Investigating prenatal hypoxia is difficult in mammals, as there are confounding factors stemming from maternal adaptations and compensatory mechanisms. We have thus established an avian model of hypoxic incubation (starting after 2 days of normoxia, 15% O2, normobaric, until the time of sampling at embryonic day 8) to study embryonic reactions to low oxygen concentration. Our previous studies have shown increased vascularization, oedema, and ventricular wall thinning preceding the lethality at mid-gestation. Analysis of the cardiac proteome after 6 days of hypoxic incubation showed strong upregulation of enzymes involved in anaerobic glycolysis as well as an increase in apoptosis-related proteins, cell adhesion proteins, and secretory activity.

• MeSH
• apoptóza MeSH
• glykolýza MeSH
• hypoxie * metabolismus   MeSH
• kuřecí embryo MeSH
• myokard metabolismus   MeSH
• proteom metabolismus   MeSH
• proteomika * metody   MeSH
• srdce * embryologie   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Maternal diabetes may influence glucose metabolism in adult offspring, an area with limited research on underlying mechanisms. Our study explored the impact of maternal hyperglycemia during pregnancy on insulin resistance development. Adult female Sprague-Dawley rats from control and diabetic mothers were mated, and their female offspring were monitored for 150 days. The rats were euthanized for blood and muscle samples. Maternal diabetes led to heightened insulin levels, increased HOMA-IR, elevated triglycerides, and a raised TyG index in adult offspring. Muscle samples showed a decreased protein expression of AMPK, PI3K, MAPK, DRP1, and MFF. These changes induced intergenerational metabolic programming in female pups, resulting in insulin resistance, dyslipidemia, and glucose intolerance by day 150. Findings highlight the offspring's adaptation to maternal hyperglycemia, involving insulin resistance, metabolic alterations, the downregulation of insulin signaling sensors, and disturbed mitochondrial morphology. Maintaining maternal glycemic control emerges as crucial in mitigating diabetes-associated disorders in adult offspring.

• MeSH
• experimentální diabetes mellitus * metabolismus   patologie   MeSH
• fenotyp * MeSH
• gestační diabetes * metabolismus   patologie   MeSH
• inzulin * metabolismus   krev   MeSH
• inzulinová rezistence * MeSH
• kosterní svaly * metabolismus   patologie   MeSH
• krevní glukóza metabolismus   MeSH
• krysa rodu rattus MeSH
• mitochondrie metabolismus   MeSH
• potkani Sprague-Dawley * MeSH
• signální transdukce * MeSH
• těhotenství MeSH
• zpožděný efekt prenatální expozice * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• 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

Our aim was to study the expression of hypoxia-related proteins as a possible regulatory pathway in the contracted side tissue of relapsed clubfoot. We compared the expression of hypoxia-related proteins in the tissue of the contracted (medial) side of relapsed clubfoot, and in the tissue of the non-contracted (lateral) side of relapsed clubfoot. Tissue samples from ten patients were analyzed by immunohistochemistry and image analysis, Real-time PCR and Mass Spectrometry to evaluate the differences in protein composition and gene expression. We found a significant increase in the levels of smooth muscle actin, transforming growth factor-beta, hypoxia-inducible factor 1 alpha, lysyl oxidase, lysyl oxidase-like 2, tenascin C, matrix metalloproteinase-2, matrix metalloproteinase-9, fibronectin, collagen types III and VI, hemoglobin subunit alpha and hemoglobin subunit beta, and an overexpression of ACTA2, FN1, TGFB1, HIF1A and MMP2 genes in the contracted medial side tissue of clubfoot. In the affected tissue, we have identified an increase in the level of hypoxia-related proteins, together with an overexpression of corresponding genes. Our results suggest that the hypoxia-associated pathway is potentially a factor contributing to the etiology of clubfoot relapses, as it stimulates both angioproliferation and fibroproliferation, which are considered to be key factors in the progression and development of relapses.

• MeSH
• hemoglobin - podjednotky MeSH
• hypoxie komplikace   genetika   MeSH
• lidé MeSH
• matrixová metaloproteinasa 2 genetika   MeSH
• pes equinovarus * genetika   MeSH
• recidiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The knowledge about proteome changes proceeding during protracted opioid withdrawal is lacking. Therefore, the aim of this work was to analyze the spectrum of altered proteins in the rat hippocampus in comparison with the forebrain cortex after 6-month morphine withdrawal. We utilized 2D electrophoretic workflow (Pro-Q® Diamond staining and Colloidal Coomassie Blue staining) which was preceded by label-free quantification (MaxLFQ). The phosphoproteomic analysis revealed six significantly altered hippocampal (Calm1, Ywhaz, Tuba1b, Stip1, Pgk1, and Aldoa) and three cortical proteins (Tubb2a, Tuba1a, and Actb). The impact of 6-month morphine withdrawal on the changes in the proteomic profiles was higher in the hippocampus-14 proteins, only three proteins were detected in the forebrain cortex. Gene Ontology (GO) enrichment analysis of differentially expressed hippocampal proteins revealed the most enriched terms related to metabolic changes, cytoskeleton organization and response to oxidative stress. There is increasing evidence that energy metabolism plays an important role in opioid addiction. However, the way how morphine treatment and withdrawal alter energy metabolism is not fully understood. Our results indicate that the rat hippocampus is more susceptible to changes in proteome and phosphoproteome profiles induced by 6-month morphine withdrawal than is the forebrain cortex.

• Publikační typ
• časopisecké články 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.

• 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

Idiopatický pes equinovarus (angl. clubfoot) je vrozená vada chodidla a bérce. Ačkoli se jedná o nejčastější vrozenou vadu pohybového aparátu, stále není jasná přesná etiopatogeneze tohoto onemocnění. Naše předchozí výsledky ukazují, že patologická tkáň na mediální straně nohy je složena převážně z fibrózních a pro-fibrózních proteinů. V tomto projektu bude aplikováno několik látek, které mají schopnost tuto fibrózní tkáň rozvolnit nebo látky, které zabraňují dalšímu sesíťování fibrózních proteinů za účelem uvolnění napětí v kontrahované tkáni nohy. Aplikace testovaných látek bude probíhat jednak přímo na vyoperovaných tkáních z kontrahované mediální strany postižené nohy a dále pak na buněčných kulturách izolovaných z těchto tkání. Poté budou u vzorků zjištěny změny v morfologii a složení mezibuněčné hmoty a viabilitě buněk a budou charakterizovány jejich mechanické vlastnosti. Výsledky budou statisticky zhodnoceny. Vybrané látky mohou být v budoucnu využity pro terapeutické působení na fibrotickou tkáň a pro vylepšení konzervativní terapie onemocnění pes equinovarus.; Idiopathic pes equinovarus (clubfoot) is a congenital deformity of the lower leg. It is the most common birth defects of the musculoskeletal system; however, the specific etiology of this disease remains unclear. Our previous results show that rigid tissue on the deformed foot is composed mainly of fibrotic and pro-fibrotic proteins. This project suggests application of several compounds which are either able to cleave the fibrous proteins or to prevent further crosslinking of the fibrous tissue in order to release the tension of foot contraction. The compounds will be applied either directly on tissue samples dissected from the contracted side of clubfoot or they will be added into the medium of cell cultures isolated from the contracted tissue. Impact of the treatments on the tissue samples and cell cultures will be analyzed comparing the changes in the extracellular matrix morphology and composition and in cell viability. Biomechanical properties of the treated samples will be also investigated by tensile tests. Selected compounds can improve conservative therapy of the clubfoot.

• MeSH
• antifibrotické látky terapeutické užití   MeSH
• enzymoterapie metody   MeSH
• fibróza farmakoterapie   MeSH
• kolagenasy terapeutické užití   MeSH
• lidé MeSH
• pes equinovarus etiologie   farmakoterapie   patofyziologie   MeSH
• separace buněk MeSH
• Check Tag
• lidé MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• farmakoterapie
• podiatrie
• genetika, lékařská genetika
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Nitrotyrosine formation is caused by presence of reactive oxygen and nitrogen species. Nitration is a very selective process leading to specific modification of only a few tyrosines in protein molecule. 2D electrophoresis and western blotting techniques coupled with mass spectrometry are common methods used in analysis of proteome. Here we describe protocol for analysis of peroxynitrite-induced protein nitration in isolated mitochondria. Mitochondrial proteins are separated by 2D electrophoresis and transferred to nitrocellulose membrane. Membranes are then incubated with antibodies against nitrotyrosine. Positive spots are compared with corresponding Coomassie-stained gels, and protein nitration is confirmed with mass spectrometry techniques.

• MeSH
• 2D gelová elektroforéza metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• imunoblotting metody   MeSH
• kyselina peroxydusitá chemie   MeSH
• mitochondriální proteiny analýza   metabolismus   MeSH
• skot MeSH
• srdeční mitochondrie chemie   metabolismus   MeSH
• tyrosin analogy a deriváty   analýza   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

AIM: Clubfoot is a congenital deformity affecting the musculoskeletal system, resulting in contracted and stiff tissue in the medial part of the foot. Minoxidil (MXD) has an inhibitory effect on lysyl hydroxylase, which influences the quality of extracellular matrix crosslinking, and could therefore be used to reduce the stiffness and to improve the flexibility of the tissue. We assessed the in vitro antifibrotic effects of minoxidil on clubfoot-derived cells. METHODS: Cell viability and proliferation were quantified by xCELLigence, MTS, and LIVE/DEAD assays. The amount of collagen I deposited into the extracellular matrix was quantified using immunofluorescence with subsequent image segmentation analysis, hydroxyproline assay, and Second Harmonic Generation imaging. Extracellular matrix contraction was studied in a 3D model of cell-populated collagen gel lattices. RESULTS: MXD concentrations of 0.25, 0.5, and 0.75 mM inhibited the cell proliferation in a concentration-dependent manner without causing a cytotoxic effect. Exposure to ≥0.5 mM MXD resulted in a decrease in collagen type I accumulation after 8 and 21 days in culture. Changes in collagen fiber assembly were observed by immunofluorescence microscopy and nonlinear optical microscopy (second harmonic generation). MXD also inhibited the contraction of cell-populated collagen lattices (0.5 mM by 22%; 0.75 mM by 28%). CONCLUSIONS: Minoxidil exerts an in vitro inhibitory effect on the cell proliferation, collagen accumulation, and extracellular matrix contraction processes that are associated with clubfoot fibrosis. This study provides important preliminary results demonstrating the potential relevance of MXD for adjuvant pharmacological therapy in standard treatment of relapsed clubfoot.

• MeSH
• kolagen typu I MeSH
• kolagen MeSH
• konzervativní terapie MeSH
• lidé MeSH
• minoxidil farmakologie   MeSH
• pes equinovarus * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

An ideal decellularized allogenic or xenogeneic cardiovascular graft should be capable of preventing thrombus formation after implantation. The antithrombogenicity of the graft is ensured by a confluent endothelial cell layer formed on its surface. Later repopulation and remodeling of the scaffold by the patient's cells should result in the formation of living autologous tissue. In the work presented here, decellularized porcine pericardium scaffolds were modified by growing a fibrin mesh on the surface and inside the scaffolds, and by attaching heparin and human vascular endothelial growth factor (VEGF) to this mesh. Then the scaffolds were seeded with human adipose tissue-derived stem cells (ASCs). While the ASCs grew only on the surface of the decellularized pericardium, the fibrin-modified scaffolds were entirely repopulated in 28 d, and the scaffolds modified with fibrin, heparin and VEGF were already repopulated within 6 d. Label free mass spectrometry revealed fibronectin, collagens, and other extracellular matrix proteins produced by ASCs during recellularization. Thin layers of human umbilical endothelial cells were formed within 4 d after the cells were seeded on the surfaces of the scaffold, which had previously been seeded with ASCs. The results indicate that an artificial tissue prepared by in vitro recellularization and remodeling of decellularized non-autologous pericardium with autologous ASCs seems to be a promising candidate for cardiovascular grafts capable of accelerating in situ endothelialization. ASCs resemble the valve interstitial cells present in heart valves. An advantage of this approach is that ASCs can easily be collected from the patient by liposuction.

• MeSH
• bioprotézy MeSH
• decelularizovaná extracelulární matrix chemie   MeSH
• endoteliální buňky pupečníkové žíly (lidské) MeSH
• endoteliální buňky cytologie   MeSH
• extracelulární matrix metabolismus   MeSH
• fibrinogen chemie   MeSH
• fibronektiny chemie   MeSH
• fluorescenční mikroskopie MeSH
• kmenové buňky MeSH
• kolagen chemie   MeSH
• lidé MeSH
• lipektomie MeSH
• perikard metabolismus   patologie   MeSH
• prasata MeSH
• proliferace buněk MeSH
• srdeční chlopně * MeSH
• techniky in vitro MeSH
• tkáňové inženýrství metody   MeSH
• tkáňové podpůrné struktury * chemie   MeSH
• trombin chemie   MeSH
• tuková tkáň cytologie   MeSH
• vaskulární endoteliální růstový faktor A metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH