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Supplemental Digital Content is available in the text., Background and Purpose: Circadian rhythms influence the extent of brain injury following subarachnoid hemorrhage (SAH), but the mechanism is unknown. We hypothesized that cerebrovascular myogenic reactivity is rhythmic and explains the circadian variation in SAH-induced injury. Methods: SAH was modeled in mice with prechiasmatic blood injection. Inducible, smooth muscle cell–specific Bmal1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1) gene deletion (smooth muscle–specific Bmal1 1 knockout [sm-Bmal1 KO]) disrupted circadian rhythms within the cerebral microcirculation. Olfactory cerebral resistance arteries were functionally assessed by pressure myography in vitro; these functional assessments were related to polymerase chain reaction/Western blot data, brain histology (Fluoro-Jade/activated caspase-3), and neurobehavioral assessments (modified Garcia scores). Results: Cerebrovascular myogenic vasoconstriction is rhythmic, with a peak and trough at Zeitgeber times 23 and 11 (ZT23 and ZT11), respectively. Histological and neurobehavioral assessments demonstrate that higher injury levels occur when SAH is induced at ZT23, compared with ZT11. In sm-Bmal1 KO mice, myogenic reactivity is not rhythmic. Interestingly, myogenic tone is higher at ZT11 versus ZT23 in sm-Bmal1 KO mice; accordingly, SAH-induced injury in sm-Bmal1 KO mice is more severe when SAH is induced at ZT11 compared to ZT23. We examined several myogenic signaling components and found that CFTR (cystic fibrosis transmembrane conductance regulator) expression is rhythmic in cerebral arteries. Pharmacologically stabilizing CFTR expression in vivo (3 mg/kg lumacaftor for 2 days) eliminates the rhythmicity in myogenic reactivity and abolishes the circadian variation in SAH-induced neurological injury. Conclusions: Cerebrovascular myogenic reactivity is rhythmic. The level of myogenic tone at the time of SAH ictus is a key factor influencing the extent of injury. Circadian oscillations in cerebrovascular CFTR expression appear to underlie the cerebrovascular myogenic reactivity rhythm.

Circadian rhythms orchestrate important functions in the cardiovascular system: the contribution of microvascular rhythms to cardiovascular disease progression/severity is unknown. This study hypothesized that (i) myogenic reactivity in skeletal muscle resistance arteries is rhythmic and (ii) that disrupting this rhythmicity would alter cardiac injury post-myocardial infarction (MI).Cremaster skeletal muscle resistance arteries were isolated and assessed using standard pressure myography. Circadian rhythmicity was globally disrupted with the ClockΔ19/Δ19 mutation or discretely through smooth muscle cell-specific Bmal1 deletion (Sm-Bmal1 KO). Cardiac structure and function were determined by echocardiographic, hemodynamic and histological assessments. Myogenic reactivity in cremaster muscle resistance arteries is rhythmic. This rhythm is putatively mediated by the circadian modulation of a mechanosensitive signalosome incorporating tumor necrosis factor and casein kinase 1. Following left anterior descending coronary artery ligation, myogenic responsiveness is locked at the circadian maximum, although circadian molecular clock gene expression cycles normally. Disrupting the molecular clock abolishes myogenic rhythmicity: myogenic tone is suspended at the circadian minimum and no longer augmented by MI. The reduced myogenic tone in ClockΔ19/Δ19 mice and Sm-Bmal1 KO mice associates with reduced total peripheral resistance (TPR), improved cardiac function and reduced infarct expansion post-MI.Augmented microvascular constriction aggravates cardiac injury post-MI. Following MI, skeletal muscle resistance artery myogenic reactivity increases specifically within the rest phase, when TPR would normally decline. Disrupting the circadian clock interrupts the MI-induced augmentation in myogenic reactivity: therapeutics targeting the molecular clock, therefore, may be useful for improving MI outcomes.A portion of peripheral vascular tone and hence, total peripheral resistance, is under circadian control. Myocardial infarction preferentially augments vascular tone in the active phase, when the heart works hardest to provide adequate tissue perfusion. Disrupting the circadian clock reduces peripheral resistance and is associated with improved cardiac function and reduced infarct expansion post-myocardial infarction. The intersection between the circadian clock and myogenic signaling could be a potential therapeutic target to manage peripheral resistance and improve cardiac outcome following an infarction. This mechanism may be safer than current vasodilators, since the therapeutic maximum does not threaten essential hemodynamic control.

Summary: Heart failure (HF) and subarachnoid hemorrhage (SAH) chronically reduce cerebral perfusion, which negatively affects clinical outcome. This work demonstrates a strong relationship between cerebral artery cystic fibrosis transmembrane conductance regulator (CFTR) expression and altered cerebrovascular reactivity in HF and SAH. In HF and SAH, CFTR corrector compounds (C18 or lumacaftor) normalize pathological alterations in cerebral artery CFTR expression, vascular reactivity, and cerebral perfusion, without affecting systemic hemodynamic parameters. This normalization correlates with reduced neuronal injury. Therefore, CFTR therapeutics have emerged as valuable clinical tools to manage cerebrovascular dysfunction, impaired cerebral perfusion, and neuronal injury. Key Words: cognitive impairment, corrector compounds, cystic fibrosis transmembrane conductance regulator (CFTR), myogenic vasoconstriction, sphingosine-1-phosphate, tumor necrosis factor, vascular smooth muscle cells

The Earth’s rotation generates environmental oscillations (e.g., in light and temperature) that have imposed unique evolutionary pressures over millions of years. Consequently, the circadian clock, a ubiquitously expressed molecular system that aligns cellular function to these environmental cues, has become an integral component of our physiology. The resulting functional rhythms optimize and economize physiological performance: perturbing these rhythms, therefore, is frequently deleterious. This perspective article focuses on circadian rhythms in resistance artery myogenic reactivity, a key mechanism governing tissue perfusion, total peripheral resistance and systemic blood pressure. Emerging evidence suggests that myogenic reactivity rhythms are locally generated in a microvascular bed-specific manner at the level of smooth muscle cells. This implies that there is a distinct interface between the molecular clock and the signalling pathways underlying myogenic reactivity in the microvascular beds of different organs. By understanding the precise nature of these molecular links, it may become possible to therapeutically manipulate microvascular tone in an organ-specific manner. This raises the prospect that interventions for vascular pathologies that are challenging to treat, such as hypertension and brain malperfusion, can be significantly improved.

Aneurysmal subarachnoid hemorrhage (SAH) is a devastating cerebral event that kills or debilitates the majority of those afflicted. The blood that spills into the subarachnoid space stimulates profound cerebral artery vasoconstriction and consequently, cerebral ischemia. Thus, once the initial bleeding in SAH is appropriately managed, the clinical focus shifts to maintaining/improving cerebral perfusion. However, current therapeutic interventions largely fail to improve clinical outcome, because they do not effectively restore normal cerebral artery function. This review discusses emerging evidence that perturbed cerebrovascular “myogenic reactivity,” a crucial microvascular process that potently dictates cerebral perfusion, is the critical element underlying cerebral ischemia in SAH. In fact, the myogenic mechanism could be the reason why many therapeutic interventions, including “Triple H” therapy, fail to deliver benefit to patients. Understanding the molecular basis for myogenic reactivity changes in SAH holds the key to develop more effective therapeutic interventions; indeed, promising recent advancements fuel optimism that vascular dysfunction in SAH can be corrected to improve outcome.

Osteocytes are proposed to be the mechanosensory cells that translate mechanical loading into biochemical signals during the process of bone adaptation. The lipid mediator sphingosine-1-phosphate (S1P) has been reported to play a role in the mechanotransduction process of blood vessels and also in the dynamic control of bone mineral homeostasis. Nevertheless, the potential role of S1P in bone mechanotransduction has yet to be elucidated. In this study, we hypothesized that a S1P cascade is involved in the activation of osteocytes in response to loading-induced oscillatory fluid flow (OFF) in bone. MLO-Y4 osteocyte-like cells express the necessary components of a functional S1P cascade. To examine the involvement of S1P signaling in osteocyte mechanotransduction, we applied OFF (1 Pa, 1 Hz) to osteocyte-like MLO-Y4 cells under conditions where the S1P signaling pathway was modulated. We found that decreased endogenous S1P levels significantly suppressed the OFF-induced intracellular calcium response. Addition of extracellular S1P to MLO-Y4 cells enhanced the synthesis and release of prostaglandin E2 (PGE2) under static cells and amplified OFF-induced PGE2 release. The stimulatory effect of OFF on the gene expression levels of osteoprotegerin (OPG) and receptor activator for nuclear factor κB ligand (RANKL) was S1P dependent. Furthermore, the S1P2 receptor subtype was shown to be involved in OFF-induced PGE2 synthesis and release, as well as down-regulation of RANKL/OPG gene expression ratio. In summary, our data suggest that S1P cascade is involved in OFF-induced mechanotransduction in MLO-Y4 cells and that extracellular S1P exerts its effect partly through S1P2 receptors.

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.

Tumour necrosis factor (TNF) is a ubiquitously expressed cytokine with functions beyond the immune system. In several diseases, the induction of TNF expression in resistance artery smooth muscle cells enhances microvascular myogenic vasoconstriction and perturbs blood flow. This pathological role prompted our hypothesis that constitutively expressed TNF regulates myogenic signalling and systemic haemodynamics under non-pathological settings. Here we show that acutely deleting the TNF gene in smooth muscle cells or pharmacologically scavenging TNF with etanercept (ETN) reduces blood pressure and resistance artery myogenic responsiveness; the latter effect is conserved across five species, including humans. Changes in transmural pressure are transduced into intracellular signals by membrane-bound TNF (mTNF) that connect to a canonical myogenic signalling pathway. Our data positions mTNF ‘reverse signalling' as an integral element of a microvascular mechanosensor; pathologic or therapeutic perturbations of TNF signalling, therefore, necessarily affect microvascular tone and systemic haemodynamics., TNF is typically viewed as an inflammatory mediator. Here the authors identify a non-inflammatory mechanism conserved across species whereby the constitutively expressed smooth muscle cell TNF mediates myogenic signal transduction in skeletal muscle resistance arteries and regulates mean arterial blood pressure.

The intrinsic ability of resistance arteries to respond to transmural pressure is the single most important determinant of their function. Despite an ever-growing catalogue of signalling pathways that underlie the myogenic response , it remains an enigmatic mechanism. The myogenic response's mechanistic diversity has largely been attributed to ‘hard-wired’ differences across species and vascular beds; however, emerging evidence suggests that the mechanistic basis for the myogenic mechanism is, in fact, ‘plastic’. This means that the myogenic response can change quantitatively (i.e. change in magnitude) and qualitatively (i.e. change in mechanistic basis) in response to environmental challenges (e.g. disease conditions). Consequently, understanding the dynamics of how the myogenic response capitalizes on its mechanistic diversity is key to unlocking clinically viable interventions. Using myogenic sphingosine-1-phosphate (S1P) signalling as an example, this review illustrates the remarkable plasticity of the myogenic response. We propose that currently unidentified ‘organizational programmes’ dictate the contribution of individual signalling pathways to the myogenic response and introduce the concept that certain signalling elements act as ‘divergence points’ (i.e. as the potential higher level regulatory sites). In the context of pressure-induced S1P signalling, the S1P-generating enzyme sphingosine kinase 1 serves as a divergence point, by orchestrating the calcium-dependent and -independent signalling pathways underlying microvascular myogenic responsiveness. By acting on divergence points, the proposed ‘organizational programmes’ could form the basis for the flexible recruitment and fine-tuning of separate signalling streams that underlie adaptive changes to the myogenic response and its distinctiveness across species and vascular beds.

Background and Purpose— We sought to demonstrate that tumor necrosis factor (TNF)-α, via sphingosine-1-phosphate signaling, has the potential to alter cochlear blood flow and thus, cause ischemic hearing loss. Methods— We performed intravital fluorescence microscopy to measure blood flow and capillary diameter in anesthetized guinea pigs. To measure capillary diameter ex vivo, capillary beds from the gerbil spiral ligament were isolated from the cochlear lateral wall and maintained in an organ bath. Isolated gerbil spiral modiolar arteries, maintained and transfected in organ culture, were used to measure calcium sensitivity (calcium-tone relationship). In a clinical study, a total of 12 adult patients presenting with typical symptoms of sudden hearing loss who were not responsive or only partially responsive to prednisolone treatment were identified and selected for etanercept treatment. Etanercept (25 mg s.c. ) was self-administered twice a week for 12 weeks. Results— TNF-α induced a proconstrictive state throughout the cochlear microvasculature, which reduced capillary diameter and cochlear blood flow in vivo. In vitro isolated preparations of the spiral modiolar artery and spiral ligament capillaries confirmed these observations. Antagonizing sphingosine-1-phosphate receptor 2 subtype signaling (by 1 μmol/L JTE013) attenuated the effects of TNF-α in all models. TNF-α activated sphingosine kinase 1 (Sk1) and induced its translocation to the smooth muscle cell membrane. Expression of a dominant-negative Sk1 mutant (Sk1 G82D ) eliminated both baseline spiral modiolar artery calcium sensitivity and TNF-α effects, whereas a nonphosphorylatable Sk1 mutant (Sk1 S225A ) blocked the effects of TNF-α only. A small group of etanercept-treated, hearing loss patients recovered according to a 1-phase exponential decay (half-life=1.56±0.20 weeks), which matched the kinetics predicted for a vascular origin. Conclusions— TNF-α indeed reduces cochlear blood flow via activation of vascular sphingosine-1-phosphate signaling. This integrates hearing loss into the family of ischemic microvascular pathologies, with implications for risk stratification, diagnosis, and treatment.

Objective : Sepsis-stimulated nitric oxide (NO) production impairs arteriolar responsiveness in skeletal muscle. Using wild type (WT), eNOS−/−, iNOS−/− and nNOS−/− mice, we aimed to determine the key nitric oxide synthase (NOS) isoenzyme(s) responsible for the arteriolar hyporesponsiveness to acetylcholine (ACh) in septic skeletal muscle. Methods : Sepsis was induced by the cecal ligation and perforation procedure (24 h model). We measured the post-ACh increase in red blood cell velocity ( V RBC) in a capillary fed by the stimulated arteriole as an index of vasodilation. NOS activity and protein expression in the muscle were measured by standard procedures. Results : In all non-septic mice, ACh increased V RBC by ∼150% from baseline. Sepsis impaired this response in WT, eNOS−/− and iNOS−/− mice, but not in nNOS−/− mice. Accordingly, pharmacological inhibition of nNOS with 7-nitroindazole reversed this impairment in WT mice. cNOS (eNOS+nNOS) activity was elevated in septic WT mice; Western blots indicated that this occurred through a post-translational mechanism. iNOS protein activity/expression was negligible. ACh caused dilation via endothelial-derived relaxing factor (EDRF) in WT mice and via endothelial-derived hyperpolarizing factor (EDHF) in eNOS−/− mice. Although exogenous NO reduced EDHF-mediated dilation in eNOS−/− mice, NOS inhibition did not reverse the sepsis-impaired dilation in these mice. Conclusions : In our 24-h mouse model of sepsis, NO in skeletal muscle is primarily derived from nNOS. Sepsis impairs both EDRF- and EDHF-mediated dilation in response to ACh. Both genetic deletion and inhibition of nNOS protect against this impairment when the dilation occurs via the EDRF but not EDHF pathway.

Diabetes strongly associates with microvascular complications that ultimately promote multiorgan failure. Altered myogenic responsiveness compromises tissue perfusion, aggravates hypertension, and sets the stage for later permanent structural changes to the microcirculation. We demonstrate that skeletal muscle resistance arteries isolated from patients with diabetes have augmented myogenic tone, despite reasonable blood glucose control. To understand the mechanisms, we titrated a standard diabetes mouse model (high-fat diet plus streptozotocin [HFD/STZ]) to induce a mild increase in blood glucose levels. HFD/STZ treatment induced a progressive myogenic tone augmentation in mesenteric and olfactory cerebral arteries; neither HFD nor STZ alone had an effect on blood glucose or resistance artery myogenic tone. Using gene deletion models that eliminate tumor necrosis factor (TNF) or sphingosine kinase 1, we demonstrate that vascular smooth muscle cell TNF drives the elevation of myogenic tone via enhanced sphingosine-1-phosphate (S1P) signaling. Therapeutically antagonizing TNF (etanercept) or S1P (JTE013) signaling corrects this defect. Our investigation concludes that vascular smooth muscle cell TNF augments resistance artery myogenic vasoconstriction in a diabetes model that induces a small elevation of blood glucose. Our data demonstrate that microvascular reactivity is an early disease marker and advocate establishing therapies that strategically target the microcirculation.

Objective: Increased nitric oxide (NO) production in sepsis precipitates microcirculatory dysfunction. We aimed (i) to determine if NO is the key water-soluble factor in the recently discovered sepsis-induced deficit in arteriolar conducted vasoconstriction, (ii) to identify which nitric oxide synthase (NOS) isoforms account for this deficit, and (iii) to examine the potential role of connexin37 (Cx37, a hypothesized signaling target of NO) in arteriolar conduction. Methods: Using intravital microscopy and the cecal ligation and perforation 24-h model of sepsis, arterioles in the cremaster muscle of male C57BL/6 wild-type (WT), iNOS−/−, eNOS−/−, nNOS−/− and Cx37−/− mice were locally stimulated with KCl to initiate conducted vasoconstriction. We used the ratio of conducted constriction (500 μm upstream) to local constriction as an index of conduction (CR500). NOS enzymatic activity and protein expression were determined in control and septic cremaster muscles. Results: Sepsis reduced CR500 in WT mice [from 0.77 ± 0.05 to 0.20 ± 0.02 (means ± SE) independent of the site of stimulation along the arteriole], in iNOS−/− and eNOS−/− mice, but not in nNOS−/− mice. The nNOS inhibitor 7-nitroindazole or NO scavenger HbO2 restored CR500 in septic WT mice, but blockade of soluble guanylate cyclase had no effect. Sepsis increased cNOS (eNOS+nNOS) activity in WT mice (from 340 ± 40 to 490 ± 30 pmol/mg/h) and in eNOS−/−, but not in nNOS−/− mice (iNOS activity was negligible in all mice). Sepsis did not alter nNOS protein expression in WT mice. CR500 in non-septic Cx37−/− mice (0.15 ± 0.1) was similar to that observed in septic WT mice. Conclusion: Increased nNOS activity and the resultant increased NO production in the septic mouse cremaster muscle are the key factors responsible for the deficit in conducted vasoconstriction along the arteriole. Deletion of Cx37 results in reduced CR500, which is consistent with the hypothesis that Cx37 in the arteriole could be a target of NO signaling.

Gap junction intercellular communication (GJIC) plays a significant role in the vascular system. Regulation of GJIC is a dynamic process, with alterations in connexin (Cx) protein expression and post-translational modification as contributing mechanisms. We hypothesized that the endothelial autacoid nitric oxide (NO) would reduce dye coupling in human umbilical vein endothelial cells (HUVECs). In our subsequent experiments, we sought to isolate the specific Cx isoform(s) targeted by NO or NO-activated signaling pathways. Since HUVEC cells variably express three Cx (Cx37, Cx40, and Cx43), this latter aim required the use of transfected HeLa cells (HeLaCx37, HeLaCx43), which do not express Cx proteins in their wild type form. Dye coupling was measured by injecting fluorescent dye (e.g., Alexa Fluor 488) into a single cell and determining the number of stained adjacent cells. Application of the NO donor SNAP (2 μM, 20 min) reduced dye coupling in HUVEC by 30%. In HeLa cells, SNAP did not reduce dye transfer of cells expressing Cx43, but decreased the dye transfer from Cx37-expressing cells to Cx43-expressing cells by 76%. The effect of SNAP on dye coupling was not mediated via cGMP. In contrast to its effect on dye coupling, SNAP had no effect on electrical coupling, measured by a double patch clamp in whole cell mode. Our results demonstrate that NO inhibits the intercellular transfer of small molecules by a specific influence on Cx37, suggesting a potential role of NO in controlling certain aspects of vascular GJIC. © 2004 Wiley-Liss, Inc.

Gap junctions are intercellular channels that provide direct passage of small molecules between adjacent cells. In pancreatic acini, the connexin26(Cx26) and connexin32 (Cx32) proteins form functional channels that coordinate the secretion of digestive enzymes. Although the function of Cx26/Cx32 gap junctions are well characterized, the regulatory circuits that control the spatial and temporal expression patterns of these connexin genes are not known. In an effort to identify the molecular pathways that regulate connexin gene expression, we examined Cx26 and Cx32 gene activities in mice lacking the basic helix-loop-helix transcription factor Mist1(Mist1KO). Mist1, Cx26 and Cx32 are co-expressed in most exocrine cell types, and acinar cells from Mist1KO mice exhibit a highly disorganized cellular architecture and an altered pattern of expression for several genes involved in regulated exocytosis. Analysis of Mist1KO mice revealed a dramatic decrease in both connexin proteins, albeit through different molecular mechanisms. Cx32 gene transcription was greatly reduced in all Mist1KO exocrine cells, while Cx26 gene expression remained unaffected. However, in the absence of Cx32 protein, Cx26 did not participate in gap junction formation, leading to a complete lack of intercellular communication among Mist1KO acinar cells. Additional studies testing Mist1 gene constructs in pancreatic exocrine cells confirmed that Mist1 transcriptionally regulates expression of the Cx32 gene. We conclude that Mist1 functions as a positive regulator of Cx32 gene expression and, in its absence, acinar cell gap junctions and intercellular communication pathways become disrupted.

The ability of an arteriole to conduct vasomotor responses along its length contributes to the control of organ perfusion. Sepsis, a systemic inflammatory response to infection, may compromise this control. We aimed to determine whether sepsis, induced by cecal ligation and perforation (CLP), reduces conducted vasoconstriction 24 h post-CLP. We locally stimulated mouse cremaster arterioles with KCl, measured the resulting local and the conducted constriction (500 µm upstream) and, based on these measurements, determined the communication ratio (CR500) as an index of the conducted response. Sepsis significantly reduced the CR500 from 0.75 to 0.20. Based on a mathematical model, this reduction was predicted to have a significant impact on blood flow control. In septic mice, either a 1-hour washout of the cremaster muscle with physiological saline or a treatment of this muscle with the tyrosine kinase inhibitor PP-2 (100 nM) restored the CR500 to the control level. Treatment of septic arterioles with the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (100 µM) partially restored the CR500 from 0.2 to 0.4. In control mice, lipopolysaccharide (LPS; 10 µg/ml) superfused over the cremaster muscle for 1 h reduced the CR500; the nitric oxide (NO) donor S-nitroso-N-acetyl-penicillamine (50 µM) also reduced the CR500. Thus, LPS and NO could be two factors mediating reduced conduction of vasoconstriction in sepsis. We conclude that sepsis reduces the KCl-induced conducted vasoconstriction in the mouse cremaster muscle by a tyrosine kinase- and nitric oxide- dependent mechanism.

Communication of agonist-induced membrane potential changes along blood vessels has been proposed to contribute to the coordination of microvascular function. Factors mediating septic shock may compromise this coordination. Using electrophysiology in a simplified in vitro model of endothelial cells grown as capillary-like structures, we aimed to determine (i) the effect of lipopolysaccharide (LPS) on endothelial cell membrane potential responses to ATP and KCl and (ii) the effect of LPS and nitric oxide (NO) on cell-to-cell communication. Treatment of ‘capillaries’ with LPS (10 µg/ml for 1 h) did not affect local responsiveness to ATP or KCl, but reduced cell communication by a tyrosine-kinase-dependent mechanism. Treatment of ‘capillaries’ with the NO donor DETA (100 µM) or the NO synthase inhibitor L-NAME (100 µM) had no effect on cell communication or the response to LPS. Endogenous NO production, stimulated by LPS + interferon-γ (100 U/ml) treatment, also had no effect on cell communication beyond that of LPS alone. We conclude that LPS, but not NO, can modulate conduction of agonist-induced electrical responses along endothelial capillary-like structures in vitro.

The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1μmol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P's inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019) and AMPK inhibition (80μmol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTRΔF508), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking.

Microvascular endothelial cells (MECs) grown in Matrigel form capillary-like structures. We hypothesized that these "capillaries" better mimic communication properties of microvessels than conventional cell monolayers. MECs were isolated from the rat hindlimb skeletal muscle. Functional communication was tested by visualizing the spread of microinjected 6-carboxyfluorescein (CF) dye and by measuring a conducted change of membrane potential after micropipette application of 500 mM KCl or 10 mM adenosine triphosphate (ATP) on the capillary and monolayer. MECs grown under both conditions were dye-coupled, as demonstrated by the spread of CF injected into a single cell. The membrane potential of cells grown in capillaries (-59 +/- 5 mV) was significantly greater than that of cells grown in monolayers (-24 +/- 2 mV). KCl and ATP caused local depolarization (18 +/- 3 mV) and hyperpolarization (21 +/- 3 mV) in capillaries that yielded conducted 13 +/- 3 mV depolarization and 15 +/- 5 mV hyperpolarization at a 300-microm distal site, respectively. In monolayers, local and distal responses to agonists were 3- to 6-fold and 9- to 10-fold less, respectively, than the corresponding responses in capillaries. Cells grown under both conditions expressed connexin 43, as demonstrated by immunohistochemistry and Western blotting. We conclude that cells grown in capillaries yield substantially larger local and communicated responses than cells in monolayers and thus offer a more sensitive model for mechanistic studies of MEC communication.

Given the roles proposed for gap junctional intercellular communication in neuronal differentiation and growth control, we examined the effects of connexin43 (Cx43) expression in a neuroblastoma cell line. A vesicular stomatitis virus G protein (VSVG)-pseudotyped retrovector was engineered to co-express the green fluorescent protein (GFP) and Cx43 in the communication-deficient neuro-2a (N2a) cell line. The 293 GPG packaging cell line was used to produce VSVG-pseudotyped retrovectors coding for GFP, Cx43, or chimeric Cx43.GFP fusion protein. The titer of viral supernatant, as measured by flow cytometry for GFP fluorescence, was approximately 2.0 x 10(7) colony form units (CFU)/ml and was free of replication-competent retroviruses. After a 7-day treatment with retinoic acid (20 microm), N2a transformants (N2a-Cx43 and N2a-Cx43.GFP) maintained the expression of Cx43 and Cx43.GFP. Expression of both constructs resulted in functional coupling, as evidenced by electrophysiological and dye-injection analysis. Suppression of cell growth correlated with expression of both Cx43 or Cx43.GFP and retinoic acid treatment. Based on morphology and immunocytochemistry for neurofilament, no difference was observed in the differentiation of N2a cells compared with cells expressing Cx43 constructs. In conclusion, constitutive expression of Cx43 in N2a cells does not alter retinoic acid-induced neuronal differentiation but does enhance growth inhibition.

Gap junction communication between microvascular endothelial cells has been proposed to contribute to the coordination of microvascular function. Septic shock may attenuate microvascular cell-to-cell communication. We hypothesized that lipopolysaccharide (LPS) attenuates communication between microvascular endothelial cells derived from rat hindlimb skeletal muscle. Endothelial cells grown in monolayers expressed mRNA for connexin 37, 40, and 43. The expression of connexin 43 protein was confirmed, but connexin 40 protein was not detected by immunocytochemistry or immunoblot analysis. Intercellular resistance between cells of the monolayer, calculated using a Bessel function model, was increased from 3.3 to 5.3 MOmega by LPS. The effect was seen after 1 h of exposure and required a minimum concentration of 10 ng/ml. Intercellular resistance returned to normal 1 h following removal of LPS. Neither the response to LPS, nor its reversal, was blocked by the protein synthesis inhibitor cycloheximide (10 microg/ml). Pretreatment of monolayers with the tyrosine kinase inhibitors PP-2 (10 nM), lavendustin-C (1 microM), and geldanamycin (200 nM) prevented this LPS response; geldanamycin was also able to reverse the response. Inhibitors of MAP kinases, PD 98059 (5 microM) and SB 202190 (5 microM), and PKC (500 nM bisindolylmaleimide I) were unable to block the LPS response. We propose that LPS attenuates cell-to-cell communication through a signaling pathway that is tyrosine kinase dependent.

Background: Subarachnoid hemorrhage (SAH) is characterized by an initial hemorrhagic and ischemic brain injury followed by delayed macro- and microvascular constriction. Large-artery vasospasm and enhanced microcirculatory myogenic tone may contribute to delayed cerebral ischemia. Although this implies that therapeutic interventions must specifically correct the SAH-induced myogenic tone enhancement, current therapeutic approaches non-selectively interfere with vasoconstriction and risk disrupting cerebral autoregulation. This may explain why most interventions do not improve clinical outcome. This study identifies the molecular basis for exacerbated cerebrovascular constriction and validates new targets for SAH treatment. Methods: Wild-type, tumor necrosis factor α (TNFα) knockout, sphingosine-1-kinase (Sphk1) knockout and inducible, smooth muscle cell-targeted TNFα knockout mice were used. SAH was created by injection of 80 μl of arterial blood into the prechiasmatic cistern. Myogenic tone in the olfactory artery was assessed with a myograph system. Standard procedures for fluorescent immunolocalization, Western blotting and assessment of apoptosis were used. Results: SAH increased myogenic tone and vascular wall TNFα expression, without enhancing overall vascular contractility in response to phenylephrine. Knockout of TNFα globally or smooth muscle cell-specifically prevented SAH-induced increased myogenic tone. Inhibition of TNFα-shedding (TAPI, 50 μmol/L) or receptor-binding (etanercept, 10 mg/ml) eliminated SAH-mediated myogenic tone augmentation. Cystic fibrosis transmembrane regulator (CFTR) protein expression was down-regulated in cerebral arteries after SAH, which was abolished by antagonism of TNFα. Genetic mouse models confirmed that S1P signaling mediates the myogenic tone augmentation in SAH. Finally, disrupting TNFα signaling attenuated neuronal apoptosis in SAH animals. Conclusion: We identify a novel smooth muscle cell autocrine/paracrine signaling network that augments myogenic tone in SAH. It links TNFα, CFTR and sphingosine-1-phosphate (S1P) signaling. Targeting TNFα and the S1P 2 receptor subtype are potential therapeutic options to improve clinical outcome in SAH.

Background— Heart failure is associated with neurological deficits, including cognitive dysfunction. However, the molecular mechanisms underlying reduced cerebral blood flow in the early stages of heart failure, particularly when blood pressure is minimally affected, are not known. Methods and Results— Using a myocardial infarction model in mice, we demonstrate a tumor necrosis factor-α (TNFα)–dependent enhancement of posterior cerebral artery tone that reduces cerebral blood flow before any overt changes in brain structure and function. TNFα expression is increased in mouse posterior cerebral artery smooth muscle cells at 6 weeks after myocardial infarction. Coordinately, isolated posterior cerebral arteries display augmented myogenic tone, which can be fully reversed in vitro by the competitive TNFα antagonist etanercept. TNFα mediates its effect via a sphingosine-1-phosphate (S1P)–dependent mechanism, requiring sphingosine kinase 1 and the S1P 2 receptor. In vivo, sphingosine kinase 1 deletion prevents and etanercept (2-week treatment initiated 6 weeks after myocardial infarction) reverses the reduction of cerebral blood flow, without improving cardiac function. Conclusions— Cerebral artery vasoconstriction and decreased cerebral blood flow occur early in an animal model of heart failure; these perturbations are reversed by interrupting TNFα/S1P signaling. This signaling pathway may represent a potential therapeutic target to improve cognitive function in heart failure.

Background— Sphingosine-1-phosphate (S1P) signaling is a central regulator of resistance artery tone. Therefore, S1P levels need to be tightly controlled through the delicate interplay of its generating enzyme sphingosine kinase 1 and its functional antagonist S1P phosphohydrolase-1. The intracellular localization of S1P phosphohydrolase-1 necessitates the import of extracellular S1P into the intracellular compartment before its degradation. The present investigation proposes that the cystic fibrosis transmembrane conductance regulator transports extracellular S1P and hence modulates microvascular S1P signaling in health and disease. Methods and Results— In cultured murine vascular smooth muscle cells in vitro and isolated murine mesenteric and posterior cerebral resistance arteries ex vivo, the cystic fibrosis transmembrane conductance regulator (1) is critical for S1P uptake; (2) modulates S1P-dependent responses; and (3) is downregulated in vitro and in vivo by tumor necrosis factor-α, with significant functional consequences for S1P signaling and vascular tone. In heart failure, tumor necrosis factor-α downregulates the cystic fibrosis transmembrane conductance regulator across several organs, including the heart, lung, and brain, suggesting that it is a fundamental mechanism with implications for systemic S1P effects. Conclusions— We identify the cystic fibrosis transmembrane conductance regulator as a critical regulatory site for S1P signaling; its tumor necrosis factor-α–dependent downregulation in heart failure underlies an enhancement in microvascular tone. This molecular mechanism potentially represents a novel and highly strategic therapeutic target for cardiovascular conditions involving inflammation.

Cells sense and respond to changes in oxygen concentration through gene regulatory processes that are fundamental to survival. Surprisingly, little is known about how anemia affects hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular responses to acute hypoxia, we defined the effects of NOS deficiency in acute anemia. In contrast to endothelial NOS or inducible NOS deficiency, neuronal NOS (nNOS) −/− mice demonstrated increased mortality during anemia. Unlike wild-type (WT) animals, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS −/− mice. At the cellular level, anemia increased expression of HIF-1α protein and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK1) in the brain of WT, but not nNOS −/− mice, despite comparable reductions in tissue PO 2 . Paradoxically, nNOS −/− mice survived longer during hypoxia, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. Real-time imaging of transgenic animals expressing a reporter HIF-α(ODD)-luciferase chimeric protein confirmed that nNOS was essential for anemia-mediated increases in HIF-α protein stability in vivo. S -nitrosylation effects the functional interaction between HIF and pVHL. We found that anemia led to nNOS-dependent S -nitrosylation of pVHL in vivo and, of interest, led to decreased expression of GSNO reductase. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo and provide essential mechanistic insight into the differences between anemia and hypoxia.

Acute β-blockade with metoprolol has been associated with increased mortality by undefined mechanisms. Since metoprolol is a relatively high affinity blocker of β2-adrenoreceptors, we hypothesized that some of the increased mortality associated with its use may be due to its abrogation of β2-adrenoreceptor-mediated vasodilation of microvessels in different vascular beds. Cardiac output (CO; pressure volume loops), mean arterial pressure (MAP), relative cerebral blood flow (rCBF; laser Doppler), and microvascular brain tissue Po2(G2 oxyphor) were measured in anesthetized mice before and after acute treatment with metoprolol (3 mg/kg iv). The vasodilatory dose responses to β-adrenergic agonists (isoproterenol and clenbuterol), and the myogenic response, were assessed in isolated mesenteric resistance arteries (MRAs; ∼200-μm diameter) and posterior cerebral arteries (PCAs ∼150-μm diameter). Data are presented as means ± SE with statistical significance applied at P < 0.05. Metoprolol treatment did not effect MAP but reduced heart rate and stroke volume, CO, rCBF, and brain microvascular Po2, while concurrently increasing systemic vascular resistance ( P < 0.05 for all). In isolated MRAs, metoprolol did not affect basal artery tone or the myogenic response, but it did cause a dose-dependent impairment of isoproterenol- and clenbuterol-induced vasodilation. In isolated PCAs, metoprolol (50 μM) impaired maximal vasodilation in response to isoproterenol. These data support the hypothesis that acute administration of metoprolol can reduce tissue oxygen delivery by impairing the vasodilatory response to β2-adrenergic agonists. This mechanism may contribute to the observed increase in mortality associated with acute administration of metoprolol in perioperative patients.

Objective— The purpose of this study was to characterize a phosphorylation motif at serine 225 as a molecular switch that regulates the pressure-dependent activation of sphingosine kinase 1 (Sk1) in resistance artery smooth muscle cells. Methods and Results— In isolated hamster gracilis muscle resistance arteries, pressure-dependent activation/translocation of Sk1 by ERK1/2 was critically dependent on its serine 225 phosphorylation site. Specifically, expression of Sk1 S225A reduced resting and myogenic tone, resting Ca 2+ , pressure-induced Ca 2+ elevations, and Ca 2+ sensitivity. The lack of function of the Sk1 S225A mutant could not be entirely overcome by forced localization to the plasma membrane via a myristoylation/palmitylation motif; the membrane anchor also significantly inhibited the function of the wild-type Sk1 enzyme. In both cases, Ca 2+ sensitivity and myogenic tone were attenuated, whereas Ca 2+ handling was normalized/enhanced. These discrete effects are consistent with cell surface receptor-mediated effects (Ca 2+ sensitivity) and intracellular effects of S1P (Ca 2+ handling). Accordingly, S1P 2 receptor inhibition (1μmol/L JTE013) attenuated myogenic tone without effect on Ca 2+ . Conclusions— Translocation and precise subcellular positioning of Sk1 is essential for full Sk1 function; and two distinct S1P pools, proposed to be intra- and extracellular, contribute to the maintenance of vascular tone.

Sphingosine-1-phosphate (S1P), which mediates pleiotropic actions within the vascular system, is a prominent regulator of microvascular tone. By virtue of its S1P-degrading function, we hypothesized that S1P-phosphohydrolase 1 (SPP1) is an important regulator of tone in resistance arteries. Hamster gracilis muscle resistance arteries express mRNA encoding SPP1. Overexpression of SPP1 (via transfection of a SPP1 wt ) reduced resting tone, Ca 2+ sensitivity, and myogenic vasoconstriction, whereas reduced SPP1 expression (antisense oligonucleotides) yielded the opposite effects. Expression of a phosphatase-dead mutant of SPP1 (SPP1 H208A ) had no effect on any parameter tested, suggesting that catalytic activity of SPP1 is critical. The enhanced myogenic tone that follows overexpression of S1P-generating enzyme sphingosine kinase 1 (Sk1 wt ) was functionally antagonized by coexpression with SPP1 wt but not SPP1 H208A . SPP1 modulated vasoconstriction in response to 1 to 100 nmol/L exogenous S1P, a concentration range that was characterized as S1P 2 -dependent, based on the effect of S1P 2 inhibition by antisense oligonucleotides and 1 μmol/L JTE013. Inhibition of the cystic fibrosis transmembrane regulator (CFTR) (1) restored S1P responses that were attenuated by SPP1 wt overexpression; (2) enhanced myogenic vasoconstriction; but (3) had no effect on noradrenaline responses. We conclude that SPP1 is an endogenous regulator of resistance artery tone that functionally antagonizes the vascular effects of both Sk1 wt and S1P 2 receptor activation. SPP1 accesses extracellular S1P pools in a manner dependent on a functional CFTR transport protein. Our study assigns important roles to both SPP1 and CFTR in the physiological regulation of vascular tone, which influences both tissue perfusion and systemic blood pressure.

Objective: Although nitric oxide (NO) is a known regulator of cardiovascular function, the effect of NO overproduction during sepsis on capillary oxygen transport and local tissue oxygen consumption is not well understood. The objectives of this study were to determine whether sepsis-induced NO overproduction increased capillary stopped-flow and modulated tissue oxygen consumption in skeletal muscle. Design: Prospective, controlled laboratory study. Setting: Animal laboratory in a university-affiliated research institute. Subjects: Male Sprague-Dawley rats, 165-180 g body weight. Interventions: Rats were made septic by cecal ligation and perforation (CLP) and were then ventilated and volume resuscitated (saline). The hind limb extensor digitorum longus (EDL) skeletal muscle was blunt dissected for in vivo microvascular imaging. The inducible NO synthase (iNOS) inhibitor L-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL) was infused (3 mg/kg body weight per hour) starting 1 hr post-CLP to maintain arterial blood and EDL tissue NO," (NO 2 - + NO 3 - ) at baseline. Measurements and Main Results: Red blood cell hemodynamics, hemoglobin oxygen saturation, capillary geometry, and functional capillary density information were used to calculate capillary oxygen flux (the rate of oxygen diffusion from capillary to tissue) and indices of local oxygen delivery and tissue oxygen consumption. Over the first 5 hrs of septic injury, mean arterial pressure decreased while capillary stopped-flow and capillary oxygen flux both increased (p

The mechanism of neuronal nitric oxide synthase (nNOS)-dependent reduction in arteriolar conducted vasoconstriction in sepsis, and the possible protection by antioxidants, are unknown. The authors hypothesized that ascorbate inhibits the conduction deficit by reducing nNOS-derived NO production.Using intravital microscopy and the cecal ligation and perforation (CLP) model of sepsis (24 h), arterioles in the cremaster muscle of male C57BL/6 wild-type mice were locally stimulated with KCl to initiate conducted vasoconstriction. The authors used the ratio of conducted constriction (500 microm upstream) to local constriction as an index of conduction (CR500). Cremaster muscle NOS enzymatic activity and protein expression, and plasma nitrite/nitrate levels were determined in control and septic mice. Intravenous ascorbate bolus (200 mg/kg in 0.1 ml of saline) was given early (0 h) or delayed at 23 h post CLP.Sepsis reduced CR500 from 0.73 +/- 0.03 to 0.21 +/- 0.03, increased nNOS activity from 87 +/- 9 to 220 +/- 29 pmol/mg/h and nitrite/nitrate from 16 +/- 1 to 39 +/- 3 microM, without affecting nNOS protein expression. Ascorbate at 0 and 23 h prevented/reversed the conduction deficit and the increases in nNOS activity and nitrite/nitrate level. NO donor SNAP (S-nitroso-N-acetylpenicillamine) reestablished the conduction deficit in ascorbate-treated septic mice. Superoxide scavenger MnTBAP (Mn(III)tetrakis(4-benzoic acid)porphyrin chloride) did not affect this deficit.These data indicate that early and delayed intravenous boluses of ascorbate prevent/reverse sepsis-induced deficit in arteriolar conducted vasoconstriction in the cremaster muscle by inhibiting nNOS-derived NO production.

Growing evidence suggests that mechanisms which regulate the Ca2+ sensitivity of the contractile apparatus in vascular smooth muscle cells form the backbone of pressure-induced myogenic vasoconstriction. The modulation of Ca2+ sensitivity is suited to partially uncouple intracellular Ca2+ from constriction, thereby allowing the maintenance of tone with fully conserved function of other Ca2+-dependent processes. Following a brief review of 'classical' Ca2+-dependent signalling pathways involved in the myogenic response, the present review describes the emerging mechanisms that promote myogenic vasoconstriction via modulation of Ca2+ sensitivity. For the purpose of this review, Ca2+ sensitivity reflects the dynamic equilibrium between myosin light-chain kinase and myosin light-chain phosphatase activities in terms of its impact on vascular tone. Several signalling pathways (PKC, RhoA/Rho kinase, ROS) which have been identified as prominent regulators of Ca2+ sensitivity will be discussed. Although Ca2+ sensitivity modulation is clearly an important component of the myogenic response, attempts to integrate it into existing mechanistic models resulted in a two-phase model, with a predominant Ca2+-dependent 'initiation/trigger' phase followed by a Ca2+-independent 'maintenance' phase. We propose that the two-phase model is rather simplistic, because the literature reviewed here demonstrates that Ca2+-dependent and -independent mechanisms do not operate in isolation and are important at all stages of the response. The regulation of Ca2+ sensitivity, as an equal and complimentary partner of Ca2+-dependent processes, significantly enhances our understanding of the complex array of signalling pathways, which ultimately mediate the myogenic response.

Growing evidence suggests that mechanisms which regulate the Ca2+ sensitivity of the contractile apparatus in vascular smooth muscle cells form the backbone of pressure-induced myogenic vasoconstriction. The modulation of Ca2+ sensitivity is suited to partially uncouple intracellular Ca2+ from constriction, thereby allowing the maintenance of tone with fully conserved function of other Ca2+-dependent processes. Following a brief review of 'classical' Ca2+-dependent signalling pathways involved in the myogenic response, the present review describes the emerging mechanisms that promote myogenic vasoconstriction via modulation of Ca2+ sensitivity. For the purpose of this review, Ca2+ sensitivity reflects the dynamic equilibrium between myosin light-chain kinase and myosin light-chain phosphatase activities in terms of its impact on vascular tone. Several signalling pathways (PKC, RhoA/Rho kinase, ROS) which have been identified as prominent regulators of Ca2+ sensitivity will be discussed. Although Ca2+ sensitivity modulation is clearly an important component of the myogenic response, attempts to integrate it into existing mechanistic models resulted in a two-phase model, with a predominant Ca2+-dependent 'initiation/trigger' phase followed by a Ca2+-independent 'maintenance' phase. We propose that the two-phase model is rather simplistic, because the literature reviewed here demonstrates that Ca2+-dependent and -independent mechanisms do not operate in isolation and are important at all stages of the response. The regulation of Ca2+ sensitivity, as an equal and complimentary partner of Ca2+-dependent processes, significantly enhances our understanding of the complex array of signalling pathways, which ultimately mediate the myogenic response.

The S1P(2) receptor is a member of a family of G protein-coupled receptors that bind the extracellular sphingolipid metabolite sphingosine 1-phosphate with high affinity. The receptor is widely expressed and linked to multiple G protein signaling pathways, but its physiological function has remained elusive. Here we have demonstrated that S1P(2) receptor expression is essential for proper functioning of the auditory and vestibular systems. Auditory brainstem response analysis revealed that S1P(2) receptor-null mice were deaf by one month of age. These null mice exhibited multiple inner ear pathologies. However, some of the earliest cellular lesions in the cochlea were found within the stria vascularis, a barrier epithelium containing the primary vasculature of the inner ear. Between 2 and 4 weeks after birth, the basal and marginal epithelial cell barriers and the capillary bed within the stria vascularis of the S1P(2) receptor-null mice showed markedly disturbed structures. JTE013, an S1P(2) receptor-specific antagonist, blocked the S1P-induced vasoconstriction of the spiral modiolar artery, which supplies blood directly to the stria vascularis and protects its capillary bed from high perfusion pressure. Vascular disturbance within the stria vascularis is a potential mechanism that leads to deafness in the S1P(2) receptor-null mice.