Subcellular fractionation of arterial smooth muscle cells laden with lipid following incubation with low density lipoproteins and chloroquine

Abstract

Lipid accumulation was induced in pig aortic smooth muscle cells in culture by incubating them with LDL and the lysosomotropic agent chloroquine, as an in vitro model of lipid accumulation in atherosclerosis. The cells were homogenized and subjected to a conventional low-speed centrifugation to prepare a postnuclear supernatant. This supernatant was then subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. This showed that in normal smooth muscle cells most of the cholesterol was in the plasma membrane. When postnuclear supernatants of cells that had been incubated with LDL, either with or without chloroquine, were applied to the density gradient, there was no change in the distribution of cholesterol. The distribution of cholesterol became denser, however, when postnuclear supernatants of cells that had been incubated with chloroquine alone were studied, whereas the equilibrium density of the plasma membrane was not increased to the same extent. The cholesterol in these postnuclear supernatants was probably contained in both the plasma membrane and in the lysosomes. The distribution of chloroquine in the density gradients was consistent with a lysosomal localization. Incubation with chloroquine alone preferentially reduced the activity of the lysosomal component of β-glucuronidase compared to its endoplasmic reticulum component. The ratio of the activity of the cytosolic to the mitochondrial forms of malate dehydrogenase in cells incubated with chloroquine, either with or without LDL, was also decreased. When the cells were incubated with LDL and chloroquine together, lipid droplets, at least some of which were membrane-bound, and large autophagic vacuoles developed in the cells. When homogenates of these cells were subjected to low-speed centrifugation to prepare a postnuclear supernatant, all of the large autophagic vacuoles were sedimented into the nuclear fraction, together with most of the cholesteryl ester and chloroquine and most of the acid hydrolase activities. Therefore whole homogenates of smooth muscle cells, rather than postnuclear supernatants, were applied to the sucrose density gradients. When the cells had been incubated with LDL and chloroquine together, most of the cholesteryl ester and chloroquine and part of the nonesterified cholesterol were then found to be associated with lysosomes. These lipid-laden lysosomes probably correspond to the autophagic vacuoles seen in the cells by electron microscopy. The density of the lipid-laden lysosomes was increased considerably compared to the lysosomes of normal cells. This may have been due to either the high chloroquine content of the lysosomes, to an enhanced permeability of the lysosomal membrane to sucrose, or to the presence of partially degraded cell organelles within the lysosomes due to the autophagy caused by chloroquine. The distributions of cholesterol and cholesteryl ester had a small shoulder of low density, which may possibly correspond to the lipid droplets seen within the cells by electron microscopy. Thus, when large-scale lipid accumulation is induced in smooth muscle cells by incubating them with LDL and chloroquine together, the large majority of the cholesterol and cholesteryl ester that accumulates in the cells is contained within the autophagic vacuoles rather than within the lipid droplets.