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Department of Medical Bioregulation, Division of Cardiovascular Surgery and Medicine, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan
Submitted 7 February 2006 ; accepted in final form 17 March 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, PDGF, and IGF-1, were produced from BM-MNCs, some of which were enhanced significantly under hypoxia stimulation. When compared with a control blank medium, the supernatants of BM-MNCs cultured under normoxic or hypoxic conditions inhibited apoptosis significantly and preserved the contractile capacity of isolated adult rat cardiomyocytes in vitro (P < 0.05). Using a rat model of acute myocardial infarction, we injected the supernatants of BM-MNCs or control medium intramyocardially on day 0 and then intraperitoneally on days 2, 4, and 6 after infarction. When compared with the control medium, the supernatants of BM-MNCs cultured under both normoxic or hypoxic conditions increased the microvessel density and decreased the fibrotic area in the infarcted myocardium significantly, contributing to remarkable improvement in cardiac function. Various cytokines were produced by BM-MNCs, and these cytokines contributed to functional improvement of the infarcted heart by directly preserving the contractile capacity of the myocardium, inhibiting apoptosis of cardiomyocytes, and inducing therapeutic angiogenesis of the infarcted heart.
ischemia; angiogenesis; bone marrow mononuclear cell; cytokine; apoptosis
It has been reported that the implanted stem cells of BM-MNCs can survive and be proliferated and differentiated into endothelial cells or cardiomyocyte in situ, contributing to functional improvement of the injured heart by rebuilding the damaged myocardium and cardiac vessels (14, 18, 26). However, there is no consensus on the plasticity of bone marrow stem cells, and most studies have found that myogenic or endothelial differentiation rarely occurs, if at all (2, 4, 22, 23, 25). Therefore, it is doubtful that the in situ differentiation or proliferation of implanted cells contributes remarkably to functional improvement of the injured heart.
Conversely, it is speculated that the cytokines produced by these implanted BM-MNCs play a more important role than their differentiation and proliferation (15). Experimental studies have shown that BM-MNCs produce many angiogenic factors, known as cytokines, in vitro and in vivo, including VEGF, basic fibroblast growth factor (bFGF), and angiopoietin-1 (11, 17, 16, 24). The implantation of BM-MNCs increases microvessel density and blood flow to the injured heart significantly (10, 11, 14, 17, 18, 24), and the resulting improvement in cardiac function is thought to be attributed to the induction of therapeutic angiogenesis by the angiogenic cytokines released from the implanted BM-MNCs. Beyond the effect of enhancing therapeutic angiogenesis, the cytokines produced by BM-MNCs may also directly improve the survival and contractile capacity of ischemic myocardium. Thus we investigated the role and mechanisms of the secretory pathway of BM-MNC implantation for myocardial repair by in vitro assessments and by using a rat model of acute myocardial infarction.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Collection and cultivation of BM-MNCs. The BM-MNCs were collected and cultivated as described previously (24). Briefly, bone marrow was collected from the femur and tibia and suspended in PBS. Mononuclear cells were separated by gradient centrifugation. Freshly collected cells were suspended at a density of 5.0 x 106 cells/ml in DMEM supplemented with 10% fetal calf serum and 1% penicillin-streptomycin. Cells were seeded on a 24-well culture plate (5.0 x 106 cells/well) and incubated under normoxia or hypoxia (1% O2) at 37°C. After 24 h of cultivation, the total number of surviving cells was counted after staining with 0.4% trypan blue solution (Sigma), and the supernatant was harvested for the following studies.
Detection of cytokine release from BM-MNCs by ELISA and Western blot analysis.
We measured the concentrations of VEGF and IL-1 in the culture supernatant with a rat IL-1 ELISA kit and a rat VEGF ELISA kit (R&D Systems), according to the manufacturer's instructions (11, 17). The expressions of IGF-1, bFGF, and transforming growth factor- (TGF-) in the culture supernatant were measured by the Western blotting method using monoclonal or polyclonal antibodies against rat IGF-1, bFGF, PDGF, and TGF-, as described previously (24).
Endothelial cell proliferation assay. To evaluate the effect of cytokines produced by BM-MNCs on the proliferation of endothelial cells, coronary endothelial cells were harvested from the Wister rat hearts by collagenase digestion, as described previously (12). Purified endothelial cells were seeded on collagen I-coated dish and expanded for 7–10 days (2 passages) by culture with Endothelial Medium Kit Liquid (Sigma). Expanded endothelial cells were then seeded in a collagen I-coated 96-well plate (1 x 104 cells/well) and cultured in DMEM containing 10% fetal calf serum and 1% penicillin-streptomycin, by the addition of 0%, 20%, or 40% of the supernatants of BM-MNCs. After 3 days of cultivation, the proliferation of endothelial cells was measured by the uptake of bromodeoxyuridine, by using a cell proliferation ELISA system (Amersham Life Science, Little Chalfont, UK) (12).
Estimation of apoptosis of isolated adult rat cardiomyocytes.
To observe if cytokines produced by BM-MNCs protected cardiomyocytes against apoptosis, adult cardiomyocytes were isolated from 15-wk-old rats by using a modified protocol described elsewhere (32). Briefly, the hearts were excised quickly and cannulated via the ascending aorta. The hearts were perfused for 2 min with Ca2+-free minimal essential medium (MEM; Sigma) containing 5 mM taurine, 3.8 mM creatine, and 10 mM 2,3-butanedione monoxime at 37°C and then with MEM containing 50 mM of CaCl2 and 0.8 mg/ml of collagenase B (Roche Applied Science, Indianapolis, IN). After 30 min of digestion, the left ventricle (LV) was removed quickly and then cut into several chunks and digested further in a shaker (60
70 rpm) for 10 min in the same enzyme solution at 37°C. Isolated cardiomyocytes were suspended in control medium, or in the supernatants of BM-MNCs collected beforehand, and then seeded on laminin-coated culture slides. After 72 h of cultivation, the apoptosis of cardiomyocytes was detected by terminal dUTP nick-end labeling (TUNEL) assay with the use of Apoptosis Detection Kits (R&D System, Minneapolis, MN). The apoptosis index was calculated as the number of apoptotic cells per 100 nucleated cells.
Measurement of contraction and Ca2+ transient of isolated adult cardiomyocytes. Adult rat cardiomyocytes were isolated and cultured on laminin-coated tissue culture dishes, as described above. After 8 h (baseline) and 72 h of cultivation, the contraction and Ca2+ transient were measured by loading with 1 mM of fura-2 (Molecular Probes, Eugene, OR) and stimulated electrically by using bipolar pulses (5-ms duration, 1 Hz). The electrical stimulation was done in HEPES buffer (37°C) containing 1.08 mM Ca2+. Only cells that remained rod shaped, without blebs or other visible morphological alterations, and responded adequately to stimulation at 1 Hz were included in the testing protocol. Fura-2 fluorescence was measured by using a dual-fluorescence, calcium ion-sensing system (Ion Optix, Milton, MA) at a sampling rate of 500 Hz. At the same time, cell length and shortening were measured by a video-based edge-detection system. The cell shortening and Ca2+ ratio after isolation were identified as the baseline.
Rat myocardial infarction model and supernatant injection.
The rat myocardial infarction model was established as described previously (17). After the left anterior descending artery was ligated, rats were randomly selected and given an intramyocardial injection immediately, with control medium (control group, n = 8), supernatant of BM-MNCs cultured in normoxic condition (normoxia group, n = 9), or supernatant of BM-MNCs cultured in hypoxic condition (hypoxic group, n = 9). With the use of a 31-gauge needle and 100-µl syringe, each rat was given four point injections (10 µl/injection) in the border zone of infarction, 2 mm apart. As our preliminary studies showed that a single intramyocardial injection failed to improve the cardiac function, the rats in each group were also matched for an additional intraperitoneal injection of 500 µl control medium or supernatants of BM-MNCs cultured in normoxic and hypoxic condition, respectively, on days 2, 4, and 6 after infarction.
Echocardiography. Cardiac function was measured before treatment and then 7, 14, and 28 days after treatment by a single observer blind to the treatment regimen, with echocardiography by using a 10-MHz annular array transducer (20, 21, 24). After the induction of light general anesthesia, the hearts were first imaged two dimensionally in long-axis views at the level of the greatest LV diameter. The systolic and diastolic LV areas were measured at the same time. This view was used to position the M-mode cursor perpendicular to the LV anterior and posterior walls. The LV dimension was measured at the end-diastolic dimension (LVEDD) and at the end-systolic dimension (LVESD). The dimensions were calculated from the average measurements of three selected beats. The LV percent fractional shortening (LVFS%) was calculated as (LVEDD – LVESD)/LVEDD x 100 (%).
Histological evaluation. After the echocardiography on postinfarction day 28, the rats were killed by an overdose of anesthesia, and the hearts were harvested. Samples were frozen in liquid nitrogen and stored at –80°C until use. To detect the development of microvessels, 5-µm-thick frozen sections were stained with alkaline phosphatase by using an indoxyl tetrazolium method and then counterstained with eosin (12, 24). The number of microvessels was counted under a microscope with x200 magnification, by a single observer blind to the treatment regimen. At least 10 different fields were selected randomly from the central infarct zone, the infarct border zone, and the remote noninfarcted zone on three independent slides of each sample, and the mean number of microvessels per field in each zone was calculated for statistical analysis.
Azan staining was also done to determine the wall thickness and the degree of collagen fiber accumulation in the infarcted region (20). By using the image analysis software NIH Image (NIH, Research Service Branch), the mean wall thickness was measured from three equidistant points of the central infarct area, and the area of fibrosis was calculated as the area of stained fibrotic tissue divided by the total area of LV wall. Measurements were done on at least two separate sections from each heart, and the averages were used for statistical analysis.
Statistical analysis. All data are expressed as means ± SD. Echocardiographical data were compared by two-way ANOVAs for repeated measures, and group differences at specific time points were then assessed by a Bonferroni t-test. Other data were assessed by one-way ANOVAs, followed by a Bonferroni t-test. A value of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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, bFGF, PDGF, IGF-1, and TGF-, were detected in the supernatants of BM-MNCs after 24-h cultivation under conditions of normoxia or hypoxia (Fig. 1). Quantitative measurement by ELISA showed that the concentrations of VEGF and IL-1 in the supernatants were significantly higher when cultured under hypoxia than normoxia, the respective values for VEGF being 18.9 ± 5.81 vs. 61.54 ± 21.2 pg/ml (P < 0.001; Fig. 1A) and for IL-1, 178.2 ± 27.7 vs. 231.9 ± 17.5 pg/ml (P < 0.01; Fig. 1B). Similarly, Western blot analysis detected the production of bFGF, PDGF, IGF-1, and TGF- from the BM-MNCs, which also seemed to be enhanced under hypoxic conditions (Fig. 1C).
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Supernatants of BM-MNCs improved function of the infarcted heart. Cardiac function was assessed by echocardiography before and then 7, 14, and 28 days after treatment (Fig. 4). The LVFS% decreased after ligation of the left anterior descending artery in all the rats, without significant differences among the groups. The LVFS% increased obviously within 7 days of treatment in the groups given the injection of both normoxic and hypoxic supernatants of the BM-MNCs but not in the group given control medium. Thereafter, the LVFS% decreased gradually when the supernatants of the BM-MNCs were stopped. However, the LVFS% was significantly higher in the hypoxia group (P < 0.01 on day 7 and P < 0.05 on day 14 after treatment) and in the normoxia group (P < 0.01 on day 7 after treatment) than in the group given control medium. Although the LVEDD did not differ significantly among the groups, the LVESD was significantly lower in the hypoxia and normoxia groups than in the control group (P < 0.05 by group x time interactions analysis).
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DISCUSSION |
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Recent experimental evidence suggests that the improvement of blood flow and cardiac function achieved by the delivery of BM-MNCs may be attributed mainly to the release of multiple cytokines and chemokines rather than to endothelial or myogenic differentiation (2, 15, 16, 23, 25). Thus it is now thought that therapeutic angiogenesis induced by the release of cytokines by the delivered BM-MNCs plays a major role in improving the function of the ischemic heart. However, there is little evidence documented on the effect of cytokine release from delivered BM-MNCs for directly protecting cardiomyocytes against ischemic injury. Using the supernatants of BM-MNCs and a rat model of acute myocardial infraction, we investigated the mechanisms of the implantation of BM-MNCs for myocardial repair through the secretory pathway.
First, we examined the production of several important cytokines from BM-MNCs in vitro, by cultivation under hypoxia and normoxia. In accordance with previous reports, many important cytokines for regulating cell proliferation, survival, and the angiogenic process were clearly detected in the supernatants of BM-MNCs after 24 h of cultivation. These cytokines included VEGF, bFGF, IL-1, PDGF, IGF-1, and TGF-, some of which were enhanced under conditions of hypoxia. We also found that the supernatants of BM-MNCs increased the proliferation of endothelial cells and inhibited the apoptosis of isolated adult cardiomyocytes in vitro. This suggests that the cytokines produced by BM-MNCs have the potential to induce therapeutic angiogenesis and inhibit cardiomyocytes apoptosis.
Second, we measured the contractile capacity and Ca2+ transient of isolated adult cardiomyocytes after cultivation in the supernatants of BM-MNCs or in the control medium. We found that the fractional shortening and amplitude of the Ca2+ ratio in isolated adult cardiomyocytes were significantly damaged after 72 h of cultivation in the control medium but that they were very well preserved when cultured in the supernatant of the BM-MNCs. This provided the first direct evidence that cytokines released by BM-MNCs have a protective effect on the contractile capacity of cardiomyocytes, providing new insight into the mechanism of how BM-MNCs improve cardiac function.
Third, using a rat model of acute myocardial infarction, we investigated the potency of cytokines produced by BM-MNCs for myocardial repair. Initially, we gave a single intramyocardial injection of supernatants after infarction. Contradictory to our in vitro findings, the single intramyocardial injection of supernatants did not increase the LVFS% significantly. It was reported previously that significant decrease in infarct size and myocyte apoptosis could be achieved by a single intramyocardial injection of the conditioned medium from mesenchymal stem cells overexpressing Akt gene but not by conditioned medium from mesenchymal stem cells without Akt overexpression (9). We speculated that the contradictory findings might be related to the insufficient dose of cytokines delivered by the single intramyocardial injection of supernatants, and we changed our in vivo protocol by giving an additional injection of supernatants. As expected, we found that the additional intraperitoneal injection of the supernatants of BM-MNCs improved the LVFS% significantly, but the administration of control medium did not. Histological analysis revealed that the administration of the supernatants of BM-MNCs increased the microvessel density in the border zone of infarcted myocardium and decreased collagen deposition significantly in the LV wall. Although we did not measure the levels of local and systemic cytokines, we speculated that the intraperitoneal injections of 500 µl of supernatants increased the levels of systemic cytokines and helped to repair the injured heart. These findings provided further evidence that cytokines produced by BM-MNCs have the capacity to protect the injured heart, which should be attributed to the augmentation of neovascularization and the improvement of remodeling after myocardial infarction.
Although we observed an increase in microvessel density and a decrease in collagen deposition in the infarcted myocardium 28 days after treatment, the LVFS% did not differ significantly among the groups 28 days after treatment. Thus regional improvements in myocardial perfusion and a decrease in collagen deposition are not enough to increase global cardiac function. According to our data, the LVFS% began to deteriorate gradually after we stopped giving the supernatants of BM-MNCs on day 7. This suggests that the continuous administration of supernatants may be necessary to maintain functional improvement of the injured heart. Our previous studies showed that BM-MNCs survived well after intramyocardial implantation and that the cytokines VEGF, IL-1, and angiopoietin were increased in the infracted heart for 1–2 wk after cell therapy (17, 24). These findings indicated that the cytokines were released continuously from the implanted cells after cell delivery. If so, cell-based therapy will become a revolutionary method for repairing the injured heart. However, recent investigations on cell-based myocardial repair found functional improvement 4 wk but not 6 mo after the implantation of mesenchymal stem cells (3). If further studies confirm that these therapeutic benefits are transient, we must find ways of extending and maintaining them.
Interestingly, the supernatants of BM-MNCs cultivated under conditions of hypoxia were more effective than those cultivated under conditions of normoxia, for preserving the contractile capacity of isolated cardiomyocytes and for improving the cardiac function of infarcted hearts. This difference might explain the higher concentration of cytokines in the hypoxic supernatant because cytokine production by BM-MNCs is enhanced under conditions of hypoxia. Although we could not conclude that the functional improvement of infarcted hearts is dependent on the quantity of cytokines produced by BM-MNCs, the mild potency of supernatant for repairing infarcted myocardium might be attributed to the fact that we used "thin" supernatant (collected after 24 h cultivation of 5 x 106/ml BM-MNCs) in this study.
Our findings showed that various cytokines are produced by BM-MNCs. Although many of these cytokines, including IGF-1 and PDGF, have been reported to prevent apoptosis of myocytes and improve cardiac function (5, 6, 8, 19), it is also possible that some of the inflammatory cytokines produced by BM-MNCs, such as TNF-
and IL-6, are detrimental to myocardial repair. However, it is clear that the mixed cytokines produced by BM-MNCs can increase the proliferation of endothelial cells, inhibit apoptosis of cardiomyocytes, preserve contractile function of isolated cardiomyocytes, and help repair the infarcted heart in vivo. We tried to identify the key cytokine in the medium that protected the heart and found that antibody perturbation to IGF-1 impaired partly (30%) the inhibitive effect of cardiomyocytes on apoptosis, induced by the supernatant of BM-MNCs (data not shown). This indicates that multiple cytokines contribute to the protective effect of the heart. However, we do not know the functional role of each cytokine and growth factor and which cytokines or factors play the most important roles in myocardial repair. Further studies must be done to investigate the roles of each cytokine and evaluate their molecular mechanisms in myocardial repair.
In conclusion, we found that BM-MNCs produce many cytokines, which contribute to functional improvement of the infarcted heart by directly preserving the contractile capacity of the myocardium, by inhibiting apoptosis of cardiomyocytes and by inducing therapeutic angiogenesis of the infarcted heart. Our data also suggest that a secretory pathway might play the key role, albeit a complicated mechanism, in myocardial repair by cell-based therapy, such as the implantation of BM-MNCs into the infarcted heart.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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-preprogrammed bone marrow stem cells. Circulation 111: 2438–2445, 2005.This article has been cited by other articles:
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  V. K. Lai, K.-L. Ang, W. Rathbone, N. J. Harvey, and M. Galinanes Randomized controlled trial on the cardioprotective effect of bone marrow cells in patients undergoing coronary bypass graft surgery Eur. Heart J., October 1, 2009; 30(19): 2354 - 2359. [Abstract] [Full Text] [PDF]
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 A. Shabbir, D. Zisa, G. Suzuki, and T. Lee Heart failure therapy mediated by the trophic activities of bone marrow mesenchymal stem cells: a noninvasive therapeutic regimen Am J Physiol Heart Circ Physiol, June 1, 2009; 296(6): H1888 - H1897. [Abstract] [Full Text] [PDF]
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 T. Kamota, T.-S. Li, N. Morikage, M. Murakami, M. Ohshima, M. Kubo, T. Kobayashi, A. Mikamo, Y. Ikeda, M. Matsuzaki, et al. Ischemic pre-conditioning enhances the mobilization and recruitment of bone marrow stem cells to protect against ischemia/reperfusion injury in the late phase. J. Am. Coll. Cardiol., May 12, 2009; 53(19): 1814 - 1822. [Abstract] [Full Text] [PDF]
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 H. Mollmann, H. Nef, A. Elsasser, and C. Hamm Stem cells in myocardial infarction: from bench to bedside Heart, March 15, 2009; 95(6): 508 - 514. [Full Text] [PDF]
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 Q. Zhao, Y. Sun, L. Xia, A. Chen, and Z. Wang Randomized Study of Mononuclear Bone Marrow Cell Transplantation in Patients With Coronary Surgery Ann. Thorac. Surg., December 1, 2008; 86(6): 1833 - 1840. [Abstract] [Full Text] [PDF]
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 M. Gnecchi, Z. Zhang, A. Ni, and V. J. Dzau Paracrine Mechanisms in Adult Stem Cell Signaling and Therapy Circ. Res., November 21, 2008; 103(11): 1204 - 1219. [Abstract] [Full Text] [PDF]
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 X. Hu, S. P. Yu, J. L. Fraser, Z. Lu, M. E. Ogle, J.-A. Wang, and L. Wei Transplantation of hypoxia-preconditioned mesenchymal stem cells improves infarcted heart function via enhanced survival of implanted cells and angiogenesis. J. Thorac. Cardiovasc. Surg., April 1, 2008; 135(4): 799 - 808. [Abstract] [Full Text] [PDF]
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P < 0.001 vs. 20%. B: detection of apoptosis of isolated adult cardiomyocytes by terminal dUTP nick-end labeling (TUNEL). B, left: representative photo, stained by 4,6-diamidino-2-phenylindole (DAPI; top) and TUNEL (bottom), of isolated cardiomyocytes after 72-h cultivation in control medium. Arrowheads, apoptotic cardiomyocytes; arrows, necrotic cardiomyocytes. Quantitative analysis revealed that the apoptosis index (%apoptotic cells) of isolated cardiomyocytes was significantly higher after 72 h of cultivation in control medium than that after 72 h of cultivation in the supernatants of BM-MNCs under both normoxic and hypoxic conditions (*P < 0.05 vs. control; data derived from 6 independent experiments).
