Vascular endothelial growth factor mediated angiogenic potential of pancreatic ductal carcinomas enhanced by hypoxia: An in vitro and in vivo study

Angiogenesis in pancreatic ductal adenocarcinomas depends on the presence of angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and is thought to be stimulated by hypoxia. We tested the angiogenic potential of 9 cell lines of pancreatic ductal carcinoma origin by screening mRNA and protein expression of VEGF and bFGF and the release of VEGF into culture medium under normoxic and hypoxic (5% or 0.2% O₂) conditions. Angiogenic activity was determined using 2- and 3-D endothelial cell assays. Furthermore, VEGF expression and tumor vascularization were studied in human pancreatic carcinoma tissues from orthotopic xenografts and resection specimens. All cell lines expressed (mRNA, protein) and secreted VEGF, whereas bFGF was only found in 3 cell lines and was secreted into the medium in low concentrations. In addition to the dominant isoforms VEGF₁₂₁,VEGF₁₆₅ and VEGF₁₈₉, 2 isoforms described recently, VEGF₁₄₅ and VEGF₁₈₃, were detected. Severe hypoxia (0.2% O₂), but not moderate hypoxia (5% O₂) raised VEGF mRNA expression and protein secretion in 7/9 and 5/9 cell lines, respectively. Conditioned media from 7/9, 6/9, 8/9 and 7/9 cell lines stimulated endothelial cell proliferation under normoxic (24 and 48 hr) or hypoxic (24 hr, 0.2% and 48 hr 5% O₂) conditions, respectively. Conditioned media from 4/9 cell lines also induced capillary-like sprouting under normoxic conditions and from 6/9 under hypoxic (0.2% O₂) conditions. In xenografted carcinoma tissues microvessel density was found not to be increased around areas of ischemic necrosis. In resected ductal carcinomas showing tumor necrosis VEGF expression and microvessel density were only increased in 3/12 and 2/13 cases, respectively. In conclusion, in vitro most pancreatic ductal carcinomas show a distinct VEGF related angiogenic potential, as demonstrated by 2- and 3-D endothelial cell proliferation, which may be promoted by severe hypoxia. Surprisingly, perinecrotic tumor areas, which are supposed to be hypoxic, only rarely showed the expected increase in microvessel density and VEGF expression.