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Cigarette smoking is not only a direct cause of lung cancer and debilitating emphysema, it is also one of the most potent risk factors in cardiovascular diseases, particularly atherosclerotic coronary heart disease (CAHD), the leading cause of death and disability by heart attack in the United States. More recently, CAHD has become recognized as epidemic in many Eastern European societies dependent upon swine foodstuffs for calories for decades. Accompanied by an increased availability of inexpensive cigarettes, this untoward set of circumstances (a high saturated fat diet and cigarette smoking) contributes to atherosclerotic CAHD and to occlusive cerebral vascular events leading to stroke.
Many researchers agree that a large proportion of smoking’s atherogenic effects result from subtle chemical alterations in the plasma lipoproteins, few of which have been identified. The most thoroughly documented of these biochemical alterations is the oxidation of the lipids in classes of lipoproteins containing a protein called apolipoprotein B (apoB), which transports the “bad” cholesterol in low density lipoproteins (LDL) in blood plasma. The precursor or source of LDL is the very low density lipoprotein (VLDL), produced by hepatocytes, the principal cells of the liver. Recent studies also show that “aged” VLDL, i.e. those that have an excessive residence time circulating in the blood, also become oxidatively changed, and are pro-atherogenic. Thus, oxidized apoB-containing particles promote damage to the arterial wall, causing lipid-laden plaques that constrict the artery diameter reducing blood flow, and often causing clotting or thrombosis, and leading to heart attack or stroke.
Despite considerable research efforts, we understand little about the molecular processes that generate apoB-containing lipoproteins (nascent VLDL particles) within the hepatocytes in the liver. This laboratory has developed a novel hypothesis based on several decades of investigation. We obtained evidence that supported the original idea that a triglyceride-rich particle of the same size as a nascent VLDL was assembled within the secretory compartments of hepatocytes completely independent of apoB. Subsequently, we and others found that small apoB particles containing small amounts of triglycerides and cholesteryl esters were assembled and secreted by hepatocytes, but only under certain experimental circumstances. This seminal observation led us to hypothesize that the assembly of nascent VLDL may require two separate stages of addition of triglycerides to the apoB protein, articulated as the “two-step core lipidation hypothesis” of VLDL assembly. The first step is the addition of a sufficient amount of triglycerides to permit the newly made apoB to dissociate from the secretory membrane, producing a small particle which subsequently fuses with the larger second-step triglyceride-rich particle formed independently in a nearby compartment. Support for this hypothesis was recently demonstrated in genetically engineered mice in which the apoB gene was regulated in in vivo (i.e. in the living animal) settings.
To test this hypothesis directly, we propose to study by electron microscopy and biochemistry, the newly secreted lipoprotein particles in synthetic blood in animal models that express a single isoform of apoB. Human liver expresses only apoB-100, whereas human intestine expresses only apoB-48, but rodent livers express both apoB forms. In spite of more than a decade of research, we don’t know why. We propose to test this directly in genetically engineered animal models expressing only apoB-48 or only apoB-100. We will also study another protein, microsomal transfer protein (MTP), to determine its role in lipoprotein assembly/secretion. Defective MTP causes a rare human disorder in which there are no circulating apoB lipoproteins. We will determine if half levels of expression of each of these proteins is regulatory in apoB lipoprotein assembly/secretion, and determine if apoE and the LDL receptor play a regulatory role in either first- or second-step particle assembly/secretion by measuring the size and number of lipoprotein particles produced by mice lacking these proteins. |
