Heparanase And Experimental Choroidal Neovascularization

Research background: Choroidal neovascularization (CNV) is a common cause of vision loss in patients, which often resulted from exudative Age-related macular degeneration, pathologic myopia, central serous chorioretinopathy,ocular histoplasmosis and angioid streaks, Rarely, CNV is seen in children, usually in association with inherited macular dystrophies. Despite extensive basic and clinical research, our knowledge of the complicated molecular mechanism of CNV is still limited, for these reasons currently used therapeutic approaches are insufficiently effective. Accumulated evidence indicates that various growth and inhibitor factors (such as VEGF,bFGF,PEDF et al.) and extracellular matrix (ECM) are major mediators of neovascularization, ECM mainly consist of collagen, fibronectin, laminin, vitronectin, and HSPG Unlike the large number of proteases that can solubilize polypeptides in the ECM, there is only heparanase used by cells to degrade heparan sulfate proteoglycan. Up to date, there is no report about the involvement of heparanase during the formation and development of CNV, the present study investigated the involvement and mechanism of it on the Krypton laser-induced rat CNV model, mainly by methods of morphology observation, cell culture in vitro, histopathology, immunohistochemistry and molecular biology.Part 1 The expression and regulation of Heparanase in experimental choroidal neovascularization Objective 1. To evaluate the dynamic characteristic of CNV model induced by krypton laser in BN rat. 2. To investigate the associativity between the expression of heparanase and the development of CNV in rat. 3. To probe the expressive significance of uPA and COX-2, they were thought to be downstream factors of HPA. Methods 1. Krypton laser photocoagulation (photocoagulation parameter: 647nm wavelength, 360mW power, 50μm spot size, 0.05second duration) weredelivered to fundus of BN rat to establish experimental CNV model, Integrating different methods of direct fundus viewing, fluorescence fundus angiography and histopathology to evaluate the dynamic characteristic of CNV. 2. In the time point of 3 days, 1 week, 2weeks, 3 weeks and 4 weeks after photocoagulation , the expression of CD31,HPA, uPA and COX-2 in photocoagulative spots was observed by immunohistochemical method of SP or SABC, and qualitative analysis was performed on the positive expressive results. 3. The levels of HPA mRNA was semi-quantified between diffirent time points after photocoagulation by RT-PCR. Results 1. Begin from acute edema, hyperplasy and reparative process accompanied by acute inflammation , subsiding edema, scarification and mussy pigment in turn were found in the photocoagulative spots ; CNV begins to emerge 1w after photocoagulation, and continuely develop during 1w～4w, the growth of CNV displayed the most productive during the time of 1w to 2w, while there is no difference between 3w and 4w, From 4w to 12w, fibrous tissue was mainly seen to increase within CNV membrane. 2. Endothelial cells to formation CNV come from the vascular lamina of choroid. 3. HPA mRNA and protin were both expressed in nomal retina and choroidal tissue, after photocoagulation, the expressive levels of HPA mRNA parallelled that of the CNV growth. 3. The expression of HPA, uPA and COX-2 presented the same pattern by immunohistochemical method during the development of CNV, they all displayed distribution at the leading edge of CNV membrane migrating toward inner retina and the vascular tissue; Within the CNV membrane, HPA and COX-2 mainly in the blood vessel tissue, while uPA was mainly expressed in the fibrous tissue. Conclusion HPA may promote the growth of CNV by direct-hydrolyzing of non-protein construction of ECM, or by up-regulating of downstream uPA and COX-2.Part 2 The involvement of heparanase during the proliferative course of HUVEC and RPE cells in vitroObjective To probe the involvement of heparanase activities with the proliferation of HUVEC and RPE cells in vitro. Methods 1. ECM and DMEM was adapted toculture HUVEC and RPE cells respectively. 2. The inhibitor of heparanase PI-88 was added to interfere with the cell culture course, Undertaking direct observation by inverted microscope and cell proliferation was indirectly reflected by MTT colorimetric assay. 3. To detect the expressive changes of heparanase after PI-88 on the growth of these two cells by immunohistochemistry method, in addition, uPA and COX-2 were also detected in RPE cells without PI-88 in the culture medium. Results PI-88 displayed evident inhibitory action on the proliferation of both vascular endothelial cells and retinal pigment epithelial cells, and both presented obvious time-effect and dose-effect relationship; Downregulation of HPA by PI-88 contributed to the inhibitory action on the cell proliferation of both HUVEC and RPE cells; Upregulation of uPA and COX-2 were found out in RPE cells without PI-88 in the culture medium. Conclusion The expression of heparanase is closely correlated with the proliferation of vascular endothelial cells and retinal pigment epithelial cells in vitro.Part 3 The study of the inhibitor of heparanase (PI-88) controling experimental choroidal neovascularization in vivoObjective To investigate the effect on the experimental CNV of PI-88 and associated molecular mechanisms in vivo. Methods 15 days continuous intraperitoneal injection of PI-88 with 25mg·kg^-1·d^-1 to interfere with the formation and growth of laser-induced rat CNV ;To combinatively evaluate the effect of PI-88 on the CNV growth by choroid-sclera stretched preparation, fluorescence fundus angiography and histopathology; The protein expression of HPA, COX-2 and uPA were surveyed by Western blot and immunohistochemistry. Results CNV area of preventive group and treated group decreased 52.1% and 53.8% respectively at the time point of 3w after photocoagulation, for the preventive group, PI-88 was administered the same day as photocoagulation, and for the treated group, PI-88 was administered 1 week after photocoagulation when CNV had been formed; The relative thickness of CNV membrane had been decreased 46% as that of control group by histopathology ; CNV occurrenced 1 week after photocongulation both in the control group and preventive group, while thefluorescein leakage of the preventive group had been inhibited significantly ; 2 weeks after photocongulation , there were 15 days continuous administration for the preventive group and 7 days continuous administration for the treated group, the fluorescein leakage of both group weakened than that of the control group; 3 weeks after photocongulation ,there had been 7 days discontinuation for the prevent group, the fluorescein leakage did not enhanced, while there had been 15 days continuous administration for the treated group,the fluorescein leakage had been decreased significantly compared to the time point of 2 week; Western blot showed that the relative expressed levels of HPA protein in both preventive and treated group decreased ,and it was more obvious in the preventive group,while the relative expressed levels of COX-2 protein in the preventive group had not decreased significantly than that of the control group; For immunohistochemistry detection, the expression of HPA and uPA were both inhibited significantly in treated group, but it seemed that the stainess of COX-2 had not been inhibited obviously. Conclusion PI-88 may not only prevent CNV in certain degree, but also make it partially subsidise for the existed CNV; Besides to downregulate the expression of HPA, the mechanisms of PI-88 inhibiting CNV also include downregulating the downstream factors of uPA and COX-2, the regulation of uPA by HPA is more identify than that of COX-2 by HPA.