Abstract Introduction Erythrocytes and pulmonary microvascular endothelial cells (PMVECs) are in continuous contact throughout lung microcirculation. Although PMVECs are not professional phagocytes, they can engulf erythrocytes, potentially leading to heme-mediated toxicity and barrier dysfunction. The endothelial glycocalyx-a thin, gel-like layer-generates an electrostatic repulsive force that prevents excessive erythrophagocytosis (EPC). The hexosamine biosynthetic pathway in PMVECs supports glycocalyx maintenance and is promoted by fructose. 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase (PFKFB3) acts as a molecular switch that suppresses fructose metabolism in PMVECs. However, how pulmonary circulation handles fructose and how PFKFB3 influences the hexosamine-glycocalyx axis, EPC, and endothelial barrier integrity remain unknown. Methods We characterized fructose metabolism in erythrocytes and PMVECs isolated from critically ill patients and rats, respectively. Stable isotope of U-13C D-glucose or D-fructose was performed by mass spectrometry on erythrocytes and on wild-type and PFKFB3 knockout PMVECs under normoxic or hypoxic conditions at 24 hours. Erythrocytes were labeled with PKH26 fluorescent dye and cocultured with PMVECs to quantify EPC. To assess physiological effects, ex-vivo isolated lung perfusion was performed using glucose- or fructose-containing perfusate. PKH26-labeled erythrocytes were circulated to evaluate EPC, and lung weight was measured following active or heat-inactivated heparinase treatment to determine the impact of glycocalyx degradation. Results Erythrocytes avidly utilized fructose and activated the sorbitol pathway, which converts glucose to fructose and provides intermediates for the hexosamine pathway. In PMVECs, PFKFB3 knockout enhanced fructose-mediated glycolysis and increased levels of hexosamine pathway final product UDP-N-acetylglucosamine. In PMVECs-erythrocyte coculture, PFKFB3 knockout reduced EPC, which may suggest enhanced hexosamine pathway activity supports glycocalyx maintenance. In ex vivo isolated lung perfusion experiments, EPC was abundant within pulmonary microcirculation under baseline conditions. In glucose-rich perfusate, heparinase-induced glycocalyx shedding increased lung permeability, whereas fructose-rich perfusate prevented this increase. Conclusion PFKFB3 suppresses the hexosamine pathway, potentially compromising glycocalyx maintenance and increasing erythrophagocytosis and lung permeability. This abstract is funded by: HL160988, HL148069
Yendrapalli et al. (Fri,) studied this question.