Age-related decrease in cerebrovascular-derived neuroprotective proteins: effect of acetaminophen

Abstract

As the population ages, the need for effective methods to maintain brain function in older adults is increasingly pressing. Vascular disease and neurodegenerative disorders commonly co-occur in older persons. Cerebrovascular products contribute to the neuronal milieu and have important consequences for neuronal viability. In this regard vascular derived neuroprotective proteins, Such as vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), and pituitary adenylate cyclase activating peptide (PACAP) are important for maintaining neuronal viability, especially in the face of injury and disease. The objective of this study is to measure and compare levels of VEGF, PEDF and PACAP released from isolated brain microvessels of Fischer 344 rats at 6, 12, 18, and 24 months of age. Addition of acetaminophen to isolated brain microvessels is employed to determine whether this drug affects vascular expression of these neuroprotective proteins. Experiments on cultured brain endothelial cells are performed to explore the mechanisms/mediators that regulate the effect of acetaminophen on endothelial cells. The data indicate cerebrovascular expression of VEGF, PEDF and PACAP significantly decreases with age. The age-associated decrease in VEGF and PEDF is ameliorated by addition of acetaminophen to isolated brain microvessels. Also, release of VEGF, PEDF, and PACAP from cultured brain endothelial cells decreases with exposure to the oxidant stressor menadione. Acetaminophen treatment upregulates VEGF, PEDF and PACAP in brain endothelial cells exposed to oxidative stress. The effect of acetaminophen on cultured endothelial cells is in part inhibited by the selective thrombin inhibitor hirudin. The results of this study suggest that acetaminophen may be a useful agent for preserving cerebrovascular function. If a low dose of acetaminophen can counteract the decrease in vascular-derived neurotrophic factors evoked by age and oxidative stress, this drug might be useful for improving brain function in the elderly.

Figure 1

Brain microvessels isolated from Fischer 344 rats (6, 12, 18 and 24 months of age) were incubated in serum-free media with 1% LAH for 8 h. Conditioned media were analyzed by ELISA to measure (a) VEGF, (b) PEDF and (c) PACAP. Results are expressed as means ± SD from three separate experiments performed in triplicate. *p<0.05, **p<0.01, ***p<0.001 vs. 6 months of age.

Figure 2

Brain microvessels isolated from Fischer 344 rats (6, 12, 18 and 24 months of age) were incubated in serum-free media with 1% LAH or in serum-free media plus 1% LAH and 100 µM of acetaminophen (APAP) for 8 h. Conditioned media were analyzed by ELISA to measure (a) VEGF and (b) PEDF . Results are expressed as means ± SD from three separate experiments performed in triplicate. #p<0.05, ##p<0.01 vs. without APAP.

Figure 3

Cultured brain endothelial cells were incubated with the oxidant stressor menadione (25 µM) in the presence or absence of increasing concentrations of acetaminophen (APAP; 0–300 µM) for 3 h and (a) VEGF, (b) PEDF and (c) PACAP released into the media determined by ELISA. Results are expressed as the mean ± SD from three separate experiments performed in triplicate. ##p<0.01, ###p<0.001 vs. cells without APAP or mendione; **p<0.01, ***p<0.001 vs. cells incubated plus mendione with and without APAP.

Figure 4

Brain endothelial cells were incubated with increasing concentrations of acetaminophen (APAP, 100—1000 µM) with and without thrombin inhibitor hirudin (12 Units/ml) for 24 h. Total RNA extracted was reverse transcribed and amplified with thrombin primers. Loading equivalency was confirmed using GAPDH. Relative intensity of the bands is shown in bar graph. Results are expressed as means ± SD from three separate experiments performed in triplicate. *p<0.05, **p <0.01 vs. control (no APAP or hirudin).

Figure 5

Brain endothelial cells were incubated with increasing concentrations of acetaminophen (APAP, 100—1000 µM) with and without thrombin inhibitor hirudin (12 Units/ml) for 24 h. Total RNA extracted was reverse transcribed and amplified with VEGF primers. Loading equivalency was confirmed using GAPDH. Relative intensity of the bands is shown in bar graph. Results are expressed as means ± SD from three separate experiments performed in triplicate. **p <0.01, ***p<0.001 vs. control (no APAP or hirudin).