HYPOXIA INDUCES VASCULAR ENDOTHELIAL GROWTH-FACTOR GENE AND PROTEIN EXPRESSION IN CULTURED RAT ISLET CELLS

The formation of new microvasculature by capillary sprouting at the si te of islet transplantation is crucial for the long-term survival and function of the graft. Vascular endothelial growth factor (VEGF), an e ndothelial cell-specific mitogen with potent angiogenic and vascular p ermeability-inducing properties, may be a key factor in modulating the revascularization of islets after transplantation. In this study, me examined the gene expression of VEGF mRNA in three tumor cell Lines an d in isolated whole and dispersed rat islets in vitro by Northern blot hybridization in normoxic (5% CO2, 95% humidified air) and hypoxic (1 % O-2, 5% CO2, 94% N-2) culture conditions. Increased expression of VE GF mRNA was observed in beta-TC3, RAW 264.7, and IC-21 tumor cell line s when subjected to hypoxia. With isolated whole islets in normoxic cu lture, a threefold increase in VEGF mRNA (P < 0.001) was seen at 48 h as compared with freshly isolated islets. This response was similar to the 3.8-fold increase observed with islets subjected to hypoxia. Disp ersed rat islet cell clusters cultured on Matrigel for 24 h under hypo xic conditions showed a 3.4-fold increase (P < 0.01) in VEGF mRNA comp ared with those cultured in normoxia. This correlated with increased V EGF secretion as determined by enzyme-linked immunosorbent assay. Immu nohistochemical studies revealed the presence of increased expression of VEGF protein near the center of islets after 24 h of normoxic cultu re. Islet cell clusters on Matrigel showed intense cellular localizati on of VEGF in both beta-cells and non-beta-cens. These findings sugges t that rat islet cells, when subjected to hypoxia during the first few days after transplantation, may act as a major source of VEGF, thereb y initiating revascularization and maintaining the vascular permeabili ty of the grafted islets.