MAB2502 Sigma-AldrichAnti-Fibrillin-1 Antibody, NT, clone 26

Matrix-based therapy restoring the cell microenvironment is a new approach in regenerative medicine successfully treating human chronic pathologies by using a heparan sulfate mimetic (ReGeneraTing agents [RGTA]). Periodontitis are inflammatory diseases destroying the tooth-supporting tissues with no satisfactory therapy. We studied in vivo RGTA ability to fully restore the tooth-supporting tissues. After periodontitis induction, hamsters were treated with RGTA (1.5 mg kg(-1) w(-1)) or saline. Bone loss was evaluated and immunohistochemical labeling of molecules expressed during cementum development was performed. RGTA treatment restored alveolar bone and the attachment apparatus where fibers were inserted in acellular decorin-negative cementum. RGTA treatment increased the epithelial rests of Malassez, previously depleted by periodontitis. Bone morphogenetic protein (BMP) expressions were compartmentalized: BMP-3 was strongly expressed by epithelial rests of Malassez; BMP-7 was expressed by cells lying on the cementum and BMP-2 by osteoprogenitors around bone formation sites but not at the root-bone interface. Cells near the cementum and bone expressed the ALK2 receptor. This is the first evidence that reconstructing the extracellular matrix scaffold with a heparan sulfate mimetic regenerated the root interface despite the persistence of the bacteria responsible for the disease The improved cellular microenvironment led to the sequential recruitment of cell populations involved in attachment apparatus regeneration.

Our aim was to obtain a viable and easily available dermal substitute (DS) for the definitive coverage of full-thickness burns. A DS composed of a collagen-glycosaminoglycan-chitosan dermal matrix (DM) colonized with foreskin fibroblasts (FF) is described. FF-colonized DS were compared to the DM seeded with adult dermal fibroblasts (DF). FF-colonized DS expressed more fibrillin and tropoelastin than that with DF. Reconstructed skin obtained with both FF- and DF-colonized DS similarly expressed laminin-5 and collagen VII at the dermal-epidermal junction. Both FF- and DF-colonized DS produced cutaneous wound healing mediators in a dose-dependent manner in the presence of platelet lysate. After freeze-thawing, the FF-colonized DS were recovered in culture and retained their ability to produce vascular endothelial growth factor. Grafting of DS into nude rats achieved a complete healing of a dermal-epidermal lesion with a good epidermalization.

Periodontitis are bacterium-driven inflammatory diseases that destroy tooth-supporting tissues whose complete restoration is not currently possible. RGTA, a new class of agents, have this capacity in an animal model. Periodontitis was induced in hamsters and, starting 8 weeks later, injected RG1503, a glycosaminoglycan synthesized from a 40 kDa dextran behaving like a heparan sulfate mimetic (1.5 mg kg(-1) w(-1)) or saline for 8 weeks. The three periodontium compartments were evaluated by immunohistochemistry and morphometry. The gingival extracellular matrix disorganized by inflammation was restoring under treatment. The collagen network was repaired and resumed its previous organization. Fibrillin-1 expression was restored so that the elastic network rebuilt at a distance from the pocket and began to reconstruct near the pocket. Apoptotic cell numbers were decreased in the pocket epithelium, and more so in the infiltrated connective tissue. The continuity and the thickness of the basement membrane were restored and testified normalization of epithelium connective tissue interaction. The amount of alveolar bone increased around the first molar, and the interradicular bone was rebuilt. The root cementum was thickened and the number of proliferating cells in the periodontal ligament was increased close to the cementum. RG1503 treatment induces potent anabolic reactions in the extracellular matrices of the different tissues of the periodontium and recruitment of progenitors. In particular, the cell proliferation close to the root surface suggests the reformation of a functional attachment apparatus. These results demonstrate that RG1503 reverses the degenerative changes induced by inflammation and favors the conditions of a regenerative process. Thus, RGTA, a known matrix component mimetic and protector, may be considered as a new therapeutic tool to regenerate the tissues destroyed by periodontitis.

Tenascin-X is a large extracellular matrix protein that is widely expressed in connective tissues during development and in the adult. Genetically determined deficiency of tenascin-X causes the connective tissue disease Ehlers-Danlos syndrome. These patients show reduced collagen density and fragmentation of elastic fibers in their skin. In vitro studies on the role of tenascin-X in elastic fiber biology are hampered because monolayers of fibroblasts do not deposit tenascin-X and elastic fibers into the extracellular matrix. Here, we applied an organotypic culture model of fibroblasts and keratinocytes to address this issue. We investigated the deposition of tenascin-X and elastin into skin-equivalent in vitro and also in vivo after transplantation onto immunodeficient mice. Whereas tenascin-C and fibrillin-1 were readily expressed in the skin-equivalents before transplantation, tenascin-X and elastin were not present. Three weeks post-grafting, a network of elastin was observed that coincided with the appearance of tenascin-X. At the ultrastructural level, microfibrils were observed, some of which were associated with elastin. Transplanted skin-equivalents containing tenascin-X-deficient fibroblasts showed deposition of immunoreactive elastin in similar quantities and distribution as those containing control fibroblasts. This suggests that tenascin-X is important for the stability and maintenance of established elastin fibers, rather than for the initial phase of elastogenesis. Thus, the transplantation of reconstructed skin on nude mice allows the study of tenascin-X and elastin expression and could be used as a model system to study the potential role of tenascin-X in matrix assembly and stability.

Deficiency of the extracellular matrix protein tenascin-X (TNX) was recently described as the molecular basis of a new, recessive type of Ehlers-Danlos syndrome. Here we report gross abnormalities of the elastic fibers and microfibrils in the dermis of these patients, and reduced dermal collagen content, as determined by quantitative image analysis. The ascending, fine elastic fibers in the papillary dermis were absent or inconspicuous and had few branches. The coarse elastic fibers of the reticular dermis were fragmented and clumped. At the ultrastructural level, irregular and immature elastin fibers and fibers devoid of microfibrils were observed. In TNX-deficient patients the dermal collagen density was reduced, but no structural abnormalities in the collagen fibrils were found. These findings suggest that both elastic fiber abnormalities and reduced collagen content contribute to the observed phenotype in TNX-deficient patients.

OBJECTIVES: Transforming growth factor-beta (TGF-beta), a potent regulator of wound healing and scar formation, is thought to have a key role in the response to arterial injury. Latent TGF-beta binding proteins (LTBPs), members of the fibrillin superfamily, govern TGF-beta1 release, targeting and activation in vitro and also play a role as structural components of fibrillin-rich microfibrils. Despite the potential of LTBPs to modulate the response to arterial injury through either or both of these mechanisms, as yet their expression and function in the injured vasculature remain poorly defined. METHODS: In this study, a porcine model of coronary angioplasty was used to investigate LTBP-1 and LTBP-2 synthesis and their association with TGF-beta 1 and fibrillin-1. RESULTS: After angioplasty, increased LTBP-1 and LTBP-2 immunostaining was detected in a similar distribution to increased TGF-beta 1 expression in the neointima and in the neoadventitia. Overnight organ cultures revealed the formation of large latent TGF-beta 1 complexes containing LTBP-1. Increased LTBP-1 proteolysis after arterial injury correlated with increased active and latent TGF-beta levels. LTBP-2 synthesis increased in response to arterial injury but was neither present in large latent complexes nor proteolytically processed. LTBP-1 and LTBP-2 both co-localised to fibrillin-rich fibrillar structures in the neointima and adventitia. CONCLUSIONS: These data suggest that LTBP-1 may have a TGF-beta 1 binding role in the arterial response to injury, and that LTBP-1 and LTBP-2 may have a structural role in association with microfibrils within the developing neointimal lesion. LTBP-1 proteolysis is potentially an important regulatory step for TGF-beta activation in the vasculature and inhibition of proteolysis could represent a novel therapeutic modality for controlling the arterial injury response.

The molecular basis for Marfan's syndrome (MS), a heritable disorder of connective tissue, is now known to reside in mutations in FBN1, the gene for fibrillin-1. Classic phenotypic manifestations of MS include several skeletal abnormalities associated primarily with overgrowth of long bones. As a first step towards understanding how mutations in FBN1 result in skeletal abnormalities, the developmental expression of fibrillin-1 (Fib-1) in human skeletal tissues is documented using immunohistochemistry and monoclonal antibodies demonstrated here to be specific for Fib-1. At around 10-11 weeks of fetal gestation, Fib-1 is limited in tissue distribution to the loose connective tissue surrounding skeletal muscle and tendon in developing limbs. By 16 weeks, Fib-1 is widely expressed in developing limbs and digits, especially in the perichondrium, but it is apparently absent within cartilage matrix. Fib-1 appears as a loose meshwork of fibers within cartilage matrix by 20 weeks of fetal gestation. Until early adolescence, Fib-1 forms loose bundles of microfibrils within cartilage. However, by late adolescence, broad banded fibers composed of Fib-1 are found accumulated pericellularly within cartilage. Because these fibers can be extracted from cartilage using dissociative conditions, we postulate that they are laterally packed and crosslinked microfibrils. On the basis of these findings, we suggest that the growth-regulating function of Fib-1 may reside persistently within the perichondrium. In addition, the accumulation of special laterally crosslinked Fib-1 microfibrils around chondrocytes during late adolescence suggests that growth-regulating activities may also be performed by Fib-1 at these sites.