Mutations In Fibrillin-1 Research Articles

Analysis of FBN1 genemutations in a pedigree with Marfan syndrome

Mutations in fibrillin-1 (FBN1) were detected in an autosomal dominant Marfan syndrome (MFS) pedigree. The related phenotypes and the significance of mutation screening were discussed. Complete medical and cardiovascular examinations for all pedigree members were performed. Whole exons sequencing (WES) was used to sequence the DNA of the patients and their relatives. The potential pathogenic mutation sites were screened by bioinformatics method. Sanger sequencing was used to verify the mutation sites in the pedigree. The results showed that FBN1 missense mutation was c.6806 T>C in exon 56, resulting in isoleucine being replaced by threonine (p. Ile2269Thr). This mutation has not been reported in Chinese Han population. The occurrence of the mutations strongly correlated with the phenotypes of the patients. The results expand the mutation spectrum of FBN1, and it is helpful to further explore the molecular pathogenesis of MFS and MFS related diseases.

Methotrexate associated with a lipid core nanoparticle prevented the dilation and dissection of the aortic arch in mice with Marfan syndrome

Background and Aims : Fibrillin-1 mutations result in rupture of the aorta, the main cause of mortality in patients with Marfan syndrome (MFS). When MTX is associated to LDE, the cell uptake is increased, which endows MTX with enhanced action mechanisms and the drug toxicity is diminished. The aims of this study was to investigate whether treatment with LDE-MTX can prevent the development of aortic arch lesions in a murine model of MFS.

Fibrillin-1 mutant mouse captures defining features of human primary open glaucoma including anomalous aqueous humor TGF beta-2

Primary open angle glaucoma (POAG) features an optic neuropathy, elevated aqueous humor (AH) TGFβ2, and major risk factors of central corneal thickness (CCT), increasing age and intraocular pressure (IOP). We examined Tight skin (Tsk) mice to see if mutation of fibrillin-1, a repository for latent TGFβ, is associated with characteristics of human POAG. We measured: CCT by ocular coherence tomography (OCT); IOP; retinal ganglion cell (RGC) and optic nerve axon counts by microscopic techniques; visual electrophysiologic scotopic threshold responses (STR) and pattern electroretinogram (PERG); and AH TGFβ2 levels and activity by ELISA and MINK epithelial cell-based assays respectively. Tsk mice had open anterior chamber angles and compared with age-matched wild type (WT) mice: 23% thinner CCT (p < 0.003); IOP that was higher (p < 0.0001), more asymmetric (p = 0.047), rose with age (p = 0.04) and had a POAG-like frequency distribution. Tsk mice also had RGCs that were fewer (p < 0.04), declined with age (p = 0.0003) and showed increased apoptosis and glial activity; fewer optic nerve axons (p = 0.02); abnormal axons and glia; reduced STR (p < 0.002) and PERG (p < 0.007) visual responses; and higher AH TGFβ2 levels (p = 0.0002) and activity (p = 1E−11) especially with age. Tsk mice showed defining features of POAG, implicating aberrant fibrillin-1 homeostasis as a pathogenic contributor to emergence of a POAG phenotype.

The critical role of the TB5 domain of fibrillin-1 in endochondral ossification.

Mutations in the fibrillin-1 (FBN1) gene are responsible for the autosomal dominant form of geleophysic dysplasia (GD), which is characterized by short stature and extremities, thick skin and cardiovascular disease. All known FBN1 mutations in patients with GD are localized within the region encoding the transforming growth factor-β binding protein-like 5 (TB5) domain of this protein. Herein, we generated a knock-in mouse model, Fbn1Y1698C by introducing the p.Tyr1696Cys mutation from a patient with GD into the TB5 domain of murine Fbn1 to elucidate the specific role of this domain in endochondral ossification. We found that both Fbn1Y1698C/+ and Fbn1Y1698C/Y1698C mice exhibited a reduced stature reminiscent of the human GD phenotype. The Fbn1 point mutation introduced in these mice affected the growth plate formation owing to abnormal chondrocyte differentiation such that mutant chondrocytes failed to establish a dense microfibrillar network composed of FBN1. This original Fbn1 mutant mouse model offers new insight into the pathogenic events underlying GD. Our findings suggest that the etiology of GD involves the dysregulation of the extracellular matrix composed of an abnormal FBN1 microfibril network impacting the differentiation of the chondrocytes.

Next-generation sequencing panel analysis in 24 Chinese patients with congenital ectopia lentis.

Congenital ectopia lentis (CEL) is a rare but serious disease. We use next-generation sequencing to detect genes associated with lens abnormalities in 24 patients with bilateral CEL and search for pathogenic genes and mutation sites. A total of 24 patients diagnosed with CEL from January 2019 to November 2019 were enrolled in this study, and their clinical data were collected and genome-wide deoxyribonucleic acid was extracted from peripheral venous blood. Targeted gene capture technology was used to obtain 188 exons of lens abnormality-related genes, which were sequenced using a high-throughput method. The mutation sites were determined through data analysis and verified by the Sanger method. According to the data from previous studies, the association between the genotype and clinical phenotype was analysed. Of the 24 patients, 23 had mutations in the fibrillin-1 (FBN1) gene, and 20 were diagnosed with Marfan syndrome. The 23 cases of FBN1 mutations were all heterozygous mutations, including 17 missense mutations, 3 splicing variants, 2 exon deletion mutations, 1 codon mutation, and 9 new mutations. A total of 17 mutations were located in the calcium-binding epidermal growth factor domain, including 16 mutations that contained missense mutations of cysteine. In addition, a heterozygous mutation of the gap junction protein alpha 8 (GJA8) gene was detected in one patient. In this study, we identified 23 FBN1 gene mutations and 1 GJA8 gene mutation in 24 patients with CEL. Of these, 9 new FBN1 mutations and 14 known mutations were found. The results expanded the mutation spectrum of the FBN1 gene, suggesting that FBN1 mutation may be the main cause of CEL in Chinese patients.

Fibrillin-1-regulated miR-122 has a critical role in thoracic aortic aneurysm formation.

Thoracic aortic aneurysms (TAA) in Marfan syndrome, caused by fibrillin-1 mutations, are characterized by elevated cytokines and fragmentated elastic laminae in the aortic wall. This study explored whether and how specific fibrillin-1-regulated miRNAs mediate inflammatory cytokine expression and elastic laminae degradation in TAA. miRNA expression profiling at early and late TAA stages using a severe Marfan mouse model (Fbn1mgR/mgR) revealed a spectrum of differentially regulated miRNAs. Bioinformatic analyses predicted the involvement of these miRNAs in inflammatory and extracellular matrix-related pathways. We demonstrate that upregulation of pro-inflammatory cytokines and matrix metalloproteinasesis a common characteristic of mouse and human TAA tissues. miR-122, the most downregulated miRNA in the aortae of 10-week-old Fbn1mgR/mgR mice, post-transcriptionally upregulated CCL2, IL-1β and MMP12. Similar data were obtained at 70weeks of age using Fbn1C1041G/+ mice. Deficient fibrillin-1-smooth muscle cell interaction suppressed miR-122 levels. The marker for tissue hypoxia HIF-1α was upregulated in the aortic wall of Fbn1mgR/mgR mice, and miR-122 was reduced under hypoxic conditions in cell and organ cultures. Reduced miR-122 was partially rescued by HIF-1α inhibitors, digoxin and 2-methoxyestradiol in aortic smooth muscle cells. Digoxin-treated Fbn1mgR/mgR mice demonstrated elevated miR-122 and suppressed CCL2 and MMP12 levels in the ascending aortae, with reduced elastin fragmentation and aortic dilation. In summary, this study demonstrates that miR-122 in the aortic wall inhibits inflammatory responses and matrix remodeling, which is suppressed by deficient fibrillin-1-cell interaction and hypoxia in TAA.

Fibrillin-1 regulates white adipose tissue development, homeostasis, and function

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 - Fbn1mgR/mgR (20–25% of normal fibrillin-1) and Fbn1C1041G/+ (missense mutation in fibrillin-1) to examine the role of fibrillin-1 in adipose tissue development and homeostasis. Fibrillin-1 was detected around mature adipocytes in both mouse and human white adipose tissues. As expected, Fbn1mgR/mgR mice displayed a significant reduction of fibrillin-1 in white adipose tissue, and no change was observed for Fbn1C1041G/+ mice, each compared to their respective littermates. Male Fbn1mgR/mgR mice had more white and brown adipose tissues, whereas female Fbn1mgR/mgR and both male and female Fbn1C1041G/+ showed no difference compared to their respective wild-type littermates. Consistent with this data, male Fbn1mgR/mgR mice displayed hyperinsulinemia and an insulin resistance phenotype with higher levels of cholesterol and high-density lipoproteins in the serum. Fibrillin-1 deficiency in male Fbn1mgR/mgR mice also promoted adipogenic gene expression and led to hypertrophic expansion of mature adipocytes. To further elucidate the fibrillin-1-dependent adipogenic mechanisms in cell culture, we used primary bone marrow derived mesenchymal stem/stromal cells (MSCs) from Fbn1mgR/mgR, Fbn1C1041G/+ and wild-type mice. Increased lipid content, adipogenic differentiation and pAKT levels were observed when MSCs from both male and female Fbn1mgR/mgR mice were differentiated. Furthermore, a recombinant fragment spanning the C-terminal half of fibrillin-1 significantly reduced adipocyte differentiation i) by binding to MSCs and inhibiting adipogenic commitment, and ii) by sequestering insulin, together suppressing the AKT signaling pathway. This fibrillin-1 fragment also rescued enhanced adipogenic differentiation of MSCs derived from Fbn1mgR/mgR mice. Overall, this study shows that altered adipose tissue homeostasis observed in fibrillin-1 deficient mice depends on the type of fibrillin-1 deficiency and the biological sex, and it shows that fibrillin-1 is a negative regulator of adipogenesis.

The Multiple Functions of Fibrillin-1 Microfibrils in Organismal Physiology.

Fibrillin-1 is the major structural component of the 10 nm-diameter microfibrils that confer key physical and mechanical properties to virtually every tissue, alone and together with elastin in the elastic fibers. Mutations in fibrillin-1 cause pleiotropic manifestations in Marfan syndrome (MFS), including dissecting thoracic aortic aneurysms, myocardial dysfunction, progressive bone loss, disproportionate skeletal growth, and the dislocation of the crystalline lens. The characterization of these MFS manifestations in mice, that replicate the human phenotype, have revealed that the underlying mechanisms are distinct and organ-specific. This brief review summarizes relevant findings supporting this conclusion.

How to Distinguish Marfan Syndrome from Marfanoid Habitus in a Physical Examination-Comparison of External Features in Patients with Marfan Syndrome and Marfanoid Habitus.

Marfan Syndrome (MFS) is a systemic disorder caused by mutations in fibrillin-1. The most common cause of mortality in MFS is dissection and rupture of the aorta. Due to a highly variable and age-dependent clinical spectrum, the diagnosis of MFS still remains sophisticated. The aim of the study was to determine if there exist phenotypic features that can play the role of “red flags” in cases of MFS suspicion. The study population included 306 patients (199 children and 107 adults) who were referred to the Department of Pediatric Cardiology due to suspicion of MFS. All patients underwent complete clinical evaluation in order to confirm the diagnosis of MFS according to the modified Ghent criteria. MFS was diagnosed in 109 patients and marfanoid habitus in 168 patients. The study excluded 29 patients with other hereditary thoracic aneurysm syndromes. Comparative analysis between patients with Marfan syndrome and marfanoid habitus was performed. Symptoms with high prevalence and high positive likelihood ratio were identified (pectus carinatum, reduced elbow extension, hindfoot deformity, gothic palate, downslanting palpebral fissures, lens subluxation, myopia ≥ 3 dioptres remarkably high stature). The differentiation between patients with MFS and marfanoid body habitus is not possible by only assessing external body features; however, “red flags” could be helpful in the screening phase.

Basigin genetic depletion reduces thoracic aortic aneurysm formation in marfan syndrome mice

Abstract Background Marfan Syndrome (MFS) is a rare genetic disease due to mutations in fibrillin 1 gene [1]. It is characterized by thoracic aortic aneurysm (TAA), whose dissection is the major cause of morbidity and mortality in MFS patients [2]. While pathological mechanisms underlying MFS extracellular matrix remodelling have started to be elucidated, an etiological treatment is not available yet [3,4]. Recently, EMMPRIN has been proposed as new actor in pro-fibrotic processes associated with TAA development in MFS patients [4,5]. Purpose To confirm the role of EMMPRIN (i.e. Basigin in mice, Bsg) in TAA formation and progression in a preclinical setting, we assessed whether Bsg genetic depletion in a mouse model of MFS (Fbn1C1039G/+) [6] had an impact on aortic phenotype. Methods We generated a double transgenic mouse model, Fbn1C1039G/+-Bsg+/−, by crossing commercially available Fbn1C1039G/+ with Bsg+/− mice (provided by Dr. Kadomatsu) [7] and we prospectively measured aortic root size in 12 Fbn1C1039G/+-Bsg+/−, 9 Fbn1C1039G/+, and 15 WT mice every 4 weeks from 12 to 20 weeks of age by 2D-echocardiography. Both qRT-PCR and Western Blot were used to evaluate EMMPRIN downstream pro-fibrotic pathway activation in thoracic aortic tissue obtained from mice sacrificed at 12 weeks. Plasma soluble EMMPRIN (sEMMPRIN) levels were assessed by ELISA. Results Echocardiographic assessment at 12 and 20 weeks of age confirmed significantly dilated aortic root in Fbn1C1039G/+ vs WT (mean±SD at 12 weeks: 1.772±0.081 vs 1.394±0.068 mm, respectively, p&amp;lt;0.0001; 20 weeks: 1.899±0.103 vs 1.520±0.083 mm, respectively, p&amp;lt;0.0001), and revealed that Fbn1C1039G/+-Bsg+/− mice had lower aortic root diameter extent compared to Fbn1C1039G/+ (12 weeks: 1.667±0.109 vs 1.772±0.081 mm, respectively, p=0.026; 20 weeks: 1.752±0.129 vs 1.899±0.103 mm, respectively, p=0.011). Pro-fibrotic gene profiling in a subset of 12 weeks old mice showed that Col1A1, Col3A1 and FN1 genes were more expressed in Fbn1C1039G/+ than in WT mice and that the up-regulation of these genes was mitigated in Fbn1C1039G/+ by Bsg depletion (ANOVA p=0.048, p=0.055, and p=0.0003, respectively). Moreover, we found a significant activation of the EMMPRIN downstream pro-fibrotic ERK1/2 pathway in Fbn1C1039G/+ compared to WT mice which was attenuated in the Fbn1C1039G/+-Bsg+/− model (p=0.030). Plasma sEMMPRIN levels were lower in Fbn1C1039G/+ than in WT mice, consistent with what we previously observed in MFS patients [5], and decreased over time in both Fbn1C1039G/+ and WT mice (2-way ANOVA disease p=0.013, time p=0.012, interaction p=ns). Conclusions Basigin genetic depletion reduces dilatation of the aortic root and pro-fibrotic pathway activation in a preclinical MFS model, confirming previous proof-of-concept findings and paving the way for the development of innovative therapies for MFS patients. Plasma sEMMPRIN levels stand as promising tool to predict the aortic enlargement progression in MFS models and patients. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Italian Ministery of Health

Correlation between FBN1 mutations and ocular features with ectopia lentis in the setting of Marfan syndrome and related fibrillinopathies.

Mutations of fibrillin-1 (FBN1) have been associated with Marfan syndrome and pleiotropic connective tissue disorders, collectively termed as "type I fibrillinopathy". However, few genotype-phenotype correlations are known in the ocular system. Patients with congenital ectopia lentis (EL) received panel-based next-generation sequencing, complemented with multiplex ligation-dependent probe amplification. In a total of 125 probands, the ocular phenotypes were compared for different types of FBN1 mutations. Premature termination codons were associated with less severe EL and a thinner central corneal thickness (CCT) than the inframe mutations. The eyes of patients with mutations in the C-terminal region had a higher incidence of posterior staphyloma than those in the middle and N-terminal regions. Mutations in the TGF-β-regulating sequence had larger horizontal corneal diameters (white-to-white [WTW]), higher incidence of posterior staphyloma, but less severe EL than those with mutations in other regions. Mutations in the neonatal region were associated with thinner CCT. Longer axial length (AL) was associated with mutations in the C-terminal region or TGF-β regulating sequence after adjusting for age, EL severity, and corneal curvature radius. FBN1 genotype-phenotype correlations were established for some ocular features, including EL severity, AL, WTW, CCT, and so forth, providing novel perspectives and directions for further mechanistic studies.

Combining clinical examination with exome sequencing for the diagnosis and treatment of Marfan syndrome: a case series of 6 families from China.

Marfan syndrome (MFS) is a rare autosomal dominant connective tissue disorder. Diagnosing MFS can be challenging as the disease's severity and clinical manifestations differ between pathogenic variants, and because a lack of published information currently exists on phenotype-genotype correlations. This report aims to underline the clinical manifestations associated with fibrillin-1 (FBN1) gene mutations by assessing MFS in 6 families from China. We diagnosed 6 patients and their relatives with MFS by combining a clinical examination (based on the 2010 revised Ghent nosology criteria) with a targeted next-generation sequencing analysis. The functional analysis of the causal mutations and clinical details of the affected patients were then assessed. We identified 6 pathogenic mutations in FBN1, including 1 novel frameshift, 1 nonsense, and 4 missense mutations. Most uniquely, mitral valve prolapses (MVP) and ectopia lentis (EL) were found in the cysteine-related mutations. Typically, facial symptoms of MFS are observed in frameshift or nonsense mutants, not in cysteine-related ones. Furthermore, the patients with premature terminal codons had a more serious skin condition than patients with missense mutations, partly indicating the important effect FBN1 has on skin. This study expands the mutation spectrum of MFS and highlights possible genotype-phenotype correlations, thereby improving the early diagnosis and symptomatic treatment of the disease.

Abstract P105: Inducible Deletion Of Fibrillin-1 In Smooth Muscle Cells Has Modest Effects On Thoracic Aortic Dilatation In Adult Mice

Introduction: Fibrillin-1 ( Fbn1 ) mutations are associated with thoracic aortic aneurysms (TAAs) in patients with Marfan syndrome. Constitutive deletion of Fbn1 leads to neonatal death due to aortic rupture in mice. However, the role of Fbn1 in maintaining aortic integrity during the adult phase remains unclear. Since smooth muscle cell (SMC) is a major constituent of the aortic media, this study examined whether deletion of Fbn1 in SMCs of adult mice leads to compromise of aortic integrity. Methods and Results: Fbn1 floxed mice were generated that included insertion of tdTomato as a reporter for gene deletion. Female Fbn1 floxed mice were bred with male Fbn1 floxed mice expressing CreER T2 driven by an Acta2 promotor. Tamoxifen was injected intraperitoneally for 5 days into male Fbn1 floxed littermate mice that were either Cre negative or positive (n=6-8/genotype, 5-week-old) to create sm Fbn1 +/+ and -/- mice, respectively. Gene recombination was confirmed by the presence of tdTomato in sm Fbn1 -/- mice using PCR and confocal microscopy. To investigate the impact of SMC-specific Fbn1 deletion, male sm Fbn1 +/+ and -/- littermates terminated at two intervals, 5 and 36 weeks after completion of tamoxifen injections. In situ aortic images were captured, and aortic diameters were measured at ascending, arch, and descending thoracic aortas. Five weeks after tamoxifen injection, aortic diameters of smFbn1+/+ and -/- mice were not different. Despite the long-term deletion of Fbn1 in SMCs, aortic dilatation was not observed in any regions of aortas in sm Fbn1 -/- mice compared to wild type littermates 36 weeks after tamoxifen injection. Next, we investigated whether Fbn1 deletion in SMCs augmented angiotensin II (AngII)-induced aortic dilatation. AngII was infused subcutaneously into male sm Fbn1 +/+ and -/- littermates for 12 weeks (n=11-12/genotype), started 2weeks after completion of tamoxifen injections. AngII-induced aortic dilatations were augmented modestly by SMC-specific Fbn1 deletion (+/+ 1.7±0.0, -/- 1.9±0.1 mm, P=0.047). Conclusion: Deletion of Fbn1 in SMCs did not cause spontaneous TAA formation and had modest effects on AngII-induced TAAs in adult mice. Fbn1 in SMCs does not exert a critical role in mice after the aortic structure has formed.

Fibrillin-1 gene mutations in a Chinese cohort with congenital ectopia lentis: spectrum and genotype–phenotype analysis

AimsTo identify the mutation spectrum and genotype–phenotype correlations of fibrillin-1 (FBN1) mutations in a Chinese cohort with congenital ectopia lentis (EL).MethodsPatients clinically suspected of congenital zonulopathy were screened using panel-based...

Genetic variants in Chinese patients with sporadic Stanford type A aortic dissection.

BackgroundGenetic disorders are strongly associated with aortic disease. However, the identities of genetic mutations in sporadic Stanford type A aortic dissection (STAAD) are not clear. The present study analysed the possible genetic mutations of the known pathogenic genes of aortic disease and the clinical characteristics in patients with sporadic STAAD.MethodsWe analysed genetic mutations in 26 genes that underlie aortic aneurysms and dissections in 100 sporadic STAAD patients and 568 healthy controls after whole-genome sequencing (WGS). Clinical features and in-hospital death were determined in all STAAD patients.ResultsIn total, 60 suspicious pathogenic mutations (56 novel and 4 previously reported) in 19 genes were identified in 50% (50/100) of patients, and 14 patients had more than 1 mutation. The ascending aortic diameter was extended in patients with mutations (49.1±12.3 vs. 43.7±11.2 mm, P=0.023), and the DeBakey type I phenotype was more common in patients with mutations in genes that coded extracellular matrix (ECM) components than in patients with mutations in other genes (96.6% vs. 66.7%, P=0.007). Patients with fibrillin-1 (FBN1) mutations were younger than patients without FBN1 mutations (44.7±11.0 vs. 53.5±12.1, P=0.030). Subgroup analyses revealed an increased risk of in-hospital mortality in mutation carriers (44.4% vs. 10.5%, P=0.029) but only in patients who received conservative treatment.ConclusionsHalf of Chinese patients with a sporadic form of STAAD may carry mutations in known pathogenic genes of aortic disease, and these patients may exhibit distinct clinical features and poor clinical outcomes with the use of conservative treatment.

Abnormal mitochondria is associated with aortic aneurysm in a mouse model of Marfan Syndrome

Aortic aneurysms, or the pathogenic remodeling and stiffening of the aorta extracellular matrix (ECM), are usually undetectable until excessive expansion, dissection, or rupture occurs. The understanding of the molecular mechanisms of aneurysm and dissection remains incomplete, but heritable syndromes such as Marfan Syndrome (MFS), where genetic mutations result in aneurysm phenotypes, offer models to discover functional pathways. Recent work in our lab and others have identified a potential association between reduced mitochondrial regulators, such as PGC1alpha, mitochondrial content, and aneurysm. These associations have not been fully described to date in a model of classical MFS driven by Fibrillin-1 (FBN1) mutation. We hypothesized that mitochondrial content and integrity would be disrupted in the MFS aorta and isolated MFS smooth muscle cell models. Western blot for several key mitochondrial proteins associated with oxidative phosphorylation (NDUFB8, SDHB, UQCRC2, MTCO1, ATP5A) demonstrated significant reduction in the aortic root of MFS mice compared to wild type (WT) mice. Consistent with these in vivo findings, we observed a reduced total quantity of mitochondria relative to total cell volume in isolated primary MFS relative to WT ascending aortic smooth muscle cells. Furthermore, analysis of mitochondrial morphology revealed that MFS primary lines have more fragmented mitochondrial networks than WT lines, indicating abnormal structural regulation of mitochondrial networks in MFS. Through these initial studies we have found indicators of potential dysfunction in the networks of MFS mitochondria. Future work will elucidate the functional consequences of these abnormalities and decipher their potential role in the pathogenesis of aneurysm progression in MFS.

The fibrillin-1 RGD motif posttranscriptionally regulates ERK1/2 signaling and fibroblast proliferation via miR-1208.

Fibrillin-1 is an extracellular matrix protein which contains one conserved RGD integrin-binding motif. It constitutes the backbone of microfibrils in many tissues, and mutations in fibrillin-1 cause various connective tissue disorders. Although it is well established that fibrillin-1 interacts with several RGD-dependent integrins, very little is known about the associated intracellular signaling pathways. Recent published evidence identified a subset of miRNAs regulated by fibrillin-1 RGD-cell adhesion, with miR-1208 among the most downregulated. The present study shows that the downregulated miR-1208 controls fibroblast proliferation. Inhibitor experiments revealed that fibrillin-1 RGD suppressed miR-1208 expression via c-Src kinase and the downstream JNK signaling. Bioinformatic prediction and experimental target sequence validation demonstrated four miR-1208 binding sites on the ERK2 mRNA and one on the MEK1 mRNA. ERK2 and MEK1 are critical proliferation-promoting kinases. Decreased miR-1208 levels elevated the total and phosphorylated ERK1/2 and MEK1/2 protein levels and the phosphorylated to total ERK1/2 ratio. Together, the data demonstrate a novel outside-in signaling mechanism explaining how fibrillin-1 RGD-cell binding regulates fibroblast proliferation.

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).

The mgΔlpn mouse model for Marfan syndrome recapitulates the ocular phenotypes of the disease

PurposeFibrillin-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. MethodsEyes 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. ResultsMutant 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. ConclusionsIn 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.

AAV-mediated AP-1 decoy oligonucleotide expression inhibits aortic elastolysis in a mouse model of Marfan syndrome.

Marfan syndrome is one of the most common inherited disorders of connective tissue caused by fibrillin-1 mutations, characterized by enhanced transcription factor AP-1 DNA binding activity and subsequently abnormally increased expression and activity of matrix-metalloproteinases (MMPs). We aimed to establish a novel adeno-associated virus (AAV)-based strategy for long-term expression of an AP-1 neutralizing RNA hairpin (hp) decoy oligonucleotide (dON) in the aorta to prevent aortic elastolysis in a murine model of Marfan syndrome. Using fibrillin-1 hypomorphic mice (mgR/mgR), aortic grafts from young (9 weeks old) donor mgR/mgR mice were transduced ex vivo with AAV vectors and implanted as infrarenal aortic interposition grafts in mgR/mgR mice. Grafts were explanted after 30 days. For in vitro studies, isolated primary aortic smooth muscle cells (SMCs) from mgR/mgR mice were used. Elastica-van-Giesson staining visualized elastolysis, reactive oxygen species (ROS) production was assessed using dihydroethidine staining. RNA F.I.S.H. verified AP-1 hp dON generation in the ex vivo transduced aortic tissue. MMP expression and activity were assessed by western blotting and immunoprecipitation combined with zymography.Transduction resulted in stable therapeutic dON expression in endothelial and SMCs. MMP expression and activity, ROS formation as well as expression of monocyte chemoattractant protein-1 were significantly reduced. Monocyte graft infiltration declined and the integrity of the elastin architecture was maintained. RNAseq analysis confirmed the beneficial effect of AP-1 neutralization on the pro-inflammatory environment in SMCs. This novel approach protects from deterioration of aortic stability by sustained delivery of nucleic acids-based therapeutics and further elucidated how to interfere with the mechanism of elastolysis.