Mass spectrometry-based proteomics identifies high glucose-induced changes in vascular smooth muscle (VSMCs) and sustained extracellular signal-regulated kinases (ERK) activation

Abstract Introduction Coexistence of diabetes and hypertension exacerbates morbidity of cardiovascular diseases, indicating a synergy/cooperativity between these pathophysiologies. Although multifactorial, hypertension is associated with vasoconstrictor-mediated arterial hyper-construction and remodelling via their cognate G protein-coupled receptors (GPCRs). Signalling via Gs-coupled GPCRs such as the β₂-adrenergic receptors (β₂ARs) opposes the actions of vasoconstrictors by inducing vasodilation and inhibiting vasoconstrictor-stimulated remodelling pathways such as ERK. As elevated glucose is known to affect multiple aspects of cellular physiology, we examined the effects of acute (6-day) glucose exposure on the proteome of rat aortic smooth muscle cells (RASM). Purposes To determine whether glucose affected protein expression by measuring differentially expressed proteins (DEPs) in RASM exposed to high (20 mM) and low (5 mM) glucose concentrations. The study also aimed to investigate the role of ERK-related protein interactions in vascular remodelling. Methods Cellular cultivation and glucose administration. RASM were isolated from male Wistar rats and cultured in Dulbecco's Modified Eagle Medium supplemented with 10% foetal bovine serum under low glucose (5 mM) or high glucose (20 mM) conditions for 6 days. Protein extraction and trypsin digestion for mass spectrometry-based proteomics were conducted prior to analysis, utilising liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for label-free quantification. A bespoke R pipeline utilising the Limma package was employed for statistical analysis and the creation of volcano plots. A string-based network analysis was utilised to show ERK-related signalling proteins. Results A total of 2,317 proteins were found, of which 21 were significantly upregulated under high glucose conditions (p 0.05, log2FC 1). RASM were cultured in high glucose showed a distinct DEP compared to those in low glucose media (Figure 1). Enrichment analysis alters targeted pathways, such as MAPK/ERK signalling, oxidative stress, and metabolic regulation (Figure 2). The main hubs of the network were MAPK1 (ERK2) and MAPK3 (ERK1), which linked to important proteins such as Hepatocyte Growth Factor (HGF), Glutathione S-Transferase Mu 1 (GSTM1), Lysine Acetyltransferase 5 (KAT5), and Y-box binding protein 3 (YBX3). These proteins all play a part in ERK-mediated cell growth and vascular remodelling. HGF strongly interacted with MAPK3, confirming its role as an upstream activator of ERK. Conclusions Short-term (6-day) exposure to high glucose (20 mM) induces massive changes in the VSMC proteome, including elevated levels of MAPK/ERK signalling proteins and oxidative stress regulators. The results point to potential molecular alterations that underlie the illness-related dysregulation of vascular physiology and provide avenues for future investigation.Volcano Plot Proteins interaction