The risk of coronary heart disease is associated with elevated plasma cholesterol and low density lipoproteins (LDL), and likely with genetic polymorphism in the structure of apolipoproteins. We have developed a strain of pigs which have hypercholesterolemia, elevated LDL and accelerated atherosclerosis, associated with three lipoprotein mutations, Lpb5, Lprl and Lpul. A long range objective is to elucidate the role of these mutations in the development of atherosclerosis and hypercholesterolemia and determine how changes in apolipoproteins lead to coronary heart disease. This may provide insights into aspects of human coronary heart disease. Investigations of this proposal will focus on 1) the question whether the Lpr1 mutant alone accompanies an increase in the level of cholesterol, LDL and /or Lpr lipoproteins and 2) further characterization of Lpr-lipoproteins an its two epitope-specific proteins, Lprl and Lpr2, determined by two allelic genes, Lpr1 and Lpr2. More specifically aims will be directed at: 1) Propagation of pigs carrying the Lpr1 allele to preserve this mutant and obtain experimental animals. 2) Reproduction of eight apolipoprotein- epitope specific antibodies. 3) Determining: a) distribution presence in complexes with apo-B, and density range of plasma Lpr in Lpr1/1, Lpr1/2 and Lpr2/2 pigs; b) levels of cholesterol, LDL and Lpr in plasma of Lpr1/1, Lpb2/2, Lpu2/2 vs Lpr2/2, Lpb2/2, Lpu2/2 genotypes; c) affinity of Lprl and Lpr2 to lipids; d) the number of Lpr-protein subunits (kD 23) per Lpr molecule in d greater than 1.21 g/ml and MW plus/minus 190,000; e) amino acid analysis and protein sequence of Lprl and Lpr2; f) immunological relationship and homology between pig apo-R and human apolipoproteins. Specific matings will be used to propagate the Lpr1 gene and to obtain pigs of proposed genotypes for all experimental purposes. Pigs of specific genotype will be injected with purified apolipoprotein-epitope antigens to obtain polyclonal alloimmune sera. Preparative ultracentrifugation, delipidation, gel affinity and gel chromatography, immunochemical and immunodiffusion techniques, native, SDS and 2D PAGE, will be employed to separate, purify, quantitate and identify Lpr lipoproteins and its protein components, Lprl and Lpr2. Affinity of Lpr for lipids will be determined by intravenous fat emulsion - Liposyn. Amino acid analysis and sequencing of proteins and peptides will be performed by HPLC. Immunological relationship of Lpr with human apolipoproteins will be determined using anti- human apolipoprotein antibodies.
