Coronary heart disease (CHD) is responsible for almost half of all deaths in western countries. A number of environmental factors have been identified as increasing the risk of this disease, i.e., smoking, high fat diet, and lack of exercise. However, a strong genetic influence exists as well. The plasma levels of lipoproteins(s), Lp(a), are highly heritable, and increased Lp(a) levels are a strong, independent risk factor atherosclerosis, the major cause of CHD. Lp(a) consist of low density lipoprotein (LDL) in which apoB is disulfide-linked to an additional high molecular weight glycoprotein, apo(a). Apo(a) is synthesized by the liver and exists as a number of genetically determined isoforms that vary in size from 400 to greater than 800 kDa. The plasma levels of Lp(a) vary from less than 1 to greater than 100 mg/dl, and there is a tendency for an inverse correlation between isoform size and plasma concentration. Despite its clinical significance, virtually nothing is known about factors which regulate rates of Lp(a) production and removal from the circulation. A great need therefore exists for an in vitro system to analyze the factors regulating the synthesis and secretion of this highly atherogenic lipoprotein. Baboons show similar characteristics to humans in terms of plasma levels of Lp(a) and apo(a) isoform sizes. We have developed a serum-free medium that maintains highly differentiated baboon hepatocytes in culture for extended times, and, recently, we have demonstrated the utility of this system for analyzing the morphogenesis of Lp(a). The first specific aim of this proposal is to examine the synthesis of apo(a) in hepatocytes obtained from genetically selected baboons expressing high and low levels of plasma Lp(a). The influence of allelic variation will be examined for the level of apo(a) mRNA transcription, the rate of apo(a) polypeptide synthesis, the kinetics of intracellular maturation of apo(a), and the level of apo(a) intracellular degradation. The second specific aim is to conduct an in depth analysis of the intracellular maturation of apo(a) including protein folding, association with chaperones in the endoplasmic reticulum, and the association of apo(a) with proteins in the trans- Golgi. The third specific aim is to examine the nature of and requirements for the interaction between apo(a) and apoB to form Lp(a). In the fourth specific aim, compounds of potential therapeutic benefit in lowering Lp(a) concentrations will be examined in vitro for their mechanism of action. The final specific aim is to develop immortalized hepatocyte cell lines that are suitable for analysis of apo(a) metabolism.
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