bluuebunny/crossref_metadata_2025 huggingface dataset:
A New Co‐micronized Composite Including Palmitoylethanolamide and Polydatin (PEA/PLD) Shows a Superior Efficacy Compared to the Simple Association on in in Vivo and in Vitro Models of Inflammation.
Authors: Siracusa Rosalba , bruschetta giuseppe , impellizzeri daniela , esposito emanuela , cuzzocrea salvatore
Published: June 2021

Palmitoylethanolamide (PEA), an endogenous fatty acid amide belonging to the N‐acylethanolamine family, has anti‐inflammatory and neuroprotective effects. However, PEA lacks direct capacity to prevent formation of free radicals. Polydatin (PLD) that is a natural precursor of resveratrol, has antioxidant activity. Thus, the combination of PEA and PLD could have beneficial effects on inflammatory process and oxidative stress. For this reason the aim of the present study was to compare the effects of micronized PEA (PEA‐m) and PLD association (PEA‐m+PLD) in the ratio 10:1 and of a new co‐micronized compound containing PEA and PLD (PEA/PLD) on the carrageenan‐induced rat paw model of inflammation. By western blot analysis, we demonstrated that the inflammatory response, oxidative stress, paw edema and thermal hyperalgesia were reduced by both compounds but the effects of co‐micronized compound PEA/PLD were superior to those of association of single compounds (PEA‐m+PLD) for several analyzed parameters. The results obtained in in vivo study were confirmed by in vitro models of inflammation. These data show a greater anti‐inflammatory and anti‐oxidant effects by using orally administration of new co‐micronized compound PEA/PLD compared to the association of PEA‐m+PLD on rat paw carrageenan–induced model of inflammatory pain and in in vitro study.
Urodynamic Measurement of Urethral Closure Function in Women with Stress Urinary Incontinence: A Single Dose Study of Duloxetine
Authors: Yono Makoto , Otani Masayuki , Ito Kazuya , Inoue Yumi , Furukawa Kohichiro , Hori Masaharu , Tsuji Shigeki , Tanaka Takanori , Sakata Yukikuni , Irie Shin , Latifpour Jamshid
Published: June 2021

This urodynamic double‐blind crossover study in women with stress urinary incontinence (SUI) was performed to evaluate the effect of duloxetine, a serotonin and noradrenaline reuptake inhibitor, on urethral function and to determine an appropriate method for making go/no‐go decisions in drug development. The urethral pressure profiles at rest and during coughing were measured before and 6 h after single oral administration of placebo or 40 mg duloxetine in 10 women aged 36 to 53 years with SUI (蠅1 episode/wk). Compared with placebo, duloxetine significantly increased the mean and maximal urethral closure pressures at rest calculated over the entire urethra. In the distal third of the urethra, during coughing, duloxetine significantly increased the mean closure pressure and the change from baseline in the maximal amplitudes of the negative closure pressure spikes compared with placebo. Our data indicate the facilitatory effects of duloxetine on both smooth muscle contractions and reflex contractions of the striated sphincters. Thus, the method for this urodynamic study in women with SUI could be used to reduce attrition rates by providing decision‐making data in early clinical studies of new drugs developed to treat SUI. Supported in part by Takeda Pharmaceutical Company Limited.
Effect of L‐cysteine on Glutamate‐Induced Excitotoxicity in Bovine Neural Retina
Authors: Mitchell Leah , Fasiku Oluwaseyi , Robinson Jenaye , Opere Catherine , Ohia Sunny , Njie‐Mbye Ya Fatou
Published: June 2021

Excessive glutamate production in the retina has been implicated in some ocular neurodegenerative diseases. There is evidence that hydrogen sulfide (H2S) can exert neuroprotective action against damage induced by oxidative stress or excitotoxicity in the central nervous system.

Purpose

To investigate the pharmacological effects of L‐cysteine, a substrate for H2S biosynthesis, on glutamate‐induced excitotoxicity in bovine neural retina.

Methods

Isolated bovine neural retinae were pretreated with L‐cysteine (10 nM ‐ 1 µM) prior to insult with 2mM glutamate, and retinal neuron survival was assessed using the methylthiazolydiphenyl‐tetrazolium bromide (MTT) assay. For studies aimed at determining the involvement of the H2S biosynthesis pathway, glutamate‐induced neurotoxic retinas were pretreated with enzyme inhibitors for H2S biosynthesis [aminooxyacetic acid (AOA, 30 µM), DL‐propargylglycine (PAG, 1mM), and ketobutyric acid (KBA, 1mM)], prior to L‐cysteine (100 nM) treatment.

Results

In the presence of glutamate (2 mM), only 68% of neurons survived when compared to control. The H2S substrate, L‐cysteine (100 nM) significantly (P<0.001) reversed glutamate (2 mM)‐induced neuron degeneration by 32%. Interestingly, the H2S biosynthetic enzyme inhibitors, AOA, PAG, and KBA did not alter the neuroprotective effects of L‐cysteine.

Conclusion

L‐cysteine can protect bovine neural retina from glutamate‐induced excitotoxic damage. However, the neuroprotective effect of L‐cysteine does not involve de novo intramural biosynthesis of H2S.
Calibration of an in‐silico Human Ventricular Action Potential Model to Detect Proarrhythmic Liabilities
Authors: Green Jonathon , Sutherland J , Gintant Gary
Published: June 2021

In‐silico models of human ventricular action potentials (APs) are useful for defining the roles of specific ionic currents on cardiac repolarization and integrating drug effects on multiple cardiac ionic currents. To provide utility in defining proarrhythmic risk, these models need to be adequately benchmarked regarding their ability to generate cellular proarrhythmia. The present studies calibrated the proarrhythmic liabilities of blocking single and multiple ionic currents using ventricular APs from the O'Hara‐Rudy (ORd) model based on delayed repolarization and early afterdepolarization (EAD) activity.

An endocardial cell model paced at a cycle length (CL) of 4 sec was used to reconstruct AP changes; the maximal conductance of IKr (hERG current), along with IKs, ICaL, INaL, and Na/K pump current were reduced (both individually and in concert with IKr).

Decreasing IKr conductance caused AP prolongation and typically required 150‐250+ beats to reach steady‐state responses when no EADs were present. EADs were first observed when IKr was reduced by 85%; additional IKr block yielded APs with patterns of 1‐6+ EADs. When concurrently blocking IKr, EADs were less prevalent when inward currents (either ICaL or INaL) were reduced, and more prominent when outward currents (either NA/K pump or IKs) were reduced.

In conclusion, in the ORd model, a) EAD activity is typically oscillatory (even after several minutes of simulated pacing), often with no stabilized, steady state rate or pattern, b) EAD activity is present only with substantial IKr block, is reduced with concomitant block of inward ICaL and INaL, and is increased by reducing IKs or Na/K pump current.

This research was supported by AbbVie.
Novel Roles for Catestatin in Cardiac Metabolism and Physiology
Authors: Schilling Jan , Pasqua Teresa , Avolio Ennio , Zemljic‐Harpf Alice , Headrick John , Kassan Adam , Bandyopadhyay Gautam , Roth David , Patel Hemal , Mahata Sushil
Published: June 2021

More than 50 million people suffer from cardiometabolic syndrome in the US. Catestatin (CST), a peptide derived form chromogranin A, acts as an antihypertensive, cardioprotective, pro‐angiogenic, and insulin‐sensitizing peptide. The role of CST in cardioprotection and substrate utilization under normal and stressed conditions is not well understood. We hypothesized that CST is a key regulator of cardiac metabolism and stress adaptation. We investigated wild‐type (WT) and CST‐KO mice to evaluate cardiac function and metabolism. CST‐KO mice display hypertension, are hyperadrenergic, insulin resistant and show increased fasting plasma insulin and leptin. Fatty acid uptake and oxidation was higher in hearts of CST‐KO mice. Ultrastructural studies revealed more glycogen deposition, decreased sarcomere length, and altered mitochondria in CST‐KO mice. We have seen that in the hearts of CST‐KO mice, pAMPK as well as insulin‐stimulated phosphorylation of Akt, and GSK‐3β were decreased. High fat diet (HFD) did not uncover differences in glycogen synthesis, but less glycogen was stored in CST‐KO‐HFD hearts. Comparison of injuries after ischemia reperfusion and ischemic preconditioning showed that protection of hearts from CST‐KO mice was not possible. The same holds true after HFD with worse recovery for CST KO mice. Our studies characterize the crucial role of CST in cardiac physiology and metabolism and suggest CST as a therapeutic target for modulating cardiometabolic disease.
The Yin and Yan of Adenosine A1 and A2A Receptors in Global Cerebral Ischemia Reperfusion Injury
Authors: Atef Reham , Agha Azza , Nassar Noha
Published: June 2021

Global cerebral ischemia reperfusion (IR) following cardiac arrest, shock and asphyxia can affect wide brain areas where new therapies are imperative. Moreover, many intracellular pathways mediating tissue response to IR are still elusive. Adenosine is a neuromodulator that greatly impacts IR through the inhibitory A1 and excitatory A2 receptors. Studies indicate that both adenosine A1 agonists and A2A antagonists are neuroprotective during ischemic insults. However, many of the signaling pathways affecting such responses are largely unknown. In the current study, bilateral carotid occlusion for 45 min. followed by 24h reperfusion period resulted in histopathological damage as well as memory impairment, motor incoordination and increased locomotor activity. Such events were attenuated by unilateral intra‐hippocampal injection of both the selective A1 agonist N6‐cyclohexyl adenosine (CHA) and the A2A antagonist 7‐(2‐phenylethyl)‐5‐amino‐2‐(2‐furyl)‐pyrazolo‐[4,3‐e]‐1,2,4‐triazolo[1,5‐c]pyrimidine (SCH58261). On the cellular level, IR increased glutamate, pERK1/2, c‐Fos, NF‐kB, TNF‐α, cytochrome c, caspase‐3, BDNF, Nrf2 and IL‐10 contents. Both CHA and SCH58261signifiantly reduced glutamate, c‐Fos, TNF‐α, cytochrome c, caspase‐3 and increased Nrf‐2 and IL‐10 contents. While only SCH58261 reduced pERK1/2, NF‐kB and BDNF contents. By the same token, iNOS and TBARS levels were increased following IR and attenuated by both CHA and SCH58261 treatments. Accordingly, these results lend insight to the neuroprotective effects of A1 and A2A agonists and antagonists respectively where pERK1/2 and NF‐kB pathways might be involved in adenosine mediated attenuation of inflammation, apoptosis and oxidative stress.
PARP Inhibition Blocks Asthma Manifestation in a Chronic House Dust Mite (HDM) Asthma Model and Differentially Modulates Human CD4+T cell Function
Authors: Ghonim Mohamed , Naura Amarjit , Pyakurel Kusma , Lammi Matthew , Rodriguez Paulo , Ibba Salomi , Al‐Khami Amir , El‐Bahrawy Ali , Rady Hamada , Zea Arnold , Mansy Moselhy , Ochoa Augusto , Boulares Hamid
Published: June 2021

One of the most essential goals for treatment of allergic asthma is the identification of therapeutic drugs that block the release or effects of Th2 cytokines. Here we show that PARP is highly activated in lung tissues and PBMCs from asthmatics demonstrating relevance to the human disease. Using HDM‐based chronic model, we show that a single administration of olaparib, a PARP inhibitor, was effective in decreasing lung eosinophilia and overall cellularity. Two additional administrations of the drug provided better protection and significantly reduced airway hyper‐responsiveness. These results were confirmed using similarly exposed PARP‐1−/− mice. Such protective effects may be mediated by a significant reduction in Th2 cytokines and a concomitant increase in T‐regulatory cells. Using human CD4+T cells, we show that PARP inhibition significantly reduced Th2 cytokine production in response to CD3/CD28 stimulation without affecting production of IFN‐γ, T cell proliferation, or the overall numbers of Th1 or Th2 cells. However and similarly to our animal studies, PARP inhibition markedly increased the number of human T‐regulatory cells suggesting that PARP inhibition may not affect Th1/Th2 differentiation but alters their ability to produce Th2 cytokines. Our findings provide additional support to the potential of PARP inhibition as a novel strategy for the treatment of asthma.

Source: NIH, ECEB funds
A Simulated Cell Reprogramming from a Pharmacological Perspective
Authors: Nadal‐Quiros Monica , Moore Leon , Marcano Mariano
Published: June 2021

By means of mathematical models, we propose a strategy for identifying the functional changes required to reprogram one type of cell into another. Here, we focus on altering the activity or expression of key transport proteins rather than manipulation of the cell nucleus or the use of embryonic stem cells. We illustrate this strategy by simulating the reprogramming of a sensory macula densa (MD) cell into a transporting cortical thick ascending limb (cTAL). Models of each of these two renal epithelial cell types were developed, and each contains sub‐models of an array of transport proteins and channels known to be expressed in both cell types although in different quantities. The MD‐to‐cTAL cell reprogramming simulations showed that just increasing (~4‐fold) basolateral Na+/K+ ATPase expression in MD cells and fixing to zero the apical membrane water permeability was sufficient to elicit behaviors exhibited by cTAL cells (low and essentially constant cytosolic [Na+] and cell volume when apical [NaCl] varies). In summary, the MD cell reprogramming only required alteration in less than 20% of the transport processes included in the models. These results illustrate how mathematical models can be used to explore possible pathways for pharmacological and genetic manipulation to functionally transform cells by augmenting or diminishing protein expression or activity without the need of reversing the cell differentiation process.

Research supported in part by the NIH MBRS‐RISE Grant 2R25GM061151 and the NIH‐NIGMS Grant SC1GM084744.
Visualization of Physiological Oscillations with Eulerian Video Magnification
Authors: Smith Jillian , Haselby Cyrus , Heinking Kurt , Henderson Kyle
Published: June 2021

Researchers at MIT developed Eulerian Video Magnification (EVM) software that uses temporal and spatial filtering to magnify subtle changes in the environment (Wu et.al.). EVM may provide a unique method for the visualization of low frequency human physiological oscillations. IRB approval was obtained to acquire physiological data from healthy graduate students. Tissue Doppler was used to measure the Traube‐Hering Mayer oscillation which is a low amplitude low frequency fluctuation in blood pressure (~0.1Hz). Respiration was controlled at 15 bpm. Videos were processed using MIT's EVM Matlab code, filtered to 0.067‐1.67 Hz (4‐100 cycles/min), gain set to 50%, using a Gaussian level = 4; and high resolution video from the upper right portion of the forehead was used for spectral analysis. To optimize the quantification of human physiological oscillations; red (R), green (G), and blue (B) spectrums were extracted from color video. These spectrums and their average (C) were auto correlated, normalized, and Fast Fourier Transformed to identify frequencies and their power. Heart rate data obtained from ECG was tightly correlated to the C, R, and G (r2=0.986), and B spectrums (r2=0.949). Low frequency changes in blood pressure were also moderately correlated to C, G and B spectrums (r2=0.75). These preliminary data (N=3) demonstrate that EVM can reveal low frequency human physiological oscillations such as the Traube‐Hering Mayer waveform.
Role of PTEN in Hypoxic Pulmonary Vasoconstriction
Authors: Krauszman Adrienn , Kuebler Wolfgang
Published: June 2021

Rationale

Hypoxic pulmonary vasoconstriciton optimizes ventilation/perfusion matching in the lung, and critically depends on transient receptor potential canonical (TRPC) 6 activation and trafficking to caveolae. However, underlying regulatory mechanisms are yet unclear. In endothelial cells, phosphatase and tensin homolog (PTEN) has been shown to serve as a scaffold for TRPC6, enabling cell surface expression of the channel and subsequent Ca2+ entry independent of its phosphatase activity. Based on these data we hypothesized that the interaction between PTEN and TRPC6 in PASMC may be required for TRPC6 transloaction to caveolae and an intact HPV response.

Methods

Interaction between PTEN and TRPC6 was tested by co‐immunoprecipitation in whole lung lysates, and by proximity ligation assay in pulmonary artery smooth muscle cells (PASMC). Caveolar recruitment of proteins was assessed in PASMC by sucrose density gradient ultracentrifugation. HPV was analyzed in isolated perfused lungs as hypoxia‐induced incresae in perfusion pressure.

Results

Hypoxia induced protein‐protein interaction between PTEN and TRPC6 in both whole lungs and PASMC. Hypoxia also translocated PTEN to caveolae (caveolin 1‐rich fraction) but inhibition of ROCK (Y27632; 5 µM) prevented this translocation. HPV response was significantly attenuated by Y27632, yet not by the PTEN phosphatase inhibitor VO‐OHpic (1‐50 µM).

Conclusion

Hypoxia induces PTEN interaction with TRPC6 in PASMC, and PTEN translocation to caveolae in a ROCK‐dependent manner. PTEN may play an important role in TRPC6 recruitment and activation downstream of ROCK and thus, mediate HPV in a manner that is independent of its phosphatase activity.

Funded by a CIHR operational grant to WMK.
The effect of an ActRIIB ligand trapping agent on the number of satellite cells and myonuclei in dystrophic (mdx) muscle fibers
Authors: Jacinto Kimberly , Borowy Christopher , Nielsen Cory , Potter Ross , Kumar Ravi , Carlson C George
Published: June 2021

Duchenne muscular dystrophy is characterized by repeated cycles of degeneration and regeneration ultimately leading to respiratory failure and death. Muscle growth is largely under the control of myostatin, which negatively influences fiber growth and regeneration by binding to the ActRIIB receptor. Ligand trapping agents for the ActRIIB receptor (RAP‐435) are therefore currently being tested as therapeutic agents using the mdx mouse model for Duchenne muscular dystrophy. The present study utilized confocal immunochemical techniques to examine the effects of in vivo RAP‐435 treatment on the number of satellite cells and myonuclei associated with isolated mdx costal diaphragm fibers. One month old mdx mice were treated with RAP435 or vehicle for one month. Following treatment, the isolated muscles were exposed to 0.2% collagenase. Satellite cells were identified with a PAX 7 primary and an AlexaFluor 488 secondary antibody. Myonuclei were identified by DAPI. Mdx costal diaphragm fibers exhibited approximately 700 myonuclei and 5 satellite cells per muscle fiber. Experiments comparing specific age‐matched nondystrophic and mdx respiratory muscles will determine if the regeneration characteristic of dystrophic muscle is associated with corresponding increases in the number of myonuclei per fiber. RAP‐435 treatment influenced neither the number of myonuclei nor satellite cells per muscle fiber. Morphometric analyses are being performed in conjunction with these confocal studies to determine if ActRIIB inhibition specifically influences myonuclear domain in promoting fiber growth and regeneration.
Cardiac output is improved in rats with myocardial infarction by enhancement of respiratory sinus arrhythmia
Authors: O'Callaghan Erin , Lataro Renata , Zhao Le , Ben‐Tal Alona , Nogaret Alain , Paton Julian
Published: June 2021

Respiratory sinus arrhythmia (RSA) is the physiological modulation of heart rate during respiration. Whilst its physiological function is debated, recent mathematical modelling predicts that RSA saves cardiac energy. Heart failure is associated with a loss of RSA. We aimed to test whether RSA would improve cardiac function in rats 3 days after left ascending coronary artery ligation (post‐MI). Cardiac dysfunction was indicated by infarct size (43±7% of left ventricle) and prolonged systole duration accompanied by reduced peak aortic flow compared to SHAM rats. We built and used a novel central pattern generator (CPG) that, utilizing real‐time diaphragm electromyogram input, generated RSA via respiratory phase‐locked stimulus output to the right cervical vagus nerve. In post‐MI rats, RSA enhancement caused a 12±3% increase in stroke volume, irrespective of the RSA amplitude. Tonic VNS at matched average heart rate increased stroke volume to a similar degree (8±1%, P =0.25 compared to RSA) (n = 7). Despite the increase in stroke volume, myocardial lactate concentrations were unchanged after 45 min of RSA (29±7bpm) or tonic VNS treatment (P = 0.35, One‐way ANOVA). Using a novel CPG device we have demonstrated that, in an acute setting, RSA increases stroke volume in post‐MI rats.

British Heart Foundation and Fundação de Amparo à Pesquisa do Estado de São Paulo
Toll‐like Receptor 4 (TLR4) Knock‐out Mice Are Partially Protected from Diabetic Bladder Dysfunction
Authors: Szasz Theodora , Nunes Kenia P , Webb R Clinton
Published: June 2021

Bladder dysfunction affects a majority of patients with diabetes and therapeutical options for diabetic bladder dysfunction (DBD) are limited. The innate immune system is now thought to be implicated in the pathogenesis of diabetic complications via inflammation and oxidative stress. We have previously shown that activation of Toll‐like receptor 4 (TLR4), a component of the innate immune system, was increased in the bladder of streptozotocin (STZ)‐induced diabetic mice, and that inhibition of TLR4 in vitro normalized the augmented bladder contraction observed in these mice. Here we hypothesized that deletion of TLR4 would ameliorate diabetic bladder dysfunction. We evaluated the development of DBD in TLR4 knock‐out (TLR4KO) and C57Bl/6 wild type (WT) mice treated with vehicle (CON) or STZ (50 mg/kg/day i.p. for 5 days). After 4 weeks of STZ treatment, we observed the same levels of glycemia in TLR4KO and WT mice (mg/dl: WT STZ=349±41, TLR4KO STZ=403±11). Bladder hypertrophy was slightly attenuated in TLR4KO compared to WT mice (% of CON bladder weight/body weight: WT STZ=145±8 TLR4KO STZ=126±11). Contraction to carbacholine (CCh) and electrical field stimulation (EFS) was increased in urothelium‐denuded bladder strips from WT STZ compared to WT CON mice, however no difference was observed between TLR4KO CON and TLR4KO STZ (CCh Emax in mN: WT CON=7.87±0.45, WT STZ=13.07±2.47, TLR4KO CON=8.01±1.35, TLR4KO STZ=9.23±3.13; mN contraction at 32 Hz EFS: WT CON=13.4±0.45, WT STZ=25.51±4.45, TLR4KO CON=14.67±1.66, TLR4KO STZ=19.07±6.45). We conclude that TLR4 contributes to the development of DBD independently of glycemia levels and the mechanism is coupled to innate immune activation in DBD. Support: NIDDK‐13GHSU302.
Regulation of scleraxis‐mediated cardiac fibrotic gene expression by serine phosphorylation
Authors: Wang Ryan , Bagchi Rushita , Czubryt Michael
Published: June 2021

Cardiac fibrosis is a major clinical problem that affects millions of people worldwide yet lacks any specific therapy. Our laboratory was the first to demonstrate the bHLH transcription factor scleraxis induces collagen Iα2 expression in cardiac fibroblasts and myofibroblasts. Scleraxis was also found to be up‐regulated in the heart post‐myocardial infarction, suggesting its role in cardiac pathogenesis. Here we identify a novel mechanism by which scleraxis activity is regulated and determine its effect on genes targeted by scleraxis. Putative phosphorylation sites on scleraxis were revealed by in silico analysis using motif prediction software. Mutation of this phosphorylatable motif to a non‐phosphorylatable form significantly attenuated the expression of fibrillar type I collagen and myofibroblast marker genes normally induced by scleraxis. Down‐regulation of collagen Iα2 expression was due to reduced binding of the non‐phosphorylated scleraxis mutant to specific DNA‐binding sites (E‐boxes) within the promoter as determined by chromatin immunoprecipitation in human cardiac fibroblast cells and electrophoretic mobility shift assay. This is the first evidence suggesting that scleraxis is likely phosphorylated under basal conditions. Analysis of the phosphorylation motif suggested the possible regulation of scleraxis activity by casein kinase 2 (CK2). Inhibition of CK2 activity disrupted the ability of scleraxis to modulate the expression of its target genes. This presents a novel mechanism for regulation of scleraxis activity and thereby provides a rationale for targeting scleraxis for development of drugs to treat cardiac fibrosis.
Enhanced Oxidative Stress Modulates Endocannabinoid Tone
Authors: Matthews Anberitha , Borazjani Abdolsamad , Mangum Lee , Ross Matthew
Published: June 2021

Overactive NADPH oxidase (Nox) in macrophages contributes to the pathology of atherosclerosis. Nox is vital in oxidizing extracellular low‐density lipoproteins (LDL), leading to the internalization of oxidized (ox)LDL by intimal macrophages. Foam cell formation is enhanced during high oxyradical flux. The increased levels of the endocannabinoid (eCB) 2‐arachidonoylglycerol (2AG) in macrophage foam cells might integrate oxidative stress and the eCB system in atherosclerosis development. We hypothesize that 2AG biosynthesis is activated following CD36‐mediated ligation of oxLDL and downstream signaling to NADPH oxidase. Treatment of human THP1 macrophages/monocytes and J774 macrophages with either acetylated LDL, phorbol 12‐myristate 13‐acetate (PMA) or xanthine/xanthine oxidase caused an increase in superoxide (O2•−) levels. 2AG biosynthesis also increased following cell activation. Further, we demonstrated an increase in O2•− production with PMA and oxLDL treated HL‐60 cells. The O2•− anion reacts with hydroethidine (HE) to form 2‐hydroxyethidium (2‐OH‐Et+), which can be chromatographically separated from ethidium, a non‐specific oxidation product. Therefore, the measurement of 2‐OH‐Et+ via LC‐MS/MS suggests a positive correlation between oxygen radical flux and 2AG biosynthesis in macrophages. These data suggest that an increase in 2AG biosynthesis might be an adaptive response to elevated oxidative stress, because of the antioxidant and anti‐inflammatory actions that are associated with this bioactive lipid. Therefore, the pathogenesis of atherosclerosis within the vessel wall intima may be reduced by enhancing eCB tone. [Supported in part by NIH R15ES015348‐02 and 1 F31 HL122082‐01A1]
Role of Oxidative Stress and Tetrahydrobiopterin Synthesis on eNOS Catalyzed Nitric Oxide and Superoxide Production: A Computational Analysis
Authors: Joshi Sheetal , Kar Saptarshi , Kavdia Mahendra
Published: June 2021

Endothelial nitric oxide synthase (eNOS) when bound to its co‐factor, tetrahydrobiopterin (BH4) produce the vasodilator nitric oxide (NO). Oxidative stress in endothelial cells causes oxidation of BH4 to dihydrobiopterin (BH2). Binding of BH2 to eNOS results in eNOS uncoupling and increased superoxide (O2•‐) production from eNOS catalysis. The extent of eNOS uncoupling depends on the biopterin ratio (the ratio of BH4 to oxidized biopterins) in endothelial cells. Changes in endothelial cell oxidative stress levels can dynamically change the biopterin ratio by oxidation of BH4 and by changes in activity of BH4 producing enzyme GTPCH. There is a lack of understanding about the individual and collective effects of oxidative stress and BH4 synthesis on biopterin ratio. In present study, we have developed a computational model for eNOS catalysis based on biochemical pathways of eNOS for NO and O2•‐ production. The model accounts for temporal changes in BH4 concentration and biopterin ratio in addition to endothelial cell based oxidative stress and GTPCH based BH4 production. The model predicts, increased endothelial cell oxidative stress causes eNOS catalyzed NO production to switch between steady state and oscillatory profiles. BH4 production from augmented GTPCH activity could mitigate these oscillations and restore eNOS coupling only under moderate oxidative stress conditions. The results indicate that oxidative stress initiates temporal perturbations in biopterin ratio that switches eNOS from coupled to an uncoupled state at moderate and high oxidative stress conditions. The model results provide valuable quantitative information about the mechanisms promoting endothelial dysfunction.
Hypertrophy Inducing Factor In Pediatric Idiopathic Dilated Cardiomyopathy Serum
Authors: Sucharov‐Costa Juliana , Sucharov Carmen , Miyamoto Shelley , Walker Lori , Stauffer Brian
Published: June 2021

Introduction

Heart failure (HF) is a devastating and costly disease that results in about 50% mortality within 5 years of diagnosis. Despite improvements in adult HF outcomes, children with HF have had no substantial improvement in survival over the past 3 decades. Our work has shown that the myocardium of children with idiopathic dilated cardiomyopathy (IDC) demonstrates a gene expression pattern consistent with pathologic remodeling. The objective of this study was to investigate whether serum from pediatric IDC patients would recapitulate this pathologic gene expression pattern in neonatal rat ventricular myocytes (NRVMs).

Methods

NRVMs were treated with: 1) whole serum from IDC and non‐failing (NF) control patients; 2) heat inactivated serum; or 3) exosomes isolated from serum. Serum protein identification was performed by mass spectrometry.

Results

Treatment of NRVMs with whole serum from pediatric IDC patients induced a pathologic hypertrophic response that was characterized by changes in gene expression and increase in cell size. These changes were prevented by heat inactivation, indicating a protein factor. Exosome treatment suggests that the observed effect is also mediated by RNAs/miRNAs.

Conclusion

We show that circulating factors (RNA and protein) present in the serum of pediatric IDC patients but not in NF controls, induce a pathologic response in NRVMs. Identification of these circulating factors could represent a novel approach to age‐specific HF drug discovery.

Source of Research Support: NIH‐NHLBI R01 HL107715 (BLS), APS Undergraduate Summer Research Fellowship (JSC)
L‐arginine Reduces Matrix Metalloproteinases Activity and Normalizes Oxidative Stress in Hypertensive Patients
Authors: Rocha Natalia , Garcia Vinicius , Rocha Helena , Cardoso Igor , Silva Gustavo , Barbosa Thales , Fernandes Igor , Secher Niels , Vianna Lauro , Nobrega Antonio
Published: June 2021

Defective L‐arginine–nitric oxide pathways and/or redox homeostasis are suggested mechanisms for impaired endothelium‐dependent vasodilation in hypertension. We determined effects of exogenous L‐arginine infusion on nitric oxide (NO) bioavailability, matrix metalloproteinases (MMPs) activity, and oxidative stress in hypertensive patients. Blood samples were drawn from seven hypertensive (HT; 45±5 yrs.) and eleven normotensive men (CT; 37±3 yrs.) before, during and after 30‐minute intravenous infusion of L‐arginine (30 g). Serum NO (NO analyzer), activity of MMP‐2 and MMP‐9 (gelatin zymography) were measured. Thiobarbituric acid reactive substances (TBARS) were quantified as a proxy of oxidative stress. At baseline, NO and MMPs activity were similar between groups, while TBARS were higher in the HT (P=0.03 vs. CT). During L‐arginine infusion, NO increased only in the CT (P=0.02 vs. baseline; P=0.02 vs. HT), while MMPs activity decreased in HT (P蠄0.02 vs. baseline). In HT, TBARS levels became similar to CT during L‐arginine infusion (P=0.21). L‐arginine infusion induced‐change (Δ infusion‐baseline) on NO was negatively associated with baseline systolic blood pressure (r=‐0.83, P=0.04) in HT. Thirty minutes after the infusion, NO was still higher in CT group (P=0.04 vs. HT) whereas TBARS and MMPs returned to baseline levels in HT. Despite impaired L‐arginine‐NO pathway, exogenous L‐arginine diminishes MMPs activity and normalizes oxidative stress in hypertensive patients.
Inflammation Mediates Adipose Derived Stem Cell Dysfunction in Hypertension
Authors: Qi YF , Rathinasabapathy A , Zhang J , Zingler M , Carvajal J , Peterson J , Horowitz A , Guzzo D , Katovich M , Raizada M , Pepine C
Published: June 2021

Adipose tissue secretes inflammation cytokines and is vital for obesity‐induced chronic inflammation caused by infiltrated macrophages. Emerging evidence implicate inflammation in obesity and hypertension (HTN), two major modifiable risk factors for cardiovascular disease. However, not all obesity patients develop HTN; the role of adipose stem cells (ASCs) in obesity‐associated HTN has not been investigated and led us to hypothesize that ASCs from obese‐HTN (OH) patients express increased inflammatory cytokines and oxidative stress, leading to diminished reparative capacity. ASCs were isolated from 12 human subjects [6 OH; 6 obese‐normal (ON)] and 8 rats [4 normotensive Wistar Kyoto (WKY) rats; 4 spontaneously hypertensive rats (SHR)]. ASCs from OH subjects have 12 fold higher TNF‐α level, 30% increase in reactive oxygen species (ROS) and 20% in proliferation than ON subjects. ASCs from SHR exhibited higher inflammatory cytokine, ROS, and proliferation than ASCs from WKY. To determine therapeutic effects of ASCs for HTN, one bolus of 5 million ASCs from WKY rats was intravenously injected into SHR and blood pressure (BP) was monitored for 3 weeks. We observed a 17mmHg decrease in systolic BP one week following cell administration. Systolic BP gradually increased to levels of SHR control. OH is associated with elevated inflammatory status in ASCs. ASCs from WKY possess transient therapeutic effects against HTN. Data suggest that anti‐inflammation treatment and multiple injections of ASCs may be employed for persistent anti‐HTN effect. Thus, inflammation status of ASCs may be a useful predictor for OH.
Sildenafil Restores Endothelial Function in Renovascular Hypertensive Mice: The Relative Contribution of Oxidative Stress and Angiotensin 1‐7
Authors: DIAS Ananda , FAHNING Bernah , CINTRA Amanda , FROSSARD JESSICA , GOMES ISABELE , MEYRELLES SILVANA , VASQUEZ ELISARDO
Published: June 2021

The aim of this study was to evaluate the effects of sildenafil on endothelial function (EF) in renovascular hypertension (RH). Male C57BL/6 mice were subjected to unilateral renal artery clipping and 14 days later were administered with sildenafil (40 mg/kg/day) or vehicle. After 14 days, the sildenafil significantly reduced the high blood pressure in the 2K1C mice. Assessment of EF through the vasodilation to acetylcholine in isolated mesenteric resistance vessels showed a significant decrease in the maximum response in 2K1C‐veh (44±3%) and it was repaired in 2K1C‐sil (67±4%). Similarly, the increased vasoconstriction (mmHg) to norepinephrine observed in 2K1C‐veh (162±14) was abolished by sildenafil in 2K1C‐sil (116±9) mice. NADPH blocker apocynin improved the EF in the 2K1C‐veh mice, without effect in the other groups. Sildenafil treatment enhanced plasma and kidney levels of Ang 1‐7 (+46%) and caused a significant reduction in the oxidative stress (TBARS assay) in the mesenteric vessels whem compared 2K1C‐veh. These data led us to conclude that sildenafil has a beneficial effect on endothelial dysfunction and oxidative stress in RH mice. Thus, sildenafil is a promising drug for the treatment endothelial dysfunction in RH. Financial support: FAPES, CNPq and CAPES.
Effect of Different Exercise Modes on Aldosterone and Sodium Retention in Rats with Myocardial Infarction
Authors: Wason Emily , Zhang John
Published: June 2021

After myocardial infarction (MI), cardiac remodeling activates Renin‐Angiotensin‐Aldosterone system (RAAS), which causes sodium (Na+)‐induced plasma expansion, a contributor to ventricular dilation. Evidence suggests Post‐MI exercise training preserve cardiac function by attenuation of RAAS. However, the effects of different exercise modes, endurance (END) versus strength (ST) training, on ALD and Na­+ in the post‐MI heart have yet to be investigated. The aim of this study was to explore the effects of ST versus END on ALD and Na+ in rats with MI. MI was surgically induced in 7‐wk‐old rats by ligation of the left anterior descending coronary artery, with sham‐operated animals serving as controls. The survivors were assigned to 4 groups: Sham (n=9,), MI+SED (n=9, MI+no exercise), MI+END (n=7), and MI+ST (n=10). One wk after surgery, MI+END were trained on a rodent treadmill (at 16m/min, 50 incline, 40min/d, 5d/wk) and MI+ST were trained on a ladder with weight attached to tail (1 m high, step loading periodization, 3d/wk) for 10 wks. Our results showed that there was no difference (P>0.05) in ALD between MI+END (605±40 pg·mL‐1) and MI+SED (656±31), but both were significantly (p<0.02) greater than MI+ST (483±19). Similarly, there was no difference (p>0.05) in plasma Na+ concentration (mMol·L‐1) between MI+END (187±3) and MI+SED (179±0.4), However, both were significantly greater (p<0.002) than MI+ST (170±4). Our data suggest that compared to MI+END, MI+ST may favorably attenuate RAAS activity and Na+ retention after MI.
Maternal obesity alters gut microbial ecology in offspring of NOD mice
Authors: Zhu Meijun , Kang Yifei , Du Min
Published: June 2021

Background

Gut microbiota (GM) are increasingly recognized as a significant factor in the pathogenesis of T1D. High Bacteroidetes/Firmicutes (B/F) is associated with the development of T1D. Previously we reported that maternal obesity (MO) increases the incidence of T1D in the offspring of nonobese diabetic (NOD) mice, and the current study investigated the possible link of GM to the exaggerated insulitis in offspring due to MO.

Methods

4‐wk‐old female NOD mice were fed either a control or a high fat diet for 8 weeks before mating. Mice were maintained in their respective diets during pregnancy and lactation. All offspring were fed the control diet to 16‐wk‐old.

Results

Pyrosequencing examination revealed that GM of offspring born to obese mothers was distinct from those of control mothers. At phylum level, MO increased Bacteroidetes, Defferibacteres and Proteobacteria, while decreased Firmicutes in offspring GM, resulting in increased B/F ratio in GM of obese offspring. Family level analysis indicated that GM of offspring from obese mothers had increased levels of Deferribacteraceae, Bacteroidaceae, Desulfovibrionaceae, Helicobacteraceaev, and Porphyromonadaceae. Reciprocally, the abundance of Rikenellaceae and Lachnospiraceae‐Ruminococcaceae were decreased in offspring from obese mothers. The analysis at the genus level revealed that Bacteriodes, Barnesiella, Desulfovibrio and Mucispirillum were more abundance, while Alistipes, Clostridium XIVa, Dorea and Osillibacter were less abundance in offspring of obese mothers.

Conclusion

MO resulted in a dysbiosis in offspring, suggesting a GM‐mediated mechanism on diabetogenesis by MO (NIH R15HD073864).
Conditional VEGF Gene Deletion in Both Endothelial Cells and Skeletal Myofibers Leads to Exercise Intolerance Accompanied by a Decrease in Body Mass and Cardiac Enlargement
Authors: Sulaeman Alexis , Fine Janelle , Wagner Peter , Breen Ellen
Published: June 2021

The mechanism by which VEGF‐dependent peripheral vascular function contributes to exercise is unclear. We hypothesized inhibition of VEGF expression in both endothelial cells (EC) and skeletal myofibers leads to capillary regression, impairs muscle function and limits exercise. To test this hypothesis, VEGF gene deletion was tamoxifen‐induced in both EC and myofibers of adult mice that carry the VEGFLoxP, HSA‐CRE‐ERT2 and PDGFb‐iCre‐ERT2 transgenes (Skm/EC‐VEGF‐/‐ mice). Additional VEGFLoxP mice carried either the HSA‐Cre‐ERT2 (SkmVEGF‐/‐) or PDGFb‐iCre‐ERT2 (EC‐VEGF‐/‐) transgene or did not express cre recombinase (WT). In treadmill running tests, Skm/EC‐VEGF‐/‐ mice showed a 46% decrease in endurance than WT mice (n= 11‐27, p < 0.01). Over the 3‐week post‐tamoxifen period, WT, SkmVEGF‐/‐, and EC‐VEGF‐/‐ mice (but not Skm/EC‐VEGF mice‐/‐) gained weight (WT, 27.4 ± 0.35 g, Skm/EC‐VEGF‐/‐, 25.7 ± 0.68 g; n=18‐48, p < 0.05). Skm/EC‐VEGF‐/‐ mice had larger heart to body mass ratios (Skm/EC‐VEGF‐/‐, 6.17 ± 0.23 g/BW; SkmVEGF‐/‐, 5.46 ± 0.14 g/BW; EC‐VEGF‐/‐, 5.46 ± 0.14 g/BW; WT, 5.63 ± 0.10 g/BW, n=18‐48,p < 0.01). Muscle mass, capillary to fiber ratio, fiber area and type in the soleus, plantaris, and gastrocnemius remained unchanged. Ex vivo muscle contractility and fatigue resistance did not differ between WT and Skm/EC‐VEGF‐/‐ soleus, and only the Skm/EC‐VEGF‐/‐ EDL was more fatigue resistant (Skm/EC‐VEGF‐/‐, 188.4 ± 6.57 s; WT, 157.48 ± 9.52 s, n= 11‐14, p < 0.01). The data suggests that endurance is limited when VEGF expression is inhibited in both EC and myofibers. This exercise intolerance is not due to muscle capillary regression, but possibly a decrease in body mass and/or cardiac function.
Prazosin Prevents the Glucocorticoid‐Induced Capillary Rarefaction in Skeletal Muscle
Authors: Mandel Erin , Dunford Emily , Tiech Trevor , Riddell Michael , Haas Tara
Published: June 2021

Prolonged exposure to elevated glucocorticoids (GC) results in skeletal muscle capillary rarefaction, which may promote insulin resistance and limb ischemia. Conversely, treatment with the a1‐adrenergic receptor inhibitor prazosin increases muscle blood flow and stimulates angiogenesis. We hypothesized that prazosin treatment would prevent GC‐induced capillary rarefaction and improve markers of insulin resistance. Corticosterone (Cort) or placebo (wax) pellets (400mg/rat) were implanted subcutaneously in young male Sprague‐Dawley rats. Two days after, prazosin was administered in the animal's drinking water (50mg/L). Plasma levels of Cort correlated significantly with fasting plasma insulin levels (r=0.76), suggesting that sustained elevations in Cort resulted in insulin resistance; this relationship was unaffected by prazosin (r=0.78). Skeletal muscle capillary‐to‐fiber ratio (C:F) correlated inversely with fasted plasma insulin levels (r=‐0.77) and with plasma Cort levels (r=‐0.62). In 16‐day Cort‐treated animals, prazosin significantly increased mRNA levels of pro‐angiogenic VEGF‐A but not levels of anti‐angiogenic thrombospondin‐1 (TSP1), thus resulting in an elevated ratio of VEGF‐A to TSP1. Most notably, the Cort‐induced capillary rarefaction (2.0+0.08 vs. 1.6+0.08) was prevented with prazosin treatment (1.6+0.08 vs. 2.0+0.11). Fasting plasma insulin increased dramatically with Cort (0.51+0.02 vs. 4.1+0.5ng/ml, P<0.05), and improved slightly with prazosin treatment (4.1+0.5 vs. 3.7+0.3ng/ml, P<0.05). This study demonstrates the potential benefits of prazosin in preventing the GC‐induced loss of capillaries and lowering fasting plasma insulin levels (Funding: NSERC and HSF Canada).
Regulation of VEGFR2 Activation in Skeletal Muscle by VEGF Isoforms with Differential ECM‐ and NRP1‐Binding: a Computational Study
Authors: Clegg Lindsay , Mac Gabhann Feilim
Published: June 2021

Tissues expressing only single isoforms of vascular endothelial growth factor (VEGF) form vascular networks that are structurally and functionally different. While the extracellular matrix (ECM)‐ and Neuropilin‐1 (NRP1)‐binding properties of the VEGF isoforms are known, the mechanism by which different isoforms lead to differential signaling downstream of VEGF receptor 2 (VEGFR2) is not well understood. Our objective is to extend a previously developed cell‐level model of VEGFR2 ligation, intracellular trafficking, and site‐specific phosphorylation to a whole‐body framework to study the implications of the ECM‐ and NRP1‐binding properties of VEGF isoforms in vivo. We include two VEGF receptors (VEGFR1 & VEGFR2), one coreceptor (NRP1), one soluble receptor (sVEGFR1), four isoforms of VEGF, and two isoforms of placental growth factor. We find that that distribution of VEGF isoforms in the body is controlled by the ECM‐binding properties of the ligands. We predict that the balance of NRP1‐binding and non‐NRP1‐binding isoforms alters the intracellular distribution of VEGFR2, and thus the relative activation on tyrosines 1175 and 1214, which control the cell behaviors of proliferation and migration/branching, respectively. We investigate changes in the predicted VEGFR2 activation pattern after application of pro‐angiogenic therapies including a VEGF isoform with very strong ECM‐binding. We show that the ECM‐ and NRP1‐ binding properties of VEGF and PlGF isoforms dictate their distribution in skeletal muscle, as well as their receptor‐binding and activating effects.

Funding: DoD NDSEG Fellowship (LEC), NIH R01HL101200 (FMG), NIH R00HL093219 (FMG), Sloan Research Fellowship (FMG)
Pregnancy modulates the expression of NOX4 in aortas of SHR.
Authors: Troiano Jessica , Potje Simone , Oliveira Priscila , Graton Murilo , Antoniali Cristina
Published: June 2021

The Phenylephrine (PE) hyporeactivity in different vessels of pregnant rats is modulated by the increased expression and activity of nitric oxide synthase (eNOS). However, the modulation of reactive oxygen species (ROS) on PE hyporeactivity in spontaneously hypertensive rats (SHR) aorta was not evaluated. We evaluated if ROS modulate the aortas reactivity to PE in pregnant (P) and non‐pregnant (NP) Wistar rats and SHR. Aortas were removed from NP and P rats to test the vascular reactivity, eNOS and NOX4 expression (Western Blot). Aortas reactivity to PE is reduced in P Wistar rats (Emax: P: 21.10 ± 1.8 mN, n=5; NP: 29.05 ± 1.4 mN, n=5) and SHR (Emax: P: 20.80 ± 2.2 mN, n=5; NP: 28.46 ± 1.7 mN, n=7). PE hyporeactivity is related to the increased expression of eNOS in aortas of P Wistar rats and SHR. NOX4 expression is similar in aortas of Wistar P and NP rats. The pre‐incubation of aortas with apocynin (100 µmol/L) decreased Emax of concentration‐effect curves to PE in aortas of P (12.17 ± 2.1 mN, n=5) and NP Wistar rats (19.05 ± 2.7 mN, n=5). These data indicate that ROS modulates the PE hyporeactivity in aortas of P Wistar rats. However, NOX4 is decreased in aortas of P compared to NP SHR. When we incubated the aortas of SHR with apocynin, Emax was not altered in aortas of P rats (16.95 ± 1.1 mN, n=5) and it was decreased in aortas of NP rats (19.03 ± 0.4 mN, n=6). Pregnancy reduces the expression of NOX4 in aorta of SHR. Our results suggest that ROS production is decreased in aorta of pregnant SHR and ROS do not participate of PE effect in aortas of pregnant SHR. Local Ethics Committee (CEEA‐FOA: Proc N 00658‐2013). Financial support: CAPES, CNPq and FAPESP (Grant: 2012/20398‐6).
Palmitate‐Induced Glycosylation of Cyclooxygenase‐2 in Primary Human Vascular Smooth Muscle Cells
Authors: Raman Puneet , Gonzales Rayna
Published: June 2021

Obesity is a major risk factor for cardiovascular health and function. One possible cause of obesity is poor nutrition, including the consumption of diets high in saturated fatty acids. Since vascular inflammation is a key etiological factor in cardiovascular disease, we investigated the effect of saturated fatty acid exposure on the modulation of COX‐2 in primary human male brain and aortic vascular smooth muscle cells. Cells were treated chronically (12 to 18 hr) with vehicle (5% BSA and 0.1% ethanol) or palmitate. Protein levels were assessed via western blotting and light microscopy was used to visualize cell density and morphology. Two distinct bands corresponding to the reported 72 and 74 kDa COX‐2 glycoforms were detected following palmitate treatment. A dose‐dependent increase in both forms was observed with the greatest increase observed in the 74 kDa glycoform (enzymatically active form). Glycosylation inhibition with tunicamycin abolished palmitate‐induced increases in the COX‐2 glycoforms and augmented band density of a lower migrating non‐glycosylated form (64 kDa). Palmitate also increased cell density and altered cell morphology. Although selective COX‐2 inhibition with NS‐398 further enhanced COX‐2 levels in the presence of palmitate, it reversed the palmitate dependent changes in cell density and morphology. In conclusion, this study suggest that palmitate alters COX‐2 function potentially by altering its post‐translation modification in vascular smooth muscle. This potential novel regulation of COX‐2 by saturated fatty acids may contribute as a mechanism in the development of cardiovascular disease in part by altering the structure, function, and health of vascular tissue. Support: University of Arizona Sarver Heart Center