Endogenous mechanisms of vascular regeneration following myocardial infarction
Solomonidis, Emmanouil Georgios
Ischaemic heart disease remains one of the leading causes of death worldwide, responsible for over 8.9 million deaths in 2017 according to the Global Burden of Disease Study. Restoring blood perfusion in the ischaemic border via a functional vascular network may enhance myocardial perfusion, limit infarct expansion and promote cardiac regeneration. However, the pathways driving endogenous vascular regeneration following myocardial infarction (MI) remain poorly understood. The aim of this thesis was twofold; First, to investigate the origin and clonal dynamics of cardiac endothelial cells (EC) post-MI. Secondly, to analyse the transcriptional profiles of pro-angiogenic EC in MI using single cell RNA sequencing in the post-ischaemic adult mouse heart, and investigate potential therapeutic targets in human patient samples. MI was induced in an EC-specific multispectral lineage-tracing mouse, “Pdgfb-iCreERT2-Brainbow2.1”, by permanent ligation of the left anterior descending coronary artery. Blood vessel formation via clonal proliferation by resident Pdgfb-lineage EC was significantly upregulated in the ischaemic border at 7 days post-MI, compared to the healthy heart. Minimal contribution from the bone marrow was observed. Bioinformatics analyses revealed 10 transcriptionally discrete heterogeneous EC states in the 7 days post-MI heart and revealed molecular pathways through which each cluster was likely to mediate neovasculogenesis following MI. Plasmalemma Vesicle–Associated Protein (Plvap) gene expression was upregulated in MI, specifically in clusters of cells from the MI group, indicating its potential relevance to neovasculogenic pathways. I validated increased Plvap expression at the protein level, which was EC-specific and was significantly higher in the infarct border of the post-ischaemic mouse heart compared to the healthy heart. PLVAP expression was also significantly increased in EC adjacent to regions of fibrosis and scarring in the ischaemic human heart, compared to healthy human hearts. Moreover, in vitro silencing using RNAi in human umbilical vein ECs showed that PLVAP may play a direct functional role in regulating EC proliferation. The single cell gene expression atlas of cardiac resident ECs presented in this thesis can be used to unravel the neovasculogenesis pathways that are activated 7 days post-MI. Cluster specific PLVAP was identified as a possible target for augmenting endogenous myocardial perfusion following ischaemia and validated in human cardiac ischaemic tissue. Future studies will involve further interrogation of the activated neovasculogenesis pathways from cardiac ECs using the single cell gene expression atlas as well as investigation of the molecular mechanisms of the role of PLVAP in EC proliferation.