Bone Marrow-Derived B Cells Preserve Ventricular Function After Acute Myocardial Infarction
Traci T. Goodchild, PhD*, ,
Keith A. Robinson, PhD,
Wenxin Pang, MD*,,
Fernando Tondato, MD, PhD,
Jianhua Cui, MD,
Johnail Arrington, MS*,
Lisa Godwin, AS,
Mark Ungs, BS, MBA*,
Nadia Carlesso, MD, PhD ,
Nadine Weich, PhD*,
Mark C. Poznansky, MD, PhD ,
Nicolas A.F. Chronos, MD*,,*
* AC Therapeutics, Inc., Norcross, Georgia
Saint Joseph's Translational Research Institute (formerly American Cardiovascular Research Institute), Atlanta, Georgia
Indiana School of Medicine, Indianapolis, Indiana
Infectious Diseases Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
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Abstract
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Objectives: In view of evidence that mature cells play a role in modulating the stem cell niche and thereby stem cell potential and proliferation, we hypothesized that a mature bone marrow (BM) mononuclear cell (MNC) infusion subfraction may have particular potency in promoting hematopoietic or resident stem cell-induced cardiac repair post-infarction.
Background: Treatment of acute myocardial infarction (MI) with BM MNC infusion has shown promise for improving patient outcomes. However, clinical data are conflicting, and demonstrate modest improvements. BM MNCs consist of different subpopulations including stem cells, progenitors, and differentiated leukocytes.
Methods: Stem cells (c-kit+) and subsets of mature cells including myeloid lineage, B and T-cells were isolated from bone marrow harvested from isogeneic donor rats. Recipient rats had baseline echocardiography then coronary artery ligation; 1 x 106 cells (enriched subpopulations or combinations of subpopulations of BM MNC) or saline was injected into ischemic and ischemic border zones. Cell subpopulations were either injected fresh or after overnight culture. After 2 weeks, animals underwent follow-up echocardiography. Cardiac tissue was assayed for cardiomyocyte proliferation and apoptosis.
Results: Fractional ventricular diameter shortening was significantly improved compared with saline (38 ± 3.2%) when B cells alone were injected fresh (44 ± 3.0%, p = 0.035), or after overnight culture (51 ± 2.9%, p < 0.001), or after culture with c-kit+ cells (44 ± 2.4%, p = 0.062). B cells reduced apoptosis at 48 h after injection compared with control cells (5.7 ± 1.2% vs. 12.6 ± 2.0%, p = 0.005).
Conclusions: Intramyocardial injection of B cells into early post-ischemic myocardium preserved cardiac function by cardiomyocyte salvage. Other BM MNC subtypes were either ineffective or suppressed cardioprotection conferred by an enriched B cell population.
Key Words: stem cells • myocardial infarction • cell therapy • regenerative medicine • myocardial repair
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Abbreviations and Acronyms
| | BM = bone marrow | | BrdU = 5-bromo-2'-deoxyuridine pellets | | HSC = hematopoietic stem cell | | LAD = left anterior descending artery | | LV = left ventricle/ventricular | | MI = myocardial infarction | | MNC = mononuclear cell | | NHSC = nonhematopoietic stem cell |
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Heart failure is the leading cause of morbidity and mortality in the Western world, affecting approximately 5 million people in the U.S. (1) and at least 10 million people in Western Europe (2). Heart transplantation remains the best available treatment option for end-stage heart disease; however, donor availability is limited. A potential alternative approach to the treatment of end stage heart disease is cell therapy in which bone marrow (BM) stem cells or mononuclear cells (MNCs) are injected into sites of cardiac injury. Currently, BM is the most frequent source of cells used for cardiac repair in clinical trials and has been shown to induce modest improvements in cardiac function in patients with acute myocardial infarction (MI) (3–5) and chronic ischemia (6–9).
While the mechanisms for improvement remain unclear, cardiac injection of autologous BM cells in recovery from an acute MI is an attractive therapeutic option. Potential mechanisms include transplanted cell differentiation into cardiac myocytes (10,11), fusion with resident myocytes (12–14), cytokine-supported recruitment of circulating progenitor cells (15,16), and secretion of beneficial paracrine factors (17–20). Clinical results from this novel approach have been mixed, and pre-clinical studies vary in terms of therapeutic cell types, administration methods, timing of cell delivery, and analytical methods. The optimal BM MNC population that induces maximal cardiac repair remains unclear.
BM contains a complex assortment of progenitor cells, including hematopoietic stem cells (HSCs), mesenchymal stem cells, and multipotential adult progenitor cells, along with lineage-positive cells such as monocytes, basophils, eosinophils, and B and T lymphocytes. Furthermore, differentiated hematopoietic cells including T and B lymphocytes have been previously shown to contribute to tissue repair and regeneration, and T cells in particular have been shown to influence the HSC niche (20). The aim of this study was to examine the possibility that a unique mature subpopulation of BM cells that, either alone or in combination with HSC subpopulations, was primarily beneficial for repair of infarcted myocardium. The objective was to evaluate left ventricular (LV) function in rats that received intramyocardial injection of various cell subpopulations from the BM, administered at the time of left anterior descending coronary artery ligation. We hypothesized that by removing certain inhibitory or noncontributory mature leukocyte populations, the beneficial effects of cell therapy in the setting of acute MI could be potentiated. Our results demonstrate that a B cell-enriched subfraction induces maximal therapeutic effect during recovery from acute MI.
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Methods
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The Saint Joseph's Research Institute is accredited by the Association for Accreditation and Advancement of Laboratory Animal Care. Experimental animal use conformed to National Institutes of Health and American Heart Association guidelines and was approved by the Institutional Animal Care and Use Committee of the Saint Joseph's Research Institute, in accordance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication no. 85-23, National Academy Press, Washington, DC, revised 1996).
BM harvest and cell preparation.
Donor male Sprague-Dawley rats were anesthetized with ketamine 80 mg/kg with xylazine 2.0 mg/kg and euthanized via exsanguination. Femurs and tibias were harvested and flushed with Dulbecco's modified essential medium (Gibco, Grand Island, New York) supplemented with 2% fetal bovine serum (Gibco), penicillin 100 U/ml (Gibco), and streptomycin (100 µg/ml Gibco). The BM was collected and diluted 1:2 with Dulbecco's phosphate-buffered saline then layered onto a Ficoll density gradient (Histopaque-1077, Sigma, St. Louis, Missouri). The buffy coat was collected and washed with Dulbecco's phosphate-buffered saline then subjected to the Miltenyi Biotech (Auburn, California) cell separation procedure. BM MNCs were washed twice in cold buffer (phosphate-buffered saline, pH 7.2, 0.5% bovine serum albumin and 2 mmol/l ethylenediaminetetraacetic acid) then depleted of non-B cells by a negative selection procedure. BM cells were incubated with antibodies specific for mature lineage (Lin) markers for: T cells (CD3, clone G4.18, BD Pharmingen, San Jose, California), helper/inducer T cells (CD4, clone OX-35, BD Pharmingen), suppressor/cytotoxic T cells (CD8, clone X8, Antigenix America, Huntington Station, New York), monocytes (CD11b/c, clone OX-42, BD Pharmingen), and neutrophils (Granulocyte, clone RP-1, BD Pharmingen) and further incubation with phycoerythrin-labeled magnetic microbeads (Miltenyi Biotech). The unlabeled BM MNC fraction (Lin negative, Lin–) was then subjected to positive selection for B cells (CD45RA, clone OX-33, BD Pharmingen) or HSCs (Lin-c-kit+, HSC, c-kit, clone H-300, Santa Cruz Biotechnologies, Santa Cruz, California). While Lin-c-kit+ consist of very primitive cells including stem cells, for simplicity we will refer to this subset as HSC. Nonhematopoietic stem cells (NHSC) were isolated by the addition of CD45RA to the negative selection cocktail followed by positive selection with c-kit and considered to be Lin-CD45RA-c-kit+-selected cells.
For B cell characterization studies, CD45RA-positive cells (21) were stained with markers for lymphocytes (CD45R, clone HIS24, BD Pharmingen), thymocytes (CD5, clone OX-19, BD Pharmingen), IgM (clone MARM-4, Serotec, Raleigh, North Carolina) and IgD (clone MARD3, Research Diagnostics, Concord, Massachusetts). Cell subpopulations were analyzed by flow cytometry (FACSCalibur, BDIS, San Jose, California). Cell viability was assessed using Trypan blue exclusion method.
To determine the impact of storage on cell-mediated cardiac effects as a prelude to possible clinical translation, we used cells that were cultured overnight. Cells were incubated at 37°C with 5% CO2 in Dulbecco's modified essential medium with 2% fetal bovine serum and penicillin/streptomycin either alone or in coculture using the Boyden chamber system (Falcon, Becton Dickinson Labware, Franklin Lakes, New Jersey). Before injection, cells were washed in phosphate-buffered saline then resuspended in sterile normal saline.
Model of MI and injections of BM cell populations.
Male Sprague-Dawley rats ( 250 to 300 g, Charles River Laboratories, Raleigh, North Carolina) were sedated, intubated, and anesthetized with inhalant isoflurane (2%). After surgical preparation and drape, using sterile technique and equipment, a left lateral thoracotomy was performed, and the left anterior descending artery (LAD) was isolated and ligated using sterile 6-0 prolene. Cardiac ischemia was confirmed by visible blanching in the anteroapical LV along with ST-segment elevation on electrocardiogram (ECG). After ligation of the LAD and confirmation of infarction, animals received a total of 106 cells (5 x 105 of each cell population) in 4 injections of 20 µl each intramyocardially in the ischemic LAD territory using a 30-G Hamilton syringe. Control animals received saline (4 injections of 20 µl each). The chest was closed using sterile sutures (4-0 vicryl), and the animal recovered post-operatively.
Assessment of wall motion by echocardiography.
Transthoracic echocardiography (ATL HD5000 with a CL 15-7 MHz probe, Phillips Technologies, Bothell, Washington) was obtained on all animals immediately before opening the chest for surgery, and repeated on all animals 2 weeks later (22–24) before sacrifice. A subset of animals underwent coronary artery ligation followed by immediate echocardiographic assessment after chest closure. A 2-dimensional short-axis view of the LV was acquired at midpapillary and apical levels, and M-mode tracings recorded to allow delineation of wall thickness and motion in infarcted and noninfarcted territories. Regional wall motion was examined per published criteria (25). The M-mode tracings were analyzed by an experienced echocardiographer who was blinded to treatment group assignment. Relative anterior and posterior wall thickness, and LV internal dimension was measured from at least 3 consecutive cardiac cycles, and an average value obtained. Endocardial and midwall, fractional LV diameter shortening were considered to represent LV systolic function.
Histological assessment of infarct size, cellular proliferation, apoptosis, and B cell retention.
After euthanization, hearts were perfusion rinsed with saline then perfusion fixed with 4% paraformaldehyde via aortic cannula. Hearts were cut into 5 transmural cardiac sections, embedded in paraffin, then processed for infarct size, cellular proliferation, or cellular apoptosis. Sections 6- to 8-µm thick were cut on a rotary microtome, adhered to glass slides, and adjacent or near-adjacent sections were stained with picric acid fuchsin to measure infarct size. For infarct measurement, prominent picrosirius red-stained LV areas were traced from low-magnification microscopic images (Image Pro Plus, Media Cybernetics, Rockville, Maryland) and expressed as a percentage of total LV wall area.
To assess cellular proliferation over a 2-week period, a subset of animals was implanted with 5-bromo-2'-deoxyuridine pellets (BrdU) (5 mg/pellet, 21-day release, Innovation Research of America, Sarasota, Florida) subcutaneously at the time of LAD ligation and B cells (n = 10), B cells cultured overnight at 37°C (n = 10), and B cells cocultured with NHSC (n = 4) transplantation. Sham-operated (no ligation, n = 5) and ligation plus saline injection (n = 6) animals were included as control animals. At 2 weeks, hearts were harvested and processed as noted in the previous text followed by deparaffinization, antigen retrieval (100°C for 30 min, antigen retrieval solution, DAKO, Carpentaria, California), and blocking with hydrogen peroxide (3%) and normal goat serum (Vector Laboratories, Burlingame, California). Sections were incubated with horseradish peroxidase-conjugated rat-specific antibody against BrdU (DAKO) and visualized using 3,3'-diaminobenzidine (Vector Laboratories). Double immunostaining was performed to determine the cellular phenotype of the proliferating cells. Sections were also stained with BrdU as described in the preceding text along with various markers for endothelial cells (von Willebrand factor, Serotec) or for cardiac myocytes (sarcomeric actin, clone 5C5, Abcam, Cambridge, Massachusetts).
In order to measure cellular apoptosis, a subset of animals underwent induction of MI followed by injections of saline (n = 6) or with B cells (n = 6) or B cell cocultured with NHSCs (n = 4). These animals were then euthanized at 48 h post-infarction. Sections were prepared as noted in the preceding text then processed according to the manufacturer's instructions by terminal nucleotidyl nick-end labeling, TUNEL, assay (DeadEnd Colorimetric TUNEL System Promega, Madison, Wisconsin).
Retention of injected B cells was assessed at 2 weeks, and tissue sections were processed as described in the preceding text. Sections were incubated with CD45RA (clone OX-33, BD Pharmingen) followed by incubation with horseradish peroxidase-labeled secondary antibody then visualized using 3,3'-diaminobenzidine (Vector Labs). All nuclei were counterstained with hematoxylin.
Statistical analysis.
Continuous variables are presented as mean ± SEM. The effect of cell treatment on dependent variables was assessed either by analysis of variance with Fisher least significant difference post hoc analysis or with Student t tests, as appropriate. A critical value of p < 0.05 was considered to indicate a significant treatment effect or difference between groups.
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Results
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Rat survival and confirmation of cardiac ischemia after coronary artery ligation.
A total of 125 male Sprague-Dawley rats (8 to 10 weeks old) underwent coronary artery ligation; 15 rats died within 24 h of surgical induction of MI (overall animal mortality was 12%). Cardiac ischemia was confirmed in all rats by visible blanching in the anteroapical LV along with ST-segment elevation on ECG (Online Fig. 1).
Flow cytometric characterization of BM-derived B cells.
The rat leukocyte-common antigen (CD45R) found on thymocytes and T and B lymphocytes are heterogenic in both molecular and antigenic structure and antibody binding. It has been shown that the OX-33 clone (CD45RA) antibody only binds to B cells and thus the CD45RA antibody was selected for separation of an enriched B cell population from rat BM. Flow cytometric analysis of BM-derived CD45RA-selected B cells (Fig. 1) illustrates that the B cell population was relatively pure as determined by staining with antibodies towards cytotoxic T cells (Fig. 1A) (CD8; 5 ± 4%), T cells (Fig. 1B) (CD3; 1 ± 0.1%), helper T cells (Fig. 1C) (CD4; 2 ± 0.4%), and neutrophils (Fig. 1D) (12 ± 11%). The purity of the other cell populations, including T cells, monocytes, neutrophils, tested was greater than 95% (data not shown).
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Figure 1 Purity and Characterization of Selected B Cells From Rat Bone Marrow
Flow cytometric analysis of bone marrow-derived CD45RA-positive B cells revealed that the injected population of CD45RA selected B cells were relatively free of other lineage-positive bone marrow cells including cytotoxic T cells (CD8+) (B), natural killer T cells (CD3) (C), helper T cells (CD4) (D), and neutrophils (E). Isotype control is shown in A. Flow cytometric analysis of bone marrow-derived CD45RA-positive B cells confirmed the majority of the B cells are CD45R-positive lymphocytes (G). IgM-positive immature B cells (H), IgG-positive mature B cells (I), and a minority of early c-kit+ positive pro-B cells (L). The bone marrow-derived B cell fraction did not contain CD5-positive marginal cells (J). Isotype control for CD45R, IgM, IgG, and CD5 antibodies is shown in F, and isotype control for c-kit is shown in K. Representative flow cytometry data are shown.
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BM-derived CD45RA+ B cells were characterized by flow cytometry and reveal that approximately one-half of the cells also express the CD45R marker (Fig. 1F) (47 ± 16%). Many of the CD45RA+ B cells were immature B cells (Fig. 1G) (IgM+; 21 ± 11%) and mature B cells (Fig. 1H) (IgG; 17 ± 11%). A very small proportion of these cells were considered to be mature marginal B cells (Fig. 1I) (CD5–; 1 ± 0.1). A very small proportion of CD45RA+ B cells were also positive for the stem cell marker c-kit (Fig. 1G) (1 ± 0.1%).
Using flow cytometric analysis, characterization of HSCs and NHSCs revealed that HSCs were positive for c-kit (34 ± 0.17%), positive for CD45RA (36 ± 0.17%), and positive for panCD45 (33 ± 0.17%). In addition, NHSCs were positive for c-kit (48 ± 0.06%), positive for CD45RA (48 ± 0.06%), and positive for panCD45 (46 ± 0.04%).
Effect of intramyocardial injections of isolated BM populations on LV diameters and fractional diametric shortening.
When saline was injected into the infarcted myocardium, LV function as reflected by LV short-axis fractional ventricular diameter shortening (Fig. 2) was reduced compared with the sham condition (38 ± 3.2%, n = 15 vs. 53 ± 2.7%, n = 9, respectively; p < 0.001). Injections of HSCs or coinjections of HSCs with B cells were not associated with functional improvement compared with saline control injections. However, a significant improvement in diametric shortening occurred after injections of B cells alone (44 ± 3.0%, n = 23, p = 0.035) and cultured B cells (51 ± 2.9%, n = 11, p < 0.001) when compared with injections of saline. Additionally, there was a functional improvement with injections of B cells after overnight coculture with NHSCs compared with injections of saline, but this did not reach statistical significance (44 ± 2.4%, n = 14, p = 0.062). No other BM cell subpopulation was associated with improved LV function including treatment with coinjections of HSCs with T cells (29 ± 4.4%, n = 6, p = 0.828), HSCs with monocytes (27 ± 6.0%, n = 6, p = 0.101), and HSCs with neutrophils (25 ± 2.2%, n = 6, p = 0.571).
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Figure 2 Echocardiographic Fractional Diametric Shortening
Two weeks after transplantation of B lineage and stem cell populations into infarcted rat myocardium, the surviving animals underwent follow-up echocardiographic assessment. Representative M-mode echocardiography is shown in A. B is a graph of normal (shaded squares, n = 9), immediate post-ligation (shaded circles, n = 9), sham operated (no ligation, open squares, n = 15), and ligation plus saline injection (open circles, n = 15) animals were included as control animals. Solid triangles represent animals injected with hematopoietic stem cells (HSC) (n = 4); solid diamonds represent animals injected with HSC coinjected with B cells (n = 5); solid circles represent B cell-injected animals (n = 24). B cells cultured overnight at 37°C (n = 11) are represented by solid squares, and B cells cocultured with nonhematopoietic stem cells (NHSCs) cultured overnight at 37°C are represented by patterned squares (n = 18). Each data point represents 1 animal, and horizontal bars are the mean ± SE. Significant differences in the mean values are denoted by *p < 0.05 using analysis of variance with post-hoc analysis using Fisher least significant difference.
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Effect of intramyocardial injections of isolated BM populations on infarct size, cellular proliferation, apoptosis, and localization of B cells.
After euthanization, hearts were perfusion-rinsed with saline, perfusion-fixed, then stained for collagen to measure infarct size (Online Fig. 2). Injection of BM-derived B cells did not affect infarct size and was not significantly different from saline-treated animals (p = 0.356): saline (18.89 ± 3.85%, n = 9); B cells (17.89 ± 1.85%, n = 23); cultured B cells (15.06 ± 2.16%, n = 12); and B cells after overnight coculture with NHSCs (14.01 ± 1.74%, n = 18). A small amount of collagen deposition was observed in the no ligation animals most probably due to the tissue trauma associated with the passage of the needle and ligature through the myocardium (2.54 ± 1.51%, n = 12).
In order to explore the mechanism(s) mediating the protective effects of B cells on myocardial function, we determined cumulative cellular proliferation over the 2-week period within infarcted regions of the myocardium. Animals received subcutaneous BrdU administration concomitant with coronary artery ligation and were terminated 2 weeks later. Immunohistochemical staining of BrdU-labeled proliferating cells was performed on the cardiac section with the greatest infarct, and positively BrdU-stained cells were counted within the infarct and border zones. There were no significant differences in cellular proliferation detected between control animals injected with saline or those in which coronary artery ligation was not performed (4.3 ± 1.2%, n = 6 vs. 4.0 ± 1.1%, n = 5, respectively; p = 0.840), B cells injected (6.3 ± 1.6%, n = 10, p = 0.345), cultured B cell injected (6.8 ± 1.9%, n = 10, p = 0.332), and B cells that had been cocultured overnight with NHSCs (7.5 ± 1.9%, n = 4 vs. 4.3 ± 1.2%, n = 6, p = 0.123) rats (Fig. 3). When the data were analyzed by using raw values rather than the mean values, significant increases were detected in cellular proliferation after transplantation of cultured B cells (6.8 ± 1.86%, n = 10, p = 0.032) and B cells that had been cocultured overnight with NHSCs (7.5 ± 1.90%, n = 4; p = 0.002), compared with controls injected with saline (4.3 ± 1.17%, n = 6). There were no significant differences in cellular proliferation detected comparing control animals injected with saline with no ligation (4.3 ± 1.20% vs. 4.0 ± 1.09%, n = 6, respectively; p = 0.565), B cell injected (6.3 ± 1.57%, p = 0.076) rats. Assessment of the cellular phenotype performed by staining with an endothelial cell marker and a cardiac myocyte marker did not show colocalization with BrdU-labeled proliferating cells (Fig. 4).
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Figure 3 Cellular Proliferation, Preservation of Cardiac Tissue, and B Cell Localization After B Cell Injection Into Infarcted Myocardium
Animals were implanted with a 5-bromo-2'-deoxyuridine pellet at the time of infarction and cell injection for determination of cumulative cellular proliferation. After 2 weeks, animals were euthanized and hearts processed for assessment of 5-bromo-2'-deoxyuridine pellet incorporation into the nuclear DNA of proliferating cells of ligation plus saline-treated animals (A), or B cells cocultured with nonhematopoietic stem cells (B), B cell (C) and cultured B cell (D) injections. Positive cells were identified using antibodies directed against a 5-bromo-2'-deoxyuridine pellet. All nuclei were stained with hematoxylin. The percentage of cells staining positive for 5-bromo-2'-deoxyuridine pellet was determined by comparison with the total number of cells visualized in a minimum of 5 fields (200x magnification). Scale bars are 100 µm. Sham operated (no ligation, open squares, n = 5) and ligation plus saline injection (open circles, n = 6) animals were included as control animals (E). Solid circles represent animals injected with B cells (n = 10). B cells cultured overnight at 37°C (n = 10) are represented by solid squares, and B cells cocultured with nonhematopoietic stem cells cultured overnight at 37°C (n = 4) are represented by patterned squares. Each data point represents the percentage of 5-bromo-2'-deoxyuridine-positive cells counted in a single field, and the horizontal bar represents the mean ± SE. Significant differences in mean values between control and experimental groups are denoted by *p < 0.05 using the Student t test, 2-tailed.
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Figure 4 Proliferating Cellular Phenotype in Cell-Treated and Saline Control Cells
Double immunohistochemical detection of proliferating 5-bromo-2'-deoxyuridine-positive cells with von Willebrand factor-positive endothelial cells (A to D) and cardiac myocytes (E to H) within the border zone of rat infarcted myocardium was determined 2 weeks after transplantation of saline control cells (A and E), B cells (B and F), cultured B cells (C and G), and B cells cocultured with nonhematopoietic stem cells (D and H). Positive 5-bromo-2'-deoxyuridine-labeled cells were visualized using 3, 3'-diaminobenzidine in which all positive cells are stained brown and von Willebrand factor and sarcomeric actin staining was visualized using Vector red. Double positive staining is denoted by white arrows. All nuclei were stained with hematoxylin. Instrument magnification 400x. Scale bars are 50 µm.
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We proposed that early myocardial salvage by B cells after infarction was another potential mechanism of cardiac protection. Animals were terminated 48 h after coronary artery ligation, and cell injection procedure and cellular apoptosis were determined by immunohistochemical staining using the TUNEL assay. Apoptotic cells were counted in each cardiac section, and the data presented as the mean generated from all cardiac sections. Animals treated with B cells cocultured with NHSCs had significantly fewer apoptotic cells 48 h after MI (Fig. 5) compared with saline-treated animals (3.5 ± 0.6%, n = 4 vs. 4.2 ± 1.2%, n = 6, respectively; p = 0.035). There was no significant difference detected in cellular apoptosis between rats treated with saline compared with animals treated with B cells (4.2 ± 1.2%, n = 6 vs. 2.8 ± 0.8%, respectively, n = 6, p = 0.580). When we compared the data from the single cardiac section that had the highest number of apoptotic cells from the saline-treated control animals (12.6 ± 2.0%, n = 6), we found that B cell treatment resulted in significantly less apoptotic cells (5.7 ± 1.2%, n = 6, p = 0.005) compared with saline control animals. In this setting, no significant difference in cellular apoptosis was detected in the cardiac sections from animals treated with B cells cocultured with NHSCs (6.8 ± 1.0%, n = 4, p = 0.060). Immunohistochemical analysis of sections of infarcted myocardium injected with saline, B cells, cultured B cells, or B cells that had been cocultured with NHSCs revealed that CD45RA+ cells are retained within the myocardial infarcted region 2 weeks after injection (Fig. 6). Staining of sections with isotype control antibody revealed no positive staining of sequential sections (data not shown).
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Figure 5 Preservation of Cardiac Tissue and B Cell Localization After B Cell Injection Into Infarcted Myocardium
Histological assessment of cellular apoptosis was performed using the TUNEL assay. Animals underwent sham (no ligation) (A) operation or ligation with B cell (B) or B cell cocultured with nonhematopoietic stem cell (NHSC) (C) injections. Animals were euthanized at 48 h after surgery, and hearts were processed for evaluation of apoptosis. Apoptotic cells within the peri-infarct domain were quantitated by visualization using 200x magnification. Scale bars are 100 µm. All nuclei were stained with hematoxylin. The percentage of cells staining positive for apoptosis was determined by comparison with the total number of cells visualized in a minimum of 5 fields. Ligation plus saline injection (open circles, n = 6) animals were included as control animals (D). Solid circles represent animals injected with B cells (n = 6), and B cells cocultured with NHSCs cultured overnight at 37°C (n = 4) are represented by solid squares. Each data point represents the percentage of TUNEL-positive cells counted in a single field in cardiac section with the largest infarction and horizontal bar is the mean ± SE. Significant differences in the mean values between control and experimental groups are denoted by *p < 0.05 using the Student t test, 2-tailed.
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Figure 6 B Cell Retention After Injection Into Infarcted Rat Myocardial Tissue
Immunohistochemical detection of CD45RA-positive cells within the border zone of rat infarcted myocardium 2 weeks after transplantation of saline control cells (A), B cells (B), cultured B cells (C), and B cells cocultured with nonhematopoietic stem cell (D). Tissue sections were stained with rat-specific antibody against CD45RA and visualized using 3, 3'-diaminobenzidine in which all positive cells are stained brown (arrows). All nuclei were stained with hematoxylin. Instrument magnification 400x. Scale bars are 50 µm.
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Discussion
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We demonstrated that rats receiving intramyocardial injections of B cells at the time of ischemic injury induced by coronary artery ligation consistently showed improved LV functional outcome compared with sham-injected control animals. This beneficial effect was maintained when B cells were cultured overnight alone or in the presence of NHSCs. No other BM cell subpopulation or unfractionated total BM cell population tested was associated with cardiac functional improvement. We do acknowledge that echocardiography parameters used to assess heart function are still controversial in small animal models; different methods such as magnetic resonance imaging to assess LV function could be used to support our results. Histological evidence supported the view that the positive effect of B cell transplantation is a form of early myocardial salvage, since apoptosis was reduced and cell proliferation was augmented during the follow-up period. These effects were modulated positively or negatively by storage conditions and coinjection of B cells with other subfractions of BM-derived cells, respectively.
It has been reported that BM-derived lineage-negative c-kit+ cells can transdifferentiate into cardiomyocyte-like cells as well as endothelial and smooth muscle phenotypes when injected into infarcted mice hearts contributing to both neovascularization and regeneration of the damaged wall (10). Similar experiments performed using the same population of BM-derived cells or unfractionated BM cells (26–28) failed to reproduce these findings. Instead, the transplanted cells adopted a mature hematopoietic phenotype in the myocardial scar (26–28) accompanied by statistically significant improvement in cardiac function (26). Of particular note, the BM-derived lineage-negative c-kit+ cells had differentiated into alternate cell types including B cells. Functionally, Balsam et al. (27) reported improved fractional shortening, decreased LV chamber dimensions at end-diastole and end-systole though no significant differences in infarct size and hemodynamic parameters in BM-derived c-kit+ cell-treated versus control mice at 6 weeks. While we did not detect these same functional results when BM-derived c-kit+ cells were injected into infarcted rat hearts, we did observe the same pattern of functional improvement in ventricular chamber dimensions without corresponding improvements in hemodynamic measurements and reduction in infarct size when B cells were injected. Thus, it is interesting to speculate that in the study by Balsam et al. (27) the small but significant improvements in cardiac function were due to the influence of the BM-derived c-kit+ differentiated B cells. These findings support the view that a mature hematopoietic cell such as a B cell can augment cardiac function possibly through the effect of paracrine factors it releases that influence the cardiac stem cell niche.
B cells play a diverse role in the pathogenesis of variety of different diseases including autoimmune disorders and have become a major therapeutic target in these illnesses. In addition to well-established contributions of B cells to antibody-mediated physiological and pathological processes, B cells also play important regulatory and potentially pathogenic roles through antibody-independent mechanisms including antigen presentation, T cell activation and polarization, dendritic cell regulation, and cytokine and chemokine production (29). Novobrantseva et al. (30) recently reported that mature B cells play a role in CCl4-induced hepatic fibrosis in mice in an antibody-independent manner. The authors demonstrated that B cell-deficient mice had decreased collagen deposition and were able to clear apoptotic cells faster than wild-type mice. In addition, they examined whether B cell regulation of fibrosis required Ig production. Mice with normal B cell numbers that lack or had low serum Ig were found to develop a similar degree of fibrosis when compared with wild-type mice indicating that B cells have an impact on liver fibrosis in an antibody-independent manner. In our studies, BM-derived B cells had no apparent effect on cardiac fibrosis as there was no difference in infarct size between cell-treated and saline control animals. In other work, adoptive transfer of B cells into mice deficient of B and T cells (recombination activating gene-1) conferred renal protection after ischemia reperfusion injury (20). B cells have also been shown to be protective against atherosclerosis progression, manifested when splenectomy severely aggravated atherosclerosis in apoE–/– mice were fed a cholesterol-enriched diet (31). The same authors reported that enhanced atherosclerosis after splenectomy could be abrogated by passive transfer of splenic cells isolated from apoE–/– mice and that this rescue was more complete if the cells were obtained from donors that were older and, therefore, had greater disease. They hypothesized that these B cells could be more "educated" and were able to transfer specific, enhanced immunocompetence that provided protection from disease. Finally, Wassmann et al. (32) reported that transfer of spleen-derived B cells significantly improved endothelium-dependent vasodilation in apoE–/– mice. These datasets support our thesis that B cells mediate antibody-independent effects, including the preservation of ventricular function after ischemic injury.
Clinical significance.
Phase I clinical trials using cell therapy for acute MI and chronic ischemia have shown reasonable improvement in cardiac function. BM cells used in the clinic include hematopoietic and mesenchymal stem cells, and endothelial progenitor cells. Stem and progenitor cells must be cultured for extended periods to generate sufficient cell numbers for therapeutic benefit thereby limiting clinical utility. Additionally, appropriate facilities for long-term culture of autologous stem/progenitor cells are not readily and widely available. BM-derived B cells are lineage-positive and can be isolated from autologous sources in large numbers; this might eliminate the need for in vitro expansion to accomplish therapeutic benefit. In the future, B cell transplantation may prove clinically useful for cardiovascular diseases.
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Conclusions
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BM-derived B cells improve cardiac function after transplantation into infarcted rat myocardium. Mechanisms for B cell-dependent improvements include a reduction in cardiac apoptosis in conjunction with increased cellular proliferation. Further, more detailed studies of cellular and molecular mechanisms, correlations to other species, and the definition of the optimal B cell subpopulation to mitigate against cardiac ischemic damage are warranted.
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Appendix
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For supplementary figures, please see the online version of this article.
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Acknowledgments
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The authors thank Melissa Moon for her technical assistance and Dr. Kevin Viel, SJTRI Senior Research Statistician, for his statistical analysis of raw datasets. They also thank The Jim Moran Foundation for their generous support of this study.
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Footnotes
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Funding for this project was provided by AC Therapeutics, Norcross, Georgia, and The Jim Moran Foundation, Deerfield Beach, Florida. Drs. Chronos and Ungs are founders of AC Therapeutics and Drs. Goodchild and Pang and Johnail Arrington are or were employees of AC Therapeutics, Inc. Drs. Carlesso, Poznansky, and Weich are paid consultants for AC Therapeutics, Inc. Grant support for research was provided to Drs. Robinson, Tondato, and Cui by AC Therapeutics, Inc. Drs. Poznansky and Chronos contributed equally to this work.
* Reprint requests and correspondence: Dr. Nicolas A. F. Chronos, Saint Joseph's Research Institute, 5673 Peachtree-Dunwoody Road, Atlanta, Georgia 30342 (Email: nchronos{at}sjha.org).
Manuscript received February 10, 2009;
revised manuscript received July 13, 2009,
accepted August 21, 2009.
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