Partial loss of endothelial nitric oxide leads to increased cerebrovascular beta amyloid.
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ABSTRACT:
SUBMITTER: Austin SA
PROVIDER: S-EPMC7370614 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundTraumatic brain injury (TBI) has been reported to increase the concentration of nitric oxide (NO) in the brain and can lead to loss of cerebrovascular tone; however, the sources, amounts, and consequences of excess NO on the cerebral vasculature are unknown. Our objective was to elucidate the mechanism of decreased cerebral artery tone after TBI.Methods and resultsCerebral arteries were isolated from rats 24 hours after moderate fluid?percussion TBI. Pressure?induced increases in vasoconstriction (myogenic tone) and smooth muscle Ca2+ were severely blunted in cerebral arteries after TBI. However, myogenic tone and smooth muscle Ca2+ were restored by inhibition of NO synthesis or endothelium removal, suggesting that TBI increased endothelial NO levels. Live native cell NO, indexed by 4,5?diaminofluorescein (DAF?2 DA) fluorescence, was increased in endothelium and smooth muscle of cerebral arteries after TBI. Clamped concentrations of 20 to 30 nmol/L NO were required to simulate the loss of myogenic tone and increased (DAF?2T) fluorescence observed following TBI. In comparison, basal NO in control arteries was estimated as 0.4 nmol/L. Consistent with TBI causing enhanced NO?mediated vasodilation, inhibitors of guanylyl cyclase, protein kinase G, and large?conductance Ca2+?activated potassium (BK) channel restored function of arteries from animals with TBI. Expression of the inducible isoform of NO synthase was upregulated in cerebral arteries isolated from animals with TBI, and the inducible isoform of NO synthase inhibitor 1400W restored myogenic responses following TBI.ConclusionsThe mechanism of profound cerebral artery vasodilation after TBI is a gain of function in vascular NO production by 60?fold over controls, resulting from upregulation of the inducible isoform of NO synthase in the endothelium.
| S-EPMC4338739 | biostudies-literature
Project description:The Phospholipase D (PLD) superfamily is linked to neurological disease, cancer, and fertility, and a recent report correlated a potential loss-of-function PLD2 polymorphism with hypotension. Surprisingly, PLD2 -/- mice exhibit elevated blood pressure accompanied by associated changes in cardiac performance and molecular markers, but do not have findings consistent with the metabolic syndrome. Instead, expression of endothelial nitric oxide synthase (eNOS), which generates the potent vasodilator nitric oxide (NO), is decreased. An eNOS inhibitor phenocopied PLD2 loss and had no further effect on PLD2 -/- mice, confirming the functional relationship. Using a human endothelial cell line, PLD2 loss of function was shown to lower intracellular free cholesterol, causing upregulation of HMG Co-A reductase, the rate-limiting enzyme in cholesterol synthesis. HMG Co-A reductase negatively regulates eNOS, and the PLD2-deficiency phenotype of decreased eNOS expression and activity could be rescued by cholesterol supplementation and HMG Co-A reductase inhibition. Together, these findings identify a novel pathway through which the lipid signaling enzyme PLD2 regulates blood pressure, creating implications for on-going therapeutic development of PLD small molecule inhibitors. Finally, we show that the human PLD2 polymorphism does not trigger eNOS loss, but rather creates another effect, suggesting altered functioning for the allele.
| S-EPMC5567230 | biostudies-literature
Project description:Rationalethe exact etiology of sporadic Alzheimer disease (AD) is unclear, but it is interesting that several cardiovascular risk factors are associated with higher incidence of AD. The link between these risk factors and AD has yet to be identified; however, a common feature is endothelial dysfunction, specifically, decreased bioavailability of nitric oxide (NO).Objectiveto determine the relationship between endothelial derived NO and the expression and processing of amyloid precursor protein (APP).Methods and resultswe used human brain microvascular endothelial cells to examine the role of NO in modulating APP expression and processing in vitro. Inhibition of endothelial nitric oxide synthase (eNOS) with the specific NOS inhibitor L-NAME (N(G)-nitro-l-arginine methyl ester) led to increased APP and BACE1 (β-site APP-cleaving enzyme1) protein levels, as well as increased secretion of the amyloidogenic peptide amyloid β (Aβ) (control 10.93 ± 0.70 pg/mL; L-NAME 168.21 ± 27.38 pg/mL; P<0.001). To examine the role of NO in modulation of APP expression and processing in vivo, we used brain and cerebral microvessels from eNOS-deficient (eNOS(-/-)) mice. Brain tissue from eNOS(-/-) mice had statistically higher APP and BACE1 protein levels, as well as increased BACE1 enzyme activity and Aβ (Aβ(1)(-)(42) wild-type control, 0.737 pg/mg; eNOS(-/-), 1.475 pg/mg; P<0.05), compared with wild-type controls (n=6 to 8 animals per background). Brain microvessels from eNOS(-/-) mice also showed statistically higher BACE1 protein levels as compared with wild-type control.Conclusionsour data suggest that endothelial NO plays an important role in modulating APP expression and processing within the brain and cerebrovasculature. The NO/cGMP pathway may be an important therapeutic target in preventing and treating mild cognitive impairment, as well as AD.
| S-EPMC3064266 | biostudies-literature
Project description:Increasing evidence implicates endothelial dysfunction in the pathogenesis of Alzheimer's disease (AD). Nitric oxide (NO) derived from endothelial NO synthase (eNOS) is essential in maintaining cerebrovascular function and can modulate the production and clearance of amyloid beta (Aβ). APPswe/PSdE1 (APP/PS1) mice display age-related Aβ accumulation and memory deficits. In order to make the model more clinically relevant with an element of endothelial dysfunction, we generated APP/PS1/eNOS+/- mice by crossing complete eNOS deficient (eNOS-/-) mice and APP/PS1 mice. APP/PS1/eNOS+/- mice at 8 months of age displayed a more severe spatial working memory deficit relative to age-matched APP/PS1 mice. Moreover, immunohistochemistry and immunoblotting revealed significantly increased Aβ plaque load in the brains of APP/PS1/eNOS+/- mice, concomitant with upregulated BACE-1 (hence increased Aβ production), downregulated insulin-degrading enzyme (hence reduced Aβ clearance) and increased immunoreactivity and expression of microglia. The present study, for the first time, demonstrated that partial eNOS deficiency exacerbated behavioral dysfunction, Aβ brain deposition, and microglial pathology in APP/PS1 mice, further implicating endothelial dysfunction in the pathogenesis of AD. The present findings also provide the scientific basis for developing preventive and/or therapeutic strategies by targeting endothelial dysfunction.
| S-EPMC9266765 | biostudies-literature
Project description:Protein-protein interactions represent an important post-translational mechanism for endothelial nitric-oxide synthase (eNOS) regulation. We have previously reported that beta-actin is associated with eNOS oxygenase domain and that association of eNOS with beta-actin increases eNOS activity and nitric oxide (NO) production. In the present study, we found that beta-actin-induced increase in NO production was accompanied by decrease in superoxide formation. A synthetic actin-binding sequence (ABS) peptide 326 with amino acid sequence corresponding to residues 326-333 of human eNOS, one of the putative ABSs, specifically bound to beta-actin and prevented eNOS association with beta-actin in vitro. Peptide 326 also prevented beta-actin-induced decrease in superoxide formation and increase in NO and L-citrulline production. A modified peptide 326 replacing hydrophobic amino acids leucine and tryptophan with neutral alanine was unable to interfere with eNOS-beta-actin binding and to prevent beta-actin-induced changes in NO and superoxide formation. Site-directed mutagenesis of the actin-binding domain of eNOS replacing leucine and tryptophan with alanine yielded an eNOS mutant that exhibited reduced eNOS-beta-actin association, decreased NO production, and increased superoxide formation in COS-7 cells. Disruption of eNOS-beta-actin interaction in endothelial cells using ABS peptide 326 resulted in decreased NO production, increased superoxide formation, and decreased endothelial monolayer wound repair, which was prevented by PEG-SOD and NO donor NOC-18. Taken together, this novel finding indicates that beta-actin binding to eNOS through residues 326-333 in the eNOS protein results in shifting the enzymatic activity from superoxide formation toward NO production. Modulation of NO and superoxide formation from eNOS by beta-actin plays an important role in endothelial function.
| S-EPMC2836036 | biostudies-literature
Project description:Alzheimer’s disease is the most common form of dementia and is associated with the accumulation of amyloid peptide β in the brain parenchyma. Vascular damage and microvascular thrombosis contribute to the neuronal degeneration and the loss of brain function typical of this disease. In this study, we utilised a murine model of Alzheimer’s disease to evaluate the neurovascular effects of this disease. Upon detection of an increase in the phosphorylation of the endothelial surface receptor VE-cadherin, we focused our attention on endothelial cells.
2022-05-19 | PXD030300 | Pride
Project description:Previous studies have demonstrated that G protein-coupled receptor kinase interacting-1 protein (GIT1) associates with endothelial nitric oxide synthase (eNOS) to regulate nitric oxide production in sinusoidal endothelial cells (SECs). Here, we hypothesized that GIT1's tightly associated binding partner, β-PIX (p21-activated kinase-interacting exchange factor β, ARHGEF7) is specifically important in the regulation of eNOS activity. We examined β-PIX expression in normal rat liver by immunohistochemistry and explored β-PIX protein-protein interactions using immunoprecipitation and immunoblotting. The role of β-PIX in regulating eNOS enzymatic activity was studied in GIT1-deficient SECs. Finally, structural analysis of interaction sites in GIT1 and β-PIX required to regulate eNOS activity were mapped. β-PIX was expressed primarily in SECs in normal liver and was either absent or expressed at extremely low levels in other liver cells (stellate cells, Kupffer cells, and hepatocytes). β-PIX interacted with GIT1 and eNOS to form a trimolecular signaling module in normal SECs and was important in stimulating eNOS activity. Of note, GIT1-β-PIX interaction led to synergistic enhancement of eNOS activity, and β-PIX-driven increase in eNOS activity was GIT1 dependent. Disruption of β-PIX or GIT1 in normal SECs using β-PIX siRNA or GIT1-deficient SECs led to reduced eNOS activity. Finally, specific GIT1 domains [Spa2 homology domain (SHD) and synaptic localization domain (SLD), aa 331-596] and the β-PIX COOH terminal (aa 496-555) appeared to be critical in the regulation eNOS activity. The data indicate that β-PIX regulates eNOS phosphorylation and function in normal SECs and highlight the importance of the GIT1/β-PIX/eNOS trimolecular complex in normal liver SEC function.NEW & NOTEWORTHY β-PIX is a multidomain protein known to be a GIT1 binding partner. We report here that in the normal liver, the distribution and cellular localization of β-PIX are restricted largely to sinusoidal endothelial cells. Furthermore, β-PIX interacts with eNOS and GIT1 promotes eNOS activity and NO production and therefore exerts a novel posttranslational regulatory function on eNOS activity in sinusoidal endothelial cells. We also have identified specific molecular domains important in GIT1 and β-PIX's interaction with eNOS, which may represent novel therapeutic targets in the control of sinusoidal blood flow and intrahepatic resistance.
| S-EPMC9639759 | biostudies-literature
Project description:Background: Moyamoya is a cerebrovascular condition of unknown mechanism characterized by a progressive stenosis of the terminal part of the internal carotid arteries (ICA) and the compensatory development of abnormal “moyamoya” vessels. It leads to ischemic and hemorrhagic stroke. We describe a novel autosomal recessive disease leading to severe moyamoya and early onset achalasia and report its cause in 3 unrelated families. Methods: We used a combination of genetic linkage and exome sequencing in 2 consanguineous to identify rare shared variants. Sanger sequencing of GUCY1A3, the sole gene mutated in both families, was then conducted in the third family. Platelets from one of the patients and controls were used to carry out functional studies. Results: Homozygous mutations of GUCY1A3 gene encoding the alpha1 subunit of soluble guanylate cyclase (sGC), the major receptor for Nitric Oxide (NO), were identified in all 3 families. Platelet analysis showed a complete loss of the mutated protein and showed an unexpected stimulatory role of sGC within platelets. Conclusion: The NO/sGC/cGMP pathway is a major pathway controlling vascular smooth muscle (VSMC) relaxation, vascular tone and vascular remodeling. Our data suggest that alterations of this pathway may lead to an abnormal vascular remodeling process in sensitive vascular areas with low blood A total of 17 samples (8 affected and 9 unaffected) were used for this study. Linkage analysis was performed in a single informative consanguine family composed of 2 unaffected parents, 4 affected siblings and 3 unaffected siblings. Two affected samples in two different families were used for the exome sequencing analysis and results were compared to 20 control exomes (in-house exomes from IntegraGen, Evry, France) and 8 HapMap exomes. All samples were used for Sanger Sequencing confirmation.
2014-06-19 | E-GEOD-48099 | biostudies-arrayexpress
Project description:Recent reports indicate that (-)-epicatechin can exert cardioprotective actions, which may involve endothelial nitric oxide synthase (eNOS)-mediated nitric oxide production in endothelial cells. However, the mechanism by which (-)-epicatechin activates eNOS remains unclear. In this study, we proposed to identify the intracellular pathways involved in (-)-epicatechin-induced effects on eNOS, using human coronary artery endothelial cells in culture. Treatment of cells with (-)-epicatechin led to time- and dose-dependent effects that peaked at 10 minutes at 1 mumol/L. (-)-Epicatechin treatment activates eNOS via serine 633 and serine 1177 phosphorylation and threonine 495 dephosphorylation. Using specific inhibitors, we have established the participation of the phosphatidylinositol 3-kinase pathway in eNOS activation. (-)-Epicatechin induces eNOS uncoupling from caveolin-1 and its association with calmodulin-1, suggesting the involvement of intracellular calcium. These results allowed us to propose that (-)-epicatechin effects may be dependent on actions exerted at the cell membrane level. To test this hypothesis, cells were treated with the phospholipase C inhibitor U73122, which blocked (-)-epicatechin-induced eNOS activation. We also demonstrated inositol phosphate accumulation in (-)-epicatechin-treated cells. The inhibitory effects of the preincubation of cells with the calmodulin-dependent kinase II (CaMKII) inhibitor KN-93 indicate that (-)-epicatechin-induced eNOS activation is at least partially mediated via the Ca(2+)/CaMKII pathway. The (-)-epicatechin stereoisomer catechin was only partially able to stimulate nitric oxide production in cells. Together, these results strongly suggest the presence of a cell surface acceptor-effector for the cacao flavanol (-)-epicatechin, which may mediate its cardiovascular effects.
| S-EPMC2874202 | biostudies-literature
Project description:Background: Moyamoya is a cerebrovascular condition of unknown mechanism characterized by a progressive stenosis of the terminal part of the internal carotid arteries (ICA) and the compensatory development of abnormal “moyamoya” vessels. It leads to ischemic and hemorrhagic stroke. We describe a novel autosomal recessive disease leading to severe moyamoya and early onset achalasia and report its cause in 3 unrelated families. Methods: We used a combination of genetic linkage and exome sequencing in 2 consanguineous to identify rare shared variants. Sanger sequencing of GUCY1A3, the sole gene mutated in both families, was then conducted in the third family. Platelets from one of the patients and controls were used to carry out functional studies. Results: Homozygous mutations of GUCY1A3 gene encoding the alpha1 subunit of soluble guanylate cyclase (sGC), the major receptor for Nitric Oxide (NO), were identified in all 3 families. Platelet analysis showed a complete loss of the mutated protein and showed an unexpected stimulatory role of sGC within platelets. Conclusion: The NO/sGC/cGMP pathway is a major pathway controlling vascular smooth muscle (VSMC) relaxation, vascular tone and vascular remodeling. Our data suggest that alterations of this pathway may lead to an abnormal vascular remodeling process in sensitive vascular areas with low blood
2014-06-19 | GSE48099 | GEO