Apolipoprotein E delivery by peritoneal implantation of encapsulated recombinant cells improves the hyperlipidaemic profile in apoE-deficient mice.

Plasma apolipoprotein E (apoE) is a 34-kDa polymorphic protein which has atheroprotective actions by clearing remnant lipoproteins and sequestering excess cellular cholesterol. Low or dysfunctional apoE is a risk factor for hyperlipidaemia and atherosclerosis, and for restenosis after angioplasty. Here, in short-term studies designed to establish proof-of-principle, we investigate whether encapsulated recombinant Chinese hamster ovary (CHO) cells can secrete wild-type apoE3 protein in vitro and then determine whether peritoneal implantation of the microcapsules into apoE-deficient (apoE(-/-)) mice reduces their hypercholesterolaemia. Recombinant CHO-E3 cells were encapsulated into either alginate poly-l-lysine or alginate polyethyleneimine/polybrene microspheres. After verifying stability and apoE3 secretion, the beads were then implanted into the peritoneal cavity of apoE(-/-) mice; levels of plasma apoE3, cholesterol and lipoproteins were monitored for up to 14 days post-implantation. Encapsulated CHO-E3 cells continued to secrete apoE3 protein throughout a 60-day study period in vitro, though levels declined after 14 days. This cell-derived apoE3 was biologically active. When conditioned medium from encapsulated CHO-E3 cells was incubated with cultured cells pre-labelled with [(3)H]-cholesterol, efflux of cholesterol was two to four times greater than with normal medium (at 8 h, for example, 7.4+/-0.3% vs. 2.4+/-0.2% of cellular cholesterol; P<0.001). Moreover, when secreted apoE3 was injected intraperitoneally into apoE(-/-) mice, apoE3 was detected in plasma and the hyperlipidaemia improved. Similarly, when alginate polyethyleneimine/polybrene capsules were implanted into the peritoneum of apoE(-/-) mice, apoE3 was secreted into plasma and at 7 days total cholesterol was reduced, while atheroprotective high-density lipoprotein (HDL) increased. In a second study, apoE was detectable in plasma of five mice treated with alginate poly-l-lysine beads, 4 and 7 days post-implantation, though not at day 14. Furthermore, their hypercholesterolaemia was reduced, while HDL was clearly elevated in all mice at days 4 and 7 (from 18.4+/-6.2% of total lipoproteins to 31.1+/-6.8% at 7 days; P<0.001); however, these had rebounded by day 14, possibly due to the emergence of anti-apoE antibodies. We conclude that microencapsulated apoE-secreting cells have the potential to ameliorate the hyperlipidaemia of apoE deficiency, but that the technology must be improved to become a feasible therapeutic to treat atherosclerosis.

Human scavenger receptor class B type II (SR-BII) and cellular cholesterol efflux.

Although studies in recombinant cells indicate that scavenger receptor class B, type I (SR-BI) can promote cholesterol efflux, investigations in transgenic mice overexpressing or deficient in SR-BI endorse its physiological function as selectively sequestering cholesteryl esters from high-density lipoproteins (HDLs). Less clear is the role of SR-BII, a splice variant of the SR-B gene that differs only in the C-terminal cytoplasmic domain. Here, we identify several putative signalling motifs in the C-terminus of human SR-BII, which are absent from SR-BI, and hypothesize that these motifs interact with signalling molecules to mobilize stored cholesteryl esters and/or promote the efflux of intracellular free cholesterol. 'Pull-down' assays using a panel of tagged SH3 (Src homology 3) domains showed that cytoplasmic SR-BII, but not cytoplasmic SR-BI, bound the SH3 domain of phospholipase C-gamma1; this interaction was not, however, detected under more physiological conditions. Specific anti-peptide antisera identified SR-BII in human monocyte/macrophage THP-1 cells and, in recombinant cells, revealed receptor localization to caveolae, a plasma membrane microdomain that concentrates signal-transducer molecules and acts as a conduit for cholesterol flux between cells and lipoproteins. Consistent with its caveolar localization, expression of human SR-BII in recombinant Chinese hamster ovary cells (CHO-SR-BII) was associated with increased HDL-mediated cholesterol efflux. Nevertheless, when CHO-SR-BII cells were pre-loaded with cholesteryl [(3)H]oleate and incubated with HDL, cholesteryl ester stores were not reduced compared with control cells. We conclude that although human SR-BII is expressed by macrophages, contains cytoplasmic signalling motifs and localizes to caveolae, its ability to stimulate cholesterol efflux does not reflect enhanced hydrolysis of stored cholesteryl esters.

ATP-binding cassette A1 protein and HDL homeostasis.

For three decades, low-density lipoprotein (LDL) dominated research into cholesterol metabolism and atherosclerosis, whereas scant attention was paid to high-density lipoprotein (HDL), an equally important risk factor for cardiovascular disease. This low interest reflected the lack of knowledge about physiological HDL receptors. As a result, our understanding of HDL-cell interactions failed to develop alongside that of LDL, and mechanisms through which atheroprotective HDL promoted clearance of cholesterol from peripheral cells remained poorly-defined. Interest was kindled with the recognition that scavenger receptor class B, type I is the cell-surface protein in hepatocytes and steroidogenic tissues which selectively extracts cholesteryl esters from HDL. Greater impetus still was given by the discovery that mutations in the gene encoding the ATP-binding cassette transporter, class A1 (ABCA1) are the cause of Tangier disease, a rare recessive disorder with near-absent plasma HDL. The ABCA1 transmembrane protein is crucial for efficient efflux of cellular cholesterol and HDL maturation and has emerged as a promising therapeutic target for cardiovascular disease. The hope is that new drugs, regulating ABCA1 activity and HDL homeostasis, will accelerate cholesterol efflux from lipid-laden foam cells and thus promote regression of atherosclerotic lesions.