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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:1871.)
© 2006 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Hypoxia Converts Human Macrophages Into Triglyceride-Loaded Foam Cells

Pontus Boström; Björn Magnusson; Per-Arne Svensson; Olov Wiklund; Jan Borén; Lena M. S. Carlsson; Marcus Ståhlman; Sven-Olof Olofsson; Lillemor Mattsson Hultén

From the Wallenberg Laboratory for Cardiovascular Research (P.B., B.M., O.W., J.B., M.S., S-O.O., L.M.H.), and the Department of Internal Medicine (P-A.S., L.M.S.C.), Division of Body Composition and Metabolism, Research Center for Endocrinology and Metabolism (RCEM), the Sahlgrenska Academy, Göteborg, Sweden.

Correspondence to Lillemor Mattsson Hultén, Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, SE-413 45 Göteborg, Sweden. E-mail Lillemor.Mattsson{at}wlab.gu.se

	Abstract

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Objectives— Atherosclerotic lesions have regions that are hypoxic. Because the lesion contains macrophages that are loaded with lipid, we investigated whether hypoxia can influence the accumulation of lipids in these cells.

Methods and Results— Exposure of human macrophages to hypoxia for 24 hours resulted in an increased formation of cytosolic lipid droplets and an increased accumulation of triglycerides. Exposure of the macrophages to oxidized low-density lipoprotein (oxLDL) increased the accumulation of cytosolic lipid droplets because of an increase in cellular cholesterol esters. The accumulation of lipid droplets in oxLDL-treated cells was further increased after hypoxia, caused by an increased level of triglycerides. Expression analyses combined with immunoblot or RT-PCR demonstrated that hypoxia increased the expression of several genes that could promote the accumulation of lipid droplets. Hypoxia increased the mRNA and protein levels of adipocyte differentiation-related protein (ADRP). It is well known that an increased expression of ADRP increases the formation of lipid droplets. Hypoxia decreased the expression of enzymes involved in ß-oxidation (acyl-coenzyme A synthetase and acyl-coenzyme A dehydrogenase) and increased the expression of stearoyl–coenzyme A desaturase, an important enzyme in the fatty acid biosynthesis. Moreover, exposure to hypoxia decreased the rate of ß-oxidation, whereas the accumulation of triglycerides increased.

Conclusions— The results demonstrate that exposure of human macrophages to hypoxia causes an accumulation of triglyceride-containing cytosolic lipid droplets. This indicates that the hypoxia present in atherosclerotic lesions can contribute to the formation of the lipid-loaded macrophages that characterize the lesion and to the accumulation of triglycerides in such lesions.

Exposure of human macrophages to hypoxia resulted in an increased accumulation of cytosolic lipid droplets containing triglycerides. This accumulation was attributable to increased triglyceride biosynthesis, reduced ß-oxidation of fatty acids, and increased expression of ADRP.

Key Words: hypoxia • macrophages • foam cells • triglycerides • ADRP

	Introduction

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Lipid-loaded macrophages, or foam cells, are characteristic of the atherosclerotic lesion. The lipids are stored in cytosolic lipid droplets,¹ which have been suggested to consist of a core of neutral lipids (cholesterol esters or triglycerides) surrounded by a monolayer of amphipathic structures such as phospholipids and proteins.² The most well known of these proteins are the PAT proteins perilipin, adipocyte differentiation-related protein (ADRP), and tail-interacting protein 47.² ADRP is the predominant PAT protein of the lipid droplets in macrophages³ and is present on newly formed droplets.⁴ It been shown to strongly influence the formation of lipid droplets.^2,5 Phospholipase D1 (PLD1), which catalyzes the formation of phosphatidic acid, has been shown to have an important role in the assembly of lipid droplets.⁶

The atherosclerotic lesion is characterized by regions of hypoxia.⁷ The role of hypoxia in the development of the lesion is unknown. However, hypoxia has been shown to reduce macrophage migration.⁸ Moreover, our previous results indicated that hypoxia resulted in an increased expression of 15-lipoxygenase-2 in macrophages, which correlated with an increased ability of the macrophage to participate in the oxidation of low-density lipoprotein (LDL).⁹ Furthermore, hypoxia caused an increase in the secretion of interleukins.^10,11 Together, these observations suggest that the influence of hypoxia on macrophages is of fundamental importance for the inflammation that characterizes the atherosclerotic lesion. It was demonstrated recently that leukocytes respond to an inflammatory stimulus by the accumulation of cytosolic lipid droplets, which may themselves have an important role in this inflammatory response.¹²

We investigated the importance of hypoxia on lipid metabolism in macrophages. Our results show that hypoxia increases the formation of triglyceride-containing lipid droplets in the cell by increasing the expression of ADRP and the rate of triglyceride biosynthesis and by reducing the rate of ß-oxidation of fatty acids.

	Materials and Methods

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For detailed Material and Methods, please see the online supplement, available at http://atvb.ahajournals.org.

	Results

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Hypoxic Treatment of Human Macrophages Results in Increased Accumulation of Triglyceride-Containing Cytosolic Lipid Droplets
Exposure of human macrophages to a hypoxic environment (1% oxygen) for 24 hours resulted in a 3.9±2.8-fold (n=3; P<0.001) increase in the size of the pool of cytosolic lipid droplets (measured as the total area of Oil Red O–stained lipid droplets) (Figure 1A and 1B). This corresponded with an increase in the amount of cellular triglycerides from 0.334±0.057 µg/µg cell protein in control cells to 0.577±0.087 µg/µg cell protein in the cells exposed to hypoxia (P<0.001; Figure 1C). Neither the amount of cholesterol nor that of cholesterol ester was influenced by hypoxia (Figure 1D and 1E).

View larger version (48K): [in this window] [in a new window]   Figure 1. Hypoxia increases the accumulation of cytosolic lipid droplets and cellular triglycerides but not cellular cholesterol esters and unesterified cholesterol in human macrophages. A, Micrograph of Oil Red O–stained control macrophages and macrophages exposed to hypoxia (1% O2 for 24 hours). B, Quantification of the pool of cytosolic lipid droplets (measured as total area of Oil Red O–stained lipid droplets per cell) in control macrophages and macrophages exposed to hypoxia. The results are mean±SD of all cells present in 20 randomly selected micrographs from 3 different donors. C through E, The effect of hypoxia on the cellular levels of triglycerides (C) cholesterol (D), and cholesterol esters (E). Results (µg/µg cellular protein) are mean±SD of an experiment based on macrophages from 3 donors (analyzed in triplicate).

Exposure to hypoxia for up to 72 hours did not have any effect on cell viability (measured by trypan blue exclusion) nor on caspase 3 activity (the principal effector caspase of apoptosis¹³; supplemental Figure IA and IB, available online at http://atvb.ahajournals.org), arguing against the possibility that accumulation of lipid in the hypoxic macrophages is the result of phagocytosis of cells that had died during the hypoxia.

To address the possibility that the accumulation of triglycerides was caused by keeping the cells in culture, we investigated the effect of incubation with oxidized LDL (oxLDL). Treatment with oxLDL under normoxic conditions resulted in a 3.4±2.9-fold (P<0.001) increase in the pool of cytosolic lipid droplets (Figure 2A and 2B), verifying the observations that such treatment can convert macrophages into foam cells.¹⁴ This increase corresponded with an increase in the cellular content of cholesterol ester from 0.0081±0.0016 to 0.0484±0.0221 µg/µg cell protein (P<0.001; Figure 2C). There were no increases in the cellular levels of triglycerides (Figure 2D).

View larger version (43K): [in this window] [in a new window]   Figure 2. Incubation of human macrophages with oxidized LDL under normoxic conditions increases the size of the pool of cytosolic lipid droplets and the cellular content of cholesterol esters but not that of triglycerides. A and B, Incubation of macrophages with 50 µg/mL of oxLDL under normoxic conditions increases the cytosolic pool of lipid droplets when compared with control cells. A, Micrograph. B, Quantification of the pool of cytosolic lipid droplets (measured as total area of Oil Red O–stained lipid droplets per cell) in control macrophages and macrophages exposed to hypoxia. The results are mean±SD of all cells present in 20 randomly selected micrographs from 3 different donors. C and D, Effect of incubation with oxLDL on the cellular levels of cholesterol esters (C) and triglycerides (D) in macrophages cultured under normoxic conditions. Results (µg/µg cellular protein) are mean±SD of an experiment based on macrophages from 3 donors (analyzed in triplicate).

When the incubation with oxLDL was performed under hypoxic conditions, there was a 1.8±1.3-fold (P<0.001) increase in the pool of cytosolic lipid droplets compared with cells incubated with oxLDL under normoxic conditions (Figure 3A and 3B). However, there was no increase in the cellular content of cholesterol esters (Figure 3C). In contrast, the level of cellular triglycerides increased from 0.423±0.056 µg/µg cell protein in cells incubated with oxLDL under normoxic conditions to 0.778±0.228 µg/µg cell protein in cells incubated with oxLDL under hypoxic conditions (Figure 3D). The level of cellular-free cholesterol after incubation with oxLDL was not affected by hypoxia (Figure 3E).

View larger version (50K): [in this window] [in a new window]   Figure 3. Hypoxia enhances the effect of oxLDL on the size of the pool of cytosolic lipid droplets but promotes the accumulation of triglycerides instead of cholesterol esters. A and B, Hypoxia increases the size of the pool of cytosolic lipid droplets in cells incubated with 50 µg/mL of oxLDL. A, Micrograph of Oil Red O–stained control macrophages incubated with oxLDL under normoxic or hypoxic (1% O2 for 24 hours) conditions. B, Quantification of the pool of cytosolic lipid droplets (measured as total area of Oil Red O–stained lipid droplets per cell) in macrophages incubated with oxLDL under normoxic and hypoxic conditions. The results are mean±SD of all cells present in 20 randomly selected micrographs from 3 different donors. C and D, Effect of hypoxia on the influence of oxLDL on cholesterol esters (C), triglycerides (D), and unesterified cholesterol (E). The cells were incubated with oxLDL under normoxic or hypoxic conditions. Results (µg/µg cellular protein) are mean±SD of an experiment based on macrophages from 3 donors (analyzed in triplicate).

Accumulation of Triglycerides in Hypoxic Human Macrophages Was Coupled to Altered Cellular Metabolism
To address the molecular mechanism behind the effect of hypoxia on the accumulation of triglyceride-containing cytosolic lipid droplets, we performed DNA microarray analyses. The microarray data discussed in this publication have been deposited in the National Cancer for Biotechnology Information Gene Expression Omnibus and are accessible through GEO Series accession number GSE4630.

Exposure of human macrophages to hypoxia increased the expression of ADRP but not of perilipin and tail-interacting protein 47 (Table). Because ADRP is known to be regulated post-translationally by proteolysis,¹⁵ we also estimated the amount of protein by immunoblot. These results confirmed increased expression of ADRP in macrophages exposed to hypoxia (Figure 4A).

View this table: [in this window] [in a new window]   DNA Microarray and Real-Time RT-PCR Analysis of Gene Expression in Macrophages Subjected to Hypoxia

View larger version (48K): [in this window] [in a new window]   Figure 4. Hypoxia increases the expression of ADRP and cellular levels of phosphatidic acid in human macrophages. A, Immunoblots of ADRP in control macrophages and macrophages exposed to hypoxia. The result is representative of 3 separate immunoblots. B, Accumulation of radiolabeled phosphatidic acid in control macrophages and macrophages exposed to hypoxia. Mean±SD of 2 different donors analyzed in triplicate (P=0.010).

The expression of PLD1 and lysophosphatidic acid acyltransferase-ß, which are known to be involved in the production of phosphatidic acid, was uninfluenced by hypoxia (Table). However, when we investigated the effects of hypoxia on incorporation of [³H]-palmitic acid into phosphatidic acid, we observed a nearly 4-fold increase in the level of phosphatidic acid in cells exposed to hypoxia compared with control cells (P=0.010; Figure 4B).

The array analyses also showed that 2 key enzymes involved in ß-oxidation (acyl-coenzyme A [CoA] synthetase and acyl-CoA dehydrogenase) were downregulated in response to hypoxia (Table). The results were confirmed by RT-PCR (0.75-fold and 0.69-fold, respectively; both P<0.05; n=5; Table). In agreement with this, we observed a significant reduction in the rate of ß-oxidation (measured as the conversion of [³H]-palmitic acid to water-soluble substances) in cells exposed to hypoxia for 24 hours (28% after 30 minutes incubation with [³H]-palmitic acid [P=0.042] and 40% after 105 minutes incubation with [³H]-palmitic acid [P=0.009; n=3; Figure 5A]). Furthermore, the expression of stearoyl-CoA desaturase, an important enzyme in the biosynthesis of fatty acids, was upregulated in cells exposed to hypoxia, as shown by the array analysis and confirmed by RT-PCR (1.65-fold; n=5; P<0.05; Table).

View larger version (23K): [in this window] [in a new window]   Figure 5. Hypoxia causes a reduction in ß-oxidation and an increase in triglyceride synthesis in human macrophages. A, Effect of hypoxia on the ß-oxidation of [3H]-palmitic acid in human macrophages. Mean±SD of 4 different donors analyzed in triplicate (P=0.042 at 30 minutes and P=0.0097 at 105 minutes). B, Effect of hypoxia on triglyceride synthesis. Mean±SD of 3 different donors analyzed in triplicate (P=0.0028 for control vs hypoxia-treated macrophages).

Together, these results indicate that hypoxia can increase the amount of fatty acid available for the biosynthesis of lipids such as triglycerides. In agreement with this, an increased incorporation of [³H]-palmitic acid into triglycerides was seen in cells exposed to hypoxia for 24 hours after 5-minute, 15-minute, and 30-minute (P=0.0028) incubation with the radiolabeled fatty acid (Figure 5B).

Hypoxia had no effect on the uptake of 2-deoxy-D-[2,6-³H]-glucose in the cell (supplemental Figure IIA). As expected, hypoxia was shown to increase anaerobic metabolism in the cell, as measured by increased lactate production (supplemental Figure IIB). Hypoxia did not influence hydrolysis of triglycerides in the cytosol (supplemental Figure IIC and IID).

	Discussion

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Our results indicate that the hypoxia present in atherosclerotic lesions has the capacity to increase the pool of cytosolic lipid droplets in human macrophages. We observed a predominant accumulation of triglycerides after exposure to hypoxia. This contrasts with macrophages isolated from human atherosclerotic plaques, which predominantly contain cholesterol esters.¹⁶ It is well known that cells in atherosclerotic lesions are exposed to oxidized lipoproteins,^14,17 preferentially oxLDL, and that such exposure is involved in the formation of foam cells. Previous studies have shown that human macrophages incubated with oxLDL accumulate cholesterol esters.¹⁸ We therefore investigated the effect of oxLDL in our cultured human macrophages to address whether such treatment results in the predominant accumulation of triglycerides or cholesterol esters. Exposure to oxLDL increased the pool of cytosolic lipid droplets and cholesterol esters in the cultured macrophages, observations that verified previous results.¹⁸

Although macrophages isolated from the atherosclerotic lesion contain predominantly cholesterol esters, they also contain substantial amounts of triglycerides.¹⁶ We observed that when human macrophages were exposed to both oxLDL and hypoxia, there was an additional increase in the pool of lipid droplets when compared with cells exposed to oxLDL alone. This additional effect could be explained by the increased accumulation of triglycerides rather than cholesterol esters. Although our results should be extrapolated carefully to the in vivo situation, they suggest that hypoxia could induce an accumulation of triglycerides in human macrophages and could partly explain why not only cholesterol esters but also triglycerides accumulate in the atherosclerotic plaque.¹⁶

Why does lipid accumulate during hypoxia? Previous studies have shown that hypoxia gives rise to an inflammatory response in macrophages, with increased production of interleukins.¹⁰ It has also been demonstrated that the inflammatory response in leukocytes involves increased production of lipid droplets, and that the enzymes involved in the biosynthesis of eicosanoids, which are mediators of inflammation, are localized around the lipid droplets.¹² Furthermore, hypoxia increases oxidation of arachidonic acid in macrophages, leading to increased levels of the eicosanoid 15-hydroxy eicosatetraenoic acid, which is the main product of 15-lipoxygenase-2 activity.⁹ Thus, the accumulation of lipids in macrophages during hypoxia may be part of the inflammatory response, in turn indicating that such a response involves increased lipid production attributable to an increase in phosphatidic acid levels in the cell.

To investigate the molecular mechanism underlying hypoxia-induced triglyceride accumulation, we performed DNA microarray analyses. We demonstrated that hypoxia increased ADRP expression, which has the potential to enhance the formation of cytosolic lipid droplets.^19,20 However, ADRP is regulated not only transcriptionally but also by post-translational proteosomal degradation.¹⁵ Thus, it was important to analyze the amount of protein using immunoblot, which confirmed an increase in ADRP levels. Post-translational degradation increases when the cell is depleted of neutral lipids,²¹ and it is therefore possible that the increase in ADRP levels reflects the increased production of triglycerides seen after exposure to hypoxia. However, previous studies have shown that an increased expression of ADRP per se results in an increased accumulation of cytosolic lipid droplets.^19,20

After exposure of macrophages to hypoxia in our studies, levels of phosphatidic acid were shown to increase. Both phosphatidic acid and PLD1 have been shown to play a role in the assembly of lipid droplets.^4,6 However, we could not detect any effect of hypoxia on the expression of PLD1 nor on lysophosphatidic acid acyltransferase-ß, enzymes that are involved in the generation of phosphatidic acid. It should be noted that PLD1 activity is highly regulated post-translationally²² and is present in signal pathways such as that from insulin.⁴

The array analyses also indicated that hypoxia could influence the expression of genes important for increasing the availability of fatty acids in the cell. We observed downregulation of 2 enzymes that are important in ß-oxidation (acyl-CoA synthetase and acyl-CoA dehydrogenase). In agreement with these findings, we measured a significant reduction in the rate of ß-oxidation in cells exposed to hypoxia. Hypoxia was also shown to increase the expression of stearoyl-CoA desaturase. Overexpression of this gene has been linked to increased accumulation of triglycerides in skeletal muscle during type 2 diabetes.²³ These findings indicate that hypoxia results in metabolic changes that have the potential to increase the amount of fatty acids available for the biosynthesis of triglycerides and cholesterol esters.

The results discussed above indicate that the hypoxia that exists in an atherosclerotic plaque can induce metabolic changes in human macrophages, resulting in an increased storage of triglycerides and the pool of lipid droplets. This opens the possibility that macrophages may be influenced not only by uptake of cholesterol from oxLDL but also by fatty acids reaching the cell by lipolysis of very low–density lipoprotein 1 (VLDL 1) and chylomicrones (or even by the lysosomal degradation of oxLDL). This may contribute to the acceleration of atherosclerosis in conditions of elevated VLDL triglycerides (production of VLDL 1).²⁴ One such situation is insulin resistance. Moreover, a direct influence of fatty acids on macrophages in the arterial wall may be one of the reasons why hypertriglyceridemia is an independent risk factor in cardiovascular disease.^25,26

The effect of hypoxia on macrophages demonstrated in this article could play a role in situations other than atherosclerotic lesions. Tissue and cellular hypoxia is suggested to be of importance in various inflamed, diseased tissues such as malignant tumors, wounds, sites of bacterial infection, and adipose tissue.^27,28 Macrophages accumulate in such sites and respond to the hypoxia present with altered gene expression and inflammation. Interestingly, more macrophages are found in ischemic hearts than in control hearts.²⁹ Moreover, there is an increased formation of lipid droplets and a predominant accumulation of triglycerides in hypoxic human hearts compared with controls.³⁰

In summary, exposure of human macrophages to hypoxia resulted in an increased accumulation of cytosolic lipid droplets containing triglycerides. This accumulation was attributable to increased triglyceride biosynthesis, reduced ß-oxidation of fatty acids, and increased expression of ADRP.

	Acknowledgments

 
Sources of Funding

This work was supported by the Swedish Research Council, the Swedish Heart Lung Foundation, Novo Nordic Foundation, the Swedish Society of Medicine, and the Swegene Foundation.

Disclosures

None.

	Footnotes

 
Original received May 30, 2005; final version accepted May 17, 2006.

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