Activation of Rho-associated kinase during augmented contraction of the basilar artery to serotonin after subarachnoid hemorrhage

Delayed cerebral vasospasm after subarachnoid hemorrhage (SAH) may be due, in part, to altered regulation of arterial smooth muscle contraction. Contraction of cerebral arteries to serotonin is augmented after experimental SAH. We hypothesized that activation of Rho-associated kinase (Rho kinase) contributes to augmented contraction of cerebral arteries to serotonin after SAH. Autologous arterial blood (SAH) or artificial cerebrospinal fluid (control) was injected into the cisterna magna of anesthetized rabbits. At 2 days after injection, the basilar artery was excised and isometric contraction of arterial rings was recorded. Maximum contraction of the basilar artery to serotonin was augmented about fourfold in SAH compared with control rabbits (P < 0.01). Contraction to histamine was similar in the two groups. Fasudil hydrochloride (3 mumol/l), an inhibitor of Rho kinase, markedly attenuated serotonin-induced contraction. Fasudil had little effect on contractions induced by histamine or phorbol 12,13-dibutyrate. In addition, phosphorylation of myosin phosphatase, a major target of Rho kinase in regulation of smooth muscle contraction, in the basilar artery was examined by Western blotting. In basilar arteries of SAH, but not control, rabbits, serotonin increased phosphorylation of myosin phosphatase about twofold at Thr(853) of the myosin-targeting subunit. These results suggest that enhanced activation of Rho kinase contributes to augmented contraction of the basilar artery to serotonin after SAH.     

Biomechanical and phenotypic changes in the vasospastic canine basilar artery after subarachnoid hemorrhage.

Because it has been argued that active myogenic tone prolongs cerebral vasospasm for >2 wk after subarachnoid hemorrhage (SAH), we attempted to identify the mechanism that plays the main role in sustaining the prolonged cerebral vasospasm. We especially focused on the roles of biomechanical and phenotypic changes in the cerebral arteries in the mechanisms of prolonged vasospasm after SAH. We used the basilar arteries from a "two-hemorrhage" canine model to make serial measurements of maximal contraction capacity and arterial stiffness (papaverine-insensitive tone) until day 28. We also examined hematoxylin-eosin-stained vasospastic canine basilar arteries for histological changes and immunohistochemically examined them for expression of myosin heavy chain isoforms (SMemb, SM1, and SM2), which are markers of smooth muscle phenotypic changes. Changes in collagen concentration in canine basilar arteries were also measured. Angiographic cerebral vasospasm persisted until day 14 and then gradually diminished; artery diameter returned to the control diameters on day 28. Maximal contraction capacity decreased until day 21 and showed some recovery by day 28. Arterial stiffness, on the other hand, progressed until day 28. Histological examination revealed medial thickening and increased connective tissue until day 21 and a return to control findings by day 28. The increased connective tissue was not accompanied by changes in collagen concentration, suggesting a role of some other protein in the increase in connective tissue. Immunohistochemical studies with anti-SMemb, anti-SM1, and anti-SM2 antibodies showed enhanced expression of SMemb from day 7 to day 21 and disappearance of SM1 and SM2 on days 14 and 21. The changes in myosin heavy chain isoform expression returned to normal on day 28. The above results indicate that biomechanical and phenotypic changes may play a pivotal role in sustaining cerebral vasospasm for >2 wk after SAH, with minimal changes in active myogenic arterial tone.     

The presence of an extractable substance in the CSF of humans with cerebral vasospasm after subarachnoid haemorrhage that correlates with phosphatase inhibition

The cellular events leading to cerebral vasospasm after subarachnoid haemorrhage are poorly understood, although an increase in smooth muscle myosin light chain phosphorylation has been observed. This study set out to determine if phosphatase inhibition may be involved in the pathological maintenance of tension observed during vasospasm. We found that 1 nM okadaic acid, a type 2A protein phosphatase inhibitor, elicited an increase in rate of O(2) consumption in the porcine carotid artery similar to that by cerebrospinal fluid (CSF) from vasospastic patients (CSF(V), n=5) (control 0.23+/-0.03, CSF(V) 0.84+/-0.16 and okadaic acid 0.85+/-0.02 micromol min(-1) g dwt(-1)). It was also observed that phosphatase inhibition with 1 nM okadaic acid significantly slowed relaxation after a stretch in a similar fashion to CSF(V) haemorrhage. CSF from vasospastic subarachnoid haemorrhage patients, but not from those without vasospasm, contains an extractable substance which modulates myosin light chain phosphorylation in vitro. A phosphatase preparation obtained from the porcine carotid artery dephosphorylated 63+/-2% of the phosphorylated (MLC(20)) substrate in vitro, and non-vasospastic CSF treated enzyme dephosphorylated 60+/-2.6%. Okadaic acid inhibited phosphatase dephosphorylated only 7.5+/-1% of the substrate where CSF(V) treated enzyme dephosphorylated 22+/-2.8% of the substrate. We conclude that inhibition of smooth muscle phosphatase may be involved in the mechanisms associated with cerebral vasospasm after subarachnoid haemorrhage.     

Calcium sensitivity of vasospastic basilar artery after experimental subarachnoid hemorrhage

Arteries that develop vasospasm after subarachnoid hemorrhage (SAH) may have altered contractility and compliance. Whether these changes are due to alterations in the smooth muscle cells or the arterial wall extracellular matrix is unknown. This study elucidated the location of such changes and determined the calcium sensitivity of vasospastic arteries. Dogs were placed under general anesthesia and underwent creation of SAH using the double-hemorrhage model. Vasospasm was assessed by angiography performed before and 4, 7, or 21 days after SAH. Basilar arteries were excised from SAH or control dogs (n = 8-52 arterial rings from 2-9 dogs per measurement) and studied under isometric tension in vitro before and after permeabilization of smooth muscle with alpha-toxin. Endothelium was removed from all arteries. Vasospastic arteries demonstrated significantly reduced contractility to KCl with a shift in the EC(50) toward reduced sensitivity to KCl 4 and 7 days after SAH (P < 0.05, ANOVA). There was reduced compliance that persisted after permeabilization (P < 0.05, ANOVA). Calcium sensitivity was decreased during vasospasm 4 and 7 days after SAH, as assessed in permeabilized arteries and in those contracted with BAY K 8644 in the presence of different concentrations of extracellular calcium (P < 0.05, ANOVA). Depolymerization of actin with cytochalasin D abolished contractions to KCl but failed to alter arterial compliance. In conclusion, it is shown for the first time that calcium sensitivity is decreased during vasospasm after SAH in dogs, suggesting that other mechanisms are involved in maintaining the contraction. Reduced compliance seems to be due to an alteration in the arterial wall extracellullar matrix rather than the smooth muscle cells themselves because it cannot be alleviated by depolymerization of smooth muscle actin.     

Erythropoietin for the Treatment of Subarachnoid Hemorrhage: A Feasible Ingredient for a Successful Medical Recipe

Subarachnoid hemorrhage (SAH) following aneurysm bleeding accounts for 6% to 8% of all cerebrovascular accidents. Although an aneurysm can be effectively managed by surgery or endovascular therapy, delayed cerebral ischemia is diagnosed in a high percentage of patients resulting in significant morbidity and mortality. Cerebral vasospasm occurs in more than half of all patients after aneurysm rupture and is recognized as the leading cause of delayed cerebral ischemia after SAH. Hemodynamic strategies and endovascular procedures may be considered for the treatment of cerebral vasospasm. In recent years, the mechanisms contributing to the development of vasospasm, abnormal reactivity of cerebral arteries and cerebral ischemia following SAH, have been investigated intensively. A number of pathological processes have been identified in the pathogenesis of vasospasm, including endothelial injury, smooth muscle cell contraction from spasmogenic substances produced by the subarachnoid blood clots, changes in vascular responsiveness and inflammatory response of the vascular endothelium. To date, the current therapeutic interventions remain ineffective as they are limited to the manipulation of systemic blood pressure, variation of blood volume and viscosity and control of arterial carbon dioxide tension. In this scenario, the hormone erythropoietin (EPO) has been found to exert neuroprotective action during experimental SAH when its recombinant form (rHuEPO) is administered systemically. However, recent translation of experimental data into clinical trials has suggested an unclear role of recombinant human EPO in the setting of SAH. In this context, the aim of the current review is to present current evidence on the potential role of EPO in cerebrovascular dysfunction following aneurysmal subarachnoid hemorrhage.     

Endothelin-1 modulates hemoglobin-mediated signaling in cerebrovascular smooth muscle via RhoA/Rho kinase and protein kinase C

Endothelin-1 (ET-1) and oxyhemoglobin (OxyHb) have been implicated in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage. However, the contribution of ET-1 to this condition has not been definitely established. In this study, we investigated whether threshold concentration of ET-1 enhances cerebrovascular smooth muscle (CVSM) contraction to OxyHb by activating the RhoA/Rho kinase and protein kinase C (PKC) pathways. CVSM contraction was measured in endothelium-denuded rabbit basilar arteries. Cytosolic and particulate fractions of CVSM cells were examined for RhoA and PKC reactivity with specific antibodies using immunoblotting procedures. ET-1 (0.1 nM) alone did not produce any significant contraction, but it markedly potentiated the magnitude (223% of control) and rate (149% of control) of contraction in response to OxyHb, which was attenuated by the inhibitors of Rho kinase Y-27632 and HA-1077. ET-1-mediated potentiation of the contraction was also inhibited by inhibitors of PKC, Ro-32-0432, and GF-109203X. BQ-123 prevented potentiation of vasoconstriction mediated by ET-1, indicating that the action of ET-1 was mediated by the endothelin type A receptor. Pretreatment with ET-1 significantly enhanced OxyHb-mediated RhoA translocation in CVSM cells and intact basilar arteries. ET-1 also caused potentiation of PKC-epsilon expression in membranes of CVSM cells exposed to OxyHb for 10 and 60 min but did not markedly change the distribution of PKC-alpha. Thus, in CVSM, threshold concentration of ET-1 potentiates contraction induced by OxyHb via RhoA/Rho kinase- and PKC-epsilon-dependent mechanisms. This process may contribute to the pathological contraction of cerebral arteries observed after subarachnoid hemorrhage.     

Vascular smooth muscle myosin light chain diphosphorylation: mechanism, function, and pathological implications

Smooth muscle contraction is activated primarily by phosphorylation at S19 of the 20-kDa regulatory light chain subunits of myosin II (LC(20) ) catalyzed by Ca(2+) /calmodulin-dependent myosin light chain kinase. Other kinases, for example, integrin-linked kinase (ILK), Rho-associated kinase (ROCK), and zipper-interacting protein kinase (ZIPK), can phosphorylate T18 in addition to S19, which increases the actin-activated myosin MgATPase activity at subsaturating actin concentrations ～3-fold. These phosphorylatable residues and the amino acid sequence surrounding them are highly conserved throughout the animal kingdom; they are also found in an LC(20) homolog within the genome of Monosiga brevicollis, the closest living relative of metazoans. LC(20) diphosphorylation has been detected in mammalian vascular smooth muscle tissues in response to specific contractile stimuli and in pathophysiological situations associated with hypercontractility. LC(20) diphosphorylation has also been observed frequently in cultured cells where it activates force generation. Kinases such as ILK, ROCK, and ZIPK, therefore, are potential therapeutic targets in the treatment of, for example, cerebral vasospasm following subarachnoid hemorrhage and atherosclerosis.     

Reactive oxygen-mediated contraction in pulmonary arterial smooth muscle: cellular mechanisms.

Reactive oxygen species (at least relatively high doses) cause contraction of pulmonary arterial smooth muscle. The objective of the present study was to elucidate the possible cellular mechanisms involved in reactive oxygen-mediated contraction. Isolated arterial rings from Sprague-Dawley rats were placed in tissue baths containing Earle's balanced salt solution. The maximum active force production (Po) in response to 80 mM KCl was obtained. All other responses were normalized as percentages of Po for comparative purposes. Exposure to reactive oxygen (generated from either the xanthine oxidase reaction (XO) or the glucose oxidase reaction) resulted in pulmonary arterial muscle developing mean active tension of 17.1 +/- 3.0% Po. This contraction was independent of extracellular calcium, since it was not affected by verapamil (a calcium channel blocker) or by placement of the arterial muscle in calcium-free media. Phentolamine (an alpha 1-receptor blocker) and propranolol (a beta-receptor blocker) did not diminish the response to XO. Ryanodine (a SR calcium release inhibitor), while reducing the response to norepinephrine, did not affect the response to XO. However, H-7 (an inhibitor of protein kinase C) decreased the XO-mediated contraction by 49%. These results indicate that while Ca2+ may not be involved as a second messenger, protein kinase C activity appears to play a role in the transduction pathway of reactive oxygen species mediated contraction of pulmonary arterial smooth muscle.     

Corticotropin releasing hormone (CRH) increases beta-endorphin (beta-end like) concentration in cerebrospinal fluid of rats with vasospasm following subarachnoid hemorrhage.

The chronic stage of vasospasm occurring several days after subarachnoid hemorrhage (SAH) is characterized by the development of histopathologic changes in cerebral arteries causing cerebral ischemia. Numerous experimental data indicate the involvement of immune mechanisms in the angiopathy caused by SAH. Endogenous opioids play also an important role in the ischemic lesions of the brain. Corticotropin releasing hormone (CRH) induces the release of beta-endorphin (beta-END) from hypothalamic neurons and also from mononuclear white blood cells. The function of CRH and beta-END in vasospasm following SAH and the interrelationship between neuroendocrine and immune changes requires further elucidation. In the present study we investigated the influence of CRH injected into cerebral cisterna magna (CM) of rats on beta-END-like level in cerebrospinal fluid (CSF) in acute and chronic phase of cerebral vasospasm following artificial SAH. Acutely CRH induced a significant rise of beta-END-like in CSF both in SAH and sham SAH rats. However, in rats subjected to SAH, a single injection of CRH caused a prolonged rise of 5-END in CSF, which was also seen 2 days after SAH, during the chronic phase of vasospasm. The obtained results indicate that CRH increases neuroendocrine changes induced by SAH, probably by an activation of immune cells involved in the patomechanism of chronic vasospasm.     

In vitro study of cerebrospinal fluid dynamics in a shaken Basal cistern after experimental subarachnoid hemorrhage

Background

Cerebral arterial vasospasm leads to delayed cerebral ischemia and constitutes the major delayed complication following aneurysmal subarachnoid hemorrhage. Cerebral vasospasm can be reduced by increased blood clearance from the subarachnoid space. Clinical pilot studies allow the hypothesis that the clearance of subarachnoid blood is facilitated by means of head shaking. A major obstacle for meaningful clinical studies is the lack of data on appropriate parameters of head shaking. Our in vitro study aims to provide these essential parameters.

Methodology/Principal Findings

A model of the basal cerebral cistern was derived from human magnetic resonance imaging data. Subarachnoid hemorrhage was simulated by addition of dyed experimental blood to transparent experimental cerebrospinal fluid (CSF) filling the model of the basal cerebral cistern. Effects of various head positions and head motion settings (shaking angle amplitudes and shaking frequencies) on blood clearance were investigated using the quantitative dye washout method. Blood washout can be divided into two phases: Blood/CSF mixing and clearance. The major effect of shaking consists in better mixing of blood and CSF thereby increasing clearance rate. Without shaking, blood/CSF mixing and blood clearance in the basal cerebral cistern are hampered by differences in density and viscosity of blood and CSF. Blood clearance increases with decreased shaking frequency and with increased shaking angle amplitude. Head shaking facilitates clearance by varying the direction of gravitational force.

Conclusions/Significance

From this in vitro study can be inferred that patient or head shaking with large shaking angles at low frequency is a promising therapeutic strategy to increase blood clearance from the subarachnoid space.     

Oxidative degradation of bilirubin produces vasoactive compounds.

Subarachnoid haemorrhage is often followed by haemolysis and concomitant oxidative stress, and is frequently complicated by pathological vasoconstriction or cerebral vasospasm. It is known that upregulation of haem oxygenase (HO-1) is induced by oxidative stress and results in release of biliverdin and bilirubin (BR), which are scavengers of reactive oxygen species (ROS). Here we report biomimetic studies aimed at modelling pathological conditions leading to oxidative degradation of BR. Oxidative degradation products of BR, formed by reaction with hydrogen peroxide (an ROS model system), demonstrated biological activity by stimulating oxygen consumption and force development in vascular smooth muscle from porcine carotid artery. Analogous biological activity was observed with vasoactive cerebrospinal fluid from subarachnoid haemorrhage patients. Three degradation products of BR were isolated: two were assigned as isomeric monopyrrole (C9H11N2O2) derivatives, 4-methyl-5-oxo-3-vinyl-(1, 5-dihydropyrrol-2-ylidene)acetamide and 3-methyl-5-oxo-4-vinyl-(1, 5-dihydropyrrol-2-ylidene)acetamide and the third was 4-methyl-3-vinylmaleimide (MVM), a previously isolated photodegradation product of biliverdin. Possible mechanisms of oxidative degradation of BR are discussed. Tentative assignment of these structures in the cerebrospinal fluid (CSF) of cerebral vasospasm patients has been made. It is proposed that one or more of the degradation products of biliverdin or bilirubin are involved in complications such as vasospasm and or pathological vasoconstriction associated with haemorrhage.     

Effects of K+ channel agonists cromakalim and pinacidil on rat basilar artery smooth muscle cells are mediated by Ca(++)-activated K+ channels

Whole-cell and cell-free inside-out patch-clamp recording techniques were used to examine the actions of potassium channel openers pinacidil and cromakalim in enzymatically isolated smooth muscle cells of rat basilar artery. Delayed rectifier and calcium-dependent potassium currents were identified from the whole-cell recordings. Only the calcium-dependent potassium current was increased by cromakalim and pinacidil. Recordings from inside-out membrane patches revealed a large conductance voltage- and calcium-dependent potassium channel, which was blocked by charybdotoxin but unaffected by ATP less than 10 mM. Cromakalim and pinacidil increased the open probability of this channel. On the basis of these results, we suggest that such drugs, acting on cerebral arterial smooth muscle cell potassium channels, may be of some benefit in the treatment of cerebral vasospasm following subarachnoid hemorrhage.     

AMP-activated protein kinase phosphorylates and desensitizes smooth muscle myosin light chain kinase

Smooth muscle contraction is initiated by a rise in intracellular calcium, leading to activation of smooth muscle myosin light chain kinase (MLCK) via calcium/calmodulin (CaM). Activated MLCK then phosphorylates the regulatory myosin light chains, triggering cross-bridge cycling and contraction. Here, we show that MLCK is a substrate of AMP-activated protein kinase (AMPK). The phosphorylation site in chicken MLCK was identified by mass spectrometry to be located in the CaM-binding domain at Ser(815). Phosphorylation by AMPK desensitized MLCK by increasing the concentration of CaM required for half-maximal activation. In primary cultures of rat aortic smooth muscle cells, vasoconstrictors activated AMPK in a calcium-dependent manner via CaM-dependent protein kinase kinase-beta, a known upstream kinase of AMPK. Indeed, vasoconstrictor-induced AMPK activation was abrogated by the STO-609 CaM-dependent protein kinase kinase-beta inhibitor. Myosin light chain phosphorylation was increased under these conditions, suggesting that contraction would be potentiated by ablation of AMPK. Indeed, in aortic rings from mice in which alpha1, the major catalytic subunit isoform in arterial smooth muscle, had been deleted, KCl- or phenylephrine-induced contraction was increased. The findings suggest that AMPK attenuates contraction by phosphorylating and inactivating MLCK. This might contribute to reduced ATP turnover in the tonic phase of smooth muscle contraction.     

Pressure-induced myogenic tone and role of 20-HETE in mediating autoregulation of cerebral blood flow

While myogenic force in response to a changing arterial pressure has been described early in the 20th century, it was not until 1984 that the effect of a sequential increase in intraluminal pressure on cannulated cerebral arterial preparations was found to result in pressure-dependent membrane depolarization associated with spike generation and reduction in lumen diameter. Despite a great deal of effort by different laboratories and investigators, the identification of the existence of a mediator of the pressure-induced myogenic constriction in arterial muscle remained a challenge. It was the original finding by our laboratory that demonstrated the capacity of cerebral arterial muscle cells to express the cytochrome P-450 4A enzyme that catalyzes the formation of the potent vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid, the production of which in cerebral arterial muscle cells increases with the elevation in intravascular pressure. 20-HETE activates protein kinase C and causes the inhibition of Ca(2+)-activated K(+) channels, depolarizes arterial muscle cell membrane, and activates L-type Ca(2+) channel to increase intracellular Ca(2+) levels and evoke vasoconstriction. The inhibition of 20-HETE formation attenuates pressure-induced arterial myogenic constriction in vitro and blunts the autoregulation of cerebral blood flow in vivo. We suggest that the formation and action of cytochrome P-450-derived 20-HETE in cerebral arterial muscle could play a critically important role in the control of cerebral arterial tone and the autoregulation of cerebral blood flow under physiological conditions.     

Interactive role of tyrosine kinase,protein kinase C,and Rho/Rho kinase systems in the mechanotransduction of vascular smooth muscles

Blood vessels are always subjected to hemodynamic stresses including blood pressure and blood flow. The cerebral artery is particularly sensitive to hemodynamic stresses such as pressure and stretch, and shows contractions that are myogenic in nature; i.e., the mechanical response is generated by the vascular smooth muscle itself. The artery constricts in response to an increase in intraluminal pressure, and dilates in response to a decrease in the intraluminal pressure. We provide herein some insights into the mechanotransduction of vascular tissue; i.e., we discuss how the tissue is receptive to mechanical force and how the latter induces the specific signals leading to myogenic contraction in terms of mechanosensor action and subsequent intracellular signaling. The interactive role of tyrosine kinase, protein kinase C, and Rho/Rho-kinase systems in the mechanotransduction process is discussed, which systems also seem to play an important role in the development of experimental cerebral vasospasm. The study of the mechanotransduction in vascular tissue may aid in clarifying the mechanisms underlying vasospastic episodes and pathologic remodeling in cardiovascular diseases, and may potentially have therapeutic consequences.     

Differential regulation of intracellular Ca2+ signalling induced by high K+ and endothelin-1 in single smooth muscle cells of intact canine basilar artery: detection by means of confocal laser microscopy

Changes in intracellular calcium concentration ([Ca2+]i) in smooth muscle cells play the key role in regulation of vascular smooth muscle tone and pathogenesis of cerebral vasospasm. In this study, we adopted the confocal laser microscopy to detect the fluorescence signals arising from the individual smooth muscle cells of canine basilar artery. Ring preparations were made, loaded with fluo-3 and changes in fluorescence induced by high K+ and endothelin-1 (ET-1) were measured by confocal laser microscopy. In some unstimulated smooth muscle cells Ca2+ waves arising from discrete region of the cell propagated to the whole cell with a velocity of approximately 10 microm/s. High K+ (80 mmol/L) induced a rapid rise in [Ca2+]i, the peak level being consistently reached approximately 10 s after stimulation. In contrast, the time to peak level of [Ca2+]i induced by ET-1 (0.3 micromol/L) varied widely between 13 and 26 s among individual cells, an indication that the extent of nonuniform coordination of increases in [Ca2+]i in individual cells may be partly responsible for the different time courses of tension development of vascular smooth muscle in response to the vasoactive stimulants. The increase in [Ca2+]i induced by ET-1 was transient but a pronounced and sustained contraction developed further in response to ET-1. Thus ET-1 has a biological property as a potential candidate to elicit cerebral vasospasm. Confocal laser microscopy could be a useful tool to measure the changes in [Ca2+]i in individual smooth muscle cells of cerebral artery.     

Effects of Tenascin-C Knockout on Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage in Mice

A matricellular protein tenascin-C (TNC) has been suggested to play a role in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH), but the direct evidence remains lacking. In this study, we examined effects of TNC knockout (TNKO) on cerebral vasospasm after experimental SAH in mice. C57BL/6 wild-type (WT) or TNKO mice were subjected to SAH by endovascular puncture. Ten WT and ten TNKO mice were randomized to WT sham (n = 4), TNKO sham (n = 4), WT SAH (n = 6), and TNKO SAH (n = 6) groups. In addition to neurobehavioral impairments and severity of SAH, cerebral vasospasm was assessed by morphometric measurements of the left internal carotid artery (ICA). Infiltration of inflammatory cells in the subarachnoid periarterial space was also assessed, and expressions of TNC and mitogen-activated protein kinases (MAPKs) in the ICA were immunohistochemically evaluated at 24 h post-surgery. TNC was induced in the smooth muscle cell layers and the adventitia in the spastic ICAs as well as the periarterial inflammatory cells in WT SAH mice. Compared with WT SAH mice, TNKO SAH mice showed better neurological scores and less severe cerebral vasospasm, as well as fewer inflammatory cell infiltration in the periarterial space. Post-SAH activation of MAPKs in the smooth muscle cell layers of the ICAs was also prevented in TNKO SAH mice. The findings in the present study suggest that TNC causes the development of cerebral vasospasm via pro-inflammatory effects and activation of MAPKs.     

Vascular mechanisms of increased arterial pressure in preeclampsia: lessons from animal models

Normal pregnancy is associated with reductions in total vascular resistance and arterial pressure possibly due to enhanced endothelium-dependent vascular relaxation and decreased vascular reactivity to vasoconstrictor agonists. These beneficial hemodynamic and vascular changes do not occur in women who develop preeclampsia; instead, severe increases in vascular resistance and arterial pressure are observed. Although preeclampsia represents a major cause of maternal and fetal morbidity and mortality, the vascular and cellular mechanisms underlying this disorder have not been clearly identified. Studies in hypertensive pregnant women and experimental animal models suggested that reduction in uteroplacental perfusion pressure and the ensuing placental ischemia/hypoxia during late pregnancy may trigger the release of placental factors that initiate a cascade of cellular and molecular events leading to endothelial and vascular smooth muscle cell dysfunction and thereby increased vascular resistance and arterial pressure. The reduction in uterine perfusion pressure and the ensuing placental ischemia are possibly caused by inadequate cytotrophoblast invasion of the uterine spiral arteries. Placental ischemia may promote the release of a variety of biologically active factors, including cytokines such as tumor necrosis factor-alpha and reactive oxygen species. Threshold increases in the plasma levels of placental factors may lead to endothelial cell dysfunction, alterations in the release of vasodilator substances such as nitric oxide (NO), prostacyclin (PGI(2)), and endothelium-derived hyperpolarizing factor, and thereby reductions of the NO-cGMP, PGI(2)-cAMP, and hyperpolarizing factor vascular relaxation pathways. The placental factors may also increase the release of or the vascular reactivity to endothelium-derived contracting factors such as endothelin, thromboxane, and ANG II. These contracting factors could increase intracellular Ca(2+) concentrations ([Ca(2+)](i)) and stimulate Ca(2+)-dependent contraction pathways in vascular smooth muscle. The contracting factors could also increase the activity of vascular protein kinases such as protein kinase C, leading to increased myofilament force sensitivity to [Ca(2+)](i) and enhancement of smooth muscle contraction. The decreased endothelium-dependent mechanisms of vascular relaxation and the enhanced mechanisms of vascular smooth muscle contraction represent plausible causes of the increased vascular resistance and arterial pressure associated with preeclampsia.     

Reactive oxygen species from smooth muscle mitochondria initiate cold-induced constriction of cutaneous arteries

Cold constricts cutaneous blood vessels by selectively increasing the activity of smooth muscle alpha2-adrenoceptors (alpha2-ARs). In mouse tail arteries, alpha2-AR constriction is mediated by alpha2A-ARs at 37 degrees C, whereas the cold-induced augmentation in alpha2-AR activity is mediated entirely by alpha2C-ARs. Cold causes translocation of alpha2C-ARs from the trans-Golgi to the plasma membrane, mediated by cold-induced activation of RhoA and Rho kinase. The present experiments analyzed the mechanisms underlying these responses. Mouse tail arteries were studied in a pressure myograph. Cooling the arteries (28 degrees C) caused a rapid increase in reactive oxygen species (ROS) in smooth muscle cells, determined by confocal microscopy of arteries loaded with the ROS-sensitive probes, dichlorodihydrofluorescein or reduced Mitotracker Red. The inhibitor of mitochondrial complex I rotenone (10 micromol/l), the antioxidant N-acetylcysteine (NAC; 20 mmol/l), or the cell-permeable mimic of superoxide dismutase MnTMPyP (50 micromol/l) did not affect vasoconstriction to alpha2-AR stimulation (UK-14304) at 37 degrees C but dramatically inhibited the response at 28 degrees C. Indeed, these ROS inhibitors abolished the cold-induced increase in alpha2-AR constrictor activity. NAC (20 mmol/l) or MnTMPyP (50 micromol/l) also abolished the cold-induced activation of RhoA in human cultured vascular smooth muscle cells and the cold-induced mobilization of alpha2C-ARs to the cell surface in human embryonic kidney 293 cells transfected with the receptor. The combined results suggest that cold-induced constriction is mediated by redox signaling in smooth muscle cells, initiated by mitochondrial generation of ROS, which stimulate RhoA/Rho kinase signaling and the subsequent mobilization of alpha2C-ARs to the cell surface. Altered activity of ROS may contribute to cold-induced vasospasm occurring in Raynaud's phenomenon.