The energy source for fibrinogen-induced formation of functional caveolae

Traumatic brain injury (TBI) results in an increase of blood fibrinogen (Fg) content, called hyperfibrinogenemia (HFg). HFg increases protein extravasation through the endothelial cell (EC) layer of blood vessels mainly through caveolar transcytosis (CT) and results in Fg deposition in brain’s interstitium, leading to neurodegeneration. We have shown that HFg-induced protein CT involves the modulation of de novo sphingolipid synthesis. However, the energy source used for caveolar motility and the dependent protein transcytosis is unknown. Mouse brain ECs (MBECs) were treated with phosphate buffered saline (control), 2 mg/ml of Fg (Fg2), 4 mg/ml of Fg (Fg4), 4 mg/ml of Fg in the presence of myriocin at 125nM (Fg4/Myriocin), fumonisin at 2.5µM (Fg4/Fumonisin), myriocin and fumonisin (Fg4/Myriocin/Fumonisin), or bedaquiline at 10µM (Fg4/Bdq). While myriocin is a specific inhibitor of serine palmitoyltransferase, fumonisin is an inhibitor of ceramide synthase, and Bdq is an inhibitor of ATP synthase. A basal glycolysis rate was assessed with a Seahorse Extracellular Flux (XF) Glycolytic Rate Test Kit using the Seahorse Bioscience XF24 Extracellular Flux Analyzer. The RealTime-Glo TM Extracellular ATP assay was performed to assess extracellular ATP with a BioTek Synergy H1 microplate reader. Intracellular ATP was calculated as a difference between the values of the total ATP and the extracellular ATP. Caveolae in ECs were defined by colocalization of antibodies against caveolin 1 and plasmalemmal vesicle-associated protein-1. We found that Fg dose-dependently reduced production of intracellular ATP and drastically decreased the basal glycolysis rate in the MBECs, which were ameliorated in the presence of myriocin and to a lesser extent fumonisin. Myriocin and fumonisin combined were more efficient in ameliorating the Fg-induced reduction in the basal glycolysis rate, than when used individually. We also found that Fg dose-dependently increased the formation of functional caveolae, which was reduced in the presence of Bdq. These results indicate that Fg-induced formation of functional caveolae requires ATP as a source of energy, leading to ATP depletion, and causing a bioenergetic shift in the EC. Supported by grants from the National Institutes of Health: HL146832 and NS129979; University of South Florida College of Medicine: HSC-18330 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.