Metabolic Responses of the Vascular Endothelium to Inflammatory Injury

The endothelial cells that line the vasculature perform essential functions during inflammation. Through interactions with the immune system, endothelial cells can drive both the extent of inflammatory injury and the rate of recovery. Although the signaling cascades that govern these processes have been well studied, the metabolic underpinnings that drive them have not been well studied. This study examines the role of endothelial metabolism in guiding both inflammatory injury and subsequent restoration. First, we examined how endothelial metabolism is reprogrammed by inflammation. We found that in response to treatment with the inflammatory mediator TNFα, endothelial cells upregulate glycolysis through activation of the glycolysis regulatory enzyme PFKFB3. Consistent with this glycolytic activation, TNFα also lead to an increase in cellular ATP, concentrated at the cell periphery. We developed a chemo-genetic tool called RapT, which locally upregulates ATP levels at the cell periphery by inducing translocation of PFKFB3 to the cell membrane on the addition of Rapamycin. Increased peripheral ATP enhances restoration of the endothelial barrier, an important factor in determining recovery. Thus, endothelial cells upregulate restorative glycolysis to drive the repair processes. We next examined how endothelial mitochondria respond to inflammation. In both in vivo and in vitro models, we found that inflamed endothelial cells upregulate mitochondrial degradation through the mitophagy pathway. TNFα also led to the stabilization of the mitophagy inducer Pink1, suggesting that inflammation induced endothelial mitophagy is driven by the Pink1/Parkin pathway. We developed a mouse model in which Pink1 is deleted specifically in endothelial cells, by combining lysosomal delivery of Pink1 sgRNA with endothelial-specific expression of Cas9. Using this mouse model, we observed that deletion of endothelial Pink1 was remarkably protective in an endotoxin-based inflammation model. Additionally, endothelial Pink1 deletion led to a significant decrease in neutrophil infiltration and activation. In response to TNFα or the mitophagy inducer FCCP, endothelial cells released the mitochondrial protein ND6, a pro-inflammatory molecule that activates neutrophil recruitment through activation of formyl-peptide receptors. Thus, endothelial Pink1-mediated mitophagy is a pro-inflammatory process involved in enhancing the neutrophil response.