Dissertation committee:
Γαϊτανάκη Αικατερίνη, Καθηγήτρια, Εθνικό & Καποδιστριακό Πανεπιστήμιο Αθηνών
Ευθυμιόπουλος Σπυρίδων, Καθηγητής, Εθνικό & Καποδιστριακό Πανεπιστήμιο Αθηνών
Μπέης Δημήτριος, Ερευνητής Β’, Ίδρυμα Ιατροβιολογικών Ερευνών της Ακαδημίας Αθηνών
Ντέντος Σκαρλάτος, Αναπληρωτής Καθηγητής, Εθνικό & Καποδιστριακό Πανεπιστήμιο Αθηνών
Παπαζαφείρη Παναγιώτα, Αναπληρώτρια Καθηγήτρια. Εθνικό & Καποδιστριακό Πανεπιστήμιο Αθηνών
Σίδερης Διαμάντης, Αναπληρωτής Καθηγητής, Εθνικό & Καποδιστριακό Πανεπιστήμιο Αθηνών
Μαυροειδής Μανώλης, Ερευνητής Γ’, Ίδρυμα Ιατροβιολογικών Ερευνών της Ακαδημίας Αθηνών
Summary:
Cardiovascular diseases are nowadays one of the leading causes of death in the developed world. Also very important is the contribution of angiogenesis to the development of cancerous tumors. It is therefore necessary to contribute to the basic research in order to better study the mechanisms of these diseases but also to discover new drugs related to angiogenesis. In recent years, the zebrafish animal model has played an important role in these conditions.
Zebrafish (Danio rerio) is widely used as an animal model to understand the pathophysiology of cardiovascular diseases. Here, we present the adult cardiac phenotype of weak atrium, myh6-/-, which carry mutations in the zebrafish atrial myosin heavy chain. Homozygous mutants survive to adulthood and are fertile despite their initial weak atrial beat. In adult mutants, the atrium remains hypoplastic and shows elastin deposition while mutant ventricles exhibit increased size. In mammals, hypertrophy is the most common mechanism resulting in cardiomegaly. Using immunohistochemistry and confocal microscopy to measure cardiomyocyte cell size, density and proliferation, we show that the enlargement of the myh6-/- ventricle is predominantly due to hyperplasia. However, we identified similar transcriptional profiles to the mammalian hypertrophy response via RT-PCR of the hyperplastic ventricles. Furthermore, we show activation of the ER-stress pathway by western blot analysis. In conclusion, we can assume, based on our model, that molecular signaling pathways associated with hypertrophy in mammals, in combination with ER-stress activation, result in hyperplasia in zebrafish. In addition, to our knowledge, this is the first time to report elastin deposition in the atrium.
In parallel, we studied small molecules that inhibit angiogenesis and are attractive drug candidates for cancer, retinopathies, and age-related macular degeneration. In vivo phenotypic screening in zebrafish (Danio rerio) emerges as a powerful methodology to identify and optimize novel compounds with pharmacological activity. Zebrafish provides several advantages for in vivo phenotypic screens especially for angiogenesis, since it develops rapidly, externally, and does not rely on a functional cardiovascular system to survive for several days during development. In this study, we utilize a transgenic line that allows the noninvasive monitoring of angiogenesis at a cellular level. The inhibition of angiogenesis can be observed under a fluorescent stereoscope and quantified. To exemplify the versatility and robustness of the zebrafish screen, we have employed a series of 60 novel compounds that were designed based on a potent VEGFR2 inhibitor. Herein, we report their structure-based design, synthesis, and in vivo zebrafish screening for optimal activity, toxicity, and off-target effects, which revealed six reversible inhibitors of angiogenesis.
Finally, in the present doctoral dissertation, genes were attempted to play a role in the morphogenesis of the heart valves and to demonstrate their relationship through targeted mutagenesis with the help of the CRISPR-Cas system and to create a transgenic zebrafish to respond to shear stress.
In conclusion, the present thesis shows the great potential of zebrafish for the reconstruction / remodeling of the heart in pathological conditions and proves its ability to be used as an ideal model for the study of cardiovascular diseases.
Keywords:
zebrafish, myh6, VEGFR2, SSRE, heart
