Biology subjects

Doran, A. C.

Publications and source records attributed to Doran, A. C..

3 recordsLinked to original sources

bioRxiv2026

N-aldehyde modified phosphatidylethanolamines formed by lipid peroxidation inhibit efferocytosis.

Objective: Impairments in efferocytosis, the phagocytic clearance of apoptotic cells, contribute to the development of chronic inflammation and promote the formation of large necrotic cores within atherosclerotic plaques. A better understanding of the factors that impair efferocytosis could lead to improved interventions. Many chronic inflammatory conditions feature excessive lipid peroxidation, which generates reactive lipid aldehydes that can potentially form N-aldehyde-modified phosphatidylethanolamines (NAMPs). We therefore investigated the extent to which levels of NAMPs are elevated under conditions of oxidative stress and whether exposure of macrophages to NAMPs inhibit efferocytosis. Approach and Results: The effects of synthetic NAMPs and lipid peroxidation on efferocytosis were measured using the Incucyte Live-Cell Analysis System to monitor rate of ingestion of CypHer-labeled apoptotic cells by cultured bone marrow-derived macrophages in real time. The extent of NAMP formation under various conditions was examined using liquid chromatography coupled with mass spectrometry. Several representative species of NAMPs (N-isolevuglandin-phosphatidylethanolamine, N-IsoLG-PE; N-4-hydroxynonenal-phosphatidylethanolamine, N-HNE-PE; and N-azeloyl-phosphatidylethanolamine, N-Aze-PE) inhibited efferocytosis in a concentration-dependent manner. Hydrolysis of NAMPs by recombinant N-acyl phosphatidylethanolamine hydrolyzing phospholipase D (NAPE-PLD) abolished their inhibitory activity, whereas genetic deletion of macrophage NAPE-PLD enhanced NAMP-mediated inhibition of efferocytosis. Blocking the inhibitor of lipid peroxidation, glutathione peroxidase 4 (GPX4), increased macrophage NAMP concentrations and impaired efferocytosis. Unmodified high-density lipoprotein (HDL) promoted efferocytosis, but exposing HDL to reactive lipid aldehydes or peroxidizing agents markedly increased NAMPs and this modified HDL inhibited efferocytosis. HDL isolated from subjects with familial hypercholesterolemia had elevated levels of multiple NAMP species. NAMPs impaired macrophage cholesterol efflux, which is required for continual efferocytosis. Conclusion: NAMPs accumulate under conditions associated with lipid peroxidation and inhibit the ability of macrophages to carry out efferocytosis.

immunology↗

bioRxiv2024

Post-transcriptional modifications on tRNA fragments confer functional changes to high-density lipoproteins in atherosclerosis

Epitranscriptomic modifications on RNA play critical roles in stability, processing, and function, partly by influencing interactions with RNA-binding proteins and receptors. The role of post-transcriptional RNA modifications on cell-free non-coding small RNA (sRNA) remains poorly understood in disease contexts. High-density lipoproteins (HDL), which transport sRNAs, can lose their beneficial properties in atherosclerosis cardiovascular disease (ASCVD). We hypothesize that changes to regulatory modifications on HDL-sRNAs contribute to this dysfunction. To assess changes in HDL-sRNA modification status, HDL-derived RNA from healthy subjects and those with atherosclerotic lesion development were analyzed using LC-MS/MS and AlkB-facilitated RNA (de)Methylation Sequencing. ASVD-HDL showed an enrichment in modified nucleosides including m1A tRNA-derived sRNAs (tDRs), particularly tDR-ArgACG-1. Functional studies revealed that ASCVD-HDL induced cell adhesion genes, including TMEM123, in primary macrophages. Recombinant HDL loaded with m1A-tDR-ArgACG-1 induced immune signaling, and similarly upregulated TMEM123. These findings suggest HDL-delivered-m1A-tDR-ArgACG-1 act on adhesion genes and immune pathways, promoting macrophage activation.

cell biology↗

bioRxiv2023

Small Molecule Activation of NAPE-PLD Enhances Efferocytosis by Macrophages

N-acyl-phosphatidylethanolamine hydrolyzing phospholipase D (NAPE-PLD) is a zinc metallohydrolase that hydrolyzes N-acyl-phosphatidylethanolamine (NAPEs) to form N-acyl-ethanolamides (NAEs) and phosphatidic acid. Several lines of evidence suggest that reduced NAPE-PLD activity could contribute to cardiometabolic diseases. For instance, NAPEPLD expression is reduced in human coronary arteries with unstable atherosclerotic lesions, defective efferocytosis is implicated in the enlargement of necrotic cores of these lesions, and NAPE-PLD products such as palmitoylethanolamide and oleoylethanolamide have been shown to enhance efferocytosis. Thus, enzyme activation mediated by a small molecule may serve as a therapeutic treatment for cardiometabolic diseases. As a proof-of-concept study, we sought to identify small molecule activators of NAPE-PLD. High-throughput screening followed by hit validation and primary lead optimization studies identified a series of benzothiazole phenylsulfonyl-piperidine carboxamides that variably increased activity of both mouse and human NAPE-PLD. From this set of small molecules, two NAPE-PLD activators (VU534 and VU533) were shown to increase efferocytosis by bone-marrow derived macrophages isolated from wild-type mice, while efferocytosis was significantly reduced in Napepld-/- BMDM or after Nape-pld inhibition. Together these studies demonstrate an essential role for NAPE-PLD in the regulation of efferocytosis and the potential value of NAPE-PLD activators as a strategy to treat cardiometabolic diseases.

biochemistry↗