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A D Tagalakis

Publications and source records attributed to A D Tagalakis.

4 recordsLinked to original sources
PubMed2001

Cell-derived apolipoprotein E (ApoE) particles inhibit vascular cell adhesion molecule-1 (VCAM-1) expression in human endothelial cells.

Sub-endothelial infiltration of monocytes occurs early in atherogenesis and is facilitated by cell adhesion molecules that are up-regulated on activated endothelium. Apolipoprotein E (apoE) helps protect against atherosclerosis, in part, because apoE particles secreted by macrophages have local beneficial effects at lesion sites. Here, we hypothesize that such protection includes anti-inflammatory actions and investigate whether cell-derived apoE can inhibit tumor necrosis factor-alpha-mediated up-regulation of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Two models were used to mimic endothelial exposure to macrophage-derived apoE. In the first, HUVECs were transiently transfected to secrete apoE; VCAM-1 induction inversely correlated with secretion of apoE into the media (r = -0.76, p < 0.001). In the second, incubation of HUVECs with media from recombinant Chinese hamster ovary (CHO) cells expressing apoE (CHO(apoE)) also reduced VCAM-1 in a dose-dependent manner (r = -0.70, p < 0.001). Characterization of CHO(apoE) cell-derived apoE revealed several similarities to apoE particles secreted by human blood monocyte-derived macrophages. The suppression of endothelial activation by apoE most likely occurs via stimulation of endothelial nitric oxide synthase; apoE increased levels of intracellular nitric oxide and its surrogate marker, cyclic guanosine monophosphate, while the nitric oxide synthase inhibitor, ethyl-isothiourea, blocked its effect. We propose that apoE secreted locally at lesion sites by macrophages may be anti-inflammatory by stimulating endothelium to release NO and suppress VCAM-1 expression.

Animals↗
PubMed2001

Gene correction of the apolipoprotein (Apo) E2 phenotype to wild-type ApoE3 by in situ chimeraplasty.

Apolipoprotein (apo) E is a polymorphic plasma protein, synthesized mainly by liver. Here, we evaluate whether synthetic DNA-RNA oligonucleotides (chimeraplasts) can convert a dysfunctional isoform, apoE2 (C --> T, R158C), which causes Type III hyperlipidemia and premature atherosclerosis, into apoE3. First, we treated recombinant Chinese hamster ovary cells stably secreting apoE2 with a 68-mer apoE2 to apoE3 chimeraplast. About one-third of apoE2 was converted to apoE3, and the repair was stable through 12 passages. Subcloning treated cells produced both apoE2 and apoE3 clones. Direct sequencing and reverse transcription polymerase chain reaction confirmed the genotype, whereas phenotypic change was verified by isoelectric focusing and immunoblotting of secreted proteins. Second, we established that the APOE2 gene can be targeted both in vivo, using transgenic mice overexpressing human apoE2, and in chromosomal context, using cultured lymphocytes from a patient homozygous for the epsilon2 allele. We conclude that chimeraplasty has the potential to convert the apoE2 mutation in patients with Type III hyperlipidemia to apoE3.

Amino Acid Substitution↗
PubMed2000

Intramuscular injection of a plasmid vector expressing human apolipoprotein E limits progression of xanthoma and aortic atheroma in apoE-deficient mice.

Apolipoprotein-E (apoE) protects against coronary artery disease via hepatic removal of atherogenic remnant lipoproteins, sequestration of cholesterol from vessel walls and local anti-oxidant, anti-platelet and anti-inflammatory actions. ApoE gene transfer may thus ameliorate a hyperlipidaemic profile and have beneficial effects at lesion sites to prevent or regress atherosclerosis, a concept endorsed by adenoviral-mediated hepatic expression studies. Here, using plasmid vectors expressing allelic human apoE2 or apoE3 isoforms, skeletal muscle was evaluated as an effective secretory platform for apoE gene augmentation. Transfected myoblasts and myotubes were found to efficiently secrete recombinant apoE in vitro as spherical 10-16 nm lipoprotein particles with pre-beta mobility. Intramuscular plasmid injection in apoE(-/-) mice, which develop spontaneous atherosclerotic plaque and xanthoma resulted in expression and secretion of apoE. Human apoE mRNA was detected by RT-PCR in injected muscles and, although concentrations of apoE3, which is rapidly cleared from plasma, were near ELISA detection limits, levels of plasma apoE2 were measurable (17.5 +/- 4.3 ng/ml). To assess whether muscle-based expression of apoE2 could inhibit atherogenesis, long-term follow-up studies were conducted. Although hyperlipidaemia was not reduced in treated animals, end-point pathology showed clear retardation of atherosclerotic and xanthomatous lesions. Up to 9 months following a single apoE2 plasmid administration, atherosclerotic lesion coverage in proximal aorta was significantly reduced by 20-30% (P < 0.01), whereas development of gross dorsal xanthoma (>5 mm diameter) was effectively reduced to zero. We conclude that expression of apoE from ectopic muscle sites has therapeutic potential to limit progression of atherosclerosis.

Animals↗