Hypoxia induces coronary artery dilation, but the responsible mechanism is largely unknown. Many stimuli induce arterial smooth muscle relaxation by reducing ser19-myosin regulatory light chain (MLC) phosphorylation. Other stimuli can induce smooth muscle relaxation without reductions in ser19-MLC phosphorylation. This form of relaxation has been termed force suppression and appears to be associated with heat shock protein 20 (HSP20) phosphorylation on ser16. We investigated whether hypoxia-induced sustained dilation in swine coronary arteries was promoted without ser19-MLC dephosphorylation and associated with ser16-HSP20 phosphorylation. Nitroglycerin vasodilation served as control.
In a pressure myograph, the tunica media of intact pre-contracted (PGF2α;10-5 m) porcine coronary artery segments were cannulated using a microdialysis catheter. Diameter responses and interstitial lactate/pyruvate ratios were studied during 90 min hypoxia, hypoxia + reoxygenation (60 min), nitroglycerin (100 μm, 90 min), and nitroglycerin + wash-out (60 min). The arterial segments were snap-frozen and analysed for ser16-HSP20 phosphorylation and ser19-MLC phosphorylation.
The normalized diameter responses to hypoxia (6.1 ± 4.3%) and nitroglycerin (12.6 ± 1.6%) were both significantly greater than normoxic control arteries (-10.5 ± 1.8%, anova, P < 0.05). Ser16-HSP20 phosphorylation was increased with hypoxia and nitroglycerin treatment and ser16-HSP20 phosphorylation correlated with changes in diameters (n = 29, r2 = 0.64, P < 0.001). Ser19-MLC phosphorylation was not significantly altered by hypoxia. The lactate/pyruvate ratio was significantly increased in hypoxic arteries but did not correlate with diameters or ser16-HSP20 phosphorylation.
Ser16-HSP20 phosphorylation is a potential regulator of hypoxia-induced dilation in coronary arteries.
hypoxia; metabolism; microdialysis; signal transduction; smooth muscle; vasodilation
Endothelial cells synthesize vasodilator nitric oxide (NO) and vasoconstrictor endothelin-1 (ET-1) from NO synthase (eNOS) and endothelin-converting enzyme-1 (ECE-1), respectively. Protein kinase C (PKC) and Rho kinase (ROCK) are major signaling molecules mediating vasoconstriction. Although endothelial cells express eNOS, ECE-1, endothelin B (ETB) receptors, PKC, and ROCK, their influences on ET-1–induced vasoconstriction remain elusive. We studied whether these endothelial signaling molecules modulate retinal arteriolar constriction to ET-1.
Porcine retinal arterioles were isolated and pressurized for vasomotor study, under conditions with intact or denuded endothelium, using videomicroscopic techniques.
Retinal arterioles developed similar resting tone (≈45% of maximum diameter) with or without endothelium. Endothelial denudation attenuated vasoconstriction to ET-1 precursor, big ET-1, by almost equal to 50%, but did not affect vasoconstrictions to ET-1, ETB agonist sarafotoxin S6c, or PKC activator phorbol-12, 13-dibutyrate (PDBu). The ROCK inhibitor H-1152 caused vasodilation, and abolished vasoconstrictions to ET-1 and PDBu independent of endothelium. With L-type voltage-operated calcium channel (L-VOCC) blocker nifedipine, PDBu-induced vasoconstriction was abolished and converted to NO-mediated vasodilation in the presence of endothelium. The ET-1–induced vasoconstriction was unaffected by NO released from endothelium during flow elevation.
Endothelial and smooth muscle ECE-1 contribute equally to synthesis of vasoactive ET-1 in retinal arterioles, with nominal role of endothelial ETB receptors in vasoconstriction to ET-1. The PKC activation leads to endothelium-dependent NO-mediated vasodilation when smooth muscle contraction is ablated by L-VOCC blockade. Endothelial cells and NO appear to have modest roles in modulating ROCK-dependent vasoconstriction, and are insufficient to counteract smooth muscle contractions to ET-1 and PKC activation.
Endothelin and protein kinase C (PKC) have been implicated in development of retinal disease, and have vasomotor roles in the retinal circulation. We report on the role of endothelium in vasomotor responses to endothelin system peptides and PKC activation in porcine retinal arterioles.
endothelins; endothelium; arterioles; PKC; retinal blood flow
Medroxyprogesterone acetate (MPA) is widely known for its use in combination hormone therapy for postmenopausal women. However, MPA is also commonly used in young women for contraception and treatment of a number of gynecological conditions. Despite its widespread use, the cardiovascular effects of MPA in young women are unclear. Therefore, the purpose of this study was to determine the acute effects of MPA when used in combination with estradiol on markers of cardiovascular risk in young women. We suppressed endogenous estrogens and progesterone in 10 premenopausal women using a gonadotropin-releasing hormone antagonist (GnRHa) for 10 days. On day 4 of GnRHa subjects received 0.1 mg of estradiol (GnRHa+E2), and on day 7 5 mg of MPA was added (GnRHa+E2+MPA). Endothelium-dependent vasodilation and endothelium-independent vasodilation of the brachial artery, lipids, homocysteine, high-sensitivity C-reactive protein, and endothelin-1 were assessed during treatment with GnRHa, GnRHa+E2, and GnRHa+E2+MPA. Four additional subjects were tested to validate the efficacy of the GnRHa model and confirm the findings. Endothelium-dependent vasodilation was greater during GnRHa+E2 than during GnRHa or GnRHa+E2+MPA (P = 0.006). Endothelin-1 was lower during GnRHa+E2 than GnRHa alone (P = 0.039). Endothelin-1 increased with the addition of MPA and was not significantly different from GnRHa alone. There were no differences in the other markers of cardiovascular risk between hormone treatment days. These data suggest that acute MPA administration negates the beneficial effects of estradiol on endothelium-dependent vasodilation in young women. In addition, these data suggest that estradiol decreases endothelin-1 concentrations and the addition of MPA may counteract the effect of estradiol on endothelin-1.
endothelium; hormones; flow-mediated vasodilation; endothelin-1; progestins
Previous studies have suggested that cerebrospinal fluid from patients with subarachnoid hemorrhage (SAH) leads to pronounced vasoconstriction in isolated arteries. We hypothesized that only cerebrospinal fluid from SAH patients with vasospasm would produce an enhanced contractile response to endothelin-1 in rat cerebral arteries, involving both endothelin ETA and ETB receptors.
Intact rat basilar arteries were incubated for 24 hours with cerebrospinal fluid from 1) SAH patients with vasospasm, 2) SAH patients without vasospasm, and 3) control patients. Arterial segments with and without endothelium were mounted in myographs and concentration-response curves for endothelin-1 were constructed in the absence and presence of selective and combined ETA and ETB receptor antagonists. Endothelin concentrations in culture medium and receptor expression were measured.
Compared to the other groups, the following was observed in arteries exposed to cerebrospinal fluid from patients with vasospasm: 1) larger contractions at lower endothelin concentrations (p<0.05); 2) the increased endothelin contraction was absent in arteries without endothelium; 3) higher levels of endothelin secretion in the culture medium (p<0.05); 4) there was expression of ETA receptors and new expression of ETB receptors was apparent; 5) reduction in the enhanced response to endothelin after ETB blockade in the low range and after ETA blockade in the high range of endothelin concentrations; 6) after combined ETA and ETB blockade a complete inhibition of endothelin contraction was observed.
Our experimental findings showed that in intact rat basilar arteries exposed to cerebrospinal fluid from patients with vasospasm endothelin contraction was enhanced in an endothelium-dependent manner and was blocked by combined ETA and ETB receptor antagonism. Therefore we suggest that combined blockade of both receptors may play a role in counteracting vasospasm in patients with SAH.
The aim of this study was to investigate whether poly(ADP-ribose) polymerase (PARP) inhibition improves endothelin-1 (ET-1)-induced endothelial dysfunction (ED).
Isolated rat thoracic aorta rings were incubated with ET-1 (10 nmol/L) in the presence or absence of either polyethylene glycol–superoxide dismutase (PEG-SOD; a cell-permeable superoxide radical scavenger, 41 U/mL) plus apocynin (a NADPH oxidase inhibitor, 300 µmol/L) or PJ34 (an inhibitor of polyADP-ribose polymerase, 3 µmol/L) for 18 h. Isometric tension studies were performed in response to acetylcholine (ACh; an endothelium-dependent vasodilator), sodium nitroprusside (SNP; an endothelium-independent vasodilator), and phenylephrine (Phe). PARP-1 and PAR (an end-product of PARP activity) expressions were evaluated by both Western blot and immunohistochemistry.
Incubation of thoracic aorta rings with ET-1 resulted in a significant inhibition of the response to ACh, while SNP-induced relaxation was unaffected. The contractile response to Phe increased in arteries that were incubated with ET-1. PARP-1 and PAR expressions increased after ET-1 incubation. The diminished vasoreactivity as well as changes in expressions of PARP-1 and PAR in ET-1-incubated vessels were improved by both PEG-SOD plus apocynin and PJ34.
Our studies demonstrate that ED induced by ET-1 seems to be effected via oxidative stress in the thoracic aorta endothelium with subsequent activation of the PARP pathway.
Endothelial dysfunction; endothelin-1; poly(ADP-ribose) polymerase (PARP)
Membrane depolarization is critical to pulmonary arterial (PA) contraction. Both L-type Ca2+ channels (CaL) and Rho-kinase are important signaling components of this process and mitochondrial and non-mitochondrial generated superoxides can be part of the signaling process. Maturation and long-term hypoxia (LTH) each can modify depolarization-dependent contraction and the role of superoxides. By the use of wire myography, we tested the hypothesis that maturation and LTH increase pulmonary arterial reactivity to high-K+-induced membrane depolarization through enhancements in the importance of CaLand Rho-kinase-dependent pathways. The data show that maturation, but not LTH, increases contraction to 125 mM KCl (high-K+) without altering the EC50. High-K+-dependent contraction was inhibited to a similar extent in fetal and adult PA by multiple CaL blockers, including 10 μM diltiazem, 10 μM verapamil, and 10 μM nifedipine. Postnatal maturation increased the role for 10 μM nifedipine-sensitive CaL, and decreased that for 10 μM Y-27632-sensitive Rho-kinase. In all groups, the combination of nifedipine and Y-27632 effectively inhibited high-K+ contraction. Tempol (3 mM) but not 100 μM apocynin slightly reduced contraction in arteries from fetal hypoxic and adult normoxic and hypoxic sheep, indicating a limited role for non-mitochondrial derived superoxide to high-K+-induced contraction. Western immunoblot for alpha smooth muscle actin indicated small increases in relative abundance in the adult. The data suggest that while CaL therapies more effectively vasodilate PA in adults and rho-kinase therapies are more effective in newborns, combination therapies would provide greater efficacy in both young and mature patients regardless of normoxic or hypoxic conditions.
K+-induced depolarization; L-type Ca2+ channel; myography; rho-kinase; superoxide
Normal pregnancy is associated with significant vascular remodeling in the uterine and systemic circulation in order to meet the metabolic demands of the mother and developing fetus. The pregnancy-associated vascular changes are largely due to alterations in the amount/activity of vascular mediators released from the endothelium, vascular smooth muscle and extracellular matrix. The endothelium releases vasodilator substances such as nitric oxide, prostacyclin and hyperpolarizing factor as well as vasoconstrictor factors such as endothelin, angiotensin II and thromboxane A2. Vascular smooth muscle contraction is mediated by intracellular free Ca2+ concentration ([Ca2+]i), and [Ca2+]i sensitization pathways such as protein kinase C, Rho-kinase and mitogen-activated protein kinase. Extracellular matrix and vascular remodeling are regulated by matrix metalloproteases. Hypertension in pregnancy and preeclampsia are major complications and life threatening conditions to both the mother and fetus, precipitated by various genetic, dietary and environmental factors. The initiating mechanism of preeclampsia and hypertension in pregnancy is unclear; however, most studies have implicated inadequate invasion of cytotrophoblasts into the uterine artery, leading to reduction in the uteroplacental perfusion pressure and placental ischemia/hypoxia. This placental hypoxic state is thought to induce the release of several circulating bioactive factors such as growth factor inhibitors, anti-angiogenic proteins, inflammatory cytokines, reactive oxygen species, hypoxia-inducible factors, and vascular receptor antibodies. Increases in the plasma levels and vascular content of these factors during pregnancy could cause an imbalance in the vascular mediators released from the endothelium, smooth muscle and extracellular matrix, and lead to severe vasoconstriction and hypertension. This review will discuss the interactions between the various circulating bioactive factors and the vascular mediators released during hypertension in pregnancy, and provide an insight into the current and future approaches in the management of preeclampsia.
cytokines; endothelium; nitric oxide; vascular smooth muscle; calcium; preeclampsia
The systemic response to decreasing oxygen levels is hypoxic vasodilation. While this mechanism has been known for more than a century, the underlying cellular events have remained incompletely understood. Nitrite signaling is critically involved in vessel relaxation under hypoxia. This can be attributed to the presence of myoglobin in the vessel wall together with other potential nitrite reductases, which generate nitric oxide, one of the most potent vasodilatory signaling molecules. Questions remain relating to the precise concentration of nitrite and the exact dose-response relations between nitrite and myoglobin under hypoxia. It is furthermore unclear whether regulatory mechanisms exist which balance this interaction. Nitrite tissue levels were similar across all species investigated. We then investigated the exact fractional myoglobin desaturation in an ex vivo approach when gassing with 1% oxygen. Within a short time frame myoglobin desaturated to 58±12%. Given that myoglobin significantly contributes to nitrite reduction under hypoxia, dose-response experiments using physiological to pharmacological nitrite concentrations were conducted. Along all concentrations, abrogation of myoglobin in mice impaired vasodilation. As reactive oxygen species may counteract the vasodilatory response, we used superoxide dismutase and its mimic tempol as well as catalase and ebselen to reduce the levels of reactive oxygen species during hypoxic vasodilation. Incubation of tempol in conjunction with catalase alone and catalase/ebselen increased the vasodilatory response to nitrite. Our study shows that modest hypoxia leads to a significant nitrite-dependent vessel relaxation. This requires the presence of vascular myoglobin for both physiological and pharmacological nitrite levels. Reactive oxygen species, in turn, modulate this vasodilation response.
Impaired endothelium-independent vasodilation is a known consequence of type-1 and 2 diabetes, and the mechanism of impaired vasodilation is not well understood. The following study investigated the effects of type-1 and 2 diabetes in endothelial-independent vasodilation associated with coronary vascular smooth muscle (VSM) relaxation and contractile signaling mechanisms.
Type-1 diabetes was induced in Yucatan mini-swine via alloxan injection and treated with or without insulin(DM and IDM). Non-diabetic swine served as controls(ND). Expression and/or phosphorylation of determinants of VSM relaxation and contraction signaling were examined in coronary arteries and microvessels. Coronary microvessel relaxation was assessed using sodium nitroprusside(SNP). In addition, SNP-induced vasodilation and myosin light chain (MLC) phosphorylation was determined in coronary microvessels isolated from ND and type-2 diabetic human atrial appendage.
Diabetic impairment in SNP-induced relaxation was completely normalized by insulin. Soluble guanylate cyclase (sGC) VSM expression decreased in both DM and IDM groups and did not correlate with vasorelaxation. Phosphorylation of MLC and myosin phosphatase increased in the DM group and MLC phosphorylation strongly correlated with impaired VSM relaxation(r=.670, p<0.01). Coronary microvessels from type-2 diabetic human patients exhibited similarly impaired vasodilation and enhanced VSM MLC phosphorylation.
Impaired vasodilation in type-1 diabetes correlates with enhanced VSM MLC phosphorylation. In addition, enhanced VSM MLC phosphorylation is associated with impaired vasodilation in type 2 diabetes in humans.
Effects of insulin on cerebral arteries have never been examined. Therefore, we determined cerebrovascular actions of insulin in rats. Both PCR and immunoblot studies identified insulin receptor expression in cerebral arteries and in cultured cerebral microvascular endothelial cells (CMVECs). Diameter measurements (% change) of isolated rat cerebral arteries showed a biphasic dose response to insulin with an initial vasoconstriction at 0.1 ng/mL (−9.7% ± 1.6%), followed by vasodilation at 1 to 100 ng/mL (31.9% ± 1.4%). Insulin also increased cortical blood flow in vivo (30% ± 8% at 120 ng/mL) when applied topically. Removal of reactive oxygen species (ROS) abolished the vasoconstriction to insulin. Endothelial denudation, inhibition of K+ channels, and nitric oxide (NO) synthase, all diminished insulin-induced vasodilation. Inhibition of cytochrome P450 enhanced vasodilation in endothelium-intact arteries, but promoted vasoconstriction after endothelial denudation. Inhibition of cyclooxygenase abolished vasoconstriction and enhanced vasodilation to insulin in all arteries. Inhibition of endothelin type A receptors enhanced vasodilation, whereas endothelin type B receptor blockade diminished vasodilation. Insulin treatment in vitro increased Akt phosphorylation in cerebral arteries and CMVECs. Fluorescence studies of CMVECs showed that insulin increased intracellular calcium and enhanced the generation of NO and ROS. Thus, cerebrovascular responses to insulin were mediated by complex mechanisms originating in both the endothelium and smooth muscle.
cerebral arteries; cyclooxygenase; endothelin; nitric oxide; P450 monooxygenase; reactive oxygen species
Estrogen receptors (ERs) mediate genomic and nongenomic vasodilator effects, but estrogen therapy may not provide systemic vascular protection. To test whether this is due to regional differences in ER distribution or vasodilator activity, cephalic (carotid), thoracic (thoracic aorta, pulmonary) and abdominal arteries (abdominal aorta, mesenteric, renal) from female Sprague-Dawley rats were prepared to measure contraction to phenylephrine (Phe), and relaxation to acetylcholine (ACh) and the ER activators 17β-estradiol (E2) (all ERs), PPT (ERα), DPN (ERβ) and G1 (GPR30). Phe caused contraction that was enhanced in endothelium-denuded aorta, supporting endothelial release of vasodilators. In cephalic and thoracic arteries, ACh relaxation was abolished by the NOS inhibitor L-NAME, suggesting a role of NO. In mesenteric vessels, ACh-induced relaxation was partly inhibited by L-NAME+COX inhibitor indomethacin and blocked by the K+ channel blocker tetraethylammonium (TEA), suggesting a hyperpolarization pathway. E2 and PPT caused similar relaxation in all vessels. DPN and G1 caused smaller relaxation that was more prominent in abdominal vessels. RT-PCR revealed variable ERα mRNA expression, and increased ERβ in carotid artery and GPR30 in abdominal arteries. Western blots revealed greater amounts of ERα, ERβ and GPR30 in abdominal arteries. In thoracic aorta, E2, PPT and DPN-induced relaxation was blocked by L-NAME, and was associated with increased nitrite/nitrate production, suggesting a role of NO. In abdominal vessels, E2, PPT, DPN and G1-induced relaxation persisted in L-NAME+indomethacin+TEA-treated or endothelium-denuded arteries, suggesting direct effect on vascular smooth muscle (VSM). E2, PPT, DPN, and G1 caused greater relaxation of KCl-induced contraction in abdominal vessels, suggesting inhibitory effects on Ca2+ entry. Thus, E2 and ERα stimulation produce similar relaxation of the cephalic, thoracic and abdominal arteries. In the cephalic and thoracic arteries, particularly the thoracic aorta, E2-induced and ERα- and ERβ-mediated vasodilation involve NO production. ERβ- and GPR30-mediated relaxation is greater in the abdominal arteries, and appears to involve hyperpolarization and inhibition of VSM Ca2+ entry. Specific ER agonists could produce vasodilation in specific vascular beds without affecting other vessels in systemic circulation.
estrogen; sex hormones; endothelium; vascular smooth muscle
The mitogenic and vasoconstrictive properties of the vascular system are attributed to endothelin-1 (ET-1). ET-1 serum concentration increases in a number of pathological conditions, particularly in those associated with blood vessel constriction. ET-1 is also associated with the underlying pathomechanisms of primary pulmonary hypertension, arterial hypertension and eclampsia. The aim of this study was to compare the vasodilating properties of selected phosphodiesterase (PDE) inhibitors and celecoxib in human mesenteric arteries constricted with ET-1, and investigate the role of the endothelium in relaxation. Perfused human mesenteric arteries were collected and stored under the same conditions as organs for transplantation. The mesenteric arteries (with and without the endothelium) were constricted by the addition of ET-1 and treated with one of the following: sildenafil (PDE5 inhibitor), zaprinast (PDE5 and 6 inhibitor), rolipram (PDE4 inhibitor) and celecoxib [cyclooxygenase-2 (COX-2) inhibitor]. Based on the observed changes of the perfusion pressure, concentration response curves (CRCs) were prepared for the respective inhibitors and the EC50 (concentration causing an effect equal to half of the maximum effect), pD2 (negative common logarithm of EC50) and relative potency (RP) were calculated. The results suggested that all the inhibitors triggered a concentration-dependent decrease in the perfusion pressure in isolated human superior mesenteric arteries with endothelium constricted by the addition of ET-1. In the arteries without endothelium, CRCs for celecoxib and rolipram were shifted to the right without a significant decrease in the maximum dilating effect. Moreover, CRCs for sildenafil and zaprinast were shifted to the right with a simultaneous significant decrease in the maximum dilating effect and with an increased inclination angle in reference to the concentration axis. In the presence of the endothelium, all of the evaluated PDE inhibitors, as well as celecoxib, reduced the reactivity of the mesenteric arteries caused by ET-1. Sildenafil indicated the lowest efficacy in the presence of the endothelium, but showed a higher potency compared to that of the other compounds. Removing the endothelium significantly reduced the vasodilating efficacy of PDE5 and 6 inhibitors and a statistically significant influence on the vasodilating efficacy of PDE4 inhibitor and celecoxib was observed. The high vasorelaxing efficacy of celecoxib at the background of the PDE inhibitors was observed, not only in the presence, but also in the absence of the endothelium and may be evidence for the relaxation induced by this COX-2 inhibitor in the cAMP- and cGMP-dependent pathways.
endothelin-1; celecoxib; phosphodiesterase inhibitors; constriction; endothelium
Systemic hypertension may be associated with an increased pulmonary vascular resistance, which we hypothesized could be, at least in part, mediated by increased leptin.
Vascular reactivity to phenylephrine (1 μmol/L), endothelin-1 (10 nmol/L) and leptin (0.001–100 nmol/L) was evaluated in endothelium-intact and -denuded isolated thoracic aorta and pulmonary arteries from spontaneously hypertensive versus control Wistar rats. Arteries were sampled for pathobiological evaluation and lung tissue for morphometric evaluation.
In control rats, endothelin-1 induced a higher level of contraction in the pulmonary artery than in the aorta. After phenylephrine or endothelin-1 precontraction, leptin relaxed intact pulmonary artery and aortic rings, while no response was observed in denuded arteries. Spontaneously hypertensive rats presented with increased reactivity to phenylephrine and endothelin-1 in endothelium-intact pulmonary arteries. After endothelin-1 precontraction, endothelium-dependent relaxation to leptin was impaired in pulmonary arteries from hypertensive rats. In both strains of rats, aortic segments were more responsive to leptin than pulmonary artery. In hypertensive rats, pulmonary arteries exhibited increased pulmonary artery medial thickness, associated with increased expressions of preproendothelin-1, endothelin-1 receptors type A and B, inducible nitric oxide synthase and decreased endothelial nitric oxide synthase, together with decreased leptin receptor and increased suppressor of cytokine signaling 3 expressions.
Altered pulmonary vascular reactivity in hypertension may be related to a loss of endothelial buffering of vasoconstriction and decreased leptin-induced vasodilation in conditions of increased endothelin-1.
Systemic hypertension; Pulmonary circulation; Vascular reactivity; Leptin; Endothelin-1
We aimed to assess age-related differences in compensatory hypoxic vasodilation during moderate-to-high dynamic exercise at absolute workloads. We hypothesized healthy older adults (n = 12, 61 ± 1 years) would exhibit impaired hypoxic vasodilation at a moderate absolute workload, and this effect would be exaggerated at a higher workload when compared to young adults (n = 17, 27 ± 2 years). Forearm blood flow (FBF) was measured with Doppler ultrasound. Dynamic forearm exercise (20 contractions/min) was completed at two absolute workloads (8 and 12 kg) under normoxic (0.21 FiO2, ~98% SpO2) and isocapnic hypoxic (~0.10 FiO2, 80% SpO2) conditions performed in random order. FBF was normalized as forearm vascular conductance (FBF / mean arterial blood pressure = FVC) to control for differences in blood pressure and to assess vasodilation. FVC increased with exercise and hypoxia (main effects, p < 0.05); vascular responses were not different between young and older adults (interaction effect exercise × group p = 0.37 and hypoxia × group p = 0.96). Results were confirmed when analyzed as either an absolute or relative change in FVC (ΔFVC and %ΔFVC, respectively). Although group responses to hypoxia were not different, individual results were highly variable (i.e., some adults constricted and others dilated to hypoxia). These data suggest (1) compensatory hypoxic vasodilation in older adults is not impaired during forearm exercise at both moderate and higher absolute exercise intensities, and (2) vascular responses to hypoxia are heterogeneous in both young and older adults. Results suggest unique individual differences exist in factors regulating vascular responses to hypoxia.
Aging; Functional hyperemia; Blood flow
Although previous studies demonstrated beneficial effects of estrogen on cardiovascular function, the Women's Health Initiative has reported an increased incidence of coronary heart disease and stroke in postmenopausal women taking hormone replacement therapy (HRT). The objective of the present study was to identify a molecular mechanism whereby estrogen, a vasodilatory hormone, could possibly increase the risk of cardiovascular disease. Isometric contractile force recordings were performed on endothelium-denuded porcine coronary arteries, while molecular and fluorescence studies identified estrogen signaling molecules in coronary smooth muscle. Estrogen (1-1000nM) relaxed arteries in an endothelium-independent fashion; however, when arteries were pretreated with agents to uncouple NO production from nitric oxide synthase (NOS), estrogen contracted coronary arteries with an EC50 of 7.3 ± 4nM. Estrogen-induced contraction was attenuated by reducing superoxide (O2−). Estrogen-stimulated O2− production was detected in NOS-uncoupled coronary myocytes. Interestingly, only the Type 1 NOS isoform (nNOS) was detected in myocytes, making this protein a likely target mediating both estrogen-induced relaxation and contraction of endothelium-denuded coronary arteries. Estrogen-induced contraction was completely inhibited by 1μM nifedipine or 10μM indomethacin, indicating involvement of dihydropyridine-sensitive calcium channels and contractile prostaglandins. We propose that a single molecular mechanism can mediate the dual and opposite effect of estrogen on coronary arteries: by stimulating Type 1 (n)NOS in coronary arteries, estrogen produces either vasodilation via NO or vasoconstriction via superoxide.
estrogens; nitric oxide; coronary circulation; smooth muscle
Alterations in human cerebral blood flow and related blood constituents were studied during exposure to acute hypoxia. Observations were made during serial inhalation of decreasing O2 concentrations with and without maintenance of normocarbia, during 8 min inhalation of 10% O2, and after hyperventilation at an arterial PO2 of about 40 mm Hg. In the range of hypoxemia studied, from normal down to arterial PO2 of about 40 mm Hg, the magnitude of the cerebral vasodilator response to hypoxia appeared to be largely dependent upon the coexisting arterial CO2 tension. The mean slope of the increase in cerebral blood flow with decreasing arterial O2 tension rose more quickly (P < 0.05) when eucapnia was maintained when compared with the slope derived under similar hypoxic conditions without maintenance of eucapnia. When 12 subjects inhaled 10% oxygen, cerebral blood flow rose to more than 135% of control in four whose mean decrease in arterial CO2 tension was - 2.0 mm Hg. The remaining eight had flows ranging from 97 to 120% of control, and their mean decrease in CO2 tension was - 5.1 mm Hg. When mean arterial PO2 was 37 mm Hg, hyperventilation was carried out in 10 subjects. Arterial PO2 increased insignificantly, arterial PCO2 declined from 34 to 27 mm Hg (P < 0.05), and cerebral blood flow which had been 143% of control decreased to 109%, a figure not significantly different from control.
These data demonstrate the powerful counterbalancing constrictor effects of modest reductions in CO2 tension on the vasodilator influence of hypoxia represented by arterial PO2 reductions to about 40 mm Hg. Indeed, mild hyperventilation completely overcame the vasodilator effect provided by an arterial O2 tension as low as 40 mm Hg. The effects of hypoxia on the control of the cerebral circulation must be analyzed in terms of the effects of any associated changes in CO2 tension.
Normal pregnancy is associated with significant hemodynamic changes and vasodilation in the uterine and systemic circulation in order to meet the metabolic demands of the mother and developing fetus. Hypertension in pregnancy (HTN-Preg) and preeclampsia (PE) are major complications and life-threatening conditions to both the mother and fetus. PE is precipitated by various genetic, dietary and environmental factors. Although the initiating events of PE are unclear, inadequate invasion of cytotrophoblasts into the uterine artery is thought to reduce uteroplacental perfusion pressure and lead to placental ischemia/hypoxia. Placental hypoxia induces the release of biologically active factors such as growth factor inhibitors, anti-angiogenic proteins, inflammatory cytokines, reactive oxygen species, hypoxia-inducible factors, and antibodies to vascular angiotensin II receptor. These bioactive factors affect the production/activity of various vascular mediators in the endothelium, smooth muscle and extracellular matrix, leading to severe vasoconstriction and HTN. As an endothelial cell disorder, PE is associated with decreased vasodilator mediators such as nitric oxide, prostacyclin and hyperpolarizing factor and increased vasoconstrictor mediators such as endothelin, angiotensin II and thromboxane A2. PE also involves enhanced mechanisms of vascular smooth muscle contraction including intracellular free Ca2+ concentration ([Ca2+]i), and [Ca2+]i sensitization pathways such as protein kinase C, Rho-kinase and mitogen-activated protein kinase. Changes in extracellular matrix composition and matrix metalloproteases activity also promote vascular remodeling and further vasoconstriction in the uterine and systemic circulation. Characterization of the predisposing risk factors, the biologically active factors, and the vascular mediators associated with PE holds the promise for early detection, and should help design specific genetic and pharmacological tools for the management of the vascular dysfunction associated with HTN-Preg.
cytokines; endothelium; growth factors; placenta; preeclampsia; vascular smooth muscle
Rationale: Impaired endothelium-dependent vasodilation has been documented in patients with sleep apnea. This impairment may result in blood flow dysregulation during apnea-induced fluctuations in arterial blood gases.
Objectives: To test the hypothesis that hypoxic and hypercapnic vasodilation in the forearm and cerebral circulation are impaired in patients with sleep apnea.
Methods: We exposed 20 patients with moderate to severe sleep apnea and 20 control subjects, to isocapnic hypoxia and hyperoxic hypercapnia. A subset of 14 patients was restudied after treatment with continuous positive airway pressure.
Measurements and Main Results: Cerebral flow velocity (transcranial Doppler), forearm blood flow (venous occlusion plethysmography), arterial pressure (automated sphygmomanometry), oxygen saturation (pulse oximetry), ventilation (pneumotachograph), and end-tidal oxygen and carbon dioxide tensions (expired gas analysis) were measured during three levels of hypoxia and two levels of hypercapnia. Cerebral vasodilator responses to hypoxia (−0.65 ± 0.44 vs. −1.02 ± 0.72 [mean ± SD] units/% saturation; P = 0.03) and hypercapnia (2.01 ± 0.88 vs. 2.57 ± 0.89 units/mm Hg; P = 0.03) were smaller in patients versus control subjects. Hypoxic vasodilation in the forearm was also attenuated (−0.05 ± 0.09 vs. −0.10 ± 0.09 unit/% saturation; P = 0.04). Hypercapnia did not elicit forearm vasodilation in either group. Twelve weeks of continuous positive airway pressure treatment enhanced hypoxic vasodilation in the cerebral circulation (−0.83 ± 0.32 vs. −0.46 ± 0.29 units/% saturation; P = 0.01) and forearm (−0.19 ± 0.15 vs. −0.02 ± 0.08 units/% saturation; P = 0.003), and hypercapnic vasodilation in the brain showed a trend toward improvement (2.24 ± 0.78 vs. 1.76 ± 0.64 units/mm Hg; P = 0.06).
Conclusions: Vasodilator responses to chemical stimuli in the cerebral circulation and the forearm are impaired in many patients with obstructive sleep apnea. Some of these impairments can be improved with continuous positive airway pressure.
hypoxia; sleep; vasodilation; cerebral vascular circulation; regional blood flow
Endothelium-derived vasodilators, i.e., nitric oxide (NO), prostacyclin (PGI2) and prostaglandin E2 (PGE2), play important roles in maintaining cardiovascular homeostasis. C-reactive protein (CRP), a biomarker of inflammation and cardiovascular disease, has been shown to inhibit NO-mediated vasodilation. The goal of this study was to determine whether CRP also affects endothelial arachidonic acid (AA)-prostanoid pathways for vasomotor regulation. Porcine coronary arterioles were isolated and pressurized for vasomotor study, as well as for molecular and biochemical analysis. AA elicited endothelium-dependent vasodilation and PGI2 release. PGI2 synthase (PGI2-S) inhibitor trans-2-phenyl cyclopropylamine blocked vasodilation to AA but not to serotonin (endothelium-dependent NO-mediated vasodilator). Intraluminal administration of a pathophysiological level of CRP (7 μg/mL, 60 minutes) attenuated vasodilations to serotonin and AA but not to nitroprusside, exogenous PGI2, or hydrogen peroxide (endothelium-dependent PGE2 activator). CRP also reduced basal NO production, caused tyrosine nitration of endothelial PGI2-S, and inhibited AA-stimulated PGI2 release from arterioles. Peroxynitrite scavenger urate failed to restore serotonin dilation, but preserved AA-stimulated PGI2 release/dilation and prevented PGI2-S nitration. NO synthase inhibitor L-NAME and superoxide scavenger TEMPOL also protected AA-induced vasodilation. Collectively, our results suggest that CRP stimulates superoxide production and the subsequent formation of peroxynitrite from basal released NO compromises PGI2 synthesis, and thus endothelium-dependent PGI2-mediated dilation, by inhibiting PGI2-S activity through tyrosine nitration. By impairing PGI2-S function, and thus PGI2 release, CRP could promote endothelial dysfunction and participate in the development of coronary artery disease.
prostaglandins; microcirculation; free radicals; vasodilation
To determine whether hydralazine, a systemic vasodilator, exerted a similar effect on the pulmonary circulation, we studied the circulatory changes in dogs during three interventions: (a) the control state during room air ventilation; (b) during continuous hypoxic ventilation with 10% oxygen, and maintaining continuous hypoxic ventilation; and (c) after 1 mg/kg hydralazine intravenously.
Ventilation with 10% oxygen caused the mean pulmonary artery pressure to increase from 10±1.2 to 23±2.4 mm Hg (P < 0.01) and the pulmonary arteriolar resistance to increase from 1.51±0.19 to 5.87±1.10 U (P < 0.01). Hydralazine significantly lowered the pulmonary artery pressure (23.0±2.4 to 14.3±1.5 mm Hg, P < 0.01) and the pulmonary arteriolar resistance (5.87±1.10 to 2.87±0.52 U, P < 0.01). Femoral artery pressure, pulmonary artery wedge pressure, heart rate, and cardiac output remained unchanged throughout.
To ascertain the contribution of the prostaglandin system to the pulmonary vasodilator effects of hydralazine, we pretreated a group of dogs with the prostaglandin synthetase inhibitor, indomethacin, 5 mg/kg s.c., twice daily for 2 d. These animals then underwent identical studies.
The pretreated dogs had comparable base-line and hypoxia hemodynamic data. However, hydralazine had no effect on pulmonary artery pressure (23.3±1.6 vs. 21.7±2.3 mm Hg, NS) or pulmonary arteriolar resistance (8.03±1.09 vs. 7.14±1.42, NS) during continuous hypoxic ventilation in the indomethacin-pretreated group. Pretreatment with indomethacin did not, however, block the pulmonary vasodilator effects of intravenous prostacyclin (PGI2). Pretreatment with meclofenamate, a cyclo-oxygenase inhibitor structurally unrelated to indomethacin, also blocked the effects of hydralazine during hypoxic ventilation. These data suggest that hydralazine exerts a pulmonary vasodilatory effect during hypoxia-induced pulmonary vasoconstriction, and that this vasodilator effect may be mediated by prostaglandins.
Endothelial membrane hyperpolarization mediated by KCa3.1 and KCa2.3 channels has been demonstrated to initiate endothelium-derived hyperpolarizing factor (EDHF)-type vasodilations. Moreover, pharmacological potentiation of KCa3.1/KCa2.3 channels has been suggested to improve EDHF-type vasodilations. Here we determined whether the KCa3.1/KCa2.3 activator SKA-31 and its derivative SKA-20 improve endothelial dysfunction in KCa3.1−/− and NOS3−/− mice.
Membrane potentials were measured using patch-clamp electrophysiology on carotid artery (CA) endothelial cells (CAEC) from wild-type (wt) and KCa3.1−/− mice. Endothelium-dependent vasodilations were determined by pressure myography in CA.
SKA-31 (1 µmol L-1) activated KCa3.1 and KCa2.3 channels and induced membrane hyperpolarization in CAEC of wt (ΔMP –45mV). These responses were significantly reduced in CAEC of KCa3.1−/− (ΔMP –8mV). SKA-31 (200 nmol L-1, 500 nmol L-1) and SKA-20 (300 nmol L-1) significantly enhanced EDHF-vasodilations in wt. SKA-20 also improved vasodilations during NO-synthesis. In KCa3.1−/−, the defective EDHF-vasodilations were unchanged at 200 nmol L-1 SKA-31, but were significantly improved at 500 nmol L-1. EDHF-vasodilations were slightly enhanced at 300 nmol L-1 SKA-20, but vasodilations during NO-synthesis were unchanged. SKA-31 (500 nmol L-1) enhanced the impaired endothelium-dependent vasodilation in NOS3−/− mice 2-fold. Pharmacological inhibition of the soluble epoxide hydrolase by t-AUCB (1 µmol L-1) in contrast did not increase ACh-induced EDHF- or NO-mediated vasodilations in wt and KCa3.1−/−.
Normal and defective endothelium-dependent vasodilations in murine carotid arteries can be improved by pharmacological enhancement of KCa3.1/KCa2.3 functions. These findings further support the concept that pharmacological activation of endothelial KCa2.3/KCa3.1 could offer a novel endothelium-specific antihypertensive strategy.
EDHF; KCa3.1; KCa2.3; soluble epoxide hydrolase; nitric oxide; endothelial dysfunction
Hypoxic vasodilation is a physiological response to low oxygen (O2) tension that increases blood supply to match metabolic demands. While this response has been characterized for more than 100 years, the underlying hypoxic sensing and effector signaling mechanisms remain uncertain. We have shown that deoxygenated myoglobin (deoxyMb) in the heart can reduce nitrite to nitric oxide (NO˙) and thereby contribute to cardiomyocyte NO˙ signaling during ischemia. Based on recent observations that Mb is expressed in the vasculature of hypoxia-tolerant fish, we hypothesized that endogenous nitrite may contribute to physiological hypoxic vasodilation via reactions with vascular Mb to form NO˙.
Methods and Results
We here show that Mb is expressed in vascular smooth muscle and contributes significantly to nitrite-dependent hypoxic vasodilation in vivo and ex vivo. The generation of NO˙ from nitrite reduction by deoxyMb activates canonical soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) signaling pathways. In vivo and ex vivo vasodilation responses, the reduction of nitrite to NO˙ and the subsequent signal transduction mechanisms were all significantly impaired in mice without myoglobin (Mb−/−). Hypoxic vasodilation studies in Mb, endothelial and inducible NO synthase knockout models (eNOS−/−, iNOS−/−) suggest that only Mb contributes to systemic hypoxic vasodilatory responses in mice.
Endogenous nitrite is a physiological effector of hypoxic vasodilation. Its reduction to NO˙ via the heme globin Mb enhances blood flow and matches O2 supply to increased metabolic demands under hypoxic conditions.
hypoxic vasodilation; myoglobin; nitrite
Rhododendron dauricum L. is an ancient Chinese traditional herb. The pharmacological effects of R. dauricum extract have been shown in chronic tracheitis. The aim of this study was to investigate the cardiovascular effects of Rhododendron dauricum L. flavonoids (RF) on rats and its mechanisms. This study was performed in isolated vascular rings and a rat model of myocardial infarction and isolated myocytes. RF (0.5 – 4 mg/mL) induced a concentration-dependent relaxant effect on the phenylephrine (10-5 M) and KCl (60 mM) contracted aortic rings, with or without intact endothelium. This effect was attenuated by pretreated with L-NAME (10-5 M) and K+ channel inhibitor 4 - AP (1 mM) and TEA (1 mM). The Ca2+-induced contraction and PE-induced contraction were obviously attenuated after pretreated with RF (2 mg/mL) for 30 min in Krebs solution, without Ca2+, containing 10-4 mol EGTA. KCl (60 mM) significantly increased the intracellular free Ca2+ concentration ([Ca2+]i) and RF inhibited the changes induced by KCl in single cardiac myocytes. RF obviously prolonged the survival time of hypoxia mice pretreated with isoprenaline and reduced the myocardial infarction size in rat coronary artery ligation. These findings suggest that RF induces concentration-dependent vasodilation and myocardial preservation.
Rhododendron dauricum flavonoids (RF); Vascular tone; Hypoxia; Myocardial preservation; Vasodilation
Most current theories for the mechanism of hypoxic pulmonary vasoconstriction (HPV) include a role for ROS and/or changes in redox regulation, but, extreme controversy exists regarding which systems and redox changes mediate the HPV response. Whereas, nitric oxide appears to help maintain low pulmonary arterial pressure, suppress HPV and to prevent the development of pulmonary hypertension. Our studies have found a key role for glucose-6-phosphate dehydrogenase in bovine pulmonary arterial smooth muscle functioning to maintain elevated levels of cytosolic NADPH which fuels the generation of vasodilator levels of hydrogen peroxide. HPV results from hypoxia removing vasodilation by peroxide. Decreased superoxide generation by Nox4 oxidase and its conversion to peroxide by Cu,Zn-SOD appear to be potential factors in sensing hypoxia, and decreased cGMP-associated vasodilation and removal of redox controlled vasodilator mechanisms by increased cytosolic NADPH may be key coordinators of the HPV response. Oxidant generation associated with vascular disease processes including the removal of NO by superoxide, and attenuation of its ability to stimulate cGMP production by oxidation of the heme and thiols of soluble guanylate cyclase attenuate potential beneficial actions of NO on pulmonary arterial function. While pulmonary hypertension appears to have multiple poorly understood effects on redox-associated processes potentially influencing responses to hypoxia and NO-cGMP signaling, much remains to be elucidated regarding how these processes maybe important factors in the progression, expression and therapeutic treatment of pulmonary hypertension.
hypoxia; nitric oxide; pulmonary hypertension; redox signaling
Xanthine oxidase is a major source of superoxide in the vascular endothelium. Previous work in humans demonstrated improved conduit artery function following xanthine oxidase inhibition in patients with obstructive sleep apnea. Objectives: To determine whether impairments in endothelium-dependent vasodilation produced by exposure to chronic intermittent hypoxia are prevented by in vivo treatment with allopurinol, a xanthine oxidase inhibitor.
Sprague-Dawley rats received allopurinol (65 mg/kg/day) or vehicle via oral gavage. Half of each group was exposed to intermittent hypoxia (FIO2 = 0.10 for 1 min, 15×/h, 12 h/day) and the other half to normoxia. After 14 days, gracilis arteries were isolated, cannulated with micropipettes, and perfused and superfused with physiological salt solution. Diameters were measured before and after exposure to acetylcholine (10−6M) and nitroprusside (10−4M).
In vehicle-treated rats, intermittent hypoxia impaired acetylcholine-induced vasodilation compared to normoxia (+4 ± 4 vs. +21 ± 6 μm, p = 0.01). Allopurinol attenuated this impairment (+26 ± 6 vs. +34 ± 9 μm for intermittent hypoxia and normoxia groups treated with allopurinol, p = 0.55). In contrast, nitroprusside-induced vasodilation was similar in all rats (p = 0.43). Neither allopurinol nor intermittent hypoxia affected vessel morphometry or systemic markers of oxidative stress. Urinary uric acid concentrations were reduced in allopurinol- versus vehicle-treated rats (p = 0.02).
These data confirm previous findings that exposure to intermittent hypoxia impairs endothelium-dependent vasodilation in skeletal muscle resistance arteries and extend them by demonstrating that this impairment can be prevented with allopurinol. Thus, xanthine oxidase appears to play a key role in mediating intermittent hypoxia-induced vascular dysfunction.
Hypoxia; Allopurinol; Endothelium; Oxidative stress