Background/Purpose: Our prior research demonstrated that epigenetic modifications are central to the pathogenesis of scleroderma (SSc)1. Using ATAC-seq, we found that chromatin accessibility was significantly reduced in diffuse cutaneous (dc) SSc dermal endothelial cells (ECs) compared to those from healthy individuals. We also observed an increase in the chromatin binding of ETV2, a key transcription factor in the ETS family, within dcSSc ECs. ETV2 is crucial for both hematopoietic and EC function and has been implicated in reprogramming non-ECs to an endothelial phenotype. Its deficiency results in developmental failure of blood vessels in embryos. Here, we explore the impact of ETV2 dysregulation on EC dysfunction in SSc.

Methods: ECs were isolated from skin biopsies of healthy subjects and dcSSc patients. We evaluated ETV2 expression using qPCR, Western blot, and immunofluorescence. ETV2 was overexpressed in ECs via viral transduction, and its expression was also profiled in scRNA-seq data from 18 healthy individuals and 23 dcSSc patients. We assessed functional outcomes like proliferation and angiogenesis. To pinpoint genes influenced by ETV2 in dcSSc ECs, CUT&RUN-seq and bulk RNA-seq were utilized. Statistical significance was set at P < 0.05.

Results: Despite low baseline levels, ETV2 was markedly upregulated in dcSSc ECs compared to controls. scRNA-seq analysis showed increased ETV2 expression in capillary and venular ECs in SSc skin. ETV2 overexpression in dcSSc ECs significantly boosted EC proliferation (n=4 patient lines, p < 0.01) and angiogenic activity, evidenced by increased tube and node formation on Matrigel (n=4 patient lines, p < 0.05). Relative to healthy ECs, dcSSc ECs showed decreased CDH5 and increased COL1A1 and CDKN1A expression, indicative of an endothelial-to-mesenchymal transition phenotype and elevated cellular senescence. ETV2 overexpression elevated EC marker CDH5 (1.385 ± 0.17, p < 0.05 vs. control, fold change) and diminished fibrotic marker COL1A1 (0.680 ± 0.147, p < 0.05 vs. control, fold change) and senescence marker CDKN1A (0.719 ± 0.175, p < 0.05 vs. control, fold change). Through CUT&RUN-seq and bulk RNA-seq, we identified specific genes and pathways significantly regulated by ETV2 in dcSSc ECs.

Conclusion: This study highlights the significant impact of ETV2 in modifying EC function in dcSSc. Upregulation of ETV2 is associated with increased cell proliferation and angiogenesis, which may contribute to disease progression. Changes in gene expression following ETV2 overexpression suggest its potential to reverse endothelial-to-mesenchymal transition and reduce senescence. Further investigation into ETV2-regulated pathways could provide valuable insights into SSc vasculopathy. 1. Tsou et al. Genome-Wide Reduction in Chromatin Accessibility and Unique Transcription Factor Footprints in Endothelial Cells and Fibroblasts in Scleroderma Skin. Arthritis Rheumatol. 2021 Aug;73(8):1501-1513.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/examining-the-impact-of-etv2-on-altered-endothelial-phenotype-in-systemic-sclerosis/