Reverse cholesterol transport: Physiology and pharmacology

Atherosclerosis, 88 (1991) 99- 107 0 1991 Elsevier Scientific Publishers ADONIS 0021915091001150

ATHERO

99 Ireland,

Ltd. OOZl-9150/91/$03.50

04640

Review article

Reverse cholesterol transport: physiology and pharmacology Guido Franceschini, Center E. Grossi Paoletti,

Institute

Paola Maderna of Pharmacological

and Cesare R. Sirtori

Sciences, Unil,ersity

of Milan,

Milan

(Italy)

(Received 18 June. 1990) (Revised, received 25 January. 1991) (Accepted 29 January. 1991)

Summary Reverse cholesterol transport identifies a series of metabolic events resulting in the transport of cholesterol from peripheral tissues to the liver and plays a major role in maintaining cholesterol homeostasis in the body. High density lipoproteins (HDL) are the vehicle of cholesterol in this reverse transport, a function believed to explain the inverse correlation between plasma HDL levels and atherosclerosis. An attempt to stimulate, by the use of drugs, this transport process seems to be of great promise in the prevention and treatment of arterial disease. Only few drugs are now known that can modify the activity of the various factors involved in the process. Clofibrate reduces cholesterol esterification, but the newer fibric acids are generally ineffective as anion-exchange resins. Probucol directly increases the activity and mass of cholesteryl ester transfer protein, thus possibly improving the physiological process of cholesterol removal from tissues. The few available data on the effects of drugs on reverse cholesterol transport should stimulate the search for new agents specifically stimulating this antiatherogenic process.

Key words: Reverse cholesterol transport; HDL Cholesteryl ester transfer protein

Introduction In most non-hepatic cells, cholesterol homeostasis is maintained by a complex balance between endogenous synthesis, uptake of extracellu-

Correspondence to: Dr. Guido Franceschini, Center E. Grossi Paoletti, Institute of Pharmacological Sciences, University of Milan, via Balzaretti 9, 20133 Milan, Italy.

subfractions;

Lecithin : cholesterol

acyltransferase;

lar cholesterol, mostly from low density lipoproteins (LDL) through the LDL receptor, and efflux of cell cholesterol to the vascular fluids. This efflux is the first step in the reverse cholesterol transport, a term originally introduced by Glomset in 1968, to define the movement of cholesterol from peripheral tissues to the liver [l]. This process appears to be dependent on high density lipoproteins (HDL) [2]: the crucial role of HDL in reverse cholesterol transport is believed to

100 explain the well established negative correlation between plasma HDL levels and atherosclerosis 131. In view of the anti-atherogenic potential of the reverse cholesterol transport, the possibility of stimulating this process has to be considered in the prevention and/or treatment of arterial disease. In this article, the physiology of reverse cholesterol transport is reviewed, and indications are provided on clinically effective tools for the modulation of this process. Physiology

of reverse cholesterol

transport

The first step in the reverse cholesterol transport pathway is the uptake of cellular cholesterol by HDL [4,5]. The mechanism/s by which HDL remove cholesterol from cells are only partially understood. Cholesterol leaves cells predominantly in the unesterified form; it may desorb from the plasma membrane into the extracellular fluids by a diffusion-limited process [6,7], being then incorporated into HDL, which have a relatively high affinity for lipids [S]. HDL can also interact with a specific surface receptor, identified in a variety of extrahepatic cell types [9-111. Cell binding of acceptor particles is not required for the transport of sterol from the plasma membranes [12-141, but may promote the translocation of excess cholesterol from intracellular to membrane pools [15,16]. Within the cell, cholesterol is continuously esterified and hydrolyzed by cytoplasmic acyl CoA : cholesterol acyltransferase (ACAT) and cholesterol esterase; in vitro inhibition of the esterification cycle results in an improved cholesterol efflux from cells [17-191, suggesting that the availability of free cholesterol is a major determinant in the first step of reverse cholesterol transport. The nature of the lipoprotein acceptor also plays a major role in the net transport of cholesterol from cells to plasma. Of the various potential acceptors tested in tissue culture experiments, small spherical HDL, and discoidal complexes of apo Al and phosphatidylcholine are the most efficient 1201. More recently, evidence has been presented supporting a primary role for a minor HDL subfraction in cell cholesterol uptake, when cultures of skin fibroblasts are incu-

bated with human plasma [21]. These acceptor particles contain only apo Al, are phospholipid enriched and cholesteryl ester (CE) poor, and display pre-beta electrophoretic mobility on agarose gels [21,22]. Finally, in cultured adipose cells, HDL particles containing only apo Al, but not those with Al and All, are able to promote cholesterol efflux from cells [23]. Once cellular free cholesterol has been incorporated into small HDL, a complex series of metabolic rearrangements takes place, involving various enzymes and plasma factors, and resulting in the formation of large HDL particles. The first step in this HDL conversion process is the esterification of cholesterol by the lecithin : cholesterol acyltransferase (LCAT) enzyme [ 11. This reaction is essential for maintaining a free cholesterol potential gradient between cells and HDL, necessary for a continuous flow of cholesterol from cells to plasma [24]. The chemical and physical properties of LCAT and the mechanisms of the LCAT catalyzed reaction have been recently reviewed [25]. The CE synthesized by LCAT in HDL, are transferred to apo B-containing lipoproteins, mainly very low density lipoproteins (VLDL) [26,27], by a specific protein called “cholesteryl ester transfer protein” (CETP or LTP-I) [28-301. CETP exchanges one mole of CE for one of triglycerides (TG) between HDL and VLDL, leading to the formation of large, CE-poor and TG-rich HDL, particles [31-331. Once the TG content in these large HDL reaches a threshold value, triglycerides and phospholipids are hydrolyzed by hepatic lipase (HL) [34], converting the large, TG-rich HDL, back into small, TGand CE-poor HDL, [31,35] (Fig. 1). Apolipoprotein composition also varies during HDL particle interconversion: apo Al is the major component of the large HDL,, whereas the small HDL, contain both apo Al and apo All [36]. The role of apolipoprotein movements between the various HDL particles, and the mechanisms of these exchanges are at present unknown. In vitro, apo All, and also other small peptides, can displace apo Al from HDL [37,38]; on the other hand, studies with synthetic model lipoproteins clearly show that the apolipoprotein content in precursor particle determines the properties of the product [39]. Other factors, i.e. lipoprotein lipase [401 and

101

Fig. 1. Interconversion of HDL in human plasma. CE, cholesteryl esters; FC, free cholesterol: PL, phospholipids. acyltransferase; TG, triglycerides; LCAT, lecithin : cholesterol CETP, cholesteryl ester transfer protein; HL. hepatic lipase. Modified from Eisenberg [31].

phospholipid transfer protein (LTP-II) [41], may be involved in the HDL conversion process [31]; their physiological role in the reverse cholesterol transport is at present unclear, although LPL and LTP-II may supply phospholipids to HDL, as substrates for the LCAT reaction. By these mechanisms, free cholesterol from peripheral cells is taken up by HDL, esterified and transferred to lower density lipoproteins; these finally deliver the cholesteryl esters to the liver [42], by interacting with LDL receptors [43]. This indirect pathway plays a major role in species with elevated CETP activity, like humans [44]. Other, direct pathways may operate in the reverse cholesterol transport, particularly in species lacking CETP [451. As HDL accumulate CE, they become enriched in apo E [46] and bind to hepatic receptors recognizing apo E [47]; it has been calculated that in the rat this apo E mediated cholesterol transport to the liver may account for approximately 10% of HDL-cholesterol [48]. Furthermore, HDL can directly deliver CE to hepatocytes, either by a process independent from particle uptake [49] or by a retroendocytic mechanism [50]. It thus appears that there may be several pathways of reverse cholesterol transport, reflecting the importance to the organism to maintain a correct balance between centripetal and centrifugal cholesterol movements. Although in humans the majority of HDL-CE seems to be delivered to hepatocytes indirectly, following transfer to other lipoproteins [51], the other pathways can compen-

sate for occasional defects in this process. Individuals with inborn CETP deficiency accumulate in plasma large, apo E-rich HDL with high affinity for the LDL receptor [52]; these can directly deliver cholesterol of hepatocytes [47]. Studies in families with CETP deficiency suggest that a low CETP activity results in an antiatherogenic lipid profile, characterized by increased plasma HDL and HDL,, and low LDL levels [531. In contrast, an elevated CETP activity should lead to the accumulation of CE-rich VLDL in plasma, possibly promoting CE deposition and foam cell formation in macrophages [54]. Other data argue for an antiatherogenic role of the CETP mediated process. A low net mass transfer of CE from HDL to VLDL has been reported in several groups of subjects at elevated risk for atherosclerotic vascular disease [55,56]; measurements of artery-hepatic vein differences of cholesterol concentrations in humans clearly showed that LDL-CE, but not CE from other lipoproteins, are extracted by splanchnic tissues [421, suggesting that CE transfer from HDL to LDL is essential for CE delivery to the liver. Evaluation man

of the reverse cholesterol

transport

in

Since reverse cholesterol transport consists of several metabolic steps, occurring both in plasma and tissues, a complete evaluation of the entire process is almost impossible. Although in vivo kinetic studies of HDL-CE transport [58,59] should be highly informative, they are of difficult clinical execution. The in vitro measurement of the individual capacity for cholesterol uptake from peripheral cells [553 and for delivery to hepatocytes [60] should also contribute to the evaluation of the reverse transport at the tissue level. At present, however, for practical reasons, the analysis of the steps occurring in plasma seems to be the most promising for evaluating reverse cholesterol transport in the individual. Among the various parameters to be considered, two are of major interest: cholesterol esterification and cholesteryl ester transfer from HDL to lower density lipoproteins. Cholesterol esterification can be measured in the presence of the endogenous substrate [61,62],

102 evaluating the cholesterol esterification rate (CER), that reflects the interaction between the LCAT enzyme and its substrate. By using a common synthetic substrate [63,64] one can evaluate LCAT activity, which is a function of the amount of enzyme and of its intrinsic activity. Finally, the LCAT mass can be quantified by immunological methods [651. The evaluation of the cholesteryl ester transport from HDL to lower density lipoproteins appears definitely more complex. Again it can be measured in the presence of endogenous lipoproteins, as the net mass transfer of CE from HDL to apo B-containing lipoproteins during incubation of whole plasma [55]. Although this assay gives a direct evaluation of the entire process in the individual, it is dependent upon (a) amount and activity of transfer protein, (b) interaction of CETP with donor and acceptor lipoproteins, and (c> amount of endogenous lipoproteins. Several substrate independent methods, using radiolabeled cholesteryl esters incorporated into donor particles [41,66-711, have been developed to overcome these obstacles, but the variety of suggested technical procedures does not allow a direct comparison between the different studies. Recently, a specific immunoassay measuring the CETP mass has been introduced, allowing a direct estimation of the amount of transfer protein [72]. Finally, characterization of HDL particles in individual plasma can also help to understand the dynamics of the HDL conversion process: an accumulation of large or small HDL particles may be the result of changes in the activity of CETP [52], LCAT [731 or HL [741. Among the various techniques for HDL subfractionation, rate zonal ultracentrifugation [75,76], nondenaturing polyacrylamide gradient gel electrophoresis [77] and immunoaffinity chromatography [78] appear as the most informative, providing both quantitative and qualitative data. Effects of drugs on the reverse cholesterol transPod Numerous pharmacological substances can modify plasma HDL levels in man; among these, several hypolipidemic drugs, antihypertensive

agents, hormones and microsomal enzyme inducers [791. The exact mechanisms responsible for these modifications are generally poorly understood, although the lipase-stimulating activity is likely to be responsible for the fibric acid induced HDL increases [79,80]. Drug-induced elevations of HDL-cholesterol levels have been associated with a reduction of cardiovascular risk [81,82], possibly secondary to an improved reverse cholesterol transport, but only few studies have examined the effect of drugs on the various steps of this process. Most studies have evaluated changes in cholesterol esterification during hypolipidemic drug treatment. Clofibrate lowers CER in both hypercholesterolemic and hypertriglyceridemic patients [83,84], but no changes have been recorded with the newer fibrate derivatives, bezafibrate [85,86] and fenofibrate [87,88]. Contrasting results have been reported on the effects of anion exchange resins, colestipol increasing CER in hypercholesterolemic patients [88,89], but not in normolipidemic individuals [87]; cholestyramine’ is apparently ineffective [90,91]. Fish oil supplementation at small dose lowers CER by 20% in mildly hypercholesterolemic patients 1921. In vitro inhibition of LCAT has been produced by addition of various antihypertensive agents [931 and of diazepam [94] to human plasma, generally at concentrations not achievable in vivo; however, propranolol treatment lowers CER in hypertensive patients [95]. Finally, stanozolol, an anabolic steroid, reduces LCAT mass in normolipidemic women with postmenopausal osteoporosis [96]. The in vitro transport of cholesteryl esters from HDL to lower density lipoproteins has been evaluated only in subjects treated with bezafibrate [86], probucol [97,98] and fish oil [92]. Bezafibrate does not modify either the net CE mass transfer (substrate-dependent), or the transport of cholesterol out of cultured cells, in normolipidemic individuals [86]. No changes in cholesterol esterification and CE transfer have been observed in patients treated with a nicotinic acid derivative, acipimox (Franceschini, G. et al., unpublished data). Fish oil lowers the substrateindependent CETP activity in hypercholesterolemic patients [921, whereas probucol stimulates the net CE mass transfer (substrate-

103 dependent) in hypercholesterolemic patients [97], possibly through a direct effect on CETP activity (Franceschini G. et al., unpublished data). By this mechanism, and by a stimulation of the uptake of cell cholesterol by HDL [99], probucol may exert the antiatherogenic effect observed in humans [loo] and experimental animals [101,102]. Finally, several studies have examined the effect of drug treatments on HDL subfraction distribution in both normo- and hyperlipidemic subjects [79,80]. The use of different techniques for HDL fractionation, often giving contrasting results [103], hampers a direct comparison of the reported data. By a combination of rate zonal ultracentrifugation and nondenaturing polyacrylamide gradient gel electrophoresis, we could demonstrate that the fibric acid derivative, gemfibrozil, increases plasma HDL, levels in both hypertriglyceridemic [104] and hypercholesterolemic [105] subjects; HDL, levels also rise during treatment with cimetidine, an H, antagonist, in patients with mild metabolic disturbances [106]. Minor changes were observed in hypertriglyceridemic patients treated with acipimox, a nicotinic acid derivative [107], whereas the HMGCoA reductase inhibitor, pravastatin, does not modify HDL subfraction distribution in hypercholesterolemic individuals [105]. Probucol instead lowers the plasma levels of both HDL subfractions, affecting mainly HDL, [97], consistent with a simulation of the CETP activity [97]. Conclusions

Reverse cholesterol transport is the result of distinct pathways, with several, different metabolic steps. There is at present, little information on the relative importance of these various mechanisms on the efficiency of the whole process in vivo, especially in man. Furthermore, a great controversy still exists on the antiatherogenic potential of the different pathways. The CETP mediated step may promote the formation of CE-rich chylomicron or VLDL remnants, eventually leading to foam cell formation [44]. Familial CETP and LCAT deficiencies are not associated with an increased incidence of premature CHD [53,108], although, due to their rarity, it is not certain that they confer protection against atherosclerosis.

LCAT activity is even increased in patients with angiographically defined CHD [109]. More and more studies, particularly in vivo studies, are necessary to definitely prove that a promotion of reverse cholesterol transport, at the plasma level, will exert an antiatherogenic effect in man. However, it seems to us that this field provides promising targets for the prevention and treatment of atherosclerotic disease. The recently proved efficacy of HDL supplementation in vivo in promoting atherosclerosis regression in experimental animals [llO] provides an extraordinary basis on which to build the pharmacology of reverse cholesterol transport. At present only a few drugs are known which can modulate a very few steps of reverse cholesterol transport. Some of these compounds, like probucol, can prevent atherosclerosis development in experimental animals [101,102] and induce a regression of tissue lipid deposits in man [loo]. These scarce and preliminary findings should stimulate the search for new agents specifically promoting the process of reverse cholesterol transport at the cellular and/or plasma level. References 1 Glomset, J.A., The plasma lecithin: cholesterol acyltransferase reaction, J. Lipid Res., 9 (19681 1.55. 2 Miller, N.E., LaVille, A. and Crook, D., Direct evidence that reverse cholesterol transport is mediated by highdensity lipoprotein in the rabbit, Nature, 314 (1985) 109. 3 Miller, G.J. and Miller, N.E., Plasma high-density lipoprotein concentration and development of ischaemic heart disease, Lancet, i (19751 16. 4 Tall, A.R. and Small, D.M., Body cholesterol removal: role of plasma high density lipoproteins, Adv. Lipid Res.. 17 (1980) 1. 5 Ho, Y.K., Brown, M.S. and Goldstein, J.L. Hydrolysis and excretion of cytoplasmic cholesteryl esters by macrophages: stimulation by high density lipoprotein and other agents, J. Lipid Res., 21 (19801 391. 6 Phillips, M.C., Johnson, W.J. and Rothblat, G.H., Mechanisms and consequences of cellular cholesterol exchange and transfer, Biochim. Biophys. Acta, 906 (1987) 223. 7 Bojesen, E., Diversity of cholesterol exchange explained by dissolution in water, Nature, 299 (1982) 276. 8 Chobanian, J.V., Tall, A.R. and Brecher, PI., Interaction between unilamellar egg yolk lecithin vesicles and human high density lipoprotein, Biochemistry, 18 (19791 180. 9 Biesbroeck, R., Oram, J.F., Albers, J.J. and Bierman, E.L., Specific high affinity binding of high density lipoprotein to cultured human skin fibroblasts and arterial smooth muscle cells, J. Clin. Invest., 71 (1983) 525.

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