Human Tissue Kallikrein Gene Transfer Accelerates Spontaneous Angiogenesis Response to Ischemia
Angiogenesis therapy is emerging as a new approach to rescue ischemic tissues not susceptible to benefit from revascularization. We investigated (1) if ischemia modulates the expression of kallikrein-kinin system (KKS) and (2) if potentiation of kinin generation by human tissue kallikrein (HK) gene transfer augments spontaneous angiogenesis and tissue perfusion in a mouse model of ischemia. Hindlimb ischemia, caused by surgical excision of femoral artery, increased capillary density (from 344±15 to 641±54 cap/mm2 at 21 d after surgery, P<.001) and expression of kinin B1 receptor gene in the adductor muscle. Chronic blockade of B1 receptors blunted the spontaneous angiogenic response to ischemia (P<.01), while B2 antagonism was ineffective. Seven days after surgery, 3.6x108 p.f.u. of adenovirus containing the HK gene under control of cytomegalovirus enhancer/promoter sequence (Ad.CMV-cHK) or the reporter gene (Ad.CMV-LacZ) was injected in the ischemic adductor. Transduction of injected muscle was demonstrated at mRNA and protein level. HK trasgene expression peaked between 3 and 7 days and was associated with increased concentration of kinins, cAMP, and cGMP. Immunoistochemical identification of endothelial cell antigen Factor VIII combined with blinded morphometric evaluation of capillary density demonstrated potentiation of angiogenesis in HK-transduced muscle (969±32 versus 541±18 cap/mm2 in LacZ at 14 d, P<.001). HK-induced angiogenesis was limited to the site of injection and was associated with 40% increase in perfusion to ischemic limb (as measured by the use of laser Doppler flowmetry or radioactive microsphere) and preserved muscular energetic charge (88±2 vs 66±4% in LacZ, P<.001). Chronic blockade of kinin B1 or B2 receptors prevented HK-induced angiogenesis. We conclude that: (1) kinin receptors are differentialy modulated by ischemia, (2) B1 receptor is implicated in spontaneous angiogenic response to ischemia, and (3) potentiation of kinin generation by HK accelerates post-ischemic recovery by stimulating angiogenesis. These results provide new insight into the role of the KKS in vascular medicine for therapeutic benefit.