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1.
Methods Mol Biol ; 2206: 27-37, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754808

RESUMO

Blood vessel formation is a key feature in physiologic and pathologic processes. Once considered a homogeneous cell population that functions as a passive physical barrier between blood and tissue, endothelial cells (ECs) are now recognized to be quite "heterogeneous." While numerous attempts to enhance endothelial repair and replacement have been attempted using so called "endothelial progenitor cells" it is now clear that a better understanding of the origin, location, and activation of stem and progenitor cells of the resident vascular endothelium is required before attempting exogenous cell therapy approaches. This chapter provides an overview for performance of single-cell clonogenic studies of human umbilical cord blood circulating endothelial colony-forming cells (ECFC) that represent distinct precursors for the endothelial lineage with vessel forming potential.

2.
Circ Res ; 127(10): 1233-1235, 2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-33090925
4.
J Alzheimers Dis ; 75(3): 959-969, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32390626

RESUMO

BACKGROUND: Aberrant angiogenesis may play a role in the development of Alzheimer's disease and related dementia. OBJECTIVE: To explore the relationship between angiogenesis activity and evidence of neurodegeneration among older adults. METHODS: Cross-sectional study of 49 older adults clinically characterized as cognitively normal, mild cognitive impairment, or early Alzheimer's disease. In addition to neuroimaging, we completed assays on peripheral blood, including: vascular endothelial growth factor, tumor necrosis factor, fibroblast growth factor, and amyloid-ß peptide 40. We used advanced polychromatic flow cytometry to phenotype circulating mononuclear cells to assess angiogenesis activity. RESULTS: Although we documented differences in cognitive performance, structural changes on neuroimaging, and burden of amyloid and tau on positron emission tomography, angiogenesis activity did not vary by group. Interestingly, VEGF levels were shown to be increased among subjects with mild cognitive impairment. In ANCOVA models controlling for age, sex, intracranial volume, and monocyte subpopulations, angiogenesis activity was correlated with increased white matter hyperintensities. CONCLUSION: We demonstrate a significant association between angiogenesis activity and cerebrovascular disease. To better understand the potential of angiogenesis as an intervention target, longitudinal studies are needed.

5.
Nat Protoc ; 15(3): 1066-1081, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32005982

RESUMO

Endothelial cells (ECs) are fundamental components of the blood vessels that comprise the vascular system; facilitate blood flow; and regulate permeability, angiogenesis, inflammatory responses and homeostatic tissue maintenance. Accumulating evidence suggests there is EC heterogeneity in vivo. However, isolation of fresh ECs from adult mice to investigate this further is challenging. Here, we describe an easy and reproducible protocol for isolation of different types of ECs and CD157+ vascular-resident endothelial stem cells (VESCs) by mechano-enzymatic tissue digestion followed by fluorescence-activated cell sorting. The procedure was established on liver tissue but can be used to isolate ECs from other organs with minimal modification. Preparation of single-cell suspensions can be completed in 2.5 h. We also describe assays for EC clonal and network formation, as well as transcriptomic analysis of isolated ECs. The protocol enables isolation of primary ECs and VESCs that can be used for a wide range of downstream analyses in vascular research.


Assuntos
Técnicas Citológicas/métodos , Células Endoteliais/fisiologia , Fígado/citologia , Células-Tronco/fisiologia , Animais , Camundongos
6.
Sci Rep ; 10(1): 1136, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980720

RESUMO

One current concept suggests that unchecked proliferation of clonally selected precursors of endothelial cells (ECs) contribute to severe pulmonary arterial hypertension (PAH). We hypothesized that clonally selected ECs expressing the progenitor marker CD117 promote severe occlusive pulmonary hypertension (PH). The remodelled pulmonary arteries of PAH patients harboured CD117+ ECs. Rat lung CD117+ ECs underwent four generations of clonal expansion to enrich hyperproliferative ECs. The resulting clonally enriched ECs behaved like ECs, as measured by in vitro and in vivo angiogenesis assays. The same primitive ECs showed a limited ability for mesenchymal lineage differentiation. Endothelial differentiation and function were enhanced by blocking TGF-ß signalling, promoting bone morphogenic protein (BMP) signalling. The transplantation of the EC clones caused arterio-occlusive PH in rats exposed to chronic hypoxia. These EC clones engrafted in the pulmonary arteries. Yet cessation of chronic hypoxia promoted lung cell apoptosis and resolution of vascular lesions. In conclusion, this is to the best of our knowledge, the first report that clonally enriched primitive ECs promote occlusive pulmonary arteriopathy and severe PH. These primitive EC clones further give rise to cells of endothelial and mesenchymal lineage as directed by BMP and TGF-ß signaling.

7.
Cardiovasc Res ; 116(2): 393-405, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30937452

RESUMO

AIMS: Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor population with established roles in vascular homeostasis and angiogenesis, which possess low immunogenicity and high potential for allogeneic therapy and are highly sensitive to regulation by reactive oxygen species (ROS). The aim of this study was to define the precise role of the major ROS-producing enzyme, NOX4 NADPH oxidase, in CB-ECFC vasoreparative function. METHODS AND RESULTS: In vitro CB-ECFC migration (scratch-wound assay) and tubulogenesis (tube length, branch number) was enhanced by phorbol 12-myristate 13-acetate (PMA)-induced superoxide in a NOX-dependent manner. CB-ECFCs highly-expressed NOX4, which was further induced by PMA, whilst NOX4 siRNA and plasmid overexpression reduced and potentiated in vitro function, respectively. Increased ROS generation in NOX4-overexpressing CB-ECFCs (DCF fluorescence, flow cytometry) was specifically reduced by superoxide dismutase, highlighting induction of ROS-specific signalling. Laser Doppler imaging of mouse ischaemic hindlimbs at 7 days indicated that NOX4-knockdown CB-ECFCs inhibited blood flow recovery, which was enhanced by NOX4-overexpressing CB-ECFCs. Tissue analysis at 14 days revealed consistent alterations in vascular density (lectin expression) and eNOS protein despite clearance of injected CB-ECFCs, suggesting NOX4-mediated modulation of host tissue. Indeed, proteome array analysis indicated that NOX4-knockdown CB-ECFCs largely suppressed tissue angiogenesis, whilst NOX4-overexpressing CB-ECFCs up-regulated a number of pro-angiogenic factors specifically-linked with eNOS signalling, in parallel with equivalent modulation of NOX-dependent ROS generation, suggesting that CB-ECFC NOX4 signalling may promote host vascular repair. CONCLUSION: Taken together, these findings indicate a key role for NOX4 in CB-ECFCs, thereby highlighting its potential as a target for enhancing their reparative function through therapeutic priming to support creation of a pro-reparative microenvironment and effective post-ischaemic revascularization.

8.
Nat Commun ; 10(1): 5649, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31827082

RESUMO

Clonal hematopoiesis of indeterminate potential (CHIP) increases with age and is associated with increased risks of hematological malignancies. While TP53 mutations have been identified in CHIP, the molecular mechanisms by which mutant p53 promotes hematopoietic stem and progenitor cell (HSPC) expansion are largely unknown. Here we discover that mutant p53 confers a competitive advantage to HSPCs following transplantation and promotes HSPC expansion after radiation-induced stress. Mechanistically, mutant p53 interacts with EZH2 and enhances its association with the chromatin, thereby increasing the levels of H3K27me3 in genes regulating HSPC self-renewal and differentiation. Furthermore, genetic and pharmacological inhibition of EZH2 decreases the repopulating potential of p53 mutant HSPCs. Thus, we uncover an epigenetic mechanism by which mutant p53 drives clonal hematopoiesis. Our work will likely establish epigenetic regulator EZH2 as a novel therapeutic target for preventing CHIP progression and treating hematological malignancies with TP53 mutations.


Assuntos
Epigênese Genética , Doenças Hematológicas/metabolismo , Hematopoese , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Doenças Hematológicas/genética , Doenças Hematológicas/fisiopatologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Metilação , Camundongos Endogâmicos C57BL , Mutação , Ligação Proteica
9.
JCI Insight ; 4(21)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31672944

RESUMO

Retinopathy of prematurity (ROP) is a disorder of the developing retina of preterm infants. ROP can lead to blindness because of abnormal angiogenesis that is the result of suspended vascular development and vaso-obliteration leading to severe retinal stress and hypoxia. We tested the hypothesis that the use of the human progenitor cell combination, bone marrow-derived CD34+ cells and vascular wall-derived endothelial colony-forming cells (ECFCs), would synergistically protect the developing retinal vasculature in a mouse model of ROP, called oxygen-induced retinopathy (OIR). CD34+ cells alone, ECFCs alone, or the combination thereof were injected intravitreally at either P5 or P12 and pups were euthanized at P17. Retinas from OIR mice injected with ECFCs or the combined treatment revealed formation of the deep vascular plexus (DVP) while still in hyperoxia, with normal-appearing connections between the superficial vascular plexus (SVP) and the DVP. In addition, the combination of cells completely prevented aberrant retinal neovascularization and was more effective anatomically and functionally at rescuing the ischemia phenotype than either cell type alone. We show that the beneficial effects of the cell combination are the result of their ability to orchestrate an acceleration of vascular development and more rapid ensheathment of pericytes on the developing vessels. Lastly, our proteomic and transcriptomic data sets reveal pathways altered by the dual cell therapy, including many involved in neuroretinal maintenance, and principal component analysis (PCA) showed that cell therapy restored OIR retinas to a state that was closely associated with age-matched normal retinas. Together, these data herein support the use of dual cell therapy as a promising preventive treatment for the development of ROP in premature infants.

11.
PLoS One ; 14(3): e0212642, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30870461

RESUMO

Previously, we showed that mesenchymal stem cells (MSC) can be mobilized into peripheral blood using electroacupuncture (EA) at acupoints, LI-4, LI-11, GV-14, and GV-20. The purpose of this study was to determine whether EA-mobilized MSC could be harvested and expanded in vitro to be used as an autologous cell therapy in horses. Peripheral blood mononuclear cells (PBMC) isolated from young and aged lame horses (n = 29) showed a marked enrichment for MSCs. MSC were expanded in vitro (n = 25) and administered intravenously at a dose of 50 x 106 (n = 24). Treatment resulted in significant improvement in lameness as assessed by the American Association of Equine Practitioners (AAEP) lameness scale (n = 23). MSCs exhibited immunomodulatory function by inhibition of lymphocyte proliferation and induction of IL-10. Intradermal testing showed no immediate or delayed immune reactions to MSC (1 x 106 to 1 x 104). In this study, we demonstrated an efficient, safe and reproducible method to mobilize and expand, in vitro, MSCs in sufficiently high concentrations for therapeutic administration. We confirm the immunomodulatory function of these cells in vitro. This non-pharmacological and non-surgical strategy for stem cell harvest has a broad range of biomedical applications and represents an improved clinically translatable and economical cell source for humans.


Assuntos
Mobilização de Células-Tronco Hematopoéticas , Imunomodulação , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/imunologia , Animais , Proliferação de Células , Separação Celular , Cavalos , Linfócitos/citologia , Linfócitos/imunologia , Células-Tronco Mesenquimais/citologia , Transplante Autólogo
12.
Stem Cell Reports ; 12(3): 572-583, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30745034

RESUMO

It is generally considered that mouse embryonic stem cell (ESC) differentiation into blood cells in vitro recapitulates yolk sac (YS) hematopoiesis. As such, similar to YS-derived B-progenitors, we demonstrate here that ESC-derived B-progenitors differentiate into B-1 and marginal zone B cells, but not B-2 cells in immunodeficient mice after transplantation. ESC-derived B-1 cells were maintained in the recipients for more than 6 months, secreting natural IgM antibodies in vivo. Gene expression profiling displayed a close relationship between ESC- and YS-derived B-1 progenitors. Because there are no hematopoietic stem cells (HSCs) detectable in our ESC differentiation culture, successful long-term engraftment of ESC-derived functional B-1 cells supports the presence of HSC-independent B-1 cell development.


Assuntos
Linfócitos B/citologia , Células-Tronco Hematopoéticas/citologia , Linfopoese/fisiologia , Células Precursoras de Linfócitos B/citologia , Animais , Diferenciação Celular/fisiologia , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Hematopoese/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Saco Vitelino/citologia
13.
Methods Mol Biol ; 1940: 97-107, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788820

RESUMO

Human umbilical cord blood contains highly proliferative circulating endothelial colony-forming cells (ECFC). These cells have promising therapeutic potential for various cardiovascular diseases by possessing robust in vitro clonal expansion potential and the ability to form functional blood vessels in vivo upon transplantation into recipient immunodeficient mice. However whether similar cells also exist in murine blood remains unresolved, which impedes the study of circulating ECFC biology using murine models. Here we describe a method to identify and culture murine embryonic peripheral blood-derived circulating ECFC through co-culture with OP9 stromal cells. Using this method, embryonic circulating ECFC can be identified by the formation of sheet-like or network-like endothelial colonies upon OP9 stromal cell monolayers.


Assuntos
Células-Tronco Embrionárias/citologia , Células Progenitoras Endoteliais/citologia , Sangue Fetal/citologia , Animais , Técnicas de Cultura de Células , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL
14.
J Cell Biol ; 218(4): 1369-1389, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30737263

RESUMO

Cell migration initiates by traction generation through reciprocal actomyosin tension and focal adhesion reinforcement, but continued motility requires adaptive cytoskeletal remodeling and adhesion release. Here, we asked whether de novo gene expression contributes to this cytoskeletal feedback. We found that global inhibition of transcription or translation does not impair initial cell polarization or migration initiation, but causes eventual migratory arrest through excessive cytoskeletal tension and over-maturation of focal adhesions, tethering cells to their matrix. The transcriptional coactivators YAP and TAZ mediate this feedback response, modulating cell mechanics by limiting cytoskeletal and focal adhesion maturation to enable persistent cell motility and 3D vasculogenesis. Motile arrest after YAP/TAZ ablation was partially rescued by depletion of the YAP/TAZ-dependent myosin phosphatase regulator, NUAK2, or by inhibition of Rho-ROCK-myosin II. Together, these data establish a transcriptional feedback axis necessary to maintain a responsive cytoskeletal equilibrium and persistent migration.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Movimento Celular , Citoesqueleto/metabolismo , Células Progenitoras Endoteliais/metabolismo , Adesões Focais/metabolismo , Mecanotransdução Celular , Neovascularização Fisiológica , Transativadores/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Citoesqueleto/genética , Retroalimentação Fisiológica , Adesões Focais/genética , Cinética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miosina Tipo II/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores/deficiência , Transativadores/genética , Transcrição Genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo
15.
Biomaterials ; 200: 25-34, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30754017

RESUMO

Whole organ engineering paradigms typically involve repopulating acellular organ scaffolds with recipient-compatible cells, to generate a neo-organ that may provide key physiological functions. In the case of whole lung engineering, functionally endothelialized pulmonary vasculature is critical for establishing a fluid-tight barrier at the level of the alveolus, so that oxygen and carbon dioxide can be exchanged in the organ. We have previously developed a protocol to efficiently seed endothelial cells into the microvascular channels of decellularized lung scaffolds, but fully functional endothelial coverage, in terms of barrier function and resistance to thrombosis, was not achieved. In this study, we investigated whether various small molecules could favorably impact endothelial functionality after seeding into decellularized lung scaffolds. We demonstrated that the Epac-selective cAMP analog 8CPT-2Me-cAMP improves endothelial barrier function in repopulated lung scaffolds. When treated with the Epac agonist, barrier function of human umbilical vein endothelial cells (HUVECs) improved, and was maintained for at least three days, whereas the effect of other tested molecules lasted for only 5 h. Treatment with the Epac agonist re-organized actin structure, and appeared to increase the continuity of junction proteins such as VE-cadherin and ZO1. Blockade of actin polymerization abolished the effect of the Epac agonist on barrier function and actin reorganization, confirming a strong actin-mediated effect. Similarly, after treatment with Epac agonist, the barrier function in iPSC-derived endothelial colony forming cells (ECFCs) was increased and the enhanced barrier was maintained for at least 60 h. After culture in lung scaffolds for 5 days, iPSC-ECFCs maintained their phenotype by expressing CD31, eNOS, vWF, and VE-Cadherin. Treatment with the Epac agonist significantly improved the barrier function of iPSC-ECFC-repopulated lung for at least 6 h. Taken together, these findings demonstrated that Epac-selective 8CPT-2Me-cAMP activation enhanced vascular barrier in iPSC-ECFC-engineered lungs, and may be useful to improve endothelial functionality for whole organ tissue engineering.

16.
J Infect Dis ; 219(7): 1076-1083, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30239747

RESUMO

BACKGROUND: Human immunodeficiency virus (HIV) may be related to cardiovascular disease through monocyte activation-associated endothelial dysfunction. METHODS: Blood samples from 15 HIV-negative participants (the uninfected group), 8 HIV-positive participants who were not receiving antiretroviral therapy (ART) (the infected, untreated group), and 15 HIV-positive participants who were receiving ART (the infected, treated group) underwent flow cytometry of endothelial colony-forming cells (ECFCs) and monocyte proportions. IncuCyte live cell imaging of 8 capillary proliferative capacity parameters were obtained from cord blood ECFCs treated with participant plasma. RESULTS: The ECFC percentage determined by flow cytometry was not different between the study groups; however, values of the majority of capillary proliferative capacity parameters (ie, cell area, network length, network branch points, number of networks, and average tube width uniformity) were significantly lower in infected, untreated participants as compared to values for uninfected participants or infected, treated participants (P < .00625 for all comparisons). CD14+CD16+ intermediate monocytes and soluble CD163 were significantly and negatively correlated with several plasma-treated, cord blood ECFC proliferative capacity parameters in the combined HIV-positive groups but not in the uninfected group. CONCLUSIONS: Cord blood ECFC proliferative capacity was significantly impaired by plasma from infected, untreated patients, compared with plasma from uninfected participants and from infected, treated participants. Several ECFC functional parameters were adversely associated with monocyte activation in the HIV-positive groups, thereby suggesting a mechanism by which HIV-related inflammation may impair vascular reparative potential and consequently increase the risk of cardiovascular disease during HIV infection.


Assuntos
Endotélio/imunologia , Soronegatividade para HIV/imunologia , Soropositividade para HIV/imunologia , Monócitos , Células-Tronco , Adulto , Fármacos Anti-HIV/uso terapêutico , Benzoxazinas/uso terapêutico , Proliferação de Células , Quimiocina CCL5/sangue , Endotélio/patologia , Feminino , Sangue Fetal , Citometria de Fluxo , Proteínas Ligadas por GPI/metabolismo , Soropositividade para HIV/sangue , Soropositividade para HIV/tratamento farmacológico , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Neovascularização Fisiológica , Plasma/imunologia , Estudos Prospectivos , Receptores de IgG/metabolismo , Células-Tronco/fisiologia , Molécula 1 de Adesão de Célula Vascular/sangue , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/sangue , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/sangue
17.
Nat Commun ; 9(1): 3518, 2018 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158520

RESUMO

While erythropoietin (EPO) constitutes the major treatment for anemia, a range of anemic disorders remain resistant to EPO treatment. The need for alternative therapeutic strategies requires the identification of mechanisms that physiologically restrain erythropoiesis. Here we show that P38α restrains erythropoiesis in mouse and human erythroblasts independently of EPO by integrating apoptotic signals during recovery from anemia. P38α deficiency promotes JNK activation through increased expression of Map3k4 via a negative feedback mechanism. JNK prevents Cdk1-mediated phosphorylation and subsequent degradation by Smurf2 of the epigenetic silencer Ezh2. Stabilized Ezh2 silences Bim expression and protects erythroblasts from apoptosis. Thus, we identify P38α/JNK signaling as a molecular brake modulating erythropoiesis through epigenetic silencing of Bim. We propose that inhibition of P38α, by enhancing erythropoiesis in an EPO-independent fashion, may provide an alternative strategy for the treatment of anemia.


Assuntos
Proteína 11 Semelhante a Bcl-2/metabolismo , Eritropoese/fisiologia , Animais , Antígenos CD34/metabolismo , Apoptose/genética , Apoptose/fisiologia , Proteína 11 Semelhante a Bcl-2/genética , Epigênese Genética/genética , Eritroblastos/metabolismo , Eritropoese/genética , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Células-Tronco/genética , Fator de Células-Tronco/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
19.
Cell Rep ; 24(4): 895-908.e6, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-30044986

RESUMO

The role of primary cilia in mechanosensation is essential in endothelial cell (EC) shear responsiveness. Here, we find that venous, capillary, and progenitor ECs respond to shear stress in vitro in a cilia-dependent manner. We then demonstrate that primary cilia assembly in human induced pluripotent stem cell (hiPSC)-derived ECs varies between different cell lines with marginal influence of differentiation protocol. hiPSC-derived ECs lacking cilia do not align to shear stress, lack stress fiber assembly, have uncoordinated migration during wound closure in vitro, and have aberrant calcium influx upon shear exposure. Transcriptional analysis reveals variation in regulatory genes involved in ciliogenesis among different hiPSC-derived ECs. Moreover, inhibition of histone deacetylase 6 (HDAC6) activity in hiPSC-ECs lacking cilia rescues cilia formation and restores mechanical sensing. Taken together, these results show the importance of primary cilia in hiPSC-EC mechano-responsiveness and its modulation through HDAC6 activity varies among hiPSC-ECs.


Assuntos
Cílios/enzimologia , Células Endoteliais/enzimologia , Desacetilase 6 de Histona/metabolismo , Células-Tronco Pluripotentes/enzimologia , Cálcio/metabolismo , Movimento Celular/fisiologia , Citoesqueleto/enzimologia , Células Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Mecanotransdução Celular , Técnicas Analíticas Microfluídicas , Células-Tronco Pluripotentes/citologia , Artérias Umbilicais/citologia , Artérias Umbilicais/enzimologia
20.
Oncotarget ; 9(31): 21831-21843, 2018 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-29774106

RESUMO

Juvenile Myelomonocytic Leukemia (JMML) is a pediatric myeloproliferative neoplasm (MPN) that has a poor prognosis. Somatic mutations in Ptpn11 are the most frequent cause of JMML and they commonly occur in utero. Animal models of mutant Ptpn11 have probed the signaling pathways that contribute to JMML. However, existing models may inappropriately exacerbate MPN features by relying on non-hematopoietic-restricted Cre-loxP strains or transplantations into irradiated recipients. In this study we generate hematopoietic-restricted models of Ptpn11E76K-mediated disease using Csf1r-MCM and Flt3Cre. We show that these animals have indolent MPN progression despite robust GM-CSF hypersensitivity and Ras-Erk hyperactivation. Rather, the dominant pathology is pronounced thrombocytopenia with expanded extramedullary hematopoiesis. Furthermore, we demonstrate that the timing of tamoxifen administration in Csf1r-MCM mice can specifically induce recombinase activity in either fetal or adult hematopoietic progenitors. We take advantage of this technique to show more rapid monocytosis following Ptpn11E76K expression in fetal progenitors compared with adult progenitors. Finally, we demonstrate that Ptpn11E76K results in the progressive reduction of T cells, most notably of CD4+ and naïve T cells. This corresponds to an increased frequency of T cell progenitors in the thymus and may help explain the occasional emergence of T-cell leukemias in JMML patients. Overall, our study is the first to describe the consequences of hematopoietic-restricted Ptpn11E76K expression in the absence of irradiation. Our techniques can be readily adapted by other researchers studying somatically-acquired blood disorders.

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