RESUMO
Fibrinogen C domain-containing protein 1 (FIBCD1) is an immune protein proposed to be involved in host recognition of chitin on the surface of pathogens. As FIBCD1 readily binds acetylated molecules, we have determined the high-resolution crystal structures of a recombinant fragment of the FIBCD1 C-terminal domain complexed with small N-acetyl-containing ligands to determine the mode of recognition. All ligands bind at the conserved N-acetyl-binding site (S1) with galactose and glucose-derived ligands rotated 180° relative to each other. One subunit of a native structure derived from protein expressed in mammalian cells binds glycosylation from a neighboring subunit, in an extended binding site. Across the various structures, the primary S1 binding pocket is occupied by N-acetyl-containing ligands or acetate, with N-acetyl, acetate, or sulfate ion in an adjacent pocket S1(2). Inhibition binding studies of N-acetylglucosamine oligomers, (GlcNAc)n, n = 1, 2, 3, 5, 11, via ELISA along with microscale thermophoresis affinity assays indicate a strong preference of FIBCD1 for longer N-acetylchitooligosaccharides. Binding studies of mutant H396A, located beyond the S1(2) site, showed no significant difference from wildtype, but K381L, within the S1(2) pocket, blocked binding to the model ligand acetylated bovine serum albumin, suggesting that S1(2) may have functional importance in ligand binding. The binding studies, alongside structural definition of diverse N-acetyl monosaccharide binding in the primary S1 pocket and of additional, adjacent binding pockets, able to accommodate both carbohydrate and sulfate functional groups, suggest a versatility in FIBCD1 to recognize chitin oligomers and other pathogen-associated carbohydrate motifs across an extended surface.
Assuntos
Receptores de Superfície Celular , Humanos , Acetatos , Sítios de Ligação/fisiologia , Carboidratos/química , Quitina/metabolismo , Hemostáticos , Ligantes , Ligação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Sulfatos , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
[Figure: see text].
Assuntos
Cardiomegalia/metabolismo , Proteínas de Transporte/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Angiotensinas/toxicidade , Animais , Aorta/cirurgia , Cardiomegalia/etiologia , Cardiomegalia/patologia , Proteínas de Transporte/genética , Células Cultivadas , Constrição , Proteínas da Matriz Extracelular/genética , Feminino , Glicoproteínas/genética , Humanos , Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Comunicação Parácrina , Fenilefrina/toxicidade , Ratos , Estresse Fisiológico , Fator de Crescimento Transformador beta/metabolismo , Remodelação VentricularRESUMO
Retinal artery occlusion (RAO) is a devastating condition with no effective treatment. The management of RAO could potentially be improved through an in-depth understanding of the molecular alterations in the condition. This study combined advanced proteomic techniques and an experimental model to uncover the retinal large-scale protein profile of RAO. In 13 pigs, RAO was induced with an argon laser and confirmed by fluorescein angiography. Left eyes serving as controls received a sham laser without inducing occlusion. Retinal samples were collected after one, three, or six days and analyzed with liquid chromatography-tandem mass spectrometry. In RAO, 36 proteins were differentially regulated on day one, 86 on day three, and 557 on day six. Upregulated proteins included clusterin, vitronectin, and vimentin, with several proteins increasing over time with a maximum on day six, including clusterin, vimentin, osteopontin, annexin-A, signal transducer, and the activator of transcription 3. On day six, RAO resulted in the upregulation of proteins involved in cellular response to stress, hemostasis, innate immune response, and cytokine signaling. Downregulated proteins were involved in transmission across chemical synapses and visual phototransduction. This study identified the upregulation of multiple inflammatory proteins in RAO and the downregulation of proteins involved in visual pathways.
Assuntos
Clusterina , Oclusão da Artéria Retiniana , Animais , Suínos , Vimentina/genética , Proteômica/métodos , RetinaRESUMO
Central retinal vein occlusion (CRVO) is a visually disabling condition resulting from a thrombus in the major outflow vessel of the eye. The inflammatory response in CRVO is effectively treated with a dexamethasone (DEX) intravitreal implant. Uncovering the proteome changes following DEX implant intervention in CRVO may identify key proteins that mediate the beneficial effects of DEX. In six Göttingen minipigs, CRVO was induced in both eyes with an argon laser using a well-established experimental model. The right eyes were treated with a DEX intravitreal implant (Ozurdex, Allergan), while the left control eyes received a sham injection. Eight weeks after DEX intervention, retinal samples were collected and analyzed with tandem mass tag-based mass spectrometry. DEX implant intervention resulted in the upregulation of peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5) and ubiquilin-4. Immunohistochemistry showed expression of FKBP5 in the nuclei in all cellular layers of the retina. Cell adhesion molecule 3, tumor necrosis factor receptor superfamily member 16, and trans-1,2-dihydrobenzene-1,2-diol dehydrogenase were downregulated following DEX intervention. The upregulation of the corticosteroid-sensitive protein FKBP5 suggests that the implant remained active at the molecular level after eight weeks of treatment. Future studies may investigate if FKBP5 regulates the efficacy and duration of the DEX implant.
Assuntos
Oclusão da Veia Retiniana , Animais , Dexametasona/farmacologia , Implantes de Medicamento , Glucocorticoides/farmacologia , Oclusão da Veia Retiniana/tratamento farmacológico , Oclusão da Veia Retiniana/metabolismo , Suínos , Porco Miniatura , Tomografia de Coerência Óptica , Resultado do Tratamento , Acuidade VisualRESUMO
Aflibercept is a frequently used inhibitor of vascular endothelial growth factor (VEGF) in the treatment of macular edema following central retinal vein occlusion (CRVO). Retinal proteome changes following aflibercept intervention in CRVO remain largely unstudied. Studying proteomic changes of aflibercept intervention may generate a better understanding of mechanisms of action and uncover aspects related to the safety profile. In 10 Danish Landrace pigs, CRVO was induced in both eyes with an argon laser. Right eyes were treated with intravitreal aflibercept while left control eyes received isotonic saline water. Retinal samples were collected 15 days after induced CRVO. Proteomic analysis by tandem mass tag-based mass spectrometry identified a total of 21 proteins that were changed in content following aflibercept intervention. In retinas treated with aflibercept, high levels of aflibercept components were reached, including the VEGF receptor-1 and VEGF receptor-2 domains. Fold changes in the additional proteins ranged between 0.70 and 1.19. Aflibercept intervention resulted in a downregulation of pigment epithelium-derived factor (PEDF) (fold change = 0.84) and endoplasmin (fold change = 0.91). The changes were slight and could thereby not be confirmed with less precise immunohistochemistry and Western blotting. Our data suggest that aflibercept had a narrow mechanism of action in the CRVO model. This may be an important observation in cases when macular edema secondary to CRVO is resistant to aflibercept intervention.
Assuntos
Edema Macular , Oclusão da Veia Retiniana , Inibidores da Angiogênese/farmacologia , Animais , Injeções Intravítreas , Edema Macular/complicações , Edema Macular/etiologia , Proteoma , Proteômica , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão/farmacologia , Oclusão da Veia Retiniana/complicações , Oclusão da Veia Retiniana/tratamento farmacológico , Oclusão da Veia Retiniana/metabolismo , Suínos , Fator A de Crescimento do Endotélio Vascular , Acuidade VisualRESUMO
BACKGROUND AND AIMS: Hepatic sinusoidal cells are known actors in the fibrogenic response to injury. Activated hepatic stellate cells (HSCs), liver sinusoidal endothelial cells, and Kupffer cells are responsible for sinusoidal capillarization and perisinusoidal matrix deposition, impairing vascular exchange and heightening the risk of advanced fibrosis. While the overall pathogenesis is well understood, functional relations between cellular transitions during fibrogenesis are only beginning to be resolved. At single-cell resolution, we here explored the heterogeneity of individual cell types and dissected their transitions and crosstalk during fibrogenesis. APPROACH AND RESULTS: We applied single-cell transcriptomics to map the heterogeneity of sinusoid-associated cells in healthy and injured livers and reconstructed the single-lineage HSC trajectory from pericyte to myofibroblast. Stratifying each sinusoidal cell population by activation state, we projected shifts in sinusoidal communication upon injury. Weighted gene correlation network analysis of the HSC trajectory led to the identification of core genes whose expression proved highly predictive of advanced fibrosis in patients with nonalcoholic steatohepatitis (NASH). Among the core members of the injury-repressed gene module, we identified plasmalemma vesicle-associated protein (PLVAP) as a protein amply expressed by mouse and human HSCs. PLVAP expression was suppressed in activated HSCs upon injury and may hence define hitherto unknown roles for HSCs in the regulation of microcirculatory exchange and its breakdown in chronic liver disease. CONCLUSIONS: Our study offers a single-cell resolved account of drug-induced injury of the mammalian liver and identifies key genes that may serve important roles in sinusoidal integrity and as markers of advanced fibrosis in human NASH.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Células Endoteliais/patologia , Redes Reguladoras de Genes , Cirrose Hepática/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Animais , Biópsia , Capilares/citologia , Capilares/patologia , Tetracloreto de Carbono/administração & dosagem , Tetracloreto de Carbono/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Modelos Animais de Doenças , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Feminino , Veias Hepáticas/citologia , Veias Hepáticas/patologia , Humanos , Fígado/irrigação sanguínea , Fígado/patologia , Cirrose Hepática/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , RNA-Seq , Análise de Célula ÚnicaRESUMO
PURPOSE: Chemotherapy-induced gastrointestinal toxicity is a common adverse event during chemotherapeutic treatment. No uniformly applicable strategies exist to predict, prevent, or treat gastrointestinal toxicity. Thus, a goal of mucositis research is to identify targets for therapeutic interventions and individualized risk prediction. Fibrinogen C domain containing 1 (FIBCD1) is a transmembrane protein expressed in human intestinal epithelial cells with functions in the innate immune system. Previous observations have shown that FIBCD1 ameliorates dextran sulfate sodium (DSS)-induced intestinal inflammation in vivo. We evaluated the effect of FIBCD1 in a murine model of chemotherapy-induced gastrointestinal toxicity and inflammation. METHODS: Transgenic (Tg) mice overexpressing FIBCD1 in the intestinal epithelium (Fibcd1Tg) and wild-type (WT) littermates (C57BL/6N) were randomized to receive an intraperitoneal injection of doxorubicin 20 mg/kg or saline and were terminated 2 or 7 days after the injection. Gastrointestinal toxicity was evaluated by weight change, intestinal length, villus height/crypt depth, and histological mucositis score. Expression of inflammatory markers (IL-6, IL-1ß, and Tnfα) was measured by quantitative real-time PCR in intestinal tissue samples. RESULTS: Following doxorubicin treatment, WT mice exhibited an increased weight loss compared with Tg littermates (p < 0.001). No differences between genotypes were seen in mucositis score, intestinal length, villus height/crypt depth, or IL-6, IL-1ß, and Tnfα expression. CONCLUSION: Our findings suggest that FIBCD1 could ameliorate chemotherapy-induced gastrointestinal toxicity by reducing weight loss; however, the mechanism of this possible protective effect remains to be defined warranting additional investigations.
Assuntos
Antineoplásicos/uso terapêutico , Mucosite/induzido quimicamente , Mucosite/tratamento farmacológico , Receptores de Superfície Celular/uso terapêutico , Redução de Peso/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Genótipo , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: Identifying biomarkers for abdominal aortic aneurysms (AAA) could prove beneficial in prognosis of AAA and thus the selection for treatment. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein that is highly expressed in aorta. MFAP4 is involved in several tissue remodeling-related diseases. We aimed to investigate the potential role of plasma MFAP4 (pMFAP4) as a biomarker of AAA. METHODS: Plasma samples and data were obtained for 504 male AAA patients and 188 controls in the Viborg Vascular (VIVA) screening trial. The pMFAP4 levels were measured by Alphalisa. The Mann-Whitney U test assessed differences in pMFAP4 levels between the presence and absence of different exposures of interest. The correlation between pMFAP4 and aorta growth rate were investigated through spearman's correlation analysis. Immunohistochemistry and multiple logistic regression adjusted for potential confounders assessed the association between pMFAP4 and AAA. Multiple linear regression assessed the correlation between pMFAP4 and aorta growth rate. Cox regression and competing risk regression were used to investigate the correlation between AAA patients with upper tertile pMFAP4 and the risk of undergoing later surgical repair. RESULTS: A significant negative correlation between pMFAP4 and aorta growth rate was observed using spearman's correlation analysis (ρ = -0.14; P = .0074). However, this finding did not reach significance when applying multiple linear regression. A tendency of decreased pMFAP4 was observed in AAA using immunohistochemistry. Competing risk regression adjusted for potential confounders indicated that patients with upper tertile pMFAP4 had a hazard ratio of 0.51 (P = .001) for risk of undergoing later surgical repair. CONCLUSIONS: High levels of pMFAP4 are associated with a decreased likelihood of receiving surgical repair in AAA. This observation warrants confirmation in an independent cohort.
Assuntos
Aneurisma da Aorta Abdominal/sangue , Aneurisma da Aorta Abdominal/cirurgia , Proteínas de Transporte/sangue , Proteínas da Matriz Extracelular/sangue , Glicoproteínas/sangue , Idoso , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Biomarcadores/sangue , Dinamarca , Progressão da Doença , Humanos , Masculino , Valor Preditivo dos Testes , Estudos Prospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Regulação para CimaRESUMO
BACKGROUND: Alcoholic liver disease (ALD) is a public health concern that is the cause of half of all cirrhosis-related deaths. Early detection of fibrosis, ideally in the precirrhotic stage, is a key strategy for improving ALD outcomes and for preventing progression to cirrhosis. Previous studies identified the blood-borne marker human microfibrillar-associated protein 4 (MFAP4) as a biomarker for detection of hepatitis C virus (HCV)-related fibrosis. AIM: To evaluate the diagnostic accuracy of MFAP4 to detect ALD-induced fibrosis. METHOD: We performed a prospective, liver biopsy-controlled study involving 266 patients with prior or current alcohol overuse. Patients were split into a training and a validation cohort. RESULTS: MFAP4 was present in fibrotic hepatic tissue and serum MFAP4 levels increased with fibrosis grade. The area under the receiver operating characteristic curve (AUROC) for detection of cirrhosis was 0.91 (95% CI 0.85-0.96) in the training cohort and 0.91 (95% CI 0.79-1.00) in the validation cohort. For detection of advanced fibrosis, the AUROC was 0.88 (95% CI 0.81-0.94) in the training cohort and 0.92 (95% CI 0.83-1.00) in the validation cohort. The diagnostic accuracy did not differ between MFAP4 and the enhanced liver fibrosis (ELF) test or transient elastography (TE) in an intention-to-diagnose analysis. MFAP4 did not predict hepatic decompensation in a time-to-decompensation analysis in a subgroup of patients with cirrhosis. CONCLUSION: MFAP4 is a novel biomarker that can detect ALD-related fibrosis with high accuracy.
Assuntos
Técnicas de Imagem por Elasticidade , Hepatopatias Alcoólicas , Biópsia , Proteínas de Transporte , Proteínas da Matriz Extracelular , Glicoproteínas , Humanos , Fígado/patologia , Cirrose Hepática/diagnóstico , Cirrose Hepática/patologia , Hepatopatias Alcoólicas/diagnóstico , Hepatopatias Alcoólicas/patologia , Estudos Prospectivos , Curva ROCRESUMO
BACKGROUND: Symptomatic peripheral artery disease (PAD) is an atherosclerotic occlusive disease affecting the lower extremities. The cause of symptomatic PAD is atherosclerosis, vascular dysfunctions, impaired angiogenesis and neointima formation. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein, which is highly expressed in the heart and arteries and recently introduced as a potential mediator of pathological vascular remodeling and neointima formation. We aimed to investigate the relationship between serum MFAP4 (sMFAP4) and symptomatic PAD outcomes. METHODS: A total of 286 PAD patients were analyzed if they had either intermittent claudication or critical lower-extremity ischemia (CLI) and followed for 7 years. The level of serum MFAP4 (sMFAP4) was measured by alphaLISA. Kaplan-Meier, Cox proportional hazard and logistic regression analysis were used to analyze the associations between upper tertile sMFAP4 and symptomatic PAD outcomes. RESULTS: Patients with upper tertile sMFAP4 had an odds ratio (OR) of 2.65 (p < 0.001) for having CLI diagnosis. Further analysis indicated that patients with upper tertile sMFAP4 had a hazard ratio (HR) of 1.97 (p = 0.04) for cardiovascular death during the 7-years follow-up. However, analysis of 2-year primary patency showed that patients with upper tertile sMFAP4 had decreased risk of vascular occlusion after reconstructive surgery with HR of 0.15 (p = 0.02). CONCLUSIONS: sMFAP4 has potential as a prognostic marker for cardiovascular death, primary patency of reconstructed vessels and CLI diagnosis in symptomatic PAD patients. Confirmation of observations in larger cohorts is warranted.
Assuntos
Proteínas de Transporte/genética , Proteínas da Matriz Extracelular/genética , Variação Genética , Glicoproteínas/genética , Doença Arterial Periférica/genética , Idoso , Proteínas de Transporte/sangue , Determinação de Ponto Final , Proteínas da Matriz Extracelular/sangue , Feminino , Seguimentos , Predisposição Genética para Doença , Glicoproteínas/sangue , Humanos , Estimativa de Kaplan-Meier , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/sangue , Doença Arterial Periférica/cirurgia , Fatores de RiscoRESUMO
MFAP4 (microfibrillar-associated protein 4) is an extracellular glycoprotein found in elastic fibers without a clearly defined role in elastic fiber assembly. In the present study, we characterized molecular interactions between MFAP4 and elastic fiber components. We established that MFAP4 primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin and fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site-directed mutagenesis disclosed residues Phe(241) and Ser(203) in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved in proper elastic fiber organization.
Assuntos
Proteínas de Transporte/metabolismo , Desmosina/metabolismo , Tecido Elástico/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Microfibrilas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Tropoelastina/metabolismo , Substituição de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Fibrilina-1 , Fibrilinas , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Ligantes , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tropoelastina/química , Tropoelastina/genéticaRESUMO
OBJECTIVE: Arterial injury stimulates remodeling responses that, when excessive, lead to stenosis. These responses are influenced by integrin signaling in vascular smooth muscle cells (VSMCs). Microfibrillar-associated protein 4 (MFAP4) is an integrin ligand localized to extracellular matrix fibers in the vascular wall. The role of MFAP4 in vascular biology is unknown. We aimed to test the hypothesis that MFAP4 would enhance integrin-dependent VSMC activation. APPROACH AND RESULTS: We produced Mfap4-deficient (Mfap4(-/-)) mice and performed carotid artery ligation to explore the role of MFAP4 in vascular biology in vivo. Furthermore, we investigated the effects of MFAP4 in neointimal formation ex vivo and in primary VSMC and monocyte cultures in vitro. When challenged with carotid artery ligation, Mfap4(-/-) mice exhibited delayed neointimal formation, accompanied by early reduction in the number of proliferating medial and neointimal cells, as well as infiltrating leukocytes. Delayed neointimal formation was associated with decreased cross-sectional area of ligated Mfap4(-/-) carotid arteries resulting in lumen narrowing 28 days after ligation. MFAP4 blockade prohibited the formation of neointimal hyperplasia ex vivo. Moreover, we demonstrated that MFAP4 is a ligand for integrin αVß3 and mediates VSMC phosphorylation of focal adhesion kinase, migration, and proliferation in vitro. MFAP4-dependent VSMC activation was reversible by treatment with MFAP4-blocking antibodies and inhibitors of focal adhesion kinase and downstream kinases. In addition, we showed that MFAP4 promotes monocyte chemotaxis in integrin αVß3-dependent manner. CONCLUSIONS: MFAP4 regulates integrin αVß3-induced VSMC proliferation and migration, as well as monocyte chemotaxis, and accelerates neointimal hyperplasia after vascular injury.
Assuntos
Doenças das Artérias Carótidas/metabolismo , Proteínas de Transporte/metabolismo , Movimento Celular , Proliferação de Células , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima , Animais , Apoptose , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Proteínas de Transporte/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiotaxia de Leucócito , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/metabolismo , Genótipo , Glicoproteínas/deficiência , Glicoproteínas/genética , Humanos , Hiperplasia , Integrina alfaVbeta3/metabolismo , Ligantes , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Fenótipo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Fatores de Tempo , Remodelação VascularRESUMO
BACKGROUND: The human microfibrillar-associated protein 4 (MFAP4) is located to extracellular matrix fibers and plays a role in disease-related tissue remodeling. Previously, we identified MFAP4 as a serum biomarker candidate for hepatic fibrosis and cirrhosis in hepatitis C patients. The aim of the present study was to elucidate the potential of MFAP4 as biomarker for hepatic fibrosis with a focus on the differentiation of no to moderate (F0-F2) and severe fibrosis stages and cirrhosis (F3 and F4, Desmet-Scheuer scoring system). METHODS: MFAP4 levels were measured using an AlphaLISA immunoassay in a retrospective study including n = 542 hepatitis C patients. We applied a univariate logistic regression model based on MFAP4 serum levels and furthermore derived a multivariate model including also age and gender. Youden-optimal cutoffs for binary classification were determined for both models without restrictions and considering a lower limit of 80 % sensitivity (correct classification of F3 and F4), respectively. To assess the generalization error, leave-one-out cross validation (LOOCV) was performed. RESULTS: MFAP4 levels were shown to differ between no to moderate fibrosis stages F0-F2 and severe stages (F3 and F4) with high statistical significance (t test on log scale, p value <2.2·10(-16)). In the LOOCV, the univariate classification resulted in 85.8 % sensitivity and 54.9 % specificity while the multivariate model yielded 81.3 % sensitivity and 61.5 % specificity (restricted approaches). CONCLUSIONS: We confirmed the applicability of MFAP4 as a novel serum biomarker for assessment of hepatic fibrosis and identification of high-risk patients with severe fibrosis stages in hepatitis C. The combination of MFAP4 with existing tests might lead to a more accurate non-invasive diagnosis of hepatic fibrosis and allow a cost-effective disease management in the era of new direct acting antivirals.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Hepatite C/complicações , Hepatite C/metabolismo , Cirrose Hepática/complicações , Cirrose Hepática/metabolismo , Adulto , Análise de Variância , Biomarcadores/metabolismo , Estudos de Coortes , Feminino , Hepatite C/diagnóstico , Humanos , Cirrose Hepática/diagnóstico , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Curva ROC , Sensibilidade e EspecificidadeRESUMO
Microfibrillar-associated protein 4 (MFAP4) is an extracellular protein belonging to the fibrinogen-related protein superfamily and is recognized as an integrin ligand with suggested functions in pulmonary and vascular tissue homeostasis. MFAP4 expression in the spleen is increased during infections; however, the significance of MFAP4 for the function of the spleen is unknown. Immunohistochemistry, morphometry and real-time RT-PCR were used to analyze wild-type and MFAP4-deficient spleens. In addition, they were compared with splenic tissue, which was newly formed 8 weeks after avascular implantation into adult mice in order to obtain information about the role of MFAP4 in the formation of splenic tissue during ontogeny and adult life. The present study shows that MFAP4 is co-localized with laminin in the B- and T-cell zones of the spleen, in addition to capsular and trabecular expression. MFAP4 is most likely produced by fibroblastic reticulum cells and follicular dendritic cells of the spleen but can also be imported via the blood from other tissues. The development of splenic tissue is not disturbed in MFAP4-deficient mice. However, in splenic tissue regenerating under MFAP4-deficient conditions, the number of FDCs is significantly decreased but is corrected by MFAP4 imported from other tissues. No differences were observed for lymphocyte numbers or splenic structure. The data indicate that MFAP4 promotes FDC development in regenerating splenic tissue and warrant further investigations regarding the MFAP4 dependency of splenic B-cell maturation.
Assuntos
Envelhecimento/metabolismo , Proteínas de Transporte/metabolismo , Embrião de Mamíferos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Baço/embriologia , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Proliferação de Células , Feminino , Centro Germinativo/citologia , Laminina/metabolismo , Camundongos Endogâmicos C57BL , Regeneração , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
The high resolution crystal structures of a recombinant fragment of the C-terminal fibrinogen-like recognition domain of FIBCD1, a vertebrate receptor that binds chitin, have been determined. The overall tetrameric structure shows similarity in structure and aggregation to the horseshoe crab innate immune protein tachylectin 5A. The high affinity ligand N-acetylmannosamine (ManNAc) binds in the S1 site, predominantly via the acetyl group with the oxygen and acetamide nitrogen hydrogen-bonded to the protein and the methyl group inserted into a hydrophobic pocket. The binding of the ManNAc pyranose ring differs markedly between the two independent subunits, but in all structures the binding of the N-acetyl group is conserved. In the native structure, a crystal contact results in one of the independent protomers binding the first GlcNAc of the Asn(340) N-linked glycan on the other independent protomer. In the ligand-bound structure this GlcNAc is replaced by the higher affinity ligand ManNAc. In addition, a sulfate ion has been modeled into the electron density at a location similar to the S3 binding site in L-ficolin, whereas in the native structure an acetate ion has been placed in the S1 N-acetyl binding site, and a sulfate ion has been placed adjacent to this site. These ion binding sites are ideally placed to receive the N-acetyl and sulfate groups of sulfated GalNAc residues of glycosaminoglycans such as chondroitin and dermatan sulfate. Together, these structures give insight into important determinants of ligand selectivity, demonstrating versatility in recognition and binding while maintaining conservation in N-acetyl and calcium binding.
Assuntos
Quitina/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular , Quitina/química , Cristalografia por Raios X , Fibrinogênio/química , Humanos , Insetos/citologia , Ligantes , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores de Superfície Celular/genética , Relação Estrutura-AtividadeRESUMO
Surfactant protein D (SP-D) is a pulmonary collectin important in lung immunity. SP-D-deficient mice (Sftpd(-/-)) are reported to be susceptible to ovalbumin (OVA)- and fungal allergen-induced pulmonary inflammation, while treatment with exogenous SP-D has therapeutic effects in such disease models. ß-Glucans are a diverse group of polysaccharides previously suggested to serve as fungal ligands for SP-D. We set out to investigate if SP-D could interact with 1,3-ß-glucan and attenuate allergic pulmonary inflammation in the presence of 1,3-ß-glucan. Allergic airway disease was induced in Sftpd(-/-) and Sftpd(+/+) mice by OVA sensitization and subsequent challenge with OVA, 1,3-ß-glucan, or OVA/1,3-ß-glucan together. Mice in the combined treatment group were further treated with a high dose of recombinant fragment of human SP-D (rfhSP-D). We demonstrated direct interaction between SP-D and 1,3-ß-glucan. OVA-induced mucous cell metaplasia was increased in Sftpd(-/-) mice, supporting previously reported protective effects of endogenous SP-D in allergy. OVA-induced parenchymal CCL11 levels and eosinophilic infiltration in bronchoalveolar lavage were unaffected by 1,3-ß-glucan, but were reversed with rfhSP-D treatment. 1,3-ß-Glucan treatment did, however, induce pulmonary neutrophilic infiltration and increased TNF-α levels in bronchoalveolar lavage, independently of OVA-induced allergy. This infiltration was also reversed by treatment with rfhSP-D. 1,3-ß-Glucan reduced OVA-induced mucous cell metaplasia, T helper 2 cytokines, and IFN-γ production. rfhSP-D treatment further reduced mucous metaplasia and T helper 2 cytokine secretion to background levels. In summary, rfhSP-D treatment resulted in attenuation of both allergic inflammation and 1,3-ß-glucan-mediated neutrophilic inflammation. Our data suggest that treatment with high-dose SP-D protects from mold-induced exacerbations of allergic asthma.
Assuntos
Hipersensibilidade/complicações , Hipersensibilidade/tratamento farmacológico , Inflamação/complicações , Inflamação/tratamento farmacológico , Substâncias Protetoras/uso terapêutico , Proteína D Associada a Surfactante Pulmonar/uso terapêutico , beta-Glucanas/metabolismo , Animais , Quimiocina CCL11/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Hipersensibilidade/patologia , Imunoglobulina E/metabolismo , Inflamação/patologia , Ligantes , Metaplasia , Camundongos Endogâmicos C57BL , Microbiota/efeitos dos fármacos , Ovalbumina , Substâncias Protetoras/farmacologia , Proteoglicanas , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Proteína D Associada a Surfactante Pulmonar/farmacologia , Hipersensibilidade Respiratória/complicaçõesRESUMO
Microfibrillar-associated protein 4 (MFAP4) is localized to elastic fibers in blood vessels and the interalveolar septa of the lungs and is further present in bronchoalveolar lavage. Mfap4 has been previously suggested to be involved in elastogenesis in the lung. We tested this prediction and aimed to characterize the pulmonary function changes and emphysematous changes that occur in Mfap4-deficient (Mfap4(-/-)) mice. Significant changes included increases in total lung capacity and compliance, which were evident in Mfap4(-/-) mice at 6 and 8 mo but not at 3 mo of age. Using in vivo breath-hold gated microcomputed tomography (micro-CT) in 8-mo-old Mfap4(-/-) mice, we found that the mean density of the lung parenchyma was decreased, and the low-attenuation area (LAA) was significantly increased by 14% compared with Mfap4(+/+) mice. Transmission electron microscopy (TEM) did not reveal differences in the organization of elastic fibers, and there was no difference in elastin content, but a borderline significant increase in elastin mRNA expression in 3-mo-old mice. Stereological analysis showed that alveolar surface density in relation to the lung parenchyma and total alveolar surface area inside of the lung were both significantly decreased in Mfap4(-/-) mice by 25 and 15%, respectively. The data did not support an essential role of MFAP4 in pulmonary elastic fiber organization or content but indicated increased turnover in young Mfap4(-/-) mice. However, Mfap4(-/-) mice developed a spontaneous loss of lung function, which was evident at 6 mo of age, and moderate air space enlargement, with emphysema-like changes.
Assuntos
Proteínas de Transporte/genética , Proteínas da Matriz Extracelular/genética , Glicoproteínas/genética , Pulmão/patologia , Enfisema Pulmonar/genética , Animais , Elastina/genética , Elastina/metabolismo , Proteínas da Matriz Extracelular/deficiência , Feminino , Glicoproteínas/deficiência , Pulmão/metabolismo , Pulmão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/fisiopatologia , Respiração , TranscriptomaRESUMO
BACKGROUND: Recently, several proteins of the extracellular matrix have been characterised as active contributors to allergic airway disease. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein abundant in the lung, whose biological functions remain poorly understood. In the current study we investigated the role of MFAP4 in experimental allergic asthma. METHODS: MFAP4-deficient mice were subjected to alum/ovalbumin and house dust mite induced models of allergic airway disease. In addition, human healthy and asthmatic primary bronchial smooth muscle cell cultures were used to evaluate MFAP4-dependent airway smooth muscle responses. RESULTS: MFAP4 deficiency attenuated classical hallmarks of asthma, such as eosinophilic inflammation, eotaxin production, airway remodelling and hyperresponsiveness. In wild-type mice, serum MFAP4 was increased after disease development and correlated with local eotaxin levels. MFAP4 was expressed in human bronchial smooth muscle cells and its expression was upregulated in asthmatic cells. Regarding the underlying mechanism, we showed that MFAP4 interacted with integrin αvß5 and promoted asthmatic bronchial smooth muscle cell proliferation and CCL11 release dependent on phosphatidyloinositol-3-kinase but not extracellular signal-regulated kinase pathway. CONCLUSIONS: MFAP4 promoted the development of asthmatic airway disease in vivo and pro-asthmatic functions of bronchial smooth muscle cells in vitro. Collectively, our results identify MFAP4 as a novel contributor to experimental asthma, acting through modulation of airway smooth muscle cells.
Assuntos
Asma/metabolismo , Proteínas de Transporte/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Pulmão/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Western Blotting , Adesão Celular , Técnicas de Cultura de Células , Proliferação de Células , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Fenótipo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Variation in surfactant protein D (SP-D) is associated with lung function in tobacco smoke-induced chronic respiratory disease. We hypothesized that the same association exists in the general population and could be used to identify individuals sensitive to smoke-induced lung damage. The association between serum SP-D (sSP-D) and expiratory lung function was assessed in a cross-sectional design in a Danish twin population (n = 1,512, 18-72 yr old). The adjusted heritability estimates for expiratory lung function, associations between SP-D gene (SFTPD) single-nucleotide polymorphisms or haplotypes, and expiratory lung function were assessed using twin study methodology and mixed-effects models. Significant inverse associations were evident between sSP-D and the forced expiratory volume in 1 s and forced vital capacity in the presence of current tobacco smoking but not in nonsmokers. The two SFTPD single-nucleotide polymorphisms, rs1923536 and rs721917, and haplotypes, including these single-nucleotide polymorphisms or rs2243539, were inversely associated with expiratory lung function in interaction with smoking. In conclusion, SP-D is phenotypically and genetically associated with lung function measures in interaction with tobacco smoking. The obtained data suggest sSP-D as a candidate biomarker in risk assessments for subclinical tobacco smoke-induced lung damage. The data and derived conclusion warrant confirmation in a longitudinal population following chronic obstructive pulmonary disease initiation and development.
Assuntos
Biomarcadores/análise , Haplótipos/genética , Pneumopatias/diagnóstico , Polimorfismo de Nucleotídeo Único/genética , Proteína D Associada a Surfactante Pulmonar/genética , Fumar/efeitos adversos , Adolescente , Adulto , Idoso , Estudos Transversais , Dinamarca , Feminino , Volume Expiratório Forçado , Estudos de Associação Genética , Humanos , Pneumopatias/induzido quimicamente , Pneumopatias/genética , Masculino , Pessoa de Meia-Idade , Prognóstico , Surfactantes Pulmonares/metabolismo , Capacidade Vital , Adulto JovemRESUMO
CD163-L1 belongs to the group B scavenger receptor cysteine-rich family of proteins, where the CD163-L1 gene arose by duplication of the gene encoding the hemoglobin scavenger receptor CD163 in late evolution. The current data demonstrate that CD163-L1 is highly expressed and colocalizes with CD163 on large subsets of macrophages, but in contrast to CD163 the expression is low or absent in monocytes and in alveolar macrophages, glia, and Kupffer cells. The expression of CD163-L1 increases when cultured monocytes are M-CSF stimulated to macrophages, and the expression is further increased by the acute-phase mediator IL-6 and the anti-inflammatory mediator IL-10 but is suppressed by the proinflammatory mediators IL-4, IL-13, TNF-α, and LPS/IFN-γ. Furthermore, we show that CD163-L1 is an endocytic receptor, which internalizes independently of cross-linking through a clathrin-mediated pathway. Two cytoplasmic splice variants of CD163-L1 are differentially expressed and have different subcellular distribution patterns. Despite its many similarities to CD163, CD163-L1 does not possess measurable affinity for CD163 ligands such as the haptoglobin-hemoglobin complex or various bacteria. In conclusion, CD163-L1 exhibits similarity to CD163 in terms of structure and regulated expression in cultured monocytes but shows clear differences compared with the known CD163 ligand preferences and expression pattern in the pool of tissue macrophages. We postulate that CD163-L1 functions as a scavenger receptor for one or several ligands that might have a role in resolution of inflammation.