Tissue kallikrein and kinin infusion promotes neovascularization in limb ischemia

Adenovirus-mediated kallikrein delivery has been shown to promote blood vessel growth in the limb under both ischemic and normoperfused conditions. Here we investigated whether a continuous supply of kallikrein and kinin peptide can induce neovascularization in a rat model of hindlimb ischemia. Rats underwent femoral artery ligation and localized injection of tissue kallikrein, bradykinin or B1 receptor agonist, followed by infusion of proteins by osmotic minipump. Regional blood flow was monitored weekly by laser Doppler perfusion imaging. Three weeks after surgery, rats receiving kallikrein and kinins showed a significant increase in the perfusion ratio of ischemic vs. normoperfused limb compared to control rats. Similarly, a microsphere assay showed that kallikrein and kinins significantly increased regional blood flow without altering blood pressure. Moreover, kallikrein and kinins significantly augmented capillary and arteriole densities, as quantified by immunostaining with CD-31 and smooth muscle a-actin. Both tissue kallikrein and bradykinin increased hemoglobin content in Matrigel implants in mice, providing further evidence of the angiogenic properties. Kinins, when delivered subcutaneously via Matrigel in rats, also increased regional perfusion. This is the first demonstration that local application of tissue kallikrein protein or kinin peptide has therapeutic value in the treatment of ischemic disease by promoting neovascularization.