During the second cycle of this Multiple Principal Investigator R01 grant, we investigated lipoprotein lipase (LPL) and GPIHBP1 (the endothelial cell protein that shuttles LPL to the capillary lumen) and the roles of both proteins in the lipolytic processing of triglyceride-rich lipoproteins (TRLs). We were productive, publishing >20 manuscripts, all in top-tier journals. Of note, we discovered a new human disease—chylomicronemia from GPIHBP1 autoantibodies. We defined the pathophysiology of that disease and with our collaborators showed that the disease can be treated quite effectively with rituximab, resulting in disappearance of autoantibodies and normalization of plasma triglyceride levels. We also explored basic physiologic mechanisms for intravascular lipolysis. We showed that LPL is active as a monomer, dispelling decades-old dogma that LPL is a homodimer. We showed that GPIHBP1 preserves the structure and activity of LPL's hydrolase domain. We solved the atomic structure of the LPL–GPIHBP1 complex, which provided insights into how GPIHBP1 stabilizes LPL and how specific LPL and GPIHBP1 mutations cause human disease. For the third grant cycle, we have two specific aims. The first is to test the hypothesis that GPIHBP1's acidic domain is crucial for the movement of LPL–GPIHBP1 complexes from the abluminal to the luminal plasma membrane (PM) of capillary endothelial cells. We recently created gene-edited mice expressing a mutant GPIHBP1 lacking the acidic domain. GPIHBP1's LU domain was unaltered; hence, the mutant GPIHBP1 was able to bind LPL. Unexpectedly, the absence of the acidic domain impeded trafficking of the GPIHBP1–LPL complex from the abluminal PM of endothelial cells to the capillary lumen. We suspect that the movement of LPL–GPIHBP1 complexes away from the abluminal plasma membrane was impeded by persistent electrostatic interactions between the GPIHBP1-bound LPL and adjacent heparan sulfate proteoglycans (HSPGs). Using a combination of surface plasmon resonance studies and in vivo GPIHBP1 transport studies, we will test the hypothesis that a crucial function of GPIHBP1's acidic domain is to disrupt interactions between GPIHBP1-bound LPL and nearby HSPGs along the abluminal plasma membrane, thereby freeing LPL to move across endothelial cells to the capillary lumen. Our second specific aim is to explore a recent discovery, using advanced microscopy, that some of the LPL that is transported into capillaries by GPIHBP1 subsequently detaches from GPIHBP1 and enters the HSPG-rich glycocalyx. Taking advantage of super-resolution confocal microscopy, electron microscopy, and NanoSIMS imaging, we will characterize GPIHBP1-bound and glycocalyx-bound pools of LPL along capillary endothelial cells. We will also examine the relevance of the glycocalyx-bound LPL to TRL margination along capillaries and TRL processing. Finally, we will develop the tools required to test the relevance of glycocalyx LPL to human disease. We are ideally positioned, with all of the reagents, experimental approaches, and expert collaborators, to address our specific aims. We anticipate that our studies will transform textbook models of intravascular triglyceride metabolism.e metabolism.