Biology subjects

den Ruijter, H. H.

Publications and source records attributed to den Ruijter, H. H..

4 recordsLinked to original sources

bioRxiv2025

Female-biased vascular smooth muscle cell gene regulatory networks predict MYH9 as a key regulator of fibrous plaque phenotype

Atherosclerosis, a chronic inflammatory condition driving coronary artery disease (CAD), manifests in two primary plaque types: unstable atheromatous plaques and stable fibrous plaques. While significant research has focused on atheromatous plaques, recent studies emphasize the growing importance of fibrous plaques, particularly in females under 50 years of age, where erosion on fibrous plaques significantly contributes to coronary thrombosis. The molecular mechanisms underlying sex differences in atherosclerotic plaque characteristics, including vascular smooth muscle cell (VSMC) contributions, remain understudied. Therefore, we utilized sex-specific gene regulatory networks (GRNs) derived from VSMC gene expression data from 119 male and 32 female heart transplant donors to identify molecular drivers of fibrous plaques. GRN analysis revealed two female-biased networks in VSMC, GRNfloralwhite and GRNyellowgreen, enriched for inflammatory signaling and actin remodeling pathways, respectively. Single-cell RNA sequencing of carotid plaques from female and male patients confirmed the sex specificity of these networks in VSMCs. Further sub cellular phenotyping of the single-cell RNA sequencing revealed a sex-specific gene expression signature within GRNyellowgreen for VSMCs enriched for contractile and vasculature development pathways. Bayesian network modeling of the GRNyellowgreen identified MYH9 as a key driver gene. Indeed, elevated MYH9 protein expression in atherosclerotic plaques was associated with higher smooth muscle cell content and lower lipid content in female plaques, suggesting its involvement in fibrous plaque formation. Further proteomic analysis confirmed MYH9s upregulation in female fibrous plaques only and its correlation with stable plaque features. These findings provide novel insights into sex-specific molecular mechanisms regulating fibrous plaque formation.

genomics↗

bioRxiv2024

Comprehensive identification of coronary artery disease-associated variants regulating vascular smooth muscle cell gene expression

Coronary artery disease (CAD) is a complex disorder with genetic and environmental influences. Genome-wide association studies (GWAS) have identified over 300 genomic loci associated with disease risk. However, identifying the functional variants within these loci has been limited in large part due to linkage disequilibrium. This represents a critical step in understanding the molecular mechanisms underlying disease risk. To identify and prioritize candidate causal CAD-associated variants, we performed lentivirus-based massively parallel reporter assays (lentiMPRAs) in primary vascular smooth muscle cells (SMCs), which play significant roles in atherosclerosis, the underlying cause of CAD. We tested 25892 CAD-associated variants for their allele-specific enhancer activity in quiescent and proliferative SMCs, modeling healthy and disease conditions. We identified 122 candidate variants showing significant enhancer activity and differences in reporter gene expression between risk and non-risk alleles. We also identified 23 variants showing condition-biased allelic imbalance and 41 variants showing sex-biased allelic imbalance. We further functionally characterized 25892 variants by performing CUT&RUN assays to identify variants in enhancer and promoter regions of SMCs. By integrating the results of these experiments, we identified a credible set of 49 CAD-associated variants in functionally relevant regions. Furthermore, by comparing these identified variants with our previously obtained expression quantitative trait loci (eQTL) data, 27 of these 49 variants were associated with SMC gene expression levels. Finally, we performed CRISPRi experiments on 8 variants comprising 9 variant-gene pairs, rs35976034 (MAP1S), rs4888409 (CFDP1), rs73193808 (MAP3K7CL), rs67631072 (INPP5B/FHL3), rs1651285 (SNHG18), rs17293632 (SMAD3), rs2238792 (ARVCF), rs4627080 (NRIP3), to confirm their regulatory potential of nearby gene expression. Taken together, our results comprehensively fine-map the causal variants that confer increased risk of CAD through their effects on vascular smooth muscle cells.

genomics↗

bioRxiv2023

IL-1β inhibition partially negates the beneficial effects of diet-induced lipid lowering

BackgroundThromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions are the leading cause of death in the world. The most common and effective means to reduce these major adverse cardiovascular events (MACE), including myocardial infarction (MI) and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, little is known regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. MethodsSmooth muscle cell (SMC)-lineage tracing Apoe-/- mice were fed a Western diet (WD) for 18 weeks and then switched to a low-fat chow diet for 12 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery (BCA) lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of SMC, and other lesion cells by SMC-lineage tracing combined with scRNA-seq, CyTOF, and immunostaining plus high resolution confocal microscopic z-stack analysis. In addition, to determine if treatment with a potent inhibitor of inflammation could augment the benefits of chow diet-induced reductions in LDL-cholesterol, SMC-lineage tracing Apoe-/- mice were fed a WD for 18 weeks and then chow diet for 12 weeks prior to treating them with an IL-1{beta} or control antibody (Ab) for 8-weeks. ResultsLipid-lowering by switching Apoe-/- mice from a WD to a chow diet reduced LDL-cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1{beta} Ab treatment resulted in multiple detrimental changes including increased plaque burden, BCA lesion size, as well as increased cholesterol crystal accumulation, intra-plaque hemorrhage, necrotic core area, and senescence as compared to IgG control Ab treated mice. Furthermore, IL-1{beta} Ab treatment upregulated neutrophil degranulation pathways but down-regulated SMC extracellular matrix pathways likely important for the protective fibrous cap. ConclusionsTaken together, IL-1{beta} appears to be required for chow diet-induced reductions in plaque burden and increases in multiple indices of plaque stability. Clinical PerspectiveAlthough one must be cautious in extrapolating results of mouse studies to humans, the current and our previous studies (Gomez et al. 2018 Nature Medicine) suggest that efforts to identify anti-inflammatory therapies for treating patients with advanced atherosclerosis should consider the possibility that inhibiting a given cytokine may have a mixture of beneficial and detrimental effects that vary between individuals. What is New?In a mouse model of advanced atherosclerosis followed by diet-induced reductions in cholesterol, IL-1{beta} inhibition unexpectedly had multiple detrimental effects including increasing overall plaque burden and decreasing various indices of plaque stability, as well as markedly increasing the number of senescent cells, and cholesterol crystal accumulation in lesions as compared to IgG control Ab treated mice. What are the clinical Implications?Results suggest that some anti-inflammatory therapies may have limited efficacy for treating patients with advanced atherosclerosis because such therapies inhibit not only detrimental pro-inflammatory responses, but also evolutionarily conserved beneficial inflammatory processes, which play a critical role in resistance to pathogenic microorganisms, in tissue repair following injury, and resolution of inflammation. We propose that the latter includes clearance of senescent cells and cholesterol from advanced atherosclerotic lesions induced by dietary-induced lipid lowering.

cell biology↗

bioRxiv2022

Genetic regulation of human aortic smooth muscle cell gene expression and splicing predict causal coronary artery disease genes

Coronary artery disease (CAD) is the leading cause of death worldwide. Recent meta-analyses of genome-wide association studies (GWAS) have identified over 175 loci associated with CAD. The majority of these loci are in non-coding regions and are predicted to regulate gene expression. Given that vascular smooth muscle cells (SMCs) play critical roles in the development and progression of CAD, we hypothesized that a subset of the CAD GWAS risk loci are associated with the regulation of transcription in distinct SMC phenotypes. Here, we measured gene expression in SMCs isolated from the ascending aortas of 151 ethnically diverse heart transplant donors in quiescent or proliferative conditions and calculated the association of their expression and splicing with [~]6.3 million imputed single nucleotide polymorphism (SNP) markers across the genome. We identified 4,910 expression and 4,412 splice quantitative trait loci (sQTL) that represent regions of the genome associated with transcript abundance and splicing. 3,660 of the eQTLs had not been observed in the publicly available Genotype-Tissue Expression dataset. Further, 29 and 880 of the eQTLs were SMC- and sex-specific, respectively. To identify the effector transcript(s) regulated by CAD GWAS loci, we used four distinct colocalization approaches and identified 84 eQTL and 164 sQTLs that colocalized with CAD loci, highlighting the importance of genetic regulation of mRNA splicing as a molecular mechanism for CAD genetic risk. Notably, 20% and 35% of the eQTLs were unique to quiescent or proliferative SMCs, respectively. Two CAD loci colocalized with a SMC sex-specific eQTL (AL160313.1 and TERF2IP) and another locus colocalized with SMC-specific eQTL (ALKBH8). Also, 27% and 37% of the sQTLs were unique to quiescent or proliferative SMCs, respectively. The most significantly associated CAD locus, 9p21, was an sQTL for the long non-coding RNA CDKN2B-AS1, also known as ANRIL, in proliferative SMCs. Collectively, these results provide evidence for the molecular mechanisms of genetic susceptibility to CAD in distinct SMC phenotypes.

genetics↗