Fibrotic changes in pediatric clubfoot provide an opportunity to improve corrective therapy and prevent relapses with targeted drugs. This study defines the parameters of clubfoot fibrosis and presents a unique analysis of a simple pseudo-3D in vitro model for disease-specific high-throughput drug screening experiments. The model combines clubfoot-derived fibroblasts with a biomimetic cultivation environment induced by the water-soluble polymers Ficoll and Polyvinylpyrrolidone, utilizing the principle of macromolecular crowding. We achieved higher conversion of soluble collagen into insoluble collagen, accelerated formation of the extracellular matrix layer and upregulated fibrosis-related genes in the mixed Ficoll environment. To test the model, we evaluated the effect of a potential antifibrotic drug, minoxidil, emphasizing collagen content and cross-linking. While the model amplified overall collagen deposition, minoxidil effectively blocked the expression of lysyl hydroxylases, which are responsible for the increased occurrence of specific collagen cross-linking in various fibrotic tissues. This limited the formation of collagen cross-link in both the model and control environments. Our findings provide a tool for expanding preclinical research for clubfoot and similar fibroproliferative conditions.

• MeSH
• biomimetika metody   MeSH
• extracelulární matrix metabolismus   účinky léků   MeSH
• fibroblasty * metabolismus   účinky léků   MeSH
• fibróza * farmakoterapie   MeSH
• kolagen * metabolismus   chemie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• pes equinovarus * metabolismus   farmakoterapie   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kolagen * MeSH

Valvular heart disease leads to ventricular pressure and/or volume overload. Pressure overload leads to fibrosis, which might regress with its resolution, but the limits and details of this reverse remodeling are not known. To gain more insight into the extent and nature of cardiac fibrosis in valve disease, we analyzed needle biopsies taken from the interventricular septum of patients undergoing surgery for valve replacement focusing on the expression and distribution of major extracellular matrix protein involved in this process. Proteomic analysis performed using mass spectrometry revealed an excellent correlation between the expression of collagen type I and III, but there was little correlation with the immunohistochemical staining performed on sister sections, which included antibodies against collagen I, III, fibronectin, sarcomeric actin, and histochemistry for wheat germ agglutinin. Surprisingly, the immunofluorescence intensity did not correlate significantly with the gold standard for fibrosis quantification, which was performed using Picrosirius Red (PSR) staining, unless multiplexed on the same tissue section. There was also little correlation between the immunohistochemical markers and pressure gradient severity. It appears that at least in humans, the immunohistochemical pattern of fibrosis is not clearly correlated with standard Picrosirius Red staining on sister sections or quantitative proteomic data, possibly due to tissue heterogeneity at microscale, comorbidities, or other patient-specific factors. For precise correlation of different types of staining, multiplexing on the same section is the best approach.

• Klíčová slova
• Collagen, Fibronectin, Fibrosis, Pressure overload, Valvular heart disease,
• MeSH
• aortální insuficience metabolismus   patologie   chirurgie   MeSH
• aortální stenóza * metabolismus   patologie   chirurgie   MeSH
• extracelulární matrix - proteiny * metabolismus   analýza   MeSH
• fibróza * metabolismus   patologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mezikomorová přepážka patologie   metabolismus   MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• extracelulární matrix - proteiny * MeSH

Congenital clubfoot is a complex musculoskeletal deformity, in which a stiff, contracted tissue forms in the medial part of the foot. Fibrotic changes are associated with increased collagen deposition and lysyl oxidase (LOX)-mediated crosslinking, which impair collagen degradation and increase the tissue stiffness. First, we studied collagen deposition, as well as the expression of collagen and the amount of pyridinoline and deoxypyridinoline crosslinks in the tissue of relapsed clubfoot by immunohistochemistry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA). We then isolated fibroblast-like cells from the contracted tissue to study the potential inhibition of these processes in vitro. We assessed the effects of a LOX inhibitor, β-aminopropionitrile (BAPN), on the cells by a hydroxyproline assay, ELISA, and Second Harmonic Generation imaging. We also evaluated the cell-mediated contraction of extracellular matrix in 3D cell-populated collagen gels. For the first time, we have confirmed significantly increased crosslinking and excessive collagen type I deposition in the clubfoot-contracted tissue. We successfully reduced these processes in vitro in a dose-dependent manner with 10-40 µg/mL of BAPN, and we observed an increasing trend in the inhibition of the cell-mediated contraction of collagen gels. The in vitro inhibitory effects indicate that BAPN has good potential for the treatment of relapsed and resistant clubfeet.

• Klíčová slova
• beta-aminopropionitrile (BAPN), collagen, congenital idiopathic Talipes equinovarus, contraction, crosslinking, fibrosis, relapsed clubfoot,
• MeSH
• aminopropionitril farmakologie   MeSH
• fibroblasty účinky léků   MeSH
• kolagen chemie   MeSH
• lidé MeSH
• lysyloxidasa antagonisté a inhibitory   MeSH
• pes equinovarus farmakoterapie   metabolismus   patologie   MeSH
• předškolní dítě MeSH
• reagencia zkříženě vázaná farmakologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminopropionitril MeSH
• kolagen MeSH
• LOX protein, human MeSH Prohlížeč
• lysyloxidasa MeSH
• reagencia zkříženě vázaná MeSH

BACKGROUND: The rumen microbiota is one of the most complex consortia of anaerobes, involving archaea, bacteria, protozoa, fungi and phages. They are very effective at utilizing plant polysaccharides, especially cellulose and hemicelluloses. The most important hemicellulose decomposers are clustered with the genus Butyrivibrio. As the related species differ in their range of hydrolytic activities and substrate preferences, Butyrivibrio fibrisolvens was selected as one of the most effective isolates and thus suitable for proteomic studies on substrate comparisons in the extracellular fraction. The B. fibrisolvens genome is the biggest in the butyrivibria cluster and is focused on "environmental information processing" and "carbohydrate metabolism". METHODS: The study of the effect of carbon source on B. fibrisolvens 3071 was based on cultures grown on four substrates: xylose, glucose, xylan, xylan with 25% glucose. The enzymatic activities were studied by spectrophotometric and zymogram methods. Proteomic study was based on genomics, 2D electrophoresis and nLC/MS (Bruker Daltonics) analysis. RESULTS: Extracellular β-endoxylanase as well as xylan β-xylosidase activities were induced with xylan. The presence of the xylan polymer induced hemicellulolytic enzymes and increased the protein fraction in the interval from 40 to 80 kDa. 2D electrophoresis with nLC/MS analysis of extracellular B. fibrisolvens 3071 proteins found 14 diverse proteins with significantly different expression on the tested substrates. CONCLUSION: The comparison of four carbon sources resulted in the main significant changes in B. fibrisolvens proteome occurring outside the fibrolytic cluster of proteins. The affected proteins mainly belonged to the glycolysis and protein synthesis cluster.

• Klíčová slova
• Butyrivibrio fibrisolvens, Carbon sources, Proteomics, Rumen,
• Publikační typ
• časopisecké články MeSH