Annick Drouin

NTPDase1 (CD39) controls nucleotide-dependent vasoconstriction in mouse

AIMS Extracellular nucleotides are vasoactive molecules. The concentrations of these molecules are regulated by ectonucleotidases. In this study, we investigated the role of the blood vessel ectonucleotidase NTPDase1, in the vasoconstrictor effect of nucleotides using Entpd1(-/-) mice. METHODS AND RESULTS Immunofluorescence, enzyme histochemistry, and HPLC analysis were used to evaluate both NTPDase expression and activity in arteries and isolated vascular smooth muscle cells (VSMCs). Vascular reactivity was evaluated in vitro and mean arterial blood pressure was recorded in anesthetized mice after nucleotide i.v. infusion. Expression of nucleotide receptors in VSMCs was determined by RT-PCR. Entpd1(-/-) mice displayed a dramatic deficit of nucleotidase activity in blood vessel wall in situ and in VSMCs in comparison to control mice. In aortic rings from Entpd1(-/-) mice, UDP and UTP induced a potent and long-lasting constriction contrasting with the weak response obtained in wild-type rings. This constriction occurred through activation of P2Y(6) receptor and was independent of other uracil nucleotide-responding receptors (P2Y(2) and P2Y(4)). UDP infusion in vivo increased blood pressure and this effect was potentiated in Entpd1(-/-) mice. In addition, pressurized mesenteric arteries from Entpd1(-/-) mice displayed an enhanced myogenic response, consistent with higher local concentrations of endogenously released nucleotides. This effect was inhibited by the P2 receptor antagonist RB-2. CONCLUSION NTPDase1 is the major enzyme regulating nucleotide metabolism at the surface of VSMCs and thus contributes to the local regulation of vascular tone by nucleotides.

Catechin treatment improves cerebrovascular flow-mediated dilation and learning abilities in atherosclerotic mice.

Severe dyslipidemia and the associated oxidative stress could accelerate the age-related decline in cerebrovascular endothelial function and cerebral blood flow (CBF), leading to neuronal loss and impaired learning abilities. We hypothesized that a chronic treatment with the polyphenol catechin would prevent endothelial dysfunction, maintain CBF responses, and protect learning abilities in atherosclerotic (ATX) mice. We treated ATX (C57Bl/6-LDLR(-/-)hApoB(+/+); 3 mo old) mice with catechin (30 mg · kg(-1) · day(-1)) for 3 mo, and C57Bl/6 [wild type (WT), 3 and 6 mo old] mice were used as controls. ACh- and flow-mediated dilations (FMD) were recorded in pressurized cerebral arteries. Basal CBF and increases in CBF induced by whisker stimulation were measured by optical coherence tomography and Doppler, respectively. Learning capacities were evaluated with the Morris water maze test. Compared with 6-mo-old WT mice, cerebral arteries from 6-mo-old ATX mice displayed a higher myogenic tone, lower responses to ACh and FMD, and were insensitive to NOS inhibition (P < 0.05), suggesting endothelial dysfunction. Basal and increases in CBF were lower in 6-mo-old ATX than WT mice (P < 0.05). A decline in the learning capabilities was also observed in ATX mice (P < 0.05). Catechin 1) reduced cerebral superoxide staining (P < 0.05) in ATX mice, 2) restored endothelial function by reducing myogenic tone, improving ACh- and FMD and restoring the sensitivity to nitric oxide synthase inhibition (P < 0.05), 3) increased the changes in CBF during stimulation but not basal CBF, and 4) prevented the decline in learning abilities (P < 0.05). In conclusion, catechin treatment of ATX mice prevents cerebrovascular dysfunctions and the associated decline in learning capacities.

Flow-Induced Dilation Is Mediated by Akt-Dependent Activation of Endothelial Nitric Oxide Synthase-Derived Hydrogen Peroxide in Mouse Cerebral Arteries

BACKGROUND AND PURPOSE Endothelial nitric oxide synthase produces superoxide under physiological conditions leading to hydrogen peroxide (H(2)O(2)) -dependent dilations to acetylcholine in isolated mouse cerebral arteries. The purpose of this study was to investigate whether H(2)O(2) was involved in flow-mediated dilation (FMD). METHODS Cerebral arteries were isolated from 12+/-2-week-old C57Bl/6 male mice. FMD (0 to 10 microL/min, 2-microL step increase at constant internal pressure) was induced in vessels preconstricted with phenylephrine (30 micromol/L). Simultaneously to diameter acquisition, H(2)O(2) or nitric oxide production was detected by the fluorescent dyes CMH(2)CFDA or 4,5-diaminofluorescein diacetate, respectively. Results are expressed as mean+/-SEM of 6 to 8 mice. RESULTS FMD (at 10 microL/min, 25+/-3% of maximal diameter) was prevented (P<0.05) by endothelium removal (6+/-1%) or endothelial nitric oxide synthase inhibition with N-nitro-L-arginine (11+/-1%) but not by the specific nitric oxide scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl3-oxide (24+/-3%). Addition of PEG-catalase and silver diethyl dithio-carbamate (superoxide dismutase inhibitor) reduced (P<0.05) FMD to 10+/-2% and 15+/-1%, respectively. Simultaneously to FMD, H(2)O(2)-associated rise in fluorescence (+133+/-19 a.u.) was prevented by N-nitro-L-arginine, PEG-catalase, and silver diethyl dithio-carbamate (+55+/-10, +64+/-4, and +50+/-10 a.u., respectively; P<0.05). Inhibition of FMD by PEG-catalase was fully restored by the addition of tetrahydrobiopterin, a cofactor of endothelial nitric oxide synthase (23+/-3%); this functional reversal in dilation was associated with the simultaneous increase in nitric oxide-associated fluorescence (+418+/-58 a.u., P<0.05), which was prevented by 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl3-oxide (+93+/-26 a.u.). Akt inhibition with triciribine prevented FMD and H(2)O(2)-associated rise in fluorescence (3+/-1% and +23+/-4% a.u., respectively; P<0.05), but not acetylcholine-induced dilation. CONCLUSIONS In healthy C57Bl/6 mouse cerebral arteries, Akt-dependent activation of endothelial nitric oxide synthase-derived H(2)O(2) mediates flow-dependent dilation.

ROS‐sensitive cytochrome P450 activity maintains endothelial dilatation in ageing but is transitory in dyslipidaemic mice

Risk factors for cardiovascular diseases (CVD) have been proposed to accelerate the vascular endothelial dysfunction that develops during the normal ageing process. The objective of this work was to study the impact of dyslipidaemia (DL) on the dilatory efficacy of the non‐NO/non‐PGI2 endothelium‐derived hyperpolarising factor (EDHF) through maturation and ageing. We isolated and pressurised (80 mmHg) gracilis arterial segments from 3, 12 and 20‐month‐old (m/o) DL mice expressing the human apolipoprotein B‐100 and wild‐type (WT) C57BL/6 mice. EDHF‐dependent dilatations to acetylcholine (ACh) were measured in the presence of L‐NNA (100 μM, NOS inhibitor) and indomethacin (INDO; 10 μM, COX inhibitor). Data are expressed as mean±s.e.m. EDHF‐mediated maximal dilatation of arteries isolated from WT mice declined by 44% with ageing, from 86±3% at 3 months to 66±8% at 12 and 48±4% at 20 months of age (P<0.05). This decline was magnified by DL to 73%, characterised by an early increased efficacy at 3 m/o (95±2%, P<0.05) and a worsening of the dysfunction at 20 m/o (26±2%, P<0.05). 17‐Octadecynoic acid (17‐ODYA), a cytochrome P450/epoxygenase inhibitor, reduced by 56% (P<0.05) ACh‐induced EDHF‐dependent dilatation of arteries isolated from 3 m/o DL – but not WT – mice, an effect of 17‐ODYA disappearing in older DL mice. 17‐ODYA, however, reduced (P<0.05) ACh‐induced EDHF‐dependent dilatation in arteries isolated from 12 m/o WT mice by 35% and from 20 m/o WT mice by 31% (P<0.05). Reactive oxygen species production was increased in arteries isolated from 12 m/o DL mice. The antioxidant N‐acetyl‐L‐cystein (NAC) restored the 17‐ODYA‐sensitive responses in arteries isolated from 12 – but not 20 – m/o DL mice (84±3% from an Emax of 57±8%; P<0.05). NAC did not affect the dilatation of arteries isolated from WT mice. Our data suggest that the decline in EDHF‐dependent dilatation is hastened by DL despite the early expression of a 17‐ODYA‐sensitive pathway increasing the efficacy of the non‐NO/non‐PGI2 endothelium‐dependent dilatation. Acute free radical production contributes to the endothelial dysfunction in the presence of DL only, by abrogating this latter pathway. This 17‐ODYA‐sensitive pathway, however, appears in 12 m/o WT mice and remains active at 20 m/o.

Heart rate-associated mechanical stress impairs carotid but not cerebral artery compliance in dyslipidemic atherosclerotic mice

The cardiac cycle imposes a mechanical stress that dilates elastic carotid arteries, while shear stress largely contributes to the endothelium-dependent dilation of downstream cerebral arteries. In the presence of dyslipidemia, carotid arteries stiffen while the endothelial function declines. We reasoned that stiffening of carotid arteries would be prevented by reducing resting heart rate (HR), while improving the endothelial function would regulate cerebral artery compliance and function. Thus we treated or not 3-mo-old male atherosclerotic mice (ATX; LDLr(-/-):hApoB(+/+)) for 3 mo with the sinoatrial pacemaker current inhibitor ivabradine (IVA), the β-blocker metoprolol (METO), or subjected mice to voluntary physical training (PT). Arterial (carotid and cerebral artery) compliance and endothelium-dependent flow-mediated cerebral dilation were measured in isolated pressurized arteries. IVA and METO similarly reduced (P < 0.05) 24-h HR by ≈15%, while PT had no impact. As expected, carotid artery stiffness increased (P < 0.05) in ATX mice compared with wild-type mice, while cerebral artery stiffness decreased (P < 0.05); this paradoxical increase in cerebrovascular compliance was associated with endothelial dysfunction and an augmented metalloproteinase-9 (MMP-9) activity (P < 0.05), without changing the lipid composition of the wall. Reducing HR (IVA and METO) limited carotid artery stiffening, but plaque progression was prevented by IVA only. In contrast, IVA maintained and PT improved cerebral endothelial nitric oxide synthase-dependent flow-mediated dilation and wall compliance, and both interventions reduced MMP-9 activity (P < 0.05); METO worsened endothelial dysfunction and compliance and did not reduce MMP-9 activity. In conclusion, HR-dependent mechanical stress contributes to carotid artery wall stiffening in severely dyslipidemic mice while cerebrovascular compliance is mostly regulated by the endothelium.

Late chronic catechin antioxidant treatment is deleterious to the endothelial function in aging mice with established atherosclerosis.

Various antioxidants, including polyphenols, prevent the development of atherosclerosis in animal models, contrasting with the failure of antioxidants to provide benefits in patients with established atherosclerosis. We therefore tested in a mouse model the hypothesis that although catechin is atheroprotective in prevention, catechin brings no global vascular protection when initiated after established atherosclerosis, because aging associated with dyslipidemia has induced irreversible dysfunctions. To this end, LDLr(-/-); hApoB(+/+) atherosclerotic (ATX, 9 mo old) and pre-ATX (3 mo old) male mice were treated with catechin (30 mg x kg(-1) x day(-1)) up to 12 mo of age. Vascular function and endothelium/leukocyte interactions were studied at 12 mo old. The renal artery endothelium-dependent dilations were impaired with age whereas adhesion of leukocytes onto the native aortic endothelium was increased (P < 0.05). Aortic oxidative stress [reactive oxygen species (ROS)] increased (P < 0.05) at 3 mo in ATX and at 12 mo in wild-type mice. Aorta mRNA expression of NADPH oxidase increased, whereas that of manganese superoxide dismutase decreased in 12-mo-old ATX mice only. In mice with established ATX, catechin (from 9 to 12 mo) reduced (P < 0.05) by approximately 60% ROS without affecting plaque burden. Notably, catechin worsened endothelial dysfunction and further increased leukocyte adhesion (P < 0.05) in ATX mice. In contrast, the same catechin treatment reversed all age-related dysfunctions in wild-type mice. On the other hand, in pre-ATX mice treated for 9 mo with catechin, plaque burden was reduced by 64% (P < 0.05) and all vascular markers were normalized to the 3-mo-old values. These results demonstrate that an antioxidant treatment is deleterious in mice with established atherosclerosis.