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The extracellular matrix is an intricate and essential network of proteins and nonproteinaceous components that provide a conducive microenvironment for cells to regulate cell function, differentiation, and survival. Fibronectin is one key component in the extracellular matrix that participates in determining cell fate and function crucial for normal vertebrate development. Fibronectin undergoes time-dependent expression patterns during stem cell differentiation, providing a unique stem cell niche. Mutations in fibronectin have been recently identified to cause a rare form of skeletal dysplasia with scoliosis and abnormal growth plates. Even though fibronectin has been extensively analyzed in developmental processes, the functional role and importance of this protein and its various isoforms in skeletal development remain less understood. This review attempts to provide a concise and critical overview of the role of fibronectin isoforms in cartilage and bone physiology and associated pathologies. This will facilitate a better understanding of the possible mechanisms through which fibronectin exerts its regulatory role on cellular differentiation during skeletal development. The review discusses the consequences of mutations in fibronectin leading to corner fracture type spondylometaphyseal dysplasia and presents a new outlook toward matrix-mediated molecular pathways in relation to therapeutic and clinical relevance.

Fibrillin-1 is a pivotal structural component of the kidney’s glomerulus and peritubular tissue. Mutations in the fibrillin-1 gene result in Marfan syndrome (MFS), an autosomal dominant disease of the connective tissue. Although the kidney is not considered a classically affected organ in MFS, several case reports describe glomerular disease in patients. Therefore, this study aimed to characterize the kidney in the mgΔlpn-mouse model of MFS. Affected animals presented a significant reduction of glomerulus, glomerulus-capillary, and urinary space, and a significant reduction of fibrillin-1 and fibronectin in the glomerulus. Transmission electron microscopy and 3D-ultrastructure analysis revealed decreased amounts of microfibrils which also appeared fragmented in the MFS mice. Increased collagen fibers types I and III, MMP-9, and α-actin were also observed in affected animals, suggesting a tissue-remodeling process in the kidney. Video microscopy analysis showed an increase of microvessel distribution coupled with reduction of blood-flow velocity, while ultrasound flow analysis revealed significantly lower blood flow in the kidney artery and vein of the MFS mice. The structural and hemodynamic changes of the kidney indicate the presence of kidney remodeling and vascular resistance in this MFS model. Both processes are associated with hypertension which is expected to worsen the cardiovascular phenotype in MFS.

A 4 month-old, 14.8 kg, male Newfoundland dog was presented for cardiovascular evaluation following detection of a heart murmur. Echocardiography revealed enlargement of the sinuses of Valsalva and marked, diffuse dilation of the ascending aorta (annuloaortic ectasia, AAE), with mild/equivocal subaortic stenosis (SAS). The dog was monitored over the duration of its lifetime, with serial echocardiograms performed at 5, 6, and 8 months and 1, 2, 3, 4, 8, and 10 years demonstrating persistent, diffuse dilation of the ascending aorta. The dog lived until it was 10 years old and died of metastatic carcinoma. Postmortem examination confirmed AAE and mild SAS. Hematoxylin and eosin and Weigert van Gieson stains were used to compare the ascending aorta to the descending aorta and left subclavian artery, and to compare aortic samples to those of three control dogs. Histopathologic evaluation revealed mild medial degeneration in the ascending aorta of all four dogs. Immunofluorescent microscopy was used for determining the deposition of proteins known to play a role in aortic aneurysms in humans: fibrillin-1 (FBN1), latent transforming growth factor beta binding protein 4 (LTBP4) and fibronectin. The ascending aorta of the AAE case demonstrated reduced deposition of FBN1, indicating that its loss may have contributed to aortic dilation. Diffuse, primary ascending aortic dilation is uncommonly reported in dogs; when it is, it carries a poor prognosis. This case provides an important example of marked dilation of the ascending aorta in a dog that lived with no associated adverse effects for 10 years.

Gene mutations in the extracellular matrix protein fibrillin-1 cause connective tissue disorders including Marfan syndrome (MFS) with clinical symptoms in the cardiovascular, skeletal, and ocular systems. Patients with MFS also exhibit alterations in adipose tissues, which in some individuals leads to lipodystrophy, whereas in others to obesity. We have recently demonstrated that fibrillin-1 regulates adipose tissue homeostasis. Here, we examined how fibrillin-1 abnormality affects metabolic adaptation to different diets. We used two MFS mouse models: hypomorph Fbn1mgR/mgR mice and Fbn1C1041G/+ mice with a fibrillin-1 missense mutation. When Fbn1mgR/mgR mice were fed with high-fat diet (HFD) for 12 wk, male mice were heavier than littermate controls (LCs), whereas female mice gained less weight compared with LCs. Female Fbn1C1041G/+ mice on an HFD for 24 wk were similarly protected from weight gain. Male Fbn1C1041G/+ mice on an HFD demonstrated higher insulin levels, insulin intolerance, circulating levels of cholesterol, and high-density lipoproteins. Moreover, male HFD-fed Fbn1C1041G/+ mice showed a higher liver weight and a fatty liver phenotype, which was reduced to LC levels after orchiectomy. Phosphorylation of protein kinase-like endoplasmic reticulum kinase (PERK) and the expression of sterol regulatory element-binding protein 1 ( Srebp1) in livers of HFD-fed male Fbn1C1041G/+ mice were elevated. In conclusion, the data demonstrate that male mice of both the MFS models are susceptible to HFD-induced obesity and diabetes. Moreover, male Fbn1C1041G/+ mice develop a fatty liver phenotype, likely mediated by a baseline increased endoplasmic reticulum stress. In contrast, female MFS mice were protected from the consequence of HFD.

The extracellular matrix microenvironment of adipose tissue is of critical importance for the differentiation, remodeling and function of adipocytes. Fibrillin-1 is one of the main components of microfibrils and a key player in this process. Furin processing of profibrillin-1 results in mature fibrillin-1 and releases the C-terminal propeptide as a circulating hunger hormone, asprosin. Mutations in the fibrillin-1 gene lead to adipose tissue dysfunction and causes Marfan syndrome, marfanoid progeroid lipodystrophy syndrome, and neonatal progeroid syndrome. Increased TGF-β signaling, altered mechanical properties and impaired adipogenesis are potential causes of adipose tissue dysfunction, mediated through deficient microfibrils. Circulating asprosin on the other hand is secreted primarily by white adipose tissue under fasting conditions and in obesity. It increases hepatic glucose production and drives insulin secretion and appetite stimulation through inter-organ cross talk. This review discusses the metabolic consequences of fibrillin-1 and fibrillin-1-derived asprosin in pathological conditions. Understanding the dynamic role of fibrillin-1 in the adipose tissue milieu and of circulating asprosin in the body can provide novel mechanistic insights into how fibrillin-1 may contribute to metabolic syndrome. This could lead to new management regimens of patients with metabolic disease.

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations in SLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicated GLUT10 in the transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygous Slc2a10 missense mutations did not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient for Slc2a10 as well as Gulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in mouse. Gulo;Slc2a10 double knock-out mice showed mild phenotypic anomalies, which were absent in single knock-out controls. While Gulo;Slc2a10 double knock-out mice did not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. Transforming growth factor beta signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived from Gulo;Slc2a10 double knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined.

Fibrillin-1 is an extracellular glycoprotein present throughout the body. Mutations in fibrillin-1 cause a wide spectrum of type I fibrillinopathies, including Marfan syndrome characterized by clinical manifestations in adipose tissues, among others. This study addresses the hypothesis that fibrillin-1 regulates adipocyte development and plays a vital role in adipose tissue homeostasis. We employed two mouse models - Fbn1

Elastogenesis is a hierarchical process by which cells form functional elastic fibers, providing elasticity and the ability to regulate growth factor bioavailability in tissues, including blood vessels, lung, and skin. This process requires accessory proteins, including fibulin-4 and -5, and latent TGF binding protein (LTBP)-4. Our data demonstrate mechanisms in elastogenesis, focusing on the interaction and functional interdependence between fibulin-4 and LTBP-4L and its impact on matrix deposition and function. We show that LTBP-4L is not secreted in the expected extended structure based on its domain composition, but instead adopts a compact conformation. Interaction with fibulin-4 surprisingly induced a conformational switch from the compact to an elongated LTBP-4L structure. This conversion was only induced by fibulin-4 multimers associated with increased avidity for LTBP-4L; fibulin-4 monomers were inactive. The fibulin-4-induced conformational change caused functional consequences in LTBP-4L in terms of binding to other elastogenic proteins, including fibronectin and fibrillin-1, and of LTBP-4L assembly. A transient exposure of LTBP-4L with fibulin-4 was sufficient to stably induce conformational and functional changes; a stable complex was not required. These data define fibulin-4 as a molecular extracellular chaperone for LTBP-4L. The altered LTBP-4L conformation also promoted elastogenesis, but only in the presence of fibulin-4, which is required to escort tropoelastin onto the extended LTBP-4L molecule. Altogether, this study provides a dual mechanism for fibulin-4 in 1) inducing a stable conformational and functional change in LTBP-4L, and 2) promoting deposition of tropoelastin onto the elongated LTBP-4L.

Supplemental Digital Content is available in the text., Objective: Marfan syndrome (MFS) is caused by mutations in FBN1 (fibrillin-1), an extracellular matrix (ECM) component, which is modified post-translationally by glycosylation. This study aimed to characterize the glycoproteome of the aortic ECM from patients with MFS and relate it to aortopathy. Approach and Results: ECM extracts of aneurysmal ascending aortic tissue from patients with and without MFS were enriched for glycopeptides. Direct N-glycopeptide analysis by mass spectrometry identified 141 glycoforms from 47 glycosites within 35 glycoproteins in the human aortic ECM. Notably, MFAP4 (microfibril-associated glycoprotein 4) showed increased and more diverse N-glycosylation in patients with MFS compared with control patients. MFAP4 mRNA levels were markedly higher in MFS aortic tissue. MFAP4 protein levels were also increased at the predilection (convexity) site for ascending aorta aneurysm in bicuspid aortic valve patients, preceding aortic dilatation. In human aortic smooth muscle cells, MFAP4 mRNA expression was induced by TGF (transforming growth factor)-β1 whereas siRNA knockdown of MFAP4 decreased FBN1 but increased elastin expression. These ECM changes were accompanied by differential gene expression and protein abundance of proteases from ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family and their proteoglycan substrates, respectively. Finally, high plasma MFAP4 concentrations in patients with MFS were associated with a lower thoracic descending aorta distensibility and greater incidence of type B aortic dissection during 68 months follow-up. Conclusions: Our glycoproteomics analysis revealed that MFAP4 glycosylation is enhanced, as well as its expression during the advanced, aneurysmal stages of MFS compared with control aneurysms from patients without MFS.

Fibulin-4 is an extracellular matrix (ECM) protein essential for elastogenesis and mutations in this protein lead to aneurysm formation. In this study, we isolated vascular smooth muscle cells (VSMCs) from mice with reduced fibulin-4 protein expression (Fibulin-4R/R) and from mice with a smooth muscle cell specific deletion of the Fibulin-4 gene (Fibulin-4f/-/SM22Cre+). We subsequently analyzed and compared the molecular consequences of reduced Fibulin-4 expression versus total ablation of Fibulin-4 expression with regard to effects on the SMC specific contractile machinery, cellular migration and TGFβ signaling. Analysis of the cytoskeleton showed that while Fibulin-4f/-/SM22Cre+ VSMCs lack smooth muscle actin (SMA) fibers, Fibulin-4R/R VSMCs were able to form SMA fibers. Furthermore, Fibulin-4f/-/SM22Cre+ VSMCs showed a decreased pCofilin to Cofilin ratio, suggesting increased actin depolymerization, while Fibulin-4R/R VSMCs did not display this decrease. Yet, both Fibulin-4 mutant VSMCs showed decreased migration. We found increased activation of TGFβ signaling in Fibulin-4R/R VSMCs. However, TGFβ signaling was not increased in Fibulin-4f/-/SM22Cre+ VSMCs. From these results we conclude that both reduction and absence of Fibulin-4 leads to structural and functional impairment of the SMA cytoskeleton. However, while reduced levels of Fibulin-4 result in increased TGFβ activation, complete absence of Fibulin-4 does not result in increased TGFβ activation. Since both mouse models show thoracic aortic aneurysm formation, we conclude that not only hampered TGFβ signaling, but also SMA cytoskeleton dynamics play an important role in aortic aneurysmal disease.

The embryonic extracellular matrix (ECM) undergoes transition to mature ECM as development progresses, yet specific transition mechanisms ensuring ECM proteostasis and their regulatory impact are poorly defined. Fibrillin microfibrils are macromolecular ECM complexes serving structural and regulatory roles. In mice, Fbn1 and Fbn2, encoding major microfibrillar components, are strongly expressed during embryogenesis, but fibrillin-1 is the major component observed in adult tissue microfibrils. Here, analysis of mouse Adamts6 and Adamts10 mutant embryos, lacking these homologous secreted metalloproteases individually and in combination, along with in vitro analysis of microfibrils, measurement of ADAMTS6-fibrillin affinities and N-terminomics identification of ADAMTS6-cleaved sites, demonstrates a transcriptionally adapted system for fibrillin-2 proteolysis that contributes to postnatal fibrillin-1 dominance. The lack of ADAMTS6, alone and in combination with ADAMTS10 led to excess fibrillin-2 in perichondrium, with impaired skeletal development resulting from a drastic reduction of aggrecan, cartilage link protein and impaired BMP, but not TGFβ signaling in cartilage. Although ADAMTS6 cleaves fibrillin-1 and fibrillin-2 as well as fibronectin, which provides the initial scaffold for microfibril assembly, primacy of the protease-substrate relationship between ADAMTS6 and fibrillin-2 was unequivocally established by reversal of these defects in Adamts6-/- embryos by genetic reduction of Fbn2, but not Fbn1.

ADAMTS proteases mediate biosynthesis and breakdown of secreted extracellular matrix (ECM) molecules in numerous physiological and disease processes. In addition to their catalytic domains, ADAMTS proteases contain ancillary domains, which mediate substrate recognition and ECM binding and confer distinctive properties and roles to individual ADAMTS proteases. Although alternative splicing can greatly expand the structural and functional diversity of ADAMTS proteases, it has been infrequently reported and functional consequences have been rarely investigated. Here, we characterize the structural and functional impact of alternative splicing of ADAMTS17, mutations in which cause Weill-Marchesani syndrome 4. Two novel ADAMTS17 splice variants, ADAMTS17A and ADAMTS17B, were investigated by structural modeling, mass spectrometry, and biochemical approaches. Our results identify a novel disulfide-bridged insertion in the ADAMTS17A spacer that originates from inclusion of a novel exon. This insertion results in differential autoproteolysis of ADAMTS17, and thus, predicts altered proteolytic activity against other substrates. The second variant, ADAMTS17B, results from an in-frame exon deletion and prevents ADAMTS17B secretion. Thus, alternative splicing of the ADAMTS spacer significantly regulates the physiologically relevant proteolytic activity of ADAMTS17, either by altering proteolytic specificity (ADAMTS17A) or by altering cellular localization (ADAMTS17B).

Marfan syndrome (MFS) is an autosomal dominant disease affecting cardiovascular, ocular and skeletal systems. It is caused by mutations in the fibrillin-1 (FBN1) gene, leading to structural defects of connective tissue and increased activation of TGF-β. Angiotensin II (ang-II) is involved in TGF-β activity and in bone mass regulation. Inhibition of TGF-β signaling by blockage of the ang-II receptor 1 (AT1R) via losartan administration leads to improvement of cardiovascular and pulmonary phenotypes, but has no effect on skeletal phenotype in the haploinsufficient mouse model of MFS mgR, suggesting a distinct mechanism of pathogenesis in the skeletal system. Here we characterized the skeletal phenotypes of the dominant-negative model for MFS mgΔlpn and tested the effect of inhibition of ang-II signaling in improving those phenotypes. As previously shown, heterozygous mice present hyperkyphosis, however we now show that only males also present osteopenia. Inhibition of ang-II production by ramipril minimized the kyphotic deformity, but had no effect on bone microstructure in male mutant animals. Histological analysis revealed increased thickness of the anterior longitudinal ligament (ALL) of the spine in mutant animals (25.8 ± 6.3 vs. 29.7 ± 7.7 μm), coupled with a reduction in type I (164.1 ± 8.7 vs. 139.0 ± 4.4) and increase in type III (86.5 ± 10.2 vs. 140.4 ± 5.6) collagen in the extracellular matrix of this ligament. In addition, we identified in the MFS mice alterations in the erector spinae muscles which presented thinner muscle fibers (1035.0 ± 420.6 vs. 655.6 ± 239.5 μm2) surrounded by increased area of connective tissue (58.17 ± 6.52 vs. 105.0 ± 44.54 μm2). Interestingly, these phenotypes were ameliorated by ramipril treatment. Our results reveal a sex-dependency of bone phenotype in MFS, where females do not present alterations in bone microstructure. More importantly, they indicate that hyperkyphosis is not a result of osteopenia in the MFS mouse model, and suggest that incompetent spine ligaments and muscles are responsible for the development of that phenotype.

Fibrillin-1 and -2 are major components of tissue microfibrils that compose the ciliary zonule and cornea. While mutations in human fibrillin-1 lead to ectopia lentis, a major manifestation of Marfan syndrome (MFS), in mice fibrillin-2 can compensate for reduced/lack of fibrillin-1 and maintain the integrity of ocular structures. Here we examine the consequences of a heterozygous dominant-negative mutation in the Fbn1 gene in the ocular system of the mgΔEyes from mgΔMutant mice presented a significantly larger distance of the ciliary body to the lens at 3 and 6 months of age when compared to wild-type, and ectopia lentis. Immunofluorescence and SEM corroborated those findings in MFS mice, revealing a disorganized mesh of microfibrils on the floor of the ciliary body. Moreover, mutant mice also had a larger volume of the anterior chamber, possibly due to excess aqueous humor. Finally, losartan treatment had limited efficacy in improving ocular phenotypes.In contrast with null or hypomorphic mutations, expression of a dominant-negative form of fibrillin-1 leads to disruption of microfibrils in the zonule of mice. This in turn causes lens dislocation and enlargement of the anterior chamber. Therefore, heterozygous mgΔ

Latent-transforming growth factor beta-binding protein 2 (LTBP-2) is a major component of arterial and lung tissue and of the ciliary zonule, the system of extracellular fibers that centers and suspends the lens in the eye. LTBP-2 has been implicated previously in the development of extracellular microfibrils, although its exact role remains unclear. Here, we analyzed the three-dimensional structure of the ciliary zonule in wild type mice and used a knockout model to test the contribution of LTBP-2 to zonule structure and mechanical properties. In wild types, zonular fibers had diameters of 0.5-1.0 micrometers, with an outer layer of fibrillin-1-rich microfibrils and a core of fibrillin-2-rich microfibrils. LTBP-2 was present in both layers. The absence of LTBP-2 did not affect the number of fibers, their diameters, nor their coaxial organization. However, by two months of age, LTBP-2-depleted fibers began to rupture, and by six months, a fully penetrant ectopia lentis phenotype was present, as confirmed by in vivo imaging. To determine whether the seemingly normal fibers of young mice were compromised mechanically, we compared zonule stress/strain relationships of wild type and LTBP-2-deficient mice and developed a quasi-linear viscoelastic engineering model to analyze the resulting data. In the absence of LTBP-2, the ultimate tensile strength of the zonule was reduced by about 50%, and the viscoelastic behavior of the fibers was altered significantly. We developed a harmonic oscillator model to calculate the forces generated during saccadic eye movement. Model simulations suggested that mutant fibers are prone to failure during rapid rotation of the eyeball. Together, these data indicate that LTBP-2 is necessary for the strength and longevity of zonular fibers, but not necessarily for their formation.

Autologous veins are the most widely used grafts for bypassing small arteries in coronary and peripheral arterial occlusive diseases. However, they have limited availability and cause donor-site morbidity. Here, we report a direct comparison of acellular biodegradable synthetic grafts and autologous veins as interposition grafts of rat carotid arteries, which is a good model for clinically relevant small arteries. Notably, extensive but transient infiltration of circulating monocytes at day 14 in synthetic grafts leads to a quickly-resolved inflammation and arterial-like tissue remodeling. The vein graft exhibits a similar inflammation phase except the prolonged presence of inflammatory monocytes. The walls of the remodeled synthetic graft contain many circumferentially aligned contractile non-proliferative smooth muscle cells (SMCs), collagen and elastin. In contrast, the walls of the vein grafts contain disorganized proliferating SMCs and thicken over time, suggesting the onset of stenosis. At 3 months, both grafts have a similar patency, extracellular matrix composition, and mechanical properties. Furthermore, synthetic grafts exhibit recruitment and re-orientation of newly synthesized collagen fibers upon mechanical loading. To our knowledge, this is the first demonstration of a biodegradable synthetic vascular graft with a performance similar to an autologous vein in small artery grafting.

Arterial tortuosity syndrome (ATS) is a recessively inherited connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutations inSLC2A10, encoding the facilitative glucose transporter GLUT10. Former studies implicate GLUT10 in transport of dehydroascorbic acid, the oxidized form of ascorbic acid (AA). Mouse models carrying homozygousSlc2a10missense mutations do not recapitulate the human phenotype. Since mice, in contrast to humans, are able to intracellularly synthesize AA, we generated a novel ATS mouse model, deficient forSlc2a10as well asGulo, which encodes for L-gulonolactone oxidase, an enzyme catalyzing the final step in AA biosynthesis in rodents.Gulo;Slc2a10knock-out mice show mild phenotypic anomalies, which were absent in single knock-out controls. WhileGulo;Slc2a10knock-out mice do not fully phenocopy human ATS, histological and immunocytochemical analysis revealed compromised extracellular matrix formation. TGFβ signaling remained unaltered, while mitochondrial function was compromised in smooth muscle cells derived fromGulo;Slc2a10knock-out mice. Altogether, our data add evidence that ATS is an ascorbate compartmentalization disorder, but additional factors underlying the observed phenotype in humans remain to be determined.

Multidomain matrix-associated zinc extracellular proteases ADAMTS and ADAMTS-like proteins have important biological activities in cells and tissues. Beyond their traditional role in procollagen and von Willebrand factor processing and proteoglycan cleavage, ADAMTS/ADAMTSL likely participate in or at least have some role in ECM assembly as some of these proteins bind ECM proteins including fibrillins, fibronectin, and LTBPs. In this chapter, we present four biophysical techniques largely used for the characterization, multimerization, and interaction of proteins: surface plasmon resonance spectroscopy, dynamic light scattering, atomic force microscopy, and circular dichroism spectroscopy.

ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs) proteins regulate tissue homeostasis and extracellular matrix (ECM)-related pathogenesis. Some ADAMTS proteins interact with or process multiple ECM proteins, including fibrillins, fibronectin, and collagens. Therefore, characterization and quantification of these ECM fiber systems is essential to understand their functional relationship with ADAMTS proteins. Here we describe unbiased methods to quantify various aspects of ADAMTS-related ECM fiber systems in cell culture and in tissues. We focus on cell counting, overall fiber intensity, fiber length, and focal adhesion analysis in cell culture, and on the quantification of immunohistochemical and immunofluorescent tissue sections. We use ImageJ/Fiji, a widely used Java-based open source software which provides efficient and customizable quantification methods for microscopy images.

Multidomain matrix-associated zinc extracellular proteases ADAMTS and ADAMTS-like proteins have important biological activities in cells and tissues. Beyond their traditional role in procollagen and von Willebrand factor processing and proteoglycan cleavage, ADAMTS/ADAMTSL likely participate in or at least have some role in ECM assembly as some of these proteins bind ECM proteins including fibrillins, fibronectin, and LTBPs. In this chapter, we present four biophysical techniques largely used for the characterization, multimerization, and interaction of proteins: surface plasmon resonance spectroscopy, dynamic light scattering, atomic force microscopy, and circular dichroism spectroscopy.

ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs) proteins regulate tissue homeostasis and extracellular matrix (ECM)-related pathogenesis. Some ADAMTS proteins interact with or process multiple ECM proteins, including fibrillins, fibronectin, and collagens. Therefore, characterization and quantification of these ECM fiber systems is essential to understand their functional relationship with ADAMTS proteins. Here we describe unbiased methods to quantify various aspects of ADAMTS-related ECM fiber systems in cell culture and in tissues. We focus on cell counting, overall fiber intensity, fiber length, and focal adhesion analysis in cell culture, and on the quantification of immunohistochemical and immunofluorescent tissue sections. We use ImageJ/Fiji, a widely used Java-based open source software which provides efficient and customizable quantification methods for microscopy images.

Mutations in extracellular matrix and smooth muscle cell contractile proteins predispose to thoracic aortic aneurysms in Marfan syndrome (MFS) and related disorders. These genetic alterations lead to a compromised extracellular matrix–smooth muscle cell contractile unit. The abnormal aortic tissue responds with defective mechanosensing under hemodynamic stress. Aberrant mechanosensing is associated with transforming growth factor-beta (TGF-β) hyperactivity, enhanced angiotensin-II (Ang-II) signaling, and perturbation of other cellular signaling pathways. The downstream consequences include enhanced proteolytic activity, expression of inflammatory cytokines and chemokines, infiltration of inflammatory cells in the aortic wall, vascular smooth muscle cell apoptosis, and medial degeneration. Mouse models highlight aortic inflammation as a contributing factor in the development of aortic aneurysms. Anti-inflammatory drugs and antioxidants can reduce aortic oxidative stress that prevents aggravation of aortic disease in MFS mice. Targeting TGF-β and Ang-II downstream signaling pathways such as ERK1/2, mTOR, PI3/Akt, P38/MAPK, and Rho kinase signaling attenuates disease pathogenesis. Aortic extracellular matrix degradation and medial degeneration were reduced upon inhibition of inflammatory cytokines and matrix metalloproteinases, but the latter lack specificity. Treating inflammation associated with aortic aneurysms in MFS and related disorders could prove to be beneficial in limiting disease pathogenesis.

Purpose Fibrillin-1 and -2 are major components of tissue microfibrils that compose the ciliary zonule and cornea. While mutations in human fibrillin-1 lead to ectopia lentis, a major manifestation of Marfan syndrome (MFS), in mice fibrillin-2 can compensate for reduced/lack of fibrillin-1 and maintain the integrity of ocular structures. Here we examine the consequences of a heterozygous dominant-negative mutation in the Fbn1 gene in the ocular system of the mgΔlpn mouse model for MFS. Methods Eyes from mgΔlpn and wild-type mice at 3 and 6 months of age were analyzed by histology. The ciliary zonule was analyzed by scanning electron microscopy (SEM) and immunofluorescence. Results Mutant mice presented a significantly larger distance of the ciliary body to the lens at 3 and 6 months of age when compared to wild-type, and ectopia lentis. Immunofluorescence and SEM corroborated those findings in MFS mice, revealing a disorganized mesh of microfibrils on the floor of the ciliary body. Moreover, mutant mice also had a larger volume of the anterior chamber, possibly due to excess aqueous humor. Finally, losartan treatment had limited efficacy in improving ocular phenotypes. Conclusions In contrast with null or hypomorphic mutations, expression of a dominant-negative form of fibrillin-1 leads to disruption of microfibrils in the zonule of mice. This in turn causes lens dislocation and enlargement of the anterior chamber. Therefore, heterozygous mgΔlpn mice recapitulate the major ocular phenotypes of MFS and can be instrumental in understanding the development of the disease.

Fibronectin is an extracellular matrix protein with pivotal physiological and pathological functions in development and adulthood. Alternative splicing of the precursor mRNA, produced from the single copy fibronectin gene, occurs at three sites coding for the EDA, EDB and IIICS domains. Fibronectin isoforms comprising the EDA or EDB domains are known as oncofetal forms due to their developmental importance and their re-expression in tumors, contrasting with restricted presence in normal adult tissues. These isoforms are also recognized as important markers of angiogenesis, a crucial physiological process in development and required by tumor cells in cancer progression. Attributed to this feature, EDA and EDB domains have been extensively used for the targeted delivery of cytokines, cytotoxic agents, chemotherapy drugs and radioisotopes to fibronectin-expressing tumors to exert therapeutic effects on primary cancers and metastatic lesions. In addition to drug delivery, the EDA and EDB domains of fibronectin have also been utilized to develop imaging strategies for tumor tissues. Furthermore, EDA and EDB based vaccines seem to be promising for the treatment and prevention of certain cancer types. In this review, we will summarize recent advances in fibronectin EDA and EDB-based therapeutic strategies developed to treat cancer.

Heterozygous pathogenic variants in the FN1 gene, encoding fibronectin (FN), have recently been shown to be associated with a skeletal disorder in some individuals affected by spondylometaphyseal dysplasia with “corner fractures” (SMD-CF). The most striking feature characterizing SMD-CF is irregularly shaped metaphyses giving the appearance of “corner fractures”. An array of secondary features, including developmental coxa vara, ovoid vertebral bodies and severe scoliosis, may also be present. FN is an important extra cellular matrix component for bone and cartilage development. Here we report five patients affected by this subtype of SMD-CF caused by five novel FN1 missense mutations: p.Cys123Tyr, p.Cys169Tyr, p.Cys213Tyr, p.Cys231Trp and p.Cys258Tyr. All individuals shared a substitution of a cysteine residue, disrupting disulfide bonds in the FN type-I assembly domains located in the N-terminal assembly region. The abnormal metaphyseal ossification and “corner fracture” appearances were the most remarkable clinical feature in these patients. In addition, generalized skeletal fragility with low-trauma bilateral femoral fractures was identified in one patient. Interestingly, the distal femoral changes in this patient healed with skeletal maturation. Our report expands the phenotypic and genetic spectrum of the FN1-related SMD-CF and emphasizes the importance of FN in bone formation and possibly also in the maintenance of bone strength.

Triggering receptor expressed on myeloid cells-2 (TREM-2), a transmembrane receptor expressed by macrophages, microglia, and osteoclasts (OCs), plays a protective role in late-onset Alzheimer Disease (AD). To validate TREM-2 as a therapeutic target in AD, its potential secondary parallel effect on bone homeostasis should be clarified. However, animal models and monolayer cultures of human cells were shown poorly predictive of TREM-2 function in human. Therefore, this study aimed to engineer a tridimensional in vitro model using human progenitors differentiated into osteoblasts and OCs, recapitulating physiological bone homeostasis. Human bone marrow-derived mesenchymal cells were seeded and cultured under perfusion inside a collagen type I scaffold for 3 weeks, generating osteoblasts and mineralized matrix. Human peripheral blood-derived CD14+ monocytes were subsequently seeded through the generated tissue, thanks to perfusion flow, and further cultured for up to 3 weeks with an inductive medium, generating mature OCs. This culture system supported collagenous matrix deposition and resorption, allowing for the investigation of kinetic of soluble TREM-2 over the coculture time. Agonistic activation of TREM-2 in this model had no effect on OC activity or on mineralized matrix turnover. In conclusion, the engineered culture system represents a tridimensional, in vitro human bone model for drug testing and suggested no effect of TREM-2 agonist on bone resorption.

Mutations in the secreted metalloproteinase ADAMTS10 cause recessive Weill-Marchesani syndrome (WMS), comprising ectopia lentis, short stature, brachydactyly, thick skin and cardiac valve anomalies. Dominant WMS caused by FBN1 mutations is clinically similar and affects fibrillin-1 microfibrils, which are a major component of the ocular zonule. ADAMTS10 was previously shown to enhance fibrillin-1 assembly in vitro. Here, Adamts10 null mice were analyzed to determine the impact of ADAMTS10 deficiency on fibrillin microfibrils in vivo. An intragenic lacZ reporter identified widespread Adamts10 expression in the eye, musculoskeletal tissues, vasculature, skin and lung. Adamts10(−/−) mice had reduced viability on the C57BL/6 background, and although surviving mice were slightly smaller and had stiff skin, they lacked brachydactyly and cardiovascular defects. Ectopia lentis was not observed in Adamts10(−/−) mice, similar to Fbn1(−/−) mice, most likely because the mouse zonule contains fibrillin-2 in addition to fibrillin-1. Unexpectedly, in contrast to wild-type eyes, Adamts10(−/−) zonule fibers were thicker and immunostained strongly with fibrillin-2 antibodies into adulthood, whereas fibrillin-1 staining was reduced. Furthermore, fibrillin-2 staining of hyaloid vasculature remnants persisted post-natally in Adamts10(−/−) eyes. ADAMTS10 was found to cleave fibrillin-2, providing an explanation for persistence of fibrillin-2 at these sites. Thus, analysis of Adamts10(−/−) mice led to identification of fibrillin-2 as a novel ADAMTS10 substrate and defined a proteolytic mechanism for clearance of ocular fibrillin-2 at the end of the juvenile period.

One of the tightest bottlenecks in vascular tissue engineering (vTE) is the lack of strength and elasticity of engineered vascular wall models caused by limited elastic fiber deposition. In this study, flat and tubular collagen gel-based scaffolds were cellularised with vascular smooth muscle cells (SMCs) and supplemented with human plasma fibronectin (FN), a known master organizer of several extracellular matrix (ECM) fiber systems. The consequences of FN on construct maturation was investigated in terms of geometrical contraction, viscoelastic mechanical properties and deposition of core elastic fiber proteins. FN was retained in the constructs and promoted deposition of elastin by SMCs as well as of several proteins required for elastogenesis such as fibrillin-1, lysyl oxidase, fibulin-4 and latent TGF-β binding protein-4. Notably, gel contraction, tensile equilibrium elastic modulus and elasticity were strongly improved in tubular engineered tissues, approaching the behaviour of native arteries. In conclusion, this study demonstrates that FN exerts pivotal roles in directing SMC-mediated remodeling of scaffolds toward the production of a physiological-like, elastin-containing ECM with excellent mechanical properties. The developed FN-supplemented systems are promising for tissue engineering applications where the generation of mature elastic tissue is desired and represent valuable advanced in vitro models to investigate elastogenesis.

Fibrillins are one of the major components of supramolecular fibrous structures in the extracellular matrix of elastic and nonelastic tissues, termed microfibrils. Microfibrils provide tensile strength in nonelastic tissues and scaffolds for the assembly of tropoelastin in elastic tissues, and act a regulator of growth factor bioavailability and activity in connective tissues. Mutations in fibrillins lead to a variety of connective tissue disorders including Marfan syndrome, stiff skin syndrome, dominant Weill-Marchesani syndrome, and others. Therefore, fibrillins are frequently studied to understand the pathophysiology of these diseases and to identify effective treatment strategies. Extraction of endogenous microfibrils from cells and tissues can aid in obtaining structural insights of microfibrils. Recombinant production of fibrillins is an important tool which can be utilized to study the properties of normal fibrillins and the consequences of disease causing mutations. Other means of studying the role of fibrillins in the context of various physiological settings is by knocking down the mRNA expression and analyzing its downstream consequences. It is also important to study the interactome of fibrillins by protein-protein interactions, which can be derailed in pathological situations. Interacting proteins can affect the assembly of fibrillins in cells and tissues or can affect the levels of growth factors in the matrix. This chapter describes important techniques in the field that facilitate answering relevant questions of fibrillin biology and pathophysiology.

Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant fibrillin-1 fragments. Those mutations affect fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated fibrillin-1.

Extracellular matrix (ECM) constituents play not only structural roles during development and tissue homeostasis, but also many biological functions throughout life. Molecular diversity and a vast interactome provide the basis for this multi-functionality. Moreover, native or processed ECM molecules interact with various receptors, thereby activating signaling pathways that control cell differentiation, proliferation, adhesion and migration, all relevant to tumor biology. Thus, there is an emerging field focused on exploiting ECM components as novel therapeutic targets in the treatment of cancer and other diseases, providing potent tools to advance drug delivery and tissue penetration. In this special issue we provide a critical appraisal of this emerging field focusing on: 1) ECM proteins (matricellular proteins, collagen, elastin, fibronectin, proteoglycans), integrins, and protease-facilitated drug delivery; 2) ECM-derived therapeutics (hyaluronan, heparin, heparan sulfate), 3) ECM-like biomaterials, and 4) ECM as critical determinant in drug efficacy, with special emphasis on applications in tumor treatment.

Fibronectin (FN) exists in two forms—plasma FN (pFN) and cellular FN (cFN). Although the role of FN in embryonic blood vessel development is well established, its function and the contribution of individual isoforms in early postnatal vascular development are poorly understood. Here, we employed a tamoxifen-dependent cFN inducible knockout (cFN iKO) mouse model to study the consequences of postnatal cFN deletion in smooth muscle cells (SMCs), the major cell type in the vascular wall. Deletion of cFN influences collagen deposition but does not affect life span. Unexpectedly, pFN translocated to the aortic wall in the cFN iKO and in control mice, possibly rescuing the loss of cFN. Postnatal pFN deletion did not show a histological aortic phenotype. Double knockout (dKO) mice lacking both, cFN in SMCs and pFN, resulted in postnatal lethality. These data demonstrate a safeguard role of pFN in vascular stability and the dispensability of the individual FN isoforms in postnatal vascular development. Complete absence of FNs in the dKOs resulted in a disorganized tunica media of the aortic wall. Matrix analysis revealed common and differential roles of the FN isoforms in guiding the assembly/deposition of elastogenic extracellular matrix (ECM) proteins in the aortic wall. In addition, we determined with two cell culture models that that the two FN isoforms acted similarly in supporting matrix formation with a greater contribution from cFN. Together, these data show that pFN exerts a critical role in safeguarding vascular organization and health, and that the two FN isoforms function in an overlapping as well as distinct manner to maintain postnatal vascular matrix integrity., Author summary Fibronectin is a protein that exists in vertebrates in two distinct forms: one present in the blood and the other in blood vessel walls. In mammals, fibronectin is important for the development of blood vessels before birth, but whether it is continuously required for blood vessel homeostasis from birth to adulthood is unknown. We present important results from three genetically modified mouse models, which show that at least one form of fibronectin is required for the proper function and integrity of blood vessels during this period. We show that fibronectin can be transferred from the blood into the vessel wall, where it can rescue the integrity of blood vessels in the absence of the vessel form. This represents an important biological mechanism to maintain the health of blood vessels. Our data also highlight the importance of both fibronectin forms in producing and organizing the microenvironment of cells, with a higher contribution from the fibronectin form residing in the blood vessel walls. Together, our findings show that fibronectin from the blood acts as a safeguard to maintain the health of blood vessels, and both fibronectin forms play crucial roles in development and support of the blood vessel microenvironment.

Item does not contain fulltext Fibronectin is a master organizer of extracellular matrices (ECMs) and promotes the assembly of collagens, fibrillin-1, and other proteins. It is also known to play roles in skeletal tissues through its secretion by osteoblasts, chondrocytes, and mesenchymal cells. Spondylometaphyseal dysplasias (SMDs) comprise a diverse group of skeletal dysplasias and often manifest as short stature, growth-plate irregularities, and vertebral anomalies, such as scoliosis. By comparing the exomes of individuals with SMD with the radiographic appearance of "corner fractures" at metaphyses, we identified three individuals with fibronectin (FN1) variants affecting highly conserved residues. Furthermore, using matching tools and the SkelDys emailing list, we identified other individuals with de novo FN1 variants and a similar phenotype. The severe scoliosis in most individuals and rare developmental coxa vara distinguish individuals with FN1 mutations from those with classical Sutcliffe-type SMD. To study functional consequences of these FN1 mutations on the protein level, we introduced three disease-associated missense variants (p.Cys87Phe [c.260G>T], p.Tyr240Asp [c.718T>G], and p.Cys260Gly [c.778T>G]) into a recombinant secreted N-terminal 70 kDa fragment (rF70K) and the full-length fibronectin (rFN). The wild-type rF70K and rFN were secreted into the culture medium, whereas all mutant proteins were either not secreted or secreted at significantly lower amounts. Immunofluorescence analysis demonstrated increased intracellular retention of the mutant proteins. In summary, FN1 mutations that cause defective fibronectin secretion are found in SMD, and we thus provide additional evidence for a critical function of fibronectin in cartilage and bone.

Bindegewebe bezeichnet verschiedene Gewebetypen im Korper, die auf unterstutzende Aufgaben spezialisiert sind. Diese Gewebetypen enthalten relativ wenige Korperzellen sowie das biologische Material zwischen den Zellen, die so genannte extrazellulare Matrix.

Finegoldia magna is a Gram-positive anaerobic commensal of the human skin microbiota, but also known to act as an opportunistic pathogen. Two primary virulence factors of F. magna are the subtilisin-like extracellular serine protease SufA and the adhesive protein FAF. This study examines the molecular mechanisms F. magna uses when colonizing or establishing an infection in the skin. FAF was found to be essential in the initial adherence of F. magna to human skin biopsies. In the upper layers of the epidermis FAF mediates adhesion through binding to galectin-7 - a keratinocyte cell marker. Once the bacteria moved deeper into the skin to the basement membrane layer, SufA was found to degrade collagen IV which forms the backbone structure of the basement membrane. It also degraded collagen V, whereby F. magna could reach deeper dermal tissue sites. In the dermis, FAF interacts with collagen V and fibrillin, which presumably helps the bacteria to establish infection in this area. The findings of this study paint a clear picture of how F. magna interacts with human skin and explain how it is such a successful opportunistic pathogen in chronic wounds and ulcers.

Novel functions of the extracellular protein fibrillin-2 were discovered in tracheal tube development: fibrillin-2 has important roles in tracheal cartilage development and smooth muscle cell orientation and polarity, all required for tracheal contractionhttp://ow.ly/rn3Z30nId3N

Fibrillins constitute the backbone of extracellular multifunctional assemblies present in elastic and non-elastic matrices, termed microfibrils. Assembly of fibrillins into microfibrils and their homoeostasis is poorly understood and is often compromised in connective tissue disorders such as Marfan syndrome and other fibrillinopathies. Using interaction mapping studies, we demonstrate that fibrillins require the complete gelatin-binding region of fibronectin for interaction, which comprises domains FNI6–FNI9. However, the interaction of fibrillin-1 with the gelatin-binding domain of fibronectin is not involved in fibrillin-1 network assembly mediated by human skin fibroblasts. We show further that the fibronectin network is essential for microfibril homoeostasis in early stages. Fibronectin is present in extracted mature microfibrils from tissue and cells as well as in some in situ microfibrils observed at the ultrastructural level, indicating an extended mechanism for the involvement of fibronectin in microfibril assembly and maturation.

Summary Mutations in the fibrillin-1 gene give rise to a number of heritable disorders, which are all characterized by various malformations of bone as well as manifestations in other tissues. However, the role of fibrillin-1 in the development and homeostasis of bone is not well understood. Here, we examined the role of fibrillin-1 in regulating osteoclast differentiation from primary bone-marrow-derived precursors and monocytic RAW 264.7 cells. The soluble N-terminal half of fibrillin-1 (rFBN1-N) strongly inhibited osteoclastogenesis, whereas the C-terminal half (rFBN1-C) did not. By contrast, when rFBN1-N was immobilized on calcium phosphate, it did not affect osteoclastogenesis but modulated osteoclast resorptive activity, which was evident by a larger number of smaller resorption pits. Using a panel of recombinant sub-fragments spanning rFBN1-N, we localized an osteoclast inhibitory activity to the 63 kDa subfragment rF23 comprising the N-terminal region of fibrillin-1. Osteoclastic resorption led to the generation of small fibrillin-1 fragments that were similar to those identified in human vertebral bone extracts. rF23, but not rFBN1-N, was found to inhibit the expression of cathepsin K, matrix metalloproteinase 9 and Dcstamp in differentiating osteoclasts. rFBN1-N, but not rF23, exhibited interaction with RANKL. Excess RANKL rescued the inhibition of osteoclastogenesis by rFBN1-N. By contrast, rF23 disrupted RANKL-induced Ca2+ signaling and activation of transcription factor NFATc1. These studies highlight a direct dual inhibitory role of N-terminal fibrillin-1 fragments in osteoclastogenesis, the sequestration of RANKL and the inhibition of NFATc1 signaling, demonstrating that osteoclastic degradation of fibrillin-1 provides a potent negative feedback that limits osteoclast formation and function.