Scripties UMCG - Rijksuniversiteit Groningen
 
English | Nederlands

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.





ID 3351
Moeder ID 3084
Volgorde Brockkötter, L.M.
Naam BrockktterLM
Publiceren yes
OAI-naam Student_thesis
Path root/geneeskunde/2016/BrockktterLM/
Gemaakt op: 2017-03-06 11:23:55
Gemodificeerd op: 2017-03-06 11:23:55
Digitaal ID 58bd46c8391ac
Afstudeerrichting opleiding/afstudeerrichting 1
Studierichting Studierichting 1
Titel The Role of TLR4 / uPAR interaction in microvascular inflammation found in Diabetes Mellitus
Ruilverkeer mogelijk no
Printen in opdracht no
Aantal pagina's 24
Publicatiejaar 2016
Taal en
Engelse samenvatting 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.
Nederlandse samenvatting Introductie Diabetes Mellitus (‘suikerziekte’) is een aandoening die op de langere termijn negatieve gevolgen heeft voor meerdere organen. De incidentie van diabetes, vooral van
type 2 diabetes, is met name in de westerse wereld enorm gestegen. Diabetische nefropathie (DN) is de belangrijkste oorzaak van chronische nierziekte en nierfalen wereldwijd en is de sterkste voorspeller voor mortaliteit bij patiënten met diabetes. Recent onderzoek heeft aangetoond dat het ontstaan en progressie van DN geassocieerd is met een Toll-like receptor 4 (TLR4)-gemedieerde ontsteking. Verder is bekend dat urokinase-type plasminogen activator receptor (uPAR) kan dienen als een co-receptor voor TLR4. Of dit ook zo is in diabetes-geassocieerde ontsteking is onbekend.
Methoden In humane microvasculaire endotheel cellen (HMEC-1) werd diabetes gesimuleerd door ze te stimuleren met hoge glucose concentraties. Inflammatoire merkers werden gemeten via PCR en ELISA. TLR4 en uPAR expressie werd gemeten middels Western blot. De interactie tussen de twee receptoren werd gekwantificeerd door co-immunoprecipitatie en immunocytochemie. Verder werd uPAR expressie verlaagd met behulp van de lentivirale siRNA, gevolgd door analyse van glucose-afhankelijke activatie van signaalroutes m.b.v. Western blot.
Resultaten Stimulatie van HMEC-1 met hoog glucose verhoogde de expressie van inflammatoire cytokines, alsmede van TLR4 en uPAR. Verder induceerde hoog glucose incubatie van HMEC-1 een afname in fysieke interactie tussen de TLR4 en uPAR. Lentivirale downregulatie van uPAR veranderde de respons op hoog glucose in HMEC-1 van verschillende TLR4-geassocieerde signaaltransductie routes, waaronder MEK en IRF3.
Discussie/Conclusie Onze resultaten suggereren dat TLR4/uPAR interactie een belangrijke rol speelt in diabetes-geassocieerde ontsteking. Deze interactie moet verder worden geverifieerd zowel in vitro studies in primaire cellen, alsmede in een vivo model in muizen. Verandering van de TLR4/uPAR interactie, b.v. door peptide-remmers, is in potentie een nieuwe therapeutische optie om diabetische nefropathie tegen te gaan.
Onderwijsinstelling Medical Sciences
Type embargo abstract openbaar, scriptie op aanvraag
Auteur(s) Brockkötter, L.M. (lucas)
Begeleider(s) opleidingsinstelling Supervisors:; Kiyan, Dr Y; Henning, Prof. Dr. R.H.; Haller, Prof. Dr. H.G.; Institution of Research: Hannover Medical School, Germany
Auteur(s) Brockkötter, L.M. (lucas)


 
To top