Effects of age on lacrimal gland bioactive lipids.

PURPOSE: Polyunsaturated fatty acids (PUFA) are a source of bioactive lipids regulating inflammation and its resolution. METHODS: Changes in PUFA metabolism were compared between lacrimal glands (LGs) from young and aged C57BL/6 J mice using a targeted lipidomics assay, as was the gene expression of enzymes involved in the metabolism of these lipids. RESULTS: Global reduction in PUFAs and their metabolites was observed in aged LGs compared to young controls, averaging between 25 and 66 % across all analytes. ꞷ-6 arachidonic acid (AA) metabolites were all reduced in aged LGs, where the changes in prostaglandin E2 (PGE2) and lipoxin A4 (LXA4) were statistically significant. Several other 5-lipoxygenase (5-LOX) mediated metabolites were significantly reduced in the aged LGs, including D-series resolvins (e.g., RvD4, RvD5, and RvD6). Along with the RvDs, several ꞷ-3 docosahexaenoic acid (DHA) metabolites such as 14-HDHA, neuroprotectin D1 (NPD1), Maresin 2 (MaR2), and MaR 1 metabolite (22-COOH-MaR1) were significantly reduced in aged LGs. Similarly, ꞷ-3 eicosapentaenoic acid (EPA) and its metabolites were significantly reduced in aged LGs, where the most significantly reduced was 18-HEPE. Using metabolite ratios (product:precursor) for specific metabolic conversions as surrogate enzymatic measures, reduced 12-LOX activity was identified in aged LGs. CONCLUSION: In this study, global reduction of PUFAs and their metabolites was found in the LGs of aged female C57BL/6 J compared to young controls. A consistent reduction was observed across all detected lipid analytes except for ꞷ-3 docosapentaenoic acid (DPA) and its special pro-resolving mediator (SPM) metabolites in aged mice, suggesting an increased risk for LG inflammation.

Unveiling Drivers of Retinal Degeneration in RCS Rats: Functional, Morphological, and Molecular Insights.

Retinal degenerative diseases, including age-related macular degeneration and retinitis pigmentosa, significantly contribute to adult blindness. The Royal College of Surgeons (RCS) rat is a well-established disease model for studying these dystrophies; however, molecular investigations remain limited. We conducted a comprehensive analysis of retinal degeneration in RCS rats, including an immunodeficient RCS (iRCS) sub-strain, using ocular coherence tomography, electroretinography, histology, and molecular dissection using transcriptomics and immunofluorescence. No significant differences in retinal degeneration progression were observed between the iRCS and immunocompetent RCS rats, suggesting a minimal role of adaptive immune responses in disease. Transcriptomic alterations were primarily in inflammatory signaling pathways, characterized by the strong upregulation of Tnfa, an inflammatory signaling molecule, and Nox1, a contributor to reactive oxygen species (ROS) generation. Additionally, a notable decrease in Alox15 expression was observed, pointing to a possible reduction in anti-inflammatory and pro-resolving lipid mediators. These findings were corroborated by immunostaining, which demonstrated increased photoreceptor lipid peroxidation (4HNE) and photoreceptor citrullination (CitH3) during retinal degeneration. Our work enhances the understanding of molecular changes associated with retinal degeneration in RCS rats and offers potential therapeutic targets within inflammatory and oxidative stress pathways for confirmatory research and development.

Loss of 15-Lipoxygenase in Retinodegenerative RCS Rats.

Retinitis pigmentosa (RP) is a retinal degenerative disease associated with a diversity of genetic mutations. In a natural progression study (NPS) evaluating the molecular changes in Royal College of Surgeons (RCS) rats using lipidomic profiling, RNA sequencing, and gene expression analyses, changes associated with retinal degeneration from p21 to p60 were evaluated, where reductions in retinal ALOX15 expression corresponded with disease progression. This important enzyme catalyzes the formation of specialized pro-resolving mediators (SPMs) such as lipoxins (LXs), resolvins (RvDs), and docosapentaenoic acid resolvins (DPA RvDs), where reduced ALOX15 corresponded with reduced SPMs. Retinal DPA RvD2 levels were found to correlate with retinal structural and functional decline. Retinal RNA sequencing comparing p21 with p60 showed an upregulation of microglial inflammatory pathways accompanied by impaired damage-associated molecular pattern (DAMP) clearance pathways. This analysis suggests that ALXR/FPR2 activation can ameliorate disease progression, which was supported by treatment with an LXA4 analog, NAP1051, which was able to promote the upregulation of ALOX12 and ALOX15. This study showed that retinal inflammation from activated microglia and dysregulation of lipid metabolism were central to the pathogenesis of retinal degeneration in RP, where ALXR/FPR2 activation was able to preserve retinal structure and function.

Lipidomics in Understanding Pathophysiology and Pharmacologic Effects in Inflammatory Diseases: Considerations for Drug Development.

The lipidome has a broad range of biological and signaling functions, including serving as a structural scaffold for membranes and initiating and resolving inflammation. To investigate the biological activity of phospholipids and their bioactive metabolites, precise analytical techniques are necessary to identify specific lipids and quantify their levels. Simultaneous quantification of a set of lipids can be achieved using high sensitivity mass spectrometry (MS) techniques, whose technological advancements have significantly improved over the last decade. This has unlocked the power of metabolomics/lipidomics allowing the dynamic characterization of metabolic systems. Lipidomics is a subset of metabolomics for multianalyte identification and quantification of endogenous lipids and their metabolites. Lipidomics-based technology has the potential to drive novel biomarker discovery and therapeutic development programs; however, appropriate standards have not been established for the field. Standardization would improve lipidomic analyses and accelerate the development of innovative therapies. This review aims to summarize considerations for lipidomic study designs including instrumentation, sample stabilization, data validation, and data analysis. In addition, this review highlights how lipidomics can be applied to biomarker discovery and drug mechanism dissection in various inflammatory diseases including cardiovascular disease, neurodegeneration, lung disease, and autoimmune disease.

NAP1051, a Lipoxin A4 Biomimetic Analogue, Demonstrates Antitumor Activity Against the Tumor Microenvironment.

Resolving tumor-associated inflammation in the tumor microenvironment (TME) may promote antitumor effects. Lipoxin A4 (LXA4) is a short-lived endogenous bioactive lipid with potent anti-inflammatory and pro-resolving properties. Here, a biomimetic of LXA4, NAP1051, was shown to have LXA4-like in vitro properties and antitumor activity in colorectal cancer xenograft models. NAP1051 inhibited neutrophil chemotaxis toward fMLP and dose-dependently promoted dTHP-1 efferocytosis which was equipotent to aspirin-triggered lipoxin A4 (ATLA). In dTHP-1 cells, NAP1051 induced strong phosphorylation on ERK1/2 and AKT similar to formyl peptide receptor 2 (FPR2/ALX) agonists. In two mouse xenograft colorectal cancer models, NAP1051 significantly inhibited tumor growth when given orally at 4.8 to 5 mg/kg/day. Flow cytometric analyses showed that NAP1051 reduced splenic and intratumoral neutrophil and myeloid-derived suppressor cell populations, which correlated to the antitumor effect. In addition, NAP1051 reduced NETosis in the TME while stimulating T-cell recruitment. Overall, these results show that NAP1051 possesses key lipoxin-like properties and has antitumor activity against colorectal cancer via modulation of neutrophils and NETosis in the TME.