HYPERCHOLESTEROLEMIA IMPACT ON PURINERGIC COMMUNICATION, INFLAMMATORY REACTION, AND BBB PERMEABILITY - RESULTS OF <EM>IN VIVO</EM> STUDIES USING DOUBLE-KNOCK-OUT APOE<SUP>-/-</SUP>/LDLR<SUP>-/-</SUP> MICE MODEL

Hypercholesterolemia is a metabolic disorder associated with increased risk of cardiovascular and neurodegenerative diseases. Elevated cholesterol levels disrupt neurovascular unit function, impair blood-brain barrier (BBB) integrity, and promote neuroinflammation. Purinergic signaling, mediated by extracellular adenine nucleotides and enzymes such as ecto-nucleoside triphosphate diphosphohydrolase (e-NTPDase), ecto-5’-nucleotidase (ecto-5’-NT), and ecto-adenosine deaminase (eADA), plays a key role in maintaining energy metabolism and regulating inflammatory responses in brain endothelial cells.
This study investigated the effects of chronic hypercholesterolemia on purinergic signaling, energy metabolism, BBB permeability, and inflammatory response in ApoE^-/-/LDLR^-/- double knockout mice.
Three-month-old ApoE^-/-/LDLR^-/- mice and age-matched C57BL/6 controls were used. Brain microvascular endothelial cells were analyzed for extracellular adenine nucleotide pathway activity using HPLC. BBB integrity was assessed with immunofluorescent detection of isothiocyanate-dextran (FD40). Pro-inflammatory cytokines IL-1β and IL-6 were quantified using ELISA. Glycolytic function and energy metabolism were evaluated with the Seahorse XFp analyzer.
Hypercholesterolemic mice exhibited elevated eADA activity, increased FD40 leakage indicating BBB disruption, and higher IL-1β and IL-6 levels compared to controls. Enhanced activity of extracellular enzymes promoted a metabolic shift toward inosine synthesis, suggesting a neuroprotective and immunomodulatory response. Glycolytic function of endothelial cells was maintained, reflecting compensatory mechanisms to preserve cellular energy homeostasis.
Chronic hypercholesterolemia disrupts BBB integrity, alters purinergic signaling, and initiates a pro-inflammatory microenvironment in the brain. The observed modulation of adenine nucleotide metabolism toward inosine synthesis may protect endothelial cells against damage and death, highlighting potential therapeutic strategies to prevent cerebrovascular complications in hypercholesterolemia.