The Effects Of Anti-VEGF Therapy On Neovascularization Fibrosis

PurposesThe current study is to observe the effects of vascular endothelial growth factor (vascular endothelial growth factor, VEGF) monoclonal antibody bevacizumab on the expresstions of cytokines related to fibrosis in human retinal pigment epithelial (ARPE-19) cells and human umbilical vein endothelial cells (HUVECs), so as to investigate whether the anti-VEGF therapy affect neovascularization fibrosis.MethodsFor hypoxia treatment, CoCl2at200μM was added to the media. According to different requirements, the ARPE-19cells and HUVEC cells were divided into three groups as follows:①normal control group:conventional culture.②hypoxia group (CoCl2):CoCl2at200μM was added to the media.②hypoxia+bevacizumab group (CoCl2+bevacizumab):CoCl2at200uM and bevacizumab at0.25mg/ml were added to the media. At6h,12h,24h and48h after cultured, cells and the supernatant were extracted. Reverse transcription polymerase chain reaction (Real-time PCR) and enzyme linked immunosorbent assay (ELISA) were used to detect the expression at gene and protein levels of connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), transforming growth factor β2(TGF-β2) in ARPE-19and HUVEC cells at various time-points. The gene and protein expressions of matrix metalloproteinase-2(MMP-2) of these two kinds of cells were detected through real-time PCR and Western blot at each time point. The expression differences of CTGF, bFGF, TGF-β2and MMP-2in ARPE-19and HUVEC cells were compared with these three different culture conditions.ResultsFor ARPE-19cells, the relative expression of CTGF, bFGF, TGF-β2and MMP-2mRNA of normal control group, hypoxia group and hypoxia+bevacizumab group at6h,12h,24h and48h time points were:①CTGF mRNA6h:1.008±0.049,2.539±0.051,4.716±0.130;12h:1.002±0.021,2.183±0.078,3.816±0.107;24h:1.010±0.127,1.849±0.120,3.330±0.141;48h:1.002±0.115,1.485±0.049,2.722±0.035;③bFGF mRNA6h:1.007±0.032,1.476±0.031,3.803±0.047;12h:1.004±0.172,1.561±0.041,3.534±0.028;24h:1.005±0.038,1.227±0.028,2.775±0.179;48h:1.001±0.116,0.965±0.112,2.722±0.060;③TGF-p2mRNA6h:1.016±0.078,3.461±0.214,4.636±0.077;12h:1.006±0.117,2.698±0.230,3.713±0.095;24h:1.007±0.163,2.196±0.217,3.048±0.198;48h:1.012±0.156,1.763±0.213,2.754±0.169;④MMP-2mRNA6h:1.013±0.091,1.998±0.117,2.717±0.103;12h:1.009±0.131,1.713±0.162,3.136±0.139;24h:1.021±0.092,1.879±0.117,3.486±0.196;48h:1.014±0.109,1.776±0.113,2.253±0.094. The expressions of CTGF, bFGF, TGF-P2and MMP-2mRNA in hypoxia group was higher compared with normal control group; compared with hypoxia group and normal control group, expressions of CTGF, bFGF, TGF-β2and MMP-2mRNA at each time point in hypoxia+bevacizumab group was increased.The differences were statistically significant (P<0.05). The ELISA results of ARPE-19cells at each time point showed that the protein expression of CTGF, bFGF and TGF-β2of hypoxia group was increased compared with the normal control group and the expression of hypoxia+bevacizumab group was significantly higher compared with those of hypoxia group and normal control group. The differences were statistically significant (P<0.05) and the concentrations of proteins were:(unit pg/ml)①CTGF6h:31.865±5.395,76.612±5.305,121.044±6.121;12h:84.225±7.165,164.173±4.905,290.827±7.091;24h:119.613±7.887,199.14±7.133,335.957±4.960,48h:95.160±9.851,133.224±8.089,217.872±8.140.②bFGF6h:27.704±4.139,31.003±6.305,89.893±5.120;12h:51.262±6.653,73.997±7.057,171.306±7.914;24h:67.148±5.088,81.759±8.339,116.435±4.096;48h:53.882±7.108,42.192±6.897,104.214±8.105;③TGF-p26h: 28.172±6.911,83.559±4.103,121.245±9.003;12h:53.96±5.131,129.203±6.639,191.712±8.106;24h:75.772±4.092,156.453±6.196,220.247±7.014;48h:42.077±6.109,70.344±7.094,117.391±7.195. For ARPE-19cells, Western blot results at each time point showed that the expression of MMP-2protein of hypoxia group was increased compared with the normal control group and the expression of hypoxic+bevacizumab group was increased compared with the hypoxia group and normal control group. The differences were statistically significant (P<0.05). For HUVEC cells, the relative expression of CTGF, bFGF, TGF-(32and MMP-2mRNA of normal control group, hypoxia group and hypoxia+bevacizumab group at6h,12h,24h and48h time points were:①CTGF mRNA6h:1.016±0.072,1.579±0.076,2.915±0.070;12h:1.104±0.073,2.280±0.070,4.357±0.064;24h:1.044±0.092,1.734±0.109,3.177±0.065;48h:0.903±0.089,1.453±0.100,2.371±0.069;②bFGF mRNA6h:0.906±0.056,1.411±0172,3.601±0.084;12h:1.106±0.092,1.623±0.113,4.034±0.106;24h:1.065±0.072,1.333±0.064,3.132±0.080;48h:1.001±0.091,1.171±0.045,2.862±0.096;③TGF-β26h:0.901±0.099,2.964±0.102,3.169±0.057;12h:0.981±0.052,2.392±0.082,4.441±0.107:24h:1.019±0.104,2.026±0.070,3.112±0.102:48h:1.072±0.123,1.762±0.120,2.685±0.080;④MMP-2mRNA6h:1.033±0.086,2.097±0.083,2.597±0.101;12h:0.933±0.092,2.879±0.125,3.298±0.087;24h:1.010±0.085,2.003±0.098,2.397±0.088;48h:1.089±0.130,1.971±0.099,2.173±0.079. By comparing the relative mRNA expression of CTGF, bFGF, TGF-02and MMP-2mRNA at each time point, the expression was higher for hypoxia group than that of normal control group and compared with hypoxia group and normal control group, the expression of hypoxia+bevacizumab group was increased obviously. These differences were statistically significant with the P values less than0.05. The ELISA results of HUVEC cells at each time point showed that CTGF, bFGF and TGF-β2proteins in hypoxia group were increased compared with the normal control group and the protein expression of hypoxia+bevacizumab group was significantly higher than those of hypoxia group and normal control group. The differences were statistically significant (P<0.05) and the protein concentrations of each group were:(unit pg/ml)①CTGF6h:25.716±5.042,36.218±4.309,76.069±6.069;12h:53.627±6.563,73.884±6.092,219.870±7.867;24h:79.618±4.873,101.451±7.104,156.523±4.516;48h:61.575±5.844,77.962±4.539,111.317±4.938;②bFGF6h:11.967±6.985,16.635±7.203,42.343±6.941; 12h:23.126±5.098,28.626±8.943,80.562±4.757;24h:34.153±7.126,40.125±7.986,103.971±4.145;48h:18.268±6.108,21.112±9.097,45.429±7.861;③TGF-p26h:17.523±4.529,57.632±8.623,68.094±7.084;12h:31.581±3.983,76.225±8.095,99.113±8.957;24h:43.415±4.079,90.462±9.106,126.457±7.627;48h:26.947±5.126,41.665±7.516,67.413±8.112. Western blot results showed that the MMP-2protein expression of HUVEC cells at four time points was increased for hypoxia group compared with the normal control group. The expression of MMP-2protein in hypoxic+bevacizumab group was also increased compared with hypoxia group and normal control group. The differences were statistically significant (P<0.05).ConclusionsIn the hypoxia environment, bevacizumab can up regulate the expressions of fibrosis related factors CTGF, bFGF, TGF-β2and MMP-2in ARPE-19and HUVEC cells. Therefore, intravitreal injection bevacizumab may increase tissue fibrosis and impact the effect of anti-VEGF thearpy, but the interaction between these cytokines associated with fibrosis and the unknown participation of other factors such as inflammation need to be further discussed and studied.