由cAMP激活的鸟苷酸交换蛋白（Epac）调控内皮细胞表面膜联蛋白A2的表达

目的 本研究旨在探讨内皮细胞内Epac1是否参与调控细胞表面ANXA2的表达。方法 本研究细胞实验以人静脉内皮细胞（HUVECs）为细胞模型，采用原子动力学检测方法，结合免疫荧光，分别从原子及蛋白水平验证Epac1是否参与细胞表面ANXA2表达的调控作用。分别提取15只Epac1基因敲除小鼠和15只C57BL6野生型小鼠血浆，检测每组样品ANXA2的表达水平。结果 （1）免疫荧光测得处理组（ESI09）相对荧光强度为17.59 ± 0.69，处理组（ESI09）相对荧光强度为6.32 ± 0.32，两组间数据结果有差异且有统计学意义（P<0.01）；原子动力显微镜法测得处理组（ESI09）测得力值为35113.07 ± 9866.65 pN，对照组（DMSO）测得力值为106277.70 ± 14233.77 pN，两组间数据结果有差异且有统计学意义（P<0.01）；由以上数据可得药物性抑制内皮细胞Epac的功能使其表面ANXA2表达量下调；（2）蛋白印迹法及酶联免疫吸附法测得处理组（ESI09）和对照组（DMSO）内皮细胞分泌ANXA2有差异，药物性抑制内皮细胞Epac1功能使内皮细胞胞外分泌ANXA2能力下降低；（3）酶联免疫吸附法测得Epac1基因敲除小鼠组血浆ANXA2平均含量为138.81 ± 38.93 ng/ml，显著低于野生型小鼠组血浆ANXA2平均含量281.46 ± 49.66 ng/ml（p<0.05）。结论 Epac1参与调控内皮细胞表面ANXA2的表达。

Exchange protein directly activated by cAMP (Epac1) regulates the expression of Annexin A2 on endothelial cells surface

Objective Taking advantage of our Epac1-null mice, and using biochemical assays, our study aimed at the critical role of Epac1 in regulating endothelial surface expression of ANXA2. Methods HUVECs as cell model, we applied novel technique atomic force microscopy (AFM) to explore ANXA2 at ECs surface, confirmed with immunofluorescence (IF). We used enzyme linked immunosorbent assay (ELISA) to determine ANXA2 concentration in plasma of Epac1-null mice (n=15) compared with that of wild-type mice (n=15). Results (1)Pharmacologically functional suppression of Epac1 in HUVECs via ESI09 decreases ANXA2 residing on EC apical surfaces: relative IF intensity shows difference between ESI09-treated group and DMSO-treated group (6.32 ± 0.32 vs 17.59 ± 0.69, p<0.01); AFM force measurement also shows difference between ESI09-treated group and DMSO-treated group (106277.70 ± 14233.77 pN vs 35113.07 ± 9866.65 pN, p<0.01); (2) Both WB and ELISA show pharmacologically functional suppression of Epac1 in HUVECs via ESI09 decreases ANXA2 secretion; Depletion of Epac1 results in decreased ANXA2 in mice plasma (138.81 ± 38.93 ng/ml in plasma of Epac1-null mice vs 281.46 ± 49.66 ng/ml in plasma of wild-type mice, p<0.05) . Conclusion Taking advantage of our Epac1-null mice, we found out that Epac1 depletion results in reduced ANXA2 concentration in mice plasma. By pharmacologically functional suppression of Epac1 in HUVECs via ESI09, we show that endothelial apical surface expression of ANXA2 is markedly decreased in ESI09 treated HUVECs.