Recent studies demonstrate that ghrelin can be an endogenous regulator of angiogenesis. We studied direct effects of human acylated (hAG) and unacylated (hUAG) ghrelin, as well as of rat acylated ghrelin (rAG) on the growth of HECa10 murine endothelial cells. Ghrelin was applied separately or together with D-Lys(3)-GHRP-6, which is commonly used as an antagonist of ghrelin receptor type 1a - GHS-R1a. The growth of HECa10 cells was assessed with Mosmann and in selected study conditions also with BrdU and TUNEL methods. Both hAG and hUAG (10(-5) M to 10(-12) M) inhibited the growth of HECa10 cells in 24h and 72 h cultures. Similarly, rAG decreased the growth of the cells after 24h (10(-7) M and 10(-11) M), and after 72 h (10(-7) M, 10(-8) M and 10(-11) M). Unexpectedly, D-Lys(3)-GHRP-6 itself also inhibited the growth of these cells at 10(-4) to 10(-6) M in 24h, 48 h (dose-response effect) and 72 h cultures. D-Lys(3)-GHRP-6 did not modify the inhibitory effect of rAG. However, D-Lys(3)-GHRP-6 at the concentration of 10(-4) M diminished, abolished or even reversed the inhibitory effect of hUAG in 72 h culture and this was dependent on ghrelin concentrations. These data indicate that both AG and UAG have antiangiogenic properties at least at the level of endothelial growth, through decreased metabolic activity of the cells or stimulation of apoptosis. D-Lys(3)-GHRP-6 (inhibitor of GHS-R1a) seems not to be an appropriate antagonist in this experimental condition. Similar effects of these substances on HECa10 cells suggest that they are not mediated by GHS-R1a.
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