A transcriptome analysis of basal and stimulated VWF release from endothelial cells derived from patients with type 1 VWD

Figures

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  Figure 1.. %VWF secretion from type 1 VWD and control ECFCs after basal and stimulated VWF release. Mean %VWF secretion from type 1 VWD and control ECFCs during basal VWF release and stimulated VWF release. VWF:Ag was measured using an ELISA. %VWF secretion was calculated using the following formula: VWF:Ag in media / (VWF:Ag in media + VWF:Ag in lysate)∗100. Data points indicate mean %VWF:Ag secretion for each cell line (performed in triplicate). Error bars represent the mean ±SD. Because of limited cell yield, this experiment could only be performed on 2/4 control-derived ECFCs and 6/8 type 1 VWD-derived ECFCs. Low cell yield also precluded statistical analysis.

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  Figure 2.. Confocal immunofluorescence microscopy of ECFCs after basal and stimulated VWF release. ECFCs derived from healthy controls (A) and patients with type 1 VWD (B). Images in the top row were taken during basal VWF release (PMA-) and images in the bottom row were taken after stimulated VWF release (PMA+). ECFC identification labels are displayed across the top of the figure. ECFCs were stained for VWF (green), VE-cadherin (red) and DAPI (blue). Images are shown at 63× magnification with white scale lines representing 40 μm. Images were taken using a Lecia TCS SP8 Confocal Microscope. (C) Pearson r correlation between VWF section measured by ELISA and WPB count quantified from confocal microscopy images of control and type 1 VWD ECFCs during basal and stimulated release. (D) The number of WPBs contained in control and type 1 VWD ECFCs after basal and stimulated secretion. ∗P<.05.

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  Figure 3.. Transcriptome analysis of basal VWF release. (A) Volcano plot outlining DEGs between type 1 VWD and control ECFCs during basal release. The horizontal dashed line represents an adjusted P value of 0.1 and the vertical dashed lines represent 1.5-fold change relative to control ECFCs. DEGs meeting the 0.1 corrected P value threshold and that exhibit a fold expression >1.5 are in red. Relative expression of VWF (B) and V2R (C). (D) Interacting miRNAs and mRNAs that were differentially regulated during basal VWF release are displayed in a chord diagram. Green indicates upregulation and red indicates downregulation relative to control ECFC expression. The outer ring indicates direction of transcript regulation, and the ribbon color represents specifically the direction of miRNA regulation.

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  Figure 4.. Transcriptome analysis of stimulated VWF release. (A) Volcano plot outlining DEGs between type 1 VWD and control ECFCs during stimulated release. The horizontal dashed line represents an adjusted P value of 0.1 and the vertical dashed lines represent 1.5-fold change relative to control ECFCs. DEGs meeting the 0.1 corrected P value threshold and that exhibit a fold expression >1.5 are in red. Relative expression of VWF (B) and V2R (C). (D) Interacting miRNAs and mRNAs that were differentially regulated during stimulated VWF release are displayed in a chord diagram. Green indicates upregulation and red indicates downregulation relative to control ECFC expression. The outer ring indicates direction of transcript regulation, and the ribbon color represents specifically the direction of miRNA regulation.