Characterization of an In Vitro Model of Elastic Fiber Assembly

Molecular and Cellular Biology

Richardson³

et al. 2007

Self Cite

Elastic fibers contribute to the structural support of tissues and to the regulation of cellular behavior. Mice deficient for the fibulin-5 gene (fbln5 ؊/؊ ) were used to further elucidate the molecular mechanism of elastic fiber assembly. Major elastic fiber components were present in the skin of fbln5 ؊/؊ mice despite a dramatic reduction of mature elastic fibers. We found that fibulin-5 preferentially bound the monomeric form of elastin through N-terminal and C-terminal elastin-binding regions and to a preexisting matrix scaffold through calcium-binding epidermal growth factor (EGF)-like (CB-EGF) domains. We further showed that adenovirusmediated gene transfer of fbln5 was sufficient to regenerate elastic fibers and increase elastic fiber-cell connections in vivo. A mutant fibulin-5 lacking the first 28 amino acids of the first CB-EGF domain, however, was unable to rescue elastic fiber defects. Fibulin-5 thus serves as an adaptor molecule between monomeric elastin and the matrix scaffold to aid in elastic fiber assembly. These results also support the potential use of fibulin-5 as a therapeutic agent for the treatment of elastinopathies.

Section: Vol 27 2007 Elastic Fiber Synthesis and Fibulin-5 1087mentioning

confidence: 99%

Molecular Analysis of Fibulin-5 Function during De Novo Synthesis of Elastic Fibers

Zheng¹,

Molecular and Cellular Biology

Richardson³

et al. 2007

Self Cite

“…Similar results were obtained with fbln-5-infected PAC-1 pulmonary vascular SMCs that express a large amount of endogenous fbln-5 (data not shown). It is unlikely that mature elastic fibers were assembled during the relatively short culture period (6-48 h) or under nonconfluent conditions that we used in our cell migration͞proliferation assays (24). We propose that the inhibitory effect of fbln-5 is most likely mediated by an elastic fiber-independent mechanism.…”

Section: Suppression Of Proliferation and Migration Phenotypes Of Fblmentioning

confidence: 90%

Altered vascular remodeling in fibulin-5-deficient mice reveals a role of fibulin-5 in smooth muscle cell proliferation and migration

Spencer

Hacker

Proc. Natl. Acad. Sci. U.S.A.

et al. 2005

Self Cite

107

101

Fibulin (fbln)-5 is an elastin-binding protein required for assembly and organization of elastic fibers. To examine the potential role of fbln-5 in vascular remodeling and neointima formation, we induced vascular injury by carotid artery ligation in fbln-5 ؊/؊ mice. Mutant mice displayed an exaggerated vascular remodeling response that was accompanied by severe neointima formation with thickened adventitia. These abnormalities were not observed in elastin ؉/؊ mice that exhibited a comparable reduction of vessel extensibility to fbln-5 ؊/؊ mice. Thus, the severe remodeling response could not be attributed to altered extensibility of the vessel wall alone. Vascular smooth muscle cells cultured from fbln-5 ؊/؊ mice displayed enhanced proliferative and migratory responses to mitogenic stimulation relative to wild-type cells, and these responses were inhibited by overexpression of fbln-5. These findings demonstrate the importance of the elastic laminae in vascular injury, and reveal an unexpected role of fbln-5 as an inhibitor of vascular smooth muscle cell proliferation and migration.elastic fibers ͉ extracellular matrix ͉ neointima ͉ elastin V ascular obstructive abnormalities, such as atherosclerosis and restenosis after percutaneous coronary intervention, are triggered by damage to the vessel wall, initiating a series of biological responses, including up-regulation of adhesion molecules, recruitment of inflammatory cells, secretion of cytokines, and activation of smooth muscle cells (SMCs). Activated SMCs secrete growth factors, extracellular matrix proteins, and matrix proteases, thereby altering the microenvironment of the injured vessel wall. Much attention has focused on the signaling pathways responsible for the proliferation and migration of SMCs associated with vascular obstruction, but relatively little is known of the potential contributions of extracellular matrix proteins to these processes (reviewed in ref. 1).Structural changes of the vessel wall induced by mechanical force or enzymatic digestion also influence the development of vascular obstructive disease (2, 3). The elastic lamina plays a critical role in maintaining the integrity of the vessel wall. Rupture of the external elastic lamina (EEL) is a more potent stimulus for neointima formation than injury involving the internal elastic lamina (IEL) alone after coronary artery stent-induced injury (4). In addition, exposure of the adventitia to the blood lumen by disruption of the EEL during percutaneous angioplasty increases the activation of adventitial myofibroblasts, leading to adventitial fibrosis that eventually constricts the vessel wall (5).The perception of the elastic lamina (or elastic fibers) as the sole structural component of the vessel wall was recently challenged by the view that elastic fibers actively modulate intercellular signaling. Elastin (eln), a major component of the elastic fibers in the arterial wall, inhibits proliferation of SMCs (6). Eln is secreted as a tropoelastin monomer and subsequently undergoes cross-linking t...

“…Transfection Constructs-The transfection constructs used for this study were derived from a full-length bovine cDNA that has been previously characterized (17). It carries a deletion of exons 13 and 14.…”

Section: Methodsmentioning

confidence: 99%

“…PE cells are a cell line derived from the pigmented epithelial cells in the ciliary body of the bovine eye (20). They are known to lay down an elaborate fibrillar matrix composed of fibrillin-1, fibrillin-2, and MAGP1 (microfibril-associated glycoprotein-1), but do not produce endogenous elastin (17). Because these cells produce all of the components necessary to form the scaffolding for elastic matrices, but not tropoelastin itself, they provide a useful system for studying the early stages of elastic fiber assembly.…”

Section: The Presence Of the Tropoelastin C Terminus Is Required For mentioning

confidence: 99%

Domains in Tropoelastin That Mediate Elastin Depositionin Vitro and in Vivo

Kozel

Wachi

Journal of Biological Chemistry

et al. 2003

Self Cite

128

138

Elastic fiber assembly is a complicated process involving multiple different proteins and enzyme activities. However, the specific protein-protein interactions that facilitate elastin polymerization have not been defined. To identify domains in the tropoelastin molecule important for the assembly process, we utilized an in vitro assembly model to map sequences within tropoelastin that facilitate its association with fibrillin-containing microfibrils in the extracellular matrix. Our results show that an essential assembly domain is located in the C-terminal region of the molecule, encoded by exons 29 -36. Fine mapping studies using an exon deletion strategy and synthetic peptides identified the hydrophobic sequence in exon 30 as a major functional element in this region and suggested that the assembly process is driven by the propensity of this sequence to form ␤-sheet structure. Tropoelastin molecules lacking the C-terminal assembly domain expressed as transgenes in mice did not assemble nor did they interfere with assembly of full-length normal mouse elastin. In addition to providing important information about elastin assembly in general, the results of this study suggest how removal or alteration of the C terminus through stop or frameshift mutations might contribute to the elastin-related diseases supravalvular aortic stenosis and cutis laxa.The inherent complexity of the elastic fiber, combined with the unique physical properties of its component proteins, has made understanding elastin structure and assembly one of the most difficult problems in matrix biology. Although technical advances in cell and molecular biology have given us new information about elastin gene expression and elastin synthesis, surprisingly little is known about how the elastic fiber is assembled at the molecular level. The major component of the mature fiber is a covalently cross-linked polymer of the protein elastin. Elastin is secreted from the cell as a soluble monomer called tropoelastin. In the extracellular space, lysine residues within tropoelastin are specifically modified to form covalent cross-links between tropoelastin chains. This cross-linked polymer has a high degree of reversible distensibility, including the ability to deform to large extensions with small forces.The tropoelastin molecule is characterized by a series of tandem repeats, each including a lysine-containing cross-linking region followed by a hydrophobic motif (1). Cross-linking is initiated by the extracellular enzyme(s) lysyl oxidase, which catalyzes the oxidative deamination of lysyl ⑀-amino groups. Under normal conditions, the cross-linking process is extremely efficient, with all but ϳ5 of the ϳ40 lysine residues in tropoelastin participating in covalent linkages that form the functional polymer. How the cross-linking sites within the monomer are aligned prior to cross-linking is unclear. It has long been assumed that microfibrils provide a scaffold or template for elastin assembly by binding and aligning tropoelastin monomers so that lysine-containin...