Mutations In Fibrillin-1 Research Articles

Marfan syndrome: whole-exome sequencing reveals de novo mutations, second gene and genotype-phenotype correlations in the Chinese population.

Marfan syndrome (MFS) is a dominant monogenic disease caused by mutations in fibrillin 1 (FBN1). Cardiovascular complications are the leading causes of mortality among MFS. In the present study, a whole-exome sequencing of MFS in the Chinese population was conducted to investigate the correlation between FBNI gene mutation and MFS. Forty-four low-frequency harmful loci were identified for the FBN1 gene in HGMD database. In addition, 38 loci were identified in the same database that have not been related to MFS before. A strict filtering and screening protocol revealed two patients of the studied group have double mutations in the FBN1 gene. The two patients harboring the double mutations expressed a prominent, highly pathological phenotype in the affected family. In addition to the FBN1 gene, we also found that 27 patients had mutations in the PKD1 gene, however these patients did not have kidney disease, and 16 of the 27 patients expressed aortic related complications. Genotype-phenotype analysis showed that patients with aortic complications are older in the family, aged between 20 and 40 years.

Health-related quality of life in Marfan syndrome: a 10-year follow-up

BackgroundMarfan syndrome, a rare hereditary connective tissue disorder caused by mutations in fibrillin-1, can affect many organ systems, especially the cardiovascular system. Previous research has paid less attention to health-related quality of life and prospective studies on this topic are needed. The aim of this study was to assess changes in health-related quality of life after 10 years in a Norwegian Marfan syndrome cohort.MethodsForty-seven Marfan syndrome patients ≥ 18 years were investigated for all organ manifestations in the 1996 Ghent nosology and completed the self-reported questionnaire, Short-Form-36 Health Survey, at baseline in 2003–2004 and at follow-up in 2014–2015. Paired sample t tests were performed to compare means and multiple regression analyses were performed with age, sex, new cardiovascular and new non-cardiovascular pathology as predictors.ResultsAt 10-year follow-up: a significant decline was found in the physical domain. The mental domain was unchanged. Older age predicted a larger decline in physical health-related quality of life. None of the chosen Marfan-related variables predicted changes in any of the subscales of the Short-Form 36 Health Survey or in the physical or the mental domain.ConclusionKnowledge of decline in the physical domain, not related to organ affections, may be important in the follow-up of Marfan syndrome patients.

Abstract 13025: Methotrexate Carried in Lipid Core Nanoparticles Prevented the Dilation and Dissection of the Aortic Arch in Mice With Marfan Syndrome by Increasing the Bioavailability of Intracellular Adenosine

Introduction: Mutations in the fibrillin-1 gene result in aneurysms and rupture of the aorta, the main cause of mortality in patients with Marfan syndrome (MFS). Inflammation is associated with dissection and rupture of the aorta. We showed that methotrexate (MTX) carried in lipid core nanoparticles (LDE) had powerful anti-inflammatory effects on atherosclerotic lesions and myocardial infarction. Hypothesis: To investigate whether treatment with LDE-MTX can prevent the development of aortic arch lesions in MFS. Methods: mgΔloxPneo MFS murine model and wild-type mice were allocated in 3 groups of treatment: LDE only; MTX in commercial formulation; LDE-MTX. The treatment occurred weekly at a dose of 1mg/kg ip, between the 3rd and 6th month of life, period in which both dilation and dissection of the aortic arch are observed. After 12 weeks, animals were submitted to analysis of the aortic arch. Results: Compared to LDE and MTX groups, LDE-MTX treatment showed smaller lumen area of ascending and descending aorta and aortic arch segments in MFS mice. LDE-MTX also decreased collagen volume fraction and the amount of aortic dissections, but did not affect the thickness of the vessel wall and the number of elastic fiber ruptures. Protein expression of the inflammatory markers CD68 (macrophages), CD3 (lymphocytes) and tumor necrosis factor alpha, of the apoptotic marker caspase 3 and of type 1 collagen were lower in LDE-MTX group when compared to LDE and MTX. Moreover, treatment with LDE-MTX reduced TGF-β, ERK and SMAD3 protein expression. Of note, CD68 and CD3 protein expression were positively correlated with the lumen area of the aortic arch (r2=0.36; p<0.001), indicating the importance of inflammation for the aortic dilation. The increase in bioavailability of intracellular adenosine was suggested by higher expression of A2a adenosine receptor and lower levels of adenosine deaminase in the aortic arch. Conclusions: Possibly by increasing the bioavailability of intracellular adenosine, LDE-MTX treatment had the ability to reduce the processes of inflammation, apoptosis and fibrosis that are consequent to fibrillin-1 mutation. By these means, LDE-MTX may conceivably prevent the development of the dilation and dissection in the aortic arch, the hallmarks of MFS.

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

Abstract Introduction Mutations in the fibrillin-1 gene result in dilation, dissection and rupture of the aorta, the main cause of mortality in patients with Marfan syndrome (MFS). Inflammation is associated with greater susceptibility to dissection and rupture of the aorta. Previously, we showed that methotrexate (MTX) carried in lipid core nanoparticles (LDE) had powerful anti-inflammatory and anti-proliferative effects on rheumatoid arthritis and atherosclerotic lesions induced in rabbits and improved myocardial infarction morphological and functional aspects in rats with ligation of left coronary artery. When MTX is associated to LDE, the uptake of the drug by the cells is increased several-fold, which conceivably endows MTX with enhanced action mechanisms and the drug toxicity is diminished. Purpose To investigate whether treatment with LDE-MTX can prevent the development of aortic arch lesions in a murine model of MFS. Methods mgΔloxPneo MFS murine model and wild-type mice were allocated in 3 groups of treatment: LDE only; MTX in commercial formulation; LDE-MTX. The treatment occurred weekly at a dose of 1mg/kg ip, between the 3rd and 6th month of life, period in which both dilation and dissection of the aortic arch are observed. After 12 weeks, the animals were submitted to echocardiography, morphometry and protein expression of the aortic arch. Results Compared to LDE and MTX groups, LDE-MTX treatment showed smaller lumen area of ascending and descending aorta and aortic arch segments in MFS mice. LDE-MTX also decreased the collagen volume fraction and the amount of aortic dissections, but did not affect the thickness of the vessel wall and the number of elastic fiber ruptures. The protein expression of the inflammatory markers CD68 (macrophages), CD3 (lymphocytes) and tumor necrosis factor alpha, of the apoptotic marker caspase 3 and of type 1 collagen were lower in MFS LDE-MTX group when compared to MFS mice treated with LDE and MTX. Moreover, treatment with LDE-MTX reduced TGF-β, ERK and the SMAD3 protein expression. Of note, CD68 and CD3 protein expression was positively correlated with the lumen area of the aortic arch (r2=0.36; p<0.001), indicating the importance of inflammation for the aortic dilation. The increase in bioavailability of intracellular adenosine in MFS animals treated with LDE-MTX was suggested by the higher expression of A2a adenosine receptor and the lower expression of adenosine deaminase in the aortic arch. Conclusion Possibly by increasing the bioavailability of intracellular adenosine, LDE-MTX treatment had the ability to reduce the processes of inflammation, apoptosis and fibrosis that are consequent to fibrillin-1 mutation. By these means, LDE-MTX may conceivably prevent the development of the dilation and dissection in the aortic arch, the hallmarks of MFS, in this animal model. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo)

Steered molecular dynamic simulations reveal Marfan syndrome mutations disrupt fibrillin-1 cbEGF domain mechanosensitive calcium binding

Marfan syndrome (MFS) is a highly variable genetic connective tissue disorder caused by mutations in the calcium binding extracellular matrix glycoprotein fibrillin-1. Patients with the most severe form of MFS (neonatal MFS; nMFS) tend to have mutations that cluster in an internal region of fibrillin-1 called the neonatal region. This region is predominantly composed of eight calcium-binding epidermal growth factor-like (cbEGF) domains, each of which binds one calcium ion and is stabilized by three highly conserved disulfide bonds. Crucially, calcium plays a fundamental role in stabilizing cbEGF domains. Perturbed calcium binding caused by cbEGF domain mutations is thus thought to be a central driver of MFS pathophysiology. Using steered molecular dynamics (SMD) simulations, we demonstrate that cbEGF domain calcium binding decreases under mechanical stress (i.e. cbEGF domains are mechanosensitive). We further demonstrate the disulfide bonds in cbEGF domains uniquely orchestrate protein unfolding by showing that MFS disulfide bond mutations markedly disrupt normal mechanosensitive calcium binding dynamics. These results point to a potential mechanosensitive mechanism for fibrillin-1 in regulating extracellular transforming growth factor beta (TGFB) bioavailability and microfibril integrity. Such mechanosensitive “smart” features may represent novel mechanisms for mechanical hemostasis regulation in extracellular matrix that are pathologically activated in MFS.

Effects of fibrillin mutations on the behavior of heart muscle cells in Marfan syndrome

Marfan syndrome (MFS) is a systemic disorder of connective tissue caused by pathogenic variants in the fibrillin-1 (FBN1) gene. Myocardial dysfunction has been demonstrated in MFS patients and mouse models, but little is known about the intrinsic effect on the cardiomyocytes (CMs). In this study, both induced pluripotent stem cells derived from a MFS-patient and the line with the corrected FBN1 mutation were differentiated to CMs. Several functional analyses are performed on this model to study MFS related cardiomyopathy. Atomic force microscopy revealed that MFS CMs are stiffer compared to corrected CMs. The contraction amplitude of MFS CMs is decreased compared to corrected CMs. Under normal culture conditions, MFS CMs show a lower beat-to-beat variability compared to corrected CMs using multi electrode array. Isoproterenol-induced stress or cyclic strain demonstrates lack of support from the matrix in MFS CMs. This study reports the first cardiac cell culture model for MFS, revealing abnormalities in the behavior of MFS CMs that are related to matrix defects. Based on these results, we postulate that impaired support from the extracellular environment plays a key role in the improper functioning of CMs in MFS.

The fibrillin microfibril/elastic fibre network: A critical extracellular supramolecular scaffold to balance skin homoeostasis.

Supramolecular networks composed of fibrillins (fibrillin-1 and fibrillin-2) and associated ligands form intricate cellular microenvironments which balance skin homoeostasis and direct remodelling. Fibrillins assemble into microfibrils which are not only indispensable for conferring elasticity to the skin, but also control the bioavailability of growth factors targeted to the extracellular matrix architecture. Fibrillin microfibrils (FMF) represent the core scaffolds for elastic fibre formation, and they also decorate the surface of elastic fibres and form independent networks. In normal dermis, elastic fibres are suspended in a three-dimensional basket-like lattice of FMF intersecting basement membranes at the dermal-epidermal junction and thus conferring pliability to the skin. The importance of FMF for skin homoeostasis is illustrated by the clinical features caused by mutations in the human fibrillin genes (FBN1, FBN2), summarized as "fibrillinopathies." In skin, fibrillin mutations result in phenotypes ranging from thick, stiff and fibrotic skin to thin, lax and hyperextensible skin. The most plausible explanation for this spectrum of phenotypic outcomes is that FMF regulate growth factor signalling essential for proper growth and homoeostasis of the skin. Here, we will give an overview about the current understanding of the underlying pathomechanisms leading to fibrillin-dependent fibrosis as well as forms of cutis laxa caused by mutational inactivation of FMF-associated ligands.

Increased Susceptibility to Glaucomatous Damage in Microfibril Deficient Mice.

PurposeTo test whether mice with microfibril deficiency due to the Tsk mutation of fibrillin-1 (Fbn1Tsk/+) have increased susceptibility to pressure-induced retinal ganglion cell (RGC) degeneration.MethodsIntraocular pressure (IOP) elevation was induced in Fbn1Tsk/+ and wild type (wt) mice by injecting microbeads into the anterior chamber. Mice were then followed up for four months, with IOP measurements every three to six days. Retinas were stained for Brn3a to determine RGC number. Optic nerve cross-sections were stained with p-phenylene diamine to determine nerve area, axon number, and caliber and thickness of the pia mater.ResultsMicrobead injection induced significant IOP elevation that was significantly less for Fbn1Tsk/+ mice compared with wt. The optic nerves and optic nerve axons were larger, and the elastic fiber-rich pia mater was thinner in Fbn1Tsk/+ mice. Microbead injection resulted in reduced optic nerve size, thicker pia mater, and a slight decrease in axon size. Fbn1Tsk/+ mice had significantly greater loss of RGCs and optic nerve axons compared with wt (14.8% vs. 5.8%, P = 0.002, and 17.0% vs. 7.5%, P = 0.002, respectively).Conclusions Fbn1Tsk/+mice had altered optic nerve structure as indicated by larger optic nerves, larger optic nerve axons and thinner pia mater, consistent with our previous findings. Despite lower IOP elevation, Fbn1Tsk/+mice had greater loss of RGCs and optic nerve axons, suggesting increased susceptibility to IOP-induced optic nerve degeneration in microfibril-deficient mice.

Three Novel Variants identified in FBN1 and TGFBR2 in seven Iranian families with suspected Marfan syndrome.

BackgroundMarfan syndrome (MFS) is a multi‐systemic autosomal dominant disease of the connective tissue characterized by the early development of thoracic aneurysms/dissections, along with various manifestations of the ocular and skeletal systems. Due to the genetic and clinical heterogeneity, the clinical diagnosis of this disorder is challenging. Loss‐of‐function mutations in FBN1 (encodes fibrillin‐1) lead to MFS type 1. Also, similar mutations in transforming growth factor β receptor 2 (TGFBR2) gene cause MFS type 2. Both proteins involve in TGF‐β signaling.MethodsIn this study, genetic screening using a panel involving 14 genes, especially FBN1 and TGFBR2, were performed on seven representatives affected members of seven unrelated Iranian families suspected with MFS. To confirm the variants, Sanger sequencing was applied to other affected/unaffected members of the families.ResultsA total of 13 patients showed MFS manifestations. Using genetic screening, two novel and three previously reported variants in FBN1 were identified. We also detected two variants (a novel and a previously reported variant) in the TGFBR2 gene.ConclusionIn this study, we introduce three novel variants identified through gene screening in seven Iranian MFS families. This report is expected to considerably improve genetic counseling for Iranian MFS families. Early precise molecular diagnosis can be helpful for better management and improving the life expectancy of these patients.

The ADAMTS/Fibrillin Connection: Insights into the Biological Functions of ADAMTS10 and ADAMTS17 and Their Respective Sister Proteases.

Secreted a disintegrin-like and metalloprotease with thrombospondin type 1 motif (ADAMTS) proteases play crucial roles in tissue development and homeostasis. The biological and pathological functions of ADAMTS proteases are determined broadly by their respective substrates and their interactions with proteins in the pericellular and extracellular matrix. For some ADAMTS proteases, substrates have been identified and substrate cleavage has been implicated in tissue development and in disease. For other ADAMTS proteases, substrates were discovered in vitro, but the role of these proteases and the consequences of substrate cleavage in vivo remains to be established. Mutations in ADAMTS10 and ADAMTS17 cause Weill–Marchesani syndrome (WMS), a congenital syndromic disorder that affects the musculoskeletal system (short stature, pseudomuscular build, tight skin), the eyes (lens dislocation), and the heart (heart valve abnormalities). WMS can also be caused by mutations in fibrillin-1 (FBN1), which suggests that ADAMTS10 and ADAMTS17 cooperate with fibrillin-1 in a common biological pathway during tissue development and homeostasis. Here, we compare and contrast the biochemical properties of ADAMTS10 and ADAMTS17 and we summarize recent findings indicating potential biological functions in connection with fibrillin microfibrils. We also compare ADAMTS10 and ADAMTS17 with their respective sister proteases, ADAMTS6 and ADAMTS19; both were recently linked to human disorders distinct from WMS. Finally, we propose a model for the interactions and roles of these four ADAMTS proteases in the extracellular matrix.

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.

DNA methylation ambiguity in the Fibrillin-1 (FBN1) CpG island shore possibly involved in Marfan syndrome

Fibrillin-1 (FBN1) is responsible for haploinsufficient and autosomal dominant Marfan syndrome. Even in the same Marfan pedigree, penetrance and expressivity in heterozygous individuals can differ and result in variable disease onset and severity. Thus, other factors in addition to mutations in FBN1 are likely to contribute to the disease. In this study, we examined the regulation of FBN1 in porcine Marfan syndrome model, focusing on DNA methylation patterns distinguishable as wild-type (WT) and FBN1 null (KO) alleles in heterozygous cells. Most importantly, the ratio of the transcriptionally active hypomethylated WT allele was altered during cellular passage and highly correlated with FBN1 mRNA level compared with that in the KO allele. Transcribed FBN1 RNA from the KO allele was abolished after splicing coupled with translational initiation, suggesting that the functional FBN1 mRNA levels were affected by DNA methylation of the WT allele.

A 45-year-old Italian male with p.(Gly1815Ser) FBN1 mutation causing a mild variant of Marfan syndrome: A case study

A 45-year-old Italian male was referred as suspected of having a heritable connective tissue disorders by clinical findings, including joint hyperlaxity and soft, smooth, velvety, and slightly elastic skin. Using a specific custom panel including genes involved in these disorders, next-generation sequencing (NGS) analysis led to the identification of the c. 5443G>A, p.(Gly1815Ser), (rs745680336) variant in fibrillin-1 (FBN1) gene, encoding the FBN1. Mutations in this protein are responsible for different connective tissue disorders, collectively known as type 1 fibrillinopathies, including Marfan syndrome (MFS). Multiple sequencing alignment of human FBN1 protein with various species revealed that the mutation occurred within a highly conserved region of the calcium-binding epidermal growth factor-like domain and affected the protein structure/function, suggesting its pathogenic role. NGS techniques successfully identified the molecular defect in this patient, clinically resembling as MFS, even if a clear genotype–phenotype correlation remains still challenging.

Weil–Marchesani syndrome: A case report and literature review

Weill–Marchesani syndrome is a rare connective tissue disorder, with a poorly understood etiology that has been primarily related to hereditary genetic factors, including mutations in ADAMTS10 and fibrillin-1. Clinically, it is characterized by a phenotype of short stature and brachydactyly, associated with joint stiffness and eye problems that begin to be noticed in childhood. There is little information about this disease in the Mexican population. This is a description of the case of a 17-year-old female patient with clinical feature compatible with this syndrome, as well as a brief review of the literature on this entity. The knowledge of this syndrome is important to achieve a timely diagnosis and prevent the complications associated with it.

Increased frequency of FBN1 frameshift and nonsense mutations in Marfan syndrome patients with aortic dissection

BackgroundMarfan syndrome (MFS) is an inherited connective tissue disease that mainly involves Fibrillin‐1 (FBN1) mutations and aortic manifestations. In this study, we investigated the correlations between the FBN1 genotype–phenotype and aortic events (aortic dissection and aortic aneurysm) in patients with Marfan syndrome.MethodsGenotype and phenotype information was evaluated in 180 patients with MFS. DNA sequencing was performed on each patient. According to the clinical manifestation, these patients were split into two groups: the aortic dissection group and the aortic aneurysm group. Aortic wall tissue was obtained from Marfan patients who underwent surgery and was used for staining.ResultsA total of 180 patients with FBN1 mutations were grouped into four categories: 90 with missense mutations, 32 with splicing mutations, 29 with frameshift mutations, and 29 with nonsense mutations. There was a significantly higher frequency of frameshift and nonsense mutations observed in aortic dissection than in aortic aneurysm (25.58% vs. 4.35%, p = .005; 25.58% vs. 8.70%, p = .033, respectively;), while missense mutations showed a higher frequency in aortic aneurysm than in aortic dissection (69.57% vs. 32.56%, respectively; p < .001) and a higher rate of lens dislocation (34.78% vs. 13.95%, respectively; p = .008). Pathological staining showed that elastic fibers were sparser in patients with a frameshift and nonsense mutations, and the smooth muscle cells were sparser and more disorganized than those observed in patients with missense mutations.ConclusionThis study showed that FBN1 gene frameshift and nonsense mutations are more common in patients with aortic dissection and may have meaningful guidance for the treatment of Marfan syndrome patients.

Mutation analysis of FBN1 gene in two Chinese families with congenital ectopia lentis in northern China.

To summarize the phenotypes and identify the underlying genetic cause of the fibrillin-1 (FBN1) gene responsible for congenital ectopia lentis (EL) in two Chinese families in northern China. A detailed family history and clinical data from all participants were collected by clinical examination. The candidate genes were captured and sequenced by targeted next-generation sequencing, and the results were confirmed by Sanger sequencing. Haplotyping was used to confirm the mutation sequence. Real-time PCR was used to determine the FBN1 messenger ribonucleic acid (mRNA) levels in patients with EL and in unaffected family members. The probands and other patients in the two families were affected with congenital isolated EL. A heterozygous FBN1 mutation in exon 21 (c.2420_IVS20-8 delTCTGAAACAinsCGAAAG) was identified in FAMILY-1. A heterozygous FBN1 mutation in exon 14 (c.1633C>T, p.R545C) was identified in FAMILY-2. Each mutation co-segregated with the affected individuals in the family and did not exist in unaffected family members and 200 unrelated normal controls. The insertion-deletion mutation (c.2420 IVS20-8delTCTGAAACA insCGAAAG) in the FBN1 gene is first identified in isolated EL. The mutation (c.1633C>T) in the FBN1 gene was a known mutation in EL patient. The variable phenotypes among the patients expand the phenotypic spectrum of EL in a different ethnic background.

A novel ADAMTS17 variant that causes Weill-Marchesani syndrome 4 alters fibrillin-1 and collagen type I deposition in the extracellular matrix

Weill-Marchesani syndrome (WMS) is a rare genetic disorder that affects the musculoskeletal system, the eye, and the cardiovascular system. Individuals with WMS present with short stature, joint contractures, thick skin, microspherophakia, small and dislocated lenses, and cardiac valve anomalies. WMS can be caused by recessive mutations in ADAMTS10 (WMS 1), ADAMTS17 (WMS 4), or LTBP2 (WMS 3), or by dominant mutations in fibrillin-1 (FBN1) (WMS 2); all genes encode secreted extracellular matrix (ECM) proteins. Individuals with WMS 4 due to ADAMTS17 mutations appear to have less severe cardiac involvement and present predominantly with the musculoskeletal and ocular features of WMS. ADAMTS17 is a member of the ADAMTS family of secreted proteases and directly binds to fibrillins. Here we report a novel pathogenic variant in ADAMTS17 that causes WMS 4 in an individual with short stature, brachydactyly, and small, spherical, and dislocated lenses. We provide biochemical and cell biological insights in the pathomechanisms of WMS 4, which also suggest potential biological functions for ADAMTS17. We show that the variant in ADAMTS17 prevents its secretion and we found intracellular accumulation of fibrillin-1 and collagen type I in patient-derived skin fibroblasts. In accordance, transmission electron microscopy revealed elastic fiber abnormalities, decreased collagen fibril diameters, and intracellular collagen accumulation in the dermis of the proband. Together, the data indicate a possible role for ADAMTS17 in the secretion of fibrillin-1 and collagen type I or in their early assembly in the pericellular matrix or the ECM.

The influence of fibrillin‐1 and physical activity upon tendon tissue morphology and mechanical properties in mice

Fibrillin‐1 mutations cause pathological changes in connective tissue that constitute the complex phenotype of Marfan syndrome. In this study, we used fibrillin‐1 hypomorphic and haploinsufficient mice (Fbn1mgr/mgR and Fbn1+/− mice, respectively) to investigate the impact of fibrillin‐1 deficiency alone or in combination with regular physical activity on tendon tissue morphology and mechanical properties. Morphological and biomechanical analyses revealed that Fbn1mgr/mgR but not Fbn1+/− mice displayed smaller tendons with physical properties that were unremarkable when normalized to tendon size. Fbn1mgR/mgR mice (n = 43) Fbn1+/−mice (n = 27) and wild‐type mice (WT, n = 25) were randomly assigned to either control cage conditions (n = 54) or to a running on a running wheel for 4 weeks (n = 41). Both fibrillin‐1‐deficient mice ran voluntarily on the running wheel in a manner similar to WT mice (3–4 km/24 h). Regular exercise did not mitigate aneurysm progression in Fbn1mgR/mgR mice (P < 0.05) as evidenced by unmodified median survival. In spite of the smaller size, tendons of fibrillin‐1‐deficient mice subjected to regular exercise showed no evidence of overt histopathological changes or tissue overload. We therefore concluded that lack of optimal fibrillin‐1 synthesis leads to a down regulation of integrated tendon formation, rather than to a loss of tendon quality, which also implies that fibrillin‐1 deficiency in combination with exercise is not a suitable animal model for studying the development of tendon overuse (tendinopathy).

Impaired vascular smooth muscle cell force-generating capacity and phenotypic deregulation in Marfan Syndrome mice

Mechanisms whereby fibrillin-1 mutations determine thoracic aorta aneurysms/dissections (TAAD) in Marfan Syndrome (MFS) are unclear. Most aortic aneurysms evolve from mechanosignaling deregulation, converging to impaired vascular smooth muscle cell (VSMC) force-generating capacity accompanied by synthetic phenotype switch. However, little is known on VSMC mechanoresponses in MFS pathophysiology. Here, we investigated traction force-generating capacity in aortic VSMC cultured from 3-month old mg∆lpn MFS mice, together with morpho-functional and proteomic data. Cultured MFS-VSMC depicted marked phenotype changes vs. wild-type (WT) VSMC, with overexpressed cell proliferation markers but either lower (calponin-1) or higher (SM alpha-actin and SM22) differentiation marker expression. In parallel, the increased cell area and its complex non-fusiform shape suggested possible transition towards a mesenchymal-like phenotype, confirmed through several markers (e.g. N-cadherin, Slug). MFS-VSMC proteomic profile diverged from that of WT-VSMC particularly regarding lower expression of actin cytoskeleton-regulatory proteins. Accordingly, MFS-VSMC displayed lower traction force-generating capacity and impaired contractile moment at physiological substrate stiffness, and markedly attenuated traction force responses to enhanced substrate rigidity. Such impaired mechanoresponses correlated with decreased number, altered morphology and delocalization of focal adhesions, as well as disorganized actin stress fiber network vs. WT-VSMC. In VSMC cultured from 6-month-old mice, phenotype changes were attenuated and both WT-VSMC and MFS-VSMC generated less traction force, presumably involving VSMC aging, but without evident senescence. In summary, MFS-VSMC display impaired force-generating capacity accompanying a mesenchymal-like phenotype switch connected to impaired cytoskeleton/focal adhesion organization. Thus, MFS-associated TAAD involves mechanoresponse impairment common to other TAAD types, but through distinct mechanisms.

P2577Losartan diminishes the expression of TGF-beta and improves cardiomyopathy in mice with Marfan syndrome

Abstract Introduction Marfan syndrome (MFS) is caused by mutations in the fibrillin-1 gene, leading to abnormal signaling of the transforming growth factor beta (TGF-β). This results in aortic root dilation, dissection and rupture, which are the main causes of morbidity and mortality of MFS patients. Current treatment with losartan, an angiotensin-II receptor-1 blocker, has shown beneficial effect on aortic disease in MFS murine models. However, the mechanisms whereby the treatment with losartan improves cardiac remodeling and function of the left ventricle (LV) in MFS are still unknown. Purpose To investigate the effects of losartan on mechanisms of the cardiomyopathy in mice with MFS. Methods mgΔloxPneo MFS murine model from C129/sv background was utilized in this study. To evaluate the effect of the treatment with losartan on the LV of MFS and wild-type mice, animals were allocated in 2 groups of treatments: Losartan group: mice were kept with water supplemented with losartan (0.6g/L); Untreated control group: mice were kept with water only. The animals received treatment from 1 month of age until completing 6 months. After five months of treatment, LV echocardiography was performed, and fragments of LV tissue were analyzed by morphometry and protein expression analysis by Western blot. Results Losartan MFS mice showed decrease in interventricular septum and posterior wall thickness and LV mass. Furthermore, losartan prevented aortic and mitral regurgitation, arrhythmia, bradycardia, septal hypokinesia and pulmonary hypertension when compared with control MFS mice. Systolic and diastolic LV function were not different between groups. Collagen volume fraction and the disorganization and disruptions of the elastic fibers in coronary arteries were lower in losartan treatment than in controls. The protein expression of pro-apoptotic factors (BAX/Bcl-2 and caspase 3 and 9), proliferating cell nuclear antigen and hypoxia-inducible factor 1 and 2α were lower in losartan treatment, whereas the expression of vascular endothelial growth factor was increased in losartan group when compared with control MFS mice. Moreover, the treatment with losartan strongly reduced the TGF-β, ERK and p38MAPK protein expression compared to controls. Conclusion In this murine model of MFS, losartan treatment has the ability to change several pathophysiological mechanisms related with the fibrillin-1 mutation, by decreasing apoptosis, cell proliferation and increasing angiogenesis. Overall, the treatment resulted in improved structural rearrangement and attenuation of the rupture of elastic fibers in the coronary arteries, of the cardiac hypertrophy and myocardial fibrosis. These effects were possibly related with the decreased TGF-β expression by ERK and p38MAPK signaling pathways by losartan. Our findings shed a new light on the mechanisms of action of losartan on MFS cardiomyopathy. Acknowledgement/Funding FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo)