Evidence That the Initial Up-regulation of Phosphatidylcholine Biosynthesis in Free Cholesterol-loaded Macrophages Is an Adaptive Response That Prevents Cholesterol-induced Cellular Necrosis

doi:10.1074/jbc.271.37.22773

Evidence That the Initial Up-regulation of Phosphatidylcholine Biosynthesis in Free Cholesterol-loaded Macrophages Is an Adaptive Response That Prevents Cholesterol-induced Cellular Necrosis

PROPOSED ROLE OF AN EVENTUAL FAILURE OF THIS RESPONSE IN FOAM CELL NECROSIS IN ADVANCED ATHEROSCLEROSIS*

From the Departments of Medicine and Anatomy & Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York 10032

^‡ To whom correspondence should be addressed:
Dept. of Medicine, Columbia University, 630 W. 168th St., New York, NY 10032
. Tel.: 212-305-9430; Fax: 212-305-5052; E-mail: iat1{at}columbia.edu.

Abstract

Macrophages in atherosclerotic lesions accumulate free cholesterol (FC) as well as cholesteryl ester and appear to have high rates of phospholipid (PL) synthesis and increased PL mass. Previous short term (i.e. ≤24 h) studies with cultured macrophages have shown that these cells respond to FC loading by up-regulating phosphatidylcholine biosynthesis. We propose that this response is adaptive by keeping the FC:PL ratio in the macrophages from reaching toxic levels. We further propose that one cause of macrophage necrosis, a prominent and important event in atherosclerosis, is an eventual decrease of this adaptive response. To explore these ideas, cultured macrophages were loaded with FC for up to 4 days and assayed for phosphatidylcholine biosynthesis, FC and PL mass, and cytotoxicity. For the first 24 h, cellular phosphatidylcholine biosynthesis and FC and PL mass increased 3-4-fold, and thus the FC:PL molar ratio was prevented from reaching very high levels; at this point, there were no overt signs of cytotoxicity. Over the next 24-48 h, however, phosphatidylcholine biosynthesis, and then phosphatidylcholine mass, began to decrease. Initially, the macrophages remained healthy and continued to accumulate FC, but eventually these macrophages, but not unloaded macrophages, became necrotic (swollen organelles and disrupted membranes). Lipoprotein dose studies indicated a close relationship between the onset of macrophage necrosis and the FC:PL ratio. To test further the causal nature of these relationships, cellular FC and PL mass were independently manipulated by using high density lipoprotein₃ (HDL₃) to decrease cellular FC and choline depletion to decrease cellular PC. As predicted by our hypotheses, HDL₃ protected FC-loaded macrophages from necrosis, whereas choline depletion accelerated cytotoxic changes. These findings support the idea that the initial increase in phosphatidylcholine biosynthesis in FC-loaded macrophages is an adaptive response that prevents cholesterol-induced macrophage necrosis. We propose that an eventual failure of the PL response in foam cells may represent one cause of macrophage necrosis in advanced atherosclerotic lesions.

Footnotes

↵* This work was supported by a Postdoctoral Training Grant in Nutrition (to S. M.), a National Research Service Award (to G. A. K.), and Grants HL-39703, HL-21006, and HL-54591 (to I. T.) from the National Institutes of Health, and a Participating Laboratory Award (to Y. S.) and a grant-in-aid (to I. T.) from the American Heart Association, New York City Affiliate. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

FC

free cholesterol

ACAT

acyl-CoA:cholesterol O-acyltransferase

CT

CTP:phosphocholine cytidylyltransferase

Con A

concanavalin A

DMEM

Dulbecco's modified Eagle's medium

FBS

fetal bovine serum

HDL₃

high density lipoprotein₃

LDH

lactate dehydrogenase

LDL

low density lipoprotein

LPDS

lipoprotein-deficient serum

PBS

phosphate-buffered saline

PC

phosphatidylcholine

PL

phospholipid

lyso-PC

lysophosphatidylcholine.
↵²Theoretically, the data in Fig. 9 could be explained by choline deficiency increasing cellular FC content instead of, or in addition to, its proven effect in decreasing cellular PL content. Unfortunately, cellular FC measurements were difficult to interpret, since a substantial amount of both cholesterol added directly to medium, in the form of microcrystals (62), as well as cholesterol-phosphatidylserine liposomes stick to the cell surface. Thus, the FC:PL ratio could not be compared with the previous experiments using acetyl-LDL as the source of cholesterol. Nonetheless, there was no evidence that choline deficiency increased cellular FC. For example, the FC contents of marcophages not exposed to exogenous cholesterol were 44.8 ± 0.1 (choline-replete) and 40.9 ± 0.1 (choline-depleted) nmol/mg cell protein; in cells exposed to cholesterol added directly to medium, the FC contents of control and choline-depleted cells were 280.5 ± 4.8 and 264.8 ± 8.7 nmol/mg cell protein, respectively.
- Received May 22, 1996.