Microfibrillar-associated protein 4 (MFAP4) is an extracellular glycoprotein found in elastic fibers with not 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, 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 F241 and S203 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.

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 α _V β ₃ 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 α _V β ₃ –dependent manner. Conclusions—: MFAP4 regulates integrin α _V β ₃ –induced VSMC proliferation and migration, as well as monocyte chemotaxis, and accelerates neointimal hyperplasia after vascular injury.

Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein belonging to the fibrinogen-related domain family. It has been localized to elastic fiber-rich regions in several tissues including the arteries, lungs, heart and skin. MFAP4 binds collagen, fibrillins and tropoelastin and contributes to the process of microfibrillar assembly and maturation of elastic fibers. MFAP4 can also bind RGD-dependent integrins, predominantly α_Vβ₃ and α_Vβ₅ through its N-terminal RGD sequence, modulating cellular behavior. Circulating MFAP4 was suggested as a robust biomarker for hepatitis C virus- and alcoholic liver disease-related liver fibrosis, cardiovascular disorders and chronic obstructive pulmonary disease. In mice, MFAP4 seems to have a widely redundant role under homeostatic conditions, as global MFAP4 deficiency results in a mild pulmonary phenotype, causing emphysema-like airspace enlargement that progresses with age. However, emerging in vivo and in vitro data suggest that MFAP4 is actively involved in the pathogenesis of remodeling-associated diseases, including fibrosis, cardiovascular disorders, aging, asthma and cancer through activation of integrin-mediated signaling as well as by modulating TGF-β pathway, thus supporting maladaptive matrix remodeling. This review summarizes the current knowledge about MFAP4 structure and localization, its mechanisms of action in disease-induced tissue remodeling as well as its potential role as a clinical biomarker.

Background Recently, several proteins of the extracellular matrix have been characterised as active contributors to allergic airway disease. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein abundant in the lung, whose biological functions remain poorly understood. In the current study we investigated the role of MFAP4 in experimental allergic asthma. Methods MFAP4-deficient mice were subjected to alum/ovalbumin and house dust mite induced models of allergic airway disease. In addition, human healthy and asthmatic primary bronchial smooth muscle cell cultures were used to evaluate MFAP4-dependent airway smooth muscle responses. Results MFAP4 deficiency attenuated classical hallmarks of asthma, such as eosinophilic inflammation, eotaxin production, airway remodelling and hyperresponsiveness. In wild-type mice, serum MFAP4 was increased after disease development and correlated with local eotaxin levels. MFAP4 was expressed in human bronchial smooth muscle cells and its expression was upregulated in asthmatic cells. Regarding the underlying mechanism, we showed that MFAP4 interacted with integrin αvβ5 and promoted asthmatic bronchial smooth muscle cell proliferation and CCL11 release dependent on phosphatidyloinositol-3-kinase but not extracellular signal-regulated kinase pathway. Conclusions MFAP4 promoted the development of asthmatic airway disease in vivo and pro-asthmatic functions of bronchial smooth muscle cells in vitro. Collectively, our results identify MFAP4 as a novel contributor to experimental asthma, acting through modulation of airway smooth muscle cells.

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.

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.