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Regulation and Function of Stem Cells in the Cardiovascular System
1Laboratory of Physiology, University of Antwerp, Antwerp, Belgium; 2Department of Haematology-Stem Cell Laboratory, Free University Brussels, Brussels, Belgium; and 3HistoGeneX, Edegem, Belgium
Submitted 19 May 2005 ; accepted in final form 17 October 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, IL-3, IL-6, stem cell factor, stromal cell-derived factor-1, or TNF-
, 10 ng/ml]. Control or TNF--treated MSCs were injected intracavitarily in rat hearts in vivo. In baseline in vitro conditions, the number of MSCs that adhered to CMVE was highly dependent on the flow rate of the superfusing medium but remained significant at venous and capillary shear stress amplitudes. Activation of both CMVE and MSCs with TNF- or IL-1 before adhesion concentration dependently increased adhesion of MSCs at each studied level of shear stress. Consistently, in vivo, activation of MSCs with TNF- before injection significantly enhanced cardiac homing of MSCs. TNF--induced adhesion could be completely blocked by pretreating either CMVE or MSCs with anti-VCAM-1 monoclonal antibodies but not by anti-ICAM-1 antibodies. Adhesion of circulating MSCs in the heart appears to be an endothelium-dependent process and is sensitive to modulation by activators of both MSCs and endothelium. Inflammation and the expression of VCAM-1 but not ICAM-1 on both cell types have a regulatory effect on MSC homing in the heart.
cardiac regeneration; heart failure
MSCs have been detected in circulating blood of mammalian species (14) and found to migrate into different organs, such as heart, liver, spleen, or lungs (3, 22). The process of homing of circulating stem cells in the bone marrow has been studied in detail. This process is clinically highly relevant as it is crucial for therapeutic transplantation of hematopoietic stem cells. In the bone marrow, homing is a multistep process that shares components with the extravasation of leukocytes at inflammatory sites with a significant role for interactions between stem cells and bone marrow endothelium (26). Only few studies, however, have examined interaction of stem cells with endothelial cells in the vasculature outside the bone marrow (12, 21, 25).
Cardiac endothelial cells in the endocardium and myocardial capillaries play a modulatory role on cardiac development and pump performance. At these locations, cardiac endothelial cells directly interact with surrounding cardiomyocytes through paracrine and nonparacrine mechanisms (5, 8). From this point of view, the heart functions as a pluricellular, multifunctional organ, in which the endothelium is a crucial functional component in cardiac metabolism, growth, contractile performance, and rhythmicity (5). In the present study we hypothesize that besides these established functions, cardiac endothelium may also have a modulatory role in cardiac regeneration. More specifically, we postulate that cardiac microvascular endothelium (CMVE) may play a role in homing and migration of circulating stem cells to myocardium and, as such, may act as a modulator of cardiac cellular homeostasis. Hence, we examined adhesion of MSCs to cardiac endothelium and determined whether a selected set of cytokines affected this process.
The inflammatory cytokines TNF- and IL-1 were selected because they increase adhesion molecule expression in endothelial cells (20) and are released after myocardial damage (7). IL-3, IL-6, and stem cell factor (SCF) were tested because these cytokines play a role in homing of hematopoietic stem cells (31). IL-6 was selected because it is markedly upregulated in cardiac failure and has a protective role in the progression of pump failure (1), whereas stromal cell-derived factor-1 (SDF-1) was tested because it has been proposed to participate in homing of stem cells to the heart (10).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Endothelial cell cultures. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publications No. 85-23, Revised 1996). All animal protocols were approved by the local ethical committee for animal research (University of Antwerp). CMVE and aortic endothelial cells (AE) were isolated and cultured from adult Sprague-Dawley rats as previously described (11).
MSC cultures. Femur and tibia of Sprague-Dawley rats were excised, and connective tissue was removed (3). Bone marrow cavity was flushed with complete culture medium. Marrow plug suspension was dispersed by passing it through subsequent pipettes of decreasing sizes. Once a homogeneous cell suspension was achieved, mononuclear cells were isolated using density gradient centrifugation (Ficoll-Paque, Amersham Biosciences), mononuclear cells were plated at 8 x 106 cells/cm2, and nonadherent cells were removed after 4 h. The mesenchymal population was isolated based on plastic adherence and was cultured in RPMI-1640 with 10% FCS of a selected batch (GIBCO, Invitrogen). At 90% confluence, the cells were trypsinized (0.25% trypsin-EDTA) and passaged at 1:9 ratios. For adhesion experiments, MSCs at passage 4 and later were used.
Adhesion Assays
Adhesion assays were performed using eight-well glass chamber slides (Falcon CultureSlide) on which 2 x 104 CMVE (passages 2–4) were plated, reaching confluence after 3 days. Unless otherwise stated, all experiments were performed with 8 x 103 MSCs labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes) and diluted in 0.15 ml RPMI. After incubation at 37°C (1–16 h), wells were gently washed three times with PBS, and adherent cells were counted in 10 fields (250-fold magnification) per well using fluorescence microscopy (Zeiss). The number of adhering cells was normalized to the total number of added cells. In some experiments, CMVE or MSCs were pretreated for adhesion studies with one of the following substances: TNF- (0.1–100 ng/ml), IL-1 (0.1–100 ng/ml), SCF (10 ng/ml), SDF-1 (10 ng/ml), IL-3 (10 ng/ml), IL-6 (10 ng/ml) (Sigma), anti-VCAM-1 antibody (mouse monoclonal anti-rat, clone 5F10, 10 µg/ml, Eurogentec), or anti-ICAM-1 antibody (mouse monoclonal anti-rat, clone 1A29, 10 µg/ml, Research Diagnostics).
Flow Chamber Adhesion Assays
Flow adhesion experiments were performed in a parallel plate flow microchamber with a slit height of 0.25 mm and slit width of 10 mm. CMVE (passages 2–4) were cultured to confluence on poly-L-lysine- and collagen (Sigma)-coated glass microscope slides. These slides were attached to the bottom of the flow chamber 15 min before flow experiments. Perfusion of flow chambers at different levels of shear stress (0.5, 1, 2, 5, or 10 dyn/cm2) was conducted for 2 h with circulating RPMI medium (37°C) containing 200 MSCs/µl. MSCs were labeled with DiI as described earlier. Thereafter, chambers were perfused for 15 min with cell-free medium at 2 dyn/cm2 to remove loose cells. Adherent MSCs were counted with fluorescence microscopy (5 fields at x100 magnification). In some experiments either MSCs or CMVE was pretreated with TNF- (10 ng/ml) for 24 h before adhesion assays. Experiments were repeated at least three times.
Rat Model of In Vivo Adhesion of MSCs
Sixteen adult Sprague-Dawley rats (150 g) were randomized into two groups. One group received intraventricular injection of control MSCs, and the other group received MSCs pretreated with TNF- (10 ng/ml, 24 h). For MSC injection, rats were anesthetized with fentanyl (0.05 mg/kg im, Janssen-Cilag), diazepam (5 mg/kg im, Roche), and haloperidol (3 mg/kg im, Janssen-Cilag) and subsequently intubated endotracheally. A left lateral thoracotomy and pericardiotomy were performed, exposing the heart and ascending aorta. In a separate group of six rats, a procedure of myocardial ischemia (30 min)-reperfusion injury was induced by temporary left anterior descending coronary artery ligation. Twenty-four hours later, MSCs were injected, following the same procedure as described below.
MSCs labeled with DiI were prepared for infusion by detaching the cells from the culture plates by 10-min incubation with 0.25% trypsin-EDTA. Cell solution was passed through a 40-µm nylon cell strainer (BD Falcon) to remove cell aggregates and centrifuged (1,000 rpm, 7 min); 1.5 x 106 MSCs were diluted in 0.5 ml PBS and were injected into the left ventricular cavity while the ascending aorta was clamped (15 s) to mimic intracoronary injection. The rats were monitored for 2 h postoperatively and received an additional subcutaneous dose of fentanyl (0.03 mg/kg) before returning to their cages. All rats survived the procedure.
Twenty-four hours after injection, hearts were removed, fixed with 4% paraformaldehyde solution through Langendorff perfusion, and left overnight in a 20% sucrose solution. Three cryosections per heart, separated by at least 10 sections, were made in the midventricular region. Three digital images were made with fluorescence microscopy (low magnification, x40), and an observer unaware of the experimental protocol (control vs. TNF--treated MSCs) counted cells in the sections.
Immunofluorescent Staining
Endothelial cells in cryosections (20 µm) were stained with mouse monoclonal anti-rat endothelial cell antigen (anti-RECA; Serotec) antibody and secondary goat anti-mouse coupled to Alexa Fluor 488 (Molecular Probes) antibody. Cryosections (10–20 µm) of infarcted hearts were stained with phalloidin-Alexa Fluor 488 for F-actin to discriminate normal from damaged cardiomyocytes. Cellular nuclei were counterstained with Hoechst. Images were captured with fluorescence microscopy (Zeiss Axioplan 2, Apotome, Axiocam HRm and Axiovision software). ZVI-images were exported as TIFF or JPG files and further processed in Adobe Photoshop.
RT-PCR
mRNA was isolated in TRIzol Reagent (Invitrogen, Life Technologies) following instructions of the manufacturer. RT-PCR was performed with One-Step RT-PCR System (Life Technologies) in 25-µl reaction volume containing 0.5 µl total RNA (10–100 ng), 12.5 µl reaction mix, 400 nM of both primers, and 0.5 µl Superscript II/Taq mix (Life Technologies). After initial incubation of 30 min at 50°C and 2 min at 94°C, 35 PCR cycles were performed, which consisted of 15-s denaturation at 94°C, 30-s annealing at 55°C, and a 1-min extension at 72°C, with a final extension step of 7 min. Primers used are listed in Table 1.
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Cells were harvested using trypsin/EDTA and incubated 10 min with 10 µg/ml FITC-labeled monoclonal antibody in RPMI per 5 x 104 cells at room temperature in the dark. Anti-rat CD34 (mouse monoclonal anti-rat, clone ICO115, Santa Cruz Biotechnology), anti-rat CD45 (leukocyte common antigen, mouse monoclonal anti-rat, clone OX-1, BD Biosciences Pharmingen), anti-rat CD29 (1-integrin, Armenian hamster monoclonal anti-rat, clone Ha2/5, BD Biosciences Pharmingen), and anti-rat CD90 (Thy-1, mouse monoclonal anti-rat, clone OX-7, Abcam) were used. Cells were washed twice with PBS-0.1% BSA. Labeled samples were examined with a Coulter Epics XL-MCL flow cytometer (Coulter). At least 10,000 events were analyzed and compared with isotype controls.
Real-Time PCR
Real-time PCR was performed for VCAM-1 and very late antigen-4 (VLA-4, 4-chain). CMVE and MSCs were cultured on six-well plates (Falcon, VWR international) for 2 days and serum starved for the last 7 h, during which some of the products used in adhesion assays were added. mRNA was isolated in TRIzol Reagent (Invitrogen). Real-Time PCR was performed with TaqMan One-Step RT-PCR System (Applied Biosystems) in 25 µl reaction volume containing 1 µl total RNA (10–100 ng), 12.5 µl One-Step RT-PCR Master Mix, 100–800 nM of both primers, 200 nM TaqMan Probe, and 0.5 µl RNase inhibitor. TaqMan probes were labeled with FAM reporter dye and TAMRA quencher dye. After initial incubation of 30 min at 48°C and 10 min at 95°C, 45 PCR cycles were performed, which consisted of 15-s denaturation at 95°C and 60-s annealing and extension at 60°C. Primers and TaqMan probes used are listed in Table 1. Expression of VCAM-1 and VLA-4 mRNA was normalized to expression of GAPDH mRNA.
Statistical Analysis
Adhesion assays were performed in duplicates. Mean values were compared with one-way ANOVA with Bonferroni correction for multiple comparisons. Real-time PCR experiments were performed four times, and differences in threshold cycle values between VCAM-1 or VLA-4 and GAPDH were compared with one-way ANOVA with Bonferroni correction for multiple comparisons. Statistical analysis was performed with SPSS software (SPSS).
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RESULTS |
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MSCs presented as a homogeneous fibroblastoid cell population. Expression of stem cell markers assessed with RT-PCR showed that after passage 2 these cells were completely negative for hematopoietic cell markers (CD34 and CD45) and positive for CD90, CD105, CD166 (ALCAM), which are markers of MSCs (RT-PCR, Fig. 1A) (22). Flow cytometric analysis of passage 4 cells confirmed that cells were negative for CD34 and CD45 and that cells were positive for CD29 (1-integrin) and CD90 (Thy-1) (Fig. 1B). Only cells at passage 4 and further passages were used for adhesion experiments.
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In static conditions, adhesion of MSCs to CMVE was dependent on the period that cells were allowed to interact, reaching a maximum after 4 h of incubation (n = 4; 1 h, 8 ± 3%; 2 h, 17 ± 1%; 4 h, 25 ± 3%). Percentage of MSCs that adhered to CMVE was fairly independent of the number of MSCs incubated, at least between 4,000 and 16,000 cells added per well (not shown). When adhesion experiments were performed in dynamic conditions in which medium containing MSCs was superfused during 2 h (200 MSCs/µl), increasing flow velocity (shear stress from 0.5 to 5 dyn/cm2) exponentially decreased adhesion of MSCs to CMVE from 2,063 ± 360 to 113 ± 24 cells/cm2 (see Fig. 3). At higher levels of shear stress, adhesion remained unchanged between 5 and 10 dyn/cm2 (75 ± 17 cells/cm2).
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A first screening experiment in static conditions, in which CMVE was treated with six different cytokines (10 ng/ml, 24 h), showed that only pretreatment of CMVE with TNF- or IL-1 increased adhesion of MSCs (Fig. 2A). Pretreating CMVE with IL-3, IL-6, SCF, or SDF-1 did not affect adhesion efficiencies (n = 6, Fig. 2A). The effect of TNF- or IL-1 was confirmed in dynamic conditions (Fig. 3) and appeared to be concentration dependent. At 100 ng/ml TNF-, for example, the percentage of MSCs that adhered to CMVE increased from 26 ± 3% to 52 ± 5% (n = 6, P < 0.001, Fig. 2B). Similar results were observed when CMVE was pretreated with IL-1 (at 100 ng/ml, the percentage of MSCs adhering to CMVE increased from 23 ± 1% to 63 ± 6%, n = 6, P = 0.002, Fig. 2C). When MSCs, instead of CMVE, were pretreated with TNF- during 24 h before incubation with CMVE, adhesion of MSCs to CMVE also increased (n = 6, increase from 28 ± 2% to 72 ± 2%, Fig. 2D). When both MSCs and CMVE were pretreated, adhesion was still a little greater, but effects were not additive. In flow-chamber experiments, pretreatment of CMVE or MSCs with TNF- (10 ng/ml, 24 h) enhanced adhesion of MSC at every studied level of shear stress (Fig. 3, values of TNF--treated groups are significantly different from control at all shear stress levels, P < 0.05, n = 3). MSCs also adhered to confluent cultures of AE, and the degree of adhesion did not differ significantly from adhesion to CMVE, either in basal conditions (24 ± 2% for CMVE, 18 ± 2% for AE, n = 6, P = 1) or after stimulation with TNF- (48 ± 4% for CMVE, 49 ± 6% for AE, P = 1, Fig. 2E).
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The in vitro experiments indicated that MSCs adhere to CMVE in flow conditions and that adhesion is sensitive to proinflammatory cytokines. As a next step, DiI-labeled MSCs were injected into the left ventricular cavity of anesthetized rats, while the ascending aorta was clamped. In 6 of the 12 rats, MSCs had been pretreated with TNF- (10 ng/ml, 24 h). Twenty-four hours after injection, MSCs residing in the myocardium were visualized by fluorescence microscopy. When compared with hearts injected with control MSCs, hearts injected with TNF--treated cells contained three times more MSCs (23 ± 7 vs. 70 ± 19 cells/field, P = 0.046, Fig. 4C). Sections for confocal microscopy were stained with anti-RECA antibody to evaluate spatial relationships between MSCs and cardiac endothelial cells (Fig. 4, A, B, D, and E). Most of the MSCs were observed inside capillaries (Fig. 4D), whereas some of them already crossed the endothelial barrier (Fig. 4E). No MSCs were observed in larger vessels. There were no obvious differences in localization between control MSCs and TNF-treated MSCs (data not shown). MSCs injected 24 h after ischemia (30 min)-reperfusion injury homed abundantly in infarcted tissue (Fig. 4F).
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The hyperbolic relation between flow velocity and MSC adhesion to CMVE suggests a role for 4-integrins (including 41-integrin = VLA-4, most important ligand of VCAM-1) in mediating cell adhesion (13). Furthermore, adhesion above 0.5 dyn/cm2 argues against involvement of 2-integrins, which are ligands to ICAM-1 (13). Therefore, monoclonal blocking antibodies against VCAM-1 were added to CMVE 1 h before MSC incubation. VCAM-1 antibody completely abolished the TNF--induced increase of MSC adhesion (n = 6, after stimulation with TNF-, from 52 ± 5% to 28 ± 4%, P = 0.013). Monoclonal blocking antibodies against ICAM-1, however, had a slight but nonsignificant effect (n = 6, after stimulation with TNF-, from 52 ± 5% to 38 ± 5%, P = 1) (Fig. 5A). Similarly, anti-VCAM-1 antibodies added to MSCs 1 h before adhesion assays inhibited the TNF--induced increase of MSC adhesion (n = 6, after stimulation with TNF-, from 66 ± 9% to 5 ± 1%, P = 0.001) and also reduced adhesion in basal conditions (n = 6, from 24 ± 4% in control to 5 ± 1%, P = 0.006). Again, anti-ICAM-1 antibodies added to MSCs before adhesion assays had no effect on MSC adhesion (Fig. 5B).
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(10 ng/ml) or IL-1 (10 ng/ml) robustly induced VCAM-1 mRNA expression levels by, respectively, 88 ± 31-fold (n = 4, P = 0.005) or 60 ± 15-fold (n = 4, P = 0.002) (Fig. 6). Similarly, treatment of MSCs with TNF- or IL-1 resulted in a 41 ± 8-fold (n = 4, P < 0.001) or 31 ± 8-fold (n = 4, P < 0.001) induction of VCAM-1 mRNA expression, respectively. When compared with VCAM-1 expression, mRNA levels of VLA-4, the principal VCAM-1 ligand, were not affected by TNF-, either in CMVE or in MSCs (data not shown). Of note, basal expression levels of VLA-4 mRNA were similar in CMVE and MSCs. However, basal expression levels of VCAM-1 mRNA was 114 times (95% confidence interval from 38 to 341 times) higher in CMVE than in MSCs.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Inflammatory cytokines (TNF- and IL-1) enhanced adhesion of MSCs to CMVE in conditions of no flow and in conditions of flow in vitro and the intact animal. These cytokines are released in myocarditis, during acute coronary syndromes, and in chronic heart failure (7, 19). The present observation that these proinflammatory cytokines promote homing of stem cells in the heart may suggest that these cytokines have a positive effect on cardiac regeneration. Albeit speculative, the potential beneficial effect of TNF- on cardiac cellular homeostasis could be an explanation for the disappointing results of recent clinical trials with TNF- inhibitors in heart failure (19). Activation of MSC adhesion does not seem to be a common feature of cytokines in general because other cytokines, including IL-6, IL-3, SCF, and SDF-1, had no effect on MSC adhesion to CMVE.
Our experiments showed that adhesion of MSCs to CMVE was dependent on the flow velocity of the superfusing fluid and rapidly decreased when flow velocities increased. This relation followed a hyperbolic curve, which bends at levels of shear stress between 1 and 2 dyn/cm2. Importantly, this range corresponds to the level of stress observed in veins and capillaries. Whether adhesion of MSCs remains still significant at arterial shear stress levels (usually assumed between 6 and 40 dyn/cm2) remains to be determined (13). Interestingly, a hyperbolic relation between adhesion and shear stress suggests a role for 4-integrins in mediating cell adhesion (13) and contrasts with the adhesion-shear stress relationship mediated by 2-integrins, which cannot support adhesion when wall shear stress is >0.5 dyn/cm2.
Consistently, VCAM-1, which interacts with 4-integrins, appeared to be the dominant adhesion molecule in the cytokine-induced adhesion of MSCs to CMVE. Interestingly, VCAM-1 was inducible in both CMVE and MSCs. Similarly, the 41-integrin VLA-4, the most important ligand of VCAM-1, was also expressed in both MSCs and CMVE. VLA-4 expression, however, was not inducible by TNF- and IL-1, indicating that MSC adhesion to CMVE is likely controlled by variations in VCAM-1 expression levels. Interestingly, in contrast to neutrophil adhesion to endothelium (17, 27), adhesion of MSCs to CMVE was less dependent on CD18-ICAM-1 interactions.
A model for the interactions between stem cells and cardiac endothelium is depicted in Fig. 7. Regeneration of cardiac tissue by adult bone marrow stem cells will require mobilization of stem cells on cardiac insult. SDF-1 is cited frequently as a candidate to attract stem cells to injured heart (10). Once MSCs circulate in the blood stream, adhesion to endothelial cells at the site of interest is the first step in homing of those stem cells to heart. Our in vitro results show that inflammatory cytokines can activate both endothelial cells and MSCs. It has been shown previously that cardiac damage augments endothelial VCAM-1 expression in vivo (16). Our in vivo data show that pretreatment of MSCs with TNF- increases homing of those cells to the heart and that MSCs home in infarcted tissue, which is consistent with previous reports (3, 30). Whether circulating MSCs express VCAM-1 in conditions of myocardial infarction for homing purposes remains to be elucidated.
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Besides interacting with CMVE, MSCs also adhered to AE, indicating that interaction of MSCs with endothelial cells may be a general endothelial feature. Adhesion of MSCs to vascular endothelial cells can be of importance for repair of arterial wall (2, 6). To what extent, however, this process may be blunted by high shear forces in the arterial vasculature remains to be determined. Furthermore, on the basis of the present study, homing of stem cells will depend on the number of adhesion molecules expressed by the endothelium. Interestingly, a recent report indicates that microvascular endothelial cells have a much higher constitutive VCAM-1 expression in the heart compared with the lungs (15).
In summary, we demonstrated for the first time that MSCs adhere to CMVE and that adherence properties of both cell types can be modified by proinflammatory cytokines (TNF- and IL-1). Cytokine-induced adhesion is, at least partly, mediated by the VCAM-1-VLA-4 pathway, with both components of this pathway expressed on both CMVE and MSCs.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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B and its proinflammatory mediator cascade in the infarcted rat heart. Biochem Biophys Res Commun 321: 879–885, 2004.[CrossRef][ISI][Medline]This article has been cited by other articles:
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) or CMVE (
) were pretreated with TNF-
P < 0.05 compared with TNF-
