Transcytosis of Lipoprotein Lipase across Cultured Endothelial Cells Requires Both Heparan Sulfate Proteoglycans and the Very Low Density Lipoprotein Receptor

Abstract

Lipoprotein lipase (LPL)1 is a 120-kDa dimeric protein that associates with the luminal surface of endothelial cells in multiple organs but especially in cardiac and skeletal muscle and in adipose tissue (1). This enzyme hydrolyzes the triglyceride in circulating lipoproteins such as chylomicrons and VLDL and produces free fatty acids that are used for metabolic energy or for fat storage. Endothelial cells do not synthesize LPL; rather myocytes and adipocytes produce it. Thus, it is a protein that requires transcytosis across the endothelial cell barrier, in this case from the interstitial fluid to the luminal side of the cells.There are several possible ways that LPL could cross the endothelial barrier. Nonspecific transport of molecules across endothelial monolayers occurs either via paracellular routes between the cells or via vesicular transit through cells (2). Alternatively, a specific transcytosis pathway could exist which requires LPL to associate with a cell surface receptor and then transports LPL through the cells. This process would be analogous to that which transfers IgA across epithelial cells (3). The first step in a specific LPL transcytosis pathway would involve LPL interaction with the basolateral side of endothelial cells. LPL binds to a number of cell surface molecules including heparan sulfate proteoglycans (HSPGs) and members of the LDL receptor family (4). In bovine endothelial cells the most highly expressed of these receptors is the VLDL receptor (VLDLr) (5). A previous study suggested that HSPGs are required for LPL transcytosis (6). It is, however, unclear whether HSPGs are sufficient for transport or whether HSPGs must operate in concert with receptors. The binding of LPL to several members of the LDL receptor family leads to uptake and degradation of LPL by cells. There are no data on whether these receptors participate in transendothelial movement of LPL or other ligands.In this report, we present data showing that LPL transcytosis across endothelial monolayers requires both HSPGs and the VLDLr. LPL transcytosis was diminished by removal of HSPGs and inhibition of receptors by RAP, a 39-kDa protein that was copurified with the LDL receptor-related protein (LRP) (7). This protein binds to members of the LDL receptor family and inhibits ligand binding and uptake by those receptors (8, 9). Furthermore, antibodies against the VLDLr blocked LPL translocation and increased expression of this receptor-increased transcytosis. Thus, LPL requires both HSPGs and receptors for translocation across endothelial cells. EXPERIMENTAL PROCEDURESPurification and Radioiodination of LPL-LPL was purified from unpasteurized bovine milk according to the method of Socorro et al. (10)

The Very Low-density Lipoprotein (VLDL) Receptor: Characterization and Functions as a Peripheral Lipoprotein Receptor

“…VLDL receptor knockout mice were always segregated with the retinal angiogenesis and subretinal neovascularization (53). On the other hand, it seems that endothelial cells are important sites for VLDL receptor because the movement of active LPL across endothelial cells involves both heparan sulfate proteoglycan and VLDL receptor, and VLDL receptor knockout mice showed lower plasma LPL activity, though the exact mechanism is under investigation (37,54).…”

The VLDL receptor plays a major role in chylomicron metabolism by enhancing LPL-mediated triglyceride hydrolysis

“…Based on these studies, it has initially been suggested that the VLDLr could play a role in the peripheral binding and subsequent internalization of TG-rich lipoproteins (4,5). More recently, Obunike et al (7) showed in vitro that the VLDLr is involved in the transcytosis of active LPL across endothelial cells. Therefore, it is now believed that the VLDLr may possibly function mainly by facilitating the binding of TG-rich lipoproteins in the capillary bed in concert with LPL, leading to the subsequent delivery of TG-derived FFAs to the underlying tissues active in FFA metabolism (5,8).…”

Effects of Lipoprotein Lipase on Uptake and Transcytosis of Low Density Lipoprotein (LDL) and LDL-associated α-Tocopherol in a Porcine in Vitro Blood-Brain Barrier Model

“…LPL is synthesized in most extrahepatic tissues where it is transported to the endothelium (1, 2), a process dependent on heparan sulfate proteoglycans (HSPG) and the very low density lipoprotein (VLDL) receptor (3). There, LPL hydrolyzes triglycerides (TGs) in chylomicrons and VLDL, thereby generating free fatty acids that enter either storage or oxidative pathways.…”