Human Tissue Kallikrein Gene Transfer Induces Angiogenesis
Angiogenesis therapy is emerging as a new strategy for treatment of vascular disease, not susceptible of revascularization. As kinins are implicated in vascular protection and repair, we hypothesized that targeted potentiation of kinin-nitric oxide (NO) pathway may stimulate neo-vascularization. CD1 mice were IM injected with 3.6x108plaque forming units of adenovirus (Ad) harboring human tissue kallikrein (HK) or β-galactosidase (LacZ) gene under the control of cytomegalovirus enhancer/promoter (CMV). Successful transduction of injected adductor skeletal muscle was documented at mRNA and protein level. HK expression reached a peak between 3 and 7 d and then declined to become undetectable at 28 d. Contralateral muscles and liver were not transduced. Immunohistochemical identification of endothelial cell antigen factor VIII combined with blinded morphometric analysis of capillary density revealed a marked angiogenic effect in HK-transduced muscle (1013±18 vs 518±15 ncap/mm2 in LacZ-injected muscle at 28 d, P<.001), whereas no angiogenic effect was seen in ipsilateral gastrocnemious or contralateral muscles. HK increased muscular kinin (60±12 vs 20±6 pg/g tissue in controls at 3 d, P<.05), cAMP (1.02±0.06 vs 0.48±0.04 ng/mg prot, P<.01) and cGMP levels (99±5 vs 68±5 fmol/mg prot, P<.01). HK-induced angiogenic response was completely prevented by pharmacologic blockade of kinin B1 or B2 receptors, or inhibition of NO synthase, but not by cycloxygenase inhibition. Neovascularization was associated with a transient increase in muscular blood flow as determined by laser Doppler flowmetry. HK-induced neo-vascularization was stable and functionally utilitarian, as surgical induction of hindlimb ischemia 28 d after gene tranfer was followed by accelerated hemodynamic recovery of HK-injected muscle (perfusion ratio: 0.84±0.05 vs 0.68±0.03 in LacZ-injected muscle at 14 d, P<.05). We conclude that (a) HK acts as an angiogenic factor via kinin-mediated stimulation of NO release, (b) neo-vascularization can preserve tissue from supervening ischemia. The discovery that HK has angiogenic activity may represent an important advancement in vascular medicine for therapeutic benefit.