Flow-induced Dilation Research Articles

Asymmetric Dimethylarginine Reduces Nitric Oxide Donor-Mediated Dilation of Arterioles by Activating the Vascular Renin-Angiotensin System and Reactive Oxygen Species

Introduction: We tested the hypothesis that asymmetric dimethylarginine (ADMA) interferes with other mechanisms in addition to inhibition of nitric oxide synthase (NOS). Thus, in skeletal muscle arterioles, in the presence of ADMA, we investigated the dilator effect of an NO donor and increases in flow and aimed to elucidate the underlying mechanisms, including the role of oxidative stress, which is known to reduce the bioavailability of NO. Methods and Results: In isolated rat gracilis skeletal muscle arterioles (∼160 µm at 80 mm Hg), ADMA (similarly to pyrogallol) reduced dilations to sodium nitroprusside (SNP), which was significantly prevented by the presence of superoxide dismutase (SOD) and catalase (CAT): SNP 10^–8M; control: 43.2 ± 3%, ADMA: 4.9 ± 1%, ADMA + SOD/CAT: 30.2 ± 9% (p < 0.05). Also, ADMA reduced basal diameter and flow-induced dilations, which were not restored by L-arginine, but prevented by SOD/CAT and by inhibition of NAD(P)H oxidase (but not xanthine oxidase) and by an angiotensin-converting enzyme inhibitor or an angiotensin type 1 receptor blocker (ARB). ADMA increased the production of reactive oxygen species detected by lucigenin-enhanced chemiluminescence, which was significantly inhibited by SNP or ARB. Conclusion: We suggest that by activating the vascular renin-angiotensin-NAD(P)H oxidase pathway, ADMA elicits oxidative stress, which interferes with the bioavailability of NO and consequently reduces NO-mediated dilations.

Microvascular reactivity in subcutaneous and visceral fat tissue in human obesity

Microvascular dysfunction contributes to cardiovascular disease risk during obesity. Prior data suggest that visceral adipose tissue (VAT) is associated to cardiometabolic risk factors and endothelial dysfunction compared subcutaneous adipose tissue (SAT). The aim of this study was to determine flow induced dilation (FID) in human VAT and SAT resistance arteries. We hypothesized that microvascular FID is impaired in VAT compared to SAT during obesity. Microvessels were obtained from VAT and SAT biopsies in lean (LN; BMI&lt;27) and during bariatric surgery (OB; BMI&gt;48). Isolated vessels were cannulated for vascular reactivity to flow (pressure gradient cmH2O, 20, 40, 60, 100) in the absence and presence of the NOS inhibitor L‐NAME (10−4 M). Dilations to papaverine (10−4M) were determined in each vessel. FID was reduced in VAT (at 60, 59±7%, p&lt;0.05) compared to SAT (at 60, 100±10%). Interestingly, FID was increased in SAT but not VAT from OB compared to LN subjects. There was no change in maximal dilation to papaverine between groups. In addition, there was no effect of L‐NAME on FID in SAT of OB and LN. However, L‐NAME reduced FID in VAT of OB (at 60, 36±8%; p&lt;0.05) vs. baseline. These data suggest that 1) FID is reduced in visceral compared to subcutaneous fat of obese patients and 2) NO mediates FID in microvessels from visceral but not subcutaneous adipose during obesity. NIH (K23HL085614, R01HL095701), UKF (87/11, 88/11).

Aging impairs flow‐induced dilation in skeletal muscle feed arteries: role of Akt‐dependent phosphorylation of eNOS

We tested the hypothesis that impaired nitric oxide (NO)‐mediated, endothelium‐dependent dilation in aged soleus muscle feed arteries (SFA) is due to an age‐related decrement in PI3K/Akt‐dependent phosphorylation of endothelial NO synthase (eNOS) on serine 1177 (p‐eNOSser1177). SFA from young and old Fischer 344 rats were cannulated for examination of flow‐induced dilation (FID). To determine how aging affected FID, dilator responses were assessed in the absence and presence of pharmacological inhibitors of NOS (L‐NNA), PI3K (LY‐294002), or Akt (AktI). Results indicate that FID was blunted in old SFA, while dilation to sodium nitroprusside was not compromised. Age‐group differences in FID were abolished in the presence of L‐NNA, LY‐294002, or AktI. In a separate experiment, FID was assessed in SFA from young and old rats. After FID assessment, SFA were then removed from the pipettes, snap frozen, and immunoblot analysis was used to assess p‐AktSer473, p‐eNOSser1177, total Akt and total eNOS protein content. FID was blunted in old SFA; however, the p‐AktSer473/Akt and p‐eNOSser1177/eNOS ratios were similar in young and old SFA. Collectively, these results indicate that NO‐mediated dilation is impaired in old SFA, but is not due to reduced PI3K/Akt‐dependent phosphorylation of eNOS.Research supported by AHA 0765043 and AHA 4150031 (CRW), and Sydney and J. L. Huffines Institute of Sports Medicine Graduate Student Research Grants (MJL, JWS, and DWT).

NADPH oxidase‐dependent reactive oxygen species are involved in flow‐induced dilation of human adipose arterioles

Hydrogen peroxide (H2O2) as an endothelium‐derived vasodilator factor mediates flow‐induced dilation (FID) in human adipose arterioles from patients with coronary artery disease (CAD). The intracellular sources responsible for H2O2 generation during FID remain unclear. This study is designed to test whether NADPH oxidase (NOX) serves as a potential source of reactive oxygen species (ROS) formation contributing to FID. Visceral adipose arterioles (~150 μm ID) obtained from patients with or without CAD were cannulated and pressurized at 60 mmHg for videomicroscopic measurement of vessel diameters. After constriction with endothelin‐1, arterioles were subjected to stepwise increase in luminal flow before and after treatment with gp91‐ ds‐tat, a cell‐permeable peptide inhibitor of NOX, or a scrambled control peptide. Flow induced a graded dilation of adipose arterioles from CAD (maximal dilation: 61±3%, n=6), and this dilation was markedly reduced by gp91‐ds‐tat (50μM; 5±4%, n=5, P&lt;0.05). FID was not affected by the scrambled peptide (67±2 vs. 61±3% of controls). In contrast to arterioles from CAD patients, gp91‐ds‐tat had no effect on FID in arterioles from patients without CAD (68±2% vs. 67±2% of controls, n=5–6). In conclusion, NOX plays an essential role in FID of human adipose arterioles.

Exercise training during human obesity protects against impaired microvascular function after acute exertion by enhancing hydrogen peroxide‐mediated flow‐induced dilation

Our recent studies suggest that increased superoxide dismutase and endothelial nitric oxide synthase (eNOS) contribute to the protective effects of chronic exercise on nitric oxide (NO) and hydrogen peroxide (H2O2)‐mediated acetylcholine (AChD) dilation after acute exertion. The goal of this study was to determine the effect of exercise training on flow‐induced dilation (FID) and AChD in resistance arteries (RAs) of obese patients. We hypothesized that H2O2 mediates the protective effect of exercise training against impaired FID and AChD induced by acute exertion. A protocol of weight lifting (WL; 8 weeks) was applied to subjects (BMI: 30–35). RAs were dissected from gluteal fat biopsies and cannulated for measurements of intraluminal diameters. NO and H2O2 production was assessed by confocal microscopy. All subjects had similar BP responses to WL. The eNOS inhibitor L‐NAME blocked FID (75% ± 12%) and AChD (60% ± 8%) in sedentary subjects. In contrast, PEG‐catalase, a H2O2 scavenger, blocked FID (68% ± 11%) and AChD (61% ± 7%) after exercise training. Fluorescence studies showed reduced NO (0.70 ± 0.05 fold) and increased H2O2 (4.55 ± 0.83 fold) production in RA post‐WL compared to pre‐WL. We conclude that H2O2 contributes to the protective effect of exercise training against reduced NO‐mediated dilation after exposure to acute exertion‐associated with hypertension in obesity. NIH (K23HL085614, R01HL095701).

11,12,20-Trihydroxy-eicosa-8(Z)-enoic acid: a selective inhibitor of 11,12-EET-induced relaxations of bovine coronary and rat mesenteric arteries

Arachidonic acid is metabolized to four regioisomeric epoxyeicosatrienoic acids (EETs) by cytochrome P-450. 5,6-, 8,9-, 11,12-, and 14,15-EET are equipotent in relaxing bovine coronary arteries (BCAs). Vasorelaxant effects of EETs are nonselectively antagonized by 14,15-epoxyeicosa-5(Z)-enoic acid. The 11,12-EET analogs, 20-hydroxy-11,12-epoxyeicosa-8(Z)-enoic acid (20-H-11,12-EE8ZE) and 11,12,20-trihydroxyeicosa-8(Z)-enoic acid (11,12,20-THE8ZE) were synthesized and tested for antagonist activity against EET-induced relaxations in BCAs. In U-46619-preconstricted arterial rings, 5,6-, 8,9-, 11,12-, and 14,15-EET caused concentration-dependent relaxations with maximal relaxations ranging from 80 to 96%. Preincubation of arteries with 20-H-11,12-EE8ZE (10(-5) M) inhibited relaxations to 14,15- and 11,12-EET, but not 5,6- and 8,9-EET; however, greatest inhibitory effect was against 11,12-EET (maximal relaxation = 80.6 ± 4.6 vs. 26.7 ± 7.4% without and with 20-H-11,12-EE8ZE, respectively). Preincubation with the soluble epoxide hydrolase inhibitor (tAUCB, 10(-6) M) significantly enhanced the antagonist effect of 20-H-11,12-EE8ZE against 14,15-EET-induced relaxations (maximal relaxation = 86.6 ± 4.4 vs. 27.8 ± 3.3%, without and with 20-H-11,12-EE8ZE and tAUCB) without any change in its effect against 11,12-EET-induced relaxations. In contrast to the parent compound, the metabolite, 11,12,20-THE8ZE (10(-5) M), significantly inhibited relaxations to 11,12-EET and was without effect on other EET regioisomers. Mass spectrometric analysis revealed conversion of 20-H-11,12-EE8ZE to 11,12,20-THE8ZE by incubation with BCA. The conversion was blocked by tAUCB. 14,15-Dihydroxy-eicosa-5Z-enoic acid (a 14,15-EET antagonist), but not 11,12,20-THE8ZE (an 11,12-EET antagonist), inhibited BCA relaxations to arachidonic acid and flow-induced dilation in rat mesenteric arteries. These results indicate that 11,12,20-THE8ZE is a selective antagonist of 11,12-EET relaxations and a useful pharmacological tool to elucidate the function of 11,12-EET in the cardiovascular system.

H 2 O 2 -Induced Dilation in Human Coronary Arterioles: Role of Protein Kinase G Dimerization and Large-Conductance Ca 2+ -Activated K + Channel Activation

Hydrogen peroxide (H(2)O(2)) serves as a key endothelium-derived hyperpolarizing factor mediating flow-induced dilation in human coronary arterioles (HCAs). The precise mechanisms by which H(2)O(2) elicits smooth muscle hyperpolarization are not well understood. An important mode of action of H(2)O(2) involves the oxidation of cysteine residues in its target proteins, including protein kinase G (PKG)-Iα, thereby modulating their activities. Here we hypothesize that H(2)O(2) dilates HCAs through direct oxidation and activation of PKG-Iα leading to the opening of the large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel and subsequent smooth muscle hyperpolarization. Flow and H(2)O(2) induced pressure gradient/concentration-dependent vasodilation in isolated endothelium-intact and -denuded HCAs, respectively. The dilation was largely abolished by iberiotoxin, a BK(Ca) channel blocker. The PKG inhibitor Rp-8-Br-PET-cGMP also markedly inhibited flow- and H(2)O(2)-induced dilation, whereas the soluble guanylate cyclase inhibitor ODQ had no effect. Treatment of coronary smooth muscle cells (SMCs) with H(2)O(2) elicited dose-dependent, reversible dimerization of PKG-Iα, and induced its translocation to the plasma membrane. Patch-clamp analysis identified a paxilline-sensitive single-channel K(+) current with a unitary conductance of 246-pS in freshly isolated coronary SMCs. Addition of H(2)O(2) into the bath solution significantly increased the probability of BK(Ca) single-channel openings recorded from cell-attached patches, an effect that was blocked by the PKG-Iα inhibitor DT-2. H(2)O(2) exhibited an attenuated stimulatory effect on BK(Ca) channel open probability in inside-out membrane patches. H(2)O(2) dilates HCAs through a novel mechanism involving protein dimerization and activation of PKG-Iα and subsequent opening of smooth muscle BK(Ca) channels.

Activation of endothelial TRPV4 channels mediates flow-induced dilation in human coronary arterioles: role of Ca2+ entry and mitochondrial ROS signaling

In human coronary arterioles (HCAs) from patients with coronary artery disease, flow-induced dilation is mediated by a unique mechanism involving the release of H(2)O(2) from the mitochondria of endothelial cells (ECs). How flow activates ECs to elicit the mitochondrial release of H(2)O(2) remains unclear. Here, we examined the role of the transient receptor potential vanilloid type 4 (TRPV4) channel, a mechanosensitive Ca(2+)-permeable cation channel, in mediating ROS formation and flow-induced dilation in HCAs. Using RT-PCR, Western blot analysis, and immunohistochemical analysis, we detected the mRNA and protein expression of TRPV4 channels in ECs of HCAs and cultured human coronary artery ECs (HCAECs). In HCAECs, 4α-phorbol-12,13-didecanoate (4α-PDD), a selective TRPV4 agonist, markedly increased (via Ca(2+) influx) intracellular Ca(2+) concentration. In isolated HCAs, activation of TRPV4 channels by 4α-PDD resulted in a potent concentration-dependent dilation, and the dilation was inhibited by removal of the endothelium and by catalase, a H(2)O(2)-metabolizing enzyme. Fluorescence ROS assays showed that 4α-PDD increased the production of mitochondrial superoxide in HCAECs. 4α-PDD also enhanced the production of H(2)O(2) and superoxide in HCAs. Finally, we found that flow-induced dilation of HCAs was markedly inhibited by different TRPV4 antagonists and TRPV4-specific small interfering RNA. In conclusion, the endothelial TRPV4 channel is critically involved in flow-mediated dilation of HCAs. TRPV4-mediated Ca(2+) entry may be an important signaling event leading to the flow-induced release of mitochondrial ROS in HCAs. Elucidation of this novel TRPV4-ROS pathway may improve our understanding of the pathogenesis of coronary artery disease and/or other cardiovascular disorders.

A novel vascular EET synthase: role of CYP2C7

We demonstrated previously that cytochrome P-450 (CYP) 2C29 is the epoxyeicosatrienoic acid (EET) synthase responsible for the EET-mediated flow/shear stress-induced dilation of vessels of female nitric oxide (NO)-deficient mice (Sun D, Yang YM, Jiang H, Wu H, Ojami C, Kaley G, Huang A. Am J Physiol Regul Integr Comp Physiol 298: R862-R869, 2010). In the present study, we aimed to identify which specific CYP isoform(s) is the source of the synthesis and release of EETs in response to stimulation by shear stress in vessels of rats. Cannulated mesenteric arteries isolated from both sexes of N(G)-nitro-L-arginine methyl ester (L-NAME)-treated rats were perfused with 2 and 10 dyn/cm(2) shear stress, followed by collection of the perfusate to determine EET concentrations and isoforms. Shear stress stimulated release of EETs in the perfusate of female (but not male) NO-deficient vessels, associated with an EET-mediated vasodilation, in which 11,12- and 14,15-EET contributed predominantly to the responses. Rat CYP cDNA array screened a total of 32 CYP genes of mesenteric arteries, indicating a significant upregulation of CYP2C7 in female L-NAME-treated rats. Endothelial RNA and protein were extracted from intact single vessels. Expression of CYP2C7 mRNA and protein in pooled extractions of endothelial lysate was identified by PCR and Western blot analyses. Transfection of the vessels with CYP2C7 short interfering RNA eliminated the release of EETs, consequently abolishing the EET-mediated flow-induced dilation; these responses, however, were maintained in vessels transfected with nonsilencing short interfering RNA. Knockdown of endothelial CYP2C7 was confirmed by PCR and Western blot analyses. In conclusion, CYP2C7 is an endothelial EET synthase in the female rat vasculature, by which, in NO deficiency, shear stress stimulates the release of EETs to initiate vasodilation.

Aging and estrogen alter endothelial reactivity to reactive oxygen species in coronary arterioles

Endothelium-dependent, nitric oxide (NO)-mediated vasodilation can be impaired by reactive oxygen species (ROS), and this deleterious effect of ROS on NO availability may increase with aging. Endothelial function declines rapidly after menopause, possibly because of loss of circulating estrogen and its antioxidant effects. The purpose of the current study was to determine the role of O(2)(-) and H(2)O(2) in regulating flow-induced dilation in coronary arterioles of young (6-mo) and aged (24-mo) intact, ovariectomized (OVX), or OVX + estrogen-treated (OVE) female Fischer 344 rats. Both aging and OVX reduced flow-induced NO production, whereas flow-induced H(2)O(2) production was not altered by age or estrogen status. Flow-induced vasodilation was evaluated before and after treatment with the superoxide dismutase (SOD) mimetic Tempol (100 μM) or the H(2)O(2) scavenger catalase (100 U/ml). Removal of H(2)O(2) with catalase reduced flow-induced dilation in all groups, whereas Tempol diminished vasodilation in intact and OVE, but not OVX, rats. Immunoblot analysis revealed elevated nitrotyrosine with aging and OVX. In young rats, OVX reduced SOD protein while OVE increased SOD in aged rats; catalase protein did not differ in any group. Collectively, these studies suggest that O(2)(-) and H(2)O(2) are critical components of flow-induced vasodilation in coronary arterioles from female rats; however, a chronic deficiency of O(2)(-) buffering by SOD contributes to impaired flow-induced dilation with aging and loss of estrogen. Furthermore, these data indicate that estrogen replacement restores O(2)(-) homeostasis and flow-induced dilation of coronary arterioles, even at an advanced age.

NAD(P)H oxidase-derived reactive oxygen species contribute to age-related impairments of endothelium-dependent dilation in rat soleus feed arteries

We tested the hypothesis that age-related endothelial dysfunction in rat soleus muscle feed arteries (SFA) is mediated in part by NAD(P)H oxidase-derived reactive oxygen species (ROS). SFA from young (4 mo) and old (24 mo) Fischer 344 rats were isolated and cannulated for examination of vasodilator responses to flow and acetylcholine (ACh) in the absence or presence of a superoxide anion (O(2)(-)) scavenger (Tempol; 100 μM) or an NAD(P)H oxidase inhibitor (apocynin; 100 μM). In the absence of inhibitors, flow- and ACh-induced dilations were attenuated in SFA from old rats compared with young rats. Tempol and apocynin improved flow- and ACh-induced dilation in SFA from old rats. In SFA from young rats, Tempol and apocynin had no effect on flow-induced dilation, and apocynin attenuated ACh-induced dilation. To determine the role of hydrogen peroxide (H(2)O(2)), dilator responses were assessed in the absence and presence of catalase (100 U/ml) or PEG-catalase (200 U/ml). Neither H(2)O(2) scavenger altered flow-induced dilation, whereas both H(2)O(2) scavengers blunted ACh-induced dilation in SFA from young rats. In old SFA, catalase improved flow-induced dilation whereas PEG-catalase improved ACh-induced dilation. Compared with young SFA, in response to exogenous H(2)O(2) and NADPH, old rats exhibited blunted dilation and constriction, respectively. Immunoblot analysis revealed that the NAD(P)H oxidase subunit gp91phox protein content was greater in old SFA compared with young. These results suggest that NAD(P)H oxidase-derived reactive oxygen species contribute to impaired endothelium-dependent dilation in old SFA.

H 2 O 2 Is the Transferrable Factor Mediating Flow-Induced Dilation in Human Coronary Arterioles

Endothelial derived hydrogen peroxide (H(2)O(2)) is a necessary component of the pathway regulating flow-mediated dilation (FMD) in human coronary arterioles (HCAs). However, H(2)O(2) has never been shown to be the endothelium-dependent transferrable hyperpolarization factor (EDHF) in response to shear stress. We examined the hypothesis that H(2)O(2) serves as the EDHF in HCAs to shear stress. Two HCAs were cannulated in series (a donor intact vessel upstream and endothelium-denuded detector vessel downstream). Diameter changes to flow were examined in the absence and presence of polyethylene glycol catalase (PEG-CAT). The open state probability of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels in smooth muscle cells downstream from the perfusate from an endothelium-intact arteriole was examined by patch clamping. In some experiments, a cyanogen bromide-activated resin column bound with CAT was used to remove H(2)O(2) from the donor vessel. When flow proceeds from donor to detector, both vessels dilate (donor:68±7%; detector: 45±11%). With flow in the opposite direction, only the donor vessel dilates. PEG-CAT contacting only the detector vessel blocked FMD in that vessel (6±4%) but not in donor vessel (61±13%). Paxilline inhibited dilation of endothelium-denuded HCAs to H(2)O(2). Effluent from donor vessels elicited K(+) channel opening in an iberiotoxin- or PEG-CAT-sensitive fashion in cell-attached patches but had little effect on channel opening on inside-out patches. Vasodilation of detector vessels was diminished when exposed to effluent from CAT-column. Flow induced endothelial production of H(2)O(2), which acts as the transferrable EDHF activating BK(Ca) channels on the smooth muscle cells.

Impaired flow-induced dilation of coronary arterioles of dogs fed a low-salt diet: roles of ANG II, PKC, and NAD(P)H oxidase

Low-salt (LS) diet has been considered to be beneficial in the prevention and treatment of hypertension; however, it also increases plasma angiotensin (ANG) II and may cause adverse cardiovascular effects, such as endothelial dysfunction. We assessed endothelial function of coronary arterioles and vascular superoxide production, as a function of LS diet. Dogs were fed with LS (0.05% NaCl) or a normal-salt (NS, 0.65% NaCl) diet for 2 wk. There were threefold increases in plasma ANG II, associated with a 60% reduction in flow-induced dilation (FID) in coronary arterioles of LS compared with NS dogs. In vessels of NS dogs, FID was primarily mediated by nitric oxide (NO), as indicated by an eliminated FID by N(ω)-nitro-l-arginine methyl ester (l-NAME). In vessels of LS dogs, however, FID was eliminated. Administration of apocynin, a NAD(P)H oxidase inhibitor, partially restored FID and additional l-NAME eliminated FID. Generation of superoxide, measured with dihydroethidium, was significantly greater in vessels of LS than in NS dogs, which was further increased in response to ANG II or phorbol 12,13-dibutyrate, an agonist of protein kinase C (PKC). The enhanced superoxide was normalized by apocynin, losartan (a blocker of angiotensin type 1 receptor), and chelerythrine chloride (an antagonist of PKC). Western blotting indicated an upregulation of gp91(phox) and p47(phox), associated with increased expression of phosphorylated PKC in vessels of LS dogs. In separate experiments, dogs were fed simultaneously with LS and losartan (LS + Losa) for 2 wk. There was a significant increase in plasma ANG II in LS + Losa dogs, which, however, was associated with normal FID and gp91(phox) expression in coronary arterioles. In conclusion, LS led to endothelial dysfunction, as indicated by an impaired flow-induced dilation caused by decreasing NO bioavailibility, a response that involves angiotensin-induced activation of PKC that, in turn, activates vascular NAD(P)H oxidase to produce superoxide.

Endothelial Dysfunction and Circulating Microparticles from Patients with Obstructive Sleep Apnea

Endothelial dysfunction is involved in vascular complications of obstructive sleep apnea (OSA). In this study, circulating microparticles (MPs) from patients with OSA-induced nocturnal desaturations were characterized and their effects on endothelial function were evaluated. Two age-matched groups of patients undergoing polysomnography for OSA were compared: 35 desaturators with a 3% oxyhemoglobin desaturation index (ODI) > or = 10 events per hour of sleep and 27 nondesaturators with ODI <10 events per hour. MPs were characterized by flow cytometry and then either used to treat in vitro human endothelial cells or to study endothelial function in mice. Circulating MPs did not differ between groups, but MPs from granulocytes and activated leukocytes (CD62L(+)) were found at higher levels in desaturators. In vitro, MPs from desaturators reduced endothelial nitric oxide (NO) production by enhancing phosphorylation of endothelial NO synthase at the site of inhibition and expression of caveolin-1. CD62L(+) MPs positively correlated with ODI. Endothelial NO production negatively correlated with both CD62L(+) MPs and ODI. MPs from desaturators increased expression of endothelial adhesion molecules including E-selectin, ICAM-1 and ITGA5, and cyclooxygenase 2. Moreover, injection of MPs from desaturators into mice impaired endothelium-dependent relaxation in aorta and flow-induced dilation in small mesenteric arteries. This study demonstrates an association between endothelial dysfunction and increased circulating levels of CD62L(+) MPs. This may initiate atherogenic processes in patients with OSA and severe nighttime hypoxia.

Vascular Arginase Contributes to Arteriolar Endothelial Dysfunction in a Rat Model of Hemorrhagic Shock

Hemorrhagic shock causes hypoperfusion of peripheral tissues and promotes endothelial dysfunction, which may lead to further tissue injury. Trauma increases extrahepatic activity of arginase, an enzyme which competes for l-arginine with nitric oxide synthase, and plays a key role in the development of endothelial dysfunction during aging, hypertension, and diabetes. However, the role of arginase in hemorrhage-induced endothelial dysfunction has not been studied. This study tests the hypothesis that arginase inhibition improves endothelial function after hemorrhage. Male Sprague-Dawley rats were implanted with indwelling arterial catheters for blood pressure measurements and blood removal. Awake animals were subjected to a 45% fixed volume controlled hemorrhage and blood pressure was monitored. Unbled rats served as controls. Skeletal muscle arterioles were isolated 24 hours after hemorrhage and cannulated in a pressure myograph system. To study endothelial function, arterioles were exposed to constant midpoint, but altered endpoint pressures, to establish graded levels of luminal flow and internal diameter was measured. Hemorrhage lowered mean arterial pressure that spontaneously recovered to 78% and 88% of baseline in 2 hours and 20 hours, respectively. Vascular arginase II and blood glucose levels were elevated, whereas hemoglobin and insulin levels were decreased 24 hours after blood loss. In posthemorrhage arterioles, flow-induced dilation was abolished. Acute in vitro treatment with an inhibitor of arginase, N-hydroxy-nor-l-arginine, restored flow-induced dilation to unbled control levels. Similarly, the arginase and nitric oxide synthase substrate, l-arginine, but not the inactive isomer, d-arginine, restored flow-induced dilation. These results indicate that arginase contributes to endothelial dysfunction in resistance vessels after significant hemorrhage.

Corrigendum

CorrigendaCorrigendumPublished Online:01 May 2010https://doi.org/10.1152/ajpregu.zh6-7190-corr.2010Original articleMoreSectionsPDF (58 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Volume 65, December 2009LeBlanc AJ, Reyes R, Kang LS, Dailey RA, Stallone JN, Moningka NC, Muller-Delp JM. Estrogen replacement restores flow-induced vasodilation in coronary arterioles of aged and ovariectomized rats. Am J Physiol Regul Integr Comp Physiol 297: , 2009. First published October 7, 2009; doi:10.1152/ajpregu.00178.2009 (http://ajpregu.physiology.org/cgi/content/full/297/6/R1713).In the original Fig. 3C, the mean data were shown for 9 old OVX rats. This has been corrected to show the mean data for 8 old OVX rats, and the corrected version of Fig. 3C is presented below with the figure legend showing old OVX (n = 8).Fig. 3.C: effects of ovariectomy (OVX) and ovariectomy with estrogen replacement (OVE) on flow-induced vasodilation of coronary arterioles from old female rats. OVE increases flow-induced dilation in old females. Values are means ± SE. ‡Significant OVE effect vs. age-matched intact. §Significant OVE effect vs. OVX (P ≤ 0.05).Download figureDownload PowerPointThis article has no references to display. Download PDF Previous Back to Top Next FiguresReferencesRelatedInformationRelated ArticlesEstrogen replacement restores flow-induced vasodilation in coronary arterioles of aged and ovariectomized rats 01 Dec 2009American Journal of Physiology-Regulatory, Integrative and Comparative Physiology More from this issue > Volume 298Issue 5May 2010Pages R1446-R1446 Copyright & PermissionsCopyright © 2010 the American Physiological Societyhttps://doi.org/10.1152/ajpregu.zh6-7190-corr.2010History Published online 1 May 2010 Published in print 1 May 2010 Metrics

Abstract 5251: H 2 O 2 -induced Protein Kinase G Dimerization and Vasodilation in Human Coronary Arteriole

Flow-induced dilation of human coronary arterioles (HCA) is mediated by a unique mechanism involving the release of H 2 O 2 from the mitochondria of endothelial cells and subsequent smooth muscle relaxation via K + -channel-dependent membrane hyperpolarization. The precise mechanisms by which H 2 O 2 induces smooth muscle hyperpolarization remain largely undefined. An important mechanism of action of H 2 O 2 involves the oxidation of key cysteine residues in its target proteins, including protein kinase G 1-alpha (PKG-1 α ). Here we hypothesize that H 2 O 2 dilates HCA through direct oxidation and activation of PKG-1 α leading to the subsequent opening of large-conductance Ca 2+ -activated K + channels (BK Ca ) in smooth muscle cells. In isolated HCA, H 2 O 2 (10 −6 −3×10 −4 M) induced dose-dependent dilations in both endothelium-intact and -denuded vessels (relaxations at 10 −4 M of 83.5±3.7% and 85.1±8.4%, respectively; n=4 – 6). The relaxations were largely abolished by iberiotoxin, a BK Ca blocker (3.4±2.1% and 19.0±10.2% in intact and denuded vessels, respectively). The PKG inhibitor Rp-8-Br-cGMP also markedly inhibited H 2 O 2 -induced dilations (4.5±6.0%; n=6), whereas the guanylyl cyclase inhibitor ODQ has no significant effects (75.6±8.4%; n=3). The expression of PKG-1 α and BK Ca channels in HCA smooth muscles were confirmed by immunohistochemistry and Western blot analysis. In cultured human coronary arterial smooth muscle cells, H 2 O 2 induced dose-dependent dimerization of PKG-1 α , which was subsequently reduced to the monomer forms by the reducing agent β -mecaptomethanol. We also examined flow-mediated dilation in HCA pretreated with iberiotoxin or Rp-8-Br-cGMP. Both agents markedly inhibited relaxation responses as compared to control (maximal relaxations of 36.0±3.8% vs. 69.2±6.8% in controls, and 30.1±4.8% vs. 57.8±3.0% in controls, respectively; n=4 –5). It is concluded that the activation of PKG-1 α via H 2 O 2 -induced protein dimerization and subsequent opening of BK Ca channels serves as a novel mechanism of flow-induced H 2 O 2 -mediated relaxations in HCA. This signaling pathway may have implications for the pathogenesis and therapy of coronary disease.

Nanoparticle Inhalation Impairs Endothelium-Dependent Vasodilation in Subepicardial Arterioles

Exposure to fine particulate matter (PM, mean aerodynamic diameter ≤2.5 μm) has been shown to be a risk factor for cardiovascular disease mortality and may contribute to acute coronary events such as myocardial infarction (MI). There is sufficient reason to believe that smaller particles, such as nanoparticles, might be even more detrimental than larger sized particles due to their increased surface area and higher pulmonary deposition. Our laboratory showed that nanoparticle inhalation impairs endothelium-dependent arteriolar vasodilation in skeletal muscle. However, it is not known whether coronary microvascular endothelial function is affected in a similar manner. Rats were exposed to filtered air (control) or TiO2 nanoparticles (primary particle diameter, ∼21 nm) via inhalation at concentrations that produced measured depositions (10 μg) relevant to ambient air pollution. Subepicardial arterioles (∼150 μm in diameter) were isolated and responses to transmural pressure, flow-induced dilation (FID), acetylcholine (ACh), the Ca2+ ionophore A23187, and sodium nitroprusside (SNP) were assessed. Myogenic responsiveness was preserved between groups. In addition, there was no difference in the vasodilation to SNP, signifying that smooth muscle sensitivity to nitric oxide (NO) is unaffected by nano-TiO2 exposure. However, inhalation of nano-TiO2 produced an increase in spontaneous tone in coronary arterioles and also impaired endothelium-dependent FID. In addition, ACh-induced and A23187-induced vasodilation was also blunted in arterioles after inhalation of nano-TiO2. Data showed that nanoparticle exposure significantly impairs endothelium-dependent vasodilation in subepicardial arterioles. Such disturbances in coronary microvascular function are consistent with the cardiac events associated with particle pollution exposure.

Estrogen replacement restores flow-induced vasodilation in coronary arterioles of aged and ovariectomized rats

The risk for cardiovascular disease (CVD) increases with advancing age; however, the age at which CVD risk increases significantly is delayed by more than a decade in women compared with men. This cardioprotection, which women experience until menopause, is presumably due to the presence of ovarian hormones, in particular, estrogen. The purpose of this study was to determine how age and ovarian hormones affect flow-induced vasodilation in the coronary resistance vasculature. Coronary arterioles were isolated from young (6 mo), middle-aged (14 mo), and old (24 mo) intact, ovariectomized (OVX), and ovariectomized + estrogen replaced (OVE) female Fischer-344 rats to assess flow-induced vasodilation. Advancing age impaired flow-induced dilation of coronary arterioles (young: 50 +/- 4 vs. old: 34 +/- 6; % relaxation). Ovariectomy reduced flow-induced dilation in arterioles from young females, and estrogen replacement restored vasodilation to flow. In aged females, flow-induced vasodilation of arterioles was unaltered by OVX; however, estrogen replacement improved flow-induced dilation by approximately 160%. The contribution of nitric oxide (NO) to flow-induced dilation, assessed by nitric oxide synthase (NOS) inhibition with N(G)-nitro-l-arginine methyl ester (l-NAME), declined with age. l-NAME did not alter flow-induced vasodilation in arterioles from OVX rats, regardless of age. In contrast, l-NAME reduced flow-induced vasodilation of arterioles from estrogen-replaced rats at all ages. These findings indicate that the age-induced decline of flow-induced, NO-mediated dilation in coronary arterioles of female rats is related, in part, to a loss of ovarian estrogen, and estrogen supplementation can improve flow-induced dilation, even at an advanced age.

Exercising an option to prevent age related decline of vascular BH4 and uncoupling of eNOS

It is well documented that skeletal muscle vascular conductance and endothelium-dependent vasodilatation are reduced with ageing. Studies utilizing isolated skeletal muscle arterioles reveal that age-related deficits involve, at least in part, alterations in the NO signalling pathway (Muller-Delp et al. 2002; Delp et al. 2008). Although manifold factors could contribute to age-related declines in NO-mediated function, recent studies highlight the potential importance of tetrahydrobiopterin (BH4), a nitric oxide synthase (NOS) cofactor. When BH4 is limited, eNOS can become uncoupled and produce superoxide instead of NO. Thus, BH4 is potentially of junctional importance in the link between NO, reactive oxygen species (ROS) and vascular function. In humans, a single oral dose of BH4 restored endothelium-dependent dilatation in the conduit brachial artery of sedentary older men (Eskurza et al. 2005). Furthermore, Delp et al. (2008) demonstrated that age-related declines in flow-mediated dilatation of rat soleus muscle arterioles resulted, at least in part, from limitations in availability of the eNOS cofactor tetrahydrobiopterin (BH4), and that administration of the BH4 precursor sepiapterin improved flow-induced dilatation in vessels from aged rats. These results collectively support the argument that interventions to preserve vascular BH4 may be a therapeutic option for age-related vascular dysfunction (Pierce & LaRocca, 2008). In the current issue of The Journal of Physiology, Sindler et al. proposed to determine if age-related declines in vascular BH4 were associated with eNOS uncoupling and ROS production, and whether these age-related changes could be reversed by a therapeutic exercise training intervention. Aerobic exercise training has previously been shown to blunt or eliminate age-dependent declines in NO-mediated endothelium-dependent dilatation and to improve skeletal muscle blood flow in a variety of models (Seals et al. 2008), and the study of Sindler et al. proposes to add novel understanding of the underlying mechanisms related to BH4. The approach is integrative, combining functional assessment of vasodilatation with biochemical measurements of BH4, NO and ROS production, all in isolated arterioles from the soleus muscle under a number of controlled pharmacological conditions. As expected, flow-mediated dilatation was impaired with ageing and restored by exercise training; novel observations point to potential underlying mechanisms: age-related reduction in BH4 occurred in conjunction with decline in flow-induced NO signalling, and increase in superoxide production (eNOS uncoupling); and exercise training prevented the age-related loss of BH4 and improved NO bio-availability by balancing accelerated NO and ROS production. These results extend the appreciation of oxidative stress impacting NO bio-availability and vasomotor function beyond the traditional, but insufficient, concept of NO destruction by ROS (Rush et al. 2005). The findings of the study of Sindler et al. are timely, reported roughly contemporaneously with identification of the need for pharmacological therapies directed toward a similar result, the restoration of BH4 in aged blood vessels. This creates a discovery environment in which potential pharmacological- and exercise-oriented approaches to the problem will undoubtedly co-evolve. The findings of Sindler et al. additionally serve as a progenitor for many hypotheses regarding BH4 in vascular adaptations to age and exercise. Fundamental questions raised include those related to identifying the cellular mechanisms accounting for age-related declines and exercise-related restoration of vascular BH4. In this regard, emerging data suggest that H2O2 is a regulatory factor controlling BH4 synthesis in vascular cells via induction of the enzyme GTP-cyclohydrolase I (GTPCH; Shimizu et al. 2003). The data of Sindler et al. indicate that H2O2 contributes to flow-induced dilatation of skeletal muscle arterioles of young sedentary animals and that, like the flow-induced dilatation functional response itself, this H2O2 signalling declines with age but is restored by exercise training. Is it possible that this same pattern of H2O2 signalling could contribute to maintenance of BH4 in young arterioles, its loss with age, and its restoration with exercise in aged arterioles, consistent with the indicated effect of H2O2 on GTPCH expression and BH4 synthesis? If so, H2O2 takes on another important role in the acute and chronic regulation of vascular function. A recurring theme in vascular biology is heterogeneity in phenotype and function depending on vessel type (conduit vs. resistance), location (vascular bed), and physiological circumstances (e.g. other conditions in vivo). While these considerations are in part cautionary regarding the global interpretation of the results of Sindler et al., they can more appropriately be seen as an invitation for additional studies designed to create a more integrative understanding of cellular mechanisms of vascular ageing and the therapeutic effects of exercise. The study of Sindler et al. creates a new paradigm justifying such an invitation with respect to BH4, vasomotor function and the regulation of skeletal muscle blood flow.