IL-10 inhibits inflammation and attenuates left ventricular remodeling after myocardial infarction via activation of STAT3 and suppression of HuR

Abstract

Persistent inflammatory response has adverse effects on left ventricular (LV) function and remodeling following acute myocardial infarction. We hypothesized that suppression of inflammation with interleukin (IL)-10 treatment attenuates LV dysfunction and remodeling after acute myocardial infarction. After the induction of acute myocardial infarction, mice were treated with either saline or recombinant IL-10, and inflammatory response and LV functional and structural remodeling changes were evaluated. IL-10 significantly suppressed infiltration of inflammatory cells and expression of proinflammatory cytokines in the myocardium. These changes were associated with IL-10-mediated inhibition of p38 mitogen-activated protein kinase activation and repression of the cytokine mRNA-stabilizing protein HuR. IL-10 treatment significantly improved LV functions, reduced infarct size, and attenuated infarct wall thinning. Myocardial infarction-induced increase in matrix metalloproteinase (MMP)-9 expression and activity was associated with increased fibrosis, whereas IL-10 treatment reduced both MMP-9 activity and fibrosis. Small interfering RNA knockdown of HuR mimicked IL-10-mediated reduction in MMP-9 expression and activity in NIH3T3 cells. Moreover, IL-10 treatment significantly increased capillary density in the infarcted myocardium which was associated with enhanced STAT3 phosphorylation. Taken together, our studies demonstrate that IL-10 suppresses inflammatory response and contributes to improved LV function and remodeling by inhibiting fibrosis via suppression of HuR/MMP-9 and by enhancing capillary density through activation of STAT3.

Figure 1

A. Immunofluorescent staining of inflammatory cells (CD68-positive, green fluorescence) in the heart at 3 days post MI. B. Quantitative analysis of infiltrating CD68-positive cells at 3 days post-MI. IL-10 inhibited CD68+ cells infiltration as compared to MI and Sham hearts. *P<0.01 vs Sham; #P<0.01 vs MI.

Figure 2

Figure 3

A. M-mode echocardiographic images obtained from baseline, MI and MI+IL-10 groups 28 days post-MI. Arrows indicate LV chamber diameter. B. Analysis of LV diameter in diastole and systole, and percent fractional shortening (%FS) and %EF calculations. LVEDD, LV end-diastolic diameter; LVESD, LV end-systolic diameter; %EF, percent ejection fraction; %FS, percent fractional shortening; *P<0.05 vs baseline; #P<0.05 vs MI.

Figure 4

A. Trichrome stained heart sections (28 days post-MI). B. Quantitative analysis of infarct wall thickness at 28 days post-MI. IL-10 treatment attenuated infarct wall thinning when compared to MI group. #P<0.01 vs MI; n=8. Figure 4. C. TUNEL staining for cardiac cell apoptosis (Red) and DAPI (blue) for nuclear staining in the border zone of LV infarct at 3 days post-MI. IL-10 treatment attenuated cardiac cell apoptosis after MI.

Figure 4

A. Trichrome stained heart sections (28 days post-MI). B. Quantitative analysis of infarct wall thickness at 28 days post-MI. IL-10 treatment attenuated infarct wall thinning when compared to MI group. #P<0.01 vs MI; n=8. Figure 4. C. TUNEL staining for cardiac cell apoptosis (Red) and DAPI (blue) for nuclear staining in the border zone of LV infarct at 3 days post-MI. IL-10 treatment attenuated cardiac cell apoptosis after MI.

Figure 5

A. Western blot for HuR protein expression B. phosphorylation of p38 MAP kinase in LV at 3 days post-MI. Equal loading of proteins in each lane is shown by β-actin and total p38, respectively. C. Western blot for phosphor-serine (activation) against Total STAT3 in LV at 28 days post-MI. Total LV lysate was immunoprecipitated with anti-STAT3 antibodies and analyzed by Western blot using anti-phospho-serine (p-ser) antibodies.

Figure 6

A. Capillary density (CD31 staining, green fluorescence) in border zone of LV infarct at 28 days post-MI. Bar graph shows quantitative analysis of CD31+ capillaries per high visual field (hvf). MI increased capillaries, which was further higher in IL-10 treated mice. *P<0.01 vs Sham; #P<0.05 vs MI. Figure 6. B. Arteriolar smooth muscle hyperplasia (smooth muscle actin stained, red fluorescence) in the LV at 28 days post-MI. Bar graph shows quantitative analysis of arteriolar wall thickness. MI increased inflammation induced SM hyperplasia, which was attenuated in IL-10 treated mice. *P<0.01 vs Sham; #P<0.01 vs MI.

Figure 6

A. Capillary density (CD31 staining, green fluorescence) in border zone of LV infarct at 28 days post-MI. Bar graph shows quantitative analysis of CD31+ capillaries per high visual field (hvf). MI increased capillaries, which was further higher in IL-10 treated mice. *P<0.01 vs Sham; #P<0.05 vs MI. Figure 6. B. Arteriolar smooth muscle hyperplasia (smooth muscle actin stained, red fluorescence) in the LV at 28 days post-MI. Bar graph shows quantitative analysis of arteriolar wall thickness. MI increased inflammation induced SM hyperplasia, which was attenuated in IL-10 treated mice. *P<0.01 vs Sham; #P<0.01 vs MI.

Figure 7

A. Quantitative analysis of mRNA expression of VEGF-A in the border zone of LV infarct at 28 days post-MI. Real time-PCR data shows that IL-10 treatment enhanced VEGF-A expression as compared to MI. *P<0.01 vs Sham; #P<0.05 vs MI B. Effect of STAT-3 inhibition on VEGF-A mRNA expression in HUVEC cells. Curcurbitacin I (Cur) treated cells inhibited IL-10 induced VEGF-A. mRNA expression normalized to 18S expression and depicted as fold change vs Sham. *P<0.01 vs control cells; #P<0.01 vs IL-10 treated cells.

Figure 8

A. Quantitative analysis of fibrosis area at 28 days post-MI. IL-10 group showed lower fibrosis area when compared to MI group. #P<0.01 vs MI. B. Quantitative analysis of mRNA expression of MMP-9 (RT-PCR) in the myocardium at 28 days post-MI. mRNA expression normalized to 18S expression and depicted as fold change vs Sham. #P<0.01 vs MI. C. Gelatin in-gel zymography of LV infarct (border zone) tissue 28 days post-MI. Densitometry analysis showed that MMP-9 was higher in MI groups and reduced with IL-10 treatment. #P<0.01 vs MI.

Figure 9

Quantitative analysis of mRNA expression of HuR (A) and MMP-9 (B) by RT-PCR in NIH3T3 cells. mRNA expression normalized to 18S expression and depicted as fold change vs Sham. IL-10 suppressed TNF-α induced HuR and MMP-9 expression (*P<0.01 vs control; #P<0.01 vs TNF-α treated cells). HuR siRNA mimicked IL-10 effects by suppressing TNF-α induced HuR and MMP-9 expression ($P<0.01, HuR siRNA+TNF-α vs scrambled siRNA+TNF-α treated cells). C. Gelatin in-gel zymography for the cell culture media. MMP-9 activity was higher in TNF-α treated cells and IL-10 and HuR siRNA inhibited TNF-α mediated increases in MMP-9 activity.