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Reverse cholesterol transport in man: promotion of fecal steroid excretion by infusion of reconstituted HDL
Atherosclerosis Supplements 3 (2002) 23 /30 www.elsevier.com/locate/atherosclerosis
Reverse cholesterol transport in man: promotion of fecal steroid excretion by infusion of reconstituted HDL Bo Angelin *, Paolo Parini, Mats Eriksson Department of Medicine, Center for Metabolism and Endocrinology, M63, Karolinska Institute at Huddinge University Hospital, S-141 86 Stockholm, Sweden
Abstract Reverse cholesterol transport is a complex process, which transfers cholesterol from peripheral cells to the liver for subsequent elimination as bile acids and neutral steroids. Although apo A-I in high density lipoproteins (HDL) is believed to have a crucial role in this process, clinical conditions with very low HDL cholesterol levels appear to maintain normal cholesterol excretion. On the other hand, infusion of ‘artificial HDL’ in the form of recombinant proapo A-I (4 g) liposome complexes results in increased fecal steroid excretion, corresponding to a removal of :/0.5 g cholesterol daily for up to 9 days. This occurs without evidence of increased cholesterol synthesis, and could not be reproduced by infusion of liposomes only. These data indicate that stimulation of reverse cholesterol transport may be induced by infusion of ‘artificial HDL’ in humans, and that a more detailed knowledge of this process may be useful in the treatment of atherosclerosis. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Artificial HDL; Cholesterol; Apo A-I
1. Introduction Reverse cholesterol transport, a concept introduced by Glomset more than 30 years ago [1], describes the flux of cellular cholesterol from peripheral tissues towards the liver and its final excretion from the body as biliary bile acids and cholesterol. The central role of high density lipoproteins (HDL) as cholesterol carriers in this process has been well established [2]. The ‘rediscovery’ of a linkage between reduced levels of plasma HDL and increased risk of coronary heart disease [3] has been supported by several prospective studies during the past decades (e.g. [4 /6]). There are now also indications that by rising plasma HDL the incidence of coronary events may be reduced, independent of changes in plasma low density lipoproteins (LDL) or triglycerides [7]. In animal models of atherosclerosis, the disease process can be retarded by infusion of HDL or apo A-I liposomes, as well as by overexpression of the human apo A-I gene [8 /11], support* Corresponding author. Tel.: /46-8-5858-2344; fax: /46-8-7110710 E-mail address: [email protected] (B. Angelin).
ing the concept of an important role for apo A-I in the prevention of atherogenesis. Mechanistic studies on the role of HDL in vivo are difficult, since HDL comprises a mixture of particles that differ in size and composition, and undergo continuous metabolic changes in the circulation [12]. Furthermore, much more information is available on the role of HDL metabolism in promoting efflux of cholesterol from peripheral tissues than on the relationship between plasma HDL cholesterol and the final excretion of biliary steroids through the liver. This presentation briefly summarizes some recent experimental studies in humans, indicating that the excretion of fecal steroids may be stimulated by the infusion of recombinant apo A-I.
2. Role of HDL in reverse cholesterol transport Cholesterol efflux, particularly studied in macrophages, is a process relying on transmembrane proteins belonging to the ATP-binding cassette transporter (ABC) family, on apoE, on the scavenger receptor SRB1, on cholesterol 27-hydroxylase activity, and on aqueous diffusion [13 /19]. The initial acceptor of
1567-5688/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 5 6 7 - 5 6 8 8 ( 0 2 ) 0 0 0 4 7 - 8
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B. Angelin et al. / Atherosclerosis Supplements 3 (2002) 23 /30
cholesterol from cells is the preb1-HDL, a discoidshaped lipid-poor particle [2,20,21]. Preb1-HDL originate mainly in the blood stream from the surface components of triglyceride-rich lipoproteins after hydrolysis of the triglyceride core. Accumulation of cholesterol from peripheral tissues into the particles transforms the preb1-HDL into larger spherical lipoprotein particles: preb2-HDL. Through the action of LCAT, free cholesterol on the surface of preb2-HDL is converted to more lipophilic cholesteryl esters, leading to an expansion of particles into spherical a3-HDL. By acquiring more cholesterol from tissue and other HDL particles, these particles enlarge into a2 -and a1-HDL [20,21]. The HDL particles finally transfer cholesterol to the liver by at least two distinct processes (Fig. 1): through docking to the SR-BI receptor, expressed on the cell membrane of the hepatocyte, or through an exchange of cholesteryl esters for triglyceride from remnant particles and LDL, a process driven by the action of cholesteryl ester transfer protein (CETP). Finally, by hydrolyzing triglyceride and phospholipids, hepatic lipase (HL) regenerates smaller a3-HDL and lipid-free apo A-I, which in turn are rapidly reloaded with cellular cholesterol and phospholipids to form new preb1-HDL.
3. Role of lipoprotein cholesterol in regulating hepatic steroid excretion As discussed above, HDL cholesterol is finally delivered to the liver, both indirectly via remnant and LDL particles, and directly by HDL docking to SR-BI (Fig. 1). Hepatic cholesterol can then be packaged into VLDL for secretion into plasma, stored as esters, or eliminated via biliary excretion either as free cholesterol or after conversion to bile acids [22 /24]. The liver is the predominant site for net excretion of cholesterol from the body (Fig. 2), and several studies have shown that cholesterol delivered by lipoproteins is a primary source of substrate for biliary lipid secretion as bile acids and cholesterol (for review, [25]). The relative importance of hepatic uptake of HDL, LDL, VLDL, and chylomicron remnant cholesterol to these pathways is still not well understood, however. The molecular details of regulation of bile acid synthesis and cholesterol secretion are now starting to be revealed [26,27], but quite a few aspects on intrahepatic trafficking of cholesterol still remain to be clarified. Although several metabolic pathways exist for the conversion of cholesterol into bile acids, it now seems clear that cholesterol 7ahydroxylase activity is the major rate-limiting step, and thus the most important regulator of bile acid production [28]. The relative contribution of the two primary bile acids synthesized, cholic acid and chenodeoxcholic acid, seems to depend on changes in the
activity of the 12a-hydroxylase [29]. The portal venous return of bile acids, which governs the activity of 7ahydroxylase (cf. Fig. 2), is under strong influence of intestinal bile acid transporters [30]. One way to try to evaluate the contribution of the individual plasma lipoproteins has been to study genetic deficiency states in humans. Thus, conditions such as lack of expression of LDL-receptors (familial hypercholesterolemia) as well as abeta-lipoproteinemia have not been shown to have diminshed bile acid synthesis [25]. In fact, it is only when bile acid synthesis is heavily stimulated (e.g. through biliary drainage, resin therapy etc.) that a reduction of bile acid excretion has been seen in situations with lowered apoB-containing lipoproteins (e.g. [31]). The impact of low HDL cholesterol for biliary steroid excretion has been less extensively studied [25]. In one patient with fish-eye disease (partial deficiency of LCAT activity and HDL cholesterol B/10% of normal), no quantitative defect in bile acid production could be demonstrated [32] (Fig. 3). In two patients with very low HDL cholesterol levels due to apo A-I/C-III deficiency, the total excretion of bile acids and neutral sterols in feces was normal [33]. However, in both situations, the ratio between the synthesis of cholic acid and chenodeoxycholic acid was reduced to less than half of normal (Fig. 3). These findings indicate that the liver is capable of maintaining a normal bile acid synthesis despite very low levels of plasma HDL cholesterol. The explanation of the clearly abnormal synthesis ratio in these patients remains to be understood. There is surprisingly little information on cholesterol excretion in patients with Tangier disease (ABCA1-deficiency). In a single case, there was no clear evidence of abnormal biliary lipid metabolism [34].
4. Infusion of recombinant apo A-I/liposome complexes in humans The need for new therapeutic approaches in the treatment of atherosclerosis has stimulated the concept of developing reconstituted HDL (rHDL) particles which could promote cholesterol efflux from foam cells. Such particles are generally discoidal complexes containing phosphatidylcholine and either apo A-I or its precursor, proapo A-I [35,36]. In vitro studies in cholesterol-loaded macrophage-derived cells have demonstrated that such discs promote cholesterol efflux from cells, whereas multilamellar phosphatidylcholine liposomes are not able to significantly induce cholesterol efflux [37]. Interestingly, reconstituted discoidal complexes containing apo A-I or proapo A-I were even more effective in promoting cholesterol efflux than HDL isolated from human plasma.
B. Angelin et al. / Atherosclerosis Supplements 3 (2002) 23 /30
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Fig. 1. Simplified scheme of lipoprotein metabolism (modified from [24]) CM, chylomicrons; CMR, chylomicron remnants; VLDL, very low density lipoprotein; IDL intermediate density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; LPL, lipoprotein lipase; HL, hepatic lipase; CETP, cholesteryl ester transfer protein; PLTP, phospholipid transfer protein; LCAT, lecithin: cholesterol acyl transferase; ABCA1, ATPbinding cassette transporter A1; ABCG1, ATP-binding cassette transporter G1: LDLR, LDL receptor; LRP, LDL receptor related protein; SR-B1; scavenger receptor class B type 1; SR-A, scavenger receptor class A. Blue arrows represent exogenous cholesterol transport; green arrows represent endogenous cholesterol transport; red arrows represent reverse cholesterol transport. The red-dashed arrows represent ways by which the cholesterol
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Fig. 2. Schematic representation of hepatic cholesterol and bile acid metabolism (modified from [24]). CM, chylomicrons; CMR, chylomicron remnants; VLDL, very low density lipoprotein; IDL intermediate density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; LDLR, LDL receptor; LRP, LDL receptor related protein; SR-B1; scavenger receptor class B type 1; ACAT, acyl CoA: cholesteryl acyl transferase; HMG CoA reductase, 3-hydroxy-3-methylglutaryl CoA reductase; IBAT, ileal bile acid transporter.
In a first study, Carlson infused 1.6 g (protein) of proapo A-I liposomes to four patients with low plasma HDL cholesterol [35]. An increase in HDL cholesterol levels persisted for several days, being maximal (:/20%)
after 1 day. From kinetic analysis of radiolabelled protein, the apparent volume of distribution of apo AI was 6.9 l and the mean residence time 134 h [35]. Nanjee et al. infused seven healthy men with apo A-I/
Fig. 3. Bile acid kinetics in patients with profoundly reduced HDL cholesterol levels (see text). Data from [32,33].
B. Angelin et al. / Atherosclerosis Supplements 3 (2002) 23 /30
phosphatidylcholine discs (25 /40 mg/kg of protein) [36]. During iv infusion for 4 h, the plasma concentration of total apo A-I increased. The increase in apo A-I was mainly in the HDL fraction corresponding to prebmigrating particles, and was paralleled by a simultaneous increase in total HDL unesterified cholesterol. After the infusion was stopped, it was also possible to observe an increase in HDL cholesteryl ester and amigrating apo A-I-containing HDL. Thus, the data support the concept that the apo A-I liposome infusion was able to promote cholesterol efflux and stimulate the production of preb-HDL.
5. Fecal steroid excretion after infusion of reconstituted HDL To test the hypothesis that reverse cholesterol transport could be could stimulated in humans, we studied four subjects with heterozygous familial hypercholesterolemia [38]. The fecal excretion of bile acids and neutral sterols was measured in 3-day pools before and after a single infusion of recombinant human proapo A-I liposome complexes. At a dose of 4 g, this would correspond to approximately 75 /100% of the calculated plasma apo A-I pool. A clear increase in plasma apo A-I levels was observed during the first 2 h after infusion; this was accompanied by a less marked elevation in HDL cholesterol (Fig. 4). In all patients, apo A-I and HDL cholesterol levels in plasma had returned to baseline after 48 h. The fecal excretion of bile acids and neutral steroids was increased by 30 and 39%, respectively (Fig. 5). To exclude the possibility that the phospholipid complexes
27
had an independent stimulatory effect on cholesterol excretion, we repeated the experiment in two of the patients with infusion of liposomes prepared without proapo A-I. The fecal excretion of cholesterol, either as neutral sterols or as bile acid, was not affected by this treatment, however. Thus, a pronounced increase in cholesterol excretion from the body was observed after intravenous infusion of recombinant proapo A-I liposomes. The lack of response in two of the patients when infused with pure liposomes indicates that, also in vivo, apo A-I plays a major role in promoting reverse cholesterol transport. In eight healthy males, Nanjee et al. [39] recently confirmed an increase in fecal bile acid excretion in response to infusion of apo A-I (40 mg/kg) lecithin discs; this was associated with a rise in prebHDL in plasma and lymph. Thus, two independent studies in humans indicate that infusion of ‘nascent’ rHDL may stimulate reverse cholesterol transport both due to stimulation of cholesterol efflux from peripheral cells and by promoting hepatic steroid excretion. The degree of stimulation of cholesterol excretion in response to proapo A-I/liposome infusion observed in our study was surprisingly large, corresponding to a removal of 0.5 g cholesterol daily for several days. It is important to realize that we cannot identify the origin of the excreted steroids. Thus, proapo A-I liposomes may stimulate hepatic cholesterol synthesis by extracting cholesterol from hepatocytes. However, repeated measurements of serum lathosterol and lathosterol/cholesterol ratio, markers of in vivo cholesterol synthesis [40,41], did not show any change in response to treatment. Plasma lathosterol was transiently increased immediately after infusion in four of the subjects studied by Nanjee et al. [41].
Fig. 4. Plasma levels of apo A-I (filled circles) and HDL cholesterol (grey squares) in response to infusion of 4 g proapo A-I/liposomes in patients with heterozygous familial hypercholesterolemia (data from [38]). Values are percent of baseline (mean9/S.E.M.).
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Fig. 5. Fecal excretion of neutral sterols, bile acids, and total steroids 9 days before (filled bars) and 9 days after (grey bars) the infusion of 4 g of proapo A-I/liposomes in patients with heterozygous familial hypercholesterolemia (data from [38]). Values are mean9/S.E.M.
6. Concluding remarks Reverse cholesterol transport is a very complex process which is difficult to study. Studies of animal models, particuarly in rodents, are currently performed, and the access to genetically modified strains of mice has considerably improved our knowledge on lipoprotein metabolism in vivo. However, species differences in lipid metabolism, e.g. lack of CETP activity and larger capacity for variation in hepatic cholesterol synthesis in response to perturbations in rodents [22], make it fundamental to include information obtained in humans. As to reverse cholesterol transport, a number of recent studies in genetically modified mice have shown, in agreement with the observations in humans, that profoundly lowered HDL cholesterol and apo A-I levels (such as in apo A-I knockout mice) do not substantially influence biliary lipid metabolism [42,43]. On the other hand, studies in mouse models have failed to demonstrate the promotion of net steroid excretion seen in humans both in response to overexpression of apo A-I and infusion of apo A-I/phospholipid discs [44]. The concept of stimulating reverse cholesterol transport in order to reduce the risk of atherosclerosis and its complications holds great clinical promise. The relation between the antiatherogenic effects and plasma levels of HDL cholesterol or apo A-I may be less clear than that to HDL cholesterol flux. The concept of infusing rHDL as a therapeutic alternative is still far from practical
reality. In addition to important clinical aspects such as the cost of production (and particularly purification), and the possible immunogenic problems with repeated protein infusions, the need for hard endpoint data (regression studies in humans) will make it a difficult project. On the other hand, the technique may provide us with new and important information on the role of apo A-I and HDL in reverse cholesterol transport which will be extremely important in the continued search for new therapeutic principles [45,46].
Acknowledgements We thank Lena Emtestam for skilful secretarial assistance. The author’s work is supported by the Swedish Research Council (03X-7137), the Old Female Servants, the Tore Nilsson, the Fredrik and Ingrid ˚ ke Wiberg, Thuring, the Ruth and Richard Julin, the A and the Swedish Heart-Lung Foundations.
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[21] Huang Y, von Eckardstein A, Assmann G. Cell-derived unesterified cholesterol cycles between different HDLs and LDL for its effective esterification in plasma. Arterioscler Thromb 1993;13:445 /58. [22] Dietschy JM, Turley SD, Spady DK. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans. J Lipid Res 1993;34:1637 /59. [23] Angelin B. Studies on the regulation of hepatic cholesterol metabolism in humans. Eur J Clin Invest 1995;25:215 /24. [24] Angelin B, Parini P. Plasma lipoprotein metabolism. In: Wass JAH, Shalet SM (Eds.), Oxford Textbook of Endocrinology and Diabetes. Oxford University Press, Oxford, 2002, pp. 1537 /1545. [25] Angelin B, Einarsson K. Bile acids and lipoprotein metabolism. Atheroscler Rev 1986;15:41 /66. [26] Edwards PA, Kast HR, Anisfeld AM. BAREing it all: the adoption of LXR and FXR and their roles in lipid homeostasis. J Lipid Res 2002;43:2 /12. [27] Chiang JYL. Bile acid regulation of gene expression: roles of nuclear hormone receptors. Endocr Rev 2002;23:443 /63. [28] Bjo¨rkhem I, Araya Z, Rudling M, Angelin B, Einarsson C, Wikvall K. Differences in the regulation of the classical and the alternative pathway for bile acid synthesis in human liver. No coordinate regulation of CYP7A1 and CYP27A1. J Biol Chem 2002;277:26804 /7. [29] Russell DW, Setchell KD. Bile acid biosynthesis. Biochemistry 1992;31:4737 /49. [30] Love MW, Dawson PA. New insights into bile acid transport. Curr Opin Lipidol 1998;9:225 /9. [31] Hillebrant CG, Nyberg B, Einarsson K, Eriksson M. The effect of plasma low density lipoprotein apheresis on the hepatic secretion of biliary lipids in humans. Gut 1997;41:700 /4. [32] Angelin B, Carlson LA. Bile acids and plasma high density lipoproteins: biliary lipid metabolism in fish eye disease. Eur J Clin Invest 1986;16:157 /62. [33] Beher WT, Gabbard A, Norum RA, Stradnieks S. Effect of blood high density lipoprotein cholesterol concentration on fecal steroid excretion in humans. Life Sci 1983;32:2933 /7. [34] Vergani CG, Plancher AC, Zuin M, et al. Bile lipid composition and haemostatic variables in a case of high density lipoprotein deficiency (Tangier disease). Eur J Clin Invest 1984;14:49 /54. [35] Carlson LA. Effect of a single infusion of recombinant human proapolipoprotein A-I liposomes (synthetic HDL) on plasma lipoproteins in patients with low high density lipoprotein cholesterol. Nutr Metab Cardiovasc Dis 1995;5:85 /91. [36] Nanjee MN, Doran JE, Lerch PG, Miller NE. Acute effects of intravenous infusion of ApoA1/phosphatidylcholine discs on plasma lipoproteins in humans. Arterioscler Thromb Vasc Biol 1999;19:979 /89. [37] Westman J, Roobol C, Heymans C, Carlson LA, Wulfert E. Efflux of cholesterol from cholesterol loaded macrophages by incubation with synthetic HDL-particles. Scand J Clin Lab Invest 1993;53:773 /82. [38] Eriksson M, Carlson LA, Miettinen TA, Angelin B. Stimulation of fecal steroid excretion after infusion of recombinant proapolipoprotein A-I. Potential reverse cholesterol transport in humans. Circulation 1999;100:594 /8. [39] Nanjee MN, Cooke CJ, Garvin R, Semeria F, Lewis G, Olszewski WL, Miller NE. Intravenous apoA-I/lecithin discs increase prebeta-HDL concentration in tissue fluid and stimulate reverse cholesterol transport in humans. J Lipid Res 2001;42:1586 /93. [40] Bjorkhem I, Reihner E, Angelin B, Ewerth S, Akerlund JE, Einarsson K. On the possible use of the serum level of 7 alphahydroxycholesterol as a marker for increased activity of the cholesterol 7 alpha-hydroxylase in humans. J Lipid Res 1987;28:889 /94.
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[41] Kempen HJ, Glatz JF, Gevers Leuven JA, van der Voort HA, Katan MB. Serum lathosterol concentration is an indicator of whole-body cholesterol synthesis in humans. J Lipid Res 1988;29:1149 /55. [42] Jolley CD, Woollett LA, Turley SD, Dietschy JM. Centripetal cholesterol flux to the liver is dictated by events in the peripheral organs and not by the plasma high density lipoprotein or apolipoprotein A-I concentration. J Lipid Res 1998;39:2143 /9. [43] Jolley CD, Dietschy JM, Turley SD. Induction of bile acid synthesis by cholesterol and cholestyramine feading is unimpaired
in mice deficient in apolipoprotein A-I. Hepatology 2000;32:1309 /16. [44] Alam K, Meidell RS, Spady DK. Effect of up-regulating individual steps in the reverse cholesterol transport pathway on reverse cholesterol transport in normolipidemic mice. J Biol Chem 2001;276:15641 /9. [45] Glass CK, Witztum JL. Atherosclerosis: the road ahead. Cell 2001;104:503 /16. [46] Angelin B, Eriksson M, Rudling M. Bile acids and lipoprotein metabolism: a renaissance for bile acids in the post-statin era. Curr Opin Lipidol 1999;10:269 /74.
Reverse cholesterol transport in man: promotion of fecal steroid excretion by infusion of reconstituted HDL Bo Angelin *, Paolo Parini, Mats Eriksson Department of Medicine, Center for Metabolism and Endocrinology, M63, Karolinska Institute at Huddinge University Hospital, S-141 86 Stockholm, Sweden
Abstract Reverse cholesterol transport is a complex process, which transfers cholesterol from peripheral cells to the liver for subsequent elimination as bile acids and neutral steroids. Although apo A-I in high density lipoproteins (HDL) is believed to have a crucial role in this process, clinical conditions with very low HDL cholesterol levels appear to maintain normal cholesterol excretion. On the other hand, infusion of ‘artificial HDL’ in the form of recombinant proapo A-I (4 g) liposome complexes results in increased fecal steroid excretion, corresponding to a removal of :/0.5 g cholesterol daily for up to 9 days. This occurs without evidence of increased cholesterol synthesis, and could not be reproduced by infusion of liposomes only. These data indicate that stimulation of reverse cholesterol transport may be induced by infusion of ‘artificial HDL’ in humans, and that a more detailed knowledge of this process may be useful in the treatment of atherosclerosis. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Atherosclerosis; Artificial HDL; Cholesterol; Apo A-I
1. Introduction Reverse cholesterol transport, a concept introduced by Glomset more than 30 years ago [1], describes the flux of cellular cholesterol from peripheral tissues towards the liver and its final excretion from the body as biliary bile acids and cholesterol. The central role of high density lipoproteins (HDL) as cholesterol carriers in this process has been well established [2]. The ‘rediscovery’ of a linkage between reduced levels of plasma HDL and increased risk of coronary heart disease [3] has been supported by several prospective studies during the past decades (e.g. [4 /6]). There are now also indications that by rising plasma HDL the incidence of coronary events may be reduced, independent of changes in plasma low density lipoproteins (LDL) or triglycerides [7]. In animal models of atherosclerosis, the disease process can be retarded by infusion of HDL or apo A-I liposomes, as well as by overexpression of the human apo A-I gene [8 /11], support* Corresponding author. Tel.: /46-8-5858-2344; fax: /46-8-7110710 E-mail address: [email protected] (B. Angelin).
ing the concept of an important role for apo A-I in the prevention of atherogenesis. Mechanistic studies on the role of HDL in vivo are difficult, since HDL comprises a mixture of particles that differ in size and composition, and undergo continuous metabolic changes in the circulation [12]. Furthermore, much more information is available on the role of HDL metabolism in promoting efflux of cholesterol from peripheral tissues than on the relationship between plasma HDL cholesterol and the final excretion of biliary steroids through the liver. This presentation briefly summarizes some recent experimental studies in humans, indicating that the excretion of fecal steroids may be stimulated by the infusion of recombinant apo A-I.
2. Role of HDL in reverse cholesterol transport Cholesterol efflux, particularly studied in macrophages, is a process relying on transmembrane proteins belonging to the ATP-binding cassette transporter (ABC) family, on apoE, on the scavenger receptor SRB1, on cholesterol 27-hydroxylase activity, and on aqueous diffusion [13 /19]. The initial acceptor of
1567-5688/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 5 6 7 - 5 6 8 8 ( 0 2 ) 0 0 0 4 7 - 8
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cholesterol from cells is the preb1-HDL, a discoidshaped lipid-poor particle [2,20,21]. Preb1-HDL originate mainly in the blood stream from the surface components of triglyceride-rich lipoproteins after hydrolysis of the triglyceride core. Accumulation of cholesterol from peripheral tissues into the particles transforms the preb1-HDL into larger spherical lipoprotein particles: preb2-HDL. Through the action of LCAT, free cholesterol on the surface of preb2-HDL is converted to more lipophilic cholesteryl esters, leading to an expansion of particles into spherical a3-HDL. By acquiring more cholesterol from tissue and other HDL particles, these particles enlarge into a2 -and a1-HDL [20,21]. The HDL particles finally transfer cholesterol to the liver by at least two distinct processes (Fig. 1): through docking to the SR-BI receptor, expressed on the cell membrane of the hepatocyte, or through an exchange of cholesteryl esters for triglyceride from remnant particles and LDL, a process driven by the action of cholesteryl ester transfer protein (CETP). Finally, by hydrolyzing triglyceride and phospholipids, hepatic lipase (HL) regenerates smaller a3-HDL and lipid-free apo A-I, which in turn are rapidly reloaded with cellular cholesterol and phospholipids to form new preb1-HDL.
3. Role of lipoprotein cholesterol in regulating hepatic steroid excretion As discussed above, HDL cholesterol is finally delivered to the liver, both indirectly via remnant and LDL particles, and directly by HDL docking to SR-BI (Fig. 1). Hepatic cholesterol can then be packaged into VLDL for secretion into plasma, stored as esters, or eliminated via biliary excretion either as free cholesterol or after conversion to bile acids [22 /24]. The liver is the predominant site for net excretion of cholesterol from the body (Fig. 2), and several studies have shown that cholesterol delivered by lipoproteins is a primary source of substrate for biliary lipid secretion as bile acids and cholesterol (for review, [25]). The relative importance of hepatic uptake of HDL, LDL, VLDL, and chylomicron remnant cholesterol to these pathways is still not well understood, however. The molecular details of regulation of bile acid synthesis and cholesterol secretion are now starting to be revealed [26,27], but quite a few aspects on intrahepatic trafficking of cholesterol still remain to be clarified. Although several metabolic pathways exist for the conversion of cholesterol into bile acids, it now seems clear that cholesterol 7ahydroxylase activity is the major rate-limiting step, and thus the most important regulator of bile acid production [28]. The relative contribution of the two primary bile acids synthesized, cholic acid and chenodeoxcholic acid, seems to depend on changes in the
activity of the 12a-hydroxylase [29]. The portal venous return of bile acids, which governs the activity of 7ahydroxylase (cf. Fig. 2), is under strong influence of intestinal bile acid transporters [30]. One way to try to evaluate the contribution of the individual plasma lipoproteins has been to study genetic deficiency states in humans. Thus, conditions such as lack of expression of LDL-receptors (familial hypercholesterolemia) as well as abeta-lipoproteinemia have not been shown to have diminshed bile acid synthesis [25]. In fact, it is only when bile acid synthesis is heavily stimulated (e.g. through biliary drainage, resin therapy etc.) that a reduction of bile acid excretion has been seen in situations with lowered apoB-containing lipoproteins (e.g. [31]). The impact of low HDL cholesterol for biliary steroid excretion has been less extensively studied [25]. In one patient with fish-eye disease (partial deficiency of LCAT activity and HDL cholesterol B/10% of normal), no quantitative defect in bile acid production could be demonstrated [32] (Fig. 3). In two patients with very low HDL cholesterol levels due to apo A-I/C-III deficiency, the total excretion of bile acids and neutral sterols in feces was normal [33]. However, in both situations, the ratio between the synthesis of cholic acid and chenodeoxycholic acid was reduced to less than half of normal (Fig. 3). These findings indicate that the liver is capable of maintaining a normal bile acid synthesis despite very low levels of plasma HDL cholesterol. The explanation of the clearly abnormal synthesis ratio in these patients remains to be understood. There is surprisingly little information on cholesterol excretion in patients with Tangier disease (ABCA1-deficiency). In a single case, there was no clear evidence of abnormal biliary lipid metabolism [34].
4. Infusion of recombinant apo A-I/liposome complexes in humans The need for new therapeutic approaches in the treatment of atherosclerosis has stimulated the concept of developing reconstituted HDL (rHDL) particles which could promote cholesterol efflux from foam cells. Such particles are generally discoidal complexes containing phosphatidylcholine and either apo A-I or its precursor, proapo A-I [35,36]. In vitro studies in cholesterol-loaded macrophage-derived cells have demonstrated that such discs promote cholesterol efflux from cells, whereas multilamellar phosphatidylcholine liposomes are not able to significantly induce cholesterol efflux [37]. Interestingly, reconstituted discoidal complexes containing apo A-I or proapo A-I were even more effective in promoting cholesterol efflux than HDL isolated from human plasma.
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Fig. 1. Simplified scheme of lipoprotein metabolism (modified from [24]) CM, chylomicrons; CMR, chylomicron remnants; VLDL, very low density lipoprotein; IDL intermediate density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; LPL, lipoprotein lipase; HL, hepatic lipase; CETP, cholesteryl ester transfer protein; PLTP, phospholipid transfer protein; LCAT, lecithin: cholesterol acyl transferase; ABCA1, ATPbinding cassette transporter A1; ABCG1, ATP-binding cassette transporter G1: LDLR, LDL receptor; LRP, LDL receptor related protein; SR-B1; scavenger receptor class B type 1; SR-A, scavenger receptor class A. Blue arrows represent exogenous cholesterol transport; green arrows represent endogenous cholesterol transport; red arrows represent reverse cholesterol transport. The red-dashed arrows represent ways by which the cholesterol
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B. Angelin et al. / Atherosclerosis Supplements 3 (2002) 23 /30
Fig. 2. Schematic representation of hepatic cholesterol and bile acid metabolism (modified from [24]). CM, chylomicrons; CMR, chylomicron remnants; VLDL, very low density lipoprotein; IDL intermediate density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; LDLR, LDL receptor; LRP, LDL receptor related protein; SR-B1; scavenger receptor class B type 1; ACAT, acyl CoA: cholesteryl acyl transferase; HMG CoA reductase, 3-hydroxy-3-methylglutaryl CoA reductase; IBAT, ileal bile acid transporter.
In a first study, Carlson infused 1.6 g (protein) of proapo A-I liposomes to four patients with low plasma HDL cholesterol [35]. An increase in HDL cholesterol levels persisted for several days, being maximal (:/20%)
after 1 day. From kinetic analysis of radiolabelled protein, the apparent volume of distribution of apo AI was 6.9 l and the mean residence time 134 h [35]. Nanjee et al. infused seven healthy men with apo A-I/
Fig. 3. Bile acid kinetics in patients with profoundly reduced HDL cholesterol levels (see text). Data from [32,33].
B. Angelin et al. / Atherosclerosis Supplements 3 (2002) 23 /30
phosphatidylcholine discs (25 /40 mg/kg of protein) [36]. During iv infusion for 4 h, the plasma concentration of total apo A-I increased. The increase in apo A-I was mainly in the HDL fraction corresponding to prebmigrating particles, and was paralleled by a simultaneous increase in total HDL unesterified cholesterol. After the infusion was stopped, it was also possible to observe an increase in HDL cholesteryl ester and amigrating apo A-I-containing HDL. Thus, the data support the concept that the apo A-I liposome infusion was able to promote cholesterol efflux and stimulate the production of preb-HDL.
5. Fecal steroid excretion after infusion of reconstituted HDL To test the hypothesis that reverse cholesterol transport could be could stimulated in humans, we studied four subjects with heterozygous familial hypercholesterolemia [38]. The fecal excretion of bile acids and neutral sterols was measured in 3-day pools before and after a single infusion of recombinant human proapo A-I liposome complexes. At a dose of 4 g, this would correspond to approximately 75 /100% of the calculated plasma apo A-I pool. A clear increase in plasma apo A-I levels was observed during the first 2 h after infusion; this was accompanied by a less marked elevation in HDL cholesterol (Fig. 4). In all patients, apo A-I and HDL cholesterol levels in plasma had returned to baseline after 48 h. The fecal excretion of bile acids and neutral steroids was increased by 30 and 39%, respectively (Fig. 5). To exclude the possibility that the phospholipid complexes
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had an independent stimulatory effect on cholesterol excretion, we repeated the experiment in two of the patients with infusion of liposomes prepared without proapo A-I. The fecal excretion of cholesterol, either as neutral sterols or as bile acid, was not affected by this treatment, however. Thus, a pronounced increase in cholesterol excretion from the body was observed after intravenous infusion of recombinant proapo A-I liposomes. The lack of response in two of the patients when infused with pure liposomes indicates that, also in vivo, apo A-I plays a major role in promoting reverse cholesterol transport. In eight healthy males, Nanjee et al. [39] recently confirmed an increase in fecal bile acid excretion in response to infusion of apo A-I (40 mg/kg) lecithin discs; this was associated with a rise in prebHDL in plasma and lymph. Thus, two independent studies in humans indicate that infusion of ‘nascent’ rHDL may stimulate reverse cholesterol transport both due to stimulation of cholesterol efflux from peripheral cells and by promoting hepatic steroid excretion. The degree of stimulation of cholesterol excretion in response to proapo A-I/liposome infusion observed in our study was surprisingly large, corresponding to a removal of 0.5 g cholesterol daily for several days. It is important to realize that we cannot identify the origin of the excreted steroids. Thus, proapo A-I liposomes may stimulate hepatic cholesterol synthesis by extracting cholesterol from hepatocytes. However, repeated measurements of serum lathosterol and lathosterol/cholesterol ratio, markers of in vivo cholesterol synthesis [40,41], did not show any change in response to treatment. Plasma lathosterol was transiently increased immediately after infusion in four of the subjects studied by Nanjee et al. [41].
Fig. 4. Plasma levels of apo A-I (filled circles) and HDL cholesterol (grey squares) in response to infusion of 4 g proapo A-I/liposomes in patients with heterozygous familial hypercholesterolemia (data from [38]). Values are percent of baseline (mean9/S.E.M.).
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Fig. 5. Fecal excretion of neutral sterols, bile acids, and total steroids 9 days before (filled bars) and 9 days after (grey bars) the infusion of 4 g of proapo A-I/liposomes in patients with heterozygous familial hypercholesterolemia (data from [38]). Values are mean9/S.E.M.
6. Concluding remarks Reverse cholesterol transport is a very complex process which is difficult to study. Studies of animal models, particuarly in rodents, are currently performed, and the access to genetically modified strains of mice has considerably improved our knowledge on lipoprotein metabolism in vivo. However, species differences in lipid metabolism, e.g. lack of CETP activity and larger capacity for variation in hepatic cholesterol synthesis in response to perturbations in rodents [22], make it fundamental to include information obtained in humans. As to reverse cholesterol transport, a number of recent studies in genetically modified mice have shown, in agreement with the observations in humans, that profoundly lowered HDL cholesterol and apo A-I levels (such as in apo A-I knockout mice) do not substantially influence biliary lipid metabolism [42,43]. On the other hand, studies in mouse models have failed to demonstrate the promotion of net steroid excretion seen in humans both in response to overexpression of apo A-I and infusion of apo A-I/phospholipid discs [44]. The concept of stimulating reverse cholesterol transport in order to reduce the risk of atherosclerosis and its complications holds great clinical promise. The relation between the antiatherogenic effects and plasma levels of HDL cholesterol or apo A-I may be less clear than that to HDL cholesterol flux. The concept of infusing rHDL as a therapeutic alternative is still far from practical
reality. In addition to important clinical aspects such as the cost of production (and particularly purification), and the possible immunogenic problems with repeated protein infusions, the need for hard endpoint data (regression studies in humans) will make it a difficult project. On the other hand, the technique may provide us with new and important information on the role of apo A-I and HDL in reverse cholesterol transport which will be extremely important in the continued search for new therapeutic principles [45,46].
Acknowledgements We thank Lena Emtestam for skilful secretarial assistance. The author’s work is supported by the Swedish Research Council (03X-7137), the Old Female Servants, the Tore Nilsson, the Fredrik and Ingrid ˚ ke Wiberg, Thuring, the Ruth and Richard Julin, the A and the Swedish Heart-Lung Foundations.
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