bluuebunny/crossref_metadata_2025 huggingface dataset:
Opposite Effect of TNF‐alpha and Activin A on CD69 Expression to Modulate Promoter Activity and Expression of Alpha‐globin and Zeta‐globin
Authors: Huang Huei‐Mei , Li Yung‐Chen , Liu Chin‐Wei
Published: June 2021

TNF‐α and activin A suppresses and induces erythroid gene expression, respectively. Previous studies showed that TNF‐α up‐regulates expression of CD69 in erythroblast K562 cells by an erythropoiesis suppressor NF‐κB. In this study, we investigated the role of CD69 in promoter activity and expression of erythroid genes, α‐globin and ζ‐globin, in response to TNF‐α and activin A. We found that activin A decreased CD69 level and increased promoter activity and expression of α‐globin and ζ‐globin in K562 cells. TNF‐α had opposite effects on CD69 level and promoter activity and expression of α‐globin and ζ‐globin in K562 cells. Knockdown of CD69 expression by shRNA abolished TNF‐α‐suppressed and activin A‐induced promoter activity and expression of α‐globin and ζ‐globin. These results suggest that TNF‐α up‐regulation of CD69 to suppress and activin A down‐regulation of CD69 to enhance promoter activity and expression of α‐globin and ζ‐globin.
SPINK5 expression in keratinocytes
Authors: Katsuyama Midori , Le Ngoc Anh , Demura Masashi , Tanii Hideji , Ota Yoko , Katsuyama Hironobu , Saijoh Kiyofumi
Published: June 2021

The balance between serine proteases, kallikreins (KLKs), and their inhibitor, Kazal type‐5 (SPINK5), is important to maintain steady desquamation. To explore regulation of SPINK5 expression in skin, its promoters (nucleotide ‐676/‐532 and ‐318/‐146 from the major transcription start site) were subjected to luciferase assay because these two sides displayed high luciferase intensity in NHEK human keratinocyte. Electrophoretic mobility shift assay (EMSA) appeared specific nuclear protein binding to these two sides with almost the same size shift. However, in DNA foot printing, ‐676/‐532 and ‐318/‐146 had no specific sequence protection. Thus, at this moment, we could not confirm the major regulator of SPNK5 gene expression in skin. We, at least, acknowledge that the exploration of the relationship between KLKs and SPINK5 will give crucial information related to skin differentiation/proliferation. (partially supported by Grant‐in‐Aid C‐264607944).
Cerium Oxide Nanoparticles Confer Protection against Severe Sepsis Induced Hepatic Inflammation and Injury in Sprague Dawley Rats
Authors: Manne Nandini , Arvapalli Ravikumar , Nepal Niraj , Rice Kevin , Blough Eric
Published: June 2021

Severe sepsis is a medical emergency that is characterized by high mortality. The acute nature of this disease is characterized by high levels of inflammatory mediators that cause multiple organ damage and death. Despite recent advances in medical care, most of the deaths due to sepsis are related to the reactive oxygen species (ROS) induced release of inflammatory mediators by host immune system in response to invading pathogens. Current treatment regime is largely supportive in nature and does not focus on the underlying cause. Studies have shown that cerium oxide (CeO2) nanoparticles have the intrinsic ability to scavenge ROS along with anti‐bacterial activity. Based on these unique properties, we hypothesized whether CeO2 nanoparticles could diminish sepsis‐induced mortality and associated hepatic damage. Male Sprague Dawley rats were subjected to a severe septic insult with 600mg/kg of cecal inoculum and treated with 0.5mg/kg of CeO2 nanoparticles intravenously. Our preliminary data suggests that the CeO2 nanoparticles improved animal survivability and attenuated hepatic superoxide levels. These changes were also accompanied by decrease in hepatic levels of iNOS and nitrotyrosine along with reduced macrophage infiltration. CeO2 nanoparticle treatment also attenuated sepsis induced increase in GST‐α and GST‐Mu by 15‐and 19‐fold respectively. Taken together, these data suggest that CeO2 nanoparticles may be used for the treatment of severe sepsis.
Effects of Ginger Juice Aflatoxin‐Induced Oxidative Stress in Rats
Authors: Rotimi Solomon , Adelani Isaacson , Okafor Adaobi , Iyanda‐Joel Wisdom , Rotimi Oluwakemi
Published: June 2021

This study was carried out to investigate the antioxidant properties of the fresh juice of ginger against aflatoxin‐exposure in rats. The preventive potential and antioxidant capacity of the juice was evaluated by assaying the activities of antioxidant enzymes and lipid peroxidation content in some organs and erythrocytes of rats. Twenty rats were randomly divided into four experimental groups thus: group 1 served as control, group 2 received ginger juice (2mL/Kg of 10% juice)alone, group 3 received aflatoxins (3.65mg/kg body weight) alone while group 4 was pre‐treated with ginger juice for 7days prior to aflatoxin administration. In the erythrocytes, aflatoxin treatment resulted in significant (p<0.05) increase in erythrocyte osmotic fragility and lipid peroxidation with a concomitant significant (p<0.05) decrease in the level of reduced glutathione. Pre‐treatment of rats with ginger juice significantly (p<0.05) prevent these changes by maintaining the activity of superoxide dismutase and preventing the increase in lipid peroxidation in the organs. Ginger juice pre‐treatment also significantly (p<0.05) increased the activities of hepatic glutathione‐S‐transferase, peroxidase and superoxide dismutase. It is concluded that ginger juice has preventive effect in rats with aflatoxicosis by promoting the antioxidant defense systems.
Metabolism of Opicapone, a Novel COMT Inhibitor: Characterization of In Vitro Glucuronidation
Authors: Loureiro Ana , Fernandes‐Lopes Carlos , Bonifácio Maria , Wright Lyndon , Soares‐da‐Silva Patricio
Published: June 2021

Opicapone, is a novel catechol‐O‐methyltransferase inhibitor, currently in phase III of clinical trials. In this study the glucuronide derivatives of opicapone, as well as the other detected opicapone metabolites, were quantified in mouse plasma and the enzymes involved in opicapone glucuronidation in humans were investigated. Following orally administration to mouse, opicapone reached plasma Cmax within 1 h and was completely eliminated from circulation by 48 h. Although opicapone sulphate and nitro reduced derivatives were detected, the primary metabolic pathway of opicapone in mouse model involved 3‐O‐glucuronidation. Opicapone glucuronidation was observed in liver and intestinal human microsomes with a Km of 27.9±6.47 µM and 41.9±26.9 µM, respectively. Among the 12 commercially available human recombinant UGT enzymes, only UGT 1A7, 1A8, 1A9 and 1A10 exhibited high opicapone glucuronidation specific activity. UGTs 1A1, 1A3 and 2B7 exhibited low glucuronidation rate, whereas no glucuronidation was observed with 1A4, 2B15, 2B17 and 2B4. UGT 1A9 had the highest formation rate of 3‐O‐glucuronide‐opicapone, with a Km similar to that obtained for UGT 1A1 and UGT 2B7 but with significantly higher intrinsic clearance. Nevertheless multiple UGT enzymes, most notable UGT 1A9 widely expressed in human liver, seems to be involved in the opicapone glucuronidation.
Metabolism of Opicapone, a Novel COMT Inhibitor, in the Rat: Characterization of In Vitro Sulfation
Authors: Loureiro Ana , Fernandes‐Lopes Carlos , Wright Lyndon , Soares‐da‐Silva Patricio
Published: June 2021

In this study, opicapone, a novel catechol‐O‐methyltransferase (COMT) inhibitor, metabolism was evaluated in the rat and the sulfotransferases (SULTs) responsible for opicapone sulfation were characterized. Opicapone 3‐O‐sulfate was present in monkey, dog, rat and human liver cellular fractions and represented the major metabolite in the rat. Regarding human kidney, liver and small intestine cellular fractions, the later displayed the highest activity towards opicapone sulfation. A systematic analysis of seven recombinant human SULTs, showed that SULT1A1*1 and SULT1A1*2 exhibited the highest catalytic activity for opicapone sulfation. The kinetic analysis of opicapone sulfation revealed that the affinity for the conjugation of opicapone by intestinal S9 fraction was 20‐fold higher than that for the liver S9 fraction. Similar apparent Km values were obtained for SULT1A1 and human liver S9. Opicapone sulfation by human S9 fractions and SULT1A1 isoform was inhibited in a concentration manner by quercetin and 2,6‐dichloro‐4‐nitrophenol, highly selective inhibitors of SULT1A1. Dopamine, the major endogenous substrate of SULT1A3, also inhibited opicapone sulfation, but acetaminophen had no effect. The enhanced catalytic activity for opicapone sulfation and the high level of expression of SULT1A1 in liver together with the similar Km values obtained for SULT1A1 and for human liver S9 fraction, suggest that SULT1A1 may play a major role in opicapone sulfation, though other SULTs, such as SULT1A3, may also be involved in opicapone sulfation, namely at the intestinal level.
Salt‐sensitive hypertension is associated with reduced urea transporter B expression in the choroid plexus and increased Na+ concentration in the cerebrospinal fluid
Authors: Guo Lirong , Sun Liankun , O'Rourke Stephen , Sun Chengwen
Published: June 2021

High salt intake causes an increase in Na+ concentration in the CSF of SHR and Dahl S rats, and increased CSF [Na+] contributes to sympothoexcitation and hypertension. However, the transporters in the choroid plexus (CP) mediating the imbalance of water and electrolytes in the CSF are still not identified. Current study demonstrated that high salt intake did not alter UT‐A expression, but there was a significant reduction in UT‐B expression in the CP of Dahl S rats. Reduced UT‐B expression was associated with increased [Na+] in the CSF and elevated MAP, as measured with radiotelemetry in Dahl S rats treated with high salt diet. We next examined the effect of selectively silencing UT‐B in the CP on CSF [Na+] and MAP in normotensive rats on normal diet versus high salt diet. ICV injection of lentiviral vector containing UT‐B shRNA (Lv‐UTB‐shRNA) significantly reduced UT‐B expression in the CP of rats as compared with rats received scramble Lv‐SCR‐shRNA ICV injection. High salt diet significantly increased MAP and HR in rats received ICV injection of Lv‐UTB‐shRNA. The elevated MAP was associated with increased [Na+] in the CSF. Chronic ICV infusion of Na+‐rich CSF resulted in significant elevations in MAP and HR. In conclusion, high salt diet significantly reduced UT‐B expression in the CP of Dahl S rats and selective silencing of UT‐B expression in the CP of normotensive rats markedly increases salt‐sensitivity in MAP and CSF [Na+]. These data indicate that reduced UT‐B expression in the CP may contribute to an imbalance of water and electrolytes in the CSF of Dahl S rats on high salt diet, thereby contributing to the development of salt‐sensitive hypertension.
Activation of the Prostanoid EP3 Receptor Mediates Central Nicotinic Acid‐Evoked Pressor Response in Conscious Rats
Authors: Rezq Samar , Abdel‐Rahman Abdel
Published: June 2021

Nicotinic acid (NA) produces prostaglandins (PGs)‐dependent flushing via GPR109A activation. Although NA crosses the blood brain barrier, there are no reports on GPR109A expression or function in blood pressure (BP) controlling nuclei; e.g. the rostral ventrolateral medulla (RVLM). We built on GPR109A expression in the RVLM by showing in conscious rats that intra‐RVLM NA (20 µg/rat) led to a biphasic BP elevation. The first pressor phase (within 1 min of NA injection) resembled intra‐RVLM L‐glutamate mediated pressor response, and was abolished by the N‐methyl‐D‐aspartate receptor (NMDAR) blocker (2‐amino‐5‐phosphonopentanoic acid; AP5). We hypothesized that the longer lasting second pressor phase (5‐15 min after NA injection) is mediated via local PGs release. The finding that the prostanoid EP3 receptor subtype blocker L‐798106 abolished the NA‐evoked delayed pressor response supported our hypothesis. NA increased L‐glutamate level in PC12 cells, which exhibit neuronal phenotype and express GPR109A. The BP and biochemical effects are GPR109A dependent because they were not reproduced by the NA isomer isonicotinic acid, which lakes the receptor activity. These novel findings suggest that both L‐glutamate and PGs release are involved in NA‐mediated pressor response and that GPR109A is the main target for NA central actions. Ongoing GPR109A knockout studies, direct measurements of PGs levels, and the effect of other EP receptor blockers on NA‐mediated pressor response will solidify the molecular mechanisms implicated in RVLM GPR109A involvement in BP regulation. The present findings are clinically relevant because they elucidate a mechanistic role for GPR109A in the central BP regulation and highlight central GPR109A blockade as a potential antihypertensive strategy.
Atypical Antipsychotic Off Label Use: Should We Worry About Cardiovascular Complications?
Authors: Packer Jonathan , Houseknecht Karen , Davidoff Amy
Published: June 2021

Despite label warnings for severe metabolic side effects causing insulin resistance and increased mortality, prescribing of atypical antipsychotic drugs (AADs) has increased significantly in recent years, particularly in children and elderly for non‐psychotic behaviors. Adverse cardiovascular (CV) events have been reported, including arrhythmias in elderly; however the underlying pharmacology is unknown. The goal of this study was to evaluate acute CV effects of several AADs. Anesthetized male adult rats were cannulated (femoral vein for drug delivery, right carotid artery for blood pressures), and sc electrodes for EKGs. Drugs selected for diverse receptor pharmacology and metabolic side effect profiles were chosen, and cumulative dose/response measurements were recorded (0.1 – 1.0 mg/kg, ~5 min intervals between doses). All drugs acutely reduced mean arterial pressure (MAP) with no compensatory increase in heart rate. Aripiprazole had the least effect on MAP (<10% reduction), while the other drugs reduced MAP by 50‐60%. Increased pulse pressure accompanied reduced MAP only with Risperidone. MAP effects were transient, recovering ~1‐2 min. No effects on EKG were detected.

Conclusions

Potency of drugs on MAP were positively correlated with Ki values for both α1‐adrenergic and D2 receptors: Risperidone<Clozapine<Olanzapine<Aripiprazole, whereas efficacy was similar for all, except reduced for Aripiprazole. Our results support the view that Aripiprazole is least likely to induce orthostatic hypotension, and its more selective D2 effects may contribute to reduced side effects.

Funded by grant DK095143 (KLH), Peter Morgane Research Fellowship (JDP)
Myeloid Differentiation Factor‐88 Deficiency Protects Against Lipopolysaccharide‐Induced Endothelial Dysfunction
Authors: Didion Sean , Sims Jerrell , Baldwin Zachary
Published: June 2021

Myeloid differentiation factor‐88 (MyD88) is an integral component of toll‐like receptor 4 signaling in response to lipopolysaccharide (LPS). The goal of this study was to test the hypothesis that homozygous MyD88‐deficiency protects against LPS‐induced endothelial dysfunction. Carotid arteries from MyD88+/+ and MyD88‐/‐ mice were incubated with either vehicle (saline) or LPS (0.05 or 0.1 µg/ml) for 22 hrs. LPS produced concentration‐dependent impairment (P<0.05) of endothelial responses to acetylcholine in MyD88+/+ mice. For example, acetylcholine (100 µM) produced 98±4, 40±6, and 20±8% relaxation in vehicle, 0.05 µg/ml LPS, and 0.1 µg/ml LPS treated vessels, respectively. Basal and NADPH‐stimulated superoxide levels were also selectively increased (P<0.05) in MyD88+/+ vessels treated with LPS. Endothelial function in MyD88+/+ vessels treated with LPS could be significantly improved in the presence of Tempol (1 mM), a superoxide scavenger. In contrast, LPS had no effect on endothelial responses or superoxide levels in MyD88‐/‐ mice. The effects of LPS were selective for endothelium as responses to nitroprusside were unaltered (P>0.05) in either vehicle‐ or LPS‐treated MyD88+/+ or MyD88‐/‐ vessels. Taken together, these findings demonstrate that MyD88 expressed in the vascular wall is a major mediator of endothelial dysfunction produced in response to LPS. Supported by NIH HL‐089884 and HL‐107632.
Endothelial Focal Adhesion Kinase Transcriptionally Regulates S1P1 to Maintain Lung Vascular Barrier Function
Authors: Yazbeck Pascal , Mehta Dolly
Published: June 2021

Formation of leaky blood vessels remains a persistent pathology in several diseases including acute lung injury. We recently demonstrated that focal adhesion kinase (FAK), a non‐receptor tyrosine kinase, canonically known to regulate focal adhesion formation plays a fundamental role in forming stable vasculature in adult mice lungs. We showed using a mouse model in which FAK was conditionally deleted only in endothelial cells (ECs), that loss of EC‐FAK spontaneously destabilized vessel formation in adult mice leading to diffuse lung hemorrhage, increased transvascular albumin influx, edema, and neutrophil accumulation in the lung mimicking acute lung injury (ALI). Sphingosine‐1‐phosphate (S1P) is a lipid mediator well known for its ability to strengthen endothelial barrier function. S1P binding to the S1P1 receptor is predominantly responsible for its barrier strengthening effect. Intriguingly, my Preliminary Data showed that S1P1 expression at the level of mRNA and protein was markedly suppressed in FAK depleted human and mouse lung ECs. I also found that KLF2 (Krϋppel‐like transcription factor 2) mRNA, which may regulate S1P1 expression, was reduced in FAK depleted cells. Based on these key findings I will address the innovative concept that FAK regulates the formation of stable endothelial barrier function by inducing S1P1 expression through KLF2 and thereby facilitate in maintaining normal vascular homeostasis following lung injury. I believe that by testing this hypothesis I will identify a novel paradigm of restoring the formation of stable endothelial barrier following lung injury potentially leading to development of novel therapeutics against ALI.
Simvastatin, Atorvastatin, and Pravastatin Equally Improve Cardiovascular Status in Diabetic Rats by Improving Endothelial Function, and Reducing Perivascular Fibrosis and Oxidative Stress
Authors: Quidgley Jose , Crespo Maria , Cruz Nildris
Published: June 2021

Statins improve cardiovascular (CV) status of diabetic patients, regardless of their cholesterol levels. It remains unclear whether this effect is drug‐specific or class‐dependent,and the underlying mechanisms involved. We compared the effects of a low dose (10 mg/kg/day) of atorvastatin (AV), simvastatin (SV), and pravastatin (PV) over a four‐week period on cardiac performance in streptozotocin‐induced diabetic rats. Age‐matched, non‐diabetic rats were used as controls (CT). Echocardiographic variables, systolic blood pressure (SBP), acetylcholine (ACh)‐induced relaxation, plasma cholesterol levels, perivascular fibrosis, and oxidative‐stress markers MDA and 4‐HAE were measured. Cholesterol levels (mg/dl) were higher in diabetic rats than in CT (248.68 ± 15.78 vs. 156.01 ± 7.32, n = 8, P<0.05), and were not modified by any statin at this low dose. All three statins significantly reduced SBP in diabetic rats. Ejection fraction, stroke volume, and cardiac output index were lower in untreated diabetic rats than in CT (n = 10, P<0.05). All three statins significantly increased these CV parameters (n = 10, P<0.05). In addition, statins improved EMax values for the ACh‐induced relaxation, decreased media thickness, perivascular fibrosis, and both MDA and 4‐HAE in the aortas of diabetic rats. Together, our results indicate that in diabetic rats, AV, SV, and PV are equally effective in improving CV performance. The observed hemodynamic benefits are cholesterol‐independent, and appear to be secondary to improved endothelial function, and to reduced vascular tone and remodeling resulting from decreased oxidative stress. Supported by MBRS‐RISE R25‐GM061838.
Increased Energy expenditure induced by Ptp1b deletion in proopiomelanocortin (POMC) neurons leads to inflammation and endothelial dysfunction
Authors: Bruder‐Nascimento Thiago , Bence Kendra , Belin de Chantemele Eric
Published: June 2021

Increased energy expenditure is associated with inflammation while genetic deletion of cytokine sensitizes mice to obesity. Deletion of Protein tyrosine phosphatase 1b (Ptp1b), a negative regulator of leptin and insulin signaling pathways, in proopiomelanocotin neurons protects mice from obesity and type 2 diabetes by increasing energy expenditure. However whether the deletion of POMC‐Ptp1b induces inflammation and impairs the cardiovascular function is unknown. We analyzed the metabolic and endothelial function of wild‐type (WT) and POMC‐Ptp1b KO mice (KO). Total body fat was reduced in KO animals as measured by EchoMRI and fat pad measurements. Plasma TNF‐α levels were doubled KO vs WT mice. Measurement of aortic rings endothelial function revealed that POMC‐Ptp1b deletion reduced ACh‐induced relaxation. Pre‐incubation with tempol [superoxide dismutase mimetic (SOD)], catalase and GKT137831 (Nox1‐4 inhibitor) restored endothelial function in KO mice. Moreover, quantitative real time RT‐PCR revealed an increase in NOXA1 and Nox4 expression and a decrease in SOD1‐3 and catalase. Pre‐incubation with indomethacin (COXs inhibitor), NS‐398 (COX2 inhibitor) and SQ28668 (TXA2 receptor antagonist) reverted the impaired relaxation for ACh in aortic rings from KO mice. POMC‐Ptp1b deletion did not affect COX1 expression, but increased COX‐2 gene and protein expression in aorta, as measured by qRT‐PCR and Western Blot respectively. These data suggest that increased energy expenditure induced by POMC‐Ptp1b deletion leads to inflammation triggering an endothelial dysfunction dependent of COX2‐derived products and NADPH‐oxidase generated ROS.
DNA‐Dependent Protein Kinase Inhibition Blocks Asthma in Mice and Modulates Human Endothelial and CD4+T Cell Function Without Causing SCID
Authors: Pyakurel Kusma , Ghonim Mohamed , Ju Jihang , Rodriguez Paulo , Lammi Matthew , Davis Christian , Abughazleh Mohammad , Naura Amarjit , Boulares Hamid
Published: June 2021

We reported that DNA‐dependent Protein Kinase (DNA‐PK) is critical for the expression of NF‐κB‐dependent genes in TNF‐α‐treated glioblastoma cells, suggesting an involvement in inflammation. Cell culture and ovalbumin (OVA) or house dust mite (HDM)‐based murine asthma models were used in this study.

Results

DNA‐PK was essential for monocyte adhesion to TNF‐α–treated‐endothelial cells. Administration of the DNA‐PK inhibitor, NU7441, reduced airway eosinophilia, mucus hypersecretion, airway hyperresponsiveness (AHR), and OVA‐specific IgE production in mice. Such effects correlated with a marked reduction in lung VCAM‐1 expression and production of several inflammatory cytokines and chemokines. DNA‐PK heterozygosity also prevented manifestation of asthma‐like traits. Remarkably, such protection occurred without causing SCID. These results were confirmed in a chronic model of asthma using HDM. Adoptive transfer of Th2‐skewed OT‐II‐WT CD4+ T cells reversed IgE and Th2 cytokine production but not AHR in ova‐challenged DNA‐PKcs+/‐ mice. DNA‐PK inhibition reduced IL‐4/IL‐5/IL‐13/eotaxin/IL‐8/MCP‐1 production without affecting IL‐2/IL‐12/IFN‐γ/IP‐10 production in CD3/CD28‐stimulated human T cells potentially by blocking expression of gata‐3. These effects occurred without significant reductions in T‐cell proliferation. In mouse T cells, DNA‐PK inhibition, in vitro, severely blocked CD3/CD28‐induced gata‐3 and t‐bet expression in T cells and prevented differentiation of Th1 and Th2 cells. Our results suggest DNA‐PK as a novel determinant of asthma and a potential target for the treatment of the disease.
Support: NIH; AHA; LCRC
Oroxylin A‐induced Endothelium‐dependent Constriction of Tail Arteries of Endotoxemic Hypotensive Rats
Authors: Tseng Tzu‐Ling , Chen Mei‐Fang , Liu Chin‐Hung , Lee Tony
Published: June 2021

Refractory hypotension and vascular hyporeactivity in severe sepsis remain major clinical concerns. Here we report the induction of endothelium‐dependent constriction of tail arteries (TA) in endotoxemic hypotensive rats. In lipopolysaccharide (LPS)‐induced endotoxemic rats, oroxylin A (OroA, 15 mg/kg, iv) administered 6 hrs after LPS‐challenge promptly reversed diminished mean arterial pressure(MAP) and heart rate. OroA (1‐10 µM), which did not constrict isolated normal TA, repeatedly induced exclusive endothelium‐dependent, sustained constriction of isolated endotoxemic TA with intact‐endothelium (EC+). OroA‐induced constriction of was not blocked by endothelin‐1 (ET‐1) receptor antagonists (BQ123 or BQ788, 10 µM), but was blocked by inhibitors for RhoA/ROCK pathway. These results suggest that OroA‐induced endothelium‐dependent constriction of endotoxemic TA is mediated by non‐ET‐1‐substance(s) which activated muscle RhoA/ROCK pathway. OroA‐post‐treatment also suppressed, via inhibition of NF‐κB, inducible‐NOS expression and circulating NO. It is concluded that LPS upregulates endothelial non‐ET‐1‐vasoconstrictor(s) in endotoxemic TA. Its release by OroA activates muscle RhoA/ROCK pathway leading to prompt and sustained vasoconstriction that partly is caused by decreased circulating NO induced by OroA. The endothelium‐dependent constriction of TA and suppression of systemic inflammation as evidenced by OroA may offer a newstrategy for acute management of endotoxemic hypotensive shock (supported by MOST, TCU, and Tzu Chi Foundation).
Mechanisms of Uridine Adenosine Tetraphosphate (Up4A)‐induced Contraction in Mice Aorta: Role of Thromboxane (TXA2) and Purinergic Receptors (P2R)
Authors: ZHOU ZHICHAO , SUN Changyan , TILLEY STEVE , MUSTAFA S JAMAL
Published: June 2021

Up4A, a novel endothelium‐derived vasoconstrictor, is proposed to play a role in cardiovascular disorders and induces aortic contraction through cyclooxygenases (COX). We demonstrated earlier that activation of A3 adenosine receptors (AR) results in aortic contraction via TXA2 production. However, the mechanisms of Up4A‐induced contraction are not understood. We hypothesize that Up4A‐induced contraction is via A3AR‐mediated TXA2 production. Concentration responses to Up4A were conducted in aorta from WT and A3AR KO. Up4A (10‐9‐10‐5 M) produced a dose‐dependent contraction (~80%), which was attenuated by non‐selective COX (indomethacin, 10 μM) and selective COX1 (SC560, 10 nM) but not by COX2 inhibition (NS398, 1 μM). Notably, Up4A‐induced contraction was blunted by TXA2 synthase inhibitor ozagrel (10 μM) and TXA2 receptor antagonist SQ29548 (1 μM). Non‐selective AR antagonist 8PST (100 μM) nor A3AR deletion affected Up4A‐induced contraction. However, both non‐selective P2R antagonist PPADS (10 μM) and P2X1R antagonist MRS2159 (30 μM) significantly attenuated Up4A‐induced contraction (~46% and 40%), respectively. Combination of ozagrel with MRS2159 further decreased this effect (~20%). Endothelial denudation almost fully reduced Up4A‐induced contraction. Furthermore, TXA2 production increased in vessels treated with Up4A (3 μM) (26.6±0.5 vs. 83.4±0.8 pg/mg), which was inhibited by either MRS2159 (49.0±0.7 pg/mg) or Ozagrel (42.4±0.5 pg/mg). In conclusion, Up4A‐induced contraction is via TXA2 production, which partially requires the activation of P2X1R but not A3AR through an endothelium‐dependent mechanism (Supported by HL 027339, HL 094447 and MEPSCKL201301).
Differential synergy with nitric oxide across a panel of soluble guanylate cyclase stimulators
Authors: Long Kimberly , Tang Kim , Sarno Renee , Solinga Rob , Masferrer Jaime
Published: June 2021

Soluble guanylate cyclase (sGC) is the endogenous receptor for nitric oxide (NO). Upon binding of NO, sGC catalyzes the synthesis of cGMP which is involved in numerous downstream processes, including vasorelaxation, fibrosis, and inflammation. Dysregulation of this pathway has been implicated in multiple pathologies. sGC stimulators are a class of molecules which are capable of increasing the activity of the enzyme. While these stimulators are capable of activating the enzyme in the absence of NO, their effect is greatly amplified in the presence of NO. However, it is unclear if all stimulators synergize with NO equally. To examine differences in NO synergy across stimulators, we assessed a panel of compounds for their ability to stimulate cGMP synthesis with or without NO in a human whole cell assay. All compounds selected for this analysis were potent in vitro under high NO conditions (with EC50 values ranging from 10 to 200nM) and were effective at lowering blood pressure in rodents at doses between 1 and 10 mg/kg. We found that all compounds tested had similar EC50 values (~5µM) when examined in the absence of NO. However, in the presence of high NO the compounds exhibited a wide range of potency shifts relative to the NO‐free state (16‐320 fold). Emax values for the compounds were also differentially affected by NO, with some compounds reaching the same Emax regardless of the presence of NO, and others exhibiting a 4‐6 fold increase in Emax upon addition of NO. These data suggest that synergy between NO and sGC stimulators is complex and varies considerably between compounds. We hypothesize that differences between compounds in NO synergy may be harnessed to maximize their therapeutic value in a variety of disease states and patient populations.
Exendin Increases Gastric Acid Secretion in the Anesthetized Rat
Authors: Shadoan Melanie , Anderson Donald , Rajpal Neetu , Numerick Mary , Dennis Steve , Young Andrew
Published: June 2021

The objective of the current study was to assess the effect of the anti‐diabetic peptide, exenatide (Byetta®), on gastric acid secretion in the rat. Fasted rats were anesthetized and cannulas placed in esophagus (timed stomach secretion sampling), femoral artery (blood pressure monitoring), carotid artery (timed blood sampling). Phenol red was injected via the esophageal cannula and recovered every 10 minutes. Stomach aspirates were assessed for phenol red content (gastric emptying), pH, and titrated to pH 7.0 to calculate umoles of acid recovered. Exendin (up to 150 ug/kg) or vehicle were administered as a subcutaneous bolus at time zero, and stomach contents and blood were collected every 10 minutes for the subsequent 90 minutes. At 90 minutes, a pentagastrin bolus (~250 ug SC) was administered to confirm integrity of parietal cell responsiveness to pharmacologic stimulation of acid secretion. Basal acid secretion rate in the anesthetized rat (~1‐3 umoles per 10 minutes) was confirmed as lower than acid secretion rates previously reported in conscious rats. Exendin increased gastric acid secretion several‐fold above basal secretion rate, with maximal effect at 20‐30 minutes post‐injection. The anesthetized rat is a suitable model for assessing acute effects of peptides on stimulation of gastric acid secretion. The low basal acid secretion provides a substantial margin to successfully observe stimulation of acid secretion above an unstimulated baseline for at least 90 minutes. This model also allows simultaneous evaluation of hemodynamics and blood samples for pharmacokinetics and biomarker analysis.
Chronic but Not Acute Exposure to Morphine Enhances nAChR Mediated Responses in Enteric Neurons
Authors: Gade Aravind , Damaj Imad , Dewey William , Akbarali Hamid
Published: June 2021

A major side‐effect of chronic opioid treatment is constipation whereas withdrawal following chronic exposure leads to diarrhea and increased GI motility. Previous studies have shown that chronic opioids enhance sensitivity to nicotine in the GI tract. In this study we examined the sensitivity to nicotine following short (10 mins) and long‐term exposure (16‐20 h) to morphine in isolated mouse single enteric neurons using patch‐clamp technique. Nicotine induced inward currents from holding potential of ‐60 mV in a dose‐dependent manner. The amplitude of the nicotine induced currents were significantly increased in neurons exposed for long‐term (16‐20h) but not short‐term (10 mins) to morphine (3 μM). The maximal current densities induced by 1mM nicotine were 139 ± 40 pA/pF, 135 ± 31 pA/pF and 274 ± 29 pA/pF (p<0.05) in control, short‐term and long‐term morphine treated cells respectively. We have previously shown that tolerance and dependence to chronic morphine occurs in the ileum but not the colon. To test if the enhanced nicotine responses are dependent on morphine tolerance and dependence, nicotine responses were determined in colonic enteric neurons. The maximal current densities induced by 1mM nicotine were 146 ± 28 pA/pF in control and 235 ± 19 pA/pF (p<0.05) in long‐term morphine treated colonic neurons. Nicotine‐induced responses were similar in neurons from α7, α5 and β2 knock out mice suggesting that these isoforms are not the principal nAChR subunits in the mouse enteric neurons. These findings indicate that enhanced nicotinic responses following prolonged morphine exposure are not associated with morphine dependence and may involve alterations in intracellular signaling pathways. Supported by NIH DA024009
Transforming Non‐Ulcerogenic Stress Stimuli into Gastric Ulcerogenic Ones under the Circumstances of Inhibition of the Hypothalamic‐Pituitary‐Adrenocortical Axis
Authors: Filaretova Ludmila , Myazina Marina , Bagaeva Tatiana
Published: June 2021

To investigate contribution of glucocorticoids to the maintenance of gastric mucosal integrity during stress we predominantly used ulcerogenic stress models. Using these models we demonstrated that glucocorticoids released in response to the ulcerogenic stimuli attenuated their harmful action on the gastric mucosa.In the present study we hypothesized that mild stressors does not damage the gastric mucosa due to gastroprotective action of glucocorticoids released in response to these stimuli. To verify the hypothesis the effects of normally non‐ulcerogenic mild stimuli (15‐30 min cold‐restraint) on the gastric mucosa have been studied under the circumstances of inhibition ofthe hypothalamic‐pituitary‐adrenocortical (HPA) axis in rats. The HPA axis was inhibited by: 1) fast inhibitory action of NBI 27914, the selective antagonist of cortricotropin‐releasing factor receptor type 1; 2) fast inhibitory action of metyrapone, inhibitor glucocoricoid synthesis; 3) delayed inhibitory action of a single pharmacological dose of cortisol injected one week before the onset of stress stimulus. Each of these pretreatments decreased 15‐30 min cold‐restraint‐produced corticosterone levels and resulted in an appearance of gastric lesions after the onset of this mild stress. The results suggest that in rats with glucocorticoid deficiency non‐ulcerogenic stimuli are transformed into ulcerogenic ones and confirm the hypothesis. The findings further support for the point of view that glucocorticoids released during acute stress are gastroprotective factors. Supported by the Russian Scientific Foundation (grant N 14‐15‐00790).
Regulation of Somatic Pain Sensitivity under the Circumstances of Indometacin‐InducedGastric Injury: Participation of Cortricotropin‐Releasing Factor
Authors: Filaretova Ludmila , Bagaeva Tatiana , Yarushkina Natalia
Published: June 2021

In the present study we investigated the somatic pain sensitivity under the circumstances of indometacin (IM)‐induced gastric ulceration and effect of cortricotropin‐releasing factor (CRF) on the somatic pain sensitivity under the circumstances in conscious rats. IM (35 mg/kg) was administered subcutaneously to preliminary (24h) fasted as well as fed rats. CRF was injected 30 min before IM administration. The somatic pain sensitivity was estimated by tail flick latencies. A single IM administration caused the gastric erosion formation 4 h after the injection only in fasted but not fed rats. IM‐induced gastric injury was accompanied by an increase in tail flick latency in fasted rats. Pretreatment with CRF prevented this increase. CRF by itself did not influence the somatic pain sensitivity. Since we did not observe any changes in tail flick latency in fed IM‐treated rats without gastric mucosal lesions, it was reasonable to assume that an increase in tail flick latency in preliminary fasted IM‐treated rats was associated with the pathological changes in the gastric mucosa, but not with IM‐caused analgesic action by itself. Thus, IM‐induced gastric injury resulted in the suppression of the somatic pain sensitivity and CRF might be involved in regulation of the somatic pain sensitivity under the circumstances in conscious rats. Supported by the Russian Scientific Foundation (grant N 14‐15‐00790).
Chemopreventative properties of β‐blockers
Authors: Huang Kevin , Cleveland Kristan , Chang Andy , Yeung Steven , Huang Ying , Andresen Bradley
Published: June 2021

Recently carvedilol, a β‐AR GRK/β‐arrestin biased agonist, was shown to have cancer preventative effects; yet, atenolol, a β2‐AR inverse agonist, was less potent and efficacious. These disparate results lead to the hypothesis that the GRK/β‐arrestin biased agonist β‐blockers act as chemopreventive agents. JB6 P+ cells are an established chemoprevention model that is transformable by epidermal growth factor (EGF). Thus, JB6 P+ cells treated with 10 ng/mL EGF were utilized to screen partial agonist, inverse agonist, neutral antagonist, and biased agonist β‐blockers for inhibition of growth in soft agar. Additionally, parallel cytotoxicity assays were conducted for each β‐blocker to ensure that any observable inhibition is not due to cell death. EGF‐mediated colony formation is fully inhibited by carvedilol with a LogIC50 of ‐6.365 ± 0.158 and partially (47.9% ± 7.6%) inhibited by atenolol with a LogIC50 of ‐5.037 ± 0.266 at non‐toxic concentrations. Preliminary studies indicate that: 1) nebivolol acts similarly to carvedilol;2) acebutolol, ICI 118,551, metoprolol, and bucindolol have little to no effect; and 3) labetalol, carazolol, and propranolol have effects similar to atenolol. Expression of only β2‐ARs can explain the changes in potency, but the differences in efficacy suggest a unique biological effect. Thus, categorization of the β‐blockers by type paradoxically indicates that β‐blockers that can activate the EGF receptor are the most potent inhibitors of EGF‐mediated transformation of JB6 P+ cells and thus my serve as chemopreventative agents.
Evaluation of CCR1 Antagonists on CCL3‐mediated receptor internalization
Authors: Gilchrist Annette , Cook Kayla
Published: June 2021

Multiple Myeloma (MM) is a cancer of plasma cells, the white blood cells that produce antibodies. MM is characterized by an accumulation of malignant plasma cells in the bone marrowand the presence of osteolytic lesions. CCR1 is a chemokine receptor expressed on a variety of cell types, including myeloma cells. While several chemokines can activate CCR1 only CCL3 has been consistently observed to be expressed and secreted in most MM cell cultures, primary MM bone marrow, and patient serum samples. Moreover, CCL3 has been shown to stimulate migration and proliferation of MM cells. We evaluated a collection of novel pyrrolidine compounds based on a previously disclosed series of CCR1 antagonists, and found that some compounds had stronger effects than others. We measured responses through the use of flow cytometry to measure surface expression of CCR1 and CCR5 on RPMI 8226 cells, a multiple myeloma cell line.
The Zinc Finger Transcription Factor Mds1‐Evi1 Forms a Novel Protein Complex in MLL leukemia
Authors: McGlynn Kelly , Zhang Yi , Perkins Archibald
Published: June 2021

The dual transcription factor‐methyltransferase Mds1‐Evi1 is frequently overexpressed in high‐risk leukemias. Mds1‐Evi1 contributes to leukemias induced by the mixed lineage leukemia (MLL) fusion protein MLL‐AF9, although the mechanism is unknown. MLL contains chromatin‐targeting and histone lysine methyltransferase domains, and fusion to AF9 can cause aberrant protein recruitment to target genes. Although Mds1‐Evi1 contains a methyltransferase domain, it does not appear to act on histones. Therefore, we are interested in how protein‐protein interactions involving Mds1‐Evi1 contribute to leukemogenesis. Many known interactors of MLL and Mds1‐Evi1 include chromatin modifying enzymes, suggesting that coordination between epigenetic modulators is key in leukemia pathogenesis.

Here, we report the biochemical purification of a novel Mds1‐Evi1‐containing protein complex using a unique murine model of leukemia. To isolate the complex, we induced acute MLL‐AF9 leukemia in mice containing a tandem affinity purification (TAP) tag knocked into the endogenous Mds1‐Evi1 gene. Leukemic spleen tissue from these mice was used to purify the TAP‐tagged protein complex. Proteomic analysis revealed several members of the SWI/SNF chromatin remodeling complex, which is involved in epigenetic control of a variety of gene programs. Association between Mds1‐Evi1 and SWI/SNF may allow the activation of cell‐cycle and differentiation programs, contributing to transformation. SWI/SNF also coordinates with MLL fusion proteins to activate the Hox family of leukemia oncogenes. Our results provide a link between Mds1‐Evi1 and the chromatin remodeling machinery, where abnormal coordination of Mds1‐Evi1, MLL‐AF9 and SWI/SNF may lead to gene activation and the development of leukemia.
Lysophosphatidic Acid Stimulation Does Not Induce a Lymphatic Identity along Blood Vessels in Intact Microvascular Networks Ex Vivo
Authors: Sweat Richard , Azimi Mohammad , Murfee Walter
Published: June 2021

The design of therapies aimed at manipulating microvascular growth requires understanding the relationships between angiogenesis and lymphangiogenesis. A critical question remains whether blood and lymphatic vessels can switch their identity in adult tissues. Lysophosphatidic acid (LPA), a lipid linked to oncogenesis, wound healing, and inflammation, has been shown to upregulate lymphatic marker expression by HUVECs in vitro. The objective of this study was to determine if LPA stimulation induces blood to lymphatic vessel phenotypic transition in intact microvascular networks. Using the rat mesentery culture model, which enables ex vivo investigation of angiogenesis and lymphangiogenesis, tissues from adult male Wistar rats were cultured for 3 days in serum‐free minimum essential media (MEM) or MEM with LPA (20 µM). Tissues were labeled for PECAM and either LYVE‐1, Prox1, or podoplanin to identify blood and lymphatic vessels, respectively. LPA stimulation caused a significant increase in blood capillary sprouting indicative of angiogenesis. Observation of LYVE‐1+ filopodia extensions suggests LPA stimulation also causes lymphangiogenesis. However, no indication of blood‐to‐lymphatic phenotype transition was observed. Blood vessels identified based on morphology and PECAM labeling intensity lacked lymphatic marker expression. These results indicate LPA stimulation is not sufficient to induce a lymphatic phenotype in blood vessels of intact microvascular networks. This emphasizes the importance of investigating lymphatic/blood vessel relationships in real microvascular network versus cell culture scenarios and motivates new questions regarding the mechanisms that maintain vessel identity.
Longitudinal Effects of Menopausal Hormone Treatments (MHT) on Platelet Characteristics and Cell‐Derived Microvesicles
Authors: Miller Virginia , Lahr Brian , Bailey Kent , Heit John , Harman S , Jayachandran Muthuvel
Published: June 2021

Objective

Activated platelets serve as a catalyst for thrombin generation. Furthermore, oral MHT may carry greater thrombotic risk than transdermal products. This study compared effects of oral and transdermal MHT on platelet characteristics including platelet‐derived microvesicles (MV) in recently menopausal women.

Methods

Platelets and MV were prepared from blood of a subset of women (n=117) enrolled in the Kronos Early Estrogen Prevention Study prior to and after 4 years of treatment with either oral conjugated equine estrogen (0.45 mg, oCEE), transdermal 17β estradiol (50 µg/day, tE2), each with intermittent progesterone (200 mg/day for 12 days a month), or placebo pills and patch.

Results

Platelet count, and activated platelet P‐selectin and fibrinogen‐binding receptor expression were similar across groups. Numbers of tissue factor positive and platelet‐derived MV increased significantly in the tE2 compared to placebo.

Conclusions

Four years of MHT did not increase thrombogenic characteristics of platelets. Thrombotic risk associated with oral MHT most likely involves liver‐derived proteins than platelets per se. Differential effects of oCEE and tE2 on circulating MV may reflect the hormonal composition of the two formulations on non‐genomic pathways in circulating platelets.

Funding

This work was supported by grants from the Aurora Foundation to the Kronos Longevity Research Institute, NIH HL90639, UL1TR000131, American Heart Association‐ Scientist Development Grant, AHA 08‐30503Z, American Heart Association, Grant‐in‐Aid, 12GRNT12050147, and the Mayo Foundation.
Advanced Glycation End Products Alters Platelet Aggregation
Authors: Soaita Ioana , Yin Wei , Rubenstein David
Published: June 2021

Diabetes mellitus is in part characterized through the presence of advanced glycation end products (AGEs) within the cardiovascular system. AGEs are a heterogeneous population of glycated proteins, lipids or other molecules. Albumin is the plasma protein that is most susceptible to glycation due to its long residence time in plasma. Thus, we aimed to evaluate the effects of reversibly and irreversibly glycated albumin on platelet aggregation towards arachadonic acid, epinephrine and ADP and to determine mechanisms responsible for altered aggregation. Previous work from our group has shown that the presence of AGEs antagonizes thrombin and collagen mediated aggregation by up‐regulating GPIIb/IIIa. We hypothesized that irreversibly glycated albumin would activate the arachadonic acid, epinephrine and ADP pathways, more so than reversibly glycated albumin and non‐glycated albumin and that this would at least be partially mediated through an enhanced expression of GPIIb/IIIa. Additionally, with longer exposure durations, we expected a further enhancement of the aggregation system as compared with shorter exposure durations. Our results suggest that irreversibly glycated albumin is more detrimental to platelet functions than reversibly or non‐glycated albumin, but in general the exposure duration, which was up to 4 hours, did not play a significant role in altered aggregation. In particular, both the rate by which platelets aggregate and the quantity of platelets participating in aggregation was enhanced after exposure to irreversibly glycated albumin. These data suggest that the presence of irreversibly glycated albumin can significant alter platelet functions in diabetics, which may serve as a link between enhanced cardiovascular disease progressions in diabetic populations.