Effect of Robo1 on Retinal Pigment Epithelial Cells and Experimental Proliferative Vitreoretinopathy

Huang, Lvzhen; Xu, Yongsheng; Yu, Wenzhen; Li, Yingjie; Liqun, Chu; Dong, Junchen; Li, Xiaoxin

doi:10.1167/iovs.09-3779

Cited by 20 publications

(14 citation statements)

References 42 publications

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“…A migration assay was performed as described previously [21]. Briefly, 2×10 4 cells were placed in the upper chamber in a final volume of 200 µl of serum-free medium, and 10% FBS then was added to the bottom chamber for a final volume of 600 µl.…”

Section: Methodsmentioning

confidence: 99%

Overexpression of HTRA1 Leads to Down-Regulation of Fibronectin and Functional Changes in RF/6A Cells and HUVECs

Jiang

Huang²,

Yu³

et al. 2012

PLoS ONE

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Multiple genetic studies have suggested that high-temperature requirement serine protease (HTRA1) is associated with polypoidal choroidal vasculopathy (PCV). To date, no functional studies have investigated the biological effect of HTRA1 on vascular endothelial cells, essential vascular components involved in polypoidal vascular abnormalities and arteriosclerosis-like changes. In vitro studies were performed to investigate the effect of HTRA1 on the regulation of fibronectin, laminin, vascular endothelial growth factor (VEGF), platelet derived growth factor receptor (PDGFR) and matrix metalloparoteinases 2 (MMP-2) and the role of HTRA1 in choroid-retina endothelial (RF/6A) and human umbilical vein endothelial (HUVEC) cells. Lentivirus-mediated overexpression of HTRA1 was used to explore effects of the protease on RF/6A and HUVEC cells in vitro. HTRA1 overexpression inhibited the proliferation, cell cycle, migration and tube formation of RF/6A and HUVEC cells, effects that might contribute to the early stage of PCV pathological lesions. Fibronectin mRNA and protein levels were significantly down-regulated following the upregulation of HTRA1, whereas the expressions of laminin, VEGF and MMP-2 were unaffected by alterations in HTRA1 expression. The decreased biological function of vascular endothelial cells and the degradation of extracellular matrix proteins, such as fibronectin, may be involved in a contributory role for HTRA1 in PCV pathogenesis.

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Section: Methodsmentioning

confidence: 99%

Overexpression of HTRA1 Leads to Down-Regulation of Fibronectin and Functional Changes in RF/6A Cells and HUVECs

Jiang

Huang²,

Yu³

et al. 2012

PLoS ONE

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“…Migration was assayed by Transwell (Corning Life Sciences, Lowell, MA) with a pore size of 8.0 m as described previously (Huang et al, 2010). In brief, 2 ϫ 10 4 cells were placed in the top part of a Transwell in 200 l of serumfree medium.…”

mentioning

confidence: 99%

Polyethylene Glycol-Modified Pigment Epithelial-Derived Factor: New Prospects for Treatment of Retinal Neovascularization

Bai

Huang²,

Xu³

et al. 2012

J Pharmacol Exp Ther

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Pathological retinal neovascularization and choroidal neovascularization are major causes of vision loss in a variety of clinical conditions, such as retinopathy of prematurity, agerelated macular degeneration, and diabetic retinopathy. Pigment epithelial-derived factor (PEDF) has been found to be the most potent natural, endogenous inhibitor of neovascularization, but its application is restricted because of its instability and short half-life. Polyethylene glycol (PEG) has been used as a drug carrier to slow clearance rate for decades. The present study investigated PEGylated-PEDF for the first time and evaluated its long-term effects on preventing angiogenesis in vitro and in vivo. PEG showed lower cytotoxicity to human umbilical vein endothelial cells (HUVECs). In vitro, PEGylated-PEDF inhibited HUVEC proliferation, migration, tube formation, and vascular endothelium growth factor secretion and induced HUVEC apoptosis in a dose-dependent manner, and it showed a statistically significant difference compared with the PEDF treatment group. In vivo, PEGylated-PEDF had a long-lasting effect in both plasma and retinal concentrations. In an oxygeninduced retinopathy model, one intravitreous injection of PEGylated-PEDF after mouse pups were moved into room air resulted in a significant difference in the inhibition of retinal neovascularization, which decreased the nonperfusion area, compared with the PEDF-treated group. Our present study demonstrated for the first time the long-term inhibitory effects of PEGylated-PEDF on the prevention of neovascularization in vitro and in vivo. These data suggest that PEGylated-PEDF could offer an innovative therapeutic strategy for preventing retinal neovascularization.

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“…Western blot analysis was performed using standard Western blotting methods as we have previously described [13]. Cells were washed twice with ice-cold phosphate-buffered saline (PBS), lysed on ice with buffer (50 m m Tris-HCl, pH 7.4, 150 m m NaCl, 1 m m EDTA, 1% NP-40, 0.5% Na deoxycholate) containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

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

Xie¹,

Zhao

Weiyan

et al. 2012

Ophthalmic Res

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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.

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Effect of Robo1 on Retinal Pigment Epithelial Cells and Experimental Proliferative Vitreoretinopathy

Cited by 20 publications

References 42 publications

Overexpression of HTRA1 Leads to Down-Regulation of Fibronectin and Functional Changes in RF/6A Cells and HUVECs

Overexpression of HTRA1 Leads to Down-Regulation of Fibronectin and Functional Changes in RF/6A Cells and HUVECs

Polyethylene Glycol-Modified Pigment Epithelial-Derived Factor: New Prospects for Treatment of Retinal Neovascularization

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

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