Proliferation Of Retinal Microvascular Endothelial Cells Research Articles

326-OR: Rod Photoreceptor Contribution to Inflammation and Angiogenesis in Diabetic Retinopathy

Purpose: Rod photoreceptors (rods) are the most numerous and metabolically active retinal cells, and are susceptible to metabolic stress in response to hyperglycemia. Yet the role of rods in diabetic retinopathy (DR) is controversial. This study aims to investigate if rods exposed to high glucose can trigger inflammatory and angiogenic responses in Müller glia (MG) and retinal microvascular endothelial cells (RMEC). Methods: The streptozocin-induced (STZ) model was used at 10 weeks of diabetes. Transcriptomic shifts in rods were analyzed by RNAseq. The mediators produced by rods were analyzed by multiplex ELISA. The effect of rod-produced mediators on RMEC permeability was assessed. Angiogenic responses of RMECs treated with rod-produced mediators were evaluated by proliferation, migration, and tube formation assays. The effect of rods on proangiogenic mediator production by RMECs and MG was evaluated by western blot. Results: Diabetes induced upregulation of transcripts for tnf (3-fold, P=0.006) and il1a (2.2-fold, P=0.001) in rods. Primary rod cultures treated with 25 mM D-glucose (high glucose) produced increased levels of VEGF (2.5-fold, p<0.05), TNF-α (2.4-fold, p<0.05), and IL-6 (2-fold, p<0.05) compared to rods treated with 5 mM D-glucose (normal glucose), or with 25 mM L-glucose (osmotic control). The mediators produced by rods treated with high glucose elicited increased RMEC proliferation (2.5 fold, p<0.001), RMEC migration (2-fold, p<0.001), and RMEC tube lengths (1.9-fold, p<0.001). The rod-produced mediators induced RMEC secretion of matrix metalloproteinase 2 (MMP-2) (8-fold increase, p<0.001) and of MMP-9 (5-fold increase, p<0.001). MG treated with rod-produced mediators increased their production of VEGF (4-fold, p<0.001) and of MMP-9 (5-fold, p<0.001). Conclusion: Rods in hyperglycemic conditions produce mediators that increase RMEC permeability, proliferation, migration, and branching and induce a proangiogenic secretory shift in RMEC and MG. Disclosure I.De la huerta: None. J.M.Poloway: None. V.Reddy: None. J.S.Penn: None. Funding National Institutes of Health (K08EY032620); International Retinal Research Foundation; Knights Templar Eye Foundation

Long Noncoding RNA PPT2-EGFL8 Regulates Pathological Retinal Neovascularization in PDR by Functioning as a Competing Endogenous RNA.

Diabetic retinopathy (DR) is a common complication in patients with diabetes, and proliferative DR (PDR) has become an important cause of blindness; however, the mechanisms involved have not been fully elucidated. miRNAs and long noncoding RNAs can play an important role in DR, and they can accurately regulate the expression of target genes through a new regulatory model: competing endogenous RNAs. We isolated total RNA of extracellular vesicles (EVs) in the serum of healthy individuals and individuals with diabetes without DR, non-PDR, or PDR, and performed deep sequencing. We found aberrantly low expression of PPT2-EGFL8 and significantly increased level of miR-423-5p. PPT2-EGFL8 adsorbs miR-423-5p as a molecular sponge and inhibits hypoxia-induced human retinal microvascular endothelial cells proliferation. In an oxygen-induced retinopathy (OIR) model and a streptozotocin-induced diabetes model, Egfl8-overexpression treatment reduces diabetes-related reactive gliosis, inflammation, and acellular capillaries and attenuates the development of pathological neovascularization. In addition, PPT2-EGFL8 targeting miR-423-5p plays an important role in hypoxia-induced peroxisome proliferator-activated receptor-β/δ (PPARD)/angiopoietin-like 4 (ANGPTL4) signaling activation, especially the expression of the C-terminal ANGPTL4 fragment. Finally, ANGPTL4 significantly induces retinal vessel breakage in the inner limiting membrane and facilitates retinal vessel sprouting into the vitreous in the OIR mice. Thus, either new biomarkers or new therapeutic targets may be identified with translation of these findings.

Long Non-coding RNA SPAG5-AS1 Attenuates Diabetic Retinal Vascular Dysfunction by Inhibiting Human Retinal Microvascular Endothelial Cell Proliferation, Migration, and Tube Formation by Regulating the MicroRNA-1224-5p/IRS-1 Axis.

Diabetic retinopathy (DR) is causal for visual impairment and blindness. The research aimed at whether and how lncRNA SPAG5-AS1 (SPAG5-AS1) is involved in retinal vascular dysfunction under diabetic conditions. After determination of SPAG5-AS1, miR-1224-5p, and IRS-1 expression in high glucose (HG)-treated human retinal microvascular endothelial cells (hRMECs), their respective influences on retinal vascular dysfunction was explored by cell counting kit-8, Transwell, wound-healing assay, and tube formation assay. SPAG5-AS1/miR-1224-5p/IRS-1 interaction was identified through bioinformatics analysis and luciferase reporter gene assays. As tested, SPAG5-AS1 and IRS-1 levels were induced, while miR-1224-5p was enhanced in HG-treated hRMECs. Up-regulating SPAG5-AS1 or downregulating miR-1224-5p could inhibit hRMECs proliferation, migration, and tube formation, and vice versa. SPAG5-AS1 can promote IRS-1 expression by miR-1224-5p, and depletion of IRS-1 was functional for the reversal of up-regulated SPAG5-AS1-modified influences on HG-treated hRMECs. Additionally, in diabetic rats, SPAG5-AS1 can alleviate retinal vascular dysfunction. All in all, SPAG5-AS1 attenuates diabetic retinal vascular dysfunction through miR-1224-5p/IRS-1 axis, providing a potential therapeutic strategy for DR.

LncRNA MALAT1 promotes diabetic retinopathy by upregulating PDE6G via miR-378a-3p

Diabetic retinopathy (DR) is the main cause of adult insomnia, which causes certain social and economic pressure. This research was to investigate the role and regulatory mechanisms of MALAT1, miR-378a-3p and PDE6g in retinal microvascular endothelial cells (RMECs) under high glucose (HG). MALAT1, Mir-378a-3p and PDE6G expressions level were detected by qRT-PCR and Western blot. The proliferation, Bax and Bcl-2 protein expression of RMECs were detected by CCK-8 and western blot. The target relationships of MALAT1, miR-378a-3p and PDE6G were determined by bioinformatics analysis, dual-luciferase reporter gene, RIP and RNA pull-down assay. HG enhanced the expression of MALAT1 and PDE6G, and inhibited the expression of miR-378a-3p. Overexpression of MALAT1 promotes the proliferation of RMECs and inhibits apoptosis under HG condition. MALAT1 competitively adsorbed miR-378a-3p, which targeted PDE6G. Data reveal that MALAT1/miR-378a-3p/PDE6G signal axis restrain the apoptosis of RMECs under HG. This finding may help to delay the development of DR.

Withdrawn: A novel microRNA-365/SIRT1/β-catenin/GLUT1 signaling axis affects the initiation of diabetic retinopathy.

Numerous studies have implicated the aberrant expression profiles of microRNAs (miRNAs or miRs) in the process of diabetic retinopathy (DR) and implied significant value as diagnostic and therapeutic targets. The current study set out to clarify the role of miR-365 in DR in relation to the sirtuin 1 (SIRT1)/β-catenin/glucose transporter 1 (GLUT1) signaling axis. DR models were established in C57BL/6 mice fed a high-fat diet by injection with streptozotocin (STZ), followed by the determination of miR-365, SIRT1, β-catenin and GLUT1 expression. The relationship between miR-365 and SIRT1, SIRT1 and β-catenin, β-catenin and GLUT1 was also verified. Retinal microvascular endothelial cells (RMECs) were cultured under high glucose conditions, and their proliferation and angiogenesis were assessed using EdU and tube formation assays. The effects of miR-365 on pre-retinal neovascular cells and newly formed blood vessels in DR mice were also explored. Up-regulated miR-365, β-catenin and GLUT1 and down-regulated SIRT1 were observed in the retinal tissues of DR mice. miR-365 targeted SIRT1, and then inhibited its expression, leading to increased β-catenin and GLUT1 expression and subsequent enhancement of RMEC proliferation and tube formation in vitro. SIRT1 deacetylated β-catenin and reduced its expression, thus inhibiting the translation of GLUT1. Furthermore, in vivo experiments demonstrated that miR-365 targeted inhibition of SIRT1 could enhance the activity of β-catenin and GLUT1, thus enhancing pre-retinal neovascular cells and newly-formed blood vessels in DR mice. Our findings highlighted a novel mechanism whereby miR-365 deteriorates DR by regulating the SIRT1-dependent β-catenin/GLUT1 signaling axis. This article is protected by copyright. All rights reserved.

The anti-angiogenesis role of FBXW7 in diabetic retinopathy by facilitating the ubiquitination degradation of c-Myc to orchestrate the HDAC2.

Diabetic retinopathy (DR) is the most prevalently occurring microvascular complication in diabetic patients that triggers severe visual impairments. The anti‐angiogenesis role of FBXW7 has been identified in breast cancer. Therefore, this study intends to decipher the mechanism of FBXW7 in angiogenesis of DR. DR model was induced on mice using high‐glucose (HG) and high‐fat diet, and retinal microvascular endothelial cells (RMECs) isolated from normal mice were induced with HG, followed by evaluation of FBXW7, Ki67, HIF‐1α and VEGF expression by immunofluorescence, immunohistochemistry or Western blot analysis. After gain‐ and loss‐of‐function assays in normal and DR mice, angiogenesis was assessed by CD31 fluorescence staining and Western blot analysis. After ectopic expression and silencing experiments in HG‐induced RMECs, RMEC proliferation, migration and angiogenesis were, respectively, determined by EdU, Transwell and in vitro angiogenesis assays. The impact of FBXW7 on the ubiquitination of c‐Myc was studied by cycloheximide chase assay and proteasome inhibition, and the binding of c‐Myc to HDAC2 promoter by dual‐luciferase reporter gene experiment. DR mice and HG‐induced RMECs possessed down‐regulated FBXW7 and up‐regulated Ki67, HIF‐1α and VEGF. Silencing FBXW7 enhanced angiogenesis in normal mouse retinal tissue, but overexpressing FBXW7 or silencing c‐Myc diminished angiogenesis in DR mouse retinal tissue. Overexpressing FBXW7 or silencing c‐Myc depressed proliferation, migration and angiogenesis in HG‐induced RMECs. FBXW7 induced c‐Myc ubiquitination degradation, and c‐Myc augmented HDAC2 expression by binding to HDAC2 promoter. Conclusively, our data provided a novel sight of anti‐angiogenesis role of FBXW7 in DR by modulating the c‐Myc/HDAC2 axis.

Overexpression of histone deacetylase SIRT1 exerts an antiangiogenic role in diabetic retinopathy via miR-20a elevation and YAP/HIF1α/VEGFA depletion.

As a basic member of the Class III histone deacetylases, SIRT1 has been implicated in the occurrence and progression of diabetic retinopathy (DR). The current study aimed to investigate the roles of SIRT1/miR-20a/Yse-associated protein (YAP)/hypoxia-inducible factor 1 α (HIF1α)/vascular endothelial growth factor A (VEGFA) in DR. The expression of SIRT1 was initially determined through quantitative RT-PCR and Western blot analysis following the successful establishment of a DR mouse model, followed by detection of SIRT1 catalytic activity. Retinal microvascular endothelial cells (RMECs) were cultured in media supplemented with normal glucose (NG) or high glucose (HG). Thereafter, SIRT1 was either silenced or overexpressed in RMECs, after which EdU staining and Matrigel-based tube formation assay were performed to assess cell proliferation and tube formation. The binding relationship between YAP, HIF1α, and VEGFA was further illustrated using dual-luciferase reporter assay. Preretinal neovascular cell number was tallied with the IB4-positive vascular endothelial cells, as determined by immunofluorescence. SIRT1 was poorly expressed in mice with DR and HG-treated RMECs with low catalytic activity. The proliferation and tube formation capabilities of RMECs were elevated under HG conditions, which could be reversed following overexpression of SIRT1. SIRT1 was identified as positively regulating the expression of miR-20a with YAP detected as the key target gene of miR-20a. Our data suggested that YAP could upregulate VEGFA via induction of HIF1α. Moreover, SIRT1 overexpression strongly repressed RMEC proliferation and angiogenesis, which could be reversed via restoration of YAP/HIF1α/VEGFA expression. Taken together, the key findings of our study suggest that upregulation of SIRT1 inhibits the development of DR via miR-20a-induced downregulation of YAP/HIF1α/VEGFA.

Inhibition of Pathological Retinal Neovascularization by a Small Peptide Derived from Human Tissue-Type Plasminogen Kringle 2.

Retinal neovascularization is a hallmark pathological process of numerous ocular diseases which comprise the most common causes of blindness and affect millions of people from infants to the elderly. Compared to large proteins, small peptides have advantages for therapeutic application in ocular diseases, especially for retinal diseases. In this study, we investigated a small peptide derived from human tissue-type plasminogen kringle 2 (t-PA kringle 2), named TKII-12, and investigated the effect of TKII-12 on various aspects of vascular endothelial growth factor (VEGF)-induced angiogenesis in vitro and in vivo. Our results showed that TKII-12 effectively inhibited VEGF-induced human retinal microvascular endothelial cell proliferation, migration and tube formation on Matrigel dose-dependently as well as sequence-dependently. TKII-12 inhibited VEGF-induced formation of actin stress fibers and focal adhesions in vascular endothelial cells. Moreover, TKII-12 effectively inhibited retinal neovascularization in a mouse oxygen-induced retinopathy (OIR) model. Our study demonstrated that TKII-12 could effectively inhibit retinal angiogenesis in vitro and in vivo by eliminating the formation of focal adhesion complexes and the organization of actin stress fibers. TKII-12 can serve as a prototype for retinal angiogenesis inhibitory drug development.

Quantitative proteomic analysis of human retinal microvascular endothelial cells stimulated with 4-Hydroxynonenal

Objective To detect the protein expression change in the proliferation of human retinal microvascular endothelial cells (hRMECs) stimulated with 4-Hydroxynonenal (4-HNE). Methods hRMECs were in a logarithmic growth phase, and then were separated into 4-HNE-stimulated group and negative control group. The concentration of 4-HNE included 5, 10, 20 and 50 μmol/L in 4-HNE-stimulated group, while the negative control group was added in the same volume of ethanol (the solvent of 4-HNE). Then the cells were stimulated with 4-HNE for 24 hours following by CCK-8 kits incubating for 4 hours to detect absorbance. It was found that 10 μmol/L 4-HNE had the most obvious effect on the proliferation of hRMECs. Therefore, the cellular proteins from 10 μmol/L 4-HNE-stimulated group and negative control group were acquired and prepared by FASP sample preparation method. Data independent acquisition was used for data acquisition, and the GO analysis and pathway enrichment were performed for analysis of differentially expressed proteins. Results CCK-8 kits detection results showed that the A value of the 10 and 20 μmol/L 4-HNE-stimulated groups were significantly higher than negative control group and 5 μmol/L 4-HNE-stimulated group (F=25.42, P 1.5, P<0.05). Seventy-two proteins were up-regulated after 4-HNE stimulation, whereas 46 proteins were down-regulated. Particularly, the expressions of Heme oxygenase-1, Sulfoxdoxin-1, Heat shock protein A1B, Thioredoxin reductase-1, Glutathione reductase, ATPase and prothrombin were increased when cells were added in 4-HNE, whereas the expressions of apolipoprotein A1 and programmed cell death protein 4 were decreased. These differentially expressed proteins were mainly involved in the biological processes such as oxidative stress, cell detoxification, and ATPase-coupled membrane transport. Conclusion After stimulated with 4-HNE, the oxidative stress, cell detoxification, and ATPase-coupled membrane transport protein expression may change in hRMECs in order to regulate oxidative stress and growth situation. Key words: Reitinal vessels/cytology; Endothelial; Cell cultured; Proteomics; 4- Hydroxynonenal

YAP1 is required for the angiogenesis in retinal microvascular endothelial cells via the inhibition of MALAT1-mediated miR-200b-3p in high glucose-induced diabetic retinopathy.

Diabetic retinopathy (DR) is one of the severest complications in the development of diabetes with a characteristic of intraretinal new vessel formation. Our present study attempts to probe into the involvement of Yes-associated protein 1 (YAP1) in retinal microvascular endothelial cells (RMECs) and the underlying mechanism. The DR mouse model was induced by streptozotocin injection and a high-glucose/high-fat diet. After that, with the utilization of the Pearson's correlation analysis, the correlation between YAP1 and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was analyzed. The relationship between microRNA-200b-3p (miR-200b-3p) and MALAT1 or vascular endothelial growth factor A (VEGFA) was then validated. Finally, the cellular processes including the abilities of proliferation, migration, as well as tube formation, were evaluated after the alteration of YAP1, MALAT1, and miR-200b-3p expression. YAP1 was observed to be highly expressed in retinas from DR mice, which promoted the cellular processes of RMECs through upregulating the MALAT1 expression. It was further confirmed that MALAT1 could sponge miR-200b-3p, and miR-200b-3p directly targeted VEGFA. When YAP1 was silenced, the RMEC proliferation, migration, and angiogenesis in the retina of DR mice were reduced. From these data, we conclude that YAP1 may exert some promotive effects on the development of DR through its regulation of the MALAT1/miR-200b-3p/VEGFA axis, highlighting that YAP1 silencing may be instrumental for the therapeutic targeting of DR.

MicroRNA-9 inhibits retinal neovascularization in rats with diabetic retinopathy by targeting vascular endothelial growth factor A.

Diabetic retinopathy (DR) is a leading cause of adult visual impairment and loss. This study aims to explore the effects of microRNA-9 (miR-9) on retinal neovascularization during DR by targeting the vascular endothelial growth factor A (VEGFA). DR rat models were successfully established. Retinal microvascular endothelial cells (RMECs) of DR rats were isolated and treated with miR-9 mimic, miR-9 inhibitor or small interfering RNA (siRNA)-VEGFA. The expressions of miR-9, VEGFA, and cluster of differentiation 31 (CD31) of the rats' tissues and cells were examined. The targeting relationship between miR-9 and VEGFA was testified. The tubule formation, the cell proliferation and the periodic distribution and apoptosis were evaluated after transfection. In the retinal tissues of DR rats, miR-9 expression decreased while the expression of VEGFA and CD31 increased. Notably, miR-9 targeted and inhibited VEGFA expression. In response to the treatment of miR-9 mimic and siRNA-VEGFA, a reduction was identified in CD31 expression, tubule formation, and proliferation of RMECs and cell ratio in the S phase, but an increase was observed in apoptosis rate of RMECs. The treatment of miR-9 inhibitor reversed the manifestations. Our study demonstrated that miR-9 could inhibit retinal neovascularization of DR and tubule formation, and promote apoptosis in RMECs by targeting VEGFA.

Microglia enhanced the angiogenesis, migration and proliferation of co-cultured RMECs

BackgroundAttention is increasingly being given to microglia-related inflammation in neovascular diseases, such as diabetic retinopathy and age-related macular disease. Evidence shows that activated microglia contribute to disruption of the blood–retinal barrier, however, the mechanism is unclear. In this study, we aimed to clarify whether and how microglia affect the function of retinal microvascular endothelial cells (RMECs).MethodsWe activated microglia by Lipopolysaccharides (LPS) stimulation. After co-culturing static or activated microglia with RMECs using the Transwell system, we evaluated the function of RMECs. Vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) levels in the supernatant from the lower chamber were evaluated by ELISA. Angiogenesis, migration, and proliferation of RMECs were assessed by tube formation, wound healing, and WST-1 assays. The expression levels of tight junction proteins (ZO-1 and occludin) and endothelial markers (CD31 and CD34) were examined by Western blot analysis.ResultsWe successfully established an LPS-activated microglia model and co-culture system of static or activated microglia with RMECs. In the co-culture system, we showed that microglia, especially activated microglia stimulated VEGF-A and PDGF-BB expression, enhanced angiogenesis, migration, proliferation, and permeability, and altered the phenotype of co-cultured RMECs.ConclusionsMicroglia, especially activated microglia, play important roles in angiogenesis and maintenance of vascular function hemostasis in the retinal microvasculature. The mechanism needs further investigation and clarification.

BH4-Mediated Enhancement of Endothelial Nitric Oxide Synthase Activity Reduces Hyperoxia-Induced Endothelial Damage and Preserves Vascular Integrity in the Neonate.

PurposeEndothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) has important vasoprotective functions that are compromised in the vasodegenerative phase of retinopathy of prematurity, owing to hyperoxia-induced depletion of the essential NOS cofactor BH4. Because modulating eNOS function can be beneficial or detrimental, our aim was to investigate the effect of BH4 supplementation on eNOS function and vascular regression in hyperoxia.MethodsEndothelial-specific eNOS-green fluorescent protein (GFP) overexpressing mice at postnatal day 7 (P7) were exposed to hyperoxia for 48 hours in the presence or absence of supplemental BH4, achieved by administration of sepiapterin, a stable BH4 precursor. Tissue was collected either for retinal flat mounts that were stained with lectin to determine the extent of vessel coverage or for analysis of BH4 by high-performance liquid chromatography, nitrotyrosine (NT) marker by Western blotting, VEGF expression by ELISA, and NOS activity by arginine-to-citrulline conversion. Primary retinal microvascular endothelial cells (RMEC) were similarly treated, and hyperoxia-induced damage was determined.ResultsSepiapterin effectively enhanced BH4 levels in hyperoxia-exposed retinas and brains, elevated NOS activity, and reduced NT-modified protein, leading to reversal of the exacerbated vasoregression observed in the presence of eNOS overexpression. In RMECs, hyperoxia-mediated depletion of BH4 dysregulated the redox balance by reducing nitrite and elevating superoxide and impaired proliferative ability. BH4 supplementation restored normal RMEC proliferation in vitro and also in vivo, providing a mechanistic link with the enhanced vascular coverage in eNOS-GFP retinas.ConclusionsThese results demonstrate that BH4 supplementation corrects hyperoxia-induced RMEC dysfunction and preserves vascular integrity by enhancing eNOS function.

Inhibition and mechanism of 15 (S)-hydroxyeicosate traenoic acid on proliferation of hypoxic retinal microvascular endothelial cells in vitro

Background Retninal neovascular diseases caused by hypoxia has become a major blinding disease,which is lack of effective chemical treatment currently,it's important to study the molecuar mechanism of the disease,so as to guide the clinical medication.Objective This study was to explore the effect of 15 (S)-hydroxyeicosate traenoic acid (15-HETE) on the proliferation of hypoxic retinal microvascular endothelial cells (RMVECs) and its probable mechanism.Methods RMVECs were isolated from C57BL/6J mice and incubated and then identified with anti-Ⅷ factor antibody by immunochemistry and immunofluorescence.The cells were divided into the normoxia group and the hypoxia group.The hypoxia cell models were established by treated with 125 μmol/L CoCl2.The cells were cultured with serum-free DMEM containing endothelial cell growth supplement (ECGS)and high glucose for 48 hours,and then different concentrations of 15-HETE (0.0,0.1,1.0,5.0 μmol/L) were added in the medium for 48 hours respectively to subgroup the groups.The proliferation of the cells (absorbance,A) was detected using MTT.The relative expression levels of protein and mRNA of hypoxia inducible factor-1α (HIF-1α),bcl-2 and caspase-3 were assayed by reverse transcription PCR (RT-RCR)and Western blot.Results The cells showed the positive response for anti-Ⅷ factor antibody with the positive rate of (94.38 ±4.25)％.No significant difference was found in the cell proliferation of various groups under the normoxia condition (F =0.283,P =0.837),but under the hypoxia condition,the proliferation values were significantly different among various groups (F =702.582,P＜0.001).The cell proliferation value in the 1.0 μmol/L 15-HETE group and 5.0 μmol/L 15-HETE group was lower than that of the simple hypoxia group respectively(both at P＜0.05).The inhibitory rates in the 0.1,1.0,5.0 μ mol/L 15-HETE groups were (1.09±0.31) ％,(21.09± 3.53) ％ and (49.86 ±4.15) ％,showing a dosedependent manner.No significant difference was seen in the expression levels of bcl-2,caspase-3 and HIF-1α mRNA in various groups under the normoxia conditions.However,compared with the simple normoxia group,the relative expressions of bcl-2 mRNA and HIF-1α mRNA in the cells were increased by 1.53 folds and 1.7 folds in the simple hypoxia group respectively,and caspasse-3 mRNA expression decreased by 70％ (all at P ＜ 0.05).Under the normoxia condition,the expression of bcl-2 and pro-caspase-3 protein in the cells were not significantly different among the various groups (P＞0.05),however,the expressions of bcl-2 and pro-caspase-3 proteins were elevated by 1.6 folds and 1.9 folds in the hypoxia group in compared with the normoxia group (P＜0.05).Compared with the simple hypoxia group,the expressions of bcl-2 and pro-caspase-3 were lowed by 40.4％ and 42.5％ in the 5.0 μmol/L 15-HETE group (P＜0.05).Conclusions 15-HETE inhibits the proliferation of RMVECs and therefore suppresses neovascularization by down-regulating the expressions of HIF-1α and bcl-2 and the activation of caspase-3 in a dose-dependent manner. Key words: Retinal neovascularization; Retinal microvascular endothelial cell; Hypoxia inducible factor1α ; Bcl-2; Caspase-3

Endoglin promotes angiogenesis in cell- and animal-based models of retinal neovascularization.

We investigated endoglin expression in hypoxic microvascular endothelial cells and retinal endoglin expression in rats that develop experimental oxygen-induced retinopathy (OIR). We also tested neutralizing antibodies (Abs) against endoglin (anti-CD105 Ab) and VEGF (anti-VEGF Ab) either alone or in combination for efficacy against serum-induced retinal microvascular endothelial cell proliferation and retinal neovascularization (NV) in OIR rats. To our knowledge, this marks the first time that a biologic agent has been used to target retinal endoglin and modulate retinal neovascularization. Induction of endoglin by hypoxia was measured by immunohistochemical analysis and ELISA. Proliferation was quantified using a colorimetric 5-bromo-2-deoxyuridine ELISA. Western blots were used to measure endoglin levels in retinas of OIR rats. Immunohistochemical staining was also preformed in OIR rats using anti-CD105 and fluorescein isothiocyanate-conjugated isolectin B4 antibodies. Anti-CD105 Ab and Anti-VEGF Ab, administered alone or in combination, reduced serum-induced retinal microvascular endothelial cell proliferation. Additionally, in a rat model of oxygen-induced retinopathy, retinal endoglin was significantly increased at 14(2), 14(3), 14(4) and 14(6) compared with retinal levels in control rats. At 14(2), immunohistochemical analysis demonstrated that endoglin was elevated in newly developed vessels at the peripheral extent of major veins, precisely where NV is expected to develop in OIR rats. Neutralizing anti-CD105 reduced retinal NV in OIR rats. Our data support other studies showing that reduction of endoglin expression inhibits retinal NV. Our findings demonstrate that retinal endoglin immunolocalization overlaps with nascent neovascular structures in OIR rats. Therefore, endoglin may serve as a useful predictor of incipient neovascular disease.

Role of Cytosolic Phospholipase A2in Retinal Neovascularization

To identify and characterize the role of cytosolic phospholipase A(2) (cPLA(2)) in retinal angiogenesis using relevant cell-based assays and a rodent model of retinopathy of prematurity. The phosphorylation states of cPLA(2) and p38 MAP kinase and the expression of COX-2 were assessed by Western blot analysis in rat Müller cells. The activities of PLA(2) enzymes in rat retinal lysates were assessed using a commercially available assay. Prostaglandin E(2) (PGE(2)) and VEGF levels in Müller cell-conditioned medium and in retinal tissue samples were measured by ELISA. Rat retinal microvascular endothelial cell proliferation was measured using a BrdU assay. Efficacy of the cPLA(2) inhibitor CAY10502 was tested using the rat model of oxygen-induced retinopathy (OIR) in which neovascularization (NV) was assessed by computer-assisted image analysis. In Müller cells, hypoxia increased the phosphorylation of cPLA(2) and p38 MAP kinase by 4-fold and 3-fold respectively. The cPLA(2) inhibitor CAY10502 decreased hypoxia-induced PGE(2) and VEGF levels in Müller cell-conditioned medium by 68.6% (P < 0.001) and 46.6% (P < 0.001), respectively. Retinal cPLA(2) activity peaked 1 day after oxygen exposure in OIR rats. CAY10502 (250 nM) decreased OIR-induced retinal PGE(2) and VEGF levels by 69% (P < 0.001) and 40.2% (P < 0.01), respectively. Intravitreal injection of 100 nM CAY10502 decreased retinal NV by 53.1% (P < 0.0001). cPLA(2) liberates arachidonic acid, the substrate for prostaglandin (PG) production by the cyclooxygenase enzymes. PGs can exert a proangiogenic influence by inducing VEGF production and by stimulating angiogenic behaviors in vascular endothelial cells. Inhibition of cPLA(2) inhibits the production of proangiogenic PGs. Thus, cPLA(2) inhibition has a significant influence on pathologic retinal angiogenesis.

Herbimycin A inhibits angiogenic activity in endothelial cells and reduces neovascularization in a rat model of retinopathy of prematurity

The pathogenesis of retinopathy of prematurity involves dysregulated angiogenesis resulting in pre-retinal growth of new vessels. Inhibition of tyrosine kinase-dependent pro-angiogenic signals may provide a rational therapeutic approach to the reduction of pre-retinal neovascularization. Vascular endothelial growth factor stimulates endothelial cell mitogenesis, differentiation and migration, by binding and activating the receptor tyrosine kinases vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2. One of the vascular endothelial growth factor receptor substrates implicated in vascular endothelial growth factor signal transduction is c-Src. The ability of herbimycin A, a c-Src-selective tyrosine kinase inhibitor, to inhibit vascular endothelial growth factor-induced bovine retinal microvascular endothelial cell proliferation and tube formation was investigated. The ability of the compound to inhibit pathologic angiogenesis was tested in a rat model of retinopathy of prematurity. Exposure of neonatal rats to oxygen concentrations cycling between 10 and 50% induced severe pre-retinal neovascularization in all rats. Some of the eyes of these variable oxygen-exposed rats were herbimycin A-injected or vehicle-injected 1 or 3 days post-oxygen exposure while some eyes were non-injected. All rats were sacrificed for assessment 6 days post-exposure. Herbimycin A inhibited both vascular endothelial growth factor-induced bovine retinal microvascular endothelial cell proliferation and capillary tube formation in a dose-dependent manner. Injection of herbimycin A into oxygen-treated rats 1day post-oxygen exposure produced a 63% decrease in pre-retinal neovascularization relative to vehicle ( P=0·0029). There was a 41% decrease in pre-retinal neovascularization in herbimycin-injected eyes relative to vehicle-injected eyes 3 days post-oxygen ( P=0·031). Pre-retinal neovascularization was reduced in vehicle-injected eyes relative to non-injected eyes at both injection times. There were no significant differences in retinal vascular area between any of the experimental groups. Based on the results of this study, herbimycin A inhibits endothelial cell proliferation and tube formation at non-toxic concentrations and reduces pre-retinal neovascularization in a rat model of retinopathy of prematurity. Reduction of angiogenesis by the inhibition of tyrosine kinase activity may be a viable route to the development of effective chemotherapies applicable to eye disease.