Matsuda Monique, Krempel Paloma Gava, Marquezini Mônica Valeria, Sholl-Franco Alfred, Lameu Amanda, Monteiro Mário Luiz R, Miguel Nádia Campos de Oliveira
Laboratory of Investigation in Ophthalmology (LIM-33), Division of Ophthalmology, University of São Paulo Medical School, Brazil.
Laboratory of Experimental Air Pollution, University of São Paulo Medical School & Pro-Sangue Foundation, São Paulo, Brazil.
Exp Eye Res. 2017 Jul;160:1-10. doi: 10.1016/j.exer.2017.04.005. Epub 2017 Apr 15.
Bevacizumab, an anti-vascular endothelial growth factor (VEGF) agent, is widely used in the treatment of retinal vascular diseases. However, due to the essential role Müller cell derived-VEGF plays in the maintenance of retinal neurons and glial cells, cell viability is likely to be affected by VEGF inhibition. We therefore evaluated the effect of bevacizumab-induced VEGF inhibition on Müller cells (MIO-M1) in vitro. MIO-M1 cells were cultured for 12 or 24 h in media containing bevacizumab at 0.25 or 0.5 mg/mL. Controls were cultured in medium only. Cell viability was determined with the trypan blue exclusion test and MTT assay. Caspase-3, beclin-1, glial fibrillary acidic protein (GFAP) and vimentin content were quantified by immunohistochemistry. Gene expression was evaluated by real-time quantitative PCR. Treatment with bevacizumab did not reduce MIO-M1 cell viability, but increased metabolic activity at 24 h (0.5 mg/mL) and induced apoptosis and autophagy, as shown by the increased caspase-3 levels at 12 h (0.25 and 0.5 mg/mL) and the increased beclin levels at 24 h (0.5 mg/mL). Caspase-3 mRNA was upregulated at 12 h and downregulated at 24 h in cells treated with bevacizumab at 0.25 mg/mL. Bevacizumab treatment was also associated with structural protein abnormalities, with decreased GFAP and vimentin content and upregulated GFAP and vimentin mRNA expression. Although bevacizumab did not significantly affect MIO-M1 cell viability, it led to metabolic and molecular changes (apoptosis, autophagy and structural abnormalities) suggestive of significant cellular toxicity.
贝伐单抗是一种抗血管内皮生长因子（VEGF）药物，广泛用于治疗视网膜血管疾病。然而，由于穆勒细胞衍生的VEGF在维持视网膜神经元和神经胶质细胞中起着至关重要的作用，细胞活力可能会受到VEGF抑制的影响。因此，我们在体外评估了贝伐单抗诱导的VEGF抑制对穆勒细胞（MIO-M1）的影响。将MIO-M1细胞在含有0.25或0.5mg/mL贝伐单抗的培养基中培养12或24小时。对照组仅在培养基中培养。用台盼蓝排斥试验和MTT法测定细胞活力。通过免疫组织化学对半胱天冬酶-3、贝林-1、胶质纤维酸性蛋白（GFAP）和波形蛋白含量进行定量。通过实时定量PCR评估基因表达。贝伐单抗治疗并未降低MIO-M1细胞活力，但在24小时（0.5mg/mL）时增加了代谢活性，并诱导了细胞凋亡和自噬，如12小时（0.25和0.5mg/mL）时半胱天冬酶-3水平升高以及24小时（0.5mg/mL）时贝林水平升高所示。在0.25mg/mL贝伐单抗处理的细胞中，半胱天冬酶-3 mRNA在12小时时上调，在24小时时下调。贝伐单抗治疗还与结构蛋白异常有关，GFAP和波形蛋白含量降低，而GFAP和波形蛋白mRNA表达上调。虽然贝伐单抗对MIO-M1细胞活力没有显著影响，但它导致了代谢和分子变化（细胞凋亡、自噬和结构异常），提示存在明显的细胞毒性。
