bluuebunny/crossref_metadata_2025 huggingface dataset:
RGS4 Differentially Regulates Antidepressant and Locomotor Behaviors In Vivo
Authors: Nielsen Cienna , Shirvani Babak , Baun Amanda , Mao Yu , DeVilbiss John , Nyangau Lily‐Ann , Chung Lena , Neubig Richard , Traynor John , Talbot Jeffery
Published: June 2021

Evidence suggests the antidepressant actions of serotonin (5‐HT) are modulated by regulators of G protein signaling (RGS) proteins. Specifically, we have shown that upregulation of cortical RGS4 (mRNA and protein) occurs in response to genetic disruption of RGS protein activity at the GTP‐binding protein Gαi2 and following chronic administration of serotonin‐selective reuptake inhibitors (SSRIs). Genetic deletion of RGS4 (RGS4(‐/‐)) had no effect on basal antidepressant‐like behaviors or generalized locomotor activity. However, RGS4(‐/‐) mice exhibited decreased antidepressant‐like responses to the SSRI fluoxetine, whereas desipramine, a selective inhibitor of norepinephrine reuptake, was fully efficacious. Conversely, the regulation of locomotor activity by desipramine was augmented in RGS4(‐/‐) animals relative to vehicle‐treated controls. RGS4 expression had no effect on locomotor activity following fluoxetine administration. Overall, these data suggest altered expression of RGS4 differentially influence antidepressant‐like behaviors in animal models of mood and potentially the therapeutic effects of SSRIs and are consistent with studies showing RGS4 positively regulates the efficacy of serotonin‐mediated antidepressants.
Nanoparticle‐Bound Cytolytic Peptide Prodrugs for Cancer Therapy
Authors: Jallouk Andrew , Palekar Rohun , Pan Hua , Schlesinger Paul , Wickline Samuel
Published: June 2021

Objective

To develop a nanoparticle platform which promotes the delivery of cytolytic peptide prodrugs to tumors and their activation by locally produced proteases.

Introduction: Melittin is a peptide toxin derived from bee venom which has been proposed as a cancer therapy due to its cytolytic activity. Yet, its therapeutic applications have been limited by its nonspecific cytotoxicity and rapid clearance from the circulation. We have previously developed a prodrug form of melittin which is activated upon cleavage by matrix metalloproteinase 9 (MMP‐9), a protease closely linked with tumor growth and metastasis. Here, we present a platform for delivery of this prodrug based on a perfluorocarbon nanoparticle system designed to enhance intratumoral accumulation while permitting prodrug activation by local MMP‐9 activity.

Methods and Results: Incubation of the melittin prodrug with perfluorocarbon nanoparticles (NPs) resulted in stable loading of 10,250 peptides per NP with no significant change in NP size. Addition of 20 nM MMP‐9 led to activation of 57.8% of NP‐bound prodrug within 3 hours. B16F10 melanoma cell viability was virtually eliminated following incubation with 50 μM free or NP‐bound prodrug in the presence of MMP‐9 (p < 0.001 compared to control). In a mouse model of B16F10 lung metastasis, NP accumulation in lung tumors was demonstrated by both fluorescence microscopy (Figure A; NPs in red) and quantitative 19F magnetic resonance spectroscopy (Figure B). Gelatin zymography confirmed production of active MMP‐9 by these tumors.

Conclusion

Perfluorocarbon nanoparticles accumulate within tumors and permit activation of bound prodrugs, making them promising delivery vehicles for cytolytic peptide agents.

Research Support: NIH R01 HL073646

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STUDIES ON THE SYNAPTIC FACILITATION EFFECT OF HYDROGEN PEROXIDE IN THE DEVELOPING NEUROMUSCULAR SYNAPSE
Authors: Liou Jau‐Cheng , Wu Yu‐Chu
Published: June 2021

We have previously demonstrated that muscle‐derived IGF‐1 is important in the development of neuromuscular synapse and hydrogen peroxide (H2O2) plays an important role in the release of IGF‐1. Here we further test the role of H2O2 in the development of neuromuscular synapse of Xenopus laevis by using the recording of whole‐cell patch clamp. Bath application of H2O2 dose‐dependently potentiates the frequency of spontaneous ACh release in Day‐1 Xenopus neuromuscular junction. The dose‐response curve shift to left while the facilitation effect of H2O2 on SSC frequency was test in Day‐3 cultures, suggesting the sensitivity of H2O2‐induced facilitating effect reduced as synapse matured. Pretreatment of the culture with H2O2 scavengers both N‐Acetylcysteine or sodium pyruvate for 1 day significantly hampered the development of synapse as the SSC frequency and amplitude both significant reduced in treated Day‐1 synapses. We next test the effect of H2O2 on Day‐1 N‐Acetylcysteine/sodium pyruvate‐pretreated synapse was test after extensively washed the culture to removed N‐Acetylcysteine/sodium pyruvate. The SSC frequency facilitating effect induced by H2O2 was significantly enhanced in treated synapse, which supports the notion that H2O2 in involved in the development of synapse. Furthermore, co‐pretreatment the culture with insulin in N‐Acetylcysteine/sodium pyruvate group reversed N‐Acetylcysteine/sodium pyruvate treatment‐induced synapse immaturity. Overall, results from our current studies provide strong supports on the involvement of H2O2 in the early development of neuromuscular synapse.
Effects of Quercetin and α‐Lipoic Acid on Acetylcholine Esterase and Paraoxonase Activities in α‐Cypermethrin Treated Rats
Authors: Rotimi Solomon , Adelani Bababode , Rotimi Oluwakemi
Published: June 2021

This study evaluated the effects of quercetin and α‐lipoic acid on the activities of acetylcholine esterase and paraoxonase in α‐Cypermethrin treated rats. 30 rats were randomly divided five experimental groups thus: (i) control group, (ii) α‐CYP alone (14.5 mg kg‐1 body weight/day), (iii) α‐CYP (14.5 mg kg‐1body weight/day) and α‐lipoic acid (20 mg kg‐1body weight/day) 1h later, (iv) α‐CYP (14.5 mg kg‐1body weight/day) and quercetin (20 mg kg‐1body weight/day) 1h later (v) α‐lipoic acid alone (vi) quercetin alone. After two weeks of treatment, the rats were sacrificed and blood collected by cardiac puncture and organs were excised for the assay of the activities of the enzymes. Arylesterase (AREase) activity was assayed in plasma, HDL1, HDL3, kidney, liver and brain while paraoxonase (PON) activity was assayed in plasma, HDL1 and HDL3 while acetylcholine esterase (AChE) activity was assayed in erythrocyte. α‐CYP treatment resulted in a significant (p<0.05) increased in the activity of erythrocyte AChE activity and both α‐lipoic acid and quercetin significantly (p<0.05) reduced this increase. There was a significant (p<0.05) decrease in AREase activity in both HDL1 and brain in α‐CYP treated groups. Treatment with α‐lipoic acid significant (p<0.05) increased AREase activity in HDL1. Rats treated with α‐CYP alone had a significant (p<0.05) increase in the activity of PON. This increase was however not observed in the groups co‐administered with α‐lipoic acid and quercetin. The results demonstrated that administration of α‐lipoic acid and quercetin to α‐CYP treated rats prevented with mild alterations of the activities of AChE and PON.
Sex Differences in Antioxidant Activity in Sub‐Chronic Low‐Level Inorganic Mercury Exposure
Authors: Wusu Adedoja , Ogunrinola Olabisi , Olaitan Samuel , Balogun Olawale , Ojo Oluwatobi , Ademuyiwa Oladipo
Published: June 2021

In order to investigate sex differences on the effects of inorganic mercury on antioxidant enzymes in different compartments, male and female albino rats were exposed to mercury (0.5, 1.0 and 1.5mg/kg) for 12 weeks. At the end of mercury exposure, total mercury in liver, kidney and whole blood was determined using Inductively‐Coupled Plasma Spectrometry (ICP‐MS) while superoxide dismutase (SOD) and catalase (CAT) activities in the liver, kidney, plasma and erythrocytes were determined by standard methods. Inhibition of CAT and SOD in plasma and erythrocyte characterised the effects of inorganic mercury in female animals with the same trend in SOD of male but vice versa in CAT respectively. Inorganic mercury exposure inhibited CAT by 23% (liver) and 84% (kidney); SOD by 30% (liver) and 16% (kidney) respectively in female animals. In the male animals, a 26% inhibition of SOD was observed in the liver whereas CAT was inhibited by 66% in the kidney. Correlation analysis shows a negative relationship between tissue mercury levels and the two antioxidant enzymes specifically in female. Our findings support sex differences in the effects of mercury on depletion of antioxidant enzymes.
Protective Effects of Resveratrol against UVA‐induced Damage in ARPE19 Cells
Authors: Hung Chi‐Feng , Chan Chi‐Ming , Su Ching‐Chieh
Published: June 2021

Exposure to solar ultraviolet (UV) radiation is believed to induce the production of reactive oxygen species (ROS) that may cause oxidative stress to retinal pigment epithelial (RPE) cells; oxidative injury to the retinal pigment epithelium has been implicated to play a contributory role in age‐related macular degeneration (AMD). Prior studies have suggested that resveratrol, an abundant and active component of red grapes, can protect several cell types from oxidative stress. This study is to evaluate if resveratrol can protect RPE cells from UVA induced damage, and be useful in the prevention of early AMD. Cell viability of adult RPE cells pretreated with different concentrations of resveratrol was determined using cell viability assay after UVA radiation. Intracellular H2O2 levels were measured by flow cytometry, and UVA induced signaling pathways were detected using Western blotting. Results show that resveratrol inhibited UVA‐induced RPE cell death. The generation of intracellular H2O2 induced by UVA irradiation in RPE cells was suppressed by resveratrol in a concentration‐dependent manner. In addition, resveratrol inhibited the activation of UVA‐induced extracellular signal‐regulated kinase, c‐jun‐NH2 terminal kinase and p38 kinase in RPE cells. There was also a reduction in UVA‐induced cyclooxygenase‐2 (COX‐2) expression in RPE cells treated with resveratrol. This study suggests that resveratrol is effective in preventing UVA‐induced damage in RPE cells and may be suitable for further developments as a chemoprotective factor for the primary prevention of early AMD.
Very High Accuracy Prediction of UDP‐Glucuronosyltransferase Sites of Metabolism
Authors: Swamidass S Joshua , Dang Lena
Published: June 2021

UDP glucuronosyltransferases (UTGs) are a family of enzymes that conjugate xenobiotics with glucuronide, in most cases detoxifying, solubilizing, and rendering them easier to excrete. In rare cases, however, UGT conjugation gives rise to toxic metabolites. Consequently, mathematical models that predict which parts of a molecule are metabolized by UGTs could be important in drug discovery. Here, we explore the feasibility of modeling UGT metabolism using a modeling system initially built to understand Cytochrome P450 metabolism. In a type of systematic review, this system builds a mathematical model to quantitatively summarize the data from hundreds of academic studies. Using a large database of over 2000 substrates in human, rat and mouse, very accurate (greater than 99% AUC) models of UGT metabolism are possible. These models predict with very high accuracy which parts of a molecule are conjugated with glucuronide by UGTs. Critically, these mathematical models can distinguish between atoms are conjugated and those of the same type of functional group (e.g. aromatic hydroxyls, carboxylic acids, nitrogens) that not conjugated. The model correctly predicts some species specific metabolism. It predicts when UGT conjugates to unexpected places. The model very accurately (approximately 98% AUC) predicts the formation of acyl‐glucuronides, an important reactive metabolites that should be avoided. However, the current model cannot accurately predict conjugation involving sulfur atoms. Nonetheless, these results are encouraging, suggesting that UGT metabolism may be effectively modeled in mathematical systems. This model may be useful to refine toxicological alerts based on problematic substructures, another goal we aim for in future work.
Identifying the Structure‐Function Activity of Human Cytosolic Sulfotransferase (SULT) 1C4
Authors: Guidry Amber , Falany Charles
Published: June 2021

Human cytosolic sulfotransferases (hSULTs) are phase II conjugation enzymes that play a critical role in the metabolism of drugs and hormones, the bioactivation of carcinogens, and the detoxification of xenobiotics. SULTs transfer a sulfuryl‐moiety from the obligate donor PAPS (3′‐phosphoadenosine 5′‐phosphosulfate) to a suitable hydroxyl or amine group of the substrate. While some of the SULTs have been studied extensively, others, such as the SULT 1C family, remain poorly characterized. The objective of this study is to determine the role of hSULT1C4 in drug metabolism by identifying novel substrates and defining the substrate binding, selectivity, and activity of the SULT1C4 enzyme using molecular modeling and kinetic analysis. SULT1C4 was bacterially expressed and purified for in vitro kinetic analysis. Steady‐state kinetic parameters for 1‐naphthol were analyzed using four concentrations of 1‐naphthol (0.5, 1, 2.5, and 7.5 uM) and four concentrations of PAPS (0.5, 1.5, 5, 15 uM). The Km of 1‐naphthol was approximately 1 uM, whereas the data for PAPS suggests the existence of two Kms (1 uM and 12 uM). To determine if these two Kms are due to SULT1C4's dimeric protein state, two different dimerization domain mutants, SULT1C4‐K272A and SULT1C4‐V277E, have been generated. Native page gel analysis has shown that these mutations prevent dimerization resulting in monomeric SULT1C4 proteins. Our data suggests that the dimeric state of SULT1C4 influences PAPS’ ability to bind to the enzyme.

Funding sources: NIH ES022606 and GM038953
sGC Stimulator Efficacy Beyond Blood Pressure in Dahl‐Salt Sensitive Model of Hypertension
Authors: Shea Courtney , Ranganath Sheila , Liu Guang , Wachtel Derek , Germano Peter , Tobin Jenny , Rivers Sam , Im Jamie , Sheppeck James , Nakai Takashi , Masferrer Jaime
Published: June 2021

Soluble guanylate cyclase (sGC) is an important intracellular receptor that can be activated by sGC stimulators to produce cyclic guanosine monophosphate (cGMP)protein kinase G (PKG) mediated effects. We have identified a novel series of sGC stimulators that decreased blood pressure (BP) in rats in a dose‐dependent manner at oral doses between 1‐10 mg/kg. These compounds possess a rat pharmacokinetic profile consistent with predicted once daily administration in humans. In this study we evaluated IWP‐121 in the Dahl Salt‐Sensitive (DSS) model of hypertension. Male DSS rats received 2 weeks of high‐salt diet prior to high salt plus compound for 6 additional weeks. Compounds were tested at doses of 1, 3, and 10 mg/kg/day eq in the chow (n=8/group) using Losartan as a positive control at a dose of 30 mg/kg/day eq in the water and compared to a high‐salt (hs) control group who received high salt diet for 8 weeks. IWP‐121 dose dependently decreased mean BP at week 8 with a maximal effect of 49 mmHg at the 10mg/kg dose. In comparison, losartan reduced mean BP by 27 mmHg. Both IWP‐121 (at all doses tested) and losartan had statistically significant effects on heart hypertrophy compared with the high‐salt (HS) group (3.5± 0.1 vs 4.5 ±0.3 g/Kg; p<0.001). IWP‐121 was efficacious at decreasing microalbuminaria in the urine (an indicator of kidney end organ protection) and serum biomarkers known to be involved in inflammatory and fibrotic processes (TIMP‐1, OPN and MCP‐1) in a dose‐dependent manner. In summary, IW‐121 demonstrated a potent anti‐hypertensive effect, additional effects in end organ protection, and effects on biomarkers consistent with anti‐fibrotic/anti‐inflammatory activity.
Metformin Inhibits Endoplasmic Reticulum Stress & Attenuates Autophagy in Glucose Starved Microvascular Endothelial Cells
Authors: Samuel Samson , Ghosh Suparna , Ding Hong , Triggle Chris
Published: June 2021

Metformin, the first choice of drug to treat diabetes, confers protection against cardiovascular disease (CVD) & also suppress tumor growth albeit at high concentrations. Since scientific evidence suggest a strong association between diabetes & CVD & more recently between concurrent diabetes & cancer, it is important to evaluate how the microvascular endothelial cells (MECs), which receive the initial impact of reduced oxygen & nutrient/glucose starvation (GS) during a myocardial infarction or in a tumor microenvironment, respond to metformin treatment. In the present study we investigated the effect of varying concentrations of metformin on GS induced endoplasmic reticulum (ER) stress & autophagy in MECs. Mouse Microvasuclar Endothelial Cells (MMECs) were subjected to GS for 24h in the presence & absence metformin (50μM & 2mM) & western blot analysis was performed to assess the induction of ER stress & autophagy. Immunofluorescence staining followed by confocal microscopy was also done to assess LC3B punctae staining in cells, indicating autophagy. ER stress activation & autophagic induction was observed in MMECs subjected to GS as evidenced by significant increase in the levels of ER stress markers (GRP78, ATF4 & CHOP) & autophagic markers (LC3A/B‐II), respectively. Accumulation of LC3B stained punctae in glucose‐starved cells also confirmed autophagic activation. Metformin treatment (2mM), independent of AMPK, significantly reversed GS induced ER stress & autophagy in MECs while lower concentrations had no effect. Our study demonstrates for the first time that metformin reverses GS induced ER stress & autophagy in the microvascular endothelium.
Cytochrome P‐450 epoxygenase 2J2 modulates adenosine receptor‐mediated vascular response in mouse mesenteric arteries
Authors: Yadav Vishal , Zeldin Darryl , Mustafa S Jamal , Xie Sherry , Nayeem Mohammed
Published: June 2021

Cytochrome P‐450 epoxygenases including CYP2J2 are expressed in vascular endothelial cells that generate epoxyeicosatrienoic acids (EETs) through epoxidation of arachidonic acid. EETs are endothelium‐derived hyperpolarizing factors which are protective in nature and are degraded by soluble epoxide hydrolase (sEH). In aorta from sEH−/− mice, we reported upregulation of A2A adenosine receptor (AR), CYP2J2, and PPARg, and the downregulation of A1AR and PPARa with enhanced vascular relaxation. In this study, we explored whether over‐expression of CYP2J2 in endothelium (Tie2‐CYP2J2 Tr) regulates AR dependent vascular responses in mouse mesenteric arteries (MAs). We performed DMT myograph experiments in WT and Tie2‐CYP2J2 Tr mice. Our findings revealed that acetylcholine‐dependent response was unchanged. Phenylephrine and KCl induced contractions were significantly higher in Tie2‐CYP2J2 Tr vs. WT mice (p<0.05). Importantly, CCPA (selective A1AR agonist)‐induced contractions were significantly reduced in Tie2‐CYP2J2 Tr vs. WT mice (p<0.05). NECA (non‐selective AR agonist)‐induced relaxation was similar Tie2‐sEH Tr vs. WT mice. Especially, pinacidil (KATP channel opener) induced relaxation was also significantly enhanced in Tie2‐CYP2J2 Tr vs. WT mice. Our data suggest that over‐expression of CYP2J2 enhances vascular relaxation possibly due to the combination of reduction in A1AR‐dependent contraction and enhancement of relaxation through KATP channel, indicating a possible interaction among CYP2J2, ARs and KATP channels (HL‐114559, Z01‐ES025034, HL027339 & HL094447).
Helicobacter heilmannii sensu lato‐induced MALT Lymphoma Formation in Lacrimal Gland as well as Fundic Gland in C57BL/6 mouse: Effect of Aprepitant
Authors: Nakamura Masahiko , Matsui Hidenori , Takahashi Tetsufumi
Published: June 2021

As to the pathogenesis of the Sjogren syndrome, Helicobacter pylori has been suggested to have some relation mostly from the epidemiological studies, while few experimental studies have clarified this point.

We have established a gastric low‐grade MALT lymphoma model in C57BL/6 mouse by the infection of Helicobacter heilmannii sensu lato obtained from cynomolgus monkey. After the long term infection, we have detected the accumulation of lymphocytes in the lacrimal glands similar to the gastric MALT lymphoma. The characteristics of this lesion were investigated as well as the effect of aprepitant, an NK‐1R antagonist on this lesion.

Materials and Methods

The characteristics of the lymphocyte accumulation were investigated by the histochemical analysis using lymphocyte marker, CD4, B220, CD10, epithelial cell marker cytokeratin, mucus marker MUC7 and aquaporin‐4 and 5. The localization of the autonomic nerves was also clarified by the choline acetyltransferase, substance P and calcitonin gene‐related peptide.

Results

By the histochemical analysis, the lymphoepithelial lesion was detected in the lymphocyte aggregation and the germinal center was formed in the center of the lesion, showing its characteristics as a mucosa‐associated lymphoid tissue (MALT) lymphoma. The lesion was also richly innervated by the autonomic nerves. Intraperitoneal administration of aprepitant induced the significant reduction in the lesion size.

Conclusion

Helicobacter heilmannii sensu lato infection to the C57BL/6 mouse induced the formation of the MALT lymphoma in the mouse lacrimal gland. This model could be one of the animal models of the Sjogren syndrome.
Involvement of Mitochondrial (mt) DNA Oxidative Damage and Circulating Fragments in Early Stages of Atherogenesis
Authors: Ruchko Mykhaylo , Sukhanov Sergiy , Gorodnya Olena , Pastukh Viktor , Danchuk Svitlana , Rachek Lyudmila , Gillespie Mark
Published: June 2021

Mitochondrial dysfunction underlies many processes in the development of atherosclerosis, including generation of reactive oxygen species, apoptosis, and inflammation. Oxidative mtDNA damage is a proximate cause of mitochondrial dysfunction and there is growing evidence that it can directly promote atherosclerosis. The mechanism of this action remains unclear, but may include initiation of apoptosis and inflammation by mtDNA oxidation. Previously, we have demonstrated protective role of mitochondria‐targeted DNA glycosylase Ogg1 against oxidative stress and apoptosis in cell culture and against vascular injury in animal models. In the present study we tested the hypothesis that transgenic modulation of Ogg1 coordinately regulates atherogenesis in mice fed a high fat diet. Wild type (WT) mice, Ogg1 knock‐out (KO) mice and KO mice transgenically overexpressing mitochondria‐targeted Ogg1 (KO‐Tg) were fed Western diet and analyzed for mtDNA damage and signs of atherogenesis. When compared to WT animals, KO mice displayed significantly higher mtDNA oxidative damage in cardiac tissue, elevated cell apoptosis and increased size of fatty streaks in aortic valve. These effects were eliminated in KO‐Tg mice. Interestingly, mtDNA oxidative damage correlated with the abundance of plasma pro‐inflammatory mtDNA fragments. These results indicate that enhanced mtDNA repair suppresses apoptosis and atherogenesis, suggesting that mtDNA and its integrity could be an important target for atheroprotection. Supported by NIH.
Time to Reach Steady State Resting Energy Expenditure in Healthy Adults Using Indirect Calorimetry
Authors: Popp Collin , Tisch Jocelyn , Bridges William , Traylor Dan , Najm Nadia , Jesch Elliot
Published: June 2021

Background

Resting energy expenditure (REE) measures the amount of calories needed for the body to maintain basic function over a 24‐hour period. Traditionally, REE protocols through indirect calorimetry take 45 minutes to complete. Generally, the first 10 to 20 minutes of data collected are excluded to allow the participants to reach steady state.

Objective

The purpose of this study was to determine a more precise time to reach a resting steady state.

Methods

Fifty healthy, adult male and female participants volunteered for this study. Each participant completed indirect calorimetry after a 12‐hour overnight fast. Participants were instructed to lay supine and awake under a plastic canopy for approximately 30‐minute, while oxygen consumption (VO2) and carbon dioxide production (VCO2) measures were collected. These values were used to determine steady state REE (kcals•day‐1) and respiratory quotient (RQ). Statistical analysis for the response of variables to time was modeled with a logistics decay function. Data are reported as mean ± SD with an alpha level of 0.05.

Results

Predicted time to steady state REE in kcals•day‐1 and in kcals•day‐1•kg‐1 was reached at 15.09 ± 0.1 minutes and 15.10 ± 0.1 minutes, respectively. There was no significant change in REE thereafter. In addition, respiratory quotient values from the first 15‐minutes (0.91) to the last 15‐minutes (0.86) were significantly different (p < 0.001).

Conclusion

Indirect calorimetry to measure steady state resting energy expenditure should exclude the first 15‐minutes in a young, healthy population.
Involvement of HCN channels in diaphragmatic lymphatic pacemaking
Authors: Negrini Daniela , Marcozzi Cristiana , Solari Eleonora , Moriondo Andrea , Bossi Elena , Cinquetti Raffaella , Reguzzoni Marcella
Published: June 2021

Pacemaking in spontaneously contracting lymphatics is thought to depend upon: a) small transient depolarisations sustained by an inward current through calcium‐dependent chloride channels, seen in mesenteric lymphatic vessels of guinea pigs, and, b) a current, observed only in bovine and sheep lymphatics, resembling cardiac If current. This study aimed at performing, a molecular, functional, and immuno‐hystological study to verify the potential involvement of hyperpolarization cyclic nucleotide gated (HCN) channels in pacing of intrinsic pumping activity. Experiments were performed in conformance with the FASEB Statement of Principles for the use of animals in research and education. After deep anesthesia, rat diaphragmatic specimens containing spontaneously contracting FITC‐dextran‐filled lymphatics were excised. Whole‐tissue perfusion with Ivabradine (300 mM) or CsCl (10 mM) caused a significant reduction in contraction frequency. CsCl also caused vasorelaxation and an increase in stroke amplitude. HCN channels were immunolocalised in the lymphatic vessel wall and this was further confirmed by RT‐PCR analysis of mRNA transcripts from the contracting vessels. Therefore, our results provide for the first time the molecular, functional, and immuno‐hystological evidences that HCN channels are responsible for pacemaking of lymphatic smooth muscle, setting the lymphatic vessel wall contractility.

Supported by the FAR2014 Research fund by the University of Insubria
Involvement of TRPM4 in Stretch‐Induced Modulation of Lymphatic Pumping
Authors: von der Weid Pierre‐Yves , Cho Eun‐Buall , Kratofil Rachel , Lee Stewart
Published: June 2021

Rhythmic and phasic contractions of collecting lymphatic vessels are critically involved in tissue fluid homeostasis and immune response. This contractile activity, or lymphatic pumping, is exquisitely regulated by luminal fluid pressure. However, the molecular contributors of stretch‐induced lymphatic pumping are not known. Some members of the TRP channel family, in particular TRPM4,have been shown to respond to stretch or pressure and to contribute to myogenic vasoconstriction and vascular response to stretch.

Using pressure myography, electrophysiology, pharmacology and quantitative real‐time PCR, we tested the hypothesis that TRPM4 is also a major contributor of the lymphatic vessel response to stretch.

We demonstrated the expression of TRPM4 mRNA in rat mesenteric lymphatic vessels and showed that the contraction frequency of the vessels pressurized at 3 mmHg was inhibited in a concentration‐dependent manner by the TRPM4 blocker 9‐phenanthrol (IC50=1.2 µM). The blocker (10 µM) also inhibited the pressure‐induce increase in contraction frequency in an endothelium‐independent manner and caused a hyperpolarization of the lymphatic muscle. These findings suggest an important role for TRPM4 in the ability of lymphatic vessels to adapt their contractile activity to changes in intraluminal lymph pressure.
INSULIN‐LIKE GROWTH FACTOR I (IGF‐1) REDUCES CHEMOKINES AND RECRUITMENT OF MONOCYTES INTO ATHEROSCLEROTIC PLAQUE: POTENTIAL MECHANISM MEDIATING IGF‐1‐INDUCED ATHEROPROTECTION
Authors: Sukhanov Sergiy , Snarski Patricia , Higashi Yusuke , Shai Shaw‐Yung , Delafontaine Patrice
Published: June 2021

We have shown that IGF‐1 decreased atherosclerosis and plaque macrophages in Apoe‐/‐ mice. We hypothesized that IGF‐1 suppressed recruitment of circulating monocytes (MN) into plaque. Apoe‐/‐ mice on a Western diet for 8 wks received IGF‐1 for 1 wk and red latex beads (2.5% wt/vol, i.v. specifically taken up by MN). IGF‐1 reduced Mac3 (macrophage marker)‐positive/red‐labeled cells in plaque (1.05±0.3, IGF‐1 vs.1.8±0.2 cells/plaque, control) without changes in labeled splenic MN. Similarly, chronic IGF‐1 (1.5 mg/kg/d, 4 wks) decreased labeled plaque MN (1.3±0.3, IGF‐1 vs. 2.6±0.4 cells/plaque, control). Conversely, mice with MN/macrophage‐specific IGF‐1 receptor deficiency (LCFIR) had increased labeled plaque MN (5.73±0.8, LCFIR vs. 2.33±0.4 cells/plaque, control). MN chemotaxis is mediated by chemokines/chemokine receptors. 6T/E mice with a 20% decline in circulating IGF‐I have increased atherosclerosis and chemokines in atherosclerotic aortas (RT array, MCP‐1, 185%; CCR1, 118%; CCR2, 99% increase vs. WT) and increased levels of circulating chemokines (ELISA, MCP‐1, 19.7 vs. 7.6 pg/ml; CXCL1, 74.4 vs. 24.9 pg/ml; E‐selectin, 70.1 vs. 48.9 pg/ml, 6T/E vs. WT). We quantified IGF‐1 effects on chemokines and on MN adhesion using human THP‐1 MN. IGF‐1 (50 ng/ml, 24h) decreased MCP‐1 (43±6% decrease), CCR1 (40±4% decrease) and CCR2 (38±5% decrease) mRNA in THP‐1 and suppressed adhesion of calcein‐labeled MN to endothelial cells (22±4% decrease). Taken together, these data suggest that IGF‐1 downregulation of chemokines reduces MN recruitment into atherosclerotic plaque mediating IGF‐1's anti‐atherosclerotic effect.
Methylene Blue Mitigates Ethanol Exacerbation of Ischemia‐Reperfusion Injury of Cardiomyocytes and Brain Cells
Authors: Ryou Myoung‐gwi , Winters Ali , Robert Mallet , Yang Shaohua
Published: June 2021

Ethanol (EtOH) intoxication is a major risk factor for myocardial and brain ischemia. Methylene blue (MB) could mitigate EtOH exacerbation of ischemia‐reperfusion injury.

Methods

HT22 cells, primary microvascular endothelial cells (MVEC), astrocytes and cardiomyocytes (CM) were treated with 100 mM EtOH ± 100 nM MB for 5 days prior to 4 h oxygen and glucose deprivation (OGD, 0.1% O2) and 24 h reoxygenation. Cell viability was assayed with Calcein AM. FITC‐Dextran leakage and transendothelial electrical resistance evaluated integrity of an in vitro blood brain barrier (BBB) model composed of MVEC and astrocytes. Generation of reactive oxygen species (ROS) was monitored with mitosox stain and DCF‐DA assay. ATP concentration was measured by spectrophotometry. Aquaporin 4 and hypoxia inducible factor‐1α (HIF‐1α) contents were assessed by immunoblot.

Results

EtOH was severely detrimental to both cell viability and BBB integrity vs. OGD control. EtOH significantly increased ROS in a manner blunted by MB. ATP depletion during OGD in HT22 cells and CM was appreciably worsened by EtOH, but MB cotreatment restored ATP content to that of EtOH‐free OGD. EtOH decreased HIF‐1α and increased aquaporin 4.

Conclusion

EtOH increased the vulnerability of all four cell types to OGD‐reoxygenation insult. MB mitigated this EtOH toxicity by minimizing ROS generation, increasing ATP availability, and maintaining HIF‐1α, a critical factor for cellular adaptation to the ischemic environment.
Preventing Nitration of Specific Tyrosine Sites in Adenine Nucleotide Translocase Differentially Protects Against Cell Oxidative Stress Injury
Authors: Yang Meiying , Kwok Wai‐Meng , Bienengraeber Martin , Camara Amadou , Heisner James , Stowe David
Published: June 2021

The adenine nucleotide translocase (ANT) is the most abundant protein in the inner mitochondrial membrane and the main transporter for ATP and ADP exchange across the membrane. Ischemia reperfusion (IR) injury causes mitochondrial accumulation of reactive O2 and N2 species (ROS/RNS), which can induce irreversible and deleterious tyrosine nitration (NTyr) of protein. In this study, we investigated IR‐induced NTyr of ANT and its impact on cell damage during oxidative stress. Isolated guinea pig hearts were subjected to 35 min ischemia and 20 min reperfusion or no‐IR (control). At the end of reperfusion, ANT was purified from IR or control heart mitochondria and subjected to Western blotting with antibodies to ANT and nitro‐Tyr. We found that IR induced NTyr of ANT. Mass spectrometry analysis identified ANT tyrosines (Y) residues Y81, Y191 and Y195 were nitrated when mitochondria were treated with 100 µM peroxynitrite, an oxidant. To determine the contribution of nitration of ANT Y81, Y191 and Y195 on cellular injury induced by oxidative stress, a single mutant Y81F (phenyalanine) and a double mutant Y192F and Y195F were constructed. After transfection of these mutants into a cardiomyocyte cell line, HL‐1, we found that overexpression of both the single and double mutants of ANT displayed less lactic acid dehydrogenase release induced by hypoxia and reoxygeneration compared to the wild type ANT. Moreover, the association of Voltage‐dependent anion channels with ANT was reduced after IR. In summary, we have identified specific NTyr residues in ANT following oxidative stress, and demonstrated that preventing nitration of these sites attenuated cell oxidative stress injury.
Macrophages Cause Reduced Biosynthesis of Hydrogen Sulfide in the Obese Microvasculature
Authors: White Carl , Velmurugan Gopal , Lin Vivian , Chang Christopher , Candela Joseph
Published: June 2021

Obesity is associated with recruitment of macrophages to the microvascular adventitia. The current objective was to define how macrophage‐derived signals contribute to microvascular dysfunction. Based on recent findings that impaired H2S signaling is associated with cardiovascular pathologies, it was hypothesized that in the diet‐induced mouse model of obesity microvascular H2S is depleted by a mechanism that involves interactions with perivascular macrophages. Mesenteric arterioles were isolated from 30 week‐old lean and obese mice and mounted for pressure myography. Vessels were loaded with the H2S indicator SF7‐AM and imaged using confocal microscopy. Obesity was associated with lower H2S levels in both the smooth muscle and endothelial layers. Generation of H2S is dependent on the substrate L‐cysteine and the enzyme cystathionine gamma‐lyase (CSE). Expression of CSE was not changed by obesity; however, L‐cysteine was less effective in evoking H2S production in these vessels, and was correlated with reduced relaxation when pre‐contracted with phenylephrine. Co‐culturing arterioles from lean mice overnight with peritoneal macrophages from obese mice resulted in vessel H2S depletion. In contrast, co‐culture of vessels from either lean or obese mice with macrophages from lean controls had no effects on vessel H2S. Taken together, these data support a model in which H2S biosynthesis is compromised in the obese microvasculature by a mechanism dependent on crosstalk with perivascular macrophages. Supported by the American Heart Association and a Chicago Medical School/Advocate Lutheran General Hospital Pilot Grant.
Role of endoplasmic reticulum stress and thrombosis in type 2 diabetes‐induced vascular dysfunction
Authors: Ait Aissa Karima , Kassan Modar , Trebak Mohamed , Belmadani Souad , Matrougui Khalid
Published: June 2021

Endoplasmic reticulum (ER) stress and thrombosis are important mechanisms that underlie many of the serious consequences of type 2 diabetes. However, the interaction between the ER stress and thrombosis in diabetes‐induced endothelial dysfunction remains unknown. Ten‐weeks‐old type 2 diabetic mice (db‐/db‐) and their controls (db‐/db+) treated with or without the ER stress inhibitor (tauroursodeoxycholic acid, TUDCA, 150 mg/kg per day, for 2 weeks) were used to determine the in vivo contribution of ER stress in vascular dysfunction. Blood pressure was similar in all groups of mice. Blood glucose and body weight were reduced in (db‐/db‐) mice treated with TUDCA. The impaired endothelium‐dependent relaxation (EDR), in response to acetylcholine, in (db‐/db‐) mice was restored after treatment with TUDCA in both aorta and mesenteric resistance arteries (MRA). Additionally, ER stress markers (CHOP and ATF4), NADPH oxidase activity and the mRNA levels of Nox2 and Nox4, which were increased in MRA and aorta from (db‐/db‐) mice, were significantly reduced after TUDCA treatment. Moreover, the vasoconstriction to thrombin and the mRNA levels of thrombin receptor (PAR1) were increased only in MRA from (db‐/db‐) mice and reduced after treatment with TUDCA.

This study unveiled novel role of ER stress in the regulation of thrombin signaling and therefore vascular dysfunction in type 2 diabetes.
Interaction of ROS and RNS with GSH and GSH/GPX Systems
Authors: Chen Juan , Bhandar Bhagyesh , Kavdia Mahendra
Published: June 2021

Rapid reactivity amongst the free radicals and its capability to inflict severe damages to cell has evolved cell redox systems with multiple pathways of counteracting and counterbalancing mechanisms. One of the important cell redox pathways is glutathione (GSH) and GSH/glutathione peroxidase (GPX) enzyme system. In addition to eliminating hydrogen peroxide (H2O2) and peroxynitrite (ONOO‐), GSH and GPX act as a buffer to protect crucial proteins against pathological modifications. In recent year, GSH and GPX have been emphasized for providing a promising treatment avenue in vascular diseases including neurovascular and cardiovascular diseases. However, complexity of cellular redox system has limited the current understanding of GSH and GPX led mechanism which prevents cell damage in pathophysiological condition. In this study, we used systems approach to model GSH and the GSH/GPX system in the presence of nitric oxide (NO) and superoxide (O2•‐) derived species. Result shows that there is an indicated dividing line between oxidative stress and nitrosative stress as the ratio of [O2•‐]/[NO] less than or greater than one. Results provide quantitative information about GSH levels that leads to the depletion of N2O3 and H2O2. In addition, reduced glutathione peroxidase (GPXr) assists GSH in removal of high levels of H2O2. The model predicted that the GPX antioxidant capabilities are dependent on the bioavailability of GSH. The levels of nitric oxide and superoxide derived species are dependent of the balance between these reactive and antioxidant species through interaction with GSH/GPX system. The results indicate that both GSH and GPX play an important role in removal of oxidative stress and GSH also plays an important role in detoxifying nitrosative stress.
Effects of Fermented Red Ginseng on High‐Fructose induced Metabolic Syndrome
Authors: Kho Min Chul , Lee Yun Jung , Kwon Oh Jeong , Lee So Heun , Kang Dae Gill , Lee Ho Sub
Published: June 2021

The red ginseng (RG), a widely used traditional herbal medicine, was reported with anti‐inflammatory and anti‐oxidant activity. Aim in the present study was to investigate that the effects of fermented red ginseng (FRG) on high‐fructose (HF) diet induced metabolic disorders, and those effects were compared to RG and losartan. Treatment with FRG was significantly suppressed the increments of body weight, liver weight, epididymal fat weight and adipocyte size. Moreover, FRG significantly prevented the development of the metabolic disturbances such as hyperlipidemia and hypertension. Staining with Oil‐red‐o demonstrated marked increase of hepatic accumulation of triglycerides, and this increases was prevented by FRG. FRG ameliorated endothelial dysfunction by down‐regulation of endothelin‐1 (ET‐1) and adhesion molecules in the aorta. In addition, FRG induced markedly up‐regulation of IRS‐1 and Glut4 in the muscle. These results indicate that FRG ameliorates obesity, dyslipidemia, hypertension and fatty liver in HF diet rats. Thus the pharmacological activity of RG was enhanced by fermentation on HF diet induced metabolic disorders. Taken together, fermentated red geinseng might be a beneficial therapeutic approach for metabolic syndrome.
Melatonin Inhibit the Angiogenesis of Gastric Cancer by Nuclear Receptor
Authors: Zhou Ruixiang , Wang Rixiong , Song Jun , Zhang Hui , Luo Jianhua , Liu Hui
Published: June 2021

This study aims to explore the relationship between melatonin (MLT) and gastric cancer angiogenesis by nuclear receptor with ELISA, real‐time quantitative RT‐PCR, IHC, Western blot, and siRNA methods.

1. The anti‐angiogenic function of MLT on the human gastric cancer cells and the impact on MLT nuclear receptor in vitro. MLT was time‐dependent in inhibiting proliferation of human gastric cancer cell lines MGC‐803, SGC‐7901, and AGS; Chemical hypoxia simulated by 100uM CoCl2 increased proliferation of SGC‐7901 cells; MLT decreased the expression of VEGF mRNA and protein of SGC‐7901; MLT decreased the expression of nuclear receptor RZR/RORγ mRNA and protein of SGC‐7901.

2. The anti‐angiogenic mechanism of MLT on human gastric cancer cells SGC‐7901 in vitro and the targets of MLT nuclear receptor RZR/RORγ. The RZR/RORγ silent by siRNA antagonized significantly MLT's inhibition on gastric cancer cell proliferation.After the 24h intervention by 3mM MLT, the mRNA and protein expression of RZR / RORγ,SENP1,HIF‐1α and VEGF was significantly decreased; but the expression of SENP1, HIF‐1α, VEGF mRNA and protein was increased.

3. The anti‐angiogenic mechanism of MLT on the gastric cancer in vivo with nude mice.We established the tumor‐bearing nude mice model of gastric cancer SGC‐7901 with intervention by different concentrations of MLT. MLT reduced the tumor volume and weight of the gastric cancer; MLT inhibited tumor proliferation and angiogenesis by decreasing the expression of RZR / RORγ ,SENP1 ,HIF‐1α, VEGF and MVD.

The present study confirmed that MLT is able to inhibit gastric cancer angiogenesis both in vivo and in vitro. It's major mechanism is reducing the expression of nuclear receptor RZR/RORγ and decreasing the expression of SENP1,HIF‐ 1α,VEGF.
Placental Growth Factor (PlGF) and Vascular Endothelial Growth Factor (VEGF) Receptors Expression in the Placenta – Correlation with Maternal Blood Hemoglobin Level and Pregnancy Outcome
Authors: Szukiewicz Dariusz , Stangret Aleksandra , Watroba Mateusz
Published: June 2021

Insufficient tissue oxygenation may be caused by lowered hemoglobin (Hb) concentration in blood. Therefore, anemia during pregnancy may stimulate angiogenesis in the placental tissues. Here we examined correlations between mean placental vascular density, local expressions of PlGF and VEGF receptors (flk‐1, flt‐1), and the birth weight and maternal Hb level. After normal full‐term deliveries ending pregnancies with maternal anemia (Group I; N = 23) and normal Hb level (Group II; N = 23), tissue samples were collected from the maternal side of each placenta (3 samples per each placenta). The mean expressions of immunostained flk‐1 and flt‐1 were analyzed by quantitative morphometry in the paraffin sections. The mean vascular/extravascular tissular index (V/EVTI) was examined as a morphometric parameter which reflects intensity of vascularization by assessing a total vascular area within standardized visual fields. Nonparametric Mann‐Whitney U‐test and Spearman's correlation were used for comparative analysis of the studied groups. Among gravidas with lowered Hb level, the local expression of flt‐1 was significantly increased (p < 0.05) and positively correlated with the mean birth weight. The same association was denoted for V/EVTI. Thus, compensatory changes in the vascular system of the human placenta evoked by low maternal Hb concentration include increased angiogenesis to maintain normal growth of the fetus. Up‐regulation of flt‐1 may play a key role.

WUM grant: 2M2‐W1‐14
Sodium Nitrite Reduces Systolic Blood Pressure in Preeclampsia
Authors: Rizzi Victor Hugo , Possomato‐Vieira José Sérgio , Mendes Gabryella , Nascimento Regina , Silva Katiussia , Pupo André , Dias‐Junior Carlos
Published: June 2021

Preeclampsia (PE) is a pregnancy disorder characterized by elevated blood pressure (>140/90 mmHg) and proteinuria. We developed PE in an animal model using nitric oxide synthase inhibitor L‐NAME (Nω‐Nitro‐L‐arginine methyl ester). L‐NAME decreases the bioavailability of nitric oxide (NO) being responsible for endothelial dysfunction. Therefore reversing the reduction of NO seems to be a promising pharmacological approach. Studies showed that oral adminstration of sodium nitrite (NaNO‐2) may increase the bioavailability of NO. Female rats (220‐250 g) (n=17) were mated with males for pregnancy. All procedures for animal experimentation were approved by the Ethics Committee, IBB/UNESP (Protocol 618/2014). From 15th to 21th day of pregnancy we applyed L‐NAME, (60mg/Kg, intraperitoneal ‐ i.p.) in all dams. We treated animals with NaNO‐2 (n=9) (15mg/Kg, once daily, by gavage). We measured pressure by tail cuff plethysmography at gestational days 14, 16, 18 and 20. Animals were killed on pregnancy day 21th. Systolic blood pressure was significantly reduced in animals treated with NaNO‐2 (148±1.47mmHg/ 149±1 mmHg PE vs. 128±1 mmHg/ 133±1 mmHg PE+NaNO‐2, on gestational days 16 and 18, respectively). Pups body weight at birth, placentas weight and number of reabsorbed pups did not change significantly. We performed a MTT assay to verify the antioxidant conditions of plasma. MTT assay showed no difference in antioxidant status in plasma of PE and treated animals. The number of viable fetuses was significantly higher in PE+NaNO‐2 (10.88±0.98 PE+NaNO‐2 vs 8.25±0.64 PE). Our data showed that NaNO‐2 has an antihypertensive effect and can also improve fetal viability.
Monophosphoryl Lipid A Induces Endotoxin Tolerance in Endothelial Cells
Authors: Stark Ryan , Koch Stephen , Lamb Fred , Sherwood Edward
Published: June 2021

Monophosphoryl lipid A (MPLA) is a lipid A TLR4 agonist that exhibits a mild inflammatory effect compared to lipopolysaccharide (LPS). As endothelial cells regulate vascular inflammation, we hypothesized that MPLA treatment would induce tolerance in endothelial cells subsequently challenged with LPS. We cultured human umbilical vein endothelial cells (HUVECs) and exposed them to MPLA or LPS for 16 hours and then allowed them to sit in fresh media for 24 hours. Afterwards, cells were exposed to LPS for an additional 6 hours. Inflammation was assessed by quantifying IL‐6 and G‐CSF levels in the culture media. Pre‐treatment with MPLA reduced LPS‐induced IL‐6 secretion by 26% ± 5% compared to LPS alone. LPS pretreatment induced a similar reduction in IL‐6 after repeat LPS exposure (25% ± 7%, n=4 per group). MPLA and LPS also induced endotoxin tolerance of GCSF secretion compared to LPS alone with a reduction of GCSF levels by 46% and 38%, respectively. MPLA alone induced only minimal secretion of IL‐6 and GCSF (227.04 ± 9.68 pg/ml and 18.81 ± 1.19 pg/ml, respectively) compared to LPS alone (1634.92 ± 76.92 pg/ml for IL‐6 and 291.29 ± 33.18 pg/ml for GCSF). These studies demonstrate the ability of MPLA to induce endotoxin tolerance in HUVECs with potency equal to that of LPS, but without the associated pro‐inflammatory response.