Date: Sunday, November 7, 2021
Session Type: Poster Session B
Session Time: 8:30AM-10:30AM
Background/Purpose: Long non-coding RNAs (lncRNAs) are non-coding transcripts involved in the regulation of gene expression; their role in disease development, to date, remains largely under investigation. Recently, we showed that lncRNA H19X is pivotal in the regulation of TGFβ driven fibrosis in SSc. Here, we aimed to characterize the functional relevance of H19X in endothelial cell (EC) activation, a crucial mechanism in SSc vasculopathy.
Methods: Single-cell RNA-sequencing (scRNA-seq) was performed on 27 dcSSc and 10 healthy controls (HC) skin biopsies. After library preparation (10X Genomics protocol) and sequencing (Illumina NextSeq-500 platform), the reads were examined by quality metrics and transcripts mapped to reference human genome (GRCh38). Data analysis was performed using Seurat package in R (v. 3.0). A total of 4981 EC (1583 from HC and 3398 from SSc patients, 59-342 EC per subject), characterized by enrichment of EC markers of CLDN5, VWF and PECAM1, were identified and analyzed for the expression of H19X and specific EC activation markers. The function of H19X was analyzed in Human Dermal Microvascular EC (HDMEC). H19X expression was analyzed by qPCR after HDMEC stimulation with different proinflammatory cytokines at biologically relevant concentrations. HDMEC were transfected using locked nucleic acid antisense oligonucleotides (LNA GapmeRs). Western Blot (WB) and qPCR were used to analyze the effects of H19X silencing on endothelial adhesion molecules.
Results: scRNA-seq data from skin biopsies showed a significant upregulation of H19X in SSc compared to healthy EC (p=0.0095), which was observed also for various EC subclusters. Identified subclusters included arterial (characterized by SEMA3G, HEY1), capillary (CA4, RGCC), venous (ACKR1, VCAM1) and lymphatic (PROX1, LYVE1) EC. Two additional clusters, proliferating (TOP2A, MKI67) and injured (HSGP2, APLNR) EC, were represented almost exclusively in SSc ECs. Specifically, the highest expression of H19X was found in injured SSc EC and capillary SSc EC. Overall, 1.5% of the SSc EC, about 51 cells, expressed detectable levels of H19X. In HDMEC (n=3), H19X was consistently induced by IFNα, IFNβ and IFNγ as well as by different IFN combination stimulations (IFNα+β+γ and α+γ). Time curve analysis demonstrated that the strongest induction was observed between 48h and 72h. Importantly, H19X knockdown lead to a consistent and significant decrease of mRNAs of the adhesion molecule VCAM1, both in untreated HDMEC (n=4, p=0.006) as well as after IFN stimulation. A decrease of VCAM1 could be also demonstrated by WB analysis (untreated, n=4, p=0.01, semiquantitative analysis). Furthermore, co-expression analysis of the scRNA-seq data from skin biopsies confirmed a higher expression of several adhesion molecules in EC expressing H19X, including ICAM, VCAM1 and JAM3.
Conclusion: H19X is overexpressed in SSc endothelium and is stimulated by IFNs. Our results point to a role of H19X as a regulator of adhesion molecules in EC, thus suggesting its potential involvement in the pathogenesis of SSc vasculopathy.
To cite this abstract in AMA style:Tirelli F, Pachera E, Lafyatis R, Huang M, Zulian F, Kania G, Distler O. Long Non-coding RNA H19X as a Regulator of Endothelial Adhesion Molecules in Systemic Sclerosis [abstract]. Arthritis Rheumatol. 2021; 73 (suppl 9). https://acrabstracts.org/abstract/long-non-coding-rna-h19x-as-a-regulator-of-endothelial-adhesion-molecules-in-systemic-sclerosis/. Accessed January 26, 2023.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/long-non-coding-rna-h19x-as-a-regulator-of-endothelial-adhesion-molecules-in-systemic-sclerosis/