Defining the Pathogenic Mechanisms of Calcific Aortic Valve Disease through Single-Cell RNA Sequencing
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Villa-Roel, Nicolas
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Abstract
Calcific Aortic Valve Disease (CAVD) is an active, multifactorial disease ranging from mild thickening and hardening of the aortic valve (AV) leaflets (sclerosis) to severe impairment of leaflet motion, inducing AV narrowing (stenosis). Currently, there are no therapeutic alternatives for CAVD aside from surgical or transcatheter AV repair or replacement with bioprosthetic or mechanical substitutes, highlighting the need for novel therapeutics to prevent and manage the progression of the disease. Interestingly, CAVD occurs preferentially in a side-dependent manner, with most of the pathology appearing on the fibrosa layer of the AV, facing the aorta, and exposed to complex, disturbed flow conditions. In contrast, the left ventricle-facing ventricularis experiences predominantly stable but pulsatile flow conditions and is spared from disease. The mechanism of the side-dependent CAVD development is unclear, but defining the underlying mechanisms could reveal potential therapeutic avenues. This dissertation research aimed to define the mechanisms by which side-dependent CAVD occurs using a single-cell RNA sequencing (scRNAseq) approach and validation studies.
Previous studies used scRNAseq and proteomics approaches to examine the physiological nature and disease development of the AV. However, those studies could not address the side-dependent mechanisms because the methods involved the digestion of the whole AV tissues. In addition, those whole tissue digestion methods led to a limited number of valvular endothelial cells (VEC) analyzed compared to other predominant cell types such as valvular interstitial cells (VIC) and immune cells, making it difficult to study the role of VECs in CAVD. To address these limitations, we developed a novel side-specific digestion method to collect VEC-enriched preparations from the ventricularis and fibrosa layers of human AV leaflets. The leftover tissues, following the side-dependent digestion, were further digested to collect additional AV cells. Human AV leaflets, excised from four non-transplantable donated hearts or an AV replacement surgery patient, ranging from non-diseased to calcified pathologies, were used in this study.
In Aim I, VEC-enriched cells from the ventricularis and fibrosa and the leftover cells were used for the scRNAseq study. From the scRNAseq analysis, we identified 27 clusters, including 7 VEC, 9 VIC, 4 transitional, and 7 immune cell populations. Differential gene expression and gene ontology analyses revealed protective genes and mechanisms, such as regulation of hemostasis and biosynthesis in clusters found predominantly in non-calcified donors. Meanwhile, clusters consisting of pro- inflammatory, pro-fibrotic, and pro-endothelial-mesenchymal transition (EndMT) cells were found in larger proportions in the more diseased donor samples.
In Aim II, scRNAseq data were analyzed in silico to identify cell types, genes, and biological pathways regulated in a side-dependent and disease-dependent manner. From two distinct analytical methods we identified a list of potential therapeutic target genes predominantly involved in inflammation, EndMT, fibrosis, extracellular matrix (ECM) remodeling, thrombosis, and apoptosis. Additionally, gene ontology analysis revealed upregulation of inflammatory, migratory, and angiogenic processes in the fibrosa and diseased VEC, relative to the ventricularis and non-diseased VEC.
In Aim III, a subset of side-dependent genes were validated in vitro and in vivo. Several of the predicted target genes from Aim II were found to be shear-sensitive, first by predictive analysis from screening microarray datasets, then by independent validation shear experiments. Furthermore, two transcription factors from the potential therapeutic gene subset, EGR1 and FOSB, which were predicted to be highly shear-sensitive, were also determined to be side-dependent by immunostaining new human AV leaflets. In addition, we tested pharmacological inhibitors for some differentially expressed genes as potential CAVD therapeutics. PLCG2, a known osteoclastogenic and potential Alzheimer’s disease protective target, was identified as a highly upregulated gene in fibrotic VIC. Due to a lack of available activators of PLCG2, we tested the effect of its pharmacological inhibition in VIC calcification and found an exacerbation and acceleration of mineralization, suggesting a therapeutic role for its activation. Meanwhile, in VEC, activator protein-1 complex inhibition with a repurposed small-molecule compound showed significant, concentration-dependent attenuation of osteogenic differentiation, also suggesting therapeutic, anti-calcific potential for this approach.
Furthermore, we used a previously identified potential CAVD therapeutic, HIF1A inhibitor PX-478, to explore its mechanisms in vivo. Due to a lack of animal models for CAVD, we used two distinct mouse models of atherosclerosis, which shares several pathogenic processes with CAVD, and found that PX-478 can regulate ECM remodeling and inflammation. In conclusion, our scRNAseq study provided us with a deeper understanding of the AV microenvironment, enabling us to study CAVD in a single-cell, side-dependent manner. Furthermore, our in silico analysis unveiled several side- dependent and disease-dependent genes which may serve as potential therapeutic targets. Future studies should expand these datasets and aim to combine the results presented here with CAVD drug screening techniques to identify novel therapeutics for this disease.
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2022-11-30
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Dissertation