The Impact of Glucose Lowering Therapies Including Dipeptidyl Peptidase-4 Inhibitor on Circulating Endothelial Progenitor Cells (EPCs) and Its Mobilising Factor Stromal Derived Factor-1α (SDF-1α) in Patients With Type 2 Diabetes

Diabetes is a chronic condition reported to affect 366 million people globally in 2011 and estimated to rise to 552 million by 2030 worldwide, i.e. one in 10 adults. In the UK alone, the prevalence is 4.5% and approximately 5 million people are projected to be affected in 2025. Of the entire population of people with diabetes, Type 2 Diabetes Mellitus (T2DM) constitutes more than 90%. In the United Kingdom Prospective Diabetes Study (UKPDS), 50% of the people with T2DM presented with micro and/or macrovascular complications at the time of diagnosis. It is well established that people with T2DM also have 4 to 5 times greater risk of cardiovascular complications compared to the general population, accounting for 80% of mortality. Thus, the burden of diabetes and its associated complications on health care services are enormous. Prescribing medications for the complications associated with diabetes alone costs 3 to 4 times more than the cost of medications for managing diabetes in the National Health Service (NHS). Thus, therapies that simultaneously target glycaemic control and diabetic complications particularly cardiovascular disease (CVD) independent of their glucose lowering effect are desirable.

The current study aims to evaluate the therapeutic effects of incretin based therapies (DPP-4 inhibitors versus GLP-1 analogues) and incretin based therapies versus non-incretin based treatments on levels of EPCs, SDF-1α and other biomarkers in patients with T2DM established on these treatments (receiving for >3 months).

Study objectives To investigate if treatment with DPP-4 inhibitors in patients with T2DM is associated with increased levels of Endothelial Progenitor Cells (EPCs) and Stromal Derived Factor-1 α (SDF-1α) compared to those receiving GLP-1 analogues in a cross sectional analysis adjusted for confounders.

Further, the study aims to determine if treatment with incretin based therapies is associated with higher circulating levels of EPC and SDF1-α compared to non-incretin based therapies.

Preparation of Peripheral Blood Mononuclear Cells (PBMCs) for subsequent EPC quantification PBMCs will be collected using standard techniques. In brief PBMCs will be isolated from venous blood collected into a 9.7ml sodium heparin blood tube (which will subsequently be stored at room temperature for up to 4 hours). The blood samples will then be centrifuged (1500g for 10 min at 4⁰C) and the plasma layer will be collected for later biomarker analysis (aliquots of plasma will be frozen at -80⁰C until use). The buffy coat layer (the white opaque layer that lies directly above the red cell pellet) will be used for PBMC isolation. To isolate the PBMCs, the buffy coat will be centrifuged at 400g on a Ficoll gradient (1.077g/ml) for 30 minutes and the resulting cellular layer will then undergo 3 further washes (300g for 10 minutes and 20g for 10 minutes X2) to wash the cells and deplete platelets. To calculate the number of PBMC isolated, the pellet will be suspended in a known volume of Phosphate Buffered Solution (PBS) (1ml) and a small quantity (10µl) will be assessed for quantity and viability under an inverted microscope using a haemocytometer and a vital stain (Trypan blue).