Interleukin-6 Mediates Post-Infarct Repair by Cardiac Explant-Derived Stem Cells

Abstract

Although patient-sourced cardiac explant-derived stem cells (EDCs) provide an exogenous source of new cardiomyocytes post-myocardial infarction, poor long-term engraftment indicates that the benefits seen in clinical trials are likely paracrine-mediated. Of the numerous cytokines produced by EDCs, interleukin-6 (IL-6) is the most abundant; however, its role in cardiac repair is uncertain. In this study, a custom short-hairpin oligonucleotide lentivirus was used to knockdown IL-6 in human EDCs, revealing an unexpected pro-healing role for the cytokine.

Methods: EDCs were cultured from atrial appendages donated by patients undergoing clinically indicated cardiac surgery. The effects of lentiviral mediated knockdown of IL-6 was evaluated using in vitro and in vivo models of myocardial ischemia.

Results: Silencing IL-6 in EDCs abrogated much of the benefits conferred by cell transplantation and revealed that IL-6 prompts cardiac fibroblasts and macrophages to reduce myocardial scarring while increasing the generation of new cardiomyocytes and recruitment of blood stem cells.

Conclusions: This study suggests that IL-6 plays a pivotal role in EDC-mediated cardiac repair and may provide a means of increasing cell-mediated repair of ischemic myocardium.

Keywords: Cell therapy; interleukin-6; myocardial infarction; stem cells.

Figure 1

All sub-populations within EDCs secrete IL-6 production while a minority express IL-6Rα. (a) Representative immunohistochemical images of magnetically sorted EDCs demonstrating the widespread expression of IL-6 by EDCs. (b) Flow cytometry demonstrating the expression of IL-6 within EDCs as a function of LTS score (n=3/LTS group). *p≤0.05. (c) ELISA of conditioned media from magnetically sorted EDCs after 48 h exposure to hypoxic (1%) low serum (1% serum) conditions (n=3/LTS group). *p≤0.05. (d) Flow cytometry of EDCs for expression of IL-6Rα (n=3/LTS group). *p≤0.05.

Figure 2

IL-6 knockdown promotes angiogenesis and reduces the recruitment of circulating cells in vitro. (a) IL-6 production in un-selected SCR and shIL-6 transduced EDCs at multiplicities of infection (MOIs) of 10, 20 and 30 compared to NT EDCs (n=3/condition). *p≤0.05. (b) Analysis of the number of CACs that migrated through a transwell filter after exposure to conditioned media sourced from NT, SCR or shIL-6 EDCs from low and high LTS score patients (n=3/group). *p≤0.05. (c) Cumulative tubular length analysis of human umbilical vein endothelial cells tubule formation within a cytokine depleted matrigel assay after exposure to conditioned media sourced from NT, SCR or shIL-6 EDCs from low and high LTS score patients (n=3/group). *p≤0.05 vs low LTS score EDCs. (d) Multiplex assay for FGF (fibroblast growth factor), HGF (hepatocyte growth factor), SCF (stem cell factor), SDF-1α (stromal-derived factor 1 alpha), VEGF (vascular endothelial growth factor) and IL-6 (interleukin-6) within media conditioned by NT, SCR or shIL-6 EDCs from low and high LTS score patients (n=3/group). *p≤0.05 vs low LTS score EDCs.

Figure 3

Effects of IL-6 production by EDCs on treatment of myocardial infarction and transplanted cell survival. (a) Comparison of the effect of IL-6 production by EDCs sourced from patients with low LTS (NT n=6 and shIL-6 n=8) and high LTS (NT n=6 and shIL-6 n=9) scores on the echocardiographic ejection fraction of immunodeficient mice as compared to myocardial injection of the negative vehicle control (PBS, n=8). *p<0.05. (b) Quantitative PCR for human alu sequences demonstrating the influence of lentiviral transduction on the modest long-term engraftment of transplanted cells (high LTS). *p≤0.05. (c) Representative images and quantification of arteriole density (isolectin B4+ cells, red) within equivalent histological sections taken from immunodeficient mice treated with NT (n=6) and shIL-6 (n=6) transduced EDCs sourced from patients with high LTS scores. Size bar = 50 µm. (d) Representative images and quantification of scar burden within histological sections of immunodeficient mice after treatment with NT EDCs sourced from low (n=5) or high (n=5) LTS score patients compared to shIL-6 transduced EDCs sourced from low (n=5) or high (n=6) LTS score patients or negative vehicle control (n=3). *p≤0.05; Size bar = 1000 µm.

Figure 4

Effect of IL-6 production by EDCs on myocytes and cardiomyocyte proliferation. (a) Representative myocardial sections from the peri-infarct region of a SCID mouse 7 days after myocardial infarction demonstrating expression of IL6Rα on cardiomyocytes (arrows, cTnT+/IL6Rα+/DAPI+). Also shown is qPCR analysis of border zone, infarct zone and distal (posterior wall) tissue from SCID mice 2 weeks post LC ligation and 1 week post injection of NT or shIL-6 EDCs for expression of Bax, p53 or Bcl-2 (n=4/group). *p<0.05 vs. NT EDCs. (b) Representative images at 40x (left) or 100x oil (right) of myocardial sections from the peri-infarct region of mice 21 days after transplant of SCR or shIL-6 high LTS score EDCs demonstrating proliferating cardiomyocytes (arrows, cTnT+/BrdU+/DAPI+). Quantification of the number of peri-infarct cTnT+/BrdU+ cells in equivalent histological sections taken from mice treated with NT or shIL-6 transduced high LTS score EDCs (n=4/group).

Figure 5

Effect of IL-6 production by EDCs on CFs and macrophages. (a) Representative myocardial sections from the peri-infarct region of mice 7 days after myocardial infarction demonstrating the expression of IL6Rα on CFs (arrows, DDR2+/IL6Rα+/DAPI+) and macrophages (arrows, CD68/IL6Rα/DAPI+). Scale bar = 50 µm. (b) Effect of co-culture of SCR, NT or shIL-6 transduced high LTS score EDCs with murine CFs on murine MMP-9 production (n=3/group). *p<0.05. (c) Effect of SCR, NT and shIL-6 EDCs on myofibroblast content within remodeled hearts (n=5/group). *p<0.05. (d) SCR, NT and shIL-6 EDCs on markers of macrophage polarization within remodeled hearts (n=5/group). *p<0.05.