Extracellular Microfibrils Research Articles

Emilin2 fosters vascular stability by promoting pericyte recruitment

Angiogenesis, the formation of the new blood vessels from pre-existing vasculature, is an essential process occurring under both normal and pathological conditions, such as inflammation and cancer. This complex process is regulated by several cytokines, growth factors and extracellular matrix components modulating endothelial cell and pericyte function. In this study, we discovered that the extracellular matrix glycoprotein Elastin Microfibril Interfacer 2 (Emilin2) plays a prominent role in pericyte physiology. This work was originally prompted by the observations that tumor-associated vessels from Emilin2−/− mice display less pericyte coverage, impaired vascular perfusion, and reduced drug efficacy, suggesting that Emilin2 could promote vessel maturation and stabilization affecting pericyte recruitment. We found that Emilin2 affects different mechanisms engaged in pericyte recruitment and vascular stabilization. First, human primary endothelial cells challenged with recombinant Emilin2 synthesized and released ∼ 2.1 and 1.2 folds more PDGF-BB and HB-EGF, two cytokines known to promote pericyte recruitment. We also discovered that Emilin2, by directly engaging α5β1 and α6β1 integrins, highly expressed in pericytes, served as an adhesion substrate and haptotactic stimulus for pericytes. Moreover, Emilin2 evoked increased NCadherin expression via the sphingosine-1-phosphate receptor, leading to enhanced vascular stability by fostering interconnection between endothelial cells and pericytes. Finally, restoring pericyte coverage in melanoma and ovarian tumor vessels developed in Emilin2−/− mice improved drug delivery to the tumors. Collectively, our results implicate Emilin2 as a prominent regulator of pericyte function and suggest that Emilin2 expression could represent a promising maker to predict the clinical outcome of patients with melanoma, ovarian, and potentially other forms of cancer.

A common variant rs2054564 in ADAMTS17 is associated with susceptibility to lumbar spondylosis

The molecular pathophysiology underlying lumbar spondylosis development remains unclear. To identify genetic factors associated with lumbar spondylosis, we conducted a genome-wide association study using 83 severe lumbar spondylosis cases and 182 healthy controls and identified 65 candidate disease-associated single nucleotide polymorphisms (SNPs). Replication analysis in 510 case and 911 control subjects from five independent Japanese cohorts identified rs2054564, located in intron 7 of ADAMTS17, as a disease-associated SNP with a genome-wide significance threshold (P = 1.17 × 10–11, odds ratio = 1.92). This association was significant even after adjustment of age, sex, and body mass index (P = 7.52 × 10–11). A replication study in a Korean cohort, including 123 case and 319 control subjects, also verified the significant association of this SNP with severe lumbar spondylosis. Immunohistochemistry revealed that fibrillin-1 (FBN1) and ADAMTS17 were co-expressed in the annulus fibrosus of intervertebral discs (IVDs). ADAMTS17 overexpression in MG63 cells promoted extracellular microfibrils biogenesis, suggesting the potential role of ADAMTS17 in IVD function through interaction with fibrillin fibers. Finally, we provided evidence of FBN1 involvement in IVD function by showing that lumbar IVDs in patients with Marfan syndrome, caused by heterozygous FBN1 gene mutation, were significantly more degenerated. We identified a common SNP variant, located in ADAMTS17, associated with susceptibility to lumbar spondylosis and demonstrated the potential role of the ADAMTS17-fibrillin network in IVDs in lumbar spondylosis development.

Fibrillin-1 and asprosin, novel players in metabolic syndrome.

Fibrillin-1 is a major component of the extracellular microfibrils, where it interacts with other extracellular matrix proteins to provide elasticity to connective tissues, and regulates the bioavailability of TGFβ family members. A peptide consisting of the C-terminal 140 amino acids of fibrillin-1 has recently been identified as a glucogenic hormone, secreted from adipose tissue during fasting and targeting the liver to release glucose. This fragment, called asprosin, also signals in the hypothalamus to stimulate appetite. Asprosin levels are correlated with many of the pathologies indicative of metabolic syndrome, including insulin resistance and obesity. Previous studies and reviews have addressed the therapeutic potential of asprosin as a target in obesity, diabetes and related conditions without considering mechanisms underlying the relationship between generation of asprosin and expression of the much larger fibrillin-1 protein. Profibrillin-1 undergoes obligatory cleavage at the cell surface as part of its assembly into microfibrils, producing the asprosin peptide as well as mature fibrillin-1. Patterns of FBN1 mRNA expression are inconsistent with the necessity for regulated release of asprosin. The asprosin peptide may be protected from degradation in adipose tissue. We present evidence for an alternative possibility, that asprosin mRNA is generated independently from an internal promoter within the 3' end of the FBN1 gene, which would allow for regulation independent of fibrillin-synthesis and is more economical of cellular resources. The discovery of asprosin opened exciting possibilities for treatment of metabolic syndrome related conditions, but there is much to be understood before such therapies could be introduced into the clinic.

Targeting of bone morphogenetic protein complexes to heparin/heparan sulfate glycosaminoglycans in bioactive conformation.

Bone morphogenetic proteins (BMP) are powerful regulators of cellular processes such as proliferation, differentiation, and apoptosis. However, the specific molecular requirements controlling the bioavailability of BMPs in the extracellular matrix (ECM) are not yet fully understood. Our previous work showed that BMPs are targeted to the ECM as growth factor-prodomain (GF-PD) complexes (CPLXs) via specific interactions of their PDs. We showed that BMP-7 PD binding to the extracellular microfibril component fibrillin-1 renders the CPLXs from an open, bioactive V-shape into a closed, latent ring shape. Here, we show that specific PD interactions with heparin/heparan sulfate glycosaminoglycans (GAGs) allow to target and spatially concentrate BMP-7 and BMP-9 CPLXs in bioactive V-shape conformation. However, targeting to GAGs may be BMP specific, since BMP-10 GF and CPLX do not interact with heparin. Bioactivity assays on solid phase in combination with interaction studies showed that the BMP-7 PD protects the BMP-7 GF from inactivation by heparin. By using transmission electron microscopy, molecular docking, and site-directed mutagenesis, we determined the BMP-7 PD-binding site for heparin. Further, fine-mapping of the fibrillin-1-binding site within the BMP-7 PD and molecular modeling showed that both binding sites are mutually exclusive in the open V- versus closed ring-shape conformation. Together, our data suggest that targeting exquisite BMP PD-binding sites by extracellular protein and GAG scaffolds integrates BMP GF bioavailability in a contextual manner in development, postnatal life, and connective tissue disease.

The Effects of Asprosin on Exercise-Intervention in Metabolic Diseases.

Fibrillin is the major constituent of extracellular microfibrils, which are distributed throughout connective tissues. Asprosin is derived from the C-terminal region of the FBN1 gene, which encodes profibrillin that undergoes cleavage by furin protein. In response to fasting with low dietary glucose, asprosin is released as a secreted factor from white adipose tissue, and is transported to the liver for the mediation of glucose release into the blood circulation. Through binding to OLFR734, an olfactory G-protein-coupled receptor in liver cells, asprosin induces a glucogenic effect to regulate glucose homeostasis. Bioinformatics analyses revealed that the FBN1 gene is abundantly expressed in human skeletal muscle-derived mesoangioblasts, osteoblast-like cells, and mesenchymal stem cells, indicating that the musculoskeletal system might play a role in the regulation of asprosin expression. Interestingly, recent studies suggest that asprosin is regulated by exercise. This timely review discusses the role of asprosin in metabolism, its receptor signalling, as well as the exercise regulation of asprosin. Collectively, asprosin may have a vital regulatory effect on the improvement of metabolic disorders such as diabetes mellitus and obesity via exercise.

Macromolecular crowding enhances fibrillin-1 deposition in the extracellular matrix.

Biochemical and biophysical factors need consideration when modelling in vivo cellular behaviour using in vitro cell culture systems. One underappreciated factor is the high concentration of macromolecules present in vivo, which is typically not simulated under standard cell culture conditions. This disparity is especially relevant when studying biochemical processes that govern extracellular matrix (ECM) deposition, which may be altered due to dilution of secreted macromolecules by the relatively large volumes of culture medium required for cell maintenance in vitro. Macromolecular crowding (MMC) utilises the addition of inert macromolecules to cell culture medium to mimic such high concentration environments found in vivo. The present study induced MMC using the sucrose polymer Ficoll and examined whether fibrillin-1 deposition by human lung fibroblasts could be augmented. Fibrillin-1 forms extracellular microfibrils, which are versatile scaffolds required for elastic fibre formation, deposition of other ECM proteins and growth factor regulation. Pathogenic variants in the fibrillin-1 gene (FBN1) cause Marfan syndrome, where ECM deposition of fibrillin-1 can be compromised. Using immunocytochemistry, significantly enhanced fibrillin-1 deposition was observed when lung fibroblasts were cultured under MMC conditions. MMC also augmented fibrillin-1 deposition in Marfan syndrome patient-derived skin fibroblasts in a cell line- and likely FBN1 variant-specific manner. The ability of MMC to increase fibrillin-1 deposition suggested potential applications for tissue-engineering approaches, e.g. to generate tendon or vascular tissues, where fibrillin-1 microfibrils and elastic fibres are key determinants of their biomechanical properties. Moreover, it suggested the potency of MMC to better mimic in vivo ECM environments in cell culture studies.

POGLUT2 and POGLUT3 O-glucosylate multiple EGF repeats in fibrillin-1, -2, and LTBP1 and promote secretion of fibrillin-1

Fibrillin-1 (FBN1) is the major component of extracellular matrix microfibrils, which are required for proper development of elastic tissues, including the heart and lungs. Through protein–protein interactions with latent transforming growth factor (TGF) β-binding protein 1 (LTBP1), microfibrils regulate TGF-β signaling. Mutations within the 47 epidermal growth factor-like (EGF) repeats of FBN1 cause autosomal dominant disorders including Marfan Syndrome, which is characterized by disrupted TGF-β signaling. We recently identified two novel protein O-glucosyltransferases, Protein O-glucosyltransferase 2 (POGLUT2) and 3 (POGLUT3), that modify a small fraction of EGF repeats on Notch. Here, using mass spectral analysis, we show that POGLUT2 and POGLUT3 also modify over half of the EGF repeats on FBN1, fibrillin-2 (FBN2), and LTBP1. While most sites are modified by both enzymes, some sites show a preference for either POGLUT2 or POGLUT3. POGLUT2 and POGLUT3 are homologs of POGLUT1, which stabilizes Notch proteins by addition of O-glucose to Notch EGF repeats. Like POGLUT1, POGLUT2 and 3 can discern a folded versus unfolded EGF repeat, suggesting POGLUT2 and 3 are involved in a protein folding pathway. In vitro secretion assays using the N-terminal portion of recombinant FBN1 revealed reduced FBN1 secretion in POGLUT2 knockout, POGLUT3 knockout, and POGLUT2 and 3 double-knockout HEK293T cells compared with wild type. These results illustrate that POGLUT2 and 3 function together to O-glucosylate protein substrates and that these modifications play a role in the secretion of substrate proteins. It will be interesting to see how disease variants in these proteins affect their O-glucosylation.

Assembly assay identifies a critical region of human fibrillin-1 required for 10-12 nm diameter microfibril biogenesis.

The human FBN1 gene encodes fibrillin-1 (FBN1); the main component of the 10-12 nm diameter extracellular matrix microfibrils. Marfan syndrome (MFS) is a common inherited connective tissue disorder, caused by FBN1 mutations. It features a wide spectrum of disease severity, from mild cases to the lethal neonatal form (nMFS), that is yet to be explained at the molecular level. Mutations associated with nMFS generally affect a region of FBN1 between domains TB3-cbEGF18-the "neonatal region". To gain insight into the process of fibril assembly and increase our understanding of the mechanisms determining disease severity in MFS, we compared the secretion and assembly properties of FBN1 variants containing nMFS-associated substitutions with variants associated with milder, classical MFS (cMFS). In the majority of cases, both nMFS- and cMFS-associated neonatal region variants were secreted at levels comparable to wild type. Microfibril incorporation by the nMFS variants was greatly reduced or absent compared to the cMFS forms, however, suggesting that nMFS substitutions disrupt a previously undefined site of microfibril assembly. Additional analysis of a domain deletion variant caused by exon skipping also indicates that register in the neonatal region is likely to be critical for assembly. These data demonstrate for the first time new requirements for microfibril biogenesis and identify at least two distinct molecular mechanisms associated with disease substitutions in the TB3-cbEGF18 region; incorporation of mutant FBN1 into microfibrils changing their integral properties (cMFS) or the blocking of wild type FBN1 assembly by mutant molecules that prevents late-stage lateral assembly (nMFS).

A new MMP-mediated prodomain cleavage mechanism to activate bone morphogenetic proteins from the extracellular matrix.

Since their discovery as pluripotent cytokines extractable from bone matrix, it has been speculated how bone morphogenetic proteins (BMPs) become released and activated from the extracellular matrix (ECM). In contrast to TGF-βs, most investigated BMPs are secreted as bioactive prodomain (PD)-growth factor (GF) complexes (CPLXs). Recently, we demonstrated that PD-dependent targeting of BMP-7 CPLXs to the extracellular fibrillin microfibril (FMF) components fibrillin-1 and -2 represents a BMP sequestration mechanism by rendering the GF latent. Understanding how BMPs become activated from ECM scaffolds such as FMF is crucial to elucidate pathomechanisms characterized by aberrant BMP activation and ECM destruction. Here, we describe a new MMP-dependent BMP-7 activation mechanism from ECM-targeted pools via specific PD degradation. Using Edman sequencing and mutagenesis, we identified a new and conserved MMP-13 cleavage site within the BMP-7 PD. A degradation screen with different BMP family PDs and representative MMP family members suggested utilization of the identified site in a general MMP-driven BMP activation mechanism. Furthermore, sandwich ELISA and solid phase cleavage studies in combination with bioactivity assays, single particle TEM, and in silico molecular docking experiments provided evidence that PD cleavage by MMP-13 leads to BMP-7 CPLX disintegration and bioactive GF release.

An integrated clinical and molecular study of a cohort of Turkish patients with Marfan syndrome harboring known and novel FBN1 variants.

Marfan syndrome (MFS) is an autosomal dominant genetic condition that mainly affects connective tissue in many parts of the body. Cardinal manifestations involve the ocular, skeletal, and cardiovascular systems. The diagnosis of MFS relies on the revised Ghent criteria, outlined by international expert opinion to facilitate accurate recognition of this syndrome as well as to improve patient management and counseling. However, it may not always be possible to make a definitive diagnosis according to these criteria in each patient and thus molecular confirmation is necessary in subjects with suspected MFS. This debilitating, if not fatal, disorder is caused by mutations in FBN1, which encodes a major constitutive element of extracellular microfibrils. Here, we present a detailed clinical and molecular analysis of 76 Turkish patients with definitive or suspected MFS diagnosed at our center between 2014 and 2019. We were able to identify a total of 51 different FBN1 variants in our cohort, 31 of which have previously been reported in the relevant scientific literature. The remaining 20 variants have not been documented to date. In one patient, we detected a large deletion including the entire FBN1 gene using the array CGH approach. Currently, there are very few studies on the genotype-phenotype correlation of patients with MFS, and no clear genotype-phenotype maps for MFS have been constructed so far, except for some cases. We believe that our findings will make a rich and peculiar contribution to the elusive genotype-phenotype relationship in MFS, especially in this large and populous ethnic group.

Latent-transforming growth factor beta-binding protein-2 (LTBP-2) is required for longevity but not for development of zonular fibers

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.

Specific miRNA and Gene Deregulation Characterize the Increased Angiogenic Remodeling of Thoracic Aneurysmatic Aortopathy in Marfan Syndrome.

Marfan syndrome (MFS) is a connective tissue disease caused by mutations in the FBN1 gene, leading to alterations in the extracellular matrix microfibril assembly and the early formation of thoracic aorta aneurysms (TAAs). Non-genetic TAAs share many clinico-pathological aspects with MFS and deregulation of some microRNAs (miRNAs) has been demonstrated to be involved in the progression of TAA. In this study, 40 patients undergoing elective ascending aorta surgery were enrolled to compare TAA histomorphological features, miRNA profile and related target genes in order to find specific alterations that may explain the earlier and more severe clinical outcomes in MFS patients. Histomorphological, ultrastructural and in vitro studies were performed in order to compare aortic wall features of MFS and non-MFS TAA. MFS displayed greater glycosaminoglycan accumulation and loss/fragmentation of elastic fibers compared to non-MFS TAA. Immunohistochemistry revealed increased CD133+ angiogenic remodeling, greater MMP-2 expression, inflammation and smooth muscle cell (SMC) turnover in MFS TAA. Cultured SMCs from MFS confirmed higher turnover and α-smooth muscle actin expression compared with non-MFS TAA. Moreover, twenty-five miRNAs, including miR-26a, miR-29, miR-143 and miR-145, were found to be downregulated and only miR-632 was upregulated in MFS TAA in vivo. Bioinformatics analysis revealed that some deregulated miRNAs in MFS TAA are implicated in cell proliferation, extracellular matrix structure/function and TGFβ signaling. Finally, gene analysis showed 28 upregulated and seven downregulated genes in MFS TAA, some of them belonging to the CDH1/APC and CCNA2/TP53 signaling pathways. Specific miRNA and gene deregulation characterized the aortopathy of MFS and this was associated with increased angiogenic remodeling, likely favoring the early and more severe clinical outcomes, compared to non-MFS TAA. Our findings provide new insights concerning the pathogenetic mechanisms of MFS TAA; further investigation is needed to confirm if these newly identified specific deregulated miRNAs may represent potential therapeutic targets to counteract the rapid progression of MFS aortopathy.

Understanding how defects in familial thoracic aneurysm and dissection (FTAAD)‐associated mutations in MAGP2 (MFAP5) affect its extracellular matrix localization and regulation of TGF‐β/SMAD signaling

In familial thoracic aneurysm and dissection (FTAAD), the aorta becomes weakened, stretching the vessel, a process known as aortic dilation, which in extreme cases can lead to tearing of the vessel wall layers, commonly referred to as aortic dissection. The direct consequences of FTAAD are stroke or heart attack that can potentially result in death of the patient. Research on Marfan Syndrome (MFS) and Loeys‐Dietz Syndrome (LDS) have shown that genes associated with similar aortic abnormalities have alterations in the TGF‐β/SMAD signaling pathway as well as extracellular matrix proteins (Faivre et al., 2007, Mizuguchi et al., 2004, and Loeys et al., 2006). Mutations in MFAP5, a gene that encodes for microfibril‐associated glycoprotein 2 (MAGP2), have been found in two FTAAD families, a nonsense mutation R158* and a missense mutation W21L (Barbier et al., 2014). The current working hypothesis is that MAGP2 normally plays an inhibitory role in TGF‐β/SMAD signaling, and that the FTAAD‐associated mutations are loss of function, since aortic tissue from a patient with the W21L mutation showed high phospho‐SMAD (p‐SMAD) levels. Moreover, MAGP2 has been shown to directly bind to active TGF‐β1 in vitro (Combs et al., 2013), and we have confirmed the results of Barbier et al. that the R158* mutant is poorly secreted. In contrast, the defect in the W21L mutant protein remains unidentified. It was secreted and bound to extracellular fibrillin microfibrils similar to wild‐type MAGP2 in our cell culture studies. What remains untested is whether FTAAD‐associated MAGP2 mutant proteins directly or indirectly affect TGF‐β/SMAD signaling. Therefore, the objective of this study is to determine whether R158* MAGP2 and W21L MAGP2 mutant proteins lose the ability to regulate TGF‐β signaling, leading to an increase in p‐SMAD2/3 levels in the cell. A TGF‐β signaling assay will be used to test soluble MAGP2 proteins, and fluorescent western blotting will then quantify the level of p‐SMAD 2/3 in whole cell lysate of MG63 cells, which expresses the TGF‐β receptor. Verification of both p‐SMAD2/3 and total SMAD antibodies have been completed via standard chemiluminescent protocols, and are currently being optimized for fluorescent detection. In parallel, stable cell lines are being produced to express wild‐type, R158*, or W21L MAGP2. A cell line expressing wild‐type MAGP2 has been generated, and the other two cell lines are being checked via immunofluorescence and western blotting to confirm that the cell lines express MAGP2. An understanding of how MAGP2 regulates the TGF‐β signaling could lead to the engineering of drugs to target mutant MAGP2 in hopes of improving the outcomes of FTAAD.Support or Funding InformationThis work was supported by the Department of Biological Science research funds (A. Miyamoto), CIRM Bridges to Stem Cell Research (A. Burcham, A. Keith), and HHMI (R. Daniels).

Are Fibrillin‐Notch Interactions Important in Development and Disease?

In humans, mutations in the gene for fibrillin‐1 (FBN1) cause both the Marfan syndrome as well as several types of acromelic dysplasias. Musculoskeletal features of the Marfan syndrome (long bone overgrowth and arachnodactyly, joint hypermobility, and a muscle wasting phenotype) are the opposite of those in acromelic dysplasias (short stature and brachydactyly, joint stiffness, and a hypermuscular build). How mutations in the same gene can result in opposite phenotypes is poorly understood. Mutations in FBN2 cause congenital contractural arachnodactyly, which features a mixture of related musculoskeletal features (muscle hypoplasia, contractures of the large and small joints and arachnodactyly).The fibrillins are multifunctional glycoproteins that polymerize into extracellular matrix microfibrils. Although we have associated fibrillin microfibril structure with the opposite musculoskeletal features of Marfan syndrome and acromelic dysplasias, we can only speculate that perturbations in growth factor signaling are involved. To begin to address this gap in our understanding, we hypothesize that direct interactions between fibrillin and cells coordinate growth factor signaling. Therefore, we searched for interactions between fibrillin and cellular receptors.Fibrillins, like Notch receptors and Notch ligands, are composed of multiple tandemly repeated cbEGF‐like domains. Each fibrillin contains 43 cbEGF‐like domains. What if Notch receptors or ligands also bind to fibrillins in the extracellular matrix? Our biochemical data imply that fibrillin interactions with Notch receptors or ligands are just as likely to occur in vivo as interactions between Notch receptors and known Notch transmembrane ligands. Human genetic evidence support significant roles for Notch signaling components in vascular and musculoskeletal diseases, tissues primarily affected in the fibrillinopathies.The current concept of Notch signaling is based strictly on cell‐cell communication. Our investigations have the potential to transform current understanding of context‐dependent Notch signaling by introducing the possibility that Notch signaling components also interact with fibrillin extracellular matrix.Support or Funding Informationgrants from the Shriners Hospitals for Children to Lynn Y. Sakai

Marfan syndrome revisited: From genetics to clinical practice

Abstract Marfan syndrome is an autosomal dominant connective tissue disease with an estimated incidence of 1 in 5000 individuals. In 90% of cases it is caused by mutations in the gene for fibrillin-1, the main constituent of extracellular microfibrils. Studies on animal models of Marfan syndrome have revealed that fibrillin-1 mutations interfere with local TGF-β signaling, in addition to impairing tissue integrity. The cardinal features involve the cardiovascular, ocular and skeletal systems. The diagnosis of Marfan syndrome is made according to the revised Ghent nosology. Early identification and appropriate management are critical for patients with Marfan syndrome, who are prone to the life-threatening cardiovascular complications of aortic aneurysms and aortic dissection. The standard treatment includes prophylactic beta-blockers in order to slow down dilation of the ascending aorta, and prophylactic aortic surgery. The success of current medical and surgical treatment of aortic disease in Marfan syndrome has substantially improved mean life expectancy, extending it above 72 years. This review aims to provide an overview of this hereditary disorder.

ADAMTSL6β promotes fibrillin-1 microfibril assembly, which is possibly mediated via binding through the third thrombospondin type I domain to fibrillin-1.

Fibrillin-1 is the major component of extracellular matrix microfibrils. Microfibrils dysfunction is responsible for the onset of various connective tissue diseases, including Marfan syndrome. Although ADAMTSL (a disintegrin and metalloproteinase with thrombospondin motifs-like) 6β is one of the fibrillin-1 binding proteins, the detailed mechanism underlying the involvement of ADAMTSL6β in microfibril formation remains unclear. In this study, we created deletion mutants of ADAMTSL6β and examined their interactions with fibrillin-1 assembly. Pull-down assay of the ADAMTSL6β deletion mutants and fibrillin-1 protein revealed that ADAMTSL6β binds to fibrillin-1 through the third thrombospondin type I domain. Furthermore, we observed that formation of fibrillin-1 matrix assembly was enhanced in MG63 cells, expressing full-length ADAMTSL6β, when compared with that of wild type MG63 cells. While MG63 cells expressing ΔTSP3-ADAMTSL6β form showed enhanced assembly formation, ΔTSP2-ADAMTSL6β form did not enhance that, indicating the difference between ΔTSP2-ΔTSP3 has a critical role for fibrillin-1 assembly. As the difference of ΔTSP2-ΔTSP3 is the third thrombospondin type I domain, we concluded that the third thrombospondin type I domain of ADAMTSL6β influence the microfibril formation. Our data are the functional presentation of the biological role of ADAMTSL6β in the process of microfibril formation.

A Review of the Type-1 Fibrillinopathies: Pathophysiology, Diagnosis and Novel Therapeutic Strategies

Type-1 fibrillinopathies are a family of connective tissue disorders with major clinical manifestations in the skeletal, ocular and cardiovascular systems. The type-1 fibrillinopathies are caused by mutations in the fibrillin-1 gene (FBN1), which encodes fibrillin-1, a large glycoprotein and a major component of the extracellular matrix microfibrils, providing both structural and regulatory support to connective tissues. The type-1 fibrillinopathies have been associated with over 1800 unique mutations within the FBN1 and demonstrate a wide range of phenotypic variability. This, in conjunction with a number of other factors has impacted on the identification of genotypephenotype correlations, pathogenesis and diagnostic tests for this family of diseases, leaving many open-ended theories. Current standard of care relies heavily on surgical intervention and lifelong use of β-blockers to slow disease progression, with research focused heavily on antagonism of transforming growth factor β, which is known to be dysregulated in patients with FBN1 mutations. Antisense oligonucleotides present a novel therapeutic strategy for the type-1 fibrillinopathies, by mediating the alteration of exon arrangement of both the normal and disease-causing mRNA transcripts, to re-establish the periodicity of fibrillin-1. The induced proteins, while internally truncated, should be homologous and thus be able to form multimer units. This treatment alone or in association with isoform switching, TGF-β antagonism or enhanced/inhibited protein degradation could facilitate the assembly of fibrillin-1 monomers into multimers and consequently a decrease in phenotypic severity. This review presents a basic overview of the past and current knowledge about the spectrum of type-1 fibrillinopathies with a particular focus on Marfan syndrome, as well as presenting novel potential therapeutic strategies.

A Mechanism for the Development of Chronic Traumatic Encephalopathy From Persistent Traumatic Brain Injury.

A mechanism that describes the progression of traumatic brain injury (TBI) to end-stage chronic traumatic encephalopathy (CTE) is offered in this article. This mechanism is based upon the observed increase in the concentration of both tau protein and of human leukocyte antigen (HLA) class I proteins; the HLA increase is expressed on the cell membrane of neural cells. These events follow the inflammatory responses caused by the repetitive TBI. Associated inflammatory changes include macrophage entry into the brain parenchyma from increased permeability of the blood-brain barrier (BBB) and microglial activation at the base of the sulci. The release of interferon gamma from the microglia and macrophages induces the marked increased expression of HLA class I proteins by the neural cells and subsequent redistribution of the tau proteins to the glial and neuronal surface. In those individuals with highly expressed HLA class I C, the high level of HLA binds tau protein electrostatically. The ionic region of HLA class I C (amino acid positions 50-90) binds to the oppositely charged ionic region of tau (amino acid positions 93-133). These interactions thereby shift the cellular localization of the tau and orient the tau spatially so that the cross-linking sites of tau (275-280 and 306-311) are aligned. This alignment facilitates the cross-linking of tau to form the intracellular and extracellular microfibrils of tau, the primary physiological characteristic of tauopathy. Following endocytosis of the membrane HLA/tau complex, these microfibrils accumulate and produce a tau-storage-like disease. Therefore, tauopathy is the secondary collateral process of brain injury, resulting from the substantial increase in tau and HLA expression on neural cells. This proposed mechanism suggests several potential targets for mitigating the clinical progression of TBI to CTE.

Abstract 284: Lrp1 Regulates Smooth Muscle Contractility by Modulating Cytoskeletal Dynamics and Ca 2+ Signaling

Objective: Mutations affecting proteins in the extracellular matrix (ECM), microfibrils, or vascular smooth muscle cells (VSMCs) that impact contractility can predispose individuals to thoracic aortic aneurysms. We reported previously that the low-density lipoprotein receptor-related protein 1 (LRP1) maintains vessel wall integrity, and smooth muscle LRP1-deficient ( smLRP1 -/- ) mice exhibited aortic dilatation. The current study focused on the descending thoracic aorta (DTA) and examined the role of LRP1 in VSMC contractility and its potential effect on the vascular ECM. Approach and Results: LRP1-deficient VSMCs exhibited a synthetic phenotype characterized by higher proliferation rates and an increase in synthetic organelles, mitochondria, multivesicular bodies, and macropinocytotic vesicles. LRP1-deficient VSMCs also displayed changes in their microfilament and actin structure that result in an inadequate interaction with the ECM. Quantitative proteomics identified proteins involved in actin polymerization and contraction that were downregulated significantly in the DTA of smLRP1 -/- mice. Further analysis by qRT-PCR revealed attenuated mRNA levels for α-1D adrenergic receptor ( adra1d ) and calcium voltage-gated channel subunit α1 C ( cacna1c ) in smLRP1 -/- aortas. Isometric contraction assays confirmed aberrant contraction of smLRP1 -/- aortic rings when stimulated with vasoconstrictors. Furthermore, intracellular calcium imaging identified defects in response to a ryanodine receptor agonist in smLRP1 -/- aortic rings. Conclusions: These results suggest that LRP1 is required for maintaining the VSMC contractile phenotype and identifies a novel role for LRP1 in calcium homeostasis that potentially protects against aneurysm development.

Alternative splicing of MAGP2 generates a non‐glycosylated variant still capable of localizing to extracellular microfibrils

Alterations in microfibril‐associated glycoprotein 2 (MAGP2) have been linked to familial thoracic aortic aneurysm and dissection (FTAAD) and ovarian cancer, suggesting that this protein has an interesting role in normal adult homeostasis. Here, a previously unstudied splice variant of MAGP2 noted in a public database search was characterized. Two mRNA isoforms of MAGP2 were expressed in a human ovarian cancer cell line (CAOV3) by RT‐PCR; sequencing revealed that the longer mRNA was the canonical MAGP2 mRNA that encodes a 173‐amino acid protein (MAGP2‐a), and the shorter variant encoded a 163‐amino acid protein (MAGP2‐b), due to the removal of the seventh exon. Interestingly, the seventh exon contains the conserved asparagine residue (Asp79) thought to be the major site of N‐glycosylation on MAGP2. As expected, MAGP2‐b was both poorly glycosylated and poorly secreted when overexpressed in either 293T cells or a mouse ovarian epithelial cell line, T3, as measured by immunoblotting analysis; this result is consistent with the canonical role of glycosylation modifications of secreted proteins. Since a small fraction of MAGP2‐b protein was secreted, studies were performed to understand what functions MAGP2‐b was capable of, if secreted. To determine if MAGP2‐b could bind and localize to extracellular microfibrils as previously reported for MAGP2‐a, immunofluorescence studies were conducted in T3 cells that generate a striking fibrillin‐2‐rich microfibril matrix in cell culture. Surprisingly, MAGP2‐b could easily be found localized to microfibrils, at levels similar to the MAGP2‐a isoform that is glycosylated and more effectively secreted than MAGP2‐b. To more clearly delineate the functional consequences of glycosylation versus the loss of ten amino acids via alternative splicing, an Asp79Gln (N79Q) mutation was generated in MAGP2‐a to block N‐glycosylation. The N79Q MAGP2‐a protein does not appear to be glycosylated, since the molecular weight of the protein on immunoblots is unchanged by PNGase F treatment. The secretion and microfibril binding properties of the N79Q mutant are very similar to MAGP2‐b; they are both poorly secreted but still able to bind to microfibrils at levels equal to or greater than the fully glycosylated MAGP2‐a variant. Together, these data indicate that glycosylation of MAGP2 is not required for microfibril binding; it also suggests that the non‐glycosylated MAGP2‐b may preferentially bind to microfibrils after cellular export. Why might it be useful for such a MAGP2 variant to exist? In this regard, there is some correlative evidence to suggest that MAGP2 binding of active TGF‐β may negatively regulate TGF‐β activity, based on studies on MAGP2 biochemistry and FTAAD‐associated mutations of MAGP2. Although speculative, the existence of a mechanism that tethers MAGP2 to microfibrils at the expense of free, soluble MAGP2 may make MAGP2 a more effective microfibril tether for active TGF‐β.Support or Funding InformationCSUF Department of Biological Science. Student research assistants funded by CIRM Bridges to Stem Cell Research (Andrew Yale, Cherie Petersen) and HHMI Undergraduate Research Program (Ryan Daniels).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.