Siedlecki Jakob, Asani Ben, Wertheimer Christian, Hillenmayer Anna, Ohlmann Andreas, Priglinger Claudia, Priglinger Siegfried, Wolf Armin, Eibl-Lindner Kirsten
Department of Ophthalmology, Ludwig-Maximilians-University, Mathildenstrasse 8, 80336, Munich, Germany.
Graefes Arch Clin Exp Ophthalmol. 2018 Jun;256(6):1141-1149. doi: 10.1007/s00417-018-3987-8. Epub 2018 May 2.
Large trials on anti-VEGF/PDGF (vascular endothelial/platelet-derived growth factor) combination therapy have been established to improve management of neovascular activity in age-related macular degeneration. Targeting pericytes, PDGF is thought to induce vessel regression and reduce fibrovascular scarring. The fate of pericytes exposed to anti-VEGF/PDGF combination therapy is not clear. Therefore, this study was designed to study the influence of anti-VEGF/PDGF on pericyte phenotype and cellular behavior.
Human pericytes from placenta (hPC-PL) were treated with axitinib, a tyrosine kinase inhibitor targeting VEGFR1-3 and PDGFR. Toxic effects were excluded using live/dead staining. Phenotypic changes were evaluated using phalloidin staining for actin cytoskeleton and the expression of stress fibers. MRNA and protein expression levels of α-smooth muscle actin (αSMA) as a marker of proto-myofibroblastic transition were evaluated with real-time PCR and Western blotting. Influences of fibrotic cellular mechanisms were evaluated with a scratch wound migration and a collagen gel contraction assay.
Treatment with 0.5, 1, and 2.5 μg/ml axitinib strongly induced a proto-myofibroblast-like actin cytoskeleton with a marked increase in stress fibers. Quantitative real-time PCR and Western blotting revealed these changes to be linked to dose-dependent increases in αSMA mRNA and protein expression. However, fibrotic cellular mechanisms were significantly reduced in the presence of axitinib (scratch wound closure: up to - 78.4%, collagen gel contraction: up to - 37.4%).
Combined anti-VEGF/PDGF inhibition seems to induce a proto-myofibroblast-like phenotype in human pericytes in vitro, but reduce profibrotic cellular mechanisms due to prolonged anti-PDGF inhibition.
已开展关于抗血管内皮生长因子/血小板衍生生长因子（VEGF/PDGF）联合治疗的大型试验，以改善年龄相关性黄斑变性中新血管活性的管理。血小板衍生生长因子靶向周细胞，被认为可诱导血管退缩并减少纤维血管瘢痕形成。暴露于抗VEGF/PDGF联合治疗的周细胞的命运尚不清楚。因此，本研究旨在探讨抗VEGF/PDGF对周细胞表型和细胞行为的影响。
用阿西替尼（一种靶向血管内皮生长因子受体1 - 3和血小板衍生生长因子受体的酪氨酸激酶抑制剂）处理来自胎盘的人周细胞（hPC - PL）。使用活/死染色排除毒性作用。使用鬼笔环肽染色评估肌动蛋白细胞骨架的表型变化以及应力纤维的表达。用实时聚合酶链反应和蛋白质印迹法评估作为原肌成纤维细胞转变标志物的α - 平滑肌肌动蛋白（αSMA）的mRNA和蛋白质表达水平。用划痕伤口迁移试验和胶原凝胶收缩试验评估纤维化细胞机制的影响。
用0.5、1和2.5μg/ml阿西替尼处理强烈诱导了原肌成纤维细胞样肌动蛋白细胞骨架，应力纤维显著增加。定量实时聚合酶链反应和蛋白质印迹显示这些变化与αSMA mRNA和蛋白质表达的剂量依赖性增加有关。然而，在阿西替尼存在的情况下，纤维化细胞机制显著降低（划痕伤口闭合：高达 - 78.4%，胶原凝胶收缩：高达 - 37.4%）。
联合抗VEGF/PDGF抑制似乎在体外诱导人周细胞出现原肌成纤维细胞样表型，但由于抗PDGF抑制时间延长而降低了促纤维化细胞机制。
