Citations

We tested hypothesis that activation of the prostacyclin (PGI 2 ) receptor (IP receptor) signaling pathway in cerebral microvessels plays an important role in the metabolism of amyloid precursor protein (APP). In human brain microvascular endothelial cells activation of IP receptor with the stable analogue of PGI 2 , iloprost, stimulated expression of amyloid precursor protein and a disintegrin and metalloprotease 10 (ADAM10), resulting in an increased production of the neuroprotective and anticoagulant molecule, soluble APPα (sAPPα). Selective agonist of IP receptor, cicaprost, and adenylyl cyclase activator, forskolin, also enhanced expression of amyloid precursor protein and ADAM10. Notably, in cerebral microvessels of IP receptor knockout mice, protein levels of APP and ADAM10 were reduced. In addition, iloprost increased protein levels of peroxisome proliferator-activated receptor δ (PPARδ) in human brain microvascular endothelial cells. PPARδ-siRNA abolished iloprost-augmented protein expression of ADAM10. In contrast, GW501516 (a selective agonist of PPARδ) upregulated ADAM10 and increased production of sAPPα. Genetic deletion of endothelial PPARδ (ePPARδ −/− ) in mice significantly reduced cerebral microvascular expression of ADAM10 and production of sAPPα. In vivo treatment with GW501516 increased sAPPα content in hippocampus of wild type mice but not in hippocampus of ePPARδ −/− mice. Our findings identified previously unrecognized role of IP-PPARδ signal transduction pathway in the production of sAPPα in cerebral microvasculature.

AbstractIn this study, we tested the hypothesis that reduced bioavailability of tetrahydrobiopterin (BH4) is a major mechanism responsible for pathogenesis of endothelial dysfunction in cerebral microvessels of transgenic mice expressing the Swedish double mutation of human amyloid precursor protein (APP) (Tg2576 mice). Endothelial nitric oxide synthase (eNOS) protein expression was significantly increased in cerebral vasculature of Tg2576 mice. In contrast, bioavailability of BH4 was significantly reduced (p < 0.05). Moreover, superoxide anion production was increased in cerebral microvessels of Tg2576 mice (p < 0.05). Incubation with NOS inhibitor, Nω‐nitro‐L‐arginine methyl ester, decreased superoxide anion indicating that uncoupled eNOS is most likely the source of superoxide anion. Increasing BH4 bioavailability either exogenously by BH4 supplementation or endogenously by treatment with the selective peroxisome proliferator‐activated receptor – delta activator GW501516 (2 mg/kg/day, 14 days) attenuated eNOS uncoupling and decreased superoxide anion production in cerebral microvessels of Tg2576 mice (p < 0.05). Treatment with GW501516 restored the biological activity of endothelial nitric oxide in cerebral microvessels of Tg2576 mice, as indicated by the increased nitrite/nitrate content and 3,5‐cyclic guanosine monophosphate levels (p < 0.05). Our studies indicate that sub‐optimal BH4 bioavailability in cerebral vasculature is an important contributor to oxidant stress and endothelial dysfunction in Tg2576 mouse model of Alzheimer's disease. imageExisting evidence suggests that Aβ peptides‐induced up‐regulation of expression and activity of NADPH oxidase causes increased production of superoxide anion (.O2−). .O2− can also be converted to hydrogen peroxide (H2O2) by enzymatic activity of superoxide dismutase (SOD) or spontaneous dismutation. Elevation of .O2− and H2O2 might cause oxidation of tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2) and subsequent uncoupling of endothelial nitric oxide synthase (eNOS) (a) thus reducing levels of nitric oxide (NO) and 3′,5′‐cyclic guanosine monophosphate (cGMP). Supplementation of BH4 or activation of PPARδ prevents detrimental effects of eNOS uncoupling by restoring bioavailability of BH4 and scavenging of .O2−, respectively (b). Activation of PPARδ also increases expression of catalase thereby inactivating H2O2. Generation of H2O2 by uncoupled eNOS in cerebral microvessels of Tg2576 mice is hypothetical.

The present study was designed to determine the effect of recombinant inducible nitric oxide (NO) synthase (iNOS) gene expression on vasomotor function in cerebral arteries. Isolated canine basilar arteries were exposed ex vivo (30 min at 37°C) to an adenoviral vector [107, 108, or 109plaque-forming units (pfu)/ml] encoding either the iNOS gene or the β-galactosidase reporter gene. Twenty-four hours after transduction, Western blot analysis demonstrated expression of iNOS protein only in iNOS (109pfu/ml)-transduced arteries. Immunohistochemical analysis localized iNOS expression predominantly in adventitia. Vascular reactivity of isolated basilar arteries was studied by isometric force recording. Concentration-response curves to UTP (10−9–10−3M) and diethylaminodiazen-1-ium-1,2-dioate (10−10–10−5M) were significantly shifted to the right in iNOS gene (109pfu/ml)-transduced rings compared with control and β-galactosidase-transduced rings ( P < 0.05, n = 5–6). Endothelium-dependent relaxation to bradykinin was significantly attenuated in iNOS-transduced rings ( P < 0.001, n = 8). The basal level of cGMP and superoxide anion (O[Formula: see text]·) production were elevated in iNOS-transduced rings ( P < 0.05, n = 7 for cGMP; P < 0.01, n = 6–9 for O[Formula: see text]· production). Our results suggest that expression of recombinant iNOS in cerebral arteries reduces vasomotor reactivity to both vasoconstrictor and vasodilator agonists. Attenuation of contractions is most likely due to functional antagonism between UTP and cGMP. Reduction of endothelium-dependent relaxation to bradykinin appears to be mediated in part by reduced reactivity of smooth muscle cells to NO.

AbstractThis study was designed to determine whether treatment with erythropoietin (EPO) could protect cerebral microvasculature against the pathological consequences of endothelial nitric oxide (NO) synthase uncoupling. Wild‐type and GTP cyclohydrolase I (GTPCH‐I)‐deficient hph1 mice were administered EPO (1000 U/kg/day, s.c., 3 days). Cerebral microvessels of hph1 mice demonstrated reduced tetrahydrobiopterin (BH4) bioavailability, increased production of superoxide anions and impaired endothelial NO signaling. Treatment of hph1 mice with EPO attenuated the levels of 7,8‐dihydrobiopterin, the oxidized product of BH4, and significantly increased the ratio of BH4 to 7,8‐dihydrobiopterin. Moreover, EPO decreased the levels of superoxide anions and increased NO bioavailability in cerebral microvessels of hph1 mice. Attenuated oxidation of BH4 and inhibition of endothelial NO synthase uncoupling were explained by the increased expression of antioxidant proteins, manganese superoxide dismutase, and catalase. The protective effects of EPO observed in cerebral microvessels of hph1 mice were also observed in GTPCH‐I siRNA‐treated human brain microvascular endothelial cells exposed to EPO (1 U/mL or 10 U/mL; 3 days). Our results suggest that EPO might protect the neurovascular unit against oxidative stress by restoring bioavailability of BH4 and endothelial NO in the cerebral microvascular endothelium. image We demonstrate that treatment with erythropoietin (EPO) could protect cerebral microvasculature against the pathological consequences of endothelial nitric oxide (NO) synthase uncoupling. Our results suggest that EPO might protect the neurovascular unit against oxidative stress by restoring bioavailability of tetrahydrobiopterin (BH4) and endothelial nitric oxide.

Peroxisome proliferator-activated receptor (PPAR)-δ is a nuclear hormone receptor that is mainly involved in lipid metabolism. Recent studies have suggested that PPAR-δ agonists exert vascular protective effects. The present study was designed to characterize vascular function in mice with genetic inactivation of PPAR-δ in the endothelium. Mice with vascular endothelial cell-specific deletion of the PPAR-δ gene (ePPARδ−/− mice) were generated using loxP/Cre technology. ePPARδ−/− mice were normotensive and did not display any sign of metabolic syndrome. Endothelium-dependent relaxations to ACh and endothelium-independent relaxations to the nitric oxide (NO) donor diethylammonium ( Z)-1-( N, N-diethylamino)diazen-1-ium-1,2-diolate were both significantly impaired in the aorta and carotid arteries of ePPARδ−/− mice ( P < 0.05). In ePPARδ−/− mouse aortas, phosphorylation of endothelial NO synthase at Ser1177 was significantly decreased ( P < 0.05). However, basal levels of cGMP were unexpectedly increased ( P < 0.05). Enzymatic activity of GTP-cyclohydrolase I and tetrahydrobiopterin levels were also enhanced in ePPARδ−/− mice ( P < 0.05). Most notably, endothelium-specific deletion of the PPAR-δ gene significantly decreased protein expressions of catalase and glutathione peroxidase 1 and resulted in increased levels of H2O2 in the aorta ( P < 0.05). In contrast, superoxide anion production was unaltered. Moreover, treatment with catalase prevented the endothelial dysfunction and elevation of cGMP detected in aortas of ePPARδ−/− mice. The findings suggest that increased levels of cGMP caused by H2O2 impair vasodilator reactivity to endogenous and exogenous NO. We speculate that chronic elevation of H2O2 predisposes PPAR-δ-deficient arteries to oxidative stress and vascular dysfunction.

Background and Purpose— Phosphorylation of eNOS, an important post-translational modulator of its enzymatic activity, is reduced in diabetes mellitus. We hypothesized that modulation of eNOS phosphorylation could overcome diabetic vascular dysfunction and improves the outcome to stroke. Methods— We used the db/db mouse model of type 2 diabetes mellitus. We mated db/db mice with eNOS knock-in mice that carry single amino acid mutations at the S1176 phosphorylation site; the phosphomimetic SD mutation (serine replaced by aspartate) shows increased eNOS enzymatic activity, whereas the unphosphorylatable SA mutation (serine replaced by alanine) shows decreased eNOS activity. We characterized the vascular anatomy, baseline physiological parameters, and vascular reactivity. We used the middle cerebral artery occlusion model of stroke and measured infarct volume and neurological deficits. Results— db/db mice showed diminished eNOS phosphorylation at S1176. eNOS SD and SA mutations do not change the vascular anatomy at the Circle of Willis, brain capillary density, heart rate, or arterial blood gases of db/db mice. The eNOS SD mutation, but not the SA mutation, lowers blood pressure and improves vascular reactivity to acetylcholine in db/db mice. The eNOS SD mutation reduces stroke size and neurological deficit after middle cerebral artery occlusion. Conclusions— Diminished eNOS phosphorylation is a mechanism of vascular dysfunction in db/db mice. We show here that modulation of the eNOS S1176 phosphorylation site in db/db mice is associated with improved vascular reactivity and improved outcome to stroke after middle cerebral artery occlusion.

Vascular access dysfunction causes morbidity in hemodialysis patients. This study examined the generation and pathobiological significance of superoxide anion in a rat femoral arteriovenous fistula (AVF). One week after AVF creation, there was increased production of superoxide anion accompanied by decreased total superoxide dismutase (SOD) and Cu/Zn SOD activities and induction of the redox-sensitive gene heme oxygenase-1. Immunohistochemical studies of nitrotyrosine formation demonstrated that peroxynitrite, a product of superoxide anion and nitric oxide, was present in increased amounts in endothelial and smooth muscle cells in the AVF. Because uncoupled NOS isoforms generate superoxide anion, and NOS coupling requires tetrahydrobiopterin (BH4) as a cofactor, we assessed NOS uncoupling by determining the ratio of BH4to dihydrobiopterin (BH2); the BH4-to-BH2ratio was markedly attenuated in the AVF. Because Src is a vasculopathic signaling species upstream and downstream of superoxide anion, such expression was evaluated; expression of Src and phosphorylated Src was both markedly increased in the AVF. Expression of NADPH oxidase (NOX) 1, NOX2, NOX4, cyclooxygenase (COX) 1, COX2, p47phox, and p67phoxwas all unchanged, as assessed by Western analyses, thereby suggesting that these proteins may not be involved in increased production of superoxide anion. Finally, administration of tempol, a superoxide anion scavenger, decreased neointima formation in the juxta-anastomotic venous segment and improved AVF blood flow. We conclude that the AVF exhibits increased superoxide anion generation that may reflect the combined effects of decreased scavenging by SOD and increased generation by uncoupled NOS, and that enhanced superoxide anion production promotes juxta-anastomotic stenosis and impairs AVF function.

Abstract —To determine whether natriuretic peptides in addition to the renin-angiotensin system are involved in functional and structural vascular changes in salt-sensitive hypertension, we compared equipotent hypotensive treatment with the dual neutral endopeptidase/ACE inhibitor omapatrilat (35 mg · kg −1 · d −1 ) or the ACE inhibitor captopril (100 mg · kg −1 · d −1 ). The reactivity and geometry of mesenteric resistance arteries from Dahl salt-sensitive rats were studied in vitro under perfused and pressurized conditions. Chronic salt administration increased systolic blood pressure by 57±4 mm Hg, whereas concentrations of atrial natriuretic peptide were reduced in heart and in plasma ( P <0.05). In addition, the medial cross-sectional area of small mesenteric arteries was increased and endothelium-dependent relaxation in response to acetylcholine and contraction in response to endothelin-1 were impaired in the mesenteric arteries of salt-sensitive rats on a high-salt diet ( P <0.05). Concomitant treatment with either omapatrilat or captopril reduced the increase in systolic blood pressure and hypertrophic remodeling to a similar degree ( P <0.05) but affected plasma and cardiac atrial natriuretic peptide levels differently ( P <0.05). In addition, omapatrilat normalized endothelium-dependent relaxations to a greater extent than captopril ( P <0.05). Furthermore, vasopeptidase inhibition increased cGMP levels compared with captopril ( P <0.05). Contractions to endothelin-1 were normalized by either antihypertensive drug. These results suggest that in the Dahl rat, with similar reductions in systolic blood pressure, omapatrilat is superior to captopril in preventing impaired endothelial function in small resistance arteries. Thus, vasopeptidase inhibition may have therapeutic advantages of the prevention of changes in vascular function and structure in salt-sensitive forms of hypertension.

Activity of the vascular large conductance Ca2+-activated K+ (BK) channel is tightly regulated by its accessory β1 subunit (BK-β1). Downregulation of BK-β1 expression in diabetic vessels is associated with upregulation of the forkhead box O subfamily transcription factor-3a (FOXO-3a)–dependent F-box–only protein (FBXO) expression. However, the upstream signaling regulating this process is unclear. Overproduction of reactive oxygen species (ROS) is a common finding in diabetic vasculopathy. We hypothesized that ROS signaling cascade facilitates the FOXO-3a/FBXO-mediated BK-β1 degradation and leads to diabetic BK channel dysfunction. Using cellular biology, patch clamp, and videomicroscopy techniques, we found that reduced BK-β1 expression in streptozotocin (STZ)-induced diabetic mouse arteries and in human coronary smooth muscle cells (SMCs) cultured with high glucose was attributable to an increase in protein kinase C (PKC)-β and NADPH oxidase expressions and accompanied by attenuation of Akt phosphorylation and augmentation of atrogin-1 expression. Treatment with ruboxistaurin (a PKCβ inhibitor) or with GW501516 (a peroxisome proliferator–activated receptor δ activator) reduced atrogin-1 expression and restored BK channel-mediated coronary vasodilation in diabetic mice. Our results suggested that oxidative stress inhibited Akt signaling and facilitated the FOXO-3a/FBXO-dependent BK-β1 degradation in diabetic vessels. Suppression of the FOXO-3a/FBXO pathway prevented vascular BK-β1 degradation and protected coronary function in diabetes.

In the present study, we used the hph-1 mouse, which displays GTP-cyclohydrolase I (GTPCH I) deficiency, to test the hypothesis that loss of tetrahydrobiopterin (BH4) in conduit and small arteries activates compensatory mechanisms designed to protect vascular wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and BH4levels were reduced in the aortas and small mesenteric arteries of hph-1 mice. However, the BH4-to-7,8-dihydrobiopterin ratio was significantly reduced only in hph-1 aortas. Furthermore, superoxide anion and 3-nitrotyrosine production were significantly enhanced in aortas but not in small mesenteric arteries of hph-1 mice. In contrast to the aorta, protein expression of copper- and zinc-containing superoxide dismutase (CuZnSOD) was significantly increased in small mesenteric arteries of hph-1 mice. Protein expression of catalase was increased in both aortas and small mesenteric arteries of hph-1 mice. Further analysis of endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) signaling demonstrated that protein expression of phosphorylated Ser1177-eNOS as well as basal cGMP levels and hydrogen peroxide was increased in hph-1 aortas. Increased production of hydrogen peroxide in hph-1 mice aortas appears to be the most likely mechanism responsible for phosphorylation of eNOS and elevation of cGMP. In contrast, upregulation of CuZnSOD and catalase in resistance arteries is sufficient to protect vascular tissue from increased production of reactive oxygen species generated by uncoupling of eNOS. The results of our study suggest that anatomical origin determines the ability of vessel wall to cope with oxidative stress induced by uncoupling of eNOS.