Medicine (St Vincent's) - Theses
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ItemMass spectrometry based examination of ADMA/DDAH modification in diabetes and cardiac disease( 2016)Nitric Oxide (NO) is an important mediator in the prevention of monocyte recruitment and endothelial dysfunction, which drive the development of atherosclerosis. Asymmetric Dimethylarginine (ADMA) but not its isomer Symmetric Dimethylarignine (SDMA) has been identified as a circulating inhibitor of Nitric Oxide Synthase (NOS), with submiromolar increases of ADMA previously been demonstrated associated with an increase in cardiovascular disease risk. Currently ADMA has been measured with low reproducibility in most analytical techniques other than the gold standard HPLC-MS/MS. This work shows the separation that can be achieved with the emerging technique of Ion Mobility Spectrometry coupled with traditional mass spectrometry. Also using a more traditional HPLC based separation we were able to develop a mass spectrometry based measurement of metabolites ADMA, SDMA, Arginine, Ornithine and Citrulline critical in the NOS/ADMA pathway and the Urea cycle. This assay was used to measure changes across cardiac surgery within the circulation as well as generation from the myocardium. We demonstrated our technique had high reproducibility (>7% CV for ADMA) and showed that ADMA but not SDMA significantly decreases after cardiac surgery. This was the first such study to perform measurements of ADMA and SDMA alongside Urea cycle metabolites and with clinical data also collected will provide the foundation to further work. Our assay was also used in a cell culture model whereby DDAH the enzyme responsible for the majority (80%) of ADMA metabolism has previously been shown to have increased expression and activity with high glucose treatment. Our results demonstrate that ADMA is highly regulated compared to SDMA and the change with high glucose treatment does not correspond to a decrease in intracellular ADMA concentration or from the treatment media. Also tested was the efficacy of the proton pump inhibitor Omeprazole which has been recently demonstrated to inhibit DDAH in endothelial cells. This work demonstrated that Omeprazole does not inhibit DDAH within our HepG2 cell culture model in low or high glucose concentrations.
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ItemContractile dysfunction of the heart in early diabetes( 2016)The prevalence of heart failure, especially heart failure with preserved ejection (HFpEF), is increasing annually in part due to an ageing population and the dramatically increased incidence of obesity, insulin resistance, prediabetes and diabetes worldwide. It is well established that patients with type-1 diabetes (T1DM) and type-2 diabetes (T2DM) are at a significantly increased risk of developing HFpEF. HFpEF is a progressive condition and its earliest manifestations are subtly impaired myocardial function that is termed diabetic cardiomyopathy (DCM). At present, there is only a limited understanding of the underlying pathophysiological mechanisms that drive the development of DCM and eventual HFpEF in early T1DM and T2DM. Therefore, the aim of this thesis was to further explore mechanisms that could drive the development of DCM and HFpEF using rat models of early T1DM and T2DM. Using synchrotron radiation as a source for small angle x-ray scattering (SAXS) in the in situ beating rat heart, we are able to measure actin-myosin cross-bridge (CB) dynamics in the entire cardiac cycle, in real time. In the first part of this thesis, we were able to demonstrate that chronic inhibition of the RhoA/Rho-kinase (ROCK) pathway with fasudil improved regionally impaired diastolic myosin head extension and depressed systolic mass transfer in the myocardium of rats with early T1DM. Further, we were also able to demonstrate that global left ventricular (LV) systolic performance was significantly improved in diabetic rats treated with fasudil. These results suggest that the activation of the ROCK pathway is involved in the development of early DCM in the context of T1DM. The Goto-Kakizaki (GK) rat is a non-obese model of spontaneous T2DM, which makes it a useful model to examine the effects of early T2DM on the myocardium without the added complication of obesity. Utilising the synchrotron radiation SAXS technique in the in situ beating heart preparation, we are able to demonstrate that young GK rats (10-12 weeks old) with early T2DM (prediabetes) exhibit impaired basal diastolic myosin head extension and reduced systolic myosin mass transfer in the deeper myocardial layer, the subendocardium. Interestingly, basal global cardiac function and β-adrenergic mediated positive inotropy was preserved in young GK rats. We speculate that a combination of cardiomyocyte hypertrophy and enhanced epicardial fibre function are the most likely mechanisms for the preserved global LV function in young GK rats. Diabetes is rarely seen in isolation in patients and commonly coexists with hypertension. The interaction of diabetes and hypertension is known to exacerbate myocardial dysfunction and accelerate the development of HFpEF, but the precise mechanisms remain elusive. The GK rat is a model of salt-sensitive hypertension induced by exposure to a high-salt diet (6% NaCl) for eight weeks. Thus, we examined if the interaction of prediabetes and salt-sensitive hypertension exacerbated myocardial dysfunction and accelerated the development of HFpEF in young GK rats. In GK rats exposed to a HS diet, we observed subtle declines in basal global diastolic and systolic LV function. Limited contractile reserve is a key feature of clinical HFpEF. Consistent with this, we were able to show that regional contractile reserve was limited at the fibre-level in the subepicardial and subendocardial fibre layers of the myocardium in GK rats maintained on a HS diet. These results suggest that limited contractile reserve at the fibre-level may be an early manifestation of HFpEF. In summary, this thesis has demonstrated that the ROCK pathway is involved in the evolution of DCM in T1DM, possibly by modulating actin-myosin interactions in the cardiac cycle. Our results also indicate that impaired CB dynamics is a feature of early T2DM DCM, although global cardiac function is preserved. Importantly, we have demonstrated that limited contractile reserve at the fibre-level may be an early manifestation of HFpEF in the presence of early T2DM and hypertension. Although further work is required to identify the specific molecular mechanisms that drive the impaired actin-myosin CB dynamics in early T1DM and T2DM, this thesis provides novel information of the pathophysiological features of contractile dysfunction of the heart in early diabetes.
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ItemAdipose-derived mesenchymal cell derivation, characterization and differentiation for potential use in cell replacement therapy for diabetes( 2013)Type 1 diabetes (T1D) is characterized by the loss of insulin-producing β-cells in the pancreas. T1D can be treated using cadaveric islet transplantation, but this therapy is severely limited by a lack of donor pancreas. To develop an alternative cell therapy, candidate populations were identified through epigenetic characterization of multiple tissues. Histone modification status at the promoter region of key endocrine pancreatic genes was assessed using chromatin immunoprecipitation sequencing (ChIP-seq) and validated using promoter-specific TaqMan-based quantitative PCR (qPCR). Visceral fat was identified as a tissue retaining epigenetic signatures similar to those observed in the pancreas. Human adipose-derived mesenchymal cells (AMCs) were characterized using flowcytometry, confocal microscopy, qPCR, in situ PCR and next generation sequencing technologies. Multiple transcription factor-encoding adenoviruses (e.g. Pdx1, MafA, Ngn3) were employed to determine the differentiation potential of these cells. Analysis of multiple pancreatic hormones and transcription factors in these samples demonstrated consistent differentiation. The differentiation potential was further explored using AMCs isolated from transgenic mice that express GFP under the regulation of Pdx1 (pancreatic and duodenal homeobox 1) or insulin-1 gene promoters. GFP expression was quantitated as an index of gene promoter activity during differentiation to insulin-producing cells, in the presence of various pro-differentiation small molecules. Human AMCs were exposed to a standard differentiation protocol and seen to migrate to form islet-like cell aggregates (ICAs), showing significant increases in islet hormone transcripts in vitro. These adipose-derived ICAs were transplanted into immunocompromised animals using two models of transplantation. Cells were transplanted in a Theracyte immunoisolation device into the peritoneum, and within a blood clot under the kidney capsule. Transplanted cells maintained expression of endocrine pancreatic transcription factors and did not undergo a regressive mesenchymal transition following surgery. Circulating blood samples collected from peripheral circulation of these mice, following a glucose injection, showed that differentiated and engrafted human AMCs could sense, transcribe, translate, package and secrete insulin in response to a glucose stimulus. These studies indicate that human AMCs can differentiate into insulin-producing cells in vitro and have potential for cell replacement therapy in diabetes.
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ItemDiabetes telephone coaching study: a randomised controlled trial and economic analysis( 2013)Aims: The randomised controlled trial (RCT) aimed to assess the efficacy of telephone coaching on glycaemic control, risk factor status and adherence to monitoring requirements in patients with poorly controlled type 2 diabetes (T2DM), both at the intervention’s conclusion (six months) and in the post-intervention period (12 months). The study also involved a cost-effectiveness analysis of the intervention, both in a within-trial setting and in a 10 year modelled scenario. Finally, this study sought to estimate the annual cost of treating Australians with poorly controlled T2DM. Method:Participants with T2DM, HbA1C >7% (n=94) were randomised to receive usual care plus six months of telephone coaching, or usual care only. Coaching involved assessment, education and goal setting regarding diet, exercise, medications and adherence to diabetes monitoring requirements. The primary outcome was HbA1C at six months. Secondary outcomes included HbA1C at 12 months and other physiological and monitoring measures. The within-trial economic analysis used cost and efficacy data collected between baseline and six months of the RCT to estimate the cost-effectiveness of the telephone coaching intervention, compared to usual diabetes care. The primary outcome was an incremental cost effectiveness ratio, expressed as cost per quality adjusted life year (QALY) saved. Items costed included those attributable to medication use, general practitioner presentations, outpatient appointments, emergency department presentations, inpatient admissions and delivery of the intervention. To estimate the annual cost of treating a person with T2DM, six-monthly costs of all participants were doubled to reflect the annual cost. The modelled economic analysis used the UKPDS Outcomes Model to extrapolate outcomes collected at six months in the RCT over 10 years, assuming the ongoing delivery of the telephone coaching intervention. Outcomes included life expectancy, quality-adjusted life expectancy (QALE) and costs. A 5% discount rate was applied to all future costs and benefits. Sensitivity analyses were conducted to reflect uncertainty surrounding key input parameters. Results: The six month intervention contributed to significant improvements in HbA1C (p=0.03), fasting glucose (p=0.02), diastolic blood pressure (BP) (p=0.03) and physical activity (p=0.02). At six months, adjusted mean HbA1C in the intervention group was 7.7% (7.4 to 8.1) compared to 8.5% (8.1 to 8.8) in controls. Intervention group participants also improved their compliance with routine foot checks and pneumococcal vaccination by six months and retinal screening by 12 months. However, the intervention’s effects on glycaemic control had disappeared by 12 months. For the within-trial economic analysis, the intervention was dominated by the control condition. This is because six monthly diabetes-related costs in the intervention group were significantly higher than the control group, $5403 versus $2260, p=0.009. Owing to one death, the intervention group also experienced a non-significant decline in health utility, -0.01 (-0.03-0.01), equating to a reduction in QALE of 0.005. The estimated mean annual cost of treating a person with poorly controlled T2DM was $7020. The intervention dominated the control condition in the modelled economic analysis, contributing to cost savings of $3327 and non-significant improvements in QALE (0.2 QALE) and life expectancy (0.3 years). The intervention also dominated the control condition in most sensitivity analyses. When the baseline imbalance in terms past history of stroke was controlled, the intervention remained highly cost-effective at a cost of $4365 per QALY saved. Conclusions: Telephone coaching improves glycaemic control and adherence to complication screening in patients with poorly controlled T2DM. The effects were not sustained in the post-intervention period, therefore strategies that facilitate the maintenance of intervention gains are required to improve long term outcomes. The intervention was not cost effective during the within-trial period, but over 10 years, the intervention’s cost was predicted to be fully recovered through cost savings from the prevention of downstream morbidity. The estimated annual cost of treating a person with T2DM updates former cost estimates that were based on data collected over 10 years ago. Collectively, these results are of major relevance to clinical practice, public health practice and health policy. Findings from this study support the need for a larger, prospective multi-centre trial of telephone coaching to confirm both the clinical and economic benefits prior to their implementation in routine clinical practice.
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ItemThe role of the coronary vasculature and myocardium in the pathogenesis of diabetic cardiomyopathy( 2012)The prevalence of diabetes is increasing worldwide. This poses a significant threat to human health, as diabetes is associated with an increased risk of mortality due to cardiovascular disease. In particular, diabetic patients develop diabetic cardiomyopathy (DCM), characterised by impaired cardiac muscle contraction and relaxation, leading to left ventricle (LV) muscle stiffness and congestive heart failure. Previous studies suggest that changes in the coronary vasculature and cardiac subcellular function may account for the progression to DCM, however as yet this has not been assessed in vivo. Synchrotron radiation (SR) now makes possible novel imaging and diffraction techniques, to investigate the role these mechanisms play in the early development of DCM, where clinical intervention is most efficacious. To assess coronary function in vivo we validated the use of SR imaging to detect and quantify regional differences in resistance microvessel calibre. In type 1 diabetic rodents we found that although endothelium-dependent and –independent vasodilatory responses in individual coronary vessels are preserved, following inhibition of NO and PGI2 production, there is evidence of localised focal and segmental constrictions. This demonstrates, for the first time, localised coronary microvascular endothelial dysfunction in early-stage type 1 diabets (T1D). Contributing to this diabetic coronary impairment is the RhoA/Rho-kinase (ROCK) pathway, which had previously been shown to play a role in endothelial dysfunction and coronary vasospasm. Our data further support a role for ROCK in early diabetic coronary dysfunction, as following nitric oxide synthase/cyclooxygenase blockade, ROCK inhibition greatly reduced regional segmental constrictions and completely alleviated persistent focal stenoses in diabetic animals. Together, these findings provide strong evidence that early vascular dysfunction may contribute to the development of DCM. In addition, although characterised by global cardiac impairment, the role subcellular changes in the sarcomere play in DCM progression is not known. SR, as a source for small-angle X-ray diffraction, allows the assessment of cardiomyocyte cross-bridge dynamics (CB) and myosin interfilament lattice spacing in situ and in real time. Using SR, our data shows that in early T1D, CB dynamics are abnormal in the beating hearts and this is directly related to impaired LV function. The change in CB dynamics is caused by myosin head displacement from actin filaments, but notably is not related to estimated sarcomere length or myofilament order. SR X-ray diffraction thus provides a robust method to assess cardiac CB dynamics in situ and for the first time we provide evidence that impairment in the regulation of myosin head extension in T1D hearts contributes to DCM. Currently 85-90% of diabetics have T2D and it is therefore critical that these coronary microvascular and cardiac subcellular impairments in T1D are explored in T2D. As such, rodent models which account for the environmental factors important in the human development of DCM are required. We conducted a comprehensive characterisation of cardiac function and structure in diet-induced rodent models of obesity, insulin resistance and T2D, and uncovered mild systolic dysfunction in fructose fed and mild diastolic dysfunction in high fat fed rodents. Furthermore, we demonstrated mild contractile dysfunction in high fat fed low dose streptozotocin rodents. The characterisation of only mild cardiac dysfunction, in spite of the lengthy time course used, suggests further refinement is required to achieve more robust DCM models. In summary, through the validation of novel SR imaging and diffraction techniques our data has confirmed a role for coronary microvascular dysfunction, via the ROCK pathway and cardiac subcellular impairment, via reduced myosin head extension, in the development of DCM. In addition, further studies investigating rodent models of T2D and DCM are required. These findings provide a strong basis for the future development of novel therapies aimed at preventing and/or reversing the decline in cardiac function associated with diabetes.
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ItemFibrosis in experimental diabetic nephropathy and cardiomyopathy: effects of FT011, a novel anti-fibrotic intervention( 2011)It is estimated that by 2025, 366 million people worldwide will be diagnosed with diabetes mellitus (DM); this brings with it the potential for an increase in the prevalence of diabetic nephropathy (DN) and cardiomyopathy (DCM). Despite current management for diabetes, DN is the leading cause of end stage of kidney disease (ESKD) for renal replacement therapy, and DCM is associated with a higher incidence of chronic heart failure (CHF). Pathological fibrosis is a hallmark of progressive renal and cardiac disease leading to ESKD and CHF. Growth factors such as transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF) have been consistently implicated in the fibrogenesis in DN and DCM. Therefore, strategies inhibiting the bioactivities of these cytokines are becoming valuable anti-fibrotic therapeutic targets. Tranilast is one of series compounds that have been shown to inhibit actions of TGF-β and PDGF. To optimise the anti-fibrotic effects of tranilast, FT011 (Fibrotech Therapeutics, Pty. Ltd, Melbourne, Australia) is a newly synthesized compound based on cinnamoyl core structure of tranilast. The hypothesis of the thesis is that targeted treatment for fibrosis with FT011 will attenuate functional and structural manifestations of injury in experimental DN and DCM. The aims of this thesis were to firstly evaluate the inhibitory effects of FT011 on collagen synthesis and cell proliferation in vitro and in vivo models, and then to explore therapeutic effects of FT011 in experimental DN and DCM. In cultured rat mesangial cells, FT011 inhibited TGF-β1 and PDGFBB induced collagen production and cell proliferation in a dose dependent manner with no evidence of cell toxicity. Consistent with these actions, treatment of anti-Thy1 nephritis with FT011 attenuated matrix accumulation, mesangial phenotypic changes, mesangial cell proliferation and glomerular macrophage infiltration. These findings suggest a promising profile of FT011 for its potential use as an anti-inflammatory and anti–fibrotic drug. Progressive DN is characterised by glomerulosclerosis and tubulointerstitial fibrosis leading to capillary rarefaction with consequent loss of renal parenchyma and function. Both early and late intervention with FT011 in diabetic Ren-2 rats, a clinically predictive experimental model of DN, prevented development of albuminuria, tubulointerstitial fibrosis, and glomerulosclerosis. In addition, treatment of diabetic Ren-2 rats with FT011 was associated with a reduction in loss of glomerular capillary endothelial cells, interstitial macrophage accumulation and tubular cell apoptosis. These findings prove the hypothesis that targeted treatment for fibrosis with FT011 would attenuate renal functional and structural injury in experimental DN. Pathologically, DCM is associated with microvascular disease and characterised by myocyte hypertrophy, apoptosis and accumulation of interstitial matrix. These structural changes ultimately lead to heart dysfunction. Treatment of diabetic Ren-2 rats with FT011 attenuated systolic and diastolic dysfunction associated with reduction in interstitial fibrosis and myocyte hypertrophy. These findings prove the hypothesis that anti-fibrotic and anti-hypertrophic therapy with FT011 would attenuate heart dysfunction in DCM. The anti-fibrotic effects of FT011 observed in diabetic kidney and heart are independent of changes in high blood pressure and glucose, and the anti-fibrotic therapeutic efficacy of FT011 is greater than previous studies with tranilast. In addition, treatment with FT011 reduced ERK1/2 MAP kinase phosphorylation in both the diabetic kidney and heart. These findings suggest that anti-fibrotic mechanism actions of FT011 are different from blood pressure and glucose lowering agents and may be attributable to events that are more down stream signalling pathways of TGF-β, although the precise mode of action for FT011 was not determined in the present thesis. In conclusion, the findings of the present thesis advocate the use of strategies that target treatment for fibrosis by inhibiting bioactivities of pro-fibrotic cytokines delays the progression of DN and DCM. These findings yield important preclinical information in terms of the potential utility of FT011 as a novel anti-fibrotic therapy in the treatment of DN and DCM.