Almalki Sami G, Agrawal Devendra K
Department of Clinical and Translational Science, Creighton University School of Medicine, Omaha, NE, 68178, USA.
Stem Cell Res Ther. 2017 May 12;8(1):113. doi: 10.1186/s13287-017-0568-4.
Cell-based therapy that can rejuvenate the endothelium with stimulated adipose-derived mesenchymal stem cells (AMSCs) is a promising therapeutic strategy for the re-endothelialization of denuded arteries at the stenting site. Previously, we have shown that silencing of MMP-2 and MMP-14 inhibits vascular endothelial growth factor receptor type 2 (VEGFR2) cleavage, and induces differentiation of AMSCs toward the endothelial cell (EC) lineage. In this study, we examined the underlying signaling pathways that regulate differentiation of AMSCs to ECs in vitro through VEGFR2.
AMSCs were isolated from porcine abdominal adipose tissue. The isolated AMSCs were characterized by positive expression of CD29, CD44, and CD90 and negative expression of CD11b and CD45. The isolated MSCs were transfected with siRNA to silence MMP-2, MMP-14, and angiotensin receptor 2 (ATR2). Cells were suspended either in endothelial basal media (EBM) or endothelial growth media (EGM) with various treatments. Flow cytometry was performed to examine the expression of EC markers, and western blot analysis was performed to examine the expression and activity of various kinases. Scratch assay was performed to examine the cell migration. Data were analyzed by ANOVA using PRISM GraphPad.
After 10 days of stimulation for EC differentiation, the morphology of AMSCs changed to a morphology similar to that of ECs. Silencing MMP-2 and MMP-14 resulted in significant decrease in the number of migrated cells compared with the EGM-only group. ATR2 siRNA transfection did not affect the migration and differentiation of AMSCs to ECs. Stimulation of AMSCs for EC differentiation with or without MMP-2 or MMP-14 siRNA resulted in significant increase in p-ERK, and significant decrease in p-JNK. There was no significant change in p-p38 in all three groups compared with the EBM group. ERK inhibition resulted in significant decrease in the expression of EC markers in the EGM, EGM + MMP-2 siRNA, and EGM + MMP-14 siRNA groups. The VEGFR2 kinase inhibitor induced a dose-dependent inhibition of ERK.
The ERK signaling pathway is critical for VEGF-A/VEGFR2-induced differentiation of AMSCs into ECs. These findings provide new insights into the role of the ERK signaling pathway in AMSC differentiation to ECs for potential clinical use in cardiovascular diseases.
利用经刺激的脂肪来源间充质干细胞（AMSCs）使内皮细胞恢复活力的细胞疗法，是一种用于支架置入部位裸露动脉再内皮化的有前景的治疗策略。此前，我们已经表明，沉默基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-14（MMP-14）可抑制血管内皮生长因子2型受体（VEGFR2）的裂解，并诱导AMSCs向内皮细胞（EC）谱系分化。在本研究中，我们通过VEGFR2研究了体外调节AMSCs向ECs分化的潜在信号通路。
从猪腹部脂肪组织中分离AMSCs。通过CD29、CD44和CD90的阳性表达以及CD11b和CD45的阴性表达对分离的AMSCs进行鉴定。将分离的间充质干细胞用小干扰RNA（siRNA）转染以沉默MMP-2、MMP-14和血管紧张素受体2（ATR2）。细胞在不同处理条件下分别悬浮于内皮细胞基础培养基（EBM）或内皮细胞生长培养基（EGM）中。进行流式细胞术检测EC标志物的表达，并进行蛋白质印迹分析检测各种激酶的表达和活性。进行划痕试验检测细胞迁移。数据采用PRISM GraphPad软件通过方差分析进行分析。
在刺激EC分化10天后，AMSCs的形态转变为与ECs相似的形态。与仅使用EGM的组相比，沉默MMP-2和MMP-14导致迁移细胞数量显著减少。ATR2 siRNA转染不影响AMSCs向ECs的迁移和分化。无论有无MMP-2或MMP-14 siRNA刺激AMSCs进行EC分化，均导致p-ERK显著增加，p-JNK显著减少。与EBM组相比，所有三组中的p-p38均无显著变化。在EGM、EGM + MMP-2 siRNA和EGM + MMP-14 siRNA组中，抑制ERK导致EC标志物的表达显著降低。VEGFR2激酶抑制剂诱导了ERK的剂量依赖性抑制。
ERK信号通路对于VEGF-A/VEGFR2诱导的AMSCs向ECs分化至关重要。这些发现为ERK信号通路在AMSCs向ECs分化中的作用提供了新的见解，有望用于心血管疾病的潜在临床应用。
