We have recently reported that two-chain high molecular weight kininogen (HKa) inhibits endothelial cell proliferation, induces endothelial cell apoptosis and inhibits angiogenesis. Recombinant high molecular weight kininogen domain 5 expresses similar activity, inhibiting endothelial cell proliferation by 50% (IC5(]) at a concentration of -60 nM. To further define the regions within kininogen domain 5 responsible for these effects, we prepared overlapping 16 amino acid peptides (each overlapping by 8 amino acids) encompassing kininogen domain 5 (aa 384-509), and measured their ability to inhibit endothelial cell proliferation and induce endothelial cell apoptosis. A similar strategy was used to assess potentially active regions within HK domain 3, which also contains regions that mediate binding of HK to endothelial cells. The most potent domain 5-derived peptides were found in the C-terminal region of the domain. H5-13 (KHGHGHGKHKNKGKKN; aa 480-495 of HK) inhibited endothelial cell proliferation by 86 ±13% at a concentration of 50 μM (IC!0 - 8 |iM), while H5-14 (HKNKGKKNGKHNGWKT; aa 488-503 of HK), used at the same concentration, inhibited proliferation by 93 ±7% (IC30 - 14 |iM). As observed with HKa and recombinant domain 5, the peptides caused endothelial cell apoptosis, and their activity was enhanced in the presence of Zn2. These results differ from a previous report, in which the domain 5 peptide most active in inhibiting endothelial cell proliferation was found to encompass amino acids 440-455 of HK. In addition, we also observed that two 16 amino acid peptides from HK domain 3 (H3-6, aa 267-282 of HK; H3-7, aa 275-290 of HK) also inhibited endothelial cell proliferation (ICJO - 1 nM) and induced apoptosis, though they were not freely soluble in aqueous buffers. Exogenous Zn2+ did not significantly affect the activity of the latter peptides. Neither the domain 3 or domain 5 peptides affected the proliferation of a number of other cell types, including primary cultures of fibroblasts and smooth muscle cells. These studies define active regions within HK domains 3 and 5 that inhibit endothelial cell proliferation and induce endothelial cell apoptosis. Current studies are focused on evaluating the antiangiogenic activity of these peptides in vitro.
|Original language||English (US)|
|Issue number||11 PART I|
|State||Published - Dec 1 2000|
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