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1.
Nat Commun ; 12(1): 66, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397952

RESUMO

IL11 is important for fibrosis in non-alcoholic steatohepatitis (NASH) but its role beyond the stroma in liver disease is unclear. Here, we investigate the role of IL11 in hepatocyte lipotoxicity. Hepatocytes highly express IL11RA and secrete IL11 in response to lipid loading. Autocrine IL11 activity causes hepatocyte death through NOX4-derived ROS, activation of ERK, JNK and caspase-3, impaired mitochondrial function and reduced fatty acid oxidation. Paracrine IL11 activity stimulates hepatic stellate cells and causes fibrosis. In mouse models of NASH, hepatocyte-specific deletion of Il11ra1 protects against liver steatosis, fibrosis and inflammation while reducing serum glucose, cholesterol and triglyceride levels and limiting obesity. In mice deleted for Il11ra1, restoration of IL11 cis-signaling in hepatocytes reconstitutes steatosis and inflammation but not fibrosis. We found no evidence for the existence of IL6 or IL11 trans-signaling in hepatocytes or NASH. These data show that IL11 modulates hepatocyte metabolism and suggests a mechanism for NAFLD to NASH transition.


Assuntos
Hepatócitos/metabolismo , Interleucina-11/metabolismo , Lipídeos/toxicidade , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Transdução de Sinais , Adulto , Animais , Comunicação Autócrina/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Comportamento Alimentar , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Subunidade alfa de Receptor de Interleucina-11/metabolismo , Interleucina-6/metabolismo , Camundongos Knockout , Modelos Biológicos , Comunicação Parácrina/efeitos dos fármacos , Fenótipo , Transdução de Sinais/efeitos dos fármacos
2.
J Surg Res ; 257: 294-305, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32871430

RESUMO

BACKGROUND: Drug-eluting stents impair post-angioplasty re-endothelialization thus compromising restenosis prevention while heightening thrombotic risks. We recently found that inhibition of protein kinase RNA-like endoplasmic reticulum kinase (PERK) effectively mitigated both restenosis and thrombosis in rodent models. This motivated us to determine how PERK inhibition impacts re-endothelialization. METHODS: Re-endothelialization was evaluated in endothelial-denuded rat carotid arteries after balloon angioplasty and periadventitial administration of PERK inhibitor in a hydrogel. To study whether PERK in smooth muscle cells (SMCs) regulates re-endothelialization by paracrinally influencing endothelial cells (ECs), denuded arteries exposing SMCs were lentiviral-infected to silence PERK; in vitro, the extracellular vesicles isolated from the medium of PDGF-activated, PERK-upregulating human primary SMCs were transferred to human primary ECs. RESULTS: Treatment with PERK inhibitor versus vehicle control accelerated re-endothelialization in denuded arteries. PERK-specific silencing in the denuded arterial wall (mainly SMCs) also enhanced re-endothelialization compared to scrambled shRNA control. In vitro, while medium transfer from PDGF-activated SMCs impaired EC viability and increased the mRNA levels of dysfunctional EC markers, either PERK inhibition or silencing in donor SMCs mitigated these EC changes. Furthermore, CXCL10, a paracrine cytokine detrimental to ECs, was increased by PDGF activation and decreased after PERK inhibition or silencing in SMCs. CONCLUSIONS: Attenuating PERK activity pharmacologically or genetically provides an approach to accelerating post-angioplasty re-endothelialization in rats. The mechanism may involve paracrine factors regulated by PERK in SMCs that impact neighboring ECs. This study rationalizes future development of PERK-targeted endothelium-friendly vascular interventions.


Assuntos
Angioplastia com Balão/efeitos adversos , Reestenose Coronária/prevenção & controle , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Reepitelização/efeitos dos fármacos , eIF-2 Quinase/antagonistas & inibidores , Angioplastia com Balão/instrumentação , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/patologia , Artérias Carótidas/cirurgia , Reestenose Coronária/etiologia , Modelos Animais de Doenças , Stents Farmacológicos/efeitos adversos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Humanos , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Comunicação Parácrina/efeitos dos fármacos , Comunicação Parácrina/genética , RNA Interferente Pequeno/metabolismo , Ratos , Reepitelização/genética , eIF-2 Quinase/genética
3.
Adv Exp Med Biol ; 1287: 183-200, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33034033

RESUMO

Notch promotes breast cancer progression through tumor initiating cell maintenance, tumor cell fate specification, proliferation, survival, and motility. In addition, Notch is recognized as a decisive mechanism in regulating various juxtacrine and paracrine communications in the tumor microenvironment (TME). In this chapter, we review recent studies on stress-mediated Notch activation within the TME and sequelae such as angiogenesis, extracellular matrix remodeling, changes in the innate and adaptive immunophenotype, and therapeutic perspectives.


Assuntos
Neoplasias da Mama , Receptores Notch/metabolismo , Transdução de Sinais , Microambiente Tumoral , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Humanos , Neovascularização Patológica , Comunicação Parácrina
4.
Nat Commun ; 11(1): 6298, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33293516

RESUMO

Immunosuppressive tumor microenvironment (TME) and ascites-derived spheroids in ovarian cancer (OC) facilitate tumor growth and progression, and also pose major obstacles for cancer therapy. The molecular pathways involved in the OC-TME interactions, how the crosstalk impinges on OC aggression and chemoresistance are not well-characterized. Here, we demonstrate that tumor-derived UBR5, an E3 ligase overexpressed in human OC associated with poor prognosis, is essential for OC progression principally by promoting tumor-associated macrophage recruitment and activation via key chemokines and cytokines. UBR5 is also required to sustain cell-intrinsic ß-catenin-mediated signaling to promote cellular adhesion/colonization and organoid formation by controlling the p53 protein level. OC-specific targeting of UBR5 strongly augments the survival benefit of conventional chemotherapy and immunotherapies. This work provides mechanistic insights into the novel oncogene-like functions of UBR5 in regulating the OC-TME crosstalk and suggests that UBR5 is a potential therapeutic target in OC treatment for modulating the TME and cancer stemness.


Assuntos
Carcinoma Epitelial do Ovário/imunologia , Macrófagos Peritoneais/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Peritoneais/imunologia , Evasão Tumoral/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Adulto , Idoso , Animais , Ascite/genética , Ascite/imunologia , Ascite/patologia , Carcinoma Epitelial do Ovário/mortalidade , Carcinoma Epitelial do Ovário/secundário , Carcinoma Epitelial do Ovário/terapia , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoterapia Adotiva/métodos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Comunicação Parácrina/imunologia , Neoplasias Peritoneais/mortalidade , Neoplasias Peritoneais/secundário , Cultura Primária de Células , Prognóstico , Receptores de Antígenos Quiméricos/imunologia , Esferoides Celulares/imunologia , Esferoides Celulares/metabolismo , Evasão Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Ubiquitina-Proteína Ligases/genética
5.
Nat Metab ; 2(11): 1350-1367, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33168981

RESUMO

Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-ß1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel-Pfkfb3 axis has potential for therapeutic applications in fibrotic disease.


Assuntos
Epitélio/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Macrófagos/patologia , Proteínas Proto-Oncogênicas c-rel/genética , Animais , Polaridade Celular/genética , Marcação de Genes , Hepatócitos/patologia , Hidroxiprolina/metabolismo , Cirrose Hepática/prevenção & controle , Regeneração Hepática/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose/genética , Comunicação Parácrina/genética , Fosfofrutoquinase-2/genética , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/metabolismo
6.
PLoS Genet ; 16(10): e1009069, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33057429

RESUMO

The genetic mechanisms that determine the size of the adult pancreas are poorly understood. Imprinted genes, which are expressed in a parent-of-origin-specific manner, are known to have important roles in development, growth and metabolism. However, our knowledge regarding their roles in the control of pancreatic growth and function remains limited. Here we show that many imprinted genes are highly expressed in pancreatic mesenchyme-derived cells and explore the role of the paternally-expressed insulin-like growth factor 2 (Igf2) gene in mesenchymal and epithelial pancreatic lineages using a newly developed conditional Igf2 mouse model. Mesenchyme-specific Igf2 deletion results in acinar and beta-cell hypoplasia, postnatal whole-body growth restriction and maternal glucose intolerance during pregnancy, suggesting that the mesenchyme is a developmental reservoir of IGF2 used for paracrine signalling. The unique actions of mesenchymal IGF2 are demonstrated by the absence of any discernible growth or functional phenotypes upon Igf2 deletion in the developing pancreatic epithelium. Additionally, increased IGF2 levels specifically in the mesenchyme, through conditional Igf2 loss-of-imprinting or Igf2r deletion, leads to pancreatic acinar overgrowth. Furthermore, ex-vivo exposure of primary acinar cells to exogenous IGF2 activates AKT, a key signalling node, and increases their number and amylase production. Based on these findings, we propose that mesenchymal Igf2, and perhaps other imprinted genes, are key developmental regulators of adult pancreas size and function.


Assuntos
Fator de Crescimento Insulin-Like II/genética , Mesoderma/crescimento & desenvolvimento , Pâncreas/crescimento & desenvolvimento , Comunicação Parácrina/genética , Células Acinares/metabolismo , Células Acinares/patologia , Aminoácidos/genética , Animais , Linhagem da Célula/genética , Cromo , Metilação de DNA/genética , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Impressão Genômica/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Camundongos , Ácidos Nicotínicos/genética , Pâncreas/citologia , Pâncreas/metabolismo , Gravidez , RNA Longo não Codificante/genética
7.
Am J Physiol Cell Physiol ; 319(6): C1141-C1150, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33026832

RESUMO

Stem cell injections are an attractive therapeutic tool. It has been demonstrated that injected stem cells promote tissue repair and regeneration via paracrine mechanisms. However, the effects of injected stem cells continue for far longer than they are present. We hypothesized that the effects of injected stem cells are prolonged because of a sequential paracrine relay mechanism. Conditioned media was collected from mesenchymal stem cells (MSCs) after 24 h. This media was then added to RAW264.7. Media was collected from the macrophages after 24 h and was then added to endothelial cells (ECs). This conditioned macrophage media, but not control media, promoted wound healing and induced EC differentiation. Similar results were observed with primary macrophages. To identify the active paracrine factors released by macrophages in response to stimulation by MSC conditioned media we used an antibody array, identifying increased expression of the angiogenesis-related proteins stromal cell-derived factor 1 (SDF1) and plasminogen activator inhibitor-1 (PAI-1). Knockdown of either protein inhibited the ability of conditioned media derived from MSC paracrine factor-stimulated macrophages to induce EC differentiation both in vitro and in vivo. Conditioned media derived from postnatal day 7 (P7) mouse macrophages induced EC differentiation. Moreover, SDF1 and PAI-1 levels were >120 higher in P7 macrophages compared with adult macrophages, suggesting that MSC paracrine factors promote adult macrophages to adopt a juvenile phenotype. These results indicate that MSC paracrine factors induce macrophages to secrete SDF1 and PAI-1, in-turn inducing endothelial cells to differentiate. Identification of a sequential paracrine mechanism opens new therapeutic avenues for stem cell therapy.


Assuntos
Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/efeitos dos fármacos , Regeneração Tecidual Guiada/métodos , Transplante de Células-Tronco Mesenquimais , Comunicação Parácrina/fisiologia , Cicatrização/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Quimiocina CXCL12/metabolismo , Macrófagos/citologia , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologia , Células RAW 264.7 , Serpina E2/metabolismo
8.
Life Sci ; 263: 118523, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33039386

RESUMO

BACKGROUND: Pancreatic stellate cells (PSCs) play key roles in the pancreatic tumor microenvironment and are considered to contribute to chemoresistance. PSCs can participate in malignant behaviors of pancreatic carcinoma (PC) by secreting hepatocyte growth factor (HGF). The objective of this research was to explore the potential molecular mechanism of HGF on gemcitabine (GEM) chemoresistance of PC. MATERIALS AND METHODS: HGF, c-Met, E-Cadherin and Vimentin levels were examined by quantitative real-time polymerase chain reaction (qRT-PCR). The changes of HGF level were detected by ELISA. The half maximal inhibitory concentration, the growth inhibitions and apoptosis of pancreatic cancer cells (PCCs) were respectively assayed using CCK-8 and flow cytometry. Associated proteins were measured using western blot and cell immunofluorescence assay. KEY FINDINGS: PSCs strongly expressed HGF, and its receptor c-Met was expressed in PCCs. PCCs exerted a positive regulative effect on HGF production. HGF neutralizing antibody AMG102 could effectively reduce the HGF level in PSC-conditioned medium (PSC-CM). PSC-CM promoted chemoresistance in PCCs. When exposed to PSC-CM, PCCs underwent epithelial-to-mesenchymal transition (EMT), and c-Met was also activated. Recombinant human HGF had the same protective effect. Blocking the HGF/c-Met axis with a c-Met inhibitor PHA665752 and AMG102 reduced the phosphorylation level of c-Met (p-c-Met) and attenuated EMT and chemoresistance. P-c-Met overexpression resulted in activation of the PI3K/Akt pathway, and inhibition of PI3K/Akt signaling with LY294002 reversed chemoresistance and EMT. SIGNIFICANCE: PSCs can activate the c-Met/PI3K/Akt pathway in PCCs via paracrine HGF, induce EMT of PCCs and inhibit cancer cell apoptosis, thus enhance chemoresistance to Gem in PCCs.


Assuntos
Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/patologia , Antimetabólitos Antineoplásicos/farmacologia , Apoptose , Proliferação de Células , Desoxicitidina/farmacologia , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Células Estreladas do Pâncreas/efeitos dos fármacos , Células Estreladas do Pâncreas/metabolismo , Comunicação Parácrina , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Células Tumorais Cultivadas
9.
Sheng Li Xue Bao ; 72(5): 651-659, 2020 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-33106835

RESUMO

The study aims to investigate the effects of cardiac fibroblast (CF) paracrine factors on murine embryonic stem cells (ESCs). Conditioned mediums from either neonatal cardiac fibroblasts (ConM-NCF) or adult cardiac fibroblasts (ConM-ACF) were diluted by 1:50 and 1:5, respectively, to investigate whether these conditioned mediums impact murine ESCs distinctly with RT-real time PCR techniques, cell proliferation essay, ELISA and by counting percentage of beating embryoid bodies (EBs) during ESCs differentiation. The data showed that the paracrine ability of CFs changed dramatically during development, in which interleukin 6 (IL6) increased with maturation. ConM-NCF 1:50 and ConM-NCF 1:5 had opposite effects on the pluripotent markers, although they both reduced mouse ESC proliferation. ConM-ACF 1:50 promoted ESCs pluripotent markers and proliferation, while ConM-ACF 1:5 exerted negative effects. All CF-derived conditioned mediums inhibited cardiac differentiation, but with distinguishable features: ConM-NCF 1:50 slightly decreased the early cardiac differentiation without altering the maturation tendency or cardiac specific markers in EBs at differentiation of day 17; ConM-ACF 1:50 had more significant inhibitory effects on early cardiac differentiation than ConM-NCF 1:50 and impeded cardiac maturation with upregulation of cardiac specific markers. In addition, IL6 neutralization antibody attenuated positive effect of ConM-ACF 1:50 on ESCs proliferation, but had no effects on ConM-NCF 1:50. Long-term IL6 neutralization reduced the percentage of beating EBs at early developmental stage, but did not alter the late cardiac differentiation. Taken together, both the quality and quantity of factors and cytokines secreted by CFs are critical for the ESC fate. IL6 could be a favorable cytokine for ESC pluripotency and the early cardiac differentiation.


Assuntos
Células-Tronco Embrionárias , Células-Tronco Embrionárias Murinas , Animais , Fibroblastos , Coração , Camundongos , Comunicação Parácrina
10.
Arterioscler Thromb Vasc Biol ; 40(11): e296-e308, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32938214

RESUMO

OBJECTIVE: Resident valvular interstitial cells (VICs) activate to myofibroblasts during aortic valve stenosis progression, which further promotes fibrosis or even differentiate into osteoblast-like cells that can lead to calcification of valve tissue. Inflammation is a hallmark of aortic valve stenosis, so we aimed to determine proinflammatory cytokines secreted from M1 macrophages that give rise to a transient VIC phenotype that leads to calcification of valve tissue. Approach and Results: We designed hydrogel biomaterials as valve extracellular matrix mimics enabling the culture of VICs in either their quiescent fibroblast or activated myofibroblast phenotype in response to the local matrix stiffness. When VIC fibroblasts and myofibroblasts were treated with conditioned media from THP-1-derived M1 macrophages, we observed robust reduction of αSMA (alpha smooth muscle actin) expression, reduced stress fiber formation, and increased proliferation, suggesting a potent antifibrotic effect. We further identified TNF (tumor necrosis factor)-α and IL (interleukin)-1ß as 2 cytokines in M1 media that cause the observed antifibrotic effect. After 7 days of culture in M1 conditioned media, VICs began differentiating into osteoblast-like cells, as measured by increased expression of RUNX2 (runt-related transcription factor 2) and osteopontin. We also identified and validated IL-6 as a critical mediator of the observed pro-osteogenic effect. CONCLUSIONS: Proinflammatory cytokines in M1 conditioned media inhibit myofibroblast activation in VICs (eg, TNF-α and IL-1ß) and promote their osteogenic differentiation (eg, IL-6). Together, our work suggests inflammatory M1 macrophages may drive a myofibroblast-to-osteogenic intermediate VIC phenotype, which may mediate the switch from fibrosis to calcification during aortic valve stenosis progression.


Assuntos
Estenose da Valva Aórtica/metabolismo , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Calcinose/metabolismo , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Miofibroblastos/metabolismo , Osteoblastos/metabolismo , Osteogênese , Comunicação Parácrina , Animais , Estenose da Valva Aórtica/patologia , Calcinose/patologia , Proliferação de Células , Matriz Extracelular/metabolismo , Fibrose , Humanos , Masculino , Miofibroblastos/patologia , Osteoblastos/patologia , Fenótipo , Via Secretória , Transdução de Sinais , Sus scrofa , Células THP-1
11.
PLoS One ; 15(9): e0238852, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32925965

RESUMO

The pathogenesis of esophageal injury in gastroesophageal reflux disease (GERD) is incompletely understood. We modeled exposure of human esophageal myofibroblasts (HEMFs) to gastroesophageal reflux by repeated treatment with pH 4.5 and pH 4.5 bile salts and determined the effects on the epithelium in a 3D organotypic-like air-liquid interface model. Total, basal and supra-basal thickness of the epithelium were measured and immunostaining for p63, for basal (CK 14) and supra-basal (CK 4) squamous differentiation markers, and for cell proliferation (PCNA) were performed. Epithelial cell proliferation in response to HEMF conditioned media was also assessed in 2D culture. In the 3D organotypic model, total epithelial thickness increased similarly with pH 4.5 and pH 4.5 bile salt treated versus untreated and bile salt treated HEMF conditioned media. Epithelial p63 immunostaining was increased and multilayered. There was expansion of the CK14+ basal and CK4+ supra-basal layers in the epithelium established with conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs versus untreated HEMF conditioned media. PCNA + cells per µm of tissue were unchanged in the basal layer across all treatment conditions while PCNA + cells per total DAPI + cells were decreased. In 2D culture, basal epithelial proliferation decreased with conditioned media from pH 4.5 and pH 4.5 bile salt treated HEMFs compared to conditioned media from untreated HEMF conditioned media. Secreted factors from HEMFs treated with acidic stimuli encountered in GERD increase epithelial thickness compared to secreted factors from untreated HEMFs and expand both basal and supra-basal layers. Our findings demonstrate for the first time paracrine regulation of the squamous epithelium from acid stimulated HEMFs. The effects of secreted factors from acid treated HEMFs on basal cell proliferation in this model and the mechanism mediating the increase in epithelial thickness merit further investigation.


Assuntos
Ácidos e Sais Biliares/farmacologia , Meios de Cultivo Condicionados/farmacologia , Células Epiteliais/patologia , Esôfago/patologia , Refluxo Gastroesofágico/patologia , Miofibroblastos/citologia , Comunicação Parácrina , Técnicas de Cultura de Células , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Esôfago/efeitos dos fármacos , Humanos , Técnicas In Vitro
12.
J Neurosci ; 40(38): 7355-7374, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32817249

RESUMO

17ß-Estradiol (E2) is produced from androgens via the action of the enzyme aromatase. E2 is known to be made in neurons in the brain, but the functions of neuron-derived E2 in the ischemic brain are unclear. Here, we used a forebrain neuron-specific aromatase KO (FBN-ARO-KO) mouse model to deplete neuron-derived E2 in the forebrain and determine its roles after global cerebral ischemia. We demonstrated that ovariectomized female FBN-ARO-KO mice exhibited significantly attenuated astrocyte activation, astrocytic aromatization, and decreased hippocampal E2 levels compared with FLOX mice. Furthermore, FBN-ARO-KO mice had exacerbated neuronal damage and worse cognitive dysfunction after global cerebral ischemia. Similar results were observed in intact male mice. RNA-seq analysis revealed alterations in pathways and genes associated with astrocyte activation, neuroinflammation, and oxidative stress in FBN-ARO-KO mice. The compromised astrocyte activation in FBN-ARO-KO mice was associated with robust downregulation of the astrocyte-derived neurotrophic factors, BDNF and IGF-1, as well as the astrocytic glutamate transporter, GLT-1. Νeuronal FGF2, which acts in a paracrine manner to suppress astrocyte activation, was increased in FBN-ARO-KO neurons. Interestingly, blocking FGF2 signaling by central injection of FGFR3-neutralizing antibody was able to reverse the diminishment in neuroprotective astrocyte reactivity, and attenuate neuronal damage in FBN-ARO-KO mice. Moreover, in vivo E2 replacement suppressed FGF2 signaling and rescued the compromised reactive astrogliosis and cognitive deficits. Collectively, our data provide novel genetic evidence for a beneficial role of neuron-derived E2 in astrocyte activation, neuroprotection, and cognitive preservation following ischemic injury to the brain.SIGNIFICANCE STATEMENT Following cerebral ischemia, astrocytes become highly reactive and can exert neuroprotection through the release of neurotrophic factors and clearance of neurotoxic glutamate. The current study advances our understanding of this process by demonstrating that neuron-derived 17ß-estradiol (E2) is neuroprotective and critical for induction of reactive astrocytes and their ability to produce astrocyte-derived neurotrophic factors, BDNF and IGF-1, and the glutamate transporter, GLT-1 after ischemic brain damage. These beneficial effects of neuron-derived E2 appear to be due, at least in part, to suppression of neuronal FGF2 signaling, which is a known suppressor of astrocyte activation. These findings suggest that neuron-derived E2 is neuroprotective after ischemic brain injury via a mechanism that involves suppression of neuronal FGF2 signaling, thereby facilitating astrocyte activation.


Assuntos
Astrócitos/metabolismo , Isquemia Encefálica/metabolismo , Estrogênios/metabolismo , Gliose/metabolismo , Neurônios/metabolismo , Comunicação Parácrina , Animais , Aromatase/genética , Aromatase/metabolismo , Isquemia Encefálica/patologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Transportador 2 de Aminoácido Excitatório/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Camundongos , Estresse Oxidativo
13.
J Neurosci ; 40(39): 7421-7435, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32847968

RESUMO

GABAergic interneurons represent a heterogenous group of cell types in neocortex that can be clustered based on developmental origin, morphology, physiology, and connectivity. Two abundant populations of cortical GABAergic interneurons include the low-threshold, somatostatin (SST)-expressing cells and the fast-spiking, parvalbumin (PV)-expressing cells. While SST+ and PV+ interneurons are both early born and migrate into the developing neocortex at similar times, SST+ cells are incorporated into functional circuits prior to PV+ cells. During this early period of neural development, SST+ cells play critical roles in the assembly and maturation of other cortical circuits; however, the mechanisms underlying this process remain poorly understood. Here, using both sexes of conditional mutant mice, we discovered that SST+ interneuron-derived Collagen XIX, a synaptogenic extracellular matrix protein, is required for the formation of GABAergic, perisomatic synapses by PV+ cells. These results, therefore, identify a paracrine mechanism by which early-born SST+ cells orchestrate inhibitory circuit formation in the developing neocortex.SIGNIFICANCE STATEMENT Inhibitory interneurons in the cerebral cortex represent a heterogenous group of cells that generate the inhibitory neurotransmitter GABA. One such interneuron type is the low-threshold, somatostatin (SST)-expressing cell, which is one of the first types of interneurons to migrate into the cerebral cortex and become incorporated into functional circuits. In addition, to contributing important roles in controlling the flow of information in the adult cerebral cortex, SST+ cells play important roles in the development of other neural circuits in the developing brain. Here, we identified an extracellular matrix protein that is released by these early-born SST+ neurons to orchestrate inhibitory circuit formation in the developing cerebral cortex.


Assuntos
Interneurônios/metabolismo , Neurogênese , Comunicação Parácrina , Somatostatina/metabolismo , Sinapses/metabolismo , Animais , Matriz Extracelular/metabolismo , Feminino , Neurônios GABAérgicos/citologia , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/fisiologia , Potenciais Pós-Sinápticos Inibidores , Interneurônios/citologia , Interneurônios/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Neocórtex/metabolismo , Neocórtex/fisiologia , Somatostatina/genética , Sinapses/fisiologia
14.
Arterioscler Thromb Vasc Biol ; 40(11): 2632-2648, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32814441

RESUMO

OBJECTIVE: We sought to identify and investigate the functional role of the major endothelial cell (EC)-derived factors that control pericyte recruitment to EC tubes and pericyte-induced tube maturation during capillary network formation. Approach and Results: We identify PDGF (platelet-derived growth factor)-BB, PDGF-DD, ET (endothelin)-1, TGF (transforming growth factor)-ß, and HB-EGF (heparin-binding epidermal growth factor), as the key individual and combined regulators of pericyte assembly around EC tubes. Using novel pericyte only assays, we demonstrate that PDGF-BB, PDGF-DD, and ET-1 are the primary direct drivers of pericyte invasion. Their addition to pericytes induces invasion as if ECs were present. In contrast, TGF-ß and HB-EGF have minimal ability to directly stimulate pericyte invasion. In contrast, TGF-ß1 can act as an upstream pericyte primer to stimulate invasion in response to PDGFs and ET-1. HB-EGF stimulates pericyte proliferation along with PDGFs and ET-1. Using EC-pericyte cocultures, individual, or combined blockade of these EC-derived factors, or their pericyte receptors, using neutralizing antibodies or chemical inhibitors, respectively, interferes with pericyte recruitment and proliferation. As individual factors, PDGF-BB and ET-1 have the strongest impact on these events. However, when the blocking reagents are combined to interfere with each of the above factors or their receptors, more dramatic and profound blockade of pericyte recruitment, proliferation, and pericyte-induced basement membrane deposition occurs. Under these conditions, ECs form tubes that become much wider and less elongated as if pericytes were absent. CONCLUSIONS: Overall, these new studies define and characterize a functional role for key EC-derived factors controlling pericyte recruitment, proliferation, and pericyte-induced basement membrane deposition during capillary network assembly.


Assuntos
Proteínas Angiogênicas/metabolismo , Encéfalo/irrigação sanguínea , Capilares/metabolismo , Movimento Celular , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica , Comunicação Parácrina , Pericitos/metabolismo , Proteínas Angiogênicas/farmacologia , Becaplermina/metabolismo , Capilares/citologia , Capilares/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Endotelina-1/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Linfocinas/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Pericitos/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
15.
Microvasc Res ; 132: 104056, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32795468

RESUMO

The dermal mesenchymal stem cells (DMSCs) from psoriasis display higher expression level of epidermal growth factor-like repeats and discoidin I-like domains 3 (EDIL3), while EDIL3 can bind integrins, including αvß3 and αvß5, to regulate angiogenesis. To assess the role of EDIL3 derived from DMSCs of psoriasis (P-DMSCs) in angiogenesis, in vitro, EDIL3 of DMSCs from psoriasis was silenced by interfering EDIL3. Then the efficacy of silencing EDIL3 was tested by fluorescent flag, qRT-PCR and western blotting. And, in vitro, the relationship of EDIL3 in DMSCs with the angiogenesis of HUVECs were investigated through co-culture system. In vivo, EDIL3 recombinant protein was injected into IMQ cream-induced psoriasis-like skin lesions of mouse and EDIL3-associated tube formation were determined using Image J software. Our results showed the capacity of the adhesion, migration and tube formation of HUVECs in all psoriatic DMSCs groups were significantly higher compared with the control and si-EDIL3 groups (all P<0.05) in vitro. Moreover, under stimulated by EDIL3 recombinant protein, EDIL3-associated tube formation was dramatically elevated in vivo (P<0.01). In this study, EDIL3 could promote the adhesion, migration and tube formation of ECs and participant in the angiogenesis pathogenesis of psoriasis through affecting biological function on ECs both in vitro and in vivo. The results suggest a potential role of the critical pro-angiogenic factor EDIL3 in psoriasis therapy.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Neovascularização Patológica , Psoríase/metabolismo , Pele/irrigação sanguínea , Animais , Proteínas de Ligação ao Cálcio/genética , Estudos de Casos e Controles , Adesão Celular , Moléculas de Adesão Celular/genética , Movimento Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Células-Tronco Mesenquimais/patologia , Camundongos Endogâmicos BALB C , Comunicação Parácrina , Psoríase/patologia , Transdução de Sinais
16.
Circ Arrhythm Electrophysiol ; 13(10): e008740, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32755466

RESUMO

BACKGROUND: The mesenchymal stem cell (MSC), known to remodel in disease and have an extensive secretome, has recently been isolated from the human heart. However, the effects of normal and diseased cardiac MSCs on myocyte electrophysiology remain unclear. We hypothesize that in disease the inflammatory secretome of cardiac human MSCs (hMSCs) remodels and can regulate arrhythmia substrates. METHODS: hMSCs were isolated from patients with or without heart failure from tissue attached to extracted device leads and from samples taken from explanted/donor hearts. Failing hMSCs or nonfailing hMSCs were cocultured with normal human cardiac myocytes derived from induced pluripotent stem cells. Using fluorescent indicators, action potential duration, Ca2+ alternans, and spontaneous calcium release (SCR) incidence were determined. RESULTS: Failing and nonfailing hMSCs from both sources exhibited similar trilineage differentiation potential and cell surface marker expression as bone marrow hMSCs. Compared with nonfailing hMSCs, failing hMSCs prolonged action potential duration by 24% (P<0.001, n=15), increased Ca2+ alternans by 300% (P<0.001, n=18), and promoted spontaneous calcium release activity (n=14, P<0.013) in human cardiac myocytes derived from induced pluripotent stem cells. Failing hMSCs exhibited increased secretion of inflammatory cytokines IL (interleukin)-1ß (98%, P<0.0001) and IL-6 (460%, P<0.02) compared with nonfailing hMSCs. IL-1ß or IL-6 in the absence of hMSCs prolonged action potential duration but only IL-6 increased Ca2+ alternans and promoted spontaneous calcium release activity in human cardiac myocytes derived from induced pluripotent stem cells, replicating the effects of failing hMSCs. In contrast, nonfailing hMSCs prevented Ca2+ alternans in human cardiac myocytes derived from induced pluripotent stem cells during oxidative stress. Finally, nonfailing hMSCs exhibited >25× higher secretion of IGF (insulin-like growth factor)-1 compared with failing hMSCs. Importantly, IGF-1 supplementation or anti-IL-6 treatment rescued the arrhythmia substrates induced by failing hMSCs. CONCLUSIONS: We identified device leads as a novel source of cardiac hMSCs. Our findings show that cardiac hMSCs can regulate arrhythmia substrates by remodeling their secretome in disease. Importantly, therapy inhibiting (anti-IL-6) or mimicking (IGF-1) the cardiac hMSC secretome can rescue arrhythmia substrates.


Assuntos
Potenciais de Ação , Arritmias Cardíacas/metabolismo , Sinalização do Cálcio , Insuficiência Cardíaca/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mediadores da Inflamação/metabolismo , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/metabolismo , Comunicação Parácrina , Adulto , Idoso , Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Estudos de Casos e Controles , Linhagem da Célula , Células Cultivadas , Técnicas de Cocultura , Feminino , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Cinética , Masculino , Células-Tronco Mesenquimais/patologia , Pessoa de Meia-Idade , Miócitos Cardíacos/patologia , Fenótipo
17.
Nat Commun ; 11(1): 3955, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769998

RESUMO

Cellular therapy to treat heart failure is an ongoing focus of intense research, but progress toward structural and functional recovery remains modest. Engineered augmentation of established cellular effectors overcomes impediments to enhance reparative activity. Such 'next generation' implementation includes delivery of combinatorial cell populations exerting synergistic effects. Concurrent isolation and expansion of three distinct cardiac-derived interstitial cell types from human heart tissue, previously reported by our group, prompted design of a 3D structure that maximizes cellular interaction, allows for defined cell ratios, controls size, enables injectability, and minimizes cell loss. Herein, mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs) and c-Kit+ cardiac interstitial cells (cCICs) when cultured together spontaneously form scaffold-free 3D microenvironments termed CardioClusters. scRNA-Seq profiling reveals CardioCluster expression of stem cell-relevant factors, adhesion/extracellular-matrix molecules, and cytokines, while maintaining a more native transcriptome similar to endogenous cardiac cells. CardioCluster intramyocardial delivery improves cell retention and capillary density with preservation of cardiomyocyte size and long-term cardiac function in a murine infarction model followed 20 weeks. CardioCluster utilization in this preclinical setting establish fundamental insights, laying the framework for optimization in cell-based therapeutics intended to mitigate cardiomyopathic damage.


Assuntos
Microambiente Celular , Miocárdio/patologia , Cicatrização , Animais , Animais Recém-Nascidos , Capilares/patologia , Agregação Celular , Morte Celular , Linhagem da Célula , Tamanho Celular , Citoproteção , Células Progenitoras Endoteliais/citologia , Feminino , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Humanos , Processamento de Imagem Assistida por Computador , Recém-Nascido , Células-Tronco Mesenquimais/citologia , Camundongos Endogâmicos NOD , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/citologia , Estresse Oxidativo , Comunicação Parácrina , Ratos Sprague-Dawley , Transcrição Genética
18.
Am J Physiol Renal Physiol ; 319(4): F664-F673, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32715764

RESUMO

Tubular changes contribute to the development of renal pathologies in diabetic kidney disease (DKD), including interstitial fibrosis. It is unclear how tubular cells relay signals to interstitial fibroblasts. Recently, exosomes have been recognized as crucial mediators of intercellular communication. We hypothesized that exosomes secreted from tubular cells may stimulate fibroblasts for interstitial fibrosis in DKD. In this study, we isolated and purified exosomes from the renal cortex of DKD mice and high glucose-treated mouse proximal tubular cells. Compared with nondiabetic mice, exosome secretion in kidney tissues decreased in DKD mice. Likewise, high glucose incubation reduced exosome secretion in mouse kidney proximal tubular BUMPT cells. To study the effect of tubular cell exosomes on fibroblasts, exosomes from BUMPT cells were added to renal fibroblast NRK-49F cell cultures. Notably, exosomes from high glucose conditioned BUMPT cells induced higher proliferation, significant morphological change, and substantial production of fibronectin, α-smooth muscle actin, and collagen type Ι in NRK-49F fibroblasts. Proteomics analysis was further performed to profile the proteins within tubular cell exosomes. Interestingly, 22 proteins were found to be differentially expressed between tubular exosomes derived from high glucose conditioned cells and those from normal glucose conditioned cells. Cytoscape analysis suggested the existence of two protein-protein interaction networks in these exosomal differentially expressed proteins. While one of the protein-protein interaction networks comprised enolase 1 (Eno1), heat shock protein family A member 8 (Hspa8), thioredoxin 1 (Txn1), peptidylprolyl isomerase A (Ppia), phosphoglycerate kinase 1 (Pgk1), DNA topoisomerase II-ß (Top2b), and ß-actin (Actb), the other had the family proteins of human leucocyte antigen F (Ywhag), a component of the ND10 nuclear body (Ywhae), interferon regulatory factor-8 (Ywhaq), and human leucocyte antigen A (Ywhaz). Gene expression analysis via Nephroseq showed a correlation of Eno1 expression with DKD clinical manifestation. In conclusion, DKD is associated with a decrease in exosome secretion in renal tubular cells. Exosomes from high glucose conditioned tubular cells may regulate the proliferation and activation of fibroblasts, contributing to the paracrine signaling mechanism responsible for the pathological onset of renal interstitial fibrosis in DKD.


Assuntos
Proliferação de Células , Nefropatias Diabéticas/metabolismo , Exossomos/metabolismo , Fibroblastos/metabolismo , Túbulos Renais Proximais/metabolismo , Comunicação Parácrina , Animais , Linhagem Celular , Técnicas de Cocultura , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Progressão da Doença , Exossomos/genética , Exossomos/patologia , Fibroblastos/patologia , Fibrose , Túbulos Renais Proximais/patologia , Masculino , Camundongos Endogâmicos C57BL , Fosfopiruvato Hidratase/metabolismo , Mapas de Interação de Proteínas , Via Secretória , Transdução de Sinais
19.
Sci Rep ; 10(1): 12350, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32704142

RESUMO

The nature of the early post-natal immune response in rodents appears to influence cardiac regeneration even though the underlying molecules remain poorly understood. Consistent with this idea, we show now significant changes in the expression of immune and cell movement gene pathways in heart samples from 1- and 7-day-old rats with ventricle resection. We then tested whether conditioned media from adult M2 anti-inflammatory macrophages target neonatal cardiac cells to a pro-regenerative like phenotype compared to the M1 pro-inflammatory macrophages. We found that M2 compared to M1 macrophage-conditioned media upregulates neonatal cardiomyocyte proliferation, suppresses myofibroblast-induced differentiation and stimulates endothelial cell tube formation. Using a cytokine array, we selected four candidate cytokine molecules uniquely expressed in M2 macrophage-conditioned media and showed that two of them (IL-4 and IL-6) induce endothelial cell proliferation whilst IL-4 promotes proliferation in neonatal cardiomyocytes and prevents myofibroblast-induced collagen type I secretion. Altogether, we provided evidence that adult M2 macrophage-conditioned media displays a paracrine beneficial pro-regenerative response in target cardiac cells and that IL-4 and IL-6 recapitulate, at least in part, these pleiotropic effects. Further characterization of macrophage immune phenotypes and their secreted molecules may give rise to novel therapeutic approaches for post-natal cardiac repair.


Assuntos
Células Endoteliais/metabolismo , Interleucina-4/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Miocárdio/metabolismo , Comunicação Parácrina , Animais , Animais Recém-Nascidos , Meios de Cultivo Condicionados , Células Endoteliais/citologia , Macrófagos/citologia , Miocárdio/citologia , Miócitos Cardíacos , Ratos
20.
Nat Commun ; 11(1): 3377, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32632100

RESUMO

The mammary gland is a highly vascularized tissue capable of expansion and regression during development and disease. To enable mechanistic insight into the coordinated morphogenic crosstalk between the epithelium and vasculature, we introduce a 3D microfluidic platform that juxtaposes a human mammary duct in proximity to a perfused endothelial vessel. Both compartments recapitulate stable architectural features of native tissue and the ability to undergo distinct forms of branching morphogenesis. Modeling HER2/ERBB2 amplification or activating PIK3CA(H1047R) mutation each produces ductal changes observed in invasive progression, yet with striking morphogenic and behavioral differences. Interestingly, PI3KαH1047R ducts also elicit increased permeability and structural disorganization of the endothelium, and we identify the distinct secretion of IL-6 as the paracrine cause of PI3KαH1047R-associated vascular dysfunction. These results demonstrate the functionality of a model system that facilitates the dissection of 3D morphogenic behaviors and bidirectional signaling between mammary epithelium and endothelium during homeostasis and pathogenesis.


Assuntos
Glândulas Mamárias Humanas/metabolismo , Morfogênese/genética , Mutação , Comunicação Parácrina/genética , Biomimética/métodos , Linhagem Celular , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Endotélio Vascular/crescimento & desenvolvimento , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Glândulas Mamárias Humanas/irrigação sanguínea , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Fenótipo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo
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