In Vitro Research On The Culture Of Human Choroidal Microvessel Endothelial Cells And The Proliferation Inhibition Of These Cells By Endostatin

Choroidal neovascularization (CNV) is an important pathobiological mechanism causing a variety of choroiretinal diseases which always result in loss of vision, and is the fundamental cause of blindness, especially in age-related macular degeneration. Although CNV is easy to be clinically diagnosed, the mechanism of CNV is not clear and no effective medicine can be administered. CNV is an abnormal occurrence of angiogenesis andvasculogenesis in choroids. The proliferation, migration and cytokine secretion of the key cells — human choroidal endothelial cells play an important role in formation, dynamic and inactive stages of CNV when the balance of angiogenesis promoters and inhibitors is broken. Endostatin, a potent endogenous angiogenesis inhibitor, can specifically inhibit the proliferation of endothelial cells. Human choroidal microvessel endothelial cells (HCMECs) are difficult to be cultured without nonendothelial cell contamination in vitro because of the complex and specific choroidal structure. The purpose of our study is to establish a rapid cell culture method of human choroidal microvessel endothelium by "two-step" digestion oftrypsin and collagenase combined with the purification technique of CD31 Dynabeads in vitro and to further study the inhibition of human choroidal endothelial cells proliferation by endostatin. Methods:1. The human choroidal tissues were digested in two steps by trypsin and collagenase combined with the purification technique of CD31 Dynabeads. The obtained HCMECs were primarily cultured and subcultured.2. Morphologic observation of HCMECs was done under the inverted phase contrast microscope.3. Ultrastructural observation of HCMECs was done with the transmission electron microscope.4. The FVBl-Ag, CD31, CD34 of HCMECs were immunohistochemically stained. The average area and average diameter of HEMCs were measured.5. HCMECs growth curve was examined by MTT method.6. Inhibition experiment of HEMECs proliferation by endostatin in vitro was carried out. We cultured the HCMECs with different medium (with serum, without serum or with VEGF), and different concentrations of ES were added to each as well. The proliferation rate of endothelial cells was assayed with MTT after 24, 48, and 72h.Result:1. We obtained sufficient number of HCMECs after digestion and purification. HCMECs presented cluster appearance.2. In primary culture. HCMECs were long and thin at initial stage and werepolygon, nearly round in shape when they grew into sheets. HCMECs maintained cobblestone morphology after HCMECs nests became confluent. It was in about 2-3 weeks that HCMECs could grow to cover the whole flask floor and form a monolayer. After a long period of confluent state, contact inhibition started. A lot of dead HCMECs were seen in the following day of subculture. The survived HCMECs maintained polygon, nearly round in shape. Cobblestone appeared after HCMECs nests became confluent again. It was about 1 week or so before the subculture occurred again after pre-subculture.3. With transmission electron microscope, we found the Weibel-Palade body which was the characteristic marker of endothelial cells.4. The FVDI-Ag, CD31, CD34 of more than 95% cultured HCMECs were positive to immunohistochemical stain. Negative control group were not stained. The average area of a single HCMEC was about 2254.37±155.94um2, and the average diameter was about 53.94±1.88um.5. The growth curve of HCMECs was S —shaped. The activity of HCMECs declined a little at the first day after subculture. HCMECs grew into proliferation after 2~3days. The growth entered plate period at the 5th day and kept growing till the 8th day, and then contact inhibition began.6. ES could inhibit the proliferation of HCMECs with different medium(compared with the control group, P<0.05) . The optimal effective concentration of ES was ^1250ng/ml. The more concentration, the more effective. We found that VEGF could induce the proliferation of HCMECs (compared with the control group, P<0.05) , and ES could obviously inhibit VEGF —induced proliferation of HCMECs at earlystage (compared with the control group, P<0.05) , which was an indication of a new treatment for antiangiogenesis of CNV. But theduration of ES inhibition was shorter than 72hr. Conclusion:Our experiments are the first success in establishing a rapid method toculture HCMECs in our country. The cultured HCMECs were EC — confirmed by morphology observation, transmission electron microscope observation, and the immunohistochemical stain of the FVI-Ag, CD31, CD34. The method is easy to apply for obtaining sufficient number of highly purified HCMECs. Under the transmission electron microscope, we found Weibel-Palade body which was the characteristic marker of endothelial cells, positive to the immunohistochemical stain of the FVl-Ag, CD31, CD34. The average area of a single HCMEC was about 2254.37±155.94um2, and the average diameter was about53.94±1.88um. ES can inhibit the proliferation of HCMECs, especially the proliferation induced by VEGF at early stage, thus ES can become a new potent treatment for antiangiogenesis of CNV.