Ephrin B2/EphB4 pathway in hepatic stellate cells stimulates Erk-dependent VEGF production and sinusoidal endothelial cell recruitment

Abstract

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.

Endocytosis of collagen by hepatic stellate cells regulates extracellular matrix dynamics

“…5300). These cells have been well characterized and used widely in prior publications (10,32,46). Wild-type mouse embryonic fibroblast (MEF) cells, MEF cells isolated form c-abl and Arg doubleknockout mice (c-abl Ϫ/Ϫ MEF), and MEF cells isolated from Yes, Src, and Fyn knockout mice (YSF Ϫ/Ϫ MEF) were kindly provided by Drs.…”

Coculture of Stem Cells from Apical Papilla and Human Umbilical Vein Endothelial Cell Under Hypoxia Increases the Formation of Three-Dimensional Vessel-Like Structuresin Vitro

“…Das et al 43 indicated that stimulation of human hepatic stellate cells with either ephrinB2 Fc or EphB4 Fc significantly increases VEGF mRNA levels, whereas small interfering RNAs for ephrinB2 and EphB4 inhibit this increase. They further observed that ephrinB2 stimulates phosphorylation of extracellular signalregulated kinases, which are key cell survival and proliferation proteins, to regulate VEGF-A expression through HIF-1 phosphorylation.…”

“…These findings indicate that VEGF might play a critical role in tissue angiogenesis under hypoxia as it does normally, but the lack of corresponding secretion of ephrinB2 protein will influence permanent angiogenesis of implanted tissues under hypoxia. 43 Therefore, we should focus on strategies that increase the protein expression of ephrinB2 in SCAP under hypoxia, and further explore the correlation between VEGF and ephrinB2 expression.…”

Acquired Lower Extremity Arteriovenous Malformation: A Rare Case in an Adult with End-Stage Alcoholic Liver Cirrhosis

“…Prior research has described the role of Ephrin-B2 and EphB4 in delineating arterial and venous boundaries in developing tissue. Their expression has been implicated in mediating angiogenesis and hepatic vascular structure formation during cirrhosis [19][20][21][22][23]. This may be an interesting avenue of future research for extremity vascular malformations.…”