Biology subjects

Rom, O.

Publications and source records attributed to Rom, O..

7 recordsLinked to original sources

bioRxiv2025

Macrophage EHD1 promotes inflammation and stabilizes sortilin to accelerate atherosclerosis

BackgroundMacrophages are key players in the pathogenesis of atherosclerosis. They trigger immune responses through their cell-surface receptors. However, how macrophages regulate those receptors in response to pro-inflammatory stimuli is not completely understood. Endocytic membrane trafficking involving receptor internalization, followed by endosomal transport and recycling of the internalized receptors, plays essential roles in balancing cell-surface receptors to meet cellular needs. Here, we explored the role of the endocytic regulator EHD1 in immune responses in macrophages and determined its contribution to atherosclerosis progression. MethodsEHD1 expression profiles in mouse and human plaques were determined by single-cell RNA sequencing (scRNA-seq) and immunofluorescence staining. Bone marrow transplantation (BMT) by transplanting bone marrow cells from Ehd1-/- or littermate wild-type mice to irradiated Ldlr-/- mice was performed to determine the effect of EHD1 deletion on atherosclerosis progression. In vitro mechanistic studies including inflammation signaling and endocytosis assays were performed in bone marrow-derived macrophages. ResultsEHD1 expression in macrophages is enhanced as atherosclerosis progresses in both mice and humans. Histological analysis of aortic root sections from BMT mice showed that EHD1 deletion reduces lesion size. ScRNA-seq of aortic CD45+ cells demonstrated that EHD1 deletion attenuates pro-inflammatory responses and cell-cell interactions. Mechanistic studies revealed that EHD1 accelerates the endocytic recycling of TNFR2 and activates NF-kB, leading to increased expression of inflammatory cytokines. Moreover, EHD1 interacts with retromer and stabilizes sortilin, a retrograde cargo of retromer and a risk factor for atherosclerosis. ConclusionsEHD1 promotes inflammation by enhancing TNFR2-NF-kB signaling and stabilizing sortilin, leading to accelerated atherosclerosis. Our study reveals novel roles for EHD1-mediated membrane trafficking in macrophage function and paves the way to innovative therapeutic strategies that aim to address dysregulated membrane trafficking in atherosclerosis.

cell biology↗

bioRxiv2025

Integrin-Specific Signaling Drives ER Stress-Dependent Atherogenic Endothelial Activation

Atherogenic endothelial activation arises from both the local arterial microenvironment--characterized by altered extracellular matrix composition and disturbed blood flow--and soluble proinflammatory stimuli such as oxidized low-density lipoprotein (oxLDL). Fibronectin, a provisional extracellular matrix protein enriched at atheroprone sites, enhances endothelial activation and inflammation triggered by oxLDL and disturbed flow. Although endoplasmic reticulum (ER) stress contributes to vascular dysfunction, the role of matrix composition in regulating ER stress remains unknown. We show that oxLDL and disturbed flow induce ER stress selectively in endothelial cells adhered to fibronectin, whereas both stimuli fail to induce ER stress in cells on basement membrane proteins. This matrix-specific ER stress response requires integrin activation, as endothelial cells deficient for integrin activation (talin1 L325R mutation) fail to activate ER stress in response to disturbed flow and oxLDL and direct stimulation of integrin activation using CHAMP peptides is sufficient to trigger ER stress. Blunting endothelial expression of fibronectin-binding integrins (5, v) using siRNA prevents ER stress in response to atherogenic stimuli in vitro, whereas endothelial 5 and v deletion reduces ER stress at atheroprone sites in vivo. The mechanisms driving integrin-dependent ER stress remain unclear, since matrix composition does not affect protein translation, unfolded protein accumulation, or superoxide production, and scavenging superoxide (TEMPOL) does not reduce integrin-dependent ER stress. Inhibiting ER stress with TUDCA reduces proinflammatory and metabolic gene expression (bulk RNAseq) but does not prevent NF-{kappa}B activation, a classic proinflammatory transcription factor. Rather, TUDCA prevents activation of c-jun N-terminal kinase (JNK) and c-jun activation, and blocking JNK (SP600126) or c-Jun activity (TAM67) prevents proinflammatory gene expression following both stimuli. Together, these findings offer new insight into how the arterial microenvironment contributes to atherogenesis, with fibronectin-binding integrin signaling promotes ER stress in response to mechanical and metabolic stressors, thereby amplying proinflammatory endothelial activation through JNK-c-Jun signaling. HighlightsO_LIFibronectin promotes endothelial ER stress in response to oxLDL and disturbed flow via integrin 5{beta}1 and v{beta}3 signaling. C_LIO_LIMatrix-specific ER stress occurs independently of oxidative stress or misfolded protein accumulation, indicating a non-canonical UPR activation. C_LIO_LIER stress amplifies proinflammatory gene expression through JNK-c-Jun signaling, without activating NF-{kappa}B. C_LIO_LITargeting the integrin-ER stress-JNK axis may offer new therapeutic strategies for early atherosclerosis. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/654582v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@ed7ca3org.highwire.dtl.DTLVardef@191c238org.highwire.dtl.DTLVardef@660f3dorg.highwire.dtl.DTLVardef@7287a4_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

pathology↗

bioRxiv2024

CD14 Blockade Does Not Improve Outcomes of Deep Vein Thrombosis Following Inferior Vena Cava Stenosis in Mice

BackgroundNeutrophil-mediated persistent inflammation and neutrophil extracellular trap formation (NETosis) promote deep vein thrombosis (DVT). CD14, a co-receptor for toll-like receptor 4 (TLR4), is actively synthesized by neutrophils, and the CD14/TLR4 signaling pathway has been implicated in proinflammatory cytokine overproduction and several aspects of thromboinflammation. The role of CD14 in the pathogenesis of DVT remains unclear. ObjectiveTo determine whether CD14 blockade improves DVT outcomes. MethodsBulk RNA sequencing and proteomic analyses were performed using isolated neutrophils following inferior vena cava (IVC) stenosis in mice. DVT outcomes (IVC thrombus weight and length, thrombosis incidence, neutrophil recruitment, and NETosis) were evaluated following IVC stenosis in mice treated with a specific anti-CD14 antibody, 4C1, or control antibody. ResultsMice with IVC stenosis exhibited increased plasma levels of granulocyte colony-stimulating factor (G-CSF) along with a higher neutrophil-to-lymphocyte ratio and increased plasma levels of cell-free DNA, elastase, and myeloperoxidase. Quantitative measurement of total neutrophil mRNA and protein expression revealed distinct profiles in mice with IVC stenosis compared to mice with sham surgery. Neutrophils of mice with IVC stenosis exhibited increased inflammatory transcriptional and proteomic responses, along with increased expression of CD14. Treatment with a specific anti-CD14 antibody, 4C1, did not result in any significant changes in the IVC thrombus weight, thrombosis incidence, or neutrophil recruitment to the thrombus. ConclusionThe results of the current study are important for understanding the role of CD14 in the regulation of DVT and suggest that CD14 lacks an essential role in the pathogenesis of DVT following IVC stenosis.

pathology↗

bioRxiv2023

Cholesterol-associated Locus EHBP1 Protects against NASH Fibrosis

SUMMARYHepatic free cholesterol contributes to fibrosis in nonalcoholic steatohepatitis (NASH), but how hepatic cholesterol metabolism becomes dysregulated in NASH is not completely understood. We show here that human fibrotic NASH livers have decreased EHBP1, a novel GWAS locus associated with LDL-cholesterol, and that the EHBP1 rs10496099 T>C variant in NASH patients is associated with decreased hepatic EHBP1 expression and augmented NASH fibrosis. Congruent with the human data, EHBP1 loss- and gain-of-function increases and decreases NASH fibrosis in mice, respectively. Mechanistic studies reveal that EHBP1 promotes sortilin (SORT1)-mediated PCSK9 secretion, leading to LDLR degradation, decreased LDL uptake, and reduced TAZ, a fibrogenic effector. Moreover, we show that the TNF/PPAR pathway suppresses EHBP1 in NASH. These data not only provide new mechanistic insight into the role of EHBP1 in cholesterol metabolism and NASH fibrosis by uncovering the interaction between EHBP1 and other cholesterol-related loci, including SORT1, PCSK9, and LDLR, but also elucidate a novel interplay between inflammation and EHBP1-mediated cholesterol metabolism. HighlightsHepatic EHBP1 is reduced in human and mouse fibrotic NASH EHBP1 rs10496099 T>C is associated with decreased hepatic EHBP1 and augmented NASH EHBP1 reduces LDLR, cholesterol uptake, and TAZ in hepatocytes EHBP1 promotes sortilin-mediated PCSK9 secretion in hepatocytes TNF suppresses EHBP1 expression in hepatocytes

pathology↗

bioRxiv2023

Fatty liver-mediated glycine restriction impairs glutathione synthesis and causes hypersensitization to acetaminophen

Non-alcoholic fatty liver disease (NAFLD) affects nearly one third of the population worldwide. Understanding metabolic pathways involved can provide insights into disease progression. Untargeted metabolomics of livers from mice with early-stage steatosis indicated a decrease in methylated metabolites suggesting altered one carbon metabolism. The levels of glycine, a central component of one carbon metabolism, were lower in steatotic mice, in line with clinical evidence. Isotope tracing studies demonstrated that increased synthesis of serine from glycine is the underlying cause for glycine limitation in fatty livers. Consequently, the low glycine availability in steatotic livers impaired glutathione (GSH) synthesis under oxidative stress induced by acetaminophen (APAP), enhancing hepatic toxicity. Glycine supplementation mitigated acute liver damage and overall toxicity caused by APAP in fatty livers by supporting de novo GSH synthesis. Thus, early metabolic changes in NAFLD that lead to glycine depletion sensitize mice to xenobiotic toxicity even at a reversible stage of NAFLD.

biochemistry↗

bioRxiv2022

Spatially Resolved Metabolites in Stable and Vulnerable Human Atherosclerotic Plaques Identified by Mass Spectrometry Imaging

Impairments in carbohydrate, lipid, and amino acid metabolism drive features of plaque instability. However, where these impairments occur within the atheroma remains largely unknown. Therefore, we sought to characterize the spatial distribution of metabolites within stable and unstable atherosclerosis in both the fibrous cap and necrotic core. Atherosclerotic tissue specimens were scored based on the Stary classification scale and subdivided into stable and unstable atheromas. After performing mass spectrometry imaging (MSI) on these samples, we identified over 850 metabolite-related peaks. Using MetaboScape, METASPACE, and HMDB, we confidently annotated 170 of these metabolites and found over 60 of these were different between stable and unstable atheroma. We then integrated these results with an RNA-sequencing dataset comparing stable and unstable human atherosclerosis. Upon integrating our MSI results with the RNA-seq dataset, we discovered that pathways related to lipid metabolism and long-chain fatty acids were enriched in stable plaques, whereas reactive oxygen species, aromatic amino acid, and tryptophan metabolism were increased in unstable plaques. Acylcarnitines and acylglycines were increased in stable plaques whereas tryptophan metabolites were enriched in unstable plaques. Evaluating spatial differences in stable plaques revealed lactic acid in the necrotic core, whereas pyruvic acid was elevated in the fibrous cap. In unstable plaques, 5-hydroxyindole-acetic acid was enriched in the fibrous cap. Our work herein represents the first step to defining an atlas of metabolic pathways involved in plaque destabilization in human atherosclerosis. We anticipate this will be a valuable resource and open new avenues of research in cardiovascular disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/494038v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@111368org.highwire.dtl.DTLVardef@1112d99org.highwire.dtl.DTLVardef@c8cdb2org.highwire.dtl.DTLVardef@13895d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2019

Loss-of-function genomic variants with impact on liver-related blood traits highlight potential therapeutic targets for cardiovascular disease

Cardiovascular diseases (CVD), and in particular cerebrovascular and ischemic heart diseases, are leading causes of death globally.1 Lowering circulating lipids is an important treatment strategy to reduce risk.2,3 However, some pharmaceutical mechanisms of reducing CVD may increase risk of fatty liver disease or other metabolic disorders.4,5,6 To identify potential novel therapeutic targets, which may reduce risk of CVD without increasing risk of metabolic disease, we focused on the simultaneous evaluation of quantitative traits related to liver function and CVD. Using a combination of low-coverage (5x) whole-genome sequencing and targeted genotyping, deep genotype imputation based on the TOPMed reference panel7, and genome-wide association study (GWAS) meta-analysis, we analyzed 12 liver-related blood traits (including liver enzymes, blood lipids, and markers of iron metabolism) in up to 203,476 people from three population-based cohorts of different ancestries. We identified 88 likely causal protein-altering variants that were associated with one or more liver-related blood traits. We identified several loss-of-function (LoF) variants reducing low-density lipoprotein cholesterol (LDL-C) or risk of CVD without increased risk of liver disease or diabetes, including variants in known lipid genes (e.g. APOB, LPL). A novel LoF variant, ZNF529:p.K405X, was associated with decreased levels of LDL-C (P=1.3x10-8) but demonstrated no association with liver enzymes or non-fasting blood glucose levels. Silencing of ZNF529 in human hepatocytes resulted in upregulation of LDL receptor (LDLR) and increased LDL-C uptake in the cells, suggesting that inhibition of ZNF529 or its gene product could be used for treating hypercholesterolemia and hence reduce the risk of CVD. Taken together, we demonstrate that simultaneous consideration of multiple phenotypes and a focus on rare protein-altering variants may identify promising therapeutic targets.

genomics↗