Park J, Ha H, Ahn H J, Kang S-W, Kim Y S, Seo J Y, Kim M S
The Research Institute for Transplantation, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-Ku, Seoul 120-752, South Korea.
Transplant Proc. 2005 Oct;37(8):3459-62. doi: 10.1016/j.transproceed.2005.09.066.
Vascular smooth muscle cell (VSMC) proliferation and extracellular matrix (ECM) accumulation play key roles in the development and the progression of vascular remodeling such as transplant arteriosclerosis and restenosis. The present study examined the effects of sirolimus (SRL) on platelet-derived growth factor (PDGF)-induced fibronectin secretion, collagen synthesis, and the related signaling pathways including reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPK) in rat VSMCs. Primary rat VSMCs were isolated from male Sprague-Dawley rats. Growth arrested, synchronized cells were treated with various concentrations of SRL before the addition of PDGF at 10 ng/mL. Proliferating cell nuclear antigen expression, fibronectin secretion, and the activation of extracellular signal-regulated protein kinase (ERK) and p38 MAPK were assessed by Western blot analysis, collagen synthesis by [(3)H]-proline incorporation, and cellular ROS by flow cytometry. PDGF (10 ng/mL) increased VSMC proliferation by 1.7-fold, fibronectin secretion by 1.5-fold, collagen synthesis by 2.1-fold, cellular ROS by 1.6-fold, and activation of ERK and p38 MAPK by 3.3- and 3.9-fold compared to controls. SRL above 1 nmol/L inhibited PDGF-induced VSMC proliferation and collagen synthesis but not PDGF-induced fibronectin secretion, cellular ROS, and activation of ERK and p38 MAPK. These data demonstrated that PDGF increased ECM synthesis as well as proliferation through cellular ROS and subsequent MAPK activation and that SRL inhibited PDGF-induced VSMC proliferation and collagen synthesis in a cellular ROS- and MAPK activation-independent way.
血管平滑肌细胞（VSMC）增殖和细胞外基质（ECM）积累在诸如移植动脉硬化和再狭窄等血管重塑的发生和发展过程中起着关键作用。本研究检测了西罗莫司（SRL）对血小板衍生生长因子（PDGF）诱导的大鼠VSMC中纤连蛋白分泌、胶原蛋白合成以及包括活性氧（ROS）和丝裂原活化蛋白激酶（MAPK）在内的相关信号通路的影响。从雄性Sprague-Dawley大鼠中分离出原代大鼠VSMC。在添加10 ng/mL PDGF之前，用不同浓度的SRL处理生长停滞、同步化的细胞。通过蛋白质免疫印迹分析评估增殖细胞核抗原表达、纤连蛋白分泌以及细胞外信号调节蛋白激酶（ERK）和p38 MAPK的激活，通过[（3）H]-脯氨酸掺入法评估胶原蛋白合成，通过流式细胞术评估细胞ROS。与对照组相比，PDGF（10 ng/mL）使VSMC增殖增加1.7倍，纤连蛋白分泌增加1.5倍，胶原蛋白合成增加2.1倍，细胞ROS增加1.6倍，ERK和p38 MAPK的激活增加3.3倍和3.9倍。1 nmol/L以上的SRL抑制PDGF诱导的VSMC增殖和胶原蛋白合成，但不抑制PDGF诱导的纤连蛋白分泌、细胞ROS以及ERK和p38 MAPK的激活。这些数据表明，PDGF通过细胞ROS以及随后的MAPK激活增加ECM合成和增殖，并且SRL以不依赖细胞ROS和MAPK激活的方式抑制PDGF诱导的VSMC增殖和胶原蛋白合成。
