Epigenetic Profiles of Primary Endothelial Cells from Patients with Low VWF Levels

Background

Von Willebrand disease (VWD) type 1 is characterized by low von Willebrand factor (VWF) levels and mucocutaneous bleeding (MCB). Approximately 50% of patients with VWD type 1 exhibit mutations in VWF. However, a large number of patients with VWF levels between 30-50 IU/dL do not show mutations in VWF indicating that other mechanisms are involved. Blood outgrowth endothelial cells (BOECs) are a source of donor-specific endothelial cells and have demonstrated impairments in VWF release and packaging in patients with VWD. BOECs have not been evaluated in individuals with low VWF levels.

Hypothesis/Objective

We hypothesize that BOECs from individuals with low VWF levels will reveal unique VWF and genome wide epigenetic signatures that may explain the altered plasma VWF levels seen in these patients.

Methods

BOEC Derivation: Patients with low VWF levels and MCB (30-50 IU/dL) were enrolled in an IRB-approved study. The mononuclear layer from whole blood was isolated and plated onto collagen coated plates. After extended incubation, the presence of BOECs was confirmed by visual morphology and flow cytometry.

VWF Transcriptional Analysis: 9 cells lines including: a) 2 BOEC cell lines from control individuals and a HUVEC cell line and c) BOECs from individuals with low VWF, were assayed via single cell RNA sequencing. Bioinformatic analysis included generalized transcriptional expression and single cell expression of VWF. RNA-sequencing expression data was filtered according to the following standardized algorithm. Cells that were defined as monocytes (TYROBP expression > 2 copies) were excluded. Following monocyte exclusions, cells were determined to be of endothelial origin if they demonstrated the presence of PECAM1, CDH5, ROBO4, ESAM, TIE1, or NOTCH4 transcripts, as previously reported by Butler et al. (Cell Reports, 2016).

Epigenetic Profiling:Genomic DNA was extracted from BOECs and from peripheral leukocytes (paired to the BOEC draw sample) and analyzed for DNA methylation via an Illumina 850K methylation array.

Results

BOEC Derivation:A total of eight BOEC lines were generated, 6 from individuals with MCB and VWF levels between 30-50 IU/dL (5:1 female: male ratio, age range 11-54 years) and 2 from healthy controls (2 female, age range 22-39 years) with normal VWF levels and no symptoms of MCB.

VWF Expression is decreased in Low VWF Samples: Overall transcript expression of VWF was significantly decreased in low VWF BOEC samples (5.341 transcripts/cell) vs. control endothelial cells (9.076 transcripts/cell), P <0.0001.

Generalized Methylation Profiling:Via adjusted P-values, there were 129 methylation sites across multiple genes that were differentially methylated in Low VWF BOECs vs. control endothelial cells. A cluster plot demonstrates that the two control BOEC samples were generally clustered as compared to the other samples (Figure 1A).

VWF Specific Methylation: The Illumina 850K array covers 70 prospective methylation sites in VWF, ranging from upstream of the transcriptional start site through the length of the gene. A previous report demonstrated that differences in 8 methylation sites in the VWF promoter correlated with VWF expression (Yuan et al. Nature Communications 2016). 7 of these sites are covered in our assay. Across all of those 7 sites, there was significant increased methylation of the CpG islands in the Low VWF BOECs when compared to the control endothelial cells (Figure 1B).

Stability of VWF Methylation:To ensure that the isolation and culture of BOECS does not significantly affect the methylation status of VWF, we conducted a Pearson correlation analysis and demonstrated that peripheral leukocyte (at time of blood draw) and BOEC methylation is highly correlated at VWF specific methylation sites (R2 0.6, P = 0.0004) (Figure 1C).

Conclusions

Single cell RNA sequencing and genome wide methylation assays of BOECs from individuals with low VWF reveal significant differences in generalized methylation status when compared to BOECs from individuals with normal VWF levels and HUVECs. There is transcriptional downregulation of VWF in low VWF BOECs that is associated with hypermethylation of 7 specific VWF CpG sites in the VWF promoter. Additional sites are being evaluated. Finally, we validated the methylation status of BOECs by demonstrating high correlation with the methylation status of leukocytes from the same individuals.

Figure 1

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Figure 1

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