The Role Of Bevacizumab In The Growth Of Fibroblasts And Modulation Of Fibrosis Post-Filtering Surgery

Objective1. To Detect mRNA expressions of Vascular Endothelial Growth Factor(VEGF), VEGF receptors (VEGFR) and inflammatory cytokines in Sprague-Dawley (S-D) rat conjunctival fibroblasts.2. To explore the influence of Bevacizumab (anti-VEGFA, Avastin) on the growth curve of rat conjunctival fibroblasts, and on the expressions of VEGF, VEGFR and inflammatory cytokines of fibroblasts.3. To evaluate the influence of filtering surgery combined with subconjunctival injection of Bevacizumab on the morphology of filtering bleb, and on the expressions of angiogenic factors and inflammatory factors.Materials and Methods1. Culture and identification of conjunctiva fibroblasts(FBs):Conjunctiva from5young S-D rats younger than4weeks was cut off into small pieces and inoculated in DMEM media with tissue culture method. FBs was continuous passage cultured and observed. After passage to the fifth generation, FBs was inoculated into96well cultureplate. Cell density was measured at24h,48h,72h,120h,7days and14days by MTT method and normal growing curve was drawn。 The fourth and fifth generations cultured FBs were identified by indirect IFA(immunofluorescence assay). The positive control was Vimentin-FITC, the negative control was Keratin-FITC.2. Expressions of growth factors and receptors of:The expression of VEGFR1was detected by VEGFR-FITC immunofluorescence for the fourth and fifth generations cultured FBs. Primers of VEGF164, TGFβ1was designed using Primer blast program (NCBI), Primers of VEGFR1(Flt-1), VEGFR2(Flkl), TGFp2and β-actin were cited. mRNAs from FBs was extracted by Trizol method. cDNA was synthesized under the action of RNA reverse transcriptase.25μl amplification system was applied in PCR. Ladder-like pattern of DNA fragmentation appeared upon2%agarose gel electrophoresis. Electrophoretic bands of designed genes (VEGF164, VEGFR1(Flt-1), VEGFR2(Flkl)1, TGFβl, TGFβ2) were cut and purified for gene sequencing. Results of sequencing were analyzed and compared using Primer Blast software(NCBI), to verify the amplification of preferred gene.3. The influence of Bevacizumab on the growth of cultured conjunctival FBs: Certain concentrations of Bevacizumab were added into the96well cultureplate of fifth generation cultured FBs. Each concentration was set in five wells. The final concentrations were12.5mg/ml,2.5mg/ml,1.25mg/ml,0.5mg/ml,0.05mg/ml,0.005mg/ml respectively. The observing time points were Dayl, Day2, Day4, Day6, Day8and Day10to detect the influence of Bevacizumab to the growth curve of FBs using MTT method. Growing curves under interventions of Bevacizumab in different concentrations were drawn. Bevacizumab was added into6well cultureplate of FBs cells which was diluted to the final concentration of2.5mg/ml (most appropriate concentration shown from previous study). mRNA from cultured FBs was extracted after48h. cDNA was synthesized by reverse transcriptase. Expressions of VEGF164, Flt-1, Flk1, TGFβ1, TGFβ2and β-actin were detected by fluorescence quantitative PCR(FQ-PCR, ABI7500). Serial dilutions of cloned genes were used to construct standard curves to test amplification efficacy, and melting curve analysis was made to test specificities. ddCt values of VEGF164, VEGFR1(Flt-1), VEGFR2(Flkl), TGFβ1, TGFβ2were recorded and statistically analyzed.4. The influence of Bevacizumab on the morphology of filtering blebs from S-D rats:55four weeks old S-D rats accepted filtering surgery and grouping. Single eyes from5rats were operated for set up standard filtering surgery model. A1group, A2group and B group were set during Bevacizumab intervention study. A1group (25eyes from25rats) represented rats which accepted subconjunctival injection of Bevacizumab. A2group(25fellow eyes from same25rats) represented rats which accepted subconjunctival injection of PBS. B group(25eyes from25rats) represented independent rats which accepted subconjunctival injection of PBS for one eye each. First, filtering surgery procedure was made and standardized with limbal-based flap on5rats. Morphologic changes of blebs and adverse events were continuously observed and recorded to14th day. Filtering surgery combined with subconjunctival injection was operated on55S-D rats.3μl of Bevacizumab or PBS were randomly injected into lower fornix subconjunctiva using blind method. Observing times were Dayl、Day2、Day3、 Day5and Day8(set up based upon the FBs growing curve and morphological changes of filtering blebs). Observing items:morphology of filtering bleb (grade0-3); congestion of filtering bleb(grade0-3). Adverse events included hyphema (grade0-3), iris changes (grade0-3) and other side effects. Statistical description and Kruskal-Wallis H tests were made.5. The influence of Bevacizumab on the expressions of growth factors and receptors in filtering blebs:Rats were sacrificed and blebs were clipped and stored in liquid nitrogen at Dayl, Day2, Day3, Day5, Day8respectively. mRNAs were extracted and cDNAs were synthesized at the same time. Expressions of VEGF164, Flt-1, Flkl, TGFβ1, TGFβ2and β-actin were detected by FQ-PCR. Expression changes of each gene at each time points from three groups were compared using One-Way ANOVA test. Total protein was simultaneously extracted during Trizol extraction of mRNA process. Expression changes of each protein at each time points from three groups were detected by Western-blotting methods.Results1. Most of cultured explants from S-D rat conjunctiva adhered to the wall within6h. FBs proliferated from the margin at day3.80%of FBs fusion was observed after3days post1:1passage culture. Length of cultured FBs was shorter than human FBs and branching, polygon and irregular shape could be observed. Logarithm stage for the growth of FBs was day2to day7, detected by MTT method.2. FBs were cultured from rat conjunctiva explants with shape of short branched, polygonal or irregular. Staining of Vimentin-FITC was positive, and that of Keratin-FITC was negative, detected by indirect IFA.3. VEGFR1-FITC staining appeared weak positive which located along the membrane. Sequencing results of RT-PCR products were consistent with mRNA sequence of VEGF164, Flt-1, Flkl, TGFβ1and TGFβ2respectively. Results of primer amplification curve showed the comparable efficiency of each RNA primer.4. Bevacizumab was added into cultured FBs in Logarithm stage according to concentration gradient, and apparent suppressive effect was observed with final concentration of2.5mg/ml Bevacizumab. FBs died within24h, under the intervention of12.5mg/ml Bevacizumab.5. Results of FQ-PCR:(1) mRNA expressions of VEGF164, VEGFR1, VEGFR2, TGFβ2in cultured FBs all increased in Logarithm stage, meanwhile, the mRNA expression of TGFβ1remained steady and appeared not affected by Bevacizumab.(2)48h after intervention of2.5mg/ml Bevacizumab, the mRNA expressions of all of the above genes decreased compared to the control group, among which expressions of VEGF164(45%) and TGFβ2(25%) appeared more suppressed.6. The uplifted diffused shape of filtering bleb of S-D rat remained in4days post filtering surgery. Bleb began to become flat from the6th day post surgery.7. Morphological change of filtering bleb and adverse events post filtering surgery combined with Bevacizumab injection:no flattened blebs observed from Bevacizumab group(less than those from two control groups), and more cases of grade three blebs observed than those from other two control groups. The significance was remarkable using Kruskal-Wallis H test(P=0.032); Less cases of hyphema were observed from Bevacizumab group compared to those from other two control groups (P=0.003); Less cases of severe hyphema were observed from Bevacizumab group compared to those from other two control groups (P=0.002); Less cases of iris reaction were observed from Bevacizumab group compared to those from other two control groups (P=0.000).8. Changes of mRNA expressions of VEGF164, Flt-1, Flkl, TGFβ1and TGFβ2 within filtering blebs post Bevacizumab intervention:The expressions of above five genes decreased in Bevacizumab group compared to those from other two control groups, the difference was statistically significant(P<0.05); Expression of VEGF164mRNA increased on dayl and reached peak value from day2to day5in independent PBS injection group(Group C). Expression of VEGF164mRNA remained low level in Bevacizumab injection group (Group A); Expression of Flt-1mRNA prolonged to increase and expression of TGFβlmRNA was lower during Logarithm stage (day3-day5) in Bevacizumab injection group compared to those in other two control groups; Expression of TGFβ2mRNA appeared much lower at each observing time in Bevacizumab group compared to those in other two control groups. Expression of TGFβ2mRNA was most enriched and the variation was most remarkable among all five genes。9. Western blots of VEGFA protein (44KD), TGFβ1protein (25KD) and TGFβ2protein (48KD) from Bevacizumab group were all weaker at most observing time compared to those from other two control groups.Conclusions1. VEGF, VEGFR (VEGFR1, VEGFR2) and inflammatory factors(TGFβ1, TGFβ2) were observed in cultured S-D rat conjunctiva fibroblasts.2. Bevacizumab could suppress the growth of cultured fibroblasts during logarithm stage, and could down regulate the expression of angiogenic factor and inflammatory factors in fibroblast.3. Morphology of filtering bleb of S-D rat remained better post filtering surgery combined with Bevacizumab subconjunctival injection. Bevacizumab could remarkably inhibit the expressions of angiogenic factor and inflammatory factors in the filtering bleb.4. The mechanism of inhibitory effect on fibrosis may be both through angiogenic pathway and non-angiogenic pathway.