Accelerated Extracellular Nucleotide Metabolism in Brain Microvascular Endothelial Cells in Experimental Hypercholesterolemia.

Hypercholesterolemia affects the neurovascular unit, including the cerebral blood vessel endothelium. Operation of this system, especially in the context of energy metabolism, is controlled by extracellular concentration of purines, regulated by ecto-enzymes, such as e-NTPDase-1/CD39, ecto-5'-NT/CD73, and eADA. We hypothesize that hypercholesterolemia, via modulation of the activity of nucleotide metabolism-regulating ecto-enzymes, deteriorates glycolytic efficiency and energy metabolism of endothelial cells, which may potentially contribute to development of neurodegenerative processes. We aimed to determine the effect of hypercholesterolemia on the concentration of purine nucleotides, glycolytic activity, and activity of ecto-enzymes in the murine brain microvascular endothelial cells (mBMECs). We used 3-month-old male LDLR-/-/Apo E-/- double knockout mice to model hypercholesterolemia and atherosclerosis. The age-matched wild-type C57/BL6 mice were a control group. The intracellular concentration of ATP and NAD and extracellular activity of the ecto-enzymes were measured by HPLC. The glycolytic function of mBMECs was assessed by means of the extracellular acidification rate (ECAR) using the glycolysis stress test. The results showed an increased activity of ecto-5'-NT and eADA in mBMECs of the hypercholesterolemic mice, but no differences in intracellular concentration of ATP, NAD, and ECAR between the hypercholesterolemic and control groups. The changed activity of ecto-5'-NT and eADA leads to increased purine nucleotides turnover and a shift in their concentration balance towards adenosine and inosine in the extracellular space. However, no changes in the energetic metabolism of the mBMECs are reported. Our results confirm the influence of hypercholesterolemia on regulation of purine nucleotides metabolism, which may impair the function of the cerebral vascular endothelium. The effect of hypercholesterolemia on the murine brain microvascular endothelial cells (mBMECs). An increased activity of ecto-5'-NT and eADA in mBMECs of the LDLR-/-/Apo E-/- mice leads to a shift in the concentration balance towards adenosine and inosine in the extracellular space with no differences in intracellular concentration of ATP. Figure was created with Biorender.com.

The extent of damage to the blood-brain barrier in the hypercholesterolemic LDLR-/-/Apo E-/- double knockout mice depends on the animal's age, duration of pathology and brain area.

One of the effects of hypercholesterolemia (Hch) exerted on the central nervous system (CNS) is damage to the blood-brain barrier (BBB). Increased permeability of BBB results from structural changes in the vascular wall, loss of the tight junctions and barrier function, as well as alterations in the concentration of proteins located in the layers of the vascular wall. These changes occur in the course of metabolic and neurodegenerative diseases. The important role in the course of these processes is attributed to agrin, matrix metalloproteinase-9, and aquaporin-4. In this study, we aimed to determine: 1) the extent of Hch-induced damage to the BBB during maturation, and 2) the distribution of the above-mentioned markers in the vascular wall. Immunohistochemical staining and confocal microscopy were used for vascular wall protein assessment. The size of BBB damage was studied based on perivascular leakage of fluorescently labeled dextran. Three- and twelve-month-old male LDLR-/-/Apo E-/- double knockout mice (EX) developing Hch were used in the study. Age-matched male wild-type (WT) C57BL/6 mice were used as a control group. Differences in the concentration of studied markers coexisted with BBB disintegration, especially in younger mice. A relationship between the maturation of the vascular system and reduction of the BBB damage was also observed. We conclude that the extent of BBB permeability depends on animal age, duration of Hch, and brain region. These may explain different susceptibility of various brain areas to Hch, and different presentation of this pathology depending on age and its duration.