Abstract 3593: Wild Type Human Tissue Kallikrein, but Not its Common Polymorphic Variant R53H, Promotes Arteriogenesis in Normoperfused and Ischemic Rodent Models.
Therapeutic angiogenesis via administration of recombinant growth factors or vector-mediated overexpression has been largely unsuccessful in part due to the inadequacy of a single growth factor to induce all aspects of vascular growth. We have previously shown that human tissue kallikrein (hK1) promotes angiogenesis by kinin-mediated activation of the Akt-eNOS pathway, independently of VEGF. To further characterise the cellular mechanisms of hK1 induced neovascularisation, we delivered adenovirus encoding the wild type kallikrein gene (Ad.KLK1, 5x106 or 5x105 pfu) or a polymorphic variant (Ad.R53H-KLK1), which is 50% less potent as a kinin generating enzyme, or control virus (Ad.eGFP) in the rat mesenteric assay, a non-ischemic model of angiogenesis. In separate experiments, Ad.KLK1 was injected into the mesentery of rats given the bradykinin-2-receptor (B2R) antagonist, Icatibant (Subc 300nmol/kg/day). Intravital and confocal imaging was used to examine vessel morphology and histology. Ad.KLK1 increased the functional vessel area (445±76 vs 32±4% in Ad.eGFP, p<0.01), conduit vessel density (193±12 vs 63±7mm−2, p<0.001) and average vessel diameter (15±1 vs 12.6±1μm, p<0.001). Ad.KLK1-induced neovasculature showed an increased pericyte (58±5% vs 31±5, p<0.05) and smooth muscle cell coverage (4±1 vs 0%, p<0.001). In addition, we found that B2R co-localises with pericytes in the mesentery. Following Icatibant, Ad.KLK1 stimulated the formation of a network of small-size vessels, without pericyte or smooth muscle cell coverage and surrounded by spots of micro-hemorrhaging. Ad.R53H-KLK1 was less efficient than Ad.KLK1 in promoting angiogenesis and recruiting vascular smooth muscle cells. In a model of limb ischemia, disruption of the kallikrein gene resulted in dysfunctional reparative arteriogenesis and delayed hemodynamic recovery, which were both corrected by local Ad.KLK1 but not by Ad.R53H-KLK1. These data provide a mechanistic explanation for the robust arteriogenic effect of Ad.KLK1, indicating a key role for the kinin B2R. We also demonstrate for the first time that a single nucleotide polymorphism in the KLK1 gene, present in 5–7% of the Caucasian population, leads to a reduced neovascularisation in-vivo. All data mean±SEM, n=6