From the Pole of Pharmacology and Therapeutics, Experimental and Clinical Research Institute (IREC), Université catholique de Louvain, Brussels, Belgium (V.J., E.L.G., L.M., H.E., J.-L.B., C.D.).
Humanitas University, Rozzano (Milan), Italy (V.D.M., G.C.).
Arterioscler Thromb Vasc Biol. 2018 Oct;38(10):2345-2357. doi: 10.1161/ATVBAHA.118.311145.
Objective- Members of the microRNA (miR)-199a family, namely miR-199a-5p and miR-199a-3p, have been recently identified as potential regulators of cardiac homeostasis. Also, upregulation of miR-199a expression in cardiomyocytes was reported to influence endothelial cells. Whether miR-199a is expressed by endothelial cells and, if so, whether it directly regulates endothelial function remains unknown. We investigate the implication of miR-199a products on endothelial function by focusing on the NOS (nitric oxide synthase)/NO pathway. Approach and Results- Bovine aortic endothelial cells were transfected with specific miRNA inhibitors (locked-nucleic acids), and potential molecular targets identified with prediction algorithms were evaluated by Western blot or immunofluorescence. Ex vivo experiments were performed with mice treated with antagomiRs targeting miR-199a-3p or -5p. Isolated vessels and blood were used for electron paramagnetic resonance or myograph experiments. eNOS (endothelial NO synthase) activity (through phosphorylations Ser1177/Thr495) is increased by miR-199a-3p/-5p inhibition through an upregulation of the PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B) and calcineurin pathways. SOD1 (superoxide dismutase 1) and PRDX1 (peroxiredoxin 1) upregulation was also observed in locked-nucleic acid-treated cells. Moreover, miR-199a-5p controls angiogenesis and VEGFA (vascular endothelial growth factor A) production and upregulation of NO-dependent relaxation were observed in vessels from antagomiR-treated mice. This was correlated with increased circulated hemoglobin-NO levels and decreased superoxide production. Angiotensin infusion for 2 weeks also revealed an upregulation of miR-199a-3p/-5p in vascular tissues. Conclusions- Our study reveals that miR-199a-3p and miR-199a-5p participate in a redundant network of regulation of the NOS/NO pathway in the endothelium. We highlighted that inhibition of miR-199a-3p and -5p independently increases NO bioavailability by promoting eNOS activity and reducing its degradation, thereby supporting VEGF-induced endothelial tubulogenesis and modulating vessel contractile tone.
目的- 微小 RNA（miR）-199a 家族的成员，即 miR-199a-5p 和 miR-199a-3p，最近被鉴定为心脏内稳态的潜在调节剂。此外，有报道称心肌细胞中 miR-199a 的表达上调会影响内皮细胞。内皮细胞是否表达 miR-199a，如果是，它是否直接调节内皮功能尚不清楚。我们通过关注 NOS（一氧化氮合酶）/NO 途径来研究 miR-199a 产物对内皮功能的影响。
方法和结果- 用特异性 miRNA 抑制剂（锁核酸）转染牛主动脉内皮细胞，并通过 Western blot 或免疫荧光评估预测算法确定的潜在分子靶标。用针对 miR-199a-3p 或 -5p 的抗miR 处理小鼠进行离体实验。分离血管和血液用于电子顺磁共振或肌动图实验。eNOS（内皮型一氧化氮合酶）活性（通过 Ser1177/Thr495 磷酸化）通过 miR-199a-3p/-5p 抑制的上调来增加，这是通过 PI3K（磷酸肌醇 3-激酶）/Akt（蛋白激酶 B）和钙调神经磷酸酶途径的上调来实现的。还观察到Locked-nucleic acid 处理的细胞中 SOD1（超氧化物歧化酶 1）和 PRDX1（过氧化物还原酶 1）的上调。此外，在抗 miR 处理的小鼠血管中观察到 miR-199a-5p 控制血管生成和 VEGFA（血管内皮生长因子 A）的产生，以及 NO 依赖性松弛的上调。这与循环血红蛋白-NO 水平的升高和超氧化物产生的减少有关。用血管紧张素灌注 2 周也揭示了血管组织中 miR-199a-3p/-5p 的上调。
结论- 我们的研究表明，miR-199a-3p 和 miR-199a-5p 参与了内皮细胞中 NOS/NO 途径的冗余调节网络。我们强调，miR-199a-3p 和 -5p 的抑制通过促进 eNOS 活性和减少其降解，独立地增加了 NO 的生物利用度，从而支持 VEGF 诱导的内皮小管形成并调节血管收缩紧张度。
