Impaired elastic-fiber assembly by fibroblasts from patients with either Morquio B disease or infantile GM1-gangliosidosis is linked to deficiency in the 67-kD spliced variant of beta-galactosidase

We have previously shown that intracellular trafficking and extracellular assembly of tropoelastin into elastic fibers is facilitated by the 67-kD elastin-binding protein identical to an enzymatically inactive, alternatively spliced variant of beta-galactosidase (S-Gal). In the present study, we investigated elastic-fiber assembly in cultures of dermal fibroblasts from patients with either Morquio B disease or GM1-gangliosidosis who bore different mutations of the beta-galactosidase gene. We found that fibroblasts taken from patients with an adult form of GM1-gangliosidosis and from patients with an infantile form, carrying a missense mutations in the beta-galactosidase gene-mutations that caused deficiency in lysosomal beta-galactosidase but not in S-Gal-assembled normal elastic fibers. In contrast, fibroblasts from two cases of infantile GM1-gangliosidosis that bear nonsense mutations of the beta-galactosidase gene, as well as fibroblasts from four patients with Morquio B who had mutations causing deficiency in both forms of beta-galactosidase, did not assemble elastic fibers. We also demonstrated that S-Gal-deficient fibroblasts from patients with either GM1-gangliosidosis or Morquio B can acquire the S-Gal protein, produced by coculturing of Chinese hamster ovary cells permanently transected with S-Gal cDNA, resulting in improved deposition of elastic fibers. The present study provides a novel and natural model validating functional roles of S-Gal in elastogenesis and elucidates an association between impaired elastogenesis and the development of connective-tissue disorders in patients with Morquio B disease and in patients with an infantile form of GM1-gangliosidosis.     

Decreased elastin deposition and high proliferation of fibroblasts from Costello syndrome are related to functional deficiency in the 67-kD elastin-binding protein

Costello syndrome is characterized by mental retardation, loose skin, coarse face, skeletal deformations, cardiomyopathy, and predisposition to numerous malignancies. The genetic origin of Costello syndrome has not yet been defined. Using immunohistochemistry and metabolic labeling with [3H]-valine, we have established that cultured skin fibroblasts obtained from patients with Costello syndrome did not assemble elastic fibers, despite an adequate synthesis of tropoelastin and normal deposition of the microfibrillar scaffold. We found that impaired production of elastic fibers by these fibroblasts is associated with a functional deficiency of the 67-kD elastin-binding protein (EBP), which is normally required to chaperone tropoelastin through the secretory pathways and to its extracellular assembly. Metabolic pulse labeling of the 67-kD EBP with radioactive serine and further chase of this tracer indicated that both normal fibroblasts and fibroblasts from patients with Costello syndrome initially synthesized comparable amounts of this protein; however, the fibroblasts from Costello syndrome patients quickly lost it into the conditioned media. Because the normal association between EBP and tropoelastin can be disrupted on contact with galactosugar-bearing moieties, and the fibroblasts from patients with Costello syndrome revealed an unusual accumulation of chondroitin sulfate-bearing proteoglycans (CD44 and biglycan), we postulate that a chondroitin sulfate may be responsible for shedding EBP from Costello cells and in turn for their impaired elastogenesis. This was further supported by the fact that exposure to chondroitinase ABC, an enzyme capable of chondroitin sulfate degradation, restored normal production of elastic fibers by fibroblasts from patients with Costello syndrome. We also present evidence that loss of EBP from fibroblasts of Costello syndrome patients is associated with an unusually high rate of cellular proliferation.     

Lysosomal sialidase (neuraminidase-1) is targeted to the cell surface in a multiprotein complex that facilitates elastic fiber assembly

We have established previously that the 67-kDa elastin-binding protein (EBP), identical to the spliced variant of beta-galactosidase, acts as a recyclable chaperone that facilitates secretion of tropoelastin. (Hinek, A., Keeley, F. W., and Callahan, J. W. (1995) Exp. Cell Res. 220, 312-324). We now demonstrate that EBP also forms a cell surface-targeted molecular complex with protective protein/cathepsin A and sialidase (neuraminidase-1), and provide evidence that this sialidase activity is a prerequisite for the subsequent release of tropoelastin. We found that treatment with sialidase inhibitors repressed assembly of elastic fibers in cultures of human skin fibroblasts, aortic smooth muscle cells, and ear cartilage chondrocytes and caused impaired elastogenesis in developing chick embryos. Fibroblasts derived from patients with congenital sialidosis (primary deficiency of neuraminidase-1) and galactosialidosis (secondary deficiency of neuraminidase-1) demonstrated impaired elastogenesis, which could be reversed after their transduction with neuraminidase-1 cDNA or after treatment with bacterial sialidase, which has a similar substrate specificity to human neuraminidase-1. We postulate that neuraminidase-1 catalyzes removal of the terminal sialic acids from carbohydrate chains of microfibrillar glycoproteins and other adjacent matrix glycoconjugates, unmasking their penultimate galactosugars. In turn, the exposed galactosugars interact with the galectin domain of EBP, thereby inducing the release of transported tropoelastin molecules and facilitating their subsequent assembly into elastic fibers.     

Significant decrease in tropoelastin gene expression in fibroblasts from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation

Costello syndrome is a connective tissue disorder associated with sparse, thin, and fragmented elastic fibers in tissues. In this study we demonstrated a significant decrease in the expression of tropoelastin mRNA in fibroblasts derived from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation. In contrast, there were no changes in expression of the Harvey ras (HRAS), fibrillin-1, fibulin-5, microfibril-associated glycoprotein-1 (MAGP-1), lysyl oxidase (LOX), or 67-kDa non-integrin elastin-binding protein (EBP) gene. The proliferative activity of the Costello fibroblasts was about 4-fold higher than that of the normal and pathological control ones. However, no mutations were detected in the coding region of HRAS mRNA. Transduction of the bovine tropoelastin (bTE) gene with the lentiviral vector restored the elastic fiber formation and decreased the growth rate in the Costello fibroblasts. These results strongly suggest that the defect of human tropoelastin (hTE) gene expression should induce the impaired elastogenesis and enhanced proliferation of Costello fibroblasts, and that a primary cause other than the HRAS gene mutation should contribute to the pathogenesis in the present Costello case.     

Induction of fibulin-5 gene is regulated by tropoelastin gene, and correlated with tropoelastin accumulation in vitro

Fibulin-5 (also known as DANCE) is an elastin-binding protein that is thought to play a role in elastogenesis. We examined the relationship between the gene expression of fibulin-5 and the gene expression and accumulation of tropoelastin by comparing elastin-producing cells (human gingival fibroblasts) with non-elastin-producing cells (human periodontal ligament fibroblasts) by Northern blot analysis. Fibulin-5 gene induction was found only in elastin-producing cells. Induction of the fibulin-5 gene in elastin-producing cells occurred after induction of the tropoelastin gene, and the fibulin-5 level was reduced upon RNA interference-mediated down-regulation of tropoelastin. Fibulin-5 gene induction was also correlated with a rapid increase of tropoelastin accumulation within the cell layer. These results may suggest that the fibulin-5 gene induction is directly or indirectly regulated by tropoelastin gene expression and plays a role in the accumulation of elastic fibers within matrices.     

Latent TGF-beta-binding protein 2 binds to DANCE/fibulin-5 and regulates elastic fiber assembly

Elastic fibers play the principal roles in providing elasticity and integrity to various types of human organs, such as the arteries, lung, and skin. However, the molecular mechanism of elastic fiber assembly that leads to deposition and crosslinking of elastin along microfibrils remains largely unknown. We have previously shown that developing arteries and neural crest EGF-like protein (DANCE) (also designated fibulin-5) is essential for elastogenesis by studying DANCE-deficient mice. Here, we report the identification of latent transforming growth factor-beta-binding protein 2 (LTBP-2), an elastic fiber-associating protein whose function in elastogenesis is not clear, as a DANCE-binding protein. Elastogenesis assays using human skin fibroblasts reveal that fibrillar deposition of DANCE and elastin is largely dependent on fibrillin-1 microfibrils. However, downregulation of LTBP-2 induces fibrillin-1-independent fibrillar deposition of DANCE and elastin. Moreover, recombinant LTBP-2 promotes deposition of DANCE onto fibrillin-1 microfibrils. These results suggest a novel regulatory mechanism of elastic fiber assembly in which LTBP-2 regulates targeting of DANCE on suitable microfibrils to form elastic fibers.     

Ultrastructural distribution of 36-kD microfibril-associated glycoprotein (MAGP-36) in human and bovine tissues.

We observed the ultrastructural distribution of MAGP-36 by immunoelectron microscopy in human and bovine tissues. MAGP-36 was present in microfibrils associated with tropoelastin in skin, aorta, and spleen. It was not detected in microfibrils from the ocular zonule and kidney mesangium that were not associated with tropoelastin. In skin, MAGP-36 was present in both early immature elastic fibers and mature elastic fibers. In mature elastic fibers, MAGP-36 was localized around amorphous elastic cores at the elastin-microfibril interface and in electron-dense bundles. Localization of MAGP-36 in elastic fibers coincided with the distribution of lysyl oxidase, an enzyme that plays a pivotal role in the deposition of tropoelastin. These findings suggest that MAGP-36 may be involved in elastogenesis.     

A Negatively Charged Residue Stabilizes the Tropoelastin N-terminal Region for Elastic Fiber Assembly

Tropoelastin is an extracellular matrix protein that assembles into elastic fibers that provide elasticity and strength to vertebrate tissues. Although the contributions of specific tropoelastin regions during each stage of elastogenesis are still not fully understood, studies predominantly recognize the central hinge/bridge and C-terminal foot as the major participants in tropoelastin assembly, with a number of interactions mediated by the abundant positively charged residues within these regions. However, much less is known about the importance of the rarely occurring negatively charged residues and the N-terminal coil region in tropoelastin assembly. The sole negatively charged residue in the first half of human tropoelastin is aspartate 72. In contrast, the same region comprises 17 positively charged residues. We mutated this aspartate residue to alanine and assessed the elastogenic capacity of this novel construct. We found that D72A tropoelastin has a decreased propensity for initial self-association, and it cross-links aberrantly into denser, less porous hydrogels with reduced swelling properties. Although the mutant can bind cells normally, it does not form elastic fibers with human dermal fibroblasts and forms fewer atypical fibers with human retinal pigmented epithelial cells. This impaired functionality is associated with conformational changes in the N-terminal region. Our results strongly point to the role of the Asp-72 site in stabilizing the N-terminal segment of human tropoelastin and the importance of this region in facilitating elastic fiber assembly.     

Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts

The formation of a mature elastic fiber is thought to proceed by the deposition of elastin on pre-existing microfibrils (10-12 nm in diameter). Immunohistochemical evidence has suggested that in developing tissues such as aorta and ligamentum nuchae, small amounts of elastin are associated with microfibrils but are not detected at the light microscopic and ultrastructural levels. Dermal tissue contains a complex elastic fiber system consisting of three types of fibers--oxytalan, elaunin, and elastic--which are believed to differ in their relative contents of microfibrils and elastin. According to ultrastructural analysis, oxytalan fibers contain only microfibrils, elaunin fibers contain small quantities of amorphous elastin, and elastic fibers are predominantly elastin. Using indirect immunofluorescence techniques, we demonstrate in this study that nonamorphous elastin is associated with the oxytalan fibers. Frozen sections of normal skin were incubated with antibodies directed against human aortic alpha elastin and against microfibrillar proteins isolated from cultured calf aortic smooth muscle cells. The antibodies to the microfibrillar proteins and elastin reacted strongly with the oxytalan fibers of the upper dermis. Oxytalan fibers therefore are composed of both microfibrils and small amounts of elastin. Elastin was demonstrated extracellularly in human skin fibroblasts in vitro by indirect immunofluorescence. The extracellular association of nonamorphous elastin and microfibrils on similar fibrils was visualized by immunoelectron microscopy. Treatment of these cultures with sodium dodecyl sulfate/mercaptoethanol (SDS/ME) solubilized tropoelastin and other proteins that reacted with the antibodies to the microfibrillar proteins. It was concluded that the association of the microfibrils with nonamorphous elastin in intact dermis and cultured human skin fibroblasts may represent the initial step in elastogenesis.     

Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo

Elastic fibers are required for the elasticity and integrity of various organs. We and others previously showed that fibulin-5 (also called developing arteries and neural crest EGF-like [DANCE] or embryonic vascular EGF-like repeat-containing protein [EVEC]) is indispensable for elastogenesis by studying fibulin-5-deficient mice, which recapitulate human aging phenotypes caused by disorganized elastic fibers (Nakamura, T., P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, et al. 2002. Nature. 415:171-175; Yanagisawa, H., E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, and E.N. Olson. 2002. Nature. 415:168-171). However, the molecular mechanism by which fiblin-5 contributes to elastogenesis remains unknown. We report that fibulin-5 protein potently induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils. Deposition of fibulin-5 on microfibrils promotes coacervation and alignment of tropoelastins on microfibrils, and also facilitates cross-linking of tropoelastin by tethering lysyl oxidase-like 1, 2, and 4 enzymes. Notably, recombinant fibulin-5 protein induced elastogenesis even in serum-free conditions, although elastogenesis in cell culture has been believed to be serum-dependent. Moreover, the amount of full-length fibulin-5 diminishes with age, while truncated fibulin-5, which cannot promote elastogenesis, increases. These data suggest that fibulin-5 could be a novel therapeutic target for elastic fiber regeneration.     

Dermal connective tissue development in mice: an essential role for tenascin-X

Deficiency of the extracellular matrix protein tenascin-X (TNX) causes a recessive form of Ehlers‐Danlos syndrome (EDS) characterized by hyperextensible skin and hypermobile joints. It is not known whether the observed alterations of dermal collagen fibrils and elastic fibers in these patients are caused by disturbed assembly and deposition or by altered stability and turnover. We used biophysical measurements and immunofluorescence to study connective tissue properties in TNX knockout and wild-type mice. We found that TNX knockout mice, even at a young age, have greatly disturbed biomechanical properties of the skin. No joint abnormalities were noted at any age. The spatio-temporal expression of TNX during normal mouse skin development, during embryonic days 13–19 (E13–E19), was distinct from tropoelastin and the dermal fibrillar collagens type I, III, and V. Our data show that TNX is not involved in the earliest phase (E10–E14) of the deposition of collagen fibrils and elastic fibers during fetal development. From E15 to E19, TNX starts partially to colocalize with the dermal collagens and elastin, and in adult mice, TNX is present in the entire dermis. In adult TNX knockout mice, we observed an apparent increase of elastin. We conclude that TNX knockout mice only partially recapitulate the phenotype of TNX-deficient EDS patients, and that TNX could potentially be involved in maturation and/or maintenance of the dermal collagen and elastin network.     

VE-statin/egfl7 regulates vascular elastogenesis by interacting with lysyl oxidases

We previously characterized VE-statin/egfl7, a protein that is exclusively secreted by endothelial cells and modulates smooth muscle cell migration. Here, we show that VE-statin/egfl7 is the first known natural negative regulator of vascular elastogenesis. Transgenic mice, expressing VE-statin/egfl7 under the control of keratin-14 promoter, showed an accumulation of VE-statin/egfl7 in arterial walls where its presence correlated with an impaired organization of elastic fibres. In vitro, fibroblasts cultured in the presence of VE-statin/egfl7 were unable to deposit elastic fibres due to a deficient conversion of soluble tropoelastin into insoluble mature elastin. VE-statin/egfl7 interacts with the catalytic domain of lysyl oxidase (LOX) enzymes and, in endothelial cells, endogenous VE-statin/egfl7 colocalizes with LoxL2 and inhibits elastic fibre deposition. In contrast, mature elastic fibres are abundantly deposited by endothelial cells that are prevented from producing endogenous VE-statin/egfl7. We propose a model where VE-statin/egfl7 produced by endothelial cells binds to the catalytic domains of enzymes of the LOX family in the vascular wall, thereby preventing the crosslink of tropoelastin molecules into mature elastin polymers and regulating vascular elastogenesis.     

Elastogenic protein expression of a highly elastic murine spinal ligament: the ligamentum flavum

Spinal ligaments, such as the ligamentum flavum (LF), are prone to degeneration and iatrogenic injury that can lead to back pain and nerve dysfunction. Repair and regeneration strategies for these tissues are lacking, perhaps due to limited understanding of spinal ligament formation, the elaboration of its elastic fibers, maturation and homeostasis. Using immunohistochemistry and histology, we investigated murine LF elastogenesis and tissue formation from embryonic to mature postnatal stages. We characterized the spatiotemporal distribution of the key elastogenic proteins tropoelastin, fibrillin-1, fibulin-4 and lysyl oxidase. We found that elastogenesis begins in utero with the microfibril constituent fibrillin-1 staining intensely just before birth. Elastic fibers were first detected histologically at postnatal day (P) 7, the earliest stage at which tropoelastin and fibulin-4 stained intensely. From P7 to P28, elastic fibers grew in diameter and became straighter along the axis. The growth of elastic fibers coincided with intense staining of tropoelastin and fibulin-4 staining, possibly supporting a chaperone role for fibulin-4. These expression patterns correlated with reported skeletal and behavioral changes during murine development. This immunohistochemical characterization of elastogenesis of the LF will be useful for future studies investigating mechanisms for elastogenesis and developing new strategies for treatment or regeneration of spinal ligaments and other highly elastic tissues.     

Elastic fiber production in cardiovascular tissue-equivalents.

Elastic fiber incorporation is critical to the success of tissue-engineered arteries and heart valves. Elastic fibers have not yet been observed in tissue-engineered replacements fabricated in vitro with smooth muscle cells. Here, rat smooth muscle cells (SMC) or human dermal fibroblasts (HDF) remodeled collagen or fibrin gels for 4 weeks as the basis for a completely biological cardiovascular tissue replacement. Immunolabeling, alkaline extraction and amino acid analysis identified and quantified elastin. Organized elastic fibers formed when neonatal SMC were cultured in fibrin gel. Fibrillin-1 deposition occurred but elastin was detected in regions without fibrillin-1, indicating that a microfibril template is not required for elastic fiber formation within fibrin. Collagen did not support substantial elastogenesis by SMC. The quantity of crosslinked elastic fibers was enhanced by treatment with TGF-beta1 and insulin, concomitant with increased collagen production. These additives overcame ascorbate's inhibition of elastogenesis in fibrin. The elastic fibers that formed in fibrin treated with TGF-beta1 and insulin contained crosslinks, as evidenced by the presence of desmosine and an altered elastin labeling pattern when beta-aminopropionitrile (BAPN) was added. These findings indicate that in vitro elastogenesis can be achieved in tissue engineering applications, and they suggest a physiologically relevant model system for the study of three-dimensional elastic structures.     

Tropoelastin regulates chemokine expression in fibroblasts in Costello syndrome

Costello syndrome is a multiple congenital anomaly associated with growth and mental retardation, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Comprehensive expression analysis revealed remarkable up-regulation of several cytokines and chemokines including Gro family proteins, interleukin-1β (IL-1β), IL-8 and MCP-1 but down-regulation of extracellular matrix components including collagens and proteoglycans of skin fibroblasts derived from a Japanese Costello syndrome patient characterized by significantly reduced tropoelastin mRNA, impaired elastogenesis and enhanced cell proliferation. In contrast, decreases in these chemokines and IL-1β expression were observed in Costello fibroblastic cell lines stably expressing the bovine tropoelastin (btEln) gene and in restored elastic fibers. These results strongly suggest that the human TE gene (ELN) transfer could be applicable for the gene therapy of a group of Costello syndrome patients with reduced ELN gene expression.     

Conformational dependence of collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in cultured fibroblasts

We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mm lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a "principal" VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII beta-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in turn exacerbate local connective tissue damage.     

Effect of monoclonal antibodies to defined regions of tropoelastin on elastogenesis in vitro.

Primary cultures of chick embryo aorta cells were grown for one week in the presence of mouse monoclonal antibodies directed against defined regions of chick tropoelastin. This treatment did not significantly alter cell proliferation, cell viability and incorporation of labeled amino acids into total protein or tropoelastin compared with control cultures in which antibodies were either omitted or substituted with an unrelated monoclonal antibody. Tropoelastin-reactive material in the cell layer was revealed by immunologic staining with rabbit antibodies against the chick protein both at the optical and ultrastructural level. Immunofluorescence of control cultures showed that tropoelastin was incorporated into thin and straight fibrils which were sometimes associated with spot-like elements. In the electron microscope tropoelastin-reactive sites were found mainly on the amorphous core of typical, small elastic fibers. The morphological picture of tropoelastin deposits in cultures exposed to anti-tropoelastin monoclonal antibodies depended on the molecular form (whole antibody or Fab fragments) and the binding specificity of the antibody used. Although alterations common to different antibodies were observed, the main structural features were peculiar for each antibody. Two antibodies which bound epitopes present in two regions of tropoelastin grossly altered the formation of amorphous elastin. Moreover, two antibodies directed against the region of tropoelastin containing the polypentapeptide-repeat (VPGVG)n stimulated the deposition of the protein into the amorphous core of normal-looking elastic fibers and disorganized the compact bundles of parallel microfibrils seen in controls. Finally, one antibody which recognized a unique epitope close to the carboxy-terminal end of tropoelastin and Fab fragments from all antibodies apparently inhibited the formation of the amorphous nuclei of elastic fibers, but not the association of tropoelastin with microfibrils. The data suggest that the association of tropoelastin molecules during fiber assembly is not random, but follows an ordered alignment process which the antibodies alter by imposing a different molecular packing.     

Microfibril-associated MAGP-2 stimulates elastic fiber assembly

Elastic fibers are complex structures composed of a tropoelastin inner core and microfibril outer mantle guiding tropoelastin deposition. Microfibrillar proteins mainly include fibrillins and microfibril-associated glycoproteins (MAGPs). MAGP-2 exhibits developmental expression peaking at elastic fiber onset, suggesting that MAGP-2 mediates elastic fiber assembly. To determine whether MAGP-2 regulates elastic fiber assembly, we used an in vitro model featuring doxycycline-regulated cells conditionally overexpressing exogenous MAGP-2 and constitutively expressing enhanced green fluorescent protein-tagged tropoelastin. Analysis by immunofluorescent staining showed that MAGP-2 overexpression dramatically increased elastic fibers levels, independently of extracellular levels of soluble tropoelastin, indicating that MAGP-2 stimulates elastic fiber assembly. This was associated with increased levels of matrix-associated MAGP-2. Electron microscopy showed that MAGP-2 specifically associates with microfibrils and that elastin globules primarily colocalize with MAGP-2-associated microfibrils, suggesting that microfibril-associated MAGP-2 facilitates elastic fiber assembly. MAGP-2 overexpression did not change levels of matrix-associated fibrillin-1, MAGP-1, fibulin-2, fibulin-5, or emilin-1, suggesting that microfibrils and other elastic fiber-associated proteins known to regulate elastogenesis do not mediate MAGP-2-induced elastic fiber assembly. Moreover, mutation analysis showed that MAGP-2 does not stimulate elastic fiber assembly through its RGD motif, suggesting that integrin receptor binding does not mediate MAGP-2-induced elastic fiber assembly. Because MAGP-2 interacts with Jagged-1 that controls cell-matrix interaction and cell motility, two key factors in elastic fiber macroassembly, microfibril-associated MAGP-2 may stimulate elastic fiber macroassembly by targeting the release of elastin globules from the cell membrane onto developing elastic fibers.