POS0482 LONG NON-CODING RNA H19X IS A MEDIATOR OF ENDOTHELIAL CELL ACTIVATION IN SYSTEMIC SCLEROSIS

Abstract

BackgroundIn one of our previous studies, we demonstrated that long non-coding RNA (lncRNA) H19X plays a crucial role in the development of TGFβ driven fibrosis in systemic sclerosis (SSc) and other fibrotic diseases1.ObjectivesTo define the functional relevance of H19X in endothelial cell (EC) activation as a decisive process in SSc vasculopathy.MethodsCorrelation of H19X expression and microvascular gene signature was computed on bulk RNA-Seq data derived from SSc skin biopsies of patients enrolled in the multicentre Prospective Registry of Early Systemic Sclerosis cohort (PRESS, n=48 SSc vs. n=33 healthy controls, HCs). Single cell RNA sequencing (scRNA-seq) data were collected from 27 diffuse cutaneous SSc (dcSSc) and 10 HC skin biopsies. Single cells were barcoded and encapsulated in droplets using a 10X Genomics system. After cDNA synthesis, the libraries were prepared and sequenced using Illumina NovaSeq-500 platform. Seurat package in R (v.3.0) was used to perform data analysis. EC were identified by enrichment of EC markers CLDN5, VWF and PECAM1. One thousand five hundred eighty-three and 3398 EC were identified from HC and SSc patients, respectively. Cells were analysed for the expression of H19X and EC activation markers. Additionally, differential expression and pathway enrichment analysis between H19X expressing cells and H19X negative cells was carried out. The function of H19X was investigated in human dermal microvascular EC (HDMEC) by silencing, using locked nucleic acid antisense oligonucleotides (LNA GapmeRs). Gene expression was measured by qPCR. Protein levels of endothelial adhesion molecules were analysed by Western Blot. Endothelial adhesion was evaluated by co-culture of HDMEC and fluorescently labelled peripheral blood mononuclear cells (PBMCs).ResultsH19X expression was found significantly upregulated in SSc skin biopsies of the PRESS cohort (p<0.0001). The expression of H19X positively correlated with the microvascular endothelial cell gene signature in all subjects (SSc and HC, R=0.43, p<0.0001), confirming that H19X is expressed in this cell type. To determine if H19X might be an important factor in SSc EC dysfunction scRNAseq was performed. This analysis revealed a significant upregulation of H19X in SSc EC as compared to HC EC (p=0.0095). H19X was found to be upregulated in several EC subclusters including arterial (SEMA3G, HEY1), capillary (CA4, RGCC), venous (ACKR1, VCAM1) and lymphatic (PROX1, LYVE1). H19X displayed highest expression in injured SSc EC and capillary SSc EC. Co-expression analysis of the scRNA-seq data revealed higher expression of several adhesion molecules in EC expressing H19X, including VCAM1, ICAM and JAM3. KEGG pathway analysis revealed that differentially expressed genes in H19X expressing cells were highly associated with the ‘Cell adhesion molecule’ pathway (p=2.209e-7). H19X silencing lead to a significant downregulation of mRNA levels of genes encoding adhesion molecules VCAM1 (n=7, p<0.05) and E-selectin (n=7, p<0.01) at 48h after transfection. VCAM1, but not E-Selectin, was also reduced at protein level as revealed by Western Blot (n=3). The functional relevance of H19X on endothelial adhesion was confirmed by PBMCs with H19X silenced HDMEC where we were able to demonstrate a significant decrease in leucocyte-to-endothelial cell adhesion (n=5, p<0.05).ConclusionOur results show that lncRNA H19X could contribute to EC activation in SSc vasculopathy, acting as a regulator of expression of adhesion molecules in EC.