Investigating gene expression patterns in the mammalian cardiovascular system
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The cardiovascular system is an essential component of mammalian biology. It is a complex network of various tissues and structures with unique functions. The function of the cardiovascular system is to supply nutrients including oxygen to the various cells, tissues and organs within the body, and remove waste products from them. Given the importance of this role, it is not surprising that there are countless regulatory mechanisms at the molecular, cellular and tissue levels that are required to support this functional system. Perturbations in parts of this system are likely to lead to abnormalities, and thus give rise to cardiovascular-related diseases. Despite the currently expanding list of genes reported to be involved in a variety of cardiovascular-related diseases, including calcific aortic valve disease (CAVD), the functions and associated pathways of these factors in both normal and pathological physiology have yet to be fully understood, such as at the transcriptomic level. In this thesis, a genome-wide transcriptomic atlas of the healthy mammalian cardiovascular system was generated using the sheep as a large animal model. This atlas was generated using RNA-seq, with the aim of further understanding normal gene expression patterns in the context of the known physiology of healthy mammalian tissues. Through this work, I identified novel gene networks and detailed functional clustering of co-expressed genes with region-specific expression and specialised cardiovascular roles. One interesting cluster was highly expressed in the cardiac valves, and shared genes found in physiological bone development, such as bone morphogenetic protein 4 (BMP4), collagen type I alpha 2 (COL1A2), Sry homeobox 8 (SOX8) and bone gamma-carboxyglutamate protein (BGLAP), some of which have been implicated in vascular calcification. Further to this work, I studied the expression profiles of these key cardiovascular genes during development in the sheep from foetal to adult stages. In addition, I investigated the gene expression patterns of various key vascular calcification genes. These studies showed differential expression of genes in the different cardiovascular tissues, demonstrating transcriptional differences between these different tissues known to have different functions. CAVD involves progressive valve leaflet thickening and severe calcification, resulting in impaired leaflet motion. The in vitro calcification of primary rat, human, porcine and bovine aortic valve interstitial cells (VICs) is commonly employed to examine the mechanisms of CAVD. However, to date, no published studies have utilised cell lines to investigate this process Thus, in this project, I generated and evaluated the calcification potential of an immortalised cell line derived from sheep aortic VICs (SAVICs). This novel large animal in vitro model of CAVD was demonstrated to calcify under high calcium and phosphate conditions. Changes in the expression of key calcification genes during VIC calcification was also observed, including increased mRNA expression of bone markers Runt-related transcription factor 2 (RUNX2) and sodium-dependent phosphate transporter 1 (PiT1), and a concomitant decrease in matrix Gla protein (MGP) mRNA expression. In addition, the role of extracellular nucleotides and their receptors (P2 receptors), which have been previously shown to be important in bone and vascular calcification, were investigated using SAVICs in vitro. This study has shown that extracellular nucleotides, particularly adenosine 5’-triphosphate (ATP) and uridine 5’-triphosphate (UTP) and other agonists of P2 receptors, reduced VIC calcification in vitro. Moreover, the cutting-edge gene-editing technology, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9), was successfully applied to generate large animal models of cardiovascular-related diseases. In this project, I applied the CRISPR/Cas9 technology to edit ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and fibrillin 1 (FBN1) to generate two models of vascular calcification and Marfan Syndrome (MFS), respectively. In the ENPP1-edited animals, soft tissue calcification has been observed in the biallelic mutant and homozygous pigs. In this project, I have developed a range of novel in vitro and in vivo tools to advance the study of cardiovascular disease. These studies demonstrate that large animal models are highly valuable in the field of cardiovascular biology. The in vivo and in vitro experimental models described should facilitate detailed analysis of cardiovascular molecular biology and ultimately lead to therapies which will minimise the morbidity and mortality currently arising from cardiovascular pathology.