A107-01 Single Cell RNA Sequencing Identifies Erythroid-like Endothelial Cells in the Lungs of a Mouse Model of Sickle Cell Disease

Abstract Rationale Patients with Sickle Cell Disease (SCD) experience lung complications due to hemolysis and inflammation, resulting in significant morbidity and mortality. These complications include acute entities such as acute chest syndrome (ACS) and vasoocclusive crises (VOC) as well as more chronic processes including pulmonary hypertension (PH). Endothelial cell dysfunction plays an important role in the pathogenesis of ACS, VOC, and PH as these cells are exposed to free heme, and related compounds, that act as damage associated molecular patterns (DAMPS) to promote lung injury and inflammation. Understanding the biological basis of the inflammation present in SCD are essential to the development of novel treatment and prevention strategies. Single cell RNA sequencing technology has allowed the identification of cell populations and transcriptional profiles involved in the pathogenesis of other disease processes. Here, we demonstrate its use in finding an interesting endothelial cell subpopulation in sickle cell mouse lungs. Methods Whole lungs from sickle cell (SCD) mice (B6;129 Hbatm1(HBA)TowHbbtm2(HBG1,HBB*)Tow/Hbbtm3(HBG1,HBB)Tow/J) and normal hemoglobin littermates, both male and female, were harvested at 10 weeks of age and the lungs prepared for single cell RNA sequencing. Data were analyzed using clustering, differential gene expression, and pathway analysis. Results Cell clustering analysis demonstrated a subpopulation of endothelial cells that is present in SCD mice but less prevalent in normal hemoglobin littermates (Figure 1A). This population of Erythroid-like Endothelial Cells (ELETs) clusters with general capillary endothelial cells, but not with aerocytes. Transcriptional analysis revealed that these ELETs express higher levels of alpha-hemoglobin than general capillary endothelial cells but lower levels of platelet endothelial cell adhesion molecule (PECAM) (CD31) (Figure 1B).Deeper single cell sequencing was performed and confirmed a large increase in ELET cells in the sickle cell mice compared to normal hemoglobin littermates (Figure 1C).Analysis of a rat model of hypoxia revealed a similar population of erythroid-like cells in the rat hypoxia model that was present in larger numbers than in the normoxia control rats (Figure 1D). Conclusions ELET cells have not been previously described in the Townes mouse model of sickle cell disease. Others have described comparable cells in fetal mouse lungs, hemogenic endothelial cells, that have hematopoietic potential, and in adult mouse lungs with cells that arise from the bone marrow and produce platelets locally in the lung. Further efforts are underway at characterizing this ELET cell population and its lineage origin, whether from endothelial cells or bone marrow progenitors. This abstract is funded by: NHLBI