University of Rijeka. Department of Biotechnology.
Abstract
Angiogenesis is the process of new blood vessel formation, crucial for the development of an embryo as well as for organ growth and wound healing later in the adulthood. A key factor responsible for the stimulation of angiogenesis is VEGF165, the most potent splicing isoform of VEGF-A. Based on recent results obtained in the host laboratory, the driving hypothesis standing beyond this work was to investigate why endothelial cells in the skeletal muscle and in the neonatal heart respond to AAV-VEGF165 inducing angiogenesis, whereas in the adult heart they do not. For this purpose, we investigated the responsiveness of adult cardiac and neonatal endothelial cells (ECs), as well as of muscle ECs to the proangiogenic factor VEGF in vitro. Based on the observation that cardiac ECs lose their proliferative capacity at day 7 after birth, we performed a transcriptome analysis of the three EC populations and identified a few differentially expressed genes (DEGs). Particularly, we focused on Dhcr24, the most interesting gene among DEG between pre-natal and post-natal ECs. We developed a genome editing strategy to investigate its functional role in angiogenesis. In the first set of experiments, the proliferation of adult and neonatal cardiac ECs and muscle ECs was assessed after VEGF administration at different time points, followed by immunofluorescence staining and image acquisition. Interestingly and different from their in vivo behaviour, adult cardiac ECs proliferated in response to VEGF at both time points, suggesting that some inhibitory factor might block their response in the adult heart in vivo. In the second set of experiments, the CRISPR/Cas9 technology was used to silence Dhcr24 in SVEC and LG cell lines. Successful knockout was confirmed by the downregulation of Dhcr24 protein expression in Western blot. Development of an efficient genome editing strategy could offer an opportunity to genetically modify Dhcr24 in quiescent ECs and test its ability to restore the pro-angiogenic phenotype.Angiogeneza je proces nastajanja novih krvnih žila, ključan tijekom razvoja embrija, te rasta organa i zacjeljivanja rana u odrasloj dobi. Ključni čimbenik koji je odgovoran za stimulaciju angiogeneze je VEGF165, najpotentnija izoforma alternativnog prekrajanja VEGF-A. Na temelju nedavno dobivenih rezultata u našem laboratoriju, glavna hipoteza ovog rada bila je istražiti zašto endotelne stanice (ES) u skeletnim mišićima i u neonatalnom srcu reagiraju na AAV-VEGF165 induciranu angiogenezu, dok one u odraslom srcu ne. U tu smo svrhu istražili odgovor odraslih i neonatalnih srčanih ES, kao i mišićnih ES mišića na proangiogeni čimbenik VEGF in vitro. Na temelju opažanja da srčane ES gube sposobnost proliferacije 7. dan nakon rođenja, izvršili smo transkripcijsku analizu tri populacije ES i identificirali nekoliko diferencijalno eksprimiranih gena (DEG). Posebno smo se usredotočili na Dhcr24, najzanimljiviji gen između DEG između prenatalnih i postnatalnih ES. Razvili smo strategiju uređivanja genoma kako bismo istražili njegovu funkcionalnu ulogu u angiogenezi. U prvoj skupini eksperimenata, proliferacija odraslih i neonatalnih srčanih ES i mišićnih ES određena je nakon administracije VEGF-a u različitim vremenskim točkama, nakon čega je slijedila imunofluorescencija i mikroskopija. Zanimljivo, za razliku od ponašanja in vivo, odrasle srčane ES su proliferirale kao odgovor na VEGF u obje vremenske točke, sugerirajući prisustvo nekog inhibitornog čimbenika koji može blokirati odgovor na VEGF u odraslom srcu in vivo. U drugoj skupini eksperimenata, CRISPR/Cas9 tehnologija korištena je za utišavanje Dhcr24 u SVEC i LG staničnim linijama. Uspješan knockout potvrđen je smanjenjem ekspresije Dhcr24 proteina u Western blotu. Razvijanje učinkovite strategije za uređivanje genoma moglo bi pružiti priliku za genetsko modificiranje Dhcr24 u ES u stanju mirovanja te ispitati ima li sposobnost vraćanja proangiogenog fenotipa
