Increase of arginase activity in old apolipoprotein-E deficient mice under Western diet associated with changes in neurovascular unit

Abstract

Aging and atherosclerosis are well-recognized risk factors for cardiac and neurovascular diseases. The Apolipoprotein E deficient (ApoE-/-) mouse on a high-fat diet is a classical model of atherosclerosis, characterized by the presence of atherosclerotic plaques in extracranial vessels but not in cerebral arteries. Increase in arginase activity was shown to participate in vascular dysfunction in the peripheral arteries of atherosclerotic mice by changing the level of nitric oxide (NO). NO plays a key role in the physiological functions of the neurovascular unit (NVU). However, the regulation of arginase expression and activity in the brain was never investigated in association with changes in the NVU, ApoE deficiency and high fat diet.Fourteen-month-old ApoE-/- mice on high-fat diet exhibited deposition of lipids in the NVU, impairment of blood-brain barrier properties, astrogliosis and an increase of aquaporin 4 staining. In association with these changes, brain arginase activity was significantly increased in the old ApoE-/- mice as compared to old wild type mice, with an increase in the level of arginase type I in the blood vessels.In conclusion, aging in this classical mouse model of atherosclerosis induces an increase in the level and activity of arginase I that may impair NO synthesis and contribute to changes in the NVU leading to blood-brain barrier leakage and inflammation.

Figure 1

Quantification of plasma cholesterol, HDL and LDL levels. Each column represents mean ± SEM (n = 5 per group). * P <0.05 as compared to age-matched WT mice and # P <0.05 as compared to young mice of the same genotype.

Figure 2

Lipid deposition in the brain of old ApoE−/− mice. Representative photomicrographs of lipid deposition in the brain of old ApoE−/− mice (A to F) as compared to age-matched WT mice (G to I, n = 9 per group). Lipid depositions were observed in the perivascular space adjacent to the lateral ventricles and third ventricle, and in the corpus callosum. A: striatum; B: corpus callosum (CC); C and I: cortex; D and G: lateral ventricle (LV); E and H: third ventricle (3rd V); F: brain vessels (BV). Scale bar = 500 μm in A, 100 μm in B to E and G to I, and 30 μm in F.

Figure 3

BBB changes in cerebral cortex of WT and ApoE−/− mice. A) Age dependent blood brain barrier impairment in ApoE−/− mice. Quantification of IgG leakage in brain sections stained with anti-IgG antibody (n = 7 to 9 per group). Each column represents mean ± SEM. # P <0.05 young ApoE−/− mice vs old ApoE−/− mice and P <0.05 old WT mice vs old ApoE−/− mice. B) Claudin 5 staining (arrow heads) along the cortical blood vessels in the old WT and ApoE−/− mice. The lumen is marked with a star. Claudin 5 staining is decreased in old ApoE−/− mice. C) Quantification of the intensity of the Claudin 5 immunoreactivity (IR) in cortical layers of young and old WT and ApoE−/− mice. Each column represents mean ± SEM (n = 4 per group). * P <0.05 young ApoE−/− mice vs old ApoE−/− mice.

Figure 4

Neurovascular alterations in cerebral cortex of WT and ApoE−/− mice. A) GFAP staining in the different brain cortical layers in young and old WT and ApoE−/− mice. Immunohistochemistry for GFAP is distributed in a gradient in cortical cross-sections of young and old WT and ApoE−/− mice as previously described. GFAP staining was increased with aging regardless of the mice strain. The increase is higher in the old ApoE−/− mice, with presence of GFAP positive astrocytes throughout all of the cortical layers. Scale bar = 50 μm. B) Quantification of the intensity of the GFAP staining in cortical layers of young and old WT and ApoE−/− mice. Each column represents mean ± SEM (n = 4 per group). * P <0.05 young WT mice vs old ApoE−/− mice and old WT mice vs old ApoE−/− mice. C) AQP4 immunohistochemistry in the cortex of the young and old WT and ApoE−/− mice. AQP4 expression is not increased in old WT. In contrast, in ApoE−/−, the AQP4 labeling is increased in the astrocyte endfeet in contact with the blood vessels. Bar = 40 μm. D) Quantification of the intensity of the AQP4 staining in cortex cross-section of young and old WT and ApoE−/− mice. Each column represents mean ± SEM (n = 4 per group). * P <0.05 ApoE−/− young vs ApoE−/− old. E) Counting of the number of positive laminin staining did not show any significant differences between the WT and ApoE−/− mice and between young and old (n = 6 per group). F) Counting of the number of positive NeuN cells did not show any significant difference between the WT and ApoE−/− mice and between young and old (n = 6 per group).

Figure 5

Arginase activity and expression in the brain of young and old WT and ApoE−/− mice. A) The highest level of arginase activity was found in old ApoE−/− mice. Each column represents mean ± SEM (n = 10 per group). * P <0.05 young WT vs old WT, young ApoE−/− vs old ApoE−/−, old ApoE−/− vs young WT, and old ApoE−/−vs old WT. B) Representative Western blots of ArgI (100 and 70 kDa bands) and quantification. Both bands of ArgI protein were significantly increased with aging in WT and ApoE−/− mice. In addition, the ArgI 100 kDa band was significantly up-regulated in ApoE−/− mice as compared to both age-matched WT. Each column represents mean ± SEM (n = 6 per group). * P <0.05 young WT vs old WT, young ApoE−/− vs old ApoE−/−, young WT vs young ApoE−/−, old ApoE−/− vs young WT, and old ApoE−/− vs old WT. C) Representative Western blots of ArgII (70 kDa band) and quantification. No significant changes in ArgII level were observed between WT and ApoE−/− mice (n = 6 per group).

Figure 6

ArgI localization in the cerebral cortex of WT and ApoE−/− mice. Representative images of ArgI staining (in red) in frontal cerebral cortex from young (A, E, I, M) and old (B, F, J, N) WT mice and from young (C, G, K, O) and old (D, H, L, P) ApoE−/− mice (n = 6 per group). Single staining is shown in the first (A-D) and last row (M-P). Arrows show a vascular localization. Arrowheads show a nuclear localization in extravascular cells. Double staining with NeuN (in blue) and laminin (in green) are shown on the second and third row, respectively. Scale bar = 100 μm in A to L and 40 μm in M to P.

Figure 7

Diagram of the potential molecular mechanisms. The combination of aging and ApoE deficiency is associated with an increase of arginase activity with no changes in eNOS. Higher arginase activity would lead to a decrease of NO availability caused by a depletion of L-arginine. One of the possible consequences would be a decrease in blood brain perfusion and facilitated lipid deposition around the microvessels. The increase of arginase activity could also contribute to an increase of urea and induce changes in the NVU homeostasis. The changes in the NVU and neuroinflammation, characterized by the opening of the BBB and increase of GFAP and AQP4 expression, might be a consequence of the decrease of NO, decrease of blood brain perfusion, and increase of urea.