Exacerbating effect of glucose on phosphate-induced endothelial calcification: analysis of lipidome signature modification

Diabetes and diabetic kidney disease are public health concerns because of the high morbidity and mortality associated with cardiovascular comorbidities. Endothelial dysfunction and calcification play a crucial role in the increase in cardiovascular risk in these patients. In the present study, we set up an in vitro model to investigate the effect of high glucose and the combination of high glucose and phosphate on endothelial cells. In this model we studied endothelial calcification and lipid profile modifications that may be relevant to plasma membrane fluidity and cellular metabolic homeostasis, with the aim of elucidating the role of alterations in the lipidome signature in endothelial dysfunction and calcification. Human aortic endothelial cells (HAEC) were treated with 30 mM glucose (HG) for up to 5 days and subsequently exposed to 3 mM phosphate (Pi) for up to an additional 7 days. At different time points, cells were processed to assess calcium deposition by Alizarin red staining, protein expression by Western blotting, differential gene expression by mRNA sequencing, and lipid profiles by untargeted mass spectrometry-based lipidomics. Changes in lipid accumulation were visualized by Oil Red O staining and transmission electron microscopy (TEM). Statistical analyses were performed on at least three independent experiments, each conducted in triplicate. Pretreatment of HAEC with HG exacerbated Pi-induced endothelial calcification, characterized by enhanced osteoblast-like differentiation. Lipid profile analysis revealed an increase in energy-storage lipids (acylglycerols) in calcified HAEC, accompanied by remodelling of the glycerophospholipid profile and a shift in the distribution of saturated acyl chains, consistent with cellular membrane rearrangement. Transcriptomic analysis of genes involved in lipid metabolism suggested an association between lipidome alterations, endothelial dysfunction, membrane reorganization, and calcification. The present study demonstrates that elevated glucose levels exacerbate phosphate-induced endothelial calcification, a process potentially linked to medial calcification, which contributes to increased cardiovascular risk in patients with diabetes and chronic kidney diseases. An association between lipidome perturbation, endothelial dysfunction, and endothelial calcification was observed. Thus, our study suggests a link between alterations in lipid metabolism under high-glucose conditions and phosphate-induced endothelial calcification, indicating that lipid dysregulation may be associated with vascular calcification beyond atherosclerosis.