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. 2011 Jun 14;57(24):2444-52.
doi: 10.1016/j.jacc.2010.11.069.

Sonic hedgehog-induced functional recovery after myocardial infarction is enhanced by AMD3100-mediated progenitor-cell mobilization

Jérôme Roncalli  1 , Marie-Ange Renault, Jörn Tongers, Sol Misener, Tina Thorne, Christine Kamide, Kentaro Jujo, Toshikazu Tanaka, Masaaki Ii, Ekaterina Klyachko, Douglas W Losordo
Affiliations

Sonic hedgehog-induced functional recovery after myocardial infarction is enhanced by AMD3100-mediated progenitor-cell mobilization

Jérôme Roncalli et al. J Am Coll Cardiol. .

Abstract

Objectives: This study was designed to compare the effectiveness of Sonic hedgehog (Shh) gene transfer, AMD3100-induced progenitor-cell mobilization, and Shh-AMD3100 combination therapy for treatment of surgically induced myocardial infarction (MI) in mice.

Background: Shh gene transfer improves myocardial recovery by up-regulating angiogenic genes and enhancing the incorporation of bone marrow-derived progenitor cells (BMPCs) in infarcted myocardium. Here, we investigated whether the effectiveness of Shh gene therapy could be improved with AMD3100-induced progenitor-cell mobilization.

Methods: Gene expression and cell function were evaluated in cells cultured with medium collected from fibroblasts transfected with plasmids encoding human Shh (phShh). MI was induced in wild-type mice, in matrix metalloproteinase (MMP)-9 knockout mice, and in mice transplanted with bone marrow that expressed green-fluorescent protein. Mice were treated with 100 μg of phShh (administered intramyocardially), 5 mg/kg of AMD3100 (administered subcutaneously), or both; cardiac function was evaluated echocardiographically, and fibrosis, capillary density, and BMPC incorporation were evaluated immunohistochemically.

Results: phShh increased vascular endothelial growth factor and stromal cell-derived factor 1 expression in fibroblasts; the medium from phShh-transfected fibroblasts increased endothelial-cell migration and the migration, proliferation, and tube formation of BMPCs. Combination therapy enhanced cardiac functional recovery (i.e., left ventricular ejection fraction) in wild-type mice, but not in MMP-9 knockout mice, and was associated with less fibrosis, greater capillary density and smooth muscle-containing vessel density, and enhanced BMPC incorporation.

Conclusions: Combination therapy consisting of intramyocardial Shh gene transfer and AMD3100-induced progenitor-cell mobilization improves cardiac functional recovery after MI and is superior to either individual treatment for promoting therapeutic neovascularization.

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Figures

Figure 1
Figure 1. Shh-conditioned medium promotes endothelial-cell migration and the migration, proliferation, and capillary morphogenesis of BMPCs
(A, B) Migration (*P<0.01), (C, D) proliferation (*P<0.01), and (E, F) tube formation (*P<0.05) were assessed for BMPCs and/or bovine aortic endothelial cells after treatment with control or Shh-conditioned medium. Three independent experiments, n=3–5 per condition for each experiment. HPF indicates high-power field; OD, optical density.
Figure 2
Figure 2. Combination therapy enhances cardiac functional recovery after MI
Mice were treated with or without phShh, AMD3100, or phShh-AMD3100 combination therapy after surgically induced MI; control mice did not undergo surgical MI. (A) Cardiac mRNA expression of the transfected phShh was confirmed 1 and 5 days after MI (*P<0.05, **P<0.0001 versus untreated, AMD3100, and controls). (B) SDF-1α mRNA expression was evaluated in non-infarcted, infarcted, and border-zone regions 5 days after MI (*P<0.05 versus untreated, AMD3100, and phShh; **P<0.01 versus untreated and phShh). (C–E) Cardiac function was evaluated via (C, D) echocardiographic assessments of (E) LVEFs (*P=0.013 versus untreated). mRNA measurements were normalized to endogenous 18S rRNA expression. Panels A, B: n=3–5 per treatment group; panel E: n=11–15 per treatment group.
Figure 3
Figure 3. Combination therapy reduces cardiac fibrosis and enhances the development of capillaries and smooth-muscle–containing vessels after MI
Mice were sacrificed 28 days after surgically induced MI and treatment with or without phShh, AMD3100, or combination phShh-AMD3100 therapy. (A–B) The proportion of fibrotic tissue (blue) in the left ventricle was determined by calculating the ratio of the area of fibrosis to the left-ventricular area (*P<0.01 versus untreated, **P<0.05 versus AMD3100 or phShh). (C–H) Sections were stained with (C–F) isolectin B4 and fluorescent anti-isolectin B4 antibodies (green) to identify capillaries or with (G–H) isolectin B4, fluorescent anti-isolectin B4 antibodies, and anti-SMA (red) antibodies to identify smooth-muscle–containing vessels; nuclei were counterstained with DAPI. Capillary density was evaluated by counting isolectin-B4–positive tubular-like structures (D) in the heart (*P<0.05 versus untreated, **P<0.05 versus AMD3100 or phShh) and in the (E) infarcted (*P<0.05 versus untreated, †P=0.051 versus AMD3100) and (F) border-zone regions (*P<0.05 versus untreated). (H) The density of smooth-muscle–containing vessels was evaluated by counting SMA-positive, isolectin-B4–positive tubular structures (*P<0.05 versus untreated, **P<0.01 versus untreated or AMD3100). n=7–10 per treatment group. HPF indicates high-power field.
Figure 4
Figure 4. Combination therapy enhances the incorporation of BMPCs
Wild-type mice transplanted with bone-marrow from GFP-expressing mice were sacrificed 28 days after surgically induced MI and treatment with or without phShh, AMD3100, or phShh-AMD3100 combination therapy; 10 minutes before sacrifice on day 28, mice were injected with FITC-conjugated BS1-lectin, which binds to endothelial tissue. (A) BMPCs were identified in the myocardial endothelium via double-positive staining (yellow-orange) for BS1-lectin (green) and GFP expression (red GFP-antibody staining); nuclei were counter-stained with DAPI (blue). (B) BMPC incorporation was evaluated by counting double-positive cells (*P<0.01 versus untreated, **P<0.001 versus untreated and single therapies). (C) Endothelial tissue (green) and BMPCs (red) are identified in cross-sectional views of individual vessels from mice treated with combination therapy. n=4–5 per treatment group. GFPab indicates GFP antibody staining; HPF, high-power field.
Figure 5
Figure 5. MMP-9 expression after MI is enhanced by combination therapy, and the benefit of combination therapy is impaired in MMP-9–knockout mice
(A) One day and (B) 5 days after surgical induction of MI and treatment with or without phShh, AMD3100, or phShh-AMD3100 combination therapy, MMP-9 mRNA expression was measured in the non-infarcted, infarcted, and border-zone regions; control mice did not undergo surgical MI (*P<0.05 versus untreated). (C) MMP-9 protein expression was summarized by counting MMP-9–positive cells (*P<0.01 versus untreated, **P<0.05 versus AMD3100 or phShh). (D) LVEFs were measured before MI and 7, 14, and 28 days afterward in wild-type FVB and MMP-9–knockout FVB mice treated with or without phShh-AMD3100 combination therapy (*P<0.05 versus MMP-9–knockout with combination therapy). mRNA measurements were normalized to endogenous 18S rRNA expression. Panels A–C: n= 3–5 per treatment group; panel D: n=7–9 per treatment group. HPF indicates high-power field.
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