Laser Doppler blood flow analysis of WT and LOX-1 KO mice.

<p>A: Representative images of LDBF just before and day 28 after surgery revealed hindlimb blood flow of the ischemic (R: right)/nonischemic (L: left) conditions in a supine position. B: Ischemic/nonischemic hindlimb blood flow ratio (mean±SD) were measured before (Pre), and on days 3, 7, 14, 21 and 28 after right femoral artery ligation. Results are expressed as the ratio of the ischemic hindlimb to nonischemic limb perfusion. ^★, P<0.05 vs. WT; ^★★, P<0.01 vs. WT; ^#, P<0.01 vs. Pre (before surgery), n = 25.</p

Ischemic/nonischemic hindlimb blood flow ratio (mean±SD) measured by laser Doppler blood flow analysis.

<p>For evaluation of blood flow recovery after ligation of the femoral artery of WT and LOX-1 KO mice, LDBF analysis was performed on the preoperative state and on days 3, 7, 14, 21 and 28. Data are expressed as the mean ± SD. n = 25.</p><p>Ischemic/nonischemic hindlimb blood flow ratio (mean±SD) measured by laser Doppler blood flow analysis.</p

Determination of capillary density (CD) and arteriole density (AD).

<p>A: Representative photographs to show CD and AD evaluated by histological examination of 20 randomly selected fields of tissue sections. CD in the ischemic tissues was immunostained with CD31 (an endothelial cell marker; brown, a1,2 and b1,2 and arrows, a2 and b2) and AD was anti-α-smooth muscle actin (α–SMA: an arteriole marker; brown and arrows, c and d) in the ischemic tissues of the gastrocnemius muscles on postoperative day 14 in high-power microscopic fields (x200). The number of vessels was reduced in LOX-1 KO mice (b1,2) compared with that in WT mice (a1,2) in CD31 staining (arrows in a2 and b2: the area of a1 and b1 surrounded by dotted line) and the number of arterioles stained with α–SMA (arrows in c and d) was also reduced in LOX-1 KO mice (d) compared with that in WT mice (c), Original bars; a1, b1: 300 µm a2, b2: 100 µm, c, d: 300 µm. B: CD and AD (mean±SD) were quantitatively assessed by histological examination of 20 randomly selected fields of tissue sections stained with CD31 staining (a) and α–SMA staining (b). The calculated capillary and arteriole density (capillaries per x200 HPF, arterioles per x200 HPF) were significantly lower in LOX-1 KO mice than in WT mice. ^★, P<0.01 vs. WT, n = 20.</p

Expression of LOX-1, VEGF VEGFR2, HIF-1α, Nox2 and generation of ROS.

<p><b>A. Effect of ischemia on expression of LOX-1.</b> a: The expression of LOX-1 was analyzed in the ischemic/nonischemic hindlimb of WT mice by Western blotting. Upregulation of LOX-1 expression in the ischemic hindlimb tissue on postoperative day 7 after ligation compared with in the nonischemic hindlimb of WT mice. Actin was used as loading control. IH: ischemia. b: Densitometric measurements (mean±SD) were performed to evaluate the fold increase in LOX-1 protein. The measured value is expressed as a value relative to the protein level of WT mice. IH: ischemia, ^★, P<0.05 vs. WT, n = 4. <b>B. Effect of deletion of LOX-1 on expression of VEGF, VEGFR2, HIF-1α, Nox2 and generation of ROS.</b> a: The expression of VEGF, VEGFR2, HIF-1α and Nox2 in the ischemic hindlimb was analyzed by Western blotting. Suppression of VEGF, HIF-1α and Nox2 expression in the ischemic hindlimb tissue of LOX-1 KO mice on postoperative day 7 after ligation compared with that in WT mice. Actin was used as loading control. b: Densitometric measurements (mean±SD) were performed to evaluate the fold increase in VEGF, VEGFR2, HIF-1α and Nox2 protein. The measured value is expressed as a value relative to the protein level of WT mice. ^★, P<0.01 vs. WT, n = 4. c: ROS generation was measured in the ischemic hindlimb of WT mice and LOX-1 KO mice. ^★, P<0.01 vs. WT, n = 4.</p

Effect of deletion of LOX-1 on signaling pathways and NF-κB dependent gene.

<p><b>A: Effect of deletion of LOX-1 on signaling pathways (p38 MAPK and NF-κB p65 subunit) and VCAM-1: NF-κB dependent adhesion molecule.</b> a: The expression of total and phosphorylated forms of p38 MAPK and NF-κB p65 subunit and VCAM-1 in the ischemic hindlimb was analyzed by Western blotting. Phosphorylation of p38 MAPK and NF-κB p65 subunit was suppressed in the ischemic hindlimb tissue of LOX-1 KO mice compared with that in WT mice as well as VCAM-1 protein, but their total proteins were equivalent between WT mice and LOX-1 KO mice. Actin was used as loading control. b: Densitometric measurements (mean±SD) were performed to evaluate the fold increase in phosphorylation of p38 MAPK and NF-κB p65 subunit and VCAM-1. The measured value is expressed as the value relative to the protein level of WT mice. Actin was used as loading control. ^★, P<0.01 vs. WT, n = 4. <b>B: Effect of deletion of LOX-1 on signaling pathways (Akt and eNOS).</b> a: The expression of total and phosphorylated forms of Akt and eNOS in the ischemic hindlimb was analyzed by Western blotting. Phosphorylation of Akt and eNOS was suppressed in LOX-1 KO mice compared with that in WT mice, but their total proteins were equivalent between WT mice and LOX-1 KO mice. Actin was used as loading control. b: Densitometric measurements (mean±SD) were performed to evaluate the fold increase in phosphorylation of Akt and eNOS. Both phospho-proteins were significantly suppressed in LOX-1KO mice compared with those in WT mice. The measured value is expressed as a value relative to the protein level of WT mice. ^★, P<0.01 vs. WT, n = 4. <b>C: Effect of deletion of LOX-1 on signaling pathways (ERK and SAPK).</b> a: The expression of total and phosphorylated forms of ERK and SAPK in the ischemic hindlimb was analyzed by Western blotting. No differences were observed in phospho-ERK and phospho-SAPK protein expression levels between KO mice and WT mice, as well as both total proteins. Actin was used as loading control. b: Densitometric measurements (mean±SD) were performed to evaluate the fold increase in phosphorylation of ERK and SAPK. Phospho-ERK and phospho-SAPK proteins were quantitatively equivalent. The measured value is expressed as a relative value relative to the protein level of WT mice. ^★, P<0.01 vs. WT, n = 4.</p

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