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.

No abstract

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 polyethylenimine/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. P<0.001).Moreover, when secreted apoE3 was injected intraperitoneally into apoE -/-mice, apoE3was detected in plasma and the hyperlipidaemia improved. Similarly, when alginate polyethylenimine/polybrene capsules were implanted into the peritoneum of apoE -/-mice, apoE3was secreted into plasma and at 7 days total cholesterol was reduced, whilst atheroprotective highdensity lipoprotein (HDL) increased. In a second study, apoE was detectable in plasma of 5 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.3