Biology subjects

Bentzon, J. F.

Publications and source records attributed to Bentzon, J. F..

7 recordsLinked to original sources

bioRxiv2026

Loss of HIF1α signaling drives oxidative stress and expansion of smooth muscle cells in murine atherosclerosis

BackgroundHypoxia develops within growing atherosclerotic lesions, inducing nuclear translocation of hypoxia-inducible factor-1 (HIF1) and metabolic reprogramming. Its role in plaque macrophages and endothelial cells has been studied, but the hypoxic plaque interior is dominated by smooth muscle cell (SMC)-derived cells, for which the role of hypoxia signaling remains unclear. Here, we investigated how loss of Hif1a in SMC lineage cells impacts plaque progression and cell phenotype in murine atherosclerosis. MethodsAtherosclerosis was induced in mice with inducible SMC-specific deletion of Hif1a (Hif1aSMC-KO) and lineage tracing of SMC-derived plaque cells. Plaque size, necrotic core size, calcification, and SMC-derived cell phenotypes were quantified in aortic root sections and gene expression changes mapped by single-cell RNA sequencing. In parallel, a cultured SMC line with or without siRNA-mediated Hif1a knockdown was exposed to hypoxia for assessments of mitochondrial function and reactive oxygen species production. ResultsHif1aSMC-KO mice developed larger plaques, with expanded necrotic cores and increased calcification, compared with littermate controls. SMC-derived plaque cells were more abundant with a higher fraction of Col2a1+ chondromyocytes, and showed elevated markers of proliferation and apoptosis, whereas macrophage and endothelial cell numbers were unaffected. Single-cell RNA sequencing analysis revealed strong dysregulation of mitochondrial genes, including electron transport chain transcripts, along with upregulation of protein folding, proteasome, and oxidative stress response pathways. In cultured SMCs subjected to hypoxia, Hif1a silencing increased cell counts, aggravated mitochondrial proton leak, and led to the accumulation of depolarized, reactive oxygen species-generating mitochondria. Further analysis of SMC-derived cells in plaques from Hif1aSMC-KO mice confirmed increased oxidative stress by 8OHdG staining. ConclusionsHIF1 maintains mitochondrial function and restrains oxidative stress in SMC-derived plaque cells in murine atherosclerosis. Its chronic loss destabilizes redox homeostasis and promotes maladaptive SMC responses, leading to SMC-driven plaque expansion, necrosis, and calcification.

pathology↗

bioRxiv2025

Mechanical stretch regulates inflammatory signaling in human smooth muscle cells

Background and aimsVascular smooth muscle cells (SMCs) cultured under standard static conditions adopt a modulated phenotype that resembles the SMC states found in atherosclerotic plaques, but the mechanical determinants of this drift are not well defined. We investigated how mechanical loading shapes SMC phenotype and inflammatory signaling. MethodsHuman aortic SMCs were maintained under static conditions, physiological cyclic stretch (10% elongation), or pathological stretch (15% elongation) and analyzed by bulk and single-cell RNA sequencing. Mechanistic experiments included siRNA-mediated knockdown of IKBKB, p65 immunofluorescence, and regulon inference from single-cell transcriptomes. ResultsStretch regulated cell cycle, contractile, and inflammatory gene programs in an intensity-dependent manner: 10% stretch suppressed basal and TNF-induced inflammatory gene expression, whereas 15% stretch did not. The anti-inflammatory effect of physiological stretch required IKBKB, yet the proportion of cells with nuclear p65 was unchanged, indicating that stretch constrains NF-{kappa}B output downstream of p65 nuclear entry rather than by blocking translocation. Single-cell RNA sequencing resolved nine states for SMCs in culture whose transcriptomes overlapped substantially with the modulated mesenchymal populations of human coronary and carotid plaques, and physiological stretch attenuated pro-inflammatory gene expression across the major clusters. Regulatory network analysis identified inflammatory transcription factors (RELB, CEBPB/D, IRF1/2, STAT2) as less active under physiological stretch, whereas mechano-lineage regulators (MEF2C, TEAD1, SMAD6) were selectively induced, providing candidate mediators of the effect. ConclusionsStatic culture represents a disease-like SMC baseline that physiological stretch attenuates, in part by constraining NF-{kappa}B transcriptional output at a step downstream of p65 nuclear entry. Graphical abstractPhysiological stretch (10%) maintains a low-inflammatory, healthy-like state in human aortic smooth muscle cells, whereas its absence (static culture) or excess (15% stretch) favors an inflammatory, plaque-like state. NF-{kappa}B-p65 enters the nucleus under all conditions; physiological stretch constrains its inflammatory output downstream of nuclear entry. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/685276v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@bbcf58org.highwire.dtl.DTLVardef@29ba95org.highwire.dtl.DTLVardef@1989aa4org.highwire.dtl.DTLVardef@1d0c253_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Mouse, pig, and human atherosclerotic lesions have common and distinct mesenchymal cell populations

The proliferation and phenotypic modulation of smooth muscle cells (SMCs) to alternative mesenchymal states is a key process by which atherosclerotic lesions grow. The underlying mechanisms can be studied in mouse and pig atherosclerosis, but it remains unclear to what extent the mesenchymal plaque cell types in these species recapitulate human disease. Here, we integrate published and new single-cell RNA sequencing data of plaque mesenchymal cells from human carotid and coronary arteries, pig aorta and coronary arteries, and mouse brachiocephalic arteries. By applying consensus across multiple integration and gene homology-matching strategies, we identify a conserved core continuum of mesenchymal plaque cells, ranging from SMCs to extracellular matrix-producing fibroblast-like cells, which is stable across species and vascular beds. Notably, several other populations differed between human and experimental lesions. Subpopulations of SMCs marked by DLX5 and RERGL expression were specific to human carotid and coronary plaques, respectively. Mesenchymal cell states with strong pro-angiogenic and inflammation-associated gene signatures were identified in pig, but not human, coronary plaque datasets, with the pro-angiogenic phenotype associated with early stages of necrotic core development. Pericytes were solely present in pig and human plaques, while chondrocyte-like cells were unique to mouse lesions. The presented interspecies maps of mesenchymal cell diversity, and their markers may inform translational research into the role of SMCs and their derived progeny in atherosclerosis.

systems biology↗

bioRxiv2025

MyD88-dependent signaling promotes smooth muscle cell phenotypic modulation and fibrous cap formation in murine atherosclerosis

Background and aimsRecruitment of fibrous cap smooth muscle cells (SMCs) is critical for stabilizing atherosclerotic plaques and preventing rupture. This study investigated the SMC-specific role of the myeloid differentiation primary response protein 88 (Myd88) gene, encoding an adaptor protein essential for signaling downstream of several cytokine and pattern-recognition receptors, in this process. MethodsThe effects of MyD88 knockdown were assessed in cultured rat aortic SMCs. SMC-specific knockout of Myd88 was induced in mice using the Cre/lox method, followed by induction of atherosclerosis by proprotein convertase subtilisin/kexin type 9 gene transfer and a high-fat diet for 12 or 20 weeks. Effects on plaque and fibrous cap formation were studied by immunofluorescence and in situ hybridization. ResultsMyd88 knockdown reduced proliferation and migration of cultured SMCs and preserved contractile gene expression under inflammatory stimulation. SMC-specific Myd88 deficiency in hyperlipidemic mice did not significantly alter plaque size in the aortic root but reduced the number of cap SMCs in advanced lesions at 20 weeks and in the most atherosclerosis-susceptible aortic sinus at 12 weeks. Other plaque features, including macrophages, necrotic core size, and collagen content, were not significantly affected. Notably, MyD88 deficiency preserved the contractile phenotype of medial SMCs beneath plaques, suggesting that impaired phenotypic modulation contributed to reduced cap SMC recruitment. ConclusionsMyD88-dependent signaling promotes medial SMC phenotypic modulation and the recruitment of fibrous cap SMCs during atherogenesis. These findings highlight MyD88 as a mediator linking inflammatory signaling to protective fibrous cap formation. HighlightsO_LIKnockdown of MyD88 preserves the contractile phenotype of cultured smooth muscle cells subjected to inflammatory cytokines. C_LIO_LITargeting MyD88 in murine atherosclerosis inhibits fibrous cap formation. C_LIO_LITargeting MyD88 in murine atherosclerosis preserves the contractile phenotype of medial smooth muscle cells beneath the plaque. C_LI

pathology↗

bioRxiv2024

Mapping atherogenesis mechanisms in smooth muscle cells by targeting genes linked to coronary artery disease

Recent genome-wide association studies (GWAS) have identified multiple vascular cell-expressed genes linked to coronary artery disease (CAD), suggesting that smooth muscle cells (SMCs) and SMC-derived metaplastic cells are promising targets for novel antiatherosclerosis therapies. However, the disease-promoting pathways of most GWAS-identified genes are unknown, hindering their translation into therapeutic targets. This study integrated public GWAS data for CAD and single-cell RNA sequencing (scRNA-seq) analyses of human atherosclerotic plaques to identify 20 GWAS risk genes with a putative mechanism of action in SMCs or SMC-derived cells. Gene perturbation experiments in SMCs coaxed to plaque-relevant phenotypes revealed that the selected risk genes, despite encoding very different types of proteins, regulated shared sets of genes associated with contractile functions, cell cycle pathways, NF{kappa}B, and type I interferon signaling. By integrating information about GWAS gene effect direction and a deep analysis of cholesterol- and stretch-induced gene modules in SMCs, we find evidence that cholesterol-induced signaling is a pro-atherogenic disease mechanism in SMCs that is upregulated by detrimental and downregulated by protective GWAS genes. Overall, our study identifies a set of candidate disease mechanisms in SMCs that are regulated by multiple GWAS genes across several SMC assays. Furthermore, it provides proof-of-concept for using GWAS gene effect directionality to predict the pathogenic effect of candidate disease mechanisms that can be extended to other GWAS genes and cell types in the future.

cell biology↗

bioRxiv2023

Conditional deletion of Ccl2 in smooth muscle cells does not reduce early atherosclerosis in mice

Background and aimsC-C motif chemokine ligand 2 (CCL2) is a pro-inflammatory chemokine important for monocyte recruitment to the arterial wall and atherosclerotic plaques. Global knockout of Ccl2 reduces plaque formation and macrophage content in mice, but the importance of different plaque cell types in mediating this effect has not been resolved. Smooth muscle cells (SMCs) can adopt a potentially pro-inflammatory function with expression of CCL2. The present study aimed to test the hypothesis that SMC-secreted CCL2 is involved in early atherogenesis in mice. MethodsSMC-restricted Cre recombinase was activated at 6 weeks of age in mice with homozygous floxed or wildtype Ccl2 alleles. Separate experiments in mice lacking the Cre recombinase transgene were conducted to control for genetic background effects. Hypercholesterolemia and atherosclerosis were induced by a tail vein injection of recombinant adeno-associated virus (rAAV) encoding proprotein convertase subtilisin /kexin type 9 (PCSK9) and a high-fat diet for 12 weeks. ResultsUnexpectedly, mice with SMC-specific Ccl2 deletion developed higher levels of plasma cholesterol and larger atherosclerotic plaques with more macrophages compared with wild-type littermates. When total cholesterol levels were incorporated into the statistical analysis, none of the effects on plaque development between groups remained significant. Importantly, changes in plasma cholesterol and atherosclerosis remained in mice lacking Cre recombinase indicating that they were not caused by SMC-specific CCL2 deletion but by effects of the floxed allele or passenger genes. ConclusionsSMC-specific deficiency of Ccl2 does not significantly affect plaque development in hypercholesterolemic mice. Bullet pointsO_LISMCs express CCL2 in human plaques and upon inflammatory activation of a murine SMC line in vitro. C_LIO_LIDeletion of the Ccl2 gene in SMCs using Cre recombinase does not influence the size or composition of atherosclerotic plaque in mice. C_LIO_LIOnly by conducting a control study in mice without Cre recombinase was an initially observed difference in atherosclerosis concluded to be a genetic background effect. C_LI

pathology↗

bioRxiv2020

Fibrous caps in atherosclerosis form by Notch-dependent mechanisms shared with arterial media development

RationaleThe rupture of the fibrous cap in atherosclerotic lesions is the underlying cause of most thrombi leading to heart attack and a frequent cause of stroke. Caps are produced by smooth muscle cells (SMCs) that are recruited to the subendothelial space. We hypothesized that the recruitment mechanisms are likely common to embryonic artery development, which relies prominently on Notch signaling to form the subendothelial layers of medial SMCs. ObjectiveTo analyze the causal roles of the Notch signaling pathway in SMCs for atherogenesis and cap formation. Methods and ResultsNotch elements involved in arterial media development were found expressed in regions of fibrous cap in mouse plaques. To assess the causal role of Notch signaling in cap formation, we studied atherosclerosis in mice in which the Notch pathway was inactivated specifically in SMCs by conditional knockout of the essential effector transcription factor RBPJ. No major effects were observed on plaque size, but the presence of cap SMCs was significantly reduced. Lineage tracing revealed that the accumulation of SMC-derived plaque cells in the cap region was unaltered but that Notch-defective cells failed to re-acquire the SMC phenotype in the cap. To analyze whether the accumulation of SMC-derived cells in atherogenesis requires down-regulation of Notch signaling, we studied atherosclerosis in mice with constitutive Notch signaling in SMCs achieved by conditional expression of the Notch intracellular domain. Forced Notch signaling inhibited the ability of medial SMCs to contribute to plaque cells, including both cap SMCs and osteochondrogenic cells, and significantly reduced atherosclerosis development. ConclusionsSequential loss and gain of Notch signaling is needed to build the cap SMC population. The shared mechanisms with embryonic arterial media assembly suggest that the fibrous cap forms as a neo-media that restores the connection between endothelium and stabilizing SMCs, which is transiently disrupted by atherogenesis.

pathology↗