Development of a versatile, endothelialized microfluidic platform to investigate vaso-occlusion in sickle cell disease

Abstract

Sickle cell disease (SCD) is an inherited autosomal recessive disorder caused by a mutation in the hemoglobin β gene, leading to hemoglobin polymerization, red blood cell sickling, and a cascade of pathophysiological events that culminate in a painful vaso-occlusive crisis (VOC). Despite its discovery over a century ago, treatment options for the millions of SCD patients worldwide remain remarkably limited, thus highlighting the need for a better understanding of its underlying mechanisms. Recently, increased attention has been directed to hypoxia/ischemia reperfusion injury as a driving mechanism of VOC in SCD pathology. While hypoxia and reperfusion are classically thought to induce harmful inflammatory responses associated with apoptosis and symptom onset, a remarkable trait of reperfusion injury is that certain mild or cyclic periods of low oxygen may instead provoke a conditioning effect that ameliorates subsequent severe reperfusion injury. A better understanding of the conditioning effect exerted by hypoxia reperfusion on the endothelium may inform our overall understanding of SCD pathophysiology and offer insight towards therapeutic intervention. Existing in vivo and in vitro models do not reliably inform clinical trials, consequently more physiologically relevant models are needed to explore these fundamental questions about SCD pathophysiology. Therefore, we developed a microfluidic platform that includes three-dimensional endothelial-lined microchannels with physiologically relevant shear stresses in an oxygen-tunable environment. These features enable simulation of hypoxia reperfusion and vasoocclusion on-chip. We utilized fluorescence microscopy to validate the biological relevance of the endothelial vascular network on-chip, demonstrating appropriate lumen formation, alignment under flow, and cellular function. This microfluidic platform is unique in its ability to recapitulate many of the physiologic components of SCD. Its applications are two-fold: (1) its use as an occlusion assay that quantifies the occlusion incidence of patient blood samples through microvasculature, and (2) its potential to leverage next generation sequencing to explore genomic changes to the endothelium. Using the platform, we demonstrated that endothelial hypoxic preconditioning initiates a protective effect that reduces incidence of sickle red blood cell occlusion on-chip. We observed average vaso-occlusion rates of 8.89% and 11.78% after cyclic and sustained hypoxia preconditioning compared to 57.93% and 55.05% for atmospheric and physiologic controls, respectively. Moreover, we investigated endothelial gene regulation via RNA sequencing to identify pathways of interest and specific genes that may directly contribute to this protective result. These results offer a better understanding of the mechanistic changes affecting the endothelium during hypoxia reperfusion injury and also support the hypothesis that hypoxia preconditioning may offer a protective effect against VOC in SCD.