Impaired recruitment of Mφ, but not of PMN, and reduced numbers of myofibroblasts and blood vessels at the wound sites of Vav3^−/− mice. (A) GR-1⁺ PMN (red; top panel) and F4/80⁺ Mφ (green; bottom panel) recruitment to wound sites assessed by immunostaining of cryosections from Vav3^−/−, Vav1^−/−/Vav3^−/− and WT mice. Quantification of GR-1⁺ PMN recruitment at 24 hours after wounding (B) and F4/80⁺ Mφ recruitment at 72 hours after wounding (C) by FACS analysis of wound cells isolated by enzymatic disruption from wound tissue. Results given as scatter plots. Bars indicate the median (n = 5). *P < .05 by Student t test. (D) Granulation tissue formation in 5 and 7 days old WT and Vav3^−/− wounds assessed by immunostaining with myofibroblasts-specific αSMA (green) and endothelial cell–specific PECAM-1 (red). Cell nuclei are counterstained with DAPI (blue). Original magnification ×20, scale bar indicates 200μm; e, eschar, he, hyperproliferative epidermis; we, wound edge, gt, granulation tissue. Quantification of PECAM-1⁺ cells in 10 high-power fields (HPFs) of 5- and 7-day-old wounds of 4 Vav3^−/− and WT mice. Data are given as mean ± SD numbers of PECAM-1⁺ cells counted per HPF. *P < .05 by Student t test. (E) Western blot analysis of snap-frozen nonwounded normal skin (NS) and of wound tissue at days 5 and 7 equilibrated to total actin and vimentin levels to measure expression of αSMA, TGFβ-RII and PECAM-1. αSMA, TGFβ-RII, PECAM-1, actin, and vimentin are expressed at identical levels in WT and Vav3^−/− nonwounded skin, excluding that these molecules are globally down-regulated in Vav3^−/− mice. (F) Semiquantitative balance analysis of immunoblots performed by densitometry of digitized Western blots. Data are given as mean ± SD. *P < .05 by Student t test.