Biology subjects

Li, D. Y.

Publications and source records attributed to Li, D. Y..

7 recordsLinked to original sources

bioRxiv2026

An in vitro System for Studying Osteochondrogenic Differentiation of Smooth Muscle Cells and Modeling Intimal Vascular Calcification

Objective: Smooth muscle cells (SMCs) undergo phenotypic transitions during atherosclerosis, including towards a chondromyocyte (CMC) state associated with intimal calcification. Although standard in vitro calcification assays robustly reproduce mineral deposition, it remains unclear how well they recapitulate these disease-associated SMC states. We sought to define the CMC transcriptional phenotype in atherosclerosis and develop an in vitro system that faithfully reproduces it. Approach and Results: We firstly identified a CMC transcriptional signature in murine and human atherosclerotic plaque through single-cell RNA-sequencing, and spatial transcriptomics. CMCs showed a conserved osteochondrogenic program which localized within plaques and adjacent to calcified regions. We then developed an osteochondrogenic differentiation (OCD) assay by combining well-established calcification components with a high-density SMC micromass culture and TGF-{beta}1 supplementation and benchmarked it against a standard calcification (SC) assay using calcium quantification and bulk RNA-sequencing. Despite comparable calcification, OCD and SC resulted in distinct transcriptional states, with OCD showing preferential upregulation of osteochondrogenic programs, and a higher CMC signature score. Additionally, OCD upregulated genes with a stronger enrichment near coronary artery disease (CAD)-associated loci. These responses were reproducible across several primary human SMC lines. Timecourse analysis also showed that chondrogenic programs preceded calcification and showed directional concordance with the inferred in vivo SMC-to-CMC trajectory. To interrogate regulatory pathways controlling this process, we overexpressed the chondrogenic regulator SOX9, which enhanced cartilage and extracellular matrix programs while repressing inflammatory pathways. Finally, we examined 552 CAD-associated genes nominated across five genome-wide association studies. Of these, 240 were differentially expressed by day 12, and included established SMC regulators as well as a number of candidates not previously characterized in osteochondrogenic SMC transition. Conclusions: The OCD assay results in a strong calcification phenotype together with a disease-associated CMC-like transcriptional state, providing a reliable in vitro model for mechanistic investigation of SMC phenotypic transition and prioritization of candidate regulators.

cell biology↗

bioRxiv2026

Damaging RBM20 E-rich domain variants are not rescued by gene replacement

The promise of precision therapeutics in genetic cardiomyopathies relies on linking specific therapies to variant mechanisms. Missense variants in the cardiac splice regulator RBM20 cause a highly penetrant and arrhythmogenic dilated cardiomyopathy. Disease-causing variants in RBM20s arginine-serine rich (RS) domain act via formation of toxic gain of function cytoplasmic granules, but this is not true for a small number of clinically adjudicated pathogenic variants in its glutamate(E)-rich domain. To better define the effects of E-rich domain variants, we developed a scalable screen based on induced pluripotent stem cell (iPSC) cardiomyocyte differentiation that identified several additional damaging variants. Several of these reduced RBM20 protein abundance and stability. We therefore hypothesized that, unlike RS domain variants, these E-rich variants might be rescued by RBM20 overexpression. To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with a pathogenic E-rich domain variant (p.E913K), and confirmed reduced RBM20 protein expression in these RBM20+/p.E913K cells after differentiation to iPSC-derived cardiomyocytes (iPSC-CM, vs. engineered isogenic RBM20+/+). These iPSC-CMs also displayed aberrant transcriptional splicing, reduced contractility, increased calcium-induced calcium release, and nuclear localization of RBM20 protein, often with more than the two expected RBM20-centric splice factories. AAV-based overexpression of RBM20 reversed some, but not all of the mis-splicing events identified in RBM20+/p.E913K iPSC-CMs, and did not improve their abnormal contractility, calcium handling or supernumerary RBM20 nuclear granules. In summary, our data indicate that pathogenic E-rich domain variants reduce RBM20 protein abundance, but that their mechanism is unlikely to be explained by haploinsufficiency alone.

genetics↗

bioRxiv2026

Transcriptomic, Specific Marker, and Pathway Analysis of Smooth Muscle Cell Foam Cells Compared to Macrophage Foam Cells in Human Atherosclerosis

BACKGROUNDSmooth muscle cells (SMCs) are reported to contribute the majority of cholesterol-overloaded foam cells in human and mouse atheromas. However, the transcriptome, specific markers, and biologic itinerary of SMC foam cells relative to macrophage foam cells have not been determined. METHODSTranscriptomic analysis by single cell RNA sequencing (scRNA-seq) was performed on fresh coronary segments from heart transplant recipients with early to intermediate stage atherosclerosis. The gene expression pattern of a putative cluster of SMC foam cells was compared to those of cultured SMCs loaded with either aggregated low density lipoprotein (agLDL) or cholesterol bound to methyl-{beta}-cyclodextrin (Chol-M{beta}CD). Candidate markers of SMC foam cells not expressed by macrophage foam cells were validated in ours and publicly available datasets, by spatial transcriptomics and by immunofluorescence microscopy of human atheromas. Pathway analysis was performed using Gene Set Enrichment Analysis Hallmark gene sets. RESULTSSMC foam cells derived from fibromyocytes were tentatively identified using a panel of markers upregulated with in vitro cholesterol loading of SMCs. agLDL loading reproduced the same transcriptional profile, whereas Chol-M{beta}CD did not reproduce any in vivo SMC state. Top genes highly represented in SMC foam cells included SERPINE1, encoding plasminogen activator inhibitor 1 (PAI-1) and CFH, complement factor H, which were validated in further human coronary scRNA-seq datasets, by Xenium spatial transcriptomics, and by immunofluorescence microscopy. Relative to macrophage foam cells, SMC foam cells exhibit a distinct biologic itinerary, including activation of extracellular matrix, coagulation and angiogenesis pathways. CONCLUSIONSSMC foam cells, which are derived from fibromyocytes ("lipomyocytes"), exhibit unique markers and biologic programs that differ markedly from macrophage foam cells in atherosclerotic plaque development. Further understanding of the role of lipomyocytes and their expression of CFH and PAI-1 expression in plaque biology may offer novel therapeutic options to reduce ischemic cardiovascular disease. Novelty and SignificanceO_ST_ABSWhat is Known?C_ST_ABSO_LISingle-cell transcriptomic studies have demonstrated vascular SMCs undergo extensive phenotypic modulation during atherosclerosis, giving rise to fibromyocytes and other intermediate cell states. C_LIO_LISMC-derived foam cells constitute a major proportion of plaque foam cells. C_LIO_LIPrevious studies have shown that SMC-derived foam cells differ from macrophage-derived foam cells in cholesterol handling and lipid metabolism, making their identification using conventional macrophage-associated foam-cell markers challenging, and highlighting the need for SMC foam cell-specific molecular markers. C_LI What New Information Does This Article ContributeO_LIBy integrating published transcriptomic datasets with bulk RNA-sequencing of agLDL-loaded human SMCs, we identify the transcriptional program of human SMC-derived foam cells and demonstrate that these cells are embedded within the fibromyocyte population rather than forming a distinct cell cluster. C_LIO_LIagLDL loading in vitro reproduces the in vivo SMC foam-cell phenotype, whereas loading with cholesterol bound to cyclodextrin induces an inflammatory state not found in any in vivo SMC state in human atherosclerosis. C_LIO_LICFH and SERPINE1 were identified as specific markers of SMC-derived foam cells and validated by immunostaining and Xenium spatial transcriptomics, providing robust markers for identification of these cells in human atherosclerotic plaques. C_LIO_LIThe top transcriptional programs activated in SMC foam cells differ completely from macrophage foam cells and include extracellular matrix remodeling, complement regulation, coagulation and angiogenesis. C_LI In this study, we mapped smooth muscle foam cells to the fibromyocyte cluster of arterial SMC subtypes in human coronary artery atheromas. In vitro loading of smooth muscle cells with aggregated low-density lipoprotein, the presumed physiologic substrate for foam cell formation during plaque development, induced a gene expression pattern that localized to the same fibromyocyte cluster. The proposed sequence of SMC foam cell formation is that fibromyocytes present in human pre-atherosclerotic intima generate the proteoglycans that bind lipoproteins and allow their uptake by fibromyocytes to generate cholesterol-loaded "lipomyocytes". Lipid loading of fibromyocytes drives expression of specific markers and transcriptional programs, but with an overall gene expression pattern that continues to resemble fibromyocytes. We demonstrate here that lipomyocytes express the specific markers CFH and SERPINE1, and exhibit activation of a completely different set of pathways relative to macrophage foam cells in the plaque. These findings provide evidence for the unique phenotype and specific markers of lipomyocytes relative to macrophage foam cells in human atherosclerosis, which will be critical in evaluating the response of these different types of foam cell to novel therapies.

genomics↗

bioRxiv2025

Vascular smooth muscle cell atherosclerosis trajectories characterized at single cell resolution identify causal transcriptomic and epigenomic mechanisms of disease risk

Vascular smooth muscle cells (SMC) contribute to heritable coronary artery disease (CAD) risk and undergo complex cell state transitions to multiple disease related phenotypes. To investigate the genetic basis of SMC state trajectories that underlie the SMC component of CAD causality we have developed a dense timecourse single cell transcriptomic and epigenetic map of atherosclerosis in a murine disease animal model. Cellular trajectories were derived from the temporal data and probabilistic fate modeling with Waddington-Optimal Transport (WOT). We created transcription factor (TF) centered regulons mapped across the developmental timeline and through network-based prioritization with WOT predicted TFs and in silico TF perturbation, identified key drivers of cell state changes associated with EMT, vascular development, and circadian clock functions. Integration of mouse disease data with human CAD genetic findings identified transition SMC phenotypes that mediate disease risk and point to causal disease mechanisms. Parallel studies using knockout of the validated CAD gene Tcf21 revealed its impact on SMC transition cellular phenotypes and disease risk genes, due in part to a role regulating the transition of SMC precursor cells in the secondary heart field. Together, these studies characterize atherosclerosis trajectories at single cell resolution and identify genetic causal transcriptomic and epigenomic mechanisms of CAD risk.

genomics↗

bioRxiv2024

Aryl-hydrocarbon receptor in smooth muscle cells protect against dioxin induced adverse remodeling of atherosclerosis

IntroductionEnvironmental exposure to dioxin has been linked to increased myocardial infarction. Smooth muscle cells (SMC) in the coronary vasculature play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity, however, the detailed mechanism linking dioxin exposure to adverse SMC modulation is not well understood. MethodsSingle-cell RNA and ATAC sequencing and histological analyses were performed on the aorta from mouse models of atherosclerosis exposed to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) or control. Primary human coronary artery SMC (HCASMC) treated in culture with TCDD were used to perform RNA-Seq, ATAC-Seq, and functional phenotypic assays. ChIP-Seq was performed with antibodies against Aryl-hydrocarbon receptor (AHR) and TCF21, two of known SMC modulating transcription factors. ResultsModulated SMC were the most transcriptionally responsive cell type to dioxin in the atherosclerotic aorta. Dioxin accelerated disease phenotype by promoting a modulated SMC phenotype early, resulting in increased lesion size, migration of SMC, and macrophage recruitment to the lesion. We found C3 expressing modulated SMCs to be likely contributing to the increased macrophage recruitment and inflammation. Analysis of the RNA-Seq data from HCASMC treated with TCDD showed differential enrichment of biological pathways related to cell migration, localization, and inflammation. Furthermore, ATAC-Seq data showed a significant activation for pathways regulating vascular development, cell migration, inflammation, and apoptosis. With TCDD treatment, there was also enrichment of AHR ChIP-Seq peaks, while the TCF21 enrichment decreased significantly. The SMC-specific Ahr knockout resulted in increased oxidative stress in SMC, increased lesion size and macrophage content, and loss of SMC lineage cells in the lesion cap when exposed to TCDD, consistent with a more vulnerable plaque phenotype. ConclusionDioxin adversely remodels atherosclerotic plaque by accelerating the SMC- phenotypic modulation, and increasing inflammation and oxidative stress resulting in increased macrophage recruitment and lesion size. Dioxin may adversely affect the SMC phenotype and disease state by affecting the TCF21 occupancy in the open chromatin regions. Furthermore, we observed that SMC-specific deletion of Ahr in mice resulted in worsening of dioxin mediated SMC modulation and atherosclerosis, suggesting that Ahr in SMC confers protection against dioxin by promoting a stable plaque phenotype and reducing dioxin induced oxidative stress. SummaryO_LIExposure to dioxin, an environmental pollutant present in tobacco smoke and air pollution, accelerates smooth muscle cell modulation, and atherosclerosis. C_LIO_LIDioxin exposure leads to inflammatory smooth muscle cell phenotype characterized by complement pathway activation and increased macrophage recruitment to plaque C_LIO_LIAryl-hydrocarbon receptor in SMC protects against oxidative stress, and promotes a stable plaque phenotype C_LI

molecular biology↗

bioRxiv2024

A cell and transcriptome atlas of the human arterial vasculature

Contiguous arterial segments show different propensities for different vascular pathologies, yet mechanisms explaining these fundamental differences remain unknown. We sought to build a transcriptomic, cellular, and spatial atlas of human arterial cells across multiple different arterial segments to understand these underlying differences. Analysis of multiple isogenic arterial segments from healthy donors reveals a significant stereotyped pattern of cell type-specific segmental heterogeneity in healthy arteries. Combining single cell analysis with spatial transcriptomic data reveals cellular heterogeneity not captured by commonly used cell-type marker genes. Determinants of arterial transcriptomic identities are predominantly encoded in fibroblasts and smooth muscle cells (SMC), and their differentially expressed genes are particularly enriched for different vascular disease-associated genetic risk- loci and risk-genes. Adventitial fibroblast-specific heterogeneity in gene expression coincides with a disproportionally large number of vascular disease genetic signals, suggesting a previously unrecognized role for this cell type in disease risk. Adult arterial cells from different segments cluster not by anatomical proximity, but by embryonic origin. Global regulon analysis of disease related segment-specific gene expression program in fibroblast and SMC enriches for binding sites of transcription factors that are developmental master regulators whose expression persists into adulthood, suggesting an important functional role of the same developmental master regulators in adult gene expression and disease. Lastly, non-coding transcriptomes across arterial cells contain extensive variation in lncRNAs expressed in cell type- and segment-specific patterns, rivaling heterogeneity in protein coding transcriptomes. Differentially expressed LncRNA demonstrate enrichment for non-coding genetic signals for vascular diseases, suggesting a potential global role of segmental specific LncRNAs in regulating inherited human vascular disease risk.

cell biology↗

bioRxiv2024

Smooth muscle expression of RNA editing enzyme ADAR1 controls vascular integrity and progression of atherosclerosis

Mapping the genomic architecture of complex disease has been predicated on the understanding that genetic variants influence disease risk through modifying gene expression. However, recent discoveries have revealed that a significant burden of disease heritability in common autoinflammatory disorders and coronary artery disease (CAD) is mediated through genetic variation modifying post-transcriptional modification of RNA through adenosine-to-inosine (A-to-I) RNA editing. This common RNA modification is catalyzed by ADAR enzymes, where ADAR1 edits specific immunogenic double stranded RNA (dsRNA) to prevent activation of the double strand RNA (dsRNA) sensor MDA5 (IFIH1) and stimulation of an interferon stimulated gene (ISG) response. Multiple lines of human genetic data indicate impaired RNA editing and increased dsRNA sensing by MDA5 to be an important mechanism of CAD risk. Here, we provide a crucial link between observations in human genetics and mechanistic cell biology leading to progression of CAD. Through analysis of human atherosclerotic plaque and culture of human coronary artery vascular smooth muscle cells (SMCs) we implicate the SMC to have a distinct requirement for RNA editing, and that MDA5 activation regulates SMC phenotypic modulation. Through generation of a conditional SMC specific Adar1 deletion mouse model on a pro-atherosclerosis background with additional constitutive deletion of MDA5 (Ifih1), and with incorporation of single cell RNA sequencing cellular profiling, we further show that Adar1 controls SMC phenotypic state by regulating Mda5 activation, is required to maintain vascular integrity, and controls progression of atherosclerosis and vascular calcification. Finally, we further corroborate our findings in a large human carotid endarterectomy dataset (Athero-Express) where we show that ISG activation is strongly associated with decreased plaque stability, increased SMC phenotypic modulation, and increased plaque calcification. Through this work, we describe a fundamental mechanism of CAD, where cell type and context specific RNA editing and sensing of dsRNA mediates disease progression, bridging our understanding of human genetics and disease causality. One Sentence SummarySmooth muscle expression of RNA editing enzyme ADAR1 regulates activation of double strand RNA sensor MDA5 in novel mechanism of atherosclerosis.

genomics↗