Will medial arterial calcification in diabetes mellitus type 2 be influenced by Glucagon-Like-Peptide 1.
1. Calcification prone DBA2J mice will be rendered type II diabetic through a combination of western diet and single injection of Streptozotocin (50mg/kg). Animals will then be injected with an AAV vector encoding for GLP-1 (7-37) or a control (LacZ). Calcium deposition in the coronary sinus, the aortic arch and the descending aorta will be assessed histologically by Alizarin Red staining and quantified by QuantiChrom Calcium Assay Kit. Expression of calcification-related genes, pro-inflammatory cytokines, receptor activator of nuclear factor-κB ligand (RANKL) system, and the RAGE system in the aorta will be analysed by qPCR.
2. Differential gene expression in the aortic tissue will be analysed by microarray and validated by qPCR. Based on the microarray data, pathway analyses will be performed.
3. Primary VSMC from DBA2 animals overexpressing GLP-1 will be isolated to investigate whether GLP-1 can influence the phenotypic switching of VSMC from contractile to synthetic VSMC using a miniaturised cell-based platform to analyse differences in proliferation, migration and invasion (xCELLigence®) (ESR3).
4. Serum of diabetic and non-diabetic DBA2J mice overexpressing GLP-1 or control (LacZ) will be sent for metabolomics analysis (10 per group) and differentially regulated metabolites will be tested for their in vitro capability to induce osteochondrogenic transformation in VSMC.
5. Metabolites found to be differentially regulated will be tested in a pre-existing Biobank containing the serum of over 1000 patients (over 300 diabetics).