Non-enzymatic modification of aminophospholipids induces angiogenesis, inflammation, and insulin signaling dysregulation in human renal glomerular endothelial cells in vitro

Abstract

Aims/hypothesis

Advanced glycation end-products (AGEs) formation in proteins are involved in healthy aging and a variety of diseases including Alzheimer's disease, atherosclerosis, and diabetic complications. However, the biological effects of the non-enzymatic modification of aminophospholipids (lipid-AGEs) at cellular level are poorly understood. This study aimed to investigate the effects of lipid-AGEs on angiogenesis, inflammation, insulin signaling, and mitochondrial function in human renal glomerular endothelial cells (HRGEC), exploring their potential role in the pathophysiology of diabetic nephropathy (DN).

Methods

HRGEC cells were exposed to non-enzymatically modified phosphatidylethanolamine (PE) by AGEs (lipid-AGEs), non-modified PE (nmPE) (aminophospholipid without modification), employed as a negative control, and lipopolysaccharides (LPS) as a positive control. Angiogenesis was assessed through vascular network formation metrics, including capillary area, junction density, and endpoints, under different extracellular matrices. Gene expression of inflammatory and angiogenic markers was quantified by RT-qPCR. Insulin signaling components, including IRS1 and AKT phosphorylation, were evaluated by immunoblotting. Mitochondrial function was assessed using high-resolution respirometry to determine ATP production rates from glycolysis and oxidative phosphorylation.

Results

Lipid-AGEs induced dose-, time-, and matrix-dependent angiogenesis, with effects comparable to LPS, particularly in Engelbreth-Holm-Swarm extracellular matrix (ECM) (capillary area increase: 25 %, p < 0.05). Lipid-AGEs significantly upregulated the expression of inflammatory genes IL8 and NFKB (p < 0.05), and the angiogenesis-related markers TGFB1 and ANGPT2 (p < 0.05). Insulin signaling was disrupted, as lipid-AGEs enhanced inhibitory phosphorylation of IRS1 (Ser-1101, 1.8-fold increase, p < 0.01) and modulated AKT (Ser-473) and p42/p44 ERK activation. At lower doses, lipid-AGEs reduced eNOS phosphorylation (p < 0.05) impairing insulin responsiveness. High-resolution respirometry revealed that lipid-AGEs reduced basal oxygen consumption rates (OCR) by 20 % (p < 0.05), with no significant changes in glycolytic ATP production.

Conclusion

Lipid-AGEs induce angiogenesis, inflammation, and insulin signaling disruption in HRGEC, contributing to endothelial dysfunction. These findings underscore the potential role of lipid-AGEs in age-related decline of renal function, as well as the pathogenic potential in DN highlighting their relevance as therapeutic targets for mitigating vascular and metabolic complications in diabetes.