Laboratory of Investigation in Ophthalmology (LIM-33), Division of Ophthalmology, School of Medicine, University of São Paulo, São Paulo, SP, Brazil.
Department of Cell & Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil.
PLoS One. 2024 Mar 27;19(3):e0300370. doi: 10.1371/journal.pone.0300370. eCollection 2024.
Anti-VEGF (vascular endothelial growth factor) drugs such as aflibercept (AFL) and bevacizumab (BVZ) inhibit pathological neo-angiogenesis and vascular permeability in retinal vascular diseases. As cytokines and growth factors are produced by Müller glial cells under stressful and pathological conditions, we evaluated the in vitro effect of AFL (Eylea®, 0.5 mg/mL) and BVZ (Avastin®, 0.5 mg/mL) on cell viability/metabolism, and cytokine/growth factor production by Müller cells (MIO-M1) under cobalt chloride (CoCl2)-induced hypoxia after 24h, 48h and 72h. Cell viability/metabolism were analyzed by Trypan Blue and MTT assays and cytokine/growth factors in supernatants by Luminex xMAP-based multiplex bead-based immunoassay. Cell viability increased with AFL at 48h and 72h and decreased with BVZ or hypoxia at 24h. BVZ-treated cells showed lower cell viability than AFL at all exposure times. Cell metabolism increased with AFL but decreased with BVZ (72h) and hypoxia (48h and72h). As expected, AFL and BVZ decreased VEGF levels. AFL increased PDGF-BB, IL-6 and TNF-α (24h) and BVZ increased PDGF-BB (72h). Hypoxia reduced IL-1β, -6, -8, TNF-α and PDGF-BB at 24h, and its suppressive effect was more prominent than AFL (EGF, PDGF-BB, IL-1β, IL-6, IL-8, and TNF-α) and BVZ (PDGF-BB and IL-6) effects. Hypoxia increased bFGF levels at 48h and 72h, even when combined with anti-VEGFs. However, the stimulatory effect of BVZ predominated over hypoxia for IL-8 and TNF-α (24h), as well as for IL-1β (72h). Thus, AFL and BVZ exhibit distinct exposure times effects on MIO-M1 cells viability, metabolism, and cytokines/growth factors. Hypoxia and BVZ decreased MIO-M1 cell viability/metabolism, whereas AFL likely induced gliosis. Hypoxia resulted in immunosuppression, and BVZ stimulated inflammation in hypoxic MIO-M1 cells. These findings highlight the complexity of the cellular response as well as the interplay between anti-VEGF treatments and the hypoxic microenvironment.
抗血管内皮生长因子（VEGF）药物如阿柏西普（AFL）和贝伐珠单抗（BVZ）可抑制视网膜血管疾病中的病理性新生血管形成和血管通透性。由于细胞因子和生长因子是在应激和病理条件下由 Müller 胶质细胞产生的，因此我们评估了 AFL（Eylea®，0.5mg/ml）和 BVZ（Avastin®，0.5mg/ml）在氯化钴（CoCl2）诱导的缺氧下对 Müller 细胞（MIO-M1）活力/代谢的体外影响，以及细胞因子/生长因子的产生，在 24h、48h 和 72h 后。细胞活力/代谢通过台盼蓝和 MTT 测定分析，细胞因子/生长因子通过基于 Luminex xMAP 的多重珠基免疫测定法在细胞上清液中测定。AFL 在 48h 和 72h 时增加细胞活力，BVZ 或缺氧在 24h 时降低细胞活力。在所有暴露时间点，BVZ 处理的细胞活力均低于 AFL。BVZ（72h）和缺氧（48h 和 72h）降低细胞代谢，AFL 增加细胞代谢。如预期的那样，AFL 和 BVZ 降低了 VEGF 水平。AFL 在 24h 时增加 PDGF-BB、IL-6 和 TNF-α，BVZ 在 72h 时增加 PDGF-BB。缺氧在 24h 时降低了 IL-1β、-6、-8、TNF-α和 PDGF-BB，其抑制作用比 AFL（EGF、PDGF-BB、IL-1β、IL-6、IL-8 和 TNF-α）和 BVZ（PDGF-BB 和 IL-6）更为显著。缺氧在 48h 和 72h 时增加 bFGF 水平，即使与抗 VEGF 联合使用也是如此。然而，BVZ 的刺激作用对于 IL-8 和 TNF-α（24h）以及 IL-1β（72h）来说，超过了缺氧的作用。因此，AFL 和 BVZ 对 MIO-M1 细胞活力、代谢和细胞因子/生长因子表现出不同的暴露时间效应。缺氧和 BVZ 降低了 MIO-M1 细胞活力/代谢，而 AFL 可能诱导了神经胶质增生。缺氧导致免疫抑制，BVZ 刺激缺氧 MIO-M1 细胞中的炎症。这些发现突出了细胞反应的复杂性以及抗 VEGF 治疗与缺氧微环境之间的相互作用。
