Effect Of RCG On Angiogenesis And The Mechanism Study

Angiogenesis is a fundamental physiological process. Angiogenic stimulus such as VEGF,bFGF,MMP and so on triggers a cascade of functional responses leading to local basement membrane dissolution, endothelial cell migration, proliferation and micro vessel morphogenesis. Homeostatic equilibrium of the vascular network within tissues depends on the balance between angiogenic and anti-angiogenic factors. Imbalance in angiogenesis regulatory mechanisms has been associated with several pathologies including chronic inflammation, psoriasis, rheumatoid arthrities, aged - related macular degeneration and cancer.Recently, a variety of synthetic, biological and natural compounds have been indentified as inhibitors of angiogenesis, the first biological regulator of neovascularization was found in cartilage. In this study, Ray cartilage has been tested. Ray which is belong to vertebrata, chondrichthyes, elasmobranchii, raji-formes, dasyatidae is ground floor fish of warm temperature. We had observed that the extraction of ray cartilage could inhibit angiogenesis of chicken embryo chorioallantoic membrane effectively. Here, Ray cartilage polysaccharide was extracted, separated and purified by guanidine hydrochloride, membrane ultrafil-trating, acetone classification precipitation and sephadex gels pillar patients, Tianqing I colorimetric method and high performance liquid chromatographic (HPLC) detected Ray cartilage polysaccharide molecular mass was 9. 7 x 10~4 and its purity exceeded ninty - nine percent. In the experiment, we explore Ray cartilage polysaccharide antiangiogenic biological activities.Objectivestudy was designed to investigate the effects of Ray Cartilage Glyco-saminoglycans ( RCG) on angiogenesis and to explore its mechanism and cultured Human Umbilical Endothelial cell ( HUVEC) , Wistar rat corneal neovas-cularization ( CNV) and transplanting tumor mouse models were used in our work.Methods1. Culture and Identification of the Endothelial Cells of Human Umbilical VeinEndothelial cells were isolated from human umbilical cords with 0. 1% I type collagenase and were digested by 0. 05% tripsin and 0. 02% EDTA when the endothelial cells confluent. The cells are cultivated in Ml99 medium containing Endothelial Cells Growth Supplement(ECGS) and fetal bovine serum. Endothelial cells were identified by morphologic and immunohistochemical assays .2. The effect of RCG on angiogenesis in vitroThe experiments were classified into normal group, RCG groups at varied concentrations (100,50,25,10, 2mg/mL ) and SCG group ( 50mg/mL ). MTT, endothelial cell migration and capillary formation experiments were adopted to e-valuate the effects of RCG on HUVEC. Meantime,we also detected the effect of RCG on HUVEC cycle by means of Flow cytometry.3. The effects of RCG on corneal neovascularization(CNV)in Wistar rats The RCG eye drop was prepared. Corneal neovascularization was inducedby sutures on Wistar rats-. The experimental rats were randomly divided into saline group,RCG groups at varied concentrations( 100,50,25mg/mL)and Hepa-rin + Corlin group. CNV area, histopathological changes and VEGF immunohisto-chemistry were used to evaluate the RCG therapeutic effects on CNV in Wistar rats.4. The effects of RCG on implanted Lewis lung carcinoma in C57BL/6J mice.The model of mouse with Lewis lung carcinoma was made;the experimental mice were randomly divided into saline group,RCG groups at varied concentrations (500,250,125mg/kg) and CTX group. Tumor growth states were observed;Tumor growth curve was described;On the 21th day, tumor was deprived, inhibitory rate of primary tumor and number of lung metastasis focus were measured. Microvessel density (MVD) was quantitated by immunohistochemistry using monoclonal antibodies of CD31.5. The effects of RCG on the expression of VEGF ,bFGF,MMP 9 mRNA and proteinThe expression of VEGF, bFGF, MMP9 were determined with RT - PCR and Western blot.Result1. The effects of RCG on angiogenesis in vitroBy observing morphologic the cells cultivated were endothelial cells typically and by immunohistochemically the cells cultured were stained positively by VI factor monoclonal antibody. 100 - lOmg/mL RCG obviously inhibited the proliferation of HUVEC, the mean MTT OD value of saline group, RCG groups at varied concentrations (100,50,25,10, 2mg/mL ) and SCG group ( 50mg/mL ) were 0.41 ±0.05,0.11 ±0.05,0. 23 ±0.06,0. 31 ±0. 02,0. 35 ±0. 07,0. 36 ±0. 03,0.21 ±0. 04, respectively,ICjo value was 62. 93mg/mL . 100 - lOmg/mL RCG strongly inhibited the migration of HUVEC, the migrated numbers cell of obove groups were 63.80 ± 10. 18,6. 60 ± 3. 58,15.40 ± 4. 61,18. 80 ± 5. 89, 41. 80 ± 3. 70,13. 60 ± 5. 98 ,respectively;100 - lOmg/mL RCG inhibited capillary formation,the mean capillary length of them were 4.63 ±0.31,1.08 ± 0.22,2.31 ±0.11,3.03 ±0. 19,3. 60 ±0. 15,2.11 ±0.12 mm,respectively. RCG could block HUVEC in G2/M phase by Flow cytometry.2. The effects of RCG on corneal neovascularization(CNV)in Wistar rats By means of RCG effect on corneal neovascularization ( CNV) in Wistarrats, We observed that the corneal neovascularization of RCG groups growed slowly and RCG groups showed smaller areas of new blood vessels and fewer inflammatory cells and lower VEGF expression than saline group, there were high expression in corneal epithelium of Saline group, and negative expression in corneal epithelium of lOOmg/mL, 50mg/mL and Heparin + corlin groups, the ex-pression of VEGF in 25mg/mL RCG group was lower .3. The effects of RCG on implanted Lewis lung carcinoma in C57BL/6J mice.The effects of RCG on the implanted Lewis lung carcinoma showed that tumor growth curves in RCG groups were smooth compared with Saline group, inhibitory rates of primary tumor in 500,250,125mg/kg RCG groups were 43. 36% ,26. 34% , 15. 10% , respectively, there were significant differences in number of lung metastasis focus between RCG groups and Saline group . In MVD assay, the MVD number of RCG groups were markedly reduced compared with Saline group, and had a clear dose - effect relationship.4. The effect of RCG on the expression of VEGF ,bFGF,MMP 9 mRNA and proteinUsing RT - PCR , RCG at various dosage groups Lewis lung cancer tissues VEGF, bFGF, MMP - 9 mRNA fluorescence intensity reduced gradually and relative AUG values were less significantly than Saline group' s (P<0.01) and there were significant differences among these dosage groups;Western blot results showed that protein strap became weaker and weaker with concentrations increased, VEGF, bFGF, MMP -9 protein expression levels in RCG groups were reduced significantly compared with Saline group (P <0.01) 0ConclusionsFrom the experiments results in vitro and in vivo, we conclude that RCG had srong antiangiogenic effect. Its mechanism in vitro might involve in the blocking of HUVEC cell cycle and its mechanism in vivo might be downregulating the expression of VEGF ,bFGF,MMP -9.