Department of Gastroenterology Surgery, Affiliated Shengjing Hospital, Medical University of China, 36 Sanhao Street, Heping District, Shenyang, China.
Mol Cell Biochem. 2012 Jan;359(1-2):217-23. doi: 10.1007/s11010-011-1016-x. Epub 2011 Aug 24.
Vascular endothelial growth factor (VEGF) is one of the major cytokines secreted by activated hepatic stellate cells (HSCs). VEGF is involved in hepatic angiogenesis and plays an important role in the development of liver fibrosis. TNP-470, an angiogenic inhibitor, attenuates the development of rat liver fibrosis with reduced angiogenesis, as demonstrated in our previous study. HSCs were prepared from specific pathogen-free Wister rat livers. The isolated HSCs were activated and stimulated with platelet-derived growth factor BB (PDGF-BB) or prostaglandin E2 with or without pretreatment with MAPK cascade inhibitors (PD98059, which inhibits MEK activation), SB203580 (a selective pharmacologic inhibitor of p38 MAPK), and SP600125 (a selective inhibitor of the c-Jun N-terminal kinase, JNK). VEGF production and those of related molecules were assayed at the protein and mRNA levels by immunostaining, western blot analysis, and real-time quantitative PCR. The activated HSCs produced more VEGF than the quiescent ones. Those that received PDGF-BB stimulation showed enhanced cyclooxygenase-2 (COX-2) expression and activation of phosphor-mitogen-activated protein kinase (MAPK) p44/p42. Pretreatment with PD98059 significantly inhibited COX-2 expression and VEGF production within the PDGF-activated HSCs, but the effect was nullified by exogenous prostaglandin E2. pJNK and p38 inhibitors do not show similar inhibitory effects on VEGF and COX-2 expression, and pJNK and p38 MAPK signals are not involved in the COX-2/MAPK signaling cascade. VEGF production in PDGF-stimulated HSCs is dependent on the overexpression of COX-2 protein via the phospho-p42/44 MAP kinase activation, based on PD98059 inhibition.
血管内皮生长因子 (VEGF) 是活化的肝星状细胞 (HSCs) 分泌的主要细胞因子之一。VEGF 参与肝血管生成，在肝纤维化的发展中发挥重要作用。在我们之前的研究中，血管生成抑制剂 TNP-470 通过减少血管生成来减轻大鼠肝纤维化的发展。HSCs 是从特定病原体无 Wister 大鼠肝脏中分离出来的。分离的 HSCs 被血小板衍生生长因子 BB (PDGF-BB) 或前列腺素 E2 激活和刺激，或用 MAPK 级联抑制剂（PD98059，抑制 MEK 激活）、SB203580（p38 MAPK 的选择性药理抑制剂）和 SP600125（c-Jun N-末端激酶，JNK 的选择性抑制剂）预处理。通过免疫染色、western blot 分析和实时定量 PCR 在蛋白质和 mRNA 水平上检测 VEGF 及其相关分子的产生。与静止状态的 HSCs 相比，激活的 HSCs 产生更多的 VEGF。接受 PDGF-BB 刺激的 HSCs 表现出增强的环氧化酶-2 (COX-2) 表达和丝裂原活化蛋白激酶 (MAPK) p44/p42 的磷酸化。PD98059 预处理可显著抑制 PDGF 激活的 HSCs 中的 COX-2 表达和 VEGF 产生，但外源性前列腺素 E2 可使该作用无效。pJNK 和 p38 抑制剂对 VEGF 和 COX-2 表达没有类似的抑制作用，并且 pJNK 和 p38 MAPK 信号不参与 COX-2/MAPK 信号级联。基于 PD98059 的抑制作用，PDGF 刺激的 HSCs 中的 VEGF 产生依赖于 COX-2 蛋白的过表达，通过磷酸化 p42/44 MAP 激酶激活。
