Assuntos
5'-Nucleotidase , Mãos , Valor Preditivo dos Testes , Calcificação Vascular , Humanos , Calcificação Vascular/diagnóstico por imagem , 5'-Nucleotidase/deficiência , 5'-Nucleotidase/genética , Mãos/irrigação sanguínea , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/metabolismo , Angiografia por Tomografia Computadorizada , Masculino , Perna (Membro)/irrigação sanguínea , FemininoRESUMO
Regulatory T (Treg) cells are one of the major immunosuppressive cell types in cancer and a potential target for immunotherapy, but targeting tumor-infiltrating (TI) Treg cells has been challenging. Here, using single-cell RNA sequencing of immune cells from renal clear cell carcinoma (ccRCC) patients, we identify two distinct transcriptional fates for TI Treg cells, Fate-1 and Fate-2. The Fate-1 signature is associated with a poorer prognosis in ccRCC and several other solid cancers. CD177, a cell surface protein normally expressed on neutrophil, is specifically expressed on Fate-1 TI Treg cells in several solid cancer types, but not on other TI or peripheral Treg cells. Mechanistically, blocking CD177 reduces the suppressive activity of Treg cells in vitro, while Treg-specific deletion of Cd177 leads to decreased tumor growth and reduced TI Treg frequency in mice. Our results thus uncover a functional CD177+ TI Treg population that may serve as a target for TI Treg-specific immunotherapy.
Assuntos
Proteínas Ligadas por GPI/metabolismo , Homeostase , Isoantígenos/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Receptores de Superfície Celular/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Sequência de Bases , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Proteínas Ligadas por GPI/deficiência , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Camundongos Knockout , Prognóstico , Receptores de Superfície Celular/deficiência , Análise de Célula Única , Transcrição GênicaRESUMO
Growth arrest-specific 1 (GAS1) acts as a co-receptor to patched 1, promoting sonic hedgehog (SHH) signaling in the developing nervous system. GAS1 mutations in humans and animal models result in forebrain and craniofacial malformations, defects ascribed to a function for GAS1 in SHH signaling during early neurulation. Here, we confirm loss of SHH activity in the forebrain neuroepithelium in GAS1-deficient mice and in induced pluripotent stem cell-derived cell models of human neuroepithelial differentiation. However, our studies document that this defect can be attributed, at least in part, to a novel role for GAS1 in facilitating NOTCH signaling, which is essential to sustain a persistent SHH activity domain in the forebrain neuroepithelium. GAS1 directly binds NOTCH1, enhancing ligand-induced processing of the NOTCH1 intracellular domain, which drives NOTCH pathway activity in the developing forebrain. Our findings identify a unique role for GAS1 in integrating NOTCH and SHH signal reception in neuroepithelial cells, and they suggest that loss of GAS1-dependent NOTCH1 activation contributes to forebrain malformations in individuals carrying GAS1 mutations.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Hedgehog/metabolismo , Prosencéfalo/metabolismo , Receptor Notch1/metabolismo , Animais , Proteínas de Ciclo Celular/deficiência , Diferenciação Celular , Embrião de Mamíferos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/metabolismo , Humanos , Camundongos , Mutação , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Receptor Patched-1/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Prosencéfalo/citologia , Prosencéfalo/embriologia , Transdução de SinaisRESUMO
Matriptase-2, a serine protease expressed in hepatocytes, is a negative regulator of hepcidin expression. The purpose of the study was to investigate the interaction of matriptase-2 with hemojuvelin protein in vivo. Mice lacking the matriptase-2 proteolytic activity (mask mice) display decreased content of hemojuvelin protein. Vice versa, the absence of hemojuvelin results in decreased liver content of matriptase-2, indicating that the two proteins interact. To further characterize the role of matriptase-2, we investigated iron metabolism in mask mice fed experimental diets. Administration of iron-enriched diet increased liver iron stores as well as hepcidin expression. Treatment of iron-overloaded mask mice with erythropoietin increased hemoglobin and hematocrit, indicating that the response to erythropoietin is intact in mask mice. Feeding of an iron-deficient diet to mask mice significantly increased spleen weight as well as the splenic content of erythroferrone and transferrin receptor proteins, indicating stress erythropoiesis. Liver hepcidin expression was decreased; expression of Id1 was not changed. Overall, the results suggest a complex interaction between matriptase-2 and hemojuvelin, and demonstrate that hepcidin can to some extent be regulated even in the absence of matriptase-2 proteolytic activity.
Assuntos
Proteínas Ligadas por GPI/fisiologia , Proteína da Hemocromatose/fisiologia , Sobrecarga de Ferro/metabolismo , Proteínas de Membrana/fisiologia , Serina Endopeptidases/fisiologia , Animais , Proteína Morfogenética Óssea 6/biossíntese , Proteína Morfogenética Óssea 6/genética , Eritropoetina/farmacologia , Feminino , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína da Hemocromatose/biossíntese , Proteína da Hemocromatose/deficiência , Proteína da Hemocromatose/genética , Hepcidinas/biossíntese , Hepcidinas/genética , Proteína 1 Inibidora de Diferenciação/biossíntese , Proteína 1 Inibidora de Diferenciação/genética , Deficiências de Ferro , Ferro da Dieta/farmacologia , Fígado/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Baço/metabolismoAssuntos
Antígenos de Superfície/genética , Comunicação Celular/genética , Tubas Uterinas/metabolismo , Fertilização/genética , Reprodução/genética , Espermatozoides/metabolismo , Animais , Copulação/fisiologia , Tubas Uterinas/anatomia & histologia , Feminino , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Tamanho da Ninhada de Vivíparos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Transdução de Sinais , Contagem de Espermatozoides , Motilidade dos Espermatozoides/genética , Útero/anatomia & histologia , Útero/metabolismoRESUMO
RATIONALE: Hypertension in obesity has become a major threat for public health. Omentin-1, a novel adipokine, is down-regulated in obesity. Tetrahydroxystilbene glycoside (TSG) is the main ingredient extracted from Polygonum multiflorum Thunb (PMT), a traditional Chinese medicinal herb safely used for protecting cardiovascular systems over bimillennium. This study aims to examine (i) the impact of omentin-1 downregulation on obesity-related hypertension in murine models and the underlying mechanisms; (ii) whether tetrahydroxystilbene glycoside (TSG) improved endothelial dysfunction and obesity-associated hypertension via the increase of omentin-1. METHODS: (TSG-treated) male Zucker diabetic fatty (ZDF) rats and omentin-1 knockout (OMT-/-) mice were used. In vitro, human umbilical vein endothelial cells (HUVECs) and mature adipocytes differentiated from human visceral preadipocyte (HPA-v) were maintained in a co-culture system. RESULTS: TSG was the main active component of PMT reducing systolic blood pressure and improving endothelial vasodilation. Fortnight-TSG treatment (100 mg/kg/day) increased serum omentin-1 level, also activated Akt/eNOS signaling and enhanced NO bioactivity; decreased expression of NOX2 and p22phox, suppressed production of superoxide and peroxynitrite anion. OMT-/- mice showed elevated blood pressure and impaired endothelial vasorelaxation, whereas hypotensive effect of TSG was blunted. In co-culture system, TSG incubation promoted binding of peroxisome proliferator-activated receptor-γ (PPAR-γ) and Itln-1 promoter in adipocytes, activated Akt/eNOS/NO signaling and attenuated oxidative/nitrative stress in HUVECs. Suppression of Itln-1 with siRNA significantly blocked the protective effect of TSG in vitro. CONCLUSIONS: Down-regulation of omentin-1 induces endothelial dysfunction and hypertension in obesity. TSG treatment (at least partially) increases omentin-1 via promoting binding of PPAR-γ and Itln-1 promoter in adipose tissues, subsequently exerts protective effects on endothelial function via activating Akt/eNOS/NO signaling and attenuating oxidative/nitrative stress. These results suggest that TSG could be developed as a promising anti-hypertension agent that protects against endothelial dysfunction and obesity-associated cardiovascular diseases.
Assuntos
Citocinas/biossíntese , Citocinas/deficiência , Endotélio Vascular/efeitos dos fármacos , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/deficiência , Glucosídeos/uso terapêutico , Hipertensão/tratamento farmacológico , Lectinas/biossíntese , Lectinas/deficiência , Estilbenos/uso terapêutico , Animais , Citocinas/genética , Endotélio Vascular/metabolismo , Proteínas Ligadas por GPI/genética , Glucosídeos/metabolismo , Glucosídeos/farmacologia , Humanos , Hipertensão/genética , Hipertensão/metabolismo , Lectinas/genética , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Zucker , Estilbenos/metabolismo , Estilbenos/farmacologiaRESUMO
The phosphatidylinositol glycan anchor biosynthesis class S protein (PIGS) gene has recently been implicated in a novel congenital disorder of glycosylation resulting in autosomal recessive inherited glycosylphosphatidylinositol-anchored protein (GPI-AP) deficiency. Previous studies described seven patients with biallelic variants in the PIGS gene, of whom two presented with fetal akinesia and five with global developmental delay and epileptic developmental encephalopathy. We present the molecular and clinical characteristics of six additional individuals from five families with unreported variants in PIGS. All individuals presented with hypotonia, severe global developmental delay, microcephaly, intractable early infantile epilepsy, and structural brain abnormalities. Additional findings include vision impairment, hearing loss, renal malformation, and hypotonic facial appearances with minor dysmorphic features but without a distinctive facial gestalt. Four individuals died due to neurologic complications. GPI anchoring studies performed on one individual revealed a significant decrease in GPI-APs. We confirm that biallelic variants in PIGS cause vitamin pyridoxine-responsive epilepsy due to inherited GPI deficiency and expand the genotype and phenotype of PIGS-related disorder. Further delineation of the molecular spectrum of PIGS-related disorders would improve management, help develop treatments, and encourage the expansion of diagnostic genetic testing to include this gene as a potential cause of neurodevelopmental disorders and epilepsy.
Assuntos
Aciltransferases/genética , Deficiências do Desenvolvimento/genética , Proteínas Ligadas por GPI/deficiência , Malformações do Sistema Nervoso/genética , Espasmos Infantis/genética , Encéfalo/anormalidades , Encéfalo/diagnóstico por imagem , Pré-Escolar , Deficiências do Desenvolvimento/fisiopatologia , Fácies , Feminino , Perda Auditiva/genética , Perda Auditiva/fisiopatologia , Humanos , Lactente , Rim/anormalidades , Masculino , Microcefalia/genética , Microcefalia/fisiopatologia , Hipotonia Muscular/genética , Hipotonia Muscular/fisiopatologia , Malformações do Sistema Nervoso/diagnóstico por imagem , Malformações do Sistema Nervoso/fisiopatologia , Fenótipo , Espasmos Infantis/fisiopatologia , Transtornos da Visão/genética , Transtornos da Visão/fisiopatologiaRESUMO
Fibrillins (FBNs) form mesh-like structures of microfibrils in various elastic tissues. RECK and FBN1 are co-expressed in many human tissues, suggesting a functional relationship. We found that dermal FBN1 fibers show atypical morphology in mice with reduced RECK expression (RECK-Hypo mice). Dermal FBN1 fibers in mice-lacking membrane-type 1-matrix metalloproteinase (MT1-MMP) show a similar atypical morphology, despite the current notion that MT1-MMP (a membrane-bound protease) and RECK (a membrane-bound protease inhibitor) have opposing functions. Our experiments using dermal fibroblasts indicated that RECK promotes pro-MT1-MMP activation, increases cell-associated gelatinase/collagenase activity, and decreases diffusible gelatinase/collagenase activity, while MT1-MMP stabilizes RECK in these cells. Experiments using purified proteins indicate that RECK and its binding partner ADAMTS10 keep the proteolytic activity of MT1-MMP within a certain range. These findings suggest that RECK, ADAMTS10, and MT1-MMP cooperate to support the formation of robust FBN1 fibers.
Assuntos
Fibrilinas/metabolismo , Proteínas Ligadas por GPI/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Proteínas ADAMTS/metabolismo , Animais , Linhagem Celular Tumoral , Colágeno/metabolismo , Elastina/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Proteínas Ligadas por GPI/deficiência , Gelatina/metabolismo , Células HEK293 , Humanos , Integrinas/metabolismo , Metaloproteinase 14 da Matriz/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Proteólise , Pele/metabolismoRESUMO
CD73 is an important ectoenzyme responsible for the production of extracellular adenosine. It is involved in regulating inflammatory responses and cell migration and is overexpressed in various cancers. The functions of CD73 in blood endothelial cells are understood in detail, but its role on afferent lymphatics remains unknown. Moreover, anti-CD73 antibodies are now used in multiple clinical cancer trials, but their effects on different endothelial cell types have not been studied. This study reveals that a previously unknown role of CD73 on afferent lymphatics is to dampen immune responses. Knocking it out or suppressing it by siRNA leads to the upregulation of inflammation-associated genes on lymphatic endothelial cells and a more pro-inflammatory phenotype of interacting dendritic cells in vitro and in vivo. In striking contrast, anti-CD73 antibodies had only negligible effects on the gene expression of lymphatic- and blood-endothelial cells. Our data thus reveal new functions of lymphatic CD73 and indicate a low likelihood of endothelial cell-related adverse effects by CD73 targeting therapeutic antibodies.
Assuntos
5'-Nucleotidase/imunologia , Células Endoteliais/imunologia , Inflamação/prevenção & controle , 5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/deficiência , 5'-Nucleotidase/genética , Animais , Anticorpos Bloqueadores/administração & dosagem , Diferenciação Celular/imunologia , Células Cultivadas , Criança , Pré-Escolar , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Técnicas de Inativação de Genes , Inativação Gênica , Humanos , Inflamação/imunologia , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regulação para CimaRESUMO
OBJECTIVE: Evidences accumulated within the past decades identified hedgehog signaling as a new regulator of endothelium integrity. More specifically, we recently identified Dhh (desert hedgehog) as a downstream effector of Klf2 (Kruppel-like factor 2) in endothelial cells (ECs). The purpose of this study is to investigate whether hedgehog coreceptors Gas1 (growth arrest-specific 1) and Cdon (cell adhesion molecule-related/downregulated by oncogenes) may be used as therapeutic targets to modulate Dhh signaling in ECs. Approach and Results: We demonstrated that both Gas1 and Cdon are expressed in adult ECs and relied on either siRNAs- or EC-specific conditional knockout mice to investigate their role. We found that Gas1 deficiency mainly phenocopies Dhh deficiency especially by inducing VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) overexpression while Cdon deficiency has opposite effects by promoting endothelial junction integrity. At a molecular level, Cdon prevents Dhh binding to Ptch1 (patched-1) and thus acts as a decoy receptor for Dhh, while Gas1 promotes Dhh binding to Smo (smoothened) and as a result potentiates Dhh effects. Since Cdon is upregulated in ECs treated by inflammatory cytokines, including TNF (tumor necrosis factor)-α and Il (interleukin)-1ß, we then tested whether Cdon inhibition would promote endothelium integrity in acute inflammatory conditions and found that both fibrinogen and IgG extravasation were decreased in association with an increased Cdh5 (cadherin-5) expression in the brain cortex of EC-specific Cdon knockout mice administered locally with Il-1ß. CONCLUSIONS: Altogether, these results demonstrate that Gas1 is a positive regulator of Dhh in ECs while Cdon is a negative regulator. Interestingly, Cdon blocking molecules may then be used to promote endothelium integrity, at least in inflammatory conditions.
Assuntos
Barreira Hematoencefálica/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neovascularização da Córnea/metabolismo , Células Endoteliais/metabolismo , Endotélio Corneano/metabolismo , Proteínas Hedgehog/metabolismo , Inflamação/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Barreira Hematoencefálica/patologia , Caderinas/genética , Caderinas/metabolismo , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/genética , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Células Cultivadas , Neovascularização da Córnea/genética , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Endotélio Corneano/patologia , Feminino , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Proteínas Hedgehog/genética , Humanos , Inflamação/genética , Inflamação/patologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Patched-1/metabolismo , Transdução de Sinais , Receptor Smoothened/metabolismoRESUMO
Stromal invasion is considered an important prognostic factor in patients with lung adenocarcinoma. The mechanisms underlying the formation of tumor stroma and stromal invasion have been studied in the lung; however, they are still unclear. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein highly expressed in several types of human malignant tumors including lung cancers. In this study, we investigated the in vivo functions of CD109 protein in malignant lung tumors. Initially, we identified an association between higher expression of CD109 protein in human lung adenocarcinoma and a significantly worse prognosis, according to immunohistochemical analysis. We also showed that CD109 deficiency significantly reduced the area of stromal invasive lesions in a genetically engineered CD109-deficient lung adenocarcinoma mouse model, which correlated with the results observed in human lung adenocarcinoma. Furthermore, we identified latent TGF-ß binding protein-1 (LTBP1) as a CD109-interacting protein using mass spectrometry and confirmed their interaction by co-immunoprecipitation. Importantly, increased CD109 expression enhanced stromal TGF-ß activation in the presence of LTBP1. Therefore, these data suggest the significance of the regulation of TGF-ß signaling through CD109 and LTBP1 interaction in tumor stroma and also reveal the importance of CD109 expression levels in promoting lung cancer cell proliferation, migration, and invasion, and thus predicting the outcome of patients suffering from lung adenocarcinoma. Therefore, CD109 protein could be a potential therapeutic target for this disease.
Assuntos
Adenocarcinoma/metabolismo , Antígenos CD/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adenocarcinoma/patologia , Idoso , Animais , Antígenos CD/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Modelos Animais de Doenças , Feminino , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Proteínas de Ligação a TGF-beta Latente/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Prognóstico , RNA Interferente Pequeno , TransfecçãoRESUMO
The understanding of protective immunity during HIV infection remains elusive. Here we showed that CD160 defines a polyfunctional and proliferative CD8+ T cell subset with a protective role during chronic HIV-1 infection. CD160+ CD8+ T cells derived from HIV+ patients correlated with slow progressions both in a cross-sectional study and in a 60-month longitudinal cohort, displaying enhanced cytotoxicity and proliferative capacity in response to HIV Gag stimulation; triggering CD160 promoted their functionalities through MEK-ERK and PI3K-AKT pathways. These observations were corroborated by studying chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. The genetic ablation of CD160 severely impaired LCMV-specific CD8+ T cell functionalities and thereby resulted in loss of virus control. Interestingly, transcriptional profiling showed multiple costimulatory and survival pathways likely to be involved in CD160+ T cell development. Our data demonstrated that CD160 acts as a costimulatory molecule positively regulating CD8+ T cells during chronic viral infections, thus representing a potential target for immune intervention.
Assuntos
Antígenos CD/imunologia , Linfócitos T CD8-Positivos/imunologia , Receptores Coestimuladores e Inibidores de Linfócitos T/imunologia , Infecções por HIV/imunologia , Coriomeningite Linfocítica/imunologia , Receptores Imunológicos/imunologia , Subpopulações de Linfócitos T/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/transplante , Doença Crônica , Progressão da Doença , Feminino , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/imunologia , Produtos do Gene gag/fisiologia , HIV-1 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores Imunológicos/deficiência , Subpopulações de Linfócitos T/transplante , TranscriptomaRESUMO
Testisin (encoded by PRSS21) is a membrane anchored serine protease, which is tethered to the cell surface via a glycosylphosphatidylinositol (GPI)-anchor. While testisin is found in abundance in spermatozoa, it is also expressed in microvascular endothelial cells where its function is unknown. Here we identify testisin as a novel regulator of physiological hormone-induced angiogenesis and microvascular endothelial permeability. Using a murine model of rapid physiological angiogenesis during corpus luteal development in the ovary, we found that mice genetically deficient in testisin (Prss21-/-) show a substantially increased incidence of hemorrhages which are significantly more severe than in littermate control Prss21+/+ mice. This phenotype was associated with increased vascular leakiness, demonstrated by a greater accumulation of extravasated Evans blue dye in Prss21-/- ovaries. Live cell imaging of in vitro cultured microvascular endothelial cells depleted of testisin by siRNA knockdown revealed that loss of testisin markedly impaired reorganization and tubule-like formation on Matrigel basement membranes. Moreover testisin siRNA knockdown increased the paracellular permeability to FITC-albumin across endothelial cell monolayers, which was associated with decreased expression of the adherens junction protein VE-cadherin and increased levels of phospho(Tyr658)-VE-cadherin, without affecting the levels of the tight junction proteins occludin and claudin-5, or ZO-1. Decreased expression of VE-cadherin in the neovasculature of Prss21-/- ovaries was also observed without marked differences in endothelial cell content, vascular claudin-5 expression or pericyte recruitment. Together, these data identify testisin as a novel regulator of VE-cadherin adhesions during angiogenesis and indicate a potential new target for regulating neovascular integrity and associated pathologies.
Assuntos
Permeabilidade Capilar/fisiologia , Corpo Lúteo/irrigação sanguínea , Neovascularização Fisiológica , Serina Endopeptidases/deficiência , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar/genética , Células Cultivadas , Corpo Lúteo/patologia , Corpo Lúteo/fisiopatologia , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/fisiologia , Técnicas de Silenciamento de Genes , Hemorragia/etiologia , Hemorragia/genética , Hemorragia/fisiopatologia , Humanos , Luteinização/genética , Luteinização/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Fisiológica/genética , Fenótipo , Serina Endopeptidases/genética , Serina Endopeptidases/fisiologiaRESUMO
OBJECTIVE: The recessive disease arterial calcification due to deficiency of CD73 (ACDC) presents with extensive nonatherosclerotic medial layer calcification in lower extremity arteries. Lack of CD73 induces a concomitant increase in TNAP (tissue nonspecific alkaline phosphatase; ALPL), a key enzyme in ectopic mineralization. Our aim was to investigate how loss of CD73 activity leads to increased ALPL expression and calcification in CD73-deficient patients and assess whether this mechanism may apply to peripheral artery disease calcification. Approach and Results: We previously developed a patient-specific disease model using ACDC primary dermal fibroblasts that recapitulates the calcification phenotype in vitro. We found that lack of CD73-mediated adenosine signaling reduced cAMP production and resulted in increased activation of AKT. The AKT/mTOR (mammalian target of rapamycin) axis blocks autophagy and inducing autophagy prevented calcification; however, we did not observe autophagy defects in ACDC cells. In silico analysis identified a putative FOXO1 (forkhead box O1 protein) binding site in the human ALPL promoter. Exogenous AMP induced FOXO1 nuclear localization in ACDC but not in control cells, and this was prevented with a cAMP analogue or activation of A2a/2b adenosine receptors. Inhibiting FOXO1 reduced ALPL expression and TNAP activity and prevented calcification. Mutating the FOXO1 binding site reduced ALPL promoter activation. Importantly, we provide evidence that non-ACDC calcified femoropopliteal arteries exhibit decreased CD73 and increased FOXO1 levels compared with control arteries. CONCLUSIONS: These data show that lack of CD73-mediated cAMP signaling promotes expression of the human ALPL gene via a FOXO1-dependent mechanism. Decreased CD73 and increased FOXO1 was also observed in more common peripheral artery disease calcification.
Assuntos
5'-Nucleotidase/deficiência , Fibroblastos/enzimologia , Proteína Forkhead Box O1/metabolismo , Doença Arterial Periférica/enzimologia , Artéria Poplítea/enzimologia , Calcificação Vascular/enzimologia , 5'-Nucleotidase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Autofagia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Fibroblastos/patologia , Proteína Forkhead Box O1/genética , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Humanos , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/genética , Doença Arterial Periférica/patologia , Artéria Poplítea/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/patologia , Adulto JovemRESUMO
Ecto-5'-nucleotidase [cluster of differentiation 73 (CD73)] is a ubiquitously expressed glycosylphosphatidylinositol-anchored glycoprotein that converts extracellular adenosine 5'-monophosphate to adenosine. Anti-CD73 inhibitory antibodies are currently undergoing clinical testing for cancer immunotherapy. However, many protective physiological functions of CD73 need to be taken into account for new targeted therapies. This review examines CD73 functions in multiple organ systems and cell types, with a particular focus on novel findings from the last 5 years. Missense loss-of-function mutations in the CD73-encoding gene NT5E cause the rare disease "arterial calcifications due to deficiency of CD73." Aside from direct human disease involvement, cellular and animal model studies have revealed key functions of CD73 in tissue homeostasis and pathology across multiple organ systems. In the context of the central nervous system, CD73 is antinociceptive and protects against inflammatory damage, while also contributing to age-dependent decline in cortical plasticity. CD73 preserves barrier function in multiple tissues, a role that is most evident in the respiratory system, where it inhibits endothelial permeability in an adenosine-dependent manner. CD73 has important cardioprotective functions during myocardial infarction and heart failure. Under ischemia-reperfusion injury conditions, rapid and sustained induction of CD73 confers protection in the liver and kidney. In some cases, the mechanism by which CD73 mediates tissue injury is less clear. For example, CD73 has a promoting role in liver fibrosis but is protective in lung fibrosis. Future studies that integrate CD73 regulation and function at the cellular level with physiological responses will improve its utility as a disease target.
Assuntos
5'-Nucleotidase/genética , Calcinose/genética , Insuficiência Cardíaca/genética , Infarto do Miocárdio/genética , Traumatismo por Reperfusão/genética , 5'-Nucleotidase/deficiência , Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Artérias/metabolismo , Artérias/patologia , Calcinose/metabolismo , Calcinose/patologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Regulação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Homeostase , Humanos , Mutação de Sentido Incorreto , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Especificidade de Órgãos , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Sistema Respiratório/metabolismo , Sistema Respiratório/patologiaRESUMO
Progressive tubulointerstitial fibrosis may occur after acute kidney injury due to persistent inflammation. Purinergic signaling by 5'-ectonucleotidase, CD73, an enzyme that converts AMP to adenosine on the extracellular surface, can suppress inflammation. The role of CD73 in progressive kidney fibrosis has not been elucidated. We evaluated the effect of deletion of CD73 from kidney perivascular cells (including pericytes and/or fibroblasts of the Foxd1+ lineage) on fibrosis. Perivascular cell expression of CD73 was necessary to suppress inflammation and prevent kidney fibrosis in Foxd1CreCD73fl/fl mice evaluated 14 days after unilateral ischemia-reperfusion injury or folic acid treatment (250 mg/kg). Kidneys of Foxd1CreCD73fl/fl mice had greater collagen deposition, expression of proinflammatory markers (including various macrophage markers), and platelet-derived growth factor recepetor-ß immunoreactivity than CD73fl/fl mice. Kidney dysfunction and fibrosis were rescued by administration of soluble CD73 or by macrophage deletion. Isolated CD73-/- kidney pericytes displayed an activated phenotype (increased proliferation and α-smooth muscle actin mRNA expression) compared with wild-type controls. In conclusion, CD73 in perivascular cells may act to suppress myofibroblast transformation and influence macrophages to promote a wound healing response. These results suggest that the purinergic signaling pathway in the kidney interstitial microenvironment orchestrates perivascular cells and macrophages to suppress inflammation and prevent progressive fibrosis.
Assuntos
5'-Nucleotidase/metabolismo , Microambiente Celular , Fibroblastos/metabolismo , Rim/metabolismo , Macrófagos/metabolismo , Nefrite Intersticial/metabolismo , Pericitos/metabolismo , Traumatismo por Reperfusão/metabolismo , 5'-Nucleotidase/deficiência , 5'-Nucleotidase/genética , Actinas/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , Modelos Animais de Doenças , Fibroblastos/patologia , Fibrose , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Mediadores da Inflamação/metabolismo , Rim/imunologia , Rim/patologia , Macrófagos/patologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nefrite Intersticial/genética , Nefrite Intersticial/imunologia , Nefrite Intersticial/patologia , Pericitos/patologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia , Transdução de Sinais , CicatrizaçãoRESUMO
Pseudoxanthoma elasticum (PXE) is a rare disorder characterized by skin, eye, and cardiovascular lesions due to ectopic mineralization and fragmentation of elastic fibers of connective tissues. We present an atypical case of PXE with diffuse vascular calcification and negligible skin and eye lesions. The patient was a 37-year-old man suffering from severe bilateral arterial calcifications in superficial femoral and posterior tibial arteries. Eye fundoscopy and skin examination were first considered normal. This phenotype suggested first the diagnosis of Arterial Calcification due to Deficiency of CD73 (ACDC) characterized by mutations in NT5E gene. However, we found two variants in ABCC6 gene, and no variant in NT5E. Skin reexamination revealed few lateral skin papules confined to the scalp. Phenotypic overlap was described in vascular calcification disorders, between GACI and PXE phenotypes, and we discuss here expansion of this overlap, including ACDC phenotype. Identification of these expanding and overlapping phenotypes was enabled by genetic screening of the corresponding genes, in a systematic approach. We propose to create a calcification next generation sequencing (NGS) panel with NT5E, GGCX, ENPP1, and ABCC6 genes to improve the molecular diagnosis of vascular calcification.
Assuntos
5'-Nucleotidase/genética , Variação Genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Doença Arterial Periférica/genética , Pseudoxantoma Elástico/genética , Calcificação Vascular/genética , 5'-Nucleotidase/deficiência , Adulto , Carbono-Carbono Ligases/genética , Diagnóstico Diferencial , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Doença Arterial Periférica/diagnóstico , Fenótipo , Diester Fosfórico Hidrolases/genética , Valor Preditivo dos Testes , Pseudoxantoma Elástico/diagnóstico , Pirofosfatases/genética , Calcificação Vascular/diagnósticoRESUMO
BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired pluripotent hematopoietic stem cell disorder associated with an increase in the number of glycosyl-phosphatidyl inositol (GPI)-deficient blood cells. We investigated PNH clonal proliferation in the three cell lineages-granulocytes, T lymphocytes, and red blood cells (RBCs)-by analyzing PIGA gene mutations and T-cell receptor (TCR) clonality. METHODS: Flow cytometry was used on peripheral blood samples from 24 PNH patients to measure the GPI-anchored protein (GPI-AP) deficient fraction in each blood cell lineage. PIGA gene mutations were analyzed in granulocytes and T lymphocytes by Sanger sequencing. A TCR clonality assay was performed in isolated GPI-AP deficient T lymphocytes. RESULTS: The GPI-AP deficient fraction among the three lineages was the highest in granulocytes, followed by RBCs and T lymphocytes. PIGA mutations were detected in both granulocytes and T lymphocytes of 19 patients (79.2%), with a higher mutation burden in granulocytes. The GPI-AP deficient fractions of granulocytes and T lymphocytes correlated moderately (rs=0.519, P=0.049) and strongly (rs=0.696, P=0.006) with PIGA mutation burden, respectively. PIGA mutations were more frequently observed in patients with clonal rearrangements in TCR genes (P=0.015). The PIGA mutation burden of T lymphocytes was higher in patients with clonal TCRB rearrangement. CONCLUSIONS: PIGA mutations were present in approximately 80% of PNH patients. PNH clone size varies according to blood cell lineage, and clonal cells may obtain proliferation potential or gain a survival advantage over normal cells.
Assuntos
Proliferação de Células , Granulócitos/citologia , Hemoglobinúria Paroxística/diagnóstico , Proteínas de Membrana/genética , Linfócitos T/citologia , Adolescente , Adulto , Idoso , Linhagem da Célula , Criança , Feminino , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Rearranjo Gênico , Granulócitos/metabolismo , Hemoglobinúria Paroxística/genética , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Adulto JovemRESUMO
Bronchopulmonary dysplasia (BPD) remains a devastating consequence of prematurity. Repeated inflammatory insults worsen lung injury, but there are no predictors for BPD-related respiratory outcomes or targeted therapies. We sought to understand inflammatory mechanisms in evolving BPD through molecular characterization of monocytes in tracheal aspirates from infants at risk for developing BPD. We performed flow cytometry targeting myeloid cell populations on prospectively collected tracheal aspirates from intubated patients born before 29 wk of gestation and <30 days old. We identified CD14+CD16+ (double-positive) and CD14+CD16- (single-positive) monocytes and characterized their gene expression profiles by RNA sequencing and quantitative PCR. We further analyzed differential gene expression between time points to evaluate changes in monocyte function over the first weeks of life. Expression of IL-1A, IL-1B, and IL-1 receptor antagonist mRNA was increased in monocytes collected at day of life (DOL) 7, DOL 14, and DOL 28 compared with those collected at DOL 3. This study suggests that early changes in monocyte-specific IL-1 cytokine pathways may be associated with evolving BPD.