HIGH-DENSITY LIPOPROTEIN TRANSPORT OF OXYLIPINS REGULATES CELLULAR INFLAMMATORY RESPONSES
Restricted (Penn State Only)
- Author:
- Harsch, Brian
- Graduate Program:
- Integrative and Biomedical Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 06, 2022
- Committee Members:
- Gregory Shearer, Chair & Dissertation Advisor
Connie Rogers, Major Field Member
Kumble Prabhu, Outside Unit & Field Member
David Proctor, Major Field Member
Donna Korzick, Program Head/Chair - Keywords:
- Oxylipins
HDL
Lipoproteins
Inflammation - Abstract:
- Oxylipins are polyunsaturated fatty acid (PUFA) derived molecules which regulate inflammatory signaling. Non-esterified oxylipins are active and potent initiators of pro- and anti-inflammatory signaling pathways and become inactivated through phospholipid membrane sequestration. Oxylipins are effluxed and transported away from sites of inflammation by high-density lipoprotein (HDL), but it remains unclear what the impact is on inflammation. In this dissertation, we investigated the cellular mechanisms of oxylipin trafficking to HDL during inflammation and how the compositional changes of oxylipins with time and treatment mediated inflammatory responses. In Study 1, we cultured murine RAW 264.7 macrophage-like cells and initiated inflammatory signaling using lipopolysaccharide (LPS) to determine how HDL mediated export of oxylipins effects on inflammatory outcomes using apolipoprotein A1 (ApoA-I) and ATP-binding cassette A-1 (ABCA1), both of which are essential for HDL formation. There were four treatment groups: ABCA1 expressing cells exposed to vehicle (Veh/ABCA1) or ApoA-I (ApoA-I/ABCA1) and silenced ABCA1 cells exposed to vehicle (Veh/Sil) or ApoA-I (ApoA-I/Sil). We measured pro-inflammatory arachidonic acid (AA) products 5-HETE, 12-HETE, and 15-HETE and found ABCA1/ApoA-I cells had a 357% [279, 436], 384% [306, 463], and 745% [667, 823] increase in HDL-HETEs (measured as esterified HETE in the media), respectively, compared to Veh/ABCA1 cells. Appearance of monocyte chemoattractant protein-1 (MCP-1), a product of 12-HETE activity through p38 MAPK activation, in the media was significantly greater 127% [65, 190] in Veh/Sil cells compared to ApoA-I/ABCA1 cells. Our results show ABCA1 and ApoA-I are both necessary for reducing cell non-esterified and esterified levels of 5-, 12-, and 15-HETE concentrations. In Study 2, we measured HDL oxylipin composition in mice with systemic deletion of free fatty acid receptor 4 (Ffar4KO) and transverse aortic constriction (TAC) induced cardiac remodeling. In TAC wild-type (WT) mice, we found a 268% [221, 369] greater concentration of cardioprotective 18-HEPE compared to TAC Ffar4KO mice. Transcriptome analysis of cardiomyocytes showed significantly higher inflammation and cell death gene expression in Ffar4KO TAC versus WT TAC mice. Ffar4 activation by TUG-891 in cultured TAC cardiomyocytes showed significantly higher production of 18-HEPE (50% [46,53]) in WT compared to Ffar4KO. Our results show increased 18-HEPE and decreased 12-HETE concentrations measured in HDL attenuates pathological cardiac remodeling and Ffar4 activity significantly impacts synthesis of the inflammatory signaling oxylipins. In Study 3, we tested the effects of prescription omega-3 ethyl esters (P-OM3; 3.4 g/d) on oxylipin composition measured in HDL after LPS was administered to healthy young men (N=17). Subjects were given placebo or P-OM3 in a randomized crossover study for 8-12 weeks followed with an endotoxin challenge with pre-determined time points for sample collection, beginning at 0 hours through 7 days. P-OM3 significantly increased omega-3 oxylipins: EpETEs by 262% [229, 319], HEPEs by 597% [432, 764], and HDoHEs by 354% [306, 433] compared to their concentrations with placebo. P-OM3 significantly reduced omega-6 HETEs by 50% [46, 53] compared to their concentrations with placebo, but no changes in EpETrEs were found. Importantly, we found an inverse relationship with 18-HEPE and 12-HETE concentrations in HDL across all time points (0-hour 53% [49, 56]; 1-hour 51% [48, 54]; 2-hour 52% [50, 55]; 4-hour 56% [53, 58]; 8-hour 56% [54, 57]; 24-hour 54% [53, 56]; 48-hour 54% [51, 56]; 72-hour 51% [48, 53]; and 168-hour 47% [45, 50]). Our results show omega-3 supplementation significantly increased 18-HEPE and decreased 12-HETE concentrations measured in HDL. Our results demonstrated HDL transport of oxylipins from inflammatory tissue occurred in macrophages, mice, and humans. We showed the intensity of macrophage response, as measured by MCP-1, to an inflammatory challenge depended on re-esterification and HDL mediated efflux of 12-HETE. In study 2, we found Ffar4 significantly increased 18-HEPE production in cardiomyocytes and increased oxylipin enzyme forming gene expression. HDL transport of oxylipins was significantly reduced in Ffar4KO TAC animals resulting in greater inflammatory signaling and exacerbated cardiac remodeling response compared to WT TAC animals. In study 3, we found significant time and treatment dependent differences unique to HDL 12-HETE and 18-HEPE concentrations demonstrating HDL transport of oxylipins during the response to an inflammatory challenge in vivo reflects our findings in cell and animal models.