Dilation Of Cerebral Arteries Research Articles

Enhanced endothelial nitric oxide production impairs cerebrovascular tone after brain trauma (1070.1)

Enhancend endothelial nitric oxide production impairs cerebrovascular tone after brain traumaNuria Villalba, Thomas Longden, Mark T. Nelson, George C. Wellman and Kalev FreemanDepartment of Pharmacology, University of Vermont, Burlington, VT Cerebral autoregulation is altered after traumatic brain injury (TBI) reflecting an impairment in myogenic tone and that may contributeing to patient morbidity in patients with TBI. We hypothesized that TBI alters nitric oxide (NO)‐dependent signaling mechanisms leading to decreased cerebral vascular tone. We studied isolated cerebral arteries from adult rats after moderate fluid percussion TBI or sham surgery. We found that both endothelial and smooth muscle (SM) NO levels—indexed by 4,5‐diaminofluorescein (DAF‐2) fluorescence—were increased in cerebral arteries from TBI animals. Arteries from TBI animals exhibited decreased cytosolic SM Ca2+ and reduced myogenic tone compared to controls. Endothelial removal restored myogenic response constriction in TBI animals. Further, inhibition of NO synthase with Nω‐L‐arginine (L‐NNA) restored both myogenic tone and SM Ca2+, and reduced DAF‐2 fluorescence in arteries from TBI animals. The guanylyl cyclase inhibitor (ODQ) and protein kinase G (PKG) inhibitor (RP‐8‐Br‐cGMPS) both elicited robust enhanced constrictions in TBI animals. Further, constriction caused by inhibition of SM large‐conductance Ca2+‐activated potassium (BK) channels with paxilline was augmented in TBI arteries. Addition of 30 nM clamped NO to control arteries provided vasodilation equivalent to that observed in TBI and increased the DAF‐2 signal to a comparable level observed in untreated TBI preparations. These data demonstrate that trauma causes persistent changes in endothelial NO production underlying profound cerebral artery dilation, and provides a quantitative measure of the degree of NO elevation, which is on the order of 30 nM.

Temporary mineralocorticoid receptor antagonism during the development of hypertension improves cerebral artery dilation

Hypertension causes cerebral artery remodeling and increases the risk of stroke. Renin angiotensin system blockade during the development of hypertension has therapeutic effects even after treatment withdrawal. Mineralocorticoid receptor (MR) activation has been implicated in artery remodeling and impaired endothelial function. The possibility that there is a critical therapeutic window for MR antagonism has not been investigated. We hypothesized that temporary MR antagonism while hypertension develops would improve middle cerebral artery (MCA) structure and function in stroke-prone spontaneously hypertensive rats (SHRSP), even after treatment withdrawal. Six-week-old SHRSP were treated with spironolactone (25 mg/kg/day) from 6 to 12 weeks and when aged to 18 weeks, these rats were compared to age-matched untreated SHRSP. Surprisingly, temporary spironolactone treatment reduced the MCA outer and lumen diameter but had no effect on the wall thickness. Temporary spironolactone treatment improved nitric oxide and endothelium-derived hyperpolarizing factor mediated dilation but had no effect on blood pressure. Spironolactone treatment caused a very small reduction in the damage caused by permanent focal cerebral ischemia. These results suggest that temporary MR antagonism during the development of hypertension has divergent effects on the MCA, in that it causes a potentially detrimental reduction in the lumen diameter while improving vasodilation.

Morphology and function of cerebral arteries in adults with pompe disease.

Cerebrovascular abnormalities have been reported in adult patients with Pompe disease. The objective was to study these abnormalities by (1) determining the diameter and mean flow velocity (MFV) of large cerebral arteries and (2) estimating cerebral blood flow (CBF), resistance index (RI) and cerebrovascular reactivity (CVR) as functions of resistance vessels. In ten adults with Pompe disease and twenty controls, the diameter, peak systolic (PSV) and end-diastolic velocities (EDV) of arteries supplying the brain were quantified by MR angiography and sonography. MFV, RI and CBF were calculated. CVR in the middle cerebral artery (MCA) was determined by hyperventilation and acetazolamide injection. MR angiography revealed dilation of cerebral arteries predominantly in the posterior circulation. Dilative arteriopathy was found in three patients; two of them showed vertebrobasilar dolichoectasia. Despite of the dilative arteriopathy, the MFV was normal, indicating increased CBF and dilated resistance vessels. RI of all examined arteries and CVR of MCA were normal. The data suggest that dilation of small and large cerebral arteries is a common feature in adults with Pompe disease. Increased CBF might be the consequence of dilated resistance vessels. However, dysfunction of resistance vessels was rarely found. In adults with Pompe disease, dilation of small and large cerebral arteries is a common feature and might be associated with increased cerebral blood flow.

Endothelial TRPV4 channels mediate dilation of cerebral arteries: impairment and recovery in cerebrovascular pathologies related to Alzheimer's disease

Transient receptor potential vanilloid type 4 (TRPV4) channels are expressed in brain endothelial cells, but their role in regulating cerebrovascular tone under physiological and pathological conditions is still largely unknown. Wild-type (WT) mice and mice that overexpress a mutated form of the human amyloid precursor protein (APP mice, model of increased amyloid β), a constitutively active form of TGF-β1 (TGF mice, model of cerebrovascular fibrosis) or both (APP/TGF mice) were used. Dilations to the selective TRPV4 channel opener GSK1016790A (GSK) or to ACh were measured in posterior cerebral artery segments. Both GSK- and ACh-induced dilations virtually disappeared following endothelium denudation in WT mice. These responses were impaired in vessels from APP, TGF and APP/TGF mice compared with WT. Pre-incubation of WT vessels with the selective TRPV4 channel blocker HC-067047, or with small-conductance (SK channel, apamin) and/or intermediate-conductance (IK channel, charybdotoxin, ChTx) Ca(2+) -sensitive K(+) channel blocker abolished GSK-induced dilations and massively decreased those induced by ACh. These treatments had no or limited effects on ACh-induced dilation in vessels from APP, TGF or APP/TGF mice, and IK and SK channel function was preserved in transgenic mice. Antioxidant superoxide dismutase or catalase normalized GSK- and ACh-mediated dilations only in APP brain arteries. We conclude that endothelial TRPV4 channels mediate ACh-induced dilation in cerebral arteries, that they are impaired in models of cerebrovascular pathology and that they are sensitive, albeit in the reversible manner, to amyloid β-induced oxidative stress.

A new rodent model for obstructive sleep apnea: effects on ATP-mediated dilations in cerebral arteries

Obstructive sleep apnea (OSA), a condition in which the upper airway collapses during sleep, is strongly associated with metabolic and cardiovascular diseases. Little is known how OSA affects the cerebral circulation. The goals of this study were 1) to develop a rat model of chronic OSA that involved apnea and 2) to test the hypothesis that 4 wk of apneas during the sleep cycle alters endothelium-mediated dilations in middle cerebral arteries (MCAs). An obstruction device, which was chronically implanted into the trachea of rats, inflated to obstruct the airway 30 times/h for 8 h during the sleep cycle. After 4 wk of apneas, MCAs were isolated, pressurized, and exposed to luminally applied ATP, an endothelial P2Y2 receptor agonist that dilates through endothelial-derived nitric oxide (NO) and endothelial-dependent hyperpolarization (EDH). Dilations to ATP were attenuated ~30% in MCAs from rats undergoing apneas compared with those from a sham control group (P < 0.04 group effect; n = 7 and 10, respectively). When the NO component of the dilation was blocked to isolate the EDH component, the response to ATP in MCAs from the sham and apnea groups was similar. This finding suggests that the attenuated dilation to ATP must occur through reduced NO. In summary, we have successfully developed a novel rat model for chronic OSA that incorporates apnea during the sleep cycle. Using this model, we demonstrate that endothelial dysfunction occurred by 4 wk of apnea, likely increasing the vulnerability of the brain to cerebrovascular related accidents.

Abstract 208: Altered Cerebrovascular Reactivity Following One Month of Obstructive Sleep Apnea

Obstructive sleep apnea (OSA) has been identified as a significant risk factor for stroke. However, little is known regarding the effects of OSA on the cerebrovascular wall. Using a novel rodent model of OSA we assessed the hypothesis that (1) OSA augments endothelin (ET-1) induced constrictions of cerebral arteries and (2) OSA attenuates dilations of cerebral arteries by agonist-induced nitric oxide (NO) release from the cerebrovascular endothelium. The repetitive airway closures associated with OSA lead to intermittent hypoxia/hypercapnia and reoxygenation, increased negative intrathoracic pressures, and arousals. In order to model the physiological consequences of OSA, we have chronically instrumented rats with inflatable endotracheal obstruction devices. Unanesthetized freely-ranging rats underwent 30 apneas/ hour for 8 hours/ day (sleep phase) for 1 month. During apnea pO 2 decreased from 122±3 to 67±3 mm Hg; pCO 2 increased from 43±1 to 51±1 mm Hg; pH decreased from 7.46±0.00 to 7.38±0.01; and hemoglobin O 2 saturation decreased from 94±1 to 82±1 % (n=5 and p&lt;0.05 for each). Following 1 month of OSA blood pressure, plasma ET-1 and NO levels were similar in sham and OSA rats. Using the pressurized cerebral artery preparation, we observed a 17.5-fold increase in sensitivity to ET-1 (n=5-6, p&lt;0.05) after 1 month of OSA. The increased sensitivity of OSA cerebral arteries to ET-1 was abolished by the ET-B receptor antagonist BQ-788 (n=6, NS). Additionally, constrictions to the ET-B specific agonist IRL-1620 were significantly greater in OSA, versus sham, cerebral arteries (n=6, p&lt;0.05). Dilations to ATP (a P2Y 2 agonist which stimulates NO production in the endothelium) were attenuated in cerebral arteries from OSA rats by 40% (n=5-8, p&lt;0.05). However dilations to the NO-donor MAHMA-NOnoate were similar between groups. In conclusion, 1 month of OSA results in (1) increased sensitivity of cerebral arteries to ET-1, likely through upregulation of ET-B receptors on the vascular smooth muscle and (2) decreased endothelial-derived NO production. These data suggest that OSA results in significant alterations to the cerebrovascular wall in the absence of hypertension.

Endogenously‐generated lipid peroxidation products dilate rat cerebral arteries by activating TRPA1 channels in the endothelium

The Ca2+‐permeable ankyrin (A) transient receptor potential (TRP) channel TRPA1 is activated by a variety of pungent and irritating electrophilic substances. TRPA1 channels are present in the cerebral artery endothelium, but physiological activators of the channel in this tissue are not known. We tested the hypothesis that lipid peroxidation products (LPP) generated by reactive oxygen species (ROS) are endogenous activators of TRPA1 in the cerebral endothelium. We found that NADPH, a substrate of the ROS‐producing enzyme NADPH oxidase (NOX), increased TRPA1 Ca2+sparklet frequency in primary cerebral artery endothelial cells (0.04 ± 0.02 Hz before vs. 0.28 ± 0.08 Hz after, n = 20–30). NADPH also dilated pressurized (80 mmHg) rat cerebral arteries in a concentration‐dependent manner (EC50 = 1.2 μM). This response was diminished by the selective TRPA1 antagonist HC‐030031 (HC) (61.3 ± 21.6% dilation (NADPH) vs. 8.7 ± 16.2% dilation (HC + NADPH), n = 3). The LPP 4‐hydroxy‐nonenal (4‐HNE) also caused cerebral artery dilation (EC50 = 8.4 μM) that was abolished by HC (51.4 ± 7.8% dilation (4‐HNE) vs. 0.0 ± 11.6 % (HC + 4‐HNE), n = 5). Immunolabeling of rat cerebral arteries demonstrates co‐localization of TRPA1, NOX2, and 4‐HNE in myoendothelial junction (MEJ) sites. Together, our findings suggest that NOX‐derived LPP dilate cerebral arteries by activating TRPA1 in MEJs. HL091905.

Remodeling and impaired dilation in middle cerebral arteries from Deoxycorticosterone acetate (DOCA)‐salt rats

Genetic hypertension causes inward remodeling and impaired dilation in the middle cerebral artery (MCA). DOCA‐salt rats are widely used model of hypertension, yet few studies have been performed on their cerebral arteries. We hypothesized that MCAs from DOCA‐salt hypertensive rats will have a reduced lumen diameter and impaired endothelium dependent dilation. Male Sprague‐Dawley rats had uninephrectomy, and received either DOCA (150mg/kg) and salt water (1% NaCl and 0.2% KCl) or tap water. MCAs were studied by pressure myography. Data are mean±SEM, SHAM vs DOCA. DOCA showed impaired dilation to 10 μM ADP (change from baseline: 41.2±5.2 vs 24.7±4.7%, p&lt; 0.05, ANOVA). Lumen diameter was reduced (295±18 vs 343±13 μm at 80 mmHg, p &lt;0.05, ANOVA) in the DOCA rats. Interestingly the wall/lumen ratio (0.06±0.02 vs 0.07±0.01 at 80 mmHg, p&gt;;0.05, ANOVA) did not change. These data suggest the MCAs from DOCA rats may exhibit inward hypotrophic remodeling. These data are contrary to the inward hypertrophic remodeling observed in MCAs from genetically hypertensive rats and imply that the mechanism of remodeling in DOCA‐salt rats may be different.

Age‐related cerebrovascular endothelial dysfunction is associated with cognitive impairment in C57BL/6 mice

Mild cognitive impairment (MCI) is an age‐related disorder that contributes to a reduced quality of life in older adults. However, the underlying mechanisms contributing to MCI with aging are incompletely understood. We tested the hypothesis that old C57 male mice demonstrate cerebrovascular endothelial dysfunction associated with MCI. Endothelium‐dependent dilation (EDD) (ex vivo dilation of mid cerebral and posterior communication arteries to acetylcholine [ACh]) was impaired in old (O: 22–26 mo, n= 17) vs. young (Y: 3–6 mo, n=11) mice (51.2 ± 5.9 % vs. 72.4 ± 5.4 %, p&lt;0.05), and this was associated with reduced L‐NAME‐sensitive dilation (O: 16.1 ± 9.1 % vs. Y: 49.5 ± 9.5 %, p&lt;0.05). Basal NO inhibition by L‐NAME had no effect on phenylephrine‐induced vasoconstriction in O (Con. 23.1 ± 2.8 % vs. L‐NAME 31.1 ± 4.1 %), but increased constriction in Y (Con 19.9 ± 3.1 % vs. L‐NAME 38.0 ± 3.9 %, p&lt;0.05). O (n = 7) had impaired spatial learning during the Morris water maze test (p&lt;0.05) and this was unrelated to differences in vision. In the probe trial, O had lower platform crossing numbers than Y (n =8) (O: 2.7 ± 0.5 vs. Y: 5.1 ± 0.7, p&lt;0.05), suggesting O had impaired spatial memory compared to Y. These results indicate that cerebrovascular endothelial dysfunction, as reflected by reduced basal NO production and NO‐dependent EDD, develops with aging and is associated with MCI in C57 mice.Work supported by NIH AG013038 and AG000279.

Recruitment of Dynamic Endothelial Ca2+ Signals by the TRPA1 Channel Activator AITC in Rat Cerebral Arteries

Stimulation of endothelial TRP channels, specifically TRPA1, promotes vasodilation of cerebral arteries through activation of Ca2+ -dependent effectors along the myoendothelial interface. However, presumed TRPA1-triggered endothelial Ca2+ signals have not been described. We investigated whether TRPA1 activation induces specific spatial and temporal changes in Ca2+ signals along the intima that correlates with incremental vasodilation. Confocal imaging, immunofluorescence staining, and custom image analysis were employed. We found that endothelial cells of rat cerebral arteries exhibit widespread basal Ca2+ dynamics (44 ± 6 events/minute from 26 ± 3 distinct sites in a 3.6 × 10(4) μm2 field). The TRPA1 activator AITC increased Ca2+ signals in a concentration-dependent manner, soliciting new events at distinct sites. Origination of these new events corresponded spatially with TRPA1 densities in IEL holes, and the events were prevented by the TRPA1 inhibitor HC-030031. Concentration-dependent expansion of Ca2+ events in response to AITC correlated precisely with dilation of pressurized cerebral arteries (p = 0.93 by F-test). Correspondingly, AITC caused rapid endothelium-dependent suppression of asynchronous Ca2+ waves in subintimal smooth muscle. Our findings indicate that factors that stimulate TRPA1 channels expand Ca2+ signal-effector coupling at discrete sites along the endothelium to evoke graded cerebral artery vasodilation.

Abstract 151: The Effects of Obstructive Sleep Apnea on the Cerebral Vascular Wall

Obstructive sleep apnea (OSA) is a significant risk factor for stroke. However, little is known regarding how OSA affects the cerebrovascular wall. We hypothesized that (A) OSA will attenuate dilations of cerebral arteries by agonist-induced NO release from cerebrovascular endothelium and (B) OSA will upregulate endothelin-1 (ET-1)-mediated constrictions in cerebral arteries. Airway obstructions were induced by chronically implanting inflatable endotracheal obstruction devices (ODs). The ODs were remotely controlled by a computer to produce 30 apneas/hr (10 sec each) during 8 hrs of the sleep cycle in free-ranging rats. During apnea pO 2 decreased from 122±3 to 67±3 mm Hg; pCO 2 increased from 43±1 to 51±1 mm Hg; pH decreased from 7.46±0.00 to 7.38±0.01; and hemoglobin O 2 saturation decreased from 94±1 to 82±1 % (n=5 and p&lt;0.003 for each). After 4 weeks of apnea blood pressure in OSA rats were similar to that in sham rats (108±1 and 103±1 mmHg respectively). Cerebral arteries were isolated after 4 weeks from sham and OSA rats, mounted on pipettes, and pressurized. Diameter changes of the cerebral arteries were measured after the addition of various agents. Dilations to ATP, a P2Y 2 agonist which stimulates NO production in the endothelium, were attenuated in cerebral arteries from OSA rats by 40% (n=5-8, P&lt;0.05). However, the dilations to the NO donor, MAHMA-NOnoate, were similar between groups. Sensitivity to ET-1, an agonist for ET A and ET B , was increased ~20 fold in OSA arteries. EC 50 s for ET-1 constrictions in cerebral arteries from sham and OSA rats were 10.7 ± 4.3 nM and 0.5 ± 0.4 nM respectively (n=5-6, p=0.01). Constrictions to IRL-1620, a selective ET B agonist, were enhanced 11-fold in OSA compared to sham cerebral arteries (n=3-4, p&lt;0.01). In preliminary studies, the increased sensitivity to ET-1in cerebral arteries from OSA rats was attenuated by the ET B antagonist, BQ-788. We conclude that OSA produces pathological changes involving decreased endothelial-derived NO production and enhances constrictor responses to ET-1 as a result of ET B upregulation. Since blood pressures were similar in the OSA and sham rats, we conclude that cerebrovascular dysfunction can occur with OSA in the absence of coexisting hypertension.

Targeting of Kir2.1 and Downregulation of Inward Rectifier K+ Current by miR-212

Downregulation of inwardly rectifying K+ channels contributes to an increased risk of cardiac arrhythmia in heart failure and to impaired cerebral arterial dilation in chronic alcohol consumption. The downregulation mechanism is unknown, although post-transcriptional regulation of gene expression by microRNAs is a strong possibility that has not been fully investigated. miR-212 is markedly augmented in heart failure and in chronic alcoholism, and is predicted by bioinformatic algorithms to target Kir2.1, the predominant inward rectifier K+ channel expressed in heart and arterial smooth muscle. We developed a fluorescence-based assay for identifying microRNA targets, using mCherry red fluorescent protein to report target sequence expression and enhanced green fluorescent protein for microRNA expression. Using this assay, we demonstrated a functional target for miR-212 in the 3′ untranslated region of Kir2.1. Red/green fluorescence intensity ratio was significantly lower in miR-212-expressing HEK293 cells compared to non-targeting control (miR-212 0.72 ± 0.024 (mean ± sem), n = 550; control 1.21 ± 0.025, n = 731; p<.001, log transformed data). The effect of miR-212 was attenuated by mutating the predicted target site (% inhibition 58.0 ± 14.51, n = 3 wild-type; 22.7 ± 1.25, n = 3 mutant). Expression of miR-212 downregulated endogenous Kir2.1 protein in HeLa cells, as shown by quantitative western blot of membrane extracts (band intensity vs Na+/K+-ATPase loading control: miR-212 0.0647 ± 0.0047; non-targeting control 0.0895 ± 0.0045; n=3, p<.05). Endogenous inward rectifier K+ current in HeLa cells was isolated by extracellular application of 100 µM Ba2+ during whole-cell patch-clamp recording. Ba2+-sensitive current density was significantly smaller in miR-212-transfected (n=13) vs control-transfected cells (n=8); p<.01. In conclusion, downregulation of inwardly rectifying K+ current and Kir2.1 expression in heart failure and alcoholic cerebrovascular dysfunction may be functionally linked to upregulation of miR-212.

Point: Counterpoint: Hypobaric hypoxia induces/does not induce different responses from normobaric hypoxia

Point: Counterpoint: Hypobaric hypoxia induces/does not induce different responses from normobaric hypoxia

Angiographic Patterns in Patients with HIV at Hahnemann University Hospital (P06.244)

Objective: To cerebral angiographic patterns of patients with HIV at Hahnemann University Hospital. Background Central nervous system vascular involvement in patients with HIV is due to infections, neoplastic disease, or abuse of toxic drugs. Nogueras et al. observed recurrent stroke as a manifestation of primary angiitis in a patient with HIV [1]. They demonstrated the presence of HIV-integrated DNA in the basal ganglionic tissues of their patient, which suggested that HIV plays a role in the vasculitic process. HIV has been demonstrated in perivascular multinucleated giant cells and endothelial cells by Gray et al. [2]. O9Chareon et al. reported a patient with HIV-associated vasculitis, demonstrated aneurysmal dilatation of cerebral arteries with alternate areas of narrowing and mural enhancement [3]. Dubrovsky et al. identified pediatric patients with AIDS who have cerebral aneurysmal arteriopathy [4]. Design/Methods: A retrospective chart review of patients who had magnetic resonance angiograms and conventional angiograms, at Hahnemann University Hospital from January 2005 to December 2010. Inclusion criteria: Patients over the age of 18 with HIV. Exclusion criteria: Patients with a known history of cerebral aneurysm prior to being diagnosed with HIV. Results: We identified 30 patients with HIV after randomization who had either an magnetic resonance angiogram or a conventional angiogram. Of those, 72% had CD4 counts Conclusions: The frequency of aneurysms in patients with HIV (3%) was not higher than that in the general population (5%-6%). In the future, we could look at subset of patients with HIV who are more likely to have aneurysms. Disclosure: Dr. Nair has nothing to disclose. Dr. Venkatesh has nothing to disclose.

Cerebrovascular Consequences of Obstructive Sleep Apnea

Obstructive sleep apnea (OSA) is an independent risk factor for stroke. However, little is known about how OSA affects the cerebral circulation. The goal of this project was to test the following hypothesis: Chronic exposure to apnea produces hypertension and alters nitric oxide (NO) mediated dilation in cerebral arteries. For these studies, we developed a novel model of OSA in rats. Airway obstruction was induced by a chronically implanted, inflatable endotracheal obstruction device. We induced 30 apneas (8 sec duration each)/hr during 8 hrs of the sleep cycle in free‐ranging rats. Apnea caused significant alterations in blood gasses [baseline vs. end apnea‐ pO2 (mmHg): 127 ± 2 vs. 67 ± 2; pCO2 (mmHg): 43 ± 1 vs. 51 ± 1; and pH: 7.46 ± 0.002 vs. 7.38 ± 0.01; n=5, p≤0.003]. Apnea decreased hemoglobin O2 saturation by 12 ± 1% (n=5, p=0.001 vs. Sham) and produced exaggerated negative airway pressures (−16 ± 0.7 mmHg; n=7, p&lt;0.001 vs. Sham). Two weeks of apnea produced a 24‐hr mean blood pressure (BP) pattern similar to non‐dipping hypertensives (i.e. ‐ &lt;10% BP dip during the sleep phase). After one month of apnea, NO‐mediated dilations to ATP in isolated cerebral arteries were attenuated by 20 ± 2% (n=4, p&lt;0.02 vs. Sham). Using a novel model of OSA, which recapitulates OSA in humans, we demonstrate that endothelial dysfunction occurs by one month, likely increasing the vulnerability of the brain during stroke.Funded by NIH R21 HL102778

Perivascular blood induces substantial constrictions of isolated basilar artery, which can be reversed by high pCO2

The vasoconstrictor mechanisms in acute intracerebral hemorrhage have not been fully clarified. We hypothesized that perivascular hemolysed blood (HB) has substantial effects on the tone and vasomotor responses of isolated cerebral arteries.Basilar artery (BA) from male rats was isolated and placed in a pressure‐myograph chamber. The diameters of vessels were measured by videomicroscopy. After administration of HB the vasomotor function of vessels was studied in responses to acetylcholine (Ach), sodium‐nitroprusside (SNP), nifedipine (Nife) and CO2, which were obtained in control condition, in the presence of HB, and after washout of HB.The active diameters of BA were 277±11 μm. HB reduced the basal diameters of BA (to 164±11 μm; 59±4% of AD). After washing out of HB, the active diameters of BA reached the control level: 284±9 μm. In control, Ach, SNP, Nife and CO2 elicited substantial dilations in cerebral arteries (20±2%, 26±2%, 32±3% and 26±2%). In contrast, HB decreased the dilation to ACh, SNP, but not to nifedipin and CO2 (7±1%, 12±2%, 28±3% and 30±2%). After washout of HB NO‐dependent dilations were significantly reduced compared to control (ACh: 6±1%, SNP:14±2%).Findings of the present study show that extraluminal HB elicits substantial constrictions and inhibits NO‐dependent dilations, but does not affect Ca‐channel blocker and CO2‐induced dilations.

Recruitment of dynamic cerebral artery endothelial Ca2+ signals by the TRPA1 channel activator AITC

Real‐time regulation of vascular tone by endothelial Ca2+ signals is a crucial determinant of blood flow. Recent findings suggest that transient receptor potential (TRP) channels, particularly TRPA1, may play a key role in activating endothelial Ca2+‐dependent effectors in cerebral arteries to promote acute vasodilation. In the current study, we employed high‐speed confocal imaging and a novel analysis algorithm to evaluate the impact of TRPA1 channel activation on Ca2+ signals in the endothelium of rat cerebral arteries and assess its relationship to vasodilation. We found that endothelial cells of rat cerebral arteries exhibit widespread basal Ca2+ dynamics (44 ± 6 events at 26 ± 3 distinct sites per minute in a 3.6×104 μm2 field). The TRPA1 activator allyl isothiocyanate (AITC) increased Ca2+ signals in a concentration dependent manner, primarily by soliciting new events at distinct sites along the intima (56 ± 5% and 52 ± 4% increase in events and sites, respectively, at 60 μM). These stimulated events corresponded spatially with TRPA1 channel densities within holes in the internal elastic lamina (IEL) and were blocked by the TRPA1‐specific inhibitor HC‐030031. The concentration‐dependent expansion of Ca2+ events in response to AITC correlated precisely with the dilation of pressurized cerebral arteries (p = 0.93 by F‐test comparison of nonlinear regression curve fits). Correspondingly, AITC caused rapid endothelium‐dependent suppression of pro‐contractile asynchronous Ca2+ waves in subintimal smooth muscle cells. The current findings suggest that factors evoking stimulation of TRPA1 channels trigger and expand discrete Ca2+ signal‐effector coupling sites along the endothelium to evoke graded cerebral artery vasodilation. Supported by NIH R01 HL085887 and R01 HL091905.

Stimulus‐specific cerebrovascular dysfunction in humans with metabolic syndrome

Metabolic syndrome (MetS) and reduced cerebrovascular reactivity are each associated with increased risk of stroke in humans. Animal models of MetS demonstrate reduced cerebral artery dilation, but cerebrovascular function in humans with MetS remains untested. We tested the hypothesis MetS humans would have reduced cerebrovascular reactivity to hypoxia and hypercapnia. Blood pressure (BP), arterial oxygen saturation (SpO2), expired carbon dioxide (PETCO2), and middle cerebral artery blood velocity (CBV, transcranial Doppler ultrasound) were measured in MetS adults (n=9, 37 ± 3 yr) and healthy controls (n=10, 32 ± 2 yr). Cerebral conductance index (CVCI = CBV / BP) was calculated to normalize CBV for higher BP in MetS adults. Cerebral vasodilation (ΔCVCI) to hypoxia (SpO2 = 90% and 80%) and hypercapnia (+10 mmHg PETCO2) were assessed. MetS adults exhibited markedly lower ΔCVCI to 90% SpO2 (1 ± 2 vs 6 ± 2 cm s−1 mmHg−1, p&lt;0.05) and 80% SpO2 (5 ± 2 vs 15 ± 3 cm s−1 mmHg−1, p&lt;0.05). In contrast, MetS adults displayed preserved cerebrovascular reactivity to hypercapnia (ΔCVCI = 18 ± 2 vs 20 ± 2 cm s−1 mmHg−1). We conclude younger MetS adults exhibit markedly reduced stimulus‐specific cerebrovascular reactivity during hypoxia. Poor hypoxia‐mediated cerebral dilation may contribute to the increased risk and severity of cerebrovascular disease in MetS humans. Support: NIH HL091397 &amp; AHA 11PRE7390038

NaHS relaxes rat cerebral artery in vitro via inhibition of l-type voltage-sensitive Ca2+ channel

H2S, a gaseous signalling molecule, relaxes blood vessels partly through activation of ATP-sensitive K+ channels. It is however unclear whether H2S or its donors could affect other ion transporting proteins. The present study examined the hypothesis that NaHS, a H2S donor inhibits voltage-sensitive Ca2+ channels and thus relaxes vascular smooth muscle cells (VSMC) in the cerebral arteries. NaHS dilated cerebral arteries from Sprague–Dawley rats with the same potency against pre-contraction by 5-HT and 60mmol/L KCl, which were unaffected by several K+ channel blockers, NG-nitro-l-arginine methyl ester or indomethacin, as assessed in wire myograph under an isometric condition. Likewise, NaHS also dilated cerebral arteries against myogenic constriction in pressurized myograph under an isobaric condition. NaHS concentration-dependently inhibited CaCl2-induced contraction in Ca2+-free, 60mM K+-containing Krebs solution. Patch clamp recordings showed that NaHS reduced the amplitude of l-type Ca2+ currents in single myocytes isolated enzymatically from the cerebral artery. Calcium fluorescent imaging using fluo-4 showed a reduced [Ca2+]i in 60mmol/L KCl-stimulated rat cerebral arteries in response to NaHS. H2S precursor l-cysteine-induced relaxation in cerebral arteries was inhibited by cystathionine γ–lyase (CSE) inhibitor dl-propargylglycine. CSE was expressed in cerebral arteries. In summary, NaHS dilates rat cerebral arteries by reducing l-type Ca2+ currents and suppressing [Ca2+]i of arterial myocyte, indicating that NaHS relaxes cerebral arteries primarily through inhibiting Ca2+ influx via Ca2+ channels

Differential vasoactive effects of sildenafil and tadalafil on cerebral arteries

Phosphodiesterase 5 (PDE5) is associated with migraine pathophysiology, stroke recovery and vasospasm treatment. The potential vascular interplay of PDE5 inhibitors sildenafil, tadalafil and UK-114,542 was studied by intra- versus extra-luminal administration in rat middle cerebral arteries in vitro and on middle meningeal arteries in vivo. By Western blot PDE5 was detected in both cerebral and meningeal arteries, though with minor variations in band intensity between vascular beds. Rat middle cerebral artery diameter was investigated using pressurised arteriography, applying UK-114,542, sildenafil, and tadalafil intra- or extra-luminally. Effects on the dural middle meningeal artery were studied in the in vivo closed cranial window model. At high concentrations, abluminal sildenafil and UK-114,542, but not tadalafil, induced dilatation of the middle cerebral artery. Luminal application elicited a contraction of 4% (sildenafil, P=0.03) and 10% (tadalafil, P=0.02). In vivo, sildenafil, but not tadalafil, dose-dependently dilated middle meningeal artery concomitant to blood pressure reduction (1–3mg/kg);1mg/kg sildenafil inducing 60±14% (P=0.04) and vehicle (DMSO) 13±6% dilatation. In conclusion, PDE5 inhibitors applied luminally had minor contractile effect, whereas abluminal sildenafil induced middle cerebral artery dilatation above therapeutic levels. In vivo, sildenafil dilated middle meningeal artery concomitant with a reduction in blood pressure. Tadalafil had no dilatory effects. PDE5 inhibitors show differential vascular activity in cerebral arteries from healthy animals; arterial dilatation is seen primarily above therapeutic levels. Such findings support clinical studies showing no vasodilator effects of sildenafil on cerebral arteries in healthy subjects.