Thesis (Ph.D.) - University of Glasgow, 2008. / Ph.D. thesis submitted to the Faculty of Medicine, Division of Cancer Sciences and Molecular Pathology, University of Glasgow, 2007. Includes bibliographical references. Print version also available.

Mutations in the gene encoding the bone morphogenetic protein type 2 receptor (BMPR2) are the most common genetic cause of heritable pulmonary arterial hypertension (PAH). However, given the reduced penetrance of BMPR2 mutations in affected families, a major outstanding question is the identity of additional factors or pathways that are responsible for the manifestation of clinical disease. Furthermore, limited human tissue is available for study and usually only from patients with end-stage disease, making it difficult to understand how PAH is established and progresses. Alternative human models of PAH are therefore required. This thesis describes the characterisation of the first human iPSC-derived smooth muscle cell (iPSC-SMC) model of PAH and elucidates the role of BMPR2 deficiency in establishing PAH-associated phenotypes in iPSC-derived SMCs. To achieve this, I used CRISPR-Cas9 gene editing to generate wild-type and BMPR2+/- iPSC lines with isogenic backgrounds which were subsequently differentiated into lineage-specific iPSC-SMCs that displayed a gene expression profile and responses to BMP signalling akin to those present in distal pulmonary artery smooth muscle cells (PASMCs). Using these cells, I found that the introduction of a single BMPR2 mutation in iPSC-SMCs was sufficient to recapitulate the pro-proliferative and anti-apoptotic phenotype of patient-derived BMPR2+/- PASMCs. However, acquisition of the mitochondrial hyperpolarisation phenotype was enhanced by inflammatory signalling and required an interaction between BMPR2 mutations and environmental stimuli provided by exposure to serum over time. Furthermore, I showed that BMPR2+/- iPSC-SMCs had an altered differentiation state and were less contractile compared to wild-type iPSC-SMCs, phenotypes which have not been observed previously in PAH-derived PASMCs. Finally, RNA sequencing analysis identified genes that were differentially expressed between wild-type and BMPR2+/- iPSC-SMCs and may hence provide further insights into PAH pathobiology. The iPSC-SMC model described in this study will be useful for identifying additional factors involved in disease penetrance and for validating therapeutic approaches that target BMPR2.

The estrogenic flavanone rac-8-prenylnaringenin (8-PN) and 3 derivatives (rac-7-(O-prenyl)naringenin-4′-acetate (7-O-PN), rac-5-(O-prenyl)naringenin-4′,7-diacetate (5-O-PN), and rac-6-(1,1-dimethylallyl)naringenin (6-DMAN) were prepared by chemical synthesis and analyzed with respect to their toxicity and possible cell cycle effects in human acute myeloid leukemia (HL-60) cells. With the exception of 5-O-PN, all the other naringenins showed only weak toxic effects at concentrations below 50 μmol/l. A cell cycle analysis over several cell generations up to 4 days was carried out using the fluorescent dye carboxyfluorescein diacetate N-succinimidyl ester (CFSE) followed by propidium iodide (PI) staining at the end of the experiment. The well-studied flavonol quercetin was included in the analysis as a reference substance. All flavonoids affected cell proliferation, but the extent and the resulting changes in the proliferation pattern were specific for each substance. In contrast to the radical scavenging activity of quercetin, the tested flavanones showed no anti-oxidative properties using several different test systems. Similarly, the mitochondrial membrane potential (ΔΨm) was hardly effected by these compounds, while both menadione and quercetin strongly reduced the potential after 1 h of treatment. The reported chemical modification of interesting lead substances (like the strongly estrogenic 8-PN) presents a promising approach to modulate the properties of a relevant substance in a pharmacologically desirable way. The low toxicity and weak cytostatic properties of the tested naringenin derivatives is encouraging for further studies on known naringenin target molecules. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.