Microfibrillar-associated protein 4 (MFAP4) is located in the extracellular matrix (ECM). We sought to identify tissues with high levels of MFAP4 mRNA and MFAP4 protein expression. Moreover, we aimed to evaluate the significance of MFAP4 as a marker of cardiovascular disease (CVD) and to correlate MFAP4 with other known ECM markers, such as fibulin-1, osteoprotegerin (OPG), and osteopontin (OPN). Quantitative real-time PCR demonstrated that MFAP4 mRNA was more highly expressed in the heart, lung, and intestine than in other elastic tissues. Immunohistochemical studies demonstrated high levels of MFAP4 protein mainly at sites rich in elastic fibers and within blood vessels in all tissues investigated. The AlphaLISA technique was used to determine serum MFAP4 levels in a clinical cohort of 172 patients consisting of 5 matched groups with varying degrees of CVD: 1: patients with ST elevation myocardial infarction (STEMI), 2: patients with non-STEMI, 3: patients destined for vascular surgery because of various atherosclerotic diseases (stable atherosclerotic disease), 4: apparently healthy individuals with documented coronary artery calcification (CAC-positive), and 5: apparently healthy individuals without signs of coronary artery calcification (CAC-negative). Serum MFAP4 levels were significantly lower in patients with stable atherosclerotic disease than CAC-negative individuals (p<0.05). Furthermore, lower serum MFAP4 levels were present in patients with stable atherosclerotic disease compared with STEMI and non-STEMI patients (p<0.05). In patients with stable atherosclerotic disease, positive correlations between MFAP4 and both fibulin-1 (ρ = 0.50; p = 0.0244) and OPG (ρ = 0.62; p = 0.0014) were found. Together, these results indicate that MFAP4 is mainly located in elastic fibers and is highly expressed in blood vessels. The present study suggests that serum MFAP4 varies in groups of patients with different cardiovascular conditions. Further studies are warranted to describe the role of serum MFAP4 as a biomarker of stable atherosclerotic disease.

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.Elastic fibers are key extracellular matrix structural elements of connective tissues that undergo repeated stretch, such as large arteries and the lung (1). The fibers consist of two major components: an amorphous elastin core surrounded by a sheath of fibrillin-rich microfibrils (2). Elastin is a highly hydrophobic polymer of the soluble precursor tropoelastin (3). Tropoelastin is known to undergo a self-assembly process known as coacervation (4), often believed to be a first step in the process of elastic fiber maturation. Because of the high content of lysine residues within the tropoelastin sequence, its assembly into a polymeric form is stabilized by formation of desmosine crosslinks, catalyzed by the lysyl oxidase (LOX) 3 enzyme family (5).Microfibrils, the other major component of elastic fibers, provide the structural scaffold for the deposition of elastin globules. They consist primarily of fibrillin-1 and fibrillin-2, large glycoproteins with a high degree of homology (6). Apart from fibrillins, numerous accessory proteins have been shown to associate with microfibrils or elastin and promote formation of mature fibers, including fibulins and microfibril-associated glycoproteins (MAGPs) (7-10). The importance of proper elastogenesis has been underscored by gene deficiency studies: mice lacking elastin, LOX, or fibrillin-1 die shortly after birth because of vascular abnormalities (11-13).MFAP4 (microfibrillar-associated protein 4) is an extracellular matrix protein belonging to the fibrinogen-related domain (FReD) family. The family includes several proteins engaged in tissue homeostasis and innate immunity, such as FIBCD1 (fibrinogen C domain-containing 1), ficolins, and angiopoietins (14 -16). The crystal structure of the FReD of several family members has been solved (17-19). The ligand-binding site, designated S1, is described in all the proteins and is located in close proximity to the calcium-binding site. MFAP4 has been reported to form homodimeric structures that further oligomerize, but its definite oligomerization pattern has not been established (20).MFAP4 is conside...

Chitin is a highly acetylated compound and the second most abundant biopolymer in the world next to cellulose. Vertebrates are exposed to chitin both through food ingestion and when infected with parasites, and fungi and chitin modulate the immune response in different directions. We have identified a novel homotetrameric 55-kDa type II transmembrane protein encoded by the FIBCD1 gene and highly expressed in the gastrointestinal tract. The ectodomain of FIBCD1 is characterized by a coiled-coil region, a polycationic region and C-terminal fibrinogen-related domain that by disulfide linkage assembles the protein into tetramers. Functional analysis showed a high-affinity and calcium-dependent binding of acetylated components to the fibrinogen domain, and a function in endocytosis was demonstrated. Screening for ligands revealed that the FIBCD1 is a high-affinity receptor for chitin and chitin fragments. FIBCD1 may play an important role in controlling the exposure of intestine to chitin and chitin fragments, which is of great relevance for the immune defense against parasites and fungi and for immune response modulation.

Hepatic cirrhosis is a life-threatening disease arising from different chronic liver disorders. One major cause for hepatic cirrhosis is chronic hepatitis C. Chronic hepatitis C is characterized by a highly variable clinical course, with at least 20% developing liver cirrhosis within 40 years. Only liver biopsy allows a reliable evaluation of the course of hepatitis C by grading inflammation and staging fibrosis, and thus serum biomarkers for hepatic fibrosis with high sensitivity and specificity are needed. To identify new candidate biomarkers for hepatic fibrosis, we performed a proteomic approach of microdissected cirrhotic septa and liver parenchyma cells. In cirrhotic septa, we detected an increasing expression of cell structure associated proteins, including actin, prolyl 4-hydroxylase, tropomyosin, calponin, transgelin, and human microfibril-associated protein 4 (MFAP-4).

V ascular smooth muscle cell (VSMC) activation and phenotypic switching are critical for remodeling processes in vascular proliferative disorders, including intimal hyperplasia. Both the migratory and proliferative activities of VSMCs, as well as the interplay between the extracellular matrix (ECM) and integrin receptors essentially, contribute to neointimal hyperplasia and restrictive remodeling processes in the vessels.1 Among integrins, the particular role of integrin α V β 3 in the induction of VSMC responses has been shown both in vivo and in vitro.

Pulmonary surfactant protein A (SP‐A) is an oligomeric collectin that recognizes lipid and carbohydrate moieties present on broad range of micro‐organisms, and mediates microbial lysis and clearance. SP‐A also modulates multiple immune‐related functions including cytokine production and chemotaxis for phagocytes. Here we describe the molecular interaction between the extracellular matrix protein microfibril‐associated protein 4 (MFAP4) and SP‐A. MFAP4 is a collagen‐binding molecule containing a C‐terminal fibrinogen‐like domain and a N‐terminal located integrin‐binding motif. We produced recombinant MFAP4 with a molecular mass of 36 and 66 kDa in the reduced and unreduced states respectively. Gel filtration chromatography and chemical crosslinking showed that MFAP4 forms oligomers of four dimers. We demonstrated calcium‐dependent binding between MFAP4 and human SP‐A1 and SP‐A2. No binding was seen to recombinant SP‐A composed of the neck region and carbohydrate recognition domain of SP‐A indicating that the interaction between MFAP4 and SP‐A is mediated via the collagen domain of SP‐A. Monoclonal antibodies directed against MFAP4 and SP‐A were used for immunohistochemical analysis, which demonstrates that the two molecules colocalize both on the elastic fibres in the interalveolar septum and in elastic lamina of pulmonary arteries of chronically inflamed lung tissue. We conclude, that MFAP4 interacts with SP‐A via the collagen region in vitro, and that MFAP4 and SP‐A colocates in different lung compartments indicating that the interaction may be operative in vivo.

Background & 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 & Results: We applied single-cell transcriptomics to map the heterogeneity of sinusoid-associated cells in healthy and injured livers and reconstructed the singlelineage HSC trajectory from pericyte to myofibroblast. Stratifying each sinusoidal cell population by activation state, we projected shifts in sinusoidal communication upon injury. Weighted Gene Co-Expression Network Analysis of the HSC trajectory led to the identification of core genes, whose expression proved highly predictive of advanced fibrosis in NASH patients. 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 advance fibrosis in human NASH.

We have purified a glycoprotein from bovine lung washings using affinity chromatography on a maltoseaffinity column. On SDS-polyacrylamide gel electrophoresis the protein showed a molecular mass of 36 kDa in the reduced state and 66 kDa in the unreduced state. On gel permeation chromatography the apparent molecular mass was 250 kDa. N-terminal sequencing showed homology to the human matrix protein microfibril-associated protein (hMFAP4), and the glycoprotein was designated bovine MFAP4 (bMFAP4) .