Phospholipid transfer protein: Studies in rabbits and humans

High HDL cholesterol protects from atherosclerosis. A phospholipid exchange protein, PLTP, affects the antiatherogenic function of HDL at several levels. Because the rabbit, like humans, is very susceptible to atherosclerosis, understanding the regulation of PLTP in rabbits is crucial. Elevated cholesterol and triglycerides in plasma (f.i occurring in obesity and diabetes) are an important cardiovascular risk factor. On the other hand, high-density lipoproteins (HDL) are called the "good cholesterol", because they afford protection from atherosclerosis. This project investigates a particular plasma protein, which, by transferring phospholipids between lipoproteins (and cells) is involved in the rearrangement of the surface layer of lipoproteins. This phospholipid transfer protein (PLTP) affords the proper disposal of surface remnants, generated during intra-vascular lipolysis of triglyceride-rich lipoproteins, thereby contributing to the formation of HDL particles. Both intravascular actions of PLTP are considered anti-atherosclerotic. On the other hand, PLTP action in liver cells stimulates the secretion of athero-genic lipoproteins, a property which promotes atherosclerosis. Before PLTP can be manipulated to prevent athero-sclerosis in humans, we have to obtain a much more detailed understanding of its tissue-specific functions and its regulation (f.i. in diabetes). Much of our current data are derived from studies of mice, which may be problematic, as mice are resistant to atherosclerosis. Thus, we have chosen to study the rabbit, which is very prone to develop atherosclerosis when fed very little amounts of cholesterol . To analyze the regulation of PLTP f.i. in diabetes tissue samples need to be obtained, which obviously cannot be done in humans. We want to answer the following questions: 1) Do entero-cytes and pancreatic cells secrete PLTP? Which cells in human brain secrete PLTP? 2) How does diabetes modify PLTP expression ? 3) Which effect will PLTP have on the lipoprotein profile in the rabbit? 4) Can the production of PLTP protect macrophages from the cytotoxicity of hyper-triglyceridemic plasma? 5) Is PLTP in plasma increased in coronary heart disease? The answer to these questions will contribute to a better understanding of the role of PLTP in atherosclerosis and have important consequences for the controversial development of PLTP inhibitors.

Our work indicates that PLTP and scavenger receptor class B, type I (SR-BI), in part by modulating HDL-function, are promising targets in antiatherosclerotic drug development. High density lipoproteins (HDL) comprise a bewildering array of protein-lipid complexes, whose function in atherogenesis, oxidation and inflammation is determined by structural variation in both lipid and protein components. We studied several factors interacting in both humans and animals to influence HDL structure and - function. This includes the handling of intravascular triglyceride (TG) metabolism, the action of endothelial lipases, the processes involved in cellular efflux of cholesterol, the lipid exchanges brought about by plasma lipid transfer proteins (such as PLTP) and, finally, the cellular uptake of HDL particles (f.i. by SR-BI) or their components. Our project has yielded 3 major important new insights in this area: First, we fed a fat-rich meal to humans and stimulated their intravascular TG-metabolism by injecting heparin, an activator of intravascular lipolysis. In contrast to fasting samples, we for the first time observed that postprandial plasma samples induced programmed cell death (apoptosis) in a number of cell lines chosen to represent components of the normal vessel wall (f.i. venous and arterial endothelial cells and macrophages) and that PLTP substantially augments this effect. To demonstrate this, we generated macrophages genetically engineered to overexpress PLTP, one of our proteins of interest, and found that apoptosis was much more pronounced. As a plausible mechanism underlying this observation, we observed that, in accordance with its primary function, PLTP increases the flux of phospholipids transported with TG-rich lipoproteins into cells. Second, we showed that low circulating activity of plasma PLTP in humans is indicative of peripheral atherosclerosis, whereas PLTP mass is not a predictor. This suggests both atheroprotective functions of PLTP and a disturbed distribution between high-activity and low-activity forms of PLTP in human atherosclerosis. Third, aspirin is used widely in clinical practice to treat atherosclerosis due to its platelet-inhibitory and anti-inflammatory properties. In our cell culture experiments, we show that low concentrations of aspirin increase the protein concentration and function of scavenger receptor class B, type I (SR- BI), an atheroprotective surface protein in human macrophages. We further demonstrate that this also applies to SR-BI levels in murine macrophages fed low-dose aspirin and in human carotid endarterectomy samples. Thus, posttranscriptional upregulation of SR-BI function causing stimulation of cholesterol efflux from macrophages identifies a novel atheroprotective property of this important drug.