bluuebunny/crossref_metadata_2025 huggingface dataset:
ANTICANCER ACTIVITY OF ANNONA MURICATA EXTRACT ON TRIPLE NEGATIVE BREAST CANCER CELLS AND ISOLATION/CHARCTERIZATION OF ACTIVE COMPOUNDS
Authors: Alshaeri Heba , Alasmari Moudi , Pino‐Figueroa Alejandro
Published: June 2021

Triple negative breast cancer (TNBC) cells are a subtype of breast cancer that lack of estrogen receptors (ERs), progesterone receptors (PRs) and human epidermal growth factor receptor 2 (HER2). This type of breast cancer has poor prognosis and accounts for 15–20% of newly diagnosed breast cancer (BC) cases. Annona muricata is a tropical plant and has been used as a folk medicine to treat several diseases such as malaria, inflammation, diabetes and recently it was known to have anticancer activity on various cancer types. However, the underlying molecular mechanisms remain to be explored. The purpose of this study was to investigate the anti‐proliferative activity and mechanism of action of Annona muricata ethyl acetate (AMEA) extract and one active fraction on BT‐20 TNBC cells.

The AMEA showed significant decrease on BT‐20 cell viability as determined by the MTS assay. Furthermore, the AMEA was subjected to preparative thin layer chromatography (TLC) and eight fractions were obtained. From the 8 fractions only fraction 4 (F4) showed significant reduction in cell viability by the MTS assay. Immunoblotting analysis revealed that AMEA and F4 produced anti‐proliferative effect via inhibiting the EGFR phosphorylation and the phosphorylation of its downstream signaling proteins including AKT and MAPK. These effects were accompanied with down‐regulation of cyclin D1 production resulting in cell cycle arrest at G1/S phase. Furthermore, NF‐κB was measured due to its involvement in the progression of cancer that overexpress EGFR as reported in several studies. It was found that AMEA and F4 decreased significantly NF‐κB p65 protein expression in the nuclear fraction, therefore, inhibiting its activation and preventing the induction of cell survival. Our data indicated that neither AMEA nor F4 had significant effect on apoptosis biomarkers when tested on Bcl‐2, Bax, cytochrome c and caspase 3/7 activities. Taken together, these findings provide a scientific basis for the molecular mechanism of action of Annona muricata extract and its active fraction F4 in the treatment of TNBC. It has been demonstrated the anti‐proliferative effect via EGFR‐mediated signaling pathways which includes AKT/MAPK/NF‐κB pathways and cyclin D1 inhibition. Further studies are required to demonstrate the applications of this natural product in breast cancer therapy.

Support or Funding Information

This work was funded by MCPHS University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
KV7.2–7.5 Channel‐Activation Contributes to Bladder Relaxation Induced by Oxybutynin or Mirabegron
Authors: Dalsgaard Thomas , Strøbæk Dorte , Christophersen Palle
Published: June 2021

The muscarinic receptor antagonist, Oxybutynin (Ox), and the β3‐receptor agonist, mirabegron (Mb), are used in the treatment of overactive bladder syndrome (OAB). However, the exact downstream mechanisms by which Ox and Mb opposes the enhanced contractility in OAB is unknown. The KCNQ genes encode five Kv7 K+ channel subunits (KV7.1–Kv7.5). Of these, KV7.2–KV7.5 channels have been proposed to be involved in regulation of smooth muscle tone, i.e. the contractile state of the bladder. In this study, we investigated the involvement of KV7.2–KV7.5 channels in Ox‐ and Mb‐induced relaxation of the bladder. Furthermore, the direct effect of KV7.2–KV7.5 channel‐activation on bladder relaxation was determined. Patch clamp recordings from HEK cells expressing KV7.1–KV7.5 channels were used to determine the relative potency of five different activators; ICA‐27243, ML213, NS15370, Retigabine, and SciFluor, on the KV7.2–KV7.5 channels. Bladder strips from rats mounted in organ baths and contracted with electrical field stimulation were used to investigate the functional effects of inhibiting and activating KV7.2–KV7.5 channels. This study found that inhibiting KV7 channels with XE991 markedly reduced Ox‐ and Mb‐induced relaxation of bladder strips. Furthermore, bladder strip EC50 values corresponded well with HEK cell EC50 values for both KV7.2–3 and Kv7.4 channels, and arranged the five activators with the most potent first accordingly: NS15370>ML213=SciFluor>Retigabine>ICA‐27243. In conclusion, this study showed that KV7.2–KV7.5 channels are probably involved in the therapeutic relaxation of the bladder induced by Ox and Mb in OAB patients. In addition, direct pharmacological activation of KV7.2–KV7.5 channels induced a potent relaxation of the bladder, suggesting that KV7.2–KV7.5 channel activators may be a new target for treating OAB ‐ either as standalone treatment or as enhancers of the effect of currently available treatment options.

Support or Funding Information

This study was funded by the Innovation Fund Denmark.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regional Haemodynamic responses to Adenosine A2A‐Receptor Agonists in Conscious Freely Moving Rats
Authors: Cooper Samantha Louise , March Julie , Hill Stephen , Woolard Jeanette
Published: June 2021

Endogenous adenosine acts as a local mediator in the cardiovascular system to protect against the influences of stress (including ischaemia and reperfusion injury). In highly metabolic tissues, such as skeletal muscle and the coronary circulation, adenosine A1 and A2 receptors help to mediate arteriolar vasodilatation [1]. The present study compared the effects of adenosine or specific A2A agonists on regional haemodynamics in the presence or absence of an A2A‐specific antagonist (SCH58261). As described previously [2], rats were instrumented with miniature pulsed Doppler flow probes (for assessment of regional haemodynamics) sutured around the renal and mesenteric arteries, and the descending abdominal aorta. Animals were also instrumented with intra‐arterial and intra‐venous catheters for measurement of heart rate (HR) and mean arterial pressure (MAP), and drug administration, respectively. Studies were conducted in conscious, freely‐moving rats. All procedures were carried out with approval of the University of Nottingham Animal Welfare Ethical Review Board, under Home Office Project and Personal License Authority. All compounds were prepared in a propylene glycol (PEG) ‐ based buffer (5% PEG, 2% Tween, 0.9% saline) or saline. Agonists were evaluated in the presence or absence of SCH58261 or vehicle (0.1 or 1.0 mg/kg; given as a bolus of 0.1 ml). For example, CGS21680 was administered as 3 min infusions of 0.1, 0.3 and 1 μg/kg/min administered over a total of 9 min at an infusion rate of 0.1ml/min, at least 10 min following the bolus injection of SCH58261 or vehicle. CGS21680 produced tachycardic and depressor effects, which were associated with renal and hindlimb vasodilatations. There was little effect in the mesenteric vascular bed. Responses were antagonised by SCH58261 at 1.0 mg/kg. Responses to adenosine and other modulators of adenosine receptors (e.g. VCP746) will also be presented.

Support or Funding Information

We thank the British Heart Foundation for financial support.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interleukin 6 plus High Salt Increases Systolic Blood Pressure
Authors: Hecht Gillian Grace , Moyer Hayley Catherine , Krishnamurthy Ashwini , van Elst Henrieke , Hoover Robert S. , Wynne Brandi Michele
Published: June 2021

Cytokines and immune cells are implicated in the development of hypertension (HTN), and clinically, interleukin 6 (IL‐6) is independently correlated with HTN. HTN is also characterized by increased sodium (Na+) reabsorption along the aldosterone‐sensitive distal nephron (ASDN), as well as increased expression and/or activity of Na+ transport proteins, such as the sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC). We have previously shown that IL‐6 can activate the mineralocorticoid receptor (MR) in vitro, and intrarenal IL‐6 perfusion significantly increases expression of total and phosphorylated (T53)‐NCC, and ENaC α/γ subunits in vivo. Here, we hypothesize that IL‐6 increases Na+ reabsorption, leading to HTN. To determine whether IL‐6 increases blood pressure (BP), mice (C57Bl6) were implanted with miniosmotic pumps and tail cuff plethysmography used to measure systolic BP during IL‐6 infusion (16 ng/hr, 0.01% BSA in saline) or sham surgery. Mice were fed normal chow (NC) or high salt (HS) food. Mice with systemic IL‐6 infusion (3 days) plus HS diet had significantly increased BP as compared to HS alone (129±6 mmHg vs. 110±5 mmHg, n=4, *p<0.05) with no increases in proteinuria (7 days). No changes in BP was observed in mice treated with IL‐6 and fed NC.

To investigate whether intra‐renal IL‐6 activates NCC via the MR, in vivo, jugular catheters were attached to a miniosmotic pump and inserted under the renal capsule. Mice were infused with IL‐6 (16 ng/hr, 0.1% BSA in saline; 3 days) or vehicle. Mice were sacrificed, tissues removed and then cryosectioned for immunofluorescence (IF) studies. We observed a significant increase in phosphor(T53)‐NCC expression (291±12 mean pixel intensity, MPI) vs. vehicle (236±9; p<0.01, n=3). Mice were then given the MR antagonist (spironolactone, [0.6 mg/day] in peanut butter); we observed a significant reduction in the IL‐6‐mediated increase in phosphor(T53)‐NCC expression (210±12 MPI; p<0.001, n=3).

Our data suggest that IL‐6 infusion plus HS increases systolic BP, beginning on day 3, without significant renal damage on day 7. Although we observed a significant increase in IL‐6‐mediated MR‐dependent phosphor(T53)‐NCC expression, our metabolic cage data suggest that pressure natriuresis may be occurring by day 3. Together, these data suggest a novel role for cytokines and high salt in the development of HTN.

Support or Funding Information

Emory SIRE to GGH and BMW; ASPET SURF to GGH; Emory University SUPERR 5R25DK101390‐05 to HCM; NIDDK085097 and VA MERIT I01BX002322 to RSH and 2T32DK7656‐2 to BMW

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
HEMODYNAMIC EFFECTS OF Gq/11‐SPECIFIC CYCLIC DEPSIPEPTIDE INHIBITOR LIGANDS IN NORMOTENSIVE AND HYPERTENSIVE MICE
Authors: Edwards Alethia J. , Meleka Matthew M. , Dahlen Shelby A. , Osei‐Owusu Patrick
Published: June 2021

Augmented vasoconstriction is a hallmark of hypertension and is mediated partly by hyper‐stimulation of G protein‐coupled receptors (GPCRs) and downstream signaling components. Although GPCR blockade is a key component of current anti‐hypertensive strategies, whether hypertension is better managed by directly targeting G proteins has not been thoroughly investigated. Here, we tested whether inhibiting Gq/11 proteins in vivo or ex vivo using cyclic depsipeptides, YM‐254890 (YM), FR‐900359 (FR) and WU‐07047 (WU) is sufficient to reduce vasoconstriction and reverse hypertension in mice. FR, YM and WU markedly reduced vasoconstriction evoked with Gq/11‐coupled GPCRs. YM and WU but not FR inhibited vasoconstriction through G protein‐dependent and independent pathways by blocking L‐type calcium channel‐mediated Ca2+ influx. Acute subcutaneous injection of FR and YM (0.3 mg/kg, s.c.) in normotensive and hypertension mice elicited marked hypotension, which was more severe and long lasting (FRt1/2 □ 12 hr vs. YMt1/2 □ 4 hr) after the injection of FR relative to YM. In DOCA‐salt hypertension mice, chronic injection of FR (0.3 mg/kg, s.c., daily for seven days) reversed hypertension (vehicle SBP: 149 ± 5 vs. FR SBP: 117 ± 7 mmHg). Incubation of cells with YM or FR caused cellular redistribution of Gq from the plasma membrane. Our results together support the hypothesis that increased vascular Gq/11 activity is involved in the pathogenesis of hypertension, and that direct systemic blockade of Gq/11 can reverse hypertension. The findings provide clear evidence for targeting Gq/11 in the cardiovascular system as an effective therapy for treating hypertension.

Support or Funding Information

This study is supported by grants from Pennsylvania Department of Health (CURE), Margaret Q. Landenberger Foundation, American Heart Association (16SDG27260276) and NIH (HL139754) to P. Osei‐Owusu.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of novel in vitro and in vivo models to evaluate antibiotic efficacy against shigellosis
Authors: Arnold Samuel , McCloskey Molly , Shaheen Shareef , Hulverson Matthew , Choi Ryan
Published: June 2021

Shigellosis is caused by oral ingestion of the gram‐negative bacteria Shigella. Symptoms of the disease include severe diarrhea and dysentery, and the impact of the disease disproportionately affects children and those in low‐income countries such as sub‐Saharan Africa, Asia, and Latin America. According to a reanalysis of the Global Enteric Multicenter Study (GEMS), Shigella has the highest attributable incidence of pathogen specific moderate to severe diarrhea in children between 12–59 months of age. Recent studies have estimated approximately 90 million shigellosis cases annually, and the Global Burden of Disease 2015 analysis suggests at least 164,000 deaths per year are due to Shigella. Interspecies‐variation with lack of cross‐protection may challenge the development of a broadly‐protective vaccine and no human vaccine candidates are currently approved for treatment. Therapeutic options for shigellosis are limited and globally emerging drug resistance to sulfonamides, tetracyclines, ampicillin, and TMP‐SMX has been reported. With resistance to many “first‐line” antibiotics, ciprofloxacin, azithromycin, and ceftriaxone have been recommended for treatment of shigellosis. However, rapidly evolving resistance has been reported for these antibiotics as well. While there is a clear need for novel therapeutics to treat this disease, current drug development efforts are hindered by a lack of fundamental in vitro and in vivo tools to assess the efficacy of pre‐clinical candidates. We have focused our efforts on establishing in vitro co‐culture systems and in vivo mouse models to evaluate small molecule therapeutics. Our aim is to use these tools to identify PK/PD relationships for established and novel therapeutics to treat Shigella infection. The gastrointestinal localization of the parasite generates a unique challenge for establishing a pharmacokinetic‐pharmacodynamic (PK/PD) relationship for an anti‐Shigella drug candidate. We have previously reported on our use of physiology based pharmacokinetic (PBPK) modeling to identify an association between gastrointestinal drug concentrations and in vivo efficacy for anti‐Cryptosporidium drugs and we plan to use a similar technique for the antibiotics used to treat Shigella infection. The presented work will be valuable for the entire research community currently engaged in the development of therapeutics for enteric diseases.

Support or Funding Information

This work was supported by the Tres Cantos Open Lab Foundation Award: TC246

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Trends and Advances on Precision Medicine through Scientometric Analysis
Authors: Sohn Eunsoo , Sohn Eun‐Hwa
Published: June 2021

Precision medicine, more advanced concepts of personalized medicine, triggered by United States' Precision Medicine Initiative has become a new paradigm of health care. Countries around the world are planning to develop precision medicine as a national strategic project, and collaborative research is being actively carried out centered on developed countries. They are making various efforts to drive toward precision medicine by establishing a large scale clinical cohort studies, etc. Despite some current limitations, many opportunities and challenges still remain in precision medicine. In this paper, we reviewed recent R&D status and trends in precision medicine by scientometric analysis methods. Total 19,403 articles retrieved from the scopus database have analyzed using KnowledgeMatrix Plus, Gephi and VOSviewer software. Especially each nation's activities and their technology position, international collaboration and hot research area in precision medicine have also been analysed and identified through network and co‐word analysis showed by visualization map. Results showed that the R&D activities on precision medicine had a dramatic increase in recent years and were located in the early stage of technology development with differentiation by country.

Support or Funding Information

This research was supported by Korea Institute of Science and Technology Information (KISTI).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phospholipids as Indicators of Castration Resistant Prostate Cancer
Authors: Ingram Lishann M. , Manosoura Maryam , Pati Sumitra , Cummings Brian
Published: June 2021

The association of circulating lipids with clinical outcomes of metastatic castration‐resistant prostate cancer (CRPC) is not fully understood. Interestingly, recent studies demonstrated a correlation between plasma lipids and CRPC prognosis, resulting in a proposed prognostic four‐lipid signature of (ceramide, sphingomyelin, phosphatidylcholine (PC) and phosphatidylserine (PS)). While the increase in these lipids correlated to decreased survival, the mechanisms mediating alterations in these lipids, and the correlation to resistance to treatment are not known. We addressed this gap‐in‐knowledge using in vitro models of non‐cancerous, hormone sensitive and CRPC cell lines and quantitative lipidomic analysis. An initial untargeted approach assessing both cells and media indicated that ceramides, sterols and glycerophospholipids had higher relative abundance in CRPC cells (PC‐3 and DU145), as well as hormone sensitive cells (LNCaP), as compared to a non‐cancer cell model (PNT2). Similar data was seen in media. A targeted approach focusing on lipid species shown to correlate to survival in recent publications showed significantly higher levels of 36:2 phosphatidylserine (PS) in all prostate cancer cell lines tested, and lower levels of 36:2 PC in the media of these same cells. Thus, both untargeted and targeted analysis of prostate cancer cells showed significant differences in lipidomic profiles as compared to non‐cancer prostate cells. These data suggest that the lipidomic profiles of prostate cancer cells lines mirror lipidomic profiles in the plasma of prostate cancer patients. As such, these cells may prove useful for understanding the molecular mechanism mediating lipid changes in prostate cancer patients and yield insight into the role of lipids in mediating CRPC.

Support or Funding Information

National Institute of Biomedical Imaging and Bioengineering NIBIB (EB0160100)Department of Defense Prostate Cancer Research Program Idea Development Award (PC150431 GRANT11996600)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Relative Neutralization of Heparin from Different Origins by Protamine, Polybrene, Platelet Factor 4, and synthetic heparin antagonist PMX
Authors: Laddu Rajan , Farooqui Ambar , Siddiqui Fakiha , Iqbal Omer , Hoppensteadt Debra , Kouta Ahmed , Fareed Jawed
Published: June 2021

Introduction

Heparin is the most widely used anticoagulant, which is derived from mammalian tissues. While porcine mucosal heparin is most commonly used clinically, sheep mucosal heparin and bovine mucosal heparin are also currently developed. Protamine Sulfate is the most commonly used antagonist of heparin, which is used clinically. Platelet Factor 4 is a platelet‐derived protein which is capable of neutralizing heparin endogenously. Polybrene and PMX are synthetic agents which can neutralize heparin. These agents are not used clinically. The purpose of this study is to compare the neutralization profile of various heparins by protamine and other heparin‐neutralizing agents.

Materials and method

Porcine heparin was obtained from Medefill Inc. (Glendale Heights, IL). Bovine Heparins were obtained from Extrasul (Sao Paolo, Brazil). Ovine Heparin was obtained from Ronssi Co. (Souzhou, China). Protamine sulfate was obtained from Sanofi Avantis Co. (Paris, France). Platelet Factor 4 was obtained from Enzyme Research (South Bend, IN). Polybrene and PMX were of synthetic origin. Pool‐citrated plasma was obtained from Loyola University Medical Center Blood Bank. Blood‐clotting tests were performed using aPPT and TT reagents. Chromogenic substrate assays were carried out on the ACL‐Elite instrument. Plasma samples were supplemented with each of the pooled heparin preparations at a concentration of 0–10 mg/ml. Protamine neutralization studies were carried out at a fixed concentration of 10 mg/ml. Neutralization profiles were determined graphically and by calculating percent neutralization at a fixed concentration of 5 mg/ml.

Results

At 5 ug/ml the protamine, the neutralization index for the different heparins were comparable in the TT, anti‐Xa and anti‐IIa assays (> 90%) however in the APTT it was around 75% for porcine and bovine pool and 66% for ovine pool. In the polybrene neutralization the all heparins comparable neutralization index in the APTT (>80%), TT (>90%) and anti‐IIa (>90%) however in the anti‐Xa the porcine and bovine were greater than 80% and bovine pool was greater than 90%.

Conclusions

Protamine sulfate was found to be the most effective neutralizing agent for the bovine, ovine and porcine heparins. Assay based variations were noted in the neutralization profiles. With polybrene and platelet factor 4. These studies suggest that titration with the heparin neutralizing agent may be useful in demonstration the biosimilarities of heparins obtained from different sources.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The association of Vitamin D levels to advanced liver fibrosis and response to hepatitis C therapy
Authors: Elhag mashair Khougali Abdelrahman , Deiab shihab , Hussain Nadia , Aziz Rasha Khougali Abdelrahman
Published: June 2021

The importance of vitamin D in many diseases has come to light recently. In the Middle East, there is a prevalence of low vitamin D levels. Evidence linking vitamin D to the parthogenesis of chronic hepatitis C is present. The aim of this study is to analyze the relationship of vitamin D levels with advanced liver fibrosis (ALF) CHC treatment‐naive patients and sustained virology response (SVR) in CHC patients on PEGylated interferon alpha plus ribavirin (pegIFNa/ribavirin) therapy.

Materials and methods

A meta‐analysis of eligible studies published from 1999 to 2014 was conducted. Databases such as PubMed, SCOPUS, Web of Science, and the Cochrane Library were used. Studies assessed included data assessment between plasma/serum vitamin D levels and its relation to ALF and/or SVR. Pooled odds ratios (ORs) were estimated by either fixed or random effects models. Eighteen studies were selected from the literature search, seven for ALF (1,099 patients) and 11 for SVR (2,580 patients). For liver fibrosis, low vitamin D status was related to a diagnosis of ALF, with the cutoffs of 10 ng/mL (OR=2.37, 95% confidence interval, [CI] = 1.20, 4.72) and 30 ng/ml (OR=2.22, 95% CI = 0.99, 3.97) being significant, and a near‐significance for 20 ng/ml.

Results

Regarding SVR, there was a significant heterogeneity among studies (P< 0.001). A significant association with SVR for a vitamin D cutoff of 20 ng/mL (OR=0.53, 95% CI=0.31, 0.91) was present. When meta‐analysis was performed excluding the outliers, significant pooled ORs were found for all patients (10 ng/mL [OR=0.48, 95% CI=0.34, 0.67] and 20 ng/mL [OR=0.58, 95% CI=0.45, 0.76]) and GT1/4 patients (10 ng/mL [OR=0.53, 95% CI=0.34, 0.81] and 20 ng/mL [OR=0.54, 95% CI=0.39, 0.74]).

Conclusion

Low vitamin D status in CHC patients is associated with a higher likelihood of having ALF and lower odds of achieving SVR following pegIFN/ribavirin therapy. Also vitamin D supplementation could improve the SVR rate in patients treated with pegIFN/ribavirin.

Support or Funding Information

This work was supported and funded by Ras Alkhaimah medical and health science university

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A novel metformin‐methylglyoxal imidazolinone metabolite (IMZ) sensitizes cells to insulin; a potential role in alleviating T2DM complications
Authors: Hargraves Tiffanie L. , Mastrandrea Nicholas J. , Lau Serrine S. , Monks Terrence J.
Published: June 2021

Reactive dicarbonyls, such as methylglyoxal (MG), are elevated in type‐two diabetes mellitus (T2DM) patients. These endogenous electrophiles covalently modify proteins, which may contribute to diabetic complications. The T2DM first‐line therapy, metformin (MF), significantly reduces adverse diabetic endpoints and mortality more effectively than other anti‐hyperglycemic agents, the mechanism(s) of which remain unclear. We previously identified and characterized the product of the MF and MG reaction as a novel five‐membered imidazolinone (IMZ) metabolite. IMZ was detected via LC/MS in all MF‐treated T2DM patients, and increased MF urinary levels directly correlated with elevations in urinary IMZ. Scavenging of MG by MF represents a possible alternative mechanism of MF drug efficacy, in addition to its antigluconeogenesis properties. Imidazoline receptor subtypes (I1R, I2R, and I3R) are novel targets for drug development in disorders associated with T2DM because they are involved in insulin sensitization, insulin secretion, and glucose homeostasis. Thus, we examined the ability of IMZ to modulate insulin‐mediated cell signaling pathways via western blot in PC12 cells, which express high levels of I1R and lack the α2‐adrenergic receptor, which many I1R agonists also activate. Combination treatment of insulin and IMZ at physiologically relevant concentrations increased AKT and ERK1/2 phosphorylation above levels seen with insulin treatment alone. This potentiation was not observed in the presence of I1R antagonists. Moreover, IMZ restores insulin sensitivity in an insulin resistant HEPG2 cell model. The results revealed that IMZ may enhance insulin action in the insulin‐dependent AKT and ERK1/2 pathways through I1R activation. Preliminary in vivo pharmacokinetic studies show that IMZ (IP; 10mg/kg and 20mg/kg) is rapidly absorbed, with an elimination half‐life of 12–16.5 minutes. In addition, IMZ enhanced hepatic pAKT 40 minutes after the 10mg/kg dose. In summary, the formation of IMZ in T2DM patients provides evidence that MF scavenges MG, potentially reducing detrimental protein modifications. This property of MF may play a role in the reduction of diabetic complications and represent a potential alternative mechanism of MF drug efficacy. Further, IMZ itself may have the ability to enhance insulin action, suggesting possible clinical utility in the treatment of T2DM.

Support or Funding Information

R24DK0090958, T32ES007091, P30ES006694

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The oxidized phospholipid POVPC induces endothelial to mesenchymal transition
Authors: Ou Zhi‐Jun , Li Yan , Chen Jing , Li Shang‐Xuan , Wang Tian‐Tian , Shi Mao‐Mao , Ou Jing‐song
Published: June 2021

The exact mechanisms of atherosclerosis remian unclear. Endothelial to mesenchymal transition (EndMT) plays an important role in atherosclerosis. 1‐Palmitoyl‐2‐(5‐oxovaleroyl)‐sn‐glycero‐3‐phosphocholine (POVPC), the oxidation product of oxidized low‐density lipoprotein, is an oxidized phospholipid and has been found in atherosclerotic lesions. We recently demonstrated that POVPC could impair endothelial function and induce endothelial cell apoptosis. However, the effects of POVPC on EndMT are unclear. Human aortic endothelial cells were treated with POVPC for long term (at least 48 h). Endothelial cell viability and morphology were observed. Nitric oxide (NO) production and superoxide anion generation (O2•−) were measured. The expression of specific endothelial markers cluster of differentiation 31 (CD31) and mesenchymal markers alpha smooth muscle actin (α‐SMA) were detected by immunofluorescence and immunoblotting. The expression of transforming growth factor beta (TGF‐β) and the expression and phosphorylation of Smad were also determined. Finally, endothelial cell migration was tested. POVPC did not affect the endothelial cell viability, but induce the cellular morphology transformation from cobblestone‐like endothelial cells into spindle‐like mesenchymal cells. POVPC decreased NO production and increased O2•− generation, which inhibited by NG‐nitro‐L‐arginine methylester (L‐NAME). POVPC also inhibited expression of CD31, but stimulated the expression of α‐SMA and TGF‐β as well as the phosphorylation of Smad, which partially inhibited by L‐NAME. Long term POVPC treatment promoted endothelial cell migration. These data demonstrated that POVPC could induce the changes in endothelial morphology and could uncouple eNOS activity to generate O2•− to increase oxidative stress, which may stimulate the expression and phosphorylation of TGF‐β/Smad to mediate the occurrence of EndMT. These effects may promote fibrosis remodel and spindle‐like mesenchymal cell migration to accelerate the process of atherosclerosis.

Support or Funding Information

National Natural Science Foundation of China (Grants 81370370, 81670392, 81600382 and Distinguished Young Scholar Grant 81325001), International cooperation project(2015DFA31070) and National Major Research Program (2016YFC0903000) from the Ministry of Science and Technology of China, Guangdong Natural Science Fund Committee (Grant 2015A030312009), the Changjiang Scholars Program from the Ministry of Education of China, the Guangdong Pearl River Scholars Program, the Sun Yat‐sen University Clinical Research 5010 Program.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Microparticle Generation from Blood and Endothelial Cells during Inflammatory Stimulation
Authors: Chatterjee Victor , Meegan Jamie , Overstreet Jonathan , Coleman Danielle , Szekeres Charles , Yuan Sarah
Published: June 2021

Microparticles (MPs) are small membrane vesicles released by activated cells in response to physical or biological stimulation. They mediate cell‐cell communication by transferring a cargo of cell surface receptors, mRNAs, and microRNAs to target cells. Recent studies have shown an altered MP production in the circulation during inflammation or sepsis. However, cell‐specific mechanisms of MP generation and their functional impact remain poorly understood. In this study, we measured MP generation from platelets, red blood cells (RBC) and leucocytes in human whole blood, as well as in cultured human umbilical vein endothelial cells (HUVEC), during stimulation by different inflammatory mediators, including tumor necrosis factor alpha (TNFα), interferon gamma (INF γ), granulocyte‐macrophage colony stimulating factor (GM‐CSF), bacterial lipopolysaccharide (LPS), thrombin, complement C5a and nitric oxide (via the NO donors DEA NONOate and SNAP). MPs isolated from blood or cell culture conditioned media were analyzed by flow cytometry using cell specific markers and Annexin V, a marker for phosphaditylserine (PS) abundant in MPs. Size‐based gating of MP was performed using a mixture of standardized beads of sub‐micron size and absolute MP numbers calculated based on reference beads of known concentration. The results showed that platelet‐derived MPs were significantly increased by C5a, thrombin, cytokines and LPS. Neutrophil‐derived MPs were increased by LPS, cytokines, GM‐CSF, C5a, and NO donors. RBC‐derived MPs were mainly increased with NO donors. In cultured endothelial cells, TNFα caused a significantly increased, whereas NO decreased production of MPs that were stained positive for ICAM‐1, VCAM‐1, PECAM‐1 and E‐selectin. Interestingly, endothelial cells were able to internalize leukocyte‐derived MPs, evidenced by confocal microscopic confirmation of their intracellular location. Furthermore, neutrophils responded to endothelial‐derived MPs by forming neutrophil extracellular traps. These findings suggest a cell‐specific production and crosstalk of microparticles with blood cell and endothelium origins during stimulation with different inflammatory mediators.

Support or Funding Information

NIH HL070752, HL126646

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of the Impact of the Traditional Medicine Formulation Goreisan and Its Components on Mesenteric Lymphatic Vessel Contraction and Lymphatic Endothelial Barrier Function
Authors: Jo Michiko , Trujillo Andrea N. , Shibahara Naotoshi , Breslin Jerome W.
Published: June 2021

Kampo medicines are traditional herbal formulas used to treat and prevent various diseases. The Kampo medicine Goreisan is a known diuretic and is currently being tested in the clinic to reduce symptoms of surgically induced lymphedema in OB/GYN patients in Japan. The present study was aimed to clarify how Goreisan and its five different components affect lymphatic pump function. Mesenteric collecting lymphatics were isolated from anesthetized male Sprague Dawley rats, and mounted on resistance‐matched glass micropipettes in a 37 °C physiological salt solution bath for studies. The luminal pressure was set at 2 cm H2O and vessels that established intrinsic phasic contractions were studied. Contraction frequency (CF), end diastolic diameter (EDD), and end systolic diameter (ESD) were measured. From these, amplitude (AMP), ejection fraction (EF), and fractional pump flow (FPF) were calculated. Goreisan was applied at concentrations of 1–30 μg/ml. Its components, Cinnamomi Cortex, Atractilodys Rhizoma, Alismatis Rhizoma, Polyporus, and Poria were also studied at concentrations of 1–30 μg/ml. To measure barrier function, human dermal lymphatic endothelial cells (LEC) were treated with 1–100 μg/ml of each compound and transendothelial electrical resistance (TER) was determined by electric cell‐substrate impedance sensing (ECIS). The results show that treatment with Goreisan causes no significant changes in all parameters compared to baseline. However, 30ug/ml Alismatis Rhizoma caused a significant reduction in CF and FPF. The other components did not affect pumping. Goreisan also does not significant affect TER. Polyporus significantly increased TER compared to baseline, while the other components elicited no change. Collectively, the results suggest that Goreisan does not directly affect lymphatic pumping or barrier function, although at its component Alismatis Rhizoma can influence pump activity and Polyporus can enhance barrier function. By extension, in the lymphedema patients treated with Goreisan, its pharmacological action is more likely to be on renal mechanism to reduce excess body fluids. Additional work is needed to determine whether Goreisan may have direct effect to rescue impaired lymphatic vessel functions under inflammatory conditions.

Support or Funding Information

This work was supported by Rotary International Global Grants Scholarship Grant Number GG1746618.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronically Increased Aldosterone In Vivo Attenuates Coronary Vasodilation to Adenosine
Authors: Khan Maloree , Brown Scott M. , Meuth Alex I. , Bender Shawn B.
Published: June 2021

Adenosine‐induced coronary vasodilation is an important cardioprotective mechanism during cardiac hypoxia and ischemia and is utilized clinically for the determination of coronary flow reserve (CFR), an independent predictor of cardiac mortality. Impaired coronary vasodilation to adenosine is more common in diseases associated with inappropriate activation of the renin‐angiotensin‐aldosterone system (RAAS) and recent evidence indicates that systemic blockade of mineralocorticoid receptors (MR) improves CFR in patients with diabetes. The mechanisms underlying RAAS‐dependent attenuation of coronary adenosine‐induced vasodilation, however, remain unclear. Therefore, we hypothesized that chronic infusion of the MR agonist aldosterone would blunt coronary adenosine‐mediated vasodilation and that this would involve reduced adenosine type 2A (A2A) receptor‐dependent vasodilation. Studies were performed using 13–15 week old male C57BL/6J mice infused with either vehicle (95% EtOH in saline) or a subpressor dose of aldosterone (250 μg/kg/d) chronically for 4 weeks via osmotic minipump. After infusion, left ventricular coronary arteries were dissected and function was assessed by wire myography. Chronic infusion of aldosterone raised circulating aldosterone concentrations but did not increase heart weight. Aldosterone infusion blunted coronary vasodilation to adenosine, particularly within the range of adenosine concentrations relevant to those in vivo during cardiac hypoxia (1–10 μM), but did not reduce maximal adenosine‐induced vasodilation. Coronary vasodilation to the endothelium‐dependent vasodilator acetylcholine was also blunted by aldosterone infusion. Additional studies revealed that coronary adenosine‐induced vasodilation in the mouse is entirely dependent on activation of K+ channels and that selective activation of adenosine type 2B (A2B) receptors with BAY 60–6583 induces greater vasodilation that activation of A2A receptors with CGS‐21680. Chronic aldosterone infusion blunted coronary vasodilation in response to selective A2A, but not selective A2B, activation. RT‐PCR revealed no difference in aortic A2A and A2B gene expression. Taken together, our data demonstrate that chronic elevations of aldosterone blunt coronary vasodilation to adenosine via impaired A2A‐dependent vasodilation. This aldosterone‐dependent blunting of adenosine‐mediated vasodilation has implications for cardiac injury in response to hypoxia in states associated with inappropriate RAAS activation such as obesity and diabetes.

Support or Funding Information

Funded by VHA BLR&D BX002030 (SBB) and a STRIDE Undergraduate Summer Research Fellowship (MK) supported by the American Physiological Society and NIH NHLBI R25 HL115473‐01.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of microRNA 15b and Its Downstream Protein Targets in the Heart by Oral Ingestion of Beetroot Wine: Potential Protective Role against Doxorubicin Cardio Toxicity
Authors: Rehman Shabina , Talukder Jamil
Published: June 2021

Background

Beetroot juice (BRJ) is a natural food product containing high levels of nitrate and antioxidant flavonoids/vitamins. Oral intake of BRJ protects against cardiotoxicity of doxorubicin (DOX) ‐ an anticancer drug. BRJ reduced serum cardiac troponin I and ventricular dysfunction caused by DOX and increased plasma nitric oxide (NO) levels. The present study aims at elucidating the effects of BRJ on microRNA 15b (miR15b) ‐ a key modulator of cellular bioenergetics and survival pathways and play an important role in cardiac ischemia‐reperfusion injury.

Methods & Results

Adult male CF‐1 mice were divided into 4 groups (n=3–4/group): 1) Saline [0.2 ml, IP]; 2) DOX [15 mg/kg, IP]; 3) BRJ [BRJ extract added in drinking water (10 g/L) for 13 days]; 4) BRJ+DOX ‐ mice ingested BRJ for 7 days before DOX injection and through the 5‐day post‐DOX period. Five days after DOX or saline injection, ventricular tissues were collected for real‐time PCR or Western blot assays. miR15b expression was significantly decreased in DOX‐treated hearts (Fig. A). BRJ intake not only enhanced the basal level of miR15b, but also completely prevented the depressive effects of DOX on miR15b. BRJ also significantly reduced the protein targets of miR15b ‐ Bcl‐2, but not Arl2 (ADP‐ribosylation factor‐like protein 2) and ANT1 (ADP/ATP translocase 1) (Fig. B, C). Interestingly, we also observed inhibitory effects of BRJ on Bax and NOSTRIN (endothelial nitric oxide synthase traffic inducer) (P<0.05), indicating its potential modulation on eNOS activity and NO production (Fig. B, C).

Conclusion

Oral ingestion of BRJ significantly enhanced miR15b expression in the heart, which is associated with down‐regulation of several protein targets that may act in concert to affect the crucial cellular bioenergetics and survival processes deranged by DOX. These novel findings may help to understand the cardioprotective mechanisms of BRJ as an effective and safe dietary supplement in combating DOX cardiotoxicity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reduction of Reperfusion Cardiac Injury in Donation After Circulatory Death Hearts Through Modulation of Electron Transport
Authors: Chen Qun , Toldo Stefano , Lesnefsky Edward , Quader Mohammed
Published: June 2021

INTODUCTION

Heart transplantation (HT) is a valuable option to prolong life in an end stage heart failure (HF) patient, but this option is limited due to the availability of donor hearts, which currently come from brain death donors only. Expansion of the heart donor pool is urgently needed. One potential source of donor hearts is donation after circulatory death (DCD) donors. Since the DCD process involves substantial ischemia (ISC) and reperfusion (REP) injury to heart, strategies are needed to reduce cardiac injury in DCD hearts for HT.

HYPOTHESIS

Amobarbital (AMO) give during REP decreases cardiac injury. Based on the current human DCD protocol, the duration of ISC prior to heart procurement varies from 25 to 40 min. Thus, we examined if AMO given at the onset of REP can decrease cardiac injury in DCD heart following 45 min of global ISC.

METHODS

After 45 min of ISC, DCD hearts received AMO (2 mM) at the onset of REP for 5 min followed by buffer perfusion for 55 min; or buffer perfusion only for 60 min. Cardiac injury was measured by the left ventricle (LV) infarct size. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria(IFM) were isolated at 10 min of REP to measure calcium retention capacity (CRC, nmol calcium/mg protein) to assess mitochondrial permeability transition pore (MPTP) opening and rate of oxidative phosphorylation (nAO/min/mg).

RESULTS

AMO given during REP decreased the infarct size by 35% compared to untreated hearts. AMO improved the CRC in both SSM and IFM (Table), indicating a decreased susceptibility to MPTP opening during early REP. AMO did not influence oxidative phosphorylation with either complex I (glutamate) or complex II substrates (succinate+rotenone), suggesting that the protection of AMO is not due to improved oxidative phosphorylation.

CONCLUSION

Modulation of mitochondrial respiration at REP is a potential strategy to decrease cardiac injury in DCD hearts through inhibition of MPTP opening.

Support or Funding Information

Scientist development grant (MQ) from American Heart Association, Merit Review Award ( 2IO1BX001355‐01 EJ), R21AG054975‐01 (QC)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intravascular pressure stimulates vasoconstriction through PKD2 channel SUMOylation in smooth muscle cells
Authors: Hasan Raquibul , Yin Wen , Bulley Simon , Leo Dennis , Jaggar Jonathan H.
Published: June 2021

Polycystin 2 (PKD2) channels are expressed in smooth muscle cells of several vascular beds, including cerebral and mesenteric arteries and aorta. Physiological functions of PKD2 channels in the vasculature are unclear. Here, to better understand physiological functions of PKD2 channels in arterial smooth muscle cells, we investigated whether this protein undergoes post‐translational modification. Using arterial biotinylation, immunoFRET and inducible, smooth muscle‐specific PKD2 knockout (PKD2sm−/−) mice, our data show that PKD2 exists as two different molecular weight proteins that are primarily plasma membrane‐localized in arterial smooth muscle cells. Coimmunoprecipitation and immunoFRET imaging indicate that PKD2 is post‐translationally modified by SUMO1 (small ubiquitin‐related modifier), but not SUMO2/3, which produces the higher molecular PKD2 protein. Physiological intravascular pressure or depolarization, through the activation of voltage‐gated Ca2+ channels and extracellular CaV1.2 influx, stimulates PKD2 SUMOylation, which promotes surface localization. 2‐D08, a SUMOylation inhibitor, abolishes PKD2 SUMOylation and prevents pressure‐induced vasoconstriction, but does not alter depolarization (60 mM K+)‐induced vasoconstriction in arteries. Collectively, data indicate that intravascular pressure stimulates PKD2 SUMOylation, which promotes channel surface localization. Intravascular pressure then activates surface PKD2 channels, leading to vasoconstriction. Thus, pressure self regulates the myogenic response by controlling PKD2 channel SUMOylation in arterial smooth muscle cells.

Support or Funding Information

NIH/NHLBI

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of 5‐month interval walking training on cognitive function in elderly people
Authors: Furihata Mayuka , Morikawa Mayuko , Hayashi Ryoichi , Tomita Wayumi , Masuki Shizue , Nose Hiroshi
Published: June 2021

Aims

Deterioration of physical fitness with advancing age is suggested to be one of the major causes for dementia. We examined whether 5‐mo interval walking training (IWT) improved cognitive function with increased physical fitness in elderly.

Methods

Residents >40 years old in Yurihonjo participated in this study. They were randomly divided into the control (CNT) and IWT groups. Subjects in the CNT group were instructed to maintain their sedentary lifestyle while those in the IWT group were instructed to perform IWT, repeating ≥5 sets of fast and slow walking for 3 min each at ≥70% and 40% peak aerobic capacity for walking (VO2peak), respectively, per day ≥4 days/week for 5 months from June to November, 2016. Before and after the intervention, we measured VO2peak and cognitive function with Urakami‐style Screening Test. Since 36 subjects dropped out during the intervention and additionally 24 subjects lacked ≥1 of the measurements, we analyzed the remaining 175 subjects (CNT, n=88 and IWT, n=87). Moreover, since subjects who marked ≤12 points in cognitive function were clinically defined as mild cognitive impairment, they were used for further analyses (CNT, n=17 and IWT, n=18).

Results

After the intervention, VO2peak and cognitive function increased by 3.0±1.1% and 3.5±3.0% in the IWT group, more than −2.0±1.3% and −7.1±2.9% in the CNT group, respectively (both, P<0.01), and these effects were enhanced in the mild cognitive impairment subjects; 5.8±2.3% and 34.1±8.7% in the IWT group vs −0.4±2.3% and 11.8±12.3% in the CNT group, respectively, (both, P<0.05).

Conclusion

The five‐month IWT improved cognitive function with increased VO2peak in elderly, especially in the mild cognitive impairment individuals.

Support or Funding Information

This study was supported by a grant from by the Cabinet Office of Japan to re‐activate local communities which would lose their activity due to advanced aging.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.