Aging-Induced Down-Regulation of PKA/BKCa Pathway in Rat Cerebral Arteries.

The incidence of cerebrovascular diseases increases significantly with aging. This study aimed to test the hypothesis that aging may influence the protein kinase A (PKA)-dependent vasodilation via RyR/BKCa pathway in the middle cerebral arteries (MCA). Male Sprague-Dawley rats were randomly divided into control (4-6 month-old) and aged (24-month-old) groups. The functions of MCA and ion channel activities in smooth muscle cells were examined using myograph system and patch-clamp. Aging decreased the isoproterenol/forskolin-induced relaxation in the MCA. Large-conductance Ca(2+)-activated-K(+) (BKCa) channel inhibitor, iberiotoxin, significantly attenuated the forskolin-induced vasodilatation and hyperpolarization in the young group, but not in the aged group. The amplitude and frequency of spontaneous transient outward currents (STOCs) were significantly decreased in the aged group. Single channel recording revealed that the mean open time of BKCa channels were decreased, while an increased mean closed time of BKCa channels were found in the aged group. The Ca(2+)/voltage sensitivity of the channels was decreased accompanied by reduced BKCa alpha and beta1-subunit, the expression of RyR2, PKA-Calpha and PKA-Cbeta subunits were also declined in the aged group. Aging induced down-regulation of PKA/BKCa pathway in cerebral artery in rats. The results provides new information on further understanding in cerebrovascular diseases resulted from age-related cerebral vascular dysfunction.

Doppler ultrasound cardiac gating of intracranial flow at 7T.

BACKGROUND: Ultra-high field magnetic resonance imaging (MR) may be used to improve intracranial blood flow measurements. However, standard cardiac synchronization methods tend to fail at ultra-high field MR. Therefore, this study aims to investigate an alternative synchronization technique using Doppler ultrasound. METHODS: Healthy subjects (n = 9) were examined with 7T MR. Flow was measured in the M1-branch of the middle cerebral artery (MCA) and in the cerebral aqueduct (CA) using through-plane phase contrast (2D flow). Flow in the circle of Willis was measured with three-dimensional, three-directional phase contrast (4D flow). Scans were gated with Doppler ultrasound (DUS) and electrocardiogram (ECG), and pulse oximetry data (POX) was collected simultaneously. False negative and false positive trigger events were counted for ECG, DUS and POX, and quantitative flow measures were compared. RESULTS: There were fewer false positive triggers for DUS compared to ECG (5.3 ± 11 vs. 25 ± 31, p = 0.031), while no other measured parameters differed significantly. Net blood flow in M1 was similar between DUS and ECG for 2D flow (1.5 ± 0.39 vs. 1.6 ± 0.41, bias ± 1.96SD: - 0.021 ± 0.36) and 4D flow (1.8 ± 0.48 vs. 9 ± 0.59, bias ± 1.96SD: - 0.086 ± 0.57 ml). Net CSF flow per heart beat in the CA was also similar for DUS and ECG (3.6 ± 2.1 vs. 3.0 ± 5.8, bias ± 1.96SD: 0.61 ± 13.6 µl). CONCLUSION: Gating with DUS produced fewer false trigger events than using ECG, with similar quantitative flow values. DUS gating is a promising technique for cardiac synchronization at 7T.

Quantitative Analysis of the Cerebral Vasculature on Magnetic Resonance Angiography.

The arterial connections in the Circle of Willis are a central source of collateral blood flow and play an important role in pathologies such as stroke and mental illness. Analysis of the Circle of Willis and its variants can shed light on optimal methods of diagnosis, treatment planning, surgery, and quantification of outcomes. We developed an automated, standardized, objective, and high-throughput approach for categorizing and quantifying the Circle of Willis vascular anatomy using magnetic resonance angiography images. This automated algorithm for processing of MRA images isolates and automatically identifies key features of the cerebral vasculature such as branching of the internal intracranial internal carotid artery and the basilar artery. Subsequently, physical features of the segments of the anterior cerebral artery were acquired on a sample and intra-patient comparisons were made. We demonstrate the feasibility of using our approach to automatically classify important structures of the Circle of Willis and extract biomarkers from cerebrovasculature. Automated image analysis can provide clinically-relevant vascular features such as aplastic arteries, stenosis, aneurysms, and vessel caliper for endovascular procedures. The developed algorithm could facilitate clinical studies by supporting high-throughput automated analysis of the cerebral vasculature.

Clinical importance of the occipital artery in vascular lesions: A review of the literature.

The occipital artery (OA) is a critical artery in vascular lesions. However, a comprehensive review of the importance of the OA is currently lacking. In this study, we used the PubMed database to perform a review of the literature on the OA to increase our understanding of its role in vascular lesions. We also provided our typical cases to illustrate the importance of the OA. The OA has several variations. For example, it may arise from the internal carotid artery or anastomose with the vertebral artery. Therefore, the OA may provide a crucial collateral vascular supply source and should be preserved in these cases. The OA is a good donor artery. Consequently, it is used in extra- to intracranial bypasses for moyamoya disease (MMD) or aneurysms. The OA can be involved in dural arteriovenous fistula (DAVF) and is a feasible artery for the embolisation of DAVF. True aneurysms and pseudoaneurysms can occur in the OA; surgical resection and embolisation are the effective treatment approaches. Direct high-flow AVF can occur in the OA; embolisation treatment is a good option in such cases. The OA can also be involved in MMD and brain arteriovenous malformation (AVM) by forming transdural collaterals. For a patient in the prone position, if occipital and suboccipital craniotomies are performed, the OA can also be used for intraoperative angiography. In brief, the OA is a very important artery in vascular lesions.

Influence of different isoflurane anesthesia protocols on murine cerebral hemodynamics measured with pseudo-continuous arterial spin labeling.

Arterial spin labeling (ASL)-MRI can noninvasively map cerebral blood flow (CBF) and cerebrovascular reactivity (CVR), potential biomarkers of cognitive impairment and dementia. Mouse models of disease are frequently used in translational MRI studies, which are commonly performed under anesthesia. Understanding the influence of the specific anesthesia protocol used on the measured parameters is important for accurate interpretation of hemodynamic studies with mice. Isoflurane is a frequently used anesthetic with vasodilative properties. Here, the influence of three distinct isoflurane protocols was studied with pseudo-continuous ASL in two different mouse strains. The first protocol was a free-breathing set-up with medium concentrations, the second a free-breathing set-up with low induction and maintenance concentrations, and the third a set-up with medium concentrations and mechanical ventilation. A protocol with the vasoconstrictive anesthetic medetomidine was used as a comparison. As expected, medium isoflurane anesthesia resulted in significantly higher CBF and lower CVR values than medetomidine (median whole-brain CBF of 157.7 vs 84.4 mL/100 g/min and CVR of 0.54 vs 51.7% in C57BL/6 J mice). The other two isoflurane protocols lowered the CBF and increased the CVR values compared with medium isoflurane anesthesia, without obvious differences between them (median whole-brain CBF of 138.9 vs 131.7 mL/100 g/min and CVR of 10.0 vs 9.6%, in C57BL/6 J mice). Furthermore, CVR was shown to be dependent on baseline CBF, regardless of the anesthesia protocol used.

Cerebrovascular reactivity after cessation of menopausal hormone treatment.

OBJECTIVE: Women who are currently using menopausal hormone therapy (MHT) have higher cerebrovascular reactivity when compared with postmenopausal women who are not taking MHT; however, the effect of cessation of MHT on cerebrovascular reactivity is not known. Given that MHT can have structural and activational effects on vascular function, this study was performed to characterize cerebrovascular reactivity following cessation of MHT in women at low risk for cerebrovascular disease. METHODS: Cerebrovascular reactivity was measured in a subset of women from the Kronos Early Estrogen Prevention Study (KEEPS) 3 years after cessation of the study drug (oral conjugated equine estrogen, transdermal 17ß-estradiol, or placebo [PLA]). RESULTS: Age, body mass index, and blood pressure were comparable among groups. At rest, the middle cerebral artery velocity (MCAv), cerebrovascular conductance index, mean arterial pressure, and cerebral pulsatility index did not differ among groups. Slope-based summary measures of cerebrovascular reactivity did not differ significantly among groups. However, utilizing repeated-measures modeling, there was a significant upward shift in MCAv responses (p = 0.029) in the combined MHT group compared with the PLA group. CONCLUSION: MHT has a marginal sustained effect on cerebrovascular reactivity when measured 3 years after cessation of hormone treatment.

Melatonin activates BKCa channels in cerebral artery myocytes via both direct and MT receptor/PKC-mediated pathway.

The pineal hormone melatonin is a neuroendocrine hormone with high membrane permeability that is involved in regulation of circadian rhythm of several biological functions. Large-conductance Ca2+-activated K+ (BKCa) channels are abundantly expressed in vascular smooth muscle cells and play an important role in vascular tone regulation. We investigated the mechanisms through which myocyte BKCa channels mediate effects of melatonin on cerebral arteries (CAs). Arterial contractility measurements showed that melatonin alone did not change vascular tone in CAs; however, it induced concentration-dependent vasodilation of phenylephrine-induced contraction in CAs. In the presence of the potent endothelial oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester, melatonin-elicited relaxation was significantly inhibited by iberiotoxin (BKCa channel blocker). Melatonin significantly increased BKCa currents but not voltage-gated K+ (KV) currents in whole-cell recordings. Melatonin decreased the amplitude of Ca2+ sparks and spontaneous transient outward currents (STOCs), however, a significant increase in open probability of BKCa channels was observed in both inside-out and cell-attached patch-clamp recordings. This melatonin-induced enhancement of BKCa channel activity was significantly suppressed by luzindole (melatonin MT1/MT2 receptor inhibitor), U73122 (phospholipase C (PLC) inhibitor), and Ro31-8220 (protein kinase C (PKC) inhibitor). Melatonin had no significant effects on sarcoplasmic reticulum release of Ca2+. These findings indicate that melatonin-induced vasorelaxation of CAs is partially attributable to direct (passing through the cell membrane) and indirect (via melatonin MT1/MT2 receptors-PLC-PKC pathway) activation of BKCa channels on CA myocytes.

Relationship Between the Ambulatory Arterial Stiffness Index and the Lower Limit of Cerebral Autoregulation During Cardiac Surgery.

BACKGROUND: Pulse pressure, the ambulatory arterial stiffness index (AASI), and the symmetric AASI are established predictors of adverse cardiovascular outcomes. However, little is known about their relationship to cerebral autoregulation. This study evaluated whether these markers of vascular properties relate to the lower limit of cerebral autoregulation (LLA). METHODS AND RESULTS: The LLA was determined during cardiac surgery with transcranial Doppler ultrasonography in 181 patients. All other variables were calculated from continuous intraoperative readings obtained before cardiopulmonary bypass. The LLA varied directly with the AASI (ß=3.12 per 0.1 change in AASI, P<0.001) and to a lesser extent the symmetric AASI (ß=2.02 per 0.1 change in symmetric AASI, P≤0.022), while peripheral pulse pressure was not significantly related (ß=0.0, P>0.99). Logistic regression revealed that the likelihood of LLA being >65 mm Hg increased by 50% (95% confidence interval, 11%-102%, P=0.008) for every 0.1 increase in the AASI. The AASI was able to predict a LLA above certain thresholds (area under the curve receiver operating characteristic for AASI predicting an LLA >65 mm Hg: 0.60; 95% confidence interval, 0.51%-0.68%, P=0.043). Incorporating additional variables improved the model's predictive ability (area under the curve for AASI predicting a LLA >65 mm Hg: 0.75; 95% confidence interval, 0.68-0.82, P=0.036). CONCLUSIONS: These data indicate that the LLA is related to the mechanical properties of the vasculature as represented by the AASI. The AASI can be used to predict LLA threshold levels during cardiac surgery. It is now possible to link elevations in the LLA with an increased AASI as determined from readily accessible intraoperative variables.

Transcranial Doppler in hemoglobin SC disease.

BACKGROUND: Stroke is a severe clinical disorder in sickle cell disease (SCD), and few studies have evaluated transcranial Doppler (TCD) flow velocities in hemoglobin SC disease (HbSC). The guidelines for stroke risk are based on evaluations in sickle cell anemia (SCA) or HbS/ß thalassemia. PROCEDURE: In this study, we compare cerebral blood flow in patients with SCD stratified by genotypes. A total of 1,664 pediatric patients with SCD underwent TCD velocity screening, and the time-averaged maximum mean velocity (TAMM) was determined in the middle cerebral artery (MCA), anterior cerebral artery (ACA), and distal intracranial internal carotid artery (ICA). RESULTS: Abnormal velocities were not identified in the ACA; therefore, we only use ICA and MCA velocities. TAMM from the left and right in the ICA and MCA was 134.3 ± 32.0 and 134.4 ± 32.6 cm/s in patients with SCA, and 105.2 ± 20.6 and 104.7 ± 20.0 cm/s in the patients with HbSC, respectively. Mean TAMM between right and left ICA/MCA was 134.5 ± 30.5 cm/s in the SCA group, and 104.9 ± 19.3 cm/s in the HbSC group. Notably, our data show that TCD velocities were significantly lower among the patients with HbSC compared to SCA. TAMM was negatively correlated with hemoglobin and hematocrit in both genotypes. CONCLUSION: These results suggest that a different cut-off value for abnormal TCD velocities could be considered for patients with HbSC. Additional studies are warranted to determine the actual risk of stroke in HbSC genotype associated with this possible TCD risk value.

Knockout of programmed cell death 5 (PDCD5) gene attenuates neuron injury after middle cerebral artery occlusion in mice.

Loss of von Hippel-Lindau tumor suppressor protein (VHL) or hypoxia results in nuclear relocalization of PDCD5 and subsequent mouse double minute 2 homolog (Mdm2) degradation. Thus, VHL may involved in the PDCD5 mediated apoptosis and autophagy after MCAO. In the present study, using PDCD5 knockout (PDCD5-/-) mice, we aimed to demonstrate that knockout of PDCD5 gene could protect the brain from ischemic injury by inhibiting the PDCD5-VHL pathway. 24h post MCAO surgery, PDCD5 gene knockout mice presented obvious improved brain blood flow, improved neurological behavior and decreased cerebral infarction compared with wild type mice. The levels of apoptotic and autophagic proteins were increased both in wild type and PDCD5 knockout mice, whereas they were more pronounced in the wild type mice. We observed decrease in the expression of VHL in wild type mice after MCAO. Reduced expression of VHL may result in increased expression of hypoxia-inducible factor 1α(HIF-1α) and (BCL2/adenovirus E1B 19kDa protein-interacting protein 3) BNIP3. However, mice lacking PDCD5 had no changes in the expression of VHL and have slighter increases in the expression of HIF-1α and BNIP3, suggesting that PDCD5 may regulate apoptosis and autophagy through VHL-HIF-1α-BNIP3 pathway.

Therapeutically Targeting Tumor Necrosis Factor-α/Sphingosine-1-Phosphate Signaling Corrects Myogenic Reactivity in Subarachnoid Hemorrhage.

BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) is a complex stroke subtype characterized by an initial brain injury, followed by delayed cerebrovascular constriction and ischemia. Current therapeutic strategies nonselectively curtail exacerbated cerebrovascular constriction, which necessarily disrupts the essential and protective process of cerebral blood flow autoregulation. This study identifies a smooth muscle cell autocrine/paracrine signaling network that augments myogenic tone in a murine model of experimental SAH: it links tumor necrosis factor-α (TNFα), the cystic fibrosis transmembrane conductance regulator, and sphingosine-1-phosphate signaling. METHODS: Mouse olfactory cerebral resistance arteries were isolated, cannulated, and pressurized for in vitro vascular reactivity assessments. Cerebral blood flow was measured by speckle flowmetry and magnetic resonance imaging. Standard Western blot, immunohistochemical techniques, and neurobehavioral assessments were also used. RESULTS: We demonstrate that targeting TNFα and sphingosine-1-phosphate signaling in vivo has potential therapeutic application in SAH. Both interventions (1) eliminate the SAH-induced myogenic tone enhancement, but otherwise leave vascular reactivity intact; (2) ameliorate SAH-induced neuronal degeneration and apoptosis; and (3) improve neurobehavioral performance in mice with SAH. Furthermore, TNFα sequestration with etanercept normalizes cerebral perfusion in SAH. CONCLUSIONS: Vascular smooth muscle cell TNFα and sphingosine-1-phosphate signaling significantly enhance cerebral artery tone in SAH; anti-TNFα and anti-sphingosine-1-phosphate treatment may significantly improve clinical outcome.

CaV1.2/CaV3.x channels mediate divergent vasomotor responses in human cerebral arteries.

The regulation of arterial tone is critical in the spatial and temporal control of cerebral blood flow. Voltage-gated Ca(2+) (CaV) channels are key regulators of excitation-contraction coupling in arterial smooth muscle, and thereby of arterial tone. Although L- and T-type CaV channels have been identified in rodent smooth muscle, little is known about the expression and function of specific CaV subtypes in human arteries. Here, we determined which CaV subtypes are present in human cerebral arteries and defined their roles in determining arterial tone. Quantitative polymerase chain reaction and Western blot analysis, respectively, identified mRNA and protein for L- and T-type channels in smooth muscle of cerebral arteries harvested from patients undergoing resection surgery. Analogous to rodents, CaV1.2 (L-type) and CaV3.2 (T-type) α1 subunits were expressed in human cerebral arterial smooth muscle; intriguingly, the CaV3.1 (T-type) subtype present in rodents was replaced with a different T-type isoform, CaV3.3, in humans. Using established pharmacological and electrophysiological tools, we separated and characterized the unique profiles of Ca(2+) channel subtypes. Pressurized vessel myography identified a key role for CaV1.2 and CaV3.3 channels in mediating cerebral arterial constriction, with the former and latter predominating at higher and lower intraluminal pressures, respectively. In contrast, CaV3.2 antagonized arterial tone through downstream regulation of the large-conductance Ca(2+)-activated K(+) channel. Computational analysis indicated that each Ca(2+) channel subtype will uniquely contribute to the dynamic regulation of cerebral blood flow. In conclusion, this study documents the expression of three distinct Ca(2+) channel subtypes in human cerebral arteries and further shows how they act together to orchestrate arterial tone.

Localized TRPA1 channel Ca2+ signals stimulated by reactive oxygen species promote cerebral artery dilation.

Reactive oxygen species (ROS) can have divergent effects in cerebral and peripheral circulations. We found that Ca(2+)-permeable transient receptor potential ankyrin 1 (TRPA1) channels were present and colocalized with NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 2 (NOX2), a major source of ROS, in the endothelium of cerebral arteries but not in other vascular beds. We recorded and characterized ROS-triggered Ca(2+) signals representing Ca(2+) influx through single TRPA1 channels, which we called "TRPA1 sparklets." TRPA1 sparklet activity was low under basal conditions but was stimulated by NOX-generated ROS. Ca(2+) entry during a single TRPA1 sparklet was twice that of a TRPV4 sparklet and ~200 times that of an L-type Ca(2+) channel sparklet. TRPA1 sparklets representing the simultaneous opening of two TRPA1 channels were more common in endothelial cells than in human embryonic kidney (HEK) 293 cells expressing TRPA1. The NOX-induced TRPA1 sparklets activated intermediate-conductance, Ca(2+)-sensitive K(+) channels, resulting in smooth muscle hyperpolarization and vasodilation. NOX-induced activation of TRPA1 sparklets and vasodilation required generation of hydrogen peroxide and lipid-peroxidizing hydroxyl radicals as intermediates. 4-Hydroxy-nonenal, a metabolite of lipid peroxidation, also increased TRPA1 sparklet frequency and dilated cerebral arteries. These data suggest that in the cerebral circulation, lipid peroxidation metabolites generated by ROS activate Ca(2+) influx through TRPA1 channels in the endothelium of cerebral arteries to cause dilation.

Cerebral blood flow reactivity in patients undergoing selective amygdalohippocampectomy for epilepsy of mesial temporal origin. A prospective randomized comparison of the trans-Sylvian and the transcortical approach.

OBJECTIVE: The aim of this study was to assess (1) whether vasoreactivity is altered in patients with epilepsy and (2) whether the two most commonly used approaches, the trans-Sylvian (TS) and the trans-cortical (TC) route, differ in their impact on cortical blood flow. METHODS: Patients were randomized to undergo selective amygdalohippocampectomy (selAH) through a TC or TS route. Before and after selAH, we recorded microcirculation parameters on the superficial cortex surrounding the surgical corridor. Blood flow and velocity were measured using laser Doppler flowmetry and micro-Doppler, respectively. Cortical oxygen saturation (SO2) was measured using remission spectrophotometry under hypocapnic and normocapnic conditions. RESULTS: Ten patients were operated using the TS approach, and eight were operated via the TC approach. Vasomotor reactivity patterns measured with micro-Doppler were physiologically prior to selAH in both groups. After completion of surgery, a significant increase in SO2-values occurred in the TS group (before: 56.7 ± 2.2, after: 65.5 ± 3.0%SO2), but not in the TC group (before: 52.9 ± 5.2, after: 53.0 ± 3.7%SO2). The rate of critical SO2 values below 25% was significantly higher after the TC approach (12.3%) compared to the TS approach (5.2%; p < 0.05). DISCUSSION: Our findings provide the first invasively measured evidence that patients with mesial temporal lobe epilepsy have preserved cerebral blood flow responses to alterations in CO2. In addition, local cortical SO2 was higher in the TS group than in the TC group after selAH. This may be a sign of reactive cortical vessel dilation after proximal vessel manipulation associated with the TS approach. In contrast, the lower values of SO2 after the TC approach indicate tissue ischaemia surrounding the surgical corridor surrounding the corticotomy.

Knockdown of angiopoietin like-2 protects against angiotensin II-induced cerebral endothelial dysfunction in mice.

Angiopoietin like-2 (angptl2) is a circulating pro-inflammatory and pro-oxidative protein, but its role in regulating cerebral endothelial function remains unknown. We hypothesized that in mice knockdown (KD) of angptl2, cerebral endothelial function would be protected against ANG II-induced damage. Subcutaneous infusion of ANG II (200 ng·kg(-1)·min(-1), n = 15) or saline (n = 15) was performed in 20-wk-old angptl2 KD mice and wild-type (WT) littermates for 14 days. In saline-treated KD and WT mice, the amplitude and the sensitivity of ACh-induced dilations of isolated cerebral arteries were similar. However, while endothelial nitric oxide (NO) synthase (eNOS)-derived O2 (-)/H2O2 contributed to dilation in WT mice, eNOS-derived NO (P < 0.05) was involved in KD mice. ANG II induced cerebral endothelial dysfunction only in WT mice (P < 0.05), which was reversed (P < 0.05) by either N-acetyl-l-cysteine, apocynin, gp91ds-tat, or indomethacin, suggesting the contribution of reactive oxygen species from Nox2 and Cox-derived contractile factors. In KD mice treated with ANG II, endothelial function was preserved, likely via Nox-derived H2O2, sensitive to apocynin and PEG-catalase (P < 0.05), but not to gp91ds-tat. In the aorta, relaxation similarly and essentially depended on NO; endothelial function was maintained after ANG II infusion in all groups, but apocynin significantly reduced aortic relaxation in KD mice (P < 0.05). Protein expression levels of Nox1/2 in cerebral arteries were similar among all groups, but that of Nox4 was greater (P < 0.05) in saline-treated KD mice. In conclusion, knockdown of angptl2 may be protective against ANG II-induced cerebral endothelial dysfunction; it favors the production of NO, likely increasing endothelial cell resistance to stress, and permits the expression of an alternative vasodilatory Nox pathway.

Pituitary adenylate cyclase activating polypeptide (PACAP) dilates cerebellar arteries through activation of large-conductance Ca(2+)-activated (BK) and ATP-sensitive (K ATP) K (+) channels.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent vasodilator of numerous vascular beds, including cerebral arteries. Although PACAP-induced cerebral artery dilation is suggested to be cyclic AMP (cAMP)-dependent, the downstream intracellular signaling pathways are still not fully understood. In this study, we examined the role of smooth muscle K(+) channels and hypothesized that PACAP-mediated increases in cAMP levels and protein kinase A (PKA) activity result in the coordinate activation of ATP-sensitive K(+) (KATP) and large-conductance Ca(2+)-activated K(+) (BK) channels for cerebral artery dilation. Using patch-clamp electrophysiology, we observed that PACAP enhanced whole-cell KATP channel activity and transient BK channel currents in freshly isolated rat cerebellar artery myocytes. The increased frequency of transient BK currents following PACAP treatment is indicative of increased intracellular Ca(2+) release events termed Ca(2+) sparks. Consistent with the electrophysiology data, the PACAP-induced vasodilations of cannulated cerebellar artery preparations were attenuated by approximately 50 % in the presence of glibenclamide (a KATP channel blocker) or paxilline (a BK channel blocker). Further, in the presence of both blockers, PACAP failed to cause vasodilation. In conclusion, our results indicate that PACAP causes cerebellar artery dilation through two mechanisms: (1) KATP channel activation and (2) enhanced BK channel activity, likely through increased Ca(2+) spark frequency.

Protein kinase A-phosphorylated KV1 channels in PSD95 signaling complex contribute to the resting membrane potential and diameter of cerebral arteries.

RATIONALE: Postsynaptic density-95 (PSD95) is a scaffolding protein that associates with voltage-gated, Shaker-type K(+) (KV1) channels and promotes the expression of KV1 channels in vascular smooth muscle cells of the cerebral (cVSMCs) circulation. However, the physiological role of PSD95 in mediating molecular signaling in cVSMCs is unknown. OBJECTIVE: We explored whether a specific interaction between PSD95 and KV1 channels enables protein kinase A phosphorylation of KV1 channels in cVSMCs to promote vasodilation. METHODS AND RESULTS: Rat cerebral arteries were used for analyses. A membrane-permeable peptide (KV1-C peptide) corresponding to the postsynaptic density-95, discs large, zonula occludens-1 binding motif in the C terminus of KV1.2α was designed as a dominant-negative peptide to disrupt the association of KV1 channels with PSD95. Application of KV1-C peptide to cannulated, pressurized cerebral arteries rapidly induced vasoconstriction and depolarized cVSMCs. These events corresponded to reduced coimmunoprecipitation of the PSD95 and KV1 proteins without altering surface expression. Middle cerebral arterioles imaged in situ through cranial window also constricted rapidly in response to local application of KV1-C peptide. Patch-clamp recordings confirmed that KV1-C peptide attenuates KV1 channel blocker (5-(4-phenylalkoxypsoralen))-sensitive current in cVSMCs. Western blots using a phospho-protein kinase A substrate antibody revealed that cerebral arteries exposed to KV1-C peptide showed markedly less phosphorylation of KV1.2α subunits. Finally, phosphatase inhibitors blunted both KV1-C peptide-mediated and protein kinase A inhibitor peptide-mediated vasoconstriction. CONCLUSIONS: These findings provide initial evidence that protein kinase A phosphorylation of KV1 channels is enabled by a dynamic association with PSD95 in cerebral arteries and suggest that a disruption of such association may compromise cerebral vasodilation and blood flow.

Cerebral angiogenesis during development: who is conducting the orchestra?

Blood vessels provide the brain with the oxygen and the nutrients it requires to develop and function. Endothelial cells (ECs) are the principal cell type forming the vascular system and driving its development and remodeling. All vessels are lined by a single EC layer. Larger blood vessels are additionally enveloped by vascular smooth muscle cells (VSMCs) and pericytes, which increase their stability and regulate their perfusion and form the blood-brain barrier (BBB). The development of the vascular system occurs by two processes: (1) vasculogenesis, the de novo assembly of the first blood vessels, and (2) angiogenesis, the creation of new blood vessels from preexisting ones by sprouting from or by division of the original vessel. The walls of maturing vessels produce a basal lamina and recruit pericytes and vascular smooth muscle cells for structural support. Whereas the process of vasculogenesis seems to be genetically programmed, angiogenesis is induced mainly by hypoxia in development and disease. Both processes and the subsequent vessel maturation are further orchestrated by a complex interplay of inhibiting and stimulating growth factors and their respective receptors, many of which are hypoxia-inducible. This chapter intends to give an overview about the array of factors directing the development and maintenance of the brain vasculature and their interdependent actions.

Pericytes and adaptive angioplasticity: the role of tumor necrosis factor-like weak inducer of apoptosis (TWEAK).

The TNF superfamily member TWEAK has emerged as a pleiotropic cytokine that regulates many cellular functions that include immune/inflammatory activity, angiogenesis, cell proliferation, and fate. TWEAK through its inducible receptor, FGF-inducible molecule 14 (Fn14), can induce both beneficial and deleterious activity that has a profound effect on cell survival. Thus it is highly likely that TWEAK and Fn14 expressed by cells of the neurovascular unit help regulate and maintain vascular and tissue homeostasis. In this chapter we discuss the expression of TWEAK and Fn14 signaling in the cerebral microvascular pericyte. Pericytes are a highly enigmatic population of microvascular cells that are important in regulatory pathways that modulate physiological angiogenesis in response to chronic mild hypoxic stress. A brief introduction will identify the microvascular pericyte. A more detailed discussion of pericyte TWEAK signaling during adaptive angioplasticity will follow.

Effects of N-acetyl-L-cysteine and hyaluronic acid on HBOC-201-induced systemic and cerebral vasoconstriction in the rat.

Hemoglobin-based oxygen carrier-201 (HBOC) was developed as a resuscitative fluid but concerns exist over potentially adverse vasoconstriction. This study evaluated whether concurrent IV (intra venous) N-acetyl-L-cysteine (NAC) or hyaluronic acid (HA) would attenuate HBOC-associated vasoconstriction, assessed by systemic blood pressures and cerebral pial microvasculature, when administered to healthy, anesthetized rats. Rats (8-9/group) received a 30 min infusion of 3 ml/kg HBOC, HBOC plus 600 mg/kg NAC (HBOC/NAC), HBOC plus 1.5 mg/kg HA (HBOC/HA) or 3 ml/kg Albumin. Mean (MAP) and systolic (SBP) blood pressures, blood chemistries and cerebral pial vessel diameters were measured at baseline, end of infusion, and intermittently for an additional 90 min. HBOC caused immediate and sustained increases in SBP and MAP (35.3 ± 3.6 and 29.1 ± 2.5 mm Hg peak increases above baseline, respectively; mean ± SEM) and immediate but progressive vasoconstriction (11 µm maximum reduction) in medium-sized (50-100 µm) pial arterioles. When NAC was co-administered, blood pressure changes were attenuated and vessel changes were abolished. Similar trends were noted with co-administration of HA but were not statistically different from HBOC-alone. Small-sized (< 50 µm) pial vessels and blood parameters showed no differences from baseline or among groups. No adverse clinical signs were observed. We demonstrated that it is possible for adjuvant drugs to reduce the vasoconstriction associated with HBOC-201. Coinfusion of the anti-oxidant NAC mitigated HBOC-201-associated increases in blood pressures and vasoconstriction in medium-sized cerebral pial vessels. The drag-reducing polymer HA may be more effective at a higher dose as a similar but non-significant trend was observed.