(607g) Red Blood Cell Adhesion to Heme-Activated Endothelial Cells in Microscale Flow

Heme is a metal complex required for aerobic metabolism. Heme serves many critical homeostatic functions, such as oxygen transfer, respiration, drug detoxification, and regulation of protein synthesis and cell differentiation. However, the accumulation of free heme in plasma, due to an imbalance of heme biosynthesis and catabolism or arising from intravascular red blood cell breakdown (hemolysis), can be toxic to surrounding tissues through the induction of heme-derived reactive oxygen species (ROS). Increased hemolysis, driven by inherited conditions, such as sickle cell disease, or infections, such as malaria, may lead to increased levels of ROS, which may result in cellular damage or apoptosis. Further, heme-enhanced oxidative stress leads to vascular inflammation and endothelial injury/activation. Heme-exposed endothelial cells (ECs) upregulate the expression of adhesion molecules, such as ICAM-1, VCAM-1, E-selectin, P-selectin, and vWF through multiple pathways, including TLR4. Recently, it was shown that heme activation of ECs played a critical role in initiating vaso-occlusion in a murine model of sickle cell disease. This activation process may be enhanced via transfer of cell-free heme within red blood cell (RBC) membrane microparticles into the endothelium. Free heme may also induce initiation of coagulation by promoting the synthesis of both endothelial- and leukocyte-derived tissue factor and thrombin generation. Here, we utilize an endothelialized microfluidic platform to assess sickle RBC adhesion to heme-activated EC in vitro. ECs were seeded into microfluidic devices and the confluent monolayers were treated with pathophysiologically relevant levels of heme. Microchannels functionalized with ECs mimic the intravascular environment and thereby allow the study of various pathological conditions in vitro. RBCs adhered to heme-activated ECs significantly more than unactivated ECs. Here, we report a direct link between heme-driven endothelial activation and RBC adhesion in a patient-specific manner. In patients with a more severe clinical phenotype, as reflected in lactate dehydrogenase, total hemoglobin, and absolute reticulocyte or recent blood transfusions, we found greater RBC adhesion to heme-activated ECs. These findings suggest that RBCs from those patients most likely to experience hemolysis in vivo may also be those RBCs most likely to adhere to heme-activated endothelium. These findings further our understanding of heme-related pathogenesis of endothelial damage in relation to RBC adhesion. We anticipate that the microfluidic approach described herein can potentially find many other applications in studies of adhesion characteristics of blood cells to immobilized ECs.