Fibrillin microfibrils: multipurpose extracellular networks in organismal physiology

Francesco Ramirez1, Lynn Y. Sakai2, Harry C. Dietz3 and Daniel B. Rifkin4

1 Laboratory of Genetics and Organogenesis, Hospital for Special Surgery, and Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York 10021
2 Department of Biochemistry and Molecular Biology, Shriners Hospital for Children, Oregon Health and Science University, Portland, Oregon 97201
3 Institute of Genetic Medicine, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
4 Department of Cell Biology, New York University School of Medicine, New York, New York 10016


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 GRANTS
 REFERENCES
 
Organismal physiology depends significantly on the proper assembly of extracellular matrix (ECM) macroaggregates that impart structural integrity to the connective tissue. Recent genetic studies in mice have unraveled unsuspected new functions of architectural matrix components in regulating signaling events that modulate patterning, morphogenesis, and growth of several organ systems. As a result, a new paradigm has emerged whereby tissue-specific organization of the ECM dictates not only the physical properties of the connective tissue, but also the ability of the matrix to direct a broad spectrum of cellular activities through the regulation of growth factor signaling. These observations pave the way to novel therapeutic approaches aimed at counteracting the deleterious consequences of perturbations of connective tissue homeostasis.

elastic fiber; growth factor signaling; Marfan syndrome


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 GRANTS
 REFERENCES
 
THE EXTRACELLULAR MATRIX (ECM) is a multifunctional determinant of organism physiology. The ECM provides the architectural framework that establishes the organizational and physical properties of different tissues; it supplies structural signals that impart positional information to the surrounding cells; and it regulates the spatiotemporal bioavailability of growth factors that modulate a variety of developmental and homeostatic events. Traditionally, each of these roles has been ascribed to specific families of ECM components. For example, elastic and collagenous networks have been viewed as serving primarily an architectural function in the connective tissue, and proteoglycans have been viewed as having both structural and instructive roles. However, recent studies using genetically targeted mice have implicated elastic networks in the control of patterning and morphogenetic programs as well. In this article we will review these studies along with the biological and clinical implications of the new findings with respect to the control of growth factor signaling.

Elastogenesis in Normal and Diseased Conditions
Elastic fibers are made of an insoluble amorphous core of cross-linked elastin and a surrounding lattice of microfibrils (10). Microfibrils are heterogeneous in composition and can also form macroaggregates devoid of elastin. Integral and associated components of the microfibrils include the superfamily of fibrillins and latent TGFß-binding proteins (LTBPs), as well as the structurally unique families of fibulins, emilins, microfibrillar-associated glycoproteins (MAGPs), and microfibril-associated proteins (MFAPs). Elastogenesis begins at midgestation and proceeds until completion of postnatal growth, and it involves the organized deposition by mesenchymal cells of several macromolecules that self-assemble into microfibrils and elastic fibers (10).

Fibrillins-1 and -2 (FBN1 and FBN2) are the major building blocks of extracellular microfibrils and the defective gene products in Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA), respectively (4, 10). Fibrillins are synthesized prior to tropoelastin deposition and polymerize into a characteristic "beads-on-a-string" structure, which gives rise to the microfibril lattice by lateral association of the polymers and probably, by inclusion of other structural components (Fig. 1). Microfibrils and elastic fibers are organized into tissue-specific architectures that reflect the mechanical demands of individual organ systems (10, 4). In the skin, microfibrils extend from the basement membrane of the dermal/epidermal junction into the reticular dermis, where they run parallel to the epidermis together with elastic fibers. This loosely organized network of microfibrils and elastic fibers confers pliability to the skin. In the eye, parallel bundles of microfibrils anchor the lens and adjust its thickness by conducting tension from the ciliary body. In the aorta, microfibrils associate with elastin in the tunica media to form the concentric lamellae that separate individual vascular smooth muscle cell (VSMC) layers and which confer elasticity to the aortic wall. Additionally, microfibrils devoid of elastin stabilize the tissue by connecting lamellar rings to one another, to VSMC, and to the subendothelial basement membrane.



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Fig. 1. Schematic representation of the major steps underlying the assembly of microfibrils and elastic fibers (top) with three examples of tissue-specific networks (bottom).

 
Recent genetic studies have revealed that elastogenesis is a more complex process than previously thought. The finding that fibulin-5 (FBLN5)-null mice exhibit phenotypic abnormalities resulting from disrupted elastogenesis has implicated this molecule in the early assembly of the elastic fiber (16, 21). Specifically, FBLN5 is believed to regulate assembly by providing the molecular bridge between tropoelastin in the pericellular space and integrins on the cell surface. The phenotype of the FBLN5-null mouse includes loose skin, pulmonary emphysema, vascular malformations, and progressive narrowing of the proximal aorta. These manifestations closely replicate the severe recessive form of cutis laxa, a human connective tissue disorder that is similarly caused by FBLN5 mutations (14).

The enzyme lysyl oxidase (LOX) mediates the extracellular conversion of tropoelastin molecules into the insoluble elastic meshwork (10). Concordant with this fact, genetic disruption of LOX activity in mice leads to reduced elastin cross-linking and fragmented elastic fiber architecture; as a result, the mutant mice manifest severe cardiovascular instability, in the form of ruptured arterial aneurysm, and perinatal death (6, 15). There are four LOX-like proteins (LOXL1–4) in the mammalian genome, and at least one of them (LOXL1) has been implicated in elastogenesis (13). Mice lacking the LOXL1 gene in fact develop abnormalities in several elastic tissues as a result of abnormal accumulation of tropoelastin. The manifestations include enlarged air space of the lung, loose skin, vascular defects, and pelvic organ prolapse. Biochemical and immunolocalization data indicate that interaction between LOXL1 and FBLN5 juxtaposes the enzyme and the tropoelastin substrate for efficient and spatially restricted polymer formation (13). That elastic fiber defects generally appear later in LOXL1- than FBLN5-null mice argues for a predominant role of the former gene product in adult elastic tissue homeostasis.

Mice without elastin (ELN) recapitulate the phenotype of ELN haploinsufficiency in human supravalvular aortic stenosis (SVAS). These features include abnormalities in the aortic wall and altered hemodynamics associated with changes in wall compliance (5, 11). ELN-null mice survive gestation but die postnatally from subendothelial accumulation of VSMC that ultimately occlude the vascular lumen. As in human SVAS, the vessel wall of ELN haploinsufficient mice displays an increased number of elastic lamellae, suggesting that the deficiency affects normal vascular development (12). The vascular phenotypes of the ELN mutant mice are accounted for by defective cell-matrix interactions that normally regulate VSMC migration, induce a quiescent phenotype, and inhibit proliferation (9).

Microfibrils and Elastogenesis
Genetically targeted strains of mice have been created that replicate the neonatal lethal or clinically progressive forms of MFS. Mice homozygous for a severely hypomorphic Fbn1 allele (mgR) die between 9 mo and 1 yr of age, due to aortic dissection and rupture, and replicate the clinically progressive form of MFS (18). That the elastic fibers of these mutant mice show normal morphology at birth suggested that aneurysm progression is largely driven by secondary cellular events (3, 18). Specifically, the first detectable alteration in mgR homozygotes is loss of the connecting filaments that normally serve as a structural interface between elastic lamellae and neighboring VSMC. Next, VSMC begin secreting multiple matrix components as well as matrix-degrading enzymes in an abortive attempt to remodel a defective tissue. The net result of the defective remodeling is the initiation of local elastolysis, which is temporally correlated with elastic fiber calcification and immediately followed by inflammatory infiltration. The latter process begins at the adventitial surface and progresses into the media and is temporally and spatially correlated with structural collapse of the vessel wall, aneurysm formation, and dissection. These observations imply that fibrillin-1 microfibrils serve an essential role in elastic fiber homeostasis during extra-uterine life. As such, they offer the potential for therapeutic intervention aimed at blunting the elastolysis that is initiated by VSMC and exacerbated by vessel wall inflammation.

Mice harboring a heterozygous cysteine substitution (C1039G allele) in a calcium-binding EGF motif of fibrillin-1 or harboring only one functional FBN1 gene show some of the above abnormalities but do not progress to the point of dissection and death (19; F. Ramirez, unpublished observations). Homozygous C1039G mutant mice and FBN1-null mice, on the other hand, complete embryonic development and die soon after birth due to failure of the vessel wall without antecedent calcification or inflammation (19; F. Ramirez, unpublished observations). Together, these data equate the consequences of qualitative and quantitative mutations in MFS pathogenesis; indicate that the relative abundance of functionally competent fibrillin-rich microfibrils determines the clinical severity of MFS; and demonstrate that elastogenesis proceeds despite severe quantitative and/or qualitative deficits in fibrillin-1.

Microfibrils and Growth Factor Signaling
FBN1-deficient mice display diffuse distal air space enlargement by 7–9 days of age in the absence of tissue destruction or inflammation (17, 1). This phenotype, which resembles the emphysema-like phenotype of some MFS patients, is associated with dysregulated TGFß signaling. TGFßs are multipotent cytokines that are synthesized as inactive precursor molecules containing an NH 2-terminal prodomain termed latency associated peptide (LAP) (20). LAP remains associated with TGFß (constituting the small latent complex) and ultimately becomes covalently linked to a latent TGFß binding protein (LTBP)-1, -3, or -4 to form the large latent complex. It is currently believed that LTBP binding facilitates growth factor secretion. The LTBPs are structurally related to the fibrillins and participate in TGFß regulation, in part by targeting TGFß complexes to the ECM (7). Hence, morphogenetic abnormalities in the microfibril-deficient state may result in incorrect latent complex sequestration and consequently, lead to excessive activation of and signaling by the growth factor. The demonstration that LTBPs bind directly to fibrillin-1 and that antagonism with TGFß-neutralizing antibody rescues lung morphogenesis is consistent with this model (1, 2).

Additional support for the involvement of microfibrils in regulating growth factor signaling derives from the analysis of FBN2-null mice (2). Like CCA patients, these mutant mice display transient contractures of small and large joints; they also exhibit a limb patterning defect in the form of bilateral syndactyly. The defect correlates with altered matrix assembly in the developing autopod and perturbed BMP signaling. The latter correlation is based on the finding that the combination of haploinsufficient FBN2 and BMP7 mutations, which are by themselves phenotypically silent, result in impaired digit formation in the absence of additional manifestation. Hence, the tissue-specific architecture of extracellular microfibrils appears to play a critical role in determining growth factor bioavailability.

Biological and Clinical Considerations
The above results have provided a conceptual framework to investigate ill-defined biological and pathological interactions between the architectural matrix and growth factor signaling (20). Although still in its infancy, this new model of elastic matrix physiology has already provided testable hypotheses to explain how the specificity of growth factor signaling may be compartmentalized within the extracellular space, and how its dysregulation may underlie the clinical variability usually observed in heritable disorders of the connective tissue.

Growth factors are one of the major classes of mediators controlling cellular responses (20). They are normally secreted by cells within the responding tissue and act over relatively restricted distances; sometimes only a few cell diameters. Because these effector molecules have such potent actions and their receptors are widely distributed, there is a need to control growth factor bioavailability in order to ensure proper spatiotemporal signaling, as well as to achieve a sufficient local concentration of the effector molecules (20). Tissue-specific organization of architectural macroaggregates, like microfibrils, properly position latent signals within an appropriate context that cells can effectively translate into productive responses. As such, these extracellular networks serve as interactive information highways with directional sign posts and embedded signals, in addition to being structural scaffolds imparting tissue integrity (Fig. 2). It follows that mutations perturbing the normal architecture of the ECM can have a wide range of deleterious consequences on tissue integrity and function, as well as organ formation and growth and tissue homeostasis. This last postulate in turn implies that additional and previously unsuspected biological targets may exist, which can be exploited with novel therapies that counteract the pathological progression of heritable and acquired diseases of the connective tissue.



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Fig. 2. A schematic model illustrating the instructive properties of the extracellular microfibrils (gray straight bars) and fibrillin polymers (green beaded strings). They include associating with LTBPs to sequester latent TGF-ß complexes (yellow curved bar); interacting with BMP molecules (red butterflies) that may in turn interact with their receptors or be released from the matrix by proteases; and providing positional information to the surrounding cells through integrin signals or other molecular cues (purple globule) scattered within the polymers.

 

    GRANTS
 TOP
 ABSTRACT
 INTRODUCTION
 GRANTS
 REFERENCES
 
We are part of the Consortium for Translational Research in Marfan Syndrome, funded by National Institutes of Health (NIH) Grant AR-49698; previous work described in this review from the our laboratories was supported by National Institutes of Health Grants AR-42044 (to F. Ramirez), AR-46811 (to L. Y. Sakai), AR-41135 (to H. C. Dietz), and CA-34282, CA-78422, and DE-13742 (to D. B. Rifkin). Additional support was received from the St. Giles Foundation (to F. Ramirez), the Shriners Hospital for Children (to L. Y. Sakai), and the Smilow and National Marfan Foundations (to H. C. Dietz).


    ACKNOWLEDGMENTS
 
We thank K. Johnson for typing the manuscript.

A. W. Cowley, Jr., served as the review editor for this manuscript submitted by Editor F. Ramirez.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: F. Ramirez, Hospital for Special Surgery, 535 East 70th St., Caspary Bldg. 7th Floor, New York, NY 10021 (E-mail: ramirezf{at}hss.edu).


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 GRANTS
 REFERENCES
 

  1. Annes JP, Munger JS, and Rifkin DB. Making sense of latent TGFß activation. J Cell Sci 116: 217–224, 2003.[Abstract/Free Full Text]
  2. Arteaga-Solis E, Gayraud B, Lee SY, Shum L, Sakai L, and Ramirez F. Regulation of limb patterning by extracellular microfibrils. J Cell Biol 154: 275–281, 2001.[Abstract/Free Full Text]
  3. Bunton T, Jensen Biery N, Gayraud B, Ramirez F and Dietz HC. Phenotypic modulation of vascular smooth muscle cells contributes to elastolysis in a mouse model of Marfan syndrome. Circ Res 88: 37–43, 2001.[Abstract/Free Full Text]
  4. Charbonneau NL, Ono RN, Corson GM, Keene DR, and Sakai LY. Fine tuning of growth factor signals depends on fibrillin microfibril networks. Birth Defects Res Part C Embryo Today 72: 37–50, 2004.[CrossRef][Medline]
  5. Curran M, Atkinson D, Ewart A, Morris C, Leppert M, and Keating MT. The elastin gene is disrupted by a translocation associated with supravalvular aortic stenosis. Cell 73: 159–168, 1993.[ISI][Medline]
  6. Hornstra IK, Birge S, Starcher B, Bailey AJ, Mecham RP, and Shapiro SD. Lysyl oxidase is required for vascular and diaphragmatic development in mice. J Biol Chem 278:14387–14393, 2003.
  7. Isogai Z, Ono RN, Ushiro S, Keene DR, Chen Y, Mazzieri R, Charbonneau NL, Reinhardt DP, Rifkin DB, and Sakai LY. Latent transforming growth factor ß-binding protein 1 interacts with fibrillin and is a microfibril-associated protein. J Biol Chem 278: 2750–2757, 2003.[Abstract/Free Full Text]
  8. Judge DP, Biery NJ, Keene DR, Geubtner J, Myers L, Huso D, Sakai LY, and Dietz HC. Evidence for haploinsufficiency-driven dominant-negative mechanism in the pathogenesis of Marfan syndrome. J Clin Invest 114: 172–181, 2004.[Abstract/Free Full Text]
  9. Karnik SK, Brooke BS, Bayes-Genis A, Sorensen L, Wythe JD, Schwartz RS, Keating MT, and Li DYA. Critical role for elastin signaling in vascular morphogenesis and disease. Development 130: 411–423, 2002.[CrossRef][ISI]
  10. Kielty CM, Sherratt MJ, and Shuttleworth CA. Elastic fibres. J Cell Sci 115: 2817–2828, 2002.[Abstract/Free Full Text]
  11. Li DY, Brooke B, Davis EC, Mecham RP, Sorensen LK, Boak BB, Eichwald E, and Keating MT. Elastin is an essential determinant of arterial morphogenesis. Nature 393: 276–280, 1998.[CrossRef][ISI][Medline]
  12. Li DY, Faury G, Taylor DG, Davis EC, Boyle WA, Mecham RP, Stenzel P, Boak B, and Keating MT. Novel arterial pathology in mice and humans hemizygous for elastin. J Clin Invest 102: 1783–1787, 1998.[Abstract/Free Full Text]
  13. Liu X, Zhao Y, Gao J, Pawlyk B, Starcher B, Spencer JA, Yanagisawa H, Zuo J, and Li T. Elastic fiber homeostasis requires lysyl oxidase-like 1 protein. Nat Genet 36: 178–182, 2004.[CrossRef][ISI][Medline]
  14. Loeys B, Van Maldergem L, Mortier G, Coucke P, Gernier S, and Naeyaert JM. Genetic heterogeneity of cutis laxa: a heterozygous tandem duplication within the fibulin-5 (FBLN5) gene. Hum Mol Genet 11: 2113–2118, 2002.[Abstract/Free Full Text]
  15. Maki JM, Rasanen J, Tikkanen H, Sormunen R, Makikallio K, Kivirikko KI, and Soininen R. Inactivation of the lysyl oxidase gene Lox leads to aortic aneurysms, cardiovascular dysfunction, and perinatal death in mice. Circulation 106: 2503–2509, 2002.[Abstract/Free Full Text]
  16. Nakamura T, Lozano PR, Ikeda Y, Iwanaga Y, Hinek A, Minamisawa S, Cheng CF, Kobuke K, Dalton N, Takada Y, Tashiro K, Ross J Jr, Honjo T, and Chien KR. Fibulin-5/DANCE is essential for elastogenesis in vivo. Nature 415: 171–175, 2002.[CrossRef][ISI][Medline]
  17. Neptune ER, Frischmeyer PA, Arking DE, Myers L, Bunton TE, Gayraud B, Ramirez F, Sakai LY, and Dietz HC. Dysregulation of TGF-ß activation contributes to pathogenesis in Marfan syndrome. Nat Genet 33: 407–411, 2003.[CrossRef][ISI][Medline]
  18. Pereira L, Lee SY, Gayraud B, Andrikopoulos K, Shapiro SD, Bunton T, Jensen Biery N, Dietz HC, Sakai LY, and Ramirez F. Pathogenetic sequence for aneurysm revealed in mice underexpressing fibrillin-1. Proc Natl Acad Sci USA 96: 3819–3823, 1999.[Abstract/Free Full Text]
  19. Pereira L, Andrikopoulos K, Tian J, Lee SY, Keene DR, Ono R, Reinhardt DP, Sakai LY, Jensen-Biery N, Bunton T, Dietz HC, and Ramirez F. Targeting of the gene coding fibrillin-1 recapitulates the vascular phenotype of Marfan syndrome in the mouse. Nat Genet 17: 218–222, 1997.[ISI][Medline]
  20. Ramirez F and Rifkin DB. Cell signaling events: a view from the matrix. Matrix Biol 22: 101–107, 2003.[CrossRef][ISI][Medline]
  21. Yanagisawa H, Davis EC, Starcher BC, Ouchi T, Yanagisawa M, Richardson JA, and Olson EN. Fibulin-5 is an elastin-binding protein essential for elastic fiber development in vivo. Nature 415: 168–171, 2002.[CrossRef][ISI][Medline]