Relationships between Plasma PEDF and Kallistatin Levels and the Vascular Complications of Type 2 Diabetes Mellitus and the Effects of Fenofibrate: A Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) Sub-study

Abstract

Diabetes in all its forms imposes unacceptably high human, social and economic costs on all countries and at all income levels, with Type 2 diabetes (T2D) consistently growing due to increasing rates of obesity, sedentary lifestyle and hypercaloric diets. The major cause of morbidity and premature mortality in people with diabetes is not so much the presence of diabetes itself, but the development of chronic diabetic complications, causing loss of vision, renal failure, amputations, coronary artery disease and stroke. In addition to hyperglycaemia, hypertension, dyslipidaemia and obesity, the related processes of inflammation, oxidative stress; and disturbed-angiogenesis, are important factors implicated in the development and progression of diabetic micro and macrovascular complications. These factors are also key therapeutic targets. The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study is a landmark study of 9795 adults with T2D aged (at baseline) 50‐75 years from Australia, New Zealand and Finland. The double blinded randomised placebo controlled FIELD study demonstrated that the oral lipid lowering drug fenofibrate reduces the occurrence of serious vascular and new complications of T2D over 5-years, including sight-threatening diabetic retinopathy by 37%, nephropathy by 15%, and lower limb amputations by 36%; and also significant reductions in some types of cardiovascular disease. Interestingly, except for some of the renoprotection, the clinical benefits were independent of fenofibrate changes to traditional lipid levels. We propose that the pleiotropic effects of fenofibrate, such as related to growth factors, inflammation and oxidative stress may contribute to fenofibrate’s effects. We conducted a clinical research project to explore this hypothesis, with an emphasis on two growth factors, Pigment Epithelium Derived Factor (PEDF) and Kallistatin. Recent literature has identified two SERPIN growth factors, PEDF and Kallistatin, to have anti-angiogenic, anti-oxidant, and anti-inflammatory effects, and small cross-sectional studies have shown promising results for their involvement in vascular function and chronic diabetes complications. The overall aims of this research project were to investigate the T2D FIELD study cohort, to determine whether plasma PEDF and Kallistatin levels were associated with T2D and its vascular and neurologic complications, their correlations with traditional and novel vascular risk factors, and if and how they are changed by fenofibrate. This thesis consists of six chapters. Chapter one is an introduction and literature review related to diabetes and risk factors; and the FIELD study and PEDF and kallistatin. Chapter two describes the FIELD study, the substudy design, and the materials and methods for both PEDF and kallistatin analyses (by ELISA). Chapter three details an issue with the identification of suboptimal quantity of the batch to batch variation of the commercially available PEDF kits and the problem resolution. Chapter four outlines the study and results of the PEDF analysis. Mean PEDF levels were significantly higher in women than men. Baseline plasma PEDF levels significantly correlated positively, however weakly, with traditional and novel vascular risk factors, including HbA1c, BMI, insulin resistance, renal dysfunction, inflammation and oxidative stress, and inversely with HDL-C levels, and with eGFR. In addition, higher PEDF levels significantly predicted the development of on-trial composite macrovascular and microvascular complications, including nephropathy and amputations. Higher baseline PEDF was also associated with a reduced risk for retinopathy development. This data suggests a role for PEDF as a potential marker to evaluate future vascular health risk and as a potential therapeutic target and agent. Plasma PEDF levels are also significantly increased by fenofibrate. We suggest that some of fenofibrate’s clinical benefits may be via its effects on PEDF; and PEDF may be a therapeutic target. Chapter five outlines the study and results of kallistatin analysis in the FIELD study T2D subjects. Baseline plasma kallistatin levels significantly correlated positively, however weakly, with traditional and novel vascular risk factors, including HbA1c, TG, total cholesterol, HDL-C, abnormal apolipoprotein profiles, and renal dysfunction. Kallistatin was also found to inversely correlate with circulating levels of markers of inflammation, MPO, and cystatin C. Significant independent determinants of baseline plasma kallistatin levels were HDL-C, TG, systolic BP, HbA1c and BMI. Unlike PEDF levels, kallistatin levels were not found to significantly correlate with vascular complications at baseline, and on-trial. Kallistatin correlated with PEDF at baseline (r=0.15), and in response to fenofibrate, circulating kallistatin levels significantly decreased, opposite to that seen in PEDF. Chapter six is a thesis summary, summarising the key findings of each chapter, and suggests research directions to extend and enhance the study findings.