Direct observation of lipoprotein cholesterol ester degradation in lysosomes

We have investigated whether pyrene-labelled cholesterol esters (PyrnCEs) (n indicates the number of aliphatic carbons in the pyrene-chain) can be used to observe the degradation of low-density lipoprotein (LDL)-derived cholesterol esters (CEs) in the lysosomes of living cells. To select the optimal substrates, hydrolysis of the PyrnCE species by lysosomal acid lipase (LAL) in detergent/phospholipid micelles was compared. The rate of hydrolysis varied markedly depending on the length of the pyrenyl chain. Pyr10CE was clearly the best substrate, while Pyr4CE was practically unhydrolysed. Pyr10CE and [3H]cholesteryl linoleate, the major CE species in LDL, were hydrolysed equally by LAL when incorporated together into reconstituted LDL (rLDL) particles, thus indicating that Pyr10CE is a reliable reporter of the lysosomal degradation of native CEs. When rLDL particles containing Pyr4CE or Pyr10CE were incubated with fibroblasts, the accumulation of bright intracellular vesicular fluorescence was observed with the former fluorescent derivative, but not with the latter. However, when the cells were treated with chloroquine, an inhibitor of lysosomal hydrolysis, or when cells with defective LAL were employed, Pyr10CE also accumulated in vesicular structures. HPLC analysis of cellular lipid extracts fully supported these imaging results. It is concluded that PyrnCEs can be used to observe degradation of CEs directly in living cells. This should be particularly useful when exploring the mechanisms responsible for the accumulation of lipoprotein-derived CEs in complex systems such as the arterial intima.