bluuebunny/crossref_metadata_2025 huggingface dataset:
Developing Pharmacological Probes Targeting exchange protein directly activated by cAMP
Authors: Cheng Xiaodong , Tsalkova Tamara , Mei Fang
Published: June 2021

Cyclic adenosine monophosphate (cAMP), a prototypical second messenger, regulates a myriad of important biological processes under both physiological and pathological conditions, including cancer, diabetes, heart failure, and neurological disorders. In eukaryotic cells, the effects of cAMP are transduced by two intracellular cAMP receptors, the classic protein kinase A (PKA) and the more recently discovered exchange proteins directly activated by cAMP (EPACs) or cAMP‐regulated guanine nucleotide exchange factors (cAMP‐GEFs). Like PKA, EPACs contain a cAMP‐binding domain, an evolutionally conserved structural motif that acts as a molecular switch for sensing intracellular second messenger cAMP levels. Depending upon the specific cellular context, EPAC and PKA can act antagonistically or synergistically in controlling various cellular functions. Recent studies have demonstrated that levels of EPAC1 expression are increased in tumors of pancreatic cancer patients and in cardiac tissue of individuals with failing hearts while EPAC2 is important for insulin secretion and for cognitive function and may play a significant role in the development of diabetes and Alzheimer's disease. Unfortunately, there are no isoform‐selective EPAC small compounds available for dissecting the physiological functions of EPAC isoforms in normal and disease states. To bridge this major gap in our knowledge, we have developed isoform‐specific EPAC pharmacological probes. These EPAC‐specific probes are complementary to our genetic approaches using knockout mice models and will be powerful pharmacological tools for elucidating the functions of EPAC isoforms in various diseases and for testing the therapeutic potential of treating cancer or diabetes by specifically targeting EPAC/cAMP‐signaling components.
Non‐raft AC2 defines a cAMP signaling compartment that selectively regulates IL‐6 expression in airway smooth muscle cells
Authors: Bogard Amy S. , Ostrom Rennolds S
Published: June 2021

The ubiquitous second messenger cAMP is produced in response to several stimuli and must initiate a variety of situation‐specific responses. The nine isoforms of the cAMP generating adenylyl cyclase (AC) enzyme differ in their tissue distribution, regulation, protein interactions, and localization in the plasma membrane. We hypothesized that these differences allow AC isoforms to differentially regulate cellular responses, including gene expression, in human bronchial smooth muscle cells (BSMC). A PCR array with 84 genes regulated by cAMP revealed that some were controlled in an AC isoform‐specific manner. Expression of pro‐inflammatory cytokine interleukin 6 (IL‐6) was selectively regulated by AC2. IL‐6 mRNA, protein and promoter activation was measured by quantitative RT‐PCR, ELISA and luciferase assay. Direct activation of AC's with Fsk, inhibition of phosphodiesterases with IBMX, or stimulation of a subset of the cell's Gs‐coupled receptors led to IL‐6 expression that was enhanced by overexpression and activation of AC2 but not AC6. Not all Gs‐coupled receptors expressed in BSMC were linked to IL‐6 expression, but AC2 specificity remained regardless of the receptor activated. BSMC from an asthmatic donor had increased basal IL‐6 expression but impaired induction by Fsk. AC2, but not AC6, produces cAMP in a compartment capable of inducing IL‐6 expression. Supported by NIH (HL079166).
Effects of D1‐like dopamine receptor stimulation and antagonism on cocaine choice in socially housed monkeys
Authors: Czoty Paul W. , Coller Michael , Nader Michael A.
Published: June 2021

Evidence supports the involvement of D2‐like and, to a lesser extent, D1‐like dopamine receptors (D2R, D1R) in the abuse‐related effects of cocaine. Effects of D2R‐acting drugs can be modulated by environmental factors including position in the social dominance hierarchy; less evidence exists for modulation of D1R‐mediated effects by social rank. Here we examined effects of acute i.v. administration of the high‐efficacy D1R agonist SKF81297 (0.1–1.0 mg/kg) and the D1R antagonist SCH23390 (0.003–0.056 mg/kg) on cocaine choice in dominant and subordinate monkeys. We used a procedure in which a complete cocaine dose‐effect curve was determined daily by making ascending cocaine doses (0.0, 0.003, 0.01, 0.03, 0.1 mg/kg/inj) available as the alternative to a food pellet under a concurrent fixed‐ratio schedule. In some subordinate monkeys, SCH23390 increased choice of lower cocaine doses. In most monkeys, however, there was no effect of either the agonist or antagonist up to a dose that decreased responding in the early components of the session. When a longer pretreatment time was used, rate‐decreasing effects were absent, and choice was no different than following saline administration. Taken together, these data suggest a lack of involvement of D1R in the relative reinforcing strength of coacine in a food‐drug choice procedure, which contrasts with previous studies using D2R agonists and antagonists. DA10584.
The effects of delta‐opioid receptor ligands on the primary and conditioned reinforcing effects of cocaine in rats
Authors: Jutkiewicz Emily M
Published: June 2021

Pharmacological and genetic studies suggest that the delta opioid receptor (DOR) system modifies addiction‐related behaviors and neurocircuitry. Therefore, the present study investigated the effects of DOR ligands on the primary and conditioned reinforcing effects of cocaine in rats. DOR agonists and antagonists were administered s.c. immediately before cocaine or food self‐administration sessions or before cue reactivity sessions. The delta opioid agonist SNC80 failed to maintain responding in naïve rats. In rats with a history of cocaine self‐administration, non‐contingent SNC80 administration failed to alter responding for cocaine injections but enhanced responding maintained by stimuli previously associated with cocaine. The delta opioid antagonist naltrindole decreased responding for cocaine‐paired stimuli in the absence of cocaine. Overall, these data suggest that the DOR system may modulate the reinforcing effectiveness of conditioned stimuli associated with drugs of abuse. These studies were supported by the Small Grants Program from the University of Michigan Substance Abuse Research Center.
Piroxicam Protects against Pentylenetetrazole Kindling: Role of Oxidative Stress and Inflammatory Pathways
Authors: Nassar Noha N , Mansour Rami N , Shoman Samia A. , Hamed Abdel Aziz A.
Published: June 2021

Increasing evidence has implicated reactive oxygen species and proinflammatory cytokines in incidence of seizures. Hence, this study investigated piroxicam (PXM), a non‐selective cyclooxygenase inhibitor in a pentylenetetrazole (PTZ) kindling model of epilepsy. To this end, male albino rats received a subconvulsive dose of PTZ (35 mg/kg IP on alternate days) for 20 days. Animals were pretreated with PXM (2 mg/kg IP) versus the standard antiepileptic drug valproate (200 mg/kg IP). Kindled rats showed elevation of hippocampal thiobarbituric acid reactive substances (TBARS) and tumor necrosis factor (TNF)‐ α versus a sharp decline in reduced non protein thiols (NPSH). Moreover, PTZ increased the expression of inducible nitric oxide (iNOS) which in turn exacerbates cellular damage. It has been demonstrated that PXM protected against PTZ‐induced seizures and delayed seizure latency onset. PXM reverted the downregulation of NPSH while unpredictably accentuating TBARS above control level. Moreover, PXM inhibited expression of iNOS consequently to the inhibition of TNF‐α thus limiting cellular damage induced by the latter. The results in the present study point that while an increase in proinflammatory cytokines is associated with a pro‐epileptogenic potential, PXM with its antiinflammatory effect showed remarkable anticonvulsive potential and cell protective properties.
Role of α3 subunit‐containing GABAA receptors in benzodiazepine (BZ)‐related anti‐conflict, reinforcing, and cognitive effects
Authors: Meng Zhiqiang , Sawyer Eileen K , Poe Michael , Wang Zhijian , Cook James M , Rowlett James K
Published: June 2021

Benzodiazepine (BZ)‐type drugs are effective in treating many disorders. However, their therapeutic use is limited by unwanted side effects including abuse potential, physical dependence, and cognitive deficits. BZ‐type drugs act by binding to GABAA receptors and facilitate the effects of GABA. As there are multiple subtypes of GABAA receptors, it may be possible to attribute the therapeutic effects and side effects of BZs to different subtypes. Here, we evaluated the behavioral effects of YT‐III‐31, a novel compound which exhibits selective efficacy at α3 subunit‐containing GABAA receptors, in rhesus monkeys. We first evaluated its anti‐anxiety effects using a conflict procedure. Pretreatment of YT‐III‐31 failed to produce robust anxiolytic effects. We then tested the reinforcing effects of YT‐III‐31 by using a progressive ratio self‐administration procedure. Significant reinforcing effects compared to vehicle control were found. Finally, we examined its effects on cognitive tasks. Using an intradimensional/extradimensional attentional set‐shift task, we found that YT‐III‐31 impaired performance similar to the reference BZ, alprazolam. Together, these results suggest that α3‐GABAA receptors play important roles in BZ‐related reinforcing effects and cognitive‐impairing effects, but not the anxiolytic effects. Funding: DA011792, DA033795, AG03536, MH046851, and OD011103
Ischemic stroke produces long‐term fiber track myelin loss that contributes to persistent sensory, motor and cognitive behavioral deficits
Authors: Poon Carrie , Zhou Jin , Zhuang Jian , Li Jie , Bloom Jonathan , Ooi Evelyn , Lax Daniel , Rosenbaum Daniel , Barone Frank
Published: June 2021

Objective

To investigate and characterize long‐term post‐ischemic stroke hemispheric and cellular loss, and fiber track injury. Also to investigate the sensory, motor and cognitive performance deficits for use in future restoration research.

Methods

Middle cerebral artery occlusion (stroke) or sham surgery was performed in male Sprague‐Dawley rats. Neurobehavioral (sensory, motor, beam balance, reflexes, hindlimb and forelimb function, and cylinder placement tests) and cognitive (complex active place avoidance; APA and simple passive avoidance retention; PA tests) performance was measured. Electroretinograms (ERG), ischemic hemisphere, hippocampus neuronal loss and fiber track injury were measured.

Results

Stroke surgery produced significant (p<0.01) sustained (10 weeks) deficits in motor, sensory, beam balance and hindlimb performance. Stroke produced large and prolonged cognitive performance deficits in APA learning (p<0.01) but smaller effects on PA retention. No effects on ERG or hippocampus neurons occurred but fiber tracts in the external capsule and striatum exhibited significant (p<0.01) damage.

Conclusion

Specific, long‐term deficits in neurobehavioral tests were verified as endpoints for stroke restorative therapeutic research. Fiber track injury contributes to these deficits and is especially important for the complex cognitive control required for APA learning.
Ubisol‐Q halts the progression of parkinsonian neurodegeneration in rodent models of Parkinson's disease
Authors: Weinstock Shelley B , Sandhu Jagdeep K , Sikorska Marianna , Pandey Siyaram , Cohen Jerome , Lanthier Patricia , Miller Harvey , Beyer Melissa , Sodja Carolyn , Facecchia Katie , Muthukumaran Krithika , Laframboise Alyson , Smith Jessica , Harrison Kate , Lopatin Daniel , Keshen Corrine
Published: June 2021

Coenzyme Q10 is a candidate for neuroprotection in Parkinson's disease (PD), in which both energy failure and oxidative stress are responsible for its progressive nature. The low therapeutic efficacy of CoQ10 in clinical trials of PD may be due to its insolubility in aqueous media, poor bioavailability and penetration into the brain. We have developed a technology to solubilize CoQ10 in water (Ubisol‐Q) in order to increase its therapeutic potential. Oral administration of Ubisol‐Q, containing PTSTM and CoQ10, elevated plasma and brain levels of both CoQ10 and vitamin E, in rats and mice. A prophylactic application of Ubisol‐Q (48 mg/kg/day or 6 mg/kg/day) in rodent models of PD (MPTP and paraquat) revealed a near complete protection of dopamine neurons. A therapeutic application of Ubisol‐Q, started immediately after the initiation of neuronal injury, protected dopamine neurons and preserved dopamine nerve terminals in both models of PD. Ubisol‐Q also resulted in a marked improvement in motor function of mice and rats performing on a beam walk test. The results of these pre‐clinical studies are highly encouraging and suggest that Ubisol‐Q, at lower doses than other CoQ10 products, has improved bioavailability and superior therapeutic efficacy and could be used as an adjuvant therapy in the management of PD. Ubisol‐Q is GRAS and will be tested clinically. Funding by MJFF and Zymes.
Suppression of Neurodegeneration in the Drosophila Models of Human Neurodegenerative Disorders
Authors: Casino Brittany A , Singh Satpal
Published: June 2021

Our lab has previously identified a mutation (levy) that provides a model of Leigh Syndrome (LS) in Drosophila melanogaster, as well as a second mutation, named Su(levy), which suppresses levy induced neurodegeneration (ND). The Su(levy) mutation confers resistance to temperature‐induced‐paralysis, a phenotypic marker of levy in Drosophila. Experiments are underway to identify and characterize which gene the suppressor mutation resides within. Preliminary experiments suggest that ND in the levy mutant may be caused by oxidative stress, a feature common to many neurodegenerative disorders such as Parkinson's Disease (PD) and Alzheimer's Disease (AD). This fact has broadened the suppressor mutation's application to possibly include PD and AD. The pesticide rotenone is known to induce Parkinson's‐like symptoms in humans and flies. Using rotenone to create a PD model, our lab is testing whether the suppressor can alleviate the phenotypic symptoms of this model. The suppressor mutation is being genetically combined with AD and PD mutations to further assess its ability to alleviate locomotor symptoms in the fly models of these disorders. The Su(levy) mutation made the levy flies resistant to paralysis. Experiments in progress suggest that the Su(levy) mutation may protect flies from the effect of the pesticide rotenone on locomotion. Suppressor's effects on PD and AD mutants will be measured through longevity, locomotion, and measurement of oxidative stress through ROS assays. If suppressor can alleviate ND in these models, this could provide leads to developing therapeutic approaches toward multiple neurodegenerative disorders. Funded by grant 1R03NS063148–01 from NINDS.
Predicting Anthracycline‐induced Cardiotoxicity in Children – Genome‐Wide Association Study
Authors: Aminkeng Folefac , Ross Colin JD , Rassekh Rod S , Brunham Liam R , Weissman Cory , Dube Marie‐Pierre , Visscher Henk , Rieder Michael J , Carleton Bruce C , Hayden Michael R
Published: June 2021

Identified genetic markers for anthracycline‐induced cardiotoxicity (ACT) explain only a small fraction of the variability of this phenotype, suggesting the presence of other, as‐of‐yet unidentified susceptibility loci. Therefore, our goal is to identify additional genetic markers with large effect size via genome‐wide association study (GWAS).

Patients were recruited and clinically characterized (age at start of treatment, cumulative dose, gender, anthracycline and tumor type, radiation therapy involving the heart, follow‐up time and assessment of LV dysfunction) via the Canadian Pharmacogenomics Network for Drug Safety. We have recruited and clinically characterized over 400 patients from across Canada to serve as our discovery cohort and over 120 patients from the Emma Children's Hospital in Amsterdam, the Netherlands, to be used as our replication cohort. More patients are currently being recruited and the clinical characterization is ongoing. We are now genotyping samples with an Illumina GWAS panel and the statistical analyses will be underway soon using SVS/Helix Tree, SPSS, Epi Info, PLINK and R.

Novel genetic predictors for ACT may become essential for screening patients before the start of treatment, comprehensive risk assessment and management, and evidence‐based treatment decisions, monitoring and prevention.

This work was supported by CIHR, CFRI, and Genome BC.
Protective effect of the RGS2‐eIF2Bε binding domain (RGS2eb) in cardiac hypertrophy
Authors: Lee Katherine Nan , Lu Sharon , Feng Qingping , Chidiac Peter
Published: June 2021

Regulator of G protein signalling 2 (RGS2) is known to play a protective role in maladaptive cardiac hypertrophy and heart failure via its ability to inhibit Gq and Gs mediated GPCR signalling. Recently, we have demonstrated that RGS2 can also inhibit protein translation, which would be expected to attenuate cell growth. This novel, G protein‐independent inhibitory effect has been mapped to a 37 a.a. domain (RGS2eb) within RGS2 that binds to eukaryotic initiation factor 2B (eIF2B). When expressed on its own in neonatal rat cardiomyocytes, RGS2eb attenuates both protein synthesis and hypertrophy induced by Gq and Gs activating agents. The objective of this study is to further elucidate the cardioprotective role of RGS2eb by determining whether mice with targeted cardiac expression of RGS2eb show resistance to the development of hypertrophy in comparison to wild‐type (WT) controls. Cardiac hypertrophy was induced by one month of transverse aortic constriction (TAC). RGS2ebtransgenic mice were found to have a significantly lower left ventricle/body weight ratio compared to WT TAC mice. In addition, the expression of hypertrophy markers, such as alpha natriuretic peptide (ANP), was decreased in RGS2eb TAC mice compared to WT TAC animals. These results suggest that RGS2eb, via its inhibition of protein synthesis, may be decreasing the severity of cardiac hypertrophy and aiding in the maintenance of cardiac function.
Decreased Telomerase Activity Sensitizes the Resistance Vasculature to Stress‐Induced Endothelial Dysfunction
Authors: Beyer Andreas M. , Hockenberry Joseph C. , Hockenberry David C.
Published: June 2021

Telomerase, an established modulator of senescence and tissue aging is traditionally described as a nuclear enzyme crucial in telomere maintenance. Recently, telomerase has been shown to co‐localize with mitochondria and to be a novel regulator of mitochondrial derived reactive oxygen species (mtROS). Previous data show that decreased that telomerase activity (TA) increases mtROS production in human and mouse resistance vessels. We hypothesized that TA has an endothelial protective role under conditions of external stress. Mouse mesenteric arteries (~200 μm) were isolated and incubated in endothelial growth medium with a subconstrictor dose of angiotensin II (A II; 10 nM) or vehicle (V) with and without telomerase inhibitor BIRB 1532 (BIRB; 10 μm) for 15–20 h. Flow‐mediated dilation (FMD) was studied in vitro (pressure gradient 0–100 cm H2O). Values expressed as % Max dilation at 50 cmH2O ± SEM; * p < 0.05 vs. control one way ANOVA RM. A II caused a trend toward a decrease in FMD (V 57.0 ±9.2 vs. A II 42.8±13.6; n≥4). BIRB did not decrease FMD, but changed the mediator from NO to mtROS (catalase and rotenone inhibitable 1.5*±6.8; 11.9*±11.1). Co‐incubation with A II significantly reduced FMD (BIRB 59.1±10.6 vs. + A II 16.3*±7.4 n≥4). These data indicate that telomerase protects the microvascular endothelium from mtROS‐induced vascular dysfunction under stress.
Microcirculatory anatomy can explain the illusory cooperativity seen in the flow response to exogenous adenosine
Authors: Bindschadler Michael David , Bassingthwaighte James
Published: June 2021

OBJECTIVE

To demonstrate that illusory cooperativity can arise in the microcirculation as a result of competition and spatial localization.

Adenosine in the myocardial interstitium binds to adenosine A2A receptors on vascular smooth muscle, leading to reduced tone and increased myocardial blood flow. The relationship between the concentration of supplied adenosine and the increase in flow is very steep (Hill coefficient 5–7), suggesting strong receptor binding site cooperativity. In fact, this apparent cooperativity may be illusory, and can arise solely from the anatomic geometry and cellular composition of myocardial tissue. Along the path from capillary to arteriolar smooth muscle cell, adenosine faces a permeability barrier (the endothelial clefts) and a diffusion distance (back from capillary to arteriole). Cellular transporters and metabolic enzymes compete for adenosine all along this path, influencing not only the apparent ED50 of arterially supplied adenosine, but also the shape of the dose‐response curve. Using a mathematical model, we demonstrate the independent influences of competition and access barriers (permeability barriers and diffusion distances), as well as how these effects can combine to make the dose‐response curve for a single binding site receptor (Hill coefficient 1) appear extremely cooperative.
Adding arginine to an essential amino acid (EAA) feed reverses age‐related impairments in vascular responsiveness
Authors: Mitchell William Kyle , Williams John , Rankin Debbie , Lund Jonathan , Larvin Mike , Phillips Bethan , Smith Kenneth , Atherton Philip J
Published: June 2021

Impaired vascular function in older age compromises the positive effects of nutrition on limb blood flow, perhaps impacting on maintenance of healthy muscle mass. Herein, we explored: (i) the effect of age upon macro/ micro‐vascular limb blood flow responses to nutrition, and (ii) the potential for arginine, the substrate of nitric oxide synthase, to improve nutrient‐mediate vascular responsiveness in older people. Using contrast enhanced ultrasound (CEUS) with Sonovue™ sulfur hexafluoride microbubbles, along with phase shift Doppler, we measured: changes in leg blood flow (LBF), quadriceps muscle microvascular perfusion (MMP) and microvascular flow velocity (MFV) in response to a 15g oral mixed EAA feeding in 8 young (~20y) and 8 older (~70y) men. An additional 8 older men (~70y) had the same 15g mixed EAA feed, supplemented with 3g L‐arginine. Young men showed marked vascular responses to EAA: LBF increased 44±11% (P<0.001), MFV increased 78±31% (P<0.01) and MMP increased 130±52% (P<0.001) from fasting. In contrast, older men fed 15g EAA had no significant changes in LBF or indices of muscle microvascular flow, while older men receiving 15g EAA with 3g arginine displayed increases in LBF (25±5%, P<0.05), MFV (88±48%, P<0.001) and MMP (110±70%, P<0.05, all 2‐way RM ANOVA). We conclude that supplementing EAA with L‐arginine restores some of the macro/ micro‐vascular responsiveness to feeding lost with aging.
Sulforaphane pre‐treatment reduces blood‐brain barrier disruption in experimental stroke by inducing heme oxygenase‐1 expression in the glio‐vascular complex of the peri‐infarct region
Authors: Alfieri Alessio , Srivastava Salil , Siow Richard , Modo Michel , Duchen Michael , Fraser Paul , Mann Giovanni
Published: June 2021

Heme oxygenase‐1 (HO‐1) serves as an inducible protective factor by its antioxidant and anti‐inflammatory actions. The isothiocyanate sulforapahane (SFN) induces HO‐1 via Nrf2. We have investigated HO‐1 induction in brain microvessels after experimental stroke, with and without SFN. Sprague‐Dawley rats (250–300g) were subjected to 70 min middle cerebral artery occlusion (MCAo) and 24 h reperfusion. SFN (5 mg/kg) was injected 1 h before MCAo. Brain sections were fluorescently stained for HO‐1 along with cell markers and IgG as marker of leak. HO‐1 expression was induced at 24h in microvessels of the peri‐infarct region, which was characterized by reduced leak compared to the infarct core. SFN reduced leakage into the brain (34±15% vs. 71±8% in untreated rats; P<0.05) and increased induction of HO‐1 in the penumbra (15±4% vs. 7±1%; P<0.05). HO‐1 upregulation was associated with perivascular astrocytes rather than the endothelium. Therefore, HO‐1 over‐expression in the glio‐vascular complex provides a protective mechanism against stroke, which can be targeted with SFN to prevent disruption of the blood‐brain barrier. Supported by the Henry Smith Charity and British Heart Foundation
TRPV4 Channel Activation Inhibits Tumor Endothelial Cell Proliferation and Migration Via Modulation of ERK1/2 pathway
Authors: Thoppil Roslin , Adapala Ravi K , Cotman Steven , Paruchuri Sailaja , Chilian William M , Meszaros J.Gary , Thodeti Charles K
Published: June 2021

We have previously shown that endothelial cells derived from tumor (TEC) exhibit aberrant Rho‐mediated mechanosensitivity that may be the underlying cause for abnormal tumor vessel formation. We recently found that TEC demonstrate significantly diminished expression and activity of a mechanosensitive ion channel, TRPV4, and exhibit abnormal angiogenesis compared to normal endothelial cells (NEC). Since angiogenesis involves endothelial cell proliferation and migration, in the present study, we investigated if activation of TRPV4 influences TEC proliferation and migration. We demonstrate higher basal proliferation and migration in TEC compared to NEC. Interestingly, activation of TRPV4 by a specific TRPV4 agonist, GSK101690A, significantly inhibited TEC proliferation. We found that basal ERK1/2 activity was higher in TEC when compared to NEC, which was significantly down regulated by GSK101690A. We further found that GSK101690A treatment inhibited abnormal TEC migration. Finally, we demonstrate that TRPV4 activation/overexpression normalized abnormal angiogenesis exhibited by TEC in 2D and 3D angiogenesis assays. Taken together, our results suggest that activation of TRPV4 normalizes angiogenesis by inhibiting abnormal proliferation and migration of TEC via modulation of ERK1/2 activity and proposes that TRPV4 is a novel target for vascular normalization therapies.
Regulation of exercise induced endothelial sprout formation by FoxO1/3/4
Authors: Liu Sammy T.K. , Slopack Dara , Roudier Emilie , Haas Tara L.
Published: June 2021

Forkhead Box “O” (FoxO) transcription factors have been shown to negatively regulated the process of capillary growth, but their influence on endothelial sprout initiation is not known. Mice with endothelial cell (EC) directed deletion of FoxO1/3/4 (FoxOΔ) have accelerated angiogenesis in response to repeated bouts of endurance exercise. We hypothesize this occurs due to FoxO regulation of genes such as Delta‐like1 (Dll1) and Tyrosine‐protein kinase receptor (Tek), which are involved in sprout formation and capillary maturation. EC with a deletion of FoxO1/3/4 showed decreases in Dll1 and Tek mRNA by 0.35 and 0.50 fold, respectively. Dll1 expression was significantly down‐regulated in mouse gastrocnemius muscle after 7 and 14 days of treadmill running, coinciding with the period of FoxO down‐regulation and capillary sprouting. In contrast, Tek expression was bi‐phasic, increasing two‐fold in animals trained for 1 and 14 days compared to sedentary counterparts. In FoxOΔ mice, basal Dll1 mRNA was reduced compared to wildtype, and there was no further decrease with training. The up‐regulation of Tek seen after 14 days of exercise was abolished in FoxOΔ animals. Our results indicate that Dll1 and Tek are novel FoxO targets, and their regulation with repeated exercise bouts may assist in the process of sprout formation and subsequent capillary maturation.
Tumor Cell Interaction with Sprouting Blood Vessels in 3D Microfluidic Platform
Authors: Ahn Song Ih , Song Sukhyun , Shin Jennifer Hyunjong
Published: June 2021

Tumor cells interact with blood vessels in the tissue in a number of different ways. For example, intravasation, tumor cell invasion into the blood vessel, is a critical step in early stage of cancer metastasis. Although previous studies have reported the tumor cell‐endothelial cell interactions in endothelial barrier, the interactions in the hyper‐sprouting tumor vasculature are not fully characterized. In this study, a novel microfluidic platform was developed to observe TC‐EC interaction between invading metastatic tumor cells and sprouting blood vessel in real‐time. Directional migration of metastatic breast tumor cells is induced by epidermal growth factor (EGF) gradient. Sprouting lumenized blood vessels are generated by the biochemical gradient of vascular endothelial growth factor (VEGF) and the secreted proteins from fibroblasts. Disruption of endothelial junction and cytoskeletal changes were observed during the tumor cell invasion into blood vessel. We also test the role of a few therapeutic biochemical reagents on the regulation of TC‐EC interactions.
Anti‐Angiogenic Factors of Tissue‐Engineered Oral Mucosal Epithelial Cell Sheets for Corneal Regeneration
Authors: Bardag‐Gorce Fawzia , Vilana Joan Oliva , Wood Andrew , Makalinao Andrew , Pan Derek , Sota Hiroyuki , Niihara Yutaka
Published: June 2021

Corneal regeneration by autologous oral mucosal epithelial cell sheet transplantation has been successfully performed in limbal stem cell deficiency (LSCD) patients. However, the molecular mechanism by which cell sheet grafting efficiently suppresses corneal vascularization, a major symptom of LSCD, is not fully understood. Here, we report the differential expression of pro‐ and anti‐angiogenic factors in cell sheets engineered from isolated rabbit oral mucosal epithelial cells, harvested on thermo‐responsive culture dish (UpCell®, CellSeed, Inc., Japan). A gene expression profiling‐directed study, followed by protein and histological analyses of cell sheets, showed a down‐regulation of pro‐angiogenic MMP‐3, and an up‐regulation of wound healing‐related proteins, MMP‐13 and collagen VIII. MMPs inhibitors, TIMP‐1 and TIMP‐3, were found to be expressed in cell sheets, thus reflecting a fine tuned balance between the pro‐ and anti‐angiogenic factors. These results indicated that tissue‐engineered cell sheets acquired a suppressive capability against corneal vascularization.
Anti‐aggregatory effects of Sulforaphane on in vitro platelet function
Authors: Gillespie Scarlett , O'Malley Aislinn , Solomon Antonia , Emerson Michael , Gavins Felicity
Published: June 2021

Introduction

Sulforaphane (SFN) is a phytochemical with proven anti‐inflammatory effects in various inflammatory models. Platelet‐leukocyte‐endothelial cell interactions are integral to the inflammatory cascade. Research has investigated SFN's effects on leukocytes and endothelial cells, but its effects on platelets are previously unknown.

Aim

Elucidate the effect of SFN on platelets in‐vitro

Methods and Results

Platelet aggregation was studied using light transmission aggregometry. Washed platelets (WP) were incubated with SFN (1, 40, 60 and 100μM) for 2/30mins, then challenged with collagen (2.5μg/ml). Incubation with SFN, 2 min (100μM) and 30min (40, 60 and 100μM), prior to collagen‐stimulated decreased platelet aggregation. Similar trends were seen following SFN incubations with platelet rich plasma (PRP) aggregations to ADP (1.56μM), however, this was not significant. Ca2+ mediated platelet signalling showed no detectable Ca2+ mobilisation following SFN addition. Lactate dehydrogenase (LDH) release following SFN incubation showed negligible differences to that associated with vehicle treatment or agonist stimulation.

Conclusion

SFN inhibited platelet aggregation to collagen in WP, and showed similar trends in PRP following ADP stimulated aggregations. SFN incubation did not appear to be associated with calcium mobilisation events, indicating these anti‐aggregatory effects were independent of intracellular Ca2+ movement, and low level LDH release suggested these effects were not via cytotoxicity. This novel work indicates an anti‐aggregatory ability of SFN in an agonist specific manner.

Work funded by the Medical Research Centre, UK.
Experimental Diabetes Impairs Endothelial IKCa Channel Function in Uteroplacental Arteries from Pregnant Rats
Authors: Gokina Natalia I. , Phillips Julie K. , Bonev Adrian D. , Gokin Alexander P. , Goloman Gabriela
Published: June 2021

Diabetic pregnancy is associated with adverse fetal complications in part due to impaired uteroplacental blood flow. In this study, we explored the role of endothelial cell (EC) SKCa and IKCa channels in impaired uteroplacental vasodilation during diabetic pregnancy. Diabetes was induced by i.p. injection of streptozotocin on 2nd day of pregnancy. We tested the effects of NS309 and CyPPA on arterial diameters, SMC [Ca2+]i and membrane potential using fura 2‐based fluorescence and glass microelectrodes. Whole‐cell patch clamp methodology was used to characterize SKCa and IKCa channel function in isolated ECs of control and diabetic rats. NS309‐induced vasodilation, SMC [Ca2+]i reduction and hyperpolarization were significantly impaired by diabetic pregnancy. Maximal responses to CyPPA were 50–60% smaller than those to NS309, and were not affected by diabetes. NS309 markedly potentiated endothelial K+ currents in ECs from control and diabetic rats. K+ currents were inhibited by appr. 80% and 20% with charybdotoxin (CTX) and apamin, respectively. Only CTX‐sensitive currents were reduced in diabetic ECs (6.5 ± 1.6 vs. 11.1 ± 1.1 pA/pF at 0 mV; n = 7).

In summary, IKCa channels play a predominant role in the control of uteroplacental dilatation. Impaired function of IKCa channels contributes to diabetes‐induced endothelial dysfunction in the maternal uteroplacental circulation.

Supported by NIH HL088245
EDHFs contribute to ACh‐mediated vasodilation in human skin in a dose‐dependent manner
Authors: Brunt Vienna E , Fujii Naoto , Minson Christopher T
Published: June 2021

The human cutaneous microcirculation has been shown to be reflective of globalized microvascular function, but the role of EDHFs in acetylcholine (ACh) mediated vasodilation has yet to be explored. In humans and animals, EDHFs appear to play only a modest role in ACh vasodilation, with a greater contribution at higher doses of ACh and under disease or NO‐deficient conditions. Five bolus doses of ACh (0.01, 0.1, 1, 10, and 100mM) were delivered via microdialysis fibers continuously infused with 1) Lactated Ringer's, 2) tetraethylammonium (TEA; calcium‐sensitive potassium channel & EDHF inhibitor), 3) L‐NNA + Ketorolac (to inhibit NO + COX), and 4) TEA + L‐NNA + Keto. The hyperemic response was characterized as area under the curve (AUC) and reported as % max cutaneous vascular conductance. In our preliminary findings (n=5), TEA had no effect on AUC compared to control at any of the lower doses of ACh, but did attenuate AUC to 100mM ACh (Control: 31,621±5,947% vs. TEA: 20,809±6,686%). We found similar results with combined NO + COX inhibition. TEA + L‐NNA + Keto did not attenuate AUC compared to L‐NNA + Keto until the 100mM dose of ACh (L‐NNA + Keto: 16,308±2831% vs. TEA + L‐NNA + Keto: 8,491±1,853%). Therefore, EDHFs appear to contribute to ACh vasodilation in the skin in a dose‐dependent manner, similarly to other vascular beds, confirming the skin to be a good model of overall microvascular function.

Supported by NIH Grant HL081671
Endothelial dynamics during sprouting morphogenesis
Authors: Bazou Despina , Song Jonathan , Munn Lance
Published: June 2021

Angiogenesis requires the coordinated growth and migration of endothelial cells (ECs), and each EC residing in a vessel wall integrates local signals to determine whether it remains quiescent or undergoes morphogenesis. Among these signals include vascular endothelial growth factor (VEGF) which induces the formation of specialized ‘tip cells’ that guide sprouts, and mechanical forces due to blood flow such as shear stress tangential to the endothelial surface and interstitial plasma flow across the vessel wall. Using a microfluidic tissue analogue of angiogenic sprouting, we found that physiological fluid shear stress (3 dyn/cm2) inhibits EC morphogenesis (sprouting and invasion into the 3‐D matrix) and interstitial flow (2.5–35 μm/s) increases the rate of morphogenesis. Surprisingly, invading ECs not only detected the direction of VEGF gradients but also showed dramatic differences in morphogenesis depending on the direction of interstitial flow. We furthermore show that these events require a multiple array of intracellular signaling pathways, including Dll4/Notch signaling as well as expression of VEGF receptor 2 (VEGFR2) at the tip cells, which are a hallmark of sprouting morphogenesis; analysis of the ultrastructural changes at the cell junctions level will further elucidate how ECs integrate signals from fluid forces and local VEGF gradients to mediate endothelial sprouting.
Post‐translational cleavage of Flt1 occurs very close to transmembrane domain releasing extracellular N‐terminal fragment which binds to PlGF
Authors: Raikwar Nandita S , Liu Kang Z , Thomas Christie P
Published: June 2021

Vascular endothelial growth factor receptor‐1/fms‐related tyrosine kinase 1 (VEGFR1/ Flt1) is a membrane bound tyrosine kinase receptor that can bind VEGF and PlGF and is involved in angiogenesis and vasculogenesis. Protein kinase C agonist phorbol myristic acid (PMA) stimulates Flt1 mRNA and protein expression in vascular endothelial cells. We studied the regulation of post translational cleavage of tagged full length Flt1 expressed in HEK 293 cells. Flt1 expression and cleavage of its extracellular (EC) domain is robustly induced by PMA and heparin treatment further enhances release of the EC domain into conditioned media. Co‐expression of the metalloproteases ADAM10 or ADAM17 significantly enhanced EC domain cleavage and reduced the abundance of uncleaved Flt1. In contrast, knock down of ADAM10 or ADAM17 reduced Flt1 EC domain cleavage. Conditioned media from Flt1 adenoviral transduced cells bound PlGF reducing free PlGF measured by ELISA. By sequential deletion analysis we identified a 5 AA region adjacent to the Flt1 transmembrane (TM) domain that is necessary to abolish the Flt1 EC domain cleavage. Thus, we demonstrate that proteolytic cleavage of Flt1 occurs close to the TM domain and is regulated by metalloproteases, with release of an EC fragment that is functionally equivalent to sFlt1.

NIDDK
Aging‐induced dysregulation of Dicer1‐dependent microRNA expression impairs angiogenic capacity of rat cerebromicrovascular endothelial cells
Authors: Ungvari Zoltan , Tucsek Zsuzsanna , Sosnowska Danuta , Toth Peter , Gautam Tripti , Valcarcel‐Ares Noa , Sonntag William , Ungvari Zoltan
Published: June 2021

Age‐related impairment of angiogenesis is likely to play a central role in cerebromicrovascular rarefaction and development of vascular cognitive impairment. To test the hypothesis that dysregulation of Dicer1 impairs endothelial angiogenic capacity in aging, primary cerebromicrovascular endothelial cells(CMVEC) were isolated from young (3 mo) and aged (24 mo) F344xBN rats. In aged CMVECs Dicer1 was down‐regulated and its expression was increased by treatment with PEG‐catalase. Compared to young cells aged CMVECs exhibited altered miRNA expression profile, which was associated with impaired proliferation, adhesion to vitronectin, collagen and fibronectin, cellular migration and impaired ability to form capillary‐like structures. Overexpression of Dicer1 in aged CMVECs partially restored miRNA expression profile and significantly improved angiogenic processes. In young CMVECs down‐regulation of Dicer1 (siRNA) resulted in altered miRNA expression profile associated with impaired proliferation, adhesion, migration and tube formation, mimicking the aging phenotype. Collectively, Dicer1 is essential for normal endothelial angiogenic processes, suggesting that age‐related dysregulation of Dicer1‐dependent microRNA expression may be a potential mechanism underlying impaired angiogenesis and cerebromicrovascular rarefaction in aging.
Role of peripheral inflammation in central cytokine signaling, depression, and fear
Authors: Tronson Natalie Celia , Radulovic Jelena
Published: June 2021

Activation of the innate immune system by chronic illness or injury is correlated with depression, anxiety and PTSD. In patients of heart attack (myocardial infarction, MI), rates of major depression and PTSD diagnoses are two‐ and three‐fold higher, respectively, than the population at large. Consistent with other patients, post‐MI incidence of PTSD and depression are correlated with increased levels of circulating pro‐ inflammatory cytokines. How circulating cytokines affect the brain remains unknown.

We used a surgical model of myocardial infarction in mice to investigate the role of sustained peripheral inflammation on alterations in (1) fear conditioning and depression‐like behaviors (2) cytokine signaling in the brain and (3) sex differences in post‐ MI behavioral and inflammatory changes.

Male, but not female mice exhibited enhanced fear conditioning soon after MI. Both males and females showed impaired memory and depression‐like behavior at later time points. 舲Within the hippocampus and prefrontal cortex, cytokine signaling and JAK/STAT3 were upregulated after MI in both female and male mice. The role of the cytokines and their downstream signaling in behavioral and emotional alterations are currently under investigation.

These findings suggest cytokine‐dependent signaling as a candidate mechanism for susceptibility to, or development of depression and PTSD.
Hypothermia through TRPV1 activation provides neuroprotection in acute cerebral ischemic stroke when initiated 90 minutes after reperfusion
Authors: Cao Zhijuan , Balasubramanian Adithya , Marrelli Sean
Published: June 2021

New treatment options are needed for ischemic stroke that are compatible with the conscious subject. We examined the therapeutic potential of producing hypothermia through activation of the transient receptor potential V1 (TRPV1) channel in a mouse model of focal stroke.

Core temperature, BP, and HR were measured in conscious TRPV1 WT and KO mice following subcutaneous dihydrocapsaicin (DHC; TRPV1 agonist) treatment. Subsequently, mild hypothermia (33 °C, >; 8 hours) by DHC infusion was initiated in conscious focal cerebral ischemia/reperfusion (I/R) mice (by distal MCA plus CCA occlusion) at 90 minutes reperfusion. Behavior testing and brain TTC staining was performed after 24 hours reperfusion.

DHC produced mild hypothermia in control and I/R mouse models, but not in TRPV1 KO mice. DHC treatment (1.25mg/kg) showed a small transient decrease (6 mmHg) in BP which could be completely blocked by atropine; there was no effect of DHC on HR. DHC‐induced hypothermia initiated at 90 minutes post‐reperfusion significantly decreased the infarct volume and improved behavioral neurofunction.

Our data indicate that activation of TRPV1 is a novel and safe method to induce sustainable and reversible hypothermia which provides neuroprotection in acute cerebral I/R mice. We propose that this method may provide a more broadly applicable means to produce therapeutic hypothermia for stroke. Funded by R21NS077413.
Subjective Tinnitus: Morphological Abnormal Features in the Inner Ear
Authors: Tanioka Hisaya , Tanioka Sayaka
Published: June 2021

Background

As many as 40 million people in the United State have tinnitus. The reported prevalence range is from 7% to 32%. The evaluation of a patient with tinnitus requires a defaulted history and comprehensive audiologic evaluation to determine the appropriate radiologic study of the choice.

Purpose

Describe the key anatomic or physiological issues, imaging findings or imaging technique the proposed paper will address. It is important to classify tinnitus as pulsatile or nonpulsatile for treatments. So, we must know how morphological features in the inner ear of subjective tinnitus.

Subjects and Methods

Imaging studies of 30 patients with clinically suspected nonvascular tinnitus were analyzed from our data. Pt not only arises from the vascular causes but can also originate from nonvascular structures and is classified as nonvascular tinnitus.

3D membranous labyrinth technique was obtained by HR‐CT with direct volume rendering by the use of current workstation soft.

Results

Unilateral tinnitus showed the lateral lesion of the affected utricle was swelling. But, the size and shape of maculae were within normal limit within early phase by virtual histological study. And, some cases revealed abnormal image of the cochlear duct such as irregular surface or discontinuity.

Conclusion

All tinnitus showed swelling of the affected utricle. This cause may be lack of uniformity of endolymphatic pressure or unbalance of metabolic process of the tissue of swelling wall of the utricle may be occurred. We thought this morphological finding was due to probably both the increased endolympatic pressure and partially decreased blood flow. Indeed, improved cases of subjective tinnitus showed improvement imaging after medical treatments and cardiovascular exercise.

Treatments

It is conceivable that increased blood flow reduce the increased endolymphatic pressure in the inner ear by achieving a balance of endolymph. Pts take some vascular medicines and cardiovascular exercise.
Mineralocorticoid receptor in NTS stimulates salt intake during 4th ventricular infusions of aldosterone
Authors: Koneru Bhuvaneswari , Bathina Chandra Sekhar , Cherry Brandon , Franzke Michelle , Mifflin Steve
Published: June 2021

The purpose of this study was to determine if neurons within the NTS that possess the mineralocorticoid receptor (MR) play a role in aldosterone stimulation of salt intake. Adult WKY rats received microinjections into the NTS of a small, hairpin RNA for the MR (shRNA; n=9) or a scrambled RNA (scRNA; n=8). After the viral construct injections, aldosterone‐filled osmotic mini‐pumps were implanted subcutaneously and connected to tubing within the 4th ventricle to infuse aldosterone at a rate of 20ng/h. Prior to and after surgeries, rats had ad libitum access to food and two graduated drinking bottles filled with distilled water and 0.3M NaCl and salt intake expressed as 100 × the ratio of 0.3M NaCl intake to total fluid intake. Prior to surgeries, basal salt intake was 4.7% ± 2.9% in the scRNA group and 3.91% ± 2.6% in the shRNA group. 24 days post‐surgery salt intake was 20.6% ± 2.9% in the scRNA group and 4.3% ± 2.8% in the shRNA group. Post‐mortem immunohistochemistry revealed a significant reduction in the number of NTS neurons exhibiting immunoreactivity for the MR (scRNA 33 ± 2 cells/section; shRNA 23 ± 1 cells/section; p=.008). These results indicate 4th ventricular infusions of aldosterone stimulate salt intake and that at least part of the stimulation of salt intake during 4th ventricular infusions of aldosterone is mediated by hindbrain NTS neurons that possess the MR.
ENaC localization in the sensory circumventricular organs
Authors: Miller Rebecca , Wang Michelle , Gray Paul , Loewy Arthur
Published: June 2021

Epithelial sodium channels (ENaCs) are widely expressed throughout the central nervous system (Amin et al., 2005). We re‐investigated this issue with a focus on the sensory circumventricular organs (CVOs) in the rat brain. Both in situ hybridization (ISH) and immunohistochemistry (IHC) were used to identify ENaC alpha‐ and gamma‐subunits in the area postrema (AP), organum vasculosum of lamina terminalis (OVLT), and subfornical organ (SFO). Using ISH, the density of ENaC alpha‐subunit expressing cells was analyzed in all three of the CVOs and found to exceed virtually all other nearby brain areas. ENaCs are present in both neurons and astrocytes in the CVOs. This was determined by double IHC procedures using antibodies against ENaCs in combination with the neuronal markers MAP‐2 or NeuN or the astrocyte marker GFAP. ENaC alpha‐subunit did not colocalize with GFAP, but the ENaC gamma‐subunit was colocalized with GFAP in all CVOs. The results suggest that an important portal of entry for sodium into the brain may be through the CVOs, which are linked to the central networks that modulate salt intake and central control of blood pressure. NHLBI NIH Grant #HL‐25449 and NINDS NIH Grant#NS057105