Fig. 4: Conditional depletion of WWP2 in macrophages protects from Angiotensin II induced cardiac fibrosis.

a Left, representative Sirius red (upper) and Masson’s Trichrome (lower) staining of short-axis sections of LV. Scale bar: 0.5 mm. Right, quantification of the area of fibrosis in transverse histological sections with Sirius red staining at the mid-ventricular level. WWP2^Mac, WWP2 conditional knockout in macrophages; Control (Ctrl), WWP2^flox/flox mice. b Collagen content in LV by hydroxy-phenyl-propionic acid (HPA) assay. n = 10 per experimental group. c Representative M-mode echocardiograms (middle LV long-axis) (left), and quantification of left ventricular internal dimension at end-diastole (LVIDed)(right). n = 12 per experimental group. d Echocardiogram-based quantification of LV ejection fraction (EF%) and fractional shortening (FS%). n = 8–11 per experimental group. e Representative WB showing fibronectin extracellular domain A (EDA-FN), vimentin (VIM), periostin (POSTN), and ACTA2 in LV tissue from control and WWP2^Mac mice. f qRT-PCR analysis of selected ECM genes in LV tissue from control and WWP2^Mac mice. n = 6 per experimental group. g Representative flow cytometry for CD11b⁺/F4/80⁺ gate for live cells from LV of control and WWP2^Mac mice (left). Quantification analysis shows reduced percentage of cardiac macrophage (live, CD45⁺CD64⁺CD11b⁺F4/80⁺Ly6G⁻) in WWP2^Mac compared with control mice (right). n = 4 for each group. h Percentage of the Ly6C^high in cardiac monocyte compartments (CD45⁺CD64⁺CD11b⁺). n = 4 for each group. i Ccl5 mRNA expression in cardiac macrophages (MΦ) sorted from LV. n = 5 per experimental group. j Relative mRNA levels of Ccl5 (upper), and representative WB for protein levels of CCL5 (lower) in LV tissue from control and WWP2^Mac mice. k–l qRT-PCR analysis of selected pro-inflammatory and homeostatic/reparatory genes in sorted cardiac macrophages (k: bar plot, n = 4–8 per group) and in LV tissue (l: heatmap, n = 6 per group). m ECM protein expression in LV from control and WWP2^Mac mice. Data in a–f refer to mice treated with Ang-II (500 ng/kg/min, 28 days). Data in g–m refer to mice treated with Ang-II (500 ng/kg/min, 7 days). Unless otherwise indicated, data are shown as dot-plots with mean ± SD, and statistical significance is assessed by the non-parametric Mann-Whitney U test. WWP2^Mac vs Ctrl: *P < 0.05; **P < 0.01; NS, not significant.