Glyoxalase 1-knockdown in human aortic endothelial cells – effect on the proteome and endothelial function estimates

Abstract

Methylglyoxal (MG), an arginine-directed glycating agent, is implicated in diabetic late complications. MG is detoxified by glyoxalase 1 (GLO1) of the cytosolic glyoxalase system. The aim was to investigate the effects of MG accumulation by GLO1-knockdown under hyperglycaemic conditions in human aortic endothelial cells (HAECs) hypothesizing that the accumulation of MG accounts for the deleterious effects on vascular function. SiRNA-mediated knockdown of GLO1 was performed and MG concentrations were determined. The impact of MG on the cell proteome and targets of MG glycation was analysed, and confirmed by Western blotting. Markers of endothelial function and apoptosis were assessed. Collagen content was assayed in cell culture supernatant. GLO1-knockdown increased MG concentration in cells and culture medium. This was associated with a differential abundance of cytoskeleton stabilisation proteins, intermediate filaments and proteins involved in posttranslational modification of collagen. An increase in fibrillar collagens 1 and 5 was detected. The extracellular concentration of endothelin-1 was increased following GLO1-knockdown, whereas the phosphorylation and amount of eNOS was not influenced by GLO1-knockdown. The expression of ICAM-1, VCAM-1 and of MCP-1 was elevated and apoptosis was increased. MG accumulation by GLO1-knockdown provoked collagen expression, endothelial inflammation and dysfunction and apoptosis which might contribute to vascular damage.

Ketone Body Acetoacetate Buffers Methylglyoxal via a Non-enzymatic Conversion during Diabetic and Dietary Ketosis

“…Still, glyoxalase I knockdown has recently been reported to slightly decrease protein MG-H1 levels in endothelial cells even though MG levels are 2-to 3-fold upregulated. This is likely due to accelerated protein turnover (Stratmann et al, 2016). Conversely, other studies report diminished plasma AGE levels following glyoxalase I expression (Brouwers et al, 2011).…”

Glyoxalase 1 Modulation in Obesity and Diabetes

“…Dicarbonyl stress may promote the development of vascular complications through protein glycation linked to: ECM protein modification leading to cell detachment and dysfunction (45,138) and changes in ECM synthesis and processing (168), mitochondrial protein modification driving increased ROS formation and oxidative stress (158), modification of p300 and p65 of HIF1α and NF-κB signalling pathways driving abnormal responses to ischaemia and persistent vascular inflammation (173,197), modification of the 20S proteasome and slowing of cellular proteolysis (139), modification of lipoproteins driving dyslipidemia and atherosclerosis (64,141), β-Klotho depletion driving inflammation, insulin resistance and nephropathy (206), and other processes.…”

“…In endothelial cells, silencing of Glo1 changed expression of over 485 genes linked to the development of CHD (107). The related increase of MG-H1 residue content of cell protein changed the levels of MG-modified and unmodified cellular and extracellular matrix (ECM) proteins (168). MG-H1 is a quantitatively the major proteinderived advanced glycation endproduct (AGE).…”

Blockage of Glyoxalase I Inhibits Colorectal Tumorigenesis and Tumor Growth via Upregulation of STAT1, p53, and Bax and Downregulation of c-Myc and Bcl-2

“…GLOI overexpression enhanced resistance to anti-tumor agents such as etoposide and doxorubicin [20]. Silencing of GLOI in tumors with high rates of glycolysis and methylglyoxal formation led to accumulation of methylglyoxal and cytotoxicity [22,23]. These findings suggest that GLOI plays a crucial role in tumorigenesis.…”