Loretha Myers

A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2

We report heterozygous mutations in the genes encoding either type I or type II transforming growth factor β receptor in ten families with a newly described human phenotype that includes widespread perturbations in cardiovascular, craniofacial, neurocognitive and skeletal development. Despite evidence that receptors derived from selected mutated alleles cannot support TGFβ signal propagation, cells derived from individuals heterozygous with respect to these mutations did not show altered kinetics of the acute phase response to administered ligand. Furthermore, tissues derived from affected individuals showed increased expression of both collagen and connective tissue growth factor, as well as nuclear enrichment of phosphorylated Smad2, indicative of increased TGFβ signaling. These data definitively implicate perturbation of TGFβ signaling in many common human phenotypes, including craniosynostosis, cleft palate, arterial aneurysms, congenital heart disease and mental retardation, and suggest that comprehensive mechanistic insight will require consideration of both primary and compensatory events.

Dysregulation of TGF-β activation contributes to pathogenesis in Marfan syndrome

Marfan syndrome is an autosomal dominant disorder of connective tissue caused by mutations in fibrillin-1 (encoded by FBN1 in humans and Fbn1 in mice), a matrix component of extracellular microfibrils. A distinct subgroup of individuals with Marfan syndrome have distal airspace enlargement, historically described as emphysema, which frequently results in spontaneous lung rupture (pneumothorax; refs. 1–3). To investigate the pathogenesis of genetically imposed emphysema, we analyzed the lung phenotype of mice deficient in fibrillin-1, an accepted model of Marfan syndrome. Lung abnormalities are evident in the immediate postnatal period and manifest as a developmental impairment of distal alveolar septation. Aged mice deficient in fibrillin-1 develop destructive emphysema consistent with the view that early developmental perturbations can predispose to late-onset, seemingly acquired phenotypes. We show that mice deficient in fibrillin-1 have marked dysregulation of transforming growth factor-β (TGF-β) activation and signaling, resulting in apoptosis in the developing lung. Perinatal antagonism of TGF-β attenuates apoptosis and rescues alveolar septation in vivo. These data indicate that matrix sequestration of cytokines is crucial to their regulated activation and signaling and that perturbation of this function can contribute to the pathogenesis of disease.

Phenotypic Alteration of Vascular Smooth Muscle Cells Precedes Elastolysis in a Mouse Model of Marfan Syndrome

Abstract— Marfan syndrome is associated with early death due to aortic aneurysm. The condition is caused by mutations in the gene (FBN1) encoding fibrillin-1, a major constituent of extracellular microfibrils. Prior observations suggested that a deficiency of microfibrils causes failure of elastic fiber assembly during late fetal development. Mice homozygous for a targeted hypomorphic allele (mgR) of Fbn1 revealed a predictable sequence of abnormalities in the vessel wall including elastic fiber calcification, excessive deposition of matrix elements, elastolysis, and intimal hyperplasia. Here we describe previously unrecognized concordant findings in elastic vessels from patients with Marfan syndrome. Furthermore, ultrastructural analysis of mgR mice revealed cellular events that initiate destructive changes. The first detectable abnormality was an unusually smooth surface of elastic laminae, manifesting the loss of cell attachments that are normally mediated by fibrillin-1. Adjacent cells adopted alteration in their expression profile accompanied by morphological changes but retained expression of vascular smooth muscle cell markers. The abnormal synthetic repertoire of these morphologically abnormal smooth muscle cells in early vascular lesions included elastin, among other matrix elements, and matrix metalloproteinase 9, a known mediator of elastolysis. Ultimately, cell processes associated with zones of elastic fiber thinning and fragmentation. These data suggest that the loss of cell attachments signals a nonproductive program to synthesize and remodel an elastic matrix. This refined understanding of the pathogenesis of vascular disease in Marfan syndrome will facilitate the development of therapeutic strategies.

Evidence for a critical contribution of haploinsufficiency in the complex pathogenesis of Marfan syndrome

Marfan syndrome is a connective tissue disorder caused by mutations in the gene encoding fibrillin-1 (FBN1). A dominant-negative mechanism has been inferred based upon dominant inheritance, mulitimerization of monomers to form microfibrils, and the dramatic paucity of matrix-incorporated fibrillin-1 seen in heterozygous patient samples. Yeast artificial chromosome-based transgenesis was used to overexpress a disease-associated mutant form of human fibrillin-1 (C1663R) on a normal mouse background. Remarkably, these mice failed to show any abnormalities of cellular or clinical phenotype despite regulated overexpression of mutant protein in relevant tissues and developmental stages and direct evidence that mouse and human fibrillin-1 interact with high efficiency. Immunostaining with a human-specific mAb provides what we believe to be the first demonstration that mutant fibrillin-1 can participate in productive microfibrillar assembly. Informatively, use of homologous recombination to generate mice heterozygous for a comparable missense mutation (C1039G) revealed impaired microfibrillar deposition, skeletal deformity, and progressive deterioration of aortic wall architecture, comparable to characteristics of the human condition. These data are consistent with a model that invokes haploinsufficiency for WT fibrillin-1, rather than production of mutant protein, as the primary determinant of failed microfibrillar assembly. In keeping with this model, introduction of a WT FBN1 transgene on a heterozygous C1039G background rescues aortic phenotype.

Loss-of-function mutations in TGFB2 cause a syndromic presentation of thoracic aortic aneurysm

Loeys-Dietz syndrome (LDS) associates with a tissue signature for high transforming growth factor (TGF)-β signaling but is often caused by heterozygous mutations in genes encoding positive effectors of TGF-β signaling, including either subunit of the TGF-β receptor or SMAD3, thereby engendering controversy regarding the mechanism of disease. Here, we report heterozygous mutations or deletions in the gene encoding the TGF-β2 ligand for a phenotype within the LDS spectrum and show upregulation of TGF-β signaling in aortic tissue from affected individuals. Furthermore, haploinsufficient Tgfb2+/− mice have aortic root aneurysm and biochemical evidence of increased canonical and noncanonical TGF-β signaling. Mice that harbor both a mutant Marfan syndrome (MFS) allele (Fbn1C1039G/+) and Tgfb2 haploinsufficiency show increased TGF-β signaling and phenotypic worsening in association with normalization of TGF-β2 expression and high expression of TGF-β1. Taken together, these data support the hypothesis that compensatory autocrine and/or paracrine events contribute to the pathogenesis of TGF-β–mediated vasculopathies.

Perturbations of Vascular Homeostasis and Aortic Valve Abnormalities in Fibulin-4 Deficient Mice

The Fibulins are a 6-member protein family hypothesized to function as intermolecular bridges that stabilize the organization of extracellular matrix structures. Here, we show that reduced expression of Fibulin-4 leads to aneurysm formation, dissection of the aortic wall and cardiac abnormalities. Fibulin-4 knockdown mice with a hypomorphic expression allele arose from targeted disruption of the adjacent Mus81 endonuclease gene. Mice homozygous for the Fibulin-4 reduced expression allele (Fibulin-4R/R) show dilatation of the ascending aorta and a tortuous and stiffened aorta, resulting from disorganized elastic fiber networks. They display thickened aortic valvular leaflets that are associated with aortic valve stenosis and insufficiency. Strikingly, already a modest reduction in expression of Fibulin-4 in the heterozygous Fibulin-4+/R mice occasionally resulted in small aneurysm formation. To get insight into the underlying molecular pathways involved in aneurysm formation and response to aortic failure, we determined the aorta transcriptome of Fibulin-4+/R and Fibulin-4R/R animals and identified distinct and overlapping biological processes that were significantly overrepresented including cytoskeleton organization, cell adhesion, apoptosis and several novel gene targets. Transcriptome and protein expression analysis implicated perturbation of TGF-&bgr; signaling in the pathogenesis of aneurysm in fibulin-4 deficient mice. Our results show that the dosage of a single gene can determine the severity of aneurysm formation and imply that disturbed TGF-&bgr; signaling underlies multiple aneurysm phenotypes.

Angiotensin II–dependent TGF-β signaling contributes to Loeys-Dietz syndrome vascular pathogenesis

Loeys-Dietz syndrome (LDS) is a connective tissue disorder that is characterized by a high risk for aneurysm and dissection throughout the arterial tree and phenotypically resembles Marfan syndrome. LDS is caused by heterozygous missense mutations in either TGF-β receptor gene (TGFBR1 or TGFBR2), which are predicted to result in diminished TGF-β signaling; however, aortic surgical samples from patients show evidence of paradoxically increased TGF-β signaling. We generated 2 knockin mouse strains with LDS mutations in either Tgfbr1 or Tgfbr2 and a transgenic mouse overexpressing mutant Tgfbr2. Knockin and transgenic mice, but not haploinsufficient animals, recapitulated the LDS phenotype. While heterozygous mutant cells had diminished signaling in response to exogenous TGF-β in vitro, they maintained normal levels of Smad2 phosphorylation under steady-state culture conditions, suggesting a chronic compensation. Analysis of TGF-β signaling in the aortic wall in vivo revealed progressive upregulation of Smad2 phosphorylation and TGF-β target gene output, which paralleled worsening of aneurysm pathology and coincided with upregulation of TGF-β1 ligand expression. Importantly, suppression of Smad2 phosphorylation and TGF-β1 expression correlated with the therapeutic efficacy of the angiotensin II type 1 receptor antagonist losartan. Together, these data suggest that increased TGF-β signaling contributes to postnatal aneurysm progression in LDS.

Mutations in a TGF-β Ligand, TGFB3, Cause Syndromic Aortic Aneurysms and Dissections

Background Aneurysms affecting the aorta are a common condition associated with high mortality as a result of aortic dissection or rupture. Investigations of the pathogenic mechanisms involved in syndromic types of thoracic aortic aneurysms, such as Marfan and Loeys-Dietz syndromes, have revealed an important contribution of disturbed transforming growth factor (TGF)-β signaling. Objectives This study sought to discover a novel gene causing syndromic aortic aneurysms in order to unravel the underlying pathogenesis. Methods We combined genome-wide linkage analysis, exome sequencing, and candidate gene Sanger sequencing in a total of 470 index cases with thoracic aortic aneurysms. Extensive cardiological examination, including physical examination, electrocardiography, and transthoracic echocardiography was performed. In adults, imaging of the entire aorta using computed tomography or magnetic resonance imaging was done. Results Here, we report on 43 patients from 11 families with syndromic presentations of aortic aneurysms caused by TGFB3 mutations. We demonstrate that TGFB3 mutations are associated with significant cardiovascular involvement, including thoracic/abdominal aortic aneurysm and dissection, and mitral valve disease. Other systemic features overlap clinically with Loeys-Dietz, Shprintzen-Goldberg, and Marfan syndromes, including cleft palate, bifid uvula, skeletal overgrowth, cervical spine instability and clubfoot deformity. In line with previous observations in aortic wall tissues of patients with mutations in effectors of TGF-β signaling (TGFBR1/2, SMAD3, and TGFB2), we confirm a paradoxical up-regulation of both canonical and noncanonical TGF-β signaling in association with up-regulation of the expression of TGF-β ligands. Conclusions Our findings emphasize the broad clinical variability associated with TGFB3 mutations and highlight the importance of early recognition of the disease because of high cardiovascular risk.

TGFβ Receptor Mutations Impose a Strong Predisposition for Human Allergic Disease

Patients with mutations in the receptors for TGFβ (Loeys-Dietz syndrome) exhibit an increased prevalence of allergic diseases. Allergy Unveiled Loeys-Dietz syndrome (LDS) is an autosomal dominant disorder closely related to Marfan syndrome caused by mutations in the genes encoding receptor subunits for transforming growth factor–β (TGFβ). Patients with LDS are predisposed to aortic aneurisms and other connective tissue disorders. Now, Frischmeyer-Guerrerio et al. report that patients with LDS are more likely to develop allergic diseases. Allergy occurs when the immune system responds to normally harmless substances. The authors observed that LDS patients had elevated incidence of allergic diseases, including asthma, food allergy, eczema, allergic rhinitis, and eosinophilic gastrointestinal disease. These patients had elevated levels of immune responses thought to contribute to allergy, including allergen-specific IgE, eosinophilia, and TH2 cytokines. Because regulatory T cell (Treg) development is regulated by TGFβ, the authors then examined Treg number and function in these patients. They found that the frequency of Tregs was increased in LDS patients, but that these cells produced TH2 effector cytokines, and in vitro studies suggested that LDS mutations promote TH2 inflammation. What’s more, children with allergic disease, but not LDS, had similar changes in Treg number and function. These data suggest that altered TGFβ signaling could promote allergic disease and support testing for U.S. Food and Drug Administration–approved drugs that affect TGFβ for treating allergy. Transforming growth factor–β (TGFβ) is a multifunctional cytokine that plays diverse roles in physiologic processes as well as human disease, including cancer, heart disease, and fibrotic disorders. In the immune system, TGFβ regulates regulatory T cell (Treg) maturation and immune homeostasis. Although genetic manipulation of the TGFβ pathway modulates immune tolerance in mouse models, the contribution of this pathway to human allergic phenotypes is not well understood. We demonstrate that patients with Loeys-Dietz syndrome (LDS), an autosomal dominant disorder caused by mutations in the genes encoding receptor subunits for TGFβ, TGFBR1 and TGFBR2, are strongly predisposed to develop allergic disease, including asthma, food allergy, eczema, allergic rhinitis, and eosinophilic gastrointestinal disease. LDS patients exhibited elevated immunoglobulin E levels, eosinophil counts, and T helper 2 (TH2) cytokines in their plasma. They had an increased frequency of CD4+ T cells that expressed both Foxp3 and interleukin-13, but retained the ability to suppress effector T cell proliferation. TH2 cytokine–producing cells accumulated in cultures of naïve CD4+ T cells from LDS subjects, but not controls, after stimulation with TGFβ, suggesting that LDS mutations support TH2 skewing in naïve lymphocytes in a cell-autonomous manner. The monogenic nature of LDS demonstrates that altered TGFβ signaling can predispose to allergic phenotypes in humans and underscores a prominent role for TGFβ in directing immune responses to antigens present in the environment and foods. This paradigm may be relevant to nonsyndromic presentations of allergic disease and highlights the potential therapeutic benefit of strategies that inhibit TGFβ signaling.

Toward an Understanding of Dural Ectasia: A Light Microscopy Study in a Murine Model of Marfan Syndrome

Study Design. Light microscopy study of the lumbar spinal meninges of a murine model of Marfan syndrome. Objective. Characterize the pathology of the lumbosacral meninges in Marfan syndrome, seeking clues to the pathophysiology behind dural ectasia. Summary of Background Data. Dural ectasia is common in Marfan syndrome. The etiology of dural ectasia is unknown, but is conjectured to be related to constitutionally weak spinal dura. The morphology of the lumbar dura in Marfan syndrome has not been described, as it has in other tissues affected by Marfan syndrome. Methods. The lumbosacral dura were removed from three 4-month-old mice, 1 homozygote (mgR/mgR) expressing the murine Marfan phenotype, 1 heterozygote expressing wild-type phenotype, and 1 homozygote wildtype. Hematoxylin and eosin, elastochrome, and immunohistochemical stains against activated transforming growth factor β, gelatinase A (matrix metalloproteinase-2), and gelatinase-B (matrix metalloproteinase-9) were used for light microscopic evaluation. Results. No difference was noted between the heterozygous and wild-type mice in dural connective tissue morphology. The homozygote (mgR/mgR) had a marked attenuation of the dura overall, in addition to elastic fiber disorganization. The homozygote dura also stained for increased presence of activated transforming growth factor β and matrix metalloproteinase-2, but not matrix metalloproteinase-9. Conclusions. These morphologic findings in the Marfan phenotype mouse mimic the findings of disordered elastic-fibers in other Marfan tissues and demonstrate gross attenuation of the tissue architecture, corroborating the theory that dural ectasia in Marfan syndrome results from hydrostatic pressure on weakened dura. These changes may be due in part to transforming growth factor β overactivation and gelatinase-A–mediated elastolysis and collagen breakdown.