A comparison of the effects of monotherapy with rosuvastatin, atorvastatin or ezetimibe versus combination treatment with rosuvastatin-ezetimibe and atorvastatin-ezetimibe on the integrity of vascular endothelial cells damaged by oxidized cholesterol

Abstract

Dyslipidemia, atherosclerosis, and cardiovascular events can be prevented, or treated, using statins, alone or in combination with ezetimibe. The aim of the study was to compare the direct pleiotropic effects of two commonly-used statins (atorvastatin, rosuvastatin), ezetimibe and their combinations on endothelial cells damaged by oxidized cholesterol. HUVEC cultures were stimulated for 20 hours with atorvastatin (5 μM; 2793 ng/mL), rosuvastatin (10 μM; 4815 ng/mL), ezetimibe (1.22 μM; 500 ng/mL), atorvastatin plus ezetimibe (5 μM + 1.22 μM; 2793 ng/mL + 500 ng/mL) and rosuvastatin plus ezetimibe (10 μM + 1.22 μM; 4815 ng/mL + 500ng/mL) in separate groups, with or without 25-hydroxycholesterol pre-incubation (24.83 μM; 10 μg/mL; four hours then washout). HUVEC integrity was measured in the RTCA-DP xCELLigence system. The mRNA expression and protein levels of ZO-1, OCLN, ICAM-1 were analyzed by real-time PCR and ELISA. Pre-incubation with 25-OHC resulted in decreased endothelial cell integrity (p<0.001), decreased expression of ZO-1 mRNA (p<0.05) and protein levels (p<0.05), OCLN mRNA (p<0.05) and protein levels (p<0.05) and increased ICAM-1 mRNA (p<0.001) and protein levels (p<0.001) compared to the control group. Incubation with rosuvastatin (12h p<0.01; 24h p<0.001) and atorvastatin (only 12h p<0.05) restored HUVEC integrity. Subsequent incubation with rosuvastatin increased ZO-1 mRNA (p<0.001) and protein (p<0.001) levels. Subsequent addition of ezetimibe increased ZO-1 mRNA level (p<0.001) but not protein level. Furthermore, only incubation with rosuvastatin increased OCLN mRNA (p<0.05) and protein (p<0.05) levels. In each drug-stimulated group, both ICAM-1 mRNA and protein levels were reduced after initial incubation with oxysterol (p<0.05). 25-hydroxycholesterol disrupts endothelial integrity, decreases the mRNA and protein levels of tight junction, and increases those of intercellular adhesion molecules. Both rosuvastatin and atorvastatin can improve endothelial integrity, but only rosuvastatin can completely abolish the effect of oxysterol. The combination of statins with ezetimibe has less direct effect on the endothelial barrier than the statins alone.

The effect of lipid-lowering therapies on the pro-inflammatory and anti-inflammatory properties of vascular endothelial cells

“…No significant changes in HUVEC cell viability were observed as a result of treatment with statins and ezetimibe at the selected concentrations, as previously presented by Niedzielski et al [ 11 ]. In each series of probes, cell viability was determined by the Trypan Blue dye exclusion test.…”

“…Following 25-OHC pre-stimulation and drug stimulation, mRNA and the supernatants were isolated from HUVECs. The specific lengths of stimulation were selected based on previous observations of the integrity of the HUVECs in the RTCA-DP system [ 11 ]. Gene expression was measured vt reverse-transcription polymerase chain reaction in real time (RT-PCR) and protein levels were measured with enzyme-linked immunosorbent assay (ELISA).…”

High cholesterol diet, oxysterols and their impact on the gut–brain axis

“…Sheng and colleagues confirmed these results in intestinal CaCo2 and HCT116 cells and suggested a role of cholesterol 25-hydroxylase in preserving ZO-1 and claudin-4 expression and synthesis (Sheng et al 2020). On the other hand, 25OHC and 27OHC were proven to substantially downregulate JAM-A protein levels in other epithelial cell lines, Hela and MA104 (Civra et al 2020), as well as both expression and synthesis of ZO-1 and occludin in HUVEC (Niedzielski et al 2021). While further studies are needed, the role of specific oxysterols in altering the intestinal barrier and their potential involvement in microbial translocation is increasingly evident.…”