A Novel Method for the Generation of Bioartificial Vascular Grafts

Abstract

ionizing radiation (LDIR) induce angiogenesis in vivo by activating the receptor 2 of VEGF but, there is no evidence that it enhances neovascularization in vascular occlusive disease, our overall objective. Methods: We used a mouse model of hind-limb ischemia. In 22-wk-old C57BL/6 mice, the distal external iliac and femoral arteries and veins were ligated and excised. After that, the mice were irradiated or not with 0.3 Gy using a linear accelerator x-rays photon beam. The laser-Doppler perfusion imager (MoorLD2-V5.x, Moor Instruments Ltd, Axminster, UK) was used to assess limb perfusion. Blood flow was measured both in the ischemic and contralateral leg preoperatively, immediately postoperatively and at post-operative day 7 and day 15. Collateral vessels were evaluated by diaphanization. Capillaries were identified by CD31 immunohistochemistry and myocytes were counterstained with haematoxylin. In order to identify the molecular targets involved in the pro-angiogenic phenotype promoted by LDIR, endothelial cells from gastrocnemius muscle sections were isolated by a Laser Capture Microdissection microscope followed by RNA extraction, cDNA synthesis, pre-amplification and qRT-PCR analysis. Results: A significant increment in perfusion in the irradiated mice when compared to the unirradiated ones (p ¼ 0.00075) was found by laser Doppler at 15 days postischemia. Accordingly, a statistically significant increase in collateral development (p ¼ 0.0198) and in capillary density (p ¼ 0.00009) was noted in irradiated mice. Interestingly, in the absence of the ischemia injury, similar capillary/myocyte ratios were found in unirradiated and irradiated gastrocnemius muscles. From an in vitro microarray data obtained in our lab, we selected the genes whose expression is significantly altered by low doses of IR and that represent the best candidates for a pro-angiogenic response. Their expression was evaluated in endothelial cells isolated from the gastrocnemius muscles from irradiated and unirradiated mice by using a laser capture microdissection microscope followed by RNA extraction and cDNA synthesis. Several pro-angiogenic genes were pre-amplified before quantitative RT-PCR analyses. Our results suggest that low-dose IR induces simultaneously the expression of VERGR1, VEGFR2, FGF2, TGFB2, ANG2, PDGFC, HGF and c-met in endothelial cells isolated from ischemic muscles. Conclusion: The outcome of these experiments performed in a mice model suggests that LDIR may have clinical use in the treatment of lower limb vascular insufficiency.