Microfibrillar-associated Protein Research Articles

MFAP5 suppression inhibits migration/invasion, regulates cell cycle and induces apoptosis via promoting ROS production in cervical cancer

Cervical cancer is one of the most lethal types of cancer among female. Microfibrillar-associated protein 5 (MFAP5) is an extracellular matrix (ECM) glycoprotein, and is confirmed to be involved in cell signaling during microfibril assembly, elastinogenesis and cell survival. However, the role of MFAP5 in cervical cancer development and progression remains poorly understood. In the study, MFAP5 was over-expressed in human cervical cancers, and in different cervical cancer cell lines. Patients suffering from cervical cancer with low MFAP5 expression exhibited better survival rate. Suppressing MFAP5 in cervical cancer cells markedly reduced the cell proliferation, migration and invasion by modulating epithelial-mesenchymal transition (EMT)-related signaling pathway. In addition, MFAP5 knockdown induced large number of cells distributed in G2/M phase, along with reduced Cyclin B1, Cyclin D1 and cyclin-dependent kinase 4 (CDK4) expressions, and enhanced p21 and p53 levels. Moreover, apoptosis was highly induced by MFAP5 silence through reducing Bcl-xl and Bcl-2 expressions, and promoting Bax, cleaved Caspase-3 and poly (ADP-Ribose) polymerase (PARP) expressions in cervical cancer cells. Reactive oxygen species (ROS) production levels were also higher in MFAP5-knockdown cells, along with Jun-N-terminal kinase (JNK) activation. Importantly, we found that MFAP5 knockdown-inhibited cervical cancer cell growth was dependent on ROS production. Finally, the depletion of MFAP5 prevented cervical cancer progression in vivo. In summary, our study identified a critical role played by MFAP5 in the progression of cervical cancer and the potential mechanisms by which exerted its effects, indicating that targeting MFAP5-related pathways could be conducive to the therapies for cervical cancer.

Potential Role of Microfibrillar-Associated Protein 4, Fibrotic Indices and Oxidative Stress in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. In an attempt to understand some potential mechanisms of persistence and oncogenicity of Hepatitis C virus (HCV)-related HCC, microfibrillar-associated protein 4 (MFAP4), fibrotic indices and oxidative status biomarkers were assessed in the sera of 50 patients with HCV-associated HCC, 25 patients with HCV-related liver cirrhosis and 15 healthy individuals. Serum oxidized Coenzyme Q10 (CoQ10) and malondialdehyde showed significant elevation in HCC patients compared to the control group (p < 0.001), as well as cirrhotic patients (p < 0.05 and p < 0.001, respectively), while serum glutathione content and superoxide dismutase activity were significantly decreased in HCC patients compared to the control group (p < 0.001). Serum MFAP4, aspartate aminotransferase to platelet ratio index (APRI), fibrosis index based on the 4 factors (FIB-4) and Forns index showed significant increase in HCC patients compared to the control group (p < 0.001), while only APRI and FIB-4 were significantly different between HCC and cirrhotic patients (p < 0.05), with a sensitivity of 86% and 92%, respectively, at cut off ≥0.7 for APRI and ≥1.57 for FIB-4. Therefore, increasing oxidative stress and fibrosis might mediate HCV induced cirrhosis and HCC. APRI and FIB-4 may be used as a simple non-expensive formula for the screening of HCC rather than MFAP4.

PO-174 Evaluation of immunohistochemical expression of microfibrillar-associated protein 5 (MFAP5) in invasive breast carcinoma of no special type

IntroductionBreast cancer is one of the most malignant cancer types which is the second leading cause of cancer-associated deaths in women. Breast cancer begins as a local disease which can then metastasize to lymph nodes and distant sites. The metastasis of breast cancer is mainly to bone, lung and liver. Being the leading cause of cancer related deaths, it is essential to understand the complex metastasis phenomenon to develop new diagnostic and therapeutic strategies. In this study, we aim to develop a lab-on-a-chip (LOC) system to detect the metastatic capacities of breast cancer cells to different target tissues.Material and methodsThe triple negative breast cancer cell line MDA-MB-231 was selected based on its high metastatic capacity, while normal lung, liver and bone fibroblasts WI-38, BRL3A and HS-5 and human umbilical vein endothelial cell line HUVEC-C were used to generate target tissue microenvironments and endothelial monolayer, respectively. A three-channel LOC system, one for endothelial monolayer formation, one for mimicking target tissue microenvironment and one for media supply, was used. The seeding cell densities, common medium composition and ECM components for microenvironment were optimised for the LOC system. Following addition of cancer cell lines to the endothelial monolayer channel, invasion and extravasation capacities to different target sites were determined by tracking 3D distribution of fluorescently labelled cancer cells up to four days.Results and discussionsMDA-MB-231 cells were shown to invade and extravasate preferentially to lung and liver microenvironments, while their movement to bone or cell-free target sites was limited. The observed results were in agreement with the previously reported phenotype of MDA-MB-231 cell lines.ConclusionOur results show that tissue- specific cell lines in a LOC system can be used to mimic target tissue microenvironments and evaluate different invasion and extravasation capacities of breast cancer cell lines to different target sites.

Microfibrillar-associated protein 4 variation in symptomatic peripheral artery disease

BackgroundSymptomatic 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.MethodsA 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.ResultsPatients 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).ConclusionssMFAP4 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.

Increased plasma microfibrillar-associated protein 4 is associated with atrial fibrillation and more advanced left atrial remodelling.

IntroductionThis study aimed to evaluate the relationship of plasma microfibrillar-associated protein 4 (MFAP4) to atrial fibrillation (AF) and atrial structural remodelling.Material and methodsPlasma MFAP4 levels were measured in 92 patients with AF (61 paroxysmal AF (PAF) patients and 31 persistent AF (PersAF) patients) and 71 control subjects without AF. Linear and logistic multivariate regression analyses were performed to determine the potential value of MFAP4 for predicting the incidence of AF and left atrial size. Then, plasma and atrial protein levels of MFAP4 and its association with atrial fibrosis ratio were analysed in an atrial-specific fibrosis rat model.ResultsThere were significant differences in MFAP4 levels based on clinical group, with a gradient from control (1.71 ±0.53 ng/ml) to PAF (1.98 ±0.53 ng/ml) to PersAF (2.09 ±0.76 ng/ml) (p < 0.01). With multivariate analyses, plasma MFAP4 was found to be an independent determinant of left atrial diameter in AF patients. In atrial fibrosis rats, both plasma MFAP4 and atrial MFAP4 protein levels increased in atrial fibrosis rats and positively correlated with atrial fibrosis severity.ConclusionsPlasma MFAP4 was increased in patients with AF and was highest in those with PersAF; both plasma MFAP4 and atrial MFAP4 protein expression were directly associated with the extent of LA structural remodelling.

Microarray analysis for delineating the gene expression in biopsies of gastrocnemius muscle of patients with chronic critical limb ischaemia compared with non-ischaemic controls.

Microarray analysis has been carried out in this pilot study to compare delineated gene expression profiles in the biopsies of skeletal muscle taken from patients with chronic critical limb ischaemia (CLI) and non-ischaemic control subjects. Biopsy of gastrocnemius muscle was obtained from six patients with unreconstructed CLI referred for surgical major amputation. As control, biopsies of six patients undergoing elective knee arthroplasty without evidence of peripheral arterial occlusive disease were taken. The differences in gene expression associated with angiogenic processes in specimens obtained from ischaemic and non-ischaemic skeletal muscle were confirmed by quantitative real-time polymerase chain reaction (PCR) analysis. Compared with non-ischaemic skeletal muscle biopsy of chronic-ischaemic skeletal muscle contained 55 significantly up-regulated and 45 down-regulated genes, out of which 64 genes had a known genetic product. Tissue samples of ischaemic muscle were characterized by increased expression of cell survival factors (e. g. tissue factor pathway inhibitor 2) in combination with reduced expression of cell proliferation effectors (e. g. microfibrillar-associated protein 5 and transferrin receptor). The expression of growth factors (e. g. early growth response 3 and chemokine receptor chemokine C-X-C motif ligand 4) which play a central role in arterial and angiogenic processes and anti-angiogenetic factors (e. g. pentraxin 3) were increased in chronic ischaemic skeletal muscle. An increased expression of extracellular matrix proteins (e. g. cysteine-rich angiogenic inducer 61) was also observed. Gene expression profiles in biopsies of gastrocnemius muscle in patients with chronic critical limb ischaemia showed an increase in pro-survival factors, extracellular matrix protein deposition, and impaired proliferation, compared with non-ischaemic controls. Further studies are required to analyse the endogenous repair mechanism.

MFAP5 promotes tumor progression and bone metastasis by regulating ERK/MMP signaling pathways in breast cancer

Breast cancer accounts for about 30% of all cancers in women, while approximately 70% breast cancer patients developed bone metastases throughout the course of their disease, highlighting the importance of exploring new therapeutic targets. Microfibrillar-associated protein 5 (MFAP5) is a component of extracellular elastic microfibril which has been confirmed to function in tissue development and cancer progression. But the role of MFAP5 in breast cancer remains unclear. The present study demonstrated that MFAP5 was up-regulated in breast cancers compared with that in normal breast tissues, and further increased in breast cancer bone metastasis. Functionally, MFAP5 overexpression accelerated breast cancer cell proliferation and migration, while an opposite effect was observed when MFAP5 was knocked down. In addition, up-regulation of MFAP5 increased the expression of MMP2 and MMP9 and activated the ERK signaling pathway. Conversely, inhibition of MFAP5 suppressed the expression of MMP2, MMP9, p-FAK, p-Erk1/2 and p-cJun. These findings may provide a better understanding about the mechanism of breast cancer and suggest that MFAP5 may be a potential prognostic biomarker and therapeutic target for breast cancer, especially for bone metastasis of breast cancer.

Preliminary analysis of proteome alterations in non-aneurysmal, internal mammary artery tissue from patients with abdominal aortic aneurysms.

ObjectiveThe pathogenesis of abdominal aortic aneurysms (AAA) involves a disturbed balance of breakdown and buildup of arterial proteins. We envision that individuals with AAA carry generalized arterial protein alterations either because of effects of genetically or environmental AAA risk factors or because of compensatory changes due to signaling molecules released from the affected aneurysmal tissue.ApproachProtein extraction and quantitative proteome analysis by LC-MS/MS (liquid chromatography-mass spectrometry) was done on individual samples from the internal mammary artery from 11 individuals with AAA and 33 sex- and age-matched controls without AAA. Samples were selected from a biobank of leftover internal mammary arterial tissue gathered at coronary by-pass operations.ResultsWe identified and quantitated 877 proteins, of which 44 were differentially expressed between the two groups (nominal p-values without correction for multiple testing). Some proteins related to the extracellular matrix displayed altered concentrations in the AAA group, particularly among elastin-related molecules [elastin, microfibrillar-associated protein 4 (MFAP4), lysyl oxidase]. In addition, several histones e.g. (e.g. HIST1H1E, HIST1H2BB) and other vascular cell proteins (e.g. versican, type VI collagen) were altered.ConclusionsOur results support the notion that generalized alterations occur in the arterial tree in patients with AAA. Elastin-related proteins and histones seem to be part of such changes, however these preliminary results require replication in an independent set of specimens and validation by functional studies.

Cancer-associated fibroblasts regulate endothelial adhesion protein LPP to promote ovarian cancer chemoresistance.

The molecular mechanism by which cancer-associated fibroblasts (CAFs) confer chemoresistance in ovarian cancer is poorly understood. The purpose of the present study was to evaluate the roles of CAFs in modulating tumor vasculature, chemoresistance, and disease progression. Here, we found that CAFs upregulated the lipoma-preferred partner (LPP) gene in microvascular endothelial cells (MECs) and that LPP expression levels in intratumoral MECs correlated with survival and chemoresistance in patients with ovarian cancer. Mechanistically, LPP increased focal adhesion and stress fiber formation to promote endothelial cell motility and permeability. siRNA-mediated LPP silencing in ovarian tumor-bearing mice improved paclitaxel delivery to cancer cells by decreasing intratumoral microvessel leakiness. Further studies showed that CAFs regulate endothelial LPP via a calcium-dependent signaling pathway involving microfibrillar-associated protein 5 (MFAP5), focal adhesion kinase (FAK), ERK, and LPP. Thus, our findings suggest that targeting endothelial LPP enhances the efficacy of chemotherapy in ovarian cancer. Our data highlight the importance of CAF-endothelial cell crosstalk signaling in cancer chemoresistance and demonstrate the improved efficacy of using LPP-targeting siRNA in combination with cytotoxic drugs.

GW28-e1196 The role of plasma microfibrillar-associated protein 4 in patients with atrial fibrillation

GW28-e1196 The role of plasma microfibrillar-associated protein 4 in patients with atrial fibrillation

Intraocular expression of microfibrillar‐associated protein 4 (MFAP4) in patients with neovascular age‐related macular degeneration (nAMD)

PurposePharmacological inhibition of the constitutively expressed extracellular matrix integrin ligand MFAP4 is previously shown with efficacy in mouse model of choroidal neovascularization. To evaluate the intraocular expression of MFAP4 in the anterior chamber (CA) and vitreous body (VB) in patients with nAMD (cases) as compared to controls.MethodsAqueous humour from 29 patients and vitreous humour of 54 patients were collected during elective cataract surgery and pars plana vitrectomy, respectively. Samples from the CA consisted of three cases and 26 controls and from the VB of 42 cases and 12 controls. The 42 samples from the VB were further divided into four subgroups depending on the subtype of nAMD (CNV with retinal hemorrhage, CNV without retinal hemorrhage, fibrosis, PE‐detachment). Samples were analyzed by the AlphaLISA technique. Wilcoxon rank‐sum test and Kruskall‐Wallis test was used to test for differences between two and multiple groups, respectively.ResultsMedian MFAP4 was significantly lower in the anterior chamber as compared to the VB (7.9 Units/ml vs. 17.9 Units/ml, p &lt; 0.0001). There was no difference in the median MFAP4 between cases and controls (CA: 8.1 Units/ml vs. 7.9 Units/ml, p = 0.33) (VB: 17.9 Units/ml vs. 17.8, p = 0.79). Neither were there any differences between nAMD‐subgroups in the VB (18.7 Units/ml vs. 24.4 Unit/ml vs 15.1 Units/ml vs. 17.5 Units/ml, p = 0.57).ConclusionsThese preliminary results demonstrated intraocular expression of MFAP4 in the human eye. Further, a higher expression of MFAP4 was demonstrated in the vitreous body as compared to the anterior chamber. Vitreal MFAP4 does not appear to be regulated with neovascular disease supporting that MFAP4 is constitutively expressed and permissive for integrin activation during neovascularization.

Association between microfibrillar-associated protein 4 (MFAP4) and micro- and macrovascular complications in long-term type 1 diabetes mellitus.

To evaluate microfibrillar-associated protein 4 (MFAP4) as a marker of micro- and macrovascular complications in patients with type 1 diabetes. This cross-sectional study included 203 persons with a long duration of type 1 diabetes from a population-based cohort ascertained in the former Funen County, Denmark. Detection of plasma-MFAP4 (pMFAP4) was performed by the AlphaLISA Technique. Diabetic retinopathy (DR) was graded in accordance with the Early Treatment Diabetic Retinopathy Study adaptation of the modified Airlie House classification. A monofilament test was used to test for neuropathy, and nephropathy was evaluated in a single spot urine sample. Data describing macrovascular disease were obtained from the Danish National Patient Register. Median age and duration of diabetes were 58.7 and 43years, respectively, and 61% were males. High levels of pMFAP4 were found in participants of old age, in women and in non-smokers (p<0.05). In a multiple logistic regression model, patients with high levels of pMFAP4 were more likely to have diabetic neuropathy (OR 2.47 for quartile 4 versus quartile 1, 95% CI 1.01-6.03). No association was found between pMFAP4 and proliferative diabetic retinopathy, nephropathy or macrovascular disease. No association between pMFAP4 and macrovascular vascular complications was found. However, high levels of pMFAP4 correlated independently with diabetic neuropathy. Further studies on the predictive value of increased circulating MFAP4 in diabetic neuropathy are warranted.

Evaluation of the biomarker candidate MFAP4 for non-invasive assessment of hepatic fibrosis in hepatitis C patients.

BackgroundThe 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).MethodsMFAP4 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.ResultsMFAP4 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).ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0952-3) contains supplementary material, which is available to authorized users.

Identification of Human Juvenile Chondrocyte-Specific Factors that Stimulate Stem Cell Growth.

Although regeneration of human cartilage is inherently inefficient, age is an important risk factor for osteoarthritis. Recent reports have provided compelling evidence that juvenile chondrocytes (from donors below 13 years of age) are more efficient at generating articular cartilage as compared to adult chondrocytes. However, the molecular basis for such a superior regenerative capability is not understood. To identify the cell-intrinsic differences between juvenile and adult cartilage, we have systematically profiled global gene expression changes between a small cohort of human neonatal/juvenile and adult chondrocytes. No such study is available for human chondrocytes although young and old bovine and equine cartilage have been recently profiled. Our studies have identified and validated new factors enriched in juvenile chondrocytes as compared to adult chondrocytes including secreted extracellular matrix factors chordin-like 1 (CHRDL1) and microfibrillar-associated protein 4 (MFAP4). Network analyses identified cartilage development pathways, epithelial-mesenchymal transition, and innate immunity pathways to be overrepresented in juvenile-enriched genes. Finally, CHRDL1 was observed to aid the proliferation and survival of bone marrow-derived human mesenchymal stem cells (hMSC) while maintaining their stem cell potential. These studies, therefore, provide a mechanism for how young cartilage factors can potentially enhance stem cell function in cartilage repair.

The role of microfibrillar-associated protein 4 (MFAP4) in the formation and function of splenic compartments during embryonic and adult life.

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 8weeks 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.

Transcriptomic analysis of stage 1 versus advanced adult granulosa cell tumors.

Ovarian granulosa cell tumors (GCT) are hormonally-active neoplasms characterized, in the adult-subtype, by a mutation in the FOXL2 gene (C134W). They exhibit an indolent course with an unexplained propensity for late recurrence; ∼80% of patients with aggressive, advanced stage tumors die from their disease; aside from surgery, therapeutic options are limited. To identify the molecular basis of advanced stage disease we have used whole transcriptome analysis of FOXL2 C134W mutation positive adult (a)GCT to identify genes that are differentially expressed between early (stage 1) and advanced (stage 3) aGCT. Transcriptome profiles for early (n = 6) and stage 3 (n = 6) aGCT, and for the aGCT-derived KGN, cell line identified 24 genes whose expression significantly differs between the early and stage 3 aGCT. Of these, 16 were more abundantly expressed in the stage 3 aGCT and 8 were higher in the stage 1 tumors. These changes were further examined for the genes which showed the greatest fold change: the cytokine CXCL14, microfibrillar-associated protein 5, insulin-like 3 and desmin. Gene Set Enrichment Analysis identified overexpression of genes on chromosome 7p15 which includes the homeobox A gene locus. The analysis therefore identifies a small number of genes with clearly discriminate patterns of expression arguing that the clinicopathological-derived distinction of the tumor stage is robust, whilst confirming the relative homogeneity of expression for many genes across the cohort and hence of aGCT. The expression profiles do however identify several overexpressed genes in both stage 1 and/or stage 3 aGCT which warrant further study as possible therapeutic targets.

Characterization of Microfibrillar-associated Protein 4 (MFAP4) as a Tropoelastin- and Fibrillin-binding Protein Involved in Elastic Fiber Formation

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.

Microfibrillar-Associated Protein 4 (MFAP4) Genes in Pelteobagrus fulvidraco play a Novel Role in Innate Immune Response

Both mannose-binding lectins (MBLs) and ficolins recognize and bind carbohydrates in pathogens and activate complement leading to opsonization, leukocyte activation, and direct pathogen clean. While MBLs have been reported in many fish species, the function of ficolins had not been identified in Pelteobagrus fulvidraco, despite their importance in invertebrate and higher vertebrate innate immunity. Although microfibrillar-associated protein 4 (MFAP4) this protein has similar fibrinogen-like domain, has expression in fish, the role of it has not been dissected. We cloned three MFAP4 genes (1MFAP4, 2MFAP4 and 3MFAP4) from the yellow catfish, P. fulvidraco. The homology similarity of MFAP4 was 78% with Astyanax mexicanus of Cypriniformes. The yellow catfish MFAP4 transcripts expression analysis revealed difference of patterns of homeostatic expression among the genes in gill, blood, muscle, gonad, liver, brain, spleen, kidney, heart, intestine from male and female, respectively. Expression of the three MFAP4 transcripts showed significant expression changes in 4 h after infection with either Edwardsiella ictaluri or Flavobacterium columnare, which has modulation of expression continuing up to 24 h or 7 d following pathogen exposure. Several different tissues and gene-specific patterns were captured and transcript expression changes of >11.45-fold were observed over the course of the bacterial challenges. This information elucidates the functions of individual MFAP4 genes in regards to pathogen recognition, binding, and their larger immune context.

MFAP4 Promotes Vascular Smooth Muscle Migration, Proliferation and Accelerates Neointima Formation.

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. 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 αVβ3 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 αVβ3-dependent manner. MFAP4 regulates integrin αVβ3-induced VSMC proliferation and migration, as well as monocyte chemotaxis, and accelerates neointimal hyperplasia after vascular injury.

Microfibrillar-Associated Protein 4: A Potential Biomarker for Screening for Liver Fibrosis in a Mixed Patient Cohort.

Background and AimsA method for assessment of liver fibrosis and cirrhosis without the need for a liver biopsy is desirable. Microfibrillar-associated protein 4 (MFAP4) is a suggested biomarker for identification of high-risk patients with severe fibrosis stages. This study aimed to examine associations between plasma MFAP4 (pMFAP4) and transient elastography or chronic hepatitis C virus infection in drug users and in a mixed patient cohort with increased risk of liver disease. Moreover, the study aimed to identify comorbidities that significantly influence pMFAP4.MethodspMFAP4 was measured in samples from 351 drug users attending treatment centres and from 248 acutely hospitalized medical patients with mixed diagnoses. Linear and logistic multivariate regression analyses were performed and nonparametric receiver operating characteristic-curves for cirrhosis were used to estimate cut-off points for pMFAP4. Univariate and subgroup analyses were performed using non-parametric methods.ResultspMFAP4 increased significantly with liver fibrosis score. pMFAP4 was significantly associated with chronic viral infection in the drug users and with transient elastography in both cohorts. In the mixed patient cohort, pMFAP4 was significantly increased among patients with a previous diagnosis of liver disease or congestive heart failure compared to patients with other diagnoses.ConclusionspMFAP4 has the potential to be used as an outreach-screening tool for liver fibrosis in drug users and in mixed medical patients. pMFAP4 level is positively associated with transient elastography, but additional studies are warranted to validate the possible use of pMFAP4 in larger cohorts and in combination with transient elastography.