Poster M1 - Single-Cell RNA Sequencing Data Informs Research on Sickle Cell Disease Arteriopathy

Introduction: Affecting approximately 100,000 people in the United States, sickle cell disease (SCD) is a genetic blood disorder that causes red blood cells to sickle in deoxygenated states. In addition to chronic pain and systemic inflammation, people with SCD are more susceptible to strokes. By age 20, 11% will suffer from a major stroke and 39% will suffer from a silent stroke. The cause for this 300-fold increase in stroke risk is unclear. Postmortem examinations of people with SCD who died of stroke showed severe elastic lamina fragmentation throughout the carotid arteries, suggesting that arterial damage may contribute to increased stroke risk for people with SCD. The mechanisms by which arterial structures are damaged and how this arteriopathy contributes to increased stroke risk is not well defined. Here, with the aim of identifying gene expression differences in the carotid artery wall related to accelerated arterial remodeling, we performed single-cell RNA sequencing on the resident vascular cells of carotid artery samples dissected from the Townes humanized sickle cell transgenic mouse.

Materials and

Methods: We performed single cell RNA sequencing on carotid artery samples from heterozygous (AS) and homozygous sickle cell disease (SS) mice. Mice were euthanized with CO2 and vessels were perfused with heparinized saline. Carotid arteries were harvested and then dissociated into single cells using the gentleMACS Octo Dissociator. Single cell RNA-sequencing was performed using the 10x Genomics Chromium platform, which employs microfluidic partitioning to capture single cells and prepare barcoded cDNA libraries. Briefly, single cells and reverse transcriptase reagents are captured in vessels called GEMS. All transcripts within a GEM are given the same barcode, facilitating efficient library preparation and accurate identification of gene expression profiles for each cell. Raw FASTQ files were processed using Cell Ranger. Statistical analyses were performed using Seurat, an R package for single-cell genomic analysis. After quality control to remove dead cells and multiplets, the data was log normalized and scaled before performing a principal component analysis and integrating AS and SS samples independently. Integrated AS data was used as a control for cell clustering and visualization. Expression of relevant cell marker genes was used to identify cell types. Differential gene expression between AS and SS samples was characterized by a log2 fold change ± 0.6, p < 0.05. Gene ontology analyses were performed using Ingenuity Pathway Analysis and DAVID. Visualization of gene networks was performed using GeneMANIA and Pathway Commons.

Results, Conclusions, and Discussions: With the goal of understanding the underlying mechanisms mediating arterial remodeling and stroke risk in patients with sickle cell disease, we developed a workflow to analyze scRNA-seq data for the carotid arteries of the Townes sickle transgenic mouse. Based on expression of cell marker genes, we identified 17 distinct cell subpopulations, which collectively represent 8 cell types. Preliminary analysis reveals hundreds of differentially expressed genes between relevant AS and SS cell types in our carotid artery samples. Cystatin C and Fos, two of the most differentially expressed genes, both play a role in the regulation of Cathepsin K, a powerful protease that breaks down elastin and collagen and may be responsible for the arterial damage seen in patients with SCD. Additional analysis is required to uncover the biological relevance of these differentially expressed genes and determine how this variable expression may be implicated in SCD arteriopathy. Following thorough analysis of our scRNA-seq data, we will use immunohistochemistry, western blot, zymography, and other lab techniques to validate differential expression.

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