Integrin β-3 is required for the attachment, retention and therapeutic benefits of human cardiospheres in myocardial infarction

Abstract

Cardiovascular diseases remain the leading causes of death worldwide. Stem cell therapy offers a promising option to regenerate injured myocardium. Among the various types of stem cells, cardiosphere cells represent a mixture of intrinsic heart stem cells and supporting cells. The safety and efficacy of cardiosphere cells have been demonstrated in recent clinical trials. Cell-matrix interaction plays an important role in mediating the engraftment of injected stem cells. Here, we studied the role of integrin β-3 in cardiosphere-mediated cell therapy in a mouse model of myocardial infarction. Our results indicated that inhibiting integrin β-3 reduced attachment, retention and therapeutic benefits of human cardiospheres in mice with acute myocardial infarction. This suggests integrin β-3 plays an important role in cardiosphere-mediated heart regeneration.

Keywords: cardiac stem cells; cardiospheres; integrin; myocardial infarction.

Figure 1

Antigenic phenotypes of the cells in cardiospheres. (A) Schematic showing the process of deriving cardiospheres from human myocardial tissue samples; (B) represent fluorescent micrographs showing the expressions of CD105 and CD90 in cardiospheres; (C) represent fluorescent micrographs showing the expressions of CD105 and ckit in cardiospheres; (D) represent fluorescent micrographs showing the expressions of CD31 and CD34 in cardiospheres; (E) represent fluorescent micrographs showing the expressions of CD31 and CD45 in cardiospheres. Scale bars = 50 μm.

Figure 2

ITB3 inhibition is non‐toxic to cardiospheres. (A, B) Cell death staining (using EthD) reveals that ITB3 inhibition does not increase the numbers of dead cells in the cardiospheres. In addition, ITB3 inhibition does not affect cardiosphere numbers (C) and sizes over the time (D). Scale bars = 20 μm. n = 3 for each experiment.

Figure 3

ITB3 inhibition reduces cardiosphere attachment in vitro and retention in vivo. (A) Schematic showing the attachment assay. (B) Treatment with ITB3 antibodies reduces cardiosphere attachment to fibronectin‐coated surface. (C) Schematic showing LAD ligation and intramyocardial injection of cardiospheres in mice. (D) Ex vivo fluorescent imaging showing DiI‐labelled cardiosphere retention in the heart. (E) Cell retention measured by sex‐mismatch PCR. n = 3 for each experiment. * indicated P < 0.05 when compared to the other group.

Figure 4

ITB3 inhibition blunts the functional benefits of cardiosphere treatment. (A) Masson's trichrome staining revealed scar (blue) versus healthy (pink) myocardium. (B, C) Cardiosphere treatment (black bars) effectively increases viable myocardium (B) but reduces scar size (C), as compared to vehicle control (white bars). Such benefits are abolished in the animals treated with ITB3‐inhibited cardiospheres (red bars). (D) LVEFs at baseline (4 hrs after MI) are indistinguishable among all groups. (E) 3 weeks after, the LVEFs for the control (white bar) and ITB3‐inhibited cardiosphere group (red bar) are still indistinguishable, while the cardiosphere‐treated group (black bar) exhibits larger LVEF. n = 5 animals per group. *indicated P < 0.05 when compared to the “AMI + PBS” group. # indicated P < 0.05 when compared to the “AMI + CS + ITB3 ab” group.

Figure 5

ITB3 inhibition impairs cardiosphere engraftment in the mouse heart. (A) Representative fluorescent micrographs showing HNA‐positive cells (green) in the mouse heart 3 weeks after injection. (B) Quantitation of the percentage of HNA‐positive cells. n = 3 for each experiment. *indicated P < 0.05 when compared to the other group. Scale bars = 100 μm.