Up-regulation of thromboxane A₂ impairs cerebrovascular eNOS function in aging atherosclerotic mice

Abstract

We previously reported that in healthy mouse cerebral arteries, endothelial nitric oxide synthase (eNOS) produces H₂O₂, leading to endothelium-dependent dilation. In contrast, thromboxane A₂ (TXA₂), a potent pro-oxidant and pro-inflammatory endogenous vasoconstrictor, is associated with eNOS dysfunction. Our objectives were to elucidate whether (1) the cerebrovascular eNOS-H₂O₂ pathway was sensitive to oxidative stress associated with aging and dyslipidemia and (2) TXA₂ contributed to cerebral eNOS dysfunction. Atherosclerotic (ATX = LDLR(-/-); hApoB(+/+)) and wild-type (WT) control mice were used at 3 and 12 months old (m/o). Three-m/o ATX mice were treated with the cardio-protective polyphenol catechin for 9 months. Dilations to ACh and the simultaneous eNOS-derived H₂O₂ production were recorded in isolated pressurized cerebral arteries. The age-associated decrease in cerebral eNOS-H₂O₂ pathway observed in WT was premature in ATX mice, decreasing at 3 m/o and abolished at 12 m/o. Thromboxane synthase inhibition by furegrelate increased dilations at 12 months in WT and at 3 and 12 months in ATX mice, suggesting an anti-dilatory role of TXA₂ with age hastened by dyslipidemia. In addition, the non-selective NADP(H) oxidase inhibitor apocynin improved the eNOS-H₂O₂ pathway only in 12-m/o ATX mice. Catechin normalized the function of this pathway, which became sensitive to L-NNA and insensitive to furegrelate or apocynin; catechin also prevented the rise in TXA₂ synthase expression. In conclusion, the age-dependent cerebral endothelial dysfunction is precocious in dyslipidemia and involves TXA₂ production that limits eNOS activity. Preventive catechin treatment reduced the impact of endogenous TXA₂ on the control of cerebral tone and maintained eNOS function.

Fig. 1

Catechin treatment reduces aortic oxidative stress. Effect of 9-month catechin treatment on a aortic and b cerebrovascular superoxide production quantified by DHE staining (n=4, in triplicate). Data are mean±SEM. ^*p<0.05 versus 3-m/o WT, ^†p<0.05 versus 3-m/o ATX, ^‡p<0.05 versus 12-m/o WT, ^§p<0.05 versus 12-m/o ATX

Fig. 2

Cerebral eNOS dysfunction with aging and atherosclerosis. a Effect of eNOS, COX, and TXA₂ synthase inhibition with L-NNA, indomethacin (INDO), and furegrelate (FUR), respectively, on cerebral myogenic tone (%) in 3- and 12-m/o WT and ATX mice treated or not with catechin (CAT). Effect of aging, atherosclerosis (ATX), and catechin treatment for 9 months (ATX+CAT) on b maximal endothelial-dependent dilations to ACh (10 μM) and c the associated eNOS–H₂O₂ production in cerebral arteries isolated from 3- and 12-m/o WT and ATX mice. Data are mean±SEM (n=8). ^*p< 0.05 versus 3-m/o WT, ^†p<0.05 versus 12-m/o WT, ^‡p<0.05 versus 12-m/o ATX, ^§p<0.05 versus MT

Fig. 3

Aging is associated with cerebral endothelial dysfunction. Effect of N-nitro-L-arginine (L-NNA; 10 μmol/l), indomethacin (INDO; 10 μmol/l), L-NNA combined with INDO, furegrelate (FUR; 10 μmol/l), and apocynin (APO; 10 μmol/l) on a dose–response curve to ACh, b maximal endothelium-dependent dilations to ACh, and c the associated eNOS-derived H₂O₂ production in cerebral arteries isolated from 3- and 12-m/o WT mice (n=6). Data are mean±SEM. ^ap<0.05 versus control, *p<0.05 versus 3-m/o WT

Fig. 4

Catechin treatment prevents cerebral endothelial dysfunction associated with aging and dyslipidemia. Effect of N-nitro-L-arginine (L-NNA; 10 μmol/l), indomethacin (INDO; 10 μmol/l), L-NNA combined with INDO, furegrelate (FUR; 10 μmol/l), apocynin (APO; 10 μmol/l), and catechin (CAT) treatment on a dose–response curve to ACh, b maximal endothelium-dependent dilations to ACh, and c the associated eNOS-derived H₂O₂ production in cerebral arteries isolated from 3- and 12-m/o ATX mice treated or not with CAT. Data are mean±SEM (n=6). ^ap<0.05 versus control, ^*p<0.05 versus 3-m/o WT, ^†p<0.05 versus 3-m/o ATX, ^‡p<0.05 versus 12-m/o WT, ^§p<0.05 versus 12-m/o ATX

Fig. 5

Evolution of the contribution of eNOS–H₂O₂ as a dilatory factor and TXA₂ as an anti-dilatory factor with age and severe dyslipidemia. The contribution of eNOS activity to the dilation induced by ACh (10 μM) is reported as the amplitude of the control dilation (%) minus the amplitude of the L-NNA-sensitive dilation (%); the inhibitory effect of TXA₂ on the dilatory response induced by ACh is reported as the amplitude of the control dilation (%) minus the amplitude of the FUR-sensitive dilation (%). Data are mean±SEM (n=6). ^*p<0.05 versus 3-m/o WT, ^§p<0.05 versus 12-m/o ATX

Fig. 6

Catechin treatment prevents cerebral up-regulation of TXA₂ synthase. Effect of aging, atherosclerosis, and catechin (CAT) treatment on whole brain vessels protein expression of a PGI₂ synthase and b TXA₂ synthase of wild-type (WT) and atherosclerotic (ATX) mice. Protein expressions are normalized to GAPDH protein expression (PGI₂ synthase) or to α smooth muscle actin protein expression (TXA₂ synthase) (n=5). Data are mean±SEM. ^*p<0.05 versus 3-m/o WT, ^†p<0.05 versus 12-m/o WT, ^‡p<0.05 versus 3-m/o ATX, ^§p<0.05 versus 12-m/o ATX

Fig. 7

Schematic representation of the effects of age and severe dyslipidemia on the regulation of the cerebrovascular function and the preventive effects of catechin. Both aging and dyslipidemia favor the contribution of contractile TXA₂ which compromises endothelium-dependent cerebral dilation mediated through eNOS-derived H₂O₂. Aging and dyslipidemia are also associated with a rise in ROS, potentially originating from NADP(H) oxidase, which further impairs endothelial-dependent dilation via inhibition of the non-NO and non-PGI₂ component of the dilation, likely cytochrome P450-derived EDHF. Chronic treatment with the cardio-protective polyphenol catechin preserves eNOS–H₂O₂ pathway, limits the implications of TXA₂, and potentially that of NADP(H) oxidase