Gastroenterology Research Unit, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905, USA.
Am J Physiol Gastrointest Liver Physiol. 2010 Jun;298(6):G908-15. doi: 10.1152/ajpgi.00510.2009. Epub 2010 Mar 25.
Chemotaxis signals between hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC) maintain hepatic vascular homeostasis and integrity and also regulate changes in sinusoidal structure in response to liver injury. Our prior studies have demonstrated that the bidirectional chemotactic signaling molecules EphrinB2 and EphB4 are expressed in HSC. The aim of our present study was to explore whether and how the EphrinB2/EphB4 system in HSC could promote SEC recruitment, which is essential for sinusoidal structure and remodeling. Stimulation of human HSC (hHSC) with chimeric agonists (2 microg/ml) of either EphrinB2 or EphB4 (EphrinB2 Fc or EphB4 Fc, respectively) significantly increased VEGF mRNA levels in hHSC as assessed by quantitative PCR, with respective small interfering RNAs for EphrinB2 and EphB4 inhibiting this increase (P < 0.05, n = 3). EphrinB2 agonist-induced increase in VEGF mRNA levels in hHSC was associated with increased phosphorylation of Erk and was significantly blocked by U0126 (20 microM), an inhibitor of MEK, which is a kinase upstream from Erk (P < 0.05, n = 3). The EphB4 agonist also significantly increased human VEGF promoter activity (P < 0.05, n = 3) as assessed by promoter reporter luciferase assay in transfected LX2-HSC. This was associated with upregulation of the vasculoprotective transcription factor, Kruppel-like factor 2 (KLF2). In Boyden chamber assays, conditioned media from hHSC stimulated with agonists of EphrinB2 or EphB4 increased SEC chemotaxis in a VEGF-dependent manner, compared with control groups that included basal media with agonists of EphrinB2, EphB4, or HSC-conditioned media from HSC in absence of agonist stimulation (P < 0.05, n = 3). EphB4 expression was detected in situ within liver sinusoidal vessels of rats after carbon tetrachloride-induced liver injury. In summary, activation of the EphrinB2/EphB4 signaling pathway in HSC promotes chemotaxis of SEC through a pathway that involves Erk, KLF2, and VEGF. These studies identify EphrinB2 or EphB4 as a key intermediary that links HSC signal transduction pathways with angiogenesis and sinusoidal remodeling.
肝星状细胞（HSC）和窦内皮细胞（SEC）之间的趋化信号维持着肝血管的稳态和完整性，并调节肝损伤时窦状结构的变化。我们之前的研究表明，双向趋化信号分子 EphrinB2 和 EphB4 在 HSC 中表达。本研究旨在探讨 HSC 中的 EphrinB2/EphB4 系统是否以及如何促进 SEC 的募集，这对于窦状结构和重塑是必不可少的。用 EphrinB2 或 EphB4 的嵌合激动剂（2μg/ml）刺激人 HSC（hHSC），通过定量 PCR 显著增加 hHSC 中的 VEGF mRNA 水平，分别用 EphrinB2 和 EphB4 的小干扰 RNA 抑制这种增加（P <0.05，n=3）。 EphrinB2 激动剂诱导 hHSC 中 VEGF mRNA 水平的增加与 Erk 的磷酸化增加有关，并用 MEK 的抑制剂 U0126（20μM）显著阻断，MEK 是 Erk 的上游激酶（P <0.05，n=3）。 EphB4 激动剂也显著增加了转染 LX2-HSC 的人 VEGF 启动子活性（P <0.05，n=3），通过转染的 LX2-HSC 的启动子报告荧光素酶测定。这与血管保护转录因子，Kruppel 样因子 2（KLF2）的上调有关。在 Boyden 室测定中，用 EphrinB2 或 EphB4 的激动剂刺激的 hHSC 的条件培养基以 VEG 依赖性方式增加 SEC 的趋化性，与包括 EphrinB2、EphB4 的基础培养基的对照组或没有激动剂刺激的 HSC 条件培养基的对照组相比（P <0.05，n=3）。在四氯化碳诱导的肝损伤后，在大鼠肝窦血管内原位检测到 EphB4 的表达。总之，HSC 中 EphrinB2/EphB4 信号通路的激活通过涉及 Erk、KLF2 和 VEGF 的途径促进 SEC 的趋化性。这些研究确定 EphrinB2 或 EphB4 是连接 HSC 信号转导途径与血管生成和窦状重塑的关键介质。
