Unraveling the role of TGFß signaling in thoracic aortic aneurysm and dissection using Fbn1 mutant mouse models.

Although abnormal TGFß signaling is observed in several heritable forms of thoracic aortic aneurysms and dissections including Marfan syndrome, its precise role in aortic disease progression is still disputed. Using a mouse genetic approach and quantitative isobaric labeling proteomics, we sought to elucidate the role of TGFß signaling in three Fbn1 mutant mouse models representing a range of aortic disease from microdissection (without aneurysm) to aneurysm (without rupture) to aneurysm and rupture. Results indicated that reduced TGFß signaling and increased mast cell proteases were associated with microdissection. In contrast, increased abundance of extracellular matrix proteins, which could be reporters for positive TGFß signaling, were associated with aneurysm. Marked reductions in collagens and fibrillins, and increased TGFß signaling, were associated with aortic rupture. Our data indicate that TGFß signaling performs context-dependent roles in the pathogenesis of thoracic aortic disease.

Molecular characterization and investigation of the role of genetic variation in phenotypic variability and response to treatment in a large pediatric Marfan syndrome cohort.

PURPOSE: In a large cohort of 373 pediatric patients with Marfan syndrome (MFS) with a severe cardiovascular phenotype, we explored the proportion of patients with MFS with a pathogenic FBN1 variant and analyzed whether the type/location of FBN1 variants was associated with specific clinical characteristics and response to treatment. Patients were recruited on the basis of the following criteria: aortic root z-score > 3, age 6 months to 25 years, no prior or planned surgery, and aortic root diameter < 5 cm. METHODS: Targeted resequencing and deletion/duplication testing of FBN1 and related genes were performed. RESULTS: We identified (likely) pathogenic FBN1 variants in 91% of patients. Ectopia lentis was more frequent in patients with dominant-negative (DN) variants (61%) than in those with haploinsufficient variants (27%). For DN FBN1 variants, the prevalence of ectopia lentis was highest in the N-terminal region (84%) and lowest in the C-terminal region (17%). The association with a more severe cardiovascular phenotype was not restricted to DN variants in the neonatal FBN1 region (exon 25-33) but was also seen in the variants in exons 26 to 49. No difference in the therapeutic response was detected between genotypes. CONCLUSION: Important novel genotype-phenotype associations involving both cardiovascular and extra-cardiovascular manifestations were identified, and existing ones were confirmed. These findings have implications for prognostic counseling of families with MFS.

Pathogenic variants in THSD4, encoding the ADAMTS-like 6 protein, predispose to inherited thoracic aortic aneurysm.

PURPOSE: Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening disease with often unrecognized inherited forms. We sought to identify novel pathogenic variants associated with autosomal dominant inheritance of TAAD. METHODS: We analyzed exome sequencing data from 35 French TAAD families and performed next-generation sequencing capture panel of genes in 1114 unrelated TAAD patients. Functional effects of pathogenic variants identified were validated in cell, tissue, and mouse models. RESULTS: We identified five functional variants in THSD4 of which two heterozygous variants lead to a premature termination codon. THSD4 encodes ADAMTSL6 (member of the ADAMTS/L superfamily), a microfibril-associated protein that promotes fibrillin-1 matrix assembly. The THSD4 variants studied lead to haploinsufficiency or impaired assembly of fibrillin-1 microfibrils. Thsd4+/- mice showed progressive dilation of the thoracic aorta. Histologic examination of aortic samples from a patient carrying a THSD4 variant and from Thsd4+/- mice, revealed typical medial degeneration and diffuse disruption of extracellular matrix. CONCLUSION: These findings highlight the role of ADAMTSL6 in aortic physiology and TAAD pathogenesis. They will improve TAAD management and help develop new targeted therapies.

MEK1/2 Inhibition in Murine Heart and Aorta After Oral Administration of Refametinib Supplemented Drinking Water.

Upregulation of the RAS-RAF-MEK-ERK-MAPK pathway is involved in the development of several human tumors, aortic aneurysms, atherosclerosis, and cardiomyopathy. Refametinib, a highly selective MEK-inhibitor, has already shown antineoplastic activity in phase II trials. Furthermore, it showed potency to attenuate aortic root growth in murine models. Current formulations of this drug however necessitate oral gavage as a delivery method for long-term studies, which is labor-intensive and induces stress and occasional injury, potentially confounding results. Therefore, we developed a novel oral administration method for refametinib. A 2-hydroxypropyl-beta-cyclodextrin (HPBCD) based drinking water preparation of refametinib was formulated, for which a selective, analytical UHPLC-UV method was developed to assess the in-use stability. Next, 16 week old male wild-type C57Bl/6J mice received either a daily dose of 50 or 75 mg/kg/day refametinib or were given regular drinking water during 7 days. In both dosage groups the refametinib plasma levels were measured (n = 10 or 7, respectively). Furthermore, pERK/total ERK protein levels were calculated in the myocardial and aortic tissue of mice receiving a daily dose of 50 mg/kg/day refametinib and untreated mice (n = 4/group). After 7 days no significant degradation of refametinib was observed when dissolved in drinking water provided that drinking bottles were protected from UV/visible light. Furthermore, a dose-dependent increase in refametinib plasma levels was found whereby active plasma levels (> 1.2 µg/mL) were obtained even in the lowest dose-group of 50 mg/kg/day. A significant reduction of pERK/total ERK protein levels compared to untreated mice was observed in aortic and myocardial tissue of mice receiving a daily dose of 50 mg/kg/day refametinib. Importantly, a relatively high mortality rate was noted in the highest dose group (n = 5). This approach provides a valid alternative oral administration method for refametinib with a reduced risk of complications due to animal manipulation and without loss of functionality, which can be implemented in future research regarding the malignant upregulation of the RAS-RAF-MEK-ERK-MAPK pathway. However, care must be taken not to exceed the toxic dose.

Spontaneous Right Ventricular Pseudoaneurysms and Increased Arrhythmogenicity in a Mouse Model of Marfan Syndrome.

Patients with Marfan syndrome (MFS), a connective tissue disorder caused by pathogenic variants in the gene encoding the extracellular matrix protein fibrillin-1, have an increased prevalence of primary cardiomyopathy, arrhythmias, and sudden cardiac death. We have performed an in-depth in vivo and ex vivo study of the cardiac phenotype of Fbn1mgR/mgR mice, an established mouse model of MFS with a severely reduced expression of fibrillin-1. Using ultrasound measurements, we confirmed the presence of aortic dilatation and observed cardiac diastolic dysfunction in male Fbn1mgR/mgR mice. Upon post-mortem examination, we discovered that the mutant mice consistently presented myocardial lesions at the level of the right ventricular free wall, which we characterized as spontaneous pseudoaneurysms. Histological investigation demonstrated a decrease in myocardial compaction in the MFS mouse model. Furthermore, continuous 24 h electrocardiographic analysis showed a decreased heart rate variability and an increased prevalence of extrasystolic arrhythmic events in Fbn1mgR/mgR mice compared to wild-type littermates. Taken together, in this paper we document a previously unreported cardiac phenotype in the Fbn1mgR/mgR MFS mouse model and provide a detailed characterization of the cardiac dysfunction and rhythm disorders which are caused by fibrillin-1 deficiency. These findings highlight the wide spectrum of cardiac manifestations of MFS, which might have implications for patient care.

Ambulatory Electrocardiographic Monitoring and Ectopic Beat Detection in Conscious Mice.

Ambulatory electrocardiography (AECG) is a primary diagnostic tool in patients with potential arrhythmic disorders. To study the pathophysiological mechanisms of arrhythmic disorders, mouse models are widely implemented. The use of a technique similar to AECG for mice is thus of great relevance. We have optimized a protocol which allows qualitative, long-term ECG data recording in conscious, freely moving mice. Automated algorithms were developed to efficiently process the large amount of data and calculate the average heart rate (HR), the mean peak-to-peak interval and heart rate variability (HRV) based on peak detection. Ectopic beats are automatically detected based on aberrant peak intervals. As we have incorporated a multiple lead configuration in our ECG set-up, the nature and origin of the suggested ectopic beats can be analyzed in detail. The protocol and analysis tools presented here are promising tools for studies which require detailed, long-term ECG characterization in mouse models with potential arrhythmic disorders.

Variants in ADRB1 and CYP2C9: Association with Response to Atenolol and Losartan in Marfan Syndrome.

OBJECTIVE: To test whether variants in ADRB1 and CYP2C9 genes identify subgroups of individuals with differential response to treatment for Marfan syndrome through analysis of data from a large, randomized trial. STUDY DESIGN: In a subset of 250 white, non-Hispanic participants with Marfan syndrome in a prior randomized trial of atenolol vs losartan, the common variants rs1801252 and rs1801253 in ADRB1 and rs1799853 and rs1057910 in CYP2C9 were analyzed. The primary outcome was baseline-adjusted annual rate of change in the maximum aortic root diameter z-score over 3 years, assessed using mixed effects models. RESULTS: Among 122 atenolol-assigned participants, the 70 with rs1801253 CC genotype had greater rate of improvement in aortic root z-score compared with 52 participants with CG or GG genotypes (Time × Genotype interaction P = .005, mean annual z-score change ± SE -0.20 ± 0.03 vs -0.09 ± 0.03). Among participants with the CC genotype in both treatment arms, those assigned to atenolol had greater rate of improvement compared with the 71 of the 121 assigned to losartan (interaction P = .002; -0.20 ± 0.02 vs -0.07 ± 0.02; P < .001). There were no differences in atenolol response by rs1801252 genotype or in losartan response by CYP2C9 metabolizer status. CONCLUSIONS: In this exploratory study, ADRB1-rs1801253 was associated with atenolol response in children and young adults with Marfan syndrome. If these findings are confirmed in future studies, ADRB1 genotyping has the potential to guide therapy by identifying those who are likely to have greater therapeutic response to atenolol than losartan.

Slc2a10 knock-out mice deficient in ascorbic acid synthesis recapitulate aspects of arterial tortuosity syndrome and display mitochondrial respiration defects.

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

Clinical Validity of Genes for Heritable Thoracic Aortic Aneurysm and Dissection.

BACKGROUND: Thoracic aortic aneurysms progressively enlarge and predispose to acute aortic dissections. Up to 25% of individuals with thoracic aortic disease harbor an underlying Mendelian pathogenic variant. An evidence-based strategy for selection of genes to test in hereditary thoracic aortic aneurysm and dissection (HTAAD) helps inform family screening and intervention to prevent life-threatening thoracic aortic events. OBJECTIVES: The purpose of this study was to accurately identify genes that predispose to HTAAD using the Clinical Genome Resource (ClinGen) framework. METHODS: We applied the semiquantitative ClinGen framework to assess presumed gene-disease relationships between 53 candidate genes and HTAAD. Genes were classified as causative for HTAAD if they were associated with isolated thoracic aortic disease and were clinically actionable, triggering routine aortic surveillance, intervention, and family cascade screening. All gene-disease assertions were evaluated by a pre-defined curator-expert pair and subsequently discussed with an expert panel. RESULTS: Genes were classified based on the strength of association with HTAAD into 5 categories: definitive (n = 9), strong (n = 2), moderate (n = 4), limited (n = 15), and no reported evidence (n = 23). They were further categorized by severity of associated aortic disease and risk of progression. Eleven genes in the definitive and strong groups were designated as "HTAAD genes" (category A). Eight genes were classified as unlikely to be progressive (category B) and 4 as low risk (category C). The remaining genes were recent genes with an uncertain classification or genes with no evidence of association with HTAAD. CONCLUSIONS: The ClinGen framework is useful to semiquantitatively assess the strength of gene-disease relationships for HTAAD. Gene categories resulting from the curation may inform clinical laboratories in the development, interpretation, and subsequent clinical implications of genetic testing for patients with aortic disease.

Tailoring the American College of Medical Genetics and Genomics and the Association for Molecular Pathology Guidelines for the Interpretation of Sequenced Variants in the FBN1 Gene for Marfan Syndrome: Proposal for a Disease- and Gene-Specific Guideline.

BACKGROUND: The introduction of next-generation sequencing techniques has substantially increased the identification of new genetic variants and hence the necessity of accurate variant interpretation. In 2015, the American College of Medical Genetics and Genomics and the Association for Molecular Pathology proposed new variant interpretation guidelines. Gene-specific characteristics were, however, not considered, sometimes leading to inconsistent variant interpretation. METHODS: To allow a more uniform interpretation of variants in the FBN1 (fibrillin-1) gene, causing Marfan syndrome, we tailored these guidelines to this gene and disease. We adapted 15 of the 28 general criteria and classified 713 FBN1 variants previously identified in our laboratory as causal mutation or variant of uncertain significance according to these adapted guidelines. We then compared the agreement between previous methods and the adapted American College of Medical Genetics and Genomics and the Association for Molecular Pathology criteria. RESULTS: Agreement between the methods was 86.4% (K-alpha, 0.6). Application of the tailored guidelines resulted in an increased number of variants of uncertain significance (14.5% to 24.2%). Of the 85 variants that were downscaled to likely benign or variant of uncertain significance, 59.7% were missense variants outside a well-established functional site. Available clinical- or segregation data, necessary to further classify these types of variants, were in many cases insufficient to aid the classification. CONCLUSIONS: Our study shows that classification of variants remains challenging and may change over time. Currently, a higher level of evidence is necessary to classify a variant as pathogenic. Gene-specific guidelines may be useful to allow a more precise and uniform interpretation of the variants to accurately support clinical decision-making.

A heart for fibrillin: spatial arrangement in adult wild-type murine myocardial tissue.

Fibrillins are major constituents of microfibrils, which are essential components of the extracellular matrix of connective tissues where they contribute to the tissue homeostasis. Although it is known that microfibrils are abundantly expressed in the left ventricle of the heart, limited data are available about the presence of microfibrils in the other parts of the myocardial tissue and whether there are age or sex-related differences in the spatial arrangement of the microfibrils. This basic knowledge is essential to better understand the impact of fibrillin-1 pathogenic variants on the myocardial tissue as seen in Marfan related cardiomyopathy. We performed histological analyses on wild-type male and female murine myocardial tissue collected at different time-points (1, 3 and 6 months). Fibrillin-1 and -2 immunofluorescence stainings were performed on cross-sections at the level of the apex, the mid-ventricles and the atria. In addition, other myocardial matrix components such as collagen and elastin were also investigated. Fibrillin-1 presented as long fibres in the apex, mid-ventricles and atria. The spatial arrangement differed between the investigated regions, but not between age groups or sexes. Collagen had a similar broad spatial arrangement to that of fibrillin-1, whereas elastic fibres were primarily present in the atria and the vessels. In contrast to fibrillin-1, limited amounts of fibrillin-2 were observed. Fibrillin-rich fibres contribute to the architecture of the myocardial tissue in a region-dependent manner in wild-type murine hearts. This knowledge is helpful for future experimental set-ups of studies evaluating the impact of fibrillin-1 pathogenic variants on the myocardial tissue.

Expressed repetitive elements are broadly applicable reference targets for normalization of reverse transcription-qPCR data in mice.

Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression analysis on mRNA level. To remove experimental variation, expression levels of the gene of interest are typically normalized to the expression level of stably expressed endogenous reference genes. Identifying suitable reference genes and determining the optimal number of reference genes should precede each quantification study. Popular reference genes are not necessarily stably expressed in the examined conditions, possibly leading to inaccurate results. Stably and universally expressed repetitive elements (ERE) have previously been shown to be an excellent alternative for normalization using classic reference genes in human and zebrafish samples. Here, we confirm that in mouse tissues, EREs are broadly applicable reference targets for RT-qPCR normalization, provided that the RNA samples undergo a thorough DNase treatment. We identified Orr1a0, Rltr2aiap, and Rltr13a3 as the most stably expressed mouse EREs across six different experimental conditions. Therefore, we propose this set of ERE reference targets as good candidates for normalization of RT-qPCR data in a plethora of conditions. The identification of widely applicable stable mouse RT-qPCR reference targets for normalization has great potential to facilitate future murine gene expression studies and improve the validity of RT-qPCR data.

Correction: Sex, pregnancy and aortic disease in Marfan syndrome.

[This corrects the article DOI: 10.1371/journal.pone.0181166.].

Sex, pregnancy and aortic disease in Marfan syndrome.

BACKGROUND: Sex-related differences as well as the adverse effect of pregnancy on aortic disease outcome are well-established phenomena in humans with Marfan syndrome (MFS). The underlying mechanisms of these observations are largely unknown. OBJECTIVES: In an initial (pilot) step we aimed to confirm the differences between male and female MFS patients as well as between females with and without previous pregnancy. We then sought to evaluate whether these findings are recapitulated in a pre-clinical model and performed in-depth cardiovascular phenotyping of mutant male and both nulliparous and multiparous female Marfan mice. The effect of 17ß-estradiol on fibrillin-1 protein synthesis was compared in vitro using human aortic smooth muscle cells and fibroblasts. RESULTS: Our small retrospective study of aortic dimensions in a cohort of 10 men and 20 women with MFS (10 pregnant and 10 non-pregnant) confirmed that aortic root growth was significantly increased in the pregnant group compared to the non-pregnant group (0.64mm/year vs. 0.12mm/year, p = 0.018). Male MFS patients had significantly larger aortic root diameters compared to the non-pregnant and pregnant females at baseline and follow-up (p = 0.002 and p = 0.007, respectively), but no significant increase in aortic root growth was observed compared to the females after follow-up (p = 0.559 and p = 0.352). In the GT-8/+ MFS mouse model, multiparous female Marfan mice showed increased aortic diameters when compared to nulliparous females. Aortic dilatation in multiparous females was comparable to Marfan male mice. Moreover, increased aortic diameters were associated with more severe fragmentation of the elastic lamellae. In addition, 17ß-estradiol was found to promote fibrillin-1 production by human aortic smooth muscle cells. CONCLUSIONS: Pregnancy-related changes influence aortic disease severity in otherwise protected female MFS mice and patients. There may be a role for estrogen in the female sex protective effect.

Nitric oxide mediates aortic disease in mice deficient in the metalloprotease Adamts1 and in a mouse model of Marfan syndrome.

Heritable thoracic aortic aneurysms and dissections (TAAD), including Marfan syndrome (MFS), currently lack a cure, and causative mutations have been identified for only a fraction of affected families. Here we identify the metalloproteinase ADAMTS1 and inducible nitric oxide synthase (NOS2) as therapeutic targets in individuals with TAAD. We show that Adamts1 is a major mediator of vascular homeostasis, given that genetic haploinsufficiency of Adamts1 in mice causes TAAD similar to MFS. Aortic nitric oxide and Nos2 levels were higher in Adamts1-deficient mice and in a mouse model of MFS (hereafter referred to as MFS mice), and Nos2 inactivation protected both types of mice from aortic pathology. Pharmacological inhibition of Nos2 rapidly reversed aortic dilation and medial degeneration in young Adamts1-deficient mice and in young or old MFS mice. Patients with MFS showed elevated NOS2 and decreased ADAMTS1 protein levels in the aorta. These findings uncover a possible causative role for the ADAMTS1-NOS2 axis in human TAAD and warrant evaluation of NOS2 inhibitors for therapy.

FBN1: The disease-causing gene for Marfan syndrome and other genetic disorders.

FBN1 encodes the gene for fibrillin-1, a structural macromolecule that polymerizes into microfibrils. Fibrillin microfibrils are morphologically distinctive fibrils, present in all connective tissues and assembled into tissue-specific architectural frameworks. FBN1 is the causative gene for Marfan syndrome, an inherited disorder of connective tissue whose major features include tall stature and arachnodactyly, ectopia lentis, and thoracic aortic aneurysm and dissection. More than one thousand individual mutations in FBN1 are associated with Marfan syndrome, making genotype-phenotype correlations difficult. Moreover, mutations in specific regions of FBN1 can result in the opposite features of short stature and brachydactyly characteristic of Weill-Marchesani syndrome and other acromelic dysplasias. How can mutations in one molecule result in disparate clinical syndromes? Current concepts of the fibrillinopathies require an appreciation of tissue-specific fibrillin microfibril microenvironments and the collaborative relationship between the structures of fibrillin microfibril networks and biological functions such as regulation of growth factor signaling.

Marfan Syndrome and Related Heritable Thoracic Aortic Aneurysms and Dissections.

In this overview we aim to address a number of recent insights and developments regarding clinical aspects, etiology, and treatment of Heritable Thoracic Aortic Disease (H-TAD). We will focus on monogenetic disorders related to aortic aneurysms. H-TADs are rare but they provide a unique basis for the study of underlying pathogenetic pathways in the complex disease process of aneurysm formation. The understanding of pathomechanisms may help us to identify medical treatment targets to improve prognosis. Among the monogenetic aneurysm disorders, Marfan syndrome is considered as a paradigm entity and many insights are derived from the study of clinical, genetic and animal models for Marfan syndrome. We will therefore first provide a detailed overview of the various aspects of Marfan syndrome after which we will give an overview of related H-TAD entities.

Intrinsic cardiomyopathy in Marfan syndrome: results from in-vivo and ex-vivo studies of the Fbn1C1039G/+ model and longitudinal findings in humans.

BACKGROUND: Mild intrinsic cardiomyopathy in patients with Marfan syndrome (MFS) has consistently been evidenced by independent research groups. So far, little is known about the long-term evolution and pathophysiology of this finding. METHODS: To gain more insights into the pathophysiology of MFS-related cardiomyopathy, we performed in-vivo and ex-vivo studies of 11 Fbn1(C1039G/+) mice and 9 wild-type (WT) littermates. Serial ultrasound findings obtained in mice were correlated to the human phenotype. We therefore reassessed left ventricular (LV) function parameters over a 6-y follow-up period in 19 previously reported MFS patients, in whom we documented mild LV dysfunction. RESULTS: Fbn1(C1039G/+) mice demonstrated LV contractile dysfunction. Subsequent ex-vivo studies of the myocardium of adult mutant mice revealed upregulation of TGFß-related pathways and consistent abnormalities of the microfibrillar network, implicating a role for microfibrils in the mechanical properties of the myocardium. Echocardiographic parameters did not indicate clinical significant deterioration of LV function during follow-up in our patient cohort. CONCLUSION: In analogy with what is observed in the majority of MFS patients, the Fbn1(C1039G/+) mouse model demonstrates mild intrinsic LV dysfunction. Both extracellular matrix and molecular alterations are implicated in MFS-related cardiomyopathy. This model may now enable us to study therapeutic interventions on the myocardium in MFS.