S100A6通过招募和活化巨噬细胞促进血管形成*

目的:探讨S100A6经由巨噬细胞介导的促血管生成作用及机制。方法:(1)用重组蛋白 GST-hS100A6处理巨噬细胞后:①收集上清制备条件培养基(简称A6-Mφ-CM),并用之重悬人脐静脉内皮细胞(HUVEC),用体外血管形成试验检测各处理因素对血管形成的影响;②分别用实时荧光定量PCR和Western blot检测巨噬细胞的M2型标志物CD163及促血管形成因子CCL2、IL-6、VEGFA的mRNA和蛋白质水平,以及JAK2和STAT3的蛋白质及其磷酸化水平;③用Transwell迁移试验检测巨噬细胞迁移能力的变化。(2)使用JAK2抑制剂(XL019)预处理巨噬细胞后再加GST-hS100A6处理,检测S100A6促巨噬细胞迁移作用的变化。以重组蛋白GST为实验对照。 结果:(1)A6-Mφ-CM组的血管分支数和血管分支长度既明显高于GST-Mφ-CM组(P值均小于0.05),也明显高于GST-hS100A6直接处理的HUVEC组(P<0.001,P<0.01),提示S100A6处理后的巨噬细胞具有促进血管形成的作用;(2)GST-hS100A6处理后的巨噬细胞中,CD163、CCL2、IL-6、VEGFA的mRNA和蛋白质水平明显高于GST组(P值均小于0.05),提示S100A6诱导巨噬细胞向促血管表型(pro-angiogenic phenotype)转化;(3)GST-hS100A6处理后,巨噬细胞的迁移数是GST组的1.4倍(P<0.01),提示S100A6具有招募巨噬细胞的作用;(4)GST-hS100A6处理组巨噬细胞的JAK2和STAT3的蛋白质及其磷酸化水平都明显高于GST组(P值均小于0.05),而JAK2抑制剂XL019可部分抑制S100A6促进巨噬细胞迁移的作用(P<0.01),提示S100A6促进巨噬细胞迁移作用机制涉及JAK2/STAT3信号通路的激活。 结论:微环境中的S100A6可通过招募巨噬细胞并进一步诱导其向促血管表型转化,进而促进新生血管形成;其招募巨噬细胞的机制涉及JAK2/STAT3信号通路的激活。

S100A6 Promotes Angiogenesis Through Recruiting and Activating Macrophages

Objective: To investigate the effect of S100A6 in microenvironment on promoting angiogenesis via macrophages and the underlying mechanism. Methods: (1)Macrophages were treated by recombinant proteins GST-hS100A6: ①Conditioned medium was collected (named A6-Mφ-CM) to resuspend human umbilical vein endothelial cells (HUVEC), and the effect on angiogenesis was examined by in vitro endothelial capillary formation assay. ②The mRNA and protein levels of CD163, CCL2, IL-6, VEGFA in macrophages were evaluated by Real-time PCR and Western blot. And JAK2 and STAT3 and their phosphorylation levels in macrophages were detected by Western blot. ③ The migration ability of macrophages was detected by Transwell migration assay. (2)The change of macrophage migration ability was detected by Transwell assay after pre-treatment of JAK2 inhibitor XL019. Results: (1) A6-Mφ-CM had obvious effects on promoting angiogenesis,the number of branches and branch length of the A6-Mφ-CM group was significantly higher than that of the GST-Mφ-CM group (P<0.05; P<0.05), and was also significantly higher than that of GST-hS100A6 group, suggesting that S100A6 treated macrophages can promote angiogenesis, while S100A6 has no direct pro-angiogenic effect. (2) After treatment with GST-hS100A6, the mRNA and protein levels of CD163, CCL2, IL-6 and VEGFA in macrophages were up-regulated compared with GST group (P<0.05; P<0.05; P<0.05; P<0.05), suggesting that S100A6 induced polarization of macrophages into a pro-angiogenic phenotype. (3) The number macrophage migrated in GST-hS100A6 group was 1.4-fold as much as that in GST group (P<0.01), suggesting that S100A6 can enhance macrophage migration. (4) GST-hS100A6 upregulated the levels of JAK2, STAT3 and their phosphorylation proteins in macrophages, indicating that JAK2/STAT3 pathway of macrophages was activated. (5) The effect of GST-hS100A6 on promoting macrophage migration was partly inhibited by JAK2 inhibitor XL019(P<0.01). Conclusion: S100A6 in the microenvironment can recruit macrophages by activating JAK2/STAT3 signaling, and further induce macrophage into a pro-angiogenic phenotype to promote angiogenesis indirectly.