Patricia G Yancey

Cholesterol efflux is defective in macrophages from atherosclerosis-susceptible White Carneau pigeons relative to resistant show racer pigeons.

White Carneau (WC) pigeons are susceptible to the development of aortic atherosclerosis, whereas Show Racer (SR) pigeons are resistant, even though there are no differences in the known risk factors, including plasma cholesterol levels, lipoproteins, blood pressure, etc. Although this suggests that the difference in atherosclerosis susceptibility between WC and SR pigeons may be mediated at the level of the arterial wall, we have yet to identify a mechanism that can account for this difference. In pigeons as in other species (including humans), macrophages play a major role in the pathogenesis of atherosclerosis. Pigeon macrophages have multiple mechanisms for the uptake of lipoproteins and the accumulation of cholesteryl esters. To date, however, no differences in lipoprotein uptake between macrophages of WC and SR pigeons have been identified that could explain the difference in atherosclerosis susceptibility. In the present study we explored the alternative hypothesis that there are differences in the rate of cholesteryl ester clearance from peritoneal macrophages isolated from the two breeds of pigeons. Cholesterol efflux studies were conducted with elicited pigeon peritoneal macrophages that were loaded with cholesteryl ester either in vitro by incubation with rabbit beta-very low density lipoprotein or in vivo by isolation of macrophages from birds fed a cholesterol-containing diet. Using these techniques we were able to load WC and SR macrophages consistently with cholesteryl esters to levels typical of arterial foam cells (150-1,150 micrograms/mg cell protein). Under these cholesterol loading conditions there was no net efflux of cholesterol from either WC or SR macrophages when incubated for up to 24 hours in the presence of pigeon or human high density lipoprotein (HDL), fetal bovine serum, or lipoprotein-deficient serum. Under the same conditions, efflux of cholesterol from mouse peritoneal macrophages was stimulated by human and pigeon HDL. Despite the inability of HDL, lipoprotein-deficient serum, or fetal bovine serum to promote net cholesterol efflux, apoprotein (apo) HDL/phosphatidylcholine (PC) vesicles stimulated cholesteryl ester clearance from both WC and SR pigeon macrophages but at a significantly slower rate from WC pigeon macrophages. When incubated in the presence of excess apoHDL/PC (400 micrograms/ml), the rate of depletion of cellular cholesteryl esters was log-linear for at least 48 hours. WC macrophages cleared an average of only 9% of their cholesteryl esters in 24 hours when incubated with excess apoHDL/PC, whereas SR macrophages reduced their cholesteryl ester content by an average of 42%.(ABSTRACT TRUNCATED AT 400 WORDS)

Uptake and Trafficking of Mildly Oxidized LDL and Acetylated LDL in THP-1 Cells Does Not Explain the Differences in Lysosomal Metabolism of These Two Lipoproteins

Foam cells in the atherosclerotic lesion have substantial cholesterol stores within large, swollen lysosomes. This feature is mimicked by incubating THP-1 macrophages with mildly oxidized low density lipoprotein (LDL). Incubation of THP-1 cells with acetylated LDL produces cytoplasmic cholesteryl ester accumulation rather than lysosomal storage. The differences could be due to differences in uptake and delivery of lipoprotein to lysosomes or to lysosomal and post-lysosomal processing events. We compared uptake and lysosomal trafficking of acetylated and oxidized LDL using colloidal gold-labeled lipoproteins. Labeling did not alter cellular cholesterol accumulation. We found that uptake and delivery to lysosomes are not different for acetylated and oxidized LDL. In fact, both oxidized and acetylated LDL can be delivered to the same lysosomes. Sequential incubation with oxidized LDL followed by acetylated LDL showed that the lipid-engorged lysosomes are long-lived structures, continuously accepting newly ingested lipoprotein. Comparison of acetylated and oxidized LDL in mouse peritoneal macrophages, a cell which does not accumulate substantial lysosomal lipid, also revealed no differences in uptake. This indicates that in THP-1 cells, the differences in metabolism of oxidized and acetylated LDL are due to cell-specific lysosomal or post-lysosomal events not present in B6C3F1 mouse macrophages.

Macrophage Cholesterol Balance

Macrophages play a central role in the pathogenesis of atherosclerosis.’ Not only do they constitute the predominant cholesteryl ester-rich foam cell of the earliest fatty streaks and of fatty plaques from atherosclerotic lesions of virtually all experimental animals that have been studied, but they also appear to play a similar role in the pathogenesis of atherosclerosis in human beings.* As atherosclerotic lesions progress, other cells such as smooth muscle cells and T lymphocytes become more prominent, but macrophages continue to be present in significant numbers particularly in areas around sites of necrosis and at the edges of atherosclerotic Since macrophage foam cells are the first abnormal cell to appear in the intima, and they secrete a variety of chemotactic and growth regulatory factor^,^ it is reasonable to speculate that it is the presence of these factors that stimulates the subsequent migration and proliferation of smooth muscle cells and the continued influx of monocyte macrophages into the developing lesion. Initially, macrophages may perform a beneficial function by removing cell debris and excess or abnormal lipoproteins that have accumulated within the extracellular matrix. Although passage of plasma lipoproteins from the blood into the arterial wall is a normal process, if the rate of influx exceeds the rate of efflux (as might occur in conditions of hyperlipidemia) or if changes in the lipoprotein or in the extracellular matrix of the arterial wall result in their prolonged retention, then abnormal amounts of lipoproteins would be expected to build up in the arterial wa1L4 Changes in lipoproteins that could result in uptake by macrophages include oxidation, aggregation, glucosylation, etc.5,h These, plus a number of other chemically modified forms of lipoproteins, are recognized by “scavenger receptors” on macrophages and rapidly internalized by receptor-mediated endocytosis.’ This results in the delivery of the lipoprotein cholesterol to the lysosomes where protein and lipid components are hydrolyzed making free cholesterol available to the cells. The free cholesterol also can efflux from the cells if there is an appropriate acceptor of cholesterol outside the cell.x High-density lipoproteins (HDL) or their precursors are thought to serve this function in viva If a cholesterol acceptor is not available, the free cholesterol is efficiently reesterified by the enzyme acyl CoA:cholesterol acyl transferase (ACAT) and stored as cholesteryl ester droplets within the cell. In a similar manner, high