Absence of autoantibodies against correctly folded recombinant fibrillin-1 protein in systemic sclerosis patients

Autoantibodies against short recombinant fragments of fibrillin-1 produced in bacterial expression systems have been found in tight-skin mouse, systemic sclerosis, mixed connective tissue disease, and primary pulmonary hypertension syndrome. In patients with scleroderma, the frequency of anti-fibrillin-1 antibodies was 42% in Caucasians. Until now it has been unclear whether this immune response has a primary function in disease pathogenesis or is a secondary phenomenon. In the present study we analyzed the frequency of autoantibodies against two overlapping recombinant polypeptides spanning the N-terminal and C-terminal halves of human fibrillin-1, which were produced in human embryonic kidney (HEK-293) cells. Correct three-dimensional structures of the recombinant fibrillin-1 polypeptides were shown by electron microscopy and immunoreactivity with antibodies. Screening of fibrillin-1 antibodies was performed in 41 sera from systemic sclerosis patients and in 44 healthy controls with a Caucasian background. Microtiter plates were coated with the recombinant polypeptides of fibrillin-1 and incubated with 1:100 diluted sera. Positive binding was defined as being more than 2 SD above the mean of the control group. ELISAs showed that none of the sera of patients with systemic sclerosis contained autoantibodies against the N-terminal or C-terminal recombinant fibrillin-1 polypeptide. The data show the absence of autoantibodies against recombinant fibrillin-1 protein in Caucasian systemic sclerosis patients. Because the correct three-dimensional folding of the recombinant proteins has been substantiated by several independent methods, we conclude that autoantibodies against correctly folded fibrillin are not a primary phenomenon in the pathogenesis of systemic sclerosis

Elastin is heterogeneously cross-linked

Elastin is an essential vertebrate protein responsible for the elasticity of force-bearing tissues such as those of the lungs, blood vessels, and skin. One of the key features required for the exceptional properties of this durable biopolymer is the extensive covalent cross-linking between domains of its monomer molecule tropoelastin. To date, elastin's exact molecular assembly and mechanical properties are poorly understood. Here, using bovine elastin, we investigated the different types of cross-links in mature elastin to gain insight into its structure. We purified and proteolytically cleaved elastin from a single tissue sample into soluble cross-linked and noncross-linked peptides that we studied by high-resolution MS. This analysis enabled the elucidation of cross-links and other elastin modifications. We found that the lysine residues within the tropoelastin sequence were simultaneously unmodified and involved in various types of cross-links with different other domains. The Lys-Pro domains were almost exclusively linked via lysinonorleucine, whereas Lys-Ala domains were found to be cross-linked via lysinonorleucine, allysine aldol, and desmosine. Unexpectedly, we identified a high number of intramolecular cross-links between lysine residues in close proximity. In summary, we show on the molecular level that elastin formation involves random cross-linking of tropoelastin monomers resulting in an unordered network, an unexpected finding compared with previous assumptions of an overall beaded structure

Nrf2-mediated fibroblast reprogramming drives cellular senescence by targeting the matrisome

Nrf2 is a key regulator of the antioxidant defense system, and pharmacological Nrf2 activation is a promising strategy for cancer prevention and promotion of tissue repair. Here we show, however, that activation of Nrf2 in fibroblasts induces cellular senescence. Using a combination of transcriptomics, matrix proteomics, chromatin immunoprecipitation and bioinformatics we demonstrate that fibroblasts with activated Nrf2 deposit a senescence-promoting matrix, with plasminogen activator inhibitor-1 being a key inducer of the senescence program. In vivo, activation of Nrf2 in fibroblasts promoted re-epithelialization of skin wounds, but also skin tumorigenesis. The pro-tumorigenic activity is of general relevance, since Nrf2 activation in skin fibroblasts induced the expression of genes characteristic for cancer-associated fibroblasts from different mouse and human tumors. Therefore, activated Nrf2 qualifies as a marker of the cancer-associated fibroblast phenotype. These data highlight the bright and the dark sides of Nrf2 and the need for time-controlled activation of this transcription factor

The Role of Duration Models and Symbolic Representation for Timing in Synthetic Speech

In order to determine priorities for the improvement of timing in synthetic speech this study looks at the role of segmental duration prediction and the role of phonological symbolic representation in the perceptual quality of a text-to-speech system. In perception experiments using German speech synthesis, two standard duration models (Klatt rules and CART) were tested. The input to these models consisted of a symbolic representation which was either derived from a database or a text-to-speech system. Results of the perception experiments show that different duration models can only be distinguished when the symbolic representation is appropriate. Considering the relative importance of the symbolic representation, post-lexical segmental rules were investigated with the outcome that listeners differ in their preferences regarding the degree of segmental reduction. As a conclusion, before fine-tuning the duration prediction, it is important to derive an appropriate phonological symbolic representation in order to improve timing in synthetic speech

On the role of duration prediction and symbolic representation for the evaluation of synthetic speech

In order to determine priorities for the improvement of timing in synthetic speech this study looks at the role of segmental duration prediction and the role of phonological symbolic representation in listeners' preferences. In perception experiments using German speech synthesis, two standard duration models (Klatt rules and CART) were tested. The input to these models consisted of symbolic strings which were either derived from a database or a text-to-speech system. Results of the perception experiments show that different duration models can only be distinguished when the symbolic string is appropriate. Considering the relative importance of the symbolic representation, "post-lexical" segmental rules were investigated with the outcome that listeners differ in their preferences regarding the degree of segmental reduction. As a conclusion, before fine-tuning the duration prediction, it is important to calculate an appropriate phonological symbolic representation in order to improve timing in synthetic speech

Fibroblast-Derived MMP-14 Regulates Collagen Homeostasis in Adult Skin

Proteolytic activities in the extracellular matrix by the matrix metalloproteinase (MMP)-14 have been implicated in the remodeling of collagenous proteins during development. To analyze the function of fibroblast-derived MMP-14 in adult skin homeostasis, we generated mice with inducible deletion of MMP-14 in the dermal fibroblast (MMP-14Sf–/–). These mice are smaller and display a fibrosis-like phenotype in the skin. The skin of these mice showed increased stiffness and tensile strength but no altered collagen cross-links. In vivo, we measured a significantly increased amount of collagen type I accumulated in the skin of MMP-14Sf–/– mice without an increase in collagen fibril diameters. However, bleomycin-induced fibrosis in skin proceeded in a comparable manner in MMP-14Sf+/+ and MMP-14Sf–/– mice, but resolution over time was impaired in MMP-14Sf–/– mice. Increased accumulation of collagen type I was detected in MMP-14Sf–/– fibroblasts in culture without significant enhancement of collagen de novo synthesis. This points to a degradative but not synthetic phenotype. In support of this, MMP-14Sf–/– fibroblasts lost their ability to process fibrillar collagen type I and to activate proMMP-2. Taken together, these data indicate that MMP-14 expression in fibroblasts plays a crucial role in collagen remodeling in adult skin and largely contributes to dermal homeostasis underlying its pathogenic role in fibrotic skin disease

Activin-mediated alterations of the fibroblast transcriptome and matrisome control the biomechanical properties of skin wounds

Matrix deposition is essential for wound repair, but when excessive, leads to hypertrophic scars and fibrosis. The factors that control matrix deposition in skin wounds have only partially been identified and the consequences of matrix alterations for the mechanical properties of wounds are largely unknown. Here, we report how a single diffusible factor, activin A, affects the healing process across scales. Bioinformatics analysis of wound fibroblast transcriptome data combined with biochemical and histopathological analyses of wounds and functional in vitro studies identify that activin promotes pro-fibrotic gene expression signatures and processes, including glycoprotein and proteoglycan biosynthesis, collagen deposition, and altered collagen cross-linking. As a consequence, activin strongly reduces the wound and scar deformability, as identified by a non-invasive in vivo method for biomechanical analysis. These results provide mechanistic insight into the roles of activin in wound repair and fibrosis and identify the functional consequences of alterations in the wound matrisome at the biomechanical level.ISSN:2041-172