|
Atherosclerosis/Molecular and Cell Biology of Plasma Apolipoproteins
Professor, Pharmacological Sciences
Ph.D., University of Illinois
Postdoctoral, University of California, San Francisco
Lab Page
Selected Publications
Receptor-mediated trafficking of cholesterol between lipoproteins and cells is a fundamental biological process at both the organismal and cellular levels. At the organismal level, these processes determine plasma cholesterol levels and are major factors in the development of atherosclerotic cardiovascular disease. High levels of low density lipoprotein (LDL, the bad cholesterol) promote atherosclerosis while high levels of high density lipoprotein (HDL, the good cholesterol) protect against atherosclerosis. At the cellular level, receptor-mediated lipoprotein trafficking of HDL and LDL provides cholesterol for membrane biogenesis during growth and cell renewal, for maintenance of membrane fluidity and function, and for the synthesis of steroid hormones and bile acids in endocrine tissues and the liver, respectively.
We study the cell and molecular biology of cell surface receptors and plasma apolipoproteins (apo) that are involved in cholesterol transport and atherosclerosis. Our studies employ a variety of approaches including transgenic and gene knockout animals, cell culture models, immunocytochemistry at the light and electron microscopic levels, and site-directed mutagenesis. The laboratory is currently focused on the following areas. Additional details can be found in recent laboratory publications and research images.
HDL is a major lipoprotein involved in the delivery of plasma cholesterol to the liver and the trafficking of cholesterol between HDL and many peripheral cells. HDL is the major cholesterol carrier that mediates the movement of cholesterol from peripheral cells, including the vascular wall, back to the liver for metabolism or elimination. This process is termed “reverse cholesterol transport” and is one of the reasons that HDL protects against the formation of atherosclerotic lesions in coronary and other arteries. HDL particles are heterogeneous in size and composition but average about 10 nm in diameter. Like other lipoproteins, HDL is composed of a surface monolayer of phospholipid and free cholesterol (FC) surrounding a core of neutral lipid, primarily cholesteryl ester (CE). Apolipoproteins are composed of amphipathic α helical repeats units that interact with the HDL surface lipids and with the aqueous phase. The major protein of HDL, apolipoprotein (apo) A-I is one focus of the laboratory. ApoA-I plays a structural role in the HDL particle, acts as a cofactor for plasma enzymes that metabolize HDL lipids, and is a ligand for scavenger receptor class B, type I, (SR-BI), which is the major cell surface receptor for HDL.
Our studies on apoA-I are focused on the role this protein plays in the cellular uptake of HDL CE via the selective uptake pathway. HDL participates in this unique selective lipid delivery system in which HDL CE and FC are transferred to the cell membrane without the uptake and degradation of the entire HDL particle. Our studies with apoA-I knockout mice showed that apoA-I is required for cellular CE accumulation and is the major HDL ligand recognized by the selective uptake receptor, SR-BI. We are studying how apoA-I interacts with SR-BI using chemical and photo cross-linking methods, mutagenesis of receptor and ligand, and protein mass spectrometry. We are now using non-specific cross-linkers as well as site-specific cross-linkers in apoA-I to define the residues in SR-BI that contact apoA-I during binding and lipid transfer.
SR-BI is the major receptor for plasma HDL in the liver and steroidogenic cells. SR-BI determines the plasma level of HDL and is protective against the development of atherosclerosis in mouse models. HDL particles bind to SR-BI via apoA-I leading to the transfer of HDL core lipids, primarily CE, to the cell membrane. We are studying the mechanism of this transfer process as well as how SR-BI stimulates the bi-directional movement of FC between cells and HDL. By accelerating the transfer of HDL lipids, SR-BI provides a conduit for the rapid mass movement of CE and FC between cells and HDL.
SR-BI is clustered on the cell surface on microvillar extensions. We are currently using light and electron microscopic methods to study this membrane domain and how it relates to the changes in plasma membrane cholesterol distribution that occur in SR-BI expressing cells. The phospholipids in the membrane domains containing SR-BI likely play an important role in cholesterol movement into and out of the plasma membrane. Tandem mass spectrometry is being used to determine the phospholipids in membranes of SR-BI expressing cells and to test whether SR-BI is associated with membrane domains of unique lipid composition.
The other research topic in the laboratory is apoE, a plasma apolipoprotein that is found on atherogenic lipoproteins and is the major ligand for the removal of these particles from plasma by the LDL receptor and other members of the LDL receptor family. Atherogenic lipoproteins are derived from the lipolytic metabolism of very low density lipoprotein (VLDL) and chylomicrons to produce CE-rich remnant particles and LDL. If these remnants are not efficiently removed by the liver and accumulate to high levels in the blood, they initiate atherosclerotic lesion formation. ApoE protects against this by promoting remnant uptake by the liver. In the absence of apoE in humans or in apoE knockout mice, atherosclerosis develops prematurely.
In addition to this role of apoE in the plasma, apoE is unusual in being expressed in many tissues and is involved in processes other than systemic lipoprotein metabolism. ApoE is expressed at high levels in steroidogenic cells, kidney, and brain, and at lower levels in many other cells. Our studies on apoE are focused on the question of why apoE is expressed in many tissues, including steroidogenic cells. Our previous work suggests that localized apoE expression modulates signal transduction pathways and cellular cholesterol metabolism. We are currently studying cultured cells in which apoE expression is inducible to examine effects on HDL and LDL interaction with cellular receptors. Recent results identified an apoE-dependent LDL selective uptake pathway in adrenal cells that involves the LRP/alpha2-macroglobulin receptor and cell surface chondroitin sulfate proteoglycans. We are also using apoE knockout mice to test the role of this protein in steroidogenic cells.
We created transgenic mouse models in which apoE expression occurs only in steroid producing cells of the adrenal gland. This model is being used to evaluate the role of apoE in adrenal cholesterol metabolism and adrenal signaling pathways.
When mice expressing apoE in the adrenal gland were crossed onto the apoE knockout background, only very low levels of apoE were detected in the plasma, as would be expected. Surprisingly, the adrenal-expressing apoE mice on the apoE knockout background are protected against atherosclerosis by these very low levels of plasma apoE that are too low to correct the hypercholesterolemia in the apoE knockout mouse. Even though these mice have high levels of atherogenic remnant particles in the blood, atherosclerosis is suppressed. These data lead to the hypothesis that apoE has actions to prevent atherosclerosis in addition to its activity to serve as a ligand for remnant particle removal by the LDL receptor. Our hypothesis is that apoE acts directly on the vascular wall to suppress early steps in atherosclerotic lesion development. Current studies are using gene profiling and immunocytochemistry to test the role of apoE in suppressing monocyte recruitment and other pathways that are involved in the early steps of lesion formation. These studies have the promise of identifying key steps in atherosclerotic lesion formation as potential therapeutic targets.
Pharmacology Home Page
Graduate Program in Molecular and Cellular Pharmacology Home Page
