Mouse models of genetic diseases resulting from mutations in elastic fiber proteins

Matrix Biology 19 Ž2000. 481᎐488

Mini review

Mouse models of genetic diseases resulting from mutations in elastic fiber proteins Harry C. Dietz a,U , Robert P. Mechamb a

Departments of Pediatrics, Medicine, and Molecular Biology and Genetics, and Howard Hughes Medical Institute, Johns Hopkins Uni¨ ersity School of Medicine, Ross 858, 720 Rutland A¨ e., Baltimore, MD 21205, USA b Department of Cell Biology and Physiology, Washington Uni¨ ersity School of Medicine, St. Louis, MO 63110, USA Accepted 10 July 2000

Abstract The inability to study appropriate human tissues at various stages of development has precluded the elaboration of a thorough understanding of the pathogenic mechanisms leading to diseases linked to mutations in genes for elastic fiber proteins. Recently, new insights have been gained by studying mice harboring targeted mutations in the genes that encode fibrillin-1 and elastin. These genes have been linked to Marfan syndrome ŽMFS. and supravalvular aortic stenosis ŽSVAS., respectively. For fibrillin-1, mouse models have revealed that phenotype is determined by the degree of functional impairment. The haploinsufficiency state or the expression of low levels of a product with dominant-negative potential from one allele is associated with mild phenotypes with a predominance of skeletal features. Exuberant expression of a dominant-negative-acting protein leads to the more severe MFS phenotype. Mice harboring targeted deletion of the elastin gene ŽELN. show many of the features of SVAS in humans, including abnormalities in the vascular wall and altered hemodynamics associated with changes in wall compliance. The genetically altered mice suggest that SVAS is predominantly a disease of haploinsufficiency. These studies have underscored the prominent role of the elastic matrix in the morphogenesis and homeostasis of the vessel wall. 䊚 2000 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved.

1. Introduction Elastic fibers are among the largest and most complex structures in the extracellular matrix. In addition to their contribution to the mechanical properties of elastic tissues, elastic fibers and constituents thereof have recently been shown to regulate critical events in development including cellular fate in developing blood vessels and branching morphogenesis in the developing lung. To a large extent, this insight has emerged through comprehensive phenotypic characterization of tissues derived from individuals or animal models with genetically determined perturbations in the biogenesis or structural integrity of elastic U

Corresponding author. Tel.: q1-410-614-0701; fax: q1-410614-2256. E-mail address: [email protected] ŽH.C. Dietz..

fibers. To date, most of the data are observational, and little is known regarding the mechanism by which the elastic matrix acts as a determinant of cellular proliferation, migration, and synthetic profile. Fortunately, correlative studies are giving rise to rational mechanistic hypotheses, which are now subject to testing and validation. Ultimately, these lessons will not only contribute to the care of individuals with MFS and SVAS, but will also apply to common conditions such as atherosclerosis, nonsyndromic aortic aneurysm, and pulmonary emphysema.

2. Mouse model of MFS The vast majority of patients with MFS show a dramatic reduction in the quantity and quality of microfibrils in the extracellular matrix. The tight tem-

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poral and spatial link between the formation of microfibrillar aggregates and subsequent deposition of amorphous elastin prompted speculation that microfibrils represent an elastin precursor or regulate elastin deposition during embryonic development ŽFahrenbach et al., 1966; Greenlee et al., 1966.. In this scenario the deficiency in elastic fiber abundance and architecture that is observed in mature vascular lesions in MFS predominantly manifests a primary failure of elastogenesis. The absence of organized elastic fibers would cause both a primary structural deficiency and a diminished capacity to modulate hemodynamic stress, perhaps predisposing to secondary damage. Since the bulk of elastic tissue is formed in late embryonic and early postnatal development, this model bodes poorly for the development of effective therapies for individuals with MFS. The inability to study human vascular lesions at various stages of development precluded full appreciation of the pathogenetic sequence leading to aneurysm in MFS. New insight has now been provided through the analysis of mice harboring targeted fibrillin-1 Ž Fbn1. alleles. One targeted mutation substituted exons 19᎐25 of Fbn1 with a neomycin resistance cassette ŽNeo r ., resulting in an in-frame deletion ŽPereira et al., 1997.. This allele Žmg⌬ . expresses a centrally truncated monomer that was predicted to have dominant-negative activity. However, due to interference from Neo r, only approximately 5᎐10% of the mutant allele is expressed compared to its wild type counterpart. Heterozygous mg⌬rq mice were born at the expected frequency and showed only occasional and subtle skeletal abnormalities late in life. There were no histopathologic vascular changes and life expectancy was normal. Homozygous mg⌬ animals were also born at the expected frequency and appeared normal. However, they die suddenly prior to weaning. Necropsy revealed aortic dilatation and dissection leading to hemopericardium or hemothorax in all homozygous mutant animals that died naturally ŽPereira et al., 1997.. The aortic media showed disruption of elastic laminae and loss of elastin content with the accumulation of amorphous matrix elements, as seen in mature human lesions. Surprisingly, at the level of histologic resolution, these abnormalities were extremely focal with the bulk of the aorta showing linear, uninterrupted and parallel elastic fibers. Thus, the mg⌬ line documented that minimal residual microfibrillar function is sufficient to support the deposition of extended elastic structures and highlighted the prominent role of fibrillin-1 in elastic fiber homeostasis. Early demise of mg⌬rmg⌬ animals precluded a comprehensive analysis of pathologic changes leading to aneurysm formation. Important new insights stem from the analysis of a second mutant mouse line that

resulted from an aberrant targeting event ŽPereira et al., 1999.. The mgR allele resulted from the insertion of Neo r into intron 18 without any corresponding rearrangement in the coding sequence. This allele is only expressed at a level approximating 15% of normal. However, in contrast to the mg⌬ line, protein derived from the R1 allele is structurally normal. Heterozygous mgRrq animals show no abnormalities throughout life. Homozygous mgRrmgR mice show normal prenatal and perinatal development, but die between 3 and 6 months of age. The longer life-span of these animals allowed the appreciation of dramatic bone overgrowth. Focal calcification of intact elastic laminae was seen as early as 6 weeks of age. Calcified segments became more numerous and coalesced over time. Intimal hyperplasia with excessive and disorganized deposition of matrix components was evident by 9 weeks of age. As a general rule, zones of calcification, intimal hyperplasia, and adventitial inflammation were spatially coincident. Infiltration of mixed inflammatory cells into the media was associated with expression of matrix metalloproteinases ŽMMPs., intense elastolysis, and structural collapse of the vessel wall. Similar changes had not been reported for human vascular lesions. A review of autopsy specimens, however, revealed diffuse calcification and intimal hyperplasia in the aorta and other muscular arteries of young individuals with MFS ŽT. Bunton and H.C.D.; manuscript submitted.. In addition, Takebayashi et al. Ž1973, 1988. had previously reported an odd appearance of fragmented elastic laminae in the media of the aorta and peripheral arteries in patients with MFS. This finding, termed ‘osmiophilic elastolysis’, included the accumulation of an electron-dense and granular material on the surface and within elastic fibers. Although the authors attributed this abnormality to the accumulation of a peculiar breakdown product of elastin, it is clear in retrospect that they were actually observing diffuse calcification of elastic structures. It is also clear that these findings in patients with MFS do not represent normal age-dependent changes. First, these changes were seen in all large arteries that were examined. More importantly, they were seen in children without an apparent correlation between distribution or severity and the age of the individual. It is important to note that calcification and intimal hyperplasia were observed in vessels that are not predisposed to dilatation or dissection in MFS. This suggests that these changes are not sufficient to commit to aneurysm formation. Ultrastructural analysis has begun to define events that initiate destructive changes in the aortic media of fibrillin-1 deficient mice. Elastic laminae connect to adjacent endothelial and smooth muscle cells ŽSMC. through an intermediate structure composed of mi-

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crofibrils ŽDavis, 1993a,b, 1994.. Furthermore, it has been shown that the RGD sequence of fibrillin-1 can support cellular adhesion via integrin ␣ ¨ ␤ 3 ŽPfaff et al., 1996; Sakamoto et al., 1996; D’Arrigo et al., 1998.. These interactions are believed to contribute to the structural integrity of the vessel wall through cell anchorage and to coordinate contractile and elastic tensions ŽDavis, 1993a,b.. Homozygous mgR mice show loss of these connections prior to elastolysis ŽT. Bunton and H.C.D.; manuscript submitted.. Attainment of a threshold loss of physical interactions and hence signals that specify context appears to initiate alteration in the morphology and synthetic program of flanking vascular SMC that are characteristic of a dedifferentiated state, perhaps in an abortive attempt to reconstitute a mature environment. In addition to multiple matrix components, the SMC elaborate mediators of early elastolysis. While the precise nature of these proteases remains unknown, these observations correlate well with the finding of increased immunoreactivity for MMPs at the margins of mature vascular lesions in patients with MFS ŽSegura et al., 1998.. Subsequent breach of the internal or external elastic laminae in fibrillin-1-deficient mice allows infiltration of inflammatory cells into the media, resulting in intense elastolysis that contributes to the structural collapse of the aortic wall.

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that appears equivalent to that achieved by the dominant-negative interaction in patients with classic MFS. Secondary events are required for this deficiency to become clinically manifest. Homozygosity for an allele that expresses low levels of a centrally deleted

3. Pathogenetic model of MFS Existing mouse models and MFS patients have partial but not complete loss of microfibrillar function due to different molecular mechanisms. While it is clear that fibrillin-1 genotype is a determinant of the degree of functional impairment, hemodynamic stress or other environmental factors may promote progressive loss of residual function. Fig. 1 reviews the various genotypes that have been studied in mouse models of MFS. The extent to which these lines recapitulate the proposed molecular mechanisms and observed phenotypic variability in the human condition is quite striking. Homozygosity for a wild-type allele Žqrq . is not associated with the attainment of a threshold loss of microfibrillar function or the development of phenotypic abnormalities. The haploinsufficiency state ŽmgRrq . or the expression of very low levels of a product with dominant-negative potential from one allele Žmg⌬rq . can be associated with mild phenotypes with a predominance of skeletal features. Human correlates include isolated dolichostenomelia in individuals that express mutant protein from one allele that cannot interact with normal fibrillin-1 or MASS phenotype in patients that express very low levels of mutant protein from heterozygous nonsense alleles. Homozygous mgR mice have a loss of function

Fig. 1. Genotypes and pathogenetic mechanisms in mouse models of MFS. Genotypes: q, wild type allele; R, mgR allele; ⌬, mg⌬ allele; Tsk, tight skin allele; Neo, neomycin resistance cassette. Percent figure indicates expression level relative to a single q allele. Parentheses indicate a deletion Žmg⌬ . or insertion ŽTsk .. Matrix cartoon indicates the abundance and character of microfibrils. Each horizontal line capped by a circle represents a fibrillin-1 monomer. A cluster of aligned monomers indicates a microfibril. Threshold column indicates the developmental timing of loss of a threshold level of microfibrillar function as manifested by phenotypic expression. Correlate column indicates the relevant pathogenetic mechanism leading to a similar phenotype in humans. Haploinsufficiency, functional haploinsufficiency; Gain fx., gain-of function; SSc, systemic sclerosis.

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monomer Žmg⌬rmg⌬ . leads to further loss of function and perinatal death due to primary structural failure of cardiovascular tissues. This can be equated to the severe and rapidly progressive neonatal phenotype that is presumed due to alleles with extreme dominant-negative potential. Even further loss of function is seen when the mgR and mg⌬ alleles occur in compound heterozygosity, resulting in prenatal vascular failure. These data suggest that homopolymers of mutant monomers retain more function than heteropolymers and that the proper alignment of selected domains in adjacent monomers is critical for microfibrillar assembly or integrity. Inheritance of two mutant human FBN1 alleles can lead to prenatal fetal loss and is incompatible with extended postnatal viability. The basis for the discordant vascular phenotypes seen in primary and secondary elastin deficiency in SVAS Žsee below. and MFS, respectively, is not fully understood. Microfibrils may have an essential structural role that is independent of their association with elastin. Probable consequences of loss of this activity include failure of cell anchorage and loss of adventitial integrity. Alternatively, there may be a narrow developmental window for the formation of the obstructive changes characteristic of SVAS. Finally, it is possible that microfibrils have nonstructural functions that are critical in the homeostasis of the vessel wall. For example, it has been proposed that microfibrils may be important in the regulation of active TGF-␤. This hypothesis stems from the homology between a repeated 8-cysteine domain in fibrillin-1 and a motif first observed in latency-inducing transforming growth factor ␤-binding proteins ŽLTBPs. ŽGleizes et al., 1996.. TGF-␤ is secreted in a latent form that results from its association with its processed propeptide dimer, called the latency associated peptide ŽLAP. ŽMunger et al., 1997.. This small latent complex binds to LTBPs via disulfide linkage to form the large latent complex ŽKanzaki et al., 1990; Munger et al., 1997.. The extracellular matrix is believed to modulate TGF-␤ activity by binding to and sequestering the large latent complex from cell surface-associated activators including plasmin and integrins ␣ ¨ ␤ 6 and ␣ ¨ ␤ 1 ŽMunger et al., 1997, 1998, 1999.. There is considerable speculation, and some direct evidence, that complexed TGF-␤ interacts with microfibrils ŽGibson et al., 1995; Hyytiainen et al., 1998; Raghunath et al., 1998.. It could, therefore, be envisioned that a genetically determined deficiency of microfibrils would allow the latent complex to be promiscuously activated by neighboring cells, resulting in an increase in the local active concentration of TGF-␤. While mesoderm-derived SMC in the descending aorta are inhibited in proliferation and migration and

have a neutral response in collagen production when exposed to TGF-␤, neural crest-derived cells in the aortic root show increased proliferation and collagen synthesis ŽRosenquist and Beall, 1990; Mii et al., 1993; Thieszen et al., 1996; Topouzis and Majesky, 1996; Gadson et al., 1997.. Further evidence of a regulatory role for microfibrils stems from studies of the tight skin ŽTsk . mouse. Homozygosity for this naturally occurring mutation causes peri-implantation developmental failure. Heterozygosity is associated with a phenotype that includes progressive dermal and visceral fibrosis, emphysema and autoimmunity ŽGreen et al., 1976; Muryoi et al., 1991.. Remarkably, Tsk mice harbor a large genomic duplication within the Fbn1 gene resulting in the tandem repeat of exons 17᎐40 in the mature message ŽSiracusa et al., 1996; Bona et al., 1997.. Subsequent analysis revealed that the transcript derived from the Tsk allele is translated ŽKielty et al., 1998; Saito et al., 1999.. Early studies suggested that mutant monomers homopolymerize but lack the ability to interact with normal fibrillin-1 ŽKielty et al., 1998.. More recent results indicate that the abnormally large proteins co-polymerizes with wild type molecules, thus exerting a limited dominant-negative potential on the resulting aggregate ŽGayraud et al., manuscript in press.. It is interesting to note that the duplication within the mutant protein spans the region of fibrillin-1 that shows greatest homology to the LTBPs ŽSiracusa et al., 1996.. This is also preliminary evidence that TGF-␤ shows increased affinity for the Tsk gene product in an in-vitro binding assay ŽSaito et al., 1999.. The lack of significant overlap between the Tsk and MFS phenotypes suggests a gain-of-function for internally duplicated fibrillin-1 that may perturb TGF-␤ regulation and result in excessive fibrosis. In addition, autoantibodies to fibrillin-1 have been identified in Tsk mice and patients with scleroderma or other related connective tissue disorders ŽMurai et al., 1998; Tan et al., 1999.. The functional significance of this finding remains to be elucidated.

4. Mouse models of SVAS and vascular remodeling Shortly after the linkage of MFS to mutations in the FBN1 gene, the obstructive vascular disease SVAS was linked to mutations in the elastin ŽELN. gene ŽCurran et al., 1993; Ewart et al., 1993; Olson et al., 1993.. Accumulating evidence suggests that the pathomechanism of SVAS is haploinsufficiency, which can result from the excision of one complete copy of the elastin gene Žas occurs in Williams-Beuren Syndrome, WBS. or through functional hemizygosity arising from the production of an unstable mutant mRNA

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transcript or a protein product that is incapable of assembling into a mature fiber Žsee accompanying review.. Recently, Li et al. Ž1998a. generated mice deficient in elastin by deleting exon 1 and 4 kb of the elastin promoter, resulting in a null mutation. The elastin null mice survive gestation but die within 4 days from a subendothelial accumulation of proliferating smc that eventually obliterate the vascular lumen. These lesions were not confined to the large elastic arteries since increased cellularity and reduced luminal diameter were found in systemic and pulmonary arteries of all sizes, including distributing arteries and arterioles. The increased cellularity observed in elastin-deficient vessels resulted from a high proliferation rate of vascular wall SMC. At E17.5, the percentage of cells that stained positive for proliferating-cell nuclear antigen in aortic sections from ELNyry mice was 88% compared to only 35% in sections from ELNqrq mice ŽLi et al., 1998a.. It is not known whether this difference is the consequence of an increase in proliferating ELNyry cells or a failure of cells to exit the cell cycle. A careful comparison of cellular proliferation rates in ELNyry and ELNqrq vessels at earlier time points will help resolve this question. Whatever the answer, the results suggest that the absence of elastin has an effect on cell proliferation. Whether this occurs directly through receptor᎐ligand signaling or indirectly through alterations in vessel compliance and the ability of vascular cells to sense and respond to wall stress is unknown. These findings raise the interesting possibility that disruption of elastin by endothelial injury, thrombosis, or inflammation may contribute to obstructive arterial pathology by altering the proliferation rate of vascular cells at the site of injury. As a model of human disease, mice hemizygous for elastin ŽELNqry . reproduce the loss-of function mutations linked to SVAS. ELNqry mice are identical to ELNqrq mice in gross appearance, behavior, and life expectancy ŽLi et al., 1998b.. Northern analysis of ELNqry vessels reveals ; 50% decrease in ELN mRNA when compared to ELNqrq mice at birth ŽLi et al., 1998b.. Elastic lamellae in ELNqry mice are approximately 50% thinner and desmosine analysis Ždesmosine is a cross-link unique to mature elastin . confirms that total elastin content in the ELNqry aorta is half the level of the ELNqrq vessel. When normalized to total protein, elastin levels are ; 35% lower in the ascending, descending and carotid arteries of ELNqry animals whereas the ratio of collagen to total protein is unchanged in all three vessel types when compared to wild type Žour unpublished results .. Interestingly, histological examination of arterial structure revealed an increased number of lamellar units in both the ascending and descending aorta

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from ELNqry animals ŽLi et al., 1998b.. These changes, apparent at birth, suggest that alterations in the vessel wall were occurring early in development. Although the ELNqry mouse does not exhibit all of the characteristics of human SVAS Žsuch as the development of stenotic lesions., the phenotype has predicted attributes of the human disease that were previously unknown. For example, the structural changes observed in the arterial wall of the ELNqry mouse were present in human aortic tissue from individuals with SVAS. In agreement with the animals studies, there were 2.5 times more lamellar units in human SVAS tissue compared to controls ŽLi et al., 1998b.. In non-syndromic SVAS, functional haploinsufficiency is produced by a spectrum of elastin gene mutations in one of two elastin alleles. Many of these mutations result in premature termination codons that likely produce mRNA products that undergo nonsense-mediated decay, resulting in a functionally inactive, null allele Ždiscussed in detail in the accompanying review.. It is likely, however, that some mRNAs are stable and produce protein products that could potentially act in a dominant-negative fashion to alter elastic fiber assembly. Most of the nonsense and frameshift mutations that lead to isolated SVAS cause the loss of the C-terminal portion of the elastin molecule. This region of the protein is responsible for initiating molecular alignment by interacting with microfibrils, which is the critical step in elastic fiber formation ŽBrown-Augsburger et al., 1996.. To determine how the loss of domains within the C-terminus influence elastin assembly, cDNAs encoding C-terminal truncations of tropoelastin were transfected into cultured cells that made microfibrils but not elastin ŽRobb et al., 1999.. The ability to the expressed elastin to associate with fibrillin-containing microfibrils was then monitored by immunofluorescence microscopy. By deleting consecutive exons beginning with the carboxy-terminus of the molecule, it was found that a hydrophobic sequence encoded by exon 30 is required for elastin association with microfibrils ŽRobert Mecham, unpublished results .. The importance of this observation is that all of the point mutations hitherto associated with to isolated SVAS cause abnormalities in the gene 5⬘ to exon 30. To test whether tropoelastin lacking exon 30 acts as a functional null protein or alters elastic fiber assembly through a dominant-negative mechanism, transgenic mice were generated which express either fulllength bovine tropoelastin or a truncated form typical of the mutated protein that would be produced in isolated SVAS. Using antibodies specific for bovine elastin, it was found that the full-length bovine protein is incorporated successfully with the mouse protein into elastic fibers. The truncated protein, which

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lacked exon 30, was produced at high levels but did not associate with the insoluble matrix. Assembly of the endogenous mouse elastin was normal in these animals, suggesting that the truncated protein did not interfere with assembly and deposition. Although preliminary, these studies suggest that protein products from mutant alleles in SVAS are non-functional; the finding is consistent with the model that SVAS is basically a disease of haploinsufficiency that can result from: Ž1. the loss of an elastin allele; Ž2. mutations that produce an unstable mRNA; or Ž3. production of mutant proteins lacking appropriate assembly domains Žsee Fig. 2..

5. Pathogenetic model of SVAS The decrease in the ratio of elastin to collagen in ELNqry arteries Žsee above. suggests that these vessels will have altered mechanical properties when compared to wild type. Because many studies have shown that embryonic vascular development is highly

sensitive to local hemodynamic forces ŽLangille, 1996., Faury et al. Žmanuscript submitted. determined whether the mechanical properties of conducting vessels in ELNqry mice are altered in ways that might explain the structural changes that occur in the vessel wall Ži.e. increased number of elastic lamellae.. In these studies, the compliance of vessels from wildtype and ELNqry mice was compared using a pressure arteriograph. To this end, vessel segment was cannulated and mounted in an organ bath placed on an inverted microscope. A computerized image analysis system was then used for measurement of alterations in inner ŽID. and outer diameter ŽOD. associated with changes in intraluminal pressure. Determination of vessel distensibility using the arteriograph showed that there were significant differences in compliance between ELNqry and ELNqrq phenotypes. Above 100 mmHg, the ELNqry vessel was stiffer than the ELNqrq vessel, but was more distensible at pressures below 100 mmHg. It was initially reported that the aorta from ELNqrq and ELNqry mice had similar extensibilities at a physiologic pressure of 100 mmHg

Fig. 2. Genotypes and pathogenetic mechanisms in mouse models of SVAS. Accumulating evidence suggests that the pathogenesis of SVAS is haploinsufficiency, which can result from the excision of one complete copy of the elastin gene as occurs in Williams syndrome or through functional hemizygosity arising from the production of either an unstable mutant mRNA transcript or a protein product that is incapable of assembling into a mature fiber. Mice hemizygous for elastin ŽELNqry . reproduce the gene deletion seen in Williams syndrome and exhibit most, but not all, of the characteristics of SVAS in humans. Transgenic mice expressing mutant forms of elastin typical of those found in isolated SVAS have been used to show that protein lacking key sequences in the C-terminal region do no incorporate into elastic fibers. Because these mutant proteins do not interfere with assembly of normal elastin, the mutations result in functional hemizygosity by produce a functionally null protein. The darker area on the end of the gene and protruding from the protein in the in the cartoon represents the critical carboxy-terminal area that is required for normal function.

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ŽLi et al., 1998b.. This subsequent and more refined analysis however, showed that ELNqry mice are hypertensive, with a mean arterial pressure approximately 25 mmHg higher than the wild type animals ŽFaury et al., manuscript submitted.. This means that circumferential wall stress, circumferential wall strain and incremental elasticity modulus at physiological mean blood pressure Žf 100 mmHg in ELNqrq and f 125 mmHg in ELNqry mice. is higher in ELNqry than in ELNqrq animals. In other words at the higher physiological pressure in the ELNqry mice, vessels are stiffer than ELNqrq vessels and are working close to their maximum strain. The higher blood pressure and altered vessel compliance found in the ELNqry animal raises the interesting possibility that the structural changes in the vessel wall occur during development in response to mechanical stress resulting from decreased elastin content and altered wall compliance. Wall stress in the ELNqry artery is different because the elastin content is lower. Because ELNqry SMC cannot make more elastin, they compensate by organizing more cell layers. In this context, hypertension may be an important adaptive response to maintain cardiovascular function. At the higher systemic pressure of the ELNqry mouse, the effective working vascular inner diameter is comparable to that of the wild type animal. If the systolic and diastolic pressures in the ELNqry animal were equivalent to those in wild type mice, the working inner diameter of the vessel would be much smaller and unable to accommodate cardiac output and tissue perfusion. It is important to note that hypertension is often correlated with SVAS in humans, which may explain why human vessels show similar structural changes to those seen in the ELNqry mice. How cells sense and adjust to changes in wall tension during development promises to be one of the more interesting areas of research in vascular biology.

6. Conclusion The study of genetic perturbation of the elastin-microfibrillar array in mice has resulted in an enhanced understanding of multiple disease processes. The events and molecules that mediate the clinical expression of a deficiency in this specialized matrix have yet to be fully defined. It is important to note that selected SMC and connective tissue abnormalities observed in SVAS and MFS are also seen in many common disease processes and are also components of normal aging. It is, therefore, interesting to speculate that an acquired deficiency of cell-elastic matrix connections contributes to all of these processes.

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References Bona, C.A., Murai, C., Casares, S., Kasturi, K., Nishimura, H., Honjo, T., Matsuda, F., 1997. Structure of the mutant fibrillin-1 gene in the tight skin ŽTSK. mouse. DNA Res. 4, 267᎐271. Brown-Augsburger, P.B., Broekelmann, T., Rosenbloom, J., Mecham, R.P., 1996. Functional domains on elastin and MAGP involved in elastic fiber assembly. Biochem. J. 318, 149᎐155. Curran, M.E., Atkinson, D.L., Ewart, A.K., Morris, C.A., Leppert, M.F., Keating, M.T., 1993. The elastin gene is disrupted by a translocation associated with supravalvular aortic-stenosis. Cell 73, 159᎐168. D’Arrigo, C., Burl, S., Withers, A.P., Dobson, H., Black, C., Boxer, M., 1998. TGF-beta1 binding protein-like modules of fibrillin-1 and -2 mediate integrin-dependent cell adhesion. Connect. Tissue. Res. 37, 29᎐51. Davis, E., 1994. Immunolocalization of microfibril and microfibril-associated proteins in the subendothelial matrix of the developing mouse aorta. J. Cell. Sci. 107, 727᎐736. Davis, E.C., 1993a. Endothelial cell connecting filaments anchor endothelial cells to the subjacent elastic lamina in the developing aortic intima of the mouse. Cell. Tissue Res. 272, 211᎐219. Davis, E.C., 1993b. Smooth muscle cell to elastic lamina connections in developing mouse aorta. Role in aortic medial organization. Lab. Invest. 68, 89᎐99. Ewart, A.K., Morris, C.A., Ensing, G.J., Loker, J., Moore, C., Leppert, M., Keating, M., 1993. A human vascular disorder, supravalvular aortic stenosis, maps to chromosome 7. Proc. Natl. Acad. Sci. USA 90, 3226᎐3230. Fahrenbach, W.H., Sandberg, L.B., Cleary, E.G., 1966. Ultrastructural studies on early elastogenesis. Anat. Rec. 155, 563᎐576. Gadson Jr., P.F., Dalton, M.L., Patterson, E., Svoboda, D.D., Hutchinson, L., Schram, D., Rosenquist, T.H., 1997. Differential response of mesoderm- and neural crest-derived smooth muscle to TGF-beta1: regulation of c-myb and alpha1 ŽI. procollagen genes. Exp. Cell. Res. 230, 169᎐180. Gibson, M.A., Hatzinikolas, G., Davis, E.C., Baker, E., Sutherland, G.R., Mecham, R.P., 1995. Bovine latent transforming growth factor beta 1-binding protein 2: molecular cloning, identification of tissue isoforms, and immunolocalization to elastin-associated microfibrils. Mol. Cell. Biol. 15, 6932᎐6942. Gleizes, P.E., Beavis, R.C., Mazzieri, R., Shen, B., Rifkin, D.B., 1996. Identification and characterization of an eight-cysteine repeat of the latent transforming growth factor-beta binding protein-1 that mediates bonding to the latent transforming growth factor-beta1. J. Biol. Chem. 271, 29891᎐29896. Green, M.C., Sweet, H.O., Bunker, L.E., 1976. Tight-skin, a new mutation of the mouse causing excessive growth of connective tissue and skeleton. Am. J. Pathol. 82, 493᎐512. Greenlee, T.K.J., Ross, R., Hartman, J.L., 1966. The fine structure of elastic fibers. J. Cell. Biol. 30, 59᎐71. Hyytiainen, M., Taipale, J., Heldin, C.H., Keski-Oja, J., 1998. Recombinant latent transforming growth factor beta-binding protein 2 assembles to fibroblast extracellular matrix and is susceptible to proteolytic processing and release. J. Biol. Chem. 273, 20669᎐20676. Kanzaki, T., Olofsson, A., Moren, A., Wernstedt, C., Hellman, U., Miyazono, K., Claesson-Welsh, L., Heldin, C.H., 1990. TGF-beta 1 binding protein: a component of the large latent complex of TGF-beta 1 with multiple repeat sequences. Cell 61, 1051᎐1061. Kielty, C.M., Raghunath, M., Siracusa, L.D., Sherratt, M.J., Peters, R., Shuttleworth, C.A., Jimenez, S.A., 1998. The Tight skin mouse: demonstration of mutant fibrillin-1 production and assembly into abnormal microfibrils. J. Cell. Biol. 140, 1159᎐1166. Langille, B.L., 1996. Arterial remodeling: relation to hemodynamics. Can. J. Physiol. Pharmacol. 74, 834᎐841.

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Li, D.Y., Brooke, B., Davis, E.C., Mecham, R.P., Sorensen, L.K., Boak, B.B., Eichwald, E., Keating, M.T., 1998aa. Elastin is an essential determinant of arterial morphogenesis. Nature 393, 276᎐280. Li, D.Y., Faury, G., Taylor, D.G., Davis, E.C., Boyle, W.A., Mecham, R.P., Stenzel, P., Boak, B., Keating, M.T., 1998bb. Novel arterial pathology in mice and humans hemizygous for elastin. J. Clin. Invest. 102, 1783᎐1787. Mii, S., Ware, J.A., Kent, K.C., 1993. Transforming growth factorbeta inhibits human vascular smooth muscle cell growth and migration. Surgery 114, 464᎐470. Munger, J.S., Harpel, J.G., Giancotti, F.G., Rifkin, D.B., 1998. Interactions between growth factors and integrins: latent forms of transforming growth factor-beta are ligands for the integrin alphavbeta1. Mol. Biol. Cell. 9, 2627᎐2638. Munger, J.S., Harpel, J.G., Gleizes, P.E., Mazzieri, R., Nunes, I., Rifkin, D.B., 1997. Latent transforming growth factor-beta: structural features and mechanisms of activation. Kidney Int. 51, 1376᎐1382. Munger, J.S., Huang, X., Kawakatsu, H., Griffiths, M.J., Dalton, S.L., Wu, J., Pittet, J.F., Kaminski, N., Garat, C., Matthay, M.A. et al., 1999. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 96, 319᎐328. Murai, C., Saito, S., Kasturi, K.N., Bona, C.A., 1998. Spontaneous occurrence of anti-fibrillin-1 autoantibodies in tight-skin mice. Autoimmunity 28, 151᎐155. Muryoi, T., Kasturi, K.N., Kafina, M.J., Saitoh, Y., Usuba, O., Perlish, J.S., Fleischmajer, R., Bona, C.A., 1991. Self reactive repertoire of tight skin mouse: immunochemical and molecular characterization of anti-topoisomerase I autoantibodies. Autoimmunity 9, 109᎐117. Olson, T.M., Michels, V.V., Lindor, N.M., Pastores, G.M., Weber, J.L., Schaid, D.J., Driscoll, D.J., Feldt, R.H., Thibodeau, S.N., 1993. Autosomal dominant supravalvular aortic stenosis: localization to chromosome 7. Hum. Mol. Genet. 2, 869᎐873. Pereira, L., Andrikopoulos, K., Tian, J., Lee, S.Y., Keene, D.R., Ono, R., Reinhardt, D.P., Sakai, L.Y., Biery, N.J., Bunton, T. et al., 1997. Targetting of the gene encoding fibrillin-1 recapitulates the vascular aspect of Marfan syndrome. Nat. Genet. 17, 218᎐222. Pereira, L., Lee, S.Y., Gayraud, B., Andrikopoulos, K., Shapiro, S.D., Bunton, T., Biery, N.J., Dietz, H.C., Sakai, L.Y., Ramirez, F., 1999. Pathogenetic sequence for aneurysm revealed in mice underexpresssing fibrillin-1. Proc. Natl. Acad. Sci. USA 96, 3819᎐3823. Pfaff, M., Reinhardt, D.P., Sakai, L.Y., Timpl, R., 1996. Cell adhesion and integrin binding to recombinant human fibrillin-1. FEBS Lett. 384, 247᎐250.

Raghunath, M., Unsold, C., Kubitscheck, U., Bruckner-Tuderman, L., Peters, R., Meuli, M., 1998. The cutaneous microfibrillar apparatus contains latent transforming growth factor-beta binding protein-1 ŽLTBP-1. and is a repository for latent TGF-beta1. J. Invest. Dermatol. 111, 559᎐564. Robb, B.W., Wachi, H., Schaub, T., Mecham, R.P., Davis, E.C., 1999. Characterization of an in vitro model of elastic fiber assembly. Mol. Biol. Cell 10, 3595᎐3605. Rosenquist, T.H., Beall, A.C., 1990. Elastogenic cells in the developing cardiovascular system. Smooth muscle, nonmuscle, and cardiac neural crest. Ann. NY Acad. Sci. 588, 106᎐119. Saito, S., Nishimura, H., Brumeanu, T.D., Casares, S., Stan, A.C., Honjo, T., Bona, C.A., 1999. Characterization of mutated protein encoded by partially duplicated fibrillin-1 gene in tight skin ŽTSK. mice. Mol. Immunol. 36, 169᎐176. Sakamoto, H., Broekelmann, T., Cheresh, D.A., Ramirez, F., Rosenbloom, J., Mecham, R.P., 1996. Cell-type specific recognition of RGD- and non-RGD-containing cell binding domains in fibrillin-1. J. Biol. Chem. 271, 4916᎐4922. Segura, A.M., Luna, R.E., Horiba, K., Stetler-Stevenson, W., McAllister, H.A.J., Willerson, J.T., and Ferrans, V.J. 1998. Immunohistochemistry of matrix metalloproteinases and their inhibitors in thoracic aortic aneurysms and aortic valves of patients with Marfan’s syndrome, Circulation 98, II331᎐337; discussion II337᎐338. Siracusa, L.D., McGrath, R., Ma, Q., Moskow, J.J., Manne, J., Christner, P.J., Buchberg, A.M., Jimenez, S.A., 1996. A tandem duplication within the fibrillin 1 gene is associated with the mouse tight skin mutation. Genome Res. 6, 300᎐313. Takebayashi, S., Kubota, I., Takagi, T., 1973. Ultrastructural and histochemical studies of vascular lesions in Marfan’s syndrome, with report of 4 autopsy cases. Acta Pathol. Jpn. 23, 847᎐866. Takebayashi, S., Taguchi, T., Kawamura, K., Sakata, N., 1988. Osmiophilic elastolysis of peripheral organ arteries in patients with Marfan’s syndrome. Acta Pathol. Jpn. 38, 1433᎐1443. Tan, F.K., Arnett, F.C., Antohi, S., Saito, S., Mirarchi, A., Spiera, H., Sasaki, T., Shoichi, O., Takeuchi, K., Pandy, J.P. et al., 1999. Autoantibodies to the extracellular matrix microfibrillar protein, fibrillin-1, in patients with scleroderma and other connective tissue diseases. J. Immunol. 163, 1066᎐1072. Thieszen, S.L., Dalton, M., Gadson, P.F., Patterson, E., Rosenquist, T.H., 1996. Embryonic lineage of vascular smooth muscle cells determines responses to collagen matrices and integrin receptor expression. Exp. Cell. Res. 227, 135᎐145. Topouzis, S., Majesky, M.W., 1996. Smooth muscle lineage diversity in the chick embryo. Two types of aortic smooth muscle cell differ in growth and receptor-mediated transcriptional responses to transforming growth factor-beta. Dev. Biol. 178, 430᎐445.