Endothelial-to-Mesenchymal Transition in Bone Marrow and Spleen of Primary Myelofibrosis
2017; Elsevier BV; Volume: 187; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2017.04.006
ISSN1525-2191
AutoresBenedetta Gaia Erba, Cristian Gruppi, Monica Corada, Federica Pisati, Vittorio Rosti, Niccolò Bartalucci, Jean‐Luc Villeval, Alessandro M. Vannucchi, Giovanni Barosi, Alessandra Balduini, Elisabetta Dejana,
Tópico(s)Acute Myeloid Leukemia Research
ResumoPrimary myelofibrosis is characterized by the development of fibrosis in the bone marrow that contributes to ineffective hematopoiesis. Bone marrow fibrosis is the result of a complex and not yet fully understood interaction among megakaryocytes, myeloid cells, fibroblasts, and endothelial cells. Here, we report that >30% of the endothelial cells in the small vessels of the bone marrow and spleen of patients with primary myelofibrosis have a mesenchymal phenotype, which is suggestive of the process known as endothelial-to-mesenchymal transition (EndMT). EndMT can be reproduced in vitro by incubation of cultured endothelial progenitor cells or spleen-derived endothelial cells with inflammatory cytokines. Megakaryocytes appear to be implicated in this process, because EndMT mainly occurs in the microvessels close to these cells, and because megakaryocyte-derived supernatant fluid can reproduce the EndMT switch in vitro. Furthermore, EndMT is an early event in a JAK2-V617F knock-in mouse model of primary myelofibrosis. Overall, these data show for the first time that microvascular endothelial cells in the bone marrow and spleen of patients with primary myelofibrosis show functional and morphologic changes that are associated to the mesenchymal phenotype. Primary myelofibrosis is characterized by the development of fibrosis in the bone marrow that contributes to ineffective hematopoiesis. Bone marrow fibrosis is the result of a complex and not yet fully understood interaction among megakaryocytes, myeloid cells, fibroblasts, and endothelial cells. Here, we report that >30% of the endothelial cells in the small vessels of the bone marrow and spleen of patients with primary myelofibrosis have a mesenchymal phenotype, which is suggestive of the process known as endothelial-to-mesenchymal transition (EndMT). EndMT can be reproduced in vitro by incubation of cultured endothelial progenitor cells or spleen-derived endothelial cells with inflammatory cytokines. Megakaryocytes appear to be implicated in this process, because EndMT mainly occurs in the microvessels close to these cells, and because megakaryocyte-derived supernatant fluid can reproduce the EndMT switch in vitro. Furthermore, EndMT is an early event in a JAK2-V617F knock-in mouse model of primary myelofibrosis. Overall, these data show for the first time that microvascular endothelial cells in the bone marrow and spleen of patients with primary myelofibrosis show functional and morphologic changes that are associated to the mesenchymal phenotype. Primary myelofibrosis (PMF) is one of the family of myeloproliferative neoplasms, which is a heterogeneous group of related diseases that also includes polycythemia vera and essential thrombocythemia.1Dameshek W. Some speculations on the myeloproliferative syndromes.Blood. 1951; 6: 372-375Crossref PubMed Google Scholar Myeloproliferative neoplasms are clonal malignant disorders that are characterized by oncogenic transformation of the hematopoietic progenitor cell compartment, which causes abnormal proliferation of the myeloid lineages driven by a hypersensitivity to regulatory growth factors.2Delhommeau F. Jeziorowska D. Marzac C. Casadevall N. Molecular aspects of myeloproliferative neoplasms.Int J Hematol. 2010; 91: 165-173Crossref PubMed Scopus (60) Google Scholar Fifty percent to 60% of patients with myeloproliferative neoplasms harbor a somatic gain-of-function mutation in the Janus kinase 2 (JAK2) gene, which results in the translation of the mutated JAK2-V617F protein. Because this mutation is in the pseudo-kinase JH2 domain, the autoinhibitory function of JAK2-V617F is impaired. This results in constitutive activation of the downstream JAK2 signaling pathways, which include the STATs, mitogen-activated kinases, and phosphatidylinositol 3-kinase–AKT.3Levine R.L. Gilliland D.G. JAK-2 mutations and their relevance to myeloproliferative disease.Curr Opin Hematol. 2007; 14: 43-47Crossref PubMed Scopus (53) Google Scholar Additional mutations in the hematopoietic compartment have also been identified, including for the thrombopoietin receptor and, as reported recently, the calreticulin genes, which lead to a similar phenotype.4Rumi E. Pietra D. Pascutto C. Guglielmelli P. Martinez-Trillos A. Casetti I. Colomer D. Pieri L. Pratcorona M. Rotunno G. Sant'Antonio E. Bellini M. Cavalloni C. Mannarelli C. Milanesi C. Boveri E. Ferretti V. Astori C. Rosti V. Cervantes F. Barosi G. Vannucchi A.M. Cazzola M. Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative InvestigatorsClinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis.Blood. 2014; 124: 1062-1069Crossref PubMed Scopus (298) Google Scholar, 5Vainchenker W. Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms.Blood. 2017; 129: 667-679Crossref PubMed Scopus (351) Google Scholar PMF clonal myeloproliferation primarily induces bone marrow fibrosis and osteosclerosis, with extramedullary hematopoiesis in the spleen, which results in splenomegaly and abnormal cytokine overexpression.6Tefferi A. Myelofibrosis with myeloid metaplasia.N Engl J Med. 2000; 342: 1255-1265Crossref PubMed Scopus (651) Google Scholar, 7Levine R.L. Wadleigh M. Cools J. Ebert B.L. Wernig G. Huntly B.J. Boggon T.J. Wlodarska I. Clark J.J. Moore S. Adelsperger J. Koo S. Lee J.C. Gabriel S. Mercher T. D'Andrea A. Frohling S. Dohner K. Marynen P. Vandenberghe P. Mesa R.A. Tefferi A. Griffin J.D. Eck M.J. Sellers W.R. Meyerson M. Golub T.R. Lee S.J. Gilliland D.G. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis.Cancer Cell. 2005; 7: 387-397Abstract Full Text Full Text PDF PubMed Scopus (2445) Google Scholar Debilitating symptoms include anemia, thrombosis, and general malaise. Ineffective erythropoiesis and extramedullary hematopoiesis can be the cause of anemia and splenomegaly. No definitive therapy is currently available for patients with PMF. The only potentially curative treatment is allogeneic stem cell transplantation, although this is associated with high rates of mortality and morbidity.8Ballen K.K. Shrestha S. Sobocinski K.A. Zhang M.J. Bashey A. Bolwell B.J. Cervantes F. Devine S.M. Gale R.P. Gupta V. Hahn T.E. Hogan W.J. Kroger N. Litzow M.R. Marks D.I. Maziarz R.T. McCarthy P.L. Schiller G. Schouten H.C. Roy V. Wiernik P.H. Horowitz M.M. Giralt S.A. Arora M. Outcome of transplantation for myelofibrosis.Biol Blood Marrow Transplant. 2010; 16: 358-367Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar Despite being a hematopoietic disease, among the myeloproliferative neoplasms, involvement of the endothelial lineage has been reported only for PMF,9Massa M. Rosti V. Ramajoli I. Campanelli R. Pecci A. Viarengo G. Meli V. Marchetti M. Hoffman R. Barosi G. Circulating CD34+, CD133+, and vascular endothelial growth factor receptor 2-positive endothelial progenitor cells in myelofibrosis with myeloid metaplasia.J Clin Oncol. 2005; 23: 5688-5695Crossref PubMed Scopus (87) Google Scholar, 10Rosti V. Bonetti E. Bergamaschi G. Campanelli R. Guglielmelli P. Maestri M. Magrini U. Massa M. Tinelli C. Viarengo G. Villani L. Primignani M. Vannucchi A.M. Frassoni F. Barosi G. AGIMM InvestigatorsHigh frequency of endothelial colony forming cells marks a non-active myeloproliferative neoplasm with high risk of splanchnic vein thrombosis.PLoS One. 2010; 5: e15277Crossref PubMed Scopus (28) Google Scholar, 11Rosti V. Villani L. Riboni R. Poletto V. Bonetti E. Tozzi L. Bergamaschi G. Catarsi P. Dallera E. Novara F. Massa M. Campanelli R. Fois G. Peruzzi B. Lucioni M. Guglielmelli P. Pancrazzi A. Fiandrino G. Zuffardi O. Magrini U. Paulli M. Vannucchi A.M. Barosi G. Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative (AGIMM) investigatorsSpleen endothelial cells from patients with myelofibrosis harbor the JAK2V617F mutation.Blood. 2013; 121: 360-368Crossref PubMed Scopus (84) Google Scholar although this has been poorly characterized to date. In the bloodstream of patients with PMF, the numbers of endothelial progenitor cells (EPCs) are significantly higher than in healthy donors and for other myeloproliferative neoplasms.9Massa M. Rosti V. Ramajoli I. Campanelli R. Pecci A. Viarengo G. Meli V. Marchetti M. Hoffman R. Barosi G. Circulating CD34+, CD133+, and vascular endothelial growth factor receptor 2-positive endothelial progenitor cells in myelofibrosis with myeloid metaplasia.J Clin Oncol. 2005; 23: 5688-5695Crossref PubMed Scopus (87) Google Scholar Moreover, a massive neoangiogenesis is observed in bone marrow12Mesa R.A. Hanson C.A. Rajkumar S.V. Schroeder G. Tefferi A. Evaluation and clinical correlations of bone marrow angiogenesis in myelofibrosis with myeloid metaplasia.Blood. 2000; 96: 3374-3380Crossref PubMed Google Scholar, 13Ni H. Barosi G. Hoffman R. Quantitative evaluation of bone marrow angiogenesis in idiopathic myelofibrosis.Am J Clin Pathol. 2006; 126: 241-247Crossref PubMed Scopus (24) Google Scholar and spleen14Barosi G. Rosti V. Massa M. Viarengo G.L. Pecci A. Necchi V. Ramaioli I. Campanelli R. Marchetti M. Bazzan M. Magrini U. Spleen neoangiogenesis in patients with myelofibrosis with myeloid metaplasia.Br J Haematol. 2004; 124: 618-625Crossref PubMed Scopus (41) Google Scholar of patients with PMF, which shows a high concentration of the proangiogenic factors vascular endothelial growth factor15Di Raimondo F. Azzaro M.P. Palumbo G.A. Bagnato S. Stagno F. Giustolisi G.M. Cacciola E. Sortino G. Guglielmo P. Giustolisi R. Elevated vascular endothelial growth factor (VEGF) serum levels in idiopathic myelofibrosis.Leukemia. 2001; 15: 976-980Crossref PubMed Scopus (76) Google Scholar and fibroblast growth factor-2.16Martyre M.C. Le Bousse-Kerdiles M.C. Romquin N. Chevillard S. Praloran V. Demory J.L. Dupriez B. Elevated levels of basic fibroblast growth factor in megakaryocytes and platelets from patients with idiopathic myelofibrosis.Br J Haematol. 1997; 97: 441-448Crossref PubMed Scopus (120) Google Scholar Although fibroblasts are believed to be directly implicated in fibrosis in PMF bone marrow, other cells might also contribute to this phenomenon, such as endothelial cells. Under inflammatory conditions, endothelial cells can undergo a biological process known as endothelial-to-mesenchymal transition (EndMT), such as during arteriosclerosis17Chen P.Y. Qin L. Tellides G. Simons M. Fibroblast growth factor receptor 1 is a key inhibitor of TGF-β signaling in the endothelium.Sci Signal. 2014; 7: ra90Crossref PubMed Scopus (76) Google Scholar or in the tumor stroma.18Kalluri R. Weinberg R.A. The basics of epithelial-mesenchymal transition.J Clin Invest. 2009; 119: 1420-1428Crossref PubMed Scopus (7271) Google Scholar This phenotypic switch is characterized by coexpression of endothelial and mesenchymal markers and by changes to some of the phenotypic characteristics, such as acquisition of an elongated fibroblastoid morphologic structure and increased migratory properties.18Kalluri R. Weinberg R.A. The basics of epithelial-mesenchymal transition.J Clin Invest. 2009; 119: 1420-1428Crossref PubMed Scopus (7271) Google Scholar, 19Zeisberg E.M. Potenta S. Xie L. Zeisberg M. Kalluri R. Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts.Cancer Res. 2007; 67: 10123-10128Crossref PubMed Scopus (683) Google Scholar Physiologically, EndMT takes place during the formation of the heart cushion in the embryo,20Armstrong E.J. Bischoff J. Heart valve development: endothelial cell signaling and differentiation.Circ Res. 2004; 95: 459-470Crossref PubMed Scopus (521) Google Scholar, 21Liebner S. Cattelino A. Gallini R. Rudini N. Iurlaro M. Piccolo S. Dejana E. β-catenin is required for endothelial-mesenchymal transformation during heart cushion development in the mouse.J Cell Biol. 2004; 166: 359-367Crossref PubMed Scopus (316) Google Scholar, 22MacGrogan D. Luna-Zurita L. de la Pompa J.L. Notch signaling in cardiac valve development and disease.Birth Defects Res A Clin Mol Teratol. 2011; 91: 449-459Crossref PubMed Scopus (57) Google Scholar but it can be reactivated in adults under pathologic conditions.23Zeisberg E.M. Tarnavski O. Zeisberg M. Dorfman A.L. McMullen J.R. Gustafsson E. Chandraker A. Yuan X. Pu W.T. Roberts A.B. Neilson E.G. Sayegh M.H. Izumo S. Kalluri R. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis.Nat Med. 2007; 13: 952-961Crossref PubMed Scopus (1620) Google Scholar, 24Zeisberg E.M. Potenta S.E. Sugimoto H. Zeisberg M. Kalluri R. Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.J Am Soc Nephrol. 2008; 19: 2282-2287Crossref PubMed Scopus (679) Google Scholar, 25Potenta S. Zeisberg E. Kalluri R. The role of endothelial-to-mesenchymal transition in cancer progression.Br J Cancer. 2008; 99: 1375-1379Crossref PubMed Scopus (405) Google Scholar The EndMT program can be induced by transforming growth factor (TGF)-β and the bone morphogenetic proteins (BMPs), and it can significantly contribute to fibrotic progression in different diseases.23Zeisberg E.M. Tarnavski O. Zeisberg M. Dorfman A.L. McMullen J.R. Gustafsson E. Chandraker A. Yuan X. Pu W.T. Roberts A.B. Neilson E.G. Sayegh M.H. Izumo S. Kalluri R. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis.Nat Med. 2007; 13: 952-961Crossref PubMed Scopus (1620) Google Scholar, 26Camenisch T.D. Molin D.G. Person A. Runyan R.B. Gittenberger-de Groot A.C. McDonald J.A. Klewer S.E. Temporal and distinct TGF-β ligand requirements during mouse and avian endocardial cushion morphogenesis.Dev Biol. 2002; 248: 170-181Crossref PubMed Scopus (231) Google Scholar, 27Medici D. Potenta S. Kalluri R. Transforming growth factor-β2 promotes Snail-mediated endothelial-mesenchymal transition through convergence of Smad-dependent and Smad-independent signalling.Biochem J. 2011; 437: 515-520Crossref PubMed Scopus (224) Google Scholar, 28Maddaluno L. Rudini N. Cuttano R. Bravi L. Giampietro C. Corada M. Ferrarini L. Orsenigo F. Papa E. Boulday G. Tournier-Lasserve E. Chapon F. Richichi C. Retta S.F. Lampugnani M.G. Dejana E. EndMT contributes to the onset and progression of cerebral cavernous malformations.Nature. 2013; 498: 492-496Crossref PubMed Scopus (334) Google Scholar In the present study, we show that endothelial cells from the microvasculature of bone marrow and spleen can undergo the EndMT switch during the development of PMF in both patients and a mouse model of PMF. This process occurs during the early stages of fibrotic degeneration and can be mediated by the release and activation of TGF-β and other inflammatory cytokines by megakaryocytes and platelets. Thus, EndMT can contribute to the development and maintenance of bone marrow fibrosis and spleen dysplasia in PMF. Bone marrow, spleen tissue, and EPCs from peripheral blood of patients with PMF were collected at the Istituto di Ricovero e Cura a Carattere Scientifico, Policlinico San Matteo (Pavia, Italy). The diagnosis of PMF was established according to the World Health Organization 2008 criteria29Thiele J. Kvasnicka H.M. The 2008 WHO diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis.Curr Hematol Malig Rep. 2009; 4: 33-40Crossref PubMed Scopus (69) Google Scholar and the Italian Consensus Conference criteria.30Barosi G. Ambrosetti A. Finelli C. Grossi A. Leoni P. Liberato N.L. Petti M.C. Pogliani E. Ricetti M. Rupoli S. Visani G. Tura S. The Italian consensus conference on diagnostic criteria for myelofibrosis with myeloid metaplasia.Br J Haematol. 1999; 104: 730-737Crossref PubMed Scopus (193) Google Scholar The clinical features of patients with PMF are summarized in Supplemental Table S1. The healthy control subjects were staff members or donors for scientific research. All of the patients and donors approved and signed the informed consent. Bone marrow sections from healthy donors were purchased from Labospace (Milano, Italy). The C57Bl/6J mice (8 to 12 weeks of age) for isolation of endothelial cells from the spleen (SECs) and of megakaryocytes and for purification of platelets were purchased from Charles Rivers Laboratories (Calco, Italy). The conditional JAK2FLEX/WT KI mice have been described previously,31Hasan S. Lacout C. Marty C. Cuingnet M. Solary E. Vainchenker W. Villeval J.L. JAK2V617F expression in mice amplifies early hematopoietic cells and gives them a competitive advantage that is hampered by IFNalpha.Blood. 2013; 122: 1464-1477Crossref PubMed Scopus (107) Google Scholar and they were crossed with transgenic mice that expressed Cre-recombinase under the Vav promoter, to obtain mice expressing the JAK2-V617F mutation in heterozygosity, namely JAK2V617F/WT KI.32Bartalucci N. Tozzi L. Bogani C. Martinelli S. Rotunno G. Villeval J.L. Vannucchi A.M. Co-targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms.J Cell Mol Med. 2013; 17: 1385-1396Crossref PubMed Scopus (78) Google Scholar The animal experimentation was approved by the FIRC Institute of Molecular Oncology Institutional Animal Care and Use Committee and was performed according to the guidelines for the regulation of animal experimentation of the Italian Ministry of Health. Murine SECs were isolated and immortalized as described previously.33Balconi G. Spagnuolo R. Dejana E. Development of endothelial cell lines from embryonic stem cells: a tool for studying genetically manipulated endothelial cells in vitro.Arterioscler Thromb Vasc Biol. 2000; 20: 1443-1451Crossref PubMed Scopus (95) Google Scholar Briefly, the mouse spleens were collected under sterile conditions and dissociated with 1.5 mg/mL collagenase type I (Roche, Mannehim, Germany) and DNase 25 μg/mL (Roche) for 3 hours at 37°C. The resulting cell suspension was filtered through a nylon screen (70-μm mesh), centrifuged, and plated. Two days later, the heterogeneous population of SECs was infected with polyoma middle T antigen to specifically immortalize them.34Garlanda C. Parravicini C. Sironi M. De Rossi M. Wainstok de Calmanovici R. Carozzi F. Bussolino F. Colotta F. Mantovani A. Vecchi A. Progressive growth in immunodeficient mice and host cell recruitment by mouse endothelial cells transformed by polyoma middle-sized T antigen: implications for the pathogenesis of opportunistic vascular tumors.Proc Natl Acad Sci U S A. 1994; 91: 7291-7295Crossref PubMed Scopus (163) Google Scholar The SECs were cultured in 0.1% gelatin-coated flasks in MCDB131 medium (Life Technologies, Paisley, UK), supplemented with 20% North American fetal bovine serum (HyClone, South Logan, UT), 50 μg/mL endothelial cell growth supplement (made from calf brain), 100 μg/mL heparin (Sigma-Aldrich, St. Louis, MO), 100 U/L penicillin/streptomycin (Sigma-Aldrich), and 2 mmol/L l-glutamine (Sigma-Aldrich). The cell-starving medium consisted of MCDB131 medium with 1% bovine serum albumin (BSA; EuroClone, Milano, Italy). EPCs from healthy donors and patients with PMF were isolated according to protocols reported previously35Ingram D.A. Mead L.E. Tanaka H. Meade V. Fenoglio A. Mortell K. Pollok K. Ferkowicz M.J. Gilley D. Yoder M.C. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.Blood. 2004; 104: 2752-2760Crossref PubMed Scopus (1312) Google Scholar and were grown on collagen-I–coated plates in Endothelial Cell Growth Medium-2 supplied with Endothelial Cell Growth Medium-2 MV SingleQuots kits (Lonza, Cologne, GmbH, Germany). To induce the EndMT phenotype, the SECs and EPCs were stimulated with a cocktail of proinflammatory cytokines that included TGF-β, IL-1β, and tumor necrosis factor (TNF)-α (Peprotech, Rocky Hill, NJ), as described previously,36Rieder F. Kessler S.P. West G.A. Bhilocha S. de la Motte C. Sadler T.M. Gopalan B. Stylianou E. Fiocchi C. Inflammation-induced endothelial-to-mesenchymal transition: a novel mechanism of intestinal fibrosis.Am J Pathol. 2011; 179: 2660-2673Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar or with BMP6 (R&D Systems, Minneapolis, MN). The SECs were also treated with the TGF-β inhibitors LY-2109761 (SelleckBio, Munich, Germany) and dorsomorphin homolog 1 (DMH1; Tocris Bioscience, Bristol, UK). Murine fetal liver cells were collected at embryonic days 13 to 15, mechanically dissociated, and cultured for 3 days in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% fetal calf serum (Life Technologies), 100 U/L penicillin/streptomycin, 2 mmol/L l-glutamine, and 10 ng/mL recombinant mouse thrombopoietin (PeproTech). The cells were then layered on a single-step gradient (1.5% to 3.0% BSA), and the megakaryocytes were allowed to settle for 30 minutes at room temperature. The megakaryocyte pellet was washed and resuspended in complete medium, to a final concentration of 0.2 × 109 cells/mL.37Drachman J.G. Sabath D.F. Fox N.E. Kaushansky K. Thrombopoietin signal transduction in purified murine megakaryocytes.Blood. 1997; 89: 483-492Crossref PubMed Google Scholar Platelet purification from peripheral blood of mice was performed as described previously.38Ruggeri Z.M. Orje J.N. Habermann R. Federici A.B. Reininger A.J. Activation-independent platelet adhesion and aggregation under elevated shear stress.Blood. 2006; 108: 1903-1910Crossref PubMed Scopus (327) Google Scholar Briefly, the blood was collected from the inferior vena cava and pooled in buffered 3.2% citrate-dextrose solution, pH 5.2, and diluted with two parts of Ca2+-free and Mg2+-free Tyrode buffer, pH 6.5, followed by centrifugation at 300 × g for 7 minutes, to obtain the platelet-rich plasma. This platelet-rich plasma was further diluted in two parts of Tyrode buffer, pH 6.5, supplemented with 0.6 U/mL apyrase α (New England BioLabs, Ipswich, MA) and centrifuged at 600 × g for 15 minutes, to obtain the platelet pellet. The platelets were then resuspended in Tyrode buffer, pH 7.4, that contained 2 mmol/L CaCl2, to a final concentration of 0.2 × 109 platelets/mL. The platelets and megakaryocytes were incubated with 2 U/mL recombinant thrombin (Sigma-Aldrich) for 15 minutes at room temperature. Then 2 U/mL recombinant hirudin (Sigma-Aldrich) was added for 15 minutes to inactivate the thrombin. An additional centrifugation at 600 × g for 15 minutes led to the separation of the supernatant fluid from the pellet. The pellet was then resuspended in Tyrode buffer, pH 7.4, and both the supernatant fluid and the pellet were supplemented with 0.5% BSA and 70 mmol/L HCl. After 30 minutes, 70 mmol/L NaOH was added to neutralize the acid pH environment of the solutions. For the TGF-β blocking experiments, the megakaryocyte supernatant fluids were preincubated with an anti–TGF-β neutralizing antibody (clone 1D11; R&D Systems) or the corresponding isotype control antibodies (R&D Systems). For immunofluorescence (IF), confluent cell monolayers were fixed with 4% formaldehyde that was freshly prepared from paraformaldehyde in phosphate-buffered saline (PBS). After 15 minutes at room temperature, the fixed cells were permeabilized with 0.5% Triton X-100 for 5 minutes. The antibodies for the blocking (1 hour), primary (2 hours), and secondary (1 hour) incubations were diluted in PBS with 2% BSA. The appropriate fluorophore-conjugated secondary antibodies were used (Alexa-488, Alexa-555, Alexa-647, respectively; Molecular Probes, Eugene, OR). The nuclei were visualized using DAPI. Frozen bone marrow and spleen biopsies were sectioned (thickness, 5 μm) using a cryotome (Leica, Wetzlar, Germany), and the sections were mounted on Superfrost glass slides (Thermo Scientific, Waltham, MA) and dried. The sections were then fixed for 15 minutes with 4% formaldehyde that was freshly prepared from paraformaldehyde in PBS at room temperature, washed with PBS, permeabilized with 0.1% TritonX-100–PBS, and blocked with PBS containing 5% donkey serum and 2% BSA for 3 hours at room temperature. Murine paraffin-embedded bone marrow and spleen were sectioned (thickness, 5 μm) using a microtome (Leica) and deparaffinized through descending concentrations of ethanol. Antigen unmasking was performed in 0.25 mmol/L EDTA, pH 8.0, for 50 minutes at 95°C. The sections were then blocked with Tris-buffered saline (TBS) containing 5% donkey serum, 0.05% Triton X-100, and 2% BSA, for 1 hour at room temperature. Incubations with primary antibodies on both the cryosections and paraffin sections were performed in blocking solution overnight at 4°C. Tissue sections were then washed and incubated with the appropriate fluorophore-conjugated secondary antibodies (Molecular Probes) for 1 hour at room temperature. The nuclei were visualized with DAPI. The specimens were mounted with Vectashield (Vector Laboratories, Burlingame, CA). Confocal microscopy was performed using a confocal microscope (TCS SP2; Leica). Image acquisition was performed using a 63×/1.4 NA oil-immersion objective (HCX PL APO 63× Lbd Bl; Leica), and with spectral detection bands and scanning modalities optimized for the removal of channel cross talk. The Leica confocal software version 2.61 and ImageJ software version 1.49 (NIH, Bethesda, MD) were used for the data analysis. Only adjustments of brightness and contrast were used in the preparation of the figures, using Photoshop software version 13.0.6 (Adobe, San Jose, CA). Hematoxylin and eosin staining of the paraffin-embedded sections was performed according to standard protocols. For immunoprecipitation (IP) cells were solubilized in JS buffer (20 mmol/L HEPES, pH 7.5, 1.5 mmol/L MgCl2, 5 mmol/L EGTA, 150 mmol/L NaCl, 1% Triton X-100, 0.5% glycerol), protease inhibitors (Roche), and phosphatase inhibitors (Sigma-Aldrich) on ice for 20 minutes. Precleared cell extracts were subjected to antibody precipitation at 4°C, and immunocomplexes were captured using protein G-Sepharose beads (GE Healthcare Europe GmbH, Milano, Italy). Immunoprecipitated material was separated using Tris-glycine sodium dodecyl sulfate-PAGE, blotted onto nitrocellulose membranes, and analyzed by standard methods. Total protein was extracted by solubilizing the cells in boiling Laemmli buffer. Lysates were incubated for 10 minutes at 100°C and then centrifuged at 10,000 × g for 5 minutes to remove the cell debris. The supernatant fluids were collected, and their protein concentrations were determined using BCA Protein Assay kits (Pierce-ThermoFisher, Rockford, IL), according to the manufacturer's instructions. Equal amounts of protein were loaded onto the acrylamide gels at different concentrations and were separated using sodium dodecyl sulfate-PAGE, transferred to Protran nitrocellulose hybridization transfer membranes (pore size, 0.2 μm; ThermoFisher, Rockford, IL), and blocked for 1 hour at room temperature in TBS with Tween-20 (TBST) and 5% BSA. The membranes were then incubated overnight at 4°C, with the primary antibodies diluted in TBST containing 5% BSA. The membranes were rinsed at least three times with TBST and then incubated with horseradish peroxidase-linked secondary antibodies (Cell Signaling Technology, Danvers, MA) for 1 hour at room temperature. After three washes, the membranes were incubated with Amersham ECL Western blot (WB) detection reagents (Amersham Biosciences, Uppsala, Sweden) for 1 minute and exposed using a ChemiDoc XRS gel imaging system (Bio-Rad, Hercules, CA), for the required time. For the gene expression analysis, total RNA was isolated using RNaesy Micro kits (Qiagen, Valencia, CA), and 500 ng of total RNA was reverse transcribed with random hexamers (High-Capacity cDNA Archive kits; Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The cDNAs were amplified using the TaqMan gene expression assay (Applied Biosystems) in a thermocycler (7900 HT; ABI/Prism, Applied Biosystems). The following antibodies were used for IF, WB, and IP analysis: anti–vascular endothelial (VE)-cadherin (IF, dilution 1:200; WB analysis, dilution 1:200; IP, dilution 1:100; Santa Cruz Biotechnology, Dallas, TX); anti-CD44 (IF, dilution 1:100; WB analysis, dilution 1:500; BD Biosciences, San Jose, CA); anti-CD41a (IF, dilution 1:100; eBioscience, San Diego, CA) anti–N-cadherin (IF, dilution 1:200; WB analysis, dilution 1:2500; IP, dilution 1:100; BD Biosciences); anti-collagen I (IF, dilution 1:200; Invitrogen, Waltham, MA); anti–fibroblast-specific protein 1 (FSP1; IF, dilution 1:200; WB analysis, dilution 1:500; Millipore-Merck, Milano, Italy), anti-claudin5 (IF, dilution 1:500; Abcam, Cambridge, UK), anti-fibronectin (WB analysis, dilution 1:1000; Abcam); anti-panTGF-β (IF, dilution 1:100; R&D Systems, Minneapolis, MN); anti–TGF-β neutralizing (clone 1D11; R&D Systems); anti-CD31 (IF, dilution 1:400; BD Biosciences); anti–junctional adhesion molecule-A (JAM-A); (IF, dilution 1:100; BV12; Santa Cruz Biotechnology); anti–α-catenin (IF, dilution 1:200; WB analysis, dilution 1:500; Cell Signaling Technology, Danvers, MA); anti–β-catenin (IF, dilution 1:200; WB analysis, dilution 1:500; Cell Signaling); anti–γ-catenin (IF, dilution 1:200; WB analysis, dilution 1:500; Cell Signaling); anti-p120 (IF, dilution 1:200; WB analysis, dilution 1:500; Cell Signaling); anti-Smad1, anti-Smad3, anti–phospho-Smad1 (WB analysis, dilution 1:1000; Cell Signaling); anti–phospho-Smad3 (IF, dilution 1:100; Santa Cruz Biotechnology; WB analysis, dilution 1:500; Abcam); anti-tubulin (WB analysis, dilution 1:2000; Sigma-Aldrich); vinculin (WB analysis, dilution 1:5000; Sigma-Aldrich). The references on the specificity of the antibodies used are reported in the brochure of the respective cited supplier companies. Statistical significance was evaluated using two-
Referência(s)