Allogeneic immunity clears latent virus following allogeneic stem cell transplantation in SIV-infected ART-suppressed macaques.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) from donors lacking C-C chemokine receptor 5 (CCR5Δ32/Δ32) can cure HIV, yet mechanisms remain speculative. To define how alloHSCT mediates HIV cure, we performed MHC-matched alloHSCT in SIV+, anti-retroviral therapy (ART)-suppressed Mauritian cynomolgus macaques (MCMs) and demonstrated that allogeneic immunity was the major driver of reservoir clearance, occurring first in peripheral blood, then peripheral lymph nodes, and finally in mesenteric lymph nodes draining the gastrointestinal tract. While allogeneic immunity could extirpate the latent viral reservoir and did so in two alloHSCT-recipient MCMs that remained aviremic >2.5 years after stopping ART, in other cases, it was insufficient without protection of engrafting cells afforded by CCR5-deficiency, as CCR5-tropic virus spread to donor CD4+ T cells despite full ART suppression. These data demonstrate the individual contributions of allogeneic immunity and CCR5 deficiency to HIV cure and support defining targets of alloimmunity for curative strategies independent of HSCT.

Overexpression of the neuronal human (pro)renin receptor mediates angiotensin II-independent blood pressure regulation in the central nervous system.

Despite advances in antihypertensive therapeutics, at least 15-20% of hypertensive patients have resistant hypertension through mechanisms that remain poorly understood. In this study, we provide a new mechanism for the regulation of blood pressure (BP) in the central nervous system (CNS) by the (pro)renin receptor (PRR), a recently identified component of the renin-angiotensin system that mediates ANG II formation in the CNS. Although PRR also mediates ANG II-independent signaling, the importance of these pathways in BP regulation is unknown. Here, we developed a unique transgenic mouse model overexpressing human PRR (hPRR) specifically in neurons (Syn-hPRR). Intracerebroventricular infusion of human prorenin caused increased BP in Syn-hPRR mice. This BP response was attenuated by a NADPH oxidase (NOX) inhibitor but not by antihypertensive agents that target the renin-angiotensin system. Using a brain-targeted genetic knockdown approach, we found that NOX4 was the key isoform responsible for the prorenin-induced elevation of BP in Syn-hPRR mice. Moreover, inhibition of ERK significantly attenuated the increase in NOX activity and BP induced by human prorenin. Collectively, our findings indicate that an ANG II-independent, PRR-mediated signaling pathway regulates BP in the CNS by a PRR-ERK-NOX4 mechanism. NEW & NOTEWORTHY This study characterizes a new transgenic mouse model with overexpression of the human (pro)renin receptor in neurons and demonstrated a novel angiotensin II-independent mechanism mediated by human prorenin and the (pro)renin receptor in the central regulation of blood pressure.

Unitary TRPV3 channel Ca2+ influx events elicit endothelium-dependent dilation of cerebral parenchymal arterioles.

Cerebral parenchymal arterioles (PA) regulate blood flow between pial arteries on the surface of the brain and the deeper microcirculation. Regulation of PA contractility differs from that of pial arteries and is not completely understood. Here, we investigated the hypothesis that the Ca(2+) permeable vanilloid transient receptor potential (TRPV) channel TRPV3 can mediate endothelium-dependent dilation of cerebral PA. Using total internal reflection fluorescence microscopy (TIRFM), we found that carvacrol, a monoterpenoid compound derived from oregano, increased the frequency of unitary Ca(2+) influx events through TRPV3 channels (TRPV3 sparklets) in endothelial cells from pial arteries and PAs. Carvacrol-induced TRPV3 sparklets were inhibited by the selective TRPV3 blocker isopentenyl pyrophosphate (IPP). TRPV3 sparklets have a greater unitary amplitude (ΔF/F0 = 0.20) than previously characterized TRPV4 (ΔF/F0 = 0.06) or TRPA1 (ΔF/F0 = 0.13) sparklets, suggesting that TRPV3-mediated Ca(2+) influx could have a robust influence on cerebrovascular tone. In pressure myography experiments, carvacrol caused dilation of cerebral PA that was blocked by IPP. Carvacrol-induced dilation was nearly abolished by removal of the endothelium and block of intermediate (IK) and small-conductance Ca(2+)-activated K(+) (SK) channels. Together, these data suggest that TRPV3 sparklets cause dilation of cerebral parenchymal arterioles by activating IK and SK channels in the endothelium.

Calcineurin/nuclear factor of activated T cells-coupled vanilliod transient receptor potential channel 4 ca2+ sparklets stimulate airway smooth muscle cell proliferation.

Proliferation of airway smooth muscle cells (ASMCs) contributes to the remodeling and irreversible obstruction of airways during severe asthma, but the mechanisms underlying this disease process are poorly understood. Here we tested the hypothesis that Ca(2+) influx through the vanilliod transient receptor potential channel (TRPV) 4 stimulates ASMC proliferation. We found that synthetic and endogenous TRPV4 agonists increase proliferation of primary ASMCs. Furthermore, we demonstrate that Ca(2+) influx through individual TRPV4 channels produces Ca(2+) microdomains in ASMCs, called "TRPV4 Ca(2+) sparklets." We also show that TRPV4 channels colocalize with the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin in ASMCs. Activated calcineurin dephosphorylates nuclear factor of activated T cells (NFAT) transcription factors cytosolic (c) to allow nuclear translocation and activation of synthetic transcriptional pathways. We show that ASMC proliferation in response to TRPV4 activity is associated with calcineurin-dependent nuclear translocation of the NFATc3 isoform tagged with green florescent protein. Our findings suggest that Ca(2+) microdomains created by TRPV4 Ca(2+) sparklets activate calcineurin to stimulate nuclear translocation of NFAT and ASMC proliferation. These findings further suggest that inhibition of TRPV4 could diminish asthma-induced airway remodeling.

Use of subcutaneous maropitant at two dosages for pain management in domestic rabbits (Oryctolagus cuniculus) undergoing elective ovariohysterectomy or orchiectomy.

OBJECTIVE: To describe the effect of two doses of maropitant on pain scores, food intake, and fecal output in domestic rabbits (Oryctolagus cuniculus) undergoing elective ovariohysterectomy or orchiectomy. ANIMALS: 26 (11 female, 15 male) rabbits from three institutions. PROCEDURES: Rabbits were randomly assigned to one of three treatment groups: low-dose maropitant (LDM; 2 mg/kg SC once; n=8), moderate-dose maropitant (MDM; 4 mg/kg SC once; n=10), and control (saline equivalent to 4 mg/kg maropitant SC once; n=8), administered prior to surgery. Following surgery, all rabbits were provided buprenorphine (0.06 mg/kg q 8 hours) and meloxicam (1 mg/kg q 24 hours) intramuscularly. Rabbits were monitored using video surveillance postoperatively until 24 hours after surgery or discharge from the hospital, whichever came first. Pain scores were assessed by three blinded observers, and results were grouped into early (0-4 hours), mid (5-8 hours), and late (12-24 hours) time frames. Food intake and fecal output were compared between groups. Statistical analysis was performed using Chi square, Fisher's exact tests, and a mixed model approach. RESULTS: There were no adverse effects with maropitant administration. Rabbits that received MDM had significantly lower pain scores in the mid-time frame and behavior scores in the late-time frame compared to controls. Male rabbits consumed more food than females and rabbits hospitalized longer than 12 hours consumed more food than those that were discharged prior. No significant differences were detected in facial grimace scale scores, food intake, or fecal production among treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE: Moderate dose maropitant decreased pain related behaviors in the mid-time frame and behavior scores in the late-time frame after surgery. Further studies are necessary to better characterize the potential use of maropitant in postoperative analgesia.

TRP channel Ca(2+) sparklets: fundamental signals underlying endothelium-dependent hyperpolarization.

Important functions of the vascular endothelium, including permeability, production of antithrombotic factors, and control of vascular tone, are regulated by changes in intracellular Ca(2+). The molecular identities and regulation of Ca(2+) influx channels in the endothelium are incompletely understood, in part because of experimental difficulties associated with application of patch-clamp electrophysiology to native endothelial cells. However, advances in confocal and total internal reflection fluorescence microscopy and the development of fast, high-affinity Ca(2+)-binding fluorophores have recently allowed for direct visualization and characterization of single-channel transient receptor potential (TRP) channel Ca(2+) influx events in endothelial cells. These events, called "TRP channel Ca(2+) sparklets," have been optically recorded from primary endothelial cells and the intact endothelium, and the biophysical properties and fundamental significance of these Ca(2+) signals in vasomotor regulation have been characterized. This review will first briefly discuss the role of endothelial cell TRP channel Ca(2+) influx in endothelium-dependent vasodilation, describe improved methods for recording unitary TRP channel activity using optical methods, and highlight discoveries regarding the regulation and physiological significance of TRPV4 Ca(2+) sparklets in the vascular endothelium enabled by this new technology. Perspectives on the potential use of these techniques to evaluate changes in TRP channel Ca(2+) influx activity associated with endothelial dysfunction are offered.

Endothelial cell TRPA1 activity exacerbates cerebral hemorrhage during severe hypertension.

Introduction: Hypoxia-induced dilation of cerebral arteries orchestrated by Ca2+-permeable transient receptor potential ankyrin 1 (TRPA1) cation channels on endothelial cells is neuroprotective during ischemic stroke, but it is unknown if the channel has a similar impact during hemorrhagic stroke. TRPA1 channels are endogenously activated by lipid peroxide metabolites generated by reactive oxygen species (ROS). Uncontrolled hypertension, a primary risk factor for the development of hemorrhagic stroke, is associated with increased ROS production and oxidative stress. Therefore, we hypothesized that TRPA1 channel activity is increased during hemorrhagic stroke. Methods: Severe, chronic hypertension was induced in control (Trpa1 fl/fl) and endothelial cell-specific TRPA1 knockout (Trpa1-ecKO) mice using a combination of chronic angiotensin II administration, a high-salt diet, and the addition of a nitric oxide synthase inhibitor to drinking water. Blood pressure was measured in awake, freely-moving mice using surgically placed radiotelemetry transmitters. TRPA1-dependent cerebral artery dilation was evaluated with pressure myography, and expression of TRPA1 and NADPH oxidase (NOX) isoforms in arteries from both groups was determined using PCR and Western blotting techniques. In addition, ROS generation capacity was evaluated using a lucigenin assay. Histology was performed to examine intracerebral hemorrhage lesion size and location. Results: All animals became hypertensive, and a majority developed intracerebral hemorrhages or died of unknown causes. Baseline blood pressure and responses to the hypertensive stimulus did not differ between groups. Expression of TRPA1 in cerebral arteries from control mice was not altered after 28 days of treatment, but expression of three NOX isoforms and the capacity for ROS generation was increased in hypertensive animals. NOX-dependent activation of TRPA1 channels dilated cerebral arteries from hypertensive animals to a greater extent compared with controls. The number of intracerebral hemorrhage lesions in hypertensive animals did not differ between control and Trpa1-ecKO animals but were significantly smaller in Trpa1-ecKO mice. Morbidity and mortality did not differ between groups. Discussion: We conclude that endothelial cell TRPA1 channel activity increases cerebral blood flow during hypertension resulting in increased extravasation of blood during intracerebral hemorrhage events; however, this effect does not impact overall survival. Our data suggest that blocking TRPA1 channels may not be helpful for treating hypertension-associated hemorrhagic stroke in a clinical setting.

Optical recording reveals novel properties of GSK1016790A-induced vanilloid transient receptor potential channel TRPV4 activity in primary human endothelial cells.

Critical functions of the vascular endothelium are regulated by changes in intracellular [Ca(2+)]. Endothelial dysfunction is tightly associated with cardiovascular disease, and improved understanding of Ca(2+) entry pathways in these cells will have a significant impact on human health. However, much about Ca(2+) influx channels in endothelial cells remains unknown because they are difficult to study using conventional patch-clamp electrophysiology. Here we describe a novel, highly efficient method for recording and analyzing Ca(2+)-permeable channel activity in primary human endothelial cells using a unique combination of total internal reflection fluorescence microscopy (TIRFM), custom software-based detection, and selective pharmacology. Our findings indicate that activity of the vanilloid (V) transient receptor potential (TRP) channel TRPV4 can be rapidly recorded and characterized at the single-channel level using this method, providing novel insight into channel function. Using this method, we show that although TRPV4 protein is evenly distributed throughout the plasma membrane, most channels are silent even during maximal stimulation with the potent TRPV4 agonist N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A). Furthermore, our findings indicate that GSK1016790A acts by recruiting previously inactive channels, rather than through increasing elevation of basal activity.

Upregulation of Superoxide Dismutase 2 by Astrocytes in the SIV/Macaque Model of HIV-Associated Neurologic Disease.

HIV-associated neurocognitive disorders (HAND) remain prevalent despite implementation of antiretroviral therapy (ART). Development of HAND is linked to mitochondrial dysfunction and oxidative stress in the brain; therefore, upregulation of antioxidant defenses is critical to curtail neuronal damage. Superoxide dismutase 2 (SOD2) is a mitochondrial antioxidant enzyme essential for maintaining cellular viability. We hypothesized that SOD2 was upregulated during retroviral infection. Using a simian immunodeficiency virus (SIV)-infected macaque model of HIV, quantitative PCR showed elevated SOD2 mRNA in cortical gray ([GM], 7.6-fold for SIV vs uninfected) and white matter ([WM], 77-fold for SIV vs uninfected) during SIV infection. Further, SOD2 immunostaining was enhanced in GM and WM from SIV-infected animals. Double immunofluorescence labeling illustrated that SOD2 primarily colocalized with astrocyte marker glial fibrillary acidic protein (GFAP) in SIV-infected animals. Interestingly, in ART-treated SIV-infected animals, brain SOD2 RNA levels were similar to uninfected animals. Additionally, using principal component analysis in a transcriptomic approach, SOD2 and GFAP expression separated SIV-infected from uninfected brain tissue. Projection of these data into a HIV dataset revealed similar expression changes, thereby validating the clinical relevance. Together, our findings suggest that novel SOD2-enhancing therapies may reduce neuroinflammation in ART-treated HIV-infected patients.

Intracerebroventricular infusion of the (Pro)renin receptor antagonist PRO20 attenuates deoxycorticosterone acetate-salt-induced hypertension.

We previously reported that binding of prorenin to the (pro)renin receptor (PRR) plays a major role in brain angiotensin II formation and the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Here, we designed and developed an antagonistic peptide, PRO20, to block prorenin binding to the PRR. Fluorescently labeled PRO20 bound to both mouse and human brain tissues with dissociation constants of 4.4 and 1.8 nmol/L, respectively. This binding was blocked by coincubation with prorenin and was diminished in brains of neuron-specific PRR-knockout mice, indicating specificity of PRO20 for PRR. In cultured human neuroblastoma cells, PRO20 blocked prorenin-induced calcium influx in a concentration- and AT(1) receptor-dependent manner. Intracerebroventricular infusion of PRO20 dose-dependently inhibited prorenin-induced hypertension in C57Bl6/J mice. Furthermore, acute intracerebroventricular infusion of PRO20 reduced blood pressure in both DOCA-salt and genetically hypertensive mice. Chronic intracerebroventricular infusion of PRO20 attenuated the development of hypertension and the increase in brain hypothalamic angiotensin II levels induced by DOCA-salt. In addition, chronic intracerebroventricular infusion of PRO20 improved autonomic function and spontaneous baroreflex sensitivity in mice treated with DOCA-salt. In summary, PRO20 binds to both mouse and human PRRs and decreases angiotensin II formation and hypertension induced by either prorenin or DOCA-salt. Our findings highlight the value of the novel PRR antagonist, PRO20, as a lead compound for a novel class of antihypertensive agents and as a research tool to establish the validity of brain PRR antagonism as a strategy for treating hypertension.

Localized TRPA1 channel Ca2+ signals stimulated by reactive oxygen species promote cerebral artery dilation.

Reactive oxygen species (ROS) can have divergent effects in cerebral and peripheral circulations. We found that Ca(2+)-permeable transient receptor potential ankyrin 1 (TRPA1) channels were present and colocalized with NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 2 (NOX2), a major source of ROS, in the endothelium of cerebral arteries but not in other vascular beds. We recorded and characterized ROS-triggered Ca(2+) signals representing Ca(2+) influx through single TRPA1 channels, which we called "TRPA1 sparklets." TRPA1 sparklet activity was low under basal conditions but was stimulated by NOX-generated ROS. Ca(2+) entry during a single TRPA1 sparklet was twice that of a TRPV4 sparklet and ~200 times that of an L-type Ca(2+) channel sparklet. TRPA1 sparklets representing the simultaneous opening of two TRPA1 channels were more common in endothelial cells than in human embryonic kidney (HEK) 293 cells expressing TRPA1. The NOX-induced TRPA1 sparklets activated intermediate-conductance, Ca(2+)-sensitive K(+) channels, resulting in smooth muscle hyperpolarization and vasodilation. NOX-induced activation of TRPA1 sparklets and vasodilation required generation of hydrogen peroxide and lipid-peroxidizing hydroxyl radicals as intermediates. 4-Hydroxy-nonenal, a metabolite of lipid peroxidation, also increased TRPA1 sparklet frequency and dilated cerebral arteries. These data suggest that in the cerebral circulation, lipid peroxidation metabolites generated by ROS activate Ca(2+) influx through TRPA1 channels in the endothelium of cerebral arteries to cause dilation.

A PLCγ1-dependent, force-sensitive signaling network in the myogenic constriction of cerebral arteries.

Maintaining constant blood flow in the face of fluctuations in blood pressure is a critical autoregulatory feature of cerebral arteries. An increase in pressure within the artery lumen causes the vessel to constrict through depolarization and contraction of the encircling smooth muscle cells. This pressure-sensing mechanism involves activation of two types of transient receptor potential (TRP) channels: TRPC6 and TRPM4. We provide evidence that the activation of the γ1 isoform of phospholipase C (PLCγ1) is critical for pressure sensing in cerebral arteries. Inositol 1,4,5-trisphosphate (IP3), generated by PLCγ1 in response to pressure, sensitized IP3 receptors (IP3Rs) to Ca(2+) influx mediated by the mechanosensitive TRPC6 channel, synergistically increasing IP3R-mediated Ca(2+) release to activate TRPM4 currents, leading to smooth muscle depolarization and constriction of isolated cerebral arteries. Proximity ligation assays demonstrated colocalization of PLCγ1 and TRPC6 with TRPM4, suggesting the presence of a force-sensitive, local signaling network comprising PLCγ1, TRPC6, TRPM4, and IP3Rs. Src tyrosine kinase activity was necessary for stretch-induced TRPM4 activation and myogenic constriction, consistent with the ability of Src to activate PLCγ isoforms. We conclude that contraction of cerebral artery smooth muscle cells requires the integration of pressure-sensing signaling pathways and their convergence on IP3Rs, which mediate localized Ca(2+)-dependent depolarization through the activation of TRPM4.

Neuron-specific (pro)renin receptor knockout prevents the development of salt-sensitive hypertension.

The (pro)renin receptor (PRR), which binds both renin and prorenin, is a newly discovered component of the renin-angiotensin system that is highly expressed in the central nervous system. The significance of brain PRRs in mediating local angiotensin II formation and regulating blood pressure remains unclear. The current study was performed to test the hypothesis that PRR-mediated, nonproteolytic activation of prorenin is the main source of angiotensin II in the brain. Thus, PRR knockout in the brain is expected to prevent angiotensin II formation and development of deoxycorticosterone acetate-salt-induced hypertension. A neuron-specific PRR (ATP6AP2) knockout mouse model was generated using the Cre-LoxP system. Physiological parameters were recorded by telemetry. PRR expression, detected by immunostaining and reverse transcription-polymerase chain reaction, was significantly decreased in the brains of knockout mice compared with wild-type mice. Intracerebroventricular infusion of mouse prorenin increased blood pressure and angiotensin II formation in wild-type mice. This hypertensive response was abolished in PRR-knockout mice in association with a reduction in angiotensin II levels. Deoxycorticosterone acetate-salt increased PRR expression and angiotensin II formation in the brains of wild-type mice, an effect that was attenuated in PRR-knockout mice. PRR knockout in neurons prevented the development of deoxycorticosterone acetate-salt-induced hypertension as well as activation of cardiac and vasomotor sympathetic tone. In conclusion, nonproteolytic activation of prorenin through binding to the PRR mediates angiotensin II formation in the brain. Neuron-specific PRR knockout prevents the development of deoxycorticosterone acetate-salt-induced hypertension, possibly through diminished angiotensin II formation.

Robust internal elastic lamina fenestration in skeletal muscle arteries.

Holes within the internal elastic lamina (IEL) of blood vessels are sites of fenestration allowing for passage of diffusible vasoactive substances and interface of endothelial cell membrane projections with underlying vascular smooth muscle. Endothelial projections are sites of dynamic Ca(2+) events leading to endothelium dependent hyperpolarization (EDH)-mediated relaxations and the activity of these events increase as vessel diameter decreases. We tested the hypothesis that IEL fenestration is greater in distal vs. proximal arteries in skeletal muscle, and is unlike other vascular beds (mesentery). We also determined ion channel protein composition within the endothelium of intramuscular and non-intramuscular skeletal muscle arteries. Popliteal arteries, subsequent gastrocnemius feed arteries, and first and second order intramuscular arterioles from rat hindlimb were isolated, cut longitudinally, fixed, and imaged using confocal microscopy. Quantitative analysis revealed a significantly larger total fenestration area in second and first order arterioles vs. feed and popliteal arteries (58% and 16% vs. 5% and 3%; N = 10 images/artery), due to a noticeably greater average size of holes (9.5 and 3.9 µm(2) vs 1.5 and 1.9 µm(2)). Next, we investigated via immunolabeling procedures whether proteins involved in EDH often embedded in endothelial cell projections were disparate between arterial segments. Specific proteins involved in EDH, such as inositol trisphosphate receptors, small and intermediate conductance Ca(2+)-activated K(+) channels, and the canonical (C) transient receptor potential (TRP) channel TRPC3 were present in both popliteal and first order intramuscular arterioles. However due to larger IEL fenestration in first order arterioles, a larger spanning area of EDH proteins is observed proximal to the smooth muscle cell plasma membrane. These observations highlight the robust area of fenestration within intramuscular arterioles and indicate that the anatomical architecture and endothelial cell hyperpolarizing apparatus for distinct vasodilatory signaling is potentially present.

Control of urinary bladder smooth muscle excitability by the TRPM4 channel modulator 9-phenanthrol.

The Ca (2+)-activated monovalent cation selective transient receptor potential melastatin 4 (TRPM4) channel has been recently identified in detrusor smooth muscle (DSM) of the urinary bladder. Two recent publications by our research group provide evidence in support of the novel hypothesis that TRPM4 channels enhance DSM excitability and contractility. This is a critical question as prior studies have primarily targeted hyperpolarizing currents facilitated by K(+) channels, but the depolarizing component in DSM cells is not well understood. For the first time, we utilized the selective TRPM4 channel inhibitor, 9-phenanthrol, to investigate TRPM4 channel functional effects in DSM at both cellular and tissue levels in rodents. Our new data presented here showed that in rat DSM cells, 9-phenanthrol attenuates spontaneous inward currents in the presence of the muscarinic receptor agonist, carbachol, thus reducing DSM cell excitability. In support of our original hypothesis, we found that TRPM4 channel mRNA levels are much higher in DSM vs. vascular smooth muscle and that inhibition of TRPM4 channels can potentially attenuate DSM excitability. Thus, we postulate the novel concept that selective pharmacological inhibition of TRPM4 channels can limit both excitability and contractility of DSM.