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Etiopathogenesis of Systemic Sclerosis: An Update

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Front. Immunol. | doi: 10.3389/fimmu.2018.02191

Single cell RNA sequencing identifies HSPG2 and APLNR as markers of endothelial cell injury in systemic sclerosis skin

 Sokratis A. Apostolidis1*,  Giuseppina Stifano2, Tracy Tabib1,  Lisa M. Rice2, Christina M. Morse1, Bashar Kahaleh3 and Robert A. Lafyatis1*
  • 1Department of Medicine, University of Pittsburgh Medical Center, United States
  • 2Department of Medicine, Boston University, United States
  • 3Department of Medicine, University of Toledo, United States

Objective: The mechanisms that lead to endothelial cell (EC) injury and propagate the vasculopathy in Systemic Sclerosis (SSc) are not well understood. Using single cell RNA sequencing (scRNA-seq), our goal was to identify EC markers and signature pathways associated with vascular injury in SSc skin.
Methods: We implemented single cell sorting and subsequent RNA sequencing of cells isolated from SSc and healthy control skin. We used t-distributed stochastic neighbor embedding (t-SNE) to identify the various cell types. We performed pathway analysis using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). Finally, we independently verified distinct markers using immunohistochemistry on skin biopsies and qPCR in primary ECs from SSc and healthy skin.
Results: By combining the t-SNE analysis with the expression of known EC markers, we positively identified ECs among the sorted cells. Subsequently, we examined the differential expression profile between the ECs from healthy and SSc skin. Using GSEA and IPA analysis, we demonstrated that the SSc endothelial cell expression profile is enriched in processes associated with extracellular matrix generation, negative regulation of angiogenesis and epithelial-to-mesenchymal transition. Two of the top differentially expressed genes, HSPG2 and APLNR, were independently verified using immunohistochemistry staining and real-time qPCR analysis.
Conclusion: ScRNA-seq, differential gene expression and pathway analysis revealed that ECs from SSc patients show a discrete pattern of gene expression associated with vascular injury and activation, extracellular matrix generation and negative regulation of angiogenesis. HSPG2 and APLNR were identified as two of the top markers of EC injury in SSc.

Keywords: ScRNA-seq, HSPG2, APLNR, systemic sclerosis, endothelial dysfunction

Received: 01 Feb 2018; Accepted: 04 Sep 2018.

Edited by:

Megan A. Cooper, Washington University in St. Louis, United States

Reviewed by:

Mirko Manetti, Università degli Studi di Firenze, Italy
Elisha D. Roberson, Washington University in St. Louis, United States  

Copyright: © 2018 Apostolidis, Stifano, Tabib, Rice, Morse, Kahaleh and Lafyatis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Dr. Sokratis A. Apostolidis, University of Pittsburgh Medical Center, Department of Medicine, Pittsburgh, United States, socrates.apostolidis@gmail.com
Dr. Robert A. Lafyatis, University of Pittsburgh Medical Center, Department of Medicine, Pittsburgh, United States, lafyatisra@upmc.edu