Published online before print December 12, 2008, doi: 10.1161/ATVBAHA.108.179457
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© 2009 American Heart Association, Inc.
Cell Biology/Signaling |
mTOR Regulates Vascular Smooth Muscle Cell Differentiation From Human Bone Marrow–Derived Mesenchymal Progenitors
From the Clinic for Nephrology and Intensive Care Medicine (B.H., M.L., A.K., K.E., M.G., D.D.), Charité Campus Virchow-Klinikum, the Center for Cardiovascular Research (B.H., M.L., A.K., D.D.), Experimental and Clinical Research Center (K.E., F.C.L., M.G.), Clinic for Hematology and Oncology (O.S.), Charité Campus Mitte, Berlin; the Department of Medicine I (E.S.-L.), Cologne General Hospital, Cologne; and Max-Delbrück Center for Molecular Medicine and HELIOS Klinikum (F.C.L.), Berlin, Germany.
Correspondence to Dr Duska Dragun, Clinic for Nephrology and Intensive Care Medicine, Charité Campus Virchow Klinihum, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail duska.dragun{at}charite.de
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Abstract |
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Objective— Vascular smooth muscle cells (VSMCs) and circulating mesenchymal progenitor cells (MSCs) with a VSMC phenotype contribute to neointima formation and lumen loss after angioplasty and during allograft arteriosclerosis. We hypothesized that phosphoinositol-Akt-mammalian target of rapamycin-p70S6 kinase (PI3K/Akt/mTOR/p70S6K) pathway activation regulates VSMC differentiation from MSCs.
Methods and Results— We studied effects of PI3K/Akt/mTOR signaling on phenotypic modulation of MSC and VSMC marker expression, including L-type Ca(2+) channels. Phosphorylation of Akt and p70S6K featured downregulation of VSMC markers in dedifferentiated MSCs. mTOR inhibition with rapamycin at below pharmacological concentrations blocked p70S6K phosphorylation and induced a differentiated contractile phenotype with smooth muscle (sm)-calponin, sm-
-actin, and SM protein 22-alpha (SM22) expression. The PI3K inhibitor Ly294002 abolished Akt and p70S6K phosphorylation and reversed the dedifferentiated phenotype via induction of sm-calponin, sm--actin, SM22, and myosin light chain kinase. Rapamycin acted antiproliferative without impairing MSC viability. In VSMCs, rapamycin increased a homing chemokine for MSCs, stromal cell–derived factor-1–alpha, at mRNA and protein levels. The CXCR4-mediated MSC migration toward conditioned medium of rapamycin-treated VSMCs was enhanced.
Conclusions— We describe novel pleiotropic effects of rapamycin at very low concentrations that stabilized differentiated contractile VSMCs from MSCs in addition to exerting antiproliferative and enhanced homing effects.
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Introduction |
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Extensive neointima formation is a primary pathophysiologic mechanism of postintervention restenosis and transplant arteriosclerosis.1 Until recently, the neointima formation was attributed to uncontrolled proliferation of vascular smooth muscle cells (VSMCs) that migrate from the media and replace the intima of affected arteries.2 A phenotypic switch from a contractile to a proliferative and less differentiated synthetic phenotype accompanies the migration process.3 Animal and human studies have indicated that neointimal cells may not only originate from the vascular media or adventitia, but also from bone marrow– and nonmarrow-derived progenitors. The cells migrate on injury to affected vasculature and actively become part of neointima formation by differentiating into VSMCs.4,5 Mesenchymal stem cells (MSCs) in particular seem to have a high potential for neointimal growth.6 The precise mechanisms responsible for differentiation and homing of VSMC precursors are unknown. The phosphoinositol-Akt-mammalian target of rapamycin-p70S6 kinase (PI3K/Akt/mTOR/p70S6K) pathway regulates cell growth and cell differentiation in response to nutrients, growth factors, and cytokines.7 Pharmacological rapamycin inhibition of mTOR markedly reduces transplant vasculopathy progression in recipients of cardiac8,9 and renal transplants.10 Comparison of rapamycin-eluting stents with paclitaxel-eluting stents documented the superiority of rapamycin-eluting stents in terms of preventing late in-stent lumen loss and a lower incidence of in-stent restenosis.11 Based on our previous findings on VSMC differentiation of human bone marrow MSCs,12 we investigated activation and inhibition of the PI3K/Akt/mTOR/p70S6K pathway in the phenotypic modulation and expression of VSMC markers in MSCs. We also sought to determine how low-dose mTOR inhibition influences MSC proliferation and migration.
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Methods |
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Cell Culture
Human MSCs were isolated by plastic adherence from aspirated iliac crest marrow from consenting normal adult donors and cultured as described previously.12 Passages 2 to 6 were used for experiments. Under these culture conditions MSCs maintain multilineage differentiation capacity,12 are positive for CD73, CD90, and CD105 and negative for CD11b, CD14, CD19, CD34, CD45, CD79alpha, and HLA-DR (supplemental Figure I, available online at http://atvb.ahajournals.org), thereby meeting the criteria for MSCs.13 Human coronary artery VSMCs were obtained from Lonza (Cambiex Bio Science, Walkersville, Md).
Ca2+ Imaging
Cells were seeded onto glass coverslips and loaded with the Ca2+ indicator fluo-4-AM (Invitrogen). Changes in intracellular calcium were expressed as relative fluorescence changes, ie, F/Fo (with Fo indicating the fluorescence before stimulation and F the time-dependent fluorescence signal after stimulation) as described previously.14
Western Blotting
Western blots were performed according to standard procedures as described previously.12
BrdU Incorporation and MTT Assays
Cell proliferation was measured as BrdU incorporation (BrdU cell proliferation kit, Roche) in MSCs grown in presence of 10% FCS and rapamycin for 24 hours. Cytotoxicity was tested in MSCs incubated with rapamycin for 24 hours and addition of 1.5 mmol/L 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) during the last 4 hours. Absorption was measured at 570 nm in an ELISA reader.
Quantitative Real-Time PCR
VSMCs and MSCs were grown serum-free for 24 hours before incubation with rapamycin for 8 hours. Total RNA was extracted using the phenol/chloroform method and transcribed into cDNA with the PCR core kit (Roche). Quantitative real-time PCR was performed using a Roche Light Cycler system.
SDF-1 ELISA
VSMCs grown with serum-free DMEM were incubated with control or rapamycin for 72 hours. SDF-1 in supernatants was measured with a human SDF-1 ELISA (R&D Systems) and normalized to total protein content.
MSC Migration on Conditioned Medium of Rapamycin-Treated VSMCs
Supernatants of VSMCs cultured for 72 hours with or without rapamycin were applied to the lower chamber of a micro chemotaxis chamber (Neuro Probe). MSCs in serum-free DMEM in presence of control IgG or anti-CXCR4 (R&D Systems) were seeded in the upper chamber and allowed to migrate for 24 hours. Migrated cells on the bottom side were stained with crystal violet. Absorbance was measured with an ELISA reader at 570 nm. Results were normalized to total protein content of the supernatants.
Statistical Analysis
Values for calcium influx are given as mean±SEM and were compared by Student t test. Band intensities of smooth muscle markers were normalized to -tubulin. Control without treatment was set 1.00. Analysis of multiple blots is expressed as mean±SD. The Kruskal—Wallis test was used for multiple comparisons, whereas pairwise comparison with control was done using Mann–Whitney U test. P<0.05 was considered statistically significant. Please see supplemental materials for expanded methods section and for detailed figure legends.
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Results |
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Resting MSCs Have Functional VSMC-Like L-Type Calcium Channels
Voltage-dependent dihydropyridine-sensitive (Cav1.2) L-type Ca2+ channels represent a specific marker of differentiated VSMCs15,16 and play a key role in excitation-contraction coupling in terminally differentiated contractile VSMCs.17 Based on our findings that resting MSCs with multilineage differentiation potential express smooth muscle specific contractile proteins,12 we investigated whether or not they would display functional L-type Cav1.2 Ca2+ channels. We studied how L-type Ca2+ channel activation relates to the expression of contractile proteins during MSC differentiation and dedifferentiation. We applied culture conditions in which MSCs maintain their capability to differentiate into several cell types such as adipocytes and osteoblasts, as previously established.12 We observed an increase in intracellular Ca2+ concentrations ([Ca2+]i) when MSCs loaded with the calcium sensitive fluorescent dye fluo-4 were depolarized with iso-osmotic 60 mmol/L KCl (Figure 1A and 1E). This increase in [Ca2+]i was largely blocked by preincubation of the cells with 1 µmol/L nimodipine (Figure 1B and 1E). MSC dedifferentiation can be reproducibly achieved by incubation with nontoxic DMSO concentrations.12 After incubation of MSCs with 1.25% DMSO for 8 days, the Ca2+ responses to KCl were significantly reduced and nimodipine did not have a significant effect on KCl-induced Ca2+ influx (Figure 1C, 1D, and 1E). The results suggest that multipotent MSCs share important features of VSMCs by expressing functional L-type Cav1.2 Ca2+ channels, which represent a specific marker of differentiated VSMCs and are required for excitation-contraction coupling.14
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The PI3K/Akt/mTOR Pathway Is Activated in Dedifferentiated MSCs
Akt links signals downstream from PI3K-coupled growth factor receptors to mTOR, whereas p70S6K, which is downstream from mTOR, governs protein synthesis.18 We tested the activation status of Akt and p70S6K in differentiated and dedifferentiated MSCs by immunoblotting with phospho-specific antibodies. Treatment with 1.25% DMSO induced a strong (10-fold) increase in phosphorylated Akt, the downstream target of PI3K (Figure 2A and 2B) without change in total Akt. The maximal phosphorylation occurred rapidly after 10 minutes and persisted for 30 minutes. Preincubation with Ly294002, a specific PI3K inhibitor, for 30 minutes completely blocked Akt phosphorylation (Figure 2C). DMSO also induced a strong (5-fold) increase in p70S6K phosphorylation at the mTOR-dependent Thr389 phosphorylation site (Figure 2D and 2E). Compared to Akt, the maximal increase in p70S6K phosphorylation occurred after 30 minutes, consistent with the more downstream location of p70S6K in the pathway. Preincubation with pharmacological 6 nmol/L rapamycin concentration for 30 minutes entirely abrogated p70S6K phosphorylation at Thr389 (Figure 2F). As expected, Akt phosphorylation was not influenced by rapamycin preincubation (data not shown). Preincubation with Ly294002, targeting PI3K upstream from mTOR, achieved the same inhibitory effect on p70S6K phosphorylation at Thr389 (data not shown).
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PI3K/Akt/mTOR-Pathway Blockade Preserves the Differentiated MSC Phenotype and Induces Expression of VSMC Antigens
To further establish the notion that VSMC differentiation and the phenotypic switch from differentiated and contractile to a less differentiated MSC phenotype is regulated via the PI3K/Akt/mTOR pathway, we performed coincubation studies with specific inhibitors of different signaling elements. Under culture conditions in which MSCs retain their pluripotent differentiation potential, VSMC-like progenitors change from large cells with prominent stress fibers to spindle-shaped cells without stress fibers after 8 days of 1.25% DMSO incubation (supplemental Figure IIA and IIB). This morphological transformation was accompanied by marked downregulation of the smooth muscle marker proteins sm-calponin, MLCK, and sm--actin, indicating dedifferentiation of MSCs.12 When we coincubated cells for 8 days with 1.25% DMSO together with the PI3K inhibitor Ly294002 at 15 µmol/L concentration or with 6 nmol/L rapamycin, MSCs preserved their VSMC-like phenotype (supplemental Figure IIC and IID).
In additional experiments, we tested whether or not PI3K inhibition mediated the switch toward differentiated contractile phenotype in MSCs and induces the expression of VSMC antigens. Quiescent MSCs with the contractile phenotype express the VSMC markers sm-calponin (Figure 3A), sm--actin (Figure 3B), SM22 (Figure 3C), and the short MLCK isoform (Figure 3D). Incubation with 1.25% DMSO downregulated all 4 markers. In contrast, blockade of PI3K with 5 and 15 µmol/L Ly294002 reversed DMSO-mediated downregulation of all smooth muscle markers, as shown by Western blot analysis.
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Rapamycin Promotes VSMC Marker Expression in Quiescent MSCs
Our data supported the hypothesis that the PI3K/Akt/mTOR pathway regulates VSMC differentiation from MSCs and that activation of this pathway induces dedifferentiation and loss of the contractile phenotype. We next studied whether or not modulation of the downstream element, mTOR, would also enhance spontaneous VSMC differentiation from quiescent MSCs in culture. Incubation of MSCs with rapamycin for 8 days at concentrations even below the recommended levels used in transplant patients, as low as 1 nmol/L, resulted in upregulation of VSMC antigens sm-calponin (supplemental Figure IIIA), -smooth muscle actin (supplemental Figure IIIB), SM22 (supplemental Figure IIIC), but had no influence on the short MLCK isoform (supplemental Figure IIID). The observed effects on VSMC antigen expression were not further enhanced by the recommended low pharmacological 6 nmol/L rapamycin concentration or at suprapharmacologic 20 and 100 nmol/L concentrations.
Rapamycin Acts Antiproliferative on MSCs Without Impairment of Viability
Beneficial effects of rapamycin and its derivatives on prevention of neointima formation during transplant vasculopathy or after angioplastic interventions were attributed to antiproliferative effects via prevention of cell cycle progression from G1 to S phase.19 We tested the antiproliferative effects in MSCs cultured in medium containing increasing rapamycin concentrations for 24 hours by measuring BrdU incorporation into newly synthesized DNA. Rapamycin exerted a potent antiproliferative effect (–50%) at below pharmacological concentrations as low as 1 nmol/L (Figure 4A). Therapeutic concentrations of rapamycin and the derivative everolimus in transplant patients are in the 4 to 10 nmol/L range.20,21 There was no additional proliferation inhibition at above pharmacological concentrations up to 100 nmol/L. To exclude the possibility that the observed decrease in DNA synthesis was related to cytotoxic effects, we analyzed cell viability by the MTT assay. MSCs were exposed to the same increasing rapamycin concentrations for 24 hours. We observed no toxic effects of rapamycin on MSCs, even at suprapharmacological concentrations (Figure 4B).
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Rapamycin Induces SDF-1 Expression in VSMCs and Enhances MSC Migration
VSMCs are target cells of injury during allograft rejection or after angioplastic interventions. Vascular cells secrete the chemokine SDF-1 as part of the response to injury and interact with target receptor CXCR4 expressed on MSCs.22 SDF-1 stimulates MSC migration. Furthermore, there is evidence that MSCs are mobilized by and directed to sites of tissue injury by a SDF-1 gradient.22 We tested how mTOR inhibition could affect VSMC SDF-1 secretion and influence mobilization and homing of MSCs. Human coronary VSMCs were incubated with pharmacological 6 nmol/L rapamycin concentrations and analyzed for SDF-1 mRNA expression and secretion of SDF-1 protein. SDF-1 mRNA was significantly upregulated after eight hours (Figure 5A), whereas secretion of SDF-1 protein (Figure 5B) increased after 72 hours of rapamycin incubation. We also documented enhanced CXCR4-mediated migration of MSCs toward conditioned medium of rapamycin-stimulated VSMCs in Boyden chamber model (Figure 5C).
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Discussion |
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Recipient-derived hematopoietic and mesenchymal progenitor cells contribute to neointima formation after extensive injury to the vascular media. We identified a critical role for PI3K/Akt/mTOR pathway activation in VSMC differentiation in mesenchymal progenitors. Pharmacological PI3K inhibition by Ly294002, as well as mTOR inhibition by rapamycin, modulated several aspects of MSC physiology including induction of a VSMC-like differentiated state and induction of a protective contractile phenotype in MSCs. In parallel, rapamycin exerted a potent antiproliferative effect on MSCs without signs of toxicity. Finally, rapamycin also interfered with mobilization and homing of MSCs by induction of SDF-1
in VSMCs and MSC migration. We describe novel effects, even at very low doses of mTOR inhibition on MSCs, and propose protective phenotypic switching together with MSC homing modulation. We believe our findings serve to explain the unique potential of mTOR inhibitors that extend beyond antiproliferatiion and T-cell suppression. The finding that rapamycin promotes a VSMC-like differentiated contractile phenotype in MSCs is compelling and may facilitate the understanding of several unexplained beneficial and adverse effects observed in transplant recipients treated with mTOR inhibitors or in patients with rapamycin-coated stents. Both rapamycin23 and the derivative everolimus8 are superior to other immunosuppressants in prevention and treatment of transplant vasculopathy in patients with cardiac allografts. Comparison of rapamycin-eluting stents with paclitaxel-eluting stents documented the superiority of rapamycin-eluting stents in terms of preventing late in-stent lumen loss and a lower incidence of in-stent restenosis.11 Similar results were reported in high-risk patients with diabetes mellitus.24 Stronger beneficial effects of rapamycin-eluting stents were thus far attributed to more prominent effects of rapamycin on VSMC proliferation and migration, lesser cytotoxicity, and stronger effect on Akt activation compared to paclitaxel.25 Similar to local drug delivery, oral rapamycin for 10 days beginning 2 days before angioplastic reintervention resulted in a significantly reduced restenosis rate in rapamycin-treated patients compared to control patients.26
Some of the rapamycin-induced effects on MSCs with the VSMC-like phenotype in terms of potent antiproliferative and lack of cytotoxic effects are consistent with studies performed in VSMCs.27 However, proliferation inhibition alone is not sufficient to promote VSMC differentiation.28 Subtle changes in signaling mechanisms may be responsible for injury-specific phenotypic changes. Loss of the contractile phenotype is faster and more prominent in transplant vasculopathy compared to atherosclerosis of native vessels.29 Transition of the contractile to the synthetic phenotype is also regulated by mTOR in VSMCs.30 Rapamycin-mediated effects on VSMC differentiation were attributed to mTOR/PI3K feedback in VSMCs.31 The authors proposed that a unique signaling pattern where S6K1, downstream from mTOR, is inhibited and Akt, upstream from mTOR but downstream from PI3K, is activated results in optimal differentiation signals and induction of contractile phenotypic markers.31 We did not focus on mTOR/PI3K feedback. Nevertheless, Akt phosphorylation was not influenced by rapamycin in quiescent MSCs. Furthermore, we documented not only reprogramming toward the differentiated state in dedifferentiated cells, but also increased VSMC marker protein expression in resting MSCs after mTOR Inhibition. Interestingly, VSMCs were described as "multifunctional mesenchymal cells" forty years ago,32 anticipating the later findings of MSCs as subpopulations of VSMCs within the vascular wall. Location and type of injury may determine the importance of MSC accumulation.33
Severe injury of the vascular media that occurs after angioplasty or during allograft vascular rejection is prerequisite for progenitor cell homing and lesion repair.33 Peritransplant ischemic injury is associated with significant SDF-1 upregulation in cardiac allografts.34 SDF-1 probably serves to recruit stem cells in response to hypoxic injury.35 Rapamycin inhibits VSMC migration.36 Rapamycin increased expression and secretion of SDF-1 in VSMCs, an effect that has not been previously described. CXCR4-dependent MSC migration to conditioned medium of rapamycin-treated VSMCs was also enhanced. Thus, rapamycin probably enhances, or at least does not impair, MSC migration and homing to target cells after vascular injury. Most studies on progenitor cell homing and differentiation in vitro and in vivo refer to cells of hematopoietic origin.22 Hematopoietic cells isolated from buffy coats cultured in platelet-derived growth factor (PDGF)-BB–enriched medium can also acquire the VSMC-like phenotype and express -smooth muscle actin and calponin.37 In mouse model of severe wire-induced femoral artery injury, locally delivered rapamycin decreased the number of bone marrow–derived CD45-positive hematopoietic cells and attenuated neointima formation.38 Induction of cell death in endothelial progenitors after rapamycin treatment may offer an explanation for this observation. Our data add to the complexity of rapamycin-mediated actions that also may help explain effects observed in other lineages. Rapamycin may influence differentiation process through opposing cell-type specific effects. For example, rapamycin induces hematopoietic differentiation,39 yet inhibits adipocyte differentiation40 and chondrogenesis.41 The PI3K/Akt/mTOR pathway is also critically involved in tumor biology. Simultaneous promotion of beneficial phenotypic switch together with antiproliferative and antiangiogenic effects may explain the reduced incidence of tumors in patients treated with mTOR inhibitors.42 Bone marrow–derived MSCs are also implicated in the initiation and progression of cancer metastasis.43
Dose-related side effects limit the use of mTOR inhibitors in transplant patients. Our finding that the pleiotropic effects were already operative at the 1 nmol/L concentration, 8-fold below the recommended trough rapamycin or everolimus levels, inspires confidence in the relevance of our findings.20,21 Poor wound healing that is associated with higher trough levels is well documented for mTOR inhibitors.44 Control of amount of drug release in eluting stents is becoming an increasingly important issue that enables application of different target drug concentrations.45 We suggest that our data may well fit to these concepts. We believe that mTOR inhibition at very low concentrations may open new treatment options with minimal side effects to suppress the development of transplant arteriosclerosis and postintervention restenosis.
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Acknowledgments |
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Sources of Funding
This work was supported by Charité intramural funds.
Disclosures
Dr Hegner and Dr Dragun received travel grants by Novartis Pharma GmbH and Wyeth Pharma GmbH.
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Footnotes |
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Received February 8, 2008; revision accepted November 27, 2008.
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