POS0332 ENDOTHELIAL ACTIVATION IN SYSTEMIC SCLEROSIS VASCULOPATHY: ROLE OF LONG NON-CODING RNA H19X

Abstract

Background:Long non-coding RNAs (lncRNA) are a class of non-coding transcripts which modulate many biological processes. Our previous studies showed that lncRNA H19X is pivotal in the regulation of TGFβ driven fibrosis in systemic sclerosis (SSc)1.Objectives:We aimed to investigate whether H19X plays a functional role in the regulation of endothelial cell (EC) activation, which is crucial in SSc vasculopathy2.Methods:Single-cell RNA sequencing (scRNA-seq) data from 27 dcSSc and 10 healthy control (HC) skin biopsies, following 10X Genomics partitioning and cDNA preparation, were analyzed for H19X expression in skin ECs, using Seurat package in R. A total of 4,981 ECs, of which 1,583 cells originated from HC and 3,398 cells originated from SSc patients, ranging from 59 to 342 ECs per subject, characterized by enrichment of EC markers of CLDN5, VWF and PECAM1.Expression of H19X in Human Dermal Microvascular ECs (HDMEC) was analyzed by qPCR. HDMEC were stimulated with different proinflammatory cytokines including IFNα, IFNβ, IFNγ, TGFβ, TNFα, IL-6, IL-1β and IL-4 at biologically relevant concentrations. In order to ascertain its effect in ECs, H19X was silenced in HDMECs using locked nucleic acid antisense oligonucleotides (LNA GapmeRs). qPCR and Western Blot (WB) were used to analyze the effects of H19X downregulation on EC activation biomarkers.Results:scRNA-seq data showed that H19X was significantly upregulated in SSc compared to healthy ECs (p=0.0095). Based on the differentially expressed gene profiles among subclusters, EC were further annotated as arterial (SEMA3G, HEY1), capillary (CA4, RGCC), venous (ACKR1, VCAM1), lymphatic (PROX1, LYVE1) ECs, as well as two aberrant clusters, proliferating (TOP2A, MKI67) and injured (HSGP2, APLNR) ECs, which were dominated by the SSc ECs. Specifically, the highest expression of H19X was found in injured SSc ECs and capillary SSc ECs. Overall, 1.5% SSc EC, about 51 cells, expressed detectable levels of H19X.In HDMEC (n=3), H19X was consistently induced by IFNα, IFNβ and IFNγ. Time curve analysis demonstrated that the strongest induction was observed at 48H (1.5±0.2, 1.6±0.4 and 2.1±0.3 – fold increase respectively). The combination of different IFNs determined stronger H19X induction after 48H stimulation, with a 2.4±0.1 increase with the combination of all IFNs and a 2.4±0.1 increase after the combination of IFNα+γ.Importantly, H19X knockdown lead to consistent and significant decrease of mRNAs of several adhesion molecules, including VCAM1, E- Selectin and P-Selectin, both in untreated HDMEC and after IFN stimulation. A decrease of VCAM and P-Selectin could be also demonstrated with WB analysis. No change was seen in other EC activation markers, including endothelin-1 and angiogenesis markers including VEGF, VEGFRA, Tie2 and thrombospondin.Conclusion:This is the first report analyzing a potential role of lncRNA H19X in SSc vasculopathy. Our results suggest that lncRNA H19X could act as a regulator of adhesion molecules expression in EC, possibly mediated by IFNs, and be therefore involved in EC activation.