Effect of heme on the expression of inflammatory mediators in HMEC-1 cells

• Grant number: 23/09206-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2023
• End date: September 30, 2024
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Nicola Amanda Conran Zorzetto
• Grantee: Vanessa Figueiredo Alberto
• Host Institution: Centro de Hematologia e Hemoterapia (HEMOCENTRO). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 19/18886-1 - Pathophysiological mechanisms and treatment of red blood cell abnormalities, AP.TEM

Abstract

Sickle cell anemia (SCA) is a genetic disease characterized by the deformation of red blood cells due to abnormal hemoglobin S polymerization. Clinical manifestations are mainly related to vaso-occlusive processes, such as painful crises and complications in various organs. In the disease there is extensive intravascular hemolysis due to the fragility of red blood cells. The release of hemoglobin and the heme group of red blood cells into the circulation causes a vascular inflammatory response that contributes to the triggering of vaso-occlusion processes in the microcirculation involving endothelial cells (EC), red blood cells, leukocytes, platelets and plasma proteins. This project aims to determine the effect of heme on microvascular EC in the expression of adhesion molecules, generation of reactive oxygen species (ROS), caspase-1 activity and production and release of the cytokine IL-1beta, in vitro. HMEC-1 cells (human microvascular EC; ATCC") will be cultured in the presence/absence of heme, and subsequently processed for cytofluorometric analysis. Flow cytometry will be used to evaluate: the expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin on the surface of HMEC-1; the generation of ROS using a fluorescent probe; and the activation of caspase-1 through the Fam-Flica assay® intracellular probe. The immunoenzymatic assay (ELISA) will be used to evaluate the release of IL-1beta in the culture supernatant. With these data, we will seek to identify possible correlations between the expression of adhesion molecules, ROS generation, pro-inflammatory cytokine production and caspase-1 activity in EC under heme-induced stimulation. We hope to find evidence that intravascular hemolysis processes, such as that found in AF, may contribute to endothelial activation in the microcirculation. Subsequently, I wish to publish and present the results found at conferences.