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Cathelicidin Related Antimicrobial Peptide (CRAMP) Enhances Bone Marrow Cell Retention and Attenuates Cardiac Dysfunction in a Mouse Model of Myocardial Infarction

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Abstract

Background

Acute myocardial infarction (MI) and the ensuing ischemic heart disease are approaching epidemic state. Unfortunately, no definitive therapies are available and human regenerative therapies have conflicting results. Limited stem cell retention following intracoronary administration has reduced the clinical efficacy of this novel therapy. Cathelicidin related antimicrobial peptides (CRAMPs) enhance chemotactic responsiveness of BMSPCs to low SDF-1 gradients, suggesting a potential role in BMSPCs engraftment. Here, we assessed the therapeutic efficacy of CRAMPs in the context of BMSPCs recruitment and retention via intracardiac delivery of CRAMP-treated BMSPCs or CRAMP-releasing hydrogels (HG) post-AMI.

Methods

For cell transplantation experiments, mice were randomized into 3 groups: MI followed by injection of PBS, BMMNCs alone, and BMMNCs pre-incubated with CRAMP. During the in vivo HG studies, BM GFP chimera mice were randomized into 4 groups: MI followed by injection of HG alone, HG + SDF-1, HG + CRAMP, HG + SDF-1 + CRAMP. Changes in cardiac function at 5 weeks after MI were assessed using echocardiography. Angiogenesis was assessed using isolectin staining for capillary density.

Results

Mice treated with BMMNCs pre-incubated with CRAMP had smaller scars, enhanced cardiac recovery and less adverse remodeling. Histologically, this group had higher capillary density. Similarly, sustained CRAMP release from hydrogels enhanced the therapeutic effect of SDF-1, leading to enhanced functional recovery, smaller scar size and higher capillary density.

Conclusion

Cathelicidins enhance BMMNC retention and recruitment after intramyocardial administration post-AMI resulting in improvements in heart physiology and recovery. Therapies employing these strategies may represent an attractive method for improving outcomes of regenerative therapies in human studies.

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Abbreviations

AMI:

Acute Myocardial Infarction

BM:

Bone Marrow

BMMNCs:

Bone Marrow mononuclear cells

CRAMP:

Cathelicidin-related antimicrobial peptides

EF:

Ejection Fraction

ESV:

End Systolic Volume

LAD:

Left Anterior Descending Artery

LV:

Left Ventricle

PB:

Peripheral Blood

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Acknowledgments

Dr. Abdel-Latif is supported by the University of Kentucky Clinical and Translational Science Pilot Award (UL1TR000117), the UK COBRE Early Career Program (P20 GM103527) and the NIH Grant R56 HL124266. Dr. Nagareddy is supported by the NIH Pathway to Independence Award (1K99HL122505-01). Dr. Ye is supported by the T32 grant (HL091812). Dr. Ratajczak is supported by NIH grants 2R01 DK074720 and R01HL112788. Christopher B. Rodell is supported by an American Heart Association Predoctoral Fellowship. Jason A. Burdick is supported by an American Heart Association Established Investigator Award.

Author information

Authors and Affiliations

  1. Gill Heart Institute and Division of Cardiovascular Medicine, University of Kentucky, Lexington, KY, USA

    Yuri M. Klyachkin, Amr Idris, Himi Tripathi, Shaojing Ye, Prabha Nagareddy, Ahmed Asfour, Rahul Annabathula & Ahmed Abdel-Latif

  2. Lexington VA Medical Center and Saha Cardiovascular Research Center, University of Kentucky, 741 South Limestone, BBSRB B349, Lexington, KY, 40536-0509, USA

    Yuri M. Klyachkin, Amr Idris, Himi Tripathi, Shaojing Ye, Prabha Nagareddy, Ahmed Asfour, Rahul Annabathula & Ahmed Abdel-Latif

  3. Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, USA

    Christopher B. Rodell & Jason A. Burdick

  4. The Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA, USA

    Erhe Gao

  5. Stem Cell Biology Institute, James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA

    Mariusz Ratajczak

Authors
  1. Yuri M. Klyachkin
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  2. Amr Idris
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Contributions

Yuri Klyachkin: conception and design, data analysis and interpretation and manuscript writing. Amr Idris: conception and design, data analysis and interpretation and manuscript writing. Christopher B. Rodell: conception and design, synthesis and preparation of hydrogels, data analysis and interpretation and manuscript writing. Himi Tripathi: collection and/or assembly of data and data analysis and interpretation. Shaojing Ye: collection and/or assembly of data and data analysis and interpretation. Prabakara Nagareddy: collection and/or assembly of data and data analysis and interpretation. Ahmed Asfour: collection and/or assembly of data. Erhe Gao: collection and/or assembly of data. Rahul Annabathula: collection and/or assembly of data. Jason A Burdick: conception and design, data interpretation and manuscript writing. Mariusz Ratajczak: conception and design, data analysis and interpretation and manuscript writing. Ahmed Abdel-Latif: conception and design, data analysis and interpretation, provision of study material or patients, financial support and manuscript writing.

Corresponding author

Correspondence to Ahmed Abdel-Latif.

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Electronic supplementary material

Supplemental Figure 1

Preincubation with CRAMP increases the retention of transplanted BM cells after myocardial infarction. Representative flow cytometry plots demonstrating the higher percentage of GFP+ BM cells in the heart 4 days after AMI and transplantation (Panel A). Quantitatively, GFP+ cells were 3–4 time higher when preincubated with CRAMP compared to controls (Panel B). Interestingly, BM cells showed higher proliferative state compared to cardiac cells as evident from the percentage of cells in G2M phase (Panel C) and BrdU positive cells (Panel D). Values are mean ± SEM (*P < 0.05, **P < 0.001 vs. HG, N = 3 animals/group). BMMNCs; bone marrow mononuclear cells, CRAMP; cathelicidin related antimicrobial peptide, GFP; green fluorescence protein. (PNG 49 kb)

High resolution image (EPS 416 kb)

Supplemental Figure 2

Limited numbers of GFP+ bone marrow cells identified in the heart 5 weeks after transplantation. Representative confocal images demonstrating single and rare GFP (green) labelled bone marrow cells in cardiac sections at 5 weeks after AMI in mice treated with CRAMP-preincubated BMMNCs. Scale bars represent 50 μm. (PNG 203 kb)

High resolution image (EPS 3158 kb)

Supplemental Figure 3

CRAMP incubation enhances CXCR4 expression on bone marrow-derived stem cells. Quantitative analysis of the median fluorescence intensity (MFI) and geometric mean of CXCR4 expression, as assessed by flow cytometry, on Lin-/cKit+ bone marrow-derived cells at various time points after CRAMP incubation. CXCR4 (APC) signal intensity peaked at 3 h after CRAMP incubation. Values are mean ± SEM (*P < 0.05, **P < 0.01, **P < 0.001 vs. 1 h, N = 3). (PNG 30 kb)

High resolution image (EPS 351 kb)

Supplemental Figure 4

HG supplementation with SDF-1, HF or both enhances cardiomyocyte turnover. Representative confocal images demonstrating BrdU+ (white) cardiomyocytes (red) in the peri-infarct border (Panel A) and remote zone (Panel B) (yellow arrows) at 5 weeks after AMI in mice treated HG alone or supplemented with SDF-1 and/or CRAMP. Panel C-Quantitative analysis of the number of BrdU+ cardiomyocytes per field. Values are mean ± SEM (*P < 0.05, **P < 0.01 vs. HG, N = 3 animals/group). Scale bars represent 50 μm. (PNG 190 kb)

High resolution image (EPS 1548 kb)

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Klyachkin, Y.M., Idris, A., Rodell, C.B. et al. Cathelicidin Related Antimicrobial Peptide (CRAMP) Enhances Bone Marrow Cell Retention and Attenuates Cardiac Dysfunction in a Mouse Model of Myocardial Infarction. Stem Cell Rev and Rep 14, 702–714 (2018). https://doi.org/10.1007/s12015-018-9833-x

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