bluuebunny/crossref_metadata_2025 huggingface dataset:
RGS2 Modulation of Angiotensin II Receptor Type 1 Trafficking and Signaling in Neuroblastoma Cells
Authors: Wiley Aidan P , Luessen Deborah J , Hagstrom Michael , Sun Haiguo , Chen Rong
Published: June 2021

Introduction

Altered expression and function of brain angiotensin II receptor type 1 (AT1R) has been implicated in the pathophysiology of various neuropsychiatric diseases such as drug addiction and Alzheimer's disease. However, there is a knowledge gap in understanding the molecular mechanisms of altered AT1R trafficking and function. Regulators of G‐protein signaling (RGS) proteins are negative modulators of G‐protein coupled receptors (GPCRs). Mechanistically, RGS proteins terminate signaling by accelerating the hydrolysis of GTP bound to active G‐alpha subunits. Among all the RGS subtypes, our lab found that RGS2 co‐localizes with AT1R in the brain, suggesting a potential role of RGS2 regulation of AT1R signaling. Additionally, our lab previously found that RGS2 regulates the constitutive and agonist‐induced trafficking of dopamine D2 receptors. Therefore, this project assessed the role of RGS2 in regulation of AT1R trafficking and signaling using a neuroblastoma 2a (N2A) cell model. Knowledge learned from this project may be relevant to the physiological function of RGS2 in the brain.

Methods

We generated stable expression of the human Angiotensin Type I receptor (HA‐AT1R) in N2A cells. N2A‐AT1R cells were then transiently transfected with RGS2 siRNA to knock down RGS2 expression. Cells were then treated with vehicle or angiotensin II (AngII, 10 μM) for 2, 5, or 15 minutes for western blotting of phosphorylated Akt and total Akt. Immunocytochemistry and confocal microscopy were performed on cells treated with AngII for various times to measure the trafficking of AT1R.

Results

We found that the knockdown of RGS2 enhanced AT1R‐mediated signaling by increasing Akt phosphorylation. RGS2 knockdown also showed to increase AngII‐induced internalization of AT1R. This is the first report on the role of RGS2 in regulation of AT1R trafficking. Further study is necessary to investigate the molecular mechanisms underlying RGS2 modulation of the signaling and trafficking of AT1R in neuronal cells.

Support or Funding Information

This work is supported by NIH R01DA042862, P50DA006634, F31AA025532

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulator of G protein Signaling 2 and 5 Coordinate Their Activity to Control Cardiac Contractility
Authors: Osei‐Owusu Patrick , Bernadyn Tyler F , Dahlen Shelby A
Published: June 2021

Fine‐tuning of signaling via heterotrimeric G proteins by regulators of G protein signaling (RGS) proteins is critical to maintaining physiological homeostasis. Abnormal G protein signaling due to the loss of G protein regulation by RGS proteins is implicated in several diseases including cardiovascular disorders such as hypertension, cardiac hypertrophy, and heart failure. RGS proteins act as GTPase activating proteins (GAPs) to control the kinetics and amplitude of G protein signaling. Multiple RGS proteins are prominently expressed in the cardiovascular system; however, whether their activities/functions are coordinated to control G protein signaling and cardiovascular function is unknown. Here, we generated mice concurrently lacking RGS2 and 5 (Rgs2/5 dbKO) to determine how the dual absence of potent GAPs for Gq/11 and Gi/oclass G proteins affects cardiovascular function. Blood pressure and heart rate were monitored in conscious, freely moving mice via radiotelemetry. Surgical implantation of the radiotelemetry device induced marked systolic blood pressure increase in Rgs2/5 dbKO mice (WT: 140 ± 6 vs. dbKO: 170 ± 2 mmHg; p<0.01) at baseline, which gradually declined but remained elevated in Rgs2/5 dbKO relative to wild type (WT) mice, several days later. Whereas all WT mice survived the surgery, ~70–80% of male Rgs2/5 dbKO mice surprisingly died 72–96 hr post‐surgery. When subjected to cardiac stress test using acute dobutamine infusion and echocardiography or the invasive pressure‐volume loop analysis of cardiac function, male Rgs2/5 dbKO mice showed hypocontractile response and decreased ejection fraction relative to WT mice. Freshly isolated ventricular cardiomyocytes from male Rgs2/5 dbKO mice showed decreased fractional shortening (WT: 16.1 ± 4.3 vs. dbKO: 7.4 ± 1.1 %; p<0.01) but high calcium transients (WT: 117 ± 20 vs. dbKO: 198 ± 50 au; p=0.07) at baseline, and application of electrical field stimulation or low concentration of the non‐selective β‐adrenergic receptor agonist, isoproterenol (ISO), triggered premature calcium transients, tachyarrhythmia and death of cells from Rgs2/5 dbKO mice. Interestingly, cells from mice harboring just one copy of Rgs2 (Rgs2+/−, Rgs5−/−) but not Rgs5 (Rgs2−/−, Rgs5+/−) were resistant to low‐dose ISO‐induced arrhythmia. These results together suggest that RGS2 and 5 coordinate their activity to control cardiomyocyte excitation‐contraction coupling and normal cardiac rhythm.

Support or Funding Information

NIH – NHLBI (1R01 HL139754‐01) and AHA Scientist Development Grant (16SDG27260276)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure Activity Relationship Optimization of a 1,3‐Disubstituted Urea‐based Inhibitor of Zika Virus Infection
Authors: Khachatoorian Ronik , Buzzanco Anthony S , Ruchala Piotr , Micewicz Ewa , French Samuel W
Published: June 2021

Zika Virus (ZIKV), a member of the Flaviviridae family of viruses, has recently led to major epidemics in South American countries and has been reported in several other countries as well. ZIKV is the cause of microcephaly and other developmental neuroimmunopathologies in fetuses of pregnant women and Guillain‐Barre Syndrome in adults, and to date, no therapeutics or vaccines are approved for ZIKV infection. In this study, we conducted rigorous structure activity relationship (SAR) studies on one of the three classes of compounds that we previously reported to display significant anti‐ZIKV activity in vitro. We synthesized and tested 50 derivatives of the parental compound (ASN07115927), a 1,3‐Disubstituted Urea‐based thiadiazole. We were able to identify a few derivatives with improved antiviral activity as well as structural features that will guide further SAR studies. Most notably, we found that the urea moiety at the center of the molecule is essential as all but one of the thiourea substituents were completely or significantly inactive. Presence of chlorine on the aromatic ring demonstrated the highest activity compared to its bromo‐, and iodo‐ counterparts. Furthermore, we tested derivatives with allylthio‐, allylamino‐, and propargylthio‐ moieties on the thiadiazole ring and found that the allylthio‐derivatives had higher activity overall. In addition, the location of the allylthio‐ moiety on the ring as well as the orientation of the thiadiazole ring affected the antiviral activity. These results identify compounds with improved antiviral activity which may be utilized/further optimized for the development of inexpensive and orally available anti‐ZIKV drugs. These compounds may also be beneficial for treatment of other viruses in the same family including Dengue virus.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New strategy of drug repositioning through anti‐fibrosis characteristic
Authors: Li Xiaoyi , Wu Dingfeng , Yang Zihuan , Jiao Na , Yuan Meifei , Zhu Lixin , Zhu Ruixin
Published: June 2021

Background

Fibrosis is a common pathogenesis in most diseases, thus different diseases may share targets and drugs related to fibrosis. For a drug that targets fibrotic process, it is likely an effective drug for treating diseases other than its original purpose. Therefore, the current study aimed to investigate the anti‐fibrosis characteristic of drugs for repositioning purpose.

Method

Anti‐fibrosis compounds identified from PubMed and ClinicalTrials.gov were selected as positive samples. FDA‐approved drugs from DrugBank and compounds from Connectivity Map (CMap) were selected as potential negative samples after excluding those compounds known as anti‐fibrosis drugs. MACCS Keys fingerprint (RDKit) and molecule‐stimulated gene signatures (CMap) are collected as the structural descriptors and transcriptomic profiles of compounds respectively. OneClassSVM was used to remove outliers. Gradient Boosting Classifier was performed to build a Structural Model based on molecular structures and to build a Gene Model based on gene signatures to predict the anti‐fibrosis potentials of the compounds. Three external molecular sets: TCMID, ChEMBL and experimental drugs from DrugBank were used for model testing.

Result

526 positive samples and 1248 negative samples were used to build the Structural Model that could predict the anti‐fibrosis potential of candidate compounds with an AUC value of 0.79. With the three molecule sets (TCMID, ChEMBL and DrugBank), 76.76%, 21.92% and 30.02% of the molecules, respectively, were identified as anti‐fibrosis molecules based on this Structural Model. As expected, most traditional chinese medicines with long history of clinical applications possess greater anti‐fibrosis potential that is consistent with their multi‐targeting effects. Among these candidates, 1‐peroxyferolide, peroxyparthenolide and formic acid are ranked to have higher success rates in drug repositioning. Similarly, the Gene Model was build with 851 positive samples and 1588 negative samples with an AUC value of 0.84. RPL30, PPIA and KLHL24 are identified as the preferred targets of drugs with anti‐fibrosis potential.

Conclusion

We first implemented a new strategy of drug repositioning through anti‐fibrosis characteristic, which may increase the success rate of novel applications of existing drugs. In addition traditional chinese medicines were found to have the highest anti‐fibrosis potentials and were considered the first choice in drugs repositioning for further studies.

Support or Funding Information

This work was supported by National Natural Science Foundation of China 81770571 (to LZ), 81774152(to RZ), 41530105 (to RZ).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nephroprotective properties of the methanol stem extract of Abrus precatorius on gentamicin‐induced renal damage through suppression of NF‐κB/CRP and enhancement of Bcl‐2 signaling pathways
Authors: Falayi Olufunke Olubunmi , Oyagbemi Ademola Adetokunbo , Omobowale Temidayo Olutayo , Ayodele Emmanuel , Adedapo Aduragbenro , Yakubu Momoh Audu , Adedapo Adeolu Alex
Published: June 2021

The nephroprotective properties of methanol stem bark extract of Abrus precatorius against gentamicin‐induced renal damage in rats was evaluated in this study. Thirty (30) male Wistar rats were divided into five equal groups. Group A was the negative control group while B was the positive control group which received gentamicin 100 mg/kg intra‐peritoneally for six days. Group C were pretreated with 100 mg/kg extract for the three days and then concurrently with gentamicin 100 mg/kg for three days and group D were pretreated with 200 mg/kg extract for three days and then concurrently with gentamicin 100mg/kg for three days. Group E received gentamicin intraperitoneally for six days followed by administration of 200 mg/kg of the extract for 3 days. Blood samples, kidneys and kidney homogenates were collected for haematological, biochemical, histopathological and immunohistochemical analysis. The results showed that no significant haematological changes were noted. The groups treated with extract exhibited significant increase in body weight gain. While group B significantly exhibited focal areas of inflammation, fatty degeneration, congestion of vessels, tubular necrosis and glomerular atrophy, the lesions were mild with the treated groups. Treated groups exhibited a dose dependent significant decrease in serum creatinine, urea, XO, NO and Myeloperoxidase, AOPP, Protein carbonyl, H2O2, MDA levels when compared with gentamicin treatment group. There were significant dose dependent improvements in SOD, GST, GSH, Protein thiol, and non‐protein thiol levels in the treated groups when compared with group B. In immunohistochemistry, gentamicin treatment group exhibited over expression of CRP and NF‐κB levels, and marked reduction in expression of Bcl‐2 while the reverse was seen in the groups treated with methanol extracts of Abrus precatorius.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glomerular Mesangial Proliferation is Mitigated by sEH/COX‐2 Dual‐Inhibition
Authors: Barnett Scott D , Khan Abdul Hye , Chesnik Marla Ann , Yeboah Michael , Imig John D
Published: June 2021

Chronic kidney disease afflicts millions across the globe, with focal segmental glomerulosclerosis (FSGS) being the most prevalent type of renal disorder. FSGS can result in irreparable damage to the kidney, and even death, if untreated. The kidney filters blood via a network of highly vascularized modules called glomeruli, of which each kidney has approximately one million, and those afflicted with FSGS exhibit damage to these filters. Intraglomerular capillaries are supported by a matrix of cells called the mesangium, and it believed that signals triggering mesangial hypertrophy and proliferation may exacerbate damage to the glomerulus in those with FSGS. Epoxyeicosatrienoic acids (EETs) are positive mediators of renal health and function. Conversely, some EET metabolites are intimated to stimulate mesangial proliferation, which is known to damage the glomerulus. EETs are most notably metabolized by soluble epoxide hydrolase (sEH), but they can also be modified by other enzymes such as cyclooxygenase‐2 (COX‐2), and we have developed a novel drug, PTUPB, that serves as a dual sEH/COX‐2 inhibitor. The objective of this research is to determine if either our non‐hydrolyzable 8,9‐EET analog, or PTUPB, reduces angiotensin II‐associated mesangial proliferation by decreasing the formation of potentially deleterious EET metabolites. Our long term goal is to mitigate the damage caused by FSGS through a better understanding of the role of EETs and their metabolites in glomerular function.

Methods

human primary mesangial cells (p4‐p8) were stimulated in serum‐free media with 100nM angiotensin II (+/−) 100nm or 300nM PTUPB, or 100nM angiotensin II (+/−) 300nM or 1000nM 8,9‐EET analog, for 72‐hours, at which point cells were counted and compared to the control values.

Results

The administration of 300nM PTUPB or 1000nM 8,9‐EET analog decreased angiotensin II‐mediated mesangial proliferation by 56.01% ± 2.5 (p = 0.0172) and 55.25% ± 5.1 (p = 0.0138), respectively, as compared to the relative increase in proliferation caused by 100nM angiotensin II alone.

Conclusion

The mesangium is an important structural component of the glomerulus, but it also serves as a more nuanced mediator or communication in the glomerulus as it directly interfaces with the endothelium and epithelial podocytes. Mesangial proliferation is a known destructive force in FSGS and we have identified two drugs that mitigate the proliferative phenotype in human mesangial cells.

Support or Funding Information

R01 DK103616‐01 (John D. Imig). Dr. Ralph and Marian Falk Medical Research Trust ‐ Phase II (John D. Imig).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ferulic Acid, A Component of Angelica Tenuissima Root Extract Induces Anti‐Melanogenic and Anti‐Oxidative Effects
Authors: Koo Hyun Jung , Park Yuna , So Gyeongseop , Kim Sung Hyeok , Ha Chang Woo , Lee Sang Eun , Namkoong Seung , Kim Tae‐Hyung , Sohn Eun‐Hwa
Published: June 2021

Angelica tenuissima Nakai (Umbelliferae) has been used as a traditional medicine in Asia. The present study investigated the anti‐oxidative and anti‐melanogenic effects of ferulic acid (FA) derived from A. tenuissima root extract (ATE). Anti‐melanogenic activity was determined by intracellular melanin content and tyrosinase activity in α‐MSH (melanin stimulating hormone)‐stimulated B16F10 cells. The anti‐oxidative property of FA was measured using the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) scavenging free radical activity assays. FA significantly suppressed the production of melanin and the tyrosinase activity in α‐MSH‐induced cells. FA also showed the significant tyrosinase inhibitory activity and the DPPH/ABTS radical scavenging activities under cell‐free conditions. These results suggest that FA is responsible for the whitening effect of ATE and could be used as an ingredient in cosmeceutical skincare products.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of the Toxicity Profile and Analgesic Property of Ethanol Extract of Lophira alata Stem Bark in Rodents
Authors: Iniaghe Loretta Oghenekome , Okpakpor Elohor Emma , Kaluaso Smart Chukwudi
Published: June 2021

Introduction

Lophira alata a woody tree found in West Africa is used in the management of pain, schizophrenia and convulsion. This study was carried out to evaluate the toxicity profile and analgesic activity of the of ethanol extract of Lophira alata in rats and mice respectively.

Methods

Stem bark of Lophira alata obtained were identified, authenticated, dried and reduced to fine powder. Phytochemical screening and determination of the mean lethal dose were carried out. Rats (n=30) in different groups were treated with 100–800 kg/mg of the extract daily for 28 days and monitored for weight loss and signs of toxicity. These animals where sacrificed at the end of the study and visceral organs isolated for histo‐pathological, biochemical and hematological assays.

Analgesic property was evaluated in mice treated with graded doses (50–800 mg/kg) of the Lophira alata and subjected to either the mouse writhing assay or hot plate test. The tail flick test was used to determine analgesic activity in rats.

Results

The mean lethal dose was determined to be 5000 mg/kg. No significant difference was observed between weights of animals treated with Lophira alata and the control groups. There was also no significant difference in organ weight ratios between treated and untreated animals. In the hematological assays, there was a decrease in erythrocytes with 800 mg/kg dose levels while a reduction in low density cholesterol was observed in the treated animals. In the mouse writhing test, treatment with Lophira alata decreased number of writhes but did not significantly affect the latency time in the tail flick test.

Conclusion

Lophira alata is relatively safe for use and reduces algesia possibly via peripherally mediated mechanisms.

Support or Funding Information

No honorarium received

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of CDK4/6 Activity Is Synthetic Lethal with VHL Inactivation in Clear Cell Renal Cell Carcinoma
Authors: Nicholson Hilary E , Housden Benjamin , Perrimon Norbert , Kaelin William G
Published: June 2021

Clear cell renal cell carcinoma (ccRCC) is the leading form of kidney cancer, constituting >70% of all cases.1 Inactivation of the von Hippel‐Lindau (VHL) tumor suppressor gene and its protein product, pVHL, occurs in ~90% of ccRCCs.2 pVHL targets the HIF2α transcription factor for degradation. Without functional pVHL, HIF2α accumulates and induces inappropriate transcription of angiogenic, invasive, and growth‐promoting genes. Drugs that inhibit HIF2α or its downstream target VEGF are active against ccRCC, but not all patients respond to these agents and none are cured by them. I hypothesized that pVHL loss might create specific dependencies that could then be targeted therapeutically. To look for such synthetic lethal targets, I used RNAi in isogenic Drosophila cells that have or lack wild‐type pVHL and used CRISPR/Cas9 and chemical compound libraries in isogenic human ccRCC cell lines that have or lack wild‐type pVHL. Analysis of the overlap amongst the hits from these screens identified an increased requirement for CDK4/6 activity in pVHL‐defective ccRCC cells compared to their pVHL‐proficient counterparts. In secondary assays I confirmed that pharmacologic inhibition of CDK4/6 by either Abemaciclib or Palbociclib preferentially reduced viability of VHL−/− cells as compared to VHL+/+ cells across a variety of ccRCC cell lines. Importantly, inhibition of VHL−/− ccRCC cells by Palbociclib was abrogated by expressing a Palbociclib‐resistant CDK6 cDNA or by knockout of the canonical CDK4/6 target pRB. Sensitivity to Palbociclib could be reversed by expressing exogenous pVHL, but not a pVHL mutant lacking its known substrate docking site. HIF2α knockout in VHL−/− cells did not eliminate the effect of CDK4/6 inhibition, and pharmacological stabilization of HIF2α in VHL+/+ cells did not sensitize them to CDK4/6 inhibition. Therefore HIF2α is neither necessary nor sufficient for the synthetic lethal relationship between VHL and CDK4/6. Moreover, the combination of the HIF2α inhibitor PT2399 and the CDK4/6 inhibitor Palbociclib synergistically suppressed proliferation of VHL−/− ccRCC in vitro. I have now confirmed that both Palbociclib and Abemaciclib suppress VHL−/− ccRCC tumor growth in nude mice, including tumors that are PT2399‐resistant. I am currently asking whether these drugs can enhance the activity of PT2399 in vivo.

Support or Funding Information

This work was supported by the Dana‐Farber Cancer Institute and the National Cancer Institute (F32 CA220849‐02) (HN), and the Howard Hughes Medical Institute (WGK). We would like to thank Nicky Persky in the Johannessen Lab at Broad Institute for the entry clone containing CDK6‐D104S, as well as Rebecca Jennings in the Signoretti Lab at DFCI for immunohistochemistry.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiomyocyte‐Specific EGFR Deletion Differentially Alters Cardiac Contractility and Remodeling
Authors: Guo Shuchi , Song Jianliang , Carter Rhonda , Lucchese Ana Maria , Gao Erhe , Cheung Joseph , Tilley Douglas
Published: June 2021

Epidermal Growth Factor Receptor (EGFR), a member of the Erbb receptor tyrosine kinase family, is essential to the development of multiple tissues and organs. Due to the embryonic lethality of global EGFR deletion, the impact of EGFR signaling in the adult heart, normally or in response to pathological stimuli, has not been well‐explored. Using mice with floxed EGFR alleles crossed with αMHC‐Cre mice, we have generated a cardiomyocyte‐specific constitutive EGFR knockout mouse model (CM‐EGFR‐KO) to address its role in the heart. Compared to their wild‐type (WT) littermate controls, CM‐EGFR‐KO mice displayed age‐related development of cardiac contractile dysfunction, marked by impaired diastolic relaxation and Ca2+ handling at the individual myocyte level, as monitored via echocardiographic, hemodynamic, myocyte contractility and Ca2+ transient analyses. RNASeq analysis of CM‐EGFR‐KO versus WT littermate left ventricular samples revealed temporally‐dependent alterations in the expression of a number of myofilament, Ca2+ handling and fetal genes. The contractile defect initially occurs between 7 and 9 weeks of age and is maintained over time without overt cardiac hypertrophic or fibrotic remodeling until 10 months of age, by which time the mice ultimately succumb to severe heart failure and reduced lifespan. Chronic administration of the β‐adrenergic receptor (βAR) agonist isoproterenol effectively compensated for the contractile dysfunction in CM‐EGFR‐KO mice, an effect that was reversible upon cessation of infusion. Notably, βAR‐mediated cardiomyocyte hypertrophy was attenuated in CM‐EGFR‐KO mice compared to WT littermate controls. Treatment of 16 week‐old CM‐EGFR‐KO mice with AAV9‐cTnT‐EGFR permanently rescued cardiomyocyte‐specific EGFR expression and contractile function, suggesting that the contractile dysfunction phenotype observed in these mice is not secondary to aberrant cardiac development, but is specific to EGFR‐dependent regulation of contractile signaling.

Support or Funding Information

NIH grant HL105414 to DGT

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The CYP4A/20‐HETE Axis Regulates Ischemia‐induced Neovascularization via Its Actions on Endothelial Progenitor and Preexisting Endothelial Cells
Authors: Tang Samantha , Chen Li , Zhang Frank F , Gotlinger Katherine H , Falck John R , Schwartzman Michal L , Arbab Ali S , Guo Austin M
Published: June 2021

20‐hydroxyeicosatetraenoic acid (20‐HETE) was recently identified as a novel contributor of ischemia‐induced neovascularization based on the key observation that pharmacological interferences of the CYP4A/20‐HETE axis decrease ischemic neovascularization. The objective of the present study is to determine whether the underlying cellular mechanisms involve endothelial progenitor cells (EPC) and pre‐existing endothelial cells (EC).

We found that ischemia leads to a time‐dependent increase in 20‐HETE that is endothelial in origin using LC/MS/MS analysis and immunofluorescent (IF) microscopy. This is accompanied by increases in SDF‐1a expressions, EPC mobilization from bone marrow, and subsequent homing to ischemic tissues. Pharmacological interferences of the CYP4A/20‐HETE axis with a 20‐HETE synthesis inhibitor Dibromo‐dodecenyl‐methylsulfimide(DDMS) or a 20‐HETE antagonist N‐(20‐hydroxyeicosa‐6(Z), 15(Z)‐dienoyl) glycine(6, 15‐20‐HEDGE) significantly attenuated these increases. Importantly, we also determined that 20‐HETE plays a novel role in maintaining EPC functions and increases in the expression of Oct4, Sox2, and Nanog expression that are indicative of increased progenitor cell stemness. Flow cytometric analysis revealed that pharmacological interferences of the CYP4A/20‐HETE axis decrease the EPC population in culture, whereas 20‐HETE increases the cultured EPC population. Furthermore, ischemia also markedly increased the proliferation, oxidative stress, and ICAM‐1 expression in preexisting EC in the hindlimb gracilis muscles. We found that these increases were markedly negated by DDMS and 6, 15‐20‐HEDGE.

Taken together, the CYP4A/20‐HETE axis regulates ischemia‐induced compensatory neovascularization via its combined actions on promoting EPC and local preexisting EC responses that are associated with increased neovascularization.

Support or Funding Information

American Heart Association [Grant 11SDG6870004]; National Institutes of Health [Grants HL34300], [Grant DK38226]; Robert A. Welch Foundation [GL625910]

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High Salt Up‐Regulates Ca2+‐Sensing Receptor and Na+/Ca2+ Exchanger Expression in Kidneys of Dahl Salt‐Sensitive Rats
Authors: Odutola Samuel O , Awumey Emmanuel M
Published: June 2021

The G protein‐coupled Ca2+‐sensing receptor (CaSR) provides the primary mechanism for Ca2+e‐mediated regulation of the release of parathyroid hormone (PTH) as a result of parathyroid cells sensing small changes in the [Ca2+]e. However, the CaSR is also involved in the fine regulation of serum Ca2+ levels and renal Ca2+ excretion, independent of PTH secretion. The mechanisms underlying these latter effects are not clear. We studied the effect of high salt on urinary ion excretion, blood pressure and expression of the CaSR and NCX1in kidneys of Dahl SR and SS rats. Our results show that four weeks of feeding a high salt diet increased BP in SS, increased urinary Na+/Ca2+ excretion in SR and SS but reduced K+ excretion. The high salt diet caused minimal structural changes in the kidneys, as determined by haematoxylin/eosin (H&E) and periodic acid‐Schiff (PAS) staining. Immunohistochemical analysis of tissue sections showed localization of the CaSR and NCX1 in the thick ascending limb of the loop of Henle, collecting ducts, cortical distal tubules, which was up‐regulated by the high salt in SS animals. NCX1 expression and localization in this study tracks closely with the CaSR. Thus, expression levels and localization patterns of these protein suggest that CaSR modulation of ion transporter and channel proteins could account for the difference in ion excretion observed between SS and SR rats as well as their responses to the high salt diet.

Support or Funding Information

NIH Grant SC1 HL136278

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Apocynin Alters Redox Signaling in Blood Vessels of Spontaneously Hypertensive Rats
Authors: Graton Murilo Eduardo , Potje Simone Regina , Troiano Jéssica Antonini , do Vale Gabriel Tavares , Benevides Priscila Scarpim , Gonçalves Emily Thalia , de Melo Stevanato Nakamune Ana Cláudia , Tirapelli Carlos Renato , Bendhack Lusiane Maria , Antoniali Cristina
Published: June 2021

Apocynin reduces blood pressure and prevents development of endothelial dysfunction in SHR. Mechanisms underlying the effects of apocynin in SHR remain unclear. Angiotensin (Ang) II regulates NOX, the major source of ROS in cardiovascular system. We hypothesized that apocynin, acting as NOX inhibitor, may alter pressor and vasoconstrictor effects of Ang II in SHR. We analyzed the effect of Ang II on blood pressure, reactivity on aorta and mesenteric resistance arteries, plasmatic antioxidant capacity, NOX and NOS expression and activity, AT1 and AT2 receptors expression in blood vessels of SHR treated with apocynin SHR treated with apocynin (30 mg/Kg/day, p.o.). In SHR, apocynin reduced pressor effect of Ang II, increased plasmatic antioxidant capacity, ablated aortic and mesenteric NOX‐dependent ROS production, NOX2 and p47phox overexpression demonstrating that apocynin acts as a NOX inhibitor in blood vessels of SHR. Moreover, apocynin increased aortic and plasmatic nitrate/nitrite levels, but did not alter mesenteric NOS activity or eNOS expression, diminished AT1 expression in aortas and mesenteric bed, and increased AT2 expression in aortas from SHR. Apocynin increased endothelial modulation on Ang II vasoconstriction in resistance arteries from SHR. All these results demonstrated that in vivo treatment with apocynin induces important alterations of several mechanisms that lead to the reduction of the pressor and vasoconstrictor effects of Ang II in SHR. Apocynin effect involves further mechanisms besides the modulation of vascular ROS, which improve NO availability in vascular cells of SHR. These integrated data could help us to understand the promising effect of apocynin as an antihypertensive drug different from those currently used clinically.

Support or Funding Information

Approved by local ethics committee CEUA FOA 450/2015.

Financial support: CNPq, CAPES and FAPESP (2016/22180‐9).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Marfan‐associated Aortic Root Dilation by Angiotensin II Receptor Blockers May Be Independent of Blood Pressure Lowering
Authors: Tehrani Arash Y , White Zoe , Milad Nadia , van Breemen Casey , Esfandiarei Mitra , Bernatchez Pascal
Published: June 2021

Background

Marfan syndrome (MFS) is a connective tissue disorder resulting in accelerated vascular aging, aortic root widening and aneurysm if unmanaged. Despite encouraging pre‐clinical data, recent clinical trials have shown underwhelming efficacy of anti‐hypertensive angiotensin II receptor type 1 (ATR1) blocker (ARB) losartan in MFS patients. We have recently reported that losartan mediates its anti‐aortic root remodeling effects in blood pressure (BP)‐ and ATR1‐independent fashions, and instead acts by improving endothelial function and protective endothelial nitric oxide (NO) synthase‐mediated NO release. Considering the unique mechanism of action of losartan in MFS, whether other ARBs can provide greater protection against MFS aortic root widening is unknown.

Objective

Here we compare the efficacy of other ARBs previously linked to endothelial function activity with losartan at blocking aortic root widening in MFS mice.

Methods and Results

Aortic root dilation, aortic vessel remodeling, BP, and aortic contractility were compared between MFS and wild‐type (WT) mice after an 18‐week treatment with losartan, telmisartan, valsartan, or vehicle. Losartan and telmisartan doses were titrated to give similar hemodynamic effects in vivo, resulting in a 20–30% decrease in BP in both groups, while valsartan was chosen at a non‐BP lowering dose. Untreated MFS mice showed increased aortic root widening compared to WT controls (1.84 ± 0.05mm vs 1.52 ± 0.03mm; p<0.05) as assessed by high frequency ultrasound imaging. Despite differences in BP lowering, all ARBs significantly reduced aortic root widening in the MFS group by 10–16% (p<0.05). Histological assessment revealed a 26–37% (p<0.05) reduction in MFS aortic root medial thickening, and a 50% reduction in elastic fiber fragmentation (p<0.0001) in all treatment groups. Using ex vivo small chamber myography, we showed that all ARBs reduced phenylephrine‐induced aortic contractility by 60–80% (p<0.05) in both MFS and WT aortas in a NO‐dependent fashion.

Conclusion

This is the first animal study to compare the effects of ARBs other than losartan in MFS. Despite differential BP effects, telmisartan and valsartan were effective at blocking MFS aortic root widening and activating endothelial function. This suggests BP lowering to be of minor therapeutic value in MFS management and thus aortic root protection could be achieved with lesser side effects for MFS patients. Future clinical trials should focus on other ARBs than losartan for the management of MFS.

Support or Funding Information

Supported by CIHR, HSFC, BCKDF, Marfan Foundation, SPH Foundation and Rare Disease Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bidirectional Effects of EZH2 Inhibitor in Endometriosis
Authors: Brunty Sarah , Ray‐Wright Kristeena , Santanam Nalini
Published: June 2021

Endometriosis is a complex disorder that afflicts many women with chronic pelvic pain and infertility. It is believed that polycomb group of proteins, such as enhancer of zeste homolog 2 (EZH2), which plays a major role in cancer, also might contribute to the etiology of endometriosis. We and others have shown upregulation of EZH2 in human endometriotic tissues. However, the factors responsible for this upregulation is unknown. This study hypothesizes that the components of peritoneal fluid (PF) plays a dynamic role in epigenetic pathways in endometriosis. To test this hypothesis we measured EZH2 expression and activity (activation of H3K27me3) in endometrial cells. Ishikawa (human endometrial) cells were treated with 1% PF from IRB‐approved and consented women with and without endometriosis (n=6–8/group age and cycle matched). A subset of cells was also treated with various concentrations of GSK126 (5 nM to 10 μM) either before or after PF treatment. Real‐time PCR was used to analyze EZH2 expression in these treated cells. Our results showed bidirectional effects of GSK126 on EZH2 and H3k27me3 expression, depending on the order of the drug treatment (before or after 24 hours of PF treatment) and the source of the PF (control vs endo). When cells were treated with endo PF, there was a 2–3 fold induction in EZH2 expression when cells were treated with endo PF compared to the media control. Surprisingly, there was a dose dependent increase in EZH2 mRNA expression with GSK126 pre‐treatment followed by the PF treatments. Almost a 9‐fold increase in EZH2 expression was observed when cells were pre‐treated with 2.5 μM GSK126 followed by control PF (*p<0.05), whereas pretreatment of GSK followed by endo PF had no such effects on EZH2 expression. However, when cells were pre‐treated with PF followed by the drug the EZH2 expression levels went down. Western blot of EZH2 and H3K27me3 showed similar findings. When MTT assay was performed to examine cell viability under the above experimental conditions, no significant changes in viability was observed at the 48 or 96 hour time points. These results suggest for an intermediate pathway modulated by the PF components that might be at play. The current studies are investigating the possible role for ARID1A mutation in the bidirectional effect of the EZH2 inhibitors in endometriosis.

Support or Funding Information

PhRMA Foundation Pre Doctoral Fellowship in Pharmacology/Toxicology

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resting Cerebral Blood Flow Immediately Post Concussion is correlated to Cognitive Performance
Authors: Iring Stephanie , Favre Michelle , Brewer Kelly , De La Cruz Leslie , Liu Jie , Breen Paul P , Falvo Michael J. , Serrador Jorge J.
Published: June 2021

Sports related concussion is estimated to affect more than 3.8 million Americans a year. Despite the large number of concussions, our understanding of the mechanisms underlying concussion are limited. We also lack an accurate diagnostic tool to provide an indication of concussion severity. Currently, most concussion assessments use cognitive testing to assess severity. Our lab has been examining use of Doppler ultrasound in the field as a possible novel objective measures of concussion severity. We recruited players from recreational rugby tournaments. Players who were seen at the medical tent and determined not to need further treatment were offered the opportunity to participate. A control group of players who had played a rugby match but did not receive a head hit were also recruited. During the tournaments, we used a portable tent based lab to obtain measures of internal carotid cerebral blood flow by imaging the artery to obtain diameter and velocity. Additionally, we measured both beat‐by‐beat blood pressure (Finometer) as well as end tidal CO2 using a nasal cannula during rest, both in the seated and supine position. In a subgroup of 47 players (28 controls, 19 concussed, Mean age= 30, range= 19–66 yrs) out of our large study (125 players, 49 concussed), we had players perform a Simple Reaction Time Test (SRTT), Trails A & B, Digit Symbol Substitution Test (DSST), Paced Auditory Serial Addition Test (PASAT) with 3 sec and 2 sec delay and a Digit Span Memory Test. Concussed players had significantly higher SCAT total number of symptoms (15 ± 11 vs 5 ± 7, P=0.029) as well as Symptom Severity Score (33 ± 18 vs 7 ± 9, P=0.002). Concussed players only performed significantly worse on the SRTT (353 ± 128 ms vs 278 ± 44 ms, P=0.046) while trending on DSST (54 ± 15 ms vs 63± 4 ms, P=0.085). Concussed players demonstrated significantly greater cerebral blood flow when seated (646 ± 180 mL/min vs 415 ± 85 mL/min, P=0.001). Cerebral blood flow was also significantly correlated to performance on SRTT (r=0.84, P=0.035), Trails B Time (r=0.94, P=0.006), DSST (r=−0.88, P=0.021) and Digit Span (r=−0.89, P=0.019). Surprisingly increased seated cerebral blood flow was found in the concussed players and associated with worse cognitive performance. This initial study suggests that cerebral blood flow immediately post‐concussion may be an objective indicator of severity. Future work is needed to examine how immediate measures of cerebral blood flow are predictive of cognitive recovery and post‐concussive symptoms.

Support or Funding Information

This work was supported by Dept. of Pharmacology, Physiology, and Neuroscience, Rutgers and the War Related Illness and Injury Study Center.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial Deacetylase Sirt3 as a New Target in Cardiovascular Diseases
Authors: Dikalova Anna , Pandey Arvind , Xiao Liang , Itani Hana , Sidorova Tatiana , Verdin Eric , Auwerx Johan , Harrison David G , Dikalov Sergey
Published: June 2021

Multiple risk factors such as aging, vascular dysfunction, metabolic disorders and hypertension contribute to the pathogenesis of heart failure. The convergent mechanism that may underlie the interplay of these major risk factors is not clear but may provide novel strategies for treatment. Clinical studies show that Sirtuin 3 (Sirt3) expression declines by 40% by age 65 and sedentary lifestyle paralleling the increased incidence of cardiovascular diseases and heart failure while aerobic exercise increases Sirt3 expression and attenuates age‐dependent Sirt3 decline. Meanwhile, the pathophysiological role of Sirt3 depletion is not clear. Mitochondrial deacetylase Sirt3 is critical in regulation of mitochondrial metabolism and it activates a key antioxidant enzyme, superoxide dismutase 2 by deacetylation of specific lysine residues. We hypothesized that Sirt3 plays critical role in vascular dysfunction and hypertension. To test this hypothesis, we have developed new tamoxifen‐inducible endothelium specific Sirt3 knockout (EcSirt3KO), endothelial Sirt3 overexpressing (EcSirt3OX), smooth muscle Sirt3 knockout (SmcSirt3KO), smooth muscle Sirt3 overexpressing (SmcSirt3OX) and global Sirt3 overexpressing (Sirt3OX) mice on C57Bl/6J background, and examined the effect of Sirt3 expression on vascular dysfunction and angiotensin II‐induced hypertension. Sirt3 depletion in EcSirt3KO and SmcSirt3KO mice raises basal vascular permeability in the heart by 2‐fold, reduces endothelial NO, increases blood pressure and accelerates vascular aging compared with wild‐type mice. Angiotensin II infusion in EcSirt3KO and SmcSirt3KO mice caused vascular hyperpermeability, increased vascular hypertrophy, exacerbated endothelial dysfunction and hypertension. Meanwhile, Sirt3 overexpression attenuates vascular aging, normalizes vascular permeability, diminishes vascular inflammation, inhibits vascular oxidative stress, preserves endothelial‐dependent relaxation, reduces vascular hypertrophy and attenuates hypertension in Sham and angiotensin II‐infused EcSirt3OX and SmcSirt3OX mice compared with the wild‐type littermates. We suggest that Sirt3 is central in redox and metabolic regulations of smooth muscle and endothelial cells. As vascular permeability increases, the access of cytokines, inflammatory cells and LDL to tissue is greater contributing to cardiac inflammation, altered tissue remodeling, hypertrophy and dysfunction. Our data support a therapeutic potential of targeting Sirt3 in cardiovascular dysfunction and hypertension which can improve treatment of the heart failure.

Support or Funding Information

This work was supported by funding from National Institute of Health (NHLBI 1R01HL124116) and American Heart Association (16GRNT31230017).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prediction of cadet basic training physical performance outcomes
Authors: Ploch Christopher , Thomas Diana M , Adams Bryan , Turner Dusty , Hug Adam , Suby Jason , Palmer John E. , Bigelman Kevin
Published: June 2021

Background

Entry to the United States Military Academy requires submission of scores on the Cadet Fitness Assessment. The cadet fitness assessment consists of push‐ups, crunches, kneeling basketball throw, shuttle run, and 1‐mile run. Upon arrival for Cadet Basic Training, cadets are assessed on the components of the Army Physical Fitness Test; push‐ups, sit‐ups and a 2‐mile run. Additionally all cadets are assessed on a swim test. Records of stress‐fractures incurred during Cadet Basic Training are also retained.

Objective

To apply supervised and unsupervised machine learning to describe Cadet Basic Training and Cadet Fitness Assessment data compiled from the graduating classes of 2012 to 2021.

Methods

Simple data‐analysis plots, linear regression models, and unsupervised k‐means cluster analysis were developed to predict Cadet Basic Training performance. Models were generated to identify the relationship between body mass index (BMI), age and gender on 2‐mile run performance to replicate past Army results.

Results

A total N=8886 (82% male) cadets were retained for analysis. The relationship between 2‐mile run performance and BMI was weak (R2=0.07), with age, BMI, and gender representing significant predictors (p<0.01). Similar results were found for all performance outcomes except pull‐ups. The relationship between pull‐up performance and BMI was stronger (R2=0.47). The cluster analysis revealed four clusters. One cluster was identified to poor 2‐mile run performance while a second cluster was identified with poor swim, push‐up, and sit‐up performance. Scatterplots did not indicate any relationship between cluster assignments and BMI.

Conclusions

BMI and even waist circumference may be poor predictors of performance on military physical fitness test outcomes. Machine learning could offer more precision for identifying poor performers early and provide novel opportunities for remediation.

Support or Funding Information

None

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.