Elaine C. Davis

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.

Elastin is an essential determinant of arterial morphogenesis

Elastin, the main component of the extracellular matrix of arteries, was thought to have a purely structural role. Disruption of elastin was believed to lead to dissection of arteries,, but we showed that mutations in one allele encoding elastin cause a human disease in which arteries are blocked, namely, supravalvular aortic stenosis,. Here we define the role of elastin in arterial development and disease by generating mice that lack elastin. These mice die of an obstructive arterial disease, which results from subendothelial cell proliferation and reorganization of smooth muscle. These cellular changes are similar to those seen in atherosclerosis. However, lack of elastin is not associated with endothelial damage, thrombosis or inflammation, which occur in models of atherosclerosis. Haemodynamic stress is not associated with arterial obstruction in these mice either, as the disease still occurred in arteries that were isolated in organ culture and therefore not subject to haemodynamic stress. Disruption of elastin is enough to induce subendothelial proliferation of smooth muscle and may contribute to obstructive arterial disease. Thus, elastin has an unanticipated regulatory function during arterial development, controlling proliferation of smooth muscle and stabilizing arterial structure.

Fibulin-5 is an elastin-binding protein essential for elastic fibre development in vivo

Extracellular elastic fibres provide mechanical elasticity to tissues and contribute towards the processes of organ remodelling by affecting cell–cell signalling. The formation of elastic fibres requires the assembly and crosslinking of tropoelastin monomers, and organization of the resulting insoluble elastin matrix into functional fibres. The molecules and mechanisms involved in this process are unknown. Fibulin-5 (also known as EVEC/DANCE) is an extracellular matrix protein abundantly expressed in great vessels and cardiac valves during embryogenesis, and in many adult tissues including the aorta, lung, uterus and skin, all of which contain abundant elastic fibres. Here we show that fibulin-5 is a calcium-dependent, elastin-binding protein that localizes to the surface of elastic fibres in vivo. fibulin-5-/- mice develop marked elastinopathy owing to the disorganization of elastic fibres, with resulting loose skin, vascular abnormalities and emphysematous lung. This phenotype, which resembles the cutis laxa syndrome in humans, reveals a critical function for fibulin-5 as a scaffold protein that organizes and links elastic fibres to cells. This function may be mediated by the RGD motif in fibulin-5, which binds to cell surface integrins, and the Ca2+-binding epidermal growth factor (EGF) repeats, which bind elastin.

Novel arterial pathology in mice and humans hemizygous for elastin.

Obstructive vascular disease is an important health problem in the industrialized world. Through a series of molecular genetic studies, we demonstrated that loss-of-function mutations in one elastin allele cause an inherited obstructive arterial disease, supravalvular aortic stenosis (SVAS). To define the mechanism of elastins effect, we generated mice hemizygous for the elastin gene (ELN +/-). Although ELN mRNA and protein were reduced by 50% in ELN +/- mice, arterial compliance at physiologic pressures was nearly normal. This discrepancy was explained by a paradoxical increase of 35% in the number of elastic lamellae and smooth muscle in ELN +/- arteries. Examination of humans with ELN hemizygosity revealed a 2. 5-fold increase in elastic lamellae and smooth muscle. Thus, ELN hemizygosity in mice and humans induces a compensatory increase in the number of rings of elastic lamellae and smooth muscle during arterial development. Humans are exquisitely sensitive to reduced ELN expression, developing profound arterial thickening and markedly increased risk of obstructive vascular disease.

Mutations in the Gene Encoding the RER Protein FKBP65 Cause Autosomal-Recessive Osteogenesis Imperfecta

Osteogenesis imperfecta is a clinically and genetically heterogeneous brittle bone disorder that results from defects in the synthesis, structure, or posttranslational modification of type I procollagen. Dominant forms of OI result from mutations in COL1A1 or COL1A2, which encode the chains of the type I procollagen heterotrimer. The mildest form of OI typically results from diminished synthesis of structurally normal type I procollagen, whereas moderately severe to lethal forms of OI usually result from structural defects in one of the type I procollagen chains. Recessively inherited OI, usually phenotypically severe, has recently been shown to result from defects in the prolyl-3-hydroxylase complex that lead to the absence of a single 3-hydroxyproline at residue 986 of the alpha1(I) triple helical domain. We studied a cohort of five consanguineous Turkish families, originating from the Black Sea region of Turkey, with moderately severe recessively inherited OI and identified a novel locus for OI on chromosome 17. In these families, and in a Mexican-American family, homozygosity for mutations in FKBP10, which encodes FKBP65, a chaperone that participates in type I procollagen folding, was identified. Further, we determined that FKBP10 mutations affect type I procollagen secretion. These findings identify a previously unrecognized mechanism in the pathogenesis of OI.

Mutations in Fibrillin-1 Cause Congenital Scleroderma: Stiff Skin Syndrome

Stiff skin syndrome, an autosomal dominant congenital form of scleroderma, is caused by mutations in the domain of fibrillin-1 that mediates integrin binding. Variation in Rare Disease Linked to Common Skin Disorder In the epic words of British physician and researcher Dr. William Harvey, “Nature is nowhere accustomed more openly to display her secret mysteries than in cases where she shows traces of her workings apart from the beaten path; nor is there any better way to advance the proper practice of medicine than to give our minds to the discovery of the usual law of nature by the careful investigation of cases of rarer forms of disease. For it has been found in almost all things, that what they contain of useful or of applicable nature, is hardly perceived unless we are deprived of them, or they become deranged in some way.” Such is the case for rare genetic diseases, which have provided the framework to understand some of the most devastating common diseases from just the simple permutation of a gene. Scleroderma, which literally means a pathological hardening of the skin, manifests as a complex phenotype. To better understand the etiology of scleroderma, Loeys and colleagues investigate four families with more than 10 affected individuals with a rare congenital form called stiff skin syndrome—which together account for more than 25% of the total cases currently documented in the literature—and identify the key mutations in a gene that encodes the connective tissue protein fibrillin-1. Fibrosis is not only seen in the context of systemic connective tissue disorders, but rather is often the major signature of the inflammatory burden in many common disorders, providing incentive to understand the factors critical in the initiation and maintenance of profibrotic programs. Mutations causing stiff skin syndrome cluster within a single domain of fibrillin-1 that mediates integin binding. Low amounts of an activated protein kinase that is triggered by ligand-integrin interactions provided further evidence for altered cell matrix interactions. This associates with evidence for activation of the TGFβ signaling cascade, a mechanism to instruct cells to deposit collagens and other matrix elements both during normal wound healing and in various fibrotic states. These data are consistent with a model in which integrins provide a means for cells to sample the matrix and to adjust their synthetic repertoire accordingly. Loss of this feedback would culminate in fibrosis. The results garnered from these patients with this rare disease prompted the authors to examine skin biopsies from five patients with common scleroderma, which surprisingly revealed that each scleroderma patient showed all of the abnormalities seen in stiff skin syndrome. These findings reinforce the power of studying rare genetic disorders to inform the underlying causes of common diseases, but also unveil a potential avenue for therapeutic intervention in common disorders that include fibrosis. The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence–encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor–β (TGFβ). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFβ concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.

Elastic fiber formation: A dynamic view of extracellular matrix assembly using timer reporters

To study the dynamics of elastic fiber assembly, mammalian cells were transfected with a cDNA construct encoding bovine tropoelastin in frame with the Timer reporter. Timer is a derivative of the DsRed fluorescent protein that changes from green to red over time and, hence, can be used to distinguish new from old elastin. Using dynamic imaging microscopy, we found that the first step in elastic fiber formation is the appearance of small cell surface‐associated elastin globules that increased in size with time (microassembly). The elastin globules are eventually transferred to pre‐existing elastic fibers in the extracellular matrix where they coalesce into larger structures (macroassembly). Mechanical forces associated with cell movement help shape the forming, extracellular elastic fiber network. Time‐lapse imaging combined with the use of Timer constructs provides unique tools for studying the temporal and spatial aspects of extracellular matrix formation by live cells. J. Cell. Physiol. 207: 87–96, 2006.

Integrin-modulating therapy prevents fibrosis and autoimmunity in mouse models of scleroderma

In systemic sclerosis (SSc), a common and aetiologically mysterious form of scleroderma (defined as pathological fibrosis of the skin), previously healthy adults acquire fibrosis of the skin and viscera in association with autoantibodies. Familial recurrence is extremely rare and causal genes have not been identified. Although the onset of fibrosis in SSc typically correlates with the production of autoantibodies, whether they contribute to disease pathogenesis or simply serve as a marker of disease remains controversial and the mechanism for their induction is largely unknown. The study of SSc is hindered by a lack of animal models that recapitulate the aetiology of this complex disease. To gain a foothold in the pathogenesis of pathological skin fibrosis, we studied stiff skin syndrome (SSS), a rare but tractable Mendelian disorder leading to childhood onset of diffuse skin fibrosis with autosomal dominant inheritance and complete penetrance. We showed previously that SSS is caused by heterozygous missense mutations in the gene (FBN1) encoding fibrillin-1, the main constituent of extracellular microfibrils. SSS mutations all localize to the only domain in fibrillin-1 that harbours an Arg-Gly-Asp (RGD) motif needed to mediate cell–matrix interactions by binding to cell-surface integrins. Here we show that mouse lines harbouring analogous amino acid substitutions in fibrillin-1 recapitulate aggressive skin fibrosis that is prevented by integrin-modulating therapies and reversed by antagonism of the pro-fibrotic cytokine transforming growth factor β (TGF-β). Mutant mice show skin infiltration of pro-inflammatory immune cells including plasmacytoid dendritic cells, T helper cells and plasma cells, and also autoantibody production; these findings are normalized by integrin-modulating therapies or TGF-β antagonism. These results show that alterations in cell–matrix interactions are sufficient to initiate and sustain inflammatory and pro-fibrotic programmes and highlight new therapeutic strategies.

Domains in tropoelastin that mediate elastin deposition in vitro and in vivo.

Elastic fiber assembly is a complicated process involving multiple different proteins and enzyme activities. However, the specific protein-protein interactions that facilitate elastin polymerization have not been defined. To identify domains in the tropoelastin molecule important for the assembly process, we utilized an in vitro assembly model to map sequences within tropoelastin that facilitate its association with fibrillin-containing microfibrils in the extracellular matrix. Our results show that an essential assembly domain is located in the C-terminal region of the molecule, encoded by exons 29–36. Fine mapping studies using an exon deletion strategy and synthetic peptides identified the hydrophobic sequence in exon 30 as a major functional element in this region and suggested that the assembly process is driven by the propensity of this sequence to form β-sheet structure. Tropoelastin molecules lacking the C-terminal assembly domain expressed as transgenes in mice did not assemble nor did they interfere with assembly of full-length normal mouse elastin. In addition to providing important information about elastin assembly in general, the results of this study suggest how removal or alteration of the C terminus through stop or frameshift mutations might contribute to the elastin-related diseases supravalvular aortic stenosis and cutis laxa.

Fibulin-4 Deficiency Results in Ascending Aortic Aneurysms. A Potential Link Between Abnormal Smooth Muscle Cell Phenotype and Aneurysm Progression

Rationale: Loss of fibulin-4 during embryogenesis results in perinatal lethality because of aneurysm rupture, and defective elastic fiber assembly has been proposed as an underlying cause for the aneurysm phenotype. However, aneurysms are never seen in mice deficient for elastin, or for fibulin-5, which absence also leads to compromised elastic fibers. Objective: We sought to determine the mechanism of aneurysm development in the absence of fibulin-4 and establish the role of fibulin-4 in aortic development. Methods and Results: We generated germline and smooth muscle cell (SMC)-specific deletion of the fibulin-4 gene in mice (Fbln4GKO and Fbln4SMKO, respectively). Fbln4GKO and Fbln4SMKO aortic walls fail to fully differentiate, exhibiting reduced expression of SM-specific contractile genes and focal proliferation of SMCs accompanied by degenerative changes of the medial wall. Marked upregulation of extracellular signal-regulated kinase 1/2 signaling pathway was observed in the aneurysmal wall of Fbln4GKO and Fbln4SMKO mice and both mutants developed aneurysm predominantly in the ascending thoracic aorta. In vitro, Fbln4GKO SMCs exhibit an immature SMC phenotype with a marked reduction of SM-myosin heavy chain and increased proliferative capacity. Conclusions: The vascular phenotype in Fbln4 mutant mice is remarkably similar to a subset of human thoracic aortic aneurysms caused by mutations in SMC contractile genes. Our study provides a potential link between the intrinsic properties of SMCs and aneurysm progression in vivo and supports the dual role of fibulin-4 in the formation of elastic fibers as well as terminal differentiation and maturation of SMCs in the aortic wall.