LIPOPROTEIN(A) SYNTHESIS IN BABOON HEPATOCYTES

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.