Post-ischemic treatment with HDL does not decrease leukocyte infiltration.

<p>Mice were submitted to LAD occlusion for 45min and hearts were reperfused for 24h. Mice were injected or not (control mice IR) with native HDL (nHDL), rHDLB (apoAI + POPC + S1P) one minute before reperfusion. <b>A.</b> Quantification of infiltrated neutrophils (Ly-6B.2⁺ cells) per area in frozen sections of infarcted hearts at 24h of reperfusion. <b>B.</b> Representative images of neutrophil (Ly-6B.2⁺ cells) infiltration. <b>C.</b> Quantification of infiltrated neutrophils (Ly6G+ cells) per area in frozen sections of infarcted hearts at 24h of reperfusion. <b>D.</b> Representative images of neutrophil (Ly6G+ cell) infiltration. <b>E.</b> Quantification of infiltrated macrophages (CD68⁺ cells) per area in frozen sections of infarcted hearts at 24h of reperfusion. <b>F.</b> Representative images of macrophage (CD68⁺ cells) infiltration. Data are expressed as scattered plots (mean±SEM, <i>n</i> = 7–10 per group). Results are expressed as percentages of stained area on total heart surface area. No significance difference between groups was found using unpaired-student t-test.</p

Activation of intracellular signaling pathways <i>in vitro</i> and <i>in vivo</i>.

<p><b>A.</b> Western blot analysis was performed with proteins extracted from neonatal cardiomyocytes treated with nHDL, rHDL (apoAI + POPC) or rHDLB (apoAI + POPC+ S1P) for 5min (n = 7–11). Specific bands corresponding to phosphorylated Akt, ERK1/2 and STAT3 were quantified by densitometry, normalized using GAPDH expression and given as a percentage of the control. <b>B.</b> Western blot analysis of proteins extracted from hearts submitted to 45min of LAD occlusion followed by 5min of reperfusion. Native HDL (nHDL) or rHDLB were injected one minute before the reperfusion (n = 9). Data are mean±SEM. *p<0.05, **p<0.01, ***p<0.001 vs control, using unpaired-student t-test.</p

Post-ischemic treatment with HDL does not decrease oxidation.

<p>Mice were submitted to LAD occlusion for 45min and hearts were reperfused for 24h. Mice were injected or not (control mice IR) with native HDL (nHDL), reHDLB (apoAI + POPC + S1P) one minute before reperfusion. <b>A.</b> Quantification of Di-BrY content of frozen sections of infarcted hearts after 24h of reperfusion. <b>B.</b> Representative images of Di-BrY stained middle heart sections. <b>C.</b> Correlation between neutrophil content and Di-BrY staining after 24h of reperfusion in the same hearts.</p

rHDL containing S1P protect against ischemia reperfusion injury <i>ex vivo</i>.

<p>Isolated hearts were submitted to global ischemia (35min) followed by reperfusion (45min). At the onset of reperfusion, hearts were treated or not (control) with native HDL (nHDL), rHDL (apoAI+POPC) or rHDLB (apoAI + POPC + S1P) during the first 7min. Infarct size is expressed as percentage of total heart area, (mean±SEM). ***p<0.001 vs control using one-way ANOVA combined with Tukey multiple comparisons post-hoc test, n≥4.</p

rHDL containing S1P protect against hypoxia <i>in vitro</i>.

<p>Cardiomyocytes were incubated in Tyrode solution, submitted to 5h hypoxia and treated during hypoxia with native HDL (nHDL), rHDL (apoAI+POPC) or rHDLB (apoAI + POPC + S1P). Cell survival was determined using the MTT assay. Cell survival is expressed in percentage of normoxia (mean±SEM). *p < 0.05 vs hypoxia, using paired-student t-test, n = 6.</p

Post-ischemic treatment with HDL protects against ischemia reperfusion injury <i>in vivo</i>.

<p>Mice were submitted to LAD occlusion for 45min and hearts were reperfused for 24h. Mice were injected or not (control mice IR) with native HDL (nHDL), POPC, rHDL (apoA1+POPC) or rHDLB (apoAI + POPC + S1P) one minute before reperfusion. <b>A.</b> Quantification of area at risk (AAR) per ventricle surface. Data are mean±SEM (n = 9–16 per group). <b>B.</b> Quantification of infarct size (IS) expressed in % of AAR. Data are mean±SEM (n = 9–16 per group), **p<0.01, vs IR, using one-way ANOVA combined with Tukey multiple comparisons post-hoc test. <b>C.</b> Representative images of TTC stained middle heart sections of control or treated mice.</p

Circulating chemokines measured 24h after reperfusion.

<p>Data are expressed as median (interquartile range [IQR]) of CCL2 or CXCL1 concentration (pg/ml). n = 7–9.</p><p>Circulating chemokines measured 24h after reperfusion.</p

Experimental protocols.

<p><b>A.</b> Hearts from mice were submitted to the ischemia reperfusion protocol as follows: 20min of stabilization, 35min of no-flow global ischemia and 45min of reperfusion. After reperfusion, infarct size was determined. Native HDL or rHDL were added for the first 7min of reperfusion. <b>B.</b> Mice were subjected to LAD occlusion for 45min and hearts were reperfused for different time periods: 5min for Western blot experiments and 24h for infarct size measurement and histological analysis. Serum from mice reperfused for 24h was collected for analysis of circulating cytokines. Native HDL or rHDL were injected one min before reperfusion.</p