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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:1296.)
© 2008 American Heart Association, Inc.

Integrative Physiology/Experimental Medicine

Cholesterol Absorption From the Intestine Is a Major Determinant of Reverse Cholesterol Transport From Peripheral Tissue Macrophages

Ephraim Sehayek; Stanley L. Hazen

From the Departments of Cell Biology and Cardiovascular Medicine, Cleveland Clinic, Ohio.

Correspondence to Ephraim Sehayek, MD, 9500 Euclid Ave, NE-10 Cleveland, OH 44195. E-mail sehayee{at}ccf.org

	Abstract

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Objective— We examined the effect of ezetimibe, a cholesterol absorption (CA) inhibitor, and genetic determinants of CA on reverse cholesterol transport (RCT) from subcutaneously injected macrophages using a new dual isotope label technique.

Methods and Results— Treatment of C57BL/6J mice with ezetimibe decreased dietary CA by 86% and increased RCT from peripheral tissue macrophages (PTM) by 6-fold (P<0.0001). Moreover, congenic 14DKK mice with a modest 41% decrease in dietary CA displayed a 67% increase in RCT from PTM (P<0.007).

Conclusions— These findings indicate that pharmacological and genetic modifiers of cholesterol absorption are major determinants of reverse cholesterol transport from peripheral tissue macrophages.

Key Words: atherosclerosis • cholesterol absorption • cholesterol efflux • ezetimibe • macrophage • reverse cholesterol transport

	Introduction

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Reverse cholesterol transport (RCT) is a multi-step process that involves the efflux of cholesterol from peripheral tissue macrophages (PTM) for ultimate excretion in the feces. In the initial step, the high-density lipoprotein (HDL) particle promotes cholesterol efflux from macrophages for delivery to the liver. Subsequently, HDL cholesterol is excreted into bile and the intestinal lumen where biliary cholesterol is partly reabsorbed, whereas the remainder is excreted into feces. Rader and colleagues provided the first direct in vivo evidence for transport of cholesterol from PTM into feces by intraperitoneal injection of [³H]-cholesterol-labeled macrophages and detection of tracer in plasma, liver, bile, and feces.¹ The presence of [³H]-cholesterol in feces suggests that the intestines may play a role in modifying RCT from PTM. Herein we used pharmacological and genetic approaches to show that cholesterol absorption (CA) from the intestine plays a major role in determining the RCT from PTM.

	Methods

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Detailed descriptions of all methods used as well as the generation and characterization of 14DKK congenic animals are available in the supplemental materials (available online at http://atvb.ahajournals.org).

	Results

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Time Course Excretion of Fecal [¹⁴C]-Cholesterol and [³H]-β-Sitostanol From PTM
Animals were injected subcutaneously with dual-labeled macrophages, and feces were collected daily for 48 hours. As shown in Figure 1, fecal excretion of the [¹⁴C]-cholesterol label significantly increased, whereas that of the [³H]-β-sitostanol label did not change from day 1 to day 2.

View larger version (16K): [in this window] [in a new window]   Figure 1. Time course of fecal [14C]-cholesterol and [3H]-β -sitostanol labels excretion from subcutaneously injected macrophages. [14C]-cholesterol and [3H]-β -sitostanol–labeled RAW 264.7 macrophages were injected subcutaneously into C57BL/6J females, and the percent of injected [14C]-cholesterol and [3H]-β-sitostanol labels excreted into feces was determined (n=5 animals).

Treatment With Ezetimibe and the Congenic 14DKK Interval Suppress the Absorption of Dietary Sterols
Feeding for 10 days with 0.005% ezetimibe largely suppressed the absorption of dietary sterols, as indicated by a decrease of 86%, 94%, and 91% in dietary CA, plasma campesterol, and plasma sitosterol levels, respectively. When compared to ezetimibe treatment, congenic 14DKK animals were characterized by only a modest decrease in dietary sterol absorption with 41%, 17%, and 27% decrease in CA, plasma campesterol, and plasma sitosterol levels, respectively (Table).

View this table: [in this window] [in a new window]   Table. Effect of Ezetimibe Treatment and Congenic 14DKK Interval on Dietary Cholesterol Absorption, Plasma Plant Sterol Levels, and Plasma Levels and Liver Content of 14[C]-Cholesterol and 3[H]-β-Sitostanol Labels

Treatment With Ezetimibe and the Congenic 14DKK Interval Increase the RCT From PTM
As shown in Figure 2A, in chow-fed animals the 48-hour fecal excretion of [¹⁴C]-cholesterol label from PTM amounted to 3.1±0.7%, whereas in ezetimibe treated animals the excretion was 6-fold higher reaching 19.4±6.6%. Concordant with these findings, as shown in Figure 2B, in C57BL/6J control animals the 48-hour fecal excretion of [¹⁴C]-cholesterol label from PTM amounted to 4.3±1.8% whereas in 14DKK congenics the excretion was 67% higher reaching 7.2±2.0%. In contrast, neither ezetimibe treatment nor the 14DKK interval displayed an effect on 24-hour and 48-hour plasma levels of [¹⁴C]-cholesterol and [³H]-β-sitostanol, or on liver content of the [¹⁴C]-cholesterol and [³H]-β-sitostanol labels (Table).

View larger version (12K): [in this window] [in a new window]   Figure 2. Ezetimibe treatment and congenic 14DKK interval increase the fecal excretion of cholesterol from peripheral tissue macrophages. A, C57BL/6J females were chow-fed with or without 0.005% ezetimibe, injected subcutaneously with dual-labeled macrophages, feces were collected for 48 hours, and percent fecal excretion of injected macrophages cholesterol was calculated. Line corresponds to mean value. B, Chow-fed C57BL/6J controls and 14DKK congenics were injected with labeled macrophages and feces processed for analysis as described for A.

	Discussion

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Treatment with bile acid sequestrants and partial ileal bypass, 2 interventions that decrease the absorption of cholesterol, are associated with decreased mortality from atherosclerotic cardiovascular disease.^2,3 Similarly, atheroprotective effects were reported in apolipoprotein E null mice treated with ezetimibe.⁴ Herein we show that potent pharmacological inhibition and moderate genetic suppression of CA markedly increased RCT from PTM. These findings raise the possibility that the atheroprotective effects reported for CA inhibitors may arise not only from reductions in plasma LDL cholesterol levels but also by impacting on net transfer of cholesterol from peripheral cells, such as macrophages in the arterial wall, for ultimate elimination in the feces.

	Acknowledgments

 
We thank Melanie Bodine and Dave Schmitt for expert technical assistance.

Sources of Funding

This study was supported by grants from the American Heart Association (0755295B) and the National Institutes of Health (P01HL087018, P01 HL076491, and P01 HL077107).

Disclosures

None.

	Footnotes

 
Original received February 27, 2008; final version accepted April 7, 2008.

	References

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1. Zhang Y, Zanotti I, Reilly MP, Glick JM, Rothblat GH, Rader DJ. Overexpression of apolipoprotein A-I promotes reverse transport of cholesterol from macrophages to feces in vivo. Circulation. 2003; 108: 661–663.[Abstract/Free Full Text]

2. The Lipid Research Clinics Coronary Primary Prevention Trial results. I. Reduction in incidence of coronary heart disease. JAMA. 1984; 251: 351–364.[Abstract/Free Full Text]

3. Buchwald H, Varco RL, Matts JP, Long JM, Fitch LL, Campbell GS, Pearce MB, Yellin AE, Edmiston WA, Smink RD Jr, et al. Effect of partial ileal bypass surgery on mortality and morbidity from coronary heart disease in patients with hypercholesterolemia. Report of the Program on the Surgical Control of the Hyperlipidemias (POSCH). N Engl J Med. 1990; 323: 946–955.[Abstract]

4. Davis HR Jr, Compton DS, Hoos L, Tetzloff G. Ezetimibe, a potent cholesterol absorption inhibitor, inhibits the development of atherosclerosis in ApoE knockout mice. Arterioscler Thromb Vasc Biol. 2001; 21: 2032–2038.[Abstract/Free Full Text]

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				D. J. Rader, E. T. Alexander, G. L. Weibel, J. Billheimer, and G. H. Rothblat The role of reverse cholesterol transport in animals and humans and relationship to atherosclerosis J. Lipid Res., April 1, 2009; 50(Supplement): S189 - S194. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 28/7/1296    most recent ATVBAHA.108.165803v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sehayek, E. Articles by Hazen, S. L. Search for Related Content PubMed PubMed Citation Articles by Sehayek, E. Articles by Hazen, S. L.