Recombinant Adeno-Associated Virus-Based Gene Transfer of Cathelicidin Induces Therapeutic Neovascularization Preferentially via Potent Collateral Growth

Therapeutic neovascularization is a concept well validated in animal models, however, without clear-cut success in clinical studies. To achieve prolonged transgene expression, recombinant adeno-associated virus (rAAV) was used in a chronic ischemic hind-limb model and the human antimicrobial peptide cathelicidin (LL-37/hCAP-18) was used as proangiogenic factor. Seven days after femoral artery excision, 0.5 x 10 11 rAAV particles encoding for green fluorescent protein ( rAAV. GFP), cathelicidin ( rAAV. cath), or vascular endothelial growth factor A ( rAAV. VEGF-A) were retroinfused into the anterior tibial vein of rabbits (n = 5 per group). In addition, one rAAV. cath-treated group obtained a constant infusion with the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin into the ischemic tissue starting on day 7. On day 7 and day 35 angiography of both hind limbs was performed for collateral quantification and frame count score (cinedensitometry). Capillary-to-muscle fiber ratios were obtained on day 35. Compared with controls, application of rAAV. cath induced a gain of perfusion ( 153 +/- 12 vs. 107+9% of day 7 controls) via increased collateral growth ( length index, 161 +/- 14 vs. 97 +/- 9%, controls), but no significant capillary growth (1.16 +/- 0.09 vs. 0.99 +/- 0.08, controls). Wortmannin application completely abolished the effects of rAAV. cath, indicating the involvement of the PI3K signal pathway. In conclusion, rAAV-mediated cathelicidin expression is capable of inducing functionally relevant neovascularization, preferentially by collateral growth. The rAAV-based vectors as long-expressing vector expression systems and cathelicidin as proangiogenic factor provide a promising new combination in the treatment of peripheral artery disease.