Figure 5.

Migration and Rac activation defects in Edg1^–/– embryonic fibroblasts. (a) RT-PCR analysis of Edg1, -3, and -5 expression was carried out with total RNA isolated from Edg1^+/+, Edg1^+/–, and Edg1^–/– embryonic fibroblasts. (b) SPP chemotactic responses of embryonic fibroblasts. Serum-starved Edg1^+/+ and Edg1^–/– fibroblasts were allowed to migrate toward a gradient produced by SPP (100 nM). Control (Cont.) indicates medium without serum was used as the chemoattractant. Chemotaxis was measured as described in Methods. Data are means ± SD of triplicate determinations. ^AStatistically significant difference compared with the control, determined by Student’s t test (P < 0.01). (c) Rac activation in fibroblasts. Edg1^+/+ and Edg1^–/– fibroblasts were serum-starved and then treated with SPP for 5 minutes. The cell lysates were used both for affinity precipitation with the PAK-1–conjugated agarose to pull down activated, GTP-bound Rac (top panel) and without fractionation to determine total Rac levels (bottom panel) by SDS-PAGE and immunoblotting. (d) Model of Edg-1 functions in blood vessel development. The Edg1 knockout demonstrates that Edg-1 is essential for vascular maturation by impairing the recruitment of smooth muscle cells to vessel walls. SPP, found in blood, may directly stimulate Edg-1 on VSMCs, facilitating their migration to vessels walls. In a second mechanism, which does not exclude the first, SPP could stimulate Edg-1 expressed on endothelial cells, which in turn recruit may VSMCs. EC, endothelial cell; SMC, smooth muscle cell.