Project description:A disease driver population within interstitial cells of human calcific aortic valves identified via single-cell and proteomic profiling Cellular heterogeneity of aortic valves complicates mechanistic evaluation of the calcification processes in calcific aortic valve disease (CAVD), and animal disease models are lacking. In this study, we identify a disease driver population (DDP) within valvular interstitial cells (VICs). Through stepwise single-cell analysis, phenotype-guided -OMIC profiling, and network-based analysis, we characterize DDP fingerprint as CD44highCD29+CD59+CD73+CD45low and discover potential key regulators of human CAVD. These DDP-VICs demonstrate multi-lineage differentiation and osteogenic properties. Temporal proteomic profiling of DDP-VICs identifies potential targets for therapy, including MAOA and CTHRC1. In vitro loss-of-function experiments confirm our targets. Such a stepwise strategy may be advantageous for therapeutic target discovery in other disease contexts.
Project description:Quiescent canine valvular interstitial cells (n=3) and activated valvular interstitial cells (n=3) were grown in 2% FBS. Quiescent cells were treated with either TGFB1 or vehicle for 3 days and activated cells were treated with SB431542 for 4 days. mRNA was extracted using the qiagen RNeasy minikit and assessed by the agilent 2200 bioanalsyser. all samples passed quality control and transcriptomic analysis was performed by edinburgh genomics using the Affymetrix GeneChip™ Canine Gene 1.1ST Array. Results were reported as CEL files and then analysed in Affymetrix expression console (version 1.4.1.46) using the canine 1.1ST library files from Affymetrix. Robust Multi-array Average (RMA) was used to perform gene level normalisation and signal level summarisation.Following this, quality control assessment was performed on the data set. This included assessment of hybridisation (spike in) controls, labelling (poly-A) controls and area under the curve (AUC) control, probe cell intensity boxplots, a signal histogram graph and principle component analysis (PCA) plot. Following this assessment, the datasets were exported with annotation as a text file or as a CHT file. Affymetrix transcriptome analysis console (version 3.1.0.5) was used to perform paired or unpaired one-way analysis of variance (ANOVA).
Project description:Expression data from valvular interstitial cells cultured in 2D or 3D PEG hydrogel systems compared to culture on tissue culture polystyrene and freshly isolated cells