctions. The formation of neovascular tufts, which are characteristic of OIR, was completely prevented in KO mice, possibly as a result of the decrease in hypoxia‐induced up‐regulation of VEGF mRNA and protein. Unexpectedly, this reduced increase in VEGF levels was paralleled by a dramatic increase in the activity of transcription factors which regulate VEGF expression indicating that in KO retinas mechanisms of VEGF transcription, such as promoter methylation, may be altered.Conclusion Together, the present results indicate a role for AQP4 in the formation of angiogenic processes in the retina and suggest that inhibiting AQP4 function may be a strategy to reduce pathologic angiogenesis in proliferative retinopathies.

Genetic deletion of β1‐ and β2‐adrenergic receptors rescues the retina from hypoxia‐induced pathologic angiogenesis

AbstractPurpose To investigate the effects of β1‐ and β2‐adrenergic receptor (AR) deletion in the vascular retinal response to hypoxia, to confirm and expand the role of β1‐ and β2‐ARs in regulating retinal angiogenesis and to get insights into the role of β3‐ARs.Methods We used wild type (WT) and β1/β2‐AR knockout (KO) mice. The retinal angiogenic response to hypoxia was studied in a mouse model of oxygen‐induced retinopathy (OIR) using real time RT‐PCR, Western blot, ELISA and immunohistochemistry.Results Our results show that β1‐ and β2‐AR deletion did not affect β3‐AR expression or norepinephrine (NE) levels. In KO mice, NE was less upregulated in response to hypoxia indicating a reduced stimulation of the β‐adrenergic system. In KO, the angiogenic response to hypoxia, including the formation of both a central avascular area and neovascular tufts, was nearly abolished while no improvements were observed in blood‐retinal barrier dysfunction typical of OIR. Unexpectedly, VEGF levels did not differ between WT and KO in spite of a recover of the angiogenic response, but a reduced activation of VEGF signaling was observed in KO, which may explain the reduced response to hypoxia. To get insights into the role of β3‐ARs, we treated OIR mice with a β3‐AR agonist, BRL37344 (2 mg/kg, s.c. injected), which resulted in an increase in VEGF levels and the formation of neovascular tufts in both WT and KO, indicating that β3‐AR stimulation may function as a proangiogenic switch.Conclusion Taken together, our results confirm the notion that β1‐ and/or β2‐ARs play a role in retinal angiogenesis and indicate that β3‐ARs may potentiate the angiogenic response in WT and sustain the angiogenic drive in KO.

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

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. 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. 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.

ENOS Overexpression Exacerbates Vascular Closure in the Obliterative Phase of OIR and Increases Angiogenic Drive in the Subsequent Proliferative Stage

In ischemic retinopathies, the misdirection of reparative angiogenesis away from the hypoxic retina leads to pathologic neovascularization. Thus, therapeutic strategies that reverse this trend would be extremely beneficial. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is an important mediator of vascular endothelial growth factor (VEGF) function facilitating vascular growth and maturation. However, in addition to NO, eNOS can also produce superoxide (O(2)(-)), exacerbating pathology. Here, our aim was to investigate the effect of eNOS overexpression on vascular closure and subsequent recovery of the ischemic retina. Mice overexpressing eNOS-GFP were subjected to oxygen-induced retinopathy (OIR) and changes in retinal vascularization quantified. Background angiogenic drive was assessed during vascular development and in aortic rings. NOS activity was measured by Griess assay or conversion of radiolabeled arginine to citrulline, nitrotyrosine (NT), and superoxide by immunolabeling and dihydroethidium fluorescence and VEGF by ELISA. In response to hyperoxia, enhanced eNOS expression led to increased NOS-derived superoxide and dysfunctional NO production, NT accumulation, and exacerbated vessel closure associated with tetrahydrobiopterin (BH₄) insufficiency. Despite worse vaso-obliteration, eNOS overexpression resulted in elevated hypoxia-induced angiogenic drive, independent of VEGF production. This correlated with increased vascular branching similar to that observed in isolated aortas and during development. Enhanced recovery was also associated with neovascular tuft formation, which showed defective NO production and increased eNOS-derived superoxide and NT levels. In hyperoxia, reduced BH₄ bioavailability causes overexpressed eNOS to become dysfunctional, exacerbating vaso-obliteration. In the proliferative phase, however, eNOS has important prorepair functions enhancing angiogenic growth potential and recovery in ischemia.

Attenuation of EphrinB2 Reverse Signaling Decreases Vascularized Area and Preretinal Vascular Tuft Formation in the Murine Model of Oxygen-Induced Retinopathy

EphB4 and ephrinB2 are known key regulators of retinal vascular development, but due to their capacity for bidirectional signaling, delineation of their individual roles in this process remains unclear. To better dissect out individual contributions, a model of proliferative retinopathy in mice with attenuated ephrinB2 reverse signaling was studied. It was hypothesized that endothelial ephrinB2 reverse signaling regulates hypoxia-induced capillary sprouting, as well as the pathologic formation of neovascular tufts in postnatal retinal microvascular networks. Genetically manipulated mice with attenuated ephrinB2 reverse signaling (ephrinB2(lacZ/+)), along with wild-type (WT) controls, were exposed to oxygen-induced retinopathy (OIR), a postnatal model of proliferative retinopathy. At peak disease (postnatal day 18), microvascular networks were analyzed to examine intraretinal revascularization, capillary sprouting, and pathologic neovascularization responses. EphB4 and phosphorylated ephrinB protein expression patterns along retinal microvessels were also assessed. EphrinB2(lacZ/+) mice exhibited reduced hypoxia-induced revascularization (P ≤ 0.04) and reduced formation of neovascular tufts (P < 0.001), as compared with WT controls. Corresponding to the observed inhibition of retinal angiogenesis, ephrinB2(lacZ/+) retinas displayed an increased number of blind-ended capillary sprout tips (P < 0.02) and endothelial filopodial processes (P = 0.001). In WT and ephrinB2(lacZ/+) OIR-exposed retinas, ephrinB was confined to endothelial cells, with expression detected along angiogenic vascular processes including neovascular tufts and blind-ended capillary sprouts. EphrinB2 reverse signaling is a regulator of key processes during retinal vascularization and controls pathologic retinal angiogenesis through direct effects on capillary sprouting and endothelial filopodia formation.

Granulocyte colony-stimulating factor attenuates oxidative stress–induced apoptosis in vascular endothelial cells and exhibits functional and morphologic protective effect in oxygen-induced retinopathy

AbstractGranulocyte colony-stimulating factor (G-CSF) is a known hematopoietic glycoprotein, and recent studies have revealed that G-CSF possesses other interesting properties. Oxidative stress is involved in many diseases, such as atherosclerosis, heart failure, myocardial infarction, Alzheimer disease, and diabetic retinopathy. This study was designed to examine whether G-CSF has a protective effect on endothelial cells against oxidative stress and to investigate whether G-CSF has a therapeutic role in ischemic vascular diseases. Expression of G-CSF (P < .01) and G-CSF receptor (P < .05) mRNA in human retinal endothelial cells (HRECs) was significantly up-regulated by oxidative stress. Treatment with 100 ng/mL G-CSF significantly reduced H2O2-induced apoptosis in HRECs from 61.7% to 41.4% (P < .05). Akt was phosphorylated in HRECs by G-CSF addition, and LY294002, a PI3K inhibitor, significantly attenuated the antiapoptotic effect of G-CSF (by 44.1%, P < .05). The rescue effect was also observed in human umbilical vein endothelial cells. In mouse oxygen-induced retinopathy model, G-CSF significantly reduced vascular obliteration (P < .01) and neovascular tuft formation (P < .01). G-CSF treatment also clearly rescued the functional and morphologic deterioration of the neural retina. A possibility of a novel therapeutic strategy for ischemic diseases through attenuating vascular regression using G-CSF was proposed.

ADAM8 is a negative regulator of retinal neovascularization and of the growth of heterotopically injected tumor cells in mice

ADAM8 is a member of the "a disintegrin and metalloproteinase" (ADAM) family of membrane-anchored metalloproteinases. ADAM8-deficient mice have no evident spontaneous developmental or pathological defects, and little is currently known about the role of ADAM8 in disease. Here, we investigated the contribution of ADAM8 to pathological neovascularization in mice using an oxygen-induced retinopathy (OIR) model and heterotopical injection of tumor cells. We found an increase in retinal re-vascularization but fewer neovascular tufts in the OIR model and increased growth of heterotopically injected tumor cells in Adam8-/- mice compared with wild-type controls. These results suggest that ADAM8 functions to limit both of these processes in wild-type mice. In cell-based assays, overexpression of ADAM8 increased the ectodomain shedding of several co-expressed membrane proteins with roles in angiogenesis (CD31, Tie-2, Flk-1, Flt-1, EphrinB2, EphB4, VE-cadherin, KL-1, E-selectin, and neuregulin-1beta2). Thus, dysregulated expression of ADAM8 in endothelial cells in vivo could potentially increase the processing of these and other substrate proteins. Taken together, our findings suggest that inhibiting ADAM8 could be useful for promoting re-vascularization and thereby preventing formation of neovascular tufts in proliferative retinopathies. On the other hand, blocking ADAM8 could be detrimental in the context of rapidly growing tumors.

Thiazolidinediones Reduce Pathological Neovascularization in Ischemic Retina Via an Adiponectin-Dependent Mechanism

Background- The insulin-sensitizing agents referred to as thiazolidinediones (TZDs) possess antiatherogenic and anti-inflammatory actions that contribute to protection against diabetic macrovascular complications. However, little is known about the effects of TZDs on retinal microvessel disorders. To investigate whether TZDs modulate retinal vessel formation in a mouse model of oxygen-induced retinopathy. Neonatal mice were subjected to ischemia-induced retinopathy to produce pathological neovascular tuft formation. Pioglitazone, 10 mg/kg per day, rosiglitazone, 10 mg/kg per day, or vehicle was given by gavage once a day from postnatal day 7 to postnatal day 17. Systemic treatment of wild-type (WT) mice with TZDs led to a significant decrease in pathological retinal neovascularization during ischemia compared with vehicle treatment, which was accompanied by increased plasma levels of the fat-derived hormone adiponectin (APN). In contrast to WT mice, TZDs had no effects on ischemia-induced pathological retinal vessel formation in APN-knockout (KO) mice. Pioglitazone reduced tumor necrosis factor (TNF) alpha expression in ischemic retina in WT mice but not in APN-KO mice. Furthermore, pioglitazone increased plasma APN levels in TNF-alpha-KO mice but did not affect ischemia-induced pathological retinal neovascularization in this strain. These data show that TZDs attenuate pathological retinal microvessel formation through APN-mediated modulation of TNF-alpha production.

Slit2–Robo4 signalling promotes vascular stability by blocking Arf6 activity

Slit-Roundabout (Robo) signalling has a well-understood role in axon guidance. Unlike in the nervous system, however, Slit-dependent activation of an endothelial-specific Robo, Robo4, does not initiate a guidance program. Instead, Robo4 maintains the barrier function of the mature vascular network by inhibiting neovascular tuft formation and endothelial hyperpermeability induced by pro-angiogenic factors. In this study, we used cell biological and biochemical techniques to elucidate the molecular mechanism underlying the maintenance of vascular stability by Robo4. Here, we demonstrate that Robo4 mediates Slit2-dependent suppression of cellular protrusive activity through direct interaction with the intracellular adaptor protein paxillin and its paralogue, Hic-5. Formation of a Robo4-paxillin complex at the cell surface blocks activation of the small GTPase Arf6 and, consequently, Rac by recruitment of Arf-GAPs (ADP-ribosylation factor- directed GTPase-activating proteins) such as GIT1. Consistent with these in vitro studies, inhibition of Arf6 activity in vivo phenocopies Robo4 activation by reducing pathologic angiogenesis in choroidal and retinal vascular disease and VEGF-165 (vascular endothelial growth factor-165)-induced retinal hyperpermeability. These data reveal that a Slit2-Robo4-paxillin-GIT1 network inhibits the cellular protrusive activity underlying neovascularization and vascular leak, and identify a new therapeutic target for ameliorating diseases involving the vascular system.

HMG-CoA Reductase Inhibitors (Statin) Prevents Retinal Neovascularization in a Model of Oxygen-Induced Retinopathy

Retinal neovascularization (RNV) is a primary cause of blindness and involves the dysfunction of retinal capillaries. Recent studies have emphasized the beneficial effects of inhibitors of HMG-CoA reductase (statins) in preventing vascular dysfunction. In the present study, the authors characterized the therapeutic effects of statins on RNV. Statin treatment (10 mg/kg/d fluvastatin) was tested in a mouse model of oxygen-induced retinopathy. Morphometric analysis was conducted to determine the extent of capillary growth. Pimonidazole hydrochloride was used to assess retinal ischemia. Western blot and immunohistochemical analyses were used to assess protein expression levels and immunolocalization. Lipid peroxidation and superoxide radical formation were determined to assess oxidative changes. Fluvastatin treatment significantly reduced the area of the capillary-free zone (P < 0.01), decreased the formation of neovascular tufts (P < 0.01), and ameliorated retinal ischemia. These morphologic and functional changes were associated with statin effects in preventing the upregulation of VEGF, HIF-1 alpha, phosphorylated STAT3, and vascular expression of the inflammatory mediator ICAM-1 (P < 0.01). Superoxide production and lipid peroxidation in the ischemic retina were also reduced by statin treatment (P < 0.01). These data suggest the beneficial effects of statin treatment in preventing retinal neovascularization. These beneficial effects appear to result from the anti-oxidant and anti-inflammatory properties of statins.