An Experimental Study Of Anti-Angiogenesis With Low Mocular Weight Heparin Endostatin

Objective: Endostatin is a widely-known endogenous angiogenesis inhibitor.It can strongly inhibit proliferation and migration of endothelial cells. It also causes apoptosis of endothelial cells. However, the results of recently phase I clinical trial indicate that its half-life is short and its bioavailability is low. If we can do something to improve its bioavailability and prolong its half-life to reduce the dose and prolong the drug cycle, the therapy effect of endostatin will be smart improved and promote its application in clinic. Our study will use low mocular weight heparin(LMWH) to modify endostatin' s structure. Moreover we are to investigate the anti-angiogenesis effects of low mocular weight heparin endostatin(LMWH- ES) on corneal neovascularization (CNV) in rabbits induced by alkali burn.Methods:1. Purification of endostatinThe culture supernatant of Pichia yeast containing human endostatin geng on the fourth day was purified through CM-Ⅱ ion exchange chromatography column and Sephadex G-50 column.The resulting of endostatin exhibited one protein band on SDS-PAGE at the position of 20 kD. We found that purified enodstatin could effiectively inhibit the growth and multiplication of human umbilical vein endothelial cell (HUECV) through MTT colorimetry.2. Chemical covalence modification of endostatin by LMWHwas oxidated by periodate. The activated LMWH solutions modified endostatin in pH 9.5, 0. 3mol/L NaC0:1- NaHC0:t carbonate buffer. The process of modification was monitored by SDS-PAGE. We compared degree of free ami no group modification, percentage of activity retention and result of electrophoresis, we assigned that the reaction time of modification by LMWH was 48h. We purified LMWH-ES through Sephadex G~50 gelfiltration column after pretreatment of the terminated solution of modification and identified by electrophoresis. Then we study the heat stability of ES and LMWH-ES in 25°C and 37°C water bath, we found that the heat stability of LMWH-ES was significant improved than ES.3. Anti-angiogenesis effects of endostatin and LMWH-ES on CNV of corneaThirty New Zealand albino rabbits were burned on the central corneas of their right eyes by ] mol/L NaOH for 30s. Animals were randomly divided into 3 groups, 10 rabbits each group. On the first day after alkali burn, 50 n g/ml LMWH- ES (0.2ml) and 50yg/ml recombinant endostatin (0.2ml) and normal saline (0. 2ml ) were respectively injected into subconjunctival tissues of rabbits,one time every other day,eight times in total.The growth state of CNV was observed,the expression of VEGF in cornea was detected with immunohistochemical assay and the microvessel quantity was counted as well.Results: 1. We successfully purified endostatin with ion exchange chromatography and gelfiltration chromatograph for the first time and obtained endostatin which had high degree of purity . With this method, it could decrease the cost of purify of endostatin. 2. The process of endostatin modification with LMWH was optimized, LMWH- ES was successfully separated by column chromatography, and the ideal modified product LMWH- ES under present conditions was obtained. 3. LMWH- ES treatment group ajid recombinant endostatin treatment group showed shorterlength of blood vessels and smaller area of blood vessels than normal saline group (p<0. 05) . LMWH- ES treatment group showed shorter length of blood vessels and smaller area of blood vessels than r'ecombinant endostatin treatment, group (p<0. 05) .The expression of VEGF and microvessel quantity in LMWH- ES treatment group and recombinant endostatin treatment group were also significantly lower than those in normal saline group (p<0. 05) .The expression of VEGF and microvessel quantity in LMWH- ES treatment group were lower than those in recombinant endostatin treatment group (p<0. 05) .Conclusion: 1. LMWH- ES was successfully separated by column chromatography, and the ideal modified product LMWH- ES under present conditions was obtained. 2. LMWH- ES and recombinant endostatin could effectively inhibit the growth of corneal neovascularization induced by alkali burn,but the former was better.Significance: In clinic, many drugs were for corneal neovascularization, however, whose effects were not ideal. Our research was aimed to observe the effects of heparin-endostatin against corneal neovascularization by animal experiments. The research can also provide a new method for the prevention and treatment of corneal neovascularization in clinic.