Glucagon like peptide-2 induces intestinal restitution through VEGF release from subepithelial myofibroblasts

Abstract

Glucagon like peptide-2 (GLP-2) exerts intestinotrophic actions, but the underlying mechanisms are still a matter of debate. Recent studies demonstrated the expression of the GLP-2 receptor on fibroblasts located in the subepithelial tissue, where it might induce the release of growth factors such as keratinocyte growth factor (KGF) or vascular endothelial growth factor (VEGF). Therefore, in the present studies we sought to elucidate the downstream mechanisms involved in improved intestinal adaptation by GLP-2. Human colonic fibroblasts (CCD-18Co), human colonic cancer cells (Caco-2 cells) and rat ileum IEC-18 cells were used. GLP-2 receptor mRNA expression was determined using real time RT-PCR. Conditioned media from CCD-18Co cells were obtained following incubation with GLP-2 (50–250 nM) for 24 h. Cell viability was assessed by a 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT)-assay, and wound healing was determined with an established migration-assay. Transforming Growth Factor beta (TGF-β), VEGF and KGF mRNA levels were determined by RT-PCR. Protein levels of VEGF and TGF-β in CCD-18Co cells following GLP-2 stimulation were determined using ELISA. Neutralizing TGF-β and VEGF-A antibodies were utilized to assess the role of TGF-β and VEGF-A in the process of wound healing. GLP-2 receptor expression was detected in CCD-18Co cells. Conditioned media from CCD-18Co cells dose-dependently induced proliferation in Caco-2 cells, but not in IEC-18 cells. Conditioned media also enhanced cell migration in IEC-18 cells ( P < 0.01), while migration was even inhibited in Caco-2 cells ( P < 0.0012). GLP-2 significantly stimulated mRNA expression of VEGF and TGF-β, but not of KGF in CCD-18Co. The migratory effects of GLP-2 were completely abolished in the presence of TGF-β and VEGF-A antibodies. GLP-2 exerts differential effects on the epithelium of the small intestine and the colon. Thus, in small intestinal cells GLP-2 stimulates wound repair, whereas no such effects were observed in colonic cells. The mechanisms underlying GLP-2 induced intestinal wound repair seem to involve the secretion of VEGF and, subsequently, TGF-β from subepithelial fibroblasts, whereas KGF appeared to be less important.