Fig. 4. Angpt2 stimulates endothelial FA uptake through integrin α5β1 signaling.

a HUVECs were integrin-motif activated using 1 mM Mn²⁺ and analyzed for FA (BODIPY C-12, 8 μM) uptake. b Representative images and comparison of FA uptake with vehicle or Angpt2 (2.5 μg/ml) treatment for 30 min in HUVECs with Mn²⁺ (1 mM). Each dot indicates a mean of quadruplicate values from three independent experiments. Scale bars, 30 μm. c Comparisons of FA uptake at different concentrations with vehicle (n = 5), Mn²⁺ (1 mM; n = 5), Angpt2 (2.5 μg/ml; n = 3 for 4 μM, 4 for 8 μM, 5 for 16 μM) or Mn²⁺ (1 mM) + Angpt2 (2.5 μg/ml) (n = 5 for 4 μM; 3 for 8, 16 μM) treatment for 15 min in HUVECs. d Comparisons of FA uptake with vehicle (n = 5) or Mn²⁺ (1 mM) + Angpt2 (2.5 μg/ml) (n = 3 for 1 min; 4 for 2, 5, 10 min; 5 for 15 min) treatment for indicated time points in HUVECs. e Comparisons of FA uptake treatment with vehicle (n = 5) or Mn²⁺ (1 mM) + Angpt2 (n = 3 for 20 ng, 1.25, 2.5, 5 μg/ml; 4 for 200, 500 ng/ml) treatment with indicated concentration for 15 min in HUVECs. f Comparisons of vascular permeability with vehicle (n = 4), Mn²⁺ (1 mM; n = 6), Angpt2 (2.5 μg/ml; n = 6) or Mn²⁺ (1 mM) + Angpt2 (2.5 μg/ml) (n = 4) treatment for 15 min in HUVECs cultured in trans-well inserts. g Comparisons of FA uptake with vehicle or Mn²⁺ (1 mM) + Angpt2 (2.5 μg/ml) treatment for 15 min in siControl (n = 4), siITGα5 (n = 3) or siITGβ1 (n = 4 for vehicle, 5 for Mn²⁺ + Angpt2) HUVECs. h Comparisons of FA uptake with or without Angpt2 (2.5 μg/ml), ITGα5β1 blocking peptide (ATN-161, 10 μM) or Mn²⁺ (1 mM) treatment for 15 min in HUVECs. Each dot indicates a mean of triplicate values from two independent experiments. i Comparisons of FA uptake with (n = 6) or without Angpt2 (2.5 μg/ml; n = 4) or ITGα5β1-specific motif activator (SNAKA-51, 10 μg/ml; n = 4) treatment for 15 min in HUVECs. Unless otherwise denoted, FA BODIPY C-12 concentration is 8 μM, horizontal bars indicate mean ± SD (b, c, f–i) or SEM (d, e) and P values versus vehicle by two-tailed Student’s t test (b, d, e, g) or one-way ANOVA followed by Tukey’s multiple comparison test (c, f, h, i). NS not significant.