Endothelium-dependent Vasodilator Acetylcholine Research Articles

Hemolysis Compromises Nitric Oxide-Dependent Vasodilatory Responses in Patients Undergoing Major Cardiovascular Surgery

The hemolytic products cell-free oxyhemoglobin (FHb) and arginase-1 reduce nitric oxide (NO) bioavailability by scavenging NO and by degrading the NO precursor arginine to ornithine, respectively. In this study we evaluated the relevance of hemolysis to NO-dependent blood flow in patients undergoing cardiovascular surgery. Plasma FHb, arginase-1, and amino acid concentrations were measured perioperatively. Forearm blood flow (FBF) responses to the intra-arterial administered NO-donor sodium nitroprusside (SNP) and the endothelium-dependent vasodilator acetylcholine (ACh) were measured by venous occlusion plethysmography. When peak values plasma FHb and arginase-1 were found, vascular dilatation to SNP, but not ACh, was significantly reduced compared with 1 day postoperatively, when FHb had returned to baseline levels (p < 0.05). Interestingly, plasma FHb concentration was inversely correlated to FBF responses to SNP (r -0.93, p < 0.001). In contrast, the increase in arginase-1 was not biologically relevant as the ratio of plasma arginine to ornithine remained constant. We conclude that hemolysis with concomitant release of FHb during cardiovascular surgery is associated with impaired NO-dependent forearm blood flow.

Abstract 106: Chronic Intermittent Hypoxia Induces Neurovascular Dysfunction through Endothelin-1 and Nox2-Derived Radical

Obstructive sleep apnea (OSA), a condition resulting in chronic intermittent hypoxia (CIH), is an independent risk factor for ischemic stroke, but the mechanisms of the effect are unknown. We hypothesized that CIH increases stroke risk by altering the regulation of cerebral blood flow (CBF) and increasing the brain’s susceptibility to ischemia. Male C57Bl/6 mice (n=5/group) were subjected to CIH (10% O 2 for 90 sec/room air for 90 sec; during sleep hours) for 14 or 35 days (d). Sham-treated mice received room air according to the same schedule. Somatosensory cortex CBF was assessed by laser-Doppler flowmetry in anesthetized mice equipped with a cranial window. CIH increased mean arterial pressure only at 35d (from 74±2 to 83±3 mmHg (p&lt;0.05). However, CIH attenuated the CBF increase produced by neocortical application of the endothelium-dependent vasodilator acetylcholine (sham: 22±1%; CIH:13±1%;p&lt;0.05) and by whisker stimulation (sham: 23±1%;CIH: 14±1%;p&lt;0.05) both at 14 and 35d. CIH did not alter the CBF increase evoked by adenosine (sham: 23±3%; CIH: 25±1%; p&gt;0.05), indicating that smooth muscle relaxation was not compromised. The effects of CIH were prevented by neocortical application of the endothelin (ET) type A (ET A ) receptor antagonist BQ123 (1µM;p&lt;0.05) and were associated with ET1 up-regulation in cerebral blood vessels (sham: 52±26;CIH: 5,486±2,466 pg/mg;p&lt;0.05;by ELISA). The angiotensin-II AT1 receptor inhibitor losartan (5µM) did not affect the dysfunction (p&gt;0.05). CIH increased reactive oxygen species (ROS) by 2.3±0.5 folds (p&lt;0.05), assessed by hydroethidine, an effect blocked by the ROS scavenger MnTBAP (100µM; p&lt;0.05) or by BQ123 (p&lt;0.05). Furthermore, the cerebrovascular dysfunction was absent in mice lacking the Nox2-/- subunit of the superoxide-producing enzyme NADPH oxidase and was reversed by neocortical application of a NADPH oxidase peptide inhibitor. The data show that CIH, induces profound alterations of key regulatory mechanisms of cerebral circulation through ET1 and Nox2-dependent ROS production, and raises the possibility that cerebrovascular dysfunction may underlie the increased stroke risk in patients with OSA.

Abstract 2305: Endothelin-1 Alters Nitric Oxide-Dependent Cerebrovascular Responses by Modulating The Phosphorylation State of eNOS via Rho Kinase

Hypertension (HTN) alters vital homeostatic mechanisms regulating cerebral blood flow (CBF) and increases the risk of stroke and dementia. HTN exerts some of its damaging effects by counteracting the beneficial vascular actions of nitric oxide (NO). The potent vasoconstrictor endothelin-1 (ET1) has been implicated in the pathogenesis of HTN, but its role in the cerebrovascular effects of HTN is unknown. We examined whether ET1 disrupts CBF regulation. CBF (laser-Doppler flowmetry) was assessed in the somatosensory cortex in anesthetized male C57Bl/6 mice (n=5/group) equipped with a cranial window. ET1 (35 pmol/kg/min; i.v. for 45 min) increased mean arterial pressure from 72±4 to 99±6 mmHg (p&lt;0.05), without reducing resting CBF (p&gt;0.05). However, ET1 attenuated the CBF increase produced by neocortical application of the endothelium-dependent vasodilator acetylcholine (ACh; -37±1%; p&lt;0.05) and by whisker stimulation (-31±1%; p&lt;0.05), responses dependent on NO. The CBF response to adenosine was intact (p&gt;0.05) indicating that ET1 did not act by compromising smooth muscle relaxation. The effects of ET1 were prevented by the ET type A (ET A ) receptor antagonist BQ123 (1µM; p&lt;0.05), by the Rho kinase (ROCK) inhibitor Y27632 (1 µM; p&lt;0.05), but not by the ET B antagonist BQ788 (100nM; p&gt;0.05). ET-1 did not affect the CBF increase produced by the NO donor SNAP and did not increase free radicals, suggesting that ET-1 did not act by reducing NO vasoactivity or bioavailability. However, in brain endothelial cell cultures ET1 (10-100nM) attenuated the NO production induced by ACh (-49±4% at 50nM; p&lt;0.05), an effect blocked by BQ123 and Y27632. ET1 increased eNOS phosphorylation at Thr495, which inhibits eNOS, and reduced phosphorylation at Ser1177, which activates eNOS, effects blocked by Y27632. These findings, collectively, suggest that ET1 alters key regulatory mechanisms of the cerebral circulation by modulating the phosphorylation state of eNOS via ROCK. The resulting downregulation of eNOS activity is responsible for the neurovascular dysregulation induced by ET1. ET A receptors may be a valuable target to counteract the deleterious cerebrovascular actions of HTN.

Diesel exhaust particulate induces pulmonary and systemic inflammation in rats without impairing endothelial function ex vivo or in vivo

BackgroundInhalation of diesel exhaust impairs vascular function in man, by a mechanism that has yet to be fully established. We hypothesised that pulmonary exposure to diesel exhaust particles (DEP) would cause endothelial dysfunction in rats as a consequence of pulmonary and systemic inflammation.MethodsWistar rats were exposed to DEP (0.5 mg) or saline vehicle by intratracheal instillation and hind-limb blood flow, blood pressure and heart rate were monitored in situ 6 or 24 h after exposure. Vascular function was tested by administration of the endothelium-dependent vasodilator acetylcholine (ACh) and the endothelium-independent vasodilator sodium nitroprusside (SNP) in vivo and ex vivo in isolated rings of thoracic aorta, femoral and mesenteric artery from DEP exposed rats. Bronchoalveolar lavage fluid (BALF) and blood plasma were collected to assess pulmonary (cell differentials, protein levels & interleukin-6 (IL-6)) and systemic (IL-6), tumour necrosis factor alpha (TNFα) and C-reactive protein (CRP)) inflammation, respectively.ResultsDEP instillation increased cell counts, total protein and IL-6 in BALF 6 h after exposure, while levels of IL-6 and TNFα were only raised in blood 24 h after DEP exposure. DEP had no effect on the increased hind-limb blood flow induced by ACh in vivo at 6 or 24 h. However, responses to SNP were impaired at both time points. In contrast, ex vivo responses to ACh and SNP were unaltered in arteries isolated from rats exposed to DEP.ConclusionsExposure of rats to DEP induces both pulmonary and systemic inflammation, but does not modify endothelium-dependent vasodilatation. Other mechanisms in vivo limit dilator responses to SNP and these require further investigation.

Abstract P132: Diabetes-Induced Cardiac Dysfunction Is Rescued by Endothelial Cationic Amino Acid Transporter 1 Overexpression in Mice

Nitric oxide (NO) bioavailability is markedly impaired in diabetes, contributing to endothelial and myocardial dysfunction. Our aim was to determine if endothelial-specific overexpression of Cationic Amino Acid Transporter 1 (CAT-1, to enhance L-arginine uptake and NO generation) prevents diabetes-induced endothelial and cardiac dysfunction in diabetic mice. Male CAT-1 transgenic and non-transgenic (Ntg) 6 week old mice were administered streptozotocin (STZ, 55mg/kg ip daily for 5 days). Sham mice received citrate buffer vehicle. Myocardial function was assessed in anaesthetised mice in vivo 12 weeks later, via echocardiography. Vascular reactivity was then assessed in vitro. Compared to their non-diabetic littermates (n=6), Ntg STZ mice (n=6) exhibited reduced early-to-late blood flow velocity ratios (E/A, 1.7±0.2 vs 2.4±0.1, p&lt;0.01) and prolonged deceleration time (DT, 40±1 vs 31±2msec, p&lt;0.002), indicative of diastolic dysfunction. Decreased E-wave velocity (109±4 vs 76±12msec, p&lt;0.01) and increased A-wave velocity (45±3 vs 55±4msec, p&lt;0.05) were also observed. Systolic dysfunction was observed in Ntg STZ mice compared to shams, on reduced fractional shortening (FS, 30±1 vs 39±1%, p&lt;0.002). In CAT-1 mice, diabetes did not impair E/A ratio (2.1±0.2), FS (40±3%) or DT (33±1msec, n=8). Thoracic aortic responses to the endothelium-dependent vasodilator acetylcholine (ACh) were unchanged by diabetes in Ntg and CAT-1 Tg mice compared to sham Ntg mice (pEC50 7.0±0.1 vs 7.2±0.1 and 7.1±0.1). There was a non-significant trend for sham CAT-1 Tg mice to show greater sensitivity to ACh than sham Ntg mice (pEC50 7.5±0.1 vs 7.1±0.1). Responses to the endothelium-independent vasodilator sodium nitroprusside and the vasoconstrictor noradrenaline were unaffected by diabetes and/or genotype. In conclusion, our results suggest that endothelial-specific CAT-1 overexpression protects against cardiac dysfunction via a mechanism independent of the systemic vasculature. The involvement of the coronary vasculature remains to be determined. Endothelial CAT-1 up-regulation as a potential therapeutic target for diabetic cardiomyopathy is an exciting prospect.

Abstract 8309: Adiponectin Deficiency Exacerbates Endothelial Dysfunction By Increasing Inflammation In High-fat Diet Induced Obese Mice

Adiponectin (APN) is a 30-kDa protein exclusively secreted by adipose tissue. Clinical data show obese and type 2 diabetic patients have decreased APN levels in the circulation. Previously, we showed that APN is deficient in the serum and in aorta protein expression in type 2 diabetic mice ( db/db , Lepr db ), which suggests APN may play an important role in vascular dysfunction and the development of type 2 diabetes. Accordingly, we hypothesized that APN deficiency will impair endothelial function in aortas in high fat diet (HF) induced obese mice. To test this hypothesis, we fed HF diet (60% calories from fat) in wild type (WT) and APN knock-out (APNKO) mice for 18 weeks and studied vascular function of aorta ex vivo. Endothelium-dependent vasodilator acetylcholine (Ach)-induced vasodilation was blunted in APNKO+HF compare to WT+HF, but sodium nitroprusside (SNP)-induced endothelium-independent vasodilation was identical. Previously, we showed that adenoviral delivery of APN (Ad-APN) improved endothelial function in diabetic mice compare to control virus treatment (Ad-βgal). Real-time PCR results showed that mRNA gene expression of TNFα, IL-6 and intercellular adhesion molecule-1(ICAM-1) was higher in db/db aortas compared to control mice, suggesting that inflammation and recruitment of immune cells may contribute to endothelial dysfunction. However, Ad-APN treatment decreased TNFα, IL-6 and ICAM-1 suggesting APN may suppress inflammation and adhesion molecules. This suggests that APN may contribute to an increase in nitric oxide bioavailability by decreasing inflammation and recruitment of immune cells in the aorta. APN appears to play an important role in the prevention of vascular dysfunction in the aortas by preventing inflammation and immune response the animal and infers a similar role may occur is in obese/diabetic patients.

Enhanced K+-channel-mediated endothelium-dependent local and conducted dilation of small mesenteric arteries from ApoE−/− mice

Agonists that evoke smooth muscle cell hyperpolarization have the potential to stimulate both local and conducted dilation. We investigated whether the endothelium-dependent vasodilators acetylcholine (ACh) and SLIGRL stimulated conducted dilation and whether this was altered by deficiency in apolipoprotein E (ApoE(-/-)). Isolated mesenteric arteries were cannulated, pressurized, and precontracted with phenylephrine. Agonists were either added to the bath to study local dilation or were restricted to one end of arteries to study conducted dilation. An enhanced sensitivity to both ACh and SLIGRL was observed in mesenteric arteries from ApoE(-/-) mice compared with wild-type controls. Inhibition of nitric oxide (NO) synthase blocked ACh responses, but had no effect on maximum dilation to SLIGRL. SLIGRL increased endothelial cell Ca(2+), hyperpolarized smooth muscle cells, and fully dilated arteries. The NO-independent dilation to SLIGRL was blocked with high [KCl] or Ca(2+)-activated K(+)-channel blockers. The hyperpolarization and dilation to SLIGRL passed through the artery to at least 2.5 mm upstream. The conducted dilation was not affected by a deficit in ApoE and could also be stimulated by ACh, suggesting NO itself could stimulate conducted dilation. In small mesenteric arteries of ApoE(-/-) mice, NO-independent dilation is enhanced. Since both NO-dependent and -independent pathways can stimulate local and conducted dilation, the potential for reducing vascular resistance is improved in these vessels.

Effects Of Aging And Exercise Training On Vasomotor Control In Spinotrapezius Resistance Arteries

INTRODUCTION: Advancing age elicits structural and functional alterations within the cardiovascular system which can compromise exercise capacity. Exercise training can mitigate these age related impairments, however, there is debate regarding the effects of exercise on vascular function in non-recruited muscles. PURPOSE: To test the hypothesis that exercise training would improve vasomotor control in first-order arterioles of the spinotrapezius muscle (not recruited during incline treadmill running) with advancing age. METHODS: Young (Y, ∼6mo) and aged (O, >24mo) male Fisher-344 rats were assigned to control sedentary (YSED; n=7, OSED; n=7) or endurance trained (YET; n=7, OET; n=7) groups. Resistance arteries were isolated and studied in vitro. First-order arterioles were exposed to cumulative additions of the endothelium-dependent vasodilator acetylcholine (ACh; 10-9 to 10-4M) and -independent vasodilator sodium nitroprusside (SNP; 10-10 to 10-4M), and subsequent vasomotor responses were recorded. RESULTS: Upon exposure to ACh, vasodilation was blunted in the aged sedentary (27 ± 9%) versus young sedentary (56 ± 10%; p<0.05) group, but was improved with exercise training to levels of younger counterparts (OET 66 ± 9% vs. YET 63 ± 6%). Responses to SNP in aged sedentary group was impaired compared to young sedentary, and improved following exercise training in both age groups versus their sedentary counterparts. CONCLUSION: The data demonstrate that endurance training can ameliorate age-associated impairments in vascular function, and furthermore improvements in vasomotor control are not isolated to recruited muscle.

N G-Nitro-l-arginine methyl ester attenuates vasodilator responses to acetylcholine but enhances those to sodium nitroprusside

The effects of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of the synthesis of the endothelium-derived relaxing factor nitric oxide, were studied in two isolated perfused vascular beds: the rat mesenteric arterial bed and the hepatic arterial bed of the rabbit liver. The tone of both preparations was raised with noradrenaline (10 and 30 microM for rabbit and rat preparations, respectively). In both preparations, L-NAME (30 microM) significantly attenuated vasodilator responses to the endothelium-dependent vasodilator acetylcholine, but enhanced responses to sodium nitroprusside (a direct smooth muscle dilator). The evidence supports the view, previously established from work carried out in isolated vessels, that in addition to acting as an inhibitor of nitric oxide, L-NAME enhances the responsiveness of smooth muscle to direct relaxation by nitrovasodilators.

Moving beyond the “Allegory of the Cave” in the assessment of pulmonary arterial hypertension

PULMONARY ARTERIAL HYPERTENSION (PAH) remains a clinically vexing problem considering the high morbidity and mortality that plagues this entity despite indication of improved survival after the introduction of more targeted therapy. While this syndrome is well described from a pathological standpoint the mechanisms involved in the pulmonary vascular remodeling are less clear but are thought to involve endothelial dysfunction and proliferation of various cell types that comprise the wall of distal pulmonary vessels (3). The specific mechanisms leading to distal vessel obliteration and increased pulmonary vascular resistance have been particularly elusive for several decades (14), and although vasoconstriction as a primary event was the focus of research and targeted therapy in early studies, the PAH research community has witnessed a paradigm shift in recent years with a strong tendency to now view the PAH lesion as a quasi-cancerous process (12). In this issue of the Journal of Applied Physiology, Schwenke et al. (8) bring a new tool for assessment of the distal pulmonary vasculature and shake an old specter, vasoconstriction. The investigators employ synchrotron radiation microangiography (SRA), a fairly novel and powerful technique, to more directly evaluate vascular tone in a classic animal model of PAH, monocrotaline (MCT)-challenged rats, providing new insight into regional specific responses of the pulmonary circulation in PAH. SRA allows for both the measurement and assessment of control of blood flow in vivo and collection of data on small arteries and arterioles in situ over time (10). Thus it can supply superior information on kinetic events relative to ex vivo techniques. Since SRA has no focal plane, it can visualize both surface and penetrating vessels simultaneously unlike intravital microscopy, which is limited to assessment of vessel caliber at a single vascular plan. In dynamic organs such as the lung and heart, which are subject to motion, temporal

Microvascular Endothelial Dysfunction in Obstructive Sleep Apnea Is Caused by Oxidative Stress and Improved by Continuous Positive Airway Pressure Therapy

Background: Endothelial dysfunction has recently been demonstrated in obstructive sleep apnea (OSA), but the underlying mechanisms are not entirely understood. Oxidative stress is a typical feature of OSA. Objectives: We investigated the influence of oxidative stress and continuous positive airway pressure (CPAP) on microvascular endothelial function in OSA. Methods: Endothelial function of forearm resistance vessels was assessed by strain gauge venous occlusion plethysmography after intra-arterial infusion of the endothelium-independent vasodilator sodium nitroprusside (1.6, 3.2, and 4.0 µg/min) and the endothelium-dependent vasodilator acetylcholine (Ach, 15, 30 and 40 µg/min) in patients with (n = 11) and without (n = 8) OSA (apnea-hypopnea index ≧15/h). These measurements have been repeated after local intra-arterial infusion of the antioxidant vitamin C (25 µg/min). Furthermore, 6 patients have been reevaluated after 6 months of OSA treatment. Results: Patients with OSA demonstrated impaired endothelial function compared to those without OSA. Thus, related to baseline flow, the increase in forearm blood flow induced by Ach was blunted in patients with OSA (148.7 ± 29.7% in OSA vs. 233.6 ± 45.7% in controls, p = 0.001). This difference, however, was abolished by co-infusion of vitamin C. Endothelial function markedly improved following treatment in 5 of 6 OSA patients. Conclusions: This study strongly suggests that microvascular endothelial function is affected by OSA predominantly through increased oxidative stress, and treatment of OSA may improve endothelial function mainly by reducing oxidative stress. The role of oxidative stress-induced endothelial dysfunction as a potential promoter of atherosclerosis and an increased cardiovascular risk in patients with OSA should be investigated in further controlled studies.

The Role of Cytokines and Inducible Nitric Oxide Synthase in Endotoxemia-Induced Endothelial Dysfunction

Sepsis is characterized by a blunted vascular responses due to impairment of endothelial function. The aim of our study was to assess endothelial function and the role of cytokines and nitric oxide (NO). Endotoxin tolerance was induced in 14 healthy volunteers by intravenous injection of 2 ng.kg.d lipopolysaccharide on 5 consecutive days. Forearm blood flow (FBF) was measured by strain-gauge plethysmography during dose-response curves of endothelium-dependent vasodilator acetylcholine and endothelium-independent vasodilator sodium nitroprusside before and 4 hours after LPS administration on days 1 and 5. In another study, 7 healthy volunteers were given selective inducible NO synthase inhibitor aminoguanidine intravenous continuously from 1 hour after a single LPS administration until 5 hours. FBF showed an attenuation of ACh-induced vasodilatory response with 67% (45%-72%) 4 hours after the first LPS administration (P = 0.01) with an unchanged dose-response curve to sodium nitroprusside. This attenuation to ACh infusion did not occur in the presence of aminoguanidine (P = 0.21) and also did not occur when tolerance was present on day 5 (P = 0.45). Our data demonstrate that endothelial dysfunction caused by endotoxemia does not occur when endotoxin tolerance develops, indicated by the absence of cytokine production and during administration of selective inducible NO synthase inhibitor aminoguanidine in vivo.

Cyclooxygenase 1–Derived Prostaglandin E 2 and EP1 Receptors Are Required for the Cerebrovascular Dysfunction Induced by Angiotensin II

Prostaglandin E(2) (PGE(2)) EP1 receptors (EP1Rs) may contribute to hypertension and related end-organ damage. Because of the key role of angiotensin II (Ang II) in hypertension, we investigated the role of EP1R in the cerebrovascular alterations induced by Ang II. Mice were equipped with a cranial window, and cerebral blood flow was monitored by laser-Doppler flowmetry. The attenuation in cerebral blood flow responses to whisker stimulation (-46+/-4%) and the endothelium-dependent vasodilator acetylcholine (-40+/-4%) induced by acute administration of Ang II (250 ng/kg per minute; IV for 30 to 40 minutes) were not observed after cyclooxygenase 1 or EP1R inhibition or in cyclooxygenase 1 or EP1-null mice. In contrast, cyclooxygenase 2 inhibition or genetic inactivation did not prevent the attenuation. Ang II-induced oxidative stress was not observed after cyclooxygenase 1 or EP1R inhibition or in EP1R-null mice. Prostaglandin E(2) reinstated the Ang II-induced cerebrovascular dysfunction and oxidative stress after cyclooxygenase 1 inhibition. Brain prostaglandin E(2) levels were not increased by Ang II but were attenuated by cyclooxygenase 1 and not cyclooxygenase 2 inhibition. The cerebrovascular dysfunction induced by 14-day administration of "slow-pressor" doses of Ang II (600 ng/kg per minute) was attenuated by neocortical application of SC51089. Cyclooxygenase 1 immunoreactivity was observed in microglia and EP1R in endothelial cells. We conclude that the cerebrovascular dysfunction induced by Ang II requires activation of EP1R by constitutive production of prostaglandin E(2) derived from cyclooxygenase 1. The findings provide the first evidence that EP1Rs are involved in the deleterious cerebrovascular effects of Ang II and suggest new therapeutic approaches to counteract them.

Sex Differences in Protection Against Angiotensin II–Induced Endothelial Dysfunction by Manganese Superoxide Dismutase in the Cerebral Circulation

Angiotensin II (Ang II) produces oxidative stress and endothelial dysfunction in blood vessels. The vasculature from females may be protected against deleterious effects of Ang II. We tested the hypothesis that manganese superoxide dismutase (MnSOD) protects against Ang II-induced endothelial dysfunction. Experiments were performed in C57Bl/6, wild-type (MnSOD(+/+)), and MnSOD-deficient (MnSOD(+/-)) mice treated systemically with vehicle or Ang II. Basilar arteries were isolated from mice treated for 1 week with a nonpressor dose of Ang II (0.28 mg/kg per day). Ang II treatment produced superoxide-mediated impairment of responses to the endothelium-dependent vasodilator acetylcholine (P<0.05). In male but not female MnSOD(+/+) mice, Ang II modestly inhibited responses to acetylcholine (P<0.05). In contrast, Ang II selectively impaired these responses by up to 70% in male MnSOD(+/-) mice (P<0.05), and this effect was reversed by Tempol (P<0.05). Ang II had no effect on acetylcholine responses in MnSOD(+/-) female mice. Vascular superoxide levels after treatment with an inhibitor of CuZn and extracellular superoxide dismutase were higher in Ang II-treated versus vehicle-treated MnSOD(+/-) mice. Thus, a nonpressor dose of Ang II produces endothelial dysfunction in male mice only, suggesting that the female vasculature is protected from Ang II. In male but not female mice, MnSOD deficiency enhanced endothelial dysfunction, suggesting that MnSOD normally protects the vasculature during disease states in which Ang II contributes to vascular dysfunction.

Endothelin attenuates endothelium-dependent platelet inhibition in man

The vascular endothelium produces several substances, including nitric oxide (NO) and endothelin-1 (ET-1), which participate in the regulation of vascular tone in humans. Both these substances may exert other actions of importance for cardiovascular disease, e.g. effects on vascular smooth muscle cell proliferation and inflammation, and NO inhibits platelet function. Experiments were designed to investigate the effect of ET-1 on endothelium-dependent vasodilatation and attenuation of platelet activation. In 25 healthy male subjects (25 +/- 1 years), forearm blood flow was measured by venous occlusion plethysmography, and platelet activity was assessed by whole blood flow cytometry (platelet fibrinogen binding and P-selectin expression) in unstimulated and adenosine diphosphate (ADP)-stimulated samples during administration of ET-1, the endothelium-dependent vasodilator acetylcholine and the NO synthase inhibitor l-NMMA. Acetylcholine increased forearm blood flow and significantly inhibited platelet activation in both unstimulated and ADP-stimulated samples. In samples stimulated with 0.3 microm ADP, fibrinogen binding decreased from 41 +/- 4% to 31 +/- 3% (P < 0.01, n = 11) after acetylcholine administration. The vasodilator response to acetylcholine was significantly impaired during infusions of ET-1 and l-NMMA. ET-1 did not affect platelet activity per se, whereas l-NMMA increased platelet P-selectin expression. Both ET-1 and l-NMMA attenuated the acetylcholine-induced inhibition of platelet activity. Our study indicates that, further to inhibiting endothelium-dependent vasodilatation, ET-1 may also attenuate endothelium-dependent inhibition of platelet activation induced by acetylcholine. An enhanced ET-1 activity, as suggested in endothelial dysfunction, may affect endothelium-dependent platelet modulation and thereby have pathophysiological implications.

Acute and Subacute Effect of Rheopheresis on Microvascular Endothelial Function in Patients Suffering From Age‐related Macular Degeneration

This study was performed on seven patients affected by the atrophic form of age-related macular degeneration (AF-ARMD). The patients under investigation belonged to a larger study aimed at evaluating the efficacy of rheopheresis treatment (RT) on the visual function of AF-ARMD patients. Following the protocol of the larger study, patients received RT twice a week, every two weeks, for a total of ten treatments, as well as high-dose supplementation with zinc and vitamins A, E and beta-carotene. Recruited patients underwent skin laser Doppler flowmetry coupled with skin iontophoresis of the endothelium-dependent vasodilator acetylcholine (ACh) and a test of skin post-ischemic reactive hyperemia, before and after the first RT (time 1: all seven patients) and the fifth RT (time 2: six patients). A significantly higher absolute (anova for repeated measures) and relative (percentage change from the baseline) skin blood flux response (SBFR) to ACh iontophoresis was observed after RT, compared to before RT at time 1 (679 +/- 43% and 436 +/- 78%, respectively; P < 0.05), as well as before RT at time 2 compared to before RT at time 1 (683 +/- 74% and 436 +/- 78%, respectively; P < 0.05). Absolute and relative SBFR to ischemia did not differ either after RT compared to before RT at time 1, or before RT at time 2 compared to before RT at time 1. These findings are consistent with an acute and subacute beneficial effect of RT on skin microvascular endothelial function in the studied AF-ARMD patients.

Coronary microcirculatory vasodilator function in relation to risk factors among patients without obstructive coronary disease and low to intermediate Framingham score

The study aim was to evaluate the relation between the Framingham risk score (FRS) and the presence of coronary risk factors to coronary microcirculatory vasodilator function in patients with early coronary atherosclerosis. We evaluated 1063 patients (age: 50 +/- 12 years, 676 (64%) females) without significant narrowing (<30%) on coronary angiography who underwent invasive assessment of coronary endothelial function. Coronary blood flow (CBF) in response to the endothelium-dependent vasodilator acetylcholine was evaluated as well as the microvascular (endothelium-independent) coronary flow reserve (CFR) in response to intracoronary adenosine. Coronary blood flow and CFR were analysed in relation to the FRS and the presence of traditional and novel risk factors. The estimated 10 years risk in this group was 5.4 +/- 5.2%. Higher FRS was associated with lower CBF in men (P = 0.008), and was a univariate predictor of lower CFR (P = 0.012) in all patients. Multivariable analysis identified a higher FRS (P < 0.001), female sex (P < 0.001) and a positive family history of coronary disease (P = 0.043) as independent predictors of reduced CFR. In patients without obstructive coronary disease, a higher FRS was an independent predictor of reduced CFR. The current study provides insight into the relation between cardiac risk profile and coronary microcirculatory function, and suggests that impaired microcirculatory vasodilator function may be present even in patients with a low to intermediate Framingham score.

3′,4′-Dihydroxyflavonol improves post-ischaemic coronary endothelial function following 7 days reperfusion in sheep

3′,4′-dihydroxyflavonol (DiOHF) is a potent antioxidant that reduces infarct size following myocardial ischaemia–reperfusion. Since oxidative stress induced by myocardial ischaemia–reperfusion impairs endothelium-dependent vasodilatation, we investigated whether DiOHF preserved coronary endothelial function following ischemia–reperfusion. One week after surgery conscious, instrumented sheep were subjected to 1 h of myocardial ischaemia followed by 7 days reperfusion. Immediately before reperfusion, sheep were injected with DiOHF (2 mg/kg iv, n = 4) or vehicle (dimethyl sulphoxide, n = 4). Coronary vascular responses to the endothelium-dependent vasodilator acetylcholine (ACh, 0.05–10.0 µg/kg/min iv), sodium nitroprusside and phenylephrine were determined. After ischaemia–reperfusion, d P/d t max decreased from 1511 ± 93 to 1094 ± 53 mmHg/s, P < 0.05) at 24 h in the vehicle group, but by 7 days had returned towards baseline (1347 ± 91 mm Hg/s). DiOHF prevented the fall in d P/d t max. Coronary conductance (CC) was increased (+ 34 ± 4%) by 10 µg/kg ACh given before ischaemia, but this vasodilatation was significantly reduced after 24 h and 7 days of reperfusion (+ 7 ± 2%, + 15 ± 2%, respectively, both P < 0.05). DiOHF partially preserved the coronary vasodilator response to ACh after 24 h reperfusion (basal 37 ± 7%, 24 h 18 ± 5%), and after 7 days reperfusion the response had recovered (31 ± 7%). DiOHF significantly decreased infarct size, expressed as a percentage of area-at-risk, by 40% after 7 days reperfusion (vehicle 80 ± 7%, DiOHF 46 ± 11%, P < 0.05). A single administration of DiOHF, during ischaemia and just prior to reperfusion, reduced infarct size, preserved ventricular contractility and caused a sustained protection against coronary endothelial dysfunction, with all these beneficial actions being preserved for 7 days reperfusion.

Role of MCP-1 in tumor necrosis factor-α-induced endothelial dysfunction in type 2 diabetic mice

Tumor necrosis factor-alpha (TNF-alpha) upregulates the expression of monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules in type 2 diabetes. We hypothesized that TNF-alpha and MCP-1 may interact to contribute to the evolution of vascular inflammation and endothelial dysfunction in coronary arterioles in type 2 diabetes. To test this hypothesis, we administered anti-MCP-1 to block MCP-1 signaling in genetically modified mice with type 2 diabetes (Lepr(db)) and in heterozygote (m Lepr(db)) lean control. Anti-MCP-1 partially restored vasodilation to the endothelium-dependent vasodilator acetylcholine in isolated, cannulated, and pressurized coronary arterioles in Lepr(db) mice but did not affect vasodilation in m Lepr(db) mice. Anti-MCP-1 attenuated superoxide production and the protein expression of nitrotyrosine, which is an indicator of peroxynitrite production, in isolated coronary arterioles of Lepr(db) mice. Immunostaining results showed that the expression of MCP-1 and vascular cellular adhesion molecule-1 is colocalized with endothelial cells and macrophages. Anti-TNF-alpha or anti-MCP-1 markedly reduced macrophage infiltration and the number of MCP-1-positive endothelium in Lepr(db) mice. The neutralization of TNF-alpha or anti-MCP-1 reduced the expression of adhesion molecules, suggesting that proinflammatory cytokines interact to amplify the signaling process that leads to vascular dysfunction. These findings demonstrate that the endothelial dysfunction occurring in type 2 diabetes is the result of the effects of the inflammatory cytokine TNF-alpha and TNF-alpha-related signaling, including the expression of MCP-1 and adhesion molecules, which further exacerbates vessel inflammation and oxidative stress.

Changes of contractility and relaxation of corpus cavernosum in hyperlipidemic rabbit in vitro

To investigate the mechanisms of erectile dysfunction induced by hyperlipidemia through studying the changes of contractility and relaxation of corpus cavernosum in hypercholesterolemic rabbit in vitro. New Zealand white rabbits were randomly divided into control and experiment groups. The control group (n = 20) received a regular diet for 8 weeks while the experimental group (n = 20) were fed a 1% cholesterol diet for 8 weeks. The authors conducted isometric tension studies with phenylephrine, endothelium-dependent vasodilators (acetylcholine), endothelium-independent vasodilators (sodium nitroprusside) and rho kinase inhibitor (Fasudil) on isolated strips of corpus cavernosum. The weight and total serum cholesterol significantly improved (P < 0.01) in experimental group as compared with the non-fed experimental group. The total serum cholesterol significantly improved (P < 0.01) in experimental group as compared with the control group. The contractility responses to phenylephrine in experiment group in doses (0.5, 1, 5, 10, 50, 100 micromol) were 4.79% +/- 2.00%, 8.84% +/-2.95%, 12.81% +/- 3.77%, 14.63% +/- 5.38%, 25.01% +/- 6.14% and 34.69% +/- 8.53% respectively. The contractility responses to phenylephrine in control group were 1.00% +/- 0.3%, 2.60% +/- 0.72%, 4.28% +/- 1.27%, 5.91% +/- 2.09%, 6.49% +/- 4.02% and 5.64% +/- 11.87% respectively. The contractility responses to phenylephrine significantly improved (P < 0.01) in experimental group in these doses as compared with the control group. The relaxation responses to acetylcholine in experimental group in doses (1, 10 and 100 micromol) were 36.28% +/- 4.71%, 48.81% +/- 4.36% and 56.27% +/- 11.93% respectively. The relaxation responses in control group were 48.04% +/- 4.78%, 69.12% +/- 5.27% and 78.23% +/- 5.30% respectively. There was significant reduction (P < 0.01) in experimental group in these doses as compared with the control group. No difference were found among the two groups in the relaxation response to sodium nitroprusside. The relaxation responses to fasudil in experimental group in doses (0.25, 1.25, 2.5 and 12.5 micromol) were 1.56% +/- 0.43%, 5.03% +/- 1.02%, 8.28% +/- 1.35% and 16.77% +/- 3.57% respectively. The relaxation responses in control group were 4.69% +/- 1.23%, 10.39% +/- 2.05%, 15.08% +/- 3.04% and 25.22% +/- 3.72% respectively. There was significant reduction (P < 0.01) in experimental group in these doses as compared with the control group. The improvement of cavernous smooth muscle contractility and the impairment of cavernous smooth muscle relaxation in response to endothelium-mediated stimuli are the mechanisms of erectile dysfunction induced by hyperlipidemia.