Biology subjects

Palmisano, B. T.

Publications and source records attributed to Palmisano, B. T..

3 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↗

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↗