不同批次的重组诱饵型受体创新药物RC28-E1与RC28-E2对视网膜新生血管的药效学比较及机制

Comparison of pharmacodynamics between different batches of recombinant decoy receptor innovative drug RC28-E1 and RC28-E2 in retinal angiogenesis and neovascularization and its mechanism

目的探讨不同批次的重组诱饵型受体创新药物RC28-E1与RC28-E2对视网膜新生血管的药效学比较及作用机制。方法选取健康清洁级4日龄C57BL/6J幼鼠60只，应用随机数字表法随机分为正常对照组、血管内皮生长因子（VEGF）＋成纤维细胞生长因子2（FGF2）组、VEGF＋FGF2＋RC28-E1组、VEGF＋FGF2＋RC28-E2组、VEGF＋FGF2＋conbercept组、VEGF＋FGF2＋FGF trap组，每组10只。取各组视网膜组织块构建培养体系，加入用饥饿培养基配置的相应因子和药物刺激，正常对照组加饥饿培养基。将各组视网膜组织块进行植物凝集素（Isolectin B4）染色并拍照，计算各组单位血管长度下视网膜血管顶细胞分叉的数量。另选96只健康清洁级7日龄C57BL/6J幼鼠，应用随机数字表法随机分为正常对照组、氧诱导视网膜病变（OIR）模型对照组、OIR＋RC28-E1组、OIR＋RC28-E2组、OIR＋conbercept组和OIR＋FGF trap组，每组16只。正常对照组在常氧下饲养10 d，其余各组在高氧环境中饲养5 d后在常氧状态下再饲养5 d。各组取17日龄鼠视网膜进行Isolectin B4染色并拍照，用计算机软件分析视网膜相对无灌注区面积和新生血管像素。Western blot法检测OIR实验各组视网膜中VEGF和FGF2的蛋白水平。最后，在VEGF和FGF2刺激下的视网膜血管内皮细胞（RF/6A）中，检测给予RC28-E1、conbercept以及FGF trap处理后，细胞的MEK-细胞外调节蛋白激酶（Erk）、蛋白激酶C（PKC）以及蛋白激酶B（Akt）信号传导通路的活性变化。结果视网膜组织块研究结果显示，VEGF＋FGF2＋RC28-E1组、VEGF＋FGF2＋RC28-E2组、VEGF＋FGF2＋conbercept组和VEGF＋FGF2＋FGF trap组中单位血管长度的顶细胞分叉数目均明显少于VEGF＋FGF2组，差异均有统计学意义（均P〈0.001），其中RC28-E1组与RC28-E2组的血管顶细胞分叉数量相似，差异无统计学意义（P＝0.15），但均明显少于VEGF＋FGF2＋conbercept组和VEGF＋FGF2＋FGF trap组，差异均有统计学意义（均P〈0.001）。OIR模型对照组无灌注区相对面积明显大于各药物干预组，差异均有统计学意义（均P〈0.05），OIR＋RC28-E1组与OIR＋RC28-E2组视网膜无灌注区相对面积的比较，差异无统计学意义（P＝0.17），但二者明显小于OIR＋conbercept组和OIR＋FGF trap组，差异均有统计学意义（均P〈0.05）。各干预组新生血管的相对像素值明显低于OIR模型对照组，差异均有统计学意义（均P〈0.001），OIR＋RC28-E1组视网膜新生血管的相对像素值明显低于VEGF＋FGF2＋conbercept组和VEGF＋FGF2＋FGF trap组，差异均有统计学意义（均P〈0.05），但与OIR＋RC28-E2组相比，差异无统计学意义（P＝0.39）。Western blot结果显示，OIR小鼠视网膜中VEGF和FGF2的蛋白表达均较正常对照组显著上调，差异均有统计学意义（均P〈0.001），而RC28-E1和RC28-E2可使其降至正常水平。VEGF和FGF2可诱导RF/6A细胞中的MEK-Erk通路活性增强，而RC28-E1可显著抑制这一通路的过度激活。结论RC28-E1和RC28-E2可在初生小鼠视网膜组织块中抑制血管生成，并减少OIR小鼠模型中视网膜的无灌注区，减轻新生血管生成。药理批次和中试批次的RC28-E在体内外模型中的效果相当，稳定性可靠，且优于临床同类药物conbercept和FGF trap。RC28-E1可能是通过抑制视网膜血管内皮细胞中MEK-Erk通路的过度激活而达到抑制病理性新生血管生成的效果的。

ObjectiveTo compare the pharmacodynamics between different batches of recombinant decoy receptor innovative drug RC28-E1 and RC28-E2 in retinal angiogenesis and neovascularization, and analyze its mechanism.MethodsSixty postnatal Day 4 （P4） C57BL/6J mice were randomly divided into normal control group, vascular endothelial growth factor （VEGF）＋ fibroblast growth factor 2 （FGF2） group, VEGF＋ FGF2＋ RC28-E1 group, VEGF＋ FGF2＋ RC28-E2 group, VEGF＋ FGF2＋ conbercept group and VEGF＋ FGF2＋ FGF trap group by using a random number table, with 10 mice in each group.The mouse retinal explant culture system was established, and stimulated with the corresponding factors and drugs prepared in the starving culture media.The normal controls were treated with the starving media.Then the retinal explants were stained with Isolectin B4 and imaged.The number of filopodia per vascular length was quantified.In addition, ninety-six P7 C57BL/6J mice were randomly divided into normal control group, oxygen-induced retinopathy （OIR） model control group, OIR＋ RC28-E1 group, OIR＋ RC28-E2 group, OIR＋ conbercept group and OIR＋ FGF trap group by using a random number table, with 16 mice in each group.The normal controls were raised under normoxia for 10 days, and the rest of the groups were raised under hyperoxia for 5 days, then returned to normoxia for another 5 days.On P17, the retinas were isolated and stained with Isolectin B4.The stained retinas were mounted on the slides and photographed.The relative vessel obliteration and neovascularization in retina were analyzed with computer software.Then the protein levels of VEGF and FGF2 were examined by Western blot in the retinas of each group in the OIR experiment.Finally, in the RF/6A cells stimulated with VEGF and FGF2, the activities of the signaling pathways, including MEK-extracellular regulated protein kinases （Erk）, protein kinase C （PKC） and protein kinase B （Akt） pathways, were examined by Western blot.All experimental procedures were evaluated and approved by the Institutional Animal Care and Use Committee of Tianjin Medical University （SYXK 2009-0001）, and were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.ResultsThe results of retinal explant cultures showed that the numbers of filopodia per vascular length in VEGF＋ FGF2＋ RC28-E1, VEGF＋ FGF2＋ RC28-E2, VEGF＋ FGF2＋ conbercept, and VEGF＋ FGF2＋ FGF trap groups were all significantly less than that in the VEGF＋ FGF2 group （all at P 〈 0.001）. The filopodia number in retinal vascular front in RC28-E1 group was similar to that in the RC28-E2 group （P=0.15）, whereas the filopodia numbers in both groups were significantly decreased as compared to those in VEGF＋ FGF2＋ conbercept group and VEGF＋ FGF2＋ FGF trap group （all at P〈0.001）. The results from the OIR mouse model showed that the relative vessel obliteration area in OIR model control group was dramatically higher than those in the drug intervention groups （all at P〈0.05）. There was no statistical significance in the relative vessel obliteration area between OIR＋ RC28-E1 group and OIR＋ RC28-E2 group （P=0.17）, while the obliteration areas in both RC28-E-intervened groups were significantly lower than those in the OIR＋ conbercept group and OIR＋ FGF trap group （all at P〈0.05）. The relative neovascular pixels in the intervention groups were significantly lower than those in the OIR model control group （all at P〈0.001）. The neovascular pixels in OIR＋ RC28-E1 group were significantly lower than those in VEGF＋ FGF2＋ conbercept group and VEGF＋ FGF2＋ FGF trap group （both at P〈0.05）, but comparable to those in OIR＋ RC28-E2 group （P =0.39）. Western blot result showed that, the protein expression of VEGF and FGF2 in the OIR mouse retinas were significantly upregulated compared to those in the normal ones （both at P〈0.001）. The upregulation of both genes were normalized by both RC28-E1 and RC28-E2.In addition, the stimulation of VEGF and FGF2 induced an enhanced activity in MEK-Erk pathway in RF/6A cells, whereas RC28-E1 inhibited the overactivation.ConclusionsRC28-E1 and RC28-E2 both can inhibit angiogenesis in the retinal explants isolated from neonatal mice; they also reduce vessel obliteration and mitigate neovascularization in the OIR mouse model.Therefore, the pharmacology batch and pilot test batch of RC28-E have similar efficacies and reliable stability, and are superior in the anti-angiogenic and anti-neovascular efficacy to the currently clinically available drugs conbercept and FGF trap.RC28-E1 may suppress pathological neovascularization through inhibiting the overactivation of MEK-Erk pathway in retinal vascular endothelial cells.Key words： Retinal neovascularization; Decoy receptor; Basic fibroblast growth factor; Vascular endothelial growth factor; Recombinant protein drugs .