Pan Sunlei, Lin Hui, Luo Hangqi, Gao Feidan, Meng Liping, Zhou Changzuan, Jiang Chengjian, Guo Yan, Ji Zheng, Chi Jufang, Guo Hangyuan
The First Clinical Medical College, Wenzhou Medical University Wenzhou 325000, Zhejiang, China.
Department of Cardiology, Shaoxing People's Hospital, Shaoxing Hospital of Zhejiang University Shaoxing 312000, Zhejiang, China.
Am J Transl Res. 2017 Mar 15;9(3):1307-1316. eCollection 2017.
Folic acid (FA) supplementation reduces the risk of atherosclerosis and stroke. Phenotypic change from differentiated to dedifferentiated vascular smooth muscle cells (VSMCs) plays an important role in atherosclerosis development; however, the exact mechanisms remain unknown. This study aimed to assess whether FA through mammalian target of rapamycin (mTOR)/P70S6K signaling inhibits platelet derived growth factor (PDGF-BB) induced VSMC dedifferentiation.
VSMCs from primary cultures were identified by morphological observation and α-smooth muscle actin (α-SM-actin, α-SMA) immunocytochemistry. Then, VSMCs were induced by PDGF-BB and treated with varying FA concentrations. Rapamycin and MHY-1485 were used to inhibit or activate the mTOR/P70S6K pathway, respectively. Next, MTT, Transwell, and wound healing assays were employed to assess proliferation and migration of VSMCs. In addition, Western blotting was used to evaluate protein levels of α-SMA, calponin, osteopontin, mTOR, p-mTOR, P70S6K and p-P70S6K in VSMCs.
VSMCs showed phenotypic alteration from differentiated to dedifferentiated cells in response to PDGF-BB. MTT, Transwell and wound healing assays showed that FA markedly inhibited proliferation and migration in PDGF-BB-induced VSMCs, in a time and concentration-dependent manner. FA treatment increased the expression levels of the contractile phenotype marker proteins α-SMA and calponin compared with VSMCs stimulated by PDGF-BB alone. Furthermore, FA significantly suppressed mTOR and P70S6K phosphorylation compared with PDGF-BB alone. Similar to FA, downregulation of mTOR signaling by rapamycin inhibited VSMC dedifferentiation. In contrast, upregulation of mTOR signaling by MHY-1485 reversed the FA-induced inhibition of VSMC dedifferentiation.
Folic acid inhibits dedifferentiation of PDGF-BB-induced VSMCs by suppressing mTOR/P70S6K signaling.
补充叶酸（FA）可降低动脉粥样硬化和中风的风险。血管平滑肌细胞（VSMC）从分化型向去分化型的表型变化在动脉粥样硬化发展中起重要作用；然而，确切机制仍不清楚。本研究旨在评估FA是否通过雷帕霉素靶蛋白（mTOR）/P70S6K信号通路抑制血小板衍生生长因子（PDGF-BB）诱导的VSMC去分化。
通过形态学观察和α-平滑肌肌动蛋白（α-SM-肌动蛋白，α-SMA）免疫细胞化学鉴定原代培养的VSMC。然后，用PDGF-BB诱导VSMC，并给予不同浓度的FA处理。分别用雷帕霉素和MHY-1485抑制或激活mTOR/P70S6K通路。接下来，采用MTT、Transwell和伤口愈合试验评估VSMC的增殖和迁移。此外，用蛋白质免疫印迹法评估VSMC中α-SMA、钙调蛋白、骨桥蛋白、mTOR、磷酸化mTOR、P70S6K和磷酸化P70S6K的蛋白水平。
VSMC在PDGF-BB作用下表现出从分化型细胞向去分化型细胞的表型改变。MTT、Transwell和伤口愈合试验表明，FA以时间和浓度依赖性方式显著抑制PDGF-BB诱导的VSMC增殖和迁移。与单独用PDGF-BB刺激的VSMC相比，FA处理增加了收缩型表型标记蛋白α-SMA和钙调蛋白的表达水平。此外，与单独用PDGF-BB相比，FA显著抑制mTOR和P70S6K磷酸化。与FA相似，雷帕霉素下调mTOR信号通路可抑制VSMC去分化。相反，MHY-1485上调mTOR信号通路可逆转FA诱导的VSMC去分化抑制。
叶酸通过抑制mTOR/P70S6K信号通路抑制PDGF-BB诱导的VSMC去分化。
