bluuebunny/crossref_metadata_2025 huggingface dataset:
Developing Burrowing as a Non‐Evoked Readout Assessment for Novel Analgesic Drug Efficacy
Authors: Lindros Kayla , Giuvelis Denise , Bilsky Edward
Published: June 2021

Traditional drug development efforts to diminish the problem of chronic pain rely heavily on pain‐evoked assays that can yield false positives, leading to confounding assessments of novel drugs. To avoid this, we are looking at burrowing as a novel readout assessment of analgesic efficacy. Preliminary data shows that induction of post‐surgical pain reduces this innate behavior by about 35%. In attempt to increase this number, we tested a variety of additional parameters. Blue pads, placed at the bottom of each chamber in order to reduce the sound made when gravel is displaced, increased baseline amounts roughly 40%. Conversely, neither sex of the animal, nor time of day the experiment was performed was found to have an affect on the amount burrowed. Further optimizations of this assay look at habituation and test session length. We found that a 5‐day habituation regiment could not be shortened, but a test session length of 15min produced comparable results to the original 60min session. Current studies are aimed at using HD webcams to quantify additional behaviors such as time spent burrowing and time spent in the tube. Ultimately, these studies could lead to the development of a beneficial assay for use in assessing novel analgesic drugs.
Marble Burying: A Novel Measure of Murine Neuropathic Pain Depressed Behavior
Authors: Wilkerson Jenny , Hsu Ku‐Lung , Niphakis Micah , van der Stelt Mario , Cravatt Benjamin , Lichtman Aron
Published: June 2021

Pathological pain states represent one of the most common reasons to seek medical attention. Most preclinical assays measure behaviors that are evoked by a nociceptive stimulus (e.g. withdrawal responses). However, not only is it difficult to distinguish between motor impairment and analgesia in these models, but also there is some question whether they reflect clinical relevance for pain treatment. Conversely, it is hypothesized that pain‐depressed behavioral models offer advantages over pain‐stimulated behaviors. In the present study, we modified the marble burying assay to explore pain‐depressed digging behavior in the chronic constriction injury of the sciatic nerve (CCI) neuropathic pain model. CCI mice displayed decreases in time spent digging and the number of marbles buried compared to sham mice. We compared a panel of drugs (i.e., morphine, valdecoxib, gabapentin, cannabinergic drugs, and diazepam) in their effectiveness in reversing CCI‐induced decreases in marble burying compared with their effectiveness in reversing CCI‐induced increases in mechanical allodynia and thermal hyperalgesia. Each of these compounds, with the exception of diazepam, dose‐responsively reversed pain‐stimulated and pain‐depressed measures. The present results indicate that the marble burying assay allows for sequential testing of candidate analgesics from multiple classes of drugs in pain‐depressed and pain‐stimulated behavior.
β‐caryophyllene‐complexed in β‐cyclodextrin Produces Antihyperalgesic Activity in Animal Model for Fibromyalgia
Authors: Quintans Jullyana , Quintans Júnior Lucindo , Araújo Adriano , Barreto Rosana , Sluka Kathleen
Published: June 2021

β‐caryophyllene is a dietary cannabinoid and it is a major plant volatile found in large amounts in the essential oils of many different spice and food plants, such as oregano (Origanum vulgare L.), cinnamon (Cinna‐momum spp.) and black pepper (Piper nigrum L.). The analgesic activity of β‐caryophyllene‐complexed in β‐cyclodextrin (CD‐CAR) was assessed in an animal model of fibromyagia (chronic non‐inflammatory muscle pain). Male Swiss mice were pretreated with CD‐CAR (10 or 20 mg/kg, p.o.) or vehicle (neutral saline). They were screened for mechanical hyperalgesia of the paw and muscle (von Frey and tweezer), motor coordination (Rota Rod) and muscle strength (Grip Strength Meter) beginning 60 min after treatment and continuing through 15 daysCD‐CAR, all doses, induced a significant reduction (p<0.05, p<0.01 or p<0.001) of mechanical hyperalgesia of the paw nd muscle in the chronic non‐inflammatory muscle pain model that lasted for 15 days. CD‐CAR produced no significant change in motor coordination or muscle strength. Therefore, we show that CD‐CAR reduces pain behaviors in an animal model for fibromyagia.

Financial Support: CNPq/Brazil, FAPITEC/Brazil and FINEP/Brazil
Effects of Incisional Pain on Food‐Maintained Responding under Progressive Ratio Schedules of Reinforcement
Authors: Warner Emily , Krivitsky Rebecca , Atherton Phillip , Pitre Travis , Cone Katherine , Lanpher Janell , Giuvelis Denise , Bilsky Edward , Stevenson Glenn
Published: June 2021

The current set of experiments characterized the effects of incisional pain on food‐maintained responding under progressive ratio operant schedules. Rats were trained to press a lever for food on a FR 1 schedule of reinforcement. The ratio requirement was gradually increased to 5, and following stable responding rats were assigned to one of four progressive ratio schedules: PR2, PR5, Log1 (j=0.17), Log2 (j=0.20). The effects of reinforcer type (grain vs. sugar pellets) limited hold duration (min), and ratio progression (logarithmic vs. arithmetic) were manipulated to develop a schedule with (1) low intra‐animal variability in breakpoint, (2) high rates of responding, and (3) session lengths shorter than 60 min to facilitate future drug testing. The Log2‐LH15 min schedule was chosen as terminal schedule. Rats received either incision + anesthesia or anesthesia only. Parallel studies assessed mechanical allodynia. For rats responding for grain, the incisional pain manipulation produced significant reductions in breakpoints and mechanical thresholds across three days. In contrast, for rats responding for sugar, incisional pain produced no change in breakpoints and decreased mechanical threshold only at post‐6hr. Given that PR schedules are interpreted to reflect workload and/or motivation, these schedules may have utility as a preclinical model for assessing pain / motivation / cognition interactions in rodents.

Funding: NIAMS/NIH 2R15AR054975‐02A1 to GWS
Interacting microRNA and mRNA genes identify potential therapeutic targets for NSCLC
Authors: Black Esther , Krentz Madeline , Wu Wenhui , Stromberg Arnold
Published: June 2021

Lung cancer patients die from drug‐resistant, metastatic disease. Lung cancers have high mutation rates. We propose that analysis of gene expression data can uncover new drug targets and strategies to improve outcomes in Non‐Small Cell Lung Cancer (NSCLC). Partial Least Squares (PLS) regression analysis coupled with bioinformatics processing can identify microRNA(miR) and mRNA interactions with druggable potential.

miR/mRNA signatures that predict NSCLC response to epidermal growth factor (EGFR) inhibitors were assembled using SAS. mRNA were regressed on miR genes for 8 samples. mRNA probesets that interacted at least 5 miR genes moved forward to biological validation by gene silencing. STRING used probeset/gene IDs to map protein‐protein interactions (PPIs). From the PPIs, the interactions of genes within two edges determined whether targets interact with the EGFR network.

After cross‐validation, 8 interactions were retained, and one physical interaction was confirmed. EIF4B, RAB8A, and PPP1CB interact in the PI3K/AKT cascade while KLK8 and PRKX interact with TGFß signaling; both pathways intersect EGFR. We silenced EIF4B, RAB8a, and PPP1CB +/‐ EGFR inhibition in NSCLC cells. Silencing of EIF4B and RAB8A did not induce cell cycle arrest or death. PPP1CB is a potential novel target. PLS modeling and bioinformatics was used to build a logical and repeatable workflow with gene expression data to identify new drug targets/strategies. Of 14000 possible interactions, 8 genes were processed, and 3 were chosen for biological validation. PLS modeling and bioinformatics analyses will be optimized.

KY Bioinformatics Research Infrastructure Network Pilot Project (NIH‐8 P20 GM103436‐12)
Inhibition of Micro‐RNA 146a Expression in Lung Fibroblasts by Fluticasone Propionate, Salmeterol Xinafoate, and Related Agents
Authors: Toews Myron , Schulte Nancy , Bang Jarrod , Rennard Stephen , Liu Xiangde
Published: June 2021

Micro‐RNA (miR) 146a is induced by inflammatory stimuli, and its pro‐inflammatory targets suggest that it may limit the inflammatory response. Fluticasone propionate (FP) and salmeterol xinafoate (SX) are used together to treat asthma, an inflammatory disease. Their effects on interleukin 1‐beta (IL‐1β)‐induced expression of miR‐146a by HFL‐1 lung fibroblasts were evaluated by quantitative RT‐PCR. Exposure of cells to the anti‐inflammatory steroid FP (1 nM) for 24 hr caused a nearly 70% inhibition. Two beta‐2 adrenergic receptor agonists, the long‐acting SX (10 nM) and the short‐acting albuterol (Alb, 10 μM) also inhibited miR‐146a expression, but only by 25‐35%. Prostaglandin E2 (100 nM) and the direct adenylyl cyclase activator forskolin (Fsk, 30 μM) both inhibited miR‐146a expression similarly to SX and Alb, suggesting cAMP elevation as the mechanism for these agents. Two primary isolates of human lung fibroblasts showed similar decreases in miR‐146a as for HFL‐1 cells when treated with FP, SX, or Fsk. FP has anti‐inflammatory effects in the airways that are augmented by concurrent SX, yet both FP and SX reduced fibroblast expression of anti‐inflammatory miR‐146a. Whether this is a direct effect of these agents or due to their overall inhibition of inflammation remains to be defined. Reduction in miR‐146a levels by these agents might limit their anti‐inflammatory actions, perhaps to prevent excess inhibition of inflammatory pathways.

Supported by Glaxo Smith Kline Respiratory Division Investigator‐Initiated Grant 200164.
Metabolic and immunologic risk factors for sulfonamide hypersensitivity in retroviral infection
Authors: Wong Yat Yee , Johnson Brian , Trepanier Lauren
Published: June 2021

HIV infected patients have a high risk of hypersensitivity (HS) reactions to trimethoprim/ sulfamethoxazole (TMP/SMX). To understand this, simian immunodeficiency virus (SIV)‐infected rhesus macaques were used to explore possible metabolic and immunologic risk factors. We hypothesized that pathways affecting sulfamethoxazole disposition might be altered by SIV infection. Liver biopsies were obtained from three SIVmac239‐infected male macaques and three non‐infected controls for a whole‐transcript gene expression array experiment. 138 genes were significantly differentially expressed (P < 0.005), with 114 genes up‐regulated and 24 genes down‐regulated. No sulfonamide biotransformation genes, to include CYP2C8, CYP2C9, CYB5R3, NAT2, or glutathione synthetic pathways, were differentially expressed. However, multiple genes involved in antigen processing, to include ubiquitination and proteasomal degradation, were significantly up‐regulated up to10‐fold. Genes involved in antigen presentation were up‐regulated by 1.7‐2.3 fold (P < 0.003). These preliminary data suggest that acquired risk of sulfonamide HS in human retroviral infection may not be due to changes in drug biotransformation, but in the antigen processing and presentation, primarily ubiquitination. Funded by R01 GM100784.
Engineering of Supramolecular Taxane Nanoparticles by Computationally Modeling Drug‐lipid Bilayer Interactions
Authors: Kulkarni Ashish , Pandey Prithviraj , Rao Poornima , Goldman Aaron , Ray Sudip , Sengupta Shiladitya
Published: June 2021

Clinical efficacy of lipid nanoparticles (NPs) depend on high drug to lipid ratio, stability during circulation and sustained drug release after tumor accumulation. Achieving this is the holy grail of paclitaxel (PTX) lipid NPs due to its incompatible physicochemical properties that leads to low drug loading efficiency and tendency to crystallize leading to significant instability. These limitations could be overcome by understanding molecular interactions of PTX with lipid bilayer at spatiotemporal level but these remain poorly understood. Using computer simulations, we have investigated the interactions of PTX with model lipid bilayer at low and high drug contents. We found that PTX at low concentrations occupied the space in both hydrophobic and hydrophilic regions of the bilayer and did not exhibit significant destabilizing effect whereas at high concentrations it exclusively accumulated at hydrophilic tail region causing significant rifts in the bilayer. In silico dynamic simulations showed that conjugation of PTX to cholesterol using a flexible spacer (Suprataxel, STX) stabilized the lipid bilayer at high concentrations. To confirm this, we engineered supramolecular NPs of STX (STX‐NP) and characterized the physicochemical and mechanical stability and found more efficacious than PTX in vitro. We showed that STX‐NPs were more efficient than PTX in inhibiting tumor growth in 4T1 breast and K‐RasLSL/+/Ptenfl/fl ovarian cancer models (p<0.01). This study shows that effective supramolecular NPs could be engineered by understanding interactions between drug and lipid layer.
Determination of the Effects of Nrf2 upon the Early Events of Jurkat T Cell Activation by Use of CRISPR‐CAS9 Mediated Mutation
Authors: Zagorski Joseph , Turley Alexandra , Maser Tyler , Rockwell Cheryl
Published: June 2021

Nuclear factor erythroid 2‐related factor 2 (Nrf2) is a transcription factor activated by cell stresses, such as reactive oxygen species and electrophilic stimuli. Upon activation Nrf2 accumulates within the nucleus, where it induces transcription of its target genes. In mice a growing number of studies indicate a role for Nrf2 in the development of immune mediated diseases, including inflammatory diseases such as allergy and autoimmunity. Recently, we have shown that the Nrf2 activator, tBHQ, modulates early events of T cell activation in human Jurkat T cells. This is evidenced by a marked inhibition of the cytokine IL‐2, as well as a reduction in the expression of CD25, the high‐affinity subunit of the IL‐2 receptor. The suppression of IL‐2 by tBHQ correlates with a concurrent suppression of the transcription factor NFκB, a transcription factor known to have multiple binding sites within the IL‐2 promoter. Although we have shown that the synthetic food additive tBHQ modulates the early events of Jurkat T cell activation, the role of Nrf2 in these effects remains unclear. Accordingly, we implemented CRISPR‐CAS9 technology to determine the Nrf2‐specific effects upon the early events of human Jurkat T cell activation. Toward this end, we have successfully targeted exon one of Nrf2 and have identified several Nrf2‐mutatnt clones. Sanger sequencing of each mutant indicates that at least two of the clones have base pair insertions, resulting in frameshift mutations. (This work is funded by NIH grants ES018885 and GM092715).
Endocannabinoid GPR18 Receptor Activation Confers Cardiovascular Protection in Diabetic Rats
Authors: Ahmed Asmaa , Abdel‐Rahman Abdel
Published: June 2021

The endocannabinoid receptor GPR18 lowers BP by reducing oxidative stress in brainstem nuclei that control sympathetic activity. We hypothesized that GPR18 activation confers cardiovascular protection in diabetes by reversing myocardial oxidative stress and dysfunction. Four weeks after diabetes induction (Streptozotocin; STZ, 55 mg/kg; i.p), diabetic rats received daily (100 ug/kg;i.p) of the GPR18 agonist, abnormal cannabidiol (Abn‐cbd) or antagonist, O‐1918, while control rats received vehicle, for 2 weeks before hemodynamic measurements in conscious rats. Abn‐cbd reversed the reduction in myocardial function by increasing left ventricular contractility index +dp/dtmax while maintaining the hypotensive response in diabetic rats, which inferred Abn‐cbd mediated reduction in vascular resistance. Ex vivo molecular findings demonstrated Abn‐cbd reversal of the diabetes‐induced: (i) reductions in plasma and myocardial nitrate /nitrite (NOX) levels; (ii) reductions in plasma adiponectin and increase in myocardial adiponectin receptor‐1 (Adipo R1) levels; (iii) reductions in myocardial protein kinase B (AKT), extracellular signaling kinase (ERK) and endothelial nitric oxide synthase (eNOS) phosphorylation; (iv) elevations in myocardial malondialdehyde (MDA) and antioxidant catalase levels; the latter might serve a counterbalancing role against diabetes‐evoked oxidative stress. The findings that Abn‐cbd enhanced, while the GPR18 antagonist O‐1918 reduced, myocardial GPR18 expression implicate GPR18 signaling in the Abn‐cbd evoked molecular and hemodynamic effects. These findings are the first to demonstrate a favorable cardiovascular role of GPR18 in diabetes and to elucidate the molecular mechanisms implicated in these clinically relevant effects.
The effects of central delivery of a positive allosteric modulator of GABAA receptors upon stress and hypertension in Schlager hypertensive mice
Authors: Stevenson Emily , Johns Esther , Jackson Kristy , Evans Roger , Marques Francine , Davern Pamela , Head Geoffrey
Published: June 2021

Hypertensive Schlager mice (BPH/2J) have neurogenic hypertension associated with abnormal reactivity of neurons in the forebrain integrating the response to aversive stress. We found that they also have functional and molecular differences in GABAA receptors compared to their normotensive counterparts (BPN/3J). Allopregnanolone is an endogenous neurosteroid reduced by chronic stress and when administered, decreases anxiety by positive allosteric modulation of GABAA receptors. We therefore compared the effect of chronic intracerebroventricular allopregnanolone and its vehicle on blood pressure and stress reactivity in BPH/2J and BPH/3J mice. Implantation of telemetric probes enabled continuous recordings of blood pressure (BP) at rest and during aversive stressors (restraint and dirty cage switch stress). Two weeks of allopregnanolone reduced MAP (‐9.8±4.3mmHg) and the depressor response to ganglionic blockade (‐10.5mmHg) in BPH/2J but had little effect in BPN/3J. Furthermore, allopregnanolone selectively reduced the pressor response to restraint stress by 27.4% in BPH/2J mice. The selective antihypertensive and stress inhibitory effects of allopregnanolone in BPH/2J mice suggests that allosteric modulation of GABAA receptors has therapeutic potential to treat stress related hypertension.
Involvement of Two Distinct Signalling Pathways in IGF‐1‐Mediated Central Control of Hypotensive Effects in Normotensive and Hypertensive Rats
Authors: Tseng Ching‐Jiunn , Cheng Pei‐Wen
Published: June 2021

Insulin‐like growth factor‐1 (IGF‐1) is abundantly expressed in the nucleus tractus solitarii (NTS). In a previous study, we revealed that the induction of nitric oxide (NO) production in the NTS reduces blood pressure (BP). It is well known that both acute and chronic administration of IGF‐I reduce BP. The aim of this study was to evaluate the short‐term hypotensive effect of IGF‐1 in the NTS and to delineate the underlying molecular mechanisms of IGF‐1 in the NTS of normotensive WKY rats and spontaneously hypertensive rats (SHRs).

Microinjections of the phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002 and the MAP kinase‐ERK kinase (MEK) inhibitor PD98059 into the NTS in WKY rats and SHRs were used to study the involvement of IGF‐1‐induced depressor effects.

An IGF‐1 (7.7 pmol) injection into the NTS resulted in a significant decrease in BP and HR in WKY rats and SHRs. Immunoblotting and immunohistochemical analysis showed that the microinjection of LY294002 (0.6 pmol) or PD98059 (3.0 pmol) into the NTS attenuated the IGF‐1‐induced depressor effects and Akt or ERK phosphorylation in WKY rats. An attenuation effect of LY294002, but not PD98059, was found in the SHRs.

These results suggest that distinct Akt and ERK signalling pathways mediated the IGF‐1 control of the central depressor effects in WKY rats and SHRs. ERK signalling defects may be associated with the development of hypertension.
Intralipid Reverses Propofol‐Mediated Hypotension in Rats
Authors: Bourque Stephane , Panahi Sareh , Mittal Rohan , Moher Conrad , Gragasin Ferrante
Published: June 2021

Introduction

Propofol is a commonly used anesthetic. Despite its favourable safety profile, propofol causes hypotension which can result in end‐organ hypoperfusion. Intralipid is a lipid emulsion that has been shown to reverse the vasodilatory effects of propofol in isolated vessels; however, whether these effects are recapitulated in vivo is not known. The objectives of this study were to determine if intralipid reverses the hypotensive and anesthetic effects of propofol in rats.

Methods

Under isoflurane anesthesia, male Sprague Dawley rats were instrumented with indwelling catheters for mean arterial pressure (MAP) assessments as well as subdural electrodes for cortical activity assessments by electroencephalography (EEG).

Results

Propofol (10 mg/kg IV) caused hypotension (55±2% drop in MAP, P<0.001) and intralipid (4mL/kg IV) caused greater reversal (80±9%) of blood pressures compared to saline (19±1%; P<0.001). Blockade of the autonomic nervous system with chlorisondamine (2.5 mg/kg IV) caused marked hypotension (56±3% lowering of MAP, P<0.001) which could be reversed with a constant infusion of phenylephrine (300 μg/kg/hr); under these conditions, propofol nevertheless caused hypotension (12±4% lowering of MAP) which was completely reversed by intralipid. Propofol‐induced cortical burst suppression was not affected by intralipid (2±3%), saline (‐4%) or 20% BSA (‐2±1%; P=0.27).

Conclusion

These results demonstrate that intralipid reverses propofol‐mediated hypotension with minimal effects on its anesthetic profile. Intralipid could be particularly useful as a rescue against propofol in patients prone to hemodynamic instability such as the elderly.

This work was funded by a grant from the University Hospital Foundation.
Therapeutic Potential of S43126 to Treat Select Patients with Type 2 Diabetes and Hypertension
Authors: Edwards Lincoln , Manaenko Anatol , Crane Louis , Baziard‐Mouysset Genevieve , Nunemaker Craig
Published: June 2021

Novel therapeutic agents with reduced side‐effect profile are needed to treat type 2 diabetes. The objective of this study is to highlight the potential of a novel imidazoline compound S43126 as a future antidiabetic drug. We hypothesize that S43126 can exert beneficial effects in type 2 diabetes by a mechanism involving insulin release and insulin sensitization. Rat tail arteries were treated in a bath system with S43126 and other compounds to determine the contractile response to these agents. Control Zucker lean rats (ZLRs) and Zucker diabetic rats (ZDRs) were treated with S43126 or moxonidine for 2 weeks, and then plasma glucose and blood pressure were measured. Insulin release by mouse islets in response to varying doses of S43126 was determined. Western blot was used to determine the effect of S43126 on PKB phosphorylation. S43126 did not contract rat tail artery, but lowered blood glucose in ZDR by 31% relative to control, with no significant effect in ZLR. S43126 significantly reduce blood pressure in ZDR only. S43126 caused a dose‐dependent insulin release of up to 4 fold from mouse islet cells, only under conditions of high glucose. S43126 increased PKB phosphorylation which was blocked by efaroxan. These data suggests that S43126 decrease blood glucose by a mechanism involving insulin release, sensitization, or both. S43126 should be further studied as a possible agent to treat some patients who have type 2 diabetes and hypertension.
P2Y2 Receptor Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: New AP‐1 Site and Negative Regulator
Authors: Liu Yiwei , Zhang Lingxin , Wang Chuan , Roy Shama , Shen Jianzhong
Published: June 2021

Recently we reported that the P2Y2 receptor (P2Y2R) is the predominant nucleotide receptor expressed in human coronary artery endothelial cells (HCAEC), and that P2Y2R activation by ATP or UTP induces dramatic up‐regulation of tissue factor (TF), key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R‐TF axis remains unclear. Here we report a role of a newly identified AP‐1 consensus sequence along with its new binding components in P2Y2R regulation of TF transcription. We identified with bioinformatics tools that a novel AP‐1 site at ‐1363 bp of human TF promoter region is highly conserved across multiple species. Activation of P2Y2R increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion and mutation of this new distal AP‐1 site all significantly supressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c‐Jun, ATF‐2 and Fra‐1, but not the typical c‐Fos, bound to the new AP‐1 site. In addition, loss‐of‐function studies using siRNAs confirmed a positive transactivation role of c‐Jun and ATF‐2, but unexpectedly revealed a strong negative role of Fra‐1 in P2Y2R‐induced TF up‐regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation, leading to Fra‐1 activation while JNK activated c‐Jun and ATF‐2. These findings reveal the basis for P2Y nucleotide receptor control of endothelial TF expression and indicate that targeting the P2Y2R‐Fra‐1‐TF pathway may be an attractive new strategy in control of vascular thrombogenicity and/or inflammation associated with endothelial dysfunction.
Increased Endothelial Expression of Human Cytochrome P450 Epoxygenase 2J2 Positively Modulates Coronary Reactive Hyperemia in Isolated Mouse Heart
Authors: Hanif Ahmad , Zeldin Darryl , Falck John , Nayeem Mohammed
Published: June 2021

Impaired coronary reactive hyperemia (RH) is associated with increased cardiovascular disease. Epoxyeicosatrienoic acids (EETs) exert cardioprotective effects in ischemia / reperfusion injury. Cytochrome P450 epoxygenase 2J2 partly generates EETs from arachidonic acid. EETs level is increased in mice with increased endothelial expression of CYP2J2 (Tie2‐CYP2J2 tr). We hypothesized that the isolated heart from Tie2‐CYP2J2 tr mouse exhibits increased RH through increased generation of EETs in response to no‐flow ischemia compared to wild type (WT). Coronary flow (CF) in isolated Tie2‐CYP2J2 Tr and WTmouse heart was measured using Langendorff system. Perfused heart was exposed to 15 second ischemia and RH was assessed. Flow repayment volume (RV) (the area under the curve normalized to heart weight (ml/g)) was significantly increased in Tie2‐CYP2J2 Tr compared to WT(9.8 ± 0.9 in Tie2‐CYP2J2 Tr vs. 6.4 ± 0.5 in WT, p<0.05). Tie2‐CYP2J2 Tr exhibited significantly longer repayment duration when compared to WT (2.5 ± 0.4 in Tie2‐CYP2J2 Tr vs. 1.7 ± 0.2 in WT, p<0.05). MS‐PPOH (CYP epoxygenase inhibitor, 1.0 mM) decreased RH in Tie2‐CYP2J2 Tr (7.96 ± 0.41 with MS‐PPOH vs. 9.8 ± 0.9 without MS‐PPOH , p<0.05), but not in WT. Our results demonstrate that increased endothelial expression of CYP2J2 increases RH compared to WT; also, MS‐PPOH decreases RH in Tie2‐CYP2J2 but not in WT. These data suggest that increased level of CYP2J2 could have a beneficial effect on myocardial recovery from short ischemia. Supported by HL‐114559 to MAN, z01‐ES025034 to DCZ and GM31278 to JRF
Aggravation by Clopidogrel, An Antiplatelet Drug, of Antral Damage Induced by NSAID in Rats: A Prophylactic Effect of Antiulcer Drugs
Authors: Takeuchi Koji , Yamasaki Kohei , Izuhara Chitose , Nishio Hikaru
Published: June 2021

Recent studies suggested a risk of gastric adverse reaction on the concomitant use of antithrombotic drugs with NSAIDs. We investigated whether clopidogrel, a P2Y12 receptor antagonist, aggravates antral damage induced in rats by indomethacin and examined the effects of several antiulcer drugs on these lesions. Male SD rats fasted for 24 h were refed for 1 h, and then administered indomethacin (30 mg/kg) s.c. 1 h after refeeding, and the stomachs were examined for lesions 6 h thereafter. Clopidogrel (3‐30 mg/kg) was given p.o. 3 times, 48, 24 h and 30 min before indomethacin. Famotidine or teprenone was given p.o. 1 h before indomethacin. Indomethacin alone produced damages, mostly nonhemorrhagic lesions, in the antrum of refed rats. Clopidogrel dose‐dependently worsened these lesions with convertion of nonhemorrhagic damages into hemorrhagic ones. The ulcerogenic response to indomethacin plus clopidogrel (30 mg/kg) was significantly suppressed by pretreatment with famotidine with a decrease in hemorrhagic lesions. The aggravating effect of clopidogrel was also attenuated by teprenone, though the effect was less pronounced than that of famotidine. These results suggest that 1) clopidogrel aggravates NSAID‐induced antral lesions, converting from nonhemorrhagic into hemorrhagic damage, and 2) these lesions are effectively prevented by both antisecretory and mucosal protective drugs.
L‐Type Calcium Channels Contribute to 5‐HT3‐Receptor‐Evoked CaMKIIα and ERK Activation and Induction of Emesis in the Least Shrew (Cryptotis parva)
Authors: Darmani Nissar , Hutchinson Tarun , Zhong Weixia , Chebolu Seetha
Published: June 2021

5‐HT3 receptor (5‐HT3Rs) activation by 2‐methyl serotonin (2‐Me‐5‐HT) induces vomiting which is sensitive to antagonists of 5‐HT3Rs (palonosetron) and L‐type calcium channels (LTCC) (amlodipine or nifedipine). Previously we had demonstrated that 5‐HT3R activation causes increases in a palonosetron‐sensitive manner, in: i) intracellular Ca2+ concentration, ii) attachment of calmodulin (CaM) to 5‐HT3R, and iii) phosphorylation of CaMKIIα and ERK1/2. Here, we investigate the role of nifedipine on 2‐Me‐5‐HT‐evoked intracellular Ca2+ increase and on downstream intracellular emetic signaling. We present evidence for the contribution of Ca2+ influx through LTCCs (sensitive to nifedipine) in 2‐Me‐5‐HT (1 µM) ‐evoked rise in cytosolic Ca2+ levels in least shrew brainstem slices. Nifedipine pretreatment (10 mg/kg) also suppressed 2‐Me‐5‐HT‐evoked interaction of 5‐HT3Rs with CaM as well as phosphorylation of CaMKIIα and ERK1/2 in the least shrew brainstem, and 5‐HT3Rs‐CaM colocalization in jejunum. In vitro exposure of isolated enterochromaffin cells to 2‐Me‐5‐HT (1 µM) caused CaMKIIα phosphorylation, which was also abrogated by nifedipine pretreatment (0.1 µM). Pretreatment with the CaMKII inhibitor KN62 (10 mg/kg, i.p.) suppressed emesis and also the activation of CaMKIIα, and ERK in brainstem caused by 2‐Me‐5‐HT (5 mg/kg). This study provides mechanistic explanation for our published findings that nifedipine can protect shrews from 2‐Me‐5‐HT‐induced vomiting.
Opposite Angiogenic Outcome of Curcumin Against Ischemia and Lewis Lung Cancer Models: in Silico, in Vitro and in Vivo Studies
Authors: LI Xuejun
Published: June 2021

We investigated the angiogenic effects of curcumin on an ischemia and lung cancer model. To induce ischemia combined with lung cancer models, unilateral femoral arteries of C57BL/6 mice were disconnected on one side of the mouse and Lewis lung carcinoma (LLC) cells were xenografted on the opposite side. Angiogenic effects and underlying mechanisms associated with curcumin were investigated. Molecular target(s), signaling cascades and binding affinities were detected by Western blot, two‐dimensional gel electrophoresis (2‐DE), computer simulations and surface plasmon resonance (SPR) techniques. Curcumin promoted post‐ischemic blood recirculation and suppressed lung cancer progression in inbred C57BL/6 mice via regulation of the HIF1α/mTOR/VEGF/VEGFR cascade oppositely. Inflammatory stimulation induced by neutrophil elastase (NE) promoted angiogenesis in lung cancer tissues, but these changes were reversed by curcumin through directly reducing NE secretion and stimulating α1‐antitrypsin (α1‐AT) and insulin receptor substrate‐1 (IRS‐1) production. Meanwhile, curcumin dose‐dependently influenced endothelial cells (EC) tube formation and chicken embryo chorioallantoic membrane (CAM) neovascularization. Curcumin had opposite effects on blood vessel regeneration under physiological and pathological angiogenesis, which was effected through negative or positive regulation of the HIF1α/mTOR/VEGF/VEGFR cascade. Curcumin had the promise as a new treatment modality for both ischemic conditions and lung cancer simultaneously in the clinic.
The Therapeutic Effect of Vitamin D3 in Conjunction with Cathelicidin for Gastric Cancer
Authors: Li Mingxing , Wu William , Zhang Lin , Shen Jing , Chan Ruby , Chan Franky , Cho Chi Hin
Published: June 2021

Objective

Our research aims to investigate the therapeutic effects of vitamin D3 in gastric cancer and its relationship with cathelicidin (LL37), a host defense and antimicrobial peptide, in mediating the anticancer action of vitamin D3 both in vitro and in vivo.

Methods

To assess the inhibitory action of vitamin D3 on gastric cancer cells (TMK1), MTT assay was used to measure the cell viability and flow cytometry was employed to detect the cell cycle distribution. The expressions of p21, vitamin D receptor (VDR), LL37 and LC3BII were determined by Western‐blot. An model of orthotopic gastric cancer was also established in nude mice to evaluate the anticancer action of vitamin D3 in vivo.

Results

1α,25(OH)2 vitamin D3 (1,25D3), the active form of vitamin D3, could significantly inhibit the proliferation and induce cell cycle arrest through stimulating the expression of p21 in TMK1 but not in the normal gastric epithelial cells (HFE145). This vitamin analogue also successfully induced the expression of LL37 and provoked autophagy in TMK1. Knockdown of LL37 by siRNA abolished the anti‐proliferative effect of 1,25D3 in gastric cancer cells. We further found that daily oral administration with vitamin D3 significantly inhibited tumor growth in the orthotopic gastric cancer model.

Conclusion

Our results suggest that vitamin D3 inhibits gastric cancer in a cathelicidin‐dependent manner and it could also function as a novel therapeutic agent for gastric cancer in the future.

Acknowledgements: This study is supported in part by the General Research Fund from the Hong Kong Research Grant Council and the Health and Medical Research Fund from the Food and Health Bureau in Hong Kong.
The Role of Neuropilin in Microglia during Glioma Progression
Authors: Miyauchi Jeremy , Nissen Jillian , Selwood David , Tsirka Stella
Published: June 2021

Malignant gliomas are the most commonly diagnosed tumors of the central nervous system (CNS). Despite management, median time of survival after diagnosis falls between 12 to 15 months. Current treatment modalities are suboptimal and there is a substantial need to develop more effective therapies. Modulation of the immune system is a promising strategy as innate and adaptive immunity play important roles in cancer progression. Neuropilin 1 (Nrp1), is responsible for amplifying pro‐angiogenic signaling within the tumor microenvironment and its expression correlates with glioma malignancy. Nrp1 also signals via additional pathways and plays a role in the behavior of innate immune cells. Microglia, the resident macrophages of the CNS, can comprise over 30% of the cells in glioma biopsies. Gliomas secrete cytokines which suppress the anti‐tumorigenicity of microglia, causing them to secrete factors that support the tumor's spread and growth. Some of the factors which are secreted by gliomas in high amounts signal via Nrp1 and its co‐receptors. The objective of this research is to identify how Nrp1‐mediated signaling in microglia affects glioma progression. Using a Cre‐lox system, we generated mice which lack expression of Nrp1 in microglia and macrophages (MG/MPs). We demonstrate in an in vivo orthotopic glioma model, that tumors exhibit less vascularity, grow at a slower pace, and are populated by MG/MPs which exhibit a more anti‐tumorigenic phenotype in mice that lack Nrp1 in MG/MPs or that are treated with a small molecule inhibitor of Nrp1. We conclude that ablation or pharmacologic inhibition of Nrp1 promotes an anti‐tumorigenic shift in the phenotype of MG/MPs, effectively reducing tumor growth.
Androgen Regulation of CXCR4/CXCR7 Chemokine Receptors: Disconnect between Transcription and Translation in Androgen‐responsive Prostate Cancer LNCaP Cells
Authors: Yu Lu , Yu Liangli , Wang Thomas
Published: June 2021

Chemokines and sex hormones are involved in prostate cancer (PCa) development. CXC‐motif ligand 12 (CXCL12) and its G‐protein‐coupled seven‐span transmembrane receptors (chemokine CXC‐motif receptor 4 and 7) are the primary regulators for PCa cell migration, invasion, and expression of metalloproteinase. The relationship between androgen and this pathway is less clear. Using real‐time PCR, we found that the androgen‐responsive LNCaP cell line had higher mRNA levels of CXCL12 and CXCR7 when compared to androgen‐unresponsive PCa cell lines such as DU145 and PC‐3. CXCR4 expression was DU145 > LNCaP > PC3. DHT (1nM) treatment of LNCaP cells for 48h inhibited CXCR7 mRNA expression by 70.70%. Moreover, the expression of CXCR4 mRNA level in LNCaP cells increased 6.29‐fold by DHT treatment. The effect of DHT on CXCR4 and CXCR7 in LNCaP cells followed a dose‐ and time‐ dependent manner at the transcriptional level. Silence of androgen receptor using siRNA significantly attenuated the effects of DHT on CXCR7 and CXCR4 mRNA by 61.32% and 72.51%, respectively. However, at protein level only a small decrease (26.75%) in CXCR7 protein level by DHT was detected using western blot. There were no significant changes found for CXCR4. In summary, DHT significantly increased CXCR4 and decreased CXCR7 at mRNA level. Effect of DHT on CXCR4 and 7 at translational level was minimal. Our study indicated that there appeared to be disconnect of the effect of DHT on CXCL12/CXCR4/CXCR7 chemokine axis between transcriptional and translation machinery in androgen‐responsive LNCaP cell line.
miR‐126 at the dawn of angiogenic defect seen in PAH right ventricular failure.
Authors: Potus Francois , Graydon Colin , Ryan Wong , Michelakis Evangelos , Provencher Steeve , Bonnet Sébastien
Published: June 2021

INTRODUCTION

Right ventricular (RV) failure is the most important predictor of both morbidity and mortality in pulmonary arterial hypertension (PAH). In PAH patients, hypertrophied RV is relatively ischemic, potentially because of suppressed angiogenesis. MicroRNAs (miRNAs) are important regulator of angiogenesis, especially miR‐126, which by inhibiting SPRED‐1, triggers the VEGF pathway and thus promoting angiogenesis. We hypothesized that specific miR‐126 downregulation in the RV promotes ischemia and triggers the transition from a compensated (CRV) to a decompensated (DRV) RV.

Results

We studied RV free wall tissues from humans with normal RV (NRV) functions, CRV and PAH (DRV), and rats with NRV and DRV. In both humans and rats, PAH DRV had decreased miR‐126 and microvessels density compared to NRV. Under the same conditions, miR‐126 were unchanged in the LV. In endothelial cells (EC) isolated from human RV, miR‐126 up‐regulation increased angiogenesis in a PAH DRV (matrigel assay), while downregulation of miR‐126 in EC from controls or CRV mimicked the PAH phenotype by decreasing angiogenesis. Artificial miR‐126 up‐regulation in PAH rat model improved RV functions, increased capillarity and decreased fibrosis specifically in RV, with no effect on PAP or lung vascular remodeling. Increasing miR‐126 levels in human PAH DRV endothelial cells also increased angiogenesis (matrigel assay).

CONCLUSION

We demonstrated that 1) exclusive RV downregulation of miR‐126 contributes to the ischemic status of the DRV; 2) in vivo up‐regulation of miR‐126 specifically improves RV functions. Thus, targeting miR‐126 represents a specific new avenue of investigation in preventing and reversing the failing RV in PAH.
Free Cholesterol‐Induced Macrophage Proliferation via Peroxisome–Proliferator Activated Receptor Gamma (PPARƳ) and Cyclin E Signaling Pathway
Authors: Xu Xiaoyang , Yuan Xinxu , Zhang Aolin , Li Ningjun , Li Pin‐lan , Zhang Fan
Published: June 2021

Our recent studies have demonstrated that the buildup of oxLDL‐derived free cholesterol (FC) in macrophages profoundly contributes to the foam cell formation and coronary artery atherosclerosis in CD38‐/‐ mice. The current study was designed to investigate the proliferative role of FC in B6 mouse bone ‐ marrow derived macrophages. With the colorimetric analysis of tetrazolium metabolite formazan, it was found that FC from (µg/ml) 0, 5, 10 and 20 to 40 concentration‐dependently increased the macrophage cell proliferation by folds of control at 1.33 , 1.41 and 1.54 to 2.24 , correspondingly. A similar macrophage cell proliferation upon FC treatment was also observed by flow cytometry cell count. Cell cycle assay with propodium iodide revealed that the increased ratio of cell population at S phrase plus G2/M over that at G0/G1 were well correlated with FC concentration enhancement as 0.28 in control; 0.31, 0.39, 0.44 and 0.48 in different FC groups as above. Both real time PCR and Western blot analyses showed that the expressions of cyclin E and PPARƳ significantly increased in the presence of FC (20 mg/ml). PPARƳ gene silencing or blockade of PPARƳ by GW9662 (20 µg/ml) markedly attenuated the expressions of cyclin E and thereby almost abolished macrophage proliferations induced by FC. These results for the first time reveal that FC acts to promote macrophage proliferation via the activation of PPARƳ and cyclin E signaling pathway. Understanding the role of FC in macrophage proliferation may facilitate the development of a novel therapeutic strategy for the prevention and treatment of atherosclerosis (Supported by NIH R01HL115068).
Considerations for Pulse Wave Analysis: Side Selection and Peripheral Arterial Blood Pressure Calibration
Authors: Martin Jeffrey , Borges Alexandra , Christy John , Beck Darren
Published: June 2021

BACKGROUND

As independent predictors of future cardiovascular morbidity and mortality, central arterial blood pressure and wave reflection characteristics derived non‐invasively have been increasingly employed in clinical and research settings. However, methods for peripheral blood pressure (PBP) calibration and pulse wave analysis (PWA) vary. We sought to evaluate the PBP assessment timing on subsequent PWA and to determine if differences in bilateral PWA exist.

METHODS

Bilateral PBP and PWA were assessed by oscillometric sphygmomanometry and a pencil‐type tonometer in 34 participants (aged 21.9±2.3 years). PBP was measured before and after PWA in each arm and bilateral PWA was performed simultaneously by two investigators.

RESULTS

No significant effect of PBP assessment prior to PWA was observed in the left arm, whereas there were moderate differences in some PWA parameters in the right arm. Simultaneous bilateral PWA demonstrated consistent, significant underestimation of clinical variables associated with wave reflection characteristics when calibrated to either within arm or equivalent PBP.

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26% (n=9) of participants presented with clinically significant differences (>10 mmHg) in bilateral PBP and contributed to the underestimates observed in the right arm.

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CONCLUSIONS

PWA in the right radial artery results in greater variability independent of the timing of PBP measurement and calibration pressures. Moreover, bilateral PBP measurement is imperative to identify subjects in whom a significant difference in bilateral PWA may exist and/or to prevent underestimation of aortic blood pressures if clinically significant differences in PBP go undetected.
Estimation of Pressure Drop Required for Lymph Flow through Initial Collecting Lymphatics
Authors: Stewart Scott , Sloas David , Murfee Walter
Published: June 2021

Lymphatic function is critical for maintaining interstitial fluid balance and is linked to multiple pathological conditions. Fluid moves through initial lymphatic networks into collecting vessels. Since initial lymphatics are not wrapped by smooth muscle cells, the mechanisms for fluid flow remain unclear. The objective of this study was to estimate the pressure drop needed for lymph flow through an intact initial lymphatic network. Pressure drops were computed for idealized networks with varying branch orders assuming Newtonian flow (1.5 cp viscosity). Vessel geometries per branch order were based on measurements from adult Wistar rat mesenteric microvascular networks. Network output velocity was equal to in vivo values reported for flow through a mesenteric collecting vessel. For vessel diameters = 50 mm and output velocity = 4 mm/s, the required pressure drop was approximately ‐1.12 mmHg. For diameters = 30 mm and velocity = 2 mm/s, the required pressure drop was approximately ‐1.55 mmHg. Our results suggest that suction pressures in collecting lymphatic vessels due to contraction and relaxation might be sufficient for drainage of proximal multi‐branched initial lymphatic networks without additional extrinsic forces.
TNF‐α Inhibits Rat Mesenteric Lymphatic Pumping via Activation of the NF‐κB and Upregulation of iNOS
Authors: von der Weid Pierre‐Yves , Chen Yingxuan
Published: June 2021

Upregulated TNF‐α production and dysfunctional lymphatic pumping are observed in inflammatory bowel disease pathogenesis. It has been previously demonstrated that TNF‐α could decrease arterial contractility via activation of the inducible nitric oxide synthase (iNOS) and that iNOS is involved in impaired mesenteric lymphatic pumping in animal model of intestinal inflammation. We investigated the effects of TNF‐α on rat mesenteric lymphatic pumping and hypothesized that the cytokine may impair lymphatic contractile function during intestinal inflammation by upregulating the synthesis of iNOS and thus the production of nitric oxide.

Using pressure myography, we examined the contractile function of isolated rat mesenteric lymphatic vessels following a 24‐hr treatment with TNF‐α. We observed that contraction frequency was significantly and concentration‐dependently decreased by TNF‐α, an effect restored following administration of inhibitors of NF‐κB (PDTC), iNOS (1400W), guanylate synthase (ODQ) and ATP‐sensitive K+ channels (glibenclamide), but not the cyclooxygenase inhibitor, indomethacin. The involvements of NF‐κB and iNOS were further confirmed by western blot analysis and quantitative real‐time PCR, which revealed upregulations of phospho‐IκB and iNOS mRNA in TNF‐α‐treated vessels that were minimized in the presence of PDTC.

These findings suggest that TNF‐α decreases lymphatic contractility via activation of the NF‐κB‐iNOS pathway.
Anti‐inflammatory Effects of Dipeptidyl Peptidase IV (DPPIV) Inhibition in Heart Failure
Authors: Salles Thiago , Zogbi Camila , Lima Thais , Soriano Francisco , Girardi Adriana
Published: June 2021

DPPIV inhibitors are antidiabetic drugs that confer cardioprotection beyond glucose lowering. We have previously demonstrated that circulating DPPIV associates with poorer cardiovascular outcomes in human and experimental heart failure (HF) and that long‐term DPPIV inhibition mitigates the development and/or progression of HF in rats. Since inflammation plays a role on HF pathophysiology, we hypothesized that the cardioprotective effects of DPPIV may be accompanied by reduction of cardiac inflammation in experimental HF. HF was induced in Wistar rats by left ventricular (LV) radiofrequency ablation. Rats were divided into three groups: Sham, HF and HF+IDPPIV inhibitor (sitagliptin 80mg/kg/day). Six‐week treatment with sitagliptin significantly improved LV ejection fraction, LV end diastolic volume, cardiac perfusion and mitigated cardiac hypertrophy. Additionally, cardiac infiltrate of macrophages increased in HF rats and treatment with sitaglipitin remarkably attenuated it. Moreover, HF rats not only showed increased cardiac expression of DPPIV, but also presented increased levels of p38 phosphorylation, interleukin 1‐beta, tumor necrosis factor alpha and interleukin‐6 compared to sitagliptin‐treated animals. Corroborating with these results, interferon regulatory factor 5, a key factor for M1 macrophage polarization, was also increased in HF rats and DPPIV inhibition was capable of preventing it. Interestingly, some macrophages presented in the lesion expressed glucagon‐like peptide 1 receptor (GLP1R) and the GLP1R agonist exendin‐4 reduced secretion of pro‐inflammatory cytokines from macrophages in vitro. Collectively, our results demonstrate that DPPIV inhibition improves cardiac function and attenuates cardiac inflammation in HF.