Abstract 100: Initial Characterization of Oxidant-Resistant Human ApoAI Transgenic Mice

Abstract

Introduction HDL functions, such as its cholesterol acceptor and anti-inflammatory activities, are impaired by myeloperoxidase (MPO), which selectively targets and oxidizes HDL and apoAI. We previously found that the 4WF apoAI isoform, in which the 4 Trp residues are substituted with Phe, is equally competent as wild type (WT) human apoAI to interact with lipids and accept cholesterol in an ABCA1-dependent manner, but is resistant to MPO mediated loss of function. Hypothesis The oxidant resistant 4WF apoAI isoform will be superior in mediating reverse cholesterol transport than WT human apoAI in vivo under conditions of elevated oxidative stress. Methods and Results We generated transgenic mice expressing the 4WF human apoAI (h-apoAI) isoform. A genomic fragment containing the h-apoAI gene was subjected to site directed mutagenesis to convert the Trp to Phe residues, and microinjected into C57BL/6 fertilized oocytes. We selected the line with the highest h-apoAI expression to compare with existing WT h-apoAI transgenics. We assessed plasma apoAI and HDL-C levels in male and female mice. Male mice have significantly higher h-apoAI levels than females for both isoforms (p<0.05). In general, the WT apoAI transgenics have higher plasma h-apoAI levels than the 4WF transgenics; however there is enough overlap that we were able to select male 4WF and WT h-apoAI transgenic mice having matched plasma h-apoAI levels of ~550 mg/dl, which are described below. HDL-C levels were slightly lower in 4WF vs. WT transgenics (157±4 vs 184±8 mg/dl, respectively, N=5 per group, p=0.03). We performed in vivo reverse cholesterol transport (RCT) assay by s.c. injection of cholesterol labeled and loaded bone-marrow macrophages. RCT to the plasma reflected the HDL-C levels and was lower in the 4WF vs. WT transgenic mice (4.5% vs 6.6%, respectively p=0.007). However, RCT to the liver and feces was equivalent in the 4WF and WT transgenics (NS). This suggests that the flux of HDL-C in the 4WF mice may be higher than in the WT apoAI transgenics, leading to equal RCT to the feces and liver despite lower HDL-C levels. Conclusion 4WF transgenic mice are now breeding with MPO transgenic mice in order to test our hypothesis that this isoform retains more HDL function under oxidative stress conditions.