bluuebunny/crossref_metadata_2025 huggingface dataset:
Title: Phosphorylation‐independent transactivation of the ERK5 by statins
Authors: Chu Uyen Bao , Yang Jay
Published: June 2021

ERK5 mitogen activated protein kinase unlike other members of the MAPK family functions both as a kinase and as a transcriptional co‐activator. It has been reported that the N‐ and C‐termini of ERK5 reciprocally inhibit the function of each segment and phosphorylation of the N‐terminal TEY motif result in an autophosphorylation of the C‐terminal tail and its translocation into the nucleus for regulation of downstream targets. We used a Gal4‐ERK5 fusion construct co‐expressed with a Gal4‐binding domain‐luciferase reporter in COS7 cells to examine whether the previously reported statin‐activation of ERK5 were mediated by kinase activation or transactivation. At 10 microM, cerivastatin, fluvastatin, pitavastatin, and simvastatin exhibited ERK5 transactivation while atorvastatin, lovastatin, mevastatin, and pravastatin did not. Western blot analysis with phospho‐specific ERK5 antibody on samples treated with statins did not reveal ERK5 TEY phosphorylation suggesting that statins may directly transactivate ERK5. If statins directly transactivate ERK5 without kinase activation, a phosphorylation‐deficient ERK5 (AEF mutant) should demonstrate transactivation. However, statins failed to transactivate the Gal4‐ERK5 (AEF) reporter indicating the likely involvement of the TEY domain in transactivation although not through phosphorylation. Further studies with other mutants and FRET assay of independently expressed N‐ and C‐terminal domains are in progress. The apparent phosphorylation‐independent activation of ERK5 by statins may provide a more fine‐tuned regulation of ERK5 and a better understanding of this process will be useful in the design of therapeutics mimicking the beneficial effects of ERK5 activation such as endothelial cell protection, enhanced recovery from myocardial injury from global ischemia ex vivo, and myocardial infarction in vivo particularly in the setting of hyperglycemia.
Localization and ERK signaling of α1‐adrenergic subtypes in HeLa cells
Authors: OˈConnell Timothy D , Schwegel Jenica S , Anderson-Daniels Jordan M , Wright Casey D
Published: June 2021

G‐protein coupled receptors (GPCRs), like α1‐adrenergic receptors (α1‐AR), are thought to localize to and signal at the plasma membrane, but many GPCRs localize to and signal at the nucleus in several cell types, including neurons, hepatocytes, and cardiac myocytes. Interestingly, α1‐ARs show differential localization based on cell type: nuclear in cardiac myocytes, but plasma membrane/intracellular in most cell lines and smooth muscle cells. Here, we examined receptor localization, oligomerization and signaling using HeLa cells as a cell model where α1‐ARs are not primarily at the nucleus. In HeLa cells, the α1A‐AR localized to the plasma membrane and nucleus, whereas the α1B‐AR localized intracellularly. Interestingly, co‐expression of both subtypes induced receptor hetero‐oligomer formation and produced a unique nuclear co‐localization. Activation of either subtype induced phosphorylation of ERK, which was unchanged by the membrane impermeant α1‐antagonist CGP‐12177a or the nuclear export inhibitor leptomycin B. These results suggest that, in contrast to other cells an intracellular, non‐nuclear α1‐AR population signals in HeLa cells. Signaling from intracellular α1‐AR and the localization differences between cell types could have implications in the treatment of hypertension and benign prostate hyperplasia. This work was supported by grants from NIH (TDO, CDW) and PhRMA Foundation (CDW).
Protective effect of resveratrol on serum deprivation induced cell death in primary fibroblasts
Authors: Tábi Tamás , Pálfi Melinda , Wagner Zsolt , Freisinger Ádám , Bagdy György , Magyar Kálmán , Szökõ Éva
Published: June 2021

Resveratrol is an active compound isolated from red grape. In the literature contradictory data are available, as both its pro‐ and antiapoptotic effect have been described. The cell type used can be one of the major differences between the various models. The compound might differentially affect tumorigenic and normal cells. In this study we aimed to study its effect in non‐transformed cells, thus primary mouse embryonic fibroblasts have been used. Apoptosis has been induced by serum deprivation resulting in significant caspase 3 activation within 3 hours.

Resveratrol showed dose dependent inhibitory effect on caspase 3 activation in primary fibroblasts with IC50 of 100 microM. Along with this effect it also ameliorated the cell death assessed by lactate dehydrogenase release. Among the major regulatory pathways, only inhibition of p38 kinase prevented the protective effect of resveratrol suggesting the role of mild stress in its effect, thus the effect of oxidative stress has also been studied.

Our results show that resveratrol exerts protective rather than proapoptotic effect on non‐transformed cells.

This work was supported by TÁMOP‐2.2.1.B‐09/1/KMR‐2010‐0001.
The aryl hydrocarbon receptor plays a role in mediating adipocyte/breast cancer interactions
Authors: Salisbury Travis , Chaudhry Ateeq , Morris Gary
Published: June 2021

The Aryl Hydrocarbon receptor (AHR) is a ligand‐activated transcription factor that mediates the effects of environmental toxicants. Obesity is a risk factor for several types of cancer. Prior reports and our data indicate that adipocytes secrete factors that induce breast cancer cell growth. Herein, we determined if adipocyte secreted factors (ASFs) regulate the expression of AHR responsive genes and induce cancer growth through AHR dependent mechanisms. We found that ASFs suppress ~90% and ~80% of basal CYP1A1 and CYP1B1 expression, respectively. In control MCF‐7 cells, ASFs induced a 3 fold increase in growth in 3 days. In AHR‐knockdown cells, ASFs induced a 2 fold increase in growth in 3 days. AHR antagonists also blocked adipocyte‐stimulated cancer growth. Collectively, these data indicate that ASFs inhibit the expression of “classic” AHR gene targets, and induce growth through AHR dependent mechanisms. This work was supported by P20RR016477 to the West Virginia IDeA Network of Biomedical Research Excellence, Cell Differentiation and Development Center at Marshall University, and Pharmacological Research and Manufactures of America Foundation (PhRMA) Research Starter Grant in Pharmacology/Toxicology.
Glucocorticoid Receptor‐Mediated Transcriptional Regulation of N‐acetyltransferase 1 Gene through Distal Promoter
Authors: Bonamassa Barbara , Ma Jongjie , Liu Dexi
Published: June 2021

Human arylamine N‐acetyltransferase 1, HUMAN(NAT1) plays important roles in drug and carcinogen biotransformation and cancer development. Its gene expression has been shown to be regulated by environmental factors. The purpose of the current study is to determine the involvement of nuclear receptors in its transcriptional regulation. We show that among the nuclear receptors, the glucocorticoid receptor (GR) plays a dominant role on the distal promoter (P3). The involvement of the GR was demonstrated by dexamethasone treatment, reporter assay using plasmid‐containing 3kbp of 5′ end region of P3, and treatment of anti‐glucocorticoid RU486 in primary human hepatocytes and transfected HepG2 cells. In addition, translation inhibition didn't affect dexamethasone‐induced gene expression through P3, suggesting that dexamethasone effect is directly mediated by GR activation. Furthermore, deletion analysis revealed the presence of multiple responsive elements. Transfection assays in mice using hydrodynamics‐based procedure and reporter gene assay in a mouse cell line revealed that GR‐induced NAT gene expression is species dependent. Dexamethasone treatment of transfected mice and mouse cell line decreased mouse NAT2 gene expression, HUMAN(NAT1) homologue. These results suggest that glucocorticoids serve as a modulator for (HUMAN)NAT1 gene expression via the P3‐containing 5′‐flanking region. (NIH grant RO1EB007357 and RO1HL098295)
Role of NMDA receptor subtypes in regulation of central amygdala
Authors: Ugale Rajesh R , Hillman Brandon G , Gupta Subhash C , Suryavanshi Pratyush , Stairs Dustin J , Dravid Shashank M
Published: June 2021

We have recently shown that mice lacking the NMDA receptor GluN2C (GluN2C KO) subunit exhibit deficits in contextual and cued fear acquisition (Hillman et al., 2011). The central amygdala (CeA) is composed of lateral (CEl) and medial (CEm) subnuclei which are involved in fear acquisition and expression respectively. Furthermore, CEloff neurons are characterized as PKCδ‐positive, tonically inhibit the CEm resulting in regulation of fear expression. We found that in comparison to wildtype, GluN2C het and KO mice have over two‐fold pERK stained neurons in the CEl which co‐localizes with PKCδ. Additionally increased frequency of sIPSCs was observed in CEm of GluN2C KO versus wildtype mice. We sought to determine if potentiation of GluN2Ccontaining receptors would alleviate fear acquisition deficit in GluN2C heterozygous mice. D‐cycloserine, which has two‐fold efficacy at GluN2C‐containing receptors compared to glycine, injected prior to fear conditioning increased fear acquisition in GluN2C heterozygous animals while it had no effect on knockout counterparts. A potential role of GluN2C receptors expressed in mediodorsal thalamus was further identified in regulating CEl activity. The findings presented herein show that GluN2C subunit may regulate basal CeA activity which may have important implication for anxiety and fear disorders.
Hirsutine, an indole alkaloid of Uncaria rhynchophylla, inhibits inflammation‐mediated neurotoxicity and microglial activation
Authors: Nam Kyong Nyon , Park Yeri , Lee Semi , Lee Eunjoo H
Published: June 2021

Uncaria rhynchophylla is a traditional oriental herb that has especially been used for treatment of disorders of the cardiovascular and central nervous systems. Hirsutine, one of the major indole alkaloids of Uncaria rhynchophylla has been shown to have neuroprotective potential. The specific aim of this study was to examine the efficacy of hirsutine in the repression of inflammation‐induced neurotoxicity and microglial cell activation. In organotypic hippocampal slice cultures, hirsutine blocked lipopolysaccharide (LPS)‐mediated hippocampal cell death and productions of nitric oxide (NO), prostaglandin (PG) E2 and interleukin (IL)‐1β. Hirsutine was shown to effectively inhibit LPS‐induced NO release from primary cultured rat brain microglia. This compound reduced the LPS‐stimulated productions of PGE2 and intracellular reactive oxygen species. Hirsutine significantly decreased LPS‐induced phosphorylation of the mitogen‐activated protein kinases and Akt signaling proteins. In conclusion, hirsutine reduces the productions of various neurotoxic factors in activated microglial cells, and possesses neuroprotective activity in a model of inflammation‐induced neurotoxicity.
Korean red ginseng prevents APAP‐induced hepatotoxicity and mortality through metabolic regulation: The role of ginsenoside Rg3, a protopanaxadiol
Authors: Cho Min Kyung , Kim Sang Geon , Gum Sang
Published: June 2021

Inappropriate use of acetaminophen (APAP) can lead to significant morbidity and mortality secondary to hepatic necrosis. We determined the beneficial effect and molecular mechanism of Korean red ginseng (KRG) on the APAP‐mediated hepatotoxicity and identified a major component of KRG for protection. Pretreatment with high doses of KRG for 1 week significantly reduced mortality at the LD50 of APAP. APAP increased AST and ALT activities, which was abrogated by low doses of KRG for 5 weeks. KRG also delayed APAP metabolism in plasma through CYP2E1 inhibition. These protective effects of KRG were consistent with the results from histopathological examinations. KRG induced antioxidant/phase 2 gene battery in rat liver and H4IIE. Exposure of KRG increased the nuclear Nrf2 and C/EBPβ, which are important transcriptional factors for those genes including GSTA2. These effects were downstream of multiple cellular signaling, including PI3K, JNK or PKA. Rg3 but not Rb1, Rc and Rg1 significantly increased those gene expressions. Rg3 resulted in the transcriptional activation of GSTA2 through Nrf2 and C/EBP downstream of the cellular signaling. These results demonstrate that KRG is efficacious in protection from APAP‐induced hepatotoxicity and mortality and that Rg3 is a major component of KRG for the hepatoprotection, implying that Rg3 should be considered to be a potential hepatoprotective agent.
Antioxidant activity of a combination of atorvastatin and lycopene in normal Sprague Dawley rats
Authors: Pina Rosa M , del Carmen Caamaño Maria , Garcia Olga P , Rosado Jorge Luis
Published: June 2021

The present study evaluated the effect of atorvastatin and lycopene on the activity of antioxidant enzymes in normal rats. Thirty male Sprague Dawley rats (200 ± 10g) received either 50 mg/kg/d of atorvastatin (AT), 20 mg/kg/d of lycopene (LYC), 50mg/kg/d of atorvastatin + 20mg/kg/d lycopene (AT50+LYC) or 20mg/kg/d of atorvastatin + 20mg/kg/d of lycopene (AT20+LYC). The control group received water; treatments were given by oral intubation during 14 days. All rats were fed with regular rodent chow. Blood samples were taken 24‐h after the last oral intubation for the determination of catalase and superoxide dismutase (SOD) enzyme activities using commercial assay kits. Catalase activity was significantly induced by all treatments by 2.1 to 4.4 fold compared with control (p<0.05). The doses of AT50+LYC and AT20+LYC had the highest effect on catalase induction compared to other treatments (p<0.05). SOD activity was significantly induced with LYC by 1.1‐fold compared to control group (p<0.05). In conclusion, the combination therapy of atorvastatin and lycopene showed a favorable antioxidant activity. The potential benefits on cardiovascular diseases remains to be evaluated. CONACYT
Sestrin2 Gene Induction by Nrf2‐ARE Pathway
Authors: Ki Sung Hwan , Shin Bo Yeon , Jin So Hee , Cho Je
Published: June 2021

The Sestrin2 (Sesn2) gene encodes a conserved antioxidant protein that is induced upon oxidative stress and protects cells against ROS. Nrf2 is an essential transcription factor that regulates expression of a variety of antioxidant genes via binding to the antioxidant‐response element (ARE), but the role of Nrf2 in Sesn2 gene expression has not been elucidated yet. The present study investigated whether the Nrf2‐ARE pathway regulates Sesn2 gene expression and the identification of the molecular mechanism. The Nrf2 activators up‐regulated Sesn2 expression in a dose‐and time‐dependent manner in hepatocytes. The specific role of Nrf2 in Sesn2 induction was verified by using Nrf2 over‐expression plasmid and Nrf2 knockout or knockdown cells. In silico analysis of the 5′ upstream region of Sesn2 gene identified a putative ARE sequence. Deletion of the putative ARE demonstrated that ARE from −550 to −539 bp in the human Sesn2 promoter was critical for the Nrf2‐mediated response. Knockdown experiments with Sesn2 siRNA showed that Sesn2 is required for the Nrf2‐mediated cytoprotective activity against hydrogen peroxide. Our results suggest that the Nrf2‐ARE pathway is critical for Sesn2 gene expression and might protect against oxidative stress.

This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. R13‐2008‐010‐00000‐0)
Protein Interactomes of Mendelian Long QT Syndrome Genes are Associated to QT Interval Variation in the General Population and Augment Genome‐Wide Association Data
Authors: Lundby Alicia , Rossin Elizabeth J. , Steffensen Annette B. , Lage Kasper , Olsen Jesper V.
Published: June 2021

Mendelian long QT syndrome (LQTS) is caused by severe mutations in genes important for cardiac ion channel function, and genome‐wide association studies (GWAS) have identified common variant loci associated with QT interval variation in the general population. We hypothesized that genes contributing to QT interval variation encode proteins that interact with ion channel complexes causal of LQTS. By label‐free quantitative proteomics we resolved cardiac protein interaction networks (interactomes) of proteins encoded by LQTS genes. The interactomes establish functional links to genes in genome wide significant loci, suggesting that these genes are responsible for the association signals. Excluding genes from the established QT interval variation loci, the genes represented in the interactomes remain strongly enriched for genetic associations. We thus identified single nucleotide polymorphisms for replication and genotyping in independent samples confirmed our finding and led to identification of two novel genome‐wide significant loci. Combining tissue‐specific high‐resolution proteomics with GWAS datasets, we show that variants causal of QT interval variation are spread across genes represented in the protein interactomes of Mendelian LQTS genes. Our approach furthermore illustrates a strategy for augmenting GWAS data using quantitative interaction proteomics.
Estrogen‐dependent regulation of endothelial connexin 43
Authors: Glinskii Olga V , Zhang Jennifer J , Huxley Virginia H , Glinsky Vladislav V
Published: June 2021

Within vascular tissue, communication between endothelial cells (EC) plays central role in physiological settings and in pathogenesis of numerous diseases. Regulation of the connexin 43 (Cx43) turnover is one of the mechanisms controlling levels of intercellular communication occurring via gap junction channels and allowing intercellular transfer of ions and molecules less than 1 kDa such as secondary messengers and small metabolites. Here we show that short (2 h) dose‐dependent treatment of brain microvascular EC bEnd3 with estrogen (E2) did not change the expression levels of Cx43. However, 24 h of treatment induced dose‐dependent decrease in Cx43 expression. E2 treatment also induced dose‐dependent increase in Cx43 phosphorylation on Ser 368 in bEnd3 cells, and this effect was not abolished by estrogen receptor (ER) inhibitor ICI182780, suggesting that Cx43 phosphorylation is ER independent. In addition, E2 mediated dose‐dependent changes in Cx43 cellular localization. Taken together, these results demonstrate that E2 alters environmental cues that critically regulate EC heterogeneity and influence EC phenotype. This work was supported in parts by the AHA SDG 0830287N (OVG) and Award Number 1I01BX000609 from BLR&D Service of the VA Office of R&D (VVG).
New assay for measuring valproic acid in plasma by conjugation to hydrazides
Authors: Darlington Daniel N. , Gonzales Mary D
Published: June 2021

Valproic acid (VA) is an anticonvulsant used to treat mania, schizophrenia, bipolar disorders, panic states and post‐traumatic stress disorder. Recently, VA has been shown to improve survival to hemorrhagic and septic shock in rats and pigs. However, the measurement of VA in plasma is difficult by standard chromatography and spectroscopy. We have developed a method that extracts VA and an internal standard (2‐phenyl butyric acid) from 10μl plasma after addition of 20μl 1M NaCl, 100μl ethyl acetate and 400μl acetonitrile. Sample is vortexed, centrifuged, and the supernatant removed and dried, and conjugated to 7‐diethylaminocoumarin‐3‐carboxylic acid, hydrazide (DCCH, Invitrogen). The conjugate is measured by Fluorometry (2020 Jasco) with λex=415nm, λem=465nm after separation by Reverse Phase High Pressure Liquid Chromatography (Beckman Coulter). A 2‐parameter exponential non‐ linear regression, y‐a(1‐bx), was fitted (r2=0.991) to the standard curve (0.033 to 10mg/ml VA, or 12.5μg to 41ng/injection). Coefficient of Variation was 3.9 and 1.8% for high and low pools. This method can be used to measure the reported dosage range of VA in human or animal plasma. Supported by the ARMY MRMC.
Oxidative stress and unfolded protein response (UPR) in advanced aortic disease from Marfan Syndrome (MFS) transgenic mice
Authors: Guido Maria Carolina , Salemi Vera , Takimura Celso , Debbas Victor , Pereira Lygia da Veiga , Laurindo Francisco Rafafel Martins
Published: June 2021

We investigated the occurrence and mechanisms of oxidative stress in aortae from MFS mouse model (mgÄFBN1). Aortic dilation, elastic fiber rupture and collagen deposition markedly increased from 3–6 months(mo). ROS production (by 3 methods) increased in aortae from 6‐mo, but not 1 or 3‐mo‐old MFS mice, mainly at crossa. Nox4 mRNA increased after 1mo, while Nox2 only at 6mo. To assess oxidative stress mechanisms, we investigated UPR markers in MFS aortae. Expression of KDEL chaperones increased at 6mo, but not 1 or 3mo. Protein disulfide isomerase(PDI), which associates with and regulates Nox(es), equally increased. Also, PDI expression topography correlated with ROS production sites (DMPO adduct antibody), mainly at adventitia in crossa and media in abdominal aorta. Adventitial PDI switch in crossa correlated with diffuse collagen accumulation. Moreover, yeast‐two‐hybrid screening disclosed PDI interaction with fibrillin‐1(FBN1); in cultured fibroblasts, such association was mainly intra, but not extracellular. FBN1 immunodetection decreased by~80% and co‐localized with PDI in MFS adventitia. PDI/UPR marker changes were not systemic and absent in kidney and liver. Losartan prevented aortic remodeling, but ROS production remained increased in aorta at 6mo. Thus, ROS production increases with MFS aortic disease progression, in close temporal and topographic correlation with PDI expression and UPR.
The Role of Beta Receptors in Olfactory Induced Learning: Morphological Changes in the Mitral Cells of Rat Olfactory Bulb after Propranolol and Isoproterenol Treatment
Authors: Jafari Fahim Haider , Khilji Muhammad S , Naveed Abdul Khaliq
Published: June 2021

Study Objectives

This study was designed to see the histological changes in the olfactory bulb after the administration of propranolol and isoproterenol.

Methods

Thirty Albino (Sprague‐Dawley) rats were divided into equal groups (n=10). Control group was treated with normal saline, experimental group I was treated with propranolol and experimental group II was treated with Isoproterenol. The morphology of the mitral cells was studied after staining with H & E and silver staining under microscope.

Results

The histology of the olfactory bulb of the control group revealed the classic six laminae. Mitral cell layer consisted of a layer of cells, which were pyramidal in shape with prominent nucleoli.

Differences in the morphological characteristics of mitral cells were observed in experimental groups. Experimental group I showed mitral cells had a vacuolar appearance; however, the nucleus was rounded in appearance with prominent nucleoli. Experimental group II showed that mitral cells present in the mitral cell layer had indistinct boundaries. Nucleus was less basophilic with prominent nucleoli.

Conclusion

We conclude that by neuromodulating norepinephrine using propranolol and isoproterenol histological changes can be observed in the rat olfactory bulb especially in the morphology of the mitral cells.

Research Support: Research was supported by University of Health Sciences, Lahore, Pakistan.
Is zinc important for the cardioprotective effect of ischemic postconditioning?
Authors: Xu Zhelong , Zhou Juan
Published: June 2021

Zinc loss may contribute the pathogenesis of myocardial ischemia/reperfusion injury. We aimed to test if ischemic postconditioning protects the heart from reperfusion injury by preventing cellular zinc loss. Isolated rat hearts were subjected to 30 min regional ischemia followed by 2 h of reperfusion. Postconditioning was elicited by six cycles of 10 second reperfusion and 10 seconds ischemia. Intracellular zinc levels were measured with inductively coupled plasma optical spectroscopy (ICPOES). Upon reperfusion cytosolic zinc levels were dramatically decreased but this was prevented by postconditioning, indicating that postconditioning can prevent reperfusion‐induced zinc loss. In agreement with these observations, the cardioprotective effect of postconditioning was reversed by the zinc chelator N,N,N′,N′‐tetrakis‐(2‐pyridylmethyl) ethylenediamine (TPEN), and exogenous zinc given at reperfusion mimicked the effect of postconditioning, suggesting that postconditioning induces cardioprotection by preventing zinc loss. Finally, postconditioning enhanced phosphorylation of Akt, ERK, and GSK‐3β upon reperfusion, and these effects of postconditioning were inhibited by TPEN, indicating that zinc may mediate the cardioprotective effect of postconditioning by activating the RISK pathway. In conclusion, these data suggest that postconditioning may protect the heart by maintaining intracellular zinc homeostasis and activation of the RISK pathway may account for zinc‐mediated cardioprotection of postconditioning.
Reduced α2 Na+ pump expression in arterial smooth muscle elevates blood pressure: lessons from smooth muscle‐specific α2‐dominant negative (α2smDN) mice
Authors: Chen Ling , Song Hong , Kotlikoff Michael I. , Lee Jane C. , Li Meng , Blaustein Mordecai P.
Published: June 2021

To test the hypothesis that arterial smooth muscle (ASM) α2 Na+ pumps help regulate blood pressure (BP), we generated α2smDN mice, which express a smooth muscle (sm)‐specific, truncated, non‐functional, dominant negative (DN), flag‐tagged α2 peptide [α2(1–120)f] (Song et al., J Biol Chem 281:12929, 2006). α2(1–120)f expression was controlled by the sm myosin heavy chain promoter. α2 Na+ pump expression is reduced in ASM and urinary bladder, but not heart or brain, of α2smDN mice. DN heterozygotes (α2smDN/−, offspring from α2smDN x WT outbreeding) have slightly reduced α2 Na+ pump expression and normal mean BP (MBP, measured with telemetry). Nevertheless, the MBP increases with chronic 4% NaCl (7 ± 1 vs 0 ± 1 mm Hg; n = 6 each), and with 0.24 mg/kg/min angiotensin II (21 ± 2 vs 11 ± 1 mm Hg, n = 6 each) are significantly greater in α2smDN/− mice than in WT littermates. In contrast, DN ‘homozygotes’ (α2smDN/DN, offspring from α2smDN x α2smDN inbreeding) have greatly reduced α2 Na+ pump expression and high MBP [telemetry: 111±3 mm Hg (n=7) vs WT = 96±1 mm Hg (n=6), P<0.01; intra‐femoral artery catheter under anesthesia: 104±5 mm Hg (n=15) vs WT = 82±3 mm Hg (n=6), P<0.01]. Thus, ASM α2 Na+ pump expression correlates inversely, and BP correlates directly, with α2(1–120)f gene dose.

Conclusion

ASM α2 Na+ pumps help control BP and the sensitivity of BP to salt and to vasoconstrictors. Supported by NHLBI.
Nonlinear parameters of heart rate variability during mental stress
Authors: Dimitriev Dmitry , Saperova Elena
Published: June 2021

Introduction

This study investigates the variations of nonlinear parameters of heart rate variability (HRV) due to an arithmetic stress test.

Material and methods

We tested 83 healthy students with a mean age of 21.12±0.21 years. The students were examining in the following sequence: recording of HRV at rest and during mental arithmetic. HRV was assessed by applying Poincare plot analysis (SD1, SD2, SD2/SD1, SS) to the R‐R interval series on a 10‐min ECG. The traditional (GI) and redefined Guzik's index (GIp) (Karmakar, 2010) was calculated for each subjects in each phase of the experiments.

Results and discussion

SD1 during mental arithmetic was significantly smaller (24.51±1.77) than that at rest (34.57±2.05; p<0.0001). SD1/SD2 ratio during mental stress was significantly lower than that at rest (0.331±0.013 vs 0.469±0.017; p<0.00001). SS was significantly lower during the test than that at rest (2756.13±313.97 vs 3822.66±370.57; p=0.008). There was no significant difference in the GI during mental stress (0.495±0.008) than that at rest (0.485±0.005; p>0.05). But after redefinition, we have found pronounced changes in this parameter with a maximum at rest day and minimum during arithmetic test (0.446±0.024 vs 0.398±0.018; p<0.05).

This findings suggest that nonlinear HRV analysis could be effective in automatically detecting functional status during mental stress.
Chronic sound stress elevates systolic blood pressure and gene expression of pituitary adenylate cyclase activating polypeptide (PACAP) and its receptor PAC1 in the PVN
Authors: Spurgin Kurt Andrew , Kaprielian Antony , Valdez Matt , Valdez Joe , Martinez Bethann Marie , Curras-Collazo Margarita
Published: June 2021

Research suggests that chronic stress and subsequent activation of the sympathetic nervous system contribute to the development of hypertension. Recent work suggests that pituitary adenylate cyclase‐activating polypeptide (PACAP)‐dependent mechanisms sustain sympathoadrenal responses to prolonged but not acute stress. Group‐housed rats were placed in a sound chamber 25 cm from speakers randomly emitting four pure tones (5, 11, 15, and 19 kHz) for 30 min. After daily sound stress for 4 weeks rats were lightly anesthetized (isoflurane: 4% induction; 1.75% maintenance) and blood pressure was measured by tail‐cuff plethysmography. Stressed rats did not exhibit changes in diastolic blood pressure but showed a significant increase in systolic blood pressure compared to age‐matched controls: 118.3 ± 1.6 and 96.9 ± 1.7 mmHg, respectively (n=8; p<0.05) suggesting increased sympathetic drive originating in central autonomic and/or pre‐autonomic nuclei. Following blood pressure measurement, brains were removed and tissue from the hypothalamic paraventricular nucleus (PVN) was subjected to qPCR. In pilot experiments we observed elevated gene expression for PACAP (normalized to ribosomal protein L30) in stressed animals (9.10 ± 4.73) versus controls (1.12 ± 0.26; n=8; p=0.1). A similar elevation was observed for the PACAP receptor PAC1R: 5.27 ± 2.47 and 1.12 ± 0.21, respectively (n=8, p=0.1).
Toll‐like receptors 2 and 4 are involved in cardiomyocyte hypertrophy in vitro
Authors: Gailser-Silva Fernanda , Carneiro-Ramos Marcela Sorelli
Published: June 2021

Objective

The innate immune response can exerts innumerous effects in vast number of tissues and organs. It's well established that cytokines and Toll‐like receptors (TLRs) participate of cardiac trophism modulation. The objective of this study is to evaluate the effect of agonists of TLR 2 and 4 modulating cardiomyocyte trophism in vitro.

Methods

Primary cultures of cardiomyocytes were prepared from neonatal ventricles of Wistar rats by collagenase/pancreatin method. Cardiomyocytes primary cultures were treated for 24hs with LPS (1μM – TLR4 agonist), Zymozan (Zym 10μM – TLR2 agonist) or LPS + Zym combined. ANF and Alpha‐actin mRNA levels were used as cardiac hypertrophy markers. Gene expression was evaluated by Real‐time PCR. The results were expressed as mean±SD and P<0.05.

Results

The LPS and Zym promoted an increase on ANF and alpha‐actin expression (p<0.05). The administration of LPS combined with Zym did not potentiate this effect. The MyD88 mRNA levels, an adaptor protein of TLR cascade, were increase after both LPS (30%) and Zym (50%) treatment compared to control cells. These results suggest an important role for TLRs in cardiomyocyte hypertrophy in vitro.

Financial Support: FAPESP.
Elevation of fetoplacental vascular resistance by chronic hypoxia in isolated blood perfused rat placenta
Authors: Kafka Petr , Vajnerova Olga , Hampl Vaclav , Herget Jan
Published: June 2021

In isolated rat placenta chronic hypoxic exposure causes a small increase in fetoplacental vascular resistance during high perfusion flows of physiological salt perfusate. No elevation was found in basal perfusion flows. We expected that perfusion with more viscous fluid will increase differences between normoxia and chronic hypoxia. Rats were exposed to normobaric hypoxia (10% O2) during the last week of the 3‐week pregnancy. One day before expected delivery the animals were anaesthetized with Thiopental (50 mg/kg i.p.). We dually perfused an isolated rat placenta with homologous blood at a constant flow rate 1 ml/min. We found significantly elevated fetoplacental basal vascular resistance to flow in chronically hypoxic rats. Vascular resistance was measured as a relationship of vascular perfusion pressure to continually rising flow (P/Q). The slope of the P/Q line was higher in the hypoxic than the normoxic rats. The pressure axis intercepts with P/Q line did not differ. We conclude that chronic hypoxia elevates resistance of fetoplacental vessels in isolated blood perfused rat placenta.

Supported by 2nd Faculty of Medicine, Charles University and Czech Science Foundation 305/08/0108.
Reactive Oxygen Species Scavenger, Tempol, Decreases Vasoconstriction after Acute Pulmonary Embolism
Authors: Mizera Roman , Hodyc Daniel , Hampl Vaclav , Herget Jan
Published: June 2021

Pulmonary vasoconstriction is an important mechanism contributing to the development of pulmonary hypertension after pulmonary embolism (PE) probably due to increased production of reactive oxygen species (ROS). Our aim was to investigate the change of NO production and to determine the effect of ROS scavenger, Tempol, on pulmonary hypertension after PE.

Using the model of in vivo pulmonary embolism and isolated, perfused rat lungs we measured the perfusion pressure and its changes after sodium nitroprusside administration and after NO synthase inhibition by L‐NAME.

We used 6 groups of Wistar male rats:

NN: control group + sodium nitroprusside (SN) (n=5), EN: PE + SN (n=5), TN: Tempol + PE + SN (n=5), NL: control group + L‐Name (n=5), EL: PE + L‐Name (n=5), TL: Tempol + PE + L‐name (n=5). Tempol was administered before PE.

PE caused higher pulmonary pressure. Tempol‐treated rats (TN, TL) had significantly lower perfusion pressure than the animals without Tempol (EN, EL). The decrease of perfusion pressure after application of SN was higher in EN than in NN. Administration of L‐Name after PE (EL) increased the pressure more than in the controls (NL). PE also caused increased NOx production.

ROS‐induced vasoconstriction contributed to the pulmonary hypertension after PE. Also, PE caused increased production of NO and NOx. Tempol decreased pulmonary hypertension after PE by inhibiting the free radicals production.
Differential impact of three β‐adrenergic receptor antagonists on the vascular dysfunction of atherosclerotic mice
Authors: Luo Xiaoyan , Bolduc Virginie , Le Gouill Christian , Bouvier Michel , Thorin Eric
Published: June 2021

β‐adrenergic receptor (β‐AR) antagonists reduce heart rate (HR) but differentially alter intracellular pathways in vitro through bias signaling. To assess the functional consequences of these diverse pharmacological properties, we treated for 3 months (n=7 per group) or not (n=6) 4‐mo atherosclerotic LDLr−/−:hApoB+/+ (ATX) male mice with carvedilol (CARV, β‐AR antagonist and partial α1‐AR antagonist; 25mg/kg/d), nebivolol (NEBI, β1‐AR antagonist with antioxidant property; 5mg/kg/d) or propranolol (PROP, β‐AR antagonist; 100mg/kg/d). Reduction (~15%) of HR at 2 weeks was similar. In pressurized cerebral arteries, CARV prevented endothelial dysfunction by improving endothelium‐dependent flow‐mediated dilations (FMD) by 57% (P<0.05) and normalized wall stiffness (P<0.05). In contrast, PROP and NEBI worsened FMD by 54% and 41% (P<0.05) and did not correct stiffness. In addition, stiffening of carotid arteries was only limited by NEBI (P<0.05), while acetylcholine‐induced endothelium‐dependent relaxation was similar in all groups. We conclude that chronic in vivo β‐AR antagonisms lead to differential vascular responses that could lead to long‐term differential clinical outcomes. CIHR MOP89733.
Aging impairs flow‐induced dilation in skeletal muscle feed arteries: role of Akt‐dependent phosphorylation of eNOS
Authors: Luttrell Meredith , Seawright John , Trott Daniel , Woodman Christopher
Published: June 2021

We tested the hypothesis that impaired nitric oxide (NO)‐mediated, endothelium‐dependent dilation in aged soleus muscle feed arteries (SFA) is due to an age‐related decrement in PI3K/Akt‐dependent phosphorylation of endothelial NO synthase (eNOS) on serine 1177 (p‐eNOSser1177). SFA from young and old Fischer 344 rats were cannulated for examination of flow‐induced dilation (FID). To determine how aging affected FID, dilator responses were assessed in the absence and presence of pharmacological inhibitors of NOS (L‐NNA), PI3K (LY‐294002), or Akt (AktI). Results indicate that FID was blunted in old SFA, while dilation to sodium nitroprusside was not compromised. Age‐group differences in FID were abolished in the presence of L‐NNA, LY‐294002, or AktI. In a separate experiment, FID was assessed in SFA from young and old rats. After FID assessment, SFA were then removed from the pipettes, snap frozen, and immunoblot analysis was used to assess p‐AktSer473, p‐eNOSser1177, total Akt and total eNOS protein content. FID was blunted in old SFA; however, the p‐AktSer473/Akt and p‐eNOSser1177/eNOS ratios were similar in young and old SFA. Collectively, these results indicate that NO‐mediated dilation is impaired in old SFA, but is not due to reduced PI3K/Akt‐dependent phosphorylation of eNOS.

Research supported by AHA 0765043 and AHA 4150031 (CRW), and Sydney and J. L. Huffines Institute of Sports Medicine Graduate Student Research Grants (MJL, JWS, and DWT).
Changes in LTCC protein expression and channel function in A7r5 cells by a truncated β subunit
Authors: Telemaque Sabine , Fausther Michel , Flynn Shawn E , Liu Meeiyueh , Singleton Terrie , Dranoff Jonathan A , Marsh James D
Published: June 2021

Cytosolic Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC) is primarily regulated by Ca2+ entry through L‐type Ca2+ channels (LTCC), via the pore‐forming α1C subunit. The intracellular β2a subunit chaperone interacts with the α1C subunit to regulate membrane localization and channel function. The aim of this study was to determine whether a truncated β2a subunit expressing the N‐terminus portion of the β subunit (N‐BID) can regulate expression of other LTCC subunits and affect channel function in VSMC. Adenoviruses were used to overexpress the truncated β subunit (N‐BID) or the native β2a subunit (Full‐β2a) fused to GFP in A7r5 cells, a rat aortic smooth muscle cell line. After 48 hr, cells were visualized under fluorescent microscopy before whole cell lysates were collected to determine total protein levels. Immunoblot analysis showed that total protein levels of the α1C subunit were significantly elevated in A7r5 cells expressing N‐BID when compared to GFP (adenovirus control). Similarly, total protein levels of the α2 subunit, a membrane‐bound subunit of the LTCC, were also increased in N‐BID‐cells when compared to GFP. In contrast, overexpression of the Full‐β2a subunit did not alter total protein expression of these membrane‐bound subunits. Importantly, neither constructs affected endogenous expression of the β2 subunit. Agonist‐induced increase in [Ca2+]i was determined by confocal microscopy and appeared to be attenuated in N‐BID cells. Our results suggest that N‐BID can act as a decoy to affect expression, processing or degradation of the LTCC subunits while decreasing LTCC function in vitro. It could potentially be used to modulate L‐type Ca2+ channel function and ultimately regulate vascular tone. This study was partially funded by a Grant in Aid from the AHA.
RENAL ISCHEMIA/REPERFUSION INDUCED CARDIAC HYPERTROPHY IN MICE: CONTRIBUTION OF CARDIAC RENIN ANGIOTENSIN SYSTEM
Authors: Abrahao Mariana Vieira , Carneiro-Ramos Marcela Sorelli
Published: June 2021

Objectives

This study aims to characterize the role of cardiac RAS in models of cardiac hypertrophy induced by ischemic renal injury in mice.

Methods

C57bl/6J mice were subjected to unilateral 60 min kidney ischemia, followed by reperfusion of 12, 15 and 20 days. The animals were randomized and equally divided into three groups: (i) untreated, (ii) treated with Losartan (Los ‐ 10mg/kg/day) for 5 days before ischemia protocol and (iii) treated with Los for 5 days after, with respective sham controls for all groups. Levels of urea were measured to confirm renal failure and gene expression were analyzed by real time PCR.

Results

The heart weight/body weight or tibia length ratios and mRNA ANF were increased on 12 and 15 days mice untreated with Los. Pre and post treatment with Los were able to prevent and reverse, respectively, the increase on mRNA ANF levels induced by renal/ischemia protocol only at the 15 days time point (p<0.01). Cardiac mRNA AT1a levels were decreased after post treatment with Los after 15 days of renal ischemia when compared to 15 days untreated mice.

Conclusion

The renal ischemia/reperfusion model was able to induce cardiac hypertrophy that was prevented and reversed with Los administration. Moreover, cardiac mRNA AT1 levels are decreased in 15 days mice post treated with Los, suggesting a local modulation of cardiac RAS in this cardiac hypertrophy model. Financial Support: FAPESP, CNPq and UFABC.
Creating of GTP Cyclohydrolase‐1 Knock in Mouse
Authors: Chen Wei , Li Li , Blinder Yelena , Zhang Helen Heju , Goldstein Anna , Harrison David
Published: June 2021

Tetrahydrobiopterin (BH4) is an essential cofactor for the nitric oxide synthase enzymes. We have shown that laminar shear stress dramatically increases BH4 levels in human aortic endothelial cells. Oscillatory shear only modestly affected BH4 levels in the cells. We also demonstrated that laminar, but not oscillatory shear stress, stimulates phosphorylation of GTP cyclohydrolase‐1 (GTPCH‐1) the rate limiting enzyme for the BH4 biosynthesis, at serine 81. To study this further, we have attempted to produce two mice in which serine 72 (the equivalent of S81 in humans) was replaced by aspartic acid or alanine, to respectively mimic or prevent phosphorylation, using knock‐in technology. The alanine substitution (GCHS72A) proved to be lethal in that no viable agouti pups were obtained from chimeric founders and analysis of spermatic plugs showed no germ‐line transmission of the knock‐in transgene. In contrast, we have successfully obtained the aspartic acid substitution (GCHS72D) heterozygotic mice, and found that they have increased aortic BH4 levels than WT mice when aortas are maintained in no‐flow conditions for 24 hours (224±10 vs. 134 ± 2 pmols/mg protein). These experiments show that GTPCH‐1 phosphorylation is essential for survival and plays a critical role in modulating GTPCH‐1 function in vivo. Supported by NIH P01 HL058000.
Cellular Mechanisms of Cardiac Dysfunction in Volume Overload Induced Heart Failure
Authors: Guggilam Anuradha , Galantowicz Maarten L , West Aaron T , Zhang Xiaojin , Lucchesi Pamela A
Published: June 2021

Left ventricular (LV) volume overload (VO) in valve regurgitation patients can be only treated by surgical intervention. The cellular mechanisms involved in cardiac dysfunction are not well known. The aim of this study was to understand the mechanical properties of myocytes isolated from VO‐induced heart failure and the possible mechanisms of dysfunction. Male Sprague‐Dawley rats underwent aorta‐caval fistula (ACF) surgery to induce VO heart failure or sham procedure. The rats with ACF went into failure at ~21wks post‐surgery. LV myocytes were isolated. Myocyte cell‐shortening fraction (CS) and Ca[i]2+ transients were measured at different frequency field stimulations to study cellular mechanical properties and molecular components of excitation‐contraction (EC) coupling. SERCA, pRYR and pPLB were reduced in ACF hearts while NCX was unchanged. Regardless of the stimulation frequency, CS of ACF myocytes was significantly lower than those of sham myocytes (~45% reduction). Interestingly, rapid stimulation at higher frequencies did not alter the Ca2+ area of relengthening in ACF myocytes, suggesting the activity of RYR and SERCA are intact despite reduction in protein levels. These data suggest that EC coupling is not a causative factor for cardiac dysfunction seen in VO and may possibly be due to reduced Ca2+ sensitivity or decreased force generation in elongated myocytes of ACF rats.
N‐terminal tyrosine phosphorylation of caveolin‐2 negates anti‐proliferative effect of transforming growth factor beta in endothelial cells
Authors: Sowa Grzegorz , Abel Britain , Willoughby Cara , Xie Leike
Published: June 2021

Here, we show that N‐terminal tyrosine phosphorylation of caveolin‐2 (Cav‐2) negatively regulates anti‐proliferative function of transforming growth factor beta (TGF‐beta) in endothelial cells. In contrast to wild type Cav‐2, retroviral expression of Y19/27F Cav‐2 in Cav‐2 knockout endothelial cells did not affect anti‐proliferative function of TGF‐beta compared to empty vector. Conversely, although less effective than wild type, re‐expression of S23/36A Cav‐2 reduced the anti‐proliferative effect of TGF‐beta. This differential effect of S23/36A Cav‐2 and Y19/27F Cav‐2 on anti‐proliferative function of TGF‐beta was documented using MTT‐ and cell count‐based proliferation assays, and correlated with TGF‐beta‐induced Smad3 phosphorylation and activation of plasminogen activator inhibitor‐1 at mRNA and protein levels. Taken together, our data suggests that phosphorylation of Cav‐2 at N‐terminal tyrosine residues 19 and/or 27 is more critical than at serine residues 23 and 36 in counteracting anti‐proliferative effect and signaling of TGF‐beta in endothelial cells. This project was supported by NIH (5R01HL081860 to GS).
KDEL Peptide Gold Nanostructures: A Promising Platform for Drug Delivery
Authors: Wang Guankui , Song Yuan , Norton Ann , Hill Rodney A.
Published: June 2021

The objective was to elucidate the mechanisms of cellular uptake and trafficking of peptide gold nanostructures (Au‐Pep). Peptide A (PepA) contains the carboxy‐terminal sequence Lys‐Asp‐Glu‐Leu (KDEL). Peptide B (PepB) is a random sequence peptide with no known receptor‐binding activity. Au‐Pep were incubated with Sol8 cells for 15 mins in DMEM without serum, followed by a 1 h chase period in DMEM plus serum. Cellular compartments including clathrin‐coated vesicles, Golgi apparatus and endoplasmic reticulum (ER) were labeled for peptide colocalization studies. In a subsequent study, cells were transfected to overexpress KDEL receptor (KDELR), followed by Au‐Pep incubation. Cell imaging was performed using confocal fluorescence microscopy. The results showed that free PepA uptake was significantly greater in KDELR overexpressing cells, whereas there was no difference in PepB uptake, suggesting that free PepA was internalized via KDELR‐mediated endocytosis. However, Au‐Pep uptake was similar in KDELR overexpressing cells and controls, suggesting that KDELR‐mediated endocytosis was not predominant for Au‐Pep internalization. Colocalization data showed that for all peptide treatments, most localized in the ER. This may suggest that these peptides are trafficked via a retrograde coat protein complex I mediated transport pathway. In conclusion, Au‐PepA may provide a nanoplatform for drug delivery. This work was supported by the University of Idaho Blue Ribbon BANTech Initiative.