Charugundla, S. | bioRxiv Science

bioRxiv2025

Trimethylamine-N-oxide affects cell type-specific pathways and networks in mouse aorta to promote atherosclerotic plaque vulnerability

BackgroundTrimethylamine-N-oxide (TMAO) has been significantly linked to atherosclerosis via several mechanisms, but its direct effect on the atherosclerosis-prone vasculature remains unclear. The objective of this study was to characterize the cell type-dependent and independent effects of TMAO on key vascular cell types involved in atherosclerosis progression in vivo. MethodsWe performed single cell RNA-sequencing (scRNAseq) on aortic athero-prone regions of female Ldlr-/- mice fed control Chow, high-cholesterol (HC), or HC+TMAO diets for three months to identify which aortic cell types, differentially expressed genes, and biological pathways are affected by TMAO. We also modeled cell-cell communications and intracellular gene regulatory networks to identify gene networks perturbed by TMAO feeding. Key genes and pathways were validated using primary human smooth muscle cells exposed to TMAO. Changes in the thickness of lesional fibrous caps in response to TMAO in female Ldlr-/- mice fed HC+TMAO versus HC diets were measured using transgelin immunostaining. ResultsOur scRNAseq analysis revealed that TMAO supplementation upregulated apoptotic gene signatures and downregulated extracellular matrix (ECM) organization and collagen formation genes in a subset of atherosclerosis-specific modulated vascular smooth muscle cells (vSMCs). We also identified "degradation of the ECM" as a top pathway for SMC-derived macrophage DEGs in response to TMAO. Network analyses support that macrophage-vSMC communication mediates ECM remodeling. Using human smooth muscle cells exposed to TMAO in vitro, we confirmed the direct effect of TMAO on regulating collagen and apoptotic genes. In agreement with the changes in these pathways that affect plaque stability, we observed a significant decrease in fibrous cap thickness in mice supplemented with TMAO. ConclusionsOur results reveal the effects of TMAO on vSMCs to promote apoptosis and decrease ECM formation, and on macrophage-mediated ECM degradation in atherosclerotic lesions to in concert enhance plaque instability. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=84 SRC="FIGDIR/small/640205v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@100254org.highwire.dtl.DTLVardef@109155aorg.highwire.dtl.DTLVardef@158d280org.highwire.dtl.DTLVardef@63d544_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIscRNAseq of the aortic athero-prone regions in female Ldlr-/- mice supplemented with TMAO in the diet revealed the effect of TMAO across cell types, particularly in SMC-derived macrophages and atheroprotective modulated vSMCs. C_LIO_LITMAO increases apoptotic gene signatures and reduces ECM organization and collagen formation gene signatures in modulated vSMCs in vivo, and in vitro exposure studies support a direct effect of TMAO on these genes. C_LIO_LIModulated vSMC-specific gene regulatory networks enriched for apoptotic genes and ECM organization genes were organized by intracellular regulators such as Ccl19 and Tnn and extracellular regulators such as Mmp9 and Spp1 from macrophages. C_LIO_LIFibrous cap thickness, a marker of atherosclerotic plaque stability, was significantly reduced in female Ldlr-/- mice fed HC+TMAO versus HC diets for five months. C_LI

physiology↗

bioRxiv2023

The Genetic Architecture of Dietary Iron Overload and Associated Pathology in Mice

Tissue iron overload is a frequent pathologic finding in multiple disease states including non-alcoholic fatty liver disease (NAFLD), neurodegenerative disorders, cardiomyopathy, diabetes, and some forms of cancer. The role of iron, as a cause or consequence of disease progression and observed phenotypic manifestations, remains controversial. In addition, the impact of genetic variation on iron overload related phenotypes is unclear, and the identification of genetic modifiers is incomplete. Here, we used the Hybrid Mouse Diversity Panel (HMDP), consisting of over 100 genetically distinct mouse strains optimized for genome-wide association studies and systems genetics, to characterize the genetic architecture of dietary iron overload and pathology. Dietary iron overload was induced by feeding male mice (114 strains, 6-7 mice per strain on average) a high iron diet for six weeks, and then tissues were collected at 10-11 weeks of age. Liver metal levels and gene expression were measured by ICP-MS/ICP-AES and RNASeq, and lipids were measured by colorimetric assays. FaST-LMM was used for genetic mapping, and Metascape, WGCNA, and Mergeomics were used for pathway, module, and key driver bioinformatics analyses. Mice on the high iron diet accumulated iron in the liver, with a 6.5 fold difference across strain means. The iron loaded diet also led to a spectrum of copper deficiency and anemia, with liver copper levels highly positively correlated with red blood cell count, hemoglobin, and hematocrit. Hepatic steatosis of various severity was observed histologically, with 52.5 fold variation in triglyceride levels across the strains. Liver triglyceride and iron mapped most significantly to an overlapping locus on chromosome 7 that has not been previously associated with either trait. Based on network modeling, significant key drivers for both iron and triglyceride accumulation are involved in cholesterol biosynthesis and oxidative stress management. To make the full data set accessible and useable by others, we have made our data and analyses available on a resource website. Author summaryThe response to a high iron diet is determined in part by genetic factors. We now report the responses to such a diet in a diverse set of inbred strains of mice, known as the Hybrid Mouse Diversity Panel, that enables high resolution genetic mapping and systems genetics analyses. The levels of iron in the liver varied about >5 fold across the strains, with genetic variation explaining up to 74% of the variation in liver iron. Pathologies included copper deficiency, anemia, and fatty liver, with liver triglycerides varying over 50 fold among the strains. Genetic mapping and network modeling identified significant genetic loci and pathways underlying the response to diet.

genetics↗