NEAT1 inhibits the angiogenic activity of cerebral arterial endothelial cells by inducing the M1 polarization of microglia through the AMPK signaling pathway.

BACKGROUND: Enhancing angiogenesis may be an effective strategy to promote functional recovery after ischemic stroke. Inflammation regulates angiogenesis. Microglia are crucial cells that initiate inflammatory responses after various brain injuries. Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) plays a role in regulating brain injury. This study aimed to explore the effects of NEAT1-regulated microglial polarization on the neovascularization capacity of cerebrovascular endothelial cells and the underlying molecular regulatory mechanisms. METHODS: Mouse cerebral arterial endothelial cells (mCAECs) were co-cultured with BV-2 cells in different groups using a Transwell system. NEAT1 expression levels were measured by fluorescence quantitative reverse transcription PCR. Levels of IL-1ß, IL-6, TNF-α, Arg-1, IL-4, and IL-10 were determined using ELISA. Expression levels of CD86 and CD163 were detected by immunofluorescence. The neovascularization capacity of mCAECs was assessed using CCK-8, Transwell, Transwell-matrigel, and tube formation assays. Label-free quantification proteomics was carried out to identify differentially expressed proteins. Protein levels were measured by Western blotting. RESULTS: NEAT1 overexpression induced M1 polarization in BV-2 cells, whereas NEAT1 knockdown blocked lipopolysaccharide-induced M1 polarization in microglia. NEAT1-overexpressing BV-2 cells suppressed the angiogenic ability of mCAECs, and NEAT1-knocking BV-2 cells promoted the angiogenic ability of mCAECs under lipopolysaccharide treatment. Label-free quantitative proteomic analysis identified 144 upregulated and 131 downregulated proteins that were induced by NEAT1 overexpression. The AMP-activated protein kinase (AMPK) signaling pathway was enriched in the Kyoto Encyclopedia of Genes and Genomes analysis of the differentially expressed proteins. Further verification showed that NEAT1 inactivated the AMPK signaling pathway. Moreover, the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide reversed the effect of NEAT1 on BV-2 polarization and the regulatory effect of NEAT1-overexpressing BV-2 cells on the angiogenic ability of mCAECs. CONCLUSIONS: NEAT1 inhibits the angiogenic activity of mCAECs by inducing M1 polarization of BV-2 cells through the AMPK signaling pathway. This study further clarified the impact and mechanism of NEAT1 on microglia and the angiogenic ability of cerebrovascular endothelial cells.

Influence of nimodipine combined with ulinastatin on neurological function and inflammatory reaction in patients with cerebral vasospasm after subarachnoid hemorrhage.

OBJECTIVE: This study aimed to discuss the influence of nimodipine+ulinastatin on the neurological function and inflammatory reaction in patients with cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH). METHODS: Overall, 90 patients with CVS after SAH who were admitted to our hospital were enrolled in this study and randomly divided into research and control groups (n = 45 for both groups). On the basis of conventional therapy, patients in the control group were injected with ulinastatin and those in the research group were injected with ulinastatin+nimodipine through an intravenous drip for 7 days with the others the same as those of the control group. RESULTS: Blood flow velocity in all cerebral arteries was lower in the research group than in the control group after treatment (P < 0.05). Calcitonin gene-related peptide and nitric oxide levels were higher in the research group than in the control group after treatment (P < 0.05). Endothelin levels were lower in the research group than in the control group (P < 0.05). The total effective rate was higher in the research group than in the control group (P < 0.05). Glasgow Coma Scale scores were higher in the research group than in the control group (P < 0.05). CONCLUSION: The drug combination of nimodipine and ulinastatin improved blood flow and neurological function in patients with CVS after SAH and enhanced the therapeutic efficacy; the underlying mechanism may be associated with the regulation of vascular endothelial dilatation function and the inhibition of relevant inflammatory factors' expression.

Embolic Stroke Model with Magnetic Nanoparticles.

Stroke models are vital tools in neuropharmacology and rehabilitation research. However, a classic and widely used model-the suture occlusion model-is not suitable for all research approaches, especially regarding thrombolysis. For embolic stroke models in thrombolytic research, the surgical procedures of thrombin injection in the middle cerebral artery or clot injection in the carotid artery involved are too sophisticated. Here, we report a new stroke model in mice that uses magnetic nanoparticle (MNP) cross-linked with thrombin to embolize. Briefly, after the magnet was positioned in the common carotid artery, MNP@Thrombin was injected from the tail vein. Within several minutes postinjection, the MNP@Thrombin accumulated in the carotid artery and induced thrombus formation. These complex clots were flushed into and subsequently blocked the cerebral artery. Collectively, these results suggested that this new method was a quick and easy stroke model that blocked hemisphere blood flow and damaged neural function. Importantly, this model had an excellent response to thrombolytic drugs. After urokinase injection, cerebral blood flow was restored and symptom scores were enhanced by nearly one. This method, including a quick synthesis of MNP and thrombin, provided an easy and minimally invasive process for a new stroke model that is usable in both pharmacological and rehabilitative research.

Cardiovascular comorbidities, inflammation, and cerebral small vessel disease.

Cerebral small vessel disease (cSVD) is the most common cause of vascular cognitive impairment and affects all levels of the brain's vasculature. Features include diverse structural and functional changes affecting small arteries and capillaries that lead to a decline in cerebral perfusion. Due to an ageing population, incidence of cSVD is continually rising. Despite its prevalence and its ability to cause multiple debilitating illnesses, such as stroke and dementia, there are currently no therapeutic strategies for the treatment of cSVD. In the healthy brain, interactions between neuronal, vascular, and inflammatory cells are required for normal functioning. When these interactions are disturbed, chronic pathological inflammation can ensue. The interplay between cSVD and inflammation has attracted much recent interest, and this review discusses chronic cardiovascular diseases, particularly hypertension, and explores how the associated inflammation may impact on the structure and function of the small arteries of the brain in cSVD. Molecular approaches in animal studies are linked to clinical outcomes in patients, and novel hypotheses regarding inflammation and cSVD are proposed that will hopefully stimulate further discussion and study in this important area.

Reversible Cerebral Vasoconstriction Syndrome Following Exposure to Oleoresin Capsicum "Pepper Spray".

OBJECTIVES: To report a case associating the use of Oleoresin Capsicum Pepper Spray (OCPS) during law enforcement training with development of Reversible Cerebral Vasoconstriction Syndrome (RCVS). MATERIALS AND METHODS: RCVS is radiographically characterized by multifocal smooth narrowing of cerebral arteries heralded by clinical manifestations of recurrent thunderclap headaches. 70% of cases with RCVS have a clear precipitating factor and agents commonly implicated were cannabis, selective serotonin reuptake inhibitors, nasal decongestants, cocaine, postpartum state, eclampsia and strenuous physical/sexual activity.1 RESULTS: 24-year-old female police officer with no past medical history who presented with thunderclap headaches after exposure to pepper spray to her face during work training. Neurological examination was unremarkable. CT angiogram (CTA) of the head and neck and subsequent conventional angiogram revealed multifocal mild arterial narrowing of bilateral middle cerebral arteries (MCA), bilateral posterior cerebral arteries (PCA) and left anterior cerebral artery (ACA) concerning for RCVS. Eight weeks later, she had a repeat MRA head and neck demonstrating complete resolution of the previously noted narrowing of her cerebral arteries. CONCLUSIONS: OCPS is widely used in law enforcement training as well as by general population as a self- defense tool. It is generally assumed to be safe, although the consequences of its use can never be predicted with certainty.2 As our case highlights, use of OCPS may be associated with development of RCVS and awareness needs to be raised regarding this rare but serious complication.

BK channel-forming slo1 proteins mediate the brain artery constriction evoked by the neurosteroid pregnenolone.

Pregnenolone is a neurosteroid that modulates glial growth and differentiation, neuronal firing, and several brain functions, these effects being attributed to pregnenolone actions on the neurons and glial cells themselves. Despite the vital role of the cerebral circulation for brain function and the fact that pregnenolone is a vasoactive agent, pregnenolone action on brain arteries remain unknown. Here, we obtained in vivo concentration response curves to pregnenolone on middle cerebral artery (MCA) diameter in anesthetized male and female C57BL/6J mice. In both male and female animals, pregnenolone (1 nM-100 µM) constricted MCA in a concentration-dependent manner, its maximal effect reaching ~22-35% decrease in diameter. Pregnenolone action was replicated in intact and de-endothelialized, in vitro pressurized MCA segments with pregnenolone evoking similar constriction in intact and de-endothelialized MCA. Neurosteroid action was abolished by 1 µM paxilline, a selective blocker of Ca2+ - and voltage-gated K+ channels of large conductance (BK). Cell-attached, patch-clamp recordings on freshly isolated smooth muscle cells from mouse MCAs demonstrated that pregnenolone at concentrations that constricted MCAs in vitro and in vivo (10 µM), reduced BK activity (NPo), with an average decrease in NPo reaching 24.2%. The concentration-dependence of pregnenolone constriction of brain arteries and inhibition of BK activity in intact cells were paralleled by data obtained in cell-free, inside-out patches, with maximal inhibition reached at 10 µM pregnenolone. MCA smooth muscle BKs include channel-forming α (slo1 proteins) and regulatory ß1 subunits, encoded by KCNMA1 and KCNMB1, respectively. However, pregnenolone-driven decrease in NPo was still evident in MCA myocytes from KCNMB1-/- mice. Following reconstitution of slo1 channels into artificial, binary phospholipid bilayers, 10 µM pregnenolone evoked slo1 NPo inhibition which was similar to that seen in native membranes. Lastly, pregnenolone failed to constrict MCA from KCNMA1-/- mice. In conclusion, pregnenolone constricts MCA independently of neuronal, glial, endothelial and circulating factors, as well as of cell integrity, organelles, complex membrane cytoarchitecture, and the continuous presence of cytosolic signals. Rather, this action involves direct inhibition of SM BK channels, which does not require ß1 subunits but is mediated through direct sensing of the neurosteroid by the channel-forming α subunit.

COVID-19 Associated Reversible Cerebral Vasoconstriction Syndrome Successfully Treated with Nimodipine and Aspirin.

There have been limited cases linking SARS-CoV-2 infection with the development of reversible cerebral vasoconstriction syndrome (RCVS). We hereby report a rare case of RCVS in the setting of mild SARS-CoV-2 respiratory infection successfully treated with nimodipine and aspirin. SARS-CoV-2 attacks the ACE2-receptors, which are expressed in various body organs including the lungs, kidneys, and blood vessels. Vasoconstriction can result from down-regulation of the ACE2-receptors that can lead to sympathetic hypertonia of the cerebral blood vessel walls and/or over-activation of the renin-angiotensin axis.

Pilot assessment of omega-3 fatty acids and potassium thiocyanate in sickle cell anemia patients with conditional peak systolic cerebral artery blood velocity.

OBJECTIVE: The purpose of this pilot study was to explore the effect of omega-3 fatty acids and potassium thiocyanate on conditional peak systolic cerebral artery blood velocity in children with sickle cell anemia (SCA). METHODS: Transcranial doppler ultrasonography (TCD) was done on 232 SCA children, and 21 found with conditional peak systolic blood velocity (PSV) of 200-249 cm/s in internal carotid, middle or anterior cerebral arteries. These were randomized to receive omega-3 fatty acids and potassium thiocyanate with standard treatment of SCA (test group, N = 14), or standard treatment only (control group, N = 7). After 3 months of treatment, PSV was measured again. RESULTS: Right middle cerebral artery PSV was significantly reduced in the test relative to the control groups (p = 0.04). PSV returned to normal in 79% of the test versus 43% of the control group; and increased to abnormal in one member of the control group, but none of the test group. CONCLUSIONS: The pilot data suggest that in SCA, omega-3 fatty acids and potassium thiocyanate might reduce conditional blood velocity to normal, or prevent progression to abnormal values. A larger, randomized, clinical trial is required to further address the current gap in management of conditional TCD blood velocity.

Interleukin-17 induces hypertension but does not impair cerebrovascular function in pregnant rats.

Preeclampsia affects 5-8% of pregnancies and is characterized by hypertension, placental ischemia, neurological impairment, and an increase in circulating inflammatory cytokines, including Interleukin-17 (IL17). While placental ischemia has also been shown to impair cerebrovascular function, it is not known which placental-associated factor(s) drive this effect. The purpose of this study was to examine the effects of IL17 on cerebrovascular function during pregnancy. To achieve this goal, pregnant rats were infused with either IL17 (150 pg/day, 5 days, osmotic minipump), or vehicle (saline/0.7% BSA osmotic minipump) starting at gestational day (GD) 14. On GD 19, the cerebral blood flow (CBF) response to increases in mean arterial pressure (MAP) was measured in vivo, and myogenic constrictor responses of the middle cerebral artery (MCA) were assessed ex vivo. IL17 increased MAP but impaired CBF responses only at the highest arterial pressure measured (190 mmHg). Myogenic constrictor responses overall were mostly unaffected by IL17 infusion; however, the intraluminal pressure at which peak myogenic tone was generated was lower in the IL17 infused group (120 vs 165 mm Hg), suggesting maximal tone is exerted at lower intraluminal pressures in IL17-treated pregnant rats. Consistent with the lack of substantial change in overall myogenic responsiveness, there was no difference in cerebral vessel expression of putative mechanosensitive protein ßENaC, but a tendency towards a decrease in ASIC2 (p = 0.067) in IL17 rats. This study suggests that infusion of IL17 independent of other placental ischemia-associated factors is insufficient to recapitulate the features of impaired cerebrovascular function during placental ischemia. Further studies to examine of the role of other pro-inflammatory cytokines, individually or a combination, are necessary to determine mechanisms of cerebral vascular dysfunction during preeclampsia.

The effects of sevoflurane anesthesia on hemodynamics and cerebral artery diameters in endovascular treatment of intracranial aneurysm: A pilot study.

BACKGROUND: Cerebral autoregulation is a steady-state of cerebral blood flow despite major changes in arterial blood pressure. Inhalation anesthetics are cerebral vasodilators. In <1 MAC values, the net effect is a moderate decrease in cerebral blood flow and maintenance of responsiveness to carbon dioxide. This study aims to investigate the effects of steady-state sevoflurane anesthesia on hemodynamic and cerebral artery diameter measurements in patients undergoing flow diverter device placement under general anesthesia. METHODS: Forty-six patients aged 18-70 years who underwent flow diverter devices under general anesthesia were included in this study. Routine monitoring was performed on the patients. Mean arterial pressure (MAP) values were recorded. Internal carotid artery, middle cerebral artery and anterior cerebral artery diameter measurements were made from digital subtraction angiography (DSA) images of patients with anterior aneurysms. Baseline artery, right posterior cerebral artery and left posterior cerebral artery diameter measurements were made from DSA images of patients with posterior aneurysms. These measurements were recorded as preoperative measurements. The same measurements were made from the DSA images performed before the Flow diverter device placement procedure performed under steady-state sevoflurane anesthesia for the same patients. These measurements were recorded as peroperative measurements. RESULTS: The average age of the patients was 56.6±15.1. The MAP of the patients before induction was 76.28±5.13 mmHg, MAP after induction was 64.36±3.23 mmHg, and MAP during sevoflurane anesthesia was 68.26±4.30 mmHg, there was a statistically significant difference. There was a statistically significant difference between the preoperative and perioperative values of anterior cerebral artery diameters. There was a strong relationship between the MAP change percentage and the ICA diameter change percentage (p<0.001, p=-0.785) and a moderate relationship between the MCA diameter change percentage (p=0.033, p=-0.338). CONCLUSION: In patients undergoing flow diverter devices, <1 MAC sevoflurane has a hemodynamic effect and creates significant vasodilation in the cerebral artery diameters.

Metoprolol Impairs ß1-Adrenergic Receptor-Mediated Vasodilation in Rat Cerebral Arteries: Implications for ß-Blocker Therapy.

The practice of prescribing ß-blockers to lower blood pressure and mitigate perioperative cardiovascular events has been questioned because of reports of an increased risk of stroke. The benefit of ß-blocker therapy primarily relies on preventing activation of cardiac ß1-adrenergic receptors (ARs). However, we reported that ß1ARs also mediate vasodilator responses of rat cerebral arteries (CAs), implying that ß-blockers may impair cerebral blood flow under some conditions. Here, we defined the impact of metoprolol (MET), a widely prescribed ß1AR-selective antagonist, on adrenergic-elicited diameter responses of rat CAs ex vivo and in vivo. MET (1-10 µmol/l) prevented ß1AR-mediated increases in diameter elicited by dobutamine in cannulated rat CAs. The ß1AR-mediated dilation elicited by the endogenous adrenergic agonist norepinephrine (NE) was reversed to a sustained constriction by MET. Acute oral administration of MET (30 mg/kg) to rats in doses that attenuated resting heart rate and dobutamine-induced tachycardia also blunted ß1AR-mediated dilation of CAs. In the same animals, NE-induced dilation of CAs was reversed to sustained constriction. Administration of MET for 2 weeks in drinking water (2 mg/ml) or subcutaneously (15 mg/kg per day) also resulted in NE-induced constriction of CAs in vivo. Thus, doses of MET that protect the heart from adrenergic stimulation also prevent ß1AR-mediated dilation of CAs and favor anomalous adrenergic constriction. Our findings raise the possibility that the increased risk of ischemic stroke in patients on ß-blockers relates in part to adrenergic dysregulation of cerebrovascular tone. SIGNIFICANCE STATEMENT: ß-Blocker therapy using second-generation, cardioselective ß-blockers is associated with an increased risk of stroke, but the responsible mechanisms are unclear. Here, we report that either acute or chronic systemic administration of a cardioselective ß-blocker, metoprolol, mitigates adrenergic stimulation of the heart as an intended beneficial action. However, metoprolol concomitantly eliminates vasodilator responses to adrenergic stimuli of rat cerebral arteries in vivo as a potential cause of dysregulated cerebral blood flow predisposing to ischemic stroke.

Cerebrovascular effects of glibenclamide investigated using high-resolution magnetic resonance imaging in healthy volunteers.

Glibenclamide inhibits sulfonylurea receptor (SUR), which regulates several ion channels including SUR1-transient receptor potential melastatin 4 (SUR1-TRPM4) channel and ATP-sensitive potassium (KATP) channel. Stroke upregulates SURl-TRPM4 channel, which causes a rapid edema formation and brain swelling. Glibenclamide may antagonize the formation of cerebral edema during stroke. Preclinical studies showed that glibenclamide inhibits KATP channel-induced vasodilation without altering the basal vascular tone. The in vivo human cerebrovascular effects of glibenclamide have not previously been investigated.In a randomized, double-blind, placebo-controlled, three-way cross-over study, we used advanced 3 T MRI methods to investigate the effects of glibenclamide and KATP channel opener levcromakalim on mean global cerebral blood flow (CBF) and intra- and extracranial artery circumferences in 15 healthy volunteers. Glibenclamide administration did not alter the mean global CBF and the basal vascular tone. Following levcromakalim infusion, we observed a 14% increase of the mean global CBF and an 8% increase of middle cerebral artery (MCA) circumference, and glibenclamide did not attenuate levcromakalim-induced vascular changes. Collectively, the findings demonstrate the vital role of KATP channels in cerebrovascular hemodynamic and indicate that glibenclamide does not inhibit the protective effects of KATP channel activation during hypoxia and ischemia-induced brain injury.

Longitudinal relation between blood pressure, antihypertensive use and cerebral blood flow, using arterial spin labelling MRI.

Consistent cerebral blood flow (CBF) is fundamental to brain function. Cerebral autoregulation ensures CBF stability. Chronic hypertension can lead to disrupted cerebral autoregulation in older people, potentially leading to blood pressure levels interfering with CBF. This study tested whether low BP and AHD use are associated with contemporaneous low CBF, and whether longitudinal change in BP is associated with change in CBF, using arterial spin labelling (ASL) MRI, in a prospective longitudinal cohort of 186 community-dwelling older individuals with hypertension (77 ± 3 years, 53% female), 125 (67%) of whom with 3-year follow-up. Diastolic blood pressure, systolic blood pressure, mean arterial pressure, and pulse pressure were assessed as blood pressure parameters. As additional cerebrovascular marker, we evaluated the ASL signal spatial coefficient of variation (ASL SCoV), a measure of ASL signal heterogeneity that may reflect cerebrovascular health. We found no associations between any of the blood pressure measures and concurrent CBF nor between changes in blood pressure measures and CBF over three-year follow-up. Antihypertensive use was associated with lower grey matter CBF (-5.49 ml/100 g/min, 95%CI = -10.7|-0.27, p = 0.04) and higher ASL SCoV (0.32 SD, 95%CI = 0.12|0.52, p = 0.002). These results warrant future research on the potential relations between antihypertensive use and cerebral perfusion.

Endothelial S1P1 Signaling Counteracts Infarct Expansion in Ischemic Stroke.

RATIONALE: Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P1 modulation in stroke. OBJECTIVE: To address roles and mechanisms of engagement of endothelial cell S1P1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy. METHODS AND RESULTS: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P1 in the mouse brain. With an S1P1 signaling reporter, we reveal that abluminal polarization shields S1P1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P1-selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion. CONCLUSIONS: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P1 agonists.

Cholesterol antagonism of alcohol inhibition of smooth muscle BK channel requires cell integrity and involves a protein kinase C-dependent mechanism(s).

Alcohol constricts cerebral arteries via inhibition of voltage/calcium-gated, large conductance potassium (BK) channels in vascular myocytes. Using a rat model of high-cholesterol (high-CLR) diet and CLR enrichment of cerebral arteries in vitro, we recently showed that CLR protected against alcohol-induced constriction of cerebral arteries. The subcellular mechanism(s) underlying CLR protection against alcohol-induced constriction of the artery is unclear. Here we use a rat model of high-CLR diet and patch-clamp recording of BK channels in inside-out patches from cerebral artery myocytes to demonstrate that this diet antagonizes inhibition of BK currents by 50 mM ethanol. High-CLR-driven protection against alcohol inhibition of BK currents is reversed following CLR depletion in vitro. Similar to CLR accumulation in vivo, pre-incubation of arterial myocytes from normocholesterolemic rats in CLR-enriching media in vitro protects against alcohol-induced inhibition of BK current. However, application of CLR-enriching media to cell-free membrane patches does not protect against the alcohol effect. These different outcomes point to the involvement of cell signaling in CLR-alcohol interaction on BK channels. Incubation of myocytes with the PKC activators phorbol 12-myristate 13-acetate or 1,2-dioctanoyl-sn-glycerol, but not with the PKC inhibitor Gouml 6983, prior to patch excision precludes CLR enrichment from antagonizing alcohol action. Thus, PKC activation either disables the CLR target(s) or competes with elevated CLR. Favoring the latter possibility, 1,2-dioctanoyl-sn-glycerol protects against alcohol-induced inhibition of BK currents in patches from myocytes with naïve CLR. Our findings document that CLR antagonism of alcohol-induced BK channel inhibition requires cell integrity and is enabled by a PKC-dependent mechanism(s).

Inhibition of big-conductance Ca2+-activated K+ channels in cerebral artery (vascular) smooth muscle cells is a major novel mechanism for tacrolimus-induced hypertension.

Tacrolimus (TAC, also called FK506), a common immunosuppressive drug used to prevent allograft rejection in transplant patients, is well known to alter the functions of blood vessels. In this study, we sought to determine whether chronic treatment of TAC could inhibit the activity of big-conductance Ca2+-activated K+ (BK) channels in vascular smooth muscle cells (SMCs), leading to hypertension. Our data reveal that the activity of BK channels was inhibited in cerebral artery SMCs (CASMCs) from mice after intraperitoneal injection of TAC once a day for 4 weeks. The voltage sensitivity, Ca2+ sensitivity, and open time of single BK channels were all decreased. In support, BK channel ß1-, but not α-subunit protein expression was significantly decreased in cerebral arteries. In TAC-treated mice, application of norepinephrine induced stronger vasoconstriction in both cerebral and mesenteric arteries as well as a larger [Ca2+]i in CASMCs. Chronic treatment of TAC, similar to BK channel ß1-subunit knockout (KO), resulted in hypertension in mice, but did not cause a further increase in blood pressure in BK channel ß1-subunit KO mice. Moreover, BK channel activity in CASMCs was negatively correlated with blood pressure. Our findings provide novel evidence that TAC inhibits BK channels by reducing the channel ß1-subunit expression and functions in vascular SMCs, leading to enhanced vasoconstriction and hypertension.

Celastrol Dilates and Counteracts Ethanol-Induced Constriction of Cerebral Arteries.

The increasing recognition of the role played by cerebral artery dysfunction in brain disorders has fueled the search for new cerebrovascular dilators. Celastrol, a natural triterpene undergoing clinical trials for treating obesity, exerts neuroprotection, which was linked to its antioxidant/anti-inflammatory activities. We previously showed that celastrol fit pharmacophore criteria for activating calcium- and voltage-gated potassium channels of large conductance (BK channels) made of subunits cloned from cerebrovascular smooth muscle (SM). These recombinant BK channels expressed in a heterologous system were activated by celastrol. Activation of native SM BK channels is well known to evoke cerebral artery dilation. Current data demonstrate that celastrol (1-100 µM) dilates de-endothelialized, ex vivo pressurized middle cerebral arteries (MCAs) from rats, with EC50 = 45 µM and maximal effective concentration (Emax)= 100 µM and with MCA diameter reaching a 10% increase over vehicle-containing, time-matched values (P < 0.05). A similar vasodilatory efficacy is achieved when celastrol is probed on MCA segments with intact endothelium. Selective BK blocking with 1 µM paxilline blunts celastrol vasodilation. Similar blunting is achieved with 0.8 mM 4-aminopirydine, which blocks voltage-gated K+ channels other than BK. Using an in vivo rat cranial window, we further demonstrate that intracarotid injections of 45 µM celastrol into pial arteries branching from MCA mimics celastrol ex vivo action. MCA constriction by ethanol concentrations reached in blood during moderate-heavy alcohol drinking (50 mM), which involves SM BK inhibition, is both prevented and reverted by celastrol. We conclude that celastrol could be an effective cerebrovascular dilator and antagonist of alcohol-induced cerebrovascular constriction, with its efficacy being uncompromised by conditions that disrupt endothelial and/or BK function. SIGNIFICANCE STATEMENT: Our study demonstrates for the first time that celastrol significantly dilates rat cerebral arteries both ex vivo and in vivo and both prevents and reverses ethanol-induced cerebral artery constriction. Celastrol actions are endothelium-independent but mediated through voltage-gated (KV) and calcium- and voltage-gated potassium channel of large conductance (BK) K+ channels. This makes celastrol an appealing new agent to evoke cerebrovascular dilation under conditions in which endothelial and/or BK channel function are impaired.

Reversible cerebral vasoconstriction syndrome and dissection in the setting of COVID-19 infection.

The current COVID-19 pandemic has recently brought to attention the myriad of neuro- logic sequelae associated with Coronavirus infection including the predilection for stroke, particularly in young patients. Reversible cerebral vasoconstriction syndrome (RCVS) is a well-described clinical syndrome leading to vasoconstriction in the intracra- nial vessels, and has been associated with convexity subarachnoid hemorrhage and oc- casionally cervical artery dissection. It is usually reported in the context of a trigger such as medications, recreational drugs, or the postpartum state; however, it has not been described in COVID-19 infection. We report a case of both cervical vertebral ar- tery dissection as well as convexity subarachnoid hemorrhage due to RCVS, in a pa- tient with COVID-19 infection and no other triggers.

Apelin inhibits an endothelium-derived hyperpolarizing factor-like pathway in rat cerebral arteries.

Apelin has complex vasomotor actions inasmuch as the peptide may cause either vasodilation or vasoconstriction depending on the vascular bed and experimental conditions. In cerebral arteries, apelin inhibits endothelium-dependent relaxations mediated by nitric oxide (NO); however, its effects on relaxation to other endothelium-derived substances (e.g. prostacyclin, endothelium-derived hyperpolarizing factors(s) (EDHF)) are unknown. The present study was designed to determine effects of apelin on endothelium-dependent relaxations that are independent of NO in rat cerebral arteries. In arterial rings contracted with 5-HT, A23187 caused endothelium-dependent relaxation that was unaffected by inhibitors of eNOS, guanylyl cyclase or cyclooxygenase, but was attenuated by MS-PPOH, a selective inhibitor of cytochrome P450 catalyzed synthesis of epoxyeicosatrienoic acids (EETs) and by 14,15-EE(Z)E, an EET-receptor antagonist. Apelin inhibited A23187-induced relaxation, as well as relaxations evoked by exogenous 11,12- and 14,15-EET. These effects of apelin were mimicked by the selective BKCa channel blocker, iberiotoxin. The APJ receptor antagonist, F13A abolished the effects of apelin on A23187-induced relaxations. Both 11,12- and 14,15-EET also increased BKCa channel current density in isolated cerebral artery smooth muscle cells, effects that were inhibited in a similar manner by apelin and iberiotoxin. These findings provide evidence that apelin impairs endothelium-dependent relaxation of cerebral arteries by inhibiting an NO-independent pathway (i.e. "EDHF-like") involving activation of smooth muscle cell BKCa channels by endothelium-derived EETs. Inhibition of such pathway may create an environment favoring vasoconstriction in cerebral arteries.

Regulation of the cerebrovascular smooth muscle cell phenotype by mitochondrial oxidative injury and endoplasmic reticulum stress in simulated microgravity rats via the PERK-eIF2α-ATF4-CHOP pathway.

Microgravity exposure results in vascular remodeling and cardiovascular dysfunction. Here, the effects of mitochondrial oxidative stress on vascular smooth muscle cells (VSMCs) in rat cerebral arteries under microgravity simulated by hindlimb unweighting (HU) was studied. Endoplasmic reticulum (ER)-resident transmembrane sensor proteins and phenotypic markers of rat cerebral VSMCs were examined. In HU rats, CHOP expression was increased gradually, and the upregulation of the PERK-eIF2α-ATF4 pathway was the most pronounced in cerebral arteries. Furthermore, PERK/p-PERK signaling, CHOP, GRP78 and reactive oxygen species were augmented by PERK overexpression but attenuated by the mitochondria-targeting antioxidant MitoTEMPO. Meanwhile, p-PI3K, p-Akt and p-mTOR protein levels in VSMCs were increased in HU rat cerebral arteries. Compared with the control, HU rats exhibited lower α-SMA, calponin, SM-MHC and caldesmon protein levels but higher OPN and elastin levels in cerebral VSMCs. The cerebral VSMC phenotype transition from a contractile to synthetic phenotype in HU rats was augmented by PERK overexpression and 740Y-P but reversed by MitoTEMPO and the ER stress inhibitors tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA). In summary, mitochondrial oxidative stress and ER stress induced by simulated microgravity contribute to phenotype transition of cerebral VSMCs through the PERK-eIF2a-ATF4-CHOP pathway in a rat model.