Abstract
In this study, the distribution of free cholesterol in cholesterol-loaded endothelial cells was examined. For these studies, cell fractionation methods were used to assess marker enzyme activity and cholesterol distribution. Treatment of rabbit aortic endothelial cells for 3 days with 50 micrograms/ml of beta-very low density lipoprotein (beta-VLDL) or malondialdehyde-low density lipoprotein (MDA-LDL) but not LDL caused a 50-100% increase in total cell unesterified cholesterol. The accumulation of free rather than esterified cholesterol in endothelial cells may be due to the ratio of hydrolysis to esterification, which we have shown in this study to be 10-fold higher in endothelial cells than in smooth muscle cells. This free cholesterol is found in the fractions enriched in plasma membrane markers and, to a lesser extent, in the Golgi-enriched fractions. The amount of cholesterol per mg of protein was increased approximately 50% in these fractions from cells treated for 3 days with 50 micrograms/ml of beta-VLDL. These increases in cholesterol content were reversible upon incubation of cells for 3 days in medium containing 15% fetal bovine serum. Alterations in several membrane functions were also observed in cholesterol-loaded cells. The activity of alkaline phosphatase, an enzyme marker for plasma membranes, was decreased by 25% and an alteration in membrane-associated microfilaments was seen with phalloidin staining. This morphological change in microfilaments was reflected in a decrease in filament ends as shown by cytochalasin binding and occurred without a change in total actin or vinculin. These microfilament changes were reversible.(ABSTRACT TRUNCATED AT 250 WORDS)
Highlights
In this study, the distribution of free cholesterol in cholesterol-loaded endothelial cells was examined
We hypothesized that in endothelial cells treated with P-very low density lipoprotein (VLDL) there is an increase in free cholesterol and that this results in an increase in the cholesterol content of the plasma membrane contributing to alterations in the function of membrane-associated enzymes and proteins
Treating cells with 0-VLDL increased the total cell cholesterol content approximately twofold; the increased total cell cholesterol was esseptially all in the form of free cholesterol (Fig. 1)
Summary
Rabbit aortic endothelial cells were isolated and cultured as described previously [9]. When they reached high density (approximately 2 x lo cells/cm2), they were treated for 3 days with or without rabbit 0-VLDL (50 pg/ml), human LDL (50 pg/ml), or human LDL modified with malondialdehyde taken up by the scavenger receptor (25 pglml) in DME containing 10% serum (4:1, calfifeta calf). Cells were rinsed four times with serum free medium and incubated for an additional hour in serum-free medium before harvesting.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
