Original article
Hyperbaric oxygenation enhances transplanted cell graft and functional recovery in the infarct heart

https://doi.org/10.1016/j.yjmcc.2009.04.005Get rights and content

Abstract

A major limitation to the application of stem-cell therapy to repair ischemic heart damage is the low survival of transplanted cells in the heart, possibly due to poor oxygenation. We hypothesized that hyperbaric oxygenation (HBO) can be used as an adjuvant treatment to augment stem-cell therapy. Therefore, the goal of this study was to evaluate the effect of HBO on the engraftment of rat bone marrow-derived mesenchymal stem cells (MSCs) transplanted in infarct rat hearts. Myocardial infarction (MI) was induced in Fisher-344 rats by permanently ligating the left-anterior-descending coronary artery. MSCs, labeled with fluorescent superparamagnetic iron oxide (SPIO) particles, were transplanted in the infarct and peri-infarct regions of the MI hearts. HBO (100% oxygen at 2 ATA for 90 min) was administered daily for 2 weeks. Four MI groups were used: untreated (MI); HBO; MSC; MSC + HBO. Echocardiography, electro-vectorcardiography, and magnetic resonance imaging were used for functional evaluations. The engraftment of transplanted MSCs in the heart was confirmed by SPIO fluorescence and Prussian-blue staining. Immunohistochemical staining was used to identify key cellular and molecular markers including CD29, troponin-T, connexin-43, VEGF, α-smooth-muscle actin, and von Willebrand factor in the tissue. Compared to MI and MSC groups, the MSC + HBO group showed a significantly increased recovery of cardiac function including left-ventricular (LV) ejection fraction, fraction shortening, LV wall thickness, and QRS vector. Further, HBO treatment significantly increased the engraftment of CD29-positive cells, expression of connexin-43, troponin-T and VEGF, and angiogenesis in the infarct tissue. Thus, HBO appears to be a potential and clinically-viable adjuvant treatment for myocardial stem-cell therapy.

Introduction

Cell transplantation shows great promise for repair and restoration of heart function following myocardial infarction [1], [2]. However, the potential of cell-based therapy for myocardial tissue repair is limited by the death of transplanted cells, mostly within the first few days after transplantation in the infarct tissue, likely from a combination of ischemia (deprivation of nutrients and oxygen), inflammation, and apoptosis [3]. Several interventions, including the use of cells over-expressing pro-survival/anti-apoptotic proteins [4], [5], and pharmacological agents [6], have been reported to augment survival, function, and homing of the transplanted cells in the hostile ischemic environment [7]. Laflamme et al. used a “cocktail” of pro-survival factors to inhibit death-promoting pathways and upregulate survival-signaling. They reported a higher proportion of engraftment when compared to simpler interventions [8]. Recently, we have shown the beneficial effect of preconditioning cells with trimetazidine, an anti-ischemic drug, prior to cardiac cell therapy [9]. Most of the above mentioned approaches involved manipulation of the cells prior to, or during, transplantation in the infarct heart. However, to date, no post-transplantation strategies have been considered for promoting cellular engraftment.

Hypoxia is a formidable factor in the ischemic tissue that can lead to the production of oxygen free radicals. The hostile environment with persistent oxidative stress ultimately leads to apoptosis of the majority of the transplanted cells. Therefore, we considered a straightforward physiological approach to reduce the severity of hypoxia by hyperoxygenating the infarct tissue following cell transplantation. Hyperbaric oxygenation (HBO) is a safe, clinically-viable treatment that has been used as a primary therapy in patients with decompression sickness, arterial gas embolism and carbon-monoxide poisoning [10]. It is also used as an adjuvant therapy to promote wound healing [11], and for the treatment of various conditions, including ischemic injury [12]. HBO involves inhalation of 100% oxygen under greater-than-one atmospheric absolute (ATA) pressure. Such doses of oxygen have a number of beneficial biochemical, cellular, and physiologic effects [13]. HBO, administered at the onset of reperfusion in an open-chest rabbit model of myocardial ischemia–reperfusion injury, showed a significant reduction in infarct size [14]. More recent studies have shown that HBO attenuates myocardial injury via nitric oxide signaling [13], improves cardiac function in patients with acute myocardial infarction [15], and helps mobilization of stem cells by enhancing CXCR4 and VEGFR-2 in humans [16]. However, the efficacy of HBO as an adjuvant to cell therapy has not yet been studied.

We therefore hypothesized that HBO, when applied in conjunction with stem-cell therapy, would improve oxygenation in the infarct heart, leading to increased cell engraftment and cardiac function. Using a rat model of myocardial infarction, induced by permanent ligation of the left-anterior-descending (LAD) coronary artery, followed by transplantation of rat bone marrow-derived mesenchymal stem cells (MSCs), we have demonstrated a substantial increase in cell engraftment, reduction in infarct size, recovery of myocardial function, restoration of electrophysiological normalcy, and angiogenesis in cell-transplanted hearts subjected to HBO treatment.

Section snippets

Reagents

Dulbecco's Modified Eagle medium (DMEM), fetal bovine serum, penicillin, streptomycin, trypsin, sodium pyruvate, and phosphate-buffered saline (PBS) were purchased from Gibco BRL (Grand Island, NY). MTT (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) colorimetric assay kit, lactate dehydrogenase (LDH) assay kit, and Nuclear Fast Red (Kernechtrot) solution were obtained from Sigma (St. Louis, MO). DAPI (4,6-diamino-2-phenylindole) was from Invitrogen.

Effect of SPIO labeling on MSCs

MSCs were labeled with Dragon-green fluorescence-conjugated SPIO particles by co-incubation in culture for 24 h. Fluorescence microscopy and phase-contrast optical images confirmed uptake of SPIO particles by MSCs (Fig. 1). The cells were then examined for possible cytotoxicity induced by the uptake of SPIO particles. The labeled cells were kept in culture for 24 h, and subjected to standard cytotoxicity tests including PI-binding assay for nuclear viability, MTT assay for mitochondrial

Discussion

The results of the present study indicated the beneficial effects of HBO treatment in enhancing the engraftment of MSCs transplanted into an ischemic heart, leading to significant improvements in cardiac function, restoration of electrophysiological normalcy, and increased angiogenesis. MSCs are pluripotent, adult stem cells residing primarily within the bone marrow. Under in vitro conditions, MSCs can be induced to differentiate into cardiomyocyte-like cells by treating with 5-azacytidine [21]

Acknowledgments

This study was supported by grants from AHA (SDG 0930181N to MK) and NIH (R01 EB006153 to PK). We thank Dr. Chandan K. Sen and Ms. Lynn Lambert for advice and technical support for using the HBO chamber. We also thank Dr. Adriana Pedraza-Toscano for help with EKG data analysis.

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