Novel roles of kallistatin in vascular remodeling and tumor angiogenesis

Kallistatin is a specific tissue kallikrein inhibitor and a unique serpin. It is expressed in endothelial and smooth muscle cells of blood vessels. The primary focus of this study was to elucidate the role of kallistatin in vascular cell regulation and in vascular diseases. We showed that kallistatin markedly increased the proliferation and migration of primary vascular smooth muscle cells (VSMC). A role of kallistatin in VSMC growth in vivo was supported by the finding that expression of rat kallistatin markedly increased in the balloon-injured vessels, and local delivery of rat kallistatin antisense cDNA significantly reduced neointima formation in rat artery after balloon angioplasty. Furthermore, adenovirus-mediated transfer of rat kallistatin antisense cDNA into VSMC inhibited both PDGF-induced cell proliferation and p42/44 MAPK activation. These results indicate that kallistatin stimulates neointima hyperplasia in injured vessels mediated via the PDGF-induced MAPK signaling pathway. In contrast, our results showed that native kallistatin, recombinant wild-type kallistatin, and kallistatin mutant lacking the inhibitory activity towards tissue kallikrein inhibited VEGF- and bFGF-induced proliferation and growth of human microvascular endothelial cells (HMVEC). Kallistatin also significantly inhibited VEGF- and bFGF-induced migration and adhesion of endothelial cells. Adenovirus mediated kallistatin gene delivery reduced regional blood flow recovery caused by spontaneous angiogenesis, and attenuated capillary formation in the ischemic hindlimb. In addition, native kallistatin inhibited new blood vessel formation in the implanted Matrigel plug in mice as identified by reduced hemoglobin levels and Masson's trichrome staining. Furthermore, kallistatin gene delivery inhibited tumor growth in the athymic mice preimplanted with human breast carcinoma cells. The inhibitory effect of kallistatin on tumor angiogenesis was verified by staining with CD31 antibody and Masson's trichrome. To elucidate the mechanism of kallistatin in regulating angiogenesis, we showed that recombinant wildtype kallistatin suppressed the binding of 125I-labeled VEGF to HMVEC, whereas neither a1-antitrypsin (a serpin without heparin-binding domain) nor a kallistatin mutant lacking heparin-binding activity interfered with VEGF binding. Consequently, kallistatin suppressed VEGF-induced phosphorylation of Akt in HMVEC. These results suggest that kallistatin inhibits angiogenesis independent of its kallikrein inhibitory activity, but dependent on its heparin-binding domain. This study demonstrated that kallistatin plays important roles in vascular remodeling and tumor angiogenesis.