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a Divisions of Cardiac Surgery and Surgical Research, McGill University Health Center, Montreal, Quebec, Canada
b Department of Anesthesia, McGill University Health Center, Montreal, Quebec, Canada
Accepted for publication February 26, 2010.
* Address correspondence to Dr Shum-Tim, Division of Cardiothoracic Surgery, McGill University Health Center, 1650 Cedar Ave, Ste C9-169, Montreal, QC H3G 1A4, Canada (Email: dshumtim{at}yahoo.ca).
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Abstract |
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Methods: Sixty-three rats that survived left coronary artery ligations were injected with culture media (group M) or MSCs (group C). Cardiac functional changes were assessed with echocardiography. Cytokine gene expressions of interleukin (IL)-1β, IL-6, IL-8, (proinflammatory) and IL-10 (antiinflammatory) were quantified by real-time polymerase chain reaction. Extracellular matrix deposition, injury score, and the matrix metallopeptidase 2/tissue inhibitor of metallopeptidase 1 ratio were also analyzed.
Results: The ratio of proinflammatory/antiinflammatory cytokine gene expression was decreased in group C at various times, particularly in the early postoperative period. In group C, the matrix metallopeptidase 2/tissue inhibitor of metallopeptidase 1 gene expression ratio was significantly lower than group M at the early phase (12 hours), which in group C was translated into significantly lower extracellular matrix deposition at 24 hours, 1, and 2 weeks. Functional recovery was also significantly better in cell therapy group C.
Conclusions: Our data demonstrate that MSC therapy decreases the proinflammatory/antiinflammatory cytokine ratio in the microenvironment early after MI. This is associated with subsequent less scar formation and improved cardiac function.
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Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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In addition, the improvement in cardiac function appeared to precede the time required for cell transdifferentiation. It has also been suggested that the MSCs could improve cardiac function through a paracrine or immunomodulatory effect, such as angiogenesis factor, secretion of various cytokines, and extracellular matrix (ECM) deposition [5–9].
A clinical study showed that a reduced ratio of proinflammatory to antiinflammatory cytokines inferred a better prognosis in patients with recent myocardial infarction (MI) [10]. Tögel and colleagues [11] suggested the role of antiinflammatory cytokines from implanted MSCs in early improvement of renal function after acute renal failure. On the basis of these observations, we sought to explore whether changes in the proinflammatory/antiinflammatory cytokine gene expression ratio in the infarct microenvironment after MSC implantations were associated with early improved cardiac function.
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Material and Methods |
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Isolation, Culture, and Labeling of Rat MSCs
Rat MSC cultures were prepared according to Caplan's method, and then MSCs were transfected as previously described [12] with pMFG-lacZ plasmid containing the β-galactosidase gene for identification of the transplanted cells in the myocardium. The resulting MSCs expressing lacZ were expanded until transplantation.
Experimental Design
Immunocompetent female syngenic Lewis rats (200 to 250 g, Charles River Canada, St. Constance, Que) were used. Proximal left coronary artery ligations were done in 72 rats and 9 rats died within 24 hours. The remaining 63 were randomly assigned to two groups, 32 to group C and 31 to group M. In group M, 150 µL of medium was directly injected into three different sites around the periinfarct area of the myocardium 10 minutes after coronary ligation. In group C, isogenic MSCs (3 x 106) suspended in medium were similarly injected after ligation. The injection of MSCs at the periinfarction area was essential because a previous study suggested that cell-to-cell contact was necessary for the phenotypic expression of MSCs in a coculture in vitro model [13]. Rats were sacrificed at various predetermined time points.
Cardiac Functional Assessment
Transthoracic echocardiography was performed on all animals in groups M and C before operation (baseline), at 12 and 24 hours, and at 1 and 2 weeks after ligation and treatment. All animals were sedated in a quite room and in a right lateral decubitus position to ensure similar views in all echocardiographic assessments. A commercial echocardiographic system was obtained that used a 12-MHz probe with a small footprint (SonoSite, Seattle, WA). Scanning was performed as previously described [12]. The heart was scanned in longitudinal view in 2-dimensions and M-mode, where it is usually possible to see a 2-chamber view. Then the probe was turned 90° to get the best possible cross-sectional view of the left ventricle (LV) just above the papillary muscles. To minimize the intraobserver error in measurements, three consecutive measurements were averaged.
The LV end-diastolic (LVEDD) and end-systolic (LVESD) diameters were measured according to the American Society of Echocardiology leading-edge method [12]. Fractional shortening (FS) was determined as [(LVEDD – LVESD)/LVEDD]. The ejection fraction (EF) was estimated as [(LVEDV – LVESV)/LVEDV]. To ensure reproducibility, all before and after intervention measurements were performed by one experienced observer (J.F.A.) who was blinded to the treatment groups.
Harvesting of Hearts and Tissue Processing
The recipient rats were sacrificed at 12 and 24 hours, and at 1 and 2 weeks after cell therapy and echocardiography. Their hearts were harvested and dissected from the surrounding tissue. The LV was cut transversely in half, evenly along the largest circumference of the infarcted area. One half was fixed and stained for β-galactosidase activity using X-gal staining, as described previously [12]. The remaining half of the heart specimen was finely minced and put into a tube with the RNALater solution (Qiagen, Valencia, CA) and stored at 4°C for RNA extraction.
Quantitative Real-Time Polymerase Chain Reaction
Total RNA was isolated with an RNeasy kit (Qiagen) according to the manufacturer's instructions. Total RNA (5 µg) was subjected to first-strand complimentary DNA synthesis with QuantiTect Reverse Transcription Kit (Qiagen).
Quantitative real-time polymerase chain reaction (RT-PCR) was performed to compare the changes in expression of different inflammatory cytokines using the following primers:
The My-iQ system (Bio-Rad, Hercules, CA) was used to monitor RT-PCR amplification using SYBR Green I. Reaction conditions were as follows: hot start for 120 seconds at 95°C, melting at 95°C for 15 seconds, annealing at 56°C for 12 seconds, and amplification at 72°C for 15 seconds. Samples were run in duplicate, and the average crossing point value was used for calculations by the method of 2-
CT.
The relative quantitation value of a target gene, normalized to β-actin as the internal control gene, was expressed as a number, which indicated the relative expression compared with that gene. The possibility of amplifying contaminating DNA and nonspecific amplification were avoided with precautions [11].
Conventional PCR and Gel Bands Semiquantification
Using the same samples as in quantitative RT-PCR assay, the complimentary DNA was also used in regular PCR to compare changes in the genes expressing matrix metallopeptidase 2 (MMP-2) and tissue inhibitor of metallopeptidase 1 (TIMP-1). We mixed the primers of β-actin and TIMP-1 together in one tube for each running, and MMP-2 was done separately with the following thermal protocol: 50°C for 30 minutes, 95°C for 15 minutes, 30 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, and the final extension step at 72°C for 10 minutes.
Primers used for amplification were:
Images of the PCR ethidium bromide-stained agarose gels were acquired with an image system (Cohu Inc, Poway, CA), and quantification of the bands was performed by ImageJ-1.41 software (National Institutes of Health, Bethesda, MD). Band intensity was expressed as relative absorbance units normalized to β-actin. The ratio between the samples was determined and compared.
Collagen Content and Tissue Injury Measurement
Five consecutive 5-µm sections were prepared from the midportion of the infracted area in all samples. Sections were stained with the collagen-specific dye Sirius red 3BA in a saturated picric acid solution to allow clear discrimination between cardiomyocytes and ECM. The slides were scanned at 2400 dpi with x4 magnification, and the pictures were red-green-blue split to the green color layer for measurement. In the digital images, scar area and the total area of myocardium were traced manually by a blinded observer and measured automatically by the computer by the ImageJ-1.41 software. Infarct size, expressed as a percentage, was calculated by dividing the sum of infarct areas by the total sum of LV areas, including those without infarct scar, and multiplying by 100.
The following morphologic criteria were applied in a blinded fashion for tissue injury assessment: score 0, no damage; score 1 (mild), interstitial edema and focal necrosis; score 2 (moderate), diffuse myocardial cell swelling and necrosis; score 3 (severe), necrosis with presence of contraction bands and neutrophil infiltrate; score 4 (highly severe), widespread necrosis with presence of contraction bands, neutrophil infiltrate, and hemorrhage.
Statistical Analysis
All data are expressed as mean ± standard deviation. Data were analyzed using SPSS 16.0 software (SPSS Inc, Chicago, IL). The t test and two-way repeated analysis of variance (ANOVA) were used to make comparisons and test the differences for continuous variables. A value of p < 0.05 was considered statistically significant.
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Results |
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Group C showed more of an increase than group M in gene expressions for all the measured proinflammatory and antiinflammatory cytokines IL-1β, IL-6, and IL-10 from 12 hours to 2 weeks and for IL-8 from 12 to 24 hours. The increase in cytokines was significant, especially during the first 12 to 24 hours after MI and gradually tapered over time. In particular, the increase in antiinflammatory IL-10 was much more exaggerated during this early phase (Table 1). Repeated measures ANOVA revealed that differences were significant over time within groups in the expression of IL-1β, IL-8, and IL-10.
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0.000001).
FS showed a similar trend with improvement in group C at 12 hours (group M = 0.283 ± 0.044 vs group C = 0.322 ± 0.082, p = 0.051) and was significantly increased at 24 hours (group M = 0.333 ± 0.055 vs group C = 0.391 ± 0.090, p = 0.038), 1 week (group M = 0.282 ± 0.037 vs group C = 0.419 ± 0.119, p = 0.001), and 2 weeks (group M = 0.198 ± 0.061 vs group C = 0.455 ± 0.111, p = 0.001), respectively. Repeated measures ANOVA also revealed that FS differences were significant over time within groups (p = 0.006) and between groups (p 0.000001). Taken together, these data suggest that the beneficial effects of stem cell therapy actually occurred quite early after myocardial injury.
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Comment |
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Although few studies have evaluated the early cardiac functional changes after MSC treatment, Francis and colleagues [14] showed improved cardiac function as early as 1 hour after transplanting MSCs, suggesting that mechanisms other than cell differentiation or cell fusion might be responsible at different stages of cell-based therapy. Tang and colleagues [19] also raised doubts about stem cell differentiation as a sole contributor to myocardial regeneration and subsequent functional recovery because very few engrafted MSCs were shown to express specific cardiac markers, such as connexin-43 and cardiac troponin I. The small number of engrafted cells in proportion to the functional improvement suggested that increased myocardial mass as a sole mechanism for improved cardiac function appeared less plausible. Some reports have suggested other mechanisms, among which the secretion of various mediators through a "paracrine" effect from the transplanted MSCs has gained increasing attention [6, 20].
Persistent proinflammatory cytokine elevation has been associated with a worse clinical outcome in patients suffering recent MI. Induction and release of the proinflammatory cytokines IL-1β, tumor necrosis factor-
(TNF-), IL-6, and IL-8 are consistently found in experimental models and clinical cases of MI [21, 22]. There is also evidence showing a dramatic reduction in infarct size with the use of specific antiinflammatory strategies [23–25]. Our current study suggests that transplanted MSCs up-regulated both proinflammatory and antiinflammatory gene expressions in the infracted myocardium early after implantation. This cytokine profile was contrary to our original hypothesis that MSCs might inhibit the expression of proinflammatory cytokines while increasing the antiinflammatory cytokine activity.
Our findings, however, are consistent with a clinical study by Kilic and colleagues [10], who showed that serum concentrations of IL-1β and IL-6 were significantly higher in patients with new coronary events. On the other hand, Tögel and colleagues [11] showed that at 24 hours after cell therapy, the expression of proinflammatory/antiinflammatory cytokines reversed significantly in favor of antiinflammatory IL-10, basic fibroblastic growth factor, transforming growth factor-β, and B-cell lymphoma 2 up-regulation in MSC treated kidneys. This was associated with measurable recovery in renal function too early to be related to neoglomerular cell formation. Interestingly, such renoprotection was not obtained with syngenic fibroblasts.
Histologic results of our study also demonstrated that acute myocardial injury caused fluxes of inflammatory cells infiltration, cytokine mediators, and tissue injury in groups M and C within the first 24 hours. However, the antiinflammatory cytokines surge exceeded that of the proinflammatory counterparts, leading to more favorable ECM remodeling and functional improvement in longer follow-up. These findings suggested that the transplanted MSCs did not change or inhibit the inflammatory reaction histopathologically after acute MI. Reactive neutrophil cells and damaged endothelial cells of blood vessels classically produce the proinflammatory cytokines, and therefore, it is not expected that MSCs implantation after MI would reverse this phenomenon immediately. The tissue trauma associated with MSC implantation per se may perhaps further aggravate the inflammatory response.
On the other hand, these findings could also reflect the insufficient number of retained MSCs to completely suppress the acute inflammation, because many injected cells might have been lost early after injection due to mechanical leakage or washout, as suggested by Teng and colleagues [26]. By 12 to 24 hours after MSC implantation, however, the retained stem cells may respond and subsequently reversed the proinflammatory/antiinflammatory ratio of the microenvironment.
In the current study, IL-10 gene expression was remarkably up-regulated relative to the proinflammatory cytokines early after MSC therapy and showed evidence of strong cytoprotective effects on the cardiomyocytes. This was associated with decreased MMP-2 and increased TIMP-1 gene expression during the repair process after MSC therapy. MMP-mediated matrix degradation is crucial in infarct healing and the pattern of left ventricular remodeling, leading to various LV functional changes in patients with heart failure. Berry and colleagues [27] showed that MSC injections limited cell apoptosis and fibrosis and thus improved LV function and reduced dilatation.
Recent work by Guo and colleagues [28] examined the cytokines and functional parameters 4 weeks after cell therapy and revealed a decrease in deposition of collagen types I and III and decreased gene and protein expression of MMP-1 and TIMP-1. These results correlated with attenuated ventricular dilatation and thickening. Interestingly, the authors also reported decreased expression of proinflammatory cytokines such as TNF-, IL-1β, and IL-6, suggesting a possible role for inflammatory mediators in the remodeling process [28, 29]. IL-1β and TNF- have been shown to increase MMP-2 and MMP-9 matrix degradation and activity [30], whereas IL-10 and transforming growth factor- enhanced TIMP synthesis, regulating the matrix degradation and producing a favorable myocardial remodeling for better functional recovery. Therefore, changes in the cytokine milieu might play a key role in regulating ECM after myocardial injury. In our study, the analysis of antiinflammatory IL-10 gene expression, ECM formation, and functional recovery correlated with result reported by others and suggests a potential mechanism of stem cell therapy in an MI model.
The temporal and spatial discrepancies of various cytokine expressions during different scenarios of tissue organ injuries have been suggested to be more important in determining the outcome rather than the individual mediator per se. One study showed IL-6 expression was enhanced early during the first 6 hours after reperfusion of the previously ischemic myocardium [31]. Others have suggested that after 24 hours of reperfusion, the IL-6 mRNA expression was down-regulated in the same ischemic segments in which IL-10 mRNA up-regulation was found [11]. On the other hand, in a non-reperfused infarct model, IL-6 over-expression lasted as long as 24 hours [32], but in such a permanent coronary occlusion model, both IL-10 and IL-6 mRNA over-expression persisted simultaneously [33]. These findings suggested that although a surge in proinflammatory cytokines invariably occurred after injury, the expression of antiinflammatory mediators reflected a counter-regulatory response that is important for survival and favorable organ function recovery.
Recent studies have supported the notion that a lack of an appropriate antiinflammatory counter-balance in response to a certain proinflammatory reaction, rather than the individual proinflammatory or antiinflammatory mechanism, might be the culprit of the increased coronary risk [34, 35]. In our study, proinflammatory and antiinflammatory cytokine gene expressions were both up-regulated after MSC therapy. However, the LV functional recovery suggested that the two opposing cytokine forces had an unbalanced effect, such that the antiinflammatory effects predominated and resulted in more favorable histopathologic remodeling and subsequent improved cardiac functional improvement. This possible paracrine mechanism of MSC therapy may at least partly explain the early functional improvement after MI after stem cell therapy, before transdifferentiation or other more protracted mechanisms take place at a later period.
The current study has some limitations that merit further investigations. First, it is possible that MSCs act through other collective multifactorial mechanisms by many complex mediators. The role of inflammatory cytokines may only be a fraction of the overall mechanisms of stem cell therapy in this MI model. It is, however, interesting that a brief and possibly small amount of retained MSCs might exert profound longer protective effects on the injured myocardium, as many others have demonstrated.
Second, we did not determine the fate, the subtype, and the actual number of MSCs required to produce the beneficial effects. This is, however, a question that is actively pursued by many others in the field, and no agreement has yet been reached at present.
Third, the current rodent model is far from clinical relevance, and a study in larger animals should be considered. On the other hand, the use of small rodents is a well accepted and more preferable model in the study of molecular mechanisms of stem cell therapy. Better understanding of the mechanisms of stem cell therapy will undoubtedly lead to future larger-animal studies and provide information for evidence-based research trials in the clinical setting. The present study proposes a new mechanism implicating antiinflammatory cytokines in the early benefit of MSC implantation after MI.
Fourth, this study did not address the relative contribution of various cytokines that surge from damaged tissue or injected MSCs, and further investigation using a knock-out rodent model will help to answer this question. However, we confirmed that the presence of MSCs modified these mediators and resulted in better functional recovery. Also, whether injection of other cell sources would produce the observed benefit is a pertinent question that we did not address. Tögel and colleagues [11], however, showed that the administration of fibroblasts did not reproduce the renoprotective effects in a renal injury model.
Finally, the cell therapy was injected 10 minutes after coronary ligation, which may not necessarily simulate clinical settings. However, the current model maximizes the effect of inflammatory cytokines after acute MI. Further study in a large-animal model will allow the introduction of MI in a noninvasive fashion, followed by cell therapy. This will overcome the current limitation with our rodent model in which creation of MI and reintervention for MSC injection at a later time will produce prohibitively high surgical mortality.
In summary, our study shows that isogeneic MSCs injected into periinfarcted regions of ischemic myocardium after coronary ligation reduces the proinflammatory/antiinflammatory cytokine gene expression ratio that is correlated with improved cardiac function. The ratio of cytokine gene expressions favoring antiinflammation with less scar formation suggests that a paracrine mechanism may at least partly explain the early functional improvement. These findings open up a new and potentially important mode of action for MSCs, which also provides further impetus to study the role of IL-10 in the pathophysiology of MI.
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Acknowledgments |
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