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

$\textbf {Background:}$ 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). $\textbf {Methods:}$ MFAP4 levels were measured using an AlphaLISA immunoassay in a retrospective study including $\it n$ = 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. $\textbf {Results:}$ MFAP4 levels were shown to differ between no to moderate fibrosis stages F0–F2 and severe stages (F3 and F4) with high statistical significance ($\it t$ test on log scale, $\it p$ 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). $\textbf {Conclusions:}$ 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

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

FIBCD1 Binds Aspergillus fumigatus and Regulates Lung Epithelial Response to Cell Wall Components

Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus of clinical importance associated with development of various pulmonary diseases and allergic hypersensitivity reactions. It is protected against environmental stress by a cell wall that contains polysaccharides such as chitin. We previously demonstrated that fibrinogen C domain-containing protein 1 (FIBCD1) is a membrane-bound protein that binds chitin through a conserved S1 binding site and is expressed in intestinal epithelium and salivary glands. Here, we further localized FIBCD1 protein expression at the surface of bronchial and alveolar human lung epithelium, observed recognition of A. fumigatus cell wall with S1 site-independent recognition. We observed FIBCD1-mediated suppression of IL-8 secretion, mucin production, and transcription of genes associated with airway inflammation and homeostasis in FIBCD1-transfected lung epithelial cells. These modulations were generally enforced by stimulation with A. fumigatus cell wall polysaccharides. In parallel, we demonstrated a FIBCD1-mediated modulation of IL-8 secretion induced by TLR2,−4, and −5. Collectively, our findings support FIBCD1 as a human lung epithelial pattern recognition receptor that recognizes the complex A. fumigatus cell wall polysaccharides and modulates the lung epithelial inflammatory response by suppressing inflammatory mediators and mucins

Surfactant Protein D in Respiratory and Non-Respiratory Diseases

Surfactant protein D (SP-D) is a multimeric collectin that is involved in innate immune defense and expressed in pulmonary, as well as non-pulmonary, epithelia. SP-D exerts antimicrobial effects and dampens inflammation through direct microbial interactions and modulation of host cell responses via a series of cellular receptors. However, low protein concentrations, genetic variation, biochemical modification, and proteolytic breakdown can induce decomposition of multimeric SP-D into low-molecular weight forms, which may induce pro-inflammatory SP-D signaling. Multimeric SP-D can decompose into trimeric SP-D, and this process, and total SP-D levels, are partly determined by variation within the SP-D gene, SFTPD. SP-D has been implicated in the development of respiratory diseases including respiratory distress syndrome, bronchopulmonary dysplasia, allergic asthma, and chronic obstructive pulmonary disease. Disease-induced breakdown or modifications of SP-D facilitate its systemic leakage from the lung, and circulatory SP-D is a promising biomarker for lung injury. Moreover, studies in preclinical animal models have demonstrated that local pulmonary treatment with recombinant SP-D is beneficial in these diseases. In recent years, SP-D has been shown to exert antimicrobial and anti-inflammatory effects in various non-pulmonary organs and to have effects on lipid metabolism and pro-inflammatory effects in vessel walls, which enhance the risk of atherosclerosis. A common SFTPD polymorphism is associated with atherosclerosis and diabetes, and SP-D has been associated with metabolic disorders because of its effects in the endothelium and adipocytes and its obesity-dampening properties. This review summarizes and discusses the reported genetic associations of SP-D with disease and the clinical utility of circulating SP-D for respiratory disease prognosis. Moreover, basic research on the mechanistic links between SP-D and respiratory, cardiovascular, and metabolic diseases is summarized. Perspectives on the development of SP-D therapy are addressed

MFAP4-Mediated Effects in Elastic Fiber Homeostasis, Integrin Signaling and Cancer, and Its Role in Teleost Fish

Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix (ECM) protein belonging to the fibrinogen-related domain superfamily. MFAP4 is highly expressed in elastin-rich tissues such as lung, blood vessels and skin. MFAP4 is involved in organization of the ECM, regulating proper elastic fiber assembly. On the other hand, during pathology MFAP4 actively contributes to disease development and progression due to its interactions with RGD-dependent integrin receptors. Both tissue expression and circulating MFAP4 levels are associated with various disorders, including liver fibrosis and cancer. In other experimental models, such as teleost fish, MFAP4 appears to participate in host defense as a macrophage-specific innate immune molecule. The aim of this review is to summarize the accumulating evidence that indicates the importance of MFAP4 in homeostasis as well as pathological conditions, discuss its known biological functions with special focus on elastic fiber assembly, integrin signaling and cancer, as well as describe the reported functions of non-mammalian MFAP4 in fish. Overall, our work provides a comprehensive overview on the role of MFAP4 in health and disease

Microfibrillar-associated protein 4 variation in symptomatic peripheral artery disease

Abstract 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

Viral aggregating and opsonizing activity in collectin trimers

Collectins are collagenous lectins present in blood, respiratory lining fluid, and other mucosal secretions that play important roles in innate defense against infection. The collectin, surfactant protein D (SP-D), limits infection by viruses and bacteria in the respiratory tract, eye, and female genital tract. Multimeric SP-D has strong antiviral activity and is a potent viral and bacterial agglutinin and opsonin; however, trimers composed of the neck and carbohydrate recognition domain (hSP-D-NCRD) of SP-D lack these activities. We now show that, in contrast, a trimeric neck and CRD construct of bovine serum collectin CL-46 induces aggregation of influenza A virus (IAV) and potently increases IAV uptake by neutrophils. CL-46-NCRD showed calcium-dependent and sugar-sensitive binding to both neutrophils and IAV. Replacement of specific residues of the CRD of human SP-D with those found in bovine serum collectins conferred opsonizing activity. The most effective substitution involved replacement of arginine 343 with valine (hSP-D-NCRD/R343V). hSP-D-NCRD/R343V greatly increased viral uptake by neutrophils and monocytes and also potentiated neutrophil respiratory burst responses. These effects were further increased by cross-linking of hSP-D-NCRD/R343V trimers with MAbs directed against areas of the hSP-D-NCRD not involved in viral binding. Unlike the wild-type human SP-D hSP-D-NCRD, hSP-D-NCRD/R343V also induced viral aggregation. These results indicate that collectins can act as opsonins for IAV even in the absence of the collagen domain or higher order multimerization. This may involve increased affinity of individual CRDs for glycoconjugates displayed on host cells or the viral envelope

Association between the surfactant protein D (SFTPD) gene and subclinical carotid artery atherosclerosis

ObjectiveSurfactant protein D (SP-D) is a defense collectin with inflammation-modulating properties. SP-D deficiency inhibits atherosclerosis in vivo, and the circulatory SP-D levels have been previously associated with cardiovascular disease mortality. We hypothesized that plasma SP-D (pSP-D) and SP-D gene (SFTPD) single nucleotide polymorphisms (SNPs) are risk factors for atherosclerosis.MethodsWe evaluated individuals who were all 60 years old and participated in The Glostrup Population Study. Subclinical atherosclerosis was diagnosed based on the ultrasonographic measurement of intima-media thickness (IMT) and protruding plaques in the right carotid artery. Associations between cardiovascular traits and the levels of pSP-D (n = 687) or two coding SFTPD SNPs rs3088308 and rs721917 (n = 396) were investigated using multiple linear regressions and logistic regressions.ResultsThere was no significant association between pSP-D and the presence of plaques or IMT. The SFTPD SNP rs3088308 was nominally associated with the presence of plaques, and rs721917 was nominally associated with IMT. The directions of effects of associations were markedly dependent on current smoking status.ConclusionsThe results do not support that pSP-D levels influence the development of subclinical atherosclerosis. However, the SFTPD SNP data support previous observations from animal studies that SP-D plays a role in the etiology of atherosclerotic disease development. The nominal significant effects are likely to be mediated by structural variant SP-D modulation of effects of tobacco smoking and are independent of pSP-D levels. The data warrant confirmation in larger cohorts