The fibrillin microfibril scaffold: A niche for growth factors and mechanosensation?

    The Fibrillin Microfibril Scaffold: Mechanosensation?

A Niche for Growth Factors And

Gerhard Sengle, Lynn Y. Sakai PII: DOI: Reference:

S0945-053X(15)00101-8 doi: 10.1016/j.matbio.2015.05.002 MATBIO 1172

To appear in:

Matrix Biology

Please cite this article as: Sengle, Gerhard, Sakai, Lynn Y., The Fibrillin Microfibril Scaffold: A Niche for Growth Factors And Mechanosensation?, Matrix Biology (2015), doi: 10.1016/j.matbio.2015.05.002

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ACCEPTED MANUSCRIPT Mini Review #1 The Fibrillin Microfibril Scaffold: A Niche for Growth Factors And

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Mechanosensation?

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Gerhard Senglea and Lynn Y. Sakaib,*

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Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany; Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany. Shriners Hospital for Children, Oregon Health & Science University, Portland, OR

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To Whom Correspondence Should be Addressed: Lynn Y. Sakai, Ph.D. Shriners Hospital for Children

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Tel: 503-221-3436

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Portland, OR 97239

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3101 SW Sam Jackson Park Road

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Email: [email protected]

Received

11 February 2015

Revised

24 March 2015

Received in re-revised form

27 March 2015

Accepted

28 March 2015

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ACCEPTED MANUSCRIPT Abstract

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The fibrillins, large extracellular matrix molecules, polymerize to form “microfibrils.” The fibrillin microfibril scaffold is populated by microfibril-associated proteins and by growth factors, which are likely to be latent. The scaffold, associated proteins, and bound growth factors, together with cellular receptors that can sense the microfibril matrix, constitute the fibrillin microenvironment. Activation of TGFβ signaling is associated with the Marfan syndrome, which is caused by mutations in fibrillin-1. Today we know that mutations in fibrillin-1 cause the Marfan syndrome as well as WeillMarchesani syndrome (and other acromelic dysplasias) and result in opposite clinical phenotypes: tall or short stature; arachnodactyly or brachydactyly; joint hypermobility or stiff joints; hypomuscularity or hypermuscularity. We also know that these different syndromes are associated with different structural abnormalities in the fibrillin microfibril scaffold and perhaps with specific cellular receptors (mechanosensors). How does the microenvironment, framed by the microfibril scaffold and populated by latent growth factors, work? We must await future investigations for the molecular and cellular mechanisms that will answer this question. However, today we can appreciate the importance of the fibrillin microfibril niche as a contextual environment for growth factor signaling and potentially for mechanosensation.

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Keywords: Fibrillin, Bone Morphogenetic Protein, TGFβ, LTBP, Marfan Syndrome, Weill-Marchesani Syndrome, Mechanosensation

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ACCEPTED MANUSCRIPT Introduction

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Fibrillin microfibrils were first identified through the electron microscope as small diameter fibrils located close to basement membranes and also at the periphery of elastic fibers [1]. Since these microfibrils were uniformly small in diameter (around 10 nm) and lacked a banding pattern, it was initially speculated that they might be immature forms of collagen or perhaps composed of a new type of collagen. However, the main component was later shown to be a glycoprotein, which was named “fibrillin”[2]. Fibrillin monomers were isolated and found to be long flexible molecules [3], whose shapes were consistent with the extended polymers that form the microfibril backbone in rotary shadowed images of microfibrils [4]. Other proteins have been identified as microfibril associated proteins. These include MAGP-1 [5] and MAGP-2 [6], biglycan and decorin [7], versican [8], perlecan [9], the fibulins [10,11], elastin [12], and Adamtslike proteins [13]. Binding sites within the fibrillin molecule are known for these associated molecules (depicted in Figure 1A). All together, these molecules constitute the fibrillin microfibril scaffold.

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Initially, discrimination between backbone “structural” proteins and microfibril “associated” or “accessory” proteins was of interest. From an evolutionary perspective, fibrillin might be viewed as the main structural protein of microfibrils, since it predates other microfibril proteins like MAGPs and fibronectin [14]. This perspective might suggest that proteins which evolve first likely perform structural roles in microfibril assembly, while associated proteins might be predicted to perform regulatory rather than structural roles [14]. For example, MAGP-1 knockout mice still have functional microfibrils, but phenotypes in these mice may be due to inappropriate regulation of TGFβ signaling [15]. LTBP-1 (Latent TGFβ Binding Protein-1), which appears in sea urchin but not in jellyfish [14], might therefore be expected to perform primarily regulatory rather than structural roles. However, a primarily structural role for LTBP-4 has been proposed [16]. It is now appreciated that fibrillin, which predates all other microfibril proteins, performs both structural as well as regulatory roles. It seems plausible, therefore, that any microfibril scaffold protein, regardless of its evolutionary history, may contribute both to the structure of the scaffold as well as to the regulation of growth factors. Cloning and sequencing cDNAs in the early 1990s revealed that fibrillin-1 is a modular protein composed primarily of epidermal growth factor like motifs interspersed by domains which contained 8 cysteines [17,18]. The similarity between fibrillin-1 and the sequence for the LTBP-1 [19] was pointed out [17,18]. This structural homology led to the hypothesis that fibrillins and LTBPs may bind and sequester different members of the TGFβ superfamily of growth factors. Also in the early 1990s, it became clear that fibrillins were critical components of the extracellular matrix. Disruption of the fibrillin microfibril scaffold was implicated in the Marfan syndrome [20], and mutations in the gene for fibrillin-1 (FBN1) were shown to cause Marfan syndrome [21]. Mutations in the gene for fibrillin-2 (FBN2) were found in congenital contractural arachnodactyly (now called Distal Arthrogryposis, Type 9 or DA9) [22]. Marfan syndrome is a connective tissue disorder with major phenotypic features in the cardiovascular, musculoskeletal, and ocular systems. Manifestations of 3

ACCEPTED MANUSCRIPT congenital contractural arachnodactyly are primarily musculoskeletal, with some features shared with Marfan syndrome (arachnodactyly, scoliosis).

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Biochemical studies of molecular interactions led to a working model of the fibrillin microfibril niche as a repository for growth factors (Figure 1B). In this model, fibrillin molecules target growth factor complexes to the microfibril niche. LTBP-1, with its covalently associated small latent TGFβ complex, interacts through its C-terminal end with fibrillin [23] and through its N-terminal end with other elements of the extracellular matrix [24]. BMP complexes, depicted as red butterflies with black prodomains, are bound directly to fibrillin [25, 26]. Investigations of the human and mouse genetic disorders caused by mutations in fibrillins support the concept that fibrillin microfibrils form a niche for growth factors and that perturbation of the fibrillin microfibril scaffold can result in abnormal growth factor signaling.

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The working model depicted in Figure 1 also contains cellular receptors and speculates on the role of these receptors. Human and mouse genetic disorders have contributed to the notion that fibrillin microfibril structure is sensed by the cell. Integrin binding to fibrillin, or binding by other cellular receptors, may play important roles in sensing the fibrillin microfibril niche. How mechanosensation of microfibril structure is transduced into signals by the cell and whether the microfibril scaffold performs a dynamic role to integrate growth factor signaling with mechanosensation are new questions open for investigation.

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Indirect Molecular Interactions Between Fibrillin and Growth Factors

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LTBP-1 was immunolocalized to fibrillin microfibrils [27]. Biochemical studies identified a direct interaction between fibrillins and LTBP-1 and LTBP-4 [23, 28], as well as between fibrillin-1 and LTBP-2 [29]. It is not clear whether LTBP-3 interacts directly with fibrillin [23, 30]. Of the 4 LTBPs, LTBP-1, -3 and -4 were reported to interact directly with TGFβ complexes, while fibrillins did not interact with TGFβ [31]. Nevertheless, it was hypothesized that fibrillin-1 deficiency would perturb TGFβ signaling, because in the absence of fibrillin-1, the large latent TGFβ complex would not be appropriately targeted and sequestered. However, it was not clear what effect this would have on TGFβ signaling. Failure to target the large latent TGFβ complex to fibrillin might lead to decreased TGFβ signaling (if appropriate location is required for activation), or to increased TGFβ signaling (if fibrillin binding is required to sequester TGFβ complexes from receptors or activators). As it turned out, TGFβ signaling was abnormally activated in the severely hypomorphic mgΔ Fbn1 mutant mouse lung [32]. The concept that fibrillin-1 deficiency results in abnormal activation of TGFβ signaling was further tested in a mouse model carrying a missense mutation (C1039G) in Fbn1. Abnormal activation of TGFβ signaling was described in the aorta [33], valves [34], and skeletal muscle [35] in heterozygous C1039G mice. However, when compared to the severe deficiency in mgΔ mice, fibrillin-1 deficiency in heterozygous C1039G mice was less obvious; the disease mechanism in C1039G mice was described as “haploinsufficiency driven” [36]. In order to test whether activation of TGFβ signaling in Fbn1 mutant mice is due to reduction in fibrillin-1 microfibrils or due to a specific inability of LTBPs to target and sequester TGFβ to the fibrillin microfibril niche, the LTBP binding site in fibrillin was 4

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located to residues within the first hybrid domain [28] and then the first hybrid domain was targeted for in vivo deletion in the mouse. Both heterozygous and homozygous hybrid-1 deleted mice (Fbn1H1Δ/+ or Fbn1H1Δ/H1Δ) assembled microfibrils and lived long lives with no signs of aortic aneurysm or dissection [37], indicating that the inability of LTBPs to bind to fibrillin-1 is not the basis for disease in Marfan mice. Further studies are required in order to elucidate the functions of LTBP interactions with fibrillin and with other components of the microfibril scaffold. However, it seems unlikely that activation of TGFβ signaling is simply due to loss of the LTBP-fibrillin-1 interaction in fibrillin-1 deficient states.

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Direct Molecular Interactions Between Fibrillin and Growth Factors

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Based on structural homologies between LTBPs and fibrillins, it was hypothesized that fibrillins may bind to BMP complexes and target these to the microfibril niche. Recombinant BMP-7 prodomain/growth factor complex was characterized biochemically and used to demonstrate that fibrillins interact with the BMP-7 complex [25]. The BMP-7 complex was dissociated in 8M urea and separated into propeptides and growth factor dimer. The BMP-7 propeptides interacted with fibrillins while the growth factor dimer did not [25].

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Bacterially expressed recombinant propeptides for BMP-2, BMP-4, BMP-5, BMP10, GDF-5, and GDF-8 were used, without their growth factors, in binding assays with fibrillins [26, 38]. With the exception of GDF-8 (also known as myostatin), these propeptides interacted with fibrillins. Therefore, the hypothesis based on structural homologies was true: both LTBPs and fibrillins interact directly with propeptides of growth factors in the TGFβ superfamily. However, BMP/GDF propeptides bind to a site close to the N-terminus of fibrillins and not to 8-cysteine domains; TGFβ propeptides bind covalently to an 8-cysteine domain in LTBPs. All members of this family have not been tested yet, but it is clear that this is not a universal mechanism, since the propeptide of GDF-8 binds to a specific glycosaminoglycan side chain present on the Cterminus of perlecan [38]. Interestingly, perlecan is part of the fibrillin microfibril niche, since perlecan interacts directly with fibrillin [9], so GDF-8 signaling may be coordinated by the microfibril niche, together with signaling from other TGFβ-like growth factors (See Figure 1). The TGFβ propeptide/growth factor complex is latent, because the propeptides place the growth factor in a kind of conformational “straitjacket” such that the growth factor is not able to bind to its receptors [39]. Unlike the latent TGFβ complex, the BMP7 complex could bind to its receptors and activate BMP signaling in cell cultures [40]. Moreover, in solution, BMP type II receptors could displace the BMP-7 propeptides and bind to the BMP-7 growth factor dimer [40]. However, BMP complexes, when targeted to fibrillins in the extracellular matrix, are not in solution. In vitro assays were performed to test whether adsorption of the BMP-7 complex to a solid support could block growth factor signaling. Recombinant fibrillin peptides adsorbed to cell culture plastic or coupled to adsorbed monoclonal antibodies were used to capture BMP-7 complexes. Monoclonal antibodies specific for different epitopes in BMP-7 propeptides were adsorbed as controls and used to capture BMP-7 complexes. Results indicated that capturing BMP-7 complex through binding to fibrillin blocked BMP signaling, when cells were added to the wells. In contrast, equivalent amounts of BMP-7 complex captured 5

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onto control wells coated with antibodies to the Histidine tag present on recombinant BMP-7 yielded clear evidence of BMP signaling. These results (Sengle G, Carlberg V, Tufa SF, Charbonneau NL, Smaldone S, Ramirez F, Keene DR, Sakai LY. Abnormal activation of BMP signaling causes myopathy in Fbn2 null mice. In revision, 2015) suggest that binding to fibrillins may change the conformation of the propeptides such that the BMP-7 complex is latent.

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Mutations in fibrillins could result in an increase in BMP signaling, if BMPs are no longer bound by fibrillin and are therefore in an active conformation. Evidence for activated BMP signaling was found in Fbn2 null skeletal muscle (Sengle, G, et al., in revision, 2015) and in Fbn1 null osteoblasts [41]. However, Fbn1 null osteoblasts also exhibited improper activation of TGFβ signaling. In contrast, Fbn2 null osteoblasts showed impaired osteoblast maturation and reduced bone formation due to improper activation of TGFβ signaling [41]. These findings indicate that effects on signaling are dependent on the tissue context.

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How Does the Scaffold Work In Vivo? Evidence from Marfan Syndrome and WeillMarchesani Syndrome

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Initially, research focused on the role of fibrillin as a structural component of microfibrils. Investigations compared the structure of fibrillin monomers with the structure of microfibrils [42, 43] and identified domains necessary for polymerization of fibrillin [44, 45, 46]. The discovery that FBN1 is the Marfan gene led naturally to questions regarding the effects of mutations in fibrillin-1 on microfibril assembly [37] and microfibril stability [47]. Later, when it was hypothesized that another function of fibrillin1 is to target and sequester growth factors, attention quickly shifted to the role of growth factor signaling in Marfan syndrome, since the challenge of correcting a structural defect in microfibrils “boded poorly for therapy” [48]. Findings over the last decade supported abnormal activation of TGFβ signaling as the main driver of pathogenesis of Marfan syndrome [48] and led to clinical trials of losartan, an angiotensin II type I receptor blocker (ARB) also thought to be an inhibitor of TGFβ signaling, as therapy for Marfan syndrome [33]. The identification of TGFβ receptors I and II and intracellular components of the TGFβ signaling pathway (SMAD3 and SKI) as disease genes for human disorders related to Marfan syndrome [49-53] seemed to clinch the case for abnormal activation of TGFβ signaling as the main driver of pathogenesis in Marfan syndrome and related disorders. It was plausible that the mechanism proposed to cause Marfan syndrome would be common to related aortopathies, especially those caused by mutations in genes for important components of the TGFβ signaling pathway. However, the case for abnormal activation of TGFβ signaling being the main driver of pathogenesis seems less convincing due to recent discoveries. Mutations in the receptor for TGFβ were originally proposed to be loss-of-function mutations [49]. This concept was replaced by findings that mutations are activating [50]. Recent genetic findings demonstrate that loss-of-function mutations in the ligand, TGFβ2, cause aortic disease [54, 55]. Thus, “paradoxical” [55] mechanisms involving both loss-offunction mutations and activation of TGFβ signaling are now proposed to underlie aortic disease. At the very least, mechanisms leading to activation of TGFβ signaling in aortopathies are today considered to be more complex than were originally thought. 6

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Recent experiments using mouse models of Marfan syndrome indicate that activation of TGFβ signaling may not be the main driver of pathogenesis. To test this hypothesis, conditional disruption of Tgfbr2 was performed in smooth muscle cells of Marfan mice (Fbn1C1039G/+, the mouse model which was used for the original hypothesis [33]). Results showed accelerated aneurysm growth, rather than rescue, indicating that TGFβ signaling promotes aortic homeostasis and impedes disease progression [56]. Therefore, activation of TGFβ signaling may in fact be a salutary response of the aorta to repair the damaged extracellular environment, and therapies aimed at inhibiting TGFβ signaling may actually worsen aortic disease. Findings [55] that haploinsufficiency of Tgfb2 lead to increased severity of aortic disease in compound heterozygous Fbn1C1039G/+;Tgfb2+/- support the latter hypotheses.

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Losartan, an ARB (angiotensin II type 1 receptor blocker), was used to treat aortic disease in the Marfan mouse model, C1039G. Since losartan treatment blocked aortic root growth and normalized pSmad2/3 levels to the same extent as TGFβ neutralizing antibodies, losartan was proposed to work as an inhibitor of abnormally active TGFβ signaling in the Marfan aorta [33]. Clinical trials of losartan treatment in more than 600 children with Marfan syndrome were completed, and results were recently published [57]. The trial compared losartan to atenolol, a β-adrenergic blocker, titrated to have an optimal hemodynamic effect. Losartan was used at recommended doses. Atenolol worked better than losartan to reduce aortic root growth with no adverse events, while losartan showed a trend toward increased risk of adverse events, but these results were not statistically significant. Hence, the clinical trials also suggest that losartan may be an effective treatment for Marfan syndrome. The question remains, however, whether losartan slows aortic root growth through its inhibitory effect on TGFβ signaling, or whether its effect is due to blocking angiotensin II signaling or mechanosensing through the AT1 receptor. Many of the effects attributed to activated TGFβ signaling may in fact be due to angiotensin II signaling [58], but this controversy remains to be resolved in future studies. Using a mouse model (Fbn1mgR/mgR) of severe aortic disease, the effects of losartan and TGFβ neutralizing antibodies were compared in a head-to-head trial [59]. Results showed that losartan improved survival of these mice; TGFβ neutralizing antibodies also improved survival; and mice treated with both losartan and TGFβ neutralizing antibodies remained alive at 3 months of age, when less than 40% of these mice treated with placebo were still alive. Treatments also revealed a “dimorphic” effect of TGFβ neutralizing antibodies: if antibodies were administered at P16, 50% survival was around 40 days, and all mice died by 60 days; if antibodies were administered at P45, survival was increased, compared to placebo. These results suggest that the effects of abnormal activation of TGFβ may be salutary (during the early course of aortic disease in Fbn1mgR/mgR) or exacerbating (during the late course of disease in Fbn1mgR/mgR). However, the contextual dependence of these salutary or exacerbating effects is not yet understood. Initially, the focus of investigations was on the effects of mutations in fibrillin-1 on the structure, assembly, and stability of fibrillin microfibrils. Indeed, the first study supporting the candidacy of fibrillin-1 as the Marfan gene showed that skin and fibroblast cultures from patients with Marfan syndrome could be identified by abnormal 7

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immunofluorescence of fibrillin microfibrils [20]. After almost two decades of research on the role of fibrillin-1 in the pathogenesis of the Marfan syndrome, it was surprising to find that not all mutations in fibrillin-1 result in Marfan syndrome. While there had been reports of mutations in FBN1 associated with isolated ectopia lentis [60], isolated tall stature [61], or with Weill-Marchesani syndrome [62], it was clearly established over the last five years that mutations in fibrillin-1 can lead to phenotypes quite different from those in the Marfan syndrome. These studies have re-established the structure of microfibrils as an important determinant in pathogenesis of Marfan syndrome and related disorders.

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Weill-Marchesani syndrome was described as the “opposite” of Marfan syndrome by Victor McKusick [63]. Instead of the tall stature and arachnodactyly typical of Marfan syndrome, short stature and brachydactyly are features of Weill-Marchesani syndrome. Similarly, hypermobility of joints and hypomuscularity are features of Marfan syndrome, whereas stiff joints and hypermuscularity are typical of Weill-Marchesani syndrome. Mutations in FBN1 were recently described in Weill-Marchesani syndrome [62], as well as in related acromelic dysplasias: geleophysic dysplasia and acromicric dysplasia [64].

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A large deletion in FBN1 was identified in a family with Weill-Marchesani syndrome [65]. A mouse model expressing this mutation was generated in order to test whether it would cause a Marfan-like or a Weill-Marchesani-like phenotype [65]. Previous criticisms of the FBN1 mutations described in humans with varying conditions, including Weill-Marchesani syndrome, were that these clinical cases might be undeveloped Marfan syndrome, but not distinct clinical entities. The Fbn1 mutant mouse with the large 3-exon deletion turned out to be a model for human WeillMarchesani syndrome. This was the second time an Fbn1 mutant mouse failed to display a Marfan phenotype. Together with mice in which the first hybrid domain is deleted, the Weill-Marchesani mice demonstrated that not every mutation in fibrillin-1 should be expected to cause Marfan syndrome. But, of most importance, studies of the Weill-Marchesani mice showed distinctive structural changes involving fibrillin microfibrils [65]. In contrast to the fragmented fibrillin microfibrils seen in human [20] and mouse [37] Marfan syndrome, fibrillin microfibrils in both human and mouse WeillMarchesani syndrome skin were accumulated into large aggregates [65]. These large aggregates of fibrillin microfibrils were similar to those present in skin from humans with Stiff Skin Syndrome, a disorder also due to mutations in FBN1 [66]. Moreover, the 3exon deletion causing Weill-Marchesani syndrome abolished a binding site for ADAMTS-Like proteins. Since ADAMTS-Like 6 had been shown to promote fibrillin-1 fibril assembly [13], loss of the binding site for ADAMTS-Like proteins further underscored an important role for structural abnormalities in Weill-Marchesani syndrome. The Fibrillin Microfibril Scaffold As A Dynamic Microenvironment An important function of fibrillin microfibrils is to form stable structures that are appropriate for tissue architecture. An equally important role for the fibrillin microfibril scaffold is to target and sequester growth factors. According to Sporn and Roberts, who first discovered TGFβ, TGFβ should be regarded as a “cellular switch”, and “its true function is to provide a mechanism for coupling a cell to its environment” [67]. Lessons 8

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from human genetic disorders demonstrate that the fibrillin microfibril scaffold can regulate skeletal growth (tall vs. short stature; arachnodactyly vs. brachydactyly), muscularity, joint mobility, and skin thickness. These opposing results of variations in the structural scaffold may be the consequence of different effects on growth factor “switches.” Hence, the fibrillin microfibril scaffold may function as part of the contextual environment regulating and integrating growth factor signaling. In this sense, fibrillin’s structural and regulatory roles work hand-in-glove.

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Bound to the fibrillin microfibril scaffold, the large latent TGFβ complex, as well as BMP prodomain/growth factor complexes, require activation to initiate growth factor signaling. For activation of TGFβ, integrin binding to the propeptide might change the conformation of the propeptide such that the growth factor is allowed to interact with its receptors [39]. In addition to this mechanism, other mechanisms of activation have been proposed (for example, proteolytic activation or activation by thrombospondin). If binding to fibrillin alters the conformation of the BMP prodomain such that its growth factor is not accessible to BMP receptors, mechanisms of activation will also be required for BMP signaling. The concept that growth factor activation occurs within a locally defined, tissue-specific microenvironment is an appealing one. With the possibility of multiple interactions between growth factors and the fibrillin microfibril scaffold, tissue-specific microenvironments can be achieved through regulation of growth factor gene expression. Coordination between different signals (for example, BMP and TGFβ signaling) could also be controlled through regulated expression of growth factors as well as activators or inhibitors. Finally, the structural organization of the scaffold itself likely participates in how growth factors are positioned, concentrated, and presented to cells and may also influence positioning of activators and inhibitors [68]. An equally important aspect of the contextual environment for growth factor signaling is cellular sensing. The concept that the fibrillin matrix should be sensed by transmembrane receptors which transduce signals that influence cell shape, gene expression, and function is an obvious one. However, little is known about how the cell senses the fibrillin matrix. In Figure 1, integrins as well as unknown receptors are included as important players in a dynamic microenvironment. It is interesting that mutations in fibrillin-1 that cause Stiff Skin Syndrome cluster close to the RGD integrin binding site66, perhaps affecting how integrins may interact with this site and implicating integrin binding in skin fibrosis. An Fbn1 RGE mutant mouse showed skin fibrosis, directly linking integrin interaction with this phenotype [69]. Integrins known to bind to fibrillin include αvβ3 [70,71], αvβ6 [72], and α5β1 [73]. Recent work has implicated important interactions between heparan sulfate and the 5th 8-cysteine domain of FBN1 [74]. Mutations in this domain cause various acromelic dysplasias [64]. Therefore, pericellular heparan sulfate containing proteoglycans (syndecans, for example) may be important for sensing the fibrillin matrix. Another possible cellular sensor is the angiotensin II type 1 receptor (AT1R). AT1R was shown to be activated by mechanical stress, independent of angiotensin II, in cardiac hypertrophy [75]. Recently, dilated cardiomyopathy was found to be a primary manifestation of fibrillin-1 deficient mice [76]. Left ventricular dysfunction is known in Marfan syndrome [76, 77], but genetic isolation of heart disease from aortic disease in 9

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conditionally mutant mice demonstrated the primary nature of cardiac dysfunction [76]. Crossing fibrillin-1 mice with mice lacking AT1R or β-arrestin 2 restored normal cardiac size and function. Consistent with the mouse genetic experiments, treatment with losartan, but not with TGFβ neutralizing antibodies, also restored normal cardiac size and function, suggesting that activation of TGFβ signaling does not account for cardiomyopathy. Interestingly, this study also suggested crosstalk between AT1R mechanosignaling and integrin β1 signaling [76]. Whether AT1R is a specific sensor of fibrillin matrix, or of mechanical stress in general, is unknown.

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Dysfunctional mechanosensing can also contribute to aortic aneurysm [78]. Mechanotransduction in the aortic wall extends from the matrix to the cell membrane to the cytoskeleton, and loss of any linkage in this chain can perturb mechanosensing [78]. Indeed, mutations causing aortic aneurysm syndromes mirror this chain. AT1R may also perform a mechanosensing role in the Marfan aorta, perhaps better explaining the efficacy of losartan on aortic root growth, but this hypothesis requires further study. Concluding Remarks

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The fibrillin microfibril scaffold regulates TGFβ and BMP signaling. Much has been learned in the last decade about fibrillin-1, TGFβ signaling, and aortic disease. However, it has become recently clear that evidence for positive TGFβ signaling in Marfan syndrome may not be sufficient proof that abnormal activation of TGFβ signaling is the main driver of pathogenesis in the Marfan syndrome. Regarding TGFβ, Sporn cautioned that it is “essential to avoid primitive, animistic thinking and to avoid ‘dressing up’ TGFβ, like a doll, with simple labels . . . . The meaning of TGFβ is always embedded in its contextual environment” [67]. This same perspective can be applied to BMP signaling. The fibrillin microfibril scaffold contributes to the contextual environment of growth factor signaling. The challenge for future research is to reveal cellular interactions that contribute to the dynamic interplay of growth factor signaling and fibrillin scaffold structure and to unravel the many mechanistic complexities of the fibrillin microfibril niche. Here, a more holistic or organismal perspective, rather than a simple, linear one (arrows pointing in only one direction), will be required. Acknowledgements

We thank the many individuals who have contributed to the results and concepts discussed in this review. We also gratefully acknowledge our funding sources (the Shriners Hospitals for Children, the National Institutes of Health, the Marfan Foundation, and the Deutsche Forschungsgemeinshaft).

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Figure 1: The Fibrillin Microfibril Niche (A) Fibrillins bind to LTBPs, which are covalently associated with the small latent TGFβ complex, BMPs, MAGP-1 and -2, elastin, fibulins, ADAMTSLike proteins, decorin, versican, and perlecan. Fibrillin molecules are depicted with color-coded domain modules (yellow: calcium-binding EGF-like domains; red: 8-cysteine containing domains; blue: hybrid domains; green: generic EGF domains; purple: proline-rich domain; black: N- and C- terminal domains). Similar color-coded domains are present in LTBP. Black broken lines indicate fibrillin monomers which have not been color coded. Approximate positions of published binding sites on fibrillin are depicted. The organization of fibrillin molecules within the microfibril is according to Kuo et al., 200779. Other organizational models have been proposed (see reference 79 for historical discussion and additional references). Domain sizes are approximate and not drawn to scale. (B) The fibrillin microfibril scaffold targets and sequesters the large latent TGFβ complex (which consists of LTBP covalently bound to the small latent TGFβ complex) as well as BMP prodomain/growth factor complexes. The cell, with receptors for BMPs and TGFβ, is depicted close to the fibrillin microfibril scaffold. Inhibitors and activators are also secreted by the cell into the niche microenvironment. Integrins bind to the RGD-containing 4th 8-cysteine (or TB) domain. The importance of this interaction was shown by the finding that mutations in this domain cause Stiff Skin Syndrome (SSkS)66,69. Acromelic dysplasia (Weill-Marchesani syndrome (WMS), geleophysic dysplasia (GD) and acromicric dysplasia (AD)) mutations cluster in the 5th 8-cysteine (or TB) domain64, which interacts with heparan sulfate74. A 3-domain deletion leading to WMS65 is also marked. Although domain sizes are estimated, the magnified area shows the proximity of these regions within the working model of fibrillin microfibril organization proposed by Kuo et al., 200779. However, it should be noted that mutations causing Marfan syndrome are also found in these domains. Also depicted are yet-to-be-identified receptors (red) and microfibril-associated proteins like MAGP-1, LTBP-1, and ADAMTS-Like proteins, which may interact with cellular receptors and transduce signals from the fibrillin microfibril scaffold.

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