| Bone marrow contains a population of progenitor cells known as mesenchymal stem cells (MSCs), which have the capability to colonize different tissues, replicate, and differentiate into multilineage cells. Because of their advantages, such as ease of obtaining by a simple routine bone marrow aspiration, ability to self-renew and pluri-potential of differentiation, MSCs have been considered as one of the most promising candidates of autologous cells for both cell therapy and gene therapy of mesenchymal tissues. MSCs can differentiate into cells of various phenotypes in the intarcted myocardium, participating in myogenesis and angiogenesis, and also secret lots of cytokines and vascular growth factors. It is reported that cardiac microenvironment might play a critical role in MSCs transdifferentiation. Interleukin-6 and tumor necrosis factor-alpha are important cytokines in the cardiac microenvironment, which can be secreted by cardiomyocytes and MSCs in some conditions. In order to understand the effect of cytokines on MSCs migration and intercellular signaling pathways, including Janus-Kinases and signal transducer and activator of transcription (JAK-STAT) and mitogen activated protein kinase/c-Jun N terminal kinase (MAPK/JNK), we simulated the inflammatory microenvironment of myocardium in vitro by adding interleukin-6 and tumor necrosis factor-alpha into MSCs culture individually. In the present study, we also investigated the effect of hypoxia/reoxygenation preconditioning medium of cardiomyocytes on MSCs differentiation into myocardial-like cells. The biological features of MSCs migration and differentiation in vitro are helpful to interpret the characteristics and mechanisms of MSCs transplantation in cardiovascular diseases.Part I Effect of cardiac inflammatory microenvironment on MSCs migration and expression of MAPK/JNK and JAK-STAT pathwayBone marrow samples were obtained from posterior superior iliac crest of healthy volunteers under general anesthesia. MSCs were isolated with a density gradient (Ficoll) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum selected for MSCs outgrowth. With two passages, homogenous MSCs devoid of hematopoietic cells were used for experiments before morphology examination and flow cytometric analysis. MSCs were divided into three groups, control group (cultured with DMEM), interleukin-6 groups (cultured with interleukin-6) and tumor necrosis factor-alpha groups (cultured with tumor necrosis factor-alpha). According to the several dilutions of interleukin-6 in the mediums, interleukin-6 groups were divided into four subgroups, lng/ml, lOng/ml, 50ng/ml and lOOng/ml. Tumor necrosis factor-alpha groups were divided into four subgroups, at the concentrations of 0. lng/ml, lng/ml, lOng/ml and 50ng/ml in the mediums. MSCs were seeded in Transwell inserts with 8.0um pore polycarbonate membrane, incubated 6 hours with the migration mediums contained several dilutions of interleukin-6 and tumor necrosis factor-alpha in the lower chamber. MSCs migration was assessed by light microscopic examination of the underside of polycarbonate membrane. Western-blotting analysis for the expression of JNK1, JNK2 and STAT1 signaling proteins was performed in MSCs co-cultured with the different mediums (as mentioned above) for 15 min, 30 min, 60 min and 120 min individually. ResultsThe present study demonstrated that interleukin-6 and tumor necrosis factor-alpha had a significant attraction on MSCs migration (/><0.05). Interleukin-6 significantly increased MSCs migration at the concentrations of lOng/ml, 50ng/ml and lOOng/ml compared to the control (PO.05). Interleukin-6 showed the greatest effect at the concentration of 50ng/ml, compared to the concentrations of lOng/ml and lOOng/ml (P<0.05). Although all four subgroups of tumor necrosis factor-alpha showed a significant attraction on MSCs migration compared to the control, tumor necrosis factor-alpha showed the greatest effect at the concentration of lOng/ml (P<0.05). Tumornecrosis factor-alpha significantly increased MSCs migration at the concentrations of lOng/ml and 50ng/ml compared to those of O.lng/ml and lng/ml (P<0.05).JNKl protein expressions in MSCs were downregulated by interleukin-6 (P<0.05), whereas JNK2 protein had no significant expressions compared to the control (P>0.05). JNKl protein levels were greatest decreased by interleukin-6 at the concentration of 50ng/ml compared to the concentrations of lng/ml, lOng/ml and lOOng/ml (P<0.05). A significant inhibition was observed at the expressions of JNKl protein in MSCs incubated with interleukin-6 for 15 min (PO.05), however the differences of JNKl expressions did not reach statistical significance in MSCs incubated with interleukin-6 for 30 min, 60 min and 120 min compared to the control (P>0.05). The expressions of JNKl and JNK2 had no significant differences between four subgroups of interleukin-6 for different culture durations (P>0.05).Tumor necrosis factor-alpha activated JNK2 protein expression in MSCs while inhibited JNKl protein expression (P<0.05). Tumor necrosis factor-alpha showed the greater activation on the expressions of JNK2 protein at the concentrations of lOng/ml and 50ng/ml, compared to the other dilutions of tumor necrosis factor-alpha and the control (P<0.05). Protein levels of JNK2 stimulated by tumor necrosis factor-alpha had no significant differences between the concentrations of lOng/ml and 50 ng/ml (P>0.05). JNK2 protein expressions were significantly elevated in MSCs incubated with tumor necrosis factor-alpha for 60 min, compared to 15 min, 30 min and 120 min (.PO.05). Expression levels of JNKl protein in MSCs were greater inhibited by tumor necrosis factor-alpha at the concentrations of O.lng/ml and lng/ml than those of lOng/ml and 50ng/ml CPO.05). Protein levels of JNKl stimulated by tumor necrosis factor-alpha had no significant differences between the concentrations of O.lng/ml and lng/ml (P>0.05). The pattern was the same at the concentrations of lOng/ml and 50ng/ml (F>0.05). A significant inhibition was observed at the expressions of JNKl protein in MSCs incubated with tumor necrosis factor-alpha for 15 min (P<0.05), while the differences did not reach statistical significance in MSCs incubated with tumor necrosis factor-alpha for 30 min, 60 min and 120 min compared to the control (.P>0.05). The expressions of JNKl and JNK2 had no significant differences between four subgroups of tumor necrosis factor-alpha for different culture durations (P>0.05).Expressions of STAT1 (p91/p84) protein in MSCs had no significant changes by interleukin-6 and tumor necrosis factor-alpha compared to the control (/">(). 05). And the pattern was the same between four subgroups of interleukin-6 or tumor necrosis factor-alpha for different culture durations (P>0.05).Part II Effect of cardiac hypoxia microenvironment on MSCs differentiation into cardiomyocyte-Iike cellsPrimary cardiac myocyte cultures were prepared by enzymatic dissociation of ventricular tissue from neonatal Sprague-Dawley rat according to methods described previously. To selectively increase the cardiac myocyte density, a two-step differential attachment technique was used. After dispersed cells were incubated on culture dishes for 60 min at 37°C in 5% CO2 incubator, non-attached viable cells were collected and incubated with DMEM supplemented with 10% fetal calf serum plus 0.1 mM bromide-oxyuridine (Brdu) to prevent low-level nonmyocardial cell proliferation. With one passage, myocardial cells were replaced with serum-free DMEM overnight and used for experiments. Ischemic injury was simulated in vitro by using a hypoxia/reoxygenation model as previously described. Briefly, myocardial cells were placed in an air-tight plexiglass humidified chamber, where air can be completely replaced by a gas mixture of 95% N2 / 5% CO2 to produce hypoxia conditions. The myocardial preconditioning medium of hypoxia for 4 hours and then reoxygenation overnight was left for following experiments and performed lactate dehydrogenase (LDH) examination.Bone marrow cells were extracted from the tibias and femurs of Sprague-Dawley rat with a modified method originally described by Dobson et al. The marrow pellet was washed in phosphate-buffered salt solution (PBS), centrifuged at 900g for 10 min and then resuspended in DMEM. Nucleated cells were isolated with a density gradient (Ficoll), then introduced into the flasks and cultured at 37°C in humid air with 5%CC>2 in DMEM containing 10% fetal calf serum, penicillin (lOOU/ml), streptomycin (lOOmg/ml). The medium was changed to remove the nonadherent cells at 48 h after seeding, andevery 4 days thereafter. Each primary culture was replated to two new flasks when MSCs grow to approximately 70% confluence. With two passages, homogeneous MSCs devoid of haematopoietic cells were used for the experiments. Cells were determined by fluorescence-activating cell sorting analysis before the experiments.MSCs were randomly divided into several groups, negative control group (MSCs incubated with DMEM for 72 h), positive control group (cardiomyocytes incubated with DMEM for 72 h), normal myocardial medium groups (MSCs incubated with normal myocardial medium) and preconditioning myocardial medium groups (MSCs incubated with hypoxia/reoxygenation preconditioning medium of cardiomyocytes). According to the culture durations (24 h, 48 h and 72 h), normal myocardial medium groups and preconditioning myocardial medium groups were divided into three subgroups respectively. Immunofluorescence detection and western-blotting analysis for cardiac myosin heavy chain, cardiac troponin and connexin 43 were performed in cultured cells of every group. ResultIt was interesting to find that hypoxia/reoxygenation preconditioning medium of cardiomyocytes could induce MSCs to express cardiac specific proteins. Immunofluorescence examination clearly detected cardiac myosin heavy chain and troponin in MSCs incubated with preconditioning myocardial mudium, whereas did not find in MSCs incubated with normal myocardial medium. Western-blotting analysis could also detect the expressions of troponin in MSCs incubated with preconditioning medium, but could not detect the expressions of connexin 43. Neither troponin nor connexin 43 could be detected in MSCs incubated with normal myocardial medium. The reliability of the analysis was confirmed with cultured cardiomyocytes under the same conditions. Semi-quantity analysis showed there was no statistical significance in the expressions of troponin among the subgroups of normal myocardial medium groups and preconditioning myocardial groups, P>0.05.ConclusionsIn the present study, interleukin-6 and tumor necrosis factor-alpha play a role as migration factors in vitro. Interleukin-6 is found to induce MSCs migration at the concentration of lOng/ml. MSCs migration was significantly attracted by interleukin-6 at the concentration of 50ng/ml compared to other dilutions and gradually inhibited at the concentration of lOOng/ml. The concentration of tumor necrosis factor -alpha is required for chemotactic migration. MSCs migration is most obvious by tumor necrosis factor-alpha at the concentration of lOng/ml while decreased at 50ng/ml.lnterleukin-6 and tumor necrosis factor-alpha have different influences on MAPK/ JNK signaling pathway. JNK1 protein expression is downregulated by interleukin-6, the higher the concentrations of interleukin-6, the greater the inhibition on JNK1 expression. It has the greatest inhibition of JNK1 expression at the concentration of 50ng/ml, decreased at lOOng/ml. Tumor necrosis factor-alpha is found to activate JNK2 expression in MSCs. the higher the concentrations of tumor necrosis factor-alpha (lOng/ml and 50ng/ml). the greater the activation on JNK2 expression. JNK1 expression in MSCs is decreased by tumor necrosis factor-alpha at the lower concerntrations (O.lng/ml and lng/ml). No significant changes can be observed at the expressions of STAT1, a signaling protein in JAK-STAT pathway, by interleukin-6 and tumor necrosis factor-alpha, which might be presumed, that other proteins of STAT family be involved in intercellular signal transductions of MSCs migration.At the same time, the study shows for the first time that the hypoxia/reoxygenation preconditioning medium of cardiomyocytes can induce MSCs to express cardiac specific contractile proteins in vitro, such as cardiac myosin heavy chain and cardiac troponin. It tends to confirm that the soluble cytokines secreted by myocardial cells in the hypoxia microenvironment are ideal inducers for MSCs differentiation into cardiomyocyte-like cells. Otherwise. MSCs in this study do not express connexin 43 when co-cultured with normal myocardial medium and hypoxia/reoxygenation preconditioning medium of cardiomyocytes. which might be presumed that the expressions of intercellular gap junction would be independent of soluble cytokines in the cardiac microenvironment.
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