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Abstract：
Diabetic retinopathy is a secondary microvascular complication of diabetes mellitus. This disease progresses from two stages, non-proliferative and proliferative diabetic retinopathy, the latter characterized by retinal abnormal angiogenesis. Pharmacological management of retinal angiogenesis employs expensive and invasive intravitreal injections of biologic drugs (anti-vascular endothelial growth factor agents). To search small molecules able to act as anti-angiogenic agents, we focused our study on axitinib, which is a tyrosine kinase inhibitor and represents the second line treatment for renal cell carcinoma. Axitinib is an inhibitor of vascular endothelial growth factor receptors, and among the others tyrosine kinase inhibitors (sunitinib and sorafenib) is the most selective towards vascular endothelial growth factor receptors 1 and 2. Besides the well-known anti-angiogenic and immune-modulatory functions, we hereby explored the polypharmacological profile of axitinib, through a bioinformatic/molecular modeling approach and in vitro models of diabetic retinopathy. We showed the anti-angiogenic activity of axitinib in two different in vitro models of diabetic retinopathy, by challenging retinal endothelial cells with high glucose concentration (fluctuating and non-fluctuating). We found that axitinib, along with inhibition of vascular endothelial growth factor receptors 1 (1.82 ± 0.10; 0.54 ± 0.13, phosphorylated protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively) and vascular endothelial growth factor receptors 2 (2.38 ± 0.21; 0.98 ± 0.20, phosphorylated protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively), was able to significantly reduce (p < 0.05) the expression of Nrf2 (1.43 ± 0.04; 0.85 ± 0.01, protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively) in retinal endothelial cells exposed to high glucose, through predicted Keap1 interaction and activation of melanocortin receptor 1. Furthermore, axitinib treatment significantly (p < 0.05) decreased reactive oxygen species production (0.90 ± 0.10; 0.44 ± 0.06, fluorescence units in high glucose vs . axitinib 1 µM, respectively) and inhibited ERK pathway (1.64 ± 0.09; 0.73 ± 0.06, phosphorylated protein levels in fluctuating high glucose vs . axitinib 1 µM, respectively) in HRECs exposed to high glucose. The obtained results about the emerging polypharmacological profile support the hypothesis that axitinib could be a valid candidate to handle diabetic retinopathy, with ancillary mechanisms of action.
摘要：
糖尿病视网膜病变是糖尿病的继发性微血管并发症。这种疾病从两个阶段发展，非增殖性和增殖性糖尿病视网膜病变，后者以视网膜异常血管生成为特征。视网膜血管生成的药理学管理采用昂贵且侵入性的生物药物(抗血管内皮生长因子剂)的玻璃体内注射。为了寻找能够作为抗血管生成剂的小分子，我们的研究重点是阿西替尼，它是一种酪氨酸激酶抑制剂，代表肾细胞癌的二线治疗。阿西替尼是一种血管内皮生长因子受体抑制剂，其中酪氨酸激酶抑制剂（舒尼替尼和索拉非尼）对血管内皮生长因子受体1和2的选择性最强。除了众所周知的抗血管生成和免疫调节功能，我们在此探索阿西替尼的多药理学概况，通过生物信息学/分子建模方法和糖尿病视网膜病变的体外模型。我们在两种不同的糖尿病视网膜病变体外模型中显示了阿西替尼的抗血管生成活性，通过挑战具有高葡萄糖浓度（波动和非波动）的视网膜内皮细胞。我们发现阿西替尼,随着血管内皮生长因子受体1的抑制（1.82±0.10；0.54±0.13，在波动的高糖vs.阿西替尼1µM，分别）和血管内皮生长因子受体2（2.38±0.21；0.98±0.20，在波动的高糖vs.阿西替尼1µM，分别)，能够显着降低（p<0.05）Nrf2的表达（1.43±0.04；0.85±0.01，在波动的高糖vs.阿西替尼1µM，分别）在暴露于高糖的视网膜内皮细胞中，通过预测Keap1相互作用和黑皮质素受体1的激活。此外，阿西替尼治疗显着（p<0.05）降低了活性氧的产生（0.90±0.10；0.44±0.06，高葡萄糖中的荧光单位vs.阿西替尼1µM，分别）并抑制ERK途径（1.64±0.09；0.73±0.06，在波动的高糖vs.阿西替尼1µM，分别）在暴露于高葡萄糖的HREC中。关于新兴的多药理作用的结果支持阿西替尼可能是治疗糖尿病性视网膜病变的有效候选者的假设。具有辅助作用机制。
