Reduced circulating CD63+ extracellular vesicle levels associate with atherosclerosis in hypercholesterolaemic mice and humans.

Abstract

Aims: The association and co-isolation of low-density lipoproteins (LDL) and extracellular vesicles (EVs) have been shown in blood plasma. Here we explore this relationship to better understand the role of EVs in atherogenesis.

Methods and results: Wild type (WT), PCSK9^-/-, and LDLR^-/- C57BL/6 mice were used in this study. Eleven week-old male mice were fed high-fat diet (HFD) for 12 weeks or kept on normal diet until old age (22-months). Cardiac function was assessed by ultrasound, cholesterol was quantified with a colorimetric kit and circulating EVs were measured using flow cytometry. Plaques were analysed post-mortem using Oil-Red-O staining of the aortic arch. EVs were measured from platelet free blood plasma samples of normal and hypercholesterolaemic clinical patients. Based on annexin V and CD63 staining, we found a significant increase in EV levels in LDLR^-/- and PCSK9^-/- mice after HFD, but CD81 showed no significant change in either group. There was no significant change in plaque formation after HFD. PCSK9^-/- mice show a favourable cardiac function after HFD. Blood cholesterol levels progressively increased during HFD, with LDLR^-/- mice showing high levels while PCSK9^-/- were significantly lowered compared to WT animals. In mice at old age, similar cholesterol levels were observed as in young mice. In old age, LDLR^-/- mice showed significantly increased plaques. At old age, ejection fraction was decreased in all groups of mice, as were CD63⁺ EVs. Similarly to mice, in patients with hypercholesterolaemia, CD63⁺ EVs were significantly depleted.

Conclusions: This research demonstrates an inverse relationship between circulating EVs and cholesterol, making EVs a potential marker for cardiovascular disease (CVD). HFD causes reduced cardiac function, but atherosclerotic development is slowly progressing in hypercholesterolaemic models and only observed with old animals. These results also bring further evidence for the benefit of using of PCSK9 inhibitors as therapeutic agents in CVD.