Assessment by transthoracic echocardiography and Millar micro-tip catheter transducer of the effect of LOX-1 deletion on LV dysfunction caused by DOX.

<p>A: Representative echocardiographic images for each of the four experimental groups. B: Time-dependent change of LV end-diastolic diameter (LVDd), LV end-systolic diameter (LVDs) and LV fractional shortening (FS) assessed by echocardiography in WT/DOX and LOX-1/DOX. Presented values are mean±SEM. ^★ P<0.01, WT/DOX vs. LOX-1 KO/DOX, ^★★ P<0.01, Day 0 vs. Day 28, n = 8. C: Effect of LOX-1 deletion on LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), maximal rate of pressure development (+dp/dt) and maximal rate of pressure relaxation (-dp/dt) in the four experimental groups. Presented values are mean±SEM. ^★P<0.01, WT/Vehicle vs. WT/DOX, ^★★P<0.01, WT/DOX vs. LOX-1 KO/DOX, n = 8; day14</p

Histopathologic findings in the four experimental groups and evaluation of LOX-1 expression in cardiomyocytes, endothelial cells, fibroblasts and inflammatory cells.

<p>A: Photographs of the histological and immunohistochemical preparations. Top: Horizontal slices of whole ventricles stained with HE (x 20), Middle: HE (x 400), Bottom: Sirius Red (x 400). Representative scaled photographic images are shown. B: Quantitative analysis of heart/body weight ratio, diameter of cardiomyocytes and % fibrosis in the four experimental groups. Presented values are mean±SEM. ^★P<0.01, WT/Vehicle vs. WT/DOX, ^★★P<0.01, WT/DOX vs. LOX-1 KO/DOX, n = 160; day14, Male LOX-1 KO mice and WT mice were used at the age of 14 weeks. C: Histological analysis and Western blotting to evaluate LOX-1 expression. Immunofluorescence images of LOX-1 expression in (a) cardiomyocytes, (b) endothelial cells and (c) fibroblasts, which were treated with vehicle or DOX. LOX-1 (green), cardiomyocyte marker: phalloidin (red), endothelial cell marker: CD31 (red), fibroblast marker: vimentin (red), nuclei: DAPI (blue). Representative scaled photographic images are shown (scale bars: 20 μm). (d) Representative Western blot and quantification of LOX-1 in the above cells in the four experimental groups. GAPDH is the internal control. Presented values (AU: arbitrary units) are mean±SEM. ^★P<0.01, Cardiomyocytes/Vehicle vs. Cardiomyocytes/DOX, ^★★P<0.01, Cardiomyocytes/Vehicle vs. Endothelial cells/Vehicle, ^★★★P<0.01, Cardiomyocytes/Vehicle vs. Fibroblasts/Vehicle, n = 4. (e) Flow cytometry analysis of the surface expression of LOX-1 on CD45-positive cells (neutrophil, monocyte and lymphocyte) in the peripheral blood of mice treated with vehicle or DOX. Plots show anti-LOX-1 Ab staining profile in the top panel and isotype control IgG staining profile in the bottom panel. Data are representative of five independent experiments (LOX-1 expression: ns, Vehicle vs. DOX, n = 5). (f) Immunohistochemical images of LOX-1 positive cardiomyocyte (Arrows indicates typical LOX-1-positive cells: brown staining.) in the hearts of mice treated with vehicle or DOX. Representative scaled photographic images are shown (scale bars: 20 μm).</p

LV functional parameters (Led, LVDs, IVSd, PWd, FS, SV, CO and LV mass) in WT mice and LOX-1 KO mice at Day 28 after DOX administration.

<p>LV functional parameters (Led, LVDs, IVSd, PWd, FS, SV, CO and LV mass) in WT mice and LOX-1 KO mice at Day 28 after DOX administration.</p

Signal transduction proteins of LOX-1 (phospho- and total NF-κB, MAPKs and Akt) and sarcomeric-related proteins (GATA-4, MHC and Trop-I).

<p>A: Representative Western blot and quantification of phospho- and total p65 (P-p65 and p65) and phospho- and total p38 (P-p38 and p38) in the four experimental groups. B: Representative Western blot and quantification of phospho- and total Akt (P-Akt and Akt) and phospho- and total ERK (P-ERK and ERK) in the four experimental groups. C: Representative Western blot and quantification of GATA-4, MHC and troponin I (Trop-I) in the four experimental groups. GAPDH is the internal control. Presented values (AU: arbitrary units) are mean±SEM. ^★P<0.05, WT/Vehicle vs. WT/DOX, ^★★P<0.05, WT/DOX vs. LOX-1 KO/DOX, n = 4; day14.</p

Effect of LOX-1 deletion on expressions of LOX-1 and VCAM-1 and productions of ROS, TNF-α and IL-1ß in the hearts after DOX treatment.

<p>A: Representative Western blot and quantification of LOX-1 and VCAM-1 in the four experimental groups. GAPDH is the internal control. Presented values (AU: arbitrary units) are mean±SEM. ^★P<0.01, WT/Vehicle vs. WT/DOX, ^★★P<0.01, WT/DOX vs. LOX-1 KO/DOX, n = 4. B: Measurement of ROS generation in the four experimental groups. Presented values (AU: arbitrary unit) are mean±SEM. ^★P<0.01, WT/Vehicle vs. WT/DOX, ^★★P<0.01, WT/DOX vs. LOX-1 KO/DOX, n = 8; day14. C: Quantification of cytokine production: TNF-α and IL-1ß in the four experimental groups. Presented values (fg/mg or pg/mg in the heart tissue) are mean±SEM. ^★P<0.01, WT/Vehicle vs. WT/DOX, ^★★P<0.01, WT/DOX vs. LOX-1 KO/DOX, n = 4; day14.</p