Úloha oxidu dusnatého během embryonálního hojení a regenerace

The study of the mechanisms that control wound healing is an attention-drawing area within the fields of biology and medicine. Wound healing can be usually defined as two basic types. The first type is adult wound healing, which is slow and results in the scar formation. The second type is referred to as embryonic wound healing, which is in contrast fast and scarless. Wound healing is a complicated process that includes many steps, which are regulated by various types of molecules. One of these important molecules is nitric oxide (NO). Its function is usually connected with the regulation of inflammation and angiogenesis during adult wound healing. However, there is currently no information on its role during embryonic wound healing, where the immune and vascular systems are not yet developed. In this work, we explore and describe the role of the NO during the healing of the early embryos. The highest concentration of the NO post wounding is produced during the first 30 minutes after injury. This applies to all developmental stages, from the blastula stage all the way to the swimming tadpole stage. The main role of the NO during embryonic wound healing is the regulation of the gene expression that is connected with the stress response and the regulation of cellular metabolism. Additionally, we...Studium mechanismů, které kontrolují hojení, je velice atraktivní oblastí biologie a medicíny. Hojení ran se obvykle dělí na dva základní typy. Prvním je hojení dospělých jedinců, které je pomalé a končí jizvou. Druhým je hojení embryí, které je velmi rychlé a bez vzniku jizvy. Hojení je složitý proces, který zahrnuje mnoho kroků a je regulován velikým množstvím různých druhů molekul. Jednou z důležitých molekul je oxid dusnatý (NO), jehož funkce je často spojována s regulací zánětu a angiogeneze v průběhu hojení u dospělých jedinců. Tato práce se zabývá popisem úlohy NO v průběhu hojení časných embryí, která ještě nemají vyvinutý imunitní a cévní systém. Bylo pozorováno, že vysoká koncentrace NO je produkována v průběhu prvních 30 minut po poranění od stádia blastuly po plovoucího pulce. Úlohou NO je hlavně regulace exprese genů, které jsou spojené s odpovědí na stres a souvisejí s buněčným metabolismem. Dopady produkce NO během hojení jsou však pozorovatelné i několik hodin po uzavření rány v námi nově popsané třetí fázi embryonálního hojení. Ve třetí fázi probíhá remodelace poraněné tkáně a NO reguluje expresi a aktivitu matrix metaloproteináz a migraci naivních buněk imunitního systému do místa poranění. Předkládaná práce ukazuje na nový mechanismus působení NO v procesu embryonálního hojení a...Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyFaculty of SciencePřírodovědecká fakult

Reprogramming of the developing heart by Hif1a-deficient sympathetic system and maternal diabetes exposure

IntroductionMaternal diabetes is a recognized risk factor for both short-term and long-term complications in offspring. Beyond the direct teratogenicity of maternal diabetes, the intrauterine environment can influence the offspring’s cardiovascular health. Abnormalities in the cardiac sympathetic system are implicated in conditions such as sudden infant death syndrome, cardiac arrhythmic death, heart failure, and certain congenital heart defects in children from diabetic pregnancies. However, the mechanisms by which maternal diabetes affects the development of the cardiac sympathetic system and, consequently, heightens health risks and predisposes to cardiovascular disease remain poorly understood.Methods and resultsIn the mouse model, we performed a comprehensive analysis of the combined impact of a Hif1a-deficient sympathetic system and the maternal diabetes environment on both heart development and the formation of the cardiac sympathetic system. The synergic negative effect of exposure to maternal diabetes and Hif1a deficiency resulted in the most pronounced deficit in cardiac sympathetic innervation and the development of the adrenal medulla. Abnormalities in the cardiac sympathetic system were accompanied by a smaller heart, reduced ventricular wall thickness, and dilated subepicardial veins and coronary arteries in the myocardium, along with anomalies in the branching and connections of the main coronary arteries. Transcriptional profiling by RNA sequencing (RNA-seq) revealed significant transcriptome changes in Hif1a-deficient sympathetic neurons, primarily associated with cell cycle regulation, proliferation, and mitosis, explaining the shrinkage of the sympathetic neuron population. DiscussionOur data demonstrate that a failure to adequately activate the HIF-1α regulatory pathway, particularly in the context of maternal diabetes, may contribute to abnormalities in the cardiac sympathetic system. In conclusion, our findings indicate that the interplay between deficiencies in the cardiac sympathetic system and subtle structural alternations in the vasculature, microvasculature, and myocardium during heart development not only increases the risk of cardiovascular disease but also diminishes the adaptability to the stress associated with the transition to extrauterine life, thus increasing the risk of neonatal death

Human bone marrow stromal cells: the impact of anticoagulants on stem cell properties

Background: Bone marrow stromal cells (BMSCs) are the source of multipotent stem cells, which are important for regenerative medicine and diagnostic purposes. The isolation of human BMSCs from the bone marrow (BM) cavity using BM aspiration applies the method with collection into tubes containing anticoagulants. Interactions with anticoagulants may affect the characteristics and composition of isolated BMSCs in the culture. Thus, we investigated how anticoagulants in isolation procedures and cultivation affect BMSC molecular characteristics.Methods: BM donors (age: 48–85 years) were recruited from the hematology clinic. BM aspirates were obtained from the iliac crest and divided into tubes coated with ethylenediaminetetraacetic acid (EDTA) or heparin anticoagulants. Isolated BMSCs were analyzed by flow cytometry and RNA-seq analysis. Further cellular and molecular characterizations of BMSCs including CFU, proliferation and differentiation assays, cytometry, bioenergetic assays, metabolomics, immunostaining, and RT-qPCR were performed.Results: The paired samples of isolated BMSCs obtained from the same patient showed increased cellular yield in heparin vs. EDTA samples, accompanied by the increased number of CFU colonies. However, no significant changes in molecular characteristics were found between heparin- and EDTA-isolated BMSCs. On the other hand, RNA-seq analysis revealed an increased expression of genes involved in nucleotide metabolism and cellular metabolism in cultivated vs. non-cultivated BMSCs regardless of the anticoagulant, while genes involved in inflammation and chromatin remodeling were decreased in cultivated vs. non-cultivated BMSCs.Conclusion: The type of anticoagulant in BMSC isolation did not have a significant impact on molecular characteristics and cellular composition, while in vitro cultivation caused the major change in the transcriptomics of BMSCs, which is important for future protocols using BMSCs in regenerative medicine and clinics

The role of nitric oxide during embryonic wound healing and regeneration

The study of the mechanisms that control wound healing is an attention-drawing area within the fields of biology and medicine. Wound healing can be usually defined as two basic types. The first type is adult wound healing, which is slow and results in the scar formation. The second type is referred to as embryonic wound healing, which is in contrast fast and scarless. Wound healing is a complicated process that includes many steps, which are regulated by various types of molecules. One of these important molecules is nitric oxide (NO). Its function is usually connected with the regulation of inflammation and angiogenesis during adult wound healing. However, there is currently no information on its role during embryonic wound healing, where the immune and vascular systems are not yet developed. In this work, we explore and describe the role of the NO during the healing of the early embryos. The highest concentration of the NO post wounding is produced during the first 30 minutes after injury. This applies to all developmental stages, from the blastula stage all the way to the swimming tadpole stage. The main role of the NO during embryonic wound healing is the regulation of the gene expression that is connected with the stress response and the regulation of cellular metabolism. Additionally, we..

The role of nitric oxide during embryonic epidermis development of Xenopus laevis

Nitric oxide (NO) is a potent radical molecule that participates in various biological processes such as vasodilation, cell proliferation, immune response and neurotransmission. NO mainly activates soluble guanylate cyclase, leading to cGMP production and activation of protein kinase G and its downstream targets. Here we report the essential role of NO during embryonic epidermis development. Xenopus embryonic epidermis has become a useful model reflecting human epithelial tissue composition. The developing epidermis of Xenopus laevis is formed from specialized ionocytes, multi-ciliated, goblet and small secretory cells. We found that NO is mainly produced in multi-ciliated cells and ionocytes. Production of NO during early developmental stages is required for formation of multi-ciliated cells, ionocytes and small secretory cells by regulation of epidermal-specific gene expression. The data from this research indicate a novel role of NO during development, which supports recent findings of NO production in human mucociliary and epithelium development

Effects of post-mortem and physical degradation on RNA integrity and quality

The precision and reliability of quantitative nucleic acid analysis depends on the quality of the sample analyzed and the integrity of the nucleic acids. The integrity of RNA is currently primarily assessed by the analysis of ribosomal RNA, which is the by far dominant species. The extrapolation of these results to mRNAs and microRNAs, which are structurally quite different, is questionable. Here we show that ribosomal and some nucleolar and mitochondrial RNAs, are highly resistant to naturally occurring post-mortem degradation, while mRNAs, although showing substantial internal variability, are generally much more prone to nucleolytic degradation. In contrast, all types of RNA show the same sensitivity to heat. Using qPCR assays targeting different regions of mRNA molecules, we find no support for 5′ or 3′ preferentiality upon post-mortem degradation

Dysregulation of hypoxia-inducible factor 1α in the sympathetic nervous system accelerates diabetic cardiomyopathy

Abstract Background An altered sympathetic nervous system is implicated in many cardiac pathologies, ranging from sudden infant death syndrome to common diseases of adulthood such as hypertension, myocardial ischemia, cardiac arrhythmias, myocardial infarction, and heart failure. Although the mechanisms responsible for disruption of this well-organized system are the subject of intensive investigations, the exact processes controlling the cardiac sympathetic nervous system are still not fully understood. A conditional knockout of the Hif1a gene was reported to affect the development of sympathetic ganglia and sympathetic innervation of the heart. This study characterized how the combination of HIF-1α deficiency and streptozotocin (STZ)-induced diabetes affects the cardiac sympathetic nervous system and heart function of adult animals. Methods Molecular characteristics of Hif1a deficient sympathetic neurons were identified by RNA sequencing. Diabetes was induced in Hif1a knockout and control mice by low doses of STZ treatment. Heart function was assessed by echocardiography. Mechanisms involved in adverse structural remodeling of the myocardium, i.e. advanced glycation end products, fibrosis, cell death, and inflammation, was assessed by immunohistological analyses. Results We demonstrated that the deletion of Hif1a alters the transcriptome of sympathetic neurons, and that diabetic mice with the Hif1a-deficient sympathetic system have significant systolic dysfunction, worsened cardiac sympathetic innervation, and structural remodeling of the myocardium. Conclusions We provide evidence that the combination of diabetes and the Hif1a deficient sympathetic nervous system results in compromised cardiac performance and accelerated adverse myocardial remodeling, associated with the progression of diabetic cardiomyopathy

Restriction of an intron size en route to endothermy

embargoed until the date of publication, presumably early in Fe

Adverse effects of Hif1a mutation and maternal diabetes on the offspring heart

Abstract Background Epidemiological studies show that maternal diabetes predisposes offspring to cardiovascular and metabolic disorders. However, the precise mechanisms for the underlying penetrance and disease predisposition remain poorly understood. We examined whether hypoxia-inducible factor 1 alpha, in combination with exposure to a diabetic intrauterine environment, influences the function and molecular structure of the adult offspring heart. Methods and results In a mouse model, we demonstrated that haploinsufficient (Hif1a +/−) offspring from a diabetic pregnancy developed left ventricle dysfunction at 12 weeks of age, as manifested by decreased fractional shortening and structural remodeling of the myocardium. Transcriptional profiling by RNA-seq revealed significant transcriptome changes in the left ventricle of diabetes-exposed Hif1a +/− offspring associated with development, metabolism, apoptosis, and blood vessel physiology. In contrast, both wild type and Hif1a +/− offspring from diabetic pregnancies showed changes in immune system processes and inflammatory responses. Immunohistochemical analyses demonstrated that the combination of haploinsufficiency of Hif1a and exposure to maternal diabetes resulted in impaired macrophage infiltration, increased levels of advanced glycation end products, and changes in vascular homeostasis in the adult offspring heart. Conclusions Together our findings provide evidence that a global reduction in Hif1a gene dosage increases predisposition of the offspring exposed to maternal diabetes to cardiac dysfunction, and also underscore Hif1a as a critical factor in the fetal programming of adult cardiovascular disease

Video_2_Reprogramming of the developing heart by Hif1a-deficient sympathetic system and maternal diabetes exposure.mp4

IntroductionMaternal diabetes is a recognized risk factor for both short-term and long-term complications in offspring. Beyond the direct teratogenicity of maternal diabetes, the intrauterine environment can influence the offspring’s cardiovascular health. Abnormalities in the cardiac sympathetic system are implicated in conditions such as sudden infant death syndrome, cardiac arrhythmic death, heart failure, and certain congenital heart defects in children from diabetic pregnancies. However, the mechanisms by which maternal diabetes affects the development of the cardiac sympathetic system and, consequently, heightens health risks and predisposes to cardiovascular disease remain poorly understood.Methods and resultsIn the mouse model, we performed a comprehensive analysis of the combined impact of a Hif1a-deficient sympathetic system and the maternal diabetes environment on both heart development and the formation of the cardiac sympathetic system. The synergic negative effect of exposure to maternal diabetes and Hif1a deficiency resulted in the most pronounced deficit in cardiac sympathetic innervation and the development of the adrenal medulla. Abnormalities in the cardiac sympathetic system were accompanied by a smaller heart, reduced ventricular wall thickness, and dilated subepicardial veins and coronary arteries in the myocardium, along with anomalies in the branching and connections of the main coronary arteries. Transcriptional profiling by RNA sequencing (RNA-seq) revealed significant transcriptome changes in Hif1a-deficient sympathetic neurons, primarily associated with cell cycle regulation, proliferation, and mitosis, explaining the shrinkage of the sympathetic neuron population. DiscussionOur data demonstrate that a failure to adequately activate the HIF-1α regulatory pathway, particularly in the context of maternal diabetes, may contribute to abnormalities in the cardiac sympathetic system. In conclusion, our findings indicate that the interplay between deficiencies in the cardiac sympathetic system and subtle structural alternations in the vasculature, microvasculature, and myocardium during heart development not only increases the risk of cardiovascular disease but also diminishes the adaptability to the stress associated with the transition to extrauterine life, thus increasing the risk of neonatal death.</p