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
Lung cancer is one of the most common cancers and its incidence is rising around the world. Various studies suggest that miR-330 acts as a tumor-suppressor microRNA (miRNA) in different types of cancers, but precisely how has remained unclear. In this study, we investigate miR-330 expression in lung cancer patient samples, as well as in vitro, by studying how normalization of miR-330 expression affects lung cancer cellular phenotypes such as viability, apoptosis, proliferation, and migration. We establish that low miR-330 expression predicts poor lung cancer prognosis. Stable restoration of reduced miR-330 expression in lung cancer cells reduces cell viability, increases the fraction of apoptotic cells, causes G2/M cell cycle arrest, and inhibits cell migration. These findings are substantiated by increased mRNA and protein expression of markers for apoptosis via the intrinsic pathway, such as caspase 9, and decreased mRNA and protein expression of markers for cell migration, such as vimentin, C-X-C chemokine receptor type 4, and matrix metalloproteinase 9. We showed that reduced miR-330 expression predicts poor lung cancer survival and that stable restoration of miR-330 expression in lung cancer cells has a broad range of tumor-suppressive effects. This indicates that miR-330 is a promising candidate for miRNA replacement therapy for lung cancer patients.
Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Células A549 , Apoptose/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor/fisiologia , Humanos , Neoplasias Pulmonares/patologia , RNA Mensageiro/genéticaRESUMO
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
MicroRNAs (miRNAs) are important molecular regulatorsof cellular signaling and behavior. They alter gene expression by targeting messenger RNAs, including those encoding transcriptional regulators, such as HMGA2. While HMGA2 is oncogenic in various tumors, miRNAs may be oncogenic or tumor suppressive. Here, we investigate the expression of HMGA2 and the miRNA miR-330 in a patient with colorectal cancer (CRC) samples and their effects on oncogenic cellular phenotypes. We found that HMGA2 expression is increased and miR-330 expression is decreased in CRCs and each predicts poor long-term patient survival. Stably increased miR-330 expression in human colorectal cancer cells (HCT116) and SW480 CRC cell lines downregulate the oncogenic expression of HMGA2, a predicted miR-330 target. Additionally, this promotes apoptosis and decreases cell migration and viability. Consistently, it also decreases protein-level expression of markers for epithelial-to-mesenchymal-transition (Snail-1, E-cadherin, and Vascular endothelial growth factor receptors) and transforming growth factor ß signaling (SMAD3), as well as phospho- Protein kinase B (AKT) and phospho-STAT3 levels. We conclude that miR-330 acts as a tumor suppressor miRNA in CRC by suppressing HMGA2 expression and reducing cell survival, proliferation, and migration. Thus, we identify miR-330 as a promising candidate for miRNA replacement therapy for patients with CRC.
Assuntos
Apoptose/genética , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/genética , Proteína HMGA2/metabolismo , MicroRNAs/genética , Antígenos CD/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes Supressores de Tumor , Células HCT116 , Proteína HMGA2/genética , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Proteína Smad3/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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
OBJECTIVES: To study circulating MFAP4 in rheumatoid arthritis (RA) and its associations with clinical phenotype. METHODS: Early RA (ERA): 47 patients with newly diagnosed, treatment naïve RA were included. Serum MFAP4, clinical and laboratory disease variables were recorded serially during 12 months of intensive synovitis suppressive treatment. Long-standing RA (LRA): 317 patients participated, all receiving DMARD treatment. Disease activity, autoantibody status, extra-articular manifestations and cardiovascular morbidity were recorded. Paired serum and synovial fluid samples were obtained from 13 untreated ERA patients. Healthy blood donors served as reference points. MFAP4 was quantified by AlphaLISA immunoassay. Univariate, multivariate and mixed effects regression models were applied in the statistical analysis. RESULTS: ERA: MFAP4 increased from baseline and was significantly elevated at the 12-month follow-up, 17.8 U/l [12.6;24.1] vs. healthy controls, 12.7 U/l [9.5;15.6], p<0.001. MFAP4 did not correlate with joint counts or C-reactive protein. LRA: MFAP4 was increased, 25.9 U/l [20.4;33.7] vs. healthy controls, 17.6 U/l [13.7;21.2], p<0.0001, but did not correlate with disease activity measures or presence of extra-articular manifestations. Notably, MFAP4 correlated inversely with smoking (p<0.0001) and presence of antibodies against cyclic citrullinated peptides (anti-CCP), p=0.005. There was a positive association with systolic blood pressure, p=0.001 and co-occurrence of three cardiovascular events and/or risk factors, p<0.0001. The serum:synovial fluid MFAP4 ratio was 2:1. CONCLUSIONS: MFAP4 increases from diagnostic baseline despite intensive treatment but does not associate with synovitis at early or late stages of RA. Correlation patterns indicate that increased MFAP4 may reflect enhanced RA-related vascular remodelling.
Assuntos
Artrite Reumatoide/sangue , Proteínas de Transporte/sangue , Proteínas da Matriz Extracelular/sangue , Glicoproteínas/sangue , Sinovite/sangue , Artrite Reumatoide/patologia , Autoanticorpos , Comorbidade , Humanos , Peptídeos Cíclicos/imunologia , Líquido Sinovial , Sinovite/patologiaRESUMO
Estrogen receptors (ERs) are involved in the development of many types of malignant tumors, in particular, breast cancer. Among others, ERs affect cell growth, proliferation, and differentiation. The microRNA (miRNA) miR-142-3p has been shown to inhibit carcinogenesis by regulating various cellular processes, including cell cycle progression, cell migration, apoptosis, and invasion. It does so via targeting molecules involved in a range of signaling pathways. We surgically collected 20 ER-positive breast cancer samples, each with matched adjacent normal breast tissue, and measured the expression of miR-142-3p via quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatics methods, luciferase reporter assay, qRT-PCR, and western blot analysis were used to assess whether miR-142-3p could target ESR1, which encodes the estrogen receptor, in ER-positive breast cancer cells and patient samples. We also restored miRNA expression and performed cell viability, cytotoxicity, and colony formation assays. Western blot analysis and qRT-PCR were used to study the expression of apoptosis and stemness markers. We found that miR-142-3p is downregulated in ER-positive breast cancers. Restoration of miR-142-3p expression in ER-positive breast cancer cells reduced cell viability, induced apoptosis via the intrinsic pathway and decreased both colony formation and the expression of stem cell markers. Bioinformatic analysis predicted miR-142-3p could bind to 3'-untranslated region ESR1 messenger RNA (mRNA). Consistently, we demonstrated that miR-142-3p reduced luciferase activity in ER-positive breast cancer cells, and decreased ESR1 expression in both mRNA and protein levels. The results revealed miR-142-3p and ESR1 expression correlated negatively in ER-positive breast cancer samples. The results suggest miR-142-3p acts as a tumor suppressor via multiple mechanisms. Thus, restoration of miR-142-3p expression, for example, via miRNA replacement therapy, may represent an effective strategy for the treatment of ER-positive breast cancer patients.
RESUMO
BACKGROUND: Breast cancer is the most common type of cancer among women, and despite improved treatments, it remains a major challenge. However, improved mechanistic insight may lead to novel therapeutic strategies. miR-142-3p belongs to the miR-142 family and is involved in pathogenesis and metastasis of various types of malignancies by targeting several important messenger RNAs (mRNAs) including Bach-1. This is especially true for breast cancer, where Bach-1 is involved in the metastatic spread by deregulation of metastasis-associated genes. METHODS: In this study, we collected 24 breast cancer tissues with 24 adjusted normal tissues to measure the expression levels of miR-142-3p and Bach-1 mRNA using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and IHC. miR-142-3p targeting of Bach-1 expression in MCF-7 and MDA-MB-468 breast cancer cells was evaluated using bioinformatics, qRT-PCR and western blot analyses. The cellular proliferation, invasion, and migration were assessed by MTT, transwell matrigel and wound healing assay and the EMT-associated proteins C-X-C chemokine receptor type 4 (CXCR-4), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor receptor (VEGFR) were analyzed by western blot analysis. Also, the expression levels of tumor suppressors including miR-330, miR-145, and miR-34a were estimated by qRT-PCR. RESULTS: Analysis of paired specimens of primary malignant and normal tissues showed that miR-142-3p was downregulated, while Bach-1 mRNA and protein both were overexpressed in the breast cancer tumors. This inverse relationship was confirmed by cell line experiments demonstrating that miR-142-3p expression reduced Bach-1 mRNA levels. Furthermore, replacement of miR-142-3p could inhibit the proliferation, invasion, and migration in breast cancer potentially by targeting of Bach-1 mRNA and subsequent inhibition of CXCR4, MMP9, and VEGFR protein expressions. In addition, induction of miR-142-3p could upregulate tumor suppressor miRNAs, including miR-330, miR-145, and miR34a. CONCLUSION: For the first time, our results revealed that miR-142-3p could target Bach-1in breast cancer cells leading to the reduction of EMT-related proteins and reduced cell proliferation, invasion, and migration. The results also demonstrated that miR-142-3p could regulate important tumor suppressor miRNAs in breast cancer cells. In conclusion, our results suggest that miR-142-3p could be a good candidate for the targeted therapy of breast cancer, especially for the invasive type.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Neoplasias da Mama/metabolismo , MicroRNAs/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Invasividade Neoplásica , RNA Mensageiro , Regulação para CimaRESUMO
Objectives: Surfactant protein-D (SP-D), an innate immune defence molecule of the collectin family, is expressed in lungs and additional extrapulmonary epithelia. SP-D has immune modulatory and anti-microbial effects depending on its oligomerization. The ratio of high molecular weight (HMW): low molecular weight (LMW) SP-D in serum is mainly determined by the Met11Thr polymorphism (SNP rs721917). We aimed to study the SP-D serum level and the molecular size distribution in patients with untreated axial spondyloarthritis (axSpA) as compared with control subjects. Methods: Thirty-four patients with disease modifier untreated axSpA according to the ASAS criteria, age 19-63 years, disease duration 3.9 (2.2-5.6) years were included. Demographics, smoking habits, HLA-B27 status, ASDAS, BASDAI, BASFI, BASMI and visual analogue scale scores were recorded. SP-D in serum was measured by ELISA. DNA was isolated from whole blood and single nucleotide polymorphism rs721917 was genotyped. SP-D molecular size distribution was determined using gel filtration chromatography. Results: SP-D in serum did not differ between patients with axSpA and healthy controls, 1177 (869, 1536) vs 910 (494, 1682) (P = 0.35) and SP-D did not correlate with disease activity. However, the HMW/LMW ratio of SP-D in serum was significantly lower in axSpA, 0.38 (0.18, 0.53) compared with controls 1.49 (0.37, 3.24) when adjusting for the Met11Thr polymorphism, gender, age, BMI and smoking (P = 0.0004). There was no correlation between HMW/LMW ratio and CRP or composite diseases outcome measures. Conclusion: We suggest that predominance of LMW oligomeric variants of SP-D may enhance local or systemic inflammatory responses in axSpA.
Assuntos
Mediadores da Inflamação/sangue , Proteína D Associada a Surfactante Pulmonar/sangue , Proteína D Associada a Surfactante Pulmonar/genética , Espondilartrite/sangue , Espondilartrite/genética , Adulto , Estudos de Casos e Controles , Feminino , Genótipo , Antígeno HLA-B27 , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Multimerização Proteica , Adulto JovemRESUMO
BACKGROUND AND OBJECTIVE: A structural single nucleotide polymorphism rs721917 in the surfactant protein D (SP-D) gene, known as Met11Thr, was reported to influence the circulating levels and degree of multimerization of SP-D and was associated with both COPD and atopy in asthma. Moreover, disease-related processes are known to degrade multimerized SP-D, however, the degree of the protein degradation in these diseases is not clarified. We aimed to determine the distribution of multimerized (high molecular weight (HMW)) and non-multimerized (low molecular weight (LMW)) species of serum SP-D and their correlation with genetic polymorphisms and presence of disease in Lebanese COPD and asthmatic patients. METHODS: Serum SP-D levels were measured by ELISA in 88 COPD, 121 asthmatic patients and 223 controls. Randomly selected subjects were chosen for genotyping of rs721917 and multimerization studies. HMW and LMW SP-D were separated by gel permeation chromatography. RESULTS: Serum SP-D levels were significantly increased in patients with COPD, but not in asthmatic patients, when compared to controls. Met11Thr variation strongly affected serum SP-D levels and the degree of multimerization, but was not associated with COPD and asthma in the study. Remarkably, HMW/LMW serum SP-D ratio was significantly lower in Met11/Met11 COPD and asthmatic patients compared to controls. CONCLUSION: Collectively, non-multimerized species of serum SP-D were dominant in COPD and asthmatic patients suggesting that degradation of SP-D takes place to a significant degree in pulmonary disease. Assays that can separate SP-D proteolytic breakdown products or modified forms from naturally occurring SP-D trimers may result in optimal disease markers for pulmonary inflammatory diseases.
Assuntos
Asma/genética , DNA/genética , Polimorfismo de Nucleotídeo Único , Multimerização Proteica/genética , Doença Pulmonar Obstrutiva Crônica/genética , Proteína D Associada a Surfactante Pulmonar/genética , Adulto , Idoso , Asma/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/sangue , Proteína D Associada a Surfactante Pulmonar/sangue , Adulto JovemRESUMO
Macrophage migration inhibitory factor (MIF), a small conserved protein, is abundant in the immune- and central nervous system (CNS). MIF has several receptors and binding partners that can modulate its action on a cellular level. It is upregulated in neurodegenerative diseases and cancer although its function is far from clear. Here, we report the finding of a new binding partner to MIF, the serine protease HTRA1. This enzyme cleaves several growth factors, extracellular matrix molecules and is implicated in some of the same diseases as MIF. We show that the function of the binding between MIF and HTRA1 is to inhibit the proteolytic activity of HTRA1, modulating the availability of molecules that can change cell growth and differentiation. MIF is therefore the first endogenous inhibitor ever found for HTRA1. It was found that both molecules were present in astrocytes and that the functional binding has the ability to modulate astrocytic activities important in development and disease of the CNS.
Assuntos
Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologia , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células/fisiologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiologia , Células HEK293 , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica/fisiologiaRESUMO
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: Surfactant protein D (SP-D) is a host defense molecule of the innate immune system that enhances pathogen clearance and modulates inflammatory responses. We hypothesized that circulating SP-D levels are associated with chemotherapy-induced mucositis and infectious morbidity in children with acute lymphoblastic leukemia (ALL). PROCEDURE: In a prospective study, 43 children receiving treatment for ALL were monitored for mucosal toxicity from diagnosis through the induction phase of treatment. Serial blood draws were taken to determine the levels of SP-D, interleukin-6 (IL-6), C-reactive protein, and white blood cells. Data on fever, antibiotics, and bacteremia were collected. Baseline levels of circulating SP-D were compared with healthy controls. RESULTS: Baseline values of circulating SP-D were similar to levels in healthy controls (median: 829 ng/ml vs. 657 ng/ml, respectively, P > 0.05). After initiation of chemotherapy, a significant reduction in SP-D levels was observed at all time points: 704 ng/ml at day 8, 413 ng/ml at day 15, 395 ng/ml at day 22, and 520 ng/ml at day 29 (all, P < 0.05). No significant associations between SP-D values, the occurrence of mucosal toxicity, or infectious morbidity were observed. However, loss of circulating SP-D from days 8 to 15 was associated with more systemic inflammation, and lower SP-D values at day 15 were associated with elevated intestinal mucositis scores (P < 0.05). CONCLUSIONS: The current study supports the hypothesis that the detrimental effect of chemotherapy on patients' immune functions includes decreased circulating levels of innate mucosal molecules such as SP-D, potentially aggravating mucosal and systemic inflammatory responses.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Mucosite/diagnóstico , Pneumonia/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Proteína D Associada a Surfactante Pulmonar/sangue , Adolescente , Proteína C-Reativa/análise , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Interleucina-6/sangue , Masculino , Mucosite/sangue , Mucosite/induzido quimicamente , Mucosa/efeitos dos fármacos , Estadiamento de Neoplasias , Pneumonia/sangue , Pneumonia/induzido quimicamente , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Estudos ProspectivosRESUMO
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.