Migration Of Retinal Endothelial Cells Research Articles

Siponimod as a novel inhibitor of retinal angiogenesis: in vitro and in vivo evidence of therapeutic efficacy.

S1P receptors control endothelial cell proliferation, migration, and survival. Evidence of the ability of S1P receptor modulators to influence multiple endothelial cell functions suggests their potential use for antiangiogenic effect. The main purpose of our study was to investigate the potential of siponimod for the inhibition of ocular angiogenesis in vitro and in vivo We investigated the effects of siponimod on the metabolic activity (MTT assay), cell toxicity (LDH release), basal proliferation and growth factor induced proliferation (BrdU assay), and migration (transwell migration assay) of human umbilical vein endothelial cells (HUVEC) and retinal microvascular endothelial cells (HRMEC). The effects of siponimod on HRMEC monolayer integrity, and barrier function under basal conditions and TNF-a induced disruption were assessed using the trans-endothelial electrical resistance (TEER) and FITC-dextran permeability assays. Siponimod's effect on TNF-α induced distribution of barrier proteins in HRMEC was investigated using immunofluorescence. Finally, the effect of siponimod on ocular neovascularization in vivo was assessed using suture-induced corneal neovascularization in albino rabbits. Our results show that siponimod did not affect endothelial cell proliferation or metabolic activity, but significantly inhibited endothelial cell migration, increased HRMEC barrier integrity, and reduced TNF-a induced barrier disruption. Siponimod also protected against TNF-a induced disruption of claudin-5, ZO-1, and VE-cadherin in HRMEC. These actions are mainly mediated by S1PR1 modulation. Finally, siponimod prevented the progression of suture-induced corneal neovascularization in albino rabbits. In conclusion, the effects of siponimod on various processes known to be involved in angiogenesis support its therapeutic potential in disorders associated with ocular neovascularization. Significance Statement Siponimod is an extensively characterized S1P receptor modulator already approved for the treatment of multiple sclerosis. It inhibits retinal endothelial cell migration, potentiates endothelial barrier function, protects against TNF-a induced barrier disruption, and also inhibits suture-induced corneal neovascularization in rabbits. These results support its use for a novel therapeutic indication in the management of ocular neovascular diseases.

PLAG1 Promotes High Glucose-Induced Angiogenesis and Migration of Retinal Endothelial Cells by Regulating the Wnt/β-Catenin Signalling Pathway

Proliferation and migration of retinal endothelial cells (RECs) contribute to the development of diabetic retinopathy. PLAG1 (pleomorphic adenoma gene 1) functions as a zinc-finger transcription factor to participate in the development of lipoblastomas or pleomorphic adenomas of the salivary glands through regulation of cell proliferation and migration. The role of PLAG1 in diabetic retinopathy was investigated in this study. Firstly, RECs were induced under high glucose conditions, which caused reduction in viability and induction of apoptosis in the RECs. Indeed, PLAG1 was elevated in high glucose-treated RECs. Functional assays showed that silence of PLAG1 increased viability and suppressed apoptosis in high glucose-induced RECs, accompanied with up-regulation of Bcl-2 and down-regulation of Bax and cleaved caspase-3. Moreover, migration of RECs was promoted by high glucose conditions, while repressed by knockdown of PLAG1. High glucose also triggered angiogenesis of RECs through up-regulation of vascular endothelial growth factor (VEGF). However, interference of PLAG1 reduced VEGF expression to retard the angiogenesis. Silence of PLAG1 also attenuated high glucose-induced up-regulation of Wnt3a, β-catenin and c-Myc in RECs. Moreover, silence of PLAG1 ameliorated histopathological changes in the retina of STZ-induced diabetic rats through down-regulation of β-catenin. In conclusion, knockdown of PLAG1 suppressed high glucose-induced angiogenesis and migration of RECs, and attenuated diabetic retinopathy by inactivation of Wnt/β-catenin signalling.

Dysregulation of miR-637 Is Involved in the Development of Retinopathy in Hypertension Patients and Serves a Regulatory Role in Retinol Endothelial Cell Proliferation

Background: MicroRNAs play an important role in the proliferation and migration of retinal endothelial cells in patients with hypertension and hypertensive retinopathy (HR). This study aimed to investigate the clinical value of miR-637 in HR and its role in retinal endothelial cell proliferation and migration. Methods: A total of 126 subjects were recruited for the study, including 42 patients with hypertension (male/female 25/17), 42 healthy individuals (male/female 20/22), and 42 cases with HR (male/female 20/22). Except SBP and DBP, there was no significant difference in other indexes among the three groups. Quantitative real-time PCR was used to detect the expression of miR-637. The receiver operating curve (ROC) was used for diagnosis value analysis. Logistic regression analysis was used to evaluate the relationship between miR-637 and HR. CCK-8 and Transwell were used to detect the effect of miR-637 on the proliferation and migration of human umbilical vein endothelial cells. Results: Compared with hypertensive patients, HR patients had the lowest expression of miR-637. The area under the curve of miR-637 detected by the ROC curve method is 0.892, which has the ability to distinguish hypertension and HR patients. Logistic regression analysis showed that miR-637 was an independent influencing factor in HR. Cell experiment results showed that overexpression of miR-637 significantly inhibited cell proliferation and migration, while downregulation of miR-637 had the opposite effect. Luciferase analysis showed that STAT3 was the target gene of miR-637. Conclusion: Our data indicate that miR-637 is a potential noninvasive marker for patients with HR. The action of miR-637 on STAT3 may inhibit the proliferation and migration of retinal endothelial cells, providing a possible target for the treatment of HR.

ANKFY1 is essential for retinal endothelial cell proliferation and migration via VEGFR2/Akt/eNOS pathway

Dysregulation of endothelial cell proliferation and migration are hallmarks of angiogenic diseases. Among them, excessive ocular angiogenesis is a major cause of blindness. Vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2) signaling plays crucial roles in angiogenesis, endothelial cell proliferation and migration. Here, we showed that ankyrin repeat and FYVE domain containing 1 (ANKFY1), a Rab5-GTP-interacting protein, is required for retinal endothelial cell proliferation and migration. ANKFY1 knockdown significantly suppressed cell growth of human retinal microvascular endothelial cells (HRMECs) in the presence or absence of VEGF. HRMEC migration was also inhibited by depletion of ANKFY1. Western blot analysis showed that ANKFY1 knockdown reduced cell surface VEGFR2 level. In contrast, qRT-PCR analysis indicated that ANKFY1 knockdown had no effect on VEGFR2 mRNA levels. We also found that the attenuation of the protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) pathway in ANKFY1 knockdown HRMECs. In conclusion, our findings revealed novel functions of ANKFY1 in cell growth and migration of retinal endothelial cells.

LncRNA MIR497HG inhibits proliferation and migration of retinal endothelial cells under high-level glucose treatment via miRNA-128-3p/SIRT1 axis.

The aim of this study was to elucidate the potential influence of MIR497HG on regulating proliferative capacity of human retinal endothelial cells (HRECs). Relative expression levels of MIR497HG, microRNA-128-3p (miRNA-128-3p) and SIRT1 in HRECs treated with different doses of glucose and mannitol were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Dual-Luciferase reporter gene assay was conducted to assess the interaction among MIR497HG, miRNA-128-3p, and SIRT1. In addition, the potential effects of MIR497HG/miRNA-128-3p/SIRT1 axis on proliferative and migratory capacities in HRECs were evaluated by Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'- deoxyuridine (EdU) and transwell assay, respectively. High-level glucose (HG) treatment significantly downregulated MIR497HG and SIRT1 expression, whereas upregulated miRNA-128-3p expression in HRECs (p<0.05). MiRNA-128-3p was the target gene binding MIR497HG, and SIRT1 was the downstream gene of miRNA-128-3p. Overexpression of MIR497HG significantly attenuated proliferative and migratory abilities of HG-induced HRECs (p<0.05). Furthermore, decreased trends were partially reversed by overexpression of miRNA-128-3p or knockdown of SIRT1. MIR497HG is downregulated after HG treatment. In addition, it suppresses the proliferation and migration of HRECs by targeting miRNA-128-3p/SIRT1 axis, thus influencing the progression of diabetic retinopathy.

597-P: Retinol Binding Protein 3 in Diabetic Retinopathy: Mechanisms of Protective Activity

The Joslin Medalist study reported that after having type 1 diabetes (T1D) for &amp;gt; 50 years, over 35% of Medalists exhibit no to mild diabetic retinopathy (DR) independent of glycemic control, suggesting presence of endogenous protective factors against DR. Proteomic analysis of Medalist retina and vitreous found that Retinol Binding Protein 3 (RBP3), secreted by photoreceptors, is elevated in individuals protected from DR. This finding was confirmed by RBP3 ELISA of replicate vitreous samples from Medalists and other T1D and T2D individuals. In contrast, vitreous VEGF concentrations increased with DR severity. Thus, elevated ratios of intravitreous RBP3/VEGF correlated with lack of progression to advanced DR. Intervention and prevention studies overexpressing RBP3 in retina/photoreceptors of nondiabetic and diabetic animals inhibited VEGF actions and normalized diabetes-induced retinal capillary permeability, neuro-retinal dysfunctions, expressions of VEGF and IL-6, and formation of acellular capillaries. Mechanistically, physiological levels of recombinant RBP3 (10-100nM), but not RBP4 or FGF, inhibited the increased in VEGFR2 tyrosine phosphorylation, hyperglycemia-induced expression of pro-inflammatory cytokines (VEGF and IL-6), and migration of retinal endothelial cells and Müller cells. RBP3, but not RBP4, physically bound to VEGFR2 in cross linkage studies. At physiological levels, RBP3 significantly inhibited glucose uptake into retinal endothelial and Müller cells, and subsequently decreased expression of VEGF and IL-6. These results suggest novel actions for RBP3 to slow glucose uptake in retinal cells and inhibit VEGF actions, thereby preventing, delaying and even arresting diabetes-induced neuro- and vascular retinal dysfunction, inflammatory abnormalities and DR pathologies. These findings strongly support therapeutic potential of RBP3 in DR. Disclosure H. Yokomizo: None. K. Park: None. A. Clermont: Consultant; Self; KalVista Pharmaceuticals, Inc. Y. Maeda: Research Support; Self; Abbott, Arkray, Inc. Speaker's Bureau; Self; Abbott, Arkray, Inc., Astellas Pharma Inc., AstraZeneca, Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Taisho Toyama Pharmaceutical Co., Ltd., Terumo Medical Corporation. W. Fickweiler: None. A. Ishikado: Employee; Self; SunStar Inc. Employee; Spouse/Partner; SunStar Inc. Q. Li: None. C. Wang: None. D.M. Pober: None. I. Wu: None. H.A. Keenan: Employee; Self; Sanofi Genzyme. L.P. Aiello: Advisory Panel; Self; Novo Nordisk A/S. Consultant; Self; Decision Resources Group, KalVista Pharmaceuticals, Inc., Mingsight, Retinal Solutions. Stock/Shareholder; Self; KalVista Pharmaceuticals, Inc. Other Relationship; Self; Optos. J.K. Sun: Research Support; Self; Adaptive Sensory Technology, Boston Micromachines Corporation, Genentech, Inc., KalVista Pharmaceuticals, Inc., Optovue, Incorporated. Other Relationship; Self; Genentech, Inc., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc. G.L. King: Research Support; Self; Sanofi. Funding National Eye Institute; National Institute of Diabetes and Digestive and Kidney Diseases; JDRF; Thomas J. Beatson, Jr. Foundation Inc.; Dianne Nunnally Hoppes Scholarship Fund

The synergistic effect of metformin and anti-vascular endothelial growth factor in the treatment of diabetic retinopathy

Objective To observe the synergistic effect of metformin and anti-vascular endothelial growth factor (VEGF) in the treatment of diabetic retinopathy. Methods This study was composed of clinical data review and in vitro cell experiment. Ten patients (12 eyes) with diabetic macular edema treated with anti- VEGF drugs were included in the study. Patients were randomly divided into the VEGF group (anti-VEGF drug therapy) and the combined treatment group (anti-VEGF drug combined with metformin). The changes of visual acuity and central retinal thickness (CRT) were compared between the two groups. As far as the in vitro experiment was concerned, vascular endothelial cells were divided into the control group (normal cells), the VEGF group (50 ng/ml VEGF), the anti-VEGF group (50 ng/ml VEGF+2.5 μg/ml of conbercept), and the combined group (50 ng/ml VEGF +2.5 μg/ml of conbercept +2.0 mmol/L of metformin). And then MTT cell viability assay, scratch assay and real-time quantitative polymerase chain reaction assay were performed to analyze the cell viability, cell migration and mRNA level of VEGFR2, protein kinase C (PKC)-α and PKC-β successively. Results Review of clinical trial shows that the CRT recovery rates in the combined treatment group were much higher than that in the VEGF group at 3 month after the operation, while the difference was statistically significant (t=−2.462, P<0.05). In vitro cell experiment results showed that VEGF induction upregulated the viability and mobility of vascular endothelial cells obviously compared with control group, at the same time, the use of anti VEGF drugs can effectively reverse the trend, in contrast, combination of metformin and anti-VEGF showed a more superior effect to some extent (P<0.05). In the VEGF group, the mRNA expression of VEGFR2, PKC-αand PKC-β were significantly increased compared with the control group (P <0.01); while the mRNA expression of VEGFR2, PKC-αand PKC-β in the combination group decreased significantly compared with the VEGF group and the control group (P<0.05). However, in the anti-VEGF group, the mRNA expression of VEGFR2, PKC-αand PKC-β were decreased, but has failed to reach the level of statistical learn the difference. Conclusions The combination of metformin and anti-VEGF drugs can reduce the CRT of diabetic retinopathy patients and inhibit the proliferation and migration of retinal vascular endothelial cells which induced by VEGF. The synergistic mechanism may be related to the inhibitory effect of metformin on the expression of VEGFR and PKC. Key words: Diabetic retinopathy/drug therapy; Metformin/therapeutic use; Angiogenesis inhibitors/therapeutic use; In vitro

Role of autophagy in angiogenesis of retinal vascular endothelial cells under hypoxia condition

Objective To study the role of autophagy in proliferation, migration and tube formation of retinal vascular endothelial cells (RVECs) under the condition of hypoxia in vitro. Methods Mouse RVECs were isolated from 50 C57BL/6J mice primarily cultured using explant culture method, and the cells were identified by immunofluorescence of CD34.Well cultured RVECs were divided into normal control group, hypoxia control group and hypoxia+ 3-methyladenine (3-MA) group.The cells in the normal control group were cultured in the normal environment for 24 hours, and the cells in the hypoxia control group were cultured 1% O2 environment for 24 hours, and the cells in the hypoxia+ 3-MA group were pretreated with 5 mmol/L 3-MA for 4 hours and then exposed to 1% O2 environment for 24 hours.Microtubule-related protein 1 light chain 3 (LC3-Ⅱ/LC3-Ⅰ) and Beclin-1 protein contents in the cells were detected by Western blot analysis; the ultrastructure of autophagosome was examined under the transmission electron microscope.The proliferation, migration and tube formation of the cells were detected by Click-iTEdU kit, scratch assay and matrigel, respectively. Results Primarily culture cells grew well with the cobblestone-like appearance 5-7 days after culture and showed positive response for CD34.The autophagosome number was increased in the hypoxia control group compared with the hypoxia+ 3-MA group and normal control group.The LC3-Ⅱ/LC3-Ⅰ ratio was 0.243±0.030, 0.658±0.032 and 0.405±0.095; the relative expression of Beclin-1 protein in the cells was 0.260±0.040, 0.650±0.071 and 0.461±0.089; the proliferation rate of the cells was (45.93±6.39)%, (22.74±2.35)% and (24.12±3.59)%; the scratch healing rate of the cells was (36.02±5.84)%, (57.26±11.98)% and (18.16±9.73)%; the number of tube formation was 29.20±6.10, 41.40±4.04 and 22.00±2.92 in the normal control group, hypoxia control group and hypoxia+ 3-MA group, respectively, with significant differences among the groups (F=35.86, 23.53, 34.28, 21.12, 23.27; all at P<0.01). Compared with the normal control group, the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression of Beclin-1 protein, scratch healing rate and the number of tube formation were significantly increased, and the proliferation rate was significantly reduced in the hypoxia control group (all at P<0.05). Compared with the hypoxia control group, the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression of Beclin-1 protein, scratch healing rate and the number of tube formation were significantly decreased in the hypoxia+ 3-MA group. Conclusions Hypoxia environment activates autophagy of mouse RVECs, which enhances the migration and tube formation abilities of the cells.3-MA inhibits the angiogenesis abilities of mouse RVECs. Key words: Autophagy; Angiogenesis; Hypoxia; Retinal neovascularization; Ischemic retinopathy; Cells, cultured; Mice, inbred C57BL

The effects of recombinant human platelet derived growth factor-BB on biological behaviours of human retinal vascular endothelial cells

Objective This study was to investigate the role of recombinant human platelet derived growth factor-BB(rhPDGF-BB) in the proliferation and migration of human retinal vascular endothelial cells (hRVECs). Methods hRVECs were cultured in DMEM with 10% fetal bovine serum.The rhPDGF-BB at the concentrations of 10, 50 and 200 ng/ml were added into the medium of exponential phase-growth cells for 24 and 48 hours, respectively, and no rhPDGF-BB was added in the normal control group.The proliferation of the cells (absorbancy) was assayed by cell counting kit 8(CCK8) method.Cell scratch test was employed to evaluate the relative migration area of cells (migrated acellular area/initial acellular area). The relative expression of rhPDGF-BB recepter (rhPDGF-BBR) mRNA in the cells was detected by reverse transcription PCR.The relative expression of VEGF mRNA and integrin mRNA in the cells was detected using real-time fluorescence quantitative PCR. Results hRVECs grew well and a expressing band according with rhPDGF-BBR prime was displayed.The absorbancy values of the cells were 1.01±0.05, 1.09±0.04, 1.10±0.02 and 1.13±0.05 in the normal control group and 10, 50, 200 ng/ml rhPDGF-BB groups at 24 hours after culture, and those in the 10, 50 and 200 ng/ml rhPDGF-BB groups were significantly increased in comparison with the normal control group (t=2.504, 3.430, 3.483, all at P<0.05). The relative migrated areas of the cells in the normal control group and 10, 50, 200 ng/ml rhPDGF-BB groups were 0.42±0.10, 0.38±0.09, 0.55±0.06 and 0.61±0.05 at 24 hours after culture, and those at 48 hours were 0.75±0.06, 0.81±0.02, 0.87±0.02 and 0.98±0.02, showing significant differences among the groups (Fgroup=16.283, P=0.000; Ftime=209.129, P=0.000), and the relative migrated areas was depended upon the rhPDGF-BB dose and time.The relative expressions of integrin mRNA were 1.06±0.02, 1.30±0.10, 1.20±0.16 and 1.27±0.08, and those of VEGF mRNA were 0.97±0.05, 1.06±0.16, 1.58±0.18 and 1.66±0.21 in the normal control group and 10, 50 ng/ml, 200 ng/ml rhPDGF-BB groups, respectively, and increased expressions of integrin mRNA and VEGF mRNA were found in the 50 and 200 ng/ml rhPDGF-BB groups compared with the normal control group (integrin mRNA: t=3.900, 4.014, both at P<0.05; VEGF mRNA: t=6.940, 7.210, both at P< 0.05). Conclusions rhPDGF-BB/rhPDGF-BBR signal promotes the proliferation and migration of hRVECs probably by up-regulating the expressions of integrin and VEGF. Key words: Endothelium, vascular/cytology; Neovascularization, pathologic/metabolism; Platelet-derived growth factor; Receptors, platelet-derived growth factor; Recombinant proteins; Humans; Cells, cultured; Integrin; Vascular endothelial growth factor

Protective effect of miR-200b/c by inhibiting vasohibin-2 in human retinal microvascular endothelial cells

AimsProliferative diabetic retinopathy (PDR), characterized by angiogenesis, can cause serve vision loss and even blindness. Recent studies have suggested a pivotal role of vasohibin-2 (VASH2) in the promotion of angiogenesis in tumor tissues. Here we further investigated the role of VASH2 in the proliferation and migration of retinal endothelial cells. Main methodsThe expression of VASH2 in vascular endothelial cells of epiretinal fibrovascular membranes (FVMs) from PDR patients were detected by immunofluorescence. VASH2 gene interfering lentiviral vectors (VASH2-shRNA) and miR-200b/c were constructed for the evaluation of the VASH2 effect on high glucose induced human retinal microvascular endothelial cell line (HRMECs). Cell proliferation, cell cycle and cell migration were carried out subsequently. The relationship between VASH2 and miR-200b/c was determined by luciferase reporter gene assays. Key findingsA positive expression of VASH2 was identified in vascular endothelial cells of FVMs from PDR patients. In HRMECs, cells transfected with shRNA or miR-200b/c mimics showed a significantly reduced VASH2 expression compared with negative control group by real time-polymerase chain reaction and western-blot analysis. Inhibition of VASH2 was demonstrated to suppress cell proliferation and migration from Day 2 to Day 4. The luciferase reporter gene assays confirmed the post-transcriptional regulation of VASH2 by miR-200b/c in HRMECs. SignificanceThe present study suggests a protective effect of miR-200b/c on high glucose induced HRMECs dysfunction by inhibiting VASH2. It could be a potential therapeutic strategy to inhibit angiogenesis for the treatment of retinal vascular disease.

The Inhibitory Effect of Mangosteen Extract on Vascular Endothelial Growth Factor Induced Retinal Endothelial Cell Migration

Objective: This study aimed to determine the effect of mangosteen extract on hypoxia induced reactive oxygen species production and vascular endothelial growth factor (VEGF) induced retinal endothelial cell migration.Material and Method: This research studied bovine retinal endothelial cells. The non-toxic concentration of mangosteen extract of water soluble part was verified using trypan blue staining. The effects of mangosteen extract on hypoxia induced reactive oxygen species production and retinal endothelial cells migration were determined using 2’, 7’ dichlorodihydrofluorescein diacetate and scrape/wound assay, respectively. The mechanism of mangosteen extract on retinal endothelial cell migration was determined using western blotting. The analysis of variance was used to determine the differences among group means.Results: The concentrations of mangosteen extract at 25, 50 and 100 mg/ml were non-toxic and these concentrations were used in further experiments. Mangosteen extract at a dose of 100 mg/ml significantly attenuated hypoxia induced reactive oxygen species formation. At all doses, mangosteen extract also significantly inhibited retinal endothelial cell migration. However, the mechanism of mangosteen extract on VEGF signaling did not affect the phosphorylation of VEGF receptor 2 (VEGFR2).Conclusion: Mangosteen extract has anti-oxidant and anti-migration effects.

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.

The effects of interleukin 17 on the proliferation, migration and apoptosis of human retinal vascular endothelial cells

Objective To address the effect and mechanism of interleukin 17 (IL-17) on the proliferation, migration and apoptosis of human retinal vascular endothelial cells (HREC). Methods IL-17 receptor (IL-17R) mRNA and protein expression in human retinal vascular endothelial cells (HREC) were quantified by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation of HREC was examined using CCK-8 assay in the presence of different concentrations of IL-17. Cell migration of HREC was detected using wound scratch assay. Flow cytometry was used to test the effect of IL-17 on the apoptosis of HREC. The effects of IL-17 on HREC expression of basic fibroblast growth factor (bFGF), Caspase-3 and thrombospondin-1 (TSP-1) were quantified by reverse transcriptase polymerase chain reaction (RT-PCR). The effect of IL-17 on HREC expression of Caspase-3 was examined using Western blot. Results IL-17 receptor (IL-17R) expressed in HREC as quantified by RT-PCR and Western blot. The proliferation of HREC in the presence of IL-17 was promoted in a dosage-dependent manner (t=-3.235, -6.276; P=0.032, 0.000). Wound scratch assay showed a significant increase in the migrated distance of HREC with IL-17 stimulation under the concentration of 100 μg/L(t=-3.551, -2.849; P=0.006, 0.019), 200 μg/L(t=-10.347, -4.519; P=0.000, 0.001) and 500 μg/L (t=-3.541, -2.607; P=0.008, 0.036). The intervention of 200 μg/L IL-17 can effectively inhibit the apoptosis of HREC, compared with the control group using flow cytometry (t=5.682, P=0.047). RT-PCR results showed that IL-17 can promote the expression of bFGF and inhibit the expression of Caspase-3 and TSP-1. Western blot result also showed that IL-17 can suppress the protein expression of Caspase-3. Conclusion The mechanism of IL-17 promoting proliferation, migration but suppress apoptosis of HREC may via regulating the expression of bFGF and Caspase-3. Key words: Interleukin-17; Endothelial cells/pathophysiology; Cell proliferation; Cell movement; Apoptosis

Ranibizumab efficiently blocks migration but not proliferation induced by growth factor combinations including VEGF in retinal endothelial cells

BackgroundProliferation and migration of retinal endothelial cells (REC) are associated with the development of proliferative diabetic retinopathy. REC proliferation is stimulated by isoforms of vascular endothelial growth factor-A (i.e., VEGF121 and VEGF165), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1) of which VEGF165 also enhances migration of REC. Effects induced by VEGF-A can be blocked with ranibizumab, a VEGF-binding Fab fragment used in therapy of diabetic macular edema. In this study, we investigated potential angiogenic effects of placental growth factors (PlGF-1, PlGF-2) as other members of the VEGF family and whether the primary action of VEGF165 is modulated in the presence of bFGF, IGF-1 and PlGF-1/-2. We also studied how effects of growth factor combinations can be attenuated with ranibizumab.MethodsEffects of single growth factors or their combinations on proliferation and migration of immortalized bovine retinal endothelial cells (iBREC) were studied with or without ranibizumab or the inhibitor of VEGF receptors KRN951.ResultsProliferation of iBREC was significantly stimulated by 1–100 ng/ml PlGF-1 or PlGF-2, but additive effects were not observed with various combinations of the tested growth factors. Ranibizumab neutralized VEGF’s effect on proliferation but was not effective when the other growth factors were used in combination with VEGF. bFGF and IGF-1 but not PlGF-1 or PlGF-2 stimulated iBREC migration as single agents, and they further enhanced VEGF-induced migration. The effects of such growth factor combinations including VEGF on migration were efficiently blocked by targeting only VEGF with ranibizumab. Migration induced by VEGF plus bFGF and IGF-1 was also almost completely inhibited by KRN951 interfering with VEGF receptor signalling.ConclusionsMigration but not proliferation of iBREC induced by combinations of bFGF, IGF-1, PlGF-1 or PlGF-2 together with VEGF is efficiently suppressed by ranibizumab. VEGF-mediated signalling through VEGFR2 seems to control REC migration dominantly in the presence of other growth factors.

Adrenomedullin-RAMP2 System Is Crucially Involved in Retinal Angiogenesis

Adrenomedullin (ADM) is an endogenous peptide first identified as a strong vasodilating molecule. We previously showed that in mice, homozygous knockout of ADM (ADM(-/-)) or its receptor regulating protein, RAMP2 (RAMP2(-/-)), is embryonically lethal due to abnormal vascular development, thereby demonstrating the importance of ADM and its receptor signaling to vascular development. ADM expression in the retina is strongly induced by ischemia; however, its role in retinal pathophysiology remains unknown. Here, we analyzed oxygen-induced retinopathy (OIR) using heterozygous ADM and RAMP2 knockout mice models (ADM(+/-) or RAMP2(+/-), respectively). In addition, we analyzed the role of the ADM-RAMP2 system during earlier stages of retinal angiogenesis using an inducible endothelial cell-specific RAMP2 knockout mouse line (DI-E-RAMP2(-/-)). Finally, we assessed the ability of antibody-induced ADM blockade to control pathological retinal angiogenesis in OIR. In OIR, neovascular tufts, avascular zones, and hypoxic areas were all smaller in ADM(+/-) retinas compared with wild-type mice. ADM(+/-) retinas also exhibited reduced levels of VEGF and eNOS expression. DI-E-RAMP2(-/-) showed abnormal retinal vascular patterns in the early stages of development. However, ADM enhanced the proliferation and migration of retinal endothelial cells. Finally, we found intravitreal injection of anti-ADM antibody reduced pathological retinal angiogenesis. In conclusion, the ADM-RAMP2 system is crucially involved in retinal angiogenesis. ADM and its receptor system are potential therapeutic targets for controlling pathological retinal angiogenesis.

Targeting of Integrin-Linked Kinase with Small Interfering RNA Inhibits VEGF-Induced Angiogenesis in Retinal Endothelial Cells

Background: The pathological angiogenesis in the retina is a major cause of vision loss at all ages. Vascular endothelial growth factor (VEGF) has been reported as the most potent inducer of retinal neovascularization. We previously demonstrated that integrin-linked kinase (ILK) regulates retinal vascular endothelial proliferation, migration and tube formation. However, little is known about the existence of cross-talk between ILK and VEGF signaling in retinal vascular endothelial cells and the probable regulatory role of ILK during VEGF-induced retinal endothelial cell migration. The purpose of this work was to investigate the role of ILK in VEGF-induced retinal neovascularization. Methods: Cultured retinal endothelial cells (RF/6A) were knocked down for ILK using a small interfering RNA (siRNA). For this, cellular ILK expression was quantified by real-time quantitative PCR, Western blot analysis and immunocytochemical assay, and cytotoxicity of transfection was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ILK siRNA-transfected RF/6A cells were induced by VEGF, and cell proliferation was determined by the MTT assay, cell migration was measured by cell counting in modified Boyden chambers and cell spreading and tube formation assays were performed. Furthermore, the impact of ILK-specific siRNA on VEGF-induced VEGF receptor 2 (VEGFR-2) phosphorylation and activation of downstream signal pathways were tested by Western blot analysis. Results: Both ILK mRNA and protein levels were virtually undetectable after transfection with ILK siRNA, and blocking the expression of ILK by siRNA significantly inhibited VEGF-induced retinal endothelial cell proliferation, attachment, spreading, migration and tube formation. Knockdown of ILK effectively suppressed VEGF-induced p38 mitogen-activated protein kinase (MAPK) and Akt phosphorylation, but had no effects on VEGFR-2, extracellular signal-regulated protein kinase and Jun N terminus kinase phosphorylation. Conclusion: We conclude that knockdown of ILK with siRNA effectively inhibited VEGF-induced retinal endothelial cell attachment, spreading, migration and tube formation. p38 MAPK and Akt are downstream signaling pathways of the ILK that regulated VEGF-induced retinal neovascularization. Targeting ILK may be a potentially useful therapeutic approach for treating ocular neovascularization.

Intra-Ophthalmic Artery Chemotherapy Triggers Vascular Toxicity through Endothelial Cell Inflammation and Leukostasis

Purpose. Super-selective intra-ophthalmic artery chemotherapy (SSIOAC) is an eye-targeted drug-delivery strategy to treat retinoblastoma, the most prevalent primary ocular malignancy in children. Unfortunately, recent clinical reports associate adverse vascular toxicities with SSIOAC using melphalan, the most commonly used chemotherapeutic. Methods. To explore reasons for the unexpected vascular toxicities, we examined the effects of melphalan, as well as carboplatin (another chemotherapeutic used with retinoblastoma), in vitro using primary human retinal endothelial cells, and in vivo using a non-human primate model, which allowed us to monitor the retina in real time during SSIOAC. Results. Both melphalan and carboplatin triggered human retinal endothelial cell migration, proliferation, apoptosis, and increased expression of adhesion proteins intracellullar adhesion molecule-1 [ICAM-1] and soluble chemotactic factors (IL-8). Melphalan increased monocytic adhesion to human retinal endothelial cells. Consistent with these in vitro findings, histopathology showed vessel wall endothelial cell changes, leukostasis, and vessel occlusion. Conclusions. These results reflect a direct interaction of chemotherapeutic drugs with both the vascular endothelium and monocytes. The vascular toxicity may be related to the pH, the pulsatile delivery, or the chemotherapeutic drugs used. Our long-term goal is to determine if changes in the drug of choice and/or delivery procedures will decrease vascular toxicity and lead to better eye-targeted treatment strategies.

High glucose promotes retinal endothelial cell migration through activation of Src, PI3K/Akt1/eNOS, and ERKs

Hyperglycemia impacts retinal vascular function and promotes the development and progression of diabetic retinopathy, which ultimately results in growth of new blood vessels and loss of vision. How high glucose affects retinal endothelial cell (EC) properties requires further investigation. Here we determined the impact of high glucose on mouse retinal EC function in vitro. High glucose significantly enhanced the migration of retinal EC without impacting their proliferation, apoptosis, adhesion, and capillary morphogenesis. The enhanced migration of retinal EC under high glucose was reversed in the presence of the antioxidant N-acetylcysteine, suggesting increased oxidative stress under high-glucose conditions. Retinal EC under high-glucose conditions also expressed increased levels of fibronectin, osteopontin, and alpha(v)beta(3)-integrin, and reduced levels of thrombospondin-1. These changes were concomitant with sustained activation of the downstream prosurvival and promigratory signaling pathways, including Src kinase, phosphatidylinositol 3-kinase/Akt1/endothelial nitric oxide synthase, and ERKs. The sustained activation of these signaling pathways was essential for enhanced migration of retinal EC under high-glucose conditions. Together, our results indicate the exposure of retinal EC to high glucose promotes a promigratory phenotype. Thus alterations in the proangiogenic properties of retinal EC during diabetes may contribute to the development and pathogenesis of diabetic retinopathy.

Attenuation of retinal endothelial cell migration and capillary morphogenesis in the absence of bcl-2

Apoptosis plays a critical role during development and in the maintenance of the vascular system. B-cell leukemia lymphoma 2 (bcl-2) protects endothelial cells (EC) from apoptosis in response to a variety of stimuli. Previous work from this laboratory demonstrated attenuation of postnatal retinal vascular development and retinal neovascularization during oxygen-induced ischemic retinopathy in bcl-2-deficient (bcl-2-/-) mice. To gain further insight into the function of bcl-2 in the endothelium, we isolated retinal EC from bcl-2+/+ and bcl-2-/- mice. Retinal EC lacking bcl-2 demonstrated reduced cell migration, tenascin-C expression, and adhesion to vitronectin and fibronectin. The bcl-2-/- retinal EC also failed to undergo capillary morphogenesis in Matrigel. In addition, using an ex vivo angiogenesis assay, we observed reduced sprouting from aortic rings grown in culture from bcl-2-/- mice compared with bcl-2+/+ mice. Furthermore, reexpression of bcl-2 was sufficient to restore migration and capillary morphogenesis defects observed in bcl-2-/- retinal EC. Mechanistically, bcl-2-/- cells expressed significantly less endothelial nitric oxide synthase, an important downstream effecter of proangiogenic signaling. This may be attributed to increased oxidative stress in the absence of bcl-2. In fact, incubation of retinal EC or aortic rings from bcl-2-/- mice with the antioxidant N-acetylcysteine rescued their capillary morphogenesis and sprouting defects. Thus, bcl-2-mediated cellular functions play important roles not only in survival but also in proangiogenic phenotype of EC with a significant impact on vascular development and angiogenesis.

Pharmacologic manipulation of sphingosine kinase in retinal endothelial cells: implications for angiogenic ocular diseases.

The increased vascular permeability and pathogenic angiogenesis observed in diabetic retinopathy are induced, at least in part, by local inflammation and vascular endothelial growth factor (VEGF). Therefore, inhibition of signaling from VEGF and tumor necrosis factor-alpha (TNFalpha) is a promising approach to the treatment of this disease, as well as ocular diseases with similar etiologies, including age-related macular degeneration. A growing body of evidence demonstrates that sphingosine kinase (SK) plays an important role in cellular proliferation and angiogenesis. This study was undertaken to examine the effects of SK inhibitors on the responses of retinal endothelial cells (RECs) to VEGF and TNFalpha and their therapeutic efficacy in a diabetic retinopathy model. The expression and function of SK in bovine and human RECs were examined by immunoblot analysis. The involvement of SK in mediating responses to VEGF and TNFalpha was examined by using pharmacologic inhibitors of SK in cellular and in vivo assays, including a 3-month streptozotocin-induced diabetic retinopathy model in rats. SK was present and active in human and bovine RECs, and SK activity in these cells was stimulated by VEGF. Inhibitors of SK blocked VEGF-induced production of sphingosine 1-phosphate and markedly attenuated VEGF-induced proliferation and migration of RECs. In addition, SK inhibitors were shown to block TNFalpha-induced expression of adhesion proteins, suppress VEGF-induced vascular leakage in an in vivo mouse model, and reduce retinal vascular leakage in the rat diabetic retinopathy model. Overall, these studies demonstrate that inhibitors of SK attenuate the effects of proliferative and inflammatory stimuli on RECs both in vitro and in vivo, and so could be significant therapeutics in the treatment of diabetic retinopathy.