Mechanisms of action of prolonged VEGF exposure on the blood-retinal barrier: Implications for the treatment of diabetic macular edema

具有高度社会经济相关性的疾病，如糖尿病性黄斑水肿，与玻璃体中血管内皮生长因子A （VEGFA）水平升高有关。玻璃体内注射vegf结合蛋白的治疗通常是成功的，但在大量患者中观察到的所谓治疗失败的原因尚不清楚。此外，玻璃体内注射必须定期重复，通常是每月一次，因此，对患者、医生和卫生保健系统的负担以及反复玻璃体内注射的累积风险是巨大的。通过更好地了解玻璃体内VEGFA水平升高如何导致黄斑水肿，可以开发出针对病因而不是疾病后期的改进治疗方法。已知VEGFA可以增加视网膜内皮细胞的通透性，这是血液-视网膜内部屏障的一部分。我们已经证明，这种屏障的损伤与紧密连接蛋白claudin-1的表达减少密切相关。此外，我们的研究表明，这种功能障碍只能通过特异性抑制VEGF诱导的信号转导来短暂地逆转——这一观察结果与VEGF拮抗剂只有暂时作用的临床表现一致。我们还观察到VEGFA改变了视网膜内皮细胞中特定microrna的表达谱，这可能有助于解释生长因子调节血液-视网膜内屏障的机制。现在我们想更详细地探索长期暴露于VEGFA如何导致视网膜内皮细胞通透性升高，以确定新的和潜在的更多因果治疗靶点。为此，我们研究了哪些蛋白激酶保持激活，哪些蛋白参与紧密连接蛋白claudin-1的降解，长期暴露于VEGFA如何改变视网膜内皮细胞中各种microrna的表达，以及miRNA沉默是否可以调节屏障损伤。在这方面，计划的实验方法模拟治疗因长期暴露于VEGFA而引起的先前存在的黄斑水肿。因此，这种方法更好地反映了临床现实，黄斑水肿的治疗通常是在它存在一段时间后才开始的。方法上采用器官芯片模型，很好地再现了视网膜内皮细胞在剪切应力下的体内情况。采用视网膜内皮细胞和原代人视网膜内皮细胞的永生化细胞系，并与视网膜周细胞共培养，以确保结果可转移到临床实践。

Diseases of high socio-economic relevance such as diabetic macular edema are associated with increased leves of vascular endothelial growth factor A (VEGFA) in the vitreous. Therapy with intravitreally injected VEGF-binding proteins is often successful, but the cause of a so-called therapy failure, which is observed in a substantial number of patients, remains unclear. Moreover intravitreal therapy must be repeated regularly, often monthly, therefore the burden on the patient, practitioner, and health care system, as well as the cumulative risks of repeated intravitreal injections, are substantial. By better understanding how elevated intravitreal VEGFA levels lead to macular edema, improved therapeutic approaches can be developed that target the cause rather than later stages of the disease. VEGFA is known to increase the permeability of retinal endothelial cells which are part of the inner blood-retinal barrier. We have shown that such an impairment of the barrier is strongly associated with decreased expression of the tight-junction protein claudin-1. Moreover, our studies have revealed that this dysfunction can be reversed only transiently by specific inhibition of VEGFA-induced signal transduction – an observation that is consistent with the clinical picture of only temporary effects of VEGF antagonists. We also observed that VEGFA alters the expression profile of specific microRNAs in retinal endothelial cells, which may help to explain the mechanism by which the growth factor modulates the inner blood-retinal barrier. Now we want to explore in more detail how long-term exposure with VEGFA results in elevated permeability of retinal endothelial cells in order to identify new and potentially more causal targets for therapy. To this end, we investigate which protein kinases remain activated, which proteins are involved in the degradation of the tight junction protein claudin-1, how the expression of various microRNAs is altered in retinal endothelial cells by long-term exposure to VEGFA and whether barrier impairment can be modulated by miRNA silencing. In this regard, the planned experimental approach mimics the therapy of preexisting macular edema caused by prolonged exposure to VEGFA. Thus, this approach better reflects the clinical reality, in which therapy for macular edema usually starts only after it has existed for some time. Methodologically, an organ-on-chip model is used, which reproduces very well the in vivo situation of retinal endothelial cells under shear stress. An immortalized cell line of retinal endothelial cells as well as primary human retinal endothelial cells, also co-cultured with retinal pericytes are used to ensure a good transferability of the results to clinical practice.