MicroRNA modulation of aldosterone production in the adrenal gland

Hypertension is the major risk factor for coronary disease worldwide. Primary hypertension is idiopathic in origin but is thought to arise from multiple risk factors including genetic, lifestyle and environmental influences. Secondary hypertension has a more definite aetiology; its major single cause is primary aldosteronism (PA), the greatest proportion of which is caused by aldosteroneproducing adenoma (APA), where aldosterone is synthesized at high levels by an adenoma of the adrenal gland. There is strong evidence to show that high aldosterone levels cause adverse effects on cardiovascular, cerebrovascular, renal and other systems. Extensive studies have been conducted to analyse the role that regulation of CYP11B2, the gene encoding the aldosterone synthase enzyme plays in determining aldosterone production and the development of hypertension. One significant regulatory factor that has only recently emerged is microRNA (miRNA). miRNAs are small non-coding RNAs, synthesized by a series of enzymatic processes, that negatively regulate gene expression at the posttranscriptional level. Detection and manipulation of miRNA is now known to be a viable method in the treatment, prevention and prognosis of certain diseases. The aim of the present study was to identify miRNAs likely to have a role in the regulation of corticosteroid biosynthesis. To achieve this, the miRNA profile of APA and normal human adrenal tissue was compared, as was the H295R adrenocortical cell line model of adrenocortical function, under both basal conditions and following stimulation of aldosterone production. Key differentially-expressed miRNAs were then identified and bioinformatic tools used to identify likely mRNA targets and pathways for these miRNAs, several of which were investigated and validated using in vitro methods. The background to this study is set out in Chapter 1 of this thesis, followed by a description of the major technical methods employed in Chapter 2. Chapter 3 presents the first of the study results, analysing differences in miRNA profile between APA and normal human adrenal tissue. Microarray was implemented to detect the expression of miRNAs in these two tissue types and several miRNAs were found to vary significantly and consistently between them. Furthermore, members of several miRNA clusters exhibited similar changes in expression pattern between the two tissues e.g. members of cluster miR-29b-1 (miR-29a-3p and miR-29b-3p) and of cluster miR-29b-2 (miR-29b-3p and miR-29c- 3p) are downregulated in APA, while members of cluster let-7a-1 (let-7a-5p and let-7d-5p), cluster let-7a-3 (let-7a-5p and let-7b-5p) and cluster miR-134 (miR- 134 and miR-382) are upregulated. Further bioinformatic analysis explored the possible biological function of these miRNAs using Ingenuity® Systems Pathway Analysis software. This led to the identification of validated mRNAs already known to be targeted by these miRNAs, as well as the prediction of other mRNAs that are likely targets and which are involved in processes relevant to APA pathology including cholesterol synthesis (HMGCR) and corticosteroidogenesis (CYP11B2). It was therefore hypothesised that increases in miR-125a-5p or miR- 335-5p would reduce HMGCR and CYP11B2 expression. Chapter 4 describes the characterisation of H295R cells of different strains and sources (H295R Strain 1, 2, 3 and HAC 15). Expression of CYP11B2 was assessed following application of 3 different stimulants: Angio II, dbcAMP and KCl. The most responsive strain to stimulation was Strain 1 at lower passage numbers. Furthermore, H295R proliferation increased following Angio II stimulation. In Chapter 5, the hypothesis that increases in miR-125a-5p or miR-335-5p reduces HMGCR and CYP11B2 expression was tested using realtime quantitative RT-PCR and transfection of miRNA mimics and inhibitors into the H295R cell line model of adrenocortical function. In this way, miR-125a-5p and miR-335-5p were shown to downregulate CYP11B2 and HMGCR expression, thereby validating certain of the bioinformatic predictions generated in Chapter 3. The study of miRNA profile in the H295R cell lines was conducted in Chapter 6, analysing how it changes under conditions that increase aldosterone secretion, including stimulation Angiotensin II, potassium chloride or dibutyryl cAMP (as a substitute for adrenocorticotropic hormone). miRNA profiling identified 7 miRNAs that are consistently downregulated by all three stimuli relative to basal cells: miR-106a-5p, miR-154-3p, miR-17-5p, miR-196b-5p, miR-19a-3p, miR-20b- 5p and miR-766-3p. These miRNAs include those derived from cluster miR-106a- 5p/miR-20b-5p and cluster miR-17-5p/miR-19a-3p, each producing a single polycistronic transcript. IPA bioinformatic analysis was again applied to identify experimentally validated and predicted mRNA targets of these miRNAs and the key biological pathways likely to be affected. This predicted several interactions between miRNAs derived from cluster miR-17-5p/miR-19a-3p and important mRNAs involved in cholesterol biosynthesis: LDLR and ABCA1. These predictions were investigated by in vitro experiment. miR-17-5p/miR-106a-p and miR-20b-5p were found to be consistently downregulated by stimulation of aldosterone biosynthesis. Moreover, miR-766-3p was upregulation throughout. Furthermore, I was able to validate the downregulation of LDLR by miR-17 transfection, as predicted by IPA. In summary, this study identified key miRNAs that are differentially-expressed in vivo in cases of APA or in vitro following stimulation of aldosterone biosynthesis. The many possible biological actions these miRNAs could have were filtered by bioinformatic analysis and selected interactions validated in vitro. While direct actions of these miRNAs on steroidogenic enzymes were identified, cholesterol handling also emerged as an important target and may represent a useful point of intervention in future therapies designed to modulate aldosterone biosynthesis and reduce its harmful effects.

Antiplatelet therapy and clinical outcomes in cardiovascular diseases

Cardiovascular diseases (CVD) is a leading cause of death in the world. Despite effective treatment regimens for ischaemic heart disease (IHD) and ischaemic stroke, mortality and recurrence rates remain high. Antiplatelet therapy is on effective treatment and reduces the risk of recurrent heart attack and stroke. Nevertheless, there are patients who stopped or interrupted their antiplatelet therapy for certain reasons or some patients may be resistant or poor responders to antiplatelet therapy. Furthermore, there is evidence of rebound effect in platelet activity after antiplatelet cessation and this may associate with increased risk of cardiovascular event. This thesis is divided into five main chapters (chapters 3 to 7) which attempt to provide data to help resolve the uncertainty. Chapter 1 highlights the background of cardiovascular diseases and the global burden of cardiovascular and cerebrovascular diseases. The metabolism of platelets, antiplatelet therapy and current antiplatelet therapy guidelines are described, followed by discussion of the risk of cardiovascular event and changes in antiplatelet therapy. Chapter 2 describes the data source from Virtual International Stroke Trial Archive (VISTA) and National Health Service Greater Glasgow and Clyde (NHSGGC) Safe Haven, followed by definition of outcome measures. In chapter 3, Virtual International Stroke Trial Archive (VISTA) data was examined to test whether continue with the same antiplatelet therapy or changing to a new antiplatelet regimen reduces the risk of subsequent events in patients who experience a stroke whilst taking antiplatelet therapy. The findings indicate that subjects who switch to a new antiplatelet regimen after stroke did not have a lower early recurrence rate than subjects who continued with the same antiplatelet therapy. Observations on bleeding complications were similar in both groups. However, changing antiplatelet regimen after stroke was associated with more favourable functional outcome across a full scale modified Rankin Scale (mRS) at 90 days. In chapter 4, association between early or later initiation of antiplatelet with a recurrent ischaemic stroke and bleeding complications was assessed using VISTA data. The findings indicate that there was no association between a recurrent ischaemic stroke and timing of initiation of antiplatelet drug after stroke. However, early initiation was associated with increased risk of bleeding. In terms of functional outcomes, this study demonstrated that the mid-time and late initiation of antiplatelet therapy after acute stroke are associated with better functional outcomes compared with early initiation. In chapter 5, a nested case-control study was performed to explore the rate of antiplatelet cessation and interruption in a sample of patients with recent ischaemic stroke and to assess the risk of cardiovascular events associated with cessation and interruption of antiplatelet. It was found that there was no increased risk of cardiovascular event among patients who had early cessation or interrupted/stopped antiplatelet therapy within 90 days following acute ischaemic stroke. In chapter 6, the incidence and predictors of cardiovascular events after DAPT cessation were evaluated. The incidence of cardiovascular event while taking DAPT and following discontinuation of DAPT was 15.7% and 16.7% respectively. This study found that increasing age was associated with an increased risk of cardiovascular event, whereas, revascularization-treated patients and longer duration of DAPT, were each associated with a decreased risk. The duration of DAPT six months and less was associated a significantly higher risk for cardiovascular event. In chapter 7, an untargeted metabolomics analysis was performed while on DAPT (aspirin plus ticagrelor) and once they stopped ticagrelor to identify metabolite changes associated with cardiovascular events after stopping DAPT. Ten ACS patients were recruited in this study and data were analysed for seven patients. Three hundred eleven putative metabolites were identified. This study found 16 putative metabolites significantly altered following ticagrelor cessation. Of these, seven metabolites were from lipid pathway and down-regulated some up to 3-fold. On the other hand, adenosine, from nucleotide metabolism was upregulated up to 2.6-fold. It concluded that there are changes in numerous pathways following DAPT discontinuation and whether these changes differ in patients who have cardiovascular event after stopping DAPT warrant further investigation. In chapter 8, a summary of the findings of this thesis are presented as well as the future directions of research in this area.