Abstract 4369247: Hemolysis-induced Endothelial Necroptotic Signaling Pathways: Insights in the Mechanisms of Acute Chest Syndrome in Sickle Cell Disease

Abstract

Backround: There is a life expectancy disparity of nearly 20 years in sickle cell disease (SCD) patients. Cardiovascular and pulmonary complications are major contributors to mortality with acute chest syndrome (ACS) being one of the most common causes of death. Hemin, the oxidized moiety of hemoglobin and a product of SCD-associated hemolysis, has been implicated in the pathogenesis of ACS in our work due to its effects on endothelial barrier integrity through induction of necroptosis. Hypothesis: We hypothesized that hemin treatment in human pulmonary arterial endothelial cells (hPAECs) would induce important transcriptomic changes that can be linked to mechanisms of ACS in SCD. Methods: HPAECs were treated with hemin (40 uM) or control saline for 4 hours. RNA was processed, purified, and submitted for RNA sequencing (Illumina NovaSeq X). 15,042 genes were counted and were determined to be significantly differentially expressed if false discovery rate was <0.1. Pathway analysis was done with STRING Protein-Protein Interaction Network and Ingenuity Pathway Analysis (IPA). Significant genes were clustered with Markov clustering (inflation parameter = 3). Results: Hemin treatment leads to upregulation of the necroptosis-associated protein MLKL and endothelial barrier dysfunction is confirmed by a drop in trans-endothelial resistance. RNA sequencing identified 147 differentially expressed genes. Heme oxygenase-1 ( HMOX1 ) was the most significantly upregulated gene (fold change >20). Caspase 8 Associated Protein 2 ( CASP8AP2 ) was a top five downregulated gene. The top Gene Ontology pathway was response to lipopolysaccharide (LPS). The top KEGG pathway and STRING cluster was TNF signaling and included key inflammatory chemokines ( CXCL1 , CXCL2 , CXCL3 , CCL2 ), cytokines ( IL-1A , IL-6 ) and transcription factors ( NRF2 ). Using IPA, oxidative stress signaling and NRF2-mediated oxidative stress response pathways were identified as key toxicology and disease process pathways. Conclusions: RNA sequencing analysis identifies multiple pathways by which hemolysis may contribute to the pathogenesis of ACS in SCD. Hemin-treated hPAECs demonstrate differential expression of key mediators in necroptosis ( CASP8AP2, NRF2 ), the inflammatory response ( CXCL1, IL1A, IL-6) and response to LPS, which is TLR4 mediated . Increased oxidative stress signaling may suggest a role for targeting necroptosis through anti-inflammatory mediators, including heme oxygenase and NRF2, in the treatment of ACS.