Since the discovery that FBN1
mutations cause MFS, the pathogenesis of the disorder has been the subject of intense investigation and controversy (4
). The Marfan phenotype is developmentally acquired and manifests the dynamic interplay between primary tissue predisposition, physiologic stress, time, and both productive and deleterious compensatory events. This and previous studies attempt to mimic this complexity using multiple strains of mice that harbor targeted fibrillin-1 gene (Fbn1
) alleles (21
). Taken together, they represent the full range of molecular mechanisms and clinical severity characteristic of MFS.
Early histopathologic studies in MFS demonstrated fragmentation of elastic fibers with a decrease in elastin content in both the skin and medial layer of the aorta (28
). Ultrastructural, biochemical, and immunohistologic analyses of forming elastic fibers revealed that the amorphous component is surrounded by a lattice of microfibrils during embryonic development (7
). The tight temporal and spatial link between the formation of microfibrillar aggregates and the subsequent deposition of amorphous elastin fueled speculation that microfibrils are required for primary formation of elastic fibers during embryogenesis (31
). Another belief was that fibrillin-1 is strictly a structural constituent of connective tissue. The implication was that individuals affected with MFS are born with an obligate predisposition for postnatal tissue failure due to failed elastogenesis and an inherent loss of biomechanical integrity. This boded poorly for the development of productive treatment strategies. Both beliefs have now been challenged through characterization of fibrillin-1–deficient mice. First, mice homozygous for a hypomorphic allele that expresses low levels of a centrally deleted fibrillin-1 monomer show normal elastin content and the ability to deposit organized linear elastic fibers during embryonic growth (21
). Fragmentation and disarray of elastic fibers is a focal and acquired event that occurs in association with abnormal production of matrix-degrading enzymes by resident VSMCs and ultimate vessel wall inflammation (22
). Second, it has been demonstrated that fibrillin-1 and microfibrils contribute to the regulated activation of members of the transforming growth factor-β (TGF-β) superfamily of cytokines (27
). Selected manifestations of MFS, including progressive pulmonary disease, manifest excessive cytokine activation and signaling, as evidenced by phenotypic rescue upon TGF-β neutralization (27
). Thus, there appear to be multiple therapeutic strategies aimed at blocking secondary events that could hold promise for the treatment of MFS.
The most effective means for primary prevention of phenotypic manifestations of MFS would be to retain or restore microfibrillar abundance and function. Although in theory this might be achieved using pharmacologic or gene-transfer strategies, enthusiasm was low given the evidence for a traditional dominant-negative pathogenetic mechanism. Indeed, the apparent lesson from the study of patients harboring nonsense alleles suggested that mutant protein in extremely low abundance had potent dominant-negative potential that would neutralize even highly efficient strategies to boost the amount of WT fibrillin-1. The favored model of microfibrillar assembly suggests that fibrillin-1 monomers self-assemble into macroaggregates characterized by parallel arrays of linear extended structures with a head-to-tail orientation (32
). In this scenario, it is easy to imagine how the incorporation of relatively few mutant monomers would be sufficient to cause global perturbation of microfibrillar assembly and function.
Data presented here suggest that reduced dosage of normal fibrillin-1, rather than the production of mutant protein, is largely responsible for primary failure of matrix deposition of fibrillin-1 and productive microfibrillar assembly in mouse models. A threshold fibrillin-1 dosage dependence for microfibrillar assembly is supported by observations in the literature. First, immunohistochemical analysis of cultured dermal fibroblasts revealed that, despite ongoing transcription and fibrillin-1 translation in low-density cell cultures, microfibrillar deposition occurs as an apparent threshold event that correlates with fibrillin-1 concentration and/or cellular confluence (13
). Second, pulse-chase analyses have demonstrated a consistent and significant time lag between fibrillin-1 synthesis and the onset of matrix deposition (34
). The critical events that regulate deposition are poorly understood, but may include binding of fibrillin-1 monomers to heparin/heparan sulfate glycosaminoglycans at the cell surface (37
). Competition with or direct inhibition of this interaction in cell culture systems resulted in a dramatic reduction of matrix-assembled microfibrils despite normal protein synthesis and secretion, suggesting that saturation of heparan sulfate chain–binding may contribute to the early coordination of macroaggregate formation (37
). The molecular basis for the apparent lack of ability of cysteine-substituted fibrillin-1 to efficiently cooperate with WT protein in early nucleation events remains unknown. In theory, this could relate to the delayed secretion of cysteine-substituted forms of fibrillin-1 seen in pulse-chase analyses (24
) or to the anticipated consequence of such mutations on the local and long-range conformation of the protein (38
The current data suggest that in the presence of a normal complement of WT protein, the production of cysteine-substituted fibrillin-1 becomes less relevant or even irrelevant to the pathogenesis of disease. It is important to avoid generalization of this observation to all mutant forms of fibrillin-1 in the absence of experimental validation. For example, multiple studies have demonstrated or implied that expression of isolated N-terminal peptides has the capacity to interfere with deposition of WT protein in cell culture systems (11
). Given our present data and the availability of improved experimental reagents, it seems advisable to revisit this issue using animal models.
If haploinsufficiency-imposed failure of microfibrillar assembly were the sole driving force in the pathogenesis of MFS, then it would be difficult to explain the wide range of clinical presentations seen in patients with MFS. The greatest variation is seen between families, including those harboring cbEGF-like domain cysteine substitutions. This observation is impossible to reconcile solely on the basis of genetic or environmental modifiers and strongly suggests the existence of additional genotype-modified events in the progression of disease. Susceptibility of established microfibrillar matrices to proteolytic degradation represents a strong candidate. For example, it has been shown that recombinant mutant fibrillin-1 peptides show enhanced proteolytic susceptibility when compared with their WT counterparts (40
) and that the severity of this effect not only depends upon the type of mutation, but also its local context within the extended monomer (41
). Calcium binding to EGF-like domains of fibrillin-1 protects the WT protein from the activity of proteases (42
). Mutations that either directly disrupt the calcium-binding consensus sequence in a given domain or that cause both local and distributed perturbation of protein conformation (e.g., cysteine substitutions) could culminate in significant (albeit variable and genotype-specified) secondary clearance. In this view, most or all patients heterozygous for missense or nonsense mutations in fibrillin-1 may show decreased protein deposition due to a dosage-dependent inhibition of assembly. Resulting clinical severity, however, titrates the inherent susceptibility of the residual microfibrils to subsequent proteolytic degradation. Indeed, the concept that clinical expression of MFS requires a threshold loss of microfibrillar function and correlates in severity with the extent of this effect is easily illustrated by previous experience with an allelic series of Fbn1
-targeted animals (21
). A natural extension of this hypothesis would be that our line expressing the mutant transgene on a normal background has accelerated turnover of microfibrils. The caveat is that given robust microfibrillar assembly, as observed in this line, the net effect never reaches the threshold loss of function needed for clinical expression in the lifetime of the animal. A second prediction from our model is that patients and experimental animals harboring nonsense alleles or heterozygous entire gene deletions should, on average, show milder manifestations of MFS than carriers of heterozygous missense mutations, but that the resultant phenotype could be especially amenable to modification by alternative sources of variation. For example, it was recently shown that a patient missing one entire FBN1
allele due to a genomic deletion had only mild and nonspecific manifestations of MFS in association with near-normal synthesis and deposition of fibrillin-1 (19
). Here it was posited that polymorphic high-expressing WT FBN1
alleles may be protective in this setting. Indeed, analysis of a family segregating a nonsense FBN1
allele showed that, among mutation carriers, only those harboring a high-expressing second allele were clinically protected. Absence of such a protective allele may explain a second patient that we have observed with an entire FBN1
allele deletion who showed striking features of MFS as an infant (H.C. Dietz, unpublished data). Informatively, preliminary analysis has revealed that mice heterozygous for a null Fbn1
allele are clinically and histologically indistinguishable from C1039G heterozygotes (Francesco Ramirez, unpublished data).
In summary, we have shown that the determinants of microfibrillar abundance and function in MFS are complex and perhaps variable in their relative significance between cases. Nevertheless, the novel insight that haploinsufficiency for WT protein can be a significant factor should prompt consideration of additional therapeutic strategies aimed at boosting protein expression. The success of our rescue experiment in which the addition of a normal allele rescued aortic phenotype in C1039G heterozygotes offers the potential that gene-transfer strategies would be productive in patients with MFS. The normal clinical and histologic presentation of mice overexpressing mutant fibrillin-1 on a normal genetic background also suggests that a pharmacologic strategy that achieves nonspecific upregulation of both WT and mutant protein expression would provide a similar benefit. Our mouse models of MFS syndrome provide an ideal system to test these hypotheses.