Abstract
Type 2 diabetes mellitus is a chronic metabolic disorder strongly associated with the development of cardiovascular diseases, collectively referred to as diabetic heart disease (DHD). DHD consists of a cluster of comorbidities driven by diabetes-induced molecular dysregulation. The diabetes-induced molecule of interest in this thesis was microRNAs (miRNAs). miRNAs are a class of non-coding RNAs that regulate physiological and pathological processes through the post-transcriptional modulation of various genes. Dysregulation of miRNAs in the type 2 diabetic heart has been associated with increased apoptosis and reduced functionality, such as angiogenesis. Additionally, miRNAs have been found to be stably expressed in various biofluids by binding to different proteins or by being enclosed within various nanovesicles. Exosomes are one such nanovesicles that are released by various cell types. These nanovesicles retain various cargos and markers from their parent cells, including miRNAs. Interestingly, exosomes have been demonstrated to exert paracrine effects on cellular functions through the transfer of their miRNA cargo.
The pericardium is a mammalian serosal cavity that hosts the heart, pericardial fluid, and ultrafiltration fluid of the lymphatic system. Although initially considered as a form of lubrication, pericardial fluid has been demonstrated to have both physiological and pathological roles through paracrine signalling in the heart. The paracrine capabilities of pericardial fluid can be explained by the presence of exosomes in the fluid. Pericardial fluid exosomes have been shown to contain cardiac-enriched miRNAs; however, the effect of diabetes on pericardial fluid exosomes and their miRNA cargo remains unknown. Therefore, the main aim of this thesis was to examine the role of pericardial fluid exosomes and their miRNAs in the diabetic heart.
The first aim was to investigate diabetes-induced miRNA alterations in the pericardial fluid exosomes. The pericardial fluid samples used for this project were collected from patients undergoing CABG surgery at Dunedin Public Hospital, New Zealand. Initially, the isolation protocol for exosomes from low-density fluids, such as pericardial fluid, was optimised. Where exosomes from pericardial fluids were first concentrated by precipitation and then purified using size exclusion chromatography. Exosomes were isolated from diabetic and matched non-diabetic pericardial fluid samples. RNA was extracted from these samples for NanoString analysis to obtain the miRNA profiles. NanoString analysis identified the expression of 798 miRNAs in pericardial fluid exosomes, of which 57 were significantly altered in diabetic pericardial fluid exosomes. Following a literature review, three miRNAs were selected for downstream validation and analysis. The downregulation of miRNA-196a-3p was the most significant change observed in the panel of miRNAs altered in diabetic pericardial fluid exosomes. However, because of the low expression profile in pericardial fluid exosomes, miRNA-196a-3p could not be validated for downstream analysis. The second miRNA chosen for this project was miRNA-181a-5p because of its association with various heart diseases, and the third was miRNA-206 because of its myocyte-specific origin. Both miRNA-181 and miRNA-206 were validated to have an increased expression in pericardial fluid exosomes from patients with a diabetes duration greater than ten years. The primary significance of the altered miRNA pattern in this project is the origin of the exosomes. This is because pericardial fluid exosomes are of cardiovascular origin, where they are released from cardiomyocytes, endothelial cells, and pericardial and epicardial adipose cells. Therefore, pericardial fluid exosomes can be used to better understand the heart and its microenvironment under diabetic conditions.
Next, the effect of the therapeutic modulation of these miRNAs on cellular functions under type 2 diabetic conditions was examined. An in vitro treatment model was optimised using an AC-16 cardiomyocyte cell line to mimic type 2 diabetic conditions. The novelty of this model is that it simulates two cardinal features of type 2 diabetes, hyperglycaemia and insulin resistance, both at the molecular and functional levels. However, studies on the therapeutic modulation of miRNA-181 have been inconclusive. Similarly, the therapeutic upregulation of miRNA-206 had no significant effect on cardiomyocyte survival or target protein expression. Interestingly, the therapeutic downregulation of miRNA-206 under in vitro type 2 diabetic conditions was associated with increased cell survival and target protein expression. Overall, the therapeutic modulation study for these miRNAs requires further optimisation to fully understand their role in the diabetic heart.
As exosomes have a complex cocktail of cargoes which work collectively to modulate cellular functions, the functional role of whole pericardial fluid exosomes in cellular function was assessed. Both diabetic and non-diabetic pericardial fluid exosomes significantly increased cardiomyocyte apoptosis under all the culture conditions. However, this could be attributed to the ischemic origin of the pericardial fluid used in this study. In contrast, diabetic and non-diabetic pericardial fluid exosomes significantly reduced endothelial cell apoptosis under hyperglycaemic conditions. Functionally, all pericardial fluid exosomes increased endothelial cell migration under all the culture conditions. However, diabetic pericardial fluid exosomes significantly downregulated endothelial cell angiogenesis compared to non-diabetic pericardial fluid exosomes and no-exosome controls.
In conclusion, this thesis provides the first evidence of an altered miRNA profile in diabetic pericardial fluid exosomes. This diabetes-induced exosomal miRNA alteration could have downstream effects on recipient cell function. However, the molecular and functional roles of individual miRNAs altered in diabetic pericardial fluid exosomes require further study. The novelty of this project is that, to my knowledge, it is the first to explore the effect of diabetic pericardial fluid exosomes on cell survival and function of cardiomyocytes and endothelial cells. This study provides new insights into the microenvironment of the diabetic heart.