Circadian Rhythmicity in Cerebral Microvascular Tone Influences Subarachnoid Hemorrhage-Induced Injury.

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.

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

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

Priming of hypoxia-inducible factor by neuronal nitric oxide synthase is essential for adaptive responses to severe anemia.

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.

Female mice display sex-specific differences in cerebrovascular function and subarachnoid haemorrhage-induced injury.

BACKGROUND: In male mice, a circadian rhythm in myogenic reactivity influences the extent of brain injury following subarachnoid haemorrhage (SAH). We hypothesized that female mice have a different cerebrovascular phenotype and consequently, a distinct SAH-induced injury phenotype. METHODS: SAH was modelled by pre-chiasmatic blood injection. Olfactory cerebral resistance arteries were functionally assessed by pressure myography; these functional assessments were related to brain histology and neurobehavioral assessments. Cystic fibrosis transmembrane conductance regulator (CFTR) expression was assessed by PCR and Western blot. We compared non-ovariectomized and ovariectomized mice. FINDINGS: Cerebrovascular myogenic reactivity is not rhythmic in females and no diurnal differences in SAH-induced injury are observed; ovariectomy does not unmask a rhythmic phenotype for any endpoint. CFTR expression is rhythmic, with similar expression levels compared to male mice. CFTR inhibition studies, however, indicate that CFTR activity is lower in female arteries. Pharmacologically increasing CFTR expression in vivo (3 mg/kg lumacaftor for 2 days) reduces myogenic tone at Zeitgeber time 11, but not Zeitgeber time 23. Myogenic tone is not markedly augmented following SAH in female mice and lumacaftor loses its ability to reduce myogenic tone; nevertheless, lumacaftor confers at least some injury benefit in females with SAH. INTERPRETATION: Female mice possess a distinct cerebrovascular phenotype compared to males, putatively due to functional differences in CFTR regulation. This sex difference eliminates the CFTR-dependent cerebrovascular effects of SAH and may alter the therapeutic efficacy of lumacaftor compared to males. FUNDING: Brain Aneurysm Foundation, Heart and Stroke Foundation and Ted Rogers Centre for Heart Research.

Tumor necrosis factor-α-mediated downregulation of the cystic fibrosis transmembrane conductance regulator drives pathological sphingosine-1-phosphate signaling in a mouse model of 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.

Proximal cerebral arteries develop myogenic responsiveness in heart failure via tumor necrosis factor-α-dependent activation of sphingosine-1-phosphate signaling.

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.

Disrupting circadian control of peripheral myogenic reactivity mitigates cardiac injury following myocardial infarction.

AIMS: 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) disrupting this rhythmicity would alter cardiac injury post-myocardial infarction (MI). METHODS AND RESULTS: 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 tumour 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 is 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. CONCLUSIONS: 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.

The emerging significance of circadian rhythmicity in microvascular resistance.

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.

Restoring myocardial infarction-induced long-term memory impairment by targeting the cystic fibrosis transmembrane regulator.

BACKGROUND: Cognitive impairment is a serious comorbidity in heart failure patients, but effective therapies are lacking. We investigated the mechanisms that alter hippocampal neurons following myocardial infarction (MI). METHODS: MI was induced in male C57Bl/6 mice by left anterior descending coronary artery ligation. We utilised standard procedures to measure cystic fibrosis transmembrane regulator (CFTR) protein levels, inflammatory mediator expression, neuronal structure, and hippocampal memory. Using in vitro and in vivo approaches, we assessed the role of neuroinflammation in hippocampal neuron degradation and the therapeutic potential of CFTR correction as an intervention. FINDINGS: Hippocampal dendrite length and spine density are reduced after MI, effects that associate with decreased neuronal CFTR expression and concomitant microglia activation and inflammatory cytokine expression. Conditioned medium from lipopolysaccharide-stimulated microglia (LCM) reduces neuronal cell CFTR protein expression and the mRNA expression of the synaptic regulator post-synaptic density protein 95 (PSD-95) in vitro. Blocking CFTR activity also down-regulates PSD-95 in neurons, indicating a relationship between CFTR expression and neuronal health. Pharmacologically correcting CFTR expression in vitro rescues the LCM-mediated down-regulation of PSD-95. In vivo, pharmacologically increasing hippocampal neuron CFTR expression improves MI-associated alterations in neuronal arborisation, spine density, and memory function, with a wide therapeutic time window. INTERPRETATION: Our results indicate that CFTR therapeutics improve inflammation-induced alterations in hippocampal neuronal structure and attenuate memory dysfunction following MI. FUNDING: Knut and Alice Wallenberg Foundation [F 2015/2112]; Swedish Research Council [VR; 2017-01243]; the German Research Foundation [DFG; ME 4667/2-1]; Hjärnfonden [FO2021-0112]; The Crafoord Foundation; Åke Wibergs Stiftelse [M19-0380], NMMP 2021 [V2021-2102]; the Albert Påhlsson Research Foundation; STINT [MG19-8469], Lund University; Canadian Institutes of Health Research [PJT-153269] and a Heart and Stroke Foundation of Ontario Mid-Career Investigator Award.

Cerebral Autoregulation in Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a devastating stroke subtype with a high rate of mortality and morbidity. The poor clinical outcome can be attributed to the biphasic course of the disease: even if the patient survives the initial bleeding emergency, delayed cerebral ischemia (DCI) frequently follows within 2 weeks time and levies additional serious brain injury. Current therapeutic interventions do not specifically target the microvascular dysfunction underlying the ischemic event and as a consequence, provide only modest improvement in clinical outcome. SAH perturbs an extensive number of microvascular processes, including the "automated" control of cerebral perfusion, termed "cerebral autoregulation." Recent evidence suggests that disrupted cerebral autoregulation is an important aspect of SAH-induced brain injury. This review presents the key clinical aspects of cerebral autoregulation and its disruption in SAH: it provides a mechanistic overview of cerebral autoregulation, describes current clinical methods for measuring autoregulation in SAH patients and reviews current and emerging therapeutic options for SAH patients. Recent advancements should fuel optimism that microvascular dysfunction and cerebral autoregulation can be rectified in SAH patients.

Tumor necrosis factor-α enhances microvascular tone and reduces blood flow in the cochlea via enhanced sphingosine-1-phosphate signaling.

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.

Role of sphingosine-1-phosphate phosphohydrolase 1 in the regulation of resistance artery 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, Ca2+ 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 S1P2-dependent, based on the effect of S1P(2) inhibition by antisense oligonucleotides and 1 mumol/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 S1P2 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.

The phosphorylation motif at serine 225 governs the localization and function of sphingosine kinase 1 in resistance arteries.

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 (1 micromol/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.

A Scientific Rationale for Using Cystic Fibrosis Transmembrane Conductance Regulator Therapeutics in COVID-19 Patients.

Several pathological manifestations in coronavirus disease 2019 (COVID-19), including thick mucus, poor mucociliary clearance, and bronchial wall thickening, overlap with cystic fibrosis disease patterns and may be indicative of "acquired" cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. Indeed, tumor necrosis factor (TNF), a key cytokine driving COVID-19 pathogenesis, downregulates lung CFTR protein expression, providing a strong rationale that acquired CFTR dysfunction arises in the context of COVID-19 infection. In this perspective, we propose that CFTR therapeutics, which are safe and generally well-tolerated, may provide benefit to COVID-19 patients. Although CFTR therapeutics are currently only approved for treating cystic fibrosis, there are efforts to repurpose them for conditions with "acquired" CFTR dysfunction, for example, chronic obstructive pulmonary disease. In addition to targeting the primary lung pathology, CFTR therapeutics may possess value-added effects: their anti-inflammatory properties may dampen exaggerated immune cell responses and promote cerebrovascular dilation; the latter aspect may offer some protection against COVID-19 related stroke.

Stabilizing Cellular Barriers: Raising the Shields Against COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its clinical manifestation (COVID-19; coronavirus disease 2019) have caused a worldwide health crisis. Disruption of epithelial and endothelial barriers is a key clinical turning point that differentiates patients who are likely to develop severe COVID-19 outcomes: it marks a significant escalation in respiratory symptoms, loss of viral containment and a progression toward multi-organ dysfunction. These barrier mechanisms are independently compromised by known COVID-19 risk factors, including diabetes, obesity and aging: thus, a synergism between these underlying conditions and SARS-CoV-2 mechanisms may explain why these risk factors correlate with more severe outcomes. This review examines the key cellular mechanisms that SARS-CoV-2 and its underlying risk factors utilize to disrupt barrier function. As an outlook, we propose that glucagon-like peptide 1 (GLP-1) may be a therapeutic intervention that can slow COVID-19 progression and improve clinical outcome following SARS-CoV-2 infection. GLP-1 signaling activates barrier-promoting processes that directly oppose the pro-inflammatory mechanisms commandeered by SARS-CoV-2 and its underlying risk factors.

Experimental Subarachnoid Hemorrhage Drives Catecholamine-Dependent Cardiac and Peripheral Microvascular Dysfunction.

Subarachnoid hemorrhage (SAH) is a devastating cerebral event caused by an aneurysmal rupture. In addition to neurological injury, SAH has significant effects on cardiac function and the peripheral microcirculation. Since these peripheral complications may exacerbate brain injury, the prevention and management of these peripheral effects are important for improving the overall clinical outcome after SAH. In this investigation, we examined the effects of SAH on cardiac function and vascular reactivity in a well-characterized blood injection model of SAH. Standard echocardiographic and blood pressure measurement procedures were utilized to assess cardiac function and hemodynamic parameters in vivo; we utilized a pressure myography approach to assess vascular reactivity in cremaster skeletal muscle resistance arteries ex vivo. We observed that elevated catecholamine levels in SAH stun the myocardium, reduce cardiac output and augment myogenic vasoconstriction in isolated cremaster arteries. These cardiac and vascular effects are driven by beta- and alpha-adrenergic receptor signaling, respectively. Clinically utilized adrenergic receptor antagonists can prevent cardiac injury and normalize vascular function. We found that tumor necrosis factor (TNF) gene deletion prevents the augmentation of myogenic reactivity in SAH: since membrane-bound TNF serves as a mechanosensor in the arteries assessed, alpha-adrenergic signaling putatively augments myogenic vasoconstriction by enhancing mechanosensor activity.

The emerging role of Ca2+ sensitivity regulation in promoting myogenic vasoconstriction.

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.

CFTR Therapeutics Normalize Cerebral Perfusion Deficits in Mouse Models of Heart Failure and Subarachnoid Hemorrhage.

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.

Inhibiting nitric oxide overproduction during hypotensive sepsis increases local oxygen consumption in rat skeletal muscle.

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(x)(-) (NO2(-) + NO3(-)) 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 < .05). Inhibiting iNOS/NO overproduction partially restored mean arterial pressure and increased arterial pH. Within the microcirculation, inhibiting NO increased capillary red cell velocity and increased local tissue oxygen consumption (p < .05). Inhibiting NO failed, however, to prevent capillary stopped-flow. CONCLUSIONS: During the onset of sepsis, concurrent with the onset of microvascular dysfunction, there is an iNOS/NO-mediated reduction in local skeletal muscle tissue oxygen consumption.

Deletion of neuronal NOS prevents impaired vasodilation in septic mouse skeletal muscle.

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 approximately 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.