Identification of Novel Glucose-Dependent Alterations Responsible for Vascular Dysfunction in Type 2 Diabetes Mellitus (T2DM) I

Bolanle, Olapeju Israel

Abstract

Despite the plethora of drugs currently available for managing coronary artery disease (CAD) patients with or without type 2 diabetes mellitus (T2DM), yet CAD frequently progresses, necessitating a coronary artery bypass graft (CABG) procedure using human saphenous vein (HSV). However, over 70 % of the procedures fail after 10 years in T2DM patients. We propose that this is due in part to glucose-dependent alterations in the function of HSV smooth muscle cell (HSVSMC), a key cell type involved in vascular dysfunction. Therefore, in this project, we examined T2DM-dependent alterations, including protein O-GlcNAcylation, a dynamic and reversible post-translational modification, in the metabolic profile and function of HSVSMC from T2DM patients versus non-diabetic control. Under NHRA ethics approval (NHS REC:15/NE/0138), HSVSMCs were explanted from surplus HSV tissues from T2DM and non-diabetic patients undergoing CABG. Expression levels of O-GlcNAcase (OGA), O-GlcNAc transferase (OGT), and Glutamine-fructose-6P amidotransferase (GFAT) were determined in lysates of HSVSMCs from T2DM versus non-diabetic patients by SDS-PAGE and immunoblotting using specific antibodies. This experiment was repeated using lysates of HSVSMCs treated with high glucose (HG) concentrations (10 mM and 25 mM) for 48 hours. Our results showed that their expression levels were not significantly different in T2DM patients versus non-diabetic controls, and after treatment with HG concentrations. Furthermore, we used the Seahorse XFP analyzer to determine real-time mitochondrial function of HSVSMCs by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) after sequential addition of modulators of respiration; oligomycin, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), and rotenone and antimycin A. Results revealed that versus unstimulated cells, IL-6/sIL-6Rα and PDGF-BB caused an increase in OCR (102.5% and 93.9% in maximal respiration, respectively) and ECAR (basal glycolysis 101.6%; glycolytic reserve 76.3%; maximal glycolysis 117.1%; glycolytic reserve 99.3%, for PDGF-BB) in HSVSMCs from T2DM patients but not in those from non-diabetic control. However, this observed increase was abolished by ruxolitinib. Similarly, versus unstimulated cells, thrombin but not Ang II caused a significant increase in OCR (85.2% in maximal respiration) of HSVSMCs from T2DM patients, and this was abolished by trametinib. Furthermore, flow cytometry assay showed that both ruxolitinib and trametinib versus unstimulated cells significantly reduced mitochondrial reactive oxygen species’ production in HSVSMCs from both T2DM (133.2% and 154.8% respectively) and non-diabetic (114.8% and 107.4% respectively) patients. These findings suggest a JAK/STAT- and MAPK/ERK-mediated regulation of mitochondrial function of HSVSMCs, hence, potential targets for regulation of the function of HSVSMCs which can be explored for drug development to limit SV graft failure.

Citation

Bolanle, O. I. (2023). Identification of Novel Glucose-Dependent Alterations Responsible for Vascular Dysfunction in Type 2 Diabetes Mellitus (T2DM) I. (Thesis). Hull York Medical School. https://hull-repository.worktribe.com/output/4420724

• Thesis Type: Thesis
• Deposit Date: Oct 18, 2023
• Publicly Available Date: Oct 18, 2023
• Keywords: Medicine
• Public URL: https://hull-repository.worktribe.com/output/4420724
• Additional Information: Hull York Medical School; University of Hull and University of York
• Award Date: Oct 10, 2023