Bone Marrow Progenitor Cell Therapy-Mediated Paracrine Regulation of Cardiac miRNA-155 Modulates Fibrotic Response in Diabetic Hearts

<div><p>Diabetes is associated with a higher incidence of myocardial infarction (MI) and increased risk for adverse vascular and fibrogenic events post-MI. Bone marrow-derived progenitor cell (BMPC) therapy has been shown to promote neovascularization, decrease infarct area and attenuate left ventricular (LV) dysfunction after MI. Unlike vascular effects, the anti-fibrosis mechanisms of BMPC, specifically under diabetic conditions, are poorly understood. We demonstrated that intramyocardial delivery of BMPCs in infarcted diabetic <i>db/db</i> mice significantly down-regulates profibrotic miRNA-155 in the myocardium and improves LV remodeling and function. Furthermore, inhibition of paracrine factor hepatocyte growth factor (HGF) signaling <i>in vivo</i> suppressed the BMPC-mediated inhibition of miR-155 expression and the associated protective effect on cardiac fibrosis and function. <i>In vitro</i> studies confirmed that the conditioned media of BMPC inhibited miR-155 expression and profibrotic signaling in mouse cardiac fibroblasts under diabetic conditions. However, neutralizing antibodies directed against HGF blocked these effects. Furthermore, miR-155 over-expression in mouse cardiac fibroblasts inhibited antifibrotic Sloan-Kettering Institute proto-oncogene (Ski) and Ski-related novel gene, non-Alu-containing (SnoN) signaling and abrogated antifibrogenic response of HGF. Together, our data demonstrates that paracrine regulation of cardiac miRNAs by transplanted BMPCs contributes to the antifibrotic effects of BMPC therapy. BMPCs release HGF, which inhibits miR-155-mediated profibrosis signaling, thereby preventing cardiac fibrosis. These data suggest that targeting miR-155 might serve as a potential therapy against cardiac fibrosis in the diabetic heart.</p> </div

miR-155 over-expression modulates fibrosis-related signaling in mouse cardiac fibroblasts (CFs).

<p>CFs were either transfected with miR-155 mimics (pre-miR-155) to over-express miR-155 or negative control mimics (control) for 72 hrs and mRNA expression of various genes were determined by qRT-PCR. miR-155 over-expression (in presence of HGF) did not affect expression of TGF-β (<b>A</b>), TGF-βR1 (<b>B</b>) and TGF-βR2 (<b>C</b>). In contrast mRNA expression of Ski (<b>D</b>) and SnoN (<b>E</b>) was decreased. *P<0.05 versus control cells. <b>F</b>. Protein expression of Ski and SnoN in cardiac fibroblasts following over-expression of miR-155 was determined by Western blotting. β-actin served as loading controls. TGF-β indicates TGF-β; TGF-βR1, TGF-beta receptor type 1; TGF-βR2, TGF-beta receptor type 2; Ski, Sloan-Kettering Institute proto-oncogene; SnoN, Ski-related novel gene, non-Alu-containing.</p

Cardiac fibrosis in <i>db/db</i> mice receiving BMPC therapy.

<p>Fibrosis area was determined at 28 days after MI by Sirius red staining. Collagen stains deep red and normal tissue stains yellowish. BMPC therapy decreased % fibrosis in comparison with saline-treated group. Administration of neutralizing antibodies against HGF (HGF-Ab) significantly aggravated fibrosis as compared to IgG treated group. Bar graph represents quantification of percent fibrosis area (percentage of LV area). HGF-Ab indicates antibodies against hepatocyte growth factor; BMPC, bone marrow-derived progenitor cell. BMPC, bone marrow-derived progenitor cell. *P<0.05 vs saline group; #P<0.05 versus IgG treated group.</p

Intramyocardial BMPC transplantation regulates cardiac miRNAs following MI in mice.

<p>miRNA expression was measured in the border zone of infarcted area at 3 days post-MI by quantitative RT-PCR. BMPC therapy decreased miR-21 (<b>A</b>) and miR-155 (<b>B</b>) and increased miR-29 (<b>C</b>) and miR-133a (<b>D</b>) expression in comparison with saline-treated or sham groups. *P<0.01 vs sham; #P<0.05 vs saline group. BMPC-bone marrow-derived progenitor cell; MI, myocardial infarction.</p

Expression of miR-155 in infarcted hearts (day 3) of <i>db/db</i> mice receiving BMPC intramyocardial.

<p>BMPC therapy marginally (although not significantly) decreased MI-induced miR-155 expression (measured by qRT-PCR) in comparison with saline-treated group. Administration of neutralizing antibodies against HGF (HGF-Ab) significantly elevated miR-155 levels as compared to IgG treated group. *<i>P<</i>0.05 versus sham; #P<0.01 versus IgG treated mice. HGF-Ab indicates antibodies against hepatocyte growth factor; Col1A1, collagen type 1 alpha 1; Col1A1, collagen type 3 alpha 1; α-SMA, alpha smooth muscle actin; BMPC, bone marrow-derived progenitor cell. BMPC, bone marrow-derived progenitor cell; MI, myocardial infarction. *P<0.01 vs sham; #P<0.05 vs IgG treated group.</p

miR-155 over-expression abrogates HGF-induced inhibition of fibrogenic response in mouse cardiac fibroblasts (CFs).

<p>CFs were either transfected with miR-155 mimics (pre-miR-155) to over-express miR-155 or with negative control mimics (control), treated with or without HGF under diabetic conditions [TGF-β (10 ug/mL+high glucose (25 mM glucose)] and mRNA expression of fibrogenesis markers like col1A1 (<b>A</b>), col3A1 (<b>B</b>) and α-SMA (<b>C</b>) were measured by qRT-PCR. HGF decreased the expression of above markers. *P<0.01 versus without HGF. Over-expression of miR-155 nullified the effects of HGF. P<0.01 versus control-transfected cells. HGF indicates hepatocyte growth factor; Col1A1, collagen type 1 alpha 1; Col1A1, collagen type 3 alpha 1; α-SMA, alpha smooth muscle actin.</p

Sch B attenuates Dox-induced myocardial ROS and oxidative stress, and enhance antioxidant enzyme.

<p>(A) MDA level, (B) Nitrite level, (C) Glutathione level and (D) Superoxide production by LV homogenates. **<i>P <</i> 0.01 vs. group N mice; ^#<i>P <</i> 0.05 and ^##<i>P <</i> 0.01 vs. group V mice.</p

Sch B suppressed the cardiac morphological changes and DNA damage.

<p>Typical photomicrographs of heart tissue from group N (A), group V (B) and group Sch B (C–E). Cytoplasmic vacuolation and myofibrillar disorganization in the doxorubicin group were effectively attenuated Sch B treatment (haematoxylin and eosin ×200). (F–J) Representative photomicrographs of DNA damage 5 days after Dox injection. DNA damage detected by single cell gel electrophoresis assay (Comet assay) in cardiomyoctes of all groups at 200x magnification. (K–L) Quantification of cardiomyocyte DNA damage analyzed by digital imaging casp-software (<a href="http://casp.sourceforge.net/" target="_blank">http://casp.sourceforge.net/</a>). **<i>P <</i> 0.01 vs. group N mice; ^#<i>P <</i> 0.05 and ^##<i>P <</i> 0.01 vs. group V mice.</p

Sch B inhibits Dox induced NADPH oxidase subunits.

<p>(A–D) Representative Western immunoblots and densitometry analysis using Scion image software for p47phox, p67phox and gp91phox in group N, group V and Sch B treated mice; Blots were normalized against GAPDH. Each bar represents means ± S.E. (n = 3–5). **<i>P <</i> 0.01 vs. group N mice; ^#<i>P <</i> 0.05 and ^##<i>P <</i> 0.01 vs. group V mice.</p

Sch B attenuates Dox-induced hydroxyl radical.

<p>(A, B) Representative ESR spectra and analysis of the hydroxyl radical signal relative to the internal standard of manganese ion. Hydroxyl radical signals were not detected (ND) in hearts of group N and group Sch B-100. Mn (3) and Mn (4) indicate the internal standard signals of manganese ion (Mn²⁺).</p