The Effect Of Survivin And Its Inhibitor YM155on Corneal Neovascularization

Background:Corneal neovascularization (CNV) is commonly associated with a number of ocular surface diseases. CNV is a major cause of blindness due to lack of sufficient anti-angiogenic treatment options. However, the pathogenesis of CNV has not been fully defined and identity. Up to now, how to effectively inhibit the occurrence and development of CNV has become a hot topic in the study of corneal disease. Hereon, the further study of the pathogenesis and treatment of CNV may provide great theoretical and clinical importance. With powerful ability to restrain the apoptosis, Survivin belongs to the anti-apoptosis protein, and plays crucial roles in maintaining cell mitosis and angiogenesis. Studies suggested that survivin played an important role in various human tumor angiogenesis. While, it’s not clear whether Survivin has close relationship with cornea angiogenesis.Objectives:This study aims to evaluate whether the survivin (a member of inhibitor-of-apoptosis proteins family) has relationship with the formation of CNV, and observe the inhibition effect of YM155(survivin specific inhibitor) on human umbilical vein endothelial cells (HUVECs) and the CNV. Methods:1. The corneal alkali-wound was made by playing a3.5mm diameter circular piece of filter paper, soaked in1mol/L NaOH, in contact with the central cornea of the right eye for50seconds. All eyes were examined under operating microscope with camera after alkali burn. Then, the length and area of the CNV were calculated. The examinations including immunohistochemistry, immunofluorescence and Western Blot were used to evaluate survivin expression in the rat cornea. RT-PCR was used to evaluate survivin and VEGF-A mRNA expression from the rat cornea.2. HUVECs were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/high-glucose medium supplemented with2%FBS and50ng/ml VEGF165at5%CO2and37℃. Observe the growth of HUVECs under this condition. The expression of survivin protein of HUVECs was detected by Western Blot at different time points. HUVECs were treated with YM155of different concentrations (0,1.25,2.5,5,10,20,40,80,160nmol/L) at different time points (12h,24h,48h,72h). The MTT assay was used to observe the effect of YM155on the proliferation of HUVECs, and also to calculate the IC50. The flow cytometry (FCM) was used to verify its effect on the apoptosis of HUVECs. The Western Blot was used to measure the survivin expression from HUVECs which were treated with20nmol/L YM155for different times.3. CNV was induced by alkali burn for the right eyes of48Sprague-Dawley rats. Then they were randomly divided into two groups, and treated with1mg/ml YM155(drug group) and PBS (control group), respectively, for14days, twice/day following the operation. Rats were anesthetized and examined under operating microscope every day after injury. The length and clocks of CNV were measured, and the areas of CNV were calculated. The survivin mRNA was detected by RT-PCR and the protein expression of survivin was measured by Western Blot.Results:1. The injured site rapidly became edematous after removed the NaOH-soaked filter disk from the cornea. On the1st day after the injury, the limbal vascular network was congestion and vasodilatation. On the3rd day, new vessels sprouted intensively from the corneal limbus and stretched to central areas. On the7th day, CNV grew to its most vigorous, with the vessels manifolding and elongating. At the same time, the backbones became thick and straight and the ends crossed like a network. Some vessels combined and stretched to the central area of cornea. On the14th day, the new vessels grew slower and approached to the central. The area of CNV continued increased maximum until on the14th day when the CNV almost covered whole cornea. The vessels stretched and slimed with branches reduced.The results of immunohistochemistry indicated that CD31and survivin positive-staining were seen very weak or absent in the control group (normal cornea). On the7th day after the injury, CD31and survivin positive-staining with brown particles increased significantly in the substrate layer of the cornea. The findings of immunofluorescence were similar to the immunohistochemistry that CD31and survivin fluorescence positive-staining increased significantly in substrate layer of cornea on the7th day after the injury comparing to the normal cornea with almost no expression of CD31and survivin fluorescence positive-staining. Furthermore, some of the CD31and survivin fluorescence positive-staining were overlapped.The results of RT-PCR showed only few survivin and VEGF-A mRNA existed in control group (normal cornea). The increase of survivin mRNA was not obvious on the1st day after injury (P>0.05). It increased over time. The increase on the3rd,7th and14th day had statistics significance (P<0.05), among which it reached to peak on the7th day. The increase of VEGF-A mRNA on the1stday had statistics significance (P<0.05) and on the3rd,7th and14th day had remarkable statistics significance (P<0.01), among which it reached to peak on the7th day. The expressions of survivin and VEGF-A mRNA from the alkali burned cornea had a significant correlation (r=0.980, P<0.01). The results of Western Blot further showed that, comparing the control group, the increase of survivin protein had remarkable statistics significance (P<0.01) on the3rd%7th and14th day.2. HUVECs proliferated well in the DMEM medium containing2%FBS and50ng/ml VEGF165, and survivin fluorescence positive-staining was observed in the cytoplasm and nucleus of HUVECs under the CLSM (Confocal Laser Scanning Microscope), and the staining in cytoplasm was more obvious. The results of Western Blot showed the survivin protein increased gradually and reached to peak on the12-18th hour and reduced from the24th hour in the medium. The results of MTT showed that YM155had significant inhibition on cell proliferationin from48and72th hours after its effect on HUVECs. The drug concentration as low as5nmol/L could significantly inhibit the proliferation of HUVECs. The inhibiting effect of YM155depended on timing and dosage. Calculated by MTT, the IC50index was8.68nmol/L at the48th hour and5.77nmol/L at the72nd hour after treatment with YM155. Comparing to the control group, the apoptosis rates of HUVECs were increased significantly after treated with YM155of5nmol/L and20nmol/L for48th hours. The results of Western Blot indicated that20nmol/L YM155could inhibit the expression of survivin protein after treated with20nmol/L YM155for12th、24th、48th and72nd hours(P<0.01).3. The corneal opacity and angiogenesis gradually increased in both groups. However, there was less angiogenesis in the YM155-treated group than that in the control group. Comparing to the control group, the scores of corneal neovascularization were significantly reduced by the treatment with YM155on the7th day and the14th day, respectively (P<0.01). The results of RT-PCR showed that the survivin mRNA was inhibited by YM155on the7th day (P<0.01) and14th day (P<0.05). The results of Western Blot further indicated that the survivin was significantly decreased by YM155on the7th day and14th day(P<0.01).Conclusions:This study is the first attempt to discuss the relationship between survivin and CNV. Our findings suggest that the survivin has a close relationship with the processes of CNV, and its inhibitor-YM155has obvious effect to restrain proliferation and promote apoptosis of HUVECs, and can effectively restrain the progress of CNV after alkali burn. This research provides theoretical and experimental basis for a new target at survivin in the treatment of CNV.