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PRETREATMENT WITH ADULT PROGENITOR CELLS IMPROVES RECOVERY AND DECREASES NATIVE MYOCARDIAL PROINFLAMMATORY SIGNALING AFTER ISCHEMIA

Wang, Meijing*,†; Tsai, Ben M.*; Crisostomo, Paul R.*; Meldrum, Daniel R.*,†,‡

doi: 10.1097/01.shk.0000209536.68682.90
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ABSTRACT Cardiogenic shock from myocardial ischemia is the leading cause of death of both men and women. Although adult progenitor cells have emerged as a potential therapy for heart disease, reports indicate that transplanted adult progenitor cells may not differentiate into heart muscle. We hypothesized that pretreatment with adult progenitor cells may protect myocardium from acute ischemic damage. Treatment immediately before an ischemic event removes the possibility that differentiation to heart muscle may account for the observed effects. In the present study, we determined that adult progenitor cells from three different sources (human bone marrow, rat bone marrow, and human adipose tissue) immediately protect native myocardium against ischemia and decrease myocardial proinflammatory and proapoptotic signaling. Postischemic recovery of adult progenitor cell-pretreated hearts was significantly better than that of control hearts. This was correlated with a 50% decrease in proinflammatory cytokine production. The use of a differentiated cell control had no such effect. Therefore, adult progenitor cell pretreatment improved postischemic myocardial function, decreased myocardial production of inflammatory mediators, and limited proapoptotic signaling. These results represent the first demonstration that pretreatment with progenitor cells is myocardial protective. These findings may not only have mechanistic implications regarding the benefit of progenitor cells but may also have clinical therapeutic implications before planned ischemic events.

Departments of *Surgery and Cellular and Integrative Physiology and the Center of Immunobiology, Indiana University Medical Center, Indianapolis, IN

Received 20 Jul 2005; first review completed 2 Aug 2005; accepted in final form 16 Dec 2005

Address reprint requests to Daniel R. Meldrum, MD, Emerson Hall, Room 215, 545 Barnhill Drive, Indianapolis, IN 46202. E-mail: dmeldrum@iupui.edu.

This research was supported by the National Institutes of Health Grant R01GM70628 (DRM).

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INTRODUCTION

Despite the rapid development of sophisticated pharmacological and revascularization strategies, heart failure persists as the major cause of mortality in developed countries. Cardiogenic shock from myocardial ischemia and reperfusion (I/R) injury is the leading cause of death of both men and women in the United States. Postinfarct injection of progenitor cells into regions of necrotic myocardium has resulted in positive remodeling and the regeneration of viable myocardium (1). Surgically induced ischemia represents the unique situation where preinsult treatment options exist. Studies have demonstrated the potential benefit of progenitor cells when administered after injury; however, the study of cell therapy pretreatment is completely novel. Although the acute use of these cells for immediate differentiation is not yet possible, progenitor cells may possess properties that protect native tissue. Thus, pretreatment with progenitor cells may not only allow the ultimate resurrection of viable tissue after injury, but they may also act as a stabilizing/protective "helper cell" during I/R. If so, cellular therapy as a pretreatment strategy may have therapeutic implications that extend beyond myocardial I/R to multiorgan and varied-insult protection.

Controversy exists concerning the ability of adult progenitor cells to regenerate myocardium after ischemia-induced cardiac myocyte loss. Orlic et al.(1) reported that adult bone marrow stem cells (BMSCs), delivered into the myocardium, dramatically improved myocardial function 9 days later. The authors presented additional evidence that the enhanced function was caused by the generation of de novo myocardium. Beltrami et al.(2) reported that the heart contains its own source of stem cells that are self-renewing, clonogenic, and multipotent. When injected into ischemic heart tissue, the cells formed well-differentiated myocardium. However, two independent laboratories (3, 4) simultaneously reported that stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcted tissue. Adult progenitor cells do appear to protect native myocardium and improve function but, perhaps, not by transdifferentiation soon after an infarct. Dernbach et al.(5) reported that circulating progenitor cells possess potent antioxidant properties. Expression profiling of adult progenitor cells revealed characteristic expression of genes (termed "stemness") associated with enhanced DNA repair, up-regulated antioxidant enzymes, and increased detoxifier systems (5-7). Pretreating myocardium with adult progenitor cells before an I/R insult may allow one to determine (1) whether adult progenitor cells possess protective properties and (2) whether transdifferentiation is required for protection. Furthermore, several clinical pretreatment opportunities exist, including coronary artery bypass grafting or stenting operations. Although recent reports (3, 4) indicated that adult progenitor cells adopt mature hematopoietic fates, those studies did not address whether those cells were nevertheless protective. A pretreatment delivery protocol immediately followed by acute ischemia would essentially eliminate the chance that any adult progenitor cell effect would be the result of transdifferentiation. Surprisingly, the effect of adult progenitor cell pretreatment has not been examined.

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MATERIALS AND METHODS

Animals

Male Sprague-Dawley rats (weighing 280-300 g, Harlan, Indianapolis, Ind) were fed a standard diet and acclimated in a quiet quarantine room for 2 weeks before the experiments. The animal protocol was reviewed and approved by the Indiana Animal Care and Use Committee of Indiana University. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1985).

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Progenitor cells

Poietic human mesenchymal stem cell and poietic preadipocyte cell (PAC) were purchased from Cambrex Bio Science (Walkersville, NJ). Mesenchymal stem cells were harvested and cultured from normal human or rat bone marrow. Cells were positive for CD105, CD166, CD29, and CD44. Cells tested negative for CD14, CD34, and CD45. Primary human PAC were isolated from subcutaneous adipose tissue.

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Chemical detection of progenitor cells in the myocardium

Fluorescent chemical detection of progenitor cells in the myocardium was performed. Vybrant DiI cell-labeling solution (Molecular Probes, Eugene, Ore) was used to stain progenitor cells according to the manufacturer's instructions. After staining, cells were examined under fluorescence microscopy. DiI-labeled progenitor cells were infused into the rat heart after 15 min of equilibration. Heart sections were embedded into Histo Prep frozen tissue embedding media (FisherChemical, Fair Lawn, NJ), and frozen sections were examined to detect DiI-labeled infused cells within the myocardium. To examine whether equal distribution occurs in the different regions of the myocardium, triplicate sections were taken from the left anterior descending coronary artery, circumflex coronary artery, and right coronary artery territories. Cells that transferred to the interstitial space were counted and recorded as the respective cell type per high-powered field. All coronary effluent was collected during and after cell infusion, and cells that exited the heart were quantified. By these two methods, there was no difference in transcoronary transfer (or coronary effluent loss) of human or rat BMSCs, human PAC, or renal tubular epithelial cells.

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Myocardial function measurements

Male Sprague-Dawley rats were anesthetized and heparinized with an i.p. injection of sodium pentobarbital (Nembutal, 60 mg/kg) and heparin sodium (500 U, SoloPak Laboratories, Inc, Elk Grove Village, Ill). After sternotomy, hearts were rapidly excised into 4°C Krebs-Henseleit. The aorta was cannulated, and the heart was perfused (37°C) with oxygenated buffer within 45 s. Hearts were perfused in the isolated isovolumetric Langendorff mode (70 mmHg) with modified Krebs-Henseleit solution (in mmol: glucose, 5.5; sodium chloride, 119; calcium chloride, 1.2; potassium chloride, 4.7; sodium bicarbonate, 25; KHPO4, 1.18; magnesium sulfate, 1.17) and saturated with 95% O2/5% CO2, to achieve a PO2 of 440 to 460 mmHg, PCO2 of 39 to 41 mmHg, and pH of 7.39 to 7.41. The perfusion buffer was continuously filtered through a 0.45-μm filter to remove particle contaminants. A pulmonary arteriotomy and left atrial resection were performed before insertion of a water-filled latex balloon through the left atrium into the left ventricle. The preload volume (balloon volume) was held constant during the entire experiment to allow continuous recording of the left ventricular developed pressure (LVDP). The balloon was adjusted to a mean left ventricular end-diastolic pressure (LVEDP) of 8 mmHg (range, 6-10 mmHg = both the peak and the plateau of the LVEDP-LVDP curve) during the initial equilibration. Pacing wires were fixed to the right atrium and pulmonary outflow tract, and hearts were paced at approximately 6 Hz, 3 V, 2 ms (350 beats/min) throughout perfusion. Data were continuously recorded using a computerized MacLab 8 preamplifier/digitizer (AD Instruments, Inc, Milford, Mass) and an Apple G5 computer (Apple Computer, Inc, Cupertino, Calif). A 3-way stopcock above the aortic root was used to create global ischemia, during which the heart was placed in a 37°C degassed organ bath. Coronary flow was measured by collecting pulmonary artery effluent. The maximal positive and negative values of the first derivative of pressure (+dP/dt and −dP/dt, respectively) were calculated using PowerLab (ADInstruments, Inc., Colorado Springs, CO) software.

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Experimental design and groups

Hearts exposed to I/R alone (I/R, n = 5) underwent 20 min of equilibration followed by 25 min of global ischemia at 37°C and 40 min of reperfusion. Human BMSCs (mesenchymal), rat BMSCs, human PAC (Cambrex Bio Science, Walkersville, NJ), or differentiated renal tubular epithelial cells (LLC-PK1 cell line) were prepared in modified Krebs-Henseleit solution with a concentration of 1.5 × 106 cells/mL (based on preliminary data that showed that greater numbers of cells caused significant myocardial depression during infusion, but lower doses did not have a significant postischemic effect). One milliliter of cell solutions, namely, human mesenchymal adult progenitor cells (I/R + BMSC, n = 6), rat BMSCs (I/R + rat BMSC, n = 7), human PAC (I/R + PAC, n = 6), and differentiated renal tubular epithelial cells (I/R + DC, n = 5) were infused before I/R. Cells were delivered through a port above the aortic root at 0.2 mL/min (not recirculated) for 5 min after 15 min of equilibration immediately before the I/R insult.

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Reverse trancriptase-polymerase chain reaction

The rat hearts were harvested after the experiments ended (after 40 min of reperfusion). Total RNA was extracted from each heart's left ventricle using RNA STAT-60 (TEL-TEST, Friendswood, Tex). Total RNA at a concentration of 0.5 μg was subjected to complementary DNA (cDNA) synthesis using cloned AMV first-strand cDNA synthesis kit (Invitrogen Life Technologies, Carlsbad, Calif). cDNA from each sample was used for multiple polymerase chain reaction (PCR) for tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-1α, and IL-6 using message screen rat inflammatory cytokine multiplex PCR kits (Biosource, Camarillo, Calif). One negative control used distilled water instead of RNA sample and a second negative control used distilled water instead of reverse transcriptase to exclude the presence of genomic contaminants. Positive controls were included in the kit to verify appropriate expression of respective markers. PCR products were separated by electrophoresis on 2% agarose gel and stained with ethidium bromide. Densitometry was performed to assess relative quantity and represented as a ratio to glyceraldehyde-3-phosphate dehydrogenase.

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Western blotting

Western blot analysis was performed to measure p38 mitogen-activated protein kinase (MAPK) pathway and apoptosis-related proteins. Each heart's left ventricle was homogenized in cold buffer containing 20 mmol Tris (pH 7.5), 150 mmol sodium chloride, 1 mmol EDTA, 1 mmol ethyleneglycol-bis (2-aminoethyl ether)-N,N,N,N-tetraacetate, 1% Triton X-100, 2.5 mmol sodium pyrophosphate, 1 mmol β-glycerophosphate, 1 mmol sodium orthovanadate, 1 μg/mL leupeptin, 1 mmol phenylmethylsulphonylfluoride, and centrifuged at 12,000 rpm for 5 min. The protein extracts (30 μg/lane) were electrophoresed on a 12% Tris-hydrochloride gel from Bio-Rad and transferred to a nitrocellulose membrane, which was stained by naphthol blue-black to confirm equal protein loading. The membranes were incubated in 5% dry milk for 1 h and then incubated with the following primary antibodies: p38 MAPK antibody, phospho-p38 MAPK (Thr180/Tyr182) antibody, phospho-heat shock protein 27 (HSP27; Ser82) antibody (Cell Signaling Technology, Beverly, Mass), caspase-3 (H-277) antibody, and caspase-8 p20 (H-134) antibody (Santa Cruz Biotechnology, Santa Cruz, Calif), followed by incubation with horseradish peroxidase-conjugated goat antirabbit IgG secondary antibody and detection using Supersignal West Pico stable peroxide solution (Pierce, Rockford, Ill).

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TNF-α, IL-1, and IL-6 enzyme-linked immunosorbent assay

Myocardial TNF-α, IL-1β, and IL-6 in the cardiac tissue were determined by enzyme-linked immunosorbent assay (ELISA) using a commercially available ELISA set (R&D Systems, Inc, Minneapolis, Minn; BD Biosciences, San Diego, Calif). ELISA was performed according to the manufacturer's instructions. All samples and standards were measured in duplicate.

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Presentation of data and statistical analysis

All reported values are mean ± SEM. Data were compared using two-way analysis of variance with post hoc Bonferroni test or Student t test. A two-tailed probability value of less than 0.05 was considered statistically significant.

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RESULTS

Chemical detection of progenitor cells in the heart

DiI-labeled human BMSCs showed red fluorescence under fluorescent microscopy (Fig. 1A). The red fluorescence on the frozen section revealed that DiI-labeled BMSCs were embedded within the myocardium (Fig. 1B). To examine whether equal distribution occurs in different regions of the myocardium, triplicate sections were taken from the left anterior descending coronary artery, circumflex coronary artery, and right coronary artery territories. Cells that transferred to the interstitial space were counted and recorded as the respective cell type per high-powered field. All coronary effluent was collected during and after cell infusion, and cells that exited the heart were quantified (>95% of cells exiting the heart did so during cell infusion or during the first minute after the completion of cell infusion). By these two methods, there was no difference in transcoronary transfer of human or rat BMSCs, human PAC, or renal tubular epithelial cells (12.4 ± 2.1, 11.3 ± 1.7, 14 ± 3.9, 10.6 ± 3.4 cells per high-power field, respectively).

Fig. 1

Fig. 1

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Myocardial function

Twenty-five minutes of ischemia and 40 min of reperfusion resulted in a marked decrease (P < 0.05) in myocardial function, as demonstrated by a decreased LVDP (mmHg) in I/R alone, I/R + DC, I/R + PAC, and I/R + BMSC groups from 114.1 ± 6.21 to 28.7 ± 1.81, 108.6 ± 4.71 to 41.9 ± 7.89, 120.2 ± 5.87 to 82.1 ± 12.75, and 114.9 ± 9.62 to 83.5 ± 8.88, respectively (Fig. 1A). Infusion of adult progenitor cells before I/R injury led to an increase in the recovery of myocardial function after I/R injury. LVDP in I/R + PAC, I/R + BMSC, and I/R + rat BMSC groups after 40 min of reperfusion was higher than the I/R alone and I/R + DC groups (82.1 ± 12.75, 83.5 ± 8.88, 71 ± 6.9 vs. 28.7 ± 1.81 and 41.9 ± 7.89, respectively, P < 0.005; Fig. 2).

Fig. 2

Fig. 2

LVEDP was elevated in response to I/R, as shown in Figure 1B. Infusion of adult progenitor cells demonstrated lower LVEDP at each time point after I/R than I/R alone and I/R + DC.

Maximum +dP/dt and −dP/dt were impaired at the start of reperfusion. I/R alone and I/R + DC hearts demonstrated more depression of +dP/dt and increase of −dP/dt compared with those of adult progenitor cell treatment groups (Fig. 1, C and D).

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Myocardial tissue cytokine messenger RNA and protein

Myocardial tissue TNF-α levels were significantly lower in the BMSC- and PAC-treated groups (Fig. 3A). Similarly, infusion of adult progenitor cells before I/R injury resulted in more than a 50% decrease (52% in I/R + BMSC and 69% in I/R + PAC) in TNF-α messenger RNA (mRNA) levels compared with I/R alone (Fig. 4). Infusion of adult progenitor cells demonstrated lower IL-1β production in the myocardium subjected to I/R (Fig. 3B). IL-1β mRNA was decreased by 30% in the BMSC and PAC groups compared with that in I/R alone (Fig. 4). IL-6 protein levels were significantly lower in the BMSC- and PAC-treated groups after I/R (Fig. 3C). Infusion of BMSC or PAC before I/R injury resulted in a 21% and 30%, respectively, decrease in myocardial IL-6 mRNA levels (Fig. 4). Similarly, rat BMSC decreased TNF, IL-1β, and IL-6 production after I/R, whereas DC did not (Fig. 3).

Fig. 3

Fig. 3

Fig. 4

Fig. 4

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p38 MAPK, HSP27, caspase-3, and caspase-8

Figure 4 shows increased p38 MAPK phosphorylation in I/R alone hearts relative to adult progenitor cell-pretreated hearts. However, total p38 MAPK was similar between all groups. Phosphorylated HSP27, a downstream phosphorylation target of activated p38 MAPK, was similarly decreased by prior adult progenitor cell infusion (Fig. 4). Adult progenitor cell pretreatment decreased active caspase-3 and caspase-8 after I/R (Fig. 4).

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DISCUSSION

These results constitute the initial demonstration that pretreatment with adult progenitor cells protects native myocardium from injury. Pretreatment of myocardium with either BMSCs or adipocyte progenitor cells resulted in improved postischemic functional recovery, decreased myocardial tissue proinflammatory cytokine production, and decreased activation of p38 MAPK, HSP27, caspase-3, and caspase-8. These effects may be mediated by insulin-like growth factor 1, inasmuch as coculture experiments revealed that the insulin-like growth factor 1 antagonist αIR3 decreased the adult progenitor cells' anti-inflammatory effects.

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Pretreatment with adult progenitor cells protects myocardial function

To determine whether pretreatment with adult progenitor cells (from either bone marrow or adipocyte source) had any protective effect on myocardial function, we delivered adult progenitor cells into the myocardium via coronary infusion immediately before global warm ischemia of isolated rat hearts (Langendorff). Functional recovery was determined during reperfusion. Adult progenitor cell-pretreated hearts demonstrated significantly enhanced myocardial functional recovery when compared with hearts from any of the control groups (Fig. 1). In this regard, the use of a differentiated cell type as a control was critically important. Indeed, the same dose (cell number) of identically delivered renal tubular epithelial cells (LLC-PK1 cell line) did not result in enhanced myocardial functional recovery after ischemia. We also considered the possibility that the enhanced recovery was caused by a "preconditioning" effect (transient ischemia induced tolerance to sustained ischemia). Cell delivery resulted in a dose-dependent decline in coronary flow and, consequently, LVDP. Indeed, some capillary trapping of delivered cells may be required for transfer from the intravascular to the interstitial compartment. However, the differentiated-cell control, which showed a similar decrease in LVDP and coronary flow during infusion, did not protect the heart similarly. Also arguing against a preconditioning explanation for these observations is the fact that preconditioning requires complete ischemia for a minimum of 2 min, which did not occur here (8).

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Adult progenitor cell anti-inflammatory effects

These findings demonstrate that adult progenitor cell differentiation is not required for adult progenitor cells to improve myocardial function after an ischemic event. Thus, by what mechanism does this protection occur? Adult progenitor cells may release protective substances that limit apoptosis. Because Orlic et al.(1) administered adult progenitor cells early after the infarct, it is possible that the protective effect observed was caused by a limitation of myocardial apoptosis, which takes many hours to days of reperfusion to occur. Similarly, the results presented here may be caused by the potentially protective properties of adult progenitor cells themselves (9, 10). The transplanted adult progenitor cell, attempting to survive in injured tissue, faces an inflammatory environment. Adult progenitor cells may release substances that limit local inflammation to enhance their survival. Indeed, shock and multiple forms of trauma have been shown to elaborate proinflammatory cytokines, and these conditions have also been shown to promote the differentiation of progenitor cells (11-20). It is possible that there is an endogenous restorative effect of circulating progenitor cells that occurs after various forms of shock and trauma (5, 18, 21, 22).

We (11, 12) and others (23, 24) have identified myocardial tissue TNF-α and other proinflammatory mediators as important contributors to cardiac myocyte functional depression and apoptosis in heart and kidney after acute ischemia. Indeed, Rauscher et al.(25) demonstrated that BMSCs decreased serum IL-6 levels in an atherosclerosis progression model. Our results reveal that BMSC pretreatment limited myocardial TNF-α, IL-1β, and IL-6 mRNA and protein levels after I/R (Figs. 3 and 4).

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Intracellular proinflammatory and proapoptotic signaling

Decreased proinflammatory monokine production was associated with decreased p38 MAPK activation, a central intracellular signaling mediator of proinflammatory monokine production (Fig. 4). HSP27 phosphorylation, a downstream indicator of p38 MAPK activation, was similarly decreased (Fig. 4). Adult progenitor cells may release substance(s) that disrupt intracellular signaling processes, leading to myocardial proinflammatory cytokine production. Proinflammatory cytokines partly mediate I/R by directly suppressing myocardial function and by inducing myocardial apoptosis. In addition to myocardial necrosis, myocardial apoptosis, which may take many hours to days to occur, has been estimated to be a significant mechanism of cell loss after I/R (12). Proapoptotic signaling enzymes activated during the acute event may serve as a surrogate of later apoptosis. Indeed, activation of the proapoptotic caspase-8 and caspase-3 was decreased after adult progenitor cell pretreatment (Fig. 5). Although currently unknown, the exact mechanism by which adult progenitor cells limit ischemia-induced inflammation and subsequent apoptosis may be related to their great capacity to release certain anti-inflammatory growth factors (26).

Fig. 5

Fig. 5

The results of the present study are somewhat surprising. The adult progenitor cell may have properties that allow it to act more as a helper cell to the native myocardium, bolstering its resistance to ischemia. It appears as though these results may help reconcile some findings that may otherwise appear contradictory. Planned ischemic events, such as those that occur during cardiac surgery, angioplasty, or transplantation, may allow an additional opportunity to observe the potential clinical benefit of adult progenitor cell pretreatment.

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ACKNOWLEDGMENTS

The authors thank Dr. Loren J. Field for his helpful suggestions during the preparation of this manuscript.

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Keywords:

Myocardial ischemia; stem cells; inflammatory signaling; cell-based therapy

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