Reduced release of active TGF-β₁ in the wound margins is causal for the wound healing defect of Vav3^−/− and Vav1^−/−/Vav3^−/− mice. (A) Immunostaining of 5-day-old wounds from Vav3^−/−, Vav1^−/−/Vav3^−/− and WT mice showing TGF-β₁ (red) localization throughout the K14⁺ epidermis (green) and the wound tissue. Blue staining indicates nuclear staining with DAPI. Original magnification ×20, scale bar indicates 200μm; he, hyperproliferative epidermis; we, wound edge, gt, granulation tissue, h, hair follicle. (B) Quantitative evaluation of total and active TGF-β₁ release from 5 days old Vav3^−/−, Vav1^−/−/Vav3^−/− and WT wound lysates by specific ELISA. Results representative of 2 independent experiments are expressed as mean ± SD (n = 4). *P < .05 by Student t test. (C) Oxidative burst of WT and Vav3^−/− Mφ upon phagocytosis of apoptotic WT or Vav3^−/− PMN measured at 3 hours and expressed as the increase in fluorescence intensity of oxidized carboxy H₂DCFDA. Data representative for at least 2 different experiments is given in RFU (relative fluorescence units) as scatter plot. Each symbol represents triplicate measurements, Mφ derived from 3 different mice of each genotype (n = 3). Bars indicate medians. *P < .05 by Student t test. (D) Representative macroscopic pictures of wounds derived from WT and Vav3^−/− mice at days 5 and 7 after wounding and repetitive injection with a physiologic concentration of rhTGF-β₁ (TGF-β₁⁺) or of NaCl (TGF-β₁⁻). (E) Statistical analysis of 16 wound areas expressed as percentage of the initial (day 0) wound size. Results presented as scatter plots. Bars indicate medians of each cohort (n = 4). *P < .05 by Mann-Whitney test. (F) Western blot analysis of snap-frozen wound tissue equilibrated to total actin and vimentin levels to assess expression levels of αSMA, TGFβ-RII and PECAM-1 in wound margins of different genotypes at different time points after injection of either rhTGF-β₁ (TGF-β₁+) or of NaCl (TGF-β₁-). (G) Quantification of PECAM-1⁺ cells in 10 high power fields (HPF) of 5- and 7-day-old wounds of WT, Vav3^−/− and TGF-β₁–treated Vav3^−/− mice. Data are given as mean ± SD numbers of PECAM-1⁺ cells counted per HPF. *P < .05 by Student t test. (H) Recruitment of F4/80⁺ Mφ assessed by FACS analysis of cells isolated by enzymatic disruption of 3- and 5-day-old wounds. Results given as scatter plots. Bars indicate the median (n = 4). *P < .05 by Student t test.