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

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

Human Apolipoprotein E Expression from Mouse Skeletal Muscle by Electrotransfer of Nonviral DNA (Plasmid) and Pseudotyped Recombinant Adeno-Associated Virus (AAV2/7)

“…In contrast, plasma from wild-type C57BL/6 mice had high levels of HDL and a relatively low proportion of clearly separated VLDL and LDL. Densitometry of the lipoprotein profiles did not reveal any difference between day 0 and day 7 samples, either in absolute amounts or in the HDL-to-total lipoprotein ratio, which can serve as a sensitive indicator of effective apoE gene transfer (Harris et al, 2002a;Tagalakis et al, 2005).…”

“…One explanation is that secreted apoE3 is promptly sequestered by the excess of remnant lipoproteins in the plasma of apoE Ϫ/Ϫ mice and, unlike binding-defective apoE2, is rapidly cleared by the liver via interaction with the LDL receptor or LDL receptorrelated protein (LRP) (Mahley et al, 1999;Mahley and Rall, 2000;Kypreos and Zannis, 2006). Such rapid removal has been reported after injection of apoE3 protein (Wu et al, 1998;Tagalakis et al, 2005). Note too that mouse apoB-containing lipoproteins (VLDL, IDL, and LDL) are almost entirely dependent on apoE for clearance; unlike their human counterparts, they contain a high proportion of apoB48, a truncated structural protein that lacks the receptor-binding domain present in fully functional apoB100 (Greeve et al, 1993).…”

Targeted In Situ Gene Correction of DysfunctionalAPOEAlleles to Produce Atheroprotective Plasma ApoE3 Protein

“…Recombinant ApoE-expressing endothelial cells embedded in Matrigel were injected intradermally into ApoE −/− mice which, 3 months later, had 50% less plasma cholesterol and reduced atherosclerotic plaque [68]. Similarly, implantation of alginate-encapsulated engineered cells into the peritoneum of ApoE −/− mice secreted sufficient ApoE to lower plasma cholesterol and increase atheroprotective HDL [69]. Transgenic mice overexpressing ApoE provide additional evidence for ApoE atheroprotection, as these animals are protected from diet-induced or diabetic hyperlipidemia [70, 71].…”

Controlled Generation of Microspheres Incorporating Extracellular Matrix Fibrils for Three‐Dimensional Cell Culture

“…Use of gravity alone to generate droplets results in large diameter microspheres after crosslinking. [8] Many manifestations have been utilised to allow smaller diameter microspheres to be produced, for example; coaxial air flow, [9] vibrating jet break-up, [10] and the rotating jet break-up and microfluidic methods. [11] …”