Master Theses UMCG - University of Groningen
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The Role of TLR4 / uPAR interaction in microvascular inflammation found in Diabetes Mellitus

(2016) Brockkötter, L.M. (lucas)

Introduction Diabetes Mellitus is a systemic disease having many adverse long-term effects on multiple organs. The incidence of both Types of Diabetes is vastly rising, affecting patients particularly in the Western World.
Diabetic nephropathy (DN) is the leading cause of chronic kidney disease and kidney failure worldwide, being the strongest mortality predictor in patients with diabetes. Recent data revealed that Toll-like receptor 4 (TLR4)-mediated inflammation is associated with initiation and progression of DN. We hypothesized that urokinase-type plasminogen activator receptor (uPAR) might serve as a co-receptor to TLR4 in diabetes-associated inflammation.
Methods Human Microvascular Endothelial Cells (HMEC-1) were stimulated with low and high concentration of glucose to simulate diabetes. Inflammatory markers were measured via PCR and ELISA. TLR4 and uPAR expression was measured via Western blot. The interaction of the two receptors was quantified by co-immunoprecipitation and immunocytochemistry. uPAR expression was downregulated using lentiviral cell infection and glucose-dependent signaling pathways were analyzed by Western blotting.
Results Inflammatory cytokines were up-regulated in HMEC-1stimulated with high glucose. TLR4 and uPAR expression was elevated when HMEC-1 were set under diabetic conditions. TLR4/uPAR interaction could be proven in glucose induced diabetic HMEC-1. Lentiviral downregulation of uPAR proved that uPAR has a functional involvement in cellular response to high glucose in endothelial cells which is probably linked to TLR4 signaling.
Discussion/Conclusion Data suggests that TLR4/uPAR interaction plays an important role in diabetes-associated inflammation. This interaction needs to be verified and established further by in vitro studies in primary cells as well as on an in vivo model in mice. TLR4/uPAR interaction could be blocked by means of peptide inhibitors, thus having a great therapeutic impact in Diabetes associated DN.

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