Vascular endothelial growth factor (VEGF) is the dominant growth factor in pathological and physiological angiogenesis. It promotes microvascular permeability, compliance and angiogenesis in vivo (Bates et al. 1999). To investigate the underlying physiological changes that occur during VEGF-mediated angiogenesis we have developed an angiogenesis model in a system amenable to functional characterisation. Adenovirus (AdV) expressing VEGF (Ad-VEGF) was produced by co-transformation of the vector pAdEasy-1 and pShuttle-CMV-VEGF₁₆₅ into BJ5183 bacteria and then transfection of the recombinant pAd-VEGF into HEK293 cells. AdV expressing β-gal (Ad-LacZ) and Ad-VEGF were purified by CsCl density gradient centrifugation, and dialysed against rat Ringer solution. Animal experiments conformed to Home Office guidelines. Male Wistar rats (300 g) were anaesthetised by halothane inhalation (5 %), the gut exposed after laparotomy, and the mesentery superfused with Ringer solution at 37 °C. A connective tissue panel with few blood vessels and no overt angiogenesis was imaged using a digital camera and light microscope. 50 µl of Ad-VEGF or Ad-LacZ of (1-3.3 X 10⁸ TCID₅₀ ml^-1) and Monastral Blue (0.6 %), diluted in rat Ringer solution, were injected into the fat pad adjacent to the panel. The mesentery was then replaced in the animal, the laparotomy sutured and the animal allowed to recover. Seven days later, the animal was anaesthetised, an incision made through the laparotomy scar, and the gut exposed as before. The same panel was identified from the Monastral Blue depot, and the tissue superfused with warm Ringer solution. The mesentery was imaged as before, and the rat killed by cervical dislocation. The degree of angiogenesis (angiogenesis index, AI) was calculated as the increase in area of vessels over 7 days (AI = 100 X (fractional vessel area on day 7 – fractional vessel area on day 1)/fractional vessel area on day 1), calculated using image analysis software (Openlab, Improvision). AdVEGF injection significantly increased AI (median ± interquartile range AI 434 ± 76 %, n = 6), compared with the control group (140 ± 39 %, n = 7, P < 0.01, Mann-Whitney U).

Immunofluorescence staining of the mesenteries with proliferating cells nuclear antigen (PCNA) showed dividing cells, confirming angiogenesis, rather than simply increased detection of vessels due to vasodilatation. These results demonstrated that AdV-mediated VEGF gene transfer was effective in inducing neovascularisation in a quantitative rat mesenteric model of angiogenesis. Since rat mesenteric microvessels have previously been characterised molecularly (e.g. using cDNA array), cellularly and physiologically, this model will allow similar characterisation during angiogenesis.