Project Grant F32DK145123

MECHANISMS OF HDL-MEDIATED LPS NEUTRALIZATION AND TRANSPORT IN THE PORTAL VEIN - PROJECT SUMMARY/ABSTRACT APOLIPOPROTEIN A1 (APOA1) IS THE CORE PROTEIN CONSTITUENT OF HIGH DENSITY LIPOPROTEIN (HDL). PREVIOUS WORK FROM OUR LAB HAS SHOWN THAT ENTERIC HDL PLAYS A CRITICAL ROLE IN THE NEUTRALIZATION OF LIPOPOLYSACCHARIDE (LPS) THAT LEAKS FROM THE GUT. LPS RAPIDLY BINDS CIRCULATING HDL AND IS NEUTRALIZED, PREVENTING CHRONIC ACTIVATION OF TLR4 WHICH LEADS TO INFLAMMATION AND LIVER FIBROSIS. HOWEVER, THE MECHANISM BY WHICH HDL NEUTRALIZES LPS IS NOT COMPLETELY UNDERSTOOD. THE LIVER DERIVED LPS BINDING PROTEIN (LBP) IS KNOWN TO ASSOCIATE WITH HDL AND PLAY A CRITICAL ROLE IN THE NEUTRALIZATION OF HDL BOUND LPS. TO ADDRESS THE MECHANISM BY WHICH LBP BOUND HDL FROM THE INTESTINE NEUTRALIZES LPS, I WILL DETERMINE THE PROTEIN CONSTITUENTS OF THE LPS NEUTRALIZING COMPLEX AND MEASURE THE CAPACITY OF DIFFERENT RECONSTITUTED LIPOPROTEIN COMPLEXES TO NEUTRALIZE LPS. TO INVESTIGATE THE MECHANISM OF HOW HDL NEUTRALIZES LPS, I WILL USE PROTEOMICS AND LIPIDOMICS TO CHARACTERIZE LBP+ HDL PARTICLES. I WILL ALSO USE A NOVEL FLOW SORTING METHOD TO DETECT PROTEINS ON INTACT HDL COMPLEXES DERIVED FROM PLASMA SOURCES. USING RECOMBINANT PROTEIN AND LIPIDS, I WILL RECONSTITUTE THIS COMPLEX AND MEASURE THE NEUTRALIZATION OF LPS USING HEK-BLUETM-4 CELL ASSAY. ANOTHER OUTSTANDING QUESTION IS HOW HDL IS TRAFFICKED FROM THE SMALL INTESTINE INTO THE PORTAL VENOUS CIRCULATION PRIOR TO REACHING THE LIVER. OUR PREVIOUS RESEARCH SUGGESTS THAT LIPIDATION OF APOA1 BY INTESTINAL ABCA1 IS NECESSARY FOR EFFICIENT TRANSPORT OF HDL TO THE PORTAL VENOUS CIRCULATION. KNOCKOUT OF ABCA1 IN THE INTESTINE LED TO A DRAMATIC REDUCTION IN THE CIRCULATING LEVELS OF HDL IN THE PORTAL VEIN AND LED TO GREATER LIVER INFLAMMATION AND FIBROSIS. ONE POSSIBILITY TO EXPLAIN THESE RESULTS IS THAT LIPIDATED HDL BINDS TO SCAVENGER RECEPTOR CLASS B TYPE 1 (SRB1) WHICH IS KNOWN TO PLAY A ROLE IN THE TRANSCYTOSIS OF HDL ACROSS THE ENDOTHELIAL MEMBRANE. AN ALTERNATIVE HYPOTHESIS IS THAT THE FENESTRATION OF THE MICROVASCULATURE IN THE VILLI OF THE SMALL INTESTINE ALLOWS FOR THE PASSIVE FILTRATION OF HDL INTO THE VENOUS CIRCULATION. TO DISTINGUISH BETWEEN THESE TWO HYPOTHESES, I WILL USE A PHOTOCONVERTIBLE FLUORESCENT PROTEIN, KIKUMEGR, TAGGED TO APOA1 TO MEASURE TRAFFICKING OF HDL FROM THE SMALL INTESTINE TO THE PORTAL VEIN. TO INVESTIGATE THE ROLE OF SRB1 ON TRAFFICKING OF ENTERIC HDL TO THE PORTAL VEIN, I WILL CROSS SRB1FL/FL MICE TO CDH5A-CREERT2 MICE, WHICH WILL REMOVE SRB1 FROM VASCULAR ENDOTHELIAL CELLS. TO TEST WHETHER HDL FROM THE SMALL INTESTINE EGRESSES THROUGH FENESTRAE IN THE MICROVASCULATURE, I WILL USE PE-CONJUGATED ANTI-PLVAP ANTIBODY (PLVAP FORMS A DIAPHRAGM REGULATING SOLUTE PASSAGE THROUGH FENESTRAE) WHICH WILL BLOCK PASSAGE OF SOLUTES THROUGH FENESTRAE. TO MEASURE HDL TRAFFICKING FROM THE SMALL INTESTINE TO THE PORTAL VEIN, I WILL TRANSDUCE MICE USING AAV EXPRESSING KIKGR-APOA1, WHICH CAN BE PHOTOCONVERTED FROM GREEN TO RED FLUORESCENCE IN THE SMALL INTESTINE USING 405NM LIGHT, WHICH CAN THEN BE DETECTED IN THE PORTAL VEIN. TOGETHER THESE EXPERIMENTS WILL HELP TO MECHANISTICALLY DEFINE THE TRAFFICKING OF HDL TO THE PORTAL VEIN AND THE NEUTRALIZATION OF LPS TO RESTRAIN LIVER INFLAMMATION AND INJURY.