缺氧诱导的视网膜新生血管中肝素酶和血管内皮生长因子的表达增加。

Heparanase and vascular endothelial growth factor expression is increased in hypoxia-induced retinal neovascularization.

机构信息

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

出版信息

Invest Ophthalmol Vis Sci. 2012 Oct 3;53(11):6810-7. doi: 10.1167/iovs.11-9144.

DOI:10.1167/iovs.11-9144

PMID:22956610

Abstract

PURPOSE

Heparanase and VEGF are related closely to angiogenesis in cancer. The purpose of our study was to evaluate the expression and correlation of heparanase and VEGF in hypoxia-induced retinal neovascularization.

METHODS

C57BL/6 oxygen-induced retinopathy (OIR) mice and human retinal microvascular endothelial cells (HRECs) were treated with the hypoxia mimetic agent cobalt chloride (CoCl₂), and in the presence of the heparanase inhibitor phosphomannopentaose sulfate (Muparfostat, PI-88). Heparanase activity was assayed in HRECs, and the expression of heparanase, VEGF protein and mRNA were evaluated by immunofluorescence, ELISA, Western blot, and real-time PCR while retinal flat mounts were used to evaluate the area of neovascularization of mice retina.

RESULTS

HREC heparanase activity was increased by treatment with CoCl₂, but was decreased by PI-88. Immunofluorescence showed that heparanase and VEGF staining was intense in hypoxia-treated HRECs and OIR mice retina, while VEGF staining was faint in the normoxia and PI-88-treated ones. Western blot and real-time PCR results indicated that the expression of heparanase and VEGF was increased under hypoxic conditions, and the increase of VEGF was inhibited by PI-88. Retinal flat mounts showed that the area of new vessels in retina of OIR mice was increased compared to the normoxic mice, and this effect was inhibited by PI-88.

CONCLUSIONS

Heparanase is upregulated and associated with the VEGF expression in hypoxia-induced retinal diseases. Heparanase is involved in hypoxia-induced neovascularization through promoting VEGF expression and may be a new therapeutic target for hypoxia-induced neovascularization retinal diseases.

摘要

目的

硫酸乙酰肝素酶（Heparanase）和血管内皮生长因子（VEGF）与癌症中的血管生成密切相关。本研究旨在评估缺氧诱导的视网膜新生血管形成中硫酸乙酰肝素酶和 VEGF 的表达及相关性。

方法

采用缺氧模拟剂氯化钴（CoCl₂）处理 C57BL/6 氧诱导视网膜病变（OIR）小鼠和人视网膜微血管内皮细胞（HRECs），并在硫酸乙酰肝素酶抑制剂磷甲戊聚糖（Muparfostat，PI-88）存在的情况下进行处理。通过酶联免疫吸附试验（ELISA）、Western blot 和实时 PCR 评估 HRECs 中硫酸乙酰肝素酶的活性以及硫酸乙酰肝素酶、VEGF 蛋白和 mRNA 的表达，同时通过视网膜铺片评估小鼠视网膜新生血管的面积。

结果

CoCl₂处理可增加 HREC 硫酸乙酰肝素酶活性，而 PI-88 则降低其活性。免疫荧光结果显示，缺氧处理的 HRECs 和 OIR 小鼠视网膜中硫酸乙酰肝素酶和 VEGF 染色强烈，而正常氧和 PI-88 处理的 HRECs 和 OIR 小鼠视网膜中 VEGF 染色较弱。Western blot 和实时 PCR 结果表明，在缺氧条件下硫酸乙酰肝素酶和 VEGF 的表达增加，而 PI-88 可抑制 VEGF 的增加。视网膜铺片结果显示，与正常氧小鼠相比，OIR 小鼠视网膜中新血管的面积增加，而 PI-88 可抑制这种作用。