Human CD34+ cells in experimental myocardial infarction: long-term survival, sustained functional improvement, and mechanism of action

Abstract

Rationale: Human CD34(+) cells have been used in clinical trials for treatment of myocardial infarction (MI). However, it is unknown how long the CD34(+) cells persist in hearts, whether the improvement in cardiac function is sustained, or what are the underlying mechanisms.

Objective: We sought to track the fate of injected human CD34(+) cells in the hearts of severe combined immune deficiency (SCID) mice after experimental MI and to determine the mechanisms of action.

Methods and results: We used multimodality molecular imaging to track the fate of injected human CD34(+) cells in the hearts of SCID mice after experimental MI, and used selective antibody blocking to determine the mechanisms of action. Bioluminescence imaging showed that injected CD34(+) cells survived in the hearts for longer than 12 months. The PET signal from the injected cells was detected in the wall of the left ventricle. Cardiac MRI showed that left ventricular ejection fraction was significantly improved in the treated mice compared to the control mice for up to 52 weeks (P<0.05). Furthermore, treatment with anti-alpha4beta1 showed that generation of human-derived cardiomyocytes was inhibited, whereas anti-vascular endothelial growth factor (VEGF) treatment blocked the production of human-derived endothelial cells. However, the improvement in cardiac function was abolished only in the anti-VEGF, but not anti-alpha4beta1, treated group.

Conclusions: Angiogenesis and/or paracrine effect, but not myogenesis, is responsible for functional improvement following CD34(+) cells therapy.

Figures

Figure 1

Long-term BLI of TGL+-CD34+ in SCID mice. A, The bioluminescent signal in the heart was superimposed on a photograph of a SCID mouse for the indicated time points after CD34+ cell injection (representative mouse). B, BLI intensity in SCID mice injected with CD34+ cells is significantly higher than the mice received PBS injection over a 52-week time period. BLI intensity was assessed by measuring the photon flux from ROI drawn over the precordium (Data are expressed in means ± SE, n=7/group **P<0.01 and *P<0.05).

Figure 2

Localization of transplanted CD34+ cells in the peri-infarct area of the heart is revealed by co-registration of MRI, micro-CT and micro-PET. A1, Three dimension rendering of micro-CT to show anatomy and viewing angle for (A2-3) micro-PET maximum intensity projections (MIP) after registration. PET MIPs demonstrate graft-related uptake and other non-specific (i.e., normal) uptake in various organs. A3, Localizer for the slice shown in B. Tissue slices using CT (B1), MRI (B3) and PET (B5) after registration. Coregistration of CT/MRI and MRI/PET are shown in (B2) and (B4), respectively.

Figure 3

Evaluation of cardiac function using MRI. A, Representative sequential images of the ES and ED volumes from a CD34+ cell-transplanted mouse and a control mouse over 25 weeks. B, Dot graph of the LVEF in control mice versus CD34+ cell-transplanted mice over a 52-week time period. There is a significant difference between groups for LVEF at each time point (Data are expressed in means ± SE, n=7/group, except for week 52, NS: no significance; **P<0.01 and *P<0.05).

Figure 4

In vivo antibody treatments inhibit myogenesis/angiogenesis and affect cardiac function induced by injection of CD34+ cells into mice after MI. A, Schematic of the experimental design. All animals underwent baseline MRI scanning before the experiments. All animals, except those in the MRI control group, which were used to monitor LVEF without any perturbation, had experimental MI on day 0. Anti-α4β1, anti-VEGF, and the respective isotype control antibodies were injected i.p. 30 min before MI. The heart was removed 8 weeks after MI and analyzed by FACS. B, Anti-α4β1, but not anti-VEGF, antibodies inhibited the formation of human-derived cardiomyocytes (HLA+ /troponin T+), as determined by FACS analysis. C, Only anti-VEGF inhibited the formation of human-derived endothelial cells (HLA+/VE-cadherin+). D, Anti-VEGF, but not anti- α4β1, antibodies diminished the effect on the improvement in the LVEF caused by the injection of human CD34+ cells. E, Treatment with anti- α4β1 or anti-VEGF antibodies did not affect LVEF following MI without cell therapy (Data are expressed in means ± SE, n=4/group, **p<0.01; *p<0.05).