Biology subjects

Wirka, R.

Publications and source records attributed to Wirka, R..

4 recordsLinked to original sources

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↗

bioRxiv2023

Ccn2 deficiency causes smooth muscle cell de-differentiation and severe atherosclerosis in hyperlipidemic mice

Cellular communication network factor 2 (CCN2/CTGF) is a matricellular protein with an established role in fibrotic diseases and cancers, and therapies targeting CCN2 is currently in Phase II and III clinical trials for idiopathic pulmonary fibrosis, pancreatic cancer and Duchenne Muscular Dystrophy. Recent studies have highlighed a protective role of CCN2 in aortic aneurysm disease, but its role in atherosclerosis remains to be investigated. We identified arteries as having the highest relative expression of CCN2 across 54 human tissues. In aortas, CCN2 was among the highest expressed genes, and in situ hybridization of human internal thoracic arteries revealed vascular smooth muscle cells (SMCs) as its principal source. Hypothesizing a role for CCN2 in SMC phenotype maintenance and athero-protection, we investigated inducible Ccn2 knockout (Ccn2{Delta}/{Delta}) mice in normo- and hyper-lipidemic settings. Induction of hyperlipidemia by single intravenous injection of 1{middle dot}1011 viral genomes of rAAV8-D377Y-mPcsk9 combined with 24 weeks of western type diet resulted in severe enlargement (3-5-fold increase of relative aorta mass compared to wildtype littermates, p < 0.0001) and whitening of Ccn2{Delta}/{Delta} aortas. Oil Red O-staining of en face prepared thoracic aortas showed a marked increase in atherosclerosis in Ccn2{Delta}/{Delta} mice as compared to wildtype littermates (75% vs. 10% Oil Red O-positive aortic area, p < 0.0001). Transcriptomic profiling of cultivated SMCs derived from aortas of normolipidemic mice showed signatures of dedifferentiation (reduced expression of e.g. Myocd, Acta2 and Myh11) and modulation toward a synthetic, pro-inflammatory phenotype of Ccn2{Delta}/{Delta} SMCs. These effects were verified in vivo and in CCN2-silenced human aortic SMCs. Taken together, we find that CCN2 plays a critical athero-protective role in artery tissues, likely through maintaining SMCs in a differentiated, contractile phenotype.

pathology↗

bioRxiv2020

The environment-sensing aryl-hydrocarbon receptor inhibits the chondrogenic fate of modulated smooth muscle cells in atherosclerotic lesions

IntroductionSmooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease (CAD) risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. MethodsWe combined RNA-Seq, ChIP-Seq, ATAC-Seq and in-vitro assays in human coronary artery SMC (HCASMC), with single-cell RNA-Seq (scRNA-Seq), histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease. ResultsGenomic studies coupled with functional assays in cultured HCASMC revealed that AHR modulates HCASMC phenotype and suppresses ossification in these cells. Lineage tracing and activity tracing studies in the mouse aortic sinus showed that the Ahr pathway is active in modulated SMC in the atherosclerotic lesion cap. Furthermore, scRNA-Seq studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMC expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term "chondromyocytes" (CMC), were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMC in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout compared to wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses. ConclusionOverall, we conclude that AHR promotes maintenance of lesion cap integrity and diminishes the disease related SMC-to-CMC transition in atherosclerotic tissues.

genomics↗

bioRxiv2020

Quantitative trait loci mapped for TCF21 binding, chromatin accessibility and chromosomal looping in coronary artery smooth muscle cells reveal molecular mechanisms of coronary disease loci

BackgroundTo investigate the epigenetic and transcriptional mechanisms of coronary artery disease (CAD) risk, as well as the functional regulation of chromatin structure and function, we have created a catalog of genetic variants associated with three stages of transcriptional cis-regulation in primary human coronary artery vascular smooth muscle cells (HCASMC). ResultsTo this end, we have used a pooling approach with HCASMC lines to map regulatory variation that mediates binding of the CAD associated transcription factor TCF21 with ChIPseq studies (bQTLs), variation that regulates chromatin accessibility with ATACseq studies (caQTLs), and chromosomal looping with HiC methods (clQTLs). We show significant overlap of the QTLs, and their relationship to smooth muscle specific genes and the binding of smooth muscle transcription factors. Further, we use multiple analyses to show that these QTLs are highly associated with CAD GWAS loci and correlated to lead SNPs in these loci where they show allelic effects. We have verified with genome editing that identified functional variants can regulate both chromatin accessibility and chromosomal looping, providing new insights into functional mechanisms regulating chromatin state and chromosomal structure. Finally, we directly link the disease associated TGF{beta}1-SMAD3 pathway to the CAD associated FN1 gene through a response QTL that modulates both chromatin accessibility and chromosomal looping. ConclusionsTogether, these studies represent the most thorough mapping of multiple QTL types in a highly disease relevant primary cultured cell type, and provide novel insights into their functional overlap and mechanisms that underlie these genomic features and their relationship to disease risk.

genomics↗