Apoptosis and proliferation of endothelial cells in early atherosclerotic lesions: possible role of oxidised LDL

Abstract

Background and aim: Evidence for apoptosis has been found in advanced atherosclerotic lesions, but the factors triggering it are poorly understood. Oxidised low-density lipoproteins (LDLs) are cytotoxic to a variety of cells and induce the apoptosis of smooth muscle cells (SMC), fibroblast, macrophages and endothelial cells in vitro. The aim of this study was to investigate apoptotic cell death in the early phases of aortic atherosclerosis in rabbits, and whether oxidised LDLs colocalize ex vivo with apoptotic cells in atherosclerotic lesions in cholesterol-fed rabbits.

Methods and results: Male albino New Zealand rabbits were fed a standard diet or a diet containing 1.2% cholesterol for 60 days. The aortic arch of each animal was sectioned and stained with antibodies against SMC, endothelial cells, macrophages and oxidised LDLs or for proteins involved in apoptotic pathways such as Fas, Bax, Bcl2, and caspase 3. The nuclei in adjacent sections were stained with Hoechst 33258, TUNEL and for the proliferating cell nuclear antigen (PCNA). Early atherosclerotic lesions were characterised by intimal thickening and the presence of SMC and macrophages. The percentage of apoptotic cells, calculated as the ratio of TUNEL-positive nuclei to total nuclei was 32.6 +/- 3.73% in the lesions and 55.9 +/- 2.36% in the endothelium. As it has been reported that nuclei undergoing active gene transcription can be TUNEL positive, we evaluated the percentage of PCNA-positive cells, which proved to be 45.2 +/- 4.68% along the endothelium and 22.3 +/- 2.7% in the intima. The true percentage of apoptotic cells was therefore about 10% in both cases. Fas, Bax and caspase3 signals were mainly located in the endothelium and SMC proximal to the lumen, whereas Bcl2 colocalized with macrophages and SMC deeper in the lesions. Abundant oxidised LDL epitopes were detected in areas of lipid accumulation and along the endothelium, mainly in the areas in which TUNEL and PCNA-positive cells were localised.

Conclusions: Our findings may be taken as ex vivo indications of an apoptotic and proliferating role of oxidised LDLs as previously shown in vitro, and may at least partially account for the endothelial dysfunction that can be rapidly induced by hypercholesterolemia.