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1.
Sci Transl Med ; 9(376)2017 02 08.
Article in English | MEDLINE | ID: mdl-28179506

ABSTRACT

The incidence of high blood pressure with advancing age is notably high, and it is an independent prognostic factor for the onset or progression of a variety of cardiovascular disorders. Although age-related hypertension is an established phenomenon, current treatments are only palliative but not curative. Thus, there is a critical need for a curative therapy against age-related hypertension, which could greatly decrease the incidence of cardiovascular disorders. We show that overexpression of human thioredoxin (TRX), a redox protein, in mice prevents age-related hypertension. Further, injection of recombinant human TRX (rhTRX) for three consecutive days reversed hypertension in aged wild-type mice, and this effect lasted for at least 20 days. Arteries of wild-type mice injected with rhTRX or mice with TRX overexpression exhibited decreased arterial stiffness, greater endothelium-dependent relaxation, increased nitric oxide production, and decreased superoxide anion (O2•-) generation compared to either saline-injected aged wild-type mice or mice with TRX deficiency. Our study demonstrates a potential translational role of rhTRX in reversing age-related hypertension with long-lasting efficacy.


Subject(s)
Aging/pathology , Blood Vessels/metabolism , Hypertension/drug therapy , Hypertension/enzymology , Nitric Oxide Synthase Type III/metabolism , Thioredoxins/therapeutic use , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/pathology , Aorta, Thoracic/physiopathology , Blood Pressure/drug effects , Blood Vessels/pathology , Blood Vessels/physiopathology , Carotid Arteries/drug effects , Carotid Arteries/pathology , Carotid Arteries/physiopathology , Endothelium, Vascular/pathology , Endothelium, Vascular/physiopathology , Glutathione/metabolism , Humans , Hypertension/physiopathology , Mesenteric Artery, Superior/drug effects , Mesenteric Artery, Superior/pathology , Mesenteric Artery, Superior/physiopathology , Mice, Inbred C57BL , Mice, Transgenic , NADPH Oxidases/metabolism , Oxidation-Reduction , Phosphorylation/drug effects , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Superoxides/metabolism , Thioredoxins/pharmacology , Vascular Resistance/drug effects , Vasodilation/drug effects
2.
J Biol Chem ; 291(45): 23374-23389, 2016 Nov 04.
Article in English | MEDLINE | ID: mdl-27587398

ABSTRACT

Reversible glutathionylation plays a critical role in protecting protein function under conditions of oxidative stress generally and for endothelial nitric-oxide synthase (eNOS) specifically. Glutathione-dependent glutaredoxin-mediated deglutathionylation of eNOS has been shown to confer protection in a model of heart damage termed ischemia-reperfusion injury, motivating further study of eNOS deglutathionylation in general. In this report, we present evidence for an alternative mechanism of deglutathionylation. In this pathway thioredoxin (Trx), a small cellular redox protein, is shown to rescue eNOS from glutathionylation during ischemia-reperfusion in a GSH-independent manner. By comparing mice with global overexpression of Trx and mice with cardiomyocyte-specific overexpression of Trx, we demonstrate that vascular Trx-mediated deglutathionylation of eNOS protects against ischemia-reperfusion-mediated myocardial infarction. Trx deficiency in endothelial cells promoted eNOS glutathionylation and reduced its enzymatic activity, whereas increased levels of Trx led to deglutathionylated eNOS. Thioredoxin-mediated deglutathionylation of eNOS in the coronary artery in vivo protected against reperfusion injury, even in the presence of normal levels of GSH. We further show that Trx directly interacts with eNOS, and we confirmed that Cys-691 and Cys-910 are the glutathionylated sites, as mutation of these cysteines partially rescued the decrease in eNOS activity, whereas mutation of a distal site, Cys-384, did not. Collectively, this study shows for the first time that Trx is a potent deglutathionylating protein in vivo and in vitro that can deglutathionylate proteins in the presence of high levels of GSSG in conditions of oxidative stress.


Subject(s)
Glutathione/metabolism , Myocardial Infarction/metabolism , Myocardium/pathology , Nitric Oxide Synthase Type III/metabolism , Thioredoxins/metabolism , Animals , Cells, Cultured , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , HEK293 Cells , Humans , Mice, Inbred C57BL , Myocardial Infarction/genetics , Myocardial Infarction/pathology , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/pathology , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , RNA Interference , RNA, Small Interfering/genetics , Thioredoxins/genetics , Up-Regulation
3.
Hypertension ; 65(1): 130-9, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25312439

ABSTRACT

Vascular thiol redox state has been shown to modulate vasodilator functions in large conductance Ca2+ -activated K+ channels and other related channels. However, the role of vascular redox in small resistance arteries is unknown. To determine how in vivo modulation of thiol redox state affects small resistance arteries relaxation, we generated a transgenic mouse strain that overexpresses thioredoxin, a small redox protein (Trx-Tg), and another strain that is thioredoxin-deficient (dnTrx-Tg). The redox state of the mesenteric arteries (MAs) in Trx-Tg mice is found to be predominantly in reduced state; in contrast, MAs from dnTrx-Tg mice remain in oxidized state. Thus, we created an in vivo redox system of mice and isolated the second-order branches of the main superior MAs from wild-type, Trx-Tg, or dnTrx-Tg mice to assess endothelium-dependent relaxing responses in a wire myograph. In MAs isolated from Trx-Tg mice, we observed an enhanced intermediate-conductance Ca2+ -activated potassium channel contribution resulting in a larger endothelium-dependent hyperpolarizing (EDH) relaxation in response to indirect (acetylcholine) and direct (NS309) opening of endothelial calcium-activated potassium channels. MAs derived from dnTrx-Tg mice showed both blunted nitric oxide-mediated and EDH-mediated relaxation compared with Trx-Tg mice. In a control study, diamide decreased EDH relaxations in MAs of wild-type mice, whereas dithiothreitol improved EDH relaxations and was able to restore the diamide-induced impairment in EDH response. Furthermore, the basal or angiotensin II-mediated systolic blood pressure remained significantly lower in Trx-Tg mice compared with wild-type or dnTrx-Tg mice, thus directly establishing redox-mediated EDH in blood pressure control.


Subject(s)
Blood Pressure/physiology , Hypertension/metabolism , Mesenteric Arteries/metabolism , Nitric Oxide/metabolism , Potassium Channels, Calcium-Activated/metabolism , Vascular Resistance/physiology , Vasodilation/physiology , Animals , Disease Models, Animal , Endothelium, Vascular/metabolism , Endothelium, Vascular/physiopathology , Hypertension/physiopathology , Mesenteric Arteries/physiopathology , Mice , Mice, Transgenic , Oxidation-Reduction
4.
Am J Physiol Heart Circ Physiol ; 305(8): H1256-64, 2013 Oct 15.
Article in English | MEDLINE | ID: mdl-23955716

ABSTRACT

Endothelial dysfunction is prevalent in chronic kidney disease. This study tested the hypothesis that transfusion of rat aortic endothelial cells (ECs) ameliorates endothelial dysfunction in a rat model of chronic kidney disease. Male Sprague-Dawley rats underwent sham surgery or 5/6 nephrectomy (Nx). Five weeks after Nx, EC (1.5 × 10(6) cells/rat) or vehicle were transfused intravenously. One week later, vascular reactivity of mesenteric artery was assessed on a wire myograph. Sensitivity of endothelium-dependent relaxation to acetylcholine and maximum vasodilation were impaired by Nx and improved by EC transfusion. Using selective pharmacological nitric oxide synthase isoform inhibitors, we demonstrated that the negative effect of Nx on endothelial function and rescue by EC transfusion are, at least in part, endothelial nitric oxide synthase mediated. Plasma asymmetric dimethylarginine was increased by Nx and decreased by EC transfusion, whereas mRNA expression of dimethylarginine dimethylaminohydrolases 1 (DDAH1) was decreased by Nx and restored by EC transfusion. Immunohistochemical staining confirmed that local expression of DDAH1 is decreased by Nx and increased by EC transfusion. In conclusion, EC transfusion attenuates Nx-induced endothelium-dependent vascular dysfunction by regulating DDAH1 expression and enhancing endothelial nitric oxide synthase activity. These results suggest that EC-based therapy could provide a novel therapeutic strategy to improve vascular function in chronic kidney disease.


Subject(s)
Endothelial Cells/transplantation , Endothelium, Vascular/physiopathology , Mesenteric Arteries/physiopathology , Renal Insufficiency, Chronic/physiopathology , Acetylcholine/pharmacology , Amidohydrolases/metabolism , Animals , Aorta/cytology , Arginine/analogs & derivatives , Disease Models, Animal , Endothelium, Vascular/drug effects , Male , Mesenteric Arteries/drug effects , Myography , Nephrectomy , Nitric Oxide Synthase Type III/metabolism , Rats , Rats, Sprague-Dawley , Vasodilation/drug effects , Vasodilation/physiology , Vasodilator Agents/pharmacology
5.
J Endocrinol ; 215(1): 97-106, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22798015

ABSTRACT

This study compared ex vivo relaxing responses to the naturally occurring human hormone estetrol (E(4)) vs 17ß-estradiol (E(2)) in eight different vascular beds. Arteries were mounted in a myograph, contracted with either phenylephrine or serotonin, and cumulative concentration-response curves (CRCs) to E(4) and E(2) (0·1-100  µmol/l) were constructed. In all arteries tested, E(4) had lower potency than E(2), although the differential effect was less in larger than smaller arteries. In uterine arteries, the nonselective estrogen receptor (ER) blocker ICI 182 780 (1  µmol/l) caused a significant rightward shift in the CRC to both E(4) and E(2), indicating that the relaxation responses were ER dependent. Pharmacological blockade of nitric oxide (NO) synthases by N(ω)-nitro-L-arginine methyl ester (L-NAME) blunted E(2)-mediated but not E(4)-mediated relaxing responses, while inhibition of prostaglandins and endothelium-dependent hyperpolarization did not alter relaxation to either E(4) or E(2) in uterine arteries. Combined blockade of NO release and action with L-NAME and the soluble guanylate cyclase (sGC) inhibitor ODQ resulted in greater inhibition of the relaxation response to E(4) compared with E(2) in uterine arteries. Endothelium denudation inhibited responses to both E(4) and E(2), while E(4) and E(2) concentration-dependently blocked smooth muscle cell Ca(2)(+) entry in K(+)-depolarized and Ca(2)(+)-depleted uterine arteries. In conclusion, E(4) relaxes precontracted rat arteries in an artery-specific fashion. In uterine arteries, E(4)-induced relaxations are partially mediated via an endothelium-dependent mechanism involving ERs, sGC, and inhibition of smooth muscle cell Ca(2)(+) entry, but not NO synthases or endothelium-dependent hyperpolarization.


Subject(s)
Arteries/drug effects , Estetrol/pharmacology , Vasodilation/drug effects , Animals , Arteries/physiology , Carotid Arteries/drug effects , Carotid Arteries/metabolism , Carotid Arteries/physiology , Cells, Cultured , Drug Evaluation, Preclinical , Female , Muscle Contraction/drug effects , Muscle Contraction/physiology , Pulmonary Artery/drug effects , Pulmonary Artery/metabolism , Pulmonary Artery/physiology , Rats , Rats, Sprague-Dawley , Receptors, Estrogen/metabolism , Receptors, Estrogen/physiology , Renal Artery/drug effects , Renal Artery/metabolism , Renal Artery/physiology , Uterine Artery/drug effects , Uterine Artery/metabolism , Uterine Artery/physiology , Vasodilator Agents/pharmacology
6.
Am J Physiol Heart Circ Physiol ; 303(5): H513-22, 2012 Sep 01.
Article in English | MEDLINE | ID: mdl-22777418

ABSTRACT

Acute increases in cellular protein O-linked N-acetyl-glucosamine (O-GlcNAc) modification (O-GlcNAcylation) have been shown to have protective effects in the heart and vasculature. We hypothesized that d-glucosamine (d-GlcN) and Thiamet-G, two agents that increase protein O-GlcNAcylation via different mechanisms, inhibit TNF-α-induced oxidative stress and vascular dysfunction by suppressing inducible nitric oxide (NO) synthase (iNOS) expression. Rat aortic rings were incubated for 3h at 37°C with d-GlcN or its osmotic control l-glucose (l-Glc) or with Thiamet-G or its vehicle control (H(2)O) followed by the addition of TNF-α or vehicle (H(2)O) for 21 h. After incubation, rings were mounted in a myograph to assess arterial reactivity. Twenty-four hours of incubation of aortic rings with TNF-α resulted in 1) a hypocontractility to 60 mM K(+) solution and phenylephrine, 2) blunted endothelium-dependent relaxation responses to ACh and substance P, and 3) unaltered relaxing response to the Ca(2+) ionophore A-23187 and the NO donor sodium nitroprusside compared with aortic rings cultured in the absence of TNF-α. d-GlcN and Thiamet-G pretreatment suppressed the TNF-α-induced hypocontractility and endothelial dysfunction. Total protein O-GlcNAc levels were significantly higher in aortic segments treated with d-GlcN or Thiamet-G compared with controls. Expression of iNOS protein was increased in TNF-α-treated rings, and this was attenuated by pretreatment with either d-GlcN or Thiamet-G. Dense immunostaining for nitrotyrosylated proteins was detected in the endothelium and media of the aortic wall, suggesting enhanced peroxynitrite production by iNOS. These findings demonstrate that acute increases in protein O-GlcNAcylation prevent TNF-α-induced vascular dysfunction, at least in part, via suppression of iNOS expression.


Subject(s)
Acetylglucosamine/metabolism , Aorta, Thoracic/metabolism , Aortic Diseases/prevention & control , Inflammation/metabolism , Protein Processing, Post-Translational , Vasoconstriction , Vasodilation , Acylation , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/immunology , Aorta, Thoracic/physiopathology , Aortic Diseases/immunology , Aortic Diseases/metabolism , Aortic Diseases/physiopathology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Immunohistochemistry , Inflammation/immunology , Inflammation/physiopathology , Inflammation Mediators/metabolism , Male , Myography , Nitric Oxide Synthase Type II/metabolism , Oxidative Stress , Peroxynitrous Acid/metabolism , Rats , Rats, Sprague-Dawley , Time Factors , Tissue Culture Techniques , Tumor Necrosis Factor-alpha/metabolism , Tyrosine/analogs & derivatives , Tyrosine/metabolism , Vasoconstriction/drug effects , Vasoconstrictor Agents/pharmacology , Vasodilation/drug effects , Vasodilator Agents/pharmacology , beta-N-Acetylhexosaminidases/antagonists & inhibitors , beta-N-Acetylhexosaminidases/metabolism
7.
Am J Physiol Heart Circ Physiol ; 299(4): H1160-7, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20639218

ABSTRACT

TNF-α is a proinflammatory cytokine and is an important mediator of maternal endothelial dysfunction leading to preeclampsia. In this study, we tested whether IL-10 protects against TNF-α-induced endothelial dysfunction in murine aorta. In in vitro experiments, aortic rings of C57BL/6 female mice were incubated in Dulbecco's modified Eagle's medium in the presence of either vehicle (distilled H(2)O), TNF-α (4 nmol/l), or recombinant mouse IL-10 (300 ng/ml) or in the presence of both TNF-α and IL-10 for 22 h at 37°C. In in vivo experiments C57BL6/IL-10 knockout female mice were treated with saline or TNF-α (220 ng·kg(-1)·day(-1)) for 14 days. Aortic rings were isolated from in vitro and in vivo experiments and mounted in a wire myograph (Danish Myotech) and stretched to a tension of 5 mN. Endothelium-dependent relaxation was assessed by constructing cumulative concentration-response curves to acetylcholine (ACh, 0.001-10 µmol/l) during phenylephrine (10 µmol/l)-induced contraction. As a result, overnight exposure of aortic rings to TNF-α resulted in significant blunted maximal relaxing responses (E(max)) to ACh compared with untreated rings (22 ± 4 vs. 82 ± 3%, respectively). IL-10 knockout mice treated with TNF-α showed significant impairment in ACh responses (E(max)) compared with C57BL/6 mice treated with TNF-α (51 ± 3 vs. 72 ± 3%, respectively). Western blot analysis showed that endothelial nitric oxide synthase (eNOS) expression was reduced by TNF-α in in vitro and in vivo experiments, whereas IL-10 restored the eNOS expression. In conclusion, the anti-inflammatory cytokine IL-10 prevents impairment in endothelium-dependent vasorelaxation caused by TNF-α by protecting eNOS expression.


Subject(s)
Aorta/drug effects , Aorta/metabolism , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Interleukin-10/deficiency , Interleukin-10/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Acetylcholine/pharmacology , Animals , Aorta/physiopathology , Endothelium, Vascular/physiopathology , Female , I-kappa B Kinase/metabolism , Interleukin-10/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , NF-kappa B/metabolism , Nitric Oxide Synthase Type III/metabolism , Phenylephrine/pharmacology , Phosphorylation , Tumor Necrosis Factor-alpha/metabolism , Vasoconstriction/drug effects , Vasoconstriction/physiology , Vasoconstrictor Agents/pharmacology , Vasodilation/drug effects , Vasodilation/physiology , Vasodilator Agents/pharmacology
8.
J Pharmacol Exp Ther ; 333(1): 210-7, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20040579

ABSTRACT

We tested the hypothesis that changes in arterial blood flow modify the function of endothelial Ca2+-activated K+ channels [calcium-activated K+ channel (K(Ca)), small-conductance calcium-activated K+ channel (SK3), and intermediate calcium-activated K+ channel (IK1)] before arterial structural remodeling. In rats, mesenteric arteries were exposed to increased [+90%, high flow (HF)] or reduced blood flow [-90%, low flow (LF)] and analyzed 24 h later. There were no detectable changes in arterial structure or in expression level of endothelial nitric-oxide synthase, SK3, or IK1. Arterial relaxing responses to acetylcholine and 3-oxime-6,7-dichlore-1H-indole-2,3-dione (NS309; activator of SK3 and IK1) were measured in the absence and presence of endothelium, NO, and prostanoid blockers, and 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-metheno-7H-dibenzo [b,n] [1,5,12,16]tetraazacyclotricosine-5,13-diium dibromide (UCL 1684; inhibitor of SK3) or 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; inhibitor of IK1). In LF arteries, endothelium-dependent relaxation was markedly reduced, due to a reduction in the endothelium-derived hyperpolarizing factor (EDHF) response. In HF arteries, the balance between the NO/prostanoid versus EDHF response was unaltered. However, the contribution of IK1 to the EDHF response was enhanced, as indicated by a larger effect of TRAM-34 and a larger residual NS309-induced relaxation in the presence of UCL 1684. Reduction of blood flow selectively blunts EDHF relaxation in resistance arteries through inhibition of the function of K(Ca) channels. An increase in blood flow leads to a more prominent role of IK1 channels in this relaxation.


Subject(s)
Endothelium, Vascular/metabolism , Intermediate-Conductance Calcium-Activated Potassium Channels/biosynthesis , Mesenteric Arteries/metabolism , Potassium Channels, Calcium-Activated/biosynthesis , Acetylcholine/pharmacology , Animals , Biological Factors/physiology , Cyclooxygenase Inhibitors/pharmacology , Endothelium, Vascular/drug effects , Guanylate Cyclase/pharmacology , Indoles/pharmacology , Intermediate-Conductance Calcium-Activated Potassium Channels/agonists , Intermediate-Conductance Calcium-Activated Potassium Channels/antagonists & inhibitors , Male , Mesenteric Arteries/drug effects , Muscle Contraction , Muscle Relaxation , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/physiology , Nitric Oxide Synthase/antagonists & inhibitors , Oximes/pharmacology , Potassium Channels, Calcium-Activated/agonists , Potassium Channels, Calcium-Activated/antagonists & inhibitors , Rats , Rats, Inbred WKY , Receptors, Cytoplasmic and Nuclear/pharmacology , Small-Conductance Calcium-Activated Potassium Channels , Soluble Guanylyl Cyclase , Splanchnic Circulation , Stress, Mechanical
9.
Can J Physiol Pharmacol ; 86(8): 557-65, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18758504

ABSTRACT

Endothelin-1 (ET-1) is implicated in the development of endothelial dysfunction through the generation of reactive oxygen species by NADPH oxidase activation. Interleukin-10 (IL-10) is an antiinflammatory cytokine that stimulates nitric oxide production, decreases superoxide production, and restores endothelial integrity after vascular injury. In this study, we tested whether IL-10 attenuates ET-1-induced endothelial dysfunction by improving acetylcholine (ACh)-induced relaxation of cultured murine aortic rings. Aortic rings (2 mm long) of C57BL/6 mice were incubated in 2 mL DMEM containing 120 U/mL penicillin and 120 microg/mL streptomycin in the presence of one of 4 treatments: vehicle (deionized water), ET-1 (100 nmol/L), recombinant mouse IL-10 (300 ng/mL), or a combination of both ET-1 and IL-10. After incubation at 37 degrees C for either 1 or 6 h (short-term exposure) or 22 h (overnight exposure), rings were mounted in a wire myograph and stretched to a passive force of 5 mN. Endothelium-dependent vasorelaxation was assessed by constructing cumulative concentration-response curves to ACh (0.001-10 micromol/L) during 10 mumol/L phenylephrine (PE)-induced contraction. Short-term exposure of ET-1 did not result in an impairment of ACh-induced relaxation. Overnight exposure of aortic rings to ET-1 resulted in a statistically significant endothelial dysfunction characterized by a reduced maximal relaxation response to ACh compared with that of untreated rings (Emax 57% +/- 3% versus 82% +/- 4%). IL-10 treatment restored ACh-induced relaxation (Emax 77% +/- 3%). Western blotting showed decreased eNOS expression in response to ET-1, whereas vessels treated with a combination of ET-1 and IL-10 showed increased expression of eNOS. Immunohistochemical analysis showed decreased eNOS expression in ET-1-treated vessels compared with those treated with both ET-1 and IL-10. We conclude that, in murine aorta, the antiinflammatory cytokine IL-10 prevents impairment in endothelium-dependent relaxation induced in response to long-term incubation with ET-1 via normalization of eNOS expression.


Subject(s)
Endothelin-1/pharmacology , Endothelium, Vascular/physiology , Interleukin-10/pharmacology , Muscle Relaxation/drug effects , Muscle, Smooth, Vascular/drug effects , Acetylcholine/pharmacology , Animals , Aorta, Thoracic/drug effects , Blotting, Western , Down-Regulation/drug effects , Fluorescent Antibody Technique , Immunohistochemistry , In Vitro Techniques , Male , Mice , Mice, Inbred C57BL , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase Type III/antagonists & inhibitors , Nitric Oxide Synthase Type III/biosynthesis , Nitroprusside/pharmacology , Phenylephrine/pharmacology , Vasoconstrictor Agents/pharmacology , Vasodilator Agents/pharmacology
10.
J Hypertens ; 25(8): 1687-97, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17620967

ABSTRACT

BACKGROUND: The phosphorylation of myosin light chain (MLC) maintains the contracted state of vascular smooth muscle. Dephosphorylation results in relaxation and is determined by the activity of myosin light chain phosphatase (MLCP), which is negatively regulated by Rho kinase. METHODS: We tested whether an increased Rho kinase activity, and hence a decreased contribution of MLCP, results in an increased contractility of small fourth-order mesenteric arteries (MA) during the early onset of angiotensin II (Ang II)-induced hypertension (Ang II-14d). RESULTS: Calcium sensitivity was similar, but contractile tension in response to [Ca]ex (5 mmol/l) in endothelium-denuded and depolarized MA was greater, in Ang II-14d rats compared to sham-operated normotensive (SHAM) and Ang II-1d. The Rho kinase inhibitor Y-27,632 caused a significantly greater inhibition of the contractile response to various agents (phenylephrine, norepinephrine, U46,619 and K) in MA of Ang II-14d compared to SHAM. Protein expression levels of the GDP/GTP exchange factor PDZ-RhoGEF, which co-immunoprecipitated with RhoA, were increased in MA of Ang II-14d compared to SHAM. RhoA translocation was greater in U46,619 (1 micromol/l)-stimulated MA of Ang II-14d compared to SHAM. Expression levels of Rho kinase beta were higher in MA of Ang II-14d. The MLCP inhibitor calyculin A (100 nmol/l) caused a greater contraction in MA of SHAM compared to Ang II-14d. Phosphorylation of the target subunit of MLCP (MYPT1) was enhanced in U46,619-stimulated MA of Ang II-14d compared to SHAM. CONCLUSION: This is the first study demonstrating enhanced PDZ-RhoGEF/RhoA/Rho kinase signaling during hypertension at the level of resistance-sized arteries. This enhanced signaling leads to increased MLCP phosphorylation, resulting in vascular hyper-reactivity.


Subject(s)
Angiotensin II/toxicity , Guanine Nucleotide Exchange Factors/metabolism , Hypertension/metabolism , Mesenteric Arteries/metabolism , Signal Transduction , rhoA GTP-Binding Protein/metabolism , Amides/pharmacology , Animals , Base Sequence , Calcium/metabolism , DNA Primers , Hypertension/chemically induced , Immunoprecipitation , Myosin-Light-Chain Phosphatase/metabolism , Protein Transport , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction
11.
Am J Physiol Heart Circ Physiol ; 292(6): H3103-8, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17322422

ABSTRACT

ANG II stimulates the production of reactive oxygen species and activates proinflammatory cytokines leading to endothelial dysfunction. We hypothesized that the anti-inflammatory cytokine IL-10 counteracts the impairment in endothelium-dependent ACh relaxation caused by ANG II. Aortic rings of C57BL/6 mice were incubated in DMEM in the presence of vehicle (deionized H(2)O), ANG II (100 nmol/l), recombinant mouse IL-10 (300 ng/ml), or both ANG II and IL-10 for 22 h at 37 degrees C. After incubation, rings were mounted in a wire myograph to assess endothelium-dependent vasorelaxation to cumulative concentrations of ACh. Overnight exposure of aortic rings to ANG II resulted in blunted ACh-induced vasorelaxation compared with that shown in untreated rings (maximal response = 44 +/- 3% vs. 64 +/- 3%, respectively; P<0.05). IL-10 treatment significantly restored this impairment in relaxation (63 +/- 2%). In addition, the NADPH oxidase inhibitor apocynin restored the impairment in relaxation (maximal response = 76 +/- 3%). Western blotting showed increased gp91(phox) expression (a subunit of NADPH oxidase) in response to ANG II. Vessels treated with a combination of ANG II and IL-10 showed decreased expression of gp91(phox). Immunohistochemical analysis showed increased gp91(phox) expression in ANG II-treated vessels compared with those treated with combined ANG II and IL-10. We found that the anti-inflammatory cytokine IL-10 prevents impairment in endothelium-dependent vasorelaxation in response to long-term incubation with ANG II via decreasing NADPH oxidase expression.


Subject(s)
Angiotensin II/metabolism , Aorta/metabolism , Interleukin-10/metabolism , Membrane Glycoproteins/metabolism , NADPH Oxidases/metabolism , Vasodilation , Acetophenones/pharmacology , Acetylcholine/pharmacology , Angiotensin II/pharmacology , Animals , Aorta/drug effects , Aorta/enzymology , Aorta/physiopathology , Dose-Response Relationship, Drug , Endothelium, Vascular/metabolism , Enzyme Inhibitors/pharmacology , Interleukin-10/pharmacology , Male , Membrane Glycoproteins/antagonists & inhibitors , Mice , Mice, Inbred C57BL , Myography , NADPH Oxidase 2 , NADPH Oxidases/antagonists & inhibitors , Organ Culture Techniques , Reactive Oxygen Species/metabolism , Recombinant Proteins/metabolism , Vasodilation/drug effects , Vasodilator Agents/pharmacology
12.
Am J Physiol Heart Circ Physiol ; 292(5): H2275-84, 2007 May.
Article in English | MEDLINE | ID: mdl-17209000

ABSTRACT

Ca(2+)-activated K(+) channels (K(Ca)), in particular, the small and intermediate K(Ca) (SK(Ca) and IK(Ca), respectively) channels, are key players in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in small arteries. Hypertension is characterized by an endothelial dysfunction, possibly via reduced EDHF release and/or function. We hypothesize that during angiotensin II (14 days)-induced hypertension (ANG II-14d), the contribution of SK(Ca) and IK(Ca) channels in ACh-induced relaxations is reduced due to decreased expression of SK(Ca) and IK(Ca) channel proteins in rat small mesenteric arteries (MAs). Nitric oxide- and prostacyclin-independent vasorelaxation to ACh was similar in small MAs of sham-operated and ANG II-14d rats. Catalase had no inhibitory effects on these relaxations. The highly selective SK(Ca) channel blocker UCL-1684 almost completely blocked these responses in MAs of sham-operated rats but partially in MAs of ANG II-14d rats. These changes were pressure dependent since UCL-1684 caused a greater inhibition in MAs of 1-day ANG II-treated normotensive rats compared with ANG II-14d rats. Expression levels of both mRNA and protein SK3 were significantly reduced in MAs of ANG II-14d rats. The IK(Ca) channel blocker 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) resulted in comparable reductions in the relaxation responses to ACh in MAs of sham-operated and ANG II-14d rats. Relative mRNA expression levels of IK1 were significantly reduced in MAs of ANG II-14d rats, whereas protein levels of IK1 were not but tended to be lower in MAs of ANG II-14d rats. The findings demonstrate that EDHF-like responses are not compromised in a situation of reduced functional activity and expression of SK3 channels in small MAs of ANG II-induced hypertensive rats. The role of IK1 channels is less clear but might compensate for reduced SK3 activity.


Subject(s)
Angiotensin II/adverse effects , Arterioles/metabolism , Hypertension/chemically induced , Hypertension/metabolism , Mesenteric Arteries/metabolism , Potassium Channels, Calcium-Activated/metabolism , Vasodilation/drug effects , Animals , Arterioles/drug effects , Gene Expression/drug effects , Mesenteric Arteries/drug effects , Rats , Rats, Sprague-Dawley
13.
J Pharmacol Exp Ther ; 318(1): 288-95, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16569756

ABSTRACT

Spontaneous tone in large arteries may contribute to the pathogenesis of hypertension. Reactive oxygen species and Ca2+ influx have been shown to stimulate the development of spontaneous tone in isolated aortic rings in several models of hypertensive rats. The aim of this study was to investigate the role of the RhoA/Rho-kinase signaling pathway in the development of spontaneous tone in angiotensin II-induced hypertension and to explore the underlying mechanisms of RhoA/Rho-kinase activation. Our results showed that spontaneous tone was greatly enhanced in endothelium-denuded aortic rings from angiotensin II-induced hypertensive rats compared with their normotensive counterparts (73+/-5 versus 7+/-3% of phenylephrine-induced maximal contraction, respectively). The Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) (0.1-10 microM) concentration dependently inhibited spontaneous tone in aortic rings from angiotensin II-treated rats. NADPH oxidase inhibitors diphenylene iodonium and apocynin also significantly reduced spontaneous tone. Chronic angiotensin II treatment markedly increased RhoA protein expression (57%) but had no effect on Rho guanine nucleotide exchange factor mRNA or Rho-kinase protein expression levels. In endothelium-denuded rings from normotensive rats, angiotensin II (100 nM) increased RhoA membrane translocation and phosphorylation of the myosin light chain phosphatase target subunit, which were both blocked by the NADPH oxidase inhibitor diphenylene iodonium (10 microM). In conclusion, these data suggest that chronic treatment with angiotensin II leads to up-regulation of the RhoA/Rho-kinase pathway, contributing to spontaneous tone development in rat aorta. Increased NADPH oxidase-dependent reactive oxygen species may be one of the mechanisms mediating the RhoA/Rho-kinase activation.


Subject(s)
Angiotensin II/toxicity , Aorta/enzymology , Hypertension/enzymology , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/physiology , Vasoconstriction/physiology , Amides/pharmacology , Animals , Aorta/drug effects , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Activation/physiology , Hypertension/chemically induced , In Vitro Techniques , Intracellular Signaling Peptides and Proteins , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Vasoconstriction/drug effects , rho-Associated Kinases
14.
Am J Physiol Heart Circ Physiol ; 291(1): H216-22, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16473954

ABSTRACT

Ca+ -activated K+ -channels (KCa) regulate vasomotor tone via smooth muscle hyperpolarization and relaxation. The relative contribution of the endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation differs depending on vessel type and size. It is unknown whether these KCa channels are differentially distributed along the same vascular bed and hence have different roles in mediating the EDHF response. We therefore assessed the role of small- (SKCa), intermediate- (IKCa), and large-conductance (BKCa) channels in mediating acetylcholine-induced relaxations in both first- and fourth-order side branches of the rat superior mesenteric artery (MA1 and MA4, respectively). Two-millimeter segments of each MA were mounted in the wire myograph, incubated with Nomega-nitro-L-arginine methyl ester (L-NAME, 100 micromol/l) and indomethacin (10 micromol/l), and precontracted with phenylephrine (10 micromol/l). Cumulative concentration-response curves to ACh (0.001-10 micromol/l) were performed in the absence or presence of selective KCa channel antagonists. Apamin almost completely abolished these relaxations in MA4 but only partially blocked relaxations in MA1. The selective IKCa channel blocker 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) caused a significantly greater inhibition of the ACh-induced relaxation in MA4 compared with MA1. Iberiotoxin had no inhibitory effect in MA4 but blunted relaxation in MA1. Relative mRNA expression levels of SKCa (rSK1, rSK3, and rSK4 = rIK1) were significantly higher in MA4 compared with MA1. BKCa (rBKalpha1 and rBKbeta1) genes were similar in both MA1 and MA4. Our data demonstrate regional heterogeneity in SKCa and IKCa function and gene expression and stress the importance of these channels in smaller resistance-sized arteries, where the role of EDHF is more pronounced.


Subject(s)
Acetylcholine/administration & dosage , Ion Channel Gating/physiology , Mesenteric Arteries/physiology , Potassium Channels, Calcium-Activated/physiology , Vasodilation/physiology , Animals , Dose-Response Relationship, Drug , In Vitro Techniques , Ion Channel Gating/drug effects , Male , Mesenteric Arteries/drug effects , Potassium Channels, Calcium-Activated/drug effects , Rats , Rats, Sprague-Dawley , Regional Blood Flow/drug effects , Regional Blood Flow/physiology , Vascular Resistance/drug effects , Vascular Resistance/physiology , Vasodilation/drug effects
15.
Exp Biol Med (Maywood) ; 230(11): 829-35, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16339747

ABSTRACT

The vascular smooth muscle cell is a highly specialized cell whose primary function is contraction and relaxation. It expresses a variety of contractile proteins, ion channels, and signalling molecules that regulate contraction. Upon contraction, vascular smooth muscle cells shorten, thereby decreasing the diameter of a blood vessel to regulate the blood flow and pressure. Contractile activity in vascular smooth muscle cells is initiated by a Ca(2+)-calmodulin interaction to stimulate phosphorylation of the light chain of myosin. Ca(2+)-sensitization of the contractile proteins is signaled by the RhoA/Rho-kinase pathway to inhibit the dephosphorylation of the light chain by myosin phosphatase, thereby maintaining force. Removal of Ca(2+) from the cytosol and stimulation of myoson phosphatase initiate the relaxation of vascular smooth muscle.


Subject(s)
Calcium Signaling/physiology , Muscle Relaxation/physiology , Muscle, Smooth, Vascular/physiology , Myocytes, Smooth Muscle/physiology , rhoA GTP-Binding Protein/metabolism , Animals , Arteries/physiology , Blood Flow Velocity/physiology , Blood Pressure/physiology , Calmodulin/metabolism , Humans , Myosin Light Chains/metabolism , Myosin-Light-Chain Phosphatase/metabolism , Phosphorylation
16.
Am J Physiol Heart Circ Physiol ; 288(3): H1022-7, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15706039

ABSTRACT

In resistance-sized arteries, a chronic increase in blood flow leads to increases in arterial structural luminal diameter and arterial wall mass. In this review, we summarize recent evidence that outward remodeling of resistance arteries 1) can help maintain and restore tissue perfusion, 2) is not intimately related to flow-induced vasodilatation, 3) involves transient dedifferentiation and turnover of arterial smooth muscle cells, and 4) is preceded by increased expression of matricellular proteins, which have been shown to promote disassembly of focal adhesion sites. Studies of experimental and physiological resistance artery remodeling involving differential gene expression analyses and the use of knockout and transgenic mouse models can help unravel the mechanisms of outward remodeling.


Subject(s)
Arteries/physiology , Gene Expression Profiling , Genomics , Vascular Resistance/genetics , Vasodilation/genetics , Animals , Humans
17.
Circ Res ; 94(12): 1623-9, 2004 Jun 25.
Article in English | MEDLINE | ID: mdl-15131008

ABSTRACT

The vascular kallikrein-kinin system contributes to about one third of flow-dependent dilation in mice carotid arteries, by activating bradykinin B2 receptors coupled to endothelial nitric oxide (NO) release. Because the bradykinin/NO pathway may mediate some of the effects of angiotensin II AT2 receptors, we examined the possible contribution of AT2 receptors to the kinin-dependent response to flow. Changes in outer diameter after increases in flow rate were evaluated in perfused arteries from wild-type animals (TK+/+) and in tissue kallikrein-deficient mice (TK-/-) in which the presence of AT2 receptor expression was verified. Saralasin, a nonselective angiotensin II receptor antagonist, impaired significantly flow-induced dilation in TK+/+, whereas it had no effect in TK-/- mice. In both groups, blockade of AT1 receptors with losartan or candesartan did not affect the response to flow. Inhibition of AT2 receptors with PD123319 reduced significantly flow-induced dilation in TK+/+ mice, but had no significant effect in TK-/- mice. Combining PD123319 with the bradykinin B2 receptor antagonist HOE-140 had no additional effect to AT2 receptor blockade alone in TK+/+ arteries. Flow-dependent-dilation was also impaired in AT2 receptor deficient mice (AT2-/-) when compared with wild-type littermates. Furthermore, HOE-140 significantly reduced the response to flow in the AT2+/+, but not in AT2-/- mice. In conclusion, this study demonstrates that the presence of functional AT2 receptors is necessary to observe the contribution of the vascular kinin-kallikrein system to flow-dependent dilation.


Subject(s)
Bradykinin/analogs & derivatives , Carotid Arteries/drug effects , Receptor, Angiotensin, Type 2/physiology , Tissue Kallikreins/physiology , Vasodilation/physiology , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Acetylcholine/pharmacology , Angiotensin II Type 1 Receptor Blockers/pharmacology , Angiotensin II Type 2 Receptor Blockers , Animals , Benzimidazoles/pharmacology , Biphenyl Compounds , Bradykinin/pharmacology , Bradykinin B2 Receptor Antagonists , Carotid Arteries/physiology , Carotid Arteries/ultrastructure , Hemorheology , Imidazoles/pharmacology , Losartan/pharmacology , Male , Mesenteric Arteries/drug effects , Mesenteric Arteries/physiology , Mesenteric Arteries/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenylephrine/pharmacology , Pyridines/pharmacology , Receptor, Angiotensin, Type 2/deficiency , Receptor, Angiotensin, Type 2/genetics , Receptor, Bradykinin B2/physiology , Tetrazoles/pharmacology , Tissue Kallikreins/deficiency , Tissue Kallikreins/genetics , Vasodilation/drug effects , Vasodilator Agents/pharmacology
18.
Arterioscler Thromb Vasc Biol ; 24(5): 892-7, 2004 May.
Article in English | MEDLINE | ID: mdl-15031129

ABSTRACT

OBJECTIVE: To test whether membrane-bound angiotensin I-converting enzyme (t-ACE) is involved in arterial remodeling, we applied unilateral carotid artery (CA) ligation and studied uterine arteries (UA) before, during, and after pregnancy in t-ACE-/- and t-ACE+/+ mice. RESULTS- In CA of t-ACE-/- mice, blood pressure, outer diameter (D), and medial cross-sectional area (mCSA) were reduced, whereas blood flow (BF) and the number of medial cells (mC) were not modified. In the ligated CA, mCSA and number of mC were increased while outer D and distensibility were reduced. These changes were significantly less pronounced in t-ACE-/- than t-ACE+/+ mice. In UA of t-ACE-/- mice, D was larger and mCSA was unaltered. At term pregnancy, D and mCSA of the UA were reversibly increased. Structural changes of UA during and after pregnancy were comparable in both strains. CONCLUSIONS: t-ACE contributes to arterial structure and remodeling. It plays a major role in hyperplastic inward remodeling of the CA imposed by blood flow cessation, but it is not essential for outward hypertrophic and subsequent inward hypotrophic remodeling of the UA during and after pregnancy.


Subject(s)
Carotid Arteries/enzymology , Hemorheology , Membrane Proteins/physiology , Peptidyl-Dipeptidase A/physiology , Uterus/blood supply , Alleles , Angiotensin II/physiology , Animals , Arteries/anatomy & histology , Arteries/enzymology , Carotid Arteries/pathology , Female , Hyperplasia , Hypertrophy , Ligation , Male , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/deficiency , Peptidyl-Dipeptidase A/genetics , Postpartum Period , Pregnancy , Protein Structure, Tertiary , Sequence Deletion , Stress, Mechanical
19.
Arterioscler Thromb Vasc Biol ; 23(10): 1826-32, 2003 Oct 01.
Article in English | MEDLINE | ID: mdl-12933530

ABSTRACT

OBJECTIVE: Tissue kallikrein (TK) participates in acute flow-induced dilatation (FID) of large arteries. We investigated whether TK deficiency blunts FID and alters chronic flow-related arterial structural and functional changes in resistance-sized muscular arteries. METHODS AND RESULTS: Vasomotor responses and structural parameters were determined in uterine arteries isolated from nonpregnant, 18- to 19-day pregnant, and 7-day postpartum TK-/- and TK+/+ littermate mice. In TK-/- mice, values of diameter, medial cross-sectional area (CSA), myogenic tone, and dilatation in response to acetylcholine were comparable to those values in TK+/+ mice, but FID (0 to 100 microL/min) was significantly reduced (55+/-4% versus 85+/-4% in TK+/+ mice). In both mouse strains, pregnancy resulted in significant increases in diameter and medial CSA and in the Nw-nitro-l-arginine methyl ester-sensitive component of FID. By 7 days after pregnancy, uterine arterial diameter and CSA values no longer differed from nonpregnant values, and FID was markedly reduced in TK-/- and TK+/+ mice. CONCLUSIONS: These observations (1) confirm at the level of resistance arteries the key role of TK in FID and (2) indicate that TK deficiency does not compromise arterial remodeling and changes in the contribution of NO to FID during and after pregnancy.


Subject(s)
Hemodynamics , Pregnancy, Animal/physiology , Tissue Kallikreins/deficiency , Uterus/blood supply , Animals , Arteries/anatomy & histology , Arteries/physiology , Female , Hemorheology , Mesenteric Arteries/anatomy & histology , Mesenteric Arteries/physiology , Mice , Mice, Knockout , Models, Animal , Nitric Oxide , Pregnancy , Tissue Kallikreins/genetics , Vascular Resistance , Vasoconstriction , Vasodilation
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