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1.
Genes (Basel) ; 10(8)2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31412643

RESUMO

BACKGROUND: Pancreatic cancer is one of the malignant tumors that threaten human health. METHODS: The gene expression profiles of GSE15471, GSE19650, GSE32676 and GSE71989 were downloaded from the gene expression omnibus database including pancreatic cancer and normal samples. The differentially expressed genes between the two types of samples were identified with the Limma package using R language. The gene ontology functional and pathway enrichment analyses of differentially-expressed genes were performed by the DAVID software followed by the construction of a protein-protein interaction network. Hub gene identification was performed by the plug-in cytoHubba in cytoscape software, and the reliability and survival analysis of hub genes was carried out in The Cancer Genome Atlas gene expression data. RESULTS: The 138 differentially expressed genes were significantly enriched in biological processes including cell migration, cell adhesion and several pathways, mainly associated with extracellular matrix-receptor interaction and focal adhesion pathway in pancreatic cancer. The top hub genes, namely thrombospondin 1, DNA topoisomerase II alpha, syndecan 1, maternal embryonic leucine zipper kinase and proto-oncogene receptor tyrosine kinase Met were identified from the protein-protein interaction network. The expression levels of hub genes were consistent with data obtained in The Cancer Genome Atlas. DNA topoisomerase II alpha, syndecan 1, maternal embryonic leucine zipper kinase and proto-oncogene receptor tyrosine kinase Met were significantly linked with poor survival in pancreatic adenocarcinoma. CONCLUSIONS: These hub genes may be used as potential targets for pancreatic cancer diagnosis and treatment.


Assuntos
Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Neoplasias Pancreáticas/genética , Transcriptoma , Algoritmos , Biomarcadores Tumorais/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Sindecana-1/genética , Sindecana-1/metabolismo , Trombospondina 1/genética , Trombospondina 1/metabolismo
2.
Neuron ; 103(4): 642-657.e7, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31255486

RESUMO

Neuronal subtypes show diverse injury responses, but the molecular underpinnings remain elusive. Using transgenic mice that allow reliable visualization of axonal fate, we demonstrate that intrinsically photosensitive retinal ganglion cells (ipRGCs) are both resilient to cell death and highly regenerative. Using RNA sequencing (RNA-seq), we show genes that are differentially expressed in ipRGCs and that associate with their survival and axon regeneration. Strikingly, thrombospondin-1 (Thbs1) ranked as the most differentially expressed gene, along with the well-documented injury-response genes Atf3 and Jun. THBS1 knockdown in RGCs eliminated axon regeneration. Conversely, RGC overexpression of THBS1 enhanced regeneration in both ipRGCs and non-ipRGCs, an effect that was dependent on syndecan-1, a known THBS1-binding protein. All structural domains of the THBS1 were not equally effective; the trimerization and C-terminal domains promoted regeneration, while the THBS type-1 repeats were dispensable. Our results identify cell-type-specific induction of Thbs1 as a novel gene conferring high regenerative capacity.


Assuntos
Regeneração Nervosa/fisiologia , Células Ganglionares da Retina/fisiologia , Trombospondina 1/fisiologia , Animais , Apoptose , Axônios/metabolismo , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Genes Reporter , Fator de Crescimento Insulin-Like I/deficiência , Fator de Crescimento Insulin-Like I/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Compressão Nervosa , Traumatismos do Nervo Óptico/genética , Traumatismos do Nervo Óptico/fisiopatologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Opsinas de Bastonetes/deficiência , Opsinas de Bastonetes/fisiologia , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/fisiologia , Trombospondina 1/biossíntese , Trombospondina 1/genética , Transcrição Genética
3.
Int J Mol Sci ; 20(13)2019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31261874

RESUMO

Esophageal squamous cell carcinoma (ESCC) is the most common primary esophageal malignancy. Telmisartan, an angiotensin II type 1 (AT1) receptor blocker (ARB) and a widely used antihypertensive, has been shown to inhibit proliferation of various cancer types. This study evaluated the effects of telmisartan on human ESCC cell proliferation in vitro and in vivo and sought to identify the microRNAs (miRNAs) involved in these antitumor effects. We examined the effects of telmisartan on three human ESCC cell lines (KYSE150, KYSE180, and KYSE850). Telmisartan inhibited proliferation of these three cell lines by inducing S-phase arrest, which was accompanied by decreased expression of cyclin A2, cyclin-dependent kinase 2, and other cell cycle-related proteins. Additionally, telmisartan reduced levels of phosphorylated ErbB3 and thrombospondin-1 in KYSE180 cells. Furthermore, expression of miRNAs was remarkably altered by telmisartan in vitro. Telmisartan also inhibited tumor growth in vivo in a xenograft mouse model. In conclusion, telmisartan inhibited cell proliferation and tumor growth in ESCC cells by inducing cell-cycle arrest.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Neoplasias Esofágicas/tratamento farmacológico , Fase S/efeitos dos fármacos , Telmisartan/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/uso terapêutico , Animais , Linhagem Celular Tumoral , Ciclina A2/genética , Ciclina A2/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Telmisartan/uso terapêutico , Trombospondina 1/genética , Trombospondina 1/metabolismo
4.
DNA Cell Biol ; 38(7): 670-677, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31188027

RESUMO

Cutis laxa represents a heterogeneous group of rare, inherited, or acquired connective tissue disorders with the common feature of loose and redundant skin with decreased elasticity. The skin of affected deer showed abnormal collagen fiber morphology. To identify the differentially expressed genes of the unusual localized skin laxity in sika deer, we performed transcriptome analysis in the affected and control sika deer. The transcriptome analysis showed 700 genes with significant differential expression in the affected skin as compared with normal skin. Pathway analysis revealed an enrichment of genes involved in tumor necrosis factor signaling, the extracellular matrix-receptor interaction, platelet activation, and Huntington's disease. A gene network was constructed, and the hub nodes such as PTGS2, THBS1, COL1A1, FOS, and NOS3 were found through PPI network analysis, which may contributed to the unusual localized skin laxity in sika deer. Abnormal expression patterns of genes during the development of the affected sika deer were successfully uncovered in the present study, which provides a reference for revealing the related mechanism underlying cutis laxa in sika deer and human beings.


Assuntos
Cútis Laxa/veterinária , Cervos/genética , Transcriptoma , Animais , Colágeno/genética , Colágeno/metabolismo , Cútis Laxa/genética , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Redes Reguladoras de Genes , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Trombospondina 1/genética , Trombospondina 1/metabolismo
5.
Anticancer Res ; 39(5): 2317-2324, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31092423

RESUMO

BACKGROUND/AIM: Thrombospondins (TSPs) play a role as inhibitors of angiogenesis under various pathological conditions. The aim of the study was to evaluate the pathological significance and prognostic role of the 4N1K-peptide (KRFYVVMWKK), which is derived from TSP-1 and -2, in bladder cancer. MATERIALS AND METHODS: Two-hundred and six bladder cancer tissues were examined for expression of TSP-1, TSP-2, and 4N1K-peptide by immunohistochemistry. Cancer cell proliferation, apoptosis, angiogenesis and matrix metalloproteinase (MMP)-9 immunoreactivity were also examined. RESULTS: Expression of TSP-2 and 4N1K-peptide was negatively associated with T stage, metastasis, and grade. TSP-2 expression was negatively associated with cancer cell proliferation and MMP-9 expression, whereas 4N1K-peptide was significantly associated with apoptosis, angiogenesis, and MMP-9 expression. Multivariate analysis showed that 4N1K-peptide expression was a significant predictor of metastasis (hazard ratio=3.90, p=0.002). CONCLUSION: TSP-2 and 4N1K peptide played important roles in malignant aggressiveness and progression of bladder cancer via complex mechanisms involving cell proliferation, apoptosis, angiogenesis, and MMP-9.


Assuntos
Metaloproteinase 9 da Matriz/genética , Neovascularização Patológica/genética , Trombospondina 1/genética , Trombospondinas/genética , Neoplasias da Bexiga Urinária/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neovascularização Patológica/patologia , Oligopeptídeos/genética , Prognóstico , Neoplasias da Bexiga Urinária/patologia
6.
Cell ; 177(5): 1280-1292.e20, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31031006

RESUMO

Hyperactivity and disturbances of attention are common behavioral disorders whose underlying cellular and neural circuit causes are not understood. We report the discovery that striatal astrocytes drive such phenotypes through a hitherto unknown synaptic mechanism. We found that striatal medium spiny neurons (MSNs) triggered astrocyte signaling via γ-aminobutyric acid B (GABAB) receptors. Selective chemogenetic activation of this pathway in striatal astrocytes in vivo resulted in acute behavioral hyperactivity and disrupted attention. Such responses also resulted in upregulation of the synaptogenic cue thrombospondin-1 (TSP1) in astrocytes, increased excitatory synapses, enhanced corticostriatal synaptic transmission, and increased MSN action potential firing in vivo. All of these changes were reversed by blocking TSP1 effects. Our data identify a form of bidirectional neuron-astrocyte communication and demonstrate that acute reactivation of a single latent astrocyte synaptogenic cue alters striatal circuits controlling behavior, revealing astrocytes and the TSP1 pathway as therapeutic targets in hyperactivity, attention deficit, and related psychiatric disorders.


Assuntos
Astrócitos/metabolismo , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Comportamento Animal , Comunicação Celular , Neurônios/metabolismo , Transdução de Sinais , Sinapses/metabolismo , Animais , Astrócitos/patologia , Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/patologia , Transtorno do Deficit de Atenção com Hiperatividade/fisiopatologia , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Sinapses/genética , Trombospondina 1/genética , Trombospondina 1/metabolismo , Ácido gama-Aminobutírico/genética , Ácido gama-Aminobutírico/metabolismo
7.
Cell Mol Biol (Noisy-le-grand) ; 65(3): 48-57, 2019 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-30942155

RESUMO

The plateau zokor (Myospalaxbaileyi) is a specialized subterranean rodent that lives on the Qinghai-Tibet Plateau, and has successfully adapted to hypoxic environment. Raised expression of vascular endothelial growth factor (VEGF) and enhanced microvessel density (MVD) in tissues enable subterranean rodents to adapt to hypoxic sealed burrows. However, the expression of VEGF is inhibited by decreases in oxygen content, which is different from what obtains in Sprague Dawley (SD)rats. Thromspondin-1(TSP-1) is the first endogenous angiogenesis inhibitor identified inp53 pathway. It has several domains that bind to different proteins which regulate cell-to-cell interactions, inhibit endothelial cell proliferation and induce endothelial cell apoptosis (anti-angiogenesis). In this study, we analyzed the coding region and the expression pattern of TSP-1 gene in plateau zokor under different oxygen partial pressures using bioinformatics and qRT-PCR, respectively. Our results showed that the base and amino acid homologies between plateau zokor and Northern Israeli blind subterranean mole rat (Nannospalaxgalili) were 95.08 and 97.61%, respectively. There were eight parallel evolution sites with Nannospalaxgalili. Evaluation by 'Sorting Tolerant From Intolerant' (SIFT) algorithm showed four sites with significant effects on the function of TSP-1. Three-dimensional (3D) structures revealed that Asp185 and Thr270 were located in the NH2 terminal domain, with Glu536 in the Type I repeat domain, and Thr1092 in the COOH terminal domain. Compared to SD rats, the polarities of these four mutation sites changed. The expression levels of TSP-1 in plateau zokor tissues increased significantly from 2 260 m(16.12kPa) to 3 300 m(14.13kPa), but there was no significant difference in TSP-1 expression in SD rats. In conclusion, due to long-term adaption to the hypoxic environment of sealed burrows, plateau zokor upregulates the expression of TSP-1 to effect anti-angiogenesis. Moreover, mutations in gene structure of TSP-1 may play an important role in inhibiting angiogenesis.


Assuntos
Evolução Molecular , Regulação da Expressão Gênica , Hipóxia/genética , Roedores/genética , Trombospondina 1/genética , Animais , DNA Mitocondrial/genética , Modelos Moleculares , Filogenia , Subunidades Proteicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Seleção Genética , Homologia de Sequência do Ácido Nucleico , Trombospondina 1/química , Trombospondina 1/metabolismo
8.
Neuroscience ; 408: 68-80, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30928339

RESUMO

Thrombospondins (TSPs) are cell adhesion molecules that play an important role in the maintenance of hearing and afferent synaptic connections. Based on their reported function in restoring synaptic connections after stroke, we tested a potential role for TSP1 and TSP2 genes in repairing cochlear synapses following noise injury. We observed a tonotopic gradient in the expression of TSP1 and TSP2 mRNA in control mouse cochleae and an upregulation of these genes following noise exposure. Examining the functional sequelae of these changes revealed that afferent synaptic counts and auditory brainstem responses (ABRs) in noise-exposed TSP1 and TSP2 knockout (-/-) mice exhibited a worst recovery when compared to controls. Consistent with their tonotopic expression, TSP1-/- mice showed greater susceptibility to noise-induced hearing loss (NIHL) at 8 kHz and 16 kHz frequencies, whereas NIHL in TSP2-/- mice occurred only at mid and high frequencies. Further analysis of the ABR waveforms indicated peripheral neuronal damage in TSP2-/- but not in TSP1-/- mice. Noise trauma affecting mid to high frequencies triggered severe seizures in the TSP2-/- mice. We found that decreased susceptibility to audiogenic seizures in TSP1-/- mice was correlated with increased TSP2 protein levels in their inner ears, suggesting that TSP2 might functionally compensate for the loss of TSP1 in these mice. Our data indicate that TSP1 and TSP2 are both involved in susceptibility to NIHL, with TSP2 playing a more prominent role.


Assuntos
Limiar Auditivo/fisiologia , Cóclea/metabolismo , Perda Auditiva Provocada por Ruído/metabolismo , Trombospondina 1/metabolismo , Trombospondinas/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Regulação da Expressão Gênica , Perda Auditiva Provocada por Ruído/fisiopatologia , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Gânglio Espiral da Cóclea/metabolismo , Trombospondina 1/genética , Trombospondinas/genética
9.
Cell Physiol Biochem ; 52(3): 532-552, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30897320

RESUMO

BACKGROUND/AIMS: Thrombospondins (TSPs) are large multi-modular proteins, identified as natural angiogenesis inhibitors that exert their activity by binding to CD36 and CD47 receptors. The anti-angiogenic effect of TSPs in luteal regression of water buffalo has not been addressed. The present study characterized the expression pattern and localization of TSPs and their receptors in ovarian corpus luteum during different stages of development in buffalo. This study also elucidated the effect of exogenous Thrombospondin1 (TSP1) or the knocking out of the endogenous protein on luteal cell viability and function. Further, the in vitro transcriptional interaction of TSP1 with hormones, LH, PGF2α and angiogenic growth factors, VEGF and FGF2 were also evaluated. METHODS: First, the CLs were classified into four groups based on macroscopic observation and progesterone concentration. mRNA expression of examined factors was measured by qPCR, localization by immunoblotting and immunohistochemistry. TSP1 was knocked out (KO) in cultured luteal cells isolated from late luteal stage CLs (day 1116) by CRISPR/Cas9 mediated gene editing technology in order to functionally validate the TSP1 gene. Isolated cells from late stage CLs were also stimulated with different doses of TSP1, LH, PGF2α, VEGF and FGF2 for various time intervals to determine transcriptional regulation of thrombospondins. RESULTS: mRNA expression of TSPs and their receptors were found to be significantly higher in late and regressed stage of CL as compared to other groups which was consistent with the findings of immunoblotting and immunolocalization experiments. It was observed that TSP1 induced apoptosis, down regulated angiogenic growth factors, VEGF and FGF2 and attenuated progesterone production in cultured luteal cells. However, knocking out of endogenous TSP1 with CRISPR/Cas9 system improved the viability of luteal cells, progesterone synthesis and upregulated the expression of VEGF and FGF2 in the KO luteal cells. PGF2α induced the upregulation of TSPs and Caspase 3 transcripts, whereas treatment with LH and angiogenic growth factors (VEGF and FGF2) down regulated the TSP system in luteal cells. CONCLUSION: Collectively, these data provide evidence that thrombospondins along with their receptors are expressed at varying levels in different stages of CL progression with maximum expression during the late and regressing stages. These results are consistent with the hypothesis that thrombospondins stimulated by PGF2α plays an essential modulatory role in bringing about structural and functional luteolysis in buffalo.


Assuntos
Sistemas CRISPR-Cas/genética , Corpo Lúteo/metabolismo , Edição de Genes , Trombospondina 1/genética , Animais , Apoptose , Búfalos/metabolismo , Antígenos CD36/genética , Antígenos CD36/metabolismo , Antígeno CD47/genética , Antígeno CD47/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Sobrevivência Celular , Corpo Lúteo/citologia , Corpo Lúteo/patologia , Dinoprosta/metabolismo , Regulação para Baixo , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Trombospondina 1/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Iran J Allergy Asthma Immunol ; 18(1): 72-79, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30848575

RESUMO

Atherosclerosis is developed due to the formation of atheroma plaques in the coronary arteries. In this process, M1 macrophages and vascular smooth muscle cells (VSMCs) are the main functional cells. Inflammatory mediators such as histamine may inflame M1 macrophages. The aim of this study was to determine the effect of M1 macrophage secretion contents on the gene and protein expression levels of focal adhesion kinase (FAK), vasodilator-stimulated phosphoprotein (VASP), and thrombospondin1 (THBS1). Whole blood samples from the six healthy subjects (stenosis<5%), and six patients (stenosis>70%) were prepared and peripheral blood mononuclear cells (PBMCs) were isolated. Then monocytes were differentiated into M1 macrophages using 100 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF). The differentiated M1 macrophages were treated with histamine (10-6 M), and their secretion contents were harvested and added to the culture medium of VSMCs. The FAK, VASP, and THBS1 gene expression and protein levels were measured using RT-qPCR and western blot techniques in VSMCs, respectively. The FAK and THBS1 gene expression levels significantly increased in VSMCs after adding secretion contents obtained from histamine-treated M1 macrophages (p=0.023 and 0.05, respectively), while significant results were not observed for VASP gene (p=0.45). In converse with the phosphorylated VASP (pVASP) (p<0.34), the phosphorylated FAK (pFAK) and THBS1 protein levels increased in VSMCs (p<0.001). We concluded that in inflammatory conditions, the immune events could affect the macrophages by histamine. The activated macrophages could locally activate signaling pathways via FAK and THBS1 genes that are effective in the proliferation and migration of VSMCs.


Assuntos
Quinase 1 de Adesão Focal/metabolismo , Macrófagos/metabolismo , Miócitos de Músculo Liso/fisiologia , Trombospondina 1/metabolismo , Idoso , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Células Cultivadas , Estenose Coronária/metabolismo , Feminino , Quinase 1 de Adesão Focal/genética , Histamina/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Músculo Liso Vascular/citologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Transdução de Sinais , Trombospondina 1/genética
11.
Nat Commun ; 10(1): 1146, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850588

RESUMO

We undertook a systematic study focused on the matricellular protein Thrombospondin-1 (THBS1) to uncover molecular mechanisms underlying the role of THBS1 in glioblastoma (GBM) development. THBS1 was found to be increased with glioma grades. Mechanistically, we show that the TGFß canonical pathway transcriptionally regulates THBS1, through SMAD3 binding to the THBS1 gene promoter. THBS1 silencing inhibits tumour cell invasion and growth, alone and in combination with anti-angiogenic therapy. Specific inhibition of the THBS1/CD47 interaction using an antagonist peptide decreases cell invasion. This is confirmed by CD47 knock-down experiments. RNA sequencing of patient-derived xenograft tissue from laser capture micro-dissected peripheral and central tumour areas demonstrates that THBS1 is one of the gene with the highest connectivity at the tumour borders. All in all, these data show that TGFß1 induces THBS1 expression via Smad3 which contributes to the invasive behaviour during GBM expansion. Furthermore, tumour cell-bound CD47 is implicated in this process.


Assuntos
Neoplasias Encefálicas/genética , Antígeno CD47/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Proteína Smad3/genética , Trombospondina 1/genética , Fator de Crescimento Transformador beta1/genética , Animais , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Antígeno CD47/antagonistas & inibidores , Antígeno CD47/metabolismo , Linhagem Celular Tumoral , Córtex Cerebral , Glioblastoma/irrigação sanguínea , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Microdissecção e Captura a Laser , Masculino , Camundongos , Camundongos Knockout , Invasividade Neoplásica , Peptídeos/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Análise de Sobrevida , Trombospondina 1/antagonistas & inibidores , Trombospondina 1/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Can J Cardiol ; 35(1): 42-50, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30595182

RESUMO

BACKGROUND: Previous studies have shown that thrombospondin 1 (TSP-1) is involved in cardiovascular diseases, such as atherosclerosis and abdominal aortic aneurysm. However, TSP-1 expression levels in human aortic dissection (AD) remain unknown. METHODS: TSP-1 levels were detected in aortas collected from control subjects and AD patients. The TSP-1, interleukin (IL) 6, matrix metalloproteinase (MMP) 2, and MMP9 levels in plasma from non-AD patients and AD patients were measured. In addition, the effects of recombinant mouse TSP-1 protein on macrophage differentiation and smooth muscle cell (SMC) apoptosis were investigated. RESULTS: Compared with the aortas from control subjects, aortas from AD patients showed a significant increase in TSP-1 expression, especially in the torn sections. SMCs and endothelial cells produced TSP-1, but SMCs were the main source. TSP-1, IL-6, MMP2, and MMP9 levels were higher in AD patients than in non-AD patients, and plasma IL-6, MMP2, and MMP9 levels were positively correlated with TSP-1 levels in AD patients. Simple linear regression analysis and multivariate linear regression analysis showed that TSP-1 levels were independently correlated with the onset of AD. In cultured cells, recombinant mouse TSP-1 further increased inducible nitric oxide synthase (iNOS) mRNA expression in angiotensin (Ang) II-treated macrophages, whereas it reduced B-cell lymphoma-2 (Bcl2) mRNA levels and increased Bcl2-associated X protein (Bax) mRNA levels in Ang II-treated SMCs. CONCLUSIONS: TSP-1 level is significantly increased in AD patients and might participate in AD via promoting classically activated macrophage (M1) macrophage differentiation and SMC apoptosis.


Assuntos
Aneurisma Dissecante/genética , Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/genética , Regulação da Expressão Gênica , Músculo Liso Vascular/metabolismo , RNA/genética , Trombospondina 1/genética , Doença Aguda , Adulto , Aneurisma Dissecante/metabolismo , Aneurisma Dissecante/patologia , Animais , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Músculo Liso Vascular/patologia , Reação em Cadeia da Polimerase , Trombospondina 1/biossíntese
13.
Nephrology (Carlton) ; 24(4): 405-413, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30129267

RESUMO

AIM: Long non-coding RNA (lncRNAs) have been shown to play a critical role in a variety of pathophysiological processes, such as cell proliferation, apoptosis and migration. However, there were few studies addressing the function of lncRNAs in renal ischaemia/reperfusion (I/R) injury. Apoptosis is an important pathogenesis during I/R injury. Here, we identified the effect of hypoxia-responsive lncRNA growth arrest-specific 5 (GAS5) on apoptosis in renal I/R injury. METHODS: Ischaemia/reperfusion injury in mice or hypoxia/re-oxygenation (H/R) in human proximal renal tubular epithelial cells (HK-2) was practiced to induce apoptosis. The kidneys and blood were collected at 24 h after reperfusion. The GAS5 messenger RNA (mRNA) expression and apoptosis-related gene mRNA and protein levels, including p53, cellular inhibitor of apoptosis protein 2 (cIAP2) and thrombospondin-1 (TSP-1), were analysed. GAS5 small-interfering RNA was transfected with H/R induced cells. Over-expression of GAS5 was performed by plasmid transfection. RESULTS: Apoptotic cells significantly increased in I/R-injured kidneys. GAS5 could be up-regulated in kidneys at 24 h after reperfusion and 3 h after re-oxygenation, combined with increased expression of its downstream apoptosis-related proteins p53 and cIAP2. GAS5 small-interfering RNA treatment down-regulated the mRNA and protein levels of p53 and TSP-1, and attenuated apoptosis induced by H/R in HK-2 cells. Conversely, over-expression of GAS5 up-regulated the mRNA and protein levels of p53 and TSP-1, and promoted apoptosis in HK-2 cells. CONCLUSION: Long non-coding RNA GAS5 induced by I/R injury could promote apoptosis in kidney. TSP-1 might be one of the downstream effectors of GAS5, which will be explored in the future.


Assuntos
Lesão Renal Aguda/metabolismo , Apoptose , Túbulos Renais Proximais/metabolismo , RNA Longo não Codificante/metabolismo , Traumatismo por Reperfusão/metabolismo , Lesão Renal Aguda/genética , Lesão Renal Aguda/patologia , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Túbulos Renais Proximais/patologia , Masculino , Camundongos Endogâmicos C57BL , RNA Longo não Codificante/genética , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Trombospondina 1/genética , Trombospondina 1/metabolismo
14.
Mol Cell Proteomics ; 18(1): 51-64, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30257878

RESUMO

Transmissible gastroenteritis virus (TGEV) is a member of Coronaviridae family. Our previous research showed that TGEV infection could induce mitochondrial dysfunction and upregulate miR-222 level. Therefore, we presumed that miR-222 might be implicated in regulating mitochondrial dysfunction induced by TGEV infection. To verify the hypothesis, the effect of miR-222 on mitochondrial dysfunction was tested and we showed that miR-222 attenuated TGEV-induced mitochondrial dysfunction. To investigate the underlying molecular mechanism of miR-222 in TGEV-induced mitochondrial dysfunction, a quantitative proteomic analysis of PK-15 cells that were transfected with miR-222 mimics and infected with TGEV was performed. In total, 4151 proteins were quantified and 100 differentially expressed proteins were obtained (57 upregulated, 43 downregulated), among which thrombospondin-1 (THBS1) and cluster of differentiation 47 (CD47) were downregulated. THBS1 was identified as the target of miR-222. Knockdown of THBS1 and CD47 decreased mitochondrial Ca2+ level and increased mitochondrial membrane potential (MMP) level. Reversely, overexpression of THBS1 and CD47 elevated mitochondrial Ca2+ level and reduced mitochondrial membrane potential (MMP) level. Together, our data establish a significant role of miR-222 in regulating mitochondrial dysfunction in response to TGEV infection.


Assuntos
Antígeno CD47/metabolismo , Gastroenterite Suína Transmissível/metabolismo , MicroRNAs/genética , Mitocôndrias/metabolismo , Trombospondina 1/metabolismo , Vírus da Gastroenterite Transmissível/patogenicidade , Animais , Antígeno CD47/genética , Cálcio/metabolismo , Linhagem Celular , Gastroenterite Suína Transmissível/genética , Regulação da Expressão Gênica , Potencial da Membrana Mitocondrial , Mapas de Interação de Proteínas , Proteômica/métodos , Suínos , Trombospondina 1/genética , Transfecção
15.
Cancer Lett ; 442: 287-298, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30439539

RESUMO

Exosomes are implicated in cancer cell development, migration and invasion. Pigment epithelium-derived factor (PEDF) is a secreted anticancer protein that can regulate lung cancer progression; however, the role of PEDF in non-small cell lung cancer (NSCLC), including metastasis and cancer cell-derived exosome secretion, is unclear. In this study, we analyzed the effects of PEDF on exosome-mediated migration, invasion, and tumorigenicity of cultured NSCLC cells. The results showed that PEDF overexpression significantly reduced NSCLC invasion and migration, while inducing cell aggregation, whereas PEDF knockdown had the opposite effects. Exosomes from NSCLC cells treated with recombinant PEDF had a significantly reduced ability to promote cancer cell motility, migration, and invasion compared to exosomes from untreated cells. Exosomes from PEDF-treated cells contained thrombospondin 1 (THBS1), which inhibited cytoskeletal remodeling and exosome-induced lung cancer cell motility, migration, and invasion. Furthermore, PEDF-overexpressing NSCLC cells formed smaller xenograft tumors with higher THBS1 expression compared to control tumors. Our findings indicate that PEDF decreases the metastatic potential of NSCLC cells through regulation of THBS1 release in cancer cell-derived exosomes, thus uncovering a new mechanism of lung cancer progression.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Movimento Celular , Exossomos/metabolismo , Proteínas do Olho/metabolismo , Neoplasias Pulmonares/metabolismo , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Trombospondina 1/metabolismo , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Exossomos/genética , Exossomos/patologia , Proteínas do Olho/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos SCID , Invasividade Neoplásica , Fatores de Crescimento Neural/genética , Serpinas/genética , Transdução de Sinais , Trombospondina 1/genética , Carga Tumoral , Regulação para Cima
16.
Pancreatology ; 19(1): 196-203, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30393009

RESUMO

OBJECTIVES: The microRNA (miRNA) let-7d is linked to the formation of pancreatic cancer-related fibrosis. In this study, the mechanism by which let-7d regulates the activation of the human pancreatic stellate cell (hPSC) was evaluated. METHODS: The transient transfection of a let-7d mimic in the hPSCs was performed, and the altered thrombospondin 1 (THBS1) expression was confirmed by western blotting and real-time qPCR. Targeting of the 3'-untranslated region (UTR) of THBS1 by let-7d was investigated by the luciferase assays. After hPSC transfection using THBS1 siRNA, the fibrosis markers (α-SMA and collagen 1A1) were evaluated by western blotting and real-time qPCR. The correlation between tumor fibrosis and let-7d or THBS1 was estimated using the data from The Cancer Genome Atlas project. Finally, the effects of genistein on the hPSCs were evaluated. RESULTS: We found that a let-7d mimic inhibits THBS1 expression by targeting its 3'-UTR. THBS1 inhibition by siRNA inhibited hPSC activation. An in silico analysis revealed that let-7d and THBS1 expression are negatively correlated. Additionally, let-7d was negatively correlated with the stromal score, while THBS1 was positively correlated with this score. Genistein substantially induced let-7d and decreased the expression of fibrosis marker along with the inhibition of THBS1. CONCLUSIONS: Let-7d inhibited hPSC activation by targeting THBS1. Genistein induced the expression of let-7d and might modulate pancreatic fibrosis.


Assuntos
MicroRNAs/metabolismo , Células Estreladas do Pâncreas/metabolismo , Trombospondina 1/metabolismo , Biomarcadores/metabolismo , Regulação para Baixo , Fibrose/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Genisteína/farmacologia , Humanos , MicroRNAs/genética , Fosfatidiletanolaminas , RNA Mensageiro , RNA Interferente Pequeno , Trombospondina 1/genética
17.
Xenotransplantation ; 26(2): e12459, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30136356

RESUMO

CD47 is a ubiquitously expressed transmembrane glycoprotein that plays a complex role in regulation of cell survival and function. We have previously shown that the interspecies incompatibility of CD47 plays an important role in triggering rejection of cellular xenografts by macrophages. However, the role of CD47 in solid organ transplantation remains undetermined. Here, we explored this question in mouse models of heart allotransplantation. We observed that the lack of CD47 in donor hearts had no deleterious effect on graft survival in syngeneic or single MHC class I-mismatched recipients, in which both wild-type (WT) and CD47 knockout (CD47 KO) mouse hearts survived long term with no sign of rejection. Paradoxically, elimination of donor CD47 was beneficial for graft survival in signal MHC class II- and class I- plus class II-mismatched combinations, in which CD47 KO donor hearts showed significantly improved survival compared to WT donor hearts. Similarly, CD47 KO donor hearts were more resistant than WT hearts to humoral rejection in α1,3-galactosyltransferase-deficient mice. Moreover, a significant prolongation of WT allografts was observed in recipient mice treated with antibodies against a CD47 ligand thrombospondin-1 (TSP1) or with TSP1 deficiency, indicating that TSP1-CD47 signaling may stimulate vascularized allograft rejection. Thus, unlike cellular transplantation, donor CD47 expression may accelerate the rejection of vascularized allografts.


Assuntos
Aloenxertos/imunologia , Antígeno CD47/imunologia , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Trombospondina 1/imunologia , Animais , Antígeno CD47/genética , Transplante de Células/métodos , Rejeição de Enxerto/genética , Sobrevivência de Enxerto/genética , Transplante de Coração/métodos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Trombospondina 1/genética , Transplante Heterólogo/métodos , Transplante Homólogo/métodos
18.
Matrix Biol ; 75-76: 300-313, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29138119

RESUMO

Thrombospondin-4 (TSP-4) belongs to the thrombospondin protein family that consists of five highly homologous members. A number of novel functions have been recently assigned to TSP-4 in cardiovascular and nervous systems, inflammation, cancer, and the motor unit, which have attracted attention to this extracellular matrix (ECM) protein. These newly discovered functions set TSP-4 apart from other thrombospondins. For example, TSP-4 promotes angiogenesis while other TSPs either prevent it or have no effect on new blood vessel growth; TSP-4 reduces fibrosis and collagen production while TSP-1 and TSP-2 promote fibrosis in several organs; unlike other TSPs, TSP-4 appears to have some structural functions in ECM. The current information about TSP-4 functions in different organs and physiological systems suggests that this evolutionary conserved protein is a major regulator of the extracellular matrix (ECM) organization and production and tissue remodeling during the embryonic development and response to injury. In this review article, we summarize the properties and functions of TSP-4 and discuss its role in tissue remodeling.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Proteínas da Matriz Extracelular/genética , Matriz Extracelular/genética , Trombospondinas/genética , Vasos Sanguíneos/metabolismo , Fibrose/genética , Fibrose/patologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Neovascularização Patológica/genética , Trombospondina 1/genética , Trombospondina 1/metabolismo , Trombospondinas/metabolismo
19.
Int J Mol Sci ; 20(1)2018 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-30577496

RESUMO

Chronic inflammation of the ocular surface poses a risk of vision impairment. The understanding of the molecular mechanisms that are involved in the inflammatory response is critical to identify novel molecular targets. Recently, thrombospondin-1 (TSP-1) has emerged as a key player in ocular surface homeostasis that efficiently activates the TGF-ß2 isoform that is predominantly expressed in the ocular mucosa. Here, the potential of the peptide derived from TSP-1 (KRFK), that can activate TGF-ß, is proposed as a potentially applicable therapeutic for chronic ocular surface inflammatory disorders. Our in vitro results confirm that the chosen peptide activates TGF-ß, reducing the expression of co-stimulatory molecules on dendritic cells, driving them towards a tolerogenic phenotype. For the in vivo studies, the TSP-1-/- mouse is used as a pre-clinical model of chronic ocular inflammation. We observe that the topical application of KRFK alters the peripheral balance of effectors by reducing the proportion of pathogenic Th1 and Th17 cells while increasing Treg cell proportion in cervical lymph nodes. In line with these findings, the development of chronic ocular surface inflammation is significantly prevented in KRFK-treated TSP-1-/- mice, as assessed by clinical parameters and inflammatory cytokine expression in conjunctival and lacrimal gland tissues. Together, our results identify the KRFK peptide as a novel therapeutic option to prevent the development of chronic inflammatory manifestations of the ocular surface.


Assuntos
Anti-Inflamatórios/farmacologia , Endoftalmite/etiologia , Endoftalmite/metabolismo , Oligopeptídeos/farmacologia , Trombospondina 1/deficiência , Fator de Crescimento Transformador beta/metabolismo , Animais , Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Doença Crônica , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Endoftalmite/tratamento farmacológico , Endoftalmite/patologia , Fibrose , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Trombospondina 1/genética , Trombospondina 1/metabolismo , Fator de Crescimento Transformador beta/química
20.
Thromb Haemost ; 118(12): 2074-2085, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30419595

RESUMO

Thrombospondin-1 (TSP-1) is primarily expressed by platelets and endothelial cells (ECs) and rapidly released upon their activation. It functions in haemostasis as a bridging molecule in platelet aggregation, by promoting platelet adhesion to collagen and by protecting von Willebrand factor strings from degradation. In blood of patients undergoing surgery and in co-cultures of neutrophils with platelets or ECs, we observed proteolysis of the 185 kDa full-length TSP-1 to a 160-kDa isoform. We hypothesized that TSP-1 processing may alter its haemostatic properties. Selective enzyme inhibitors in co-cultures revealed that neutrophil proteases elastase and cathepsin G mediate TSP-1 processing. The cut site of cathepsin G was mapped to TSP-1 amino acids R237/T238 by Edman sequencing. Formation of neutrophil extracellular traps protected TSP-1 from complete degradation and promoted controlled processing to the 160-kDa isoform. Haemostatic properties were tested by platelet aggregation, adhesion, coagulation and string formation under flow. Platelets from TSP-1 deficient mice did not differ from wild-type in platelet aggregation but showed severe impairment of platelet adhesion to collagen and string formation under flow. Reconstitution experiments revealed that the 160-kDa TSP-1 isoform was markedly more potent than the 185-kDa full-length molecule in restoring function. Thus, TSP-1 processing by neutrophil proteases yields a 160-kDa isoform which shows enhanced potency to promote platelet adhesion and string formation. This finding reveals a novel mechanism of neutrophil-mediated thrombus formation and provides first evidence for the impact of TSP-1 proteolysis on its haemostatic properties.


Assuntos
Plaquetas/fisiologia , Endotélio Vascular/fisiologia , Neutrófilos/fisiologia , Trombospondina 1/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Hemostasia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Adesividade Plaquetária , Agregação Plaquetária , Multimerização Proteica , Proteólise , Trombospondina 1/genética , Trombospondina 1/imunologia , Fator de von Willebrand/metabolismo
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