Pulmonary arterial hypertension (PAH) is a rare fatal disease characterised by endothelial dysfunction and obliteration of small pulmonary arteries. The resulting increase in pulmonary arterial pressure and right ventricular afterload ultimately causes death by right heart failure. Current available treatments show little impact on patient mortality and there is thus an urgent need to better understand the causes of PAH pathology to derive new transformative therapies.

Recently, a whole-genome sequencing study of >1000 idiopathic PAH patients identified pathogenic mutations in several new genes. In a small subset of patients, rare heterozygous variants in the SOX17 gene, predicted to be functionally deleterious, were significantly associated with PAH. SOX17 is an essential transcription factor involved in the development of the pulmonary vasculature and is highly expressed in endothelial cells. The aims of the studies underlying this thesis were to explore how SOX17 deficiency may compromise endothelial integrity and cause dysregulated vascular functions.

To achieve this, I used SOX17-specific silencing RNA (siSOX17) to knock down SOX17 in pulmonary arterial endothelial cells (PAECs). siSOX17-transfected PAECs express only 10-20% of endogenous SOX17, without significant effects on the expression levels of other SOX family members including SOX2, SOX4, SOX7 and SOX18. SOX17-deficient PAECs exhibited decreased proliferation rates and transcriptional analysis suggested this to be a result of decreased cyclin A (CCNA1) and cyclin B (CCNB1) expression and increased expression of p15 (CDKN2B), a cyclin-dependent kinase inhibitor. Additionally, siSOX17-transfected PAECs showed reduced monolayer permeability and impaired cellular angiogenesis, namely through horseradish peroxidase (HRP) permeability assay as well as tube-network and bead-sprouting assays, respectively.

In parallel, to recapitulate the discrete manner in which a patient-specific mutation in SOX17 may initiate PAH disease development, a heterozygous mutation found in patients was introduced into human wild-type (C2 SOX17+/+) induced pluripotent stem cells (iPSCs), resulting in the generation of the C2 SOX17+/R140P iPSC line via CRISPR-Cas9 and homologous recombination. The R140P mutation, replacing the amino acid arginine (R) to proline (P) at position 140, was chosen as it is predicted to substantially impair the DNA binding capacity of SOX17. To assess the impact on endothelial cell development, the gene-edited iPSCs were subsequently differentiated into iPSC-derived endothelial cells (iPSC-ECs). In mutant C2 SOX17+/R140P iPSC-ECs, the SOX17 mRNA expression level was substantially lower than in C2 SOX17+/+ iPSC-ECs, suggesting the mutation may impair a SOX17 autoregulatory positive feedback loop. Furthermore, transcriptional analysis of arteriovenous gene expression revealed a significant reduction in the arterial markers, NOTCH, HEY1 and EPHNB2 in mutant C2 SOX17+/R140P iPSC-ECs compared to C2 SOX17+/+ cells. Additionally, two SOX17 iPSC lines, SOX17147Δ8del and SOX17145Δ18del, containing an 8bp and 18bp deletion at amino acid position 147 and 145 respectively, were obtained via non-homologous end joining and studied in parallel to the C2 SOX17+/R140P iPSCs.

Moreover, the effects of SOX17 mutations on protein biochemistry, namely SOX17 DNA binding capability and localisation, were also investigated in this study. WT SOX17 localised to the nucleus of both PAECs and C2 iPSC-ECs. Interestingly, C2 SOX17+/R140P iPSC-ECs exhibited nuclear SOX17 staining, while SOX17147Δ8del and SOX17145Δ18del iPSC-ECs demonstrated partial non-nuclear staining. Nuclear localisation site (NLS) prediction algorithms consistently identified a monopartite NLS within the SOX17 transcript at amino acid position 146-151. The deleted regions in both SOX17147Δ8del and SOX17145Δ18del overlapped with this predicted NLS, supporting the localisation data. To assess SOX17 DNA binding ability, a SOX17 luciferase reporter system was generated, though current data suggest further optimisations are required to increase specificity and remove confounding factors.

Lastly, in vivo assessment of pulmonary haemodynamics in Sox17+/GFP mice showed Sox17 haploinsufficiency alone did not lead to PAH but may require pulmonary-artery specific deletion of Sox17 or additional stimulus such as hypoxia to precipitate PAH. Total RNA extraction for transcriptional analysis and immunohistochemistry for assessment of vascular remodelling are currently underway.