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Keywords:

  • Bone marrow stromal cells;
  • Homing;
  • Chemokine receptors;
  • Chemokine

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

MSCs have received attention for their therapeutic potential in a number of disease states, including bone formation, diabetes, stem cell engraftment after marrow transplantation, graft-versus-host disease, and heart failure. Despite this diverse interest, the molecular signals regulating MSC trafficking to sites of injury are unclear. MSCs are known to transiently home to the freshly infarcted myocardium. To identify MSC homing factors, we determined chemokine expression pattern as a function of time after myocardial infarction (MI). We merged these profiles with chemokine receptors expressed on MSCs but not cardiac fibroblasts, which do not home after MI. This analysis identified monocyte chemotactic protein-3 (MCP-3) as a potential MSC homing factor. Overexpression of MCP-3 1 month after MI restored MSC homing to the heart. After serial infusions of MSCs, cardiac function improved in MCP-3-expressing hearts (88.7%, p < .001) but not in control hearts (8.6%, p = .47). MSC engraftment was not associated with differentiation into cardiac myocytes. Rather, MSC engraftment appeared to result in recruitment of myofibroblasts and remodeling of the collagen matrix. These data indicate that MCP-3 is an MSC homing factor; local overexpression of MCP-3 recruits MSCs to sites of injured tissue and improves cardiac remodeling independent of cardiac myocyte regeneration.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

MSCs are under active investigation as a potential therapy for a number of disease states with the hope of restoring disparate organ systems such as the pancreas [1], heart [2], bone marrow [3], and brain [4, [5]6]. MSCs appear not only to be able to differentiate into a number of organ-specific cell types, but also to modulate the local microenvironment of injured tissues [6] and modulate the immune system [7, 8]. Although this cell population holds great promise, little is known about how MSCs traffic/home to injured tissue. Recently, a subpopulation of MSCs was shown to express the stromal-derived factor-1 (SDF-1) chemokine receptor CXCR4 [1, 3], and some studies have suggested that this population may therefore home in response to SDF-1 expression [1, 9]. Interestingly, the relatively low level of inhibition of MSC homing to the bone marrow by blocking CXCR4 in these studies and that fact that the majority of human MSCs do not express CXCR4 suggest that other chemokines are involved [3].

We have previously demonstrated that there is transient homing of hematopoietic stem cells (HSCs) to the heart after myocardial infarction (MI). The transient nature of HSC homing is due, at least in part, to the transient expression of SDF-1 [10]. Whereas HSCs seem not to transdifferentiate into cardiac tissue [11, 12], MSCs can acquire some properties of cardiomyocytes in vitro [13]. Because MSCs have also been shown to home to the heart early after MI [9], we hypothesized that, similarly, there are chemokine(s) temporally secreted by the myocardium which can attract MSCs. The current study was undertaken to identify potential MSC homing factor(s) and to test their effect on myocardial function if stably expressed within the border zone at a time remote from MI.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

Left Anterior Descending Ligation

The Animal Research Committee approved all animal protocols, and all animals were housed in the AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) animal facility of the Cleveland Clinic Foundation. Ligation of the left anterior descending (LAD) artery in an inbred strain of rat (Lewis rat) was performed as described previously [10, 14]. Briefly, animals were anesthetized with intraperitoneal ketamine and xylazine and intubated and ventilated with room air at 80 breaths per minute using a pressure-cycled rodent ventilator (RSP1002; Kent Scientific Corporation, Torrington, CT, http://www.kentscientific.com). Anterior wall MI was achieved with the aid of a surgical microscope (M500; Leica Microsystems GmbH, Wetzlar, Germany, http://www.leica-microsystems.com).

Cell Preparation and Delivery

Rat bone marrow was isolated by flushing the femurs with 0.6 ml of Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Clumps of bone marrow were gently minced with a 20-gauge needle. Cells were separated by Percoll density gradient. The cells were centrifuged for 10 minutes at 260g and washed with three changes of phosphate-buffered saline (PBS) containing 100 U/ml penicillin and 100 g/ml streptomycin (Invitrogen). The washed cells were then resuspended and plated in DMEM-LG (low glucose) (Invitrogen) with 10% fetal bovine serum (FBS) and 1% antibiotic and antimycotic (Invitrogen). The cells were incubated at 37°C. Nonadherent cells were removed by replacing the medium after 3 days. Fourteen days later (passage 4), cells were harvested by incubation with 0.05% trypsin and 2 mM EDTA (Invitrogen) for 5 minutes. MSC cultures were depleted of CD45+ cells by negative selection using 10 μl each of primary phycoerythrin (PE)-conjugated mouse anti-rat CD45 antibodies per 106 cells (catalog number 554,878; BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). PE-positive cells were negatively selected using the EasySep PE selection kit according to the manufacturer's instructions (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). The resulting MSCs (passages 6–12) were used for our studies. Three days before infusion, the cells were freshly plated out at a 1:3 ratio and incubated in complete medium with 10 μM 5-bromo 2-deoxyuridine (BrdU) to label those cells in the S phase of the cell cycle. BrdU-labeled MSCs were harvested at 106 cells per 100 μl of PBS.

The status of our MSC phenotype was validated by staining the cells with oil red (adipogenic lineage), alcian blue (chondrogenic lineage), or alkaline phosphatase (osteogenic lineage) after culture under specific differentiation conditions. The BrdU labeling had no effect on MSC proliferation or differentiation capacity.

Syngeneic rat cardiac fibroblasts (CFs) were obtained from a donor Lewis rat heart stably transfected with rat monocyte chemotactic protein-3 (MCP-3) expression vector or pcDNA3.1 (control vector) as described previously [10]. The expression of MCP-3 was confirmed by real-time polymerase chain reaction (PCR). Confluent cells were passaged and plated out at 1:2–1:3 dilutions until passage 11.

Gene Array Analysis

We used a chemokine/chemokine receptor array nylon membrane array system that contained 67 distinct targets (SuperArray Bioscience Corporation, Frederick, MD, http://www.superarray.com). One microgram of total RNA was used to make cDNA by reverse transcription (RT) using random primers. cRNA was generated and hybridization was performed using company-supplied protocols. Chemiluminescent signals were measured using a cooled charge-coupled device camera with a 20-second exposure time. Each filter was used once. Three individual animals were studied at each time point. Time points studied were 1 hour and 1, 3, 7, and 10 days after LAD ligation. Control groups included no surgery and 1 hour and 7 days after sham LAD ligation in which a suture was placed but not tightened over the LAD.

Myocardial Chemokine Expression as a Function of Time After Acute MI

A positive result for a specific chemokine in myocardial tissue was a threefold increase in expression of one experimental animal compared with all controls (sham and no surgery) and at least a twofold increase in expression in the remaining experimental animals compared with each of the controls at that time point. Furthermore, all other time points had to be increased or there had to be no change from controls.

Identification of Differential Receptor on MSCs Compared with CFs

Because there is less variability in expression profiles from cells in cultures compared with tissue, we increased the stringency of a positive result in arrays performed on cells in culture. In this case, a significant difference in receptor expression levels was defined as a 10-fold increase in expression in MSCs compared with CFs. Three separate cultures of each cell type were studied. All positive results were confirmed by PCR or real-time PCR.

Real-Time PCR

RT-PCR was performed after isolation of RNA from 6 × 106 cells by using an RNeasy Mini Kit (Qiagen Inc., Valencia, CA, http://www1.qiagen.com) according to manufacturer instructions. Quantitative real-time PCR was performed using the ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA, https://www2.appliedbiosystems.com). The reaction mixture contained SYBR Green PCR master mix (Applied Biosystems), each primer at 300 nM, and 10 ìl of cDNA. After activation of the AmpliTaq Gold (Applied Biosystems) for 10 minutes at 95°C, we carried out 45 cycles with each cycle consisting of 15 seconds at 95°C followed by 1 minute at 60°C. The dissociation curve for each amplification was analyzed to confirm that there were no nonspecific PCR products.

Immunostaining

Animals were sacrificed 72 hours or 4 weeks after MI. Tissues were fixed in formalin and embedded in paraffin blocks according to established protocols. Antigen retrieval was performed using 10 mM sodium citrate buffer (pH 6.0) and heat at 95°C for 5 minutes. The buffer was replaced with fresh buffer and reheated for an additional 5 minutes and then cooled for approximately 20 minutes. The slides were then washed in deionized water three times for 2 minutes each. Specimens were then incubated with 1% normal blocking serum in PBS for 60 minutes to suppress nonspecific binding of immunoglobulin G. Slides were then incubated for 60 minutes with the mouse anti-BrdU primary antibody (BD Biosciences). Optimal antibody concentration was determined by titration. Slides were then washed with PBS, incubated for 45 minutes with fluorescein isothiocyanate-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com) diluted to 1.5 ìg/ml in PBS with 1% serum, and incubated in a dark chamber. After washing extensively with PBS, coverslips were mounted with aqueous mounting medium (Vectashield mounting medium with 4,6-diamidino-2-phenylindole [DAPI], H-1200; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com).

Confocal Immunofluorescence Microscopy

Tissues were analyzed using an upright spectral laser scanning confocal microscope (Model TCS-SP; Leica Microsystems GmbH) equipped with blue argon (for DAPI), green argon (for Alexa Fluor 488), and red krypton (for Alexa Fluor 594) lasers. Data were collected by sequential excitation to minimize “bleed-through.” Image processing, analysis, and the extent of colocalization were evaluated using the Leica Confocal software. Optical sectioning was averaged over four frames, and the image size was set at 1,024 × 1,024 pixels. No digital adjustments were made to the images.

Quantification of MSC Engraftment and Vascular Density

Engrafted MSCs were quantified as the number of BrdU-positive cells per high-power field. The number of vessels was quantified as the number of von Willebrand factor-positive vessels per high-power field. At least eight high-power fields across the infarct zone were randomly counted by two observers blinded to the treatment of the animals. The number of cells or vessels per high-power field was averaged and normalized by the calibrated area per high-power field.

Echocardiography

Two-dimensional (2D) echocardiography was performed at 2 and 5 weeks after LAD ligation and MSC transplantation using a 15-MHz linear array transducer interfaced with a Sequoia C256 (Siemens Medical Solutions, Inc., Malvern, PA, http://www.smed.com) and GE Vision 7 (GE Healthcare, New York, http://www.gehealthcare.com) as previously described [10, 14]. Left ventricle (LV) dimensions and wall thickness were quantified by digitally recorded 2D clips and M-mode images in a short-axis view from the mid-LV just below the papillary muscles to allow for consistent measurements from the same anatomical location in different rats. The ultrasonographer was blinded to treatment group. Measurements were made by two independent blinded observers offline using ProSolv (ProSolv Cardiovascular, Indianapolis, http://www.prosolv.com) echocardiography software. Measurements in each animal were made six times from three of five randomly chosen M-mode clips recorded by an observer blinded to the treatment arm. The shortening fraction was calculated from the M-mode recordings. Shortening fraction (percentage) = (LVEDD − LVESD)/LVEDD × 100, where LVEDD is the left ventricular end diastolic dimension, and LVESD is the left ventricular end systolic dimension.

Determination of Collagen Content

Paraffin sections (5 μm) of the heart tissue were prepared. Sections were stained with collagen-specific Masson's trichrome stain and observed by light microscopy. A quantitative estimation of collagen content was performed to assess fibrillar collagen accumulation (stained blue) using Image-Pro Plus version 5.1 (MediaCybernetics, Inc., Silver Spring, MD, http://www.mediacy.com) image analysis software. Fibrosis size was quantified by percentage LV area containing collagen tissue (blue). Because the hearts were at 8 weeks after MI and the anterior wall had significantly thinned, the percentage of the LV cavity circumference that had collagen tissue was also quantified as a measure of infarct size after remodeling [14].

In Vitro Migration Assay

MSCs were detached with trypsin-EDTA, counted, and resuspended in complete media. Cells (1 × 105 in 400 μl) were then plated onto Millicell culture inserts (8-μm pore size; Millipore, Billerica, MA, http://www.millipore.com) in a 24-well plate and allowed to adhere overnight at 37°C. To initiate migration, DMEM containing 1% FBS (600 μl) without or with the chemoattractant factor MCP-3 (R&D Systems, Inc., Minneapolis, http://www.rndsystems.com) was added to the lower wells (in triplicate). Cells were allowed to migrate through the insert membrane for 4 hours at 37°C. The inserts were then washed with PBS, and the nonmigrating cells remaining on the upper surface of the insert were removed with a cotton swab. Migrating cells were fixed with 4% paraformaldehyde, stained with 0.25% crystal violet, and counted using a microscope (×10). The mean number of cells (± SEM) of four randomly chosen fields was calculated for each treatment.

Statistical Analysis

Data are presented as mean ± SD. Comparisons between groups were by unpaired Student's t test (cell engraftment, collagen content) or by analysis of variance with Bonferroni correction (echocardiographic data) for multiple comparisons where appropriate.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

MSCs Transiently Home to Injured Myocardium

Two million BrdU-labeled MSCs were infused into the tail vein of each rat at 1 day or 14 days after LAD ligation. Three days after MSC infusion, the rats were killed, and the heart was harvested. MSCs were quantified as the number of BrdU-positive cells per mm2. The data in Figure 1 demonstrate that our MSC preparation transiently homes to the myocardium after acute MI (AMI). One day after LAD ligation, a significant number of MSCs were identified per unit area, whereas 14 days after LAD ligation, the infusion of MSCs did not result in significant MSC engraftment within the infarct zone.

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Figure Figure 1.. MSCs transiently home to the myocardium after acute myocardial infarction. Two million 5-bromo-2-deoxyuridine (BrdU)-labeled MSCs were infused via the tail vein 1 day or 14 days after LAD ligation. The number of BrdU-positive cells was quantified per square millimeter by immunohistochemistry 3 days after MSC infusion. Data represent mean ± SD, n = 5 per group. Abbreviations: d, days; LAD, left anterior descending.

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Identification of Candidate MSC Homing Factors

Figure 2A depicts the strategy we implemented to identify candidate MSC homing factors. We used the chemokine and chemokine receptor array to identify two distinct lists. The first list was the population of chemokines that were expressed as early as 1 hour after LAD ligation, with an expression that was gone by 10 days after LAD ligation and with a peak expression at least threefold over that of sham-operated animals (light gray grouping on left, Fig. 2A). The second list represented chemokine receptors that were expressed at least 10-fold more on MSCs compared with CFs (circle on right, Fig. 2A). The intersection of the candidate MSC homing factors consists of those chemokines that were contained in the circle on the left (Fig. 2A) (transiently expressed by myocardial tissue after LAD ligation) and that bound receptors contained in the circle on the right (expressed by MSCs and not CFs) and is presented in the open nonshaded area. As depicted in the open area of Figure 2A, only two families of chemokines were identified, MCP-1 and MCP-3 via receptors CCR-1 and CCR-2 and macrophage inflammatory protein (MIP)-1α and MIP-1β via the receptor CCR-5.

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Figure Figure 2.. Monocyte chemotactic protein-3 (MCP-3) is a candidate MSC homing factor. (A): Schematic representation of the strategy and findings of array analysis identifying chemokines (circle on left) expressed in the myocardium after left anterior descending ligation and chemokine receptors (circle on right) expressed by MSCs and not expressed by cardiac fibroblasts (CFs). Matched chemokine and chemokine receptor pairs of interest are delineated in the area of overlap represented by the middle area. (B): Representative agarose gel of polymerase chain reaction (PCR) products (40 cycles) for identified chemokine receptors in MSCs at passages 6 and 20, CFs, and spleen (positive control). GAPDH was used as a loading control. PCR study was repeated with at least five samples per cell type/passage per receptor target. Abbreviations: bp, base pairs; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MI, myocardial infarction.

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To validate and refine the findings from our array studies, we performed PCR to further assess the presence of CCR-1, CCR-2, and CCR-5. Figure 2B shows PCR products from MSCs at passages 6 and 20, CFs, and rat spleen (positive control). These results indicate that expression of CCR-1 and CCR-5 is significantly greater than CF in young MSCs and that the expression of CCR-5 by MSCs is lost with passage.

Effect of MCP-3 Expression on MSC Homing

Based on the observation that (a) CCR-1 expression appears to be maintained in MSCs and (b) the ability of MSCs to home over time is not lost, we chose to focus on MCP-3. An additional predefined criterion for identifying an MSC homing factor is that MSCs do not express the chemokine of interest. We performed real-time PCR analysis for MCP-3 expression in MSCs and CFs which showed that MSCs do not express significant levels of MCP-3 (data not shown).

To test whether MCP-3 can induce MSC homing, we performed in vitro cell migration studies to test the ability of MSCs to migrate in response to varying concentrations of MCP-3. The data in Figure 3 show that there was an increase in MSC migration in a concentration-dependent manner.

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Figure Figure 3.. Monocyte chemotactic protein-3 (MCP-3) causes mesenchymal stem cell (MSC) chemotaxis in vitro. MSCs migrated in response to MCP-3 in a concentration-dependent manner in an in vitro chemotaxis assay. Data represent mean ± SD, n = 10 per MCP-3 concentration.

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To test the ability of MCP-3 to recruit MSCs to remotely injured myocardium, we transplanted control or MCP-3-expressing CFs into the infarct border zone 1 month after LAD ligation. Three days later, we infused 1 × 106 BrdU-labeled MSCs via the tail vein and quantified MSC engraftment 3 days later (6 days after CF transplantation). The data in Figure 4(single infusion) demonstrate that reestablishment of MCP-3 expression in myocardial tissue restores the ability of MSCs to home to myocardial tissue. Although these data are consistent with MCP-3 having a role in MSC homing, the level of MSC engraftment was low compared with HSC engraftment in response to chronic SDF-1 expression in the same model [10].

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Figure Figure 4.. MCP-3 expression leads to MSC homing to the myocardium in vivo. One month after LAD ligation, 1 × 106 control or MCP-3-expressing CFs were transplanted into the infarct border zone. Three days later, the animals received saline or one dose (single infusion) or six doses (multiple infusions for 12 days) of 1 × 106 BrdU-labeled MSCs. Single-infusion animals were sacrificed 1 week after MSC infusion, and multiple infusion animals were sacrificed 1 month after MSC infusions (10 weeks after LAD ligation). (A): The number of engrafted MSCs in each treatment group was quantified per square millimeter by immunofluorescence using an antibody against BrdU. Data represent mean ± SD, n = 7–10 animals per group. (B): Representative photomicrographs of infarct zone after staining for BrdU (green, center images) and counterstaining for nuclei (DAPI, blue, leftmost images). Merged images of BrdU and nuclei are on the right. ∗, p < .05; #, p < .001 compared with infusion-matched control CF group. Abbreviations: BrdU, 5-bromo 2-deoxyuridine; CF, cardiac fibroblasts; DAPI, 4,6-diamidino-2-phenylindole; LAD, left anterior descending; MCP-3, monocyte chemotactic protein-3; NT, not treated.

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We reasoned that among the causes of the relatively low engraftment of MSCs in response to MCP-3 were that (unlike HSCs [15]) MSCs are not constitutively released by the bone marrow, that some MSCs are trapped in the lung when given i.v. [16], and that the half-life of MSCs in the blood stream after i.v. infusion is short (<1 hour) [17]. We hypothesized that serial infusions of MSCs into animals transplanted with MCP-3-expressing CFs would lead to greater MSC engraftment. The data in Figure 4 (multiple infusions) show that after six i.v. infusions of 1 × 106 MSCs per infusion over the course of 12 days, there were significantly more MSCs engrafted in the myocardium of animals that received MCP-3-expressing CFs compared with control CFs (Fig. 4A, 4B).

Effect of Reestablishing MSC Homing on Cardiac Function

We transplanted control and MCP-3-expressing CFs 1 month after LAD ligation. After CF transplantation, animals received six infusions of 1 × 106 MSCs per infusion every other day for 12 days or saline beginning 3 days after CF transplantation. Cardiac function and dimensions were quantified by echocardiography 1 month after MI, before CF transplantation (baseline), and 1 month after CF transplantation (2 months after MI). The data in Figure 5A demonstrate that cardiac function as measured by shortening fraction was significantly increased in those animals that received MCP-3-expressing CF and MSC infusions. No significant benefit was seen when animals received MCP-3-expressing CFs without MSC infusions (Fig. 5C). There was evidence of reverse remodeling with a decrease in LVEDD 1 month after infusion of MSCs into animals that received MCP-3-expressing CF and MSC infusions. Further dilation of the left ventricular cavity was observed in those animals that received either control CFs despite serial infusions of MSCs or MCP-3-expressing CFs without serial infusions of MSCs (Fig. 5B, 5D).

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Figure Figure 5.. MCP-3 expression combined with MSC infusions results in improved cardiac function and remodeling. One month after LAD ligation, cardiac function (shortening fraction [percentage]) (A, C) and left ventricular end diastolic dimension (LVEDD) (B, D) were quantified by echocardiography (□, ○). After echocardiography, 1 × 106 control (•) or MCP-3-expressing cardiac fibroblasts (▪) were transplanted into the infarct border zone. Beginning 3 days after cardiac fibroblast injections, the animals received the first of six doses of 1 × 106 BrdU-labeled MSCs (A, B) or saline (C, D). Successive doses were given every other day in the ensuing 12 days. Echocardiography was repeated 6 weeks after cardiac fibroblast transplantation (10 weeks after LAD ligation, •, ▪). Data represent individual animals. Solid lines represent the mean for the group, n = 7–10 per group. ∗, p < .05; #, p < .001 compared with baseline parameter measured at 1 month after myocardial infarction. Abbreviations: BrdU, 5-bromo-2-deoxyuridine; LAD, left anterior descending; MCP-3, monocyte chemotactic protein-3.

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The engrafted MSCs did not differentiate into cardiac myocytes. Costaining for BrdU and cardiac myosin, troponin I, or connexin 43 revealed that none of the engrafted MSCs expressed cardiac markers in vivo (data not shown). We hypothesized that MSC engraftment resulted in remodeling of the infarct zone leading to improvement in cardiac function. Masson's trichrome staining revealed a significant difference in collagen content in the infarct-infarct border zone between animals that were treated with control and MCP-3-expressing CFs prior to serial MSC infusion (Fig. 6A and 6B, respectively). No changes were observed with the injection of MCP-3-expressing CFs without MSC infusion (data not shown). Injection of CFs with or without MCP-3 expression and with or without MSC infusions had no effect on vascular density (data not shown). The percentage of the LV that stained positive for collagen was significantly decreased by 25.4% (p < .02; Fig. 6C) in the animals that received MCP-3-expressing CFs and serial MSC infusions. In these animals, we observed a 35.3% (p < .01; Fig. 6D) decrease in the LV circumference that stained positive for collagen. These data are consistent with our observation that there was a significant decrease in LVEDD (Fig. 5) in animals that received MCP-3-expressing CFs and serial MSC infusions.

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Figure Figure 6.. MCP expression combined with MSC infusions causes ventricular remodeling and myofibroblast recruitment. Representative photomicrographs of Masson's trichrome-stained cross-sections of the midventricular segments from animals that received (A) MCP-3-expressing or (B) control cardiac fibroblasts 4 weeks after left anterior descending (LAD) ligation followed by serial infusions of MSCs. (C) The percentage area the ventricle containing collagen fibriles or (D) the percentage of the endocardial circumference in which there was collagen fibriles was quantified in five animals per group. Data represent mean ± SD (n = 5 per group, ∗, p < .05). Representative confocal micrographs of myofibroblasts in the infarct border zone in animals that received serial infusions of MSCs after transplantation of (E) MCP-3-expressing or (F) control cardiac fibroblasts. Tissue was stained 10 weeks after LAD ligation using immunofluorescence with an antibody that recognizes vimentin (green). The nuclei were counterstained with DAPI (blue), and the cardiac myocytes were identified using an antibody that recognizes ventricular myosin heavy chain (red). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; LV, left ventricular; MCP-3, monocyte chemotactic protein-3.

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Myofibroblasts have been associated with improved cardiac remodeling and function [18, 19]; therefore, we wanted to determine whether the favorable collagen remodeling was associated with a greater number of myofibroblasts in the infarct zone. Staining with an antibody to vimentin and α-smooth muscle cell actin [20, 21] demonstrated a greater number of myofibroblasts in the infarct border zone of animals that received MCP-3 and serial MSC infusions compared with those that received control CF and serial MSC infusions (Fig. 6E, 6F). The vimentin-positive cells were rarely BrdU-positive, suggesting that the majority of these cells were recruited to the infarct border zone in response to MSC engraftment given that MCP-3 expression alone did not result in an increase in myofibroblasts.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

MSCs are under active investigation as a stem cell source for tissue repair. MSCs are known to home to injured tissue of multiple organs [6, 22, 23]; however, the biological signals responsible for MSC homing have not been previously described. In this study, we identified MCP-3 as a homing factor for MSCs.

Some studies have suggested that MSCs home in response to SDF-1 [1, [2]3]. Others, however, demonstrated that local SDF-1 expression is only sufficient to attract MSCs early after (but not in the absence of) organ injury, perhaps due to the transient upregulation of other chemokines or adhesion molecules early after MI [24]. Moreover, SDF-1 seems important for growth and survival of MSCs, perhaps due to autocrine mechanisms (because MSCs themselves express SDF-1), but these effects of SDF-1 are distinct from SDF-1 being responsible for MSC homing [25]. Consistent with the idea that SDF-1 overexpression at a time remote from MI does not induce significant homing of MSCs, we only encountered HSC recruitment and engraftment in previous studies that defined SDF-1 as a myocardial stem cell homing factor [10].

MCP-3 belongs to the family of CC chemokines with potent chemotactic activities for several cell types, including monocytes, leukocytes, and dendritic cells. These chemokines exert their effects through interaction with the chemokine receptors CCR-1, CCR-2, CCR-3, and CCR-5. MCP-3 has been shown to be expressed at multiple sites of inflammation [26, [27], [28]29], although its role in wound healing has not been fully elucidated. In this study, we show that MCP-3 is transiently expressed by myocardial tissue after AMI. Because MSCs are not known to be mobilized in response to MI, the utility of MCP-3 expression as an MSC homing factor for the intrinsic repair of the heart at the time of MI is unclear. However, as shown by our study, exploiting the MSC homing effects of MCP-3 may have therapeutic potential.

Our data demonstrate that after MI there is a transient upregulation and release of multiple chemokines that may impact on stem cell trafficking to sites of injury. Identification and re-expression of these stem cell homing factors weeks to months after MI appears to reestablish the ability of stem cells to traffic to and engraft in the infarct zone. Furthermore, injecting the heart with cells that reestablish stem cell homing in the myocardial tissue could be a potential strategy for increasing stem cell content in the heart over time. Future studies are necessary to determine whether this strategy is as efficacious or more efficacious than multiple invasive injections over time and/or what can be achieved with a single injection of stem cells.

The recruitment of MSCs to the heart 1 month after MI did not result in regeneration of cardiac myocytes. Rather, as has been shown with MSC injections in the peri-infarct period, MSC engraftment results in beneficial remodeling in the infarct zone [30]. The lack of new cardiac myocyte formation could be due to the inability of MSCs to differentiate into cardiac myocytes or the lack of critical mediators of cell signaling required for cardiac differentiation in the myocardial tissue beyond the peri-infarct period [31]. MSCs are known to release multiple factors, including vascular endothelial growth factor, SDF-1, fibroblast growth factor, and insulin-like growth factor-1 [32, [33], [34], [35]36]. Although beyond the scope of our current study, it is interesting to note that we observed improved cardiac function in the absence of vasculogenesis or angiogenesis. Thus, the effects of recruiting MSCs via the overexpression of MCP-3 appear distinct from those observed after overexpression of an HSC homing factor [10, 37] or injection of HSCs themselves [38, 39]. This observation suggests that the mechanism of benefit after reestablishment of MSC homing and engraftment of MSCs at a time remote from MI for MSC transplantation at a time remote from AMI is related to improved cardiac remodeling and perhaps to trophic effects on surviving myocardium rather than to improved tissue perfusion.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References

This work was supported by the National Institutes of Health (HL74400), the Shalom Foundation, the Wilson Foundation, and the State of Ohio. N.M. and S.S. contributed equally to this work.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References