ABCA1 and apoA-I dependent 12-hydroxyeicosatetraenoic acid efflux regulates macrophage inflammatory signaling.

Aberrant high-density lipoprotein (HDL) function is implicated in inflammation-associated pathologies. While HDL ABCA1-mediated reverse cholesterol and phospholipid transport are well described, the movement of pro-/anti-inflammatory lipids has not been explored. HDL phospholipids are the largest reservoir of circulating arachidonic acid-derived oxylipins. Endotoxin-stimulation activates inflammatory cells leading to hydroxyeicosatetraenoic acid (HETE) production, oxylipins which are involved in inflammatory response coordination. Active signaling in the non-esterified (NE) pool is terminated by sequestration of HETEs as esterified (Es) forms and degradation. We speculate that an ABCA1-apoA-I-dependent efflux of HETEs from stimulated cells could regulate intracellular HETE availability. Here we test this hypothesis both in vitro and in vivo. In endotoxin-stimulated RAW-264.7 macrophages preloaded with d8-arachidonic acid we use compartmental tracer modeling to characterize the formation of HETEs, and their efflux into HDL. We found that in response to endotoxin: I) Cellular NE 12-HETE is positively associated with MCP-1 secretion (p<0.001); II) HETE transfer from NE to Es pools is ABCA1-depedent (p<0.001); III) Cellular Es HETEs are transported into media when both apoA-I and ABCA1 are present (p<0.001); IV) The stimulated efflux of HETEs >> arachidonate (p<0.001). Finally, in endotoxin challenged humans (n=17), we demonstrate that intravenous lipopolysaccharide (0.6 ng/kg body weight) resulted in accumulation of 12-HETE in HDL over a 168-hour follow-up. Therefore, HDL can suppress inflammatory responses in macrophages by regulating intracellular HETE content in an apoA-I/ABCA1 dependent manner. The described mechanism may apply to other oxylipins and explain anti-inflammatory properties of HDL. This newly defined HDL property opens new doors for the study of lipoprotein interactions in metabolic diseases.

Oxylipin transport by lipoprotein particles and its functional implications for cardiometabolic and neurological disorders.

Lipoprotein metabolism is critical to inflammation. While the periphery and central nervous system (CNS) have separate yet connected lipoprotein systems, impaired lipoprotein metabolism is implicated in both cardiometabolic and neurological disorders. Despite the substantial investigation into the composition, structure and function of lipoproteins, the lipoprotein oxylipin profiles, their influence on lipoprotein functions, and their potential biological implications are unclear. Lipoproteins carry most of the circulating oxylipins. Importantly, lipoprotein-mediated oxylipin transport allows for endocrine signaling by these lipid mediators, long considered to have only autocrine and paracrine functions. Alterations in plasma lipoprotein oxylipin composition can directly impact inflammatory responses of lipoprotein metabolizing cells. Similar investigations of CNS lipoprotein oxylipins are non-existent to date. However, as APOE4 is associated with Alzheimer's disease-related microglia dysfunction and oxylipin dysregulation, ApoE4-dependent lipoprotein oxylipin modulation in neurological pathologies is suggested. Such investigations are crucial to bridge knowledge gaps linking oxylipin- and lipoprotein-related disorders in both periphery and CNS. Here, after providing a summary of existent literatures on lipoprotein oxylipin analysis methods, we emphasize the importance of lipoproteins in oxylipin transport and argue that understanding the compartmentalization and distribution of lipoprotein oxylipins may fundamentally alter our consideration of the roles of lipoprotein in cardiometabolic and neurological disorders.

Effect of omega-3 ethyl esters on the triglyceride-rich lipoprotein response to endotoxin challenge in healthy young men.

Oxylipins are produced enzymatically from polyunsaturated fatty acids, are abundant in triglyceride-rich lipoproteins (TGRLs), and mediate inflammatory processes. Inflammation elevates TGRL concentrations, but it is unknown if the fatty acid and oxylipin compositions change. In this study, we investigated the effect of prescription ω-3 acid ethyl esters (P-OM3; 3.4 g/d EPA + DHA) on the lipid response to an endotoxin challenge (lipopolysaccharide; 0.6 ng/kg body weight). Healthy young men (N = 17) were assigned 8-12 weeks of P-OM3 and olive oil control in a randomized order crossover study. Following each treatment period, subjects received endotoxin challenge, and the time-dependent TGRL composition was observed. Postchallenge, arachidonic acid was 16% [95% CI: 4%, 28%] lower than baseline at 8 h with control. P-OM3 increased TGRL ω-3 fatty acids (EPA 24% [15%, 34%]; DHA 14% [5%, 24%]). The timing of ω-6 oxylipin responses differed by class; arachidonic acid-derived alcohols peaked at 2 h, while linoleic acid-derived alcohols peaked at 4 h (pint = 0.006). P-OM3 increased EPA alcohols by 161% [68%, 305%] and DHA epoxides by 178% [47%, 427%] at 4 h compared to control. In conclusion, this study shows that TGRL fatty acid and oxylipin composition changes following endotoxin challenge. P-OM3 alters the TGRL response to endotoxin challenge by increasing availability of ω-3 oxylipins for resolution of the inflammatory response.

Signaling through Free Fatty Acid Receptor 4 Attenuates Cardiometabolic Disease.

A surge in the prevalence of obesity and metabolic syndrome, which promote systemic inflammation, underlies an increase in cardiometabolic disease. Free fatty acid receptor 4 is a nutrient sensor for long-chain fatty acids, like ω3-polyunsaturated fatty acids (ω3-PUFAs), that attenuates metabolic disease and resolves inflammation. Clinical trials indicate ω3-PUFAs are cardioprotective, and this review discusses the mechanistic links between ω3-PUFAs, free fatty acid receptor 4, and attenuation of cardiometabolic disease.

Free fatty acid receptor 4 responds to endogenous fatty acids to protect the heart from pressure overload.

AIMS: Free fatty acid receptor 4 (Ffar4) is a G-protein-coupled receptor for endogenous medium-/long-chain fatty acids that attenuates metabolic disease and inflammation. However, the function of Ffar4 in the heart is unclear. Given its putative beneficial role, we hypothesized that Ffar4 would protect the heart from pathologic stress. METHODS AND RESULTS: In mice lacking Ffar4 (Ffar4KO), we found that Ffar4 is required for an adaptive response to pressure overload induced by transverse aortic constriction (TAC), identifying a novel cardioprotective function for Ffar4. Following TAC, remodelling was worsened in Ffar4KO hearts, with greater hypertrophy and contractile dysfunction. Transcriptome analysis 3-day post-TAC identified transcriptional deficits in genes associated with cytoplasmic phospholipase A2α signalling and oxylipin synthesis and the reduction of oxidative stress in Ffar4KO myocytes. In cultured adult cardiac myocytes, Ffar4 induced the production of the eicosapentaenoic acid (EPA)-derived, pro-resolving oxylipin 18-hydroxyeicosapentaenoic acid (18-HEPE). Furthermore, the activation of Ffar4 attenuated cardiac myocyte death from oxidative stress, while 18-HEPE rescued Ffar4KO myocytes. Systemically, Ffar4 maintained pro-resolving oxylipins and attenuated autoxidation basally, and increased pro-inflammatory and pro-resolving oxylipins, including 18-HEPE, in high-density lipoproteins post-TAC. In humans, Ffar4 expression decreased in heart failure, while the signalling-deficient Ffar4 R270H polymorphism correlated with eccentric remodelling in a large clinical cohort paralleling changes observed in Ffar4KO mice post-TAC. CONCLUSION: Our data indicate that Ffar4 in cardiac myocytes responds to endogenous fatty acids, reducing oxidative injury, and protecting the heart from pathologic stress, with significant translational implications for targeting Ffar4 in cardiovascular disease.

Subcellular compartmentalization of proximal Gαq-receptor signaling produces unique hypertrophic phenotypes in adult cardiac myocytes.

G protein-coupled receptors that signal through Gαq (Gq receptors), such as α1-adrenergic receptors (α1-ARs) or angiotensin receptors, share a common proximal signaling pathway that activates phospholipase Cß1 (PLCß1), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. Despite these common proximal signaling mechanisms, Gq receptors produce distinct physiological responses, yet the mechanistic basis for this remains unclear. In the heart, Gq receptors are thought to induce myocyte hypertrophy through a mechanism termed excitation-transcription coupling, which provides a mechanistic basis for compartmentalization of calcium required for contraction versus IP3-dependent intranuclear calcium required for hypertrophy. Here, we identified subcellular compartmentalization of Gq-receptor signaling as a mechanistic basis for unique Gq receptor-induced hypertrophic phenotypes in cardiac myocytes. We show that α1-ARs co-localize with PLCß1 and PIP2 at the nuclear membrane. Further, nuclear α1-ARs induced intranuclear PLCß1 activity, leading to histone deacetylase 5 (HDAC5) export and a robust transcriptional response (i.e. significant up- or down-regulation of 806 genes). Conversely, we found that angiotensin receptors localize to the sarcolemma and induce sarcolemmal PLCß1 activity, but fail to promote HDAC5 nuclear export, while producing a transcriptional response that is mostly a subset of α1-AR-induced transcription. In summary, these results link Gq-receptor compartmentalization in cardiac myocytes to unique hypertrophic transcription. They suggest a new model of excitation-transcription coupling in adult cardiac myocytes that accounts for differential Gq-receptor localization and better explains distinct physiological functions of Gq receptors.