Endothelial Cells In Vitro Synergism With Calcitonin Gene-Related Peptide, Insulin, and Plasma Factors

The purpose of this study was to determine the effects of the vasoactive perivascular neuropeptide substance P (SP) on the growth and function of vascular endothelial cells in serum-free culture conditions with cells quiescent in the GO-G, phase of the cell cycle and to characterize the response. In addition, interactions between SP and other growth factors and neuropeptides including insulin, platelet factors, neurokinin A, neurokinin B, and calcitonin gene-related peptide (CGRP) were studied on endothelial cell growth and compared. Growth effects were determined by stimulation of tritiated thymidine incorporation into DNA and cell proliferation. SP exhibited differential effects on cell growth that were a function of concentration, incubation time, interaction with other growth factors, and cell culture conditions. DNA synthesis in response to SP showed a bell-shaped distribution with a maximal effect that was 10.5-fold over control at 500 ,ug/mL of SP after 48 hours of incubation. The effect showed marked synergism with insulin (10 ,ug/mL) and with CGRP (0.01 to 10 ,ug/mL), which is colocalized with SP in vivo. Insulin and CGRP alone had no significant effect on endothelial cell growth. Furthermore, no synergism was observed between SP and platelet-derived growth factor or platelet-derived endothelial cell growth factor. Endothelial cell proliferation increased in response to SP to 2.6-fold over control at 48 hours, was maximal at 10 ,tg/mL SP, and also demonstrated synergism with insulin (10 ,ug/mL). Our studies indicate that neuropeptides play a significant role in regulating endothelial cell growth and proliferation. The demonstrated synergism between SP and other known growth factors and neuropeptides implies that synergism between differing signal transduction pathways may be significant in the total observed response. These findings suggest that SP may have an important role in the normal and pathophysiological states of the vasculature. (Circ Res. 1994;75:1113-1120.)