Abstract：
BACKGROUND: Excessive angiogenesis of the retina is a key component of irreversible causes of blindness in many ocular diseases. Pitavastatin is a cholesterol-lowering drug used to reduce the risk of cardiovascular diseases. Various studies have shown the effects of pitavastatin on angiogenesis but the conclusions are contradictory. The effects of pitavastatin on retinal angiogenesis have not been revealed. This study investigated the effects of pitavastatin at clinically relevant concentrations on retinal angiogenesis and its underlying mechanisms using retinal microvascular endothelial cells (RMECs).
METHODS: The effects of pitavastatin on retinal angiogenesis were determined using in vitro model of retinal angiogenesis, endothelial cell migration, adhesion, proliferation, and apoptosis assays. The mechanism studies were conducted using immunoblotting and stress fiber staining.
RESULTS: Pitavastatin stimulated capillary network formation of RMECs in a similar manner as vascular endothelial growth factor (VEGF) and lipopolysaccharide (LPS). Pitavastatin also increased RMEC migration, adhesion to Matrigel, growth, and survival. The combination of pitavastatin with VEGF or LPS was more effective than VEGF or LPS alone in stimulating biological activities of RMECs, suggesting that pitavastatin can enhance the stimulatory effects of VEGF and LPS on retinal angiogenesis. Pitavastatin acted on RMECs in a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-independent manner. In contrast, pitavastatin activated pro-angiogenic microenvironment via promoting the secretion of VEGF and stimulated retinal angiogenesis via multiple mechanisms including activation of RhoA-mediated pathways, induction of focal adhesion complex formation, and activation of ERK pathway.
CONCLUSIONS: Our work provides a preclinical evidence on the pro-angiogenic effect of pitavastatin in retina via multiple mechanisms that are irrelevant to mevalonate pathway.
METHODS: The effects of pitavastatin on retinal angiogenesis were determined using in vitro model of retinal angiogenesis, endothelial cell migration, adhesion, proliferation, and apoptosis assays. The mechanism studies were conducted using immunoblotting and stress fiber staining.
RESULTS: Pitavastatin stimulated capillary network formation of RMECs in a similar manner as vascular endothelial growth factor (VEGF) and lipopolysaccharide (LPS). Pitavastatin also increased RMEC migration, adhesion to Matrigel, growth, and survival. The combination of pitavastatin with VEGF or LPS was more effective than VEGF or LPS alone in stimulating biological activities of RMECs, suggesting that pitavastatin can enhance the stimulatory effects of VEGF and LPS on retinal angiogenesis. Pitavastatin acted on RMECs in a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-independent manner. In contrast, pitavastatin activated pro-angiogenic microenvironment via promoting the secretion of VEGF and stimulated retinal angiogenesis via multiple mechanisms including activation of RhoA-mediated pathways, induction of focal adhesion complex formation, and activation of ERK pathway.
CONCLUSIONS: Our work provides a preclinical evidence on the pro-angiogenic effect of pitavastatin in retina via multiple mechanisms that are irrelevant to mevalonate pathway.
摘要：
背景：视网膜的过度血管生成是许多眼部疾病中导致失明的不可逆原因的关键组成部分。匹伐他汀是一种降胆固醇药物，用于降低心血管疾病的风险。各种研究已经显示匹伐他汀对血管生成的影响,但是结论是矛盾的。尚未揭示匹伐他汀对视网膜血管生成的影响。这项研究使用视网膜微血管内皮细胞（RMEC）研究了临床相关浓度的匹伐他汀对视网膜血管生成的影响及其潜在机制。
方法：使用体外视网膜血管生成模型测定匹伐他汀对视网膜血管生成的影响，内皮细胞迁移，附着力,扩散,和细胞凋亡测定。使用免疫印迹和应力纤维染色进行机制研究。
结果：匹伐他汀以与血管内皮生长因子（VEGF）和脂多糖（LPS）相似的方式刺激RMEC的毛细血管网络形成。匹伐他汀还增加了RMEC迁移，对Matrigel的粘附力,增长,和生存。匹伐他汀与VEGF或LPS的组合在刺激RMEC的生物学活性方面比单独的VEGF或LPS更有效。提示匹伐他汀可以增强VEGF和LPS对视网膜血管生成的刺激作用。匹伐他汀以不依赖3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶的方式作用于RMEC。相比之下,匹伐他汀通过促进VEGF的分泌激活促血管生成微环境，并通过多种机制刺激视网膜血管生成，包括激活RhoA介导的途径，诱导粘着斑复合物的形成，ERK通路的激活。
结论：我们的工作提供了关于匹伐他汀通过与甲羟戊酸途径无关的多种机制在视网膜中的促血管生成作用的临床前证据。
方法：使用体外视网膜血管生成模型测定匹伐他汀对视网膜血管生成的影响，内皮细胞迁移，附着力,扩散,和细胞凋亡测定。使用免疫印迹和应力纤维染色进行机制研究。
结果：匹伐他汀以与血管内皮生长因子（VEGF）和脂多糖（LPS）相似的方式刺激RMEC的毛细血管网络形成。匹伐他汀还增加了RMEC迁移，对Matrigel的粘附力,增长,和生存。匹伐他汀与VEGF或LPS的组合在刺激RMEC的生物学活性方面比单独的VEGF或LPS更有效。提示匹伐他汀可以增强VEGF和LPS对视网膜血管生成的刺激作用。匹伐他汀以不依赖3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶的方式作用于RMEC。相比之下,匹伐他汀通过促进VEGF的分泌激活促血管生成微环境，并通过多种机制刺激视网膜血管生成，包括激活RhoA介导的途径，诱导粘着斑复合物的形成，ERK通路的激活。
结论：我们的工作提供了关于匹伐他汀通过与甲羟戊酸途径无关的多种机制在视网膜中的促血管生成作用的临床前证据。
