An adenovirus-mediated gene-transfer model of angiogenesis in rat mesentery

Objective: To develop an adenovirus-mediated angiogenesis model, dependent on VEGF, in a system amenable to functional characterization. Methods: Adenovirus (AdV) expressing VEGF (Ad-VEGF) or GFP (Ad-EGFP) (1-3.3 x 10(8) TCID50/mL), and Monastral blue were injected into the fat pad on either side of a mesenteric connective tissue panel of halothane-anesthetized male Wistar rats after intravital microscopic imaging. The intestine was replaced in the animal, the laparotomy was sutured, and the animal was allowed to recover. Six days later, the same connective tissue panel was identified from the Monastral blue depot and the mesentery was imaged as before, and then excised, fixed, and stained for endothelial cells (GSL isolectin-1B4), proliferating cells, VEGF, VEGF-R2, and actin. The increase in fractional microvascular area (FVA) was measured, and proliferating cell density, sprout density, vessel branch point density, vessel density, and mean vessel length were determined. Results: AdVEGF injection significantly increased the fractional vessel area (2.9 +/- 0.4-fold), proliferating endothelial cell density (1.7 +/- 0.1-fold), sprout density (3.1 +/- 0.3-fold), branch point density (1.9 +/- 0.1-fold), and the microvessel density (1.4 +/- 0.1-fold), and decreased the mean vessel length (0.69 +/- 0.04-fold). VEGF-R2 staining was evident near sprouting tips of endothelial cells, but not on the tip itself, and evidence for arteriogenesis was observed as well as clear evidence of angiogenesis. Conclusions: Ad-VEGF injection into the fat pad of the rat induced significant angiogenesis in the mesentery. This two-dimensional model of VEGF-induced angiogenesis is amenable to physiological, biochemical, and molecular assessment and may be a useful tool to help understand mechanisms of angiogenesis.