Search

Atherosclerosis is a disease of blood vessel walls that is thought to be initiated as a reaction of insults to the endothelium. The complex sequence of cellular events that begins with focal inflammation leads to the accumulation of leukocytes in the subendothelial layer and unrestricted uptake of oxidized lipoproteins by macrophages and smooth muscle cells, leading to foam cell formation. Vascular endothelial cells do not undergo the foam cell transformation and do not accumulate cholesterol in atherosclerotic plaques to the same extent as macrophages or smooth muscle cells. However, vascular endothelial cells express receptors for oxidized lipoproteins, and have the biochemical pathways for sterol synthesis and receptor-mediated endocytosis of lipoproteins. Data from the authors' laboratory show that high density lipoproteins but not lipid-free apolipoprotein A-I promote cellular cholesterol efflux in human umbilical vascular endothelial cells and human aortic endothelial cells. Gene expression microarrays were used to examine the differential expression of genes after cholesterol loading. While sterol regulatory element-binding protein-sensitive genes were downregulated, the authors identified a novel transporter, the ATP-binding cassette G1 (ABCG1) to be highly expressed in response to both cellular cholesterol loading and stimulation with the liver X receptor agonist 22-hydroxycholesterol. The ABCA1 gene and protein, the major modulator of cellular cholesterol efflux in macrophages and in peripheral and hepatic tissues, are only weakly expressed in human umbilical vascular endothelial cells and human aortic endothelial cells. These data suggest that endothelial cells maintain cholesterol homeostasis by downregulating cholesterol synthesis and low density lipoprotein receptors and by a cellular cholesterol efflux mechanism onto low-affinity but high-capacity high density lipoproteins. The role of ABC-type transporters, including ABCG1, requires further examination.

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither prebeta(1)-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from alpha-HDL to prebeta-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma prebeta(1)-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.

Background: Prospective studies have examined the relationship between coronary artery disease and low plasma levels of high-density lipoprotein cholesterol (HDL-C)., Methods and Results: We investigated the causes of hypoalphalipoproteinemia (HypoA; HDL-C <5th percentile) in 64 subjects (12 women and 52 men). Apolipoprotein AI-mediated cellular cholesterol and phospholipid efflux were measured in fibroblasts from HypoA subjects, 9 controls, 2 patients with Tangier disease, and 5 patients with hyperalphalipoproteinemia. A phospholipid efflux defect was defined as <70% of controls. Mean HDL-C was 0.49+/-0.21 mmol/L. Cholesterol and phospholipid efflux correlated strongly (r=0.72, P<0.001). Phospholipid efflux and HDL-C (r=0.64, P<0.001) correlated in HypoA subjects. However, phospholipid or cholesterol efflux was no longer a determinant of HDL-C levels at higher levels (> approximately 1.0 mmol/L) of HDL-C. In HypoA subjects, 4 cases of Tangier disease and 6 of familial HDL deficiency (heterozygous Tangier disease) were identified (10 of 64; 16%). In the remaining 54 subjects, mean lipid efflux was not significantly different from controls and subjects with hyperalphalipoproteinemia. A phospholipid efflux defect was identified in 7 additional HypoA subjects, and a cholesterol efflux defect was detected in 11 subjects. In 2 of these subjects, the ABCA1 gene was ruled out as the cause of the efflux defect, while in 3, the low HDL-C trait segregated with the ABCA1 gene locus., Conclusions: Lipidation of lipid-poor apolipoprotein AI may not be a major determinant of cholesterol accumulation within more mature HDL particles and increasing cholesterol or phospholipid efflux beyond normal levels may not lead to increase in plasma HDL-C levels. ABCA1 is essential in the initial steps of HDL formation but other plasma events are major modulators of HDL-C levels.

ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in apoA-I lipidation, a key step in reverse cholesterol transport. cAMP induces apoA-I binding activity and promotes cellular cholesterol efflux. We investigated the role of the cAMP/protein kinase A (PKA) dependent pathway in the regulation of cellular cholesterol efflux. Treatment of normal fibroblasts with 8-bromo-cAMP (8-Br-cAMP) increased significantly apoA-I-mediated cholesterol efflux, with specificity for apoA-I, but not for cyclodextrin. Concomitantly, 8-Br-cAMP increased ABCA1 phosphorylation in a time-dependent manner. Maximum phosphorylation was reached in <10 min, representing a 260% increase compared to basal ABCA1 phosphorylation level. Forskolin, a known cAMP regulator, increased both cellular cholesterol efflux and ABCA1 phosphorylation. In contrast, H-89 PKA inhibitor reduced cellular cholesterol efflux by 70% in a dose-dependent manner and inhibited almost completely ABCA1 phosphorylation. To determine whether naturally occurring mutants of ABCA1 may affect its phosphorylation activity, fibroblasts from subjects with familial HDL deficiency (FHD, heterozygous ABCA1 defect) and Tangier disease (TD, homozygous/compound heterozygous ABCA1 defect) were treated with 8-Br-cAMP or forskolin. Cellular cholesterol efflux and ABCA1 phosphorylation were increased in FHD but not in TD cells. Taken together, these findings provide evidence for a link between the cAMP/PKA-dependent pathway, ABCA1 phosphorylation, and apoA-I mediated cellular cholesterol efflux.

Familial high density lipoprotein (HDL) deficiency (FHD) is a genetic lipoprotein disorder characterized by a severe decrease in the plasma HDL cholesterol (-C) level (less than the fifth percentile). Unlike Tangier disease, FHD is transmitted as an autosomal dominant trait. FHD subjects have none of the clinical manifestations of Tangier disease (lymphoid tissue infiltration with cholesteryl esters and/or neurological manifestations). Plasmas from FHD subjects contain pre-beta-migrating HDLs but are deficient in alpha-migrating HDLs. We hypothesized that a reduced HDL-C level in FHD is due to abnormal transport of cellular cholesterol to the plasma membrane, resulting in reduced cholesterol efflux onto nascent HDL particles, leading to lipid-depleted HDL particles that are rapidly catabolized. Cellular cholesterol metabolism was investigated in skin fibroblasts from FHD and control subjects. HDL3- and apolipoprotein (apo) A-I-mediated cellular cholesterol and phosphatidylcholine efflux was examined by labeling cells with [3H]cholesterol and [3H]choline, respectively, during growth and cholesterol loading during growth arrest. FHD cells displayed an approximately 25% reduction in HDL3-mediated cellular cholesterol efflux and an approximately 50% to 80% reduction in apoA-I-mediated cholesterol and phosphatidylcholine efflux compared with normal cells. Cellular cholesterol ester levels were decreased when cholesterol-labeled cells were incubated with HDL3 in normal cells, but cholesterol ester mobilization was significantly reduced in FHD cells. HDL3 binding to fibroblasts and the possible role of the HDL binding protein/vigilin in FHD were also investigated. No differences were observed in 125I-HDL3 binding to LDL-loaded cells between FHD and control cells. HDL binding protein/vigilin mRNA levels and its protein expression were constitutively expressed in FHD cells and could be modulated ( approximately 2-fold increase) by elevated cellular cholesterol in normal cells. In conclusion, FHD is characterized by reduced HDL3- and apoA-I-mediated cellular cholesterol efflux. It is not associated with abnormal cellular HDL3 binding or a defect in a putative HDL binding protein.

Remnant-like particle (RLP) lipid and apolipoprotein (apo) levels were determined in the plasma of normolipidemic and hyperlipidemic subjects, in order to investigate the relationship between RLP levels and the concentration of other plasma lipoprotein parameters. Plasma RLP fractions were isolated with the use of an immunoaffinity gel (RLP-Cholesterol Jimro II, Japan Immunoresearch Lab.), containing specific anti-apoB-100 and anti-apoA-I antibodies. Four groups of human subjects were selected, who had either matching or significantly different levels of plasma triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C): (1) normolipidemic control (NC) subjects (n = 10), (2) patients with elevated levels of LDL-C (type IIa, LDL-C (mean +/- S.E.), 4.65 +/- 0.09 mmol/l, n = 10), (3) hypertriglyceridemic (HTG) patients with elevated LDL-C (type IIb, TG: 3.86 +/- 0.36; LDL-C: 4.67 +/- 0.21 mmol/l, n = 10), and (4) HTG patients with normal LDL-C (type IV, TG: 3.71 +/- 0.39 mmol/l, n = 10). NC subjects (RLP-C: 0.22 +/- 0.01; RLP-TG: 0.24 +/- 0.03 mmol/l) had RLP apoB, apoC-III and apoE levels of 3.2 +/- 0.3, 1.8 +/- 0.3, and 1.4 +/- 0.1 mg/dl, representing 3.2 +/- 0.4, 14.5 +/- 1.4 and 32.1 +/- 2.1% of total plasma levels, respectively. RLP lipid and apolipoprotein concentrations were significantly higher in HTG groups (type IIb and IV) compared to NTG groups (NC and type IIa) (e.g. RLP-C: 0.50 +/- 0.07 and 0.58 +/- 0.11 vs. 0.22 +/- 0.01 and 0.21 +/- 0.01 mmol/l, respectively (P < 0.01); RLP apoB: 8.4 +/- 1.6 and 8.2 +/- 0.9 vs. 3.2 +/- 0.3 and 3.4 +/- 0.2 mg/dl, respectively (P < 0.01)). No significant difference in RLP levels was observed between groups having different LDL levels, and thus no correlation existed between RLP-C and LDL-C levels (r = 0.24, n.s.). RLP-C and RLP apoB levels were, however, correlated with VLDL-C and VLDL apoB (r = 0.86, P < 0.001 and r = 0.70, P < 0.001, respectively). These results demonstrate that elevated levels of both RLP lipids and apolipoproteins are characteristic of patients with increased levels of plasma triglyceride, and not patients with increased levels of LDL.