The role of microRNA 21 in the development of in-stent restenosis

Coronary heart disease is a major cause of morbidity and mortality worldwide. Percutaneous coronary intervention (PCI) has become the most widely used method of coronary artery revascularisation. The use of stents to hold open atherosclerosis induced arterial narrowing has significantly reduced elastic recoil and acute vessel occlusion following balloon angioplasty. However, bare metal stents have been associated with in-stent restenosis attributed to vascular smooth muscle cell (VSMC) hyperplasia and excessive neointimal formation. The resultant luminal renarrowing may manifest clinically with the return of symptoms such as chest pain or shortness of breath. The development of drug eluting stents has significantly reduced the incidence of in-stent restenosis (ISR). Unfortunately the antiproliferative medications used not only inhibit VSMC proliferation but also re-endothelialisation of the stented vessel. In addition, the drug impregnated polymer coating has been associated with a chronic inflammatory response within the vessel wall predisposing patients to stent thrombosis. Thus the identification of novel therapies which promote vessel healing without excessive proliferative or inflammatory response may improve long term outcome and reduce the need for repeated revascularisation. MicroRNAs (miRs) are short (18-25 nucleotide) non-coding RNAs acting to regulate gene expression. By binding to the 3’untranslated region of mRNA they act to fine tune gene expression either by mRNA degradation or translational repression. Originally identified in coordinating tissue development microRNAs have also been shown to play important roles coordinating the inflammatory response and in numerous cardiovascular diseases. MiR-21 has been identified in human atherosclerotic plaques, arteriosclerosis obliterans and abdominal aortic aneurysms. In addition, its up regulation has been documented in preclinical models of vascular injury. This study sought to identify the role of miR-21 in the development of ISR. Utilising a small animal model of stenting and in vitro techniques, we sought to investigate its influence upon VSMC and immune cell response following stenting. 19 The refinement of a murine stenting model within the Baker laboratory and the electrochemical dissolution of the metal stent from within harvested vascular tissues significantly improved the ability to perform detailed histological analysis. In addition, identification of miRNAs using in situ hybridisation was achieved for the first time within stented tissue. Neointimal formation and ISR was significantly reduced in mice in which miR-21 had been genetically deleted. In addition, neointimal composition was found to be altered in miR-21 KO mice with reductions in VSMC and elastin content demonstrated. Importantly, no difference in re-endothelialisation was observed. In vitro analysis demonstrated that VSMCs from miR-21 KO mice had both reduced proliferative and migratory capacity following platelet derived growth factor stimulation. Molecular analysis revealed that these differences may, at least in part, be due to de-repression of programmed cell death 4 (PDCD4). PDCD4 is a known miR-21 target within VSMCs implicated in the suppression of proliferation and promotion of apoptosis. Unfortunately, initial attempts at antimiR mediated knockdown of miR-21 in vivo, failed to produce a similar change in the suppression of ISR. Furthermore, a significant alteration in macrophage polarisation state within the neointima of miR-21 WT and KO mice was noted. Immunohistochemical staining revealed a preponderance of anti-inflammatory M2 macrophages in KO mice. Analysis of bone marrow derived macrophages from miR-21 KO mice demonstrated an increased level of the peroxisome proliferation activating receptor-γ (PPARγ) which facilitates M2 polarisation. Importantly, significant alterations in numerous pro-inflammatory cytokines, which also have mitogenic effects, were also found following genetic deletion of miR-21. In Summary, this is the first study to look at miRs in the development of ISR. MiR-21 plays an important role in the development of ISR by influencing the proliferative response of VSMCs and modulating the immune response following stent deployment. Further attempts to modulate miR-21 expression following PCI may reduce ISR and the need for repeat revascularisation while also reducing the risk of stent thrombosis.

Identifying health inequalities in individuals with major mental illness (MMI) using routine data

Abstract and Summary of Thesis: Background: Individuals with Major Mental Illness (such as schizophrenia and bipolar disorder) experience increased rates of physical health comorbidity compared to the general population. They also experience inequalities in access to certain aspects of healthcare. This ultimately leads to premature mortality. Studies detailing patterns of physical health comorbidity are limited by their definitions of comorbidity, single disease approach to comorbidity and by the study of heterogeneous groups. To date the investigation of possible sources of healthcare inequalities experienced by individuals with Major Mental Illness (MMI) is relatively limited. Moreover studies detailing the extent of premature mortality experienced by individuals with MMI vary both in terms of the measure of premature mortality reported and age of the cohort investigated, limiting their generalisability to the wider population. Therefore local and national data can be used to describe patterns of physical health comorbidity, investigate possible reasons for health inequalities and describe mortality rates. These findings will extend existing work in this area. Aims and Objectives: To review the relevant literature regarding: patterns of physical health comorbidity, evidence for inequalities in physical healthcare and evidence for premature mortality for individuals with MMI. To examine the rates of physical health comorbidity in a large primary care database and to assess for evidence for inequalities in access to healthcare using both routine primary care prescribing data and incentivised national Quality and Outcome Framework (QOF) data. Finally to examine the rates of premature mortality in a local context with a particular focus on cause of death across the lifespan and effect of International Classification of Disease Version 10 (ICD 10) diagnosis and socioeconomic status on rates and cause of death. Methods: A narrative review of the literature surrounding patterns of physical health comorbidity, the evidence for inequalities in physical healthcare and premature mortality in MMI was undertaken. Rates of physical health comorbidity and multimorbidity in schizophrenia and bipolar disorder were examined using a large primary care dataset (Scottish Programme for Improving Clinical Effectiveness in Primary Care (SPICE)). Possible inequalities in access to healthcare were investigated by comparing patterns of prescribing in individuals with MMI and comorbid physical health conditions with prescribing rates in individuals with physical health conditions without MMI using SPICE data. Potential inequalities in access to health promotion advice (in the form of smoking cessation) and prescribing of Nicotine Replacement Therapy (NRT) were also investigated using SPICE data. Possible inequalities in access to incentivised primary healthcare were investigated using National Quality and Outcome Framework (QOF) data. Finally a pre-existing case register (Glasgow Psychosis Clinical Information System (PsyCIS)) was linked to Scottish Mortality data (available from the Scottish Government Website) to investigate rates and primary cause of death in individuals with MMI. Rate and primary cause of death were compared to the local population and impact of age, socioeconomic status and ICD 10 diagnosis (schizophrenia vs. bipolar disorder) were investigated. Results: Analysis of the SPICE data found that sixteen out of the thirty two common physical comorbidities assessed, occurred significantly more frequently in individuals with schizophrenia. In individuals with bipolar disorder fourteen occurred more frequently. The most prevalent chronic physical health conditions in individuals with schizophrenia and bipolar disorder were: viral hepatitis (Odds Ratios (OR) 3.99 95% Confidence Interval (CI) 2.82-5.64 and OR 5.90 95% CI 3.16-11.03 respectively), constipation (OR 3.24 95% CI 3.01-3.49 and OR 2.84 95% CI 2.47-3.26 respectively) and Parkinson’s disease (OR 3.07 95% CI 2.43-3.89 and OR 2.52 95% CI 1.60-3.97 respectively). Both groups had significantly increased rates of multimorbidity compared to controls: in the schizophrenia group OR for two comorbidities was 1.37 95% CI 1.29-1.45 and in the bipolar disorder group OR was 1.34 95% CI 1.20-1.49. In the studies investigating inequalities in access to healthcare there was evidence of: under-recording of cardiovascular-related conditions for example in individuals with schizophrenia: OR for Atrial Fibrillation (AF) was 0.62 95% CI 0.52 - 0.73, for hypertension 0.71 95% CI 0.67 - 0.76, for Coronary Heart Disease (CHD) 0.76 95% CI 0.69 - 0.83 and for peripheral vascular disease (PVD) 0.83 95% CI 0.72 - 0.97. Similarly in individuals with bipolar disorder OR for AF was 0.56 95% CI 0.41-0.78, for hypertension 0.69 95% CI 0.62 - 0.77 and for CHD 0.77 95% CI 0.66 - 0.91. There was also evidence of less intensive prescribing for individuals with schizophrenia and bipolar disorder who had comorbid hypertension and CHD compared to individuals with hypertension and CHD who did not have schizophrenia or bipolar disorder. Rate of prescribing of statins for individuals with schizophrenia and CHD occurred significantly less frequently than in individuals with CHD without MMI (OR 0.67 95% CI 0.56-0.80). Rates of prescribing of 2 or more anti-hypertensives were lower in individuals with CHD and schizophrenia and CHD and bipolar disorder compared to individuals with CHD without MMI (OR 0.66 95% CI 0.56-0.78 and OR 0.55 95% CI 0.46-0.67, respectively). Smoking was more common in individuals with MMI compared to individuals without MMI (OR 2.53 95% CI 2.44-2.63) and was particularly increased in men (OR 2.83 95% CI 2.68-2.98). Rates of ex-smoking and non-smoking were lower in individuals with MMI (OR 0.79 95% CI 0.75-0.83 and OR 0.50 95% CI 0.48-0.52 respectively). However recorded rates of smoking cessation advice in smokers with MMI were significantly lower than the recorded rates of smoking cessation advice in smokers with diabetes (88.7% vs. 98.0%, p<0.001), smokers with CHD (88.9% vs. 98.7%, p<0.001) and smokers with hypertension (88.3% vs. 98.5%, p<0.001) without MMI. The odds ratio of NRT prescription was also significantly lower in smokers with MMI without diabetes compared to smokers with diabetes without MMI (OR 0.75 95% CI 0.69-0.81). Similar findings were found for smokers with MMI without CHD compared to smokers with CHD without MMI (OR 0.34 95% CI 0.31-0.38) and smokers with MMI without hypertension compared to smokers with hypertension without MMI (OR 0.71 95% CI 0.66-0.76). At a national level, payment and population achievement rates for the recording of body mass index (BMI) in MMI was significantly lower than the payment and population achievement rates for BMI recording in diabetes throughout the whole of the UK combined: payment rate 92.7% (Inter Quartile Range (IQR) 89.3-95.8 vs. 95.5% IQR 93.3-97.2, p<0.001 and population achievement rate 84.0% IQR 76.3-90.0 vs. 92.5% IQR 89.7-94.9, p<0.001 and for each country individually: for example in Scotland payment rate was 94.0% IQR 91.4-97.2 vs. 96.3% IQR 94.3-97.8, p<0.001. Exception rate was significantly higher for the recording of BMI in MMI than the exception rate for BMI recording in diabetes for the UK combined: 7.4% IQR 3.3-15.9 vs. 2.3% IQR 0.9-4.7, p<0.001 and for each country individually. For example in Scotland exception rate in MMI was 11.8% IQR 5.4-19.3 compared to 3.5% IQR 1.9-6.1 in diabetes. Similar findings were found for Blood Pressure (BP) recording: across the whole of the UK payment and population achievement rates for BP recording in MMI were also significantly reduced compared to payment and population achievement rates for the recording of BP in chronic kidney disease (CKD): payment rate: 94.1% IQR 90.9-97.1 vs.97.8% IQR 96.3-98.9 and p<0.001 and population achievement rate 87.0% IQR 81.3-91.7 vs. 97.1% IQR 95.5-98.4, p<0.001. Exception rates again were significantly higher for the recording of BP in MMI compared to CKD (6.4% IQR 3.0-13.1 vs. 0.3% IQR 0.0-1.0, p<0.001). There was also evidence of differences in rates of recording of BMI and BP in MMI across the UK. BMI and BP recording in MMI were significantly lower in Scotland compared to England (BMI:-1.5% 99% CI -2.7 to -0.3%, p<0.001 and BP: -1.8% 99% CI -2.7 to -0.9%, p<0.001). While rates of BMI and BP recording in diabetes and CKD were similar in Scotland compared to England (BMI: -0.5 99% CI -1.0 to 0.05, p=0.004 and BP: 0.02 99% CI -0.2 to 0.3, p=0.797). Data from the PsyCIS cohort showed an increase in Standardised Mortality Ratios (SMR) across the lifespan for individuals with MMI compared to the local Glasgow and wider Scottish populations (Glasgow SMR 1.8 95% CI 1.6-2.0 and Scotland SMR 2.7 95% CI 2.4-3.1). Increasing socioeconomic deprivation was associated with an increased overall rate of death in MMI (350.3 deaths/10,000 population/5 years in the least deprived quintile compared to 794.6 deaths/10,000 population/5 years in the most deprived quintile). No significant difference in rate of death for individuals with schizophrenia compared with bipolar disorder was reported (6.3% vs. 4.9%, p=0.086), but primary cause of death varied: with higher rates of suicide in individuals with bipolar disorder (22.4% vs. 11.7%, p=0.04). Discussion: Local and national datasets can be used for epidemiological study to inform local practice and complement existing national and international studies. While the strengths of this thesis include the large data sets used and therefore their likely representativeness to the wider population, some limitations largely associated with using secondary data sources are acknowledged. While this thesis has confirmed evidence of increased physical health comorbidity and multimorbidity in individuals with MMI, it is likely that these findings represent a significant under reporting and likely under recognition of physical health comorbidity in this population. This is likely due to a combination of patient, health professional and healthcare system factors and requires further investigation. Moreover, evidence of inequality in access to healthcare in terms of: physical health promotion (namely smoking cessation advice), recording of physical health indices (BMI and BP), prescribing of medications for the treatment of physical illness and prescribing of NRT has been found at a national level. While significant premature mortality in individuals with MMI within a Scottish setting has been confirmed, more work is required to further detail and investigate the impact of socioeconomic deprivation on cause and rate of death in this population. It is clear that further education and training is required for all healthcare staff to improve the recognition, diagnosis and treatment of physical health problems in this population with the aim of addressing the significant premature mortality that is seen. Conclusions: Future work lies in the challenge of designing strategies to reduce health inequalities and narrow the gap in premature mortality reported in individuals with MMI. Models of care that allow a much more integrated approach to diagnosing, monitoring and treating both the physical and mental health of individuals with MMI, particularly in areas of social and economic deprivation may be helpful. Strategies to engage this “hard to reach” population also need to be developed. While greater integration of psychiatric services with primary care and with specialist medical services is clearly vital the evidence on how best to achieve this is limited. While the National Health Service (NHS) is currently undergoing major reform, attention needs to be paid to designing better ways to improve the current disconnect between primary and secondary care. This should then help to improve physical, psychological and social outcomes for individuals with MMI.

Exploring the potential role of allostatic load biomarkers in risk assessment of patients presenting with depressive symptoms

Background: Depression is a major health problem worldwide and the majority of patients presenting with depressive symptoms are managed in primary care. Current approaches for assessing depressive symptoms in primary care are not accurate in predicting future clinical outcomes, which may potentially lead to over or under treatment. The Allostatic Load (AL) theory suggests that by measuring multi-system biomarker levels as a proxy of measuring multi-system physiological dysregulation, it is possible to identify individuals at risk of having adverse health outcomes at a prodromal stage. Allostatic Index (AI) score, calculated by applying statistical formulations to different multi-system biomarkers, have been associated with depressive symptoms. Aims and Objectives: To test the hypothesis, that a combination of allostatic load (AL) biomarkers will form a predictive algorithm in defining clinically meaningful outcomes in a population of patients presenting with depressive symptoms. The key objectives were: 1. To explore the relationship between various allostatic load biomarkers and prevalence of depressive symptoms in patients, especially in patients diagnosed with three common cardiometabolic diseases (Coronary Heart Disease (CHD), Diabetes and Stroke). 2 To explore whether allostatic load biomarkers predict clinical outcomes in patients with depressive symptoms, especially in patients with three common cardiometabolic diseases (CHD, Diabetes and Stroke). 3 To develop a predictive tool to identify individuals with depressive symptoms at highest risk of adverse clinical outcomes. Methods: Datasets used: ‘DepChron’ was a dataset of 35,537 patients with existing cardiometabolic disease collected as a part of routine clinical practice. ‘Psobid’ was a research data source containing health related information from 666 participants recruited from the general population. The clinical outcomes for 3 both datasets were studied using electronic data linkage to hospital and mortality health records, undertaken by Information Services Division, Scotland. Cross-sectional associations between allostatic load biomarkers calculated at baseline, with clinical severity of depression assessed by a symptom score, were assessed using logistic and linear regression models in both datasets. Cox’s proportional hazards survival analysis models were used to assess the relationship of allostatic load biomarkers at baseline and the risk of adverse physical health outcomes at follow-up, in patients with depressive symptoms. The possibility of interaction between depressive symptoms and allostatic load biomarkers in risk prediction of adverse clinical outcomes was studied using the analysis of variance (ANOVA) test. Finally, the value of constructing a risk scoring scale using patient demographics and allostatic load biomarkers for predicting adverse outcomes in depressed patients was investigated using clinical risk prediction modelling and Area Under Curve (AUC) statistics. Key Results: Literature Review Findings. The literature review showed that twelve blood based peripheral biomarkers were statistically significant in predicting six different clinical outcomes in participants with depressive symptoms. Outcomes related to both mental health (depressive symptoms) and physical health were statistically associated with pre-treatment levels of peripheral biomarkers; however only two studies investigated outcomes related to physical health. Cross-sectional Analysis Findings: In DepChron, dysregulation of individual allostatic biomarkers (mainly cardiometabolic) were found to have a non-linear association with increased probability of co-morbid depressive symptoms (as assessed by Hospital Anxiety and Depression Score HADS-D≥8). A composite AI score constructed using five biomarkers did not lead to any improvement in the observed strength of the association. In Psobid, BMI was found to have a significant cross-sectional association with the probability of depressive symptoms (assessed by General Health Questionnaire GHQ-28≥5). BMI, triglycerides, highly sensitive C - reactive 4 protein (CRP) and High Density Lipoprotein-HDL cholesterol were found to have a significant cross-sectional relationship with the continuous measure of GHQ-28. A composite AI score constructed using 12 biomarkers did not show a significant association with depressive symptoms among Psobid participants. Longitudinal Analysis Findings: In DepChron, three clinical outcomes were studied over four years: all-cause death, all-cause hospital admissions and composite major adverse cardiovascular outcome-MACE (cardiovascular death or admission due to MI/stroke/HF). Presence of depressive symptoms and composite AI score calculated using mainly peripheral cardiometabolic biomarkers was found to have a significant association with all three clinical outcomes over the following four years in DepChron patients. There was no evidence of an interaction between AI score and presence of depressive symptoms in risk prediction of any of the three clinical outcomes. There was a statistically significant interaction noted between SBP and depressive symptoms in risk prediction of major adverse cardiovascular outcome, and also between HbA1c and depressive symptoms in risk prediction of all-cause mortality for patients with diabetes. In Psobid, depressive symptoms (assessed by GHQ-28≥5) did not have a statistically significant association with any of the four outcomes under study at seven years: all cause death, all cause hospital admission, MACE and incidence of new cancer. A composite AI score at baseline had a significant association with the risk of MACE at seven years, after adjusting for confounders. A continuous measure of IL-6 observed at baseline had a significant association with the risk of three clinical outcomes- all-cause mortality, all-cause hospital admissions and major adverse cardiovascular event. Raised total cholesterol at baseline was associated with lower risk of all-cause death at seven years while raised waist hip ratio- WHR at baseline was associated with higher risk of MACE at seven years among Psobid participants. There was no significant interaction between depressive symptoms and peripheral biomarkers (individual or combined) in risk prediction of any of the four clinical outcomes under consideration. Risk Scoring System Development: In the DepChron cohort, a scoring system was constructed based on eight baseline demographic and clinical variables to predict the risk of MACE over four years. The AUC value for the risk scoring system was modest at 56.7% (95% CI 55.6 to 57.5%). In Psobid, it was not possible to perform this analysis due to the low event rate observed for the clinical outcomes. Conclusion: Individual peripheral biomarkers were found to have a cross-sectional association with depressive symptoms both in patients with cardiometabolic disease and middle-aged participants recruited from the general population. AI score calculated with different statistical formulations was of no greater benefit in predicting concurrent depressive symptoms or clinical outcomes at follow-up, over and above its individual constituent biomarkers, in either patient cohort. SBP had a significant interaction with depressive symptoms in predicting cardiovascular events in patients with cardiometabolic disease; HbA1c had a significant interaction with depressive symptoms in predicting all-cause mortality in patients with diabetes. Peripheral biomarkers may have a role in predicting clinical outcomes in patients with depressive symptoms, especially for those with existing cardiometabolic disease, and this merits further investigation.

Studies of antihypertensive drug persistence and adherence in the Glasgow Blood Pressure Clinic

Hypertension (HTN) is a major risk factor for cardiovascular diseases including stroke, coronary heart disease (CHD), chronic renal failure, peripheral vascular disease, myocardial infarction, congestive heart failure and premature death. The prevalence of HTN in Scotland is very high and although a high proportion of the patients receive antihypertensive medications, blood pressure (BP) control is very low. Recommendations for starting a specific antihypertensive class have been debated between various guidelines over the years. Some guidelines and HTN studies have preferred to start with a combination of an antihypertensive class instead of using a single therapy, and they have found greater BP reductions with combination therapies than with monotherapy. However, it has been shown in several clinical trials that 20% to 35% of hypertensive patients could not achieve the target BP, even though they received more than three antihypertensive medications. Several factors were found to affect BP control. Adherence and persistence were considered as the factors contributing the most to uncontrolled hypertension. Other factors such as age, sex, body mass index (BMI), alcohol intake, baseline systolic BP (SBP), and the communication between physicians and patients have been shown to be associated with uncontrolled BP and resistant hypertension. Persistence, adherence and compliance are interchangeable terms and have been used in the literature to describe a patient’s behaviour with their antihypertensive drugs and prescriptions. The methods used to determine persistence and adherence, as well as the inclusion and exclusion criteria, vary between persistence and adherence studies. The prevalence of persistence and adherence have varied between these studies, and were determined to be high in some studies and low in others. The initiation of a specific antihypertensive class has frequently been associated with an increase or decrease in adherence and persistence. The tolerability and efficacy of the initial antihypertensive class have been the most common methods of explaining this association. There are also many factors that suggest a relationship with adherence and persistence. Some factors in previous studies, such as age, were frequently associated with adherence and persistence. On the other hand, relationships with certain factors have varied between the studies. The associations of age, sex, alcohol use, smoking, baseline systolic blood pressure (SBP) and diastolic BP (DBP), the presence of comorbidities, an increase in the number of pills and the relationship between patients and physicians with adherence and persistence have been the most commonly investigated factors. Most studies have defined persistence in terms of a patient still taking medication after a period of time. A medication possession ratio (MPR) ≥ 80 has been used to define compliance. Either of these terminologies, or both, have been used to estimate adherence. In this study, I used the same definition for persistence to identify patients who have continued with their initial treatment, and used persistence and MPR to define patients who adhered to their initial treatment. The aim of this study was to estimate the prevalence of persistence and adherence in Scotland. Also, factors that could have had an effect on persistence and adherence were studied. The number of antihypertensive drugs taken by patients during the study and factors that led to an increase in patients being on a combination therapy were also evaluated. The prevalence of resistance and BP control were determined by taking the BP after the last drug had been taken by persistent patients during five follow-up studies. The relationship of factors such as age, sex, BMI, alcohol use, smoking, estimated glomerular filtration rate (eGFR), and albumin levels with BP reductions for each antihypertensive class were determined. Information Services Division (ISD) data, which includes all antihypertensive drugs, were collected from pharmacies in Scotland and linked to the Glasgow Blood Pressure Clinic (GBPC) database. This database also includes demographic characteristics, BP readings and clinical results for all patients attending the GBPC. The case notes for patients who attended the GBPC were reviewed and all new antihypertensive drugs that were prescribed between visits, BP before and after taking drugs, and any changes in the hypertensive drugs were recorded. A total of 4,232 hypertensive patients were included in the first study. The first study showed that angiotensin converting enzyme inhibitor (ACEI) and beta-blockers (BB) were the most prescribed antihypertensive classes between 2004 and 2013. Calcium channel blockers (CCB), thiazide diuretics and angiotensin receptor blockers (ARB) followed ACEI and BB as the most prescribed drugs during the same period. The prescription trend of the antihypertensive class has changed over the years with an increase in prescriptions for ACEI and ARB and a decrease in prescriptions for BB and diuretics. I observed a difference in antihypertensive class prescriptions by age, sex, SBP and BMI. For example, CCB, thiazide diuretics and alpha-blockers were more likely to be prescribed to older patients, while ACEI, ARB or BB were more commonly prescribed for younger patients. In a second study, 4,232 and 3,149 hypertensive patients were included to investigate the prevalence of persistence in the Scottish population in 1- and 5-year studies, respectively. The prevalence of persistence in the 1-year study was 72.9%, while it was only 62.8% in the 5-year study. Those patients taking ARB and ACEI showed high rates of persistence and those taking diuretics and alpha blockers had low rates of persistence. The association of persistence with clinical characteristics was also investigated. Younger patients were more likely to totally stop their treatment before restarting their treatment with other antihypertensive drugs. Furthermore, patients who had high SBP tended to be non-persistent. In a third study, 3,085 and 1,979 patients who persisted with their treatment were included. In the first part of the study, MPR was calculated, and patients with an MPR ≥ 80 were considered as adherent. Adherence rates were 29.9% and 23.4% in the 1- and 5-year studies, respectively. Patients who initiated the study with ACEI were more likely to adhere to their treatments. However, patients who initiated the study with thiazide diuretics were less likely to adhere to their treatments. Sex, age and BMI were different between the adherence and non-adherence groups. Age was an independent factor affecting adherence rates during both the 1- and 5-year studies with older patients being more likely to be adherent. In the second part of the study, pharmacy databases were checked with patients' case notes to compare antihypertensive drugs that were collected from the pharmacy with the antihypertensive prescription given during the patient’s clinical visit. While 78.6% of the antihypertensive drugs were collected between clinical visits, 21.4% were not collected. Patients who had more days to see the doctor in the subsequent visit were more likely to not collect their prescriptions. In a fourth study, 3,085 and 1,979 persistent patients were included to calculate the number of antihypertensive classes that were added to the initial drug during the 1-year and 5-year studies, respectively. Patients who continued with treatment as a monotherapy and who needed a combination therapy were investigated during the 1- and 5-year studies. In all, 55.8% used antihypertensive drugs as a monotherapy and 44.2% used them as a combination therapy during the 1-year study. While 28.2% of patients continued with treatment without the required additional therapy, 71.8% of the patients needed additional therapy. In all, 20.8% and 46.5% of patients required three different antihypertensive classes or more during the 1-year and 5-year studies, respectively. Patients who started with ACEI, ARB and BB were more likely to continue as monotherapy and less likely to need two more antihypertensive drugs compared with those who started with alpha-blockers, non-thiazide diuretics and CCB. Older ages, high BMI levels, high SBP and high alcohol intake were independent factors that led to an increase in the probability of patients taking combination therapies. In the first part of the final study, BPs were recorded after the last drug had been taken during the 5 year study. There were 815 persistent patients who were assigned for this purpose. Of these, 39% had taken one, two or three antihypertensive classes and had controlled BP (controlled hypertension [HTN]), 29% of them took one or two antihypertensive classes and had uncontrolled BP (uncontrolled HTN), and 32% of the patients took three antihypertensive classes or more and had uncontrolled BP (resistant HTN). The initiation of an antihypertensive drug and the factors affecting BP pressure were compared between the resistant and controlled HTN groups. Patients who initiated the study with ACEI were less likely to be resistant compared with those who started with alpha blockers and non-thiazide diuretics. Older patients, and high BMI tended to result in resistant HTN. In the second part of study, BP responses for patients who initiated the study with ACEI, ARB, BB, CCB and thiazide diuretics were compared. After adjusting for risk factors, patients who initiated the study with ACEI and ARB were more respondent than those who took CCB and thiazide diuretics. In the last part of this study, the association between BP reductions and factors affecting BP were tested for each antihypertensive drug. Older patients responded better to alpha blockers. Younger patients responded better to ACEI and ARB. An increase in BMI led to a decreased reduction in patients on ACEI and diuretics (thiazide and non-thiazide). An increase in albumin levels and a decrease in eGFR led to decreases in BP reductions in patients on thiazide diuretics. An increase in eGFR decreased the BP response with ACEI. In conclusion, although a high percentage of hypertensive patients in Scotland persisted with their initial drug prescription, low adherence rates were found with these patients. Approximately half of these patients required three different antihypertensive classes during the 5 years, and 32% of them had resistant HTN. Although this study was observational in nature, the large sample size in this study represented a real HTN population, and the large pharmacy data represented a real antihypertensive population, which were collected through the support of prescription data from the GBPC database. My findings suggest that ACEI, ARB and BB are less likely to require additional therapy. However, ACEI and ARB were better tolerated than BB in that they were more likely to be persistent than BB. In addition, users of ACEI, and ARB have good BP response and low resistant HTN. Linkage patients who participated in these studies with their morbidity and mortality will provide valuable information concerning the effect of adherence on morbidity and mortality and the potential benefits of using ACEI or ARB over other drugs.

Investigating Angiotensin II in cardiac remodelling and the counter-regulatory actions of Angiotensin-(1-9)

Cardiovascular diseases (CVDs) including, hypertension, coronary heart disease and heart failure are the leading cause of death worldwide. Hypertension, a chronic increase in blood pressure above 140/90 mmHg, is the single main contributor to deaths due to heart disease and stroke. In the heart, hypertension results in adaptive cardiac remodelling, including LV hypertrophy to normalize wall stress and maintain cardiac contractile function. However, chronic increases in BP results in the development of hypertensive heart disease (HHD). HHD describes the maladaptive changes during cardiac remodelling which result in reduced systolic and diastolic function and eventually heart failure. This includes ventricular dilation due to eccentric hypertrophy, cardiac fibrosis which stiffens the ventricular wall and microvascular rarefaction resulting in a decrease in coronary blood flow albeit an increase in energy demand. Chronic activation of the renin-angiotensin-system (RAS) with its effector peptide angiotensin (Ang)II plays a key role in the development of hypertension and the maladaptive changes in HHD. Ang II acts via the angiotensin type 1 receptor (AT1R) to mediate most of its pathological actions during HHD, including stimulation of cardiomyocyte hypertrophy, activation of cardiac fibroblasts and increased collagen deposition. The counter-regulatory axis of the RAS which is centred on the ACE2/Ang-(1-7)/Mas axis has been demonstrated to counteract the pathological actions of Ang II in the heart and vasculature. Ang-(1-7) via the Mas receptor prevents Ang II-induced cardiac hypertrophy and fibrosis and improves cardiac contractile function in animal models of HHD. In contrast, less is known about Ang-(1-9) although evidence has demonstrated that Ang-(1-9) also antagonises Ang II and is anti-hypertrophic and anti-fibrotic in animal models of acute cardiac remodelling. However, so far it is not well documented whether Ang-(1-9) can reverse established cardiac dysfunction and remodelling and whether it is beneficial when administered chronically. Therefore, the main aim of this thesis was to assess the effects of chronic Ang-(1-9) administration on cardiac structure and function in a model of Ang II-induced cardiac remodelling. Furthermore, this thesis aimed to investigate novel pathways contributing to the pathological remodelling in response to Ang II. First, a mouse model of chronic Ang II infusion was established and characterised by comparing the structural and functional effects of the infusion of a low and high dose of Ang II after 6 weeks. Echocardiographic measurements demonstrated that low dose Ang II infusion resulted in a gradual decline in cardiac function while a high dose of Ang II induced acute cardiac contractile dysfunction. Both doses equally induced the development of cardiac hypertrophy and cardiac fibrosis characterised by an increase in the deposition of collagen I and collagen III. Moreover, increases in gene expression of fibrotic and hypertrophic markers could be detected following high dose Ang II infusion over 6 weeks. Following this characterisation, the high dose infusion model was used to assess the effects of Ang-(1-9) on cardiac structural and functional remodelling in established disease. Initially, it was evaluated whether Ang-(1-9) can reverse Ang II-induced cardiac disease by administering Ang-(1-9) for 2-4 weeks following an initial 2 week infusion of a high dose of Ang II to induce cardiac contractile dysfunction. The infusion of Ang-(1-9) for 2 weeks was associated with a significant improvement of LV fractional shortening compared to Ang II infusion. However, after 4 weeks fractional shortening declined to Ang II levels. Despite the transient improvement in cardiac contractile function, Ang-(1-9) did not modulate blood pressure, LV hypertrophy or cardiac fibrosis. To further investigate the direct cardiac effects of Ang-(1-9), cardiac contractile performance in response to Ang-(1-9) was evaluated in the isolated Langendorff-perfused rat heart. Perfusion of Ang-(1-9) in the paced and spontaneously beating rat heart mediated a positive inotropic effect characterised by an increase in LV developed pressure, cardiac contractility and relaxation. This was in contrast to Ang II and Ang-(1-7). Furthermore, the positive inotropic effect to Ang-(1-9) was blocked by the AT1R antagonist losartan and the protein kinase A inhibitor H89. Next, endothelial-to-mesenchymal transition (EndMT) as a novel pathway that may contribute to Ang II-induced cardiac remodelling was assessed in Ang II-infused mice in vivo and in human coronary artery endothelial cells (HCAEC) in vitro. Infusion of Ang II to mice for 2-6 weeks resulted in a significant decrease in myocardial capillary density and this was associated with the occurrence of dual labelling of endothelial cells for endothelial and mesenchymal markers. In vitro stimulation of HCAEC with TGFβ and Ang II revealed that Ang II exacerbated TGF-induced gene expression of mesenchymal markers. This was not correlated with any changes in SMAD2 or ERK1/2 phosphorylation with co-stimulation of TGFβ and Ang II. However, superoxide production was significantly increased in HCAEC stimulated with Ang II but not TGFβ. Finally, the role of Ang II in microvesicle (MV)-mediated cardiomyocyte hypertrophy was investigated. MVs purified from neonatal rat cardiac fibroblasts were found to contain detectable Ang II and this was increased by stimulation of fibroblasts with Ang II. Treatment of cardiomyocytes with MVs derived from Ang II-stimulated fibroblasts induced cardiomyocyte hypertrophy which could be blocked by the AT1R antagonist losartan and an inhibitor of MV synthesis and release brefeldin A. Furthermore, Ang II was found to be present in MVs isolated from serum and plasma of Ang II-infused mice and SHRSP and WKY rats. Overall, the findings of this thesis demonstrate for the first time that the actions of Ang-(1-9) in cardiac pathology are dependent on its time of administration and that Ang-(1-9) can reverse Ang II-induced cardiac contractile dysfunction by acting as a positive inotrope. Furthermore, this thesis demonstrates evidence for an involvement of EndMT and MV signalling as novel pathways contributing to Ang II-induced cardiac fibrosis and hypertrophy, respectively. These findings provide incentive to further investigate the therapeutic potential of Ang-(1-9) in the treatment of cardiac contractile dysfunction in heart disease, establish the importance of novel pathways in Ang II-mediated cardiac remodelling and evaluate the significance of the presence of Ang II in plasma-derived MVs.