Signals Through Glycoprotein 130 Regulate the Endothelial Differentiation of Cardiac Stem Cells
Objective— Cardiac Sca-1+ cells were originally identified as multipotent stem cells. To address the regulation of their differentiation, we investigated the effects of the proinflammatory cytokines on their endothelial differentiation.
Methods and Results— We examined the effects of the proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-11, and cardiotrophin-1 (CT-1) on the cardiac Sca-1+ cell differentiation. IL-11 and CT-1, whose receptor systems use glycoprotein 130 (gp130), induced endothelial-specific genes in Sca-1+ cells, but not TNF-α, IL-1β, or IL-6, analyzed by RT-PCR and by immunocytochemistry. Immnunoblot analyses showed that IL-11 and CT-1 activated signal transducer and activator of transcription 3 (STAT3), a downstream target of gp130, but not other cytokines. Though IL-6 receptor is not endogenously expressed in Sca-1+ cells, IL-6 exhibited the activity to induce the endothelial markers in the presence of soluble IL-6 receptor, an agonistic receptor, associated with STAT3 phosphorylation. Moreover, the inhibition of STAT3, by its dominant-negative form or siRNA, suppressed the induction of endothelial specific genes by IL-11 and CT-1. Finally, LIF and IL-11 transcripts were upregulated in postinfarct myocardium, accompanied by the induction of Sca-1+/VE-cadherin+ cells.
Conclusions— Gp130/STAT3 pathway plays critical roles in the regulation of endothelial differentiation of cardiac Sca-1+ cells.
Cardiac functions are regulated by various kinds of neurohumoral factors through paracrine/autocrine systems. Cardiac cells produce a wide range of the proinflammatory cytokines and their family, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, leukemia inhibitory factor (LIF), cardiotrophin-1 (CT-1), and IL-11, under pathological conditions.1 These cytokines transduce their signals in cardiac myocytes, vascular cells, or infiltrating inflammatory cells, modulating cardiac remodeling, repair, and regeneration after myocardial injury as a result.1–4
Based on the signaling pathways, these cytokines can be classified into 3 families; TNF-α family, IL-1β family, and IL-6 family. TNF-α exerts its cellular effects through 2 distinct receptors, TNF receptor type1 (TNFR1) and type 2 (TNFR2), and shares its signaling pathways with death signals such as Fas.5 IL-1β signaling is exclusively transduced through IL-1 receptor type 1 (IL-1RI), a member of Toll-like receptor/IL-1 receptor superfamily. Toll-like receptor/IL-1 receptor superfamily members are essential for innate immune and inflammatory responses.6,7 IL-6, LIF, CT-1, and IL-11 belong to IL-6 family. IL-6 family cytokines bind to their specific receptor subunit (α subunit), such as IL-6 receptor (IL-6R), LIF receptor (LIFR), and IL-11 receptor (IL-11R). The IL-6 family cytokine-its receptor α subunit complex makes a dimer with glycoprotein 130 (gp130), a common receptor subunit, followed by the activation of signal transducer and activator of transcription 3 (STAT3).8
Recent studies have revealed that one of the most important roles of cardiac cytokines is the regulation of cardiomyocyte–endothelial cell interaction. LIF and CT-1 upregulate the expression of vascular endothelial growth factor (VEGF) in cardiac myocytes.9 Importantly, VEGF, secreted from cardiomyocytes, is required to maintain endothelial cell survival, which is critical for preserving myocardium.10 These findings strongly propose the importance of the cytokine network between cardiac myocytes and endothelial cells; however, the effects of these cytokines on the tissue resident stem or progenitor cells, which have the potential to differentiate into endothelial cells, remain to be fully addressed.
So far, several kinds of cardiac stem cells have been identified in the adult hearts. These cardiac stem cells differentiate into 3 lines of cardiac cells including cardiomyocytes, smooth muscle cells, and endothelial cells.11–13 Interestingly, the injection of cardiac stem cells into injured hearts promotes cardiac repair and regeneration,11,12 with the increase in capillary density, though the signals responsible for the differentiation of cardiac stem cells are unknown. Recently, we have revealed that LIF regulates the endothelial differentiation of cardiac Sca-1+ cells,14 proposing that the activation of LIF signaling in Sca-1+ cells might promote vessel formation.
In the present study, we investigated the effects of the cardiac proinflammatory cytokines and their family on endothelial differentiation of cardiac Sca-1+ cells and demonstrated that signals through gp130 play critical roles in regulating the endothelial differentiation of Sca-1+ cells. Moreover, IL-6 family cytokines are upregulated in postinfarct myocardium, associated with the induction of Sca-1+/VE-cadherin+ cells, suggesting pathophysiological significances of the IL-6 family cytokines in the regulation of cardiac stem cell differentiation. The understanding of the biological effects of cardiac cytokines on stem cell functions might provide the insights into the development for novel targeted therapies to tissue resident stem cells.
Materials and Methods
An expanded Materials and Methods section is available in the online Data Supplement at http://atvb.ahajournals.org.
The care of all animals was in compliance with the Osaka University animal care guideline. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication No. 85-23, revised 1996).
Preparation of Cardiac Sca-1+ Cells
Cardiac Sca-1+ cells were isolated by magnetic cell sorting (MACS) from C57Bl/6 mice (10- to 12-week-old) with about 98% purity, as described previously.14
RT-PCR was performed as previously described.15 Gene-specific primers used for PCR amplification were shown in supplemental Table I.
Cell lysates were prepared in RIPA buffer, and incubated with anti–p-Tyr antibody (Santa Cruz Biotechnology) and Protein A agarose (Santa Cruz Biotechnology) overnight at 4°C. After washing with RIPA buffer, immunoprecipitates were resuspended in SDS-PAGE buffer, and immunoblotted with anti-gp130 antibody (Santa Cruz Biotechnology).
Immunoblotting was performed as previously described.16
Immunocytochemical analyses were performed as previously described.14
Construction and Infection of Adenoviral Vectors
Adenoviral vectors expressing dominant negative STAT3 (dnSTAT3) and β-galacotosidase were previously described.17 Adenoviral infection was performed at a multiplicity of infection of 100 for 24 hours. Forty-eight hours after infection, cells were cultured in the medium containing each cytokine.
Gene Silencing of STAT3 With siRNA
Cells were transfected with control or STAT3 siRNA using Lipofectamine RNAi MAX (Invitrogen). Forty-eight hours after transfection, cells were cultured in the medium containing each cytokine.
Generation of Myocardial Infarction
Myocardial infarction (MI) was generated by coronary artery ligation according to previous report18 with minor modification. In immunohistological analyses, the hearts were excised 14 days after surgery and embedded in OCT compound. In experiments for the estimation of cytokine expression, total RNA was prepared from hearts 0, 1, 4, 7, and 14 days after surgery.
Frozen sections (5 μm thick) embedded in OCT compound were prepared from normal or MI hearts (n=3 mice), and fixed with 4% paraformaldehyde. After blocking with 3% BSA/0.1% Triton X-100 in TBS, sections were incubated with anti–Sca-1 (BD Bioscience) and anti–VE-cadherin antibodies (Santa Cruz Biotechnology) followed by the incubation with Alexa Fluor 488–conjugated anti-rat IgG (Molecular Probes) and Alexa Fluor 546–conjugated anti-goat IgG (Molecular Probes).
Statistical significance was determined by paired t test or Student t test. Data were presented as mean±SE or mean±SD. Probability value <0.05 was considered to be statistically significant.
IL-11 and CT-1 Induced the Expression of Endothelial Cell Specific Markers in Cardiac Sca-1+ Cells
To address the effects of the proinflammatory cytokines and their families on the differentiation of cardiac Sca-1+ cells, we first examined the expression of the receptors for these cytokines in cardiac Sca-1+ cells by RT-PCR. The transcripts of the receptors for TNF-α, IL-1β, and IL-11 were expressed in cardiac Sca-1+ cells (supplemental Figure I). LIFR, which functions as the ligand-specific receptor for both CT-1 and LIF, was also expressed. In contrast, IL-6R was not detected in cardiac Sca-1+ cells. We also confirmed that gp130, common receptor subunit for IL-6 family cytokines, was expressed in cardiac Sca-1+ cells.
Next, we examined whether these proinflammatory cytokines induce the endothelial differentiation in cardiac Sca-1+ cells. Cardiac Sca-1+ cells were cultured with TNF-α, IL-1β, IL-6, IL-11, or CT-1 for 14 days, and the expression of the marker genes for endothelial cells was analyzed by RT-PCR (Figure 1A and 1B). Cells, cultured with LIF, were used as positive control.14 Though both TNF-α and IL-1β activated their downstream signaling pathways (supplemental Figure II), they did not induce the endothelial specific marker genes in cardiac Sca-1+ cells. In contrast, endothelial cell-specific markers, VE-cadherin, CD31, and Flk-1, were upregulated in response to IL-11 (VE-cadherin: 14.3-fold, CD31: 13.5-fold, Flk-1: 11.0-fold compared with control, n=4, analyzed by real time RT-PCR) and CT-1 (VE-cadherin: 8.1-fold, CD31: 7.5-fold, Flk-1: 7.9-fold compared with control, n=4, by real time RT-PCR), but not in response to IL-6 (Figure 1A and 1B and supplemental Figure III). Although cardiac Sca-1+ cells have also been demonstrated to be differentiated into other cardiac cell lineages,19 neither IL-11 nor CT-1 induced the differentiation into cardiomyocytes or smooth muscle cells (supplemental Figure IV). These findings suggest that IL-11 receptor and LIF receptor can functionally transduce their signals and induce endothelial markers in cardiac Sca-1+ cells. Indeed, the stimulation of IL-11 receptor or LIF receptor, with IL-11 or LIF, respectively, resulted in the rapid phosphorylation of gp130 (Figure 1C).
Recently, c-kit+ cells have also been reported to be another population of cardiac stem cells.11 Therefore, we prepared c-kit+ cells from adult murine hearts by MACS and examined the effects of LIF on their differentiation. RT-PCR analyses demonstrated that c-kit+ cells also differentiated into endothelial cells (supplemental Figure V). However, the repeated experiments using MACS methods achieved the preparation of lower number of c-kit+ cells than Sca-1+ cells. Thus further experiments focused on Sca-1+ cells in this study.
Next, to examine the expression of endothelial marker proteins in cytokines-treated cardiac Sca-1+ cells, immunocytochemical analyses were performed. Cells were cultured with cytokines for 14 days and stained with anti-VE-cadherin antibody (Figure 1D and supplemental Figure VI). The cells cultured with IL-11 or CT-1 showed the expression of VE-cadherin at higher frequency than those with TNF-α, IL-1β, or IL-6. Consistently, the frequency of CD31 positive cells was also increased in the cells cultured with IL-11 or CT-1 (data not shown). The uptake of acetylated low-density lipoprotein (Ac-LDL) is characteristic of endothelial cell lineage.20,21 To investigate the functional relevance of the newly-differentiated endothelial cells derived from cardiac Sca-1+ cells, we examined whether the CD31-positive cells incorporated DiI-labeled Ac-LDL and confirmed that the endothelial marker-positive cells from cardiac Sca-1+ cells have a functional property of endothelial lineage cells (supplemental Figure VII).
IL-11 and CT-1 Activated STAT3 in Cardiac Sca-1+ Cells
To address the signaling pathways of IL-11 and CT-1, we examined whether IL-11 or CT-1 activates STAT3 and ERK1/2 in cardiac Sca-1+ cells. Cardiac Sca-1+ cells were stimulated with IL-11 or CT-1 for the indicated time, and then cell lysates were immunoblotted with antiphospho-specific antibodies (Figure 2). STAT3 was prominently phosphorylated by IL-11 or CT-1, reaching a peak within 15 minutes of stimulation, whereas the phosphorylation of ERK1/2 was not remarkable. In contrast, IL-6, TNF-α and IL-1β, which did not show the capacity of induction of endothelial markers, did not activate STAT3. Because much attention has been paid to Akt-mediated vessel formation,22,23 we also examined the effects of IL-6 family cytokines on the activation of Akt. IL-6 family cytokines did not lead to the activation of Akt in cardiac Sca-1+ cells (data not shown).
Soluble Form of IL-6R Conferred the Potential to Induce the Sca-1+ Cell Commitment to Endothelial Cell Lineage on IL-6
Because the data presented above demonstrated the importance of gp130 signals in the endothelial differentiation, we examined the effects of sIL-6R on the Sca-1+ cells differentiation, because sIL-6R has the potential for activating gp130 in an IL-6-dependent manner. The cells were cultured in medium containing IL-6 in the presence or absence of sIL-6R for 14 days and stained with anti-VE-cadherin antibody (Figure 3A). The cells cultured with IL-6 in the presence of sIL-6R expressed VE-cadherin, confirming the critical roles of signals through gp130 activation in the endothelial lineage commitment. Consistently, IL-6 remarkably activated STAT3 in the presence of sIL-6R (Figure 3B and 3C). Thus, these data suggest that activation of gp130/STAT3 signals is a critical event for the induction of cardiac Sca-1+ cell differentiation to endothelial cells.
STAT3 Was Required for IL-6 Family–Mediated Commitment of Cardiac Sca-1+ Cells to Endothelial Cell Lineage
To examine whether STAT3 activation is essential for the endothelial differentiation of cardiac Sca-1+ cells by IL-6 family cytokines, we analyzed the effects of the inhibition of STAT3 by adenoviral vectors expressing dnSTAT3 (Figure 4A). IL-11 and CT-1 did not induce the endothelial differentiation in dnSTAT3-expressing cells, whereas endothelial-specific genes were upregulated by IL-11 and CT-1 in the cells expressing β-galactosidase, a control. Similarly, gene silencing experiments using siRNA for STAT3 demonstrated that the inhibition of STAT3 pathways also suppressed the induction of endothelial-specific genes by IL-6 family cytokines (Figure 4B and 4C). Further, to characterize STAT3-knockdown cardiac Sca-1+ cells, DiI-labeled Ac-LDL uptake was examined (Figure 4D). As shown in supplemental Figure VII, LIF treatment increased the frequency of Ac-LDL-positive cells. Importantly, compared with cells transfected with control siRNA, Ac-LDL uptake was reduced in cells transfected with siRNA for STAT3. These data propose the additional evidence for the requirement of STAT3 for gp130-mediated endothelial differentiation in Sca-1+ cells. We have also noticed that control siRNA have a tendency to inhibit Ac-LDL uptake in comparison with nontreated cells, though not significantly. The precise mechanisms remain to be known; however, the transfection procedure might have affected Ac-LDL uptake.
The Upregulation of IL-6 Family Cytokines Was Associated by the Commitment of Sca-1+ Cells to Endothelial Lineage After Myocardial Infarction
To explore the pathophysiological significances of the IL-6 family cytokines in cardiac stem cell fate in vivo, we generated MI model and, first, we examined the gene expression profiles of IL-6 family cytokines in the hearts (Figure 5A). Real time RT-PCR analyses have demonstrated that the transcripts of IL-11 and LIF were elevated 1 day after MI (IL-11: 32.9-fold, LIF: 19.9-fold compared with no operation) and the expression levels were gradually reduced, whereas CT-1 was transiently decreased and recovered to the basal level. IL-6R was also immediately upregulated, and the induction was sustained up to 14 days after infarction.
Because these data suggest that IL-6 family signals are activated in the postinfarct myocardium, we investigated the dynamics of cardiac Sca-1+ cells by immunofluorescent microscopy using anti–Sca-1 and anti-VE-cadherin antibodies (Figure 5B). Consistent with the previous report,24 a significant number of Sca-1+/VE-cadherin+ cells were detected in myocardium. Importantly, the double positive cells were increased in number at the border zones of postinfarct myocardium (normal: 4.9±1.9 cells/high power field [HPF], MI: 7.9±1.8 cells/HPF, P<0.01, n=9 fields from 3 mice). Moreover, Sca-1+ cells expressed VE-cadherin more abundantly at border zone in the infarct hearts than in normal hearts.
In the present study, we examined the effects of the proinflammatory cytokines and their family on cardiac Sca-1+ cell commitment to endothelial cell lineage. CT-1, IL-11, and LIF induced the endothelial cell lineage commitment of the Sca-1+ cells, although not TNF-α, IL-1β, or IL-6. CT-1 and IL-11 activated STAT3 in cardiac Sca-1 cells, as is the case with LIF. IL-6, whose receptor is not expressed in cardiac Sca-1+ cells, gained the potential to induce the endothelial markers in the presence of sIL-6R. Moreover, we demonstrated that the inhibition of STAT3 abrogated the endothelial commitment of cardiac Sca-1+ cells. Finally, by generating MI model, we proposed the association between the upregulation of LIF and IL-11 and the induction of Sca-1+/VE-cadherin+ cells in postinfarct myocardium.
The inflammation is closely associated with neovascularization. Accumulating evidences have demonstrated that proinflammatory cytokines mediate angiogenic signals in vascular cells. In skeletal muscles, IL-1β is required for neovascularlization through the mononuclear cells-skeletal muscle interaction.25 Although TNF-α has reported to have both proangiogenic and antiangiogenic properties, TNFR2-deficient mice show impaired capacity of blood flow recovery after hindlimb ischemia surgery, accompanied with reduction of endothelial cell/endothelial progenitor cell survival and migration.26,27 Similarly, IL-6 upregulates VEGF expression in rheumatoid arthritis, leading to the promotion of vessel formation in the inflamed joints.28 In cardiovascular system, we previously demonstrated that LIF and CT-1 increased VEGF expression in cardiac myocytes through STAT3 activation, which enhanced the capillary density in the heart.9,29 In addition to these cytokine-mediated angiogenic effects, here we have demonstrated that not only LIF14 but also a wide range of IL-6 family cytokines, which are produced from cardiac cells in response to cardiac stress, can induce the endothelial cell lineage commitment of cardiac stem cells, suggesting that cardiac stem cells may function as endothelial progenitors under diverse pathological conditions. Interestingly, Flk-1, one of VEGF receptors, was induced by IL-6 family cytokines in cardiac Sca-1+ cells. Moreover we have confirmed that VEGF activated its downstream signaling pathway in LIF-treated Sca-1+ cells, but not in nontreated Sca-1+ cells (data not shown). Therefore, it is possible that IL-6 family-induced VEGF in myocardium stimulates not only preexisting endothelial cells but also Sca-1+ cell-derived differentiating endothelial cells through paracrine systems.
In this study, we characterized the differentiating Sca-1+ cells by RT-PCR, because RT-PCR is much reliable to analyze the stem cell fate than immunofluorescence.30 To estimate the maturity of newly-differentiating endothelial cells from Sca-1+ cells, we compared their expression level of CD31 with those in matured mouse microvascular cell line by FACS (data not shown). Compared with the fully-differentiated endothelial cell line, only less than 20% of CD31 protein was expressed in newly-differentiating endothelial cells induced by LIF, suggesting that IL-6 family cytokines promote the commitment of Sca-1+ cells to endothelial cells, but that these cytokines might not achieve full differentiation. In this context, further analyses on the expression profile of cytokine receptors, such as flk-1, might provide more information on molecular mechanisms of Sca-1+ cell–derived endothelial differentiation.
IL-6 family cytokines stimulate gp130, a common receptor subunit, leading to activation of STAT3. Therefore, the redundancy among LIF, CT-1, IL-11, and IL-6/sIL-6R in Sca-1+ cell commitment to endothelial cells should be derived from the common signaling pathways through gp130/STAT3. Pathologically it should be emphasized that IL-6 exhibited the potential to induce the endothelial differentiation in the presence of sIL-6R, because the expression of IL-6R was elevated after MI, as reported previously.31 Unexpectedly, CT-1 exhibited a lower potency than LIF, though LIF and CT-1 share LIFR as their receptor α subunit. The relative potency of CT-1 to LIF may depend on cell lineage. LIF and CT-1 have similar relative potencies in inhibiting the growth of the mouse M1 cells and in inducing the hypertrophy in cultured cardiomyocytes.32 Considering the previous reports that CT-1 requires the third receptor component in neuronal cells,33,34 the expression level of the third receptor may be low in Sca-1+ cells, though the third receptor has not been identified.
IL-6 family cytokines, such as LIF, CT-1, and IL-11, activate the downstream signaling pathways in cardiac myocytes and contribute to cytoprotection and vessel formation in the heart.4 Based on these beneficial effects of gp130/STAT3 activation in cardiomyocytes, the molecular target therapies toward gp130 are being explored as a novel therapeutic strategy against heart disease.35,36 The data presented here propose the possibility that the therapeutic strategy toward gp130 could achieve additional outcomes by inducing neovascularization through differentiation of cardiac stem cells into endothelium.
In summary, activation of gp130/STAT3 is a critical event for the endothelial linage commitment of cardiac Sca-1+ cells. Our findings suggest that cardiac cytokine network, especially IL-6 family, could regulate the cell fate of cardiac stem cells.
We thank Yasuko Murao for her excellent secretary work.
Sources of Funding
This work was supported by Grant-in-Aid for Research Fellows of Japan Society for the Promotion of Science (to T.M.). This study was also supported by Grant-in-Aid for Scientific Research (B) from Japan Society for the Promotion of Science, by Mitsubishi Pharma Research Foundation, by Takeda Science Foundation, and by the Osaka Foundation for Promotion of Clinical Immunology. This study was partially supported by Grant-in-Aid from Knowledge Cluster Initiative (2nd Stage) of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
Received July 28, 2008; revision accepted February 1, 2009.
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