Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY Sickle cell disease (SCD) is caused by abnormal polymerization of deoxygenated sickle hemoglobin, which damages red blood cells (RBCs), resulting in hemolysis and thromboinflammation. Sickled-RBCs lead to heterogeneous clinical complications in SCD and induce challenges in determining the exact therapeutic interventions. Extracellular vesicles (EVs) are released from cells, and can serve as vehicles for exchanging biomolecules and reflect the parent cell’s functional state. Complement system (CS) activation plays important roles in intravascular hemolysis, red blood cell EV (REV) generation, and thromboinflammation. In SCD, REVs express phosphatidylserine and transport heme that are capable of promoting thrombosis, microvascular inflammation triggering thrombus formation and cell adhesion. I hypothesize that in SCD: REVs will serve as a biomarker to predict CS activation and CS-mediated thromboinflammation, evaluate disease activity, and assist in preliminary evaluation of responses to therapeutic intervention. Here, I propose to use microfluidic systems and functional assays I developed to enhance the mechanistic understanding of stress-induced CS activation and REV generation, and the roles of REV in CS activation and thromboinflammation in heterogeneous population of patients with SCD. First, I will determine the responses of SCD RBC and complement system after exposure to individual and cumulated stresses including shear stress, hypoxia, and cycles of hypoxia- reoxygenation using microfluidic system. I will determine the role of stresses in CS activation, hemolysis, and REV generation. Autologous REVs generated under in vitro stresses and endogenous REVs isolated from plasma will be characterized for defining their surface protein expression, proteomic content, and effect on amplifying CS activation. I will refine the applied stresses to generate autologous REVs using samples at baseline states for mimicking endogenous REVs in samples at elevated disease state. Second, I will elucidate the roles of REV and CS activation in thromboinflammation. REV-mediated endothelial activation through Kruppel Like Factor 4 and complement activation will be assessed using both analytical and function endpoints including abnormal cellular adhesion and thrombus formation. Third, I will utilize analytical and functional assays to assess (using endogenous REVs) and predict (using autologous REVs) clinical outcome and efficacies of therapeutic intervention for individual patients. I will establish a temporal and progressive database of REV- initiated CS activation and thromboinflammation for assist patient-specific diagnosis and prognosis. This project will improve the understanding of the roles of CS and REV in hemolysis and thromboinflammation in SCD, and has the potential to establish a REV database to enable accurate evaluation of disease outcomes. Given my background as a chemical engineer, the K25 award will uniquely enable me to gain experience and become an independent scientist in basic and translational biomedical research.
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