Hepatic stellate cells produce vascular endothelial growth factor via phospho-p44/42 mitogen–activated protein kinase/cyclooxygenase-2 pathway

Abstract

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.