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A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here, we examined the mechanisms underlying this susceptibility. We found that monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1, and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1), which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD, OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD.
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Antígeno CD47/metabolismo , Cromossomos Humanos Par 10/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Degeneração Macular/genética , Osteopontina/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Sítios de Ligação/fisiologia , Células COS , Linhagem Celular , Chlorocebus aethiops , Olho/patologia , Predisposição Genética para Doença/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Humanos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Transdução de Sinais/genéticaRESUMO
Fucosylation plays a critical role in cell-to-cell interactions and disease progression. However, the effects of fucosylation on splenocytes and their interactions with T cells remain unclear. In this study, we aimed to explore the transcriptome profiles of splenocytes deficient in fucosyltransferase (FUT) 1, an enzyme that mediates fucosylation, and investigate their impact on the proliferation and differentiation of T cells. We analysed and compared the transcriptomes of splenocytes isolated from Fut1 knockout (KO) mice and those from wild-type (WT) mice using RNA-seq. Additionally, we examined the effects of Fut1 KO splenocytes on CD4 T cell proliferation and differentiation, in comparison to WT splenocytes, and elucidated the mechanisms involved. The comparative analysis of transcriptomes between Fut1 KO and WT splenocytes revealed that thrombospondin-1, among the genes related to immune response and inflammation, was the most highly downregulated gene in Fut1 KO splenocytes. The reduced expression of thrombospondin-1 was further confirmed using qRT-PCR and flow cytometry. In coculture experiments, Fut1 KO splenocytes promoted the proliferation of CD4 T cells and drove their differentiation toward Th1 and Th17 cells, compared with WT splenocytes. Moreover, the levels of IL-2, IFN-γ and IL-17 were increased, while IL-10 was decreased, in T cells cocultured with Fut1 KO splenocytes compared with those with WT splenocytes. These effects of Fut1 KO splenocytes on T cells were reversed when thrombospondin-1 was replenished. Taken together, our results demonstrate that splenocytes with Fut1 deficiency promote CD4 T cell proliferation and Th1/Th17 differentiation at least in part through thrombospondin-1 downregulation.
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Linfócitos T CD4-Positivos , Baço , Animais , Camundongos , Regulação para Baixo , Diferenciação Celular , Proliferação de Células , Trombospondinas/genética , Camundongos Knockout , Camundongos Endogâmicos C57BLRESUMO
Osimertinib induces a marked response in non-small-cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR) gene mutations. However, acquired resistance to osimertinib remains an inevitable problem. In this study, we aimed to investigate osimertinib-resistant mechanisms and evaluate the combination therapy of afatinib and chemotherapy. We established osimertinib-resistant cell lines (PC-9-OR and H1975-OR) from EGFR-mutant lung adenocarcinoma cell lines PC-9 and H1975 by high exposure and stepwise method. Combination therapy of afatinib plus carboplatin (CBDCA) and pemetrexed (PEM) was effective in both parental and osimertinib-resistant cells. We found that expression of thrombospondin-1 (TSP-1) was upregulated in resistant cells using cDNA microarray analysis. We demonstrated that TSP-1 increases the expression of matrix metalloproteinases through integrin signaling and promotes tumor invasion in both PC-9-OR and H1975-OR, and that epithelial-to-mesenchymal transition (EMT) was involved in H1975-OR. Afatinib plus CBDCA and PEM reversed TSP-1-induced invasion ability and EMT changes in resistant cells. In PC-9-OR xenograft mouse models (five female Balb/c-Nude mice in each group), combination therapy strongly inhibited tumor growth compared with afatinib monotherapy (5 mg/kg, orally, five times per week) or CBDCA (75 mg/kg, intraperitoneally, one time per week) + PEM (100 mg/kg, intraperitoneally, one time per week) over a 28-day period. These results suggest that the combination of afatinib plus CBDCA and PEM, which effectively suppresses TSP-1 expression, may be a promising option in EGFR-mutated NSCLC patients after the acquisition of osimertinib resistance.
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Acrilamidas , Afatinib , Compostos de Anilina , Protocolos de Quimioterapia Combinada Antineoplásica , Carcinoma Pulmonar de Células não Pequenas , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB , Neoplasias Pulmonares , Mutação , Trombospondina 1 , Humanos , Trombospondina 1/genética , Trombospondina 1/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Animais , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Afatinib/farmacologia , Afatinib/uso terapêutico , Receptores ErbB/genética , Camundongos , Linhagem Celular Tumoral , Acrilamidas/farmacologia , Acrilamidas/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Carboplatina/farmacologia , Carboplatina/uso terapêutico , Feminino , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Camundongos Nus , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Indóis , PirimidinasRESUMO
BACKGROUND: The key role of thrombospondin 1 (THBS1) in the pathogenesis of acute-on-chronic liver failure (ACLF) is unclear. Here, we present a transcriptome approach to evaluate THBS1 as a potential biomarker in ACLF disease pathogenesis. METHODS: Biobanked peripheral blood mononuclear cells (PBMCs) from 330 subjects with hepatitis B virus (HBV)-related etiologies, including HBV-ACLF, liver cirrhosis (LC), and chronic hepatitis B (CHB), and normal controls (NC) randomly selected from the Chinese Group on the Study of Severe Hepatitis B (COSSH) prospective multicenter cohort underwent transcriptome analyses (ACLF = 20; LC = 10; CHB = 10; NC = 15); the findings were externally validated in participants from COSSH cohort, an ACLF rat model and hepatocyte-specific THBS1 knockout mice. RESULTS: THBS1 was the top significantly differentially expressed gene in the PBMC transcriptome, with the most significant upregulation in ACLF, and quantitative polymerase chain reaction (ACLF = 110; LC = 60; CHB = 60; NC = 45) was used to verify that THBS1 expression corresponded to ACLF disease severity outcome, including inflammation and hepatocellular apoptosis. THBS1 showed good predictive ability for ACLF short-term mortality, with an area under the receiver operating characteristic curve (AUROC) of 0.8438 and 0.7778 at 28 and 90 days, respectively. Enzyme-linked immunosorbent assay validation of the plasma THBS1 using an expanded COSSH cohort subjects (ACLF = 198; LC = 50; CHB = 50; NC = 50) showed significant correlation between THBS1 with ALT and γ-GT (P = 0.01), and offered a similarly good prognostication predictive ability (AUROC = 0.7445 and 0.7175) at 28 and 90 days, respectively. ACLF patients with high-risk short-term mortality were identified based on plasma THBS1 optimal cut-off value (< 28 µg/ml). External validation in ACLF rat serum and livers confirmed the functional association between THBS1, the immune response and hepatocellular apoptosis. Hepatocyte-specific THBS1 knockout improved mouse survival, significantly repressed major inflammatory cytokines, enhanced the expression of several anti-inflammatory mediators and impeded hepatocellular apoptosis. CONCLUSIONS: THBS1 might be an ACLF disease development-related biomarker, promoting inflammatory responses and hepatocellular apoptosis, that could provide clinicians with a new molecular target for improving diagnostic and therapeutic strategies.
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Insuficiência Hepática Crônica Agudizada , Trombospondina 1 , Animais , Humanos , Camundongos , Ratos , Biomarcadores , Vírus da Hepatite B , Inflamação , Leucócitos Mononucleares , Cirrose Hepática , Estudos Prospectivos , Trombospondina 1/genéticaRESUMO
Inappropriate endometrial stromal decidualization has been implied as an important reason of many pregnancy-related complications, such as unexplained recurrent spontaneous abortion, preeclampsia, and intrauterine growth restriction. Here, we observed that thrombospondin-1, an adhesive glycoprotein, was significantly downregulated in endometrial decidual cells from patients with unexplained recurrent spontaneous abortion. The immortalized human endometrial stromal cell line was used to investigate the possible THBS1-mediated regulation of decidualization. In vitro experiments found that the expression level of THBS1 increased with the normal decidualization process. Knockdown of THBS1 could decrease the expression levels of prolactin and insulin-like growth factor binding protein-1, two acknowledged human decidualization markers, whereas THBS1 overexpression could reverse these effects. The RNA sequencing results demonstrated that the extracellular regulated protein kinases signaling pathway was potentially affected by the knockdown of THBS1. We further confirmed that the regulation of THBS1 on decidualization was achieved through the ERK signaling pathway by the treatment of inhibitors. Moreover, knockdown of THBS1 in pregnant mice could impair decidualization and result in an increased fetus resorption rate. Altogether, our study demonstrated a crucial role of THBS1 in the pathophysiological process of unexplained recurrent spontaneous abortion and provided some new insights into the research of pregnancy-related complications.
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Aborto Habitual , Decídua , Endométrio , Células Estromais , Trombospondina 1 , Adulto , Animais , Feminino , Humanos , Camundongos , Gravidez , Aborto Habitual/genética , Aborto Habitual/metabolismo , Decídua/metabolismo , Endométrio/metabolismo , Endométrio/patologia , Células Estromais/metabolismo , Trombospondina 1/genética , Trombospondina 1/metabolismo , MasculinoRESUMO
During pregnancy, it is necessary to create appropriate conditions for the development of the placenta and the fetus. However, during parturition, the placenta must be separated and subsequently removed as soon as possible to not expose the female to the possibility of infection. In this study, the relationship between thrombospondin-1 (THBS1) and transforming growth factor beta1 (TGFß1) concentrations was described during bovine pregnancy (second, fourth, and sixth months; n = 3/each month), at normal parturition (NR) and parturition with fetal membrane retention (R). The presence of THBS1 and TGFß1 was confirmed in bovine placental tissues of both maternal and fetal parts. Enzyme-linked immunosorbent assay showed statistically significant differences (p < 0.05) in THBS1 concentrations (pg/mg protein) between examined parturient samples (maternal part: 5.76 ± 1.61 in R vs. 2.26 ± 1.58 in NR; fetal part: 2.62 ± 1.94 in R vs. 1.70 ± 0.23 in NR). TGFß1 concentrations (pg/mg protein) were significantly lower (p < 0.05) in the retained fetal membranes compared to the released fetal membranes in the maternal part of the placenta (26.22 ± 7.53 in NR vs. 17.80 ± 5.01 in R). The participation of THBS1 in the activation of TGFß1 in parturient bovine placental tissues leading to the normal release of fetal membranes may be suggested.
Assuntos
Placenta Retida , Gravidez , Feminino , Bovinos , Animais , Humanos , Placenta Retida/veterinária , Placenta Retida/metabolismo , Placenta/metabolismo , Projetos Piloto , Fator de Crescimento Transformador beta1/metabolismo , Parto , Trombospondinas/metabolismoRESUMO
The matrix glycoprotein thrombospondin-1 (THBS1) modulates nitric oxide (NO) signaling in endothelial cells. A high-salt diet induces deficiencies of NO production and bioavailability, thereby leading to endothelial dysfunction. In this study we investigated the changes of THBS1 expression and its pathological role in the dysfunction of mesenteric artery endothelial cells (MAECs) induced by a high-salt diet. Wild-type rats, and wild-type and Thbs1-/- mice were fed chow containing 8% w/w NaCl for 4 weeks. We showed that a high salt diet significantly increased THBS1 expression and secretion in plasma and MAECs, and damaged endothelium-dependent vasodilation of mesenteric resistance arteries in wild-type animals, but not in Thbs1-/- mice. In rat MAECs, we demonstrated that a high salt environment (10-40 mM) dose-dependently increased THBS1 expression accompanied by suppressed endothelial nitric oxide synthase (eNOS) and phospho-eNOS S1177 production as well as NO release. Blockade of transforming growth factor-ß1 (TGF-ß1) activity by a TGF-ß1 inhibitor SB 431542 reversed THBS1 up-regulation, rescued the eNOS decrease, enhanced phospho-eNOS S1177 expression, and inhibited Smad4 translocation to the nucleus. By conducting dual-luciferase reporter experiments in HEK293T cells, we demonstrated that Smad4, a transcription promoter, upregulated Thbs1 transcription. We conclude that THBS1 contributes to endothelial dysfunction in a high-salt environment and may be a potential target for treatment of high-salt-induced endothelium dysfunction.
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Células Endoteliais , Cloreto de Sódio , Humanos , Ratos , Camundongos , Animais , Cloreto de Sódio/metabolismo , Células Endoteliais/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Células HEK293 , Endotélio Vascular/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Vasodilatação , Artérias Mesentéricas , Trombospondinas/metabolismo , Óxido Nítrico/metabolismoRESUMO
BACKGROUND: Thrombospondin-1 (THBS1) is a secretory adhesive glycoprotein involved in the progression of multiple malignancies, including breast cancer. However, the clinical significance and prognostic role of plasma THBS1 in breast cancer have yet to be clarified. METHODS: Plasma THBS1 levels in 627 breast cancer patients were analyzed by enzyme-linked immunosorbent assay. Bone marrow blood was drawn from the anterior/posterior superior iliac spine to detect the presence of disseminated tumor cells (DTCs). The effects of plasma THBS1 on the clinicopathological characteristics and survival prediction of breast cancer patients were explored. RESULTS: Plasma THBS1 did not correlate with overall survival, breast cancer-specific survival (BCSS), and distant disease-free survival (DDFS) in the entire breast cancer cohort. Notably, HER2-enriched patients with high-plasma THBS1 levels had significantly shorter BCSS (P = 0.027) and DDFS (P = 0.011) than those with low levels. Multivariate analyses revealed that plasma THBS1 was an independent prognostic marker of BCSS (P = 0.026) and DDFS (P = 0.007) in HER2-enriched patients. THBS1 levels were 24% higher in positive DTC patients than in negative DTC patients (P = 0.031), and high levels were significantly associated with poor BCSS in positive DTC patients (HR 2.08, 95% CI 1.17-3.71; P = 0.019). Moreover, high-plasma THBS1 levels were specifically associated with an increased occurrence of brain metastasis in HER2-enriched patients (P = 0.041). CONCLUSION: These findings suggest that plasma THBS1 may be serving as an unfavorable prognosis predictor for HER2-enriched breast cancer and justifies the need for further research.
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Neoplasias Encefálicas , Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Prognóstico , Biomarcadores Tumorais , Intervalo Livre de Doença , Receptor ErbB-2RESUMO
PURPOSE OF REVIEW: Fibromyalgia syndrome (FMS) is a disease of unknown pathophysiology, with the diagnosis being based on a set of clinical criteria. Proteomic analysis can provide significant biological information for the pathophysiology of the disease but may also reveal biomarkers for diagnosis or therapeutic targets. The present systematic review aims to synthesize the evidence regarding the proteome of adult patients with FMS using data from observational studies. RECENT FINDINGS: An extensive literature search was conducted in MEDLINE/PubMed, CENTRAL, and clinicaltrials.gov from inception until November 2022. The study protocol was published in OSF. Two independent reviewers evaluated the studies and extracted data. The quality of studies was assessed using the modified Newcastle-Ottawa scale adjusted for proteomic research. Ten studies fulfilled the protocol criteria, identifying 3328 proteins, 145 of which were differentially expressed among patients with FMS against controls. The proteins were identified in plasma, serum, cerebrospinal fluid, and saliva samples. The control groups included healthy individuals and patients with pain (inflammatory and non-inflammatory). The most important proteins identified involved transferrin, α-, ß-, and γ-fibrinogen chains, profilin-1, transaldolase, PGAM1, apolipoprotein-C3, complement C4A and C1QC, immunoglobin parts, and acute phase reactants. Weak correlations were observed between proteins and pain sensation, or quality of life scales, apart from the association of transferrin and a2-macroglobulin with moderate-to-severe pain sensation. The quality of included studies was moderate-to-good. FMS appears to be related to protein dysregulation in the complement and coagulation cascades and the metabolism of iron. Several proteins may be dysregulated due to the excessive oxidative stress response.
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Fibromialgia , Estudos Observacionais como Assunto , Proteômica , Humanos , Biomarcadores/sangue , Biomarcadores/metabolismo , Fibromialgia/metabolismo , Fibromialgia/sangue , Proteômica/métodosRESUMO
Non-union fractures pose a significant clinical challenge, often leading to prolonged pain and disability. Understanding the molecular mechanisms underlying non-union fractures is crucial for developing effective therapeutic interventions. This study integrates bioinformatics analysis and experimental validation to unravel key genes and pathways associated with non-union fractures. We identified differentially expressed genes (DEGs) between non-union and fracture healing tissues using bioinformatics techniques. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to elucidate the biological processes and pathways involved. Common DEGs were identified, and a protein-protein interaction (PPI) network was constructed. Fibronectin-1 (FN1), Thrombospondin-1 (THBS1), and Biglycan (BGN) were pinpointed as critical target genes for non-union fracture treatment. Experimental validation involved alkaline phosphatase (ALP) and Alizarin Red staining to confirm osteogenic differentiation. Our analysis revealed significant alterations in pathways related to cell behavior, tissue regeneration, wound healing, infection, and immune responses in non-union fracture tissues. FN1, THBS1, and BGN were identified as key genes, with their upregulation indicating potential disruptions in the bone remodeling process. Experimental validation confirmed the induction of osteogenic differentiation. The study provides comprehensive insights into the molecular mechanisms of non-union fractures, emphasizing the pivotal roles of FN1, THBS1, and BGN in extracellular matrix dynamics and bone regeneration. The findings highlight potential therapeutic targets and pathways for further investigation. Future research should explore interactions between these genes, validate results using in vivo fracture models, and develop tailored treatment strategies for non-union fractures, promising significant advances in clinical management.
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OBJECTIVES: Stroke is a devastating clinical outcome that significantly contributes to the morbidity and mortality of sickle cell anemia (SCA) patients. Despite its advantages in predicting stroke risk, transcranial Doppler screening has limitations that restrict its applicability, highlighting the need for emerging prognostic tools. Thrombospondin-1 plays a crucial role in endothelial injury, platelet adhesion, and nitric oxide metabolism and may be implicated in stroke pathophysiology. Here, we aimed to evaluate the association of THBS1 genetic variations with the occurrence of stroke in SCA patients MATERIALS AND METHODS: By real-time PCR, 512 SCA patients were fully genotyped for THBS1 A-296G (rs1478605) polymorphism RESULTS: THBS1 GG genotype was associated with a lower risk for stroke occurrence [odds ratio (OR): 0.30; 95% confidence interval (CI): 0.11-0.78; P = 0.011], although these findings were not consistent with multivariate logistic regression analysis (OR: 0.73, 95% CI: 0.12 - 4.37; P = 0.736). In agreement, the cumulative incidence of stroke for patients with AG/AA genotypes was higher when compared to the GG genotype (P = 0.018). However, the association was not maintained in the multivariate proportional hazards model (hazard ratio: 0.67, 95% CI: 0.12-3.61; P = 0.643) CONCLUSIONS: In summary, the present study shows that the THBS1 A-296G (rs1478605) polymorphism may be a potential modifier for stroke in SCA.
Assuntos
Anemia Falciforme , Acidente Vascular Cerebral , Humanos , Anemia Falciforme/complicações , Anemia Falciforme/diagnóstico , Anemia Falciforme/epidemiologia , Brasil/epidemiologia , Genótipo , Polimorfismo Genético , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/genéticaRESUMO
Alterations in angiogenic properties play a pivotal role in the manifestation and onset of various pathologies, including vascular diseases and cancer. Thrombospondin-1 (TSP1) protein is one of the master regulators of angiogenesis. This study unveils a novel aspect of TSP1 regulation through reversible phosphorylation. The silencing of the B55α regulatory subunit of protein phosphatase 2A (PP2A) in endothelial cells led to a significant decrease in TSP1 expression. Direct interaction between TSP1 and PP2A-B55α was confirmed via various methods. Truncated TSP1 constructs were employed to identify the phosphorylation site and the responsible kinase, ultimately pinpointing PKC as the enzyme phosphorylating TSP1 on Ser93. The biological effects of B55α-TSP1 interaction were also analyzed. B55α silencing not only counteracted the increase in TSP1 expression during wound closure but also prolonged wound closure time. Although B55α silenced cells initiated tube-like structures earlier than control cells, their spheroid formation was disrupted, leading to disintegration. Cells transfected with phosphomimic TSP1 S93D exhibited smaller spheroids and reduced effectiveness in tube formation, revealing insights into the effects of TSP1 phosphorylation on angiogenic properties. In this paper, we introduce a new regulatory mechanism of angiogenesis by reversible phosphorylation on TSP1 S93 by PKC and PP2A B55α.
Assuntos
Células Endoteliais , Proteína Fosfatase 2 , Angiogênese , Células Endoteliais/metabolismo , Fosforilação , Proteína Fosfatase 2/metabolismo , Processamento de Proteína Pós-Traducional , Trombospondina 1/genética , Trombospondina 1/metabolismo , HumanosRESUMO
This study aimed to investigate the role of macrophage polarization in the treatment of liver fibrosis by Fuzheng Huayu Tablets(FZHY) through single-cell, transcriptome sequencing and in vitro and in vivo experiments. Liver fibrosis-related datasets, transcriptomic datasets, and single-cell sequencing datasets were obtained from the Gene Expression Omnibus(GEO) database to screen differential genes. Liver fibrosis-related genes were obtained from GeneCards, DisGeNET, NCBI, PharmgKB, TTD and OMIM databases. Macrophage polarization-related genes were obtained from the GeneCards database. The above three gene sets were intersected to construct a protein-protein interaction(PPI) network. Cytoscape software was used to screen core proteins, and the expression pattern of core proteins was visualized by single-cell sequencing. A mouse model of liver fibrosis was constructed using carbon tetrachloride(CCl_4). Hematoxylin-eosin(HE) staining and Masson staining were used to observe the pathological morphology of liver tissues. The expressions of α-smooth muscle actin(α-SMA) and transforming growth factor-ß1(TGF-ß1) were detected by immunohistochemistry. The levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were detected by colorimetry. The le-vels of inflammatory factors in serum were detected by the enzyme-linked immunosorbent assay(ELISA). Furthermore, the expressions of α-SMA, TGF-ß1, cluster of differentiation 86(CD86) and thrombospondin 1(THBS1) in liver tissues were detected by Western blot(WB). Lipopolysaccharide(LPS) was used to stimulate RAW264.7 cells to construct the M1 macrophage polarization model. The cell counting kit-8(CCK-8) method was used to detect cell viability. WB was used to detect the protein expressions of CD86 and THBS1 in cells, and the messenger ribonucleic acid(mRNA) expression levels of tumor necrosis factor-α(TNF-α) and interleukin(IL)-1ß by real-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-qPCR). The results showed that a total of 26 potential genes related to the polarization of liver fibrosis macrophages were obtained, and 10 core proteins related to the polarization of liver fibrosis macrophages such as THBS1, lumican(LUM) and fibulin-5(FBLN5) were screened. Single-cell data analysis indicated that THBS1, ranking highest, may be expressed by M1 macrophages. Animal experiments demonstrated that FZHY reduced inflammatory cell infiltration and collagen deposition in CCl_4-induced mouse liver, relieved liver injury and inflammation levels, and inhibited the expressions of α-SMA, TGF-ß1, CD86, and THBS1 proteins. Cell experiments revealed that FZHY significantly reduced intracellular expression of CD86 and THBS1 proteins and mRNA levels of TNF-α and IL-1ß. In conclusion, FZHY may ameliorate liver fibrosis by inhibiting THBS1 protein expression, suppressing M1 macrophage polarization, and reducing inflammation.
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Medicamentos de Ervas Chinesas , Cirrose Hepática , Transcriptoma , Animais , Medicamentos de Ervas Chinesas/farmacologia , Camundongos , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Transcriptoma/efeitos dos fármacos , Masculino , Análise de Célula Única , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Thrombospondin 1 (THBS1) is a secreted extracellular matrix glycoprotein that regulates a variety of cellular and physiological processes. THBS1's diverse functions are attributed to interactions between the modular domains of THBS1 with an array of proteins found in the extracellular matrix. THBS1's three Thrombospondin type 1 repeats (TSRs) are modified with O-linked glucose-fucose disaccharide and C-mannose. It is unknown whether these modifications impact trafficking and/or function of THBS1 in vivo. The O-fucose is added by Protein O-fucosyltransferase 2 (POFUT2) and is sequentially extended to the disaccharide by ß3glucosyltransferase (B3GLCT). The C-mannose is added by one or more of four C-mannosyltransferases. O-fucosylation by POFUT2/B3GLCT in the endoplasmic reticulum has been proposed to play a role in quality control by locking TSR domains into their three-dimensional fold, allowing for proper secretion of many O-fucosylated substrates. Prior studies showed the siRNA knockdown of POFUT2 in HEK293T cells blocked secretion of TSRs 1-3 from THBS1. Here we demonstrated that secretion of THBS1 TSRs 1-3 was not reduced by CRISPR-Cas9-mediated knockout of POFUT2 in HEK293T cells and demonstrated that knockout of Pofut2 or B3glct in mice did not reduce the trafficking of endogenous THBS1 to secretory granules of platelets, a major source of THBS1. Additionally, we demonstrated that all three TSRs from platelet THBS1 were highly C-mannosylated, which has been shown to stabilize TSRs in vitro. Combined, these results suggested that POFUT2 substrates with TSRs that are also modified by C-mannose may be less susceptible to trafficking defects resulting from the loss of the glucose-fucose disaccharide.
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Fucosiltransferases , Trombospondina 1 , Animais , Humanos , Camundongos , Fucose/metabolismo , Fucosiltransferases/metabolismo , Glucose , Células HEK293 , Manose , Vesículas Secretórias/metabolismo , Trombospondina 1/genética , Trombospondina 1/metabolismo , Trombospondinas/genéticaRESUMO
Thrombospondin-1 (TSP1) is a secreted protein minimally expressed in health but increased in disease and age. TSP1 binds to the cell membrane receptor CD47, which itself engages signal regulatory protein α (SIRPα), and the latter creates a checkpoint for immune activation. Individuals with cancer administered checkpoint-blocking molecules developed insulin-dependent diabetes. Relevant to this, CD47 blocking antibodies and SIRPα fusion proteins are in clinical trials. We characterized the molecular signature of TSP1, CD47, and SIRPα in human islets and pancreata. Fresh islets and pancreatic tissue from nondiabetic individuals were obtained. The expression of THBS1, CD47, and SIRPA was determined using single-cell mRNA sequencing, immunofluorescence microscopy, Western blot, and flow cytometry. Islets were exposed to diabetes-affiliated inflammatory cytokines and changes in protein expression were determined. CD47 mRNA was expressed in all islet cell types. THBS1 mRNA was restricted primarily to endothelial and mesenchymal cells, whereas SIRPA mRNA was found mostly in macrophages. Immunofluorescence staining showed CD47 protein expressed by ß cells and present in the exocrine pancreas. TSP1 and SIRPα proteins were not seen in islets or the exocrine pancreas. Western blot and flow cytometry confirmed immunofluorescent expression patterns. Importantly, human islets produced substantial quantities of secreted TSP1. Human pancreatic exocrine and endocrine tissue expressed CD47, whereas fresh islets displayed cell surface CD47 and secreted TSP1 at baseline and in inflammation. These findings suggest unexpected effects on islets from agents that intersect TSP1-CD47-SIRPα.NEW & NOTEWORTHY CD47 is a cell surface receptor with two primary ligands, soluble thrombospondin-1 (TSP1) and cell surface signal regulatory protein alpha (SIRPα). Both interactions provide checkpoints for immune cell activity. We determined that fresh human islets display CD47 and secrete TSP1. However, human islet endocrine cells lack SIRPα. These gene signatures are likely important given the increasing use of CD47 and SIRPα blocking molecules in individuals with cancer.
Assuntos
Antígeno CD47 , Neoplasias , Humanos , Antígeno CD47/genética , Antígeno CD47/metabolismo , Macrófagos/metabolismo , Neoplasias/metabolismo , Receptores de Superfície Celular/metabolismo , Trombospondinas/metabolismo , Trombospondinas/uso terapêutico , Trombospondina 1/genética , Trombospondina 1/metabolismoRESUMO
Microglial necroptosis exacerbates neurodegenerative diseases, central nervous system (CNS) injury, and demonstrates a proinflammatory process, but its contribution to subarachnoid hemorrhage (SAH) is poorly characterized. BCL-2 homologous antagonist-killer protein (Bak1), a critical regulatory molecule of endogenous apoptosis, can be involved in the pathologic process of necroptosis by regulating mitochondrial permeability. In this study, we revealed microglia undergo necroptosis after SAH in vivo and vitro. Western blot revealed that Bak1 was elevated at 24 h after SAH. Knocked down of Bak1 by adeno-associated virus attenuates microglial necroptosis, alleviates neuroinflammation, and improves neurologic function after SAH in mice. Furthermore, oxyhemoglobin (10 µM) induced necroptosis in BV2 microglia, increasing Bak1 expression and mediating proinflammatory phenotype transformation, exacerbating oxidative stress and neuroinflammation. Abrogating BV2 Bak1 could reduce necroptosis by down-regulating the expression of phosphorylated pseudokinase mixed lineage kinase domain-like protein (p-MLKL), then down-regulating proinflammatory phenotype gene expression. RNA-Seq showed that disrupting BV2 Bak1 down-regulates multiple immune and inflammatory pathways and ameliorates cell injury by elevating thrombospondin 1 (THBS1) expression. In summary, we identified a critical regulatory role for Bak1 in microglial necroptosis and neuroinflammation after SAH. Bak1 is expected to be a potential target for the treatment strategy of SAH.
Assuntos
Doenças Neuroinflamatórias , Hemorragia Subaracnóidea , Camundongos , Animais , Microglia/metabolismo , Hemorragia Subaracnóidea/metabolismo , Necroptose , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Levels of neutrophil extracellular traps (NETs) were measured in plasma of healthy controls (HC, n = 30) and patients with granulomatosis with polyangiitis (GPA, n = 123), microscopic polyangiitis (MPA, n = 61), Takayasu's arteritis (TAK, n = 58), and giant cell arteritis (GCA, n = 68), at times of remission or activity and correlated with levels of the platelet-derived thrombospondin-1 (TSP-1). Levels of NETs were elevated during active disease in patients with GPA (p < 0.0001), MPA (p = 0.0038), TAK (p < 0.0001), and GCA (p < 0.0001), and in remission for GPA, p < 0.0001, MPA, p = 0.005, TAK, p = 0.03, and GCA, p = 0.0009. All cohorts demonstrated impaired NET degradation. Patients with GPA (p = 0.0045) and MPA (p = 0.005) had anti-NET IgG antibodies. Patients with TAK had anti-histone antibodies (p < 0.01), correlating with presence of NETs. Levels of TSP-1 were increased in all patients with vasculitis, and associated with NET formation. NET formation is a common process in vasculitides. Targeting NET formation or degradation could be potential therapeutic approaches for vasculitides.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos , Armadilhas Extracelulares , Arterite de Células Gigantes , Granulomatose com Poliangiite , Poliangiite Microscópica , Arterite de Takayasu , Trombospondina 1 , Humanos , Masculino , Feminino , Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Armadilhas Extracelulares/metabolismo , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Estudos de Casos e Controles , Granulomatose com Poliangiite/metabolismo , Arterite de Células Gigantes/metabolismo , Poliangiite Microscópica/metabolismo , Arterite de Takayasu/metabolismo , Neutrófilos , Trombospondina 1/metabolismoRESUMO
The use of treatments, such as programmed death protein 1 (PD1) or cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibodies, that loosen the natural checks upon immune cell activity to enhance cancer killing have shifted clinical practice and outcomes for the better. Accordingly, the number of antibodies and engineered proteins that interact with the ligand-receptor components of immune checkpoints continue to increase along with their use. It is tempting to view these molecular pathways simply from an immune inhibitory perspective. But this should be resisted. Checkpoint molecules can have other cardinal functions relevant to the development and use of blocking moieties. Cell receptor CD47 is an example of this. CD47 is found on the surface of all human cells. Within the checkpoint paradigm, non-immune cell CD47 signals through immune cell surface signal regulatory protein alpha (SIRPα) to limit the activity of the latter, the so-called trans signal. Even so, CD47 interacts with other cell surface and soluble molecules to regulate biogas and redox signaling, mitochondria and metabolism, self-renewal factors and multipotency, and blood flow. Further, the pedigree of checkpoint CD47 is more intricate than supposed. High-affinity interaction with soluble thrombospondin-1 (TSP1) and low-affinity interaction with same-cell SIRPα, the so-called cis signal, and non-SIRPα ectodomains on the cell membrane suggests that multiple immune checkpoints converge at and through CD47. Appreciation of this may provide latitude for pathway-specific targeting and intelligent therapeutic effect.
Assuntos
Antígeno CD47 , Neoplasias , Humanos , Antígenos de Diferenciação/farmacologia , Receptores Imunológicos/metabolismo , Neoplasias/terapia , Anticorpos/farmacologia , Proteínas de Transporte , FagocitoseRESUMO
BACKGROUND: Indoxyl sulfate (IS) is a protein-bound uremic toxin with vascular toxicity. The primary cause of death in uremic patients on maintenance hemodialysis is vascular disease, and it had been reported that vascular smooth muscle cells (VSMCs) trans-differentiation (VT) plays a vital role in the context of vascular diseases, but the underlying mechanisms remain obscure. Thrombospondin-1 (TSP-1) participates in vascular calcification by keeping the balance of extracellular matrix, but its role in IS-induced VT is unclear. METHODS: In this study, clinical specimens, animal models, and in vitro VSMCs were used to investigate the role of TSP-1 in IS induced VT and the potential therapeutic methods. RESULTS: We found that TSP-1 was significantly decreased in arterial samples from uremic patients, animal models, and in VSMCs after IS treatment. Downregulation of TSP-1 sufficiently induced the trans-differentiation genotypes of VSMCs. CONCLUSION: Emodin, the main monomer extracted from rhubarb, could alleviate IS-induced VT in vitro by upregulating TSP-1. Taken together, IS induces VT by downregulating TSP-1. Emodin might be a candidate drug to alleviate VT under IS treatment.
Assuntos
Emodina , Músculo Liso Vascular , Animais , Humanos , Indicã/toxicidade , Emodina/farmacologia , Trombospondina 1 , Transdiferenciação Celular , Miócitos de Músculo Liso , Células CultivadasRESUMO
Intercellular communication is pivotal in various stages of cancer progression. For smart and effective communication, cancer cells employ diverse modes of messaging that may be further fine-tuned by the microenvironmental changes. Extracellular matrix (ECM) stiffening due to excess deposition and crosslinking of collagen is one of the crucial tumor-microenvironmental changes that influence a plethora of cellular processes, including cell-cell communication. We herein studied the crosstalk between exosomes and tunneling nanotubes (TNT), the two distinct means of cell-cell communication under varying ECM-stiffness conditions. We show that exosomes promote the formation of tunneling nanotubes in breast cancer cells, which results in cellular internet. Interestingly, exosomes drastically increased the fraction of cells connected by TNT; however, they elicited no effect on the number of TNTs per pair of connected cells or the length of TNT. The observed pro-TNT effects of exosomes were found to be ECM-stiffness dependent. ECM-stiffness tuned exosomes were found to promote TNT formation predominantly via the 'cell dislodgment model'. At the molecular level, exosomal thrombospondin-1 was identified as a critical pro-TNT factor. These findings underline the influence of ECM stiffening on two diverse modes of cell communication and their interdependence, which may have significant implications in cancer biomedical research.