Fibulin-4 is essential for maintaining arterial wall integrity in conduit but not muscular arteries.

Homozygous or compound heterozygous mutations in fibulin-4 (FBLN4) lead to autosomal recessive cutis laxa type 1B (ARCL1B), a multisystem disorder characterized by significant cardiovascular abnormalities, including abnormal elastin assembly, arterial tortuosity, and aortic aneurysms. We sought to determine the consequences of a human disease-causing mutation in FBLN4 (E57K) on the cardiovascular system and vascular elastic fibers in a mouse model of ARCL1B. Fbln4E57K/E57K mice were hypertensive and developed arterial elongation, tortuosity, and ascending aortic aneurysms. Smooth muscle cell organization within the arterial wall of large conducting vessels was abnormal, and elastic fibers were fragmented and had a moth-eaten appearance. In contrast, vessel wall structure and elastic fiber integrity were normal in resistance/muscular arteries (renal, mesenteric, and saphenous). Elastin cross-linking and total elastin content were unchanged in large or small arteries, whereas elastic fiber architecture was abnormal in large vessels. While the E57K mutation did not affect Fbln4 mRNA levels, FBLN4 protein was lower in the ascending aorta of mutant animals compared to wild-type arteries but equivalent in mesenteric arteries. We found a differential role of FBLN4 in elastic fiber assembly, where it functions mainly in large conduit arteries. These results suggest that elastin assembly has different requirements depending on vessel type. Normal levels of elastin cross-links in mutant tissue call into question FBLN4's suggested role in mediating lysyl oxidase-elastin interactions. Future studies investigating tissue-specific elastic fiber assembly may lead to novel therapeutic interventions for ARCL1B and other disorders of elastic fiber assembly.

Functional consequence of fibulin-4 missense mutations associated with vascular and skeletal abnormalities and cutis laxa.

Fibulin-4 is a 60kDa calcium binding glycoprotein that has an important role in development and integrity of extracellular matrices. It interacts with elastin, fibrillin-1 and collagen IV as well as with lysyl oxidases and is involved in elastogenesis and cross-link formation. To date, several mutations in the fibulin-4 gene (FBLN4/EFEMP2) are known in patients whose major symptoms are vascular deformities, aneurysm, cutis laxa, joint laxity, or arachnodactyly. The pathogenetic mechanisms how these mutations translate into the clinical phenotype are, however, poorly understood. In order to elucidate these mechanisms, we expressed fibulin-4 mutants recombinantly in HEK293 cells, purified the proteins in native forms and analyzed alterations in protein synthesis, secretion, matrix assembly, and interaction with other proteins in relation to wild type fibulin-4. Our studies show that different mutations affect these properties in multiple ways, resulting in fibulin-4 deficiency and/or impaired ability to form elastic fibers. The substitutions E126K and C267Y impaired secretion of the protein, but not mRNA synthesis. Furthermore, the E126K mutant showed less resistance to proteases, reduced binding to collagen IV and fibrillin-1, as well as to LTBP1s and LTBP4s. The A397T mutation introduced an extra O-glycosylation site and deleted binding to LTBP1s. We show that fibulin-4 binds stronger than fibulin-3 and -5 to LTBP1s, 3, and 4s, and to the lysyl oxidases LOX and LOXL1; the binding of fibulin-4 to the LOX propeptide was strongly reduced by the mutation E57K. These findings show that different mutations in the fibulin-4 gene result in different molecular defects affecting secretion rates, protein stability, LOX-induced cross-linking, or binding to other ECM components and molecules of the TGF-ß pathway, and thus illustrate the complex role of fibulin-4 in connective tissue assembly.

Fibulin-2 is essential for angiotensin II-induced myocardial fibrosis mediated by transforming growth factor (TGF)-ß.

Fibrosis is an ominous pathological process in failing myocardium, but its pathogenesis is poorly understood. We recently reported that loss of an extracellular matrix (ECM) protein, fibulin-2, protected against ventricular dysfunction after myocardial infarction (MI) in association with absence of activation of transforming growth factor (TGF)-ß signaling and suppressed upregulation of ECM protein expression during myocardial remodeling. Here we investigated the role of fibulin-2 in the development of myocardial hypertrophy and fibrosis induced by continuous pressor-dosage of angiotensin II (Ang II) infusion. Both wild type (WT) and fibulin-2 null (Fbln2KO) mice developed comparable hypertension and myocardial hypertrophy by Ang II infusion. However, myocardial fibrosis with significant upregulation of collagen type I and III mRNA was only seen in WT but not in Fbln2KO mice.Transforming growth factor (TGF)-ß1 mRNA and its downstream signal, Smad2, were significantly upregulated in WT by Ang II, whereas there were no Ang II-induced changes in Flbn2KO, suggesting fibulin-2 is necessary for Ang II-induced TGF-ß signaling that induces myocardial fibrosis. To test whether fibulin-2 is sufficient for Ang II-induced TGF-ß upregulation, isolated Flbn2KO cardiac fibroblasts were treated with Ang II after transfecting with fibulin-2 expression vector or pretreating with recombinant fibulin-2 protein. Ang II-induced TGF-ß signaling in Fbln2KO cells was partially rescued by exogenous fibulin-2, suggesting that fibulin-2 is required and probably sufficient for Ang II-induced TGF-ß activation. Smad2 phosphorylation was induced just by adding recombinant fibulin-2 to KO cells, suggesting that extracellular interaction between fibulin-2 and latent TGF-ß triggered initial TGF-ß activation. Our study indicates that Ang II cannot induce TGF-ß activation without fibulin-2 and that fibulin-2 has an essential role in Ang II-induced TGF-ß signaling and subsequent myocardial fibrosis. Fibulin-2 can be considered as a critical regulator of TGF-ß that induces myocardial fibrosis.

Forelimb contractures and abnormal tendon collagen fibrillogenesis in fibulin-4 null mice.

Fibulin-4 is an extracellular matrix glycoprotein essential for elastic fiber formation. Mice deficient in fibulin-4 die perinatally because of severe pulmonary and vascular defects associated with the lack of intact elastic fibers. Patients with fibulin-4 mutations demonstrate similar defects, and a significant number die shortly after birth or in early childhood from cardiopulmonary failure. The patients also demonstrate skeletal and other systemic connective tissue abnormalities, including joint laxity and flexion contractures of the wrist. A fibulin-4 null mouse strain was generated and used to analyze the roles of fibulin-4 in tendon fibrillogenesis. This mouse model displayed bilateral forelimb contractures, in addition to pulmonary and cardiovascular defects. The forelimb and hindlimb tendons exhibited disruption in collagen fibrillogenesis in the absence of fibulin-4 as analyzed by transmission electron microscopy. Fewer fibrils were assembled, and fibrils were disorganized compared with wild-type controls. The organization of developing tenocytes and compartmentalization of the extracellular space was also disrupted. Fibulin-4 was co-localized with fibrillin-1 and fibrillin-2 in limb tendons by using immunofluorescence microscopy. Thus, fibulin-4 seems to play a role in regulating tendon collagen fibrillogenesis, in addition to its essential function in elastogenesis.

Loss of fibulin-4 results in abnormal collagen fibril assembly in bone, caused by impaired lysyl oxidase processing and collagen cross-linking.

The extracellular matrix protein fibulin-4 has been shown to be indispensable for elastic fiber assembly, but there is also evidence from human mutations that it is involved in controlling skeletal development and bone stability. Fibulin-4 mutations were identified in patients suffering from vascular abnormality and/or cutis laxa, and some of these patients exhibited bone fragility, arachnodactyly and joint laxity. In order to elucidate the role of fibulin-4 in bone structure and skeletal development, we analyzed structural changes in skeletal tissues of Fbln4(-/-) mice. Immunostaining confirmed that fibulin-4 is highly expressed in cartilage, bone, ligaments and tendons. No morphological abnormalities were found in the skeleton of Fbln4(-/-) mice as compared to wild type littermates except forelimb contractures as well as unusually thick collagen fibrils. Furthermore, fibulin-4 deficiency caused enhanced susceptibility of bone collagen for acid extraction, consistent with significantly reduced lysylpyridinoline and hydroxylysylpyridinoline cross-links in bone. In accordance with that, the amount of lysyl oxidase in long bones and calvaria was strongly decreased and proteolytic activation of lysyl oxidase was reduced in fibulin-4 deficient osteoblasts, while addition of recombinant fibulin-4 rescued the activation. The finding suggested that fibulin-4 is important for the proteolytic activation of lysyl oxidase which has a pivotal role in cross-linking of collagen and elastin.

Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities.

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.

Human adipose-derived stem cell transplantation as a potential therapy for collagen VI-related congenital muscular dystrophy.

INTRODUCTION: Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders characterized by muscle weakness within the first two years of life. Collagen VI-related muscle disorders have recently emerged as one of the most common types of CMD. COL6 CMD is caused by deficiency and/or dysfunction of extracellular matrix (ECM) protein collagen VI. Currently, there is no specific treatment for this disabling and life-threatening disease. The primary cellular targets for collagen VI CMD therapy are fibroblasts in muscle, tendon and skin, as opposed to muscle cells for other types of muscular dystrophies. However, recent advances in stem cell research have raised the possibility that use of adult stem cells may provide dramatic new therapies for treatment of COL6 CMD. METHODS: Here, we developed a procedure for isolation of human stem cells from the adipose layer of neonatal skin. The adipose-derived stem cells (ADSC) were examined for expression of ECM and related genes using gene expression array analysis. The therapeutic potential of ADSC was assessed after a single intramuscular transplantation in collagen VI-deficient mice. RESULTS: Analysis of primary cultures confirmed that established ADSC represent a morphologically homogenous population with phenotypic and functional features of adult mesenchymal stem cells. A comprehensive gene expression analysis showed that ADSC express a vast array of ECM genes. Importantly, it was observed that ADSC synthesize and secrete all three collagen VI chains, suggesting suitability of ADSC for COL6 CMD treatment. Furthermore, we have found that a single intramuscular transplantation of ADSC into Col6a1-/-Rag1-/- mice under physiological and cardiotoxin-induced injury/regeneration conditions results in efficient engraftment and migration of stem cells within the skeletal muscle. Importantly, we showed that ADSC can survive long-term and continuously secrete the therapeutic collagen VI protein missing in the mutant mice. CONCLUSIONS: Overall, our findings suggest that stem cell therapy can potentially provide a new avenue for the treatment of COL6 CMD and other muscular disorders and injuries.

A mouse model for dominant collagen VI disorders: heterozygous deletion of Col6a3 Exon 16.

Dominant and recessive mutations in collagen VI genes, COL6A1, COL6A2, and COL6A3, cause a continuous spectrum of disorders characterized by muscle weakness and connective tissue abnormalities ranging from the severe Ullrich congenital muscular dystrophy to the mild Bethlem myopathy. Herein, we report the development of a mouse model for dominant collagen VI disorders by deleting exon 16 in the Col6a3 gene. The resulting heterozygous mouse, Col6a3(+/d16), produced comparable amounts of normal Col6a3 mRNA and a mutant transcript with an in-frame deletion of 54 bp of triple-helical coding sequences, thus mimicking the most common molecular defect found in dominant Ullrich congenital muscular dystrophy patients. Biosynthetic studies of mutant fibroblasts indicated that the mutant α3(VI) collagen protein was produced and exerted a dominant-negative effect on collagen VI microfibrillar assembly. The distribution of the α3(VI)-like chains of collagen VI was not altered in mutant mice during development. The Col6a3(+/d16) mice developed histopathologic signs of myopathy and showed ultrastructural alterations of mitochondria and sarcoplasmic reticulum in muscle and abnormal collagen fibrils in tendons. The Col6a3(+/d16) mice displayed compromised muscle contractile functions and thereby provide an essential preclinical platform for developing treatment strategies for dominant collagen VI disorders.

Reduced fibulin-2 contributes to loss of basement membrane integrity and skin blistering in mice lacking integrin α3ß1 in the epidermis.

Deficient epidermal adhesion is a hallmark of blistering skin disorders and chronic wounds, implicating integrins as potential therapeutic targets. Integrin α3ß1, a major receptor in the epidermis for adhesion to laminin-332 (LN-332), has critical roles in basement membrane (BM) organization during skin development. In the current study we identify a role for α3ß1 in promoting stability of nascent epidermal BMs through induction of fibulin-2, a matrix-associated protein that binds LN-332. We demonstrate that mice lacking α3ß1 in the epidermis display ruptured BM beneath neo-epidermis of wounds, characterized by extensive blistering. This junctional blistering phenocopies defects reported in newborn α3-null mice, as well as in human patients with α3 gene mutations, indicating that the developmental role of α3ß1 in BM organization is recapitulated during wound healing. Mice lacking epidermal α3ß1 also have reduced fibulin-2 expression, and fibulin-2-null mice display perinatal skin blisters similar to those in α3ß1-deficient mice. Interestingly, α3-null wound epidermis or keratinocytes also show impaired processing of the LN-332 γ2 chain, although this defect was independent of reduced fibulin-2 and did not appear to cause blistering. Our findings indicate a role for integrin α3ß1 in BM stability through fibulin-2 induction, both in neonatal skin and in adult wounds.

Clinical significance of serum COL6A3 in pancreatic ductal adenocarcinoma.

Type VI collagen (COL6) forms a microfibrillar network often associated with type I collagen and constitutes a major component of the desmoplastic reaction in pancreatic ductal adenocarcinoma (PDA). We have demonstrated recently that the α3 chain of COL6, COL6A3, is highly expressed in PDA tissue and undergoes tumor-specific alternative splicing. In this study, we investigated the diagnostic value and clinical significance of circulating COL6A3 protein and mRNA in PDA. COL6A3 levels in sera from patients with PDA (n = 44), benign lesions (n = 46) and age-matched healthy volunteers (n = 30) were analyzed by enzyme-linked immunosorbent assays (ELISA). Predictive abilities of COL6A3 were examined using receiver operating characteristic (ROC) curves from logistic regression models for PDA versus normal or benign serum levels. Expression levels were correlated with clinicopathological parameters. Real-time PCR was used to analyze the presence of COL6A3 mRNA containing alternative spliced exons E3, E4, and E6. Circulating COL6A3 protein levels were significantly elevated in PDA patients when compared to healthy sera (p = 0.0001) and benign lesions (p = 0.0035). The overall area under the ROC was 0.975. Log(COL6A3) alone provided good discrimination between PDA and benign lesions (area under the curve (AUC) = 0.817), but combined with CA19-9 provided excellent discrimination (AUC = 0.904). Interestingly, high COL6A3 serum levels were significantly associated with perineural invasion and cigarette smoking. Combined E3, E4, and E6 serum RNA values provided good sensitivity but low specificity. Our data demonstrate for the first time the potential clinical significance of circulating COL6A3 in the diagnosis of pancreatic malignancy.

Fibulin-2 deficiency attenuates angiotensin II-induced cardiac hypertrophy by reducing transforming growth factor-ß signalling.

AngII (angiotensin II) is a potent neurohormone responsible for cardiac hypertrophy, in which TGF (transforming growth factor)-ß serves as a principal downstream mediator. We recently found that ablation of fibulin-2 in mice attenuated TGF-ß signalling, protected mice against progressive ventricular dysfunction, and significantly reduced the mortality after experimental MI (myocardial infarction). In the present study, we investigated the role of fibulin-2 in AngII-induced TGF-ß signalling and subsequent cardiac hypertrophy. We performed chronic subcutaneous infusion of AngII in fibulin-2 null (Fbln2-/-), heterozygous (Fbln2+/-) and WT (wild-type) mice by a mini-osmotic pump. After 4 weeks of subpressor dosage of AngII infusion (0.2 µg/kg of body weight per min), WT mice developed significant hypertrophy, whereas the Fbln2-/- showed no response. In WT, AngII treatment significantly up-regulated mRNAs for fibulin-2, ANP (atrial natriuretic peptide), TGF-ß1, Col I (collagen type I), Col III (collagen type III), MMP (matrix metalloproteinase)-2 and MMP-9, and increased the phosphorylation of TGF-ß-downstream signalling markers, Smad2, TAK1 (TGF-ß-activated kinase 1) and p38 MAPK (mitogen-activated protein kinase), which were all unchanged in AngII-treated Fbln2-/- mice. The Fbln2+/- mice consistently displayed AngII-induced effects intermediate between WT and Fbln2-/-. Pressor dosage of AngII (2 mg/kg of body weight per min) induced significant fibrosis in WT but not in Fbln2-/- mice with comparable hypertension and hypertrophy in both groups. Isolated CFs (cardiac fibroblasts) were treated with AngII, in which direct AngII effects and TGF-ß-mediated autocrine effects was observed in WT. The latter effects were totally abolished in Fbln2-/- cells, suggesting that fibulin-2 is essential for AngII-induced TGF-ß activation. In conclusion our data indicate that fibulin-2 is essential for AngII-induced TGF-ß-mediated cardiac hypertrophy via enhanced TGF-ß activation and suggest that fibulin-2 is a potential therapeutic target to inhibit AngII-induced cardiac remodelling.

COL6A3 protein deficiency in mice leads to muscle and tendon defects similar to human collagen VI congenital muscular dystrophy.

Collagen VI is a ubiquitously expressed extracellular microfibrillar protein. Its most common molecular form is composed of the α1(VI), α2(VI), and α3(VI) collagen α chains encoded by the COL6A1, COL6A2, and COL6A3 genes, respectively. Mutations in any of the three collagen VI genes cause congenital muscular dystrophy types Bethlem and Ullrich as well as intermediate phenotypes characterized by muscle weakness and connective tissue abnormalities. The α3(VI) collagen α chain has much larger N- and C-globular domains than the other two chains. Its most C-terminal domain can be cleaved off after assembly into microfibrils, and the cleavage product has been implicated in tumor angiogenesis and progression. Here we characterize a Col6a3 mutant mouse that expresses a very low level of a non-functional α3(VI) collagen chain. The mutant mice are deficient in extracellular collagen VI microfibrils and exhibit myopathic features, including decreased muscle mass and contractile force. Ultrastructurally abnormal collagen fibrils were observed in tendon, but not cornea, of the mutant mice, indicating a distinct tissue-specific effect of collagen VI on collagen I fibrillogenesis. Overall, the mice lacking normal α3(VI) collagen chains displayed mild musculoskeletal phenotypes similar to mice deficient in the α1(VI) collagen α chain, suggesting that the cleavage product of the α3(VI) collagen does not elicit essential functions in normal growth and development. The Col6a3 mouse mutant lacking functional α3(VI) collagen chains thus serves as an animal model for COL6A3-related muscular dystrophy.

Loss of fibulin-2 protects against progressive ventricular dysfunction after myocardial infarction.

Remodeling of the cardiac extracellular matrix (ECM) is an integral part of wound healing and ventricular adaptation after myocardial infarction (MI), but the underlying mechanisms remain incompletely understood. Fibulin-2 is an ECM protein upregulated during cardiac development and skin wound healing, yet mice lacking fibulin-2 do not display any identifiable phenotypic abnormalities. To investigate the effects of fibulin-2 deficiency on ECM remodeling after MI, we induced experimental MI by permanent coronary artery ligation in both fibulin-2 null and wild-type mice. Fibulin-2 expression was up-regulated at the infarct border zone of the wild-type mice. Acute myocardial tissue responses after MI, including inflammatory cell infiltration and ECM protein synthesis and deposition in the infarct border zone, were markedly attenuated in the fibulin-2 null mice. However, the fibulin-2 null mice had significantly better survival rate after MI compared to the wild-type mice as a result of less frequent cardiac rupture and preserved left ventricular function. Up-regulation of TGF-ß signaling and ECM remodeling after MI were attenuated in both ischemic and non-ischemic myocardium of the fibulin-2 null mice compared to the wild type counterparts. Increase in TGF-ß signaling in response to angiotensin II was also lessened in cardiac fibroblasts isolated from the fibulin-2 null mice. The studies provide the first evidence that absence of fibulin-2 results in decreased up-regulation of TGF-ß signaling after MI and protects against ventricular dysfunction, suggesting that fibulin-2 may be a potential therapeutic target for attenuating the progression of ventricular remodeling.

Tumor-specific expression and alternative splicing of the COL6A3 gene in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease; a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagens III and V, comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked with inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events. METHODS: We analyzed paired PDA-normal (n = 18), intraductal papillary mucinous neoplasms (IPMN; n = 5), pancreatic cystadenoma (n = 5), and 8 PDA cell lines with reverse transcriptase polymerase chain reaction, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in 2 animal models of PDA. RESULTS: COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA-adjacent tissues. Inclusion of exon 4 was exclusively tumor specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in 6 PDA cell lines, but only 2 cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma and some PDA cells. CONCLUSION: We have described, for the first time, a dynamic process of tumor-specific alternative splicing in several exons of stromal COL6A3. Alternatively spliced proteins may contribute to the etiology or progression of cancer and may serve as markers for cancer diagnosis. Identification of COL6A3 isoforms as PDA-specific provides the basis for future studies to explore the oncogenic and diagnostic potential of these alternative splicing events.

Dysfunctional tendon collagen fibrillogenesis in collagen VI null mice.

Tendons are composed of fibroblasts and collagen fibrils. The fibrils are organized uniaxially and grouped together into fibers. Collagen VI is a non-fibrillar collagen expressed in developing and adult tendons. Human collagen VI mutations result in muscular dystrophy, joint hyperlaxity and contractures. The purpose of this study is to determine the functional roles of collagen VI in tendon matrix assembly. During tendon development, collagen VI was expressed throughout the extracellular matrix, but enriched around fibroblasts and their processes. To analyze the functional roles of collagen VI a mouse model with a targeted inactivation of Col6a1 gene was utilized. Ultrastructural analysis of Col6a1-/- versus wild type tendons demonstrated disorganized extracellular micro-domains and associated collagen fibers in the Col6a1-/- tendon. In Col6a1-/- tendons, fibril structure and diameter distribution were abnormal compared to wild type controls. The diameter distributions were shifted significantly toward the smaller diameters in Col6a1-/- tendons compared to controls. An analysis of fibril density (number/µm(2)) demonstrated a ~2.5 fold increase in the Col6a1-/- versus wild type tendons. In addition, the fibril arrangement and structure were aberrant in the peri-cellular regions of Col6a1-/- tendons with frequent very large fibrils and twisted fibrils observed restricted to this region. The biomechanical properties were analyzed in mature tendons. A significant decrease in cross-sectional area was observed. The percent relaxation, maximum load, maximum stress, stiffness and modulus were analyzed and Col6a1-/- tendons demonstrated a significant reduction in maximum load and stiffness compared to wild type tendons. An increase in matrix metalloproteinase activity was suggested in the absence of collagen VI. This suggests alterations in tenocyte expression due to disruption of cell-matrix interactions. The changes in expression may result in alterations in the peri-cellular environment. In addition, the absence of collagen VI may alter the sequestering of regulatory molecules such as leucine rich proteoglycans. These changes would result in dysfunctional regulation of tendon fibrillogenesis indirectly mediated by collagen VI.

Recessive COL6A2 C-globular missense mutations in Ullrich congenital muscular dystrophy: role of the C2a splice variant.

Ullrich congenital muscular dystrophy (UCMD) is a disabling and life-threatening disorder resulting from either recessive or dominant mutations in genes encoding collagen VI. Although the majority of the recessive UCMD cases have frameshift or nonsense mutations in COL6A1, COL6A2, or COL6A3, recessive structural mutations in the COL6A2 C-globular region are emerging also. However, the underlying molecular mechanisms have remained elusive. Here we identified a homozygous COL6A2 E624K mutation (C1 subdomain) and a homozygous COL6A2 R876S mutation (C2 subdomain) in two UCMD patients. The consequences of the mutations were investigated using fibroblasts from patients and cells stably transfected with the mutant constructs. In contrast to expectations based on the clinical severity of these two patients, secretion and assembly of collagen VI were moderately affected by the E624K mutation but severely impaired by the R876S substitution. The E624K substitution altered the electrostatic potential of the region surrounding the metal ion-dependent adhesion site, resulting in a collagen VI network containing thick fibrils and spots with densely packed microfibrils. The R876S mutation prevented the chain from assembling into triple-helical collagen VI molecules. The minute amount of collagen VI secreted by the R876S fibroblasts was solely composed of a faster migrating chain corresponding to the C2a splice variant with an alternative C2 subdomain. In transfected cells, the C2a splice variant was able to assemble into short microfibrils. Together, the results suggest that the C2a splice variant may functionally compensate for the loss of the normal COL6A2 chain when mutations occur in the C2 subdomain.

Fibulin-2 and fibulin-5 cooperatively function to form the internal elastic lamina and protect from vascular injury.

OBJECTIVE: Recent findings on the role of fibulin-5 (Fbln5) have provided substantial progress in understanding the molecular mechanism of elastic fiber assembly in vitro. However, little is known about differential roles of fibulins in the elastogenesis of blood vessels. Here, we generated double knockout mice for Fbln5 and Fbln2 (termed DKO) and examined the role of fibulins-2 and -5 in development and injury response of the blood vessel wall. METHODS AND RESULTS: Fibulin-2 is distinctly located in the subendothelial matrix, whereas fibulin-5 is observed throughout the vessel wall. All of the elastic laminae, including the internal elastic lamina (IEL), were severely disorganized in DKO mice, which was not observed in single knockout mice for Fbln2 or Fbln5. Furthermore, DKO vessels displayed upregulation of vascular adhesion molecules, tissue factor expression, and thrombus formation with marked dilation and thinning of the vessel wall after carotid artery ligation-injury. CONCLUSIONS: Fibulin-2 and fibulin-5 cooperatively function to form the IEL during postnatal development by directing the assembly of elastic fibers, and are responsible for maintenance of the adult vessel wall after injury. The DKO mouse will serve as a unique animal model to test the effect of vessel integrity during various pathological insults.

Latent transforming growth factor beta-binding proteins and fibulins compete for fibrillin-1 and exhibit exquisite specificities in binding sites.

Latent transforming growth factor (TGF) beta-binding proteins (LTBPs) interact with fibrillin-1. This interaction is important for proper sequestration and extracellular control of TGFbeta. Surface plasmon resonance interaction studies show that residues within the first hybrid domain (Hyb1) of fibrillin-1 contribute to interactions with LTBP-1 and LTBP-4. Modulation of binding affinities by fibrillin-1 polypeptides in which residues in the third epidermal growth factor-like domain (EGF3) are mutated demonstrates that the binding sites for LTBP-1 and LTBP-4 are different and suggests that EGF3 may also contribute residues to the binding site for LTBP-4. In addition, fibulin-2, fibulin-4, and fibulin-5 bind to residues contained within EGF3/Hyb1, but mutated polypeptides again indicate differences in their binding sites in fibrillin-1. Results demonstrate that these protein-protein interactions exhibit "exquisite specificities," a phrase commonly used to describe monoclonal antibody interactions. Despite these differences, interactions between LTBP-1 and fibrillin-1 compete for interactions between fibrillin-1 and these fibulins. All of these proteins have been immunolocalized to microfibrils. However, in fibrillin-1 (Fbn1) null fibroblast cultures, LTBP-1 and LTBP-4 are not incorporated into microfibrils. In contrast, in fibulin-2 (Fbln2) null or fibulin-4 (Fbln4) null cultures, fibrillin-1, LTBP-1, and LTBP-4 are incorporated into microfibrils. These data show for the first time that fibrillin-1, but not fibulin-2 or fibulin-4, is required for appropriate matrix assembly of LTBPs. These studies also suggest that the fibulins may affect matrix sequestration of LTBPs, because in vitro interactions between these proteins are competitive.

Retinoic acid receptors are required for skeletal growth, matrix homeostasis and growth plate function in postnatal mouse.

The retinoic acid receptors alpha, beta and gamma (RARalpha, RARbeta and RARgamma) are nuclear hormone receptors that regulate fundamental processes during embryogenesis, but their roles in skeletal development and growth remain unclear. To study skeletal-specific RAR function, we created conditional mouse mutants deficient in RAR expression in cartilage. We find that mice deficient in RARalpha and RARgamma (or RARbeta and RARgamma) exhibit severe growth retardation obvious by about 3 weeks postnatally. Their growth plates are defective and, importantly, display a major drop in aggrecan expression and content. Mice deficient in RARalpha and RARbeta, however, are virtually normal, suggesting that RARgamma is essential. In good correlation, we find that RARgamma is the most strongly expressed RAR in mouse growth plate and its expression characterizes the proliferative and pre-hypertrophic zones where aggrecan is strongly expressed also. By being avascular, those zones lack endogenous retinoids as indicated by previous RARE reporter mice and our direct biochemical measurements and thus, RARgamma is likely to exert ligand-less repressor function. Indeed, our data indicate that: aggrecan production is enhanced by RARgamma over-expression in chondrocytes under retinoid-free culture conditions; production is further boosted by co-repressor Zac1 or pharmacologic agents that enhance RAR repressor function; and RAR/Zac1 function on aggrecan expression may involve Sox proteins. In sum, our data reveal that RARs, and RARgamma in particular, exert previously unappreciated roles in growth plate function and skeletal growth and regulate aggrecan expression and content. Since aggrecan is critical for growth plate function, its deficiency in RAR-mutant mice is likely to have contributed directly to their growth retardation.

Muscle interstitial fibroblasts are the main source of collagen VI synthesis in skeletal muscle: implications for congenital muscular dystrophy types Ullrich and Bethlem.

Mutations in the extracellular matrix molecule collagen VI underlie the congenital muscular dystrophy types Ullrich and Bethlem. Establishing the origin of collagen VI in muscle is important for understanding the pathophysiology of these diseases and for developing future treatment approaches involving cell-specific delivery. Because the cells that produce collagen VI cannot be identified by histologic analysis, we examined the production of collagen VI in pure cultures of primary myogenic cells and muscle interstitial fibroblasts from limb muscle of neonatal mice. Immunofluorescence staining and Western blot analysis revealed secretion and matrix deposition of collagen VI by interstitial fibroblasts but not by myogenic cells in vitro. Using Northern blot and real-time reverse-transcriptase-polymerase chain reaction analysis for the collagen VI genes col6a1, col6a2, col6a3, transcript levels for the 3 mRNAs were high in interstitial fibroblasts, whereas in primary myogenic cells, they were indistinguishable from background. Furthermore, retention of mutant collagen VI in muscle from 3 patients with collagen VI mutation was identified in interstitial fibroblastic cells but not in their myofibers. These results suggest that interstitial fibroblasts but not myogenic cells contribute significantly to the deposition of collagen VI in the extracellular matrix in skeletal muscle and imply major roles of this cell type and the extracellular matrix in the pathogenesis of these diseases.