IN VITRO EFFECTS OF HEME ON THE ACTIVATION OF MICROVASCULAR ENDOTHELIAL CELLS, HMEC-1

Abstract

Introduction: Sickle cell anemia (SCA) is a hereditary condition characterized by morphological changes in erythrocytes, which increase their fragility and susceptibility to rupture, releasing intracellular components, including the heme molecule. Release of heme during intravascular hemolysis is closely associated with the vascular inflammatory response in this condition, contributing to the development of vaso-occlusive processes in the microcirculation. Objective: To evaluate the influence of heme on the activation of human microvascular endothelial cells, particularly with regard to the expression of surface adhesion molecules, the production of reactive oxygen species (ROS), and activation of caspase-1. Materials and methods: HMEC-1 (Human Microvascular Endothelial Cells) were used and incubated with varying concentrations of heme (0, 25, 50, and 100 μM) for 3 hours (37°C). Specific antibodies were used to assess the expression of adhesion molecules ICAM-1 (CD54), VCAM-1 (CD106), and E-selectin (CD62E) on the cells using flow cytometry. ROS production was measured using the 2,7-dichlorofluorescein diacetate (DCFH-DA) probe and caspase-1 activation was evaluated using the FAM-FLICA probe, and both were analyzed by flow cytometry. Data were analyzed using FlowJo, and statistical analysis was performed using ANOVA with Sidak's multiple comparisons post-tests, using Prism software. Results: Heme induced significant and dose-dependent increases in the expressions of ICAM-1 ([25 μM] p = 0.011; [50 μM] p = 0.002; [100 μM] p = 0.007, N = 5), VCAM-1 ([25 μM] p = 0.023; [50 μM] p = 0.011; [100 μM] p = 0.02, N = 5), and E-selectin ([25 μM] p = 0.024; [50 μM] p = 0.011; [100 μM] p = 0.001, N = 5) on the HMEC-1 cell surface. The evaluation of ROS production demonstrated a significant and progressive increase with increasing heme concentrations ([25 μM] p < 0.001; [50 μM] p = 0.023; [100 μM] p = 0.020, N = 6). Additionally, the analysis of caspase-1 activation revealed a significant and dose-dependent increase in response to heme concentration ([25 μM] p = 0.023; [50 μM] p < 0.001; [100 μM] p = 0.015, N = 5). Discussion and conclusion: The heme molecule was found to induce the expression of the adhesion molecules ICAM-1, VCAM-1, and E-selectin by HMEC-1 cells. Additionally, heme prompted oxidative stress, with the generation of ROS, and activation of caspase-1, indicating the involvement of the inflammasome in this process of cell activation. Similar results have been observed by our group, previously, in macrovascular Human Umbilical Vein Endothelial Cells (HUVEC). However, this study is novel in that we have investigated this effect specifically in microvascular HMEC-1 cells. These data suggest that heme, released during intravascular hemolysis and present in sickle cell anemia, may activate microvascular endothelial cells in small blood vessels such as venules, thereby contributing to the recruitment of circulating cells and the development of vaso-occlusive processes.