Elucidating the roles of endothelial PTBP1 and PKM2 in Pulmonary Arterial Hypertension: implications for therapy

Abstract

Pulmonary Arterial Hypertension (PAH) is a rare, life-limiting disease with no cure, and is regarded the most severe form of pulmonary hypertension (PH). The disease is characterised by remodelling of peripheral pulmonary arteries, leading to increased mean pulmonary arterial pressure, right ventricular afterload, and heart failure. Pulmonary artery endothelial cell (PAEC) dysfunction is regarded as a key driver of the disease and is characterised by heightened proliferation and development of apoptosis resistance. Dysfunctional metabolism also observed within the PAH pulmonary vasculature and in systemic tissues. PAECs display a hyperglycolytic metabolic signature, fuelled by increased expression of splicing factor Poly-pyrimidine tract binding protein 1 (PTBP1) and its gene product, the glycolytic enzyme Pyruvate Kinase M2 (PKM2). Moreover, normal EC function and metabolism is restored by PTBP1 silencing, highlighting the therapeutic potential of targeting PTBP1 and PKM2. In this thesis, I have explored the therapeutic potential of pharmacological and genetic inhibition of PTBP1 and PKM2 in BOECs, a progenitor EC subset isolated from the peripheral blood, with high transcriptome homology to PAECs. Crucially, I have explored their influences in BOECs isolated from non-carriers, and PAH patient carriers of causal missense or truncating BMPR2 mutations. In doing so, my work sheds light on the causal and therapeutic roles of PTBP1 and PKM2 in several patient contexts. In this thesis, I have shown that treatment with the anti-tumorigenic compound apigenin, inhibits PTBP1 and PKM2 expression, in addition to lactate production, and functional parameters including cell proliferation and apoptosis susceptibility. I also confirm the therapeutic relevance of dimeric PKM2 to BOEC function, by demonstrating inhibition of proliferation and apoptosis susceptibility following TEPP-46 treatment. In the Sugen-Hypoxia rodent model of PAH I also demonstrated amelioration of right ventricular systolic pressure following oral gavage TEPP-46 treatment, further supporting the therapeutic potential of PTBP1 and PKM2 inhibition in PAH. Therapeutic benefit was not observed following apigenin treatment, however exploration of alternative routes of delivery and formulation strategies are needed. Ultimately, the results presented in this thesis improve our understanding of the molecular mechanisms orchestrating EC dysfunction in PAH and may guide the development of novel metabolically-targeted therapeutic strategies.