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Das A, Shergill U, Thakur L, Sinha S, Urrutia R, Mukhopadhyay D, Shah VH: EphrinB2/EphB4 pathway in hepatic stellate cells stimulates ERK dependent VEGF production and sinusoidal endothelial cell recruitment. Am J Physiol Gastrointest Liver Physiol. 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 bi-directional chemotactic signaling molecules EphrinB2 and EphB4 are expressed in HSC. The Aim of our present study was to explore if 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 significantly increased VEGF mRNA levels in hHSC as assessed by qPCR, with respective siRNAs 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 muM), 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, KLF2. In Boyden chamber assays, conditioned media from hHSC stimulated with agonists of EphrinB2 or EphB4 increased SEC chemotaxis, compared to 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/EphB4 as a key intermediary that links HSC signal transduction pathways with angiogenesis and sinusoidal remodeling. |
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