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Role of endothelial and vascular smooth muscle cells in the origin, progression and therapy of vascular diseases
Chlupáč, Jaroslav ; Bačáková, Lucie (advisor) ; Piťha, Jan (referee) ; Sedmera, David (referee)
Introduction: Vascular surgery for atherosclerosis is confronted by the lack of a suitable bypass material. Synthetic vascular prostheses include polyethylene terephthalate (PET) and expanded polytetrafluoroethylene (ePTFE). However, these materials become thrombosed in small-caliber applications (<6 mm) because of the lack of an endothelium. The objectives of this study were to make modifications to clinically-used PET vascular prostheses with tissue-engineered surfaces to improve their bio-compatibility towards vascular smooth muscle cells (VSMC) and endothelial cells (EC). Methods: Blood coagulation protein fibrin (Fb) and extracellular matrix proteins collagen (Co), laminin (LM) and fibronectin (FN) were used. Cell adhesive assemblies were prepared: Co, Co/LM, Co/FN, Co/Fb, Co/Fb/FN. Cell culture experiments were performed: (1) planar static, (2) planar dynamic with simulation of blood flow, (3) tubular dynamic, and (4) animal porcine implantation. Results: The growth of EC and VSMC on commercial prostheses (ePTFE, PET and PET/Co) was low. The growth of both cell types was lower on PET/Co than on PET. After modification with protein assemblies, the highest numbers of EC were reached on PET/Co and on PET/Co +Co/Fb. There was no difference in the densities of VSMC among various assemblies. The...
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Role of Nkx2.5 in development and electrophysiology of the mouse heart
Hámor, Peter ; Sedmera, David (advisor) ; Neckář, Jan (referee)
Role of Nkx2.5 in development and electrophysiology of the mouse heart Prague 2016 Peter Hámor, B.S. ABSTRACT The objective of this thesis is to investigate the role of Nkx2.5 gene dosage on electrophysiology of the mouse heart in prenatal stage of its development. The main goal of this work is to search for differences in conduction of electric impulses through the embryonic mouse hearts of different genotype. Special method of capturing the conduction of electric impulse through myocardium, called optical mapping, was used to visualize the electrical activity. Thanks to this method I was able to construct images and videos capturing the spread of the impulse with identification of the beginning of the activation and its direction in the heart. These outputs, or optical maps, help to define anomalies and defects in mutants compared with a normal functioning heart. The thesis focuses on the expression of the transcription factor Nkx2.5 and regulatory components related with the correct formation and physiology of the heart until 9.5 days post coitum. Embryos at this developmental stage were optically mapped and analysed according to their genotype. While the wild type and heterozygote mouse embryos exhibited high degree of similarity, the homozygous mutants were dramatically different. Considering this work...
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Multipotent mesenchymal stromal cells in orthopedic: Potentiation of bone healing
Stehlík, David ; Trč, Tomáš (advisor) ; Janíček, Pavel (referee) ; Sedmera, David (referee)
The aim of the thesis was development of an innovative treatment of bone defects. Human multipotent mesenchymal stromal cells (MSC) play a crucial role in bone healing. Clinical applications of MSC require large amount of cells, which could be obtained by autologous expansion of MSC harvested from bone marrow. As a first step, the standard protocol of MSC expansion based on αMEM medium and fetal bovine serum (FBS) was used. Experiments replacing FBS by pooled human serum (HS) in the culture medium concluded in patenting of a new MSC cultivation protocol (EU 1999250, CR 301141). This one-step cultivation protocol and xenogeneic protein-free cultivation medium is based on CellGro® for Hematopoietic Cells' Medium, HS, human recombinant growth factors, dexamethasone, insulin and ascorbic acid. The preclinical in vitro and in vivo experiments with MSC from both expansion protocols were carried out. Fibrillar polylactic scaffolds were seeded with MSC, cultured, differentiated and implanted in immunodeficient mice (NOD/LtSz-Rag1-). Bone-like mineralized tissue containing vessels was observed. The MSC cultured according to patented method were classified as Advanced-therapy Medicinal Product and has to fulfil the European Medicines Agency regulations to enter the clinical trials. Nevertheless the use of MSC seems...
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Role of Nkx2.5 on development and electrophysiology of the mouse heart
Hámor, Peter ; Sedmera, David (advisor) ; Neckář, Jan (referee)
Role of Nkx2.5 on development and electrophysiology of the mouse heart Prague 2015 Bc. Peter Hámor ABSTRACT The objective of this thesis is to investigate the role of Nkx2.5 gene dosage on electrophysiology of the mouse heart in prenatal stage of its development, in which the physiological functions of the heart fail to function properly. The main goal of this work is to search for differences in conduction of electric impulses through the embryonic mouse heart according to their genotype. Special method of capturing the conduction of electric impulse through myocardium was used for this purpose, called optical mapping. Thanks to this method I was able to construct images and videos capturing transition of the impulse with marked beginning of the activation and its direction in the heart. These outputs, or optical maps, help to define anomalies and defects compared with a normal functioning heart. The thesis focuses on the expression of the transcription factor Nkx2.5 and regulatory components related with the correct formation and physiology of the heart until 9.5 days post coitum. Individuals in this developmental stage were optically mapped and compared according to their genotypes - homozygous non-mutant, heterozygote and homozygous mutant mouse embryos exhibited some degree of similarity, while other...
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Mechanisms of growth of the cardiac conduction system
Skuhrová, Kristýna ; Sedmera, David (advisor) ; Elsnicová, Barbara (referee)
The ability to produce regular rhythm and independence of nervous system and are some of the features of the cardiac conduction system. The conduction system comprises the sinoatrial node, internodal tracts, the atrioventricular node, the atrioventricular (His) bundle, its right and left branches, and the terminal network of Purkinje fibers. However, this system is frequently the cause of the cardiac rhythm disorders, i.e., arrhythmias. There are many unanswered questions about the conduction system, even though its development is closely connected to the growth of the whole heart. The heart undergoes many dramatic changes during its development, such as modification of linear heart tube into the mature four-chamber organ. Looping and forming chambers cause change of localization first "pacemaker" from the caudal end of the heart tube to the area of the right atrium. Prenatal growth of the heart is based upon cell proliferation or hyperplasia. The cell divisions are rapidly stopped soon after birth and the cells start to grow by increase in volume, i.e., hypertrophy. The cells of some species can expand hyperplasia or hypertrophy in early postnatal period. The mouse is one of the organisms with the cell expansion provided by combination of proliferation and hypertrophy. Most of the adult...
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Analysis of heart rhythm disturbances in fetuses
Tomek, Viktor ; Ošťádal, Bohuslav (advisor) ; Sedmera, David (referee) ; Calda, Pavel (referee)
Analysis of heart rhythm distrubances V.Tomek Abstract Objective: The dissertation was focused on prenatal cardiology. Prenatal cardiology is a relatively young field, which is focused on diagnosis and treatment of fetal heart. In the thesis, I focused on the issue of cardiac arrhythmias: pathophysiology, diagnosis and therapeutic management of prenatally heart rhythm disturbances. The most frequent type of prenatal arrhythmias are fetal isolated complete atrioventricular (AV) block and supraventricular tachycardia (SVT). Although these rhythm heart disorders are relatively rare, but can lead to heart failure and are important causes of fetal mortality. Methods: We performed measurement of mechanical atrioventricular conduction time intervals in human foetuses assessed by Doppler echocardiography and provided reference values. We compared reference values with foetuses of mothers with anti-SSA Ro/SSB La autoantibodies, being in risk of isolated congenital heart block development. We collaborated in a multinational, multicenter retrospective study of 175 fetuses diagnosed with AV block (2000 -2007) to analyse the influence of the treatment. We compared 2 treatment protocols (Prague and London) to find out the optimal treatment strategy of fetuses with supraventricular tachycardia. The severity of heart...
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Developmental mechanisms of arrhythmias - role of connexins in arrhythmogenesis
Beneš, Jiří ; Sedmera, David (advisor) ; Kolář, František (referee) ; Král, Jiří (referee)
Developmental mechanisms of arrhythmias - role of connexins in arrhythmogenesis MUDr.Jiří Beneš Abstract: Objective: The aim of this study is an improvement of our knowledge concerning the role of connexins in arrhythmogenesis. The main focus is on the role of connexin40 (Cx40) in heart development in mice and changes in connexin43 expression in volume overload heart failure rat model. Methods: The infuence of Cx40 on heart develoment was studied on transgenic mouse Cx40:GFP model using the method of optical mapping. Volume overload heart failure was examined in the rats with aortocaval shunt. Morphological changes in hearts were examined using imunofluorescence microscopical techniques. Results: In the atria, Cx40 is important especially during the early stages. Cx43 can partially substitute its function from 12.5 embryonic day on. Cx40 deficiency leads to decreased conduction velocity and ectopic sites of activation. Absence of Cx40 in ventricular conduction system leads to the development of right bunde branch block. Volume overload in rats leads to excentric hypertrofy and later to heart failure. We described morphological as well as microscopical changes in failing hearts. Without the presence of fibrosis, the main arrhythmogenic substrate we found was reduced amount of Cx43 and its dephosphorylation....
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Effects of Mechanical Loading on Early Conduction System Differentiation
Machálek, Jakub ; Sedmera, David (advisor) ; Neckář, Jan (referee)
6 ABSTRAKT Kardiovaskulární onemocnění představují celosvětově nejčastější příčinu úmrtí. Arytmie neboli poruchy srdečního rytmu k této mortalitě přispívají nemalou měrou. Výzkum v oblasti morfologie převodního systému srdečního má více jak stoletou historii, ale oblast ontogeneze není ani v dnešní době dostatečně prozkoumána. Přitom pochopení mechanismů indukce pracovního myokardu ve specializovanou tkáň by mohl být jeden z klíčů k otevření dveří nových možností diagnostiky a terapie onemocnění v této oblasti. Cílem této práce je pochopení vlivu mechanické zátěže na vznik a vývoj převodního systému srdečního. Chci prokázat, že mechanické zatížení srdce krevním oběhem hraje v embryonálním vývoji nezastupitelnou roli v diferenciaci převodního systému srdečního (PSS). Jako model pozorování jsem si vybral kuřecí srdce. Při porovnání dat získaných na srdci ptáků a savců můžeme najít drobné odlišnosti v detailech, nicméně hlavní principy a mechanismy se zdají být stejné. Kuřecí embryo se vyvíjí 21 dní, přičemž toto časové údobí lze podle Hamburgera a Hamiltona rozdělit do 46 stádií. Zpočátku představuje srdce pouze primitivní trubici, která se stáčí v srdeční kličku. Posléze je přeměněna na zralý čtyřoddílový orgán charakteristický pro vyšší obratlovce. Tyto morfologické změny korelují i se změnami šíření...
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Effect of hypoxia on embryonic development of the heart and coronary circulation
Křížová, Petra ; Naňka, Ondřej (advisor) ; Sedmera, David (referee)
Human, as all multicelullar organisms, is dependant on oxygen availability. Hypoxia is a stimulus which must the organism adapt from early development. In prenatal life fundaments of circulation originate separately and first than they interconnect in complete system of vessel tubes incorporating heart. Haemodynamic - as one of the factors - soon takes part in vessels system development. Restricted blood flow results in vascular bed regression; on the contrary higher flow promotes its development in prenatal life as well as in adulthood. The aim of our study was to investigate the effect of hypoxia on coronary arterial tree formation and myocardial development and to explain embryonic lethality. Previous studies found ventricular dilatation and increased mortality with signs of heart failure. We used quail embryos (Coturnix coturnix japonica). Eggs were incubated under hypoxic conditions (16% O2). Controls were incubated in normal oxygen tension (21% O2). The effect of hypoxia was analyzed 5 - 9 day of embryo development. Coronary system was visualized by injecting Indian ink. Proliferation was measured using BrdU labelling. For histological analysis we used standard haematoxylin and eosin labelling. For further analysis we used immunohistochemical staining with antibodies against sarcomeric actin...
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