Cholesteryl ester-loaded macrophages (foam cells), a prominent and early feature of atherosclerotic lesions, form by the interaction of atherogenic lipoproteins with macrophages and the stimulation of intracellular cholesterol esterification by acyl-CoA:cholesterol acyl transferase (ACAT). Two atherogenic lipoprotein pathways which stimulate macrophage ACAT and induce foam cells are Beta-VLDL, internalized via the LDL receptor, and modified forms of LDL (e.g. acetyl-LDL and oxidized LDL), internalized via the scavenger receptor. The long-term objective of this proposal is to explore the cellular and molecular mechanisms responsible for ACAT stimulation and foam cell formation by these two atherogenic pathways. In particular, the Beta-VLDL pathway is characterized by endocytic targeting to a distinct set of widely- distributed vesicles that may be related to its relative potency as an ACAT stimulator.
Specific Aim #1 of the proposal will use a combination of fluorescence microscopy and biochemical techniques to explore several key properties of the Beta-VLDL-containing vesicles, including intravesicular Ph, presence of the LDL receptor, evidence of intravesicular particle disruption, cellular fate of the vesicles, and the presence of relevant enzymatic activities (especially cholesteryl ester hydrolase) and protein markers. The results of these studies will elucidate when and how endocytosed Beta-VLDL lipid (especially Beta- VLDL-cholesteryl ester) and protein are degraded and thus will be important for understanding how the special Beta-VLDL endocytic pathway is related to ACAT stimulation and other aspects of Beta-VLDL metabolism by macrophages.
Specific Aim #2 will focus on the properties of Beta- VLDL particles that lead to their targeting to the peripheral vesicles and their potency as ACAT stimulators. In particular, the role of multi- valent apoprotein E on the surface of large Beta-VLDL particles in endocytic targeting and ACAT stimulation will be addressed. The approach will be to determine the effect of apo E depletion, repletion, and modification of Beta-VLDL particles and lipid emulsions on patterns of endocytosis and ACAT stimulation. Lastly, the proposal will also focus on the other major pathway of foam cell formation--the scavenger receptor. Preliminary work has disclosed that a factor likely to be present in atherosclerotic lesions, transforming growth factor Beta1 (TGF-Beta1), inhibits the induction of scavenger receptors as THP-1 human monocytes differentiate into macrophages.
In Specific Aim #3, the mechanisms (e.g. transcriptional vs. post-transcriptional), extent (relationship to other means of scavenger receptor regulation), and possible physiological consequences (e.g. applicability to an in vivo model of foam cell formation) of this effect will be explored. In summary, we will explore several specific hypotheses and findings regarding two major pathways of macrophage ACAT stimulation and foam cell formation. Knowledge gained from these studies will provide important information regarding macrophage intracellular lipoprotein metabolism in particular and atherogenesis in general.
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