Combinatorial G-CSF/AMD3100 treatment in cardiac repair after myocardial infarction

Abstract

Aims: Several studies suggest that circulating bone marrow derived stem cells promote the regeneration of ischemic tissues. For hematopoietic stem cell transplantation combinatorial granulocyte-colony stimulating factor (G-CSF)/Plerixafor (AMD3100) administration was shown to enhance mobilization of bone marrow derived stem cells compared to G-CSF monotherapy. Here we tested the hypothesis whether combinatorial G-CSF/AMD3100 therapy has beneficial effects in cardiac recovery in a mouse model of myocardial infarction.

Methods: We analyzed the effect of single G-CSF (250 µg/kg/day) and combinatorial G-CSF/AMD3100 (100 µg/kg/day) treatment on cardiac morphology, vascularization, and hemodynamics 28 days after permanent ligation of the left anterior descending artery (LAD). G-CSF treatment started directly after induction of myocardial infarction (MI) for 3 consecutive days followed by a single AMD3100 application on day three after MI in the G-CSF/AMD3100 group. Cell mobilization was assessed by flow cytometry of blood samples drawn from tail vein on day 0, 7, and 14.

Results: Peripheral blood analysis 7 days after MI showed enhanced mobilization of white blood cells (WBC) and endothelial progenitor cells (EPC) upon G-CSF and combinatorial G-CSF/AMD3100 treatment. However, single or combinatorial treatment showed no improvement in survival, left ventricular function, and infarction size compared to the saline treated control group 28 days after MI. Furthermore, no differences in histology and vascularization of infarcted hearts could be observed.

Conclusion: Although the implemented treatment regimen caused no adverse effects, our data show that combinatorial G-CSF/AMD therapy does not promote myocardial regeneration after permanent LAD occlusion.

Figure 1. Mobilized peripheral blood cells after MI.

Circulating white blood cells were counted before (day 0), 7 and 14 days after MI. (upper panel) G-CSF and combinatorial G-CSF/AMD treatment enhances white blood cell numbers 7 days after MI (*p<0.05 G-CSF day 7 vs. day 0, p<0.075 G-CSF/AMD day 7 vs. day 0). Flow cytometry analysis on peripheral blood mononuclear cells was done before (day 0) and 7 and 14 days after induction of MI in control MI, G-CSF and G-CSF/AMD treated mice. (middle panel) The absolute numbers of circulating c-Kit⁺Sca-1⁺ double positive HPC were not different between control MI and drug treated groups. The HSC fraction was significantly increased in the control and G-CSF/AMD group (*p<0.05 vs. day 0), but not in G-CSF treated mice (middle panel inset). (lower panel) Flk-1⁺Sca-1⁺ double positive EPC mobilization peaked 7 days after MI in drug treated mice (p<0.054 G-CSF day 7 vs. day 0; p<0.071 G-CSF/AMD day 7 vs. day 0). EPC fractions were increased upon drug treatment, but did not reach statistical significance (lower panel inset). Data represent means ± SEM. (n>10 per group).*p<0.05.

Figure 2. Cumulative Kaplan-Meier survival analysis.

Kaplan-Meier survival curve of control MI and drug-treated mice during the observation period of 28 days after MI. Treatment of mice with G-CSF or G-CSF/AMD did not improve the (A) overall survival and did not alter (B) the mortality of mice that survived the first 4 days after MI.

Figure 3. TTC and Masson trichrome staining of infarcted cardiac tissue for assessment of infarction size and fibrosis.

Infarction size expressed as percentage of left ventricular area of control MI and drug treated mice assessed by TTC (A, B) and Masson trichrome (C) staining 28 days after MI. (D, E) Masson trichrome staining of sequential heart sections of control MI, G-CSF and G-CSF/AMD treated mice reveals no difference in left ventricular dilation, infarction size and fibrosis.

Figure 4. Cardiac histology of infarcted hearts 28 days after MI.

Overview of Masson trichrome stained heart section (upper panel) and higher magnification images (lower panels) of border zone (BZ), infarcted region (IF) and remote area (RA). Images show no evident alteration of collagen deposition in designated areas between treatment groups. Bar: 100 µm.

Figure 5. Cardiac vascularization 28 days after MI.

Blood vessel formation was analyzed by immunfluorescence staining with specific antibodies against endothelial (CD31/PECAM-1) and smooth muscle (α-smooth muscle actin) cells (A). Comparable density of (B) CD31/PECAM-1 and (C) smooth muscle actin positive vascular structures in the remote area (RA, upper panel), border zone (BZ, middle panel) and infarcted area (IF, lower panel) among control MI and drug-treated groups. Data represent means ± SEM. (n = 5−8 hearts per group).