Cerebral Vascular Smooth Muscle Cells Research Articles

The expression of human brain vascular smooth muscle cell AT receptor after the UL83 gene of HCMV inhibition by small interfering RNAs

Objectives: To explore the role of angiotensin receptors (AT1 and AT2) and human cytomegalovirus (HCMV) infection in arthrosclerosis, and to observe effect of HCMV UL83 gene on angiotensin receptor AT receptors.Methods: Cell model of RNA interference (RNAi) HCMV UL83 gene was set up by transfection RNAi into HCMV infected human cerebral artery vascular smooth muscle cells cultured in vitro by application of transfection technique and adoption of RT-PCR, etc. Effect of HCMV on gene expression of AT1 and AT2 receptors in human cerebral artery vascular smooth muscle cells cultured in vitro under the action of Ganciclovir (GCV), AT1 and AT2 receptor antagonists by immunofluorescence technique, RT-PCR and protein electrophoresis.Results: The duplication and protein expression of HCMV UL83 gene mRNA was successfully interfered using RNAi technique. After silencing HCMV UL83 gene, gene expression of AT1 receptor was down-regulated while that of AT2 was up-regulated. AT1 and AT2 receptor antagonists and GCV, under certain condition, could inhibit the activation on AT1 and AT2 receptor in human cerebral artery vascular smooth muscle cells induced by HCMV infection.Conclusion: Cell model of HCMV infected UL83 silence gene was successfully set up in human cerebral artery vascular smooth muscle cells cultured in vitro. HCMV UL83 might play a role by activating AT1 receptor and inhibiting expression of AT2 receptor.

Solubilized cystatin C amyloid is cytotoxic to cultured human cerebrovascular smooth muscle cells

Cerebral amyloid angiopathy (CAA) is characterized by the accumulation of amyloid within arteries of the cerebral cortex and leptomeninges. This condition is age related, especially prevalent in Alzheimer's disease (AD) and the main feature of certain hereditary disorders. The vascular smooth muscle cells (VSMC) appear to play a vital role in the development of CAA and have been found to produce the amyloid β precursor protein (AβPP) and process it to Aβ the major component of most CAA amyloid. Moreover, synthesized Aβ has proven to be toxic to cerebral VSMC in culture possibly explaining the disintegration and disappearance of the muscle cells from affected cerebral blood vessels seen in CAA. An aggressive and extremely rare form of CAA, known as Hereditary Cerebral Hemorrhage With Amyloidosis–Icelandic Type (HCHWA-I), exhibits this withdrawal of VSMC as amyloid accumulates in the vessel wall. However, the amyloid in HCHWA-I is made from a variant of cystatin C (L68Q) instead of the more common Aβ. To evaluate possible cytotoxicity in this condition solubilized cystatin C amyloid extracted from HCHWA-I leptomeninges was applied to cerebral smooth muscle cells in culture and was found to kill the cells.

The vasodilator 17,18‐epoxyeicosatetraenoic acid targets the pore‐forming BK α channel subunit in rodents

17,18-Epoxyeicosatetraenoic acid (17,18-EETeTr) stimulates vascular large-conductance K(+) (BK) channels. BK channels are composed of the pore-forming BK alpha and auxiliary BK beta1 subunits that confer an increased sensitivity for changes in membrane potential and calcium to BK channels. Ryanodine-sensitive calcium-release channels (RyR3) in the sarcoplasmic reticulum (SR) control the process. To elucidate the mechanism of BK channel activation, we performed whole-cell and perforated-patch clamp experiments in freshly isolated cerebral and mesenteric artery vascular smooth muscle cells (VSMC) from Sprague-Dawley rats, BK beta1 gene-deficient (-/-), BK alpha (-/-), RyR3 (-/-) and wild-type mice. The 17,18-EETeTr (100 nm) increased tetraethylammonium (1 mm)-sensitive outward K(+) currents in VSMC from wild-type rats and wild-type mice. The effects were not inhibited by the epoxyeicosatrienoic acid (EET) antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 mum). BK channel currents were increased 3.5-fold in VSMC from BK beta1 (-/-) mice, whereas a 2.9-fold stimulation was observed in VSMC from RyR3 (-/-) mice (at membrane voltage 60 mV). The effects were similar compared with those observed in cells from wild-type mice. The BK current increase was neither influenced by strong internal calcium buffering (Ca(2)(+), 100 nm), nor by external calcium influx. The 17,18-EETeTr did not induce outward currents in VSMC BK alpha (-/-) cells. We next tested the vasodilator effects of 17,18-EETeTr on isolated arteries of BK alpha-deficient mice. Vasodilatation was largely inhibited in cerebral and mesenteric arteries isolated from BK alpha (-/-) mice compared with that observed in wild-type and BK beta1 (-/-) arteries. We conclude that 17,18-EETeTr represents an endogenous BK channel agonist and vasodilator. Since 17,18-EETeTr is active in small arteries lacking BK beta1, the data further suggest that BK alpha represents the molecular target for the principal action of 17,18-EETeTr. Finally, the action of 17,18-EETeTr is not mediated by changes of the internal global calcium concentration or local SR calcium release events.

Oxidative stress triggers the amyloidogenic pathway in human vascular smooth muscle cells

Cerebral amyloid angiopathy, associated to most cases of Alzheimer's disease (AD), is characterized by the deposition of amyloid ß-peptide (Aß) in brain vessels, although the origin of the vascular amyloid deposits is still controversial: neuronal versus vascular. In the present work, we demonstrate that primary cultures of human cerebral vascular smooth muscle cells (HC-VSMCs) have all the secretases involved in amyloid ß-protein precursor (APP) cleavage and produce Aß 1–40 and Aß 1–42. Oxidative stress, a key factor in the etiology and pathophysiology of AD, up-regulates ß-site APP cleaving enzyme 1 (BACE1) expression, as well as Aß 1–40 and Aß 1–42 secretion in HC-VSMCs. This process is mediated by c-Jun N-terminal Kinase and p38 MAPK signaling and appears restricted to BACE1 regulation as no changes in the other secretases were observed. In conclusion, oxidative stress-mediated up-regulation of the amyloidogenic pathway in human cerebral vascular smooth muscle cells may contribute to the overall cerebrovascular amyloid angiopathy observed in AD patients.

Serum response factor and myocardin mediate arterial hypercontractility and cerebral blood flow dysregulation in Alzheimer's phenotype

Cerebral angiopathy contributes to cognitive decline and dementia in Alzheimer's disease (AD) through cerebral blood flow (CBF) reductions and dysregulation. We report vascular smooth muscle cells (VSMC) in small pial and intracerebral arteries, which are critical for CBF regulation, express in AD high levels of serum response factor (SRF) and myocardin (MYOCD), two interacting transcription factors that orchestrate a VSMC-differentiated phenotype. Consistent with this finding, AD VSMC overexpressed several SRF-MYOCD-regulated contractile proteins and exhibited a hypercontractile phenotype. MYOCD overexpression in control human cerebral VSMC induced an AD-like hypercontractile phenotype and diminished both endothelial-dependent and -independent relaxation in the mouse aorta ex vivo. In contrast, silencing SRF normalized contractile protein content and reversed a hypercontractile phenotype in AD VSMC. MYOCD in vivo gene transfer to mouse pial arteries increased contractile protein content and diminished CBF responses produced by brain activation in wild-type mice and in two AD models, the Dutch/Iowa/Swedish triple mutant human amyloid beta-peptide (Abeta)-precursor protein (APP)- expressing mice and APPsw(+/-) mice. Silencing Srf had the opposite effect. Expression of SRF did not change in VSMC subjected to Alzheimer's neurotoxin, Abeta. Thus, SRF-MYOCD overexpression in small cerebral arteries appears to initiate independently of Abeta a pathogenic pathway mediating arterial hypercontractility and CBF dysregulation, which are associated with Alzheimer's dementia.

Evidence for modulation of Shaker ‐type (K v 1) channels in rat small cerebral arteries by ancillary K v β 2 subunits

Voltage-gated K+ (Kv) channels in vascular smooth muscle cells (VSMCs) mediate vasodilation, and their loss is linked to abnormal vascular reactivity. We recently demonstrated that Kv channels in rat small cerebral arteries represent pore-forming Kv1.2/1.5α tetramers, which associate with ancillary Kvβ2 subunits. Based on a chaperoning role for Kvβ2 in nonvascular tissues, we hypothesized that Kvβ2 is required for the normal expression of Kv channels in cerebral VSMCs. Cultured rat cerebral VSMCs were electroporated with 10μg of Kvβ2-specific siRNA for 48 hours. Kvβ2 was knocked down in VSMCs treated with siRNA compared to scrambled sequence. This event was associated with a concomitant decrease in the mature glycosylated Kv1.2α subunits that compose the Kv channel tetramers; an increase in immature Kv1.2α was detected. In further studies, we evaluated if the reduced expression of Kv channels in abnormally reactive cerebral arteries from hypertensive rats is associated with a loss of Kvβ2 subunits. Indeed, cerebral arteries from two different rat models of hypertension showed abnormally low levels of Kvβ2. Collectively, these results suggest that Kvβ2 subunits promote anterograde trafficking and expression of Kv channels in cerebral VSMCs, and that a loss of Kvβ2 is associated with reduced Kv channel expression and cerebral vasoconstriction during hypertensive disease. (Supported by HL-59238-08).

Evidence for two-pore domain potassium channels in rat cerebral arteries

Little is known about the presence and function of two-pore domain K(+) (K(2P)) channels in vascular smooth muscle cells (VSMCs). Five members of the K(2P) channel family are known to be directly activated by arachidonic acid (AA). The purpose of this study was to determine 1) whether AA-sensitive K(2P) channels are expressed in cerebral VSMCs and 2) whether AA dilates the rat middle cerebral artery (MCA) by increasing K+ currents in VSMCs via an atypical K+ channel. RT-PCR revealed message for the following AA-sensitive K(2P) channels in rat MCA: tandem of P domains in weak inward rectifier K+ (TWIK-2), TWIK-related K+ (TREK-1 and TREK-2), TWIK-related AA-stimulated K+ (TRAAK), and TWIK-related halothane-inhibited K+ (THIK-1) channels. However, in isolated VSMCs, only message for TWIK-2 was found. Western blotting showed that TWIK-2 is present in MCA, and immunohistochemistry further demonstrated its presence in VSMCs. AA (10-100 microM) dilated MCAs through an endothelium-independent mechanism. AA-induced dilation was not affected by inhibition of cyclooxygenase, epoxygenase, or lipoxygenase or inhibition of classical K+ channels with 10 mM TEA, 3 mM 4-aminopyridine, 10 microM glibenclamide, or 100 microM Ba2+. AA-induced dilations were blocked by 50 mM K+, indicating involvement of a K+ channel. AA (10 microM) increased whole cell K+ currents in dispersed cerebral VSMCs. AA-induced currents were not affected by inhibitors of the AA metabolic pathways or blockade of classical K+ channels. We conclude that AA dilates the rat MCA and increases K+ currents in VSMCs via an atypical K+ channel that is likely a member of the K(2P) channel family.

Arachidonic acid (AA)‐sensitive tandem pore domain K (K2P) channels in rat cerebral arteries

K2P, a new family of K channels, has over a dozen members that are expressed in various tissues throughout the body. Very little is known about the presence and function of these channels in vascular smooth muscle cells (VSMC). Five members of the K2P family are directly activated by AA. The purpose of this study was to determine (a) if AA dilates the rat middle cerebral artery (MCA) and increases K+ currents in VSMC via a non-conventional K channel, and (b) if AA-sensitive K2P are expressed in cerebral VSMC. AA (10−5 M) dilated isolated and pressurized MCAs through a mechanism independent of endothelium. The dilation to AA was not affected by inhibiting AA metabolic pathways (cyclooxygenase, epoxygenase, or lipoxygenase). The dilation elicited by AA was not affected following inhibition of KCa, Kv, KATP, or Kir using 10 mM TEA, 3 mM 4AP, 10 uM gilbenclamide, or 100 uM Ba respectively. However, the dilations were blocked by 50 mM K indicating involvement of a K channel. AA (10−5 M) increased whole cell K+ currents in dispersed cerebral VSMC. The AA-induced currents were not affected by inhibitors of the AA metabolic pathways or blockade of conventional K channels (TEA, 4AP, glibenclamide, or Ba). RT-PCR in MCA revealed message for the AA-sensitive K2Ps, TREK-1, −2, TRAAK, THIK-1, and TWIK-2. Western blot and immunohistochemistry showed the presence of TWIK-2 and TRAAK in VSMC. We conclude that AA dilates rat middle cerebral arteries through activation of an AA-sensitive K2P channel. (NIH NS46666 & AHA 027011N)

K IR channels mediate parenchymal arteriole dilation to extracellular potassium

Astrocytes, in response to increases in neuron activity, release vasoactive substance(s) from perivascular endfeet to dilate cortical parenchymal arterioles (PAs) and drive local changes in cerebral blood flow. Recent evidence indicates astrocytic endfeet exhibit preferential expression of Ca2+-sensitive K+ (BKCa) channels, and we have measured significant BKCa currents from endfeet. However, potential PA smooth muscle targets for localized release of K+ from endfeet BKCa channels remain unclear. Elevation of [K+]o could dilate PAs by activating Na+/K+ATPases and/or inward rectifier K+ (KIR) channels to cause membrane hyperpolarization and a reduction in cytosolic Ca2+ concentration ([Ca2+]c). To investigate this, PAs were dissected from rat cerebral cortex and vascular smooth muscle cells were isolated for patch clamp studies. KIR currents were observed, as well as BKCa and KV currents. Ba2+–sensitive (100 μM) KIR current density at −100 mV was more pronounced for PA versus pial arteries (> 5 pA/pF difference at 60 mM [K+]o), with some PA cells exhibiting large KIR currents. Furthermore, we investigated the effects of [K+]o on pressurized (40 mm Hg) PA diameter, Em and [Ca2+]c. Increasing [K+]o from 3 to 8 mM hyperpolarized the smooth muscle, reduced [Ca2+]c and elicited a 38% dilation that could be blocked by exposure to 30 μM Ba2+, but not by the selective Na+/K+ATPase inhibitor dihydro-ouabain (30 μM). These data indicate KIR are responsible for [K+]o–induced dilations in PAs and suggest a key role for smooth muscle KIR channels in neurovascular coupling by mediating arteriolar dilations in response to neuron activation. Supported by NIH HL44455.

The Effect of Superoxide Anion on Autoregulation of Cerebral Blood Flow

Recent studies have suggested that autoregulation of cerebral blood flow (CBF) is impaired after traumatic and ischemic brain injury. Given that the levels of superoxide anion (O2*-) are increased in these conditions, we postulate that O2*- contributes to the impairment of CBF autoregulation. CBF was monitored with laser Doppler flowmetry during increases in blood pressure. During the control period, CBF was well autoregulated after the increase in mean arterial pressure (MAP) from 98+/-3 to 140+/-6 mm Hg. The autoregulation index (AI; DeltaCBF/DeltaMAP) averaged 0.25+/-0.02 (n=6). O2*- in the brain was then increased by subdural perfusion of xanthine/xanthine oxidase (different concentrations) and catalase. Low concentrations of O2*- decreased basal CBF by 10+/-1.6% but had no effect on autoregulation (AI, 0.19+/-0.02; n=6). Higher concentrations of O2*- (0.2 mmol/L xanthine and either 3 or 20 mU xanthine oxidase) increased basal CBF by 30+/-2% and 42+/-4%, respectively, and impaired autoregulation of CBF (AI, 0.55+/-0.03 and 0.76+/-0.02; n=6). Inclusion of superoxide dismutase in the O2*(-)-generating system restored autoregulation (AI, 0.28+/-0.05; n=6). Neither inhibition of NO synthase nor the addition of deferioxamine had any effect on the ability of higher concentrations of O2*- to impair autoregulation of CBF (AI, 0.65+/-0.07 and 0.72+/-0.05 respectively; n=6). O2*- also increased the activity of KCa channels in cerebral vascular smooth muscle cells (VSMCs; n=8). These results suggest that O2*- increases basal CBF and impairs autoregulation of CBF, likely through the activation of KCa channels in cerebral VSMCs.

Role for the α7β1 integrin in vascular development and integrity

The alpha7beta1 integrin is a laminin receptor that has been implicated in muscle disease and the development of neuromuscular and myotendinous junctions. Studies have shown the alpha7beta1 integrin is also expressed in nonskeletal muscle tissues. To identify the expression pattern of the alpha7 integrin in these tissues during embryonic development, alpha7 integrin chain knockout mice were generated by a LacZ knockin strategy. In these mice, expression from the alpha7 promoter is reported by beta-galactosidase. From embryonic day (ED) 11.5 to ED14.5, beta-galactosidase was detected in the developing central and peripheral nervous systems and vasculature. The loss of the alpha7 integrin gene resulted in partial embryonic lethality. Several alpha7 null embryos were identified with cerebrovascular hemorrhages and showed reduced vascular smooth muscle cells and cerebral vascularization. The alpha7 null mice that survived to birth exhibited vascular smooth muscle defects, including hyperplasia and hypertrophy. In addition, altered expression of alpha5 and alpha6B integrin chains was detected in the cerebral arteries of alpha7 null mice, which may contribute to the vascular phenotype. Our results demonstrate for the first time that the alpha7beta1 integrin is important for the recruitment or survival of cerebral vascular smooth muscle cells and that this integrin plays an important role in vascular development and integrity.

Functional alterations in cerebrovascular K+ and Ca2+ channels are comparable between simulated microgravity rat and SHR

Exposure to microgravity leads to a sustained elevation in transmural pressure across the cerebral vasculature due to removal of hydrostatic pressure gradients. We hypothesized that ion channel remodeling in cerebral vascular smooth muscle cells (VSMCs) similar to that associated with hypertension may occur and play a role in upward autoregulation of cerebral vessels during microgravity. Sprague-Dawley rats were subjected to 4-wk tail suspension (Sus) to simulate the cardiovascular effect of microgravity. Large-conductance Ca(2+)-activated K(+) (BK(Ca)), voltage-gated K(+) (K(V)), and L-type voltage-dependent Ca(2+) (Ca(L)) currents of Sus and control (Con) rat cerebral VSMCs were investigated with a whole cell voltage-clamp technique. Under the same experimental conditions, K(V), BK(Ca), and Ca(L) currents of cerebral VSMCs from adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were also investigated. K(V) current density decreased in Sus rats vs. Con rats [1.07 +/- 0.14 (n = 22) vs. 1.31 +/- 0.28 (n = 16) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 1.70 +/- 0.37 (n = 23) vs. 0.88 +/- 0.22 (n = 19) pA/pF at +20 mV (P < 0.05); Ca(L): -2.17 +/- 0.21 (n = 35) vs. -1.31 +/- 0.10 (n = 26) pA/pF at +10 mV (P < 0.05)]. Similar changes were also observed in SHR vs. WKY cerebral VSMCs: K(V) current density decreased [1.03 +/- 0.33 (n = 9) vs. 1.62 +/- 0.64 (n = 9) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 2.54 +/- 0.47 (n = 11) vs. 1.12 +/- 0.33 (n = 12) pA/pF at +20 mV (P < 0.05); Ca(L): -3.99 +/- 0.53 (n = 12) vs. -2.28 +/- 0.20 (n = 10) pA/pF at +20 mV (P < 0.05)]. These findings support our hypothesis, and their impact on space cardiovascular research is discussed.

U-46619 but not serotonin increases endocannabinoid content in middle cerebral artery: evidence for functional relevance

Cerebral vascular smooth muscle cells express the CB(1) cannabinoid receptor, and CB(1) receptor agonists produce vasodilation of cerebral arteries. The purpose of this study was to determine whether vasoconstriction of rat middle cerebral artery (MCA) results in the local formation of endocannabinoids (eCBs), which, via activation of CB(1) receptors, oppose the vasoconstriction in a feedback manner. The thromboxane A(2) (TXA(2)) mimetic U-46619 significantly increased N-arachidonylethanolamine (AEA) and 2-arachidonylglycerol (2-AG) content of isolated MCA, whereas 5-hydroxytrypamine (5-HT) decreased AEA and 2-AG content. If eCBs play a feedback role in the regulation of MCA tone, then CB(1) receptor antagonists should enhance the constriction of MCA produced by U-46619 but not 5-HT. U-46619 caused concentration-dependent constrictions of endothelium-denuded MCA. Two CB(1) receptor antagonists SR-141716 and AM-251 decreased the EC(50) value for U-46619 to constrict endothelium-denuded MCA without affecting the maximal effect. A low concentration of CB(1) receptor agonist Win-55212-2 (30 nM) produced vasodilation of MCAs constricted with low but not saturating concentrations of U-46619. SR-141716 had no effect on the 5-HT concentration-contraction relationship. These data suggest that TXA(2) receptor activation increases MCA eCB content, which, via activation of CB(1) receptors, reduces the constriction produced by moderate concentrations of the TXA(2) agonist. Although 5-HT-induced vasoconstriction is reduced by exogenous CB(1) receptor agonist, activation of 5-HT receptors does not increase eCB content. These results suggest that MCA production of eCBs is not regulated by constriction per se but likely via a signaling pathway that is specific for TXA(2) receptors and not 5-HT receptors.

EP1- and EP3-Receptors Mediate Prostaglandin E2–Induced Constriction of Porcine Large Cerebral Arteries

Prostaglandin E2 (PGE2) has been shown to dilate and constrict the systemic vascular beds, including cerebral vessels. The exact mechanism of PGE2-induced cerebral vasoconstriction, however, is less clarified. The authors' preliminary studies showed that PGE2 exclusively constricted the adult porcine basilar arteries. The present study, therefore, was designed to examine the receptor mechanisms involved in PGE2-induced constriction of large cerebral arteries in the adult pig. Results from an in vitro tissue-bath study indicated that PGE2 and its agonists 17-phenyl trinor PGE2 (17-PGE2), sulprostone (EP1/EP3 receptor agonists), and 11-deoxy-16,16-dimethyl PGE2 (11-PGE2, an EP2/EP3-receptor agonist) induced exclusive constriction, which was not affected by endothelium denudation or cold-storage denervation of perivascular nerves. The constriction induced by PGE2, 17-PGE2, and sulprostone, but not by potassium chloride, was blocked by SC-19220 (a selective EP1-receptor antagonist), AH-6809 (an EP1/EP2-receptor antagonist), and U-73122 and neomycin (phospholipase C inhibitors). AH-6809, however, did not affect 11-PGE2-induced contraction. These results suggest that the contraction was not mediated by the EP2-receptor, but was mediated by EP1- and EP3-receptors. Furthermore, EP1-receptor immunoreactivities were found across the entire medial smooth muscle layers, whereas EP3-receptor immunoreactivities were limited to the outer smooth muscle layer toward the adventitia. Western blotting also showed the presence of EP1- and EP3-receptor proteins in cultured primary cerebral vascular smooth muscle cells. In conclusion, PGE2 exclusively constricts the adult porcine large cerebral arteries. This constriction is mediated by phosphatidyl-inositol pathway via activation of EP1- and EP3-receptors located on the smooth muscle cells. These two receptor subtypes may play important roles in physiologic and pathophysiologic control of cerebral vascular tone.

Degenerin/epithelial Na+ channel proteins: components of a vascular mechanosensor.

Mechanosensitive ion channels are thought to mediate stretch-induced contraction in vascular smooth muscle cells (VSMCs); however, the molecular identity of the mechanosensitive ion channel complex is unknown. Although recent reports suggest degenerin/epithelial Na+ channel (DEG/ENaC) proteins may be mechanosensors in sensory neurons, their role as mechanosensors in vascular tissue has not been examined. We first tested whether DEG/ENaC subunits are expressed in cerebral blood vessels and VSMCs and then examined their role as mechanosensors in mediating the myogenic response in intact blood vessels. Using RT-PCR, we found ENaC transcripts expressed in rat cerebral arteries and freshly dissociated rat cerebral VSMCs. We also detected ENaC expression in isolated blood vessels and VSMCs by immunoblotting and immunolocalization. Moreover, inhibition of ENaC with amiloride (1 micromol/L) and benzamil (30 nmol/L, 1 micromol), an amiloride analog, blocked myogenic constriction in isolated rat cerebral arteries. These data suggest that DEG/ENaC proteins are required for vessel responses to pressure and are consistent with the evolutionary conservation of mechanosensory function of DEG/ENaC proteins.

CAMP modulates cGMP-mediated cerebral arteriolar relaxation in vivo.

No studies have specifically addressed whether cAMP can influence nitric oxide (NO)/cGMP-induced cerebral vasodilation. In this study, we examined whether cAMP can enhance or reduce NO-induced cerebral vasodilation in vivo via interfering with cGMP efflux or through potentiating phosphodiesterase 5 (PDE5)-mediated cGMP breakdown, respectively, in cerebral vascular smooth muscle cells (CVSMCs). To that end, we evaluated, in male rats, the effects of knockdown [via antisense oligodeoxynucleotide (ODN) applications] of the cGMP efflux protein multidrug resistance protein 5 (MRP5) and PDE5 inhibition on pial arteriolar NO donor [S-nitroso-N-acetyl penicillamine (SNAP)]-induced dilations in the absence and presence of cAMP elevations via forskolin. Pial arteriolar diameter changes were measured using well-established protocols in anesthetized rats. In control (missense ODN treated) rats, forskolin elicited a leftward shift in the SNAP dose-response curves (approximately 50% reduction in SNAP EC50). However, in MRP5 knockdown rats, cAMP increases were associated with a substantial reduction in SNAP-induced vasodilations (reflected as a significant 35-50% lower maximal response). In the presence of the PDE5 inhibitor MY-5445, the repression of the NO donor response accompanying forskolin was prevented. These findings suggest that cAMP has opposing effects on NO-stimulated cGMP increases. On the one hand, cAMP limits CVSMC cGMP loss by restricting cGMP efflux. On the other, cAMP appears to enhance PDE5-mediated cGMP breakdown. However, because increased endogenous cAMP seems to potentiate NO/cGMP-induced arteriolar relaxation when MRP5 expression is normal, the effect of cAMP to reduce cGMP efflux appears to predominate over cAMP stimulation of cGMP hydrolysis.

Differential activation of potassium channels in cerebral and hindquarter arteries of rats during simulated microgravity.

The purpose of this study was to test the hypothesis that differential autoregulation of cerebral and hindquarter arteries during simulated microgravity is mediated or modulated by differential activation of K(+) channels in vascular smooth muscle cells (VSMCs) of arteries in different anatomic regions. Sprague-Dawley rats were subjected to 1- and 4-wk tail suspension to simulate the cardiovascular deconditioning effect due to short- and medium-term microgravity. K(+) channel function of VSMCs was studied by pharmacological methods and patch-clamp techniques. Large-conductance Ca(2+)-activated K(+) (BK(Ca)) and voltage-gated K(+) (K(v)) currents were determined by subtracting the current recorded after applications of 1 mM tetraethylammonium (TEA) and 1 mM TEA + 3 mM 4-aminopyridine (4-AP), respectively, from that of before. For cerebral vessels, the normalized contractility of basilar arterial rings to TEA, a BK(Ca) blocker, and 4-AP, a K(v) blocker, was significantly decreased after 1- and 4-wk simulated microgravity, respectively. VSMCs isolated from the middle cerebral artery branches of suspended rats had a more depolarized membrane potential (E(m)) and a smaller K(+) current density compared with those of control rats. Furthermore, the reduced total current density was due to smaller BK(Ca) and smaller K(v) current density in cerebral VSMCs after 1- and 4-wk tail suspension, respectively. For hindquarter vessels, VSMCs isolated from second- to sixth-order small mesenteric arteries of both 1- and 4-wk suspended rats had a more negative E(m) and larger K(+) current densities for total, BK(Ca), and K(v) currents. These results indicate that differential activation of K(+) channels occur in cerebral and hindquarter VSMCs during short- and medium-term simulated microgravity. It is further suggested that different profiles of channel remodeling might occur in VSMCs as one of the important underlying cellular mechanisms to mediate and modulate differential vascular adaptation during microgravity.

Alcohol-induced apoptosis of canine cerebral vascular smooth muscle cells: role of extracellular and intracellular calcium ions

Exposure of canine cerebral vascular smooth muscle cells (VSMCs) to ethanol (10, 25 and 100 mM) for 1, 3 and 5 days induced apoptosis with its typical characteristics of nuclear shrinkage, condensation, and DNA breakage as well as formation of apoptotic bodies observed by fluorescence staining, terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling and comet assays. Such effects of alcohol on cerebral VSMCs were time- and concentration-dependent. The threshold ethanol concentration for induction of the apoptotic process was found to be 10 mM. Extracellular and intracellular Ca 2+ chelators, i.e. ethylglycol-bisβ-aminoethylether- N, N, N′ N′-tetraacetic acid (EGTA, 5 mM) and 1,2-bis(2-aminophenoxy)-ethane- N, N, N′, N′-tetra-acetic acid AM (BAPTA, 10 −6 M), respectively, ameliorated greatly the number of cerebral VSMCs which underwent apoptosis. Verapamil, however, failed to inhibit apoptosis of cerebral VSMCs. From these new findings, we suggest that alcohol-induced apoptosis may contribute to alcohol-induced brain-vascular damage and stroke. In addition, our findings point to potential caution for humans who imbibe two or more standard drinks per day or who undergo ‘binge drinking’.

Cocaine induces apoptosis in cerebral vascular muscle cells: potential roles in strokes and brain damage

Cocaine abuse is known to induce different types of brain-microvascular damage and many adverse cerebrovascular effects, including cerebral vasculitis, intracranial hemorrhage, cerebral infarction and stroke. A major physiological event leading to these pathophysiological actions of cocaine could be apoptosis. Whether cocaine can cause brain-microvascular pathology and vascular toxicity by inducing apoptosis of cerebral vascular smooth muscle cells is not known. This study, using several different methods to discern apoptosis, was designed to investigate if primary cultured canine cerebral vascular smooth muscle cells can undergo apoptosis when treated with cocaine. After treatment with cocaine (10 −6–10 −3 M) for 12–24 h, the death rates of cerebral vascular smooth muscle cells increased in a concentration-dependent manner compared with controls. Morphological analysis of cerebral vascular smooth muscle cells using confocal fluoresence microscopy showed that the percentage of apoptotic cerebral vascular smooth muscle cells increased after cocaine (10 −6–10 −3 M) treatment in a concentration-dependent manner. TUNEL assays also showed positive results for cerebral vascular smooth muscle cells treated with cocaine. These results clearly demonstrate that cerebral vascular smooth muscle cells can undergo rapid apoptosis in response to cocaine in a concentration-dependent manner. Cocaine-induced apoptosis may thus play a major role in brain-microvascular damage, cerebral vascular toxicity and strokes.

Hydrogen peroxide induces apoptosis in cerebral vascular smooth muscle cells: possible relation to neurodegenerative diseases and strokes

Recently, reactive oxygen species (ROS) have been suggested as important mediators of brain damage in a number of disease states, including traumatic brain injury, neurodegenerative diseases and strokes. Apoptosis has been suggested to play an important role in neurodegenerative diseases, traumatic brain injury and strokes. The aim of this study was to determine whether or not cerebral vascular smooth muscle cells (CVSMCs) undergo apoptosis following treatment with hydrogen peroxide (H 2O 2). Herein, we demonstrate, for the first time, that H 2O 2 can induce apoptosis in a concentration-dependent manner in primary cultured CVSMCs, as measured by several morphological and biochemical criteria. H 2O 2-induced apoptosis may be initiated by stimulating Ca 2+-dependent endonuclease activity. The present new data suggest that apoptosis in cerebral VSMCs, induced by ROS, such as H 2O 2, could play important roles in neruodegenerative processes, traumatic brain injury and strokes.