Oxygen-tunable endothelialized microvascular chip to assess hypoxia-reperfusion in sickle cell disease.

Abstract

A better understanding of hypoxia reperfusion (H/R) injury is needed to gain deeper insight into the mechanisms driving sickle cell disease (SCD) pathophysiology. Existing <i>in vivo</i> and <i>in vitro</i> models have yet to fully explain H/R, which is typically associated with harmful inflammatory processes but has also been linked to a protective effect ameliorating subsequent severe vaso-occlusion. To address this need, we developed a novel microfluidic platform that includes three-dimensional endothelial-lined microchannels within an oxygen-tunable environment. These features enable simulation of H/R, red blood cell (RBC) sickling, and vaso-occlusion on-chip. The endothelial network cultured on-chip is physiologically relevant and expresses crucial microvascular features such as 3D lumen structure and expression of functional endothelial markers. We utilized this platform to perform an occlusion assay, evaluating the effects of hypoxic preconditioning on RBC-endothelial interactions contributing to occlusion. Our results demonstrate that both sustained mild hypoxia and cyclic hypoxia endothelial treatment reduce the likelihood of SCD occlusion on-chip. Specifically, average vaso-occlusion rates of 8.89% and 11.78% were observed among endothelialized devices preconditioned to cyclic and sustained hypoxia, respectively, compared to 57.93% and 55.05% for the control groups. Additionally, we leveraged RNA sequencing to identify differential regulation of specific genes contributing to this protective outcome. Of note, hypoxia preconditioning resulted in significant modulation of <i>CYBB</i>, <i>RELN</i>, and <i>SERPINA1</i>. These results offer a better understanding of the mechanistic changes affecting the endothelium during H/R and also offer potential targets for further exploration and therapeutic intervention in SCD.