Reverse cholesterol transport and cholesterol efflux in atherosclerosis

Reverse cholesterol transport (RCT) is a pathway by which accumulated cholesterol is transported from the vessel wall to the liver for excretion, thus preventing atherosclerosis. Major constituents of RCT include acceptors such as high-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I), and enzymes such as lecithin:cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), hepatic lipase (HL) and cholesterol ester transfer protein (CETP). A critical part of RCT is cholesterol efflux, in which accumulated cholesterol is removed from macrophages in the subintima of the vessel wall by ATP-binding membrane cassette transporter A1 (ABCA1) or by other mechanisms, including passive diffusion, scavenger receptor B1 (SR-B1), caveolins and sterol 27-hydroxylase, and collected by HDL and apoA-I. Esterified cholesterol in the HDL is then delivered to the liver for excretion. In patients with mutated ABCA1 genes, RCT and cholesterol efflux are impaired and atherosclerosis is increased. In studies with transgenic mice, disruption of ABCA1 genes can induce atherosclerosis. Levels of HDL are inversely correlated with incidences of cardiovascular disease. Supplementation with HDL or apoA-I can reverse atherosclerosis by accelerating RCT and cholesterol efflux. On the other hand, pro-inflammatory factors such as interferon-gamma (IFN-gamma), endotoxin, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta), can be atherogenic by impairing RCT and cholesterol efflux, according to in vitro studies. RCT and cholesterol efflux play a major role in anti-atherogenesis, and modification of these processes may provide new therapeutic approaches to cardiovascular disease. Further research on new modifying factors for RCT and cholesterol efflux is warranted.     

Intracellular transport of recombinant adeno-associated virus vectors

Recombinant adeno-associated viral vectors (rAAVs) have been widely used for gene delivery in animal models, and are currently evaluated for human gene therapy after successful clinical trials in the treatment of inherited, degenerative or acquired diseases, such as Leber congenital amaurosis, Parkinson disease or heart failure. However, limitations in vector tropism, such as limited tissue specificity and insufficient transduction efficiencies of particular tissues and cell types, still preclude therapeutic applications in certain tissues. Wild-type adeno-associated viruses (AAVs) are defective viruses that require the presence of a helper virus to complete their life cycle. On the one hand, this unique property makes AAV vectors one of the safest available viral vectors for gene delivery. On the other, it also represents a potential obstacle because rAAV vectors have to overcome several biological barriers in the absence of a helper virus to transduce successfully a cell. Consequently, a better understanding of the cellular roadblocks that limit rAAV gene delivery is crucial and, during the last 15 years, numerous studies resulted in an expanding body of knowledge of the intracellular trafficking pathways of rAAV vectors. This review describes our current understanding of the mechanisms involved in rAAV attachment to target cells, endocytosis, intracellular trafficking, capsid processing, nuclear import and genome release with an emphasis on the most recent discoveries in the field and the emerging strategies used to improve the efficiency of AAV-derived vectors.     

Putting cholesterol in its place: apoE and reverse cholesterol transport

To avoid toxic overload of cholesterol in peripheral cells, the reverse cholesterol transport pathway directs excess cholesterol through HDL acceptors to the liver for elimination. In this issue of the JCI, a study by Matsuura et al. reveals new features of this pathway, including the importance of the ATP-binding cassette transporter G1 in macrophages and apoE in cholesteryl efflux from cells to cholesterol ester-rich (CE-rich) HDL(2) acceptors (see the related article beginning on page 1435). One proposal for boosting reverse cholesterol transport has been to elevate plasma HDL levels by inhibiting CE transfer protein (CETP), which transfers CE from HDL to lower-density lipoproteins. However, there has been concern that large, CE-rich HDL(2) generated by CETP inhibition might impair reverse cholesterol transport. ApoE uniquely facilitates reverse cholesterol transport by allowing CE-rich core expansion in HDL. In lower species, these large HDLs are not atherogenic. Thus, CETP might not be essential for reverse cholesterol transport in humans, raising hope of using a CETP inhibitor to elevate HDL levels.     

Plasma cholesterol transport in anhepatic rats.

The plasma appearance of newly synthesized cholesterol in anhepatic laboratory diet-fed rats was 10% of the intact rat. In intact rats this cholesterol was mainly ester in lower density lipoproteins, but for anhepatic rats it was virtually only free in high density lipoprotein. Chylomicron cholesterol ester was removed much more slowly from anhepatic than control plasma and returned primarily as free in high density lipoproteins, with the control return 10 times the anhepatic return. Lower density lipoprotein cholesterol ester transfer to an extravascular pool in anhepatic rats was less than 10% of controls. The liver was responsible for 95% of the extravascular lower density lipoprotein ester pool and only 50% of the for high density lipoprotein ester. Despite decreased anhepatic lipoprotein catabolism, the mass of both plasma low and high density lipoproteins progressively decreased indicating an even greater decrease in influx. The anhepatic fractional catabolic rate of apo A1 was similar to controls, but that of apo E was considerably less. Despite the unchanged catabolism of apo A1 and the reduced catabolism of apo E, plasma apo A1 decreased less than apo E after hepatectomy. The anhepatic data confirm the pivotal role of the liver in maintaining plasma low and high density lipoprotein cholesterol concentrations. They suggest that, in addition to its anabolic and catabolic functions, the liver also acts as a reservoir buffering changes in plasma concentration.     

The ins and outs of reverse cholesterol transport

It is generally assumed that HDL is the obligate transport vehicle for 'reverse cholesterol transport', the pathway for removal of excess cholesterol from peripheral tissues via the liver into bile and subsequent excretion via the feces. During the last few years, intensive research has generated exciting new data on the separate processes involved in reverse cholesterol transport. Many 'new' proteins, particularly members of the ABC transporter and nuclear receptor subfamilies, that mediate or influence cholesterol fluxes have been identified and characterized. An important role of the intestine in regulation of cholesterol homeostasis is emerging. In this paper, new insights into mechanisms of reverse cholesterol are reviewed.     

Lymphatic and portal venous transport of alpha-tocopherol and cholesterol

The absorption of ³H-α-tocopherol was studied in rats after intraduodenal administration in micellar solutions. Net absorption from mixed micelles in lymph fistula rats was 66%, but only 42% appeared in lymph and 3% was excreted in urine. Administration of ³H-α-tocopherol in taurocholate micelles reduced both the rate of absorption into lymph and the proportion carried in chylomicrons. In studies with lymph and bile duct cannulated rats, up to 8% of the radioactivity was excreted in bile, irrespective of whether the cannula was in the mesenteric or thoracic lymph duct. These findings suggested that radioactivity was being absorbed via the portal vein, which was confirmed by demonstrating a higher concentration in portal than in aortic plasma. Radioactivity in plasma partly consisted of polar metabolites, in contrast to lymph where over 90% was free α-tocopherol. In parallel experiments with ¹⁴C-cholesterol, significant amounts of radioactivity were excreted in bile and the ratio of portal: aortic radioactivity again suggested absorption via the portal route. The majority of ¹⁴C-cholesterol in portal plasma was free sterol, in contrast to lymph where it was mainly esterified. These results suggest that small amounts of both α-tocopherol and free cholesterol can be transported from the intestine via the portal vein, although under normal circumstances absorption of both compounds occurs mainly via the lymphatic pathway.     

Defect in cholesterol transport in patients receiving maintenance hemodialysis

A defect in cholesterol transport was detected in patients with uremia who were receiving long-term hemodialysis when the rate of cholesterol transfer (RCT) from high-density lipoprotein (HDL) to very low-density (VLDL) and low-density lipoproteins (LDL) was compared with that in controls. The RCT (mean +/- SD) in 29 men with uremia (1.85 +/- 1.29 mg/hr/100 ml) and 11 women with uremia (1.84 +/- 1.00 mg/hr/100 ml) was significantly lower (P less than 0.001) than values in 55 healthy men (4.50 +/- 2.61 mg/hr/100 ml) and 23 healthy women (3.72 +/- 1.92 mg/hr/100 ml), respectively. Six patients, but none of the controls, totally lacked the ability for cholesterol transfer. The decreased RCT of the patients could not be completely accounted for by their decreased HDL cholesterol levels, because patients matched with controls for HDL cholesterol within 1 mg/100 ml also had lower RCT (P less than 0.0025). Recombination and crossover of serum fractions of patients and controls separated by ultracentrifugation revealed that the defect in cholesterol transfer of the patients was in the d greater than 1.063 gm/ml fraction (containing HDL and other serum proteins), which not only contained less HDL cholesterol, but was also qualitatively inferior as donor for cholesterol transfer. In one of four patients studied, the d less than 1.063 gm/ml fraction (VLDL and LDL) also had deficient ability to accept cholesteryl esters in the transfer. These in vitro data indicate a defect in cholesterol transport in the patients who are undergoing hemodialysis. Whether this defect exists in vivo and creates the risk of accelerated atherosclerosis warrants further study.     

Intracellular cystine loading inhibits transport in the rabbit proximal convoluted tubule.

Cystinosis is an autosomal recessive disorder characterized by a high intracellular cystine concentration. To establish an in vitro model of this disorder and examine the mechanism of the proximal tubule transport defect seen with elevated intracellular cystine concentrations, rabbit proximal convoluted tubules (PCT) were perfused in vitro. PCTs were loaded with cystine using cystine dimethyl ester, a permeative methyl ester derivative. Bath cystine dimethyl ester (0.5 mM) reduced volume absorption (Jv) (0.67 +/- 0.07 to 0.15 +/- 0.09 nl/mm.min, P less than 0.01), bicarbonate transport (JTCO2) (47.2 +/- 4.9 to 11.1 +/- 2.8 pmol/mm.min, P less than 0.001) and glucose transport (JGLU) (34.1 +/- 1.5 to 19.7 +/- 1.5 pmol/mm.min, P less than 0.001). The methyl esters of leucine (0.5 mM), and tryptophan (0.5 and 2.0 mM) had no effect on these parameters. To examine if intracellular reduction of cystine to cysteine could contribute to the inhibition in transport, the effect of bath cysteine methyl ester on proximal tubular transport was examined. Bath cysteine methyl ester (2 but not 0.5 mM) resulted in an inhibition in Jv, JGLU, and JTCO2. Cystine dimethyl ester had no effect on mannitol or bicarbonate permeability. These data are consistent with intracellular proximal tubular cystine accumulation resulting in an inhibition of active transport.     

Advances in understanding of the role of lecithin cholesterol acyltransferase (LCAT) in cholesterol transport.

We review the structure and function of lecithin cholesterol acyl transferase (LCAT), the advances in the studies of molecular genetics of LCAT and its deficiency states as well as the developments in assessment of LCAT activity particularly the concept of measurement of fractional esterification rate of plasma cholesterol in the absence of apoB lipoproteins (FER(HDL)) as an indication of atherogenic risk. We discuss LCAT reaction from two points of view: one that is consistent with the general belief in LCAT antiatherogenic potential and another, namely, a proposed concept of potentially opposing roles of LCAT in normal and dyslipidemic plasmas. While other plasma lipoproteins can (in addition to HDL) provide unesterified cholesterol (UC) for LCAT reaction, HDL may play an unique role in trafficking of newly formed cholesteryl esters (CE) rather than as a primary acceptor of cellular cholesterol. Thus, the plasma HDL, specifically the larger (HDL2b) particles, direct the efflux of most of (LCAT produced) CE to its specific catabolic sites rather than to potentially atherogenic VLDLs and back to LDLs.     

Reverse cholesterol transport: relationship between free cholesterol uptake and HDL3 in normolipidaemic and hyperlipidaemic subjects

Abstract. High density lipoproteins (HDL) are responsible for the Reverse Cholesterol Transport (RCT). The role of the composition of the HDL particle in RCT, involving free cholesterol (chol) uptake from cell membranes, is not completely understood. We have therefore studied the uptake capacity from subjects with a wide variety of plasma HDL cholesterol concentrations in an HDL-receptor free model consisting of bovine heart mitochondrial membranes labeled with [¹⁴C]cholesterol. HDL were isolated by molecular sieve chromatography from fresh plasma samples of eight subjects with low plasma HDL chol concentrations (≤ 1.0 mmol L-¹) and 15 subjects with normal HDL chol concentrations. The latter were subdivided into an intermediate (HDL chol: 1.0–1.4 mmol L^-1; n= 9) and a high HDL chol group (>1.4 mmol L-¹; n= 6). In the HDL fractions isolated by chromatography (cHDL), total chol and apolipopro-tein (apo) A1 were measured. Free chol uptake was significantly decreased by 32% in the tertile with the lowest plasma HDL chol (49 1 ± 15.8 arbitrary units; mean ± SD), compared to the tertile with high HDL chol (72.1 ± 16.6 au). Linear regression analysis showed a positive correlation between the free choi uptake and plasma HDL3 concentrations (r= 0.61; P<0.01), HDL chol (r= 0.56; P<0.01), HDL associated apo A1 (r = 0.46; P<0.05), cHDL apo AT (r = 0.56; P<0.05) and cHDL chol (r = 0.46; P<0.05) in all subjects combined. Stepwise multiple-regression analysis confirmed the association of [¹⁴C]cholesterol uptake with plasma HDL3 concentrations (β, 061; P= 0.004). No correlations were found between free chol uptake and total plasma apoAI (r = 0.26; ns) or HDL2 (r = 0.27; ns). After an oral fat load in four FCH patients, free chol uptake parallelled the changes in plasma HDL3 chol concentrations. We conclude that HDL3 is involved in the early steps of RCT and low HDL3 levels may result in less efficient RCT in hypertriglyceridemia.