bluuebunny/crossref_metadata_2025 huggingface dataset:
MicroRNA‐21 Antagonism Attenuates Inflammation and Endothelial‐Mesenchymal Transition in Aged Kidney Ischemia Reperfusion Injury
Authors: Pushpakumar Sathnur , Weber Gregory , Majumder Suravi , Sen Utpal
Published: June 2021

Ischemia‐reperfusion (IR) is one of the most common causes of acute kidney injury (AKI) in the aging population. MicroRNAs (miRs) are endogenous non‐coding RNA which have the ability to target the expression of multiple genes involved in the inflammatory and repair processes, thus influencing the course of disease. Although elevation of miR‐21 following renal IR has been observed in several studies involving young animals including humans, its role in the aged kidney remains unknown. The purpose of this study was to determine whether miR‐21 contributes directly to persistent inflammation in the aged kidney following IR injury and whether its inhibition attenuates inflammation to promote repair. Because profibrotic cues originating from inflammatory cells are known to favor endothelial‐mesenchymal transition (endoMT) by activation of myofibroblasts, we also investigated whether miR‐21 inhibition reduces endoMT. Wild type (WT, C57BL/6J) mice aged 12–14 wk and 75–78 wk were used in this study. After treatment without or with Locked Nucleic Acid Anti‐miR‐21 (20 mg/kg b.w.), mice underwent bilateral renal ischemia (27 min.) followed by reperfusion for 7 days. Aged mice exhibited severe renal IR injury and anti‐miR‐21 treatment improved glomerular filtration rate, renal blood flow and decreased renal resistive index. Anti‐miR‐21 treated mice demonstrated predominance of M2 macrophages associated with increased expression of arginase‐1 and IL‐4 and reduction of inflammatory cytokines. Further, anti‐miR‐21 reduced TGF‐β, matrix metalloproteinase‐9 and collagen expression in the aged kidney. Double labeling for endothelial marker, Tie2, and fibroblast markers, FSP‐1/α‐SMA showed significant reduction in their coexpression suggesting reduction in endoMT. Our findings suggest that miR‐21 inhibition reduces macrophage mediated inflammation and endoMT to promote reparative process following IR injury in the aged kidney.

Support or Funding Information

AHA grant to SP: 15SDG25840013NIH grant to US: DK104653

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Twist2 is a Novel Pro‐Fibrotic Mediator in the Aging Kidney
Authors: Parrish Alan R. , Grunz‐Borgmann Elizabeth
Published: June 2021

Twist2 (also known as Dermo‐1) is a basic helix‐loop‐helix transcription factor that binds E‐boxes (CAnnTG) to regulate gene expression. We identified Twist2 as pro‐fibrogenic in a study testing the effects of a soy protein diet on renal dysfunction in aging Fischer 344 rats; the soy diet decreased Twist 2 expression and markedly attenuated fibrosis and loss of renal function. Furthermore Twist2, but not Twist1, expression is significantly increased in aging rats and Twist2 overexpression was inhibited by lifelong caloric restriction, which prevents renal fibrosis and dysfunction. Twist2 expression is also increased in several other models of renal fibrosis, including the mouse unilateral ureteral obstruction (UUO) model of fibrosis, and Zucker obese rats. Twist2 is predominantly expressed in the distal tubular epithelium of both mice and rats. In an effort to delineate the mechanism of Twist2 overexpression, we have shown that hypoxia increases Twist2 in mouse distal convoluted tubule cells (DCT209 cells). In addition, RNAseq analysis of a cell line constitutively overexpressing Twist2 (DCT209‐Twist2) identified overexpression of CCL2, a pro‐fibrotic mediator that has been shown to recruit fibrocytes and promote extracellular matrix production in fibroblasts, and osteopontin. Based on these data, we hypothesize that hypoxia induces Twist2 which leads to fibrosis and renal dysfunction.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
THE REGULATION OF SKELETAL MUSCLE FUNCTION BY MUSCLE GP130 SIGNALING
Authors: VanderVeen Brandon , Fix Dennis , Counts Brittany , Hardee Justin , Carson James
Published: June 2021

Chronic inflammation is integral to the pathology of several chronic diseases and has been linked to muscle wasting, weakness, and fatigue resulting in reduced life quality and survival. Interleukin‐6 (IL‐6) signaling through the membrane gp130 receptor has been implicated as a regulator of skeletal muscle mass in both atrophic and hypertrophic conditions through the regulation of STAT3, MAPK, and AKT signaling. Additionally, elevated plasma IL‐6 and associative increases in IL‐6/gp130 signaling have been demonstrated to contribute to disrupted metabolic homeostasis and increased perceived fatigue during chronic disease; however, the regulation of skeletal muscle function by IL‐6/gp130 signaling has not been well described. The purpose of this study was to determine the regulation of skeletal muscle strength and fatigability by gp130 signaling. To test the role of muscle gp130 signaling on muscle strength and fatigability, male gp130 floxed (WT) mice were bred with Cre‐expressing mice driven by myosin light chain to obtain a skeletal muscle specific gp130 knockout (gp130 KO). Both WT and gp130 KO mice were electroporated with an IL‐6 plasmid into the quad to increase circulating IL‐6 for 2 weeks. Littermate controls were electroporated with an empty vector. The functional properties of the Tibialis Anterior (TA) were assessed in situ. Neither gp130 nor elevated plasma IL‐6 (>40pg/mL) had an effect on body or muscle weight. IL‐6 overexpression increased (215%) spleen weight regardless of genotype. While specific force (kN/m2) was reduced (9.8%) in gp130 KO compared to WT, elevated plasma IL‐6 had no effect on TA maximal force across all groups. Muscle fatigability was not different between WT and gp130 KO. Interestingly, elevated plasma IL‐6 increased (15.2%) muscle fatigability in the WT, but had no effect on muscle fatigability in the gp130 KO. Together these results demonstrate that increased levels of IL‐6 accelerates skeletal muscle fatigability through muscle gp130 signaling independent of changes in body weight or skeletal muscle mass.

Support or Funding Information

This work was supported by National Institute of Health (NIH) #NCI‐R01CA121249 (JAC)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Soleus and gastrocnemius tenotomy causes fiber type specific muscle atrophy in mouse tibialis anterior
Authors: Bott Kirsten N. , Watson Colton J.F. , Leveille Cameron F. , MacNeil Adam J. , Peters Sandra J. , LeBlanc Paul J. , Fajardo Val Andrew
Published: June 2021

Examining the molecular mechanisms regulating muscle mass is critical in determining potential therapeutic strategies for preventing muscle loss in a variety of conditions. We have previously utilized a simultaneous muscle overload/unload model, whereby tenotomy of soleus and gastrocnemius tendons mechanically overloads plantaris leading to hypertrophy while unloading soleus leading to atrophy after two weeks. In this model, the foot of the tenotomized leg appears to be held in dorsiflexion, likely due to the loss of soleus and gastrocnemius muscle function, which synergistically act towards plantar flexion. Previous research has shown that dorsiflexion immobilization results in significant muscle atrophy of tibialis anterior (TA) due to an increase in inflammation and polyubiquitylation. Therefore, the present study examined whether TA muscles from the tenotimized leg would also display significant muscle atrophy, and whether increased inflammation and polyubiquitylation would contribute to the muscle loss. We also examined glycogen synthase kinase 3 (GSK3) activation, given this enzyme's role in promoting polyubiquitylation and muscle atrophy. Ten male C57BL/6N mice (28.1 ± 0.8g) were subjected to soleus and gastrocnemius tenotomy (TN) in one leg, while the other leg was subjected to a sham surgery to serve as an internal control (CON). Two‐weeks post‐surgery the mice were euthanized and the TA muscles were collected. On average, TA muscles in the TN leg were smaller compared to the CON leg (CON, 60.0 +/− 2.0 mg vs TNY 52.8 +/− 2.2 mg, p<0.01), suggesting muscle atrophy was occurring in the TN leg. Using immunofluorescent fiber typing, it was found that this was due to a selective reduction in myofiber cross‐sectional area of type IIB (CON 4091± 275 μm2 vs. TN 3599± 193 μm2, p<0.01) and type IIX‐IIB (CON 3281± 243 μm2 vs. TN 2933± 262 μm2, p<0.05), without any significant reductions in total number of myofibers or fiber type distribution. H&E staining showed some centralized nuclei with no signs of mononuclear cell infiltration in the TN TA. Correspondingly, no significant changes in mRNA expression of IL‐6 and TNFa inflammatory cytokines were observed in TN TA. However, we did observe a 1.9‐fold (p<0.05) increase in polyubiquitylated proteins and higher GSK3 activation as revealed by a 30% reduction in phosphorylated GSK3 (relative to total GSK3; p<0.05) in the TN TA compared to CON. In conclusion, TA muscle undergoes a selective atrophy of fast‐glycolytic fibers in response to soleus and gastrocnemius TN, and this may be due to GSK3 activation and polyubiquitylation. Future studies should investigate whether GSK3 represents a viable therapeutic target in this model.

Support or Funding Information

NSERC

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Precipitation patterns and hydration status of a unique insular snake population.
Authors: Sandfoss Mark , Lillywhite Harvey
Published: June 2021

Water is essential to survival, and thus invasion of habitats having limitations of water necessitates adaptations to maintain water balance. Climate change is expected to alter precipitation patterns worldwide. A unique population of Florida cottonmouth, Agkistrodon conanti, inhabits Seahorse Key (SHK) near coastal Levy County, Florida where they are entirely terrestrial, scavenge fish carrion, and are without permanent sources of fresh water. Insofar as cottonmouths are generally associated with freshwater habitats, this insular population of snakes is likely to exhibit adaptations for living with limited resources, especially water. Our objective is to measure the hydration status in free‐ranging snakes to better understand the possible stresses related to dehydration. We hypothesize that hydration status of cottonmouths on SHK will follow patterns of precipitation. We tested dehydration and drinking behavior of cottonmouth snakes inhabiting SHK from 2001–2017. Snakes were captured and tested for thirst immediately following capture. They were provided access to fresh water for at least 12 hours while we noted drinking and weighed snakes before and after. Mass gains reflected drinking and therefore hydration status. These data were compared to satellite data for date of last rainfall event (peak >2.5 mm/hr) preceding capture (NASA TRMM & GPM). The percentage of captured cottonmouths found to drink was found to increase with an increase in the number of days since a rainfall event. Results support the prediction that hydration status of cottonmouths on SHK correlates with rainfall patterns, suggesting that snakes rely on periodic rainfall to remain hydrated. Insular cottonmouth populations are of conservation concern as annual precipitation rates have declined over the past century and may continue to decline due to climate change.

Support or Funding Information

U.S. Fish & Wildlife Service, Sigma Xi, Chicago Herpetological Society, & SSAR

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial ROS activates PKC alpha translocation to the membrane of vagal sensory neurons.
Authors: Bahia Parmvir K. , Barannikov Ivan , Taylor‐Clark Thomas E.
Published: June 2021

Hyperexcitability of airway sensory nerves can trigger disease‐associated reflexes resulting in cough and dyspnea. We have observed that mitochondrial ROS production increases hyperexcitability in nociceptive bronchopulmonary C‐fibers. We also found that this increased excitability can be blocked by the PKC inhibitor bisindolylmaleimide (BIM) I, but not the inactive PKC analog BIM V. In previous work we have shown, using a pan‐PKC antibody, that mitochondrial ROS production via Antimycin A (AntA; mitochondrial complex III Qi site inhibitor) causes translocation to the cell membrane. However, there are several PKC isoforms expressed in nociceptive neurons. We wanted to determine which isoforms were present, their distribution in nociceptive cells and whether specific isoforms were activated by AntA. Studies looking at mRNA expression have highlighted PKC alpha, beta1, beta2, delta and epsilon as abundant in nociceptors so our studies have focused on these particular isoforms. Sensory neuron cell bodies were isolated from mouse vagal ganglia. To identify nociceptive cells, we used a mouse strain that co‐expresses a red fluorescent protein (tdTomato) with the Transient Receptor Potential V1 ion channel. Cells were treated for 2 min with: vehicle, 10 μM AntA or 300 nM PMA then immediately fixed. We performed immunocytochemistry to determine cellular location of different isoforms. Of the TRPV1 expressing nociceptive cells, more than 50% were positive for any given isoform of PKC. All isoforms translocated to the membrane in response to PMA treatment. However, only PKC alpha exhibited translocation to the membrane in response to AntA treatment. In the case of PKC delta, AntA appeared to cause translocation of this isoform to the nucleus. Our experiments suggest that nociceptor hyperexcitability, in response to ROS produced via mitochondrial dysfunction, may occur through the translocation and activation of the PKC alpha isoform.

Support or Funding Information

This work was supported by the National Heart Lung and Blood Institute in Bethesda, USA(R01‐HL119802)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of Salt Sensing Kinase, SGK1, in T cells abrogates Memory Cell Formation, Hypertension and End‐Organ Damage
Authors: Itani Hana A. , Pandey Arvind , Norlander Allison E. , Harrison David G.
Published: June 2021

Accumulating evidence indicates that high salt‐intake, aging and hypertension promote NaCl accumulation in tissues. Elevated NaCl has been shown to promote TH17 cell formation in an SGK1‐dependent fashion. We have previously shown that memory T cells play a major role in the genesis of hypertension. These long‐lived cells remain responsive to repeated hypertensive stimuli, such as repeated salt feeding, and can be mobilized to enter the kidney where they release cytokines that promote renal dysfunction. To examine mechanisms by which T cells sense salt and contribute to salt‐sensitivity, we tested the hypothesis that SGK1 in T cells is necessary for formation of memory T cells and their activation in salt‐sensitive hypertension. We produced mice with T cell specific deletion of SGK1, SGK1fl/fl × tgCD4cre mice and used SGK1fl/fl mice as controls. To impose repeated episodes of hypertension, we treated these mice with L‐NAME (0.5mg/ml) in drinking water for two weeks, allowed a two‐week normotensive interval and a then fed high salt (4% NaCl) for three weeks. L‐NAME followed by high salt increased memory T cells in the kidney, aorta and bone marrow of SGK1fl/fl control mice but not in SGK1fl/fl × tgCD4cre mice, as identified by the surface marker CD44hi. To assess markers of renal injury, we measured albumin in 24‐hour urine samples collected at the end of the L‐NAME/high salt. L‐NAME/high salt caused striking albuminuria in SGK1fl/fl mice and this was prevented in SGK1fl/fl × tgCD4cre mice. In additional studies, we found that loss of SGK1 in T cells abrogates renal and vascular inflammation and protects against hypertensive renal and vascular injury in the L‐NAME/high salt model. Thus, our data indicate that SGK1 in T cells promotes their development of salt sensitivity and mediates their ability to cause renal and vascular dysfunction in hypertension.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Colonic Indole And Hydrogen Sulfide, Gut Bacterial Metabolites, Affect Portal Blood Pressure in Healthy And Cirrhotic Rats
Authors: Ufnal Marcin , Huc Tomasz , Jurkowska Halina , Wróbel Maria , Jaworska Kinga , Onyszkiewicz Maksymilian
Published: June 2021

Liver cirrhosis is a leading cause of portal hypertension, and is often accompanied by systemic hypotension. Accumulating evidence suggests that gut bacteria may affect hemodynamics, however, the mechanisms are not clear. We investigated the effect of colon‐administered gut bacteria metabolites on portal blood pressure (PBP), portal blood flow (PBF) and systemic arterial blood pressure (ABP) in healthy (controls) and cirrhotic rats (CRH).

10‐week‐old, male, Sprague Dawley rats were maintained either on tap water (controls) or water solution of thioacetamide for 18 weeks to induce liver cirrhosis (CRH). PBP, PBF and ABP were measured in anaesthetized rats at baseline and after the intracolonic administration of either a vehicle, indole, or Na2S, a hydrogen sulfide (H2S) donor into the colon.

CRH showed significantly higher PBP but lower ABP than controls. The intracolonic administration of indole increased portal blood level of indole, and peripheral blood level of indoxyl sulfate, a liver metabolite of indole. Rats treated with Na2S showed increased portal blood levels of thiosulfate and sulfane sulfur, products of H2S oxidation. The intracolonic administration of vehicle did not affect hemodynamic parameters. In controls intracolonic H2S increased PBP but decreased PBF and ABP. In contrast, intracolonic indole increased both BPB and PBF, whereas did not affect significantly ABP. In comparison to controls, CRH showed significantly greater hemodynamic responses to H2S but not to indole. Cirrhotic livers had a significantly higher concentration of H2S but lower expression of rhodanese, an enzyme converting H2S to sulfate.

Colon‐administered indole and H2S increase portal blood pressure via distinct hemodynamic mechanisms. Indole increases, whereas H2S decreases portal blood flow. Indole and H2S signaling in the gut‐liver axis may be involved in the control of portal blood pressure and in the etiology of portal hypertension. Further studies on the effects of gut bacterial metabolites on portal circulation are needed.

Support or Funding Information

This work was supported by the National Science Centre, Poland grant no. UMO‐2016/22/E/NZ5/00647

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Therapeutic Administration of Probiotic VSL#3 on Dextran Sodium Sulfate‐ Induced Colitis
Authors: Prest Rebecca , Sapp Madison , Glise Joe , Moore Jaime , Walton Kristen LW.
Published: June 2021

Inflammatory bowel disease (IBD) affects as many as 1.6 million Americans every year. This condition is a chronic intestinal disease that includes Crohn's disease and ulcerative colitis. Probiotic bacteria, including specific species of Lactobacillus and Bacteroides, have previously been shown to have beneficial effects in some intestinal disorders such as pouchitis, irritable bowel syndrome, and some models of IBD. We examined the effects of the probiotic cocktail VSL#3 administration for 3 days of recovery after a 5‐day treatment with 3% dextran sodium sulfate (DSS). Clinical signs of colitis, including weight loss, fecal occult blood, and fecal consistency, were monitored daily during the treatment and recovery period. At the end of the experiment, samples were collected for histological scoring, fecal bacterial analysis by quantitative PCR, and RNA isolation and analysis of colonic pro‐inflammatory cytokine expression by quantitative PCR. As expected, clinical signs of colitis were observed more frequently in DSS treated mice than in non‐DSS treated mice. However, probiotic‐treated colitic mice had clinical signs of disease similar to those of non‐probiotic treated colitic mice. Histological scoring of colitis showed a trend towards decreased histological colitis with VSL#3 treatment compared to colitic mice. Quantitative PCR for the pro‐inflammatory cytokine KC suggested decreased expression in probiotic‐treated mice compared to colitic mice. In conclusion, 3 days of treatment with VSL#3 following acute DSS‐induced colitis did not significantly improve recovery from colitis at the macroscopic level, but may improve inflammation at this time point.

Support or Funding Information

Supported by the PORTAL program, Missouri Western State University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The impact of cognitive fatigue on aerobic output in a 6‐minute walk test among older adults
Authors: Goodwin Aurora , Avolio Abby , Boolani Ali
Published: June 2021

Purpose

The purpose of this study was to determine the impact of cognitive fatigue on aerobic output during a 6‐minute walk test (6MWT) in older adults.

Methods

Healthy older adults (N=9, age= 67.39±4.58 yrs, height=168.01±55.82 cm, weight=60.80±12.41 kg) were involved in 2 days of testing. One day was a 2‐hour protocol of cognitively fatiguing tasks (S3 and S7, continuous performance task (CPT), rapid visual input processing (RVIP),) after which they were tasked to complete a 6MWT at their normal speed and VO2 was measured using the Cosmed Fitmate Pro. The other day, a baseline 6MWT was performed and VO2 were measured. A One‐Sample Kolmogorov‐Smirnov Test to determine distribution of peak VO2 scores prior to performing an independent sample T‐test to determine differences between cognitive fatigue and non‐cognitive fatigue days.

Results

Results yielded a normal distribution (Mean=871,33, SD=265.89) and T‐test analysis yielded no significant differences (p=.734) between cognitive fatigue and non‐cognitive fatigue days.

Conclusion

Although analysis yielded no difference in peak VO2 scores between days, it should be noted that 6 out of the 9 subjects had lower peak VO2 on cognitive fatigue days than on non‐cognitive fatigue days. Further research needs to be conducted to determine whether cognitive fatigue does decrease aerobic performance in older adults.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alpha adrenergic inputs to PVN do not play a role in the sympathetic, phrenic or cardiovascular responses to intravenous sodium cyanide
Authors: Beig Irfan , Schreihofer Ann , Mifflin Steve
Published: June 2021

Previous studies from our lab indicate that catecholaminergic inputs to the hypothalamic paraventricular nucleus (PVN) mediate at least part of the hypertension induced by exposure to intermittent hypoxia. The present study was designed to examine the role of alpha adrenergic inputs to PVN in the cardiovascular and phrenic nerve responses to chemoreceptor activation induced by sodium cyanide (NaCN). Rats were anesthetized with chloralose and urethane and vagotomized. Mean arterial pressure (MAP), heart rate (HR), renal sympathetic nerve activity (RSNA), phrenic nerve activity (PNA) and end tidal CO2 recorded. The rat was placed in a stereotaxic frame for PVN microinjections, ventilated with O2‐enriched room air and paralyzed with pancuronium. Chemoreceptor activation was induced by iv. NaCN (200mcg/kg) to obtain baseline levels of responses. After return of MAP to baseline, bilateral injection of vehicle or either α1 antagonist prazosin (500nmol) or α2 antagonist idazoxin (5nMol) was made into PVN. A 2nd and 3rd iv. injection of NaCN was given 15 minutes and 1 hour after PVN microinjections of vehicle or drug. At the end of experiment brain was removed for localization of PVN injection sites. NaCN induced significant increases in MAP (Δ 32.5±4.5 mmHg, n=9), RSNA (Δ63.5±8.6% n=7) and PNA frequency (Δ 28.4±4.7%, n=7) and no change in HR. Bilateral administration of either prazosin or idazoxin into PVN did not alter the MAP, HR, RSNA or PNA responses to iv. NaCN. In separate set of experiments, bilateral injection of the α1–adrenergic agonist phenylephrine (200 ng) into the PVN increased RSNA within 5min. RSNA remained elevated by 36±12% for at least 60min after the injections, which did not change MAP and HR. Our study indicates that alpha adrenergic inputs to PVN do not appear to play a significant role in the MAP, RSNA, PNA and HR responses to chemoreceptor activation.

Support or Funding Information

This work supported by HL088052

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ventilatory, Arterial Blood Gas, pH, and Electrolyte Adaptations to Chronic Hypercapnia in Healthy Goats
Authors: Buchholz Kirstyn , Burgraff Nicholas , Neumueller Suzanne , Hodges Matthew , Pan Lawrence , Langer Thomas , Forster Hubert
Published: June 2021

Respiratory diseases, such as chronic obstructive pulmonary disease (COPD), affect ~24 million U.S adults and cost $49B annually. Many of these patients hypoventilate, leading to chronic hypercapnia, cognitive dysfunction, and high mortality rates. To gain insight into hypercapnia induced changes in neural and physiological function, we studied the breathing, arterial blood gases, pH, electrolytes, and cognitive function in goats during 30 days of chronic hypercapnia (inspired CO2 (InCO2) of 6%) or normocapnia. PaCO2 increased 10 mmHg during the first day of hypercapnia, continued to rise to a maximum of 16 mmHg above control between days 11–12, and remained near this level thereafter. PaO2 remained ~20 mmHg above control throughout the exposure to hypercapnia. Minute ventilation increased by 319% above control on day 1 of hypercapnia (n=7), but declined to 249% above control by day 2 of hypercapnia (n=7). Thereafter, ventilation progressively decreased to a nadir around day 12 at 211% above control (n=6) and remained near this level for the remainder of CO2 exposure. The initial hyperpnea during hypercapnic exposure was due to increases in both tidal volume and breathing frequency. However, breathing frequency returned to near control levels during the first week of hypercapnia, while tidal volume remained elevated. Arterial blood was acidic (−0.05) during day 1 of hypercapnia, followed by a partial compensation by day 8 due to increased arterial [HCO3+] (+7.4 mEq/L). VI/PaCO2 and VI/H+ at 6% InCO2 initially increased ~250% above control and remained elevated for the duration of hypercapnic exposure. Arterial [Cl−] decreased ~4 mmol/L during the first day of CO2 exposure and remained below control (−4–5 mmol/L) for the remainder of the 30 days. Arterial [K+] increased ~0.5 mmol/L during the first week of hypercapnia, and remained above control. No changes were found in arterial [Na+] or [Ca2+]. Accuracy of shape selection (index of cognitive function) decreased nearly 50% during hypercapnia. There were no apparent changes in any physiologic measurement for up to 30 days in goats maintained in room air conditions. In conclusion, we have established a model for future study of neuroadaptive responses, that potentially underlie compromised phenotypic/cognitive function, in COPD patients.

Support or Funding Information

Department of Veterans Affairs; NIH HL007852

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CAROM, a novel gene suppressing endothelial cell migration.
Authors: Xia Jixiang , Xiong Xinyu , Saredy Jason Jonathan , Xu Yanjie , Liu Suxuan , Yang Xiao‐Feng , Wang Hong
Published: June 2021

Hyperhomocysteinemia (HHcy), a syndrome displayed by high concentration of homocysteine (Hcy) in blood has been demonstrated as a significant risk factor for cardiovascular disease (CVD). We have shown that Hcy leaded to endothelial cells (EC) injuries through inhibition of proliferation and migration. However, the comprehensive disclosure of mechanisms accounted for Hcy mediated EC injuries are still lacking up to date. It's compelling to discover more molecules that participate in the pathogenesis of diseases involving HHcy. Through differential display screening of cDNA from Hcy stimulated cultured EC, we discovered a highly induced gene and it was identified as CAROM, which was also called FCHSD2 (FCH and double SH3 domains 2). CAROM protein belongs to Bin‐Amphiphysin‐Rvs (BAR) superfamily and specifically it is a FBAR protein since it displays Fes/CIP homology (FCH) domain on N‐terminus. Proteins in this family are usually involved in endocytosis and other cellular trafficking regulation. The function of CAROM has been poorly characterized. Three major areas were focused for this study: 1) Genomic alignment among species and prediction of interacting partners through bioinformatics; 2) Mass Spectrometry (MS) analysis for discovering interacting proteins; 3) Characterizing its role of regulating EC function. This gene displays highly conservative homology among mammalian species, with 96.5% identity of amino acids between human and mouse homologue. Information collected from multiple interactome databases showed CAROM could bind to more than thirty proteins, although most of them haven't been experimentally verified. We used samples from CAROM overexpression EC for MS analysis. Our data disclosed that CAROM complexes covered a lot proteins regulating endocytosis, vesicle trafficking and cytoskeleton dynamics. These partners included EEA1(Early Endosome Antigen 1), Filamin, Vinculin, Importin‐5, Dynein et al, suggesting it could regulate motility, migration and other cell behaviors. CAROM expression was reduced more than 80% by siRNA targeted at different regions of its transcript. With gain and loss of function strategies, we tested its potential effect on EC function. Our preliminary data showed that inhibition on EC migration by Hcy were partially reversed after CAROM downregulation, indicating its interference with EC repair after vascular injury. Further studies are needed to unveil the underlying mechanisms mediated by CAROM.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessment of Cardiac Function of Rbm 20−/− Rats Using Pressure‐Volume Loop Analysis After 28 Days of Angiotensin II Treatment
Authors: Ahuja Hanna , Zhu Chaoqun , Willoughby Ashley , Pedrei Filipa Monteiro , Guo Wei , Chew Herbert G.
Published: June 2021

RNA binding motif 20 (Rbm20), a muscle specific splicing factor for the sarcomeric protein titin, affects diastolic and systolic properties of the heart per the Frank‐Starling mechanism. Failure to alter titin expression from the predominant fetal isoform N2BA to the shorter, adult N2B isoform in Rbm20−/− (KO) rats, results in impaired cardiomyocyte relaxation, increased compliance of the ventricular walls, and dilated cardiomyopathy. These pathologies are similar to those seen in humans with this mutation, which often results in early, sudden death in both species. Angiotensin II (AII) acutely causes vasoconstriction, and chronically results in cardiac hypertrophy, characterized by an increase in cardiac wall stiffness. We hypothesized that 28 days of treatment with AII would increase the stiffness of the myocardium, resulting in improved cardiac function in KO rats. KO rats and age‐matched wild type (WT) controls were fitted with subdermal osmotic pumps, delivering AII continuously (0.25 μL·hr −1) for 28 days. After AII exposure, cardiac function was assessed in vivo using pressure‐volume loop analysis. Under isoflurane anesthesia, loops were recorded under baseline, brief inferior vena cava occlusion, and hypertonic saline infusion (20–40 μL) conditions. The rat was then heparinized, and blood collected for cuvette calibration. No group differences (p<0.05) were found in cardiac output (23.3±0.8 KO; 26.9±2.5 mL·min−1 WT), heart rate (295±21 KO; 341±16 b·min−1 WT), and stroke volume (80.8±9.1 KO; 79.5±7.6 μL WT). However, significant reductions were found in contractility (preload recruitable stroke work; 41.6±10.8 KO; 86.9±8.2 mmHg WT), stroke work (5.13±0.73; 8.13±0.86 mmHg·min WT), and ejection fraction (49.8±4.2 KO; 74.7±7.4 % WT) in KO rats. KO rats were able to maintain stroke volume, despite impaired contractility, due to a significant increase in preload (169±16 KO; 113±7 μL WT) and decrease in afterload (84.4±8.7 KO; 113±9 mmHg WT). We conclude that KO rats failed to improve contractility with 28 days of AII treatment, but maintained normal cardiac output through compensatory changes in preload and afterload.

Support or Funding Information

This project is supported in part by a grant from the National Institute of General Medical Sciences (2P20GM103432) from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acid‐Base Compensation During Incremental Ascent to High Altitude
Authors: Zouboules Shaelynn M. , Nysten Heidi , Brutsaert Tom , Nysten Cassandra , O'Halloran Ken D. , Steinback Craig D. , Sherpa Mingma , Day Trevor A.
Published: June 2021

Ascent to high altitude, and the associated hypoxic ventilatory response, imposes an acid‐base challenge, namely chronic hypocapnia and respiratory alkalosis. The kidneys act to compensate for this respiratory alkalosis via bicarbonate (HCO3−) excretion in urine to induce a compensatory metabolic acidosis. Although the importance of renal responsiveness through acid‐base modifications is well‐defined, the time course and extent of plasticity in this response during incremental ascent to altitude is unclear. Thus, we developed a practical index of renal reactivity (RR), indexing the relative change in arterial HCO3− concentration ([HCO3−]a; i.e., response) against the relative change in arterial partial pressure of CO2 (PaCO2; i.e., stimulus) during incremental ascent to high altitude (RR=Δ[HCO3−]a/ΔPaCO2). We sought to assess (1) if RR increased over time and with incremental ascent to high altitude and (2) if RR was correlated with relative changes in arterial pH (ΔpHa) throughout ascent. During ascent to 5160 m over 10 days in the Nepal Himalaya, arterial blood was drawn from the radial artery for measurement of acid‐base variables (Abbott i‐STAT portable blood gas/electrolyte analyzer; CG4+ and CHEM8+ cartridges) in lowlanders at 1045/1400 m (baseline) and at four different altitudes following one‐night sleep: 3440 m (n=18), 3820 m (n=17), 4370 m (n=15) and 5160 m (n=13). At 3820 m (day five of altitude exposure) and higher, RR significantly increased and plateaued in comparison to 3440 m (day three of altitude exposure; P<0.04), suggesting plasticity in renal acid‐base compensation. At all four altitudes, we observed a strong correlation (range: r=−0.71 to −0.98; P<0.001) between RR and relative ΔpHa from baseline, suggesting that the RR index accurately quantified acid‐base responsiveness via renal mechanisms throughout ascent. In conclusion, renal acid‐base compensation mechanisms demonstrate plasticity and a unique time course during incremental ascent to high altitude, which was detected using a novel RR index. The extent of plasticity and plateau in renal responsiveness may predict severity of altitude illness or acclimatization at higher or more prolonged stays at altitude.

Support or Funding Information

This work was supported by (a) Alberta Government Student Temporary Employment Program, (b) Alberta Innovates Health Solutions Summer Studentship, and (c) Natural Sciences and Engineering Research Council of Canada Discovery grant.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Superior Cervical Ganglionectomy Affects the Cardiorespiratory Network of the In Situ Arterially Perfused Brainstem Preparation
Authors: Kola Gjinovefa , Coffee Gregory A. , Getsy Paulina M. , Dick Thomas E. , Hsieh Yee‐Hsee , Dutschmann Mathias , Lewis Stephen J.
Published: June 2021

Recently we found that the superior cervical ganglia projects to the nucleus tractus solitarii. Here, we report the effect of superior cervical ganglionectomy (SCGx) on cardiorespiratory activity in the in situ, arterially perfused brainstem preparation. For these studies, we used male Sprague‐Dawley rat pups (P25). Five days after either bilateral removal of superior cervical ganglia (n=14) or sham surgery (n=14), we recorded efferent activities of the phrenic nerve, cervical vagus nerve, and thoracic sympathetic chain and monitored heart rate and perfusion pressure. Baseline cardio‐respiratory patterns were recorded for 10 min to assess differences in patterning between the SCGx and sham groups. Then, we evoked the peripheral chemoreceptor reflex by injecting (intra‐arterially) sodium cyanide (100 μL of a 0.03% solution) and the baroreceptor reflex by increasing perfusion pressure (increasing the speed of the roller pump to its maximum). Baseline respiratory frequency was similar in the SCGx and sham group. However, the SCGx group had a greater but delayed response to chemoreceptor stimulation. Similarly, the SCGx group had a greater response to baroreceptor stimulation. Baseline heart rates (HR) were similar in both groups. For the SCGx group, HR decreased less than sham group during chemoreceptor stimulation; but, decreased more than the sham group during baroreceptor stimulation. Finally, the SCGx group required a greater perfusion pressure (PP) than the sham group during baseline; and had smaller increase in PP during chemoreceptor stimulation. These data indicate that superior cervical ganglia modulate vasomotor tone at rest and buffer cardio‐respiratory responses to chemoreceptor and baroreceptor reflexes.

Support or Funding Information

NIH‐SPARC 1OT2OD023860‐02SJLewis

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nutrient Regulation of Signaling and Transcription
Authors: Hart Gerald W.
Published: June 2021

O‐GlcNAc cycles on and off thousands of nucleocytoplasmic proteins and has extensive crosstalk with protein phosphorylation. O‐GlcNAc cycles on nearly all proteins involved in transcription, where it regulates gene expression in response to nutrients. O‐GlcNAc also regulates the cycling of the TATA‐binding (TBP) protein on and off DNA during the transcription cycle.

Targeted, inducible, deletion of the O‐GlcNAc Transferase (OGT) in aCAMKII positive (excitatory) neurons of adult mice results in a morbidly obese mouse with a satiety defect. Thus, O‐GlcNAcylation not only serves as a nutrient sensor in all cells, but also is directly involved in appetite regulation. O‐GlcNAcylation also plays an important role in the trafficking of the AMPA receptors in neurons and in the development of functional synaptic spines. Recent studies have shown that more than one‐half of all human protein kinases are modified by O‐GlcNAc and all kinases that have been tested are indeed regulated in some way by the sugar. Abnormal O‐GlcNAcylation of CAMKII contributes directly to diabetic cardiomyopathy and to arrhythmias associated with diabetes. Prolonged elevation of O‐GlcNAc, as occurs in diabetes, contributes directly to diabetic complications and is a major mechanism of glucose toxicity. Drugs that elevate O‐GlcNAcylation in the brain, which, in‐turn, prevents hyperphosphorylation, appear to be of benefit for the treatment of Alzheimer's disease in animal models. To date, all cancers have elevated O‐GlcNAc cycling, which may play a key role in the regulation of metabolism in cancer cells.

Support or Funding Information

Supported by NIH P01HL107153, R01GM116891, R01DK61671, and N01‐HV‐00240. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genome‐wide analysis of single nucleotide variants on phosphorylation motifs
Authors: Yoshizaki Hisayoshi , Ling Yiwei , Kohno Miyuki , Okuda Shujiro
Published: June 2021

In order to conveniently characterize enormous phosphorylation sites, we attempted to summarize them as phosphorylation motifs, extracting the motif structure from the features of the surrounding amino acid sequence of the phosphorylation sites. Approximately 100,000 known phosphorylation sites registered in PhosphoSitePlus (http://www.phosphosite.org) were clustered with the features of amino acids and were organized into about 200 phosphorylation motifs based on the distribution of amino acids in each cluster. The determined motifs were analyzed by signal transduction pathway analysis, GeneOntrogy analysis and comparative evolutionary analysis to determine gene function, signal pathway and physiological importance parameters (Yoshizaki H et al. BMC genomics 2014). In addition, a database was created in which the phosphorylation motifs, motif structures for approximately one million of serine, threonine and tyrosine residues on the human genome, and kinase‐substrate information for kinase prediction were stored (Yoshizaki H Gigascience 2015). Using these data, we investigated associations between cancer‐specific mutations and phosphorylation motifs. Of more than 16 million cancer specific mutations registered in the International Cancer Genome Consortium (ICGC) (https://icgc.org/), about 100,000 mutations were found on the phosphorylation motif sequences with amino acid substitutions. Distributions with these mutations and cancer tissues for these mutations and the evolutionary conservation of each phosphorylation motif were investigated. As a result, it was revealed that the frequency of cancer specific mutagenesis on the phosphorylation motif has a positive correlation with the evolutionary conservation of the motif. In addition, this correlation was not confirmed by healthy subject SNP data. This means that in cancer tissues, mutations tend to accumulate in physiologically important phosphorylation motifs, mutations on the phosphorylation motif in the cancer tissue are not random mutations due to genomic instability, suggesting spontaneously selective mutations. Furthermore, in cancer specific mutations, the position and frequency of mutations in the motif were biased due to the type of phosphorylation motif, and biases in the motif sequence change by amino acid substitution were also observed. Our results suggest that mutations on the phosphorylation motif affect not only the destruction of the motif structure but also the signal disruption due to the appearance of a new motif by amino acid substitution. This study combining tissue specific gene mutation information with our own phosphorylation motif data is expected to lead to predict the effect on canceration by gene mutation, to functionally unknown phosphorylation sites.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epigenome‐Wide Association Study of Incident Cardiovascular Disease
Authors: Westerman Kenneth , Ordovas Jose
Published: June 2021

DNA methylation is an ideal biomarker for many applications, because it has both the stability for prognostic use and the plasticity to be altered by environmental variables including diet. Investigations of relationships between DNA methylation and cardiovascular disease (CVD) have been performed, but these cross‐sectional analyses are susceptible to reverse causation, making causal inference difficult. Thus, we hypothesized that analysis of methylation in large‐scale cohorts with follow‐up for CVD development could provide potential risk biomarkers as well as mechanistic insights into disease risk. We performed an epigenome‐wide association study for incident CVD in individuals from the Framingham Heart Study Offspring Cohort and Women's Health Initiative studies. Methylation data were previously collected from whole blood samples using the Illumina Infinium HumanMethylation450k platform. Cox regression methods were used to identify relationships between relative methylation levels and incident CVD both at specific CpG sites and using a region‐based approach. We identified 10 single CpG sites and 4 regions demonstrating suggestive associations with incident CVD. Mendelian randomization approaches provided preliminary evidence of a causal relationship between methylation within the APOB gene and CVD. Investigations of cell‐type specific relationships using available epigenomic annotations highlighted the importance of monocytes in mediating the risk captured by methylation data in blood. Finally, relationships between these CpG sites and known traditional cardiovascular risk factors were examined to understand how they might interact to increase CVD risk. Based on these results, DNA methylation may provide a useful complement to existing cardiovascular risk prediction methods, and provides insights into genomic processes that may mediate the effects of genetics and environmental factors on disease risk.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proteomic profiling of exosomes leads to the identification of novel biomarkers for prostate cancer progression
Authors: Shin Kyeong Jin , Seo Young‐kyo , Yoo Youngbum
Published: June 2021

Exosomes are vesicles which have garnered interest due to their diagnostic and therapeutic potential. Isolation of cancer‐derived exosomes is a prerequisite for their better understanding. We have investigated a new biomarker for prostate cancer progression via quantitative proteomics. Exosomes from androgen‐independent PC‐3 and the androgen‐dependent LNCaP prostate cancer cells were isolated by ultracentrifugation. After tryptic digestion, proteomic analyses utilized a nanoLC coupled with an LTQ‐Orbitrap operated in tandem MS (MS/MS) mode. Accurate Mass and Time (AMT) tag approach was employed for peptide identification and quantitation. Proteomic characterization showed that 105 proteins from PC‐3 exosomes were 10‐fold higher than those from LNCap. Validation by Western blotting and qRT‐PCR confirmed a higher abundance of PFKL, NRP1 and MCAM(CD146) in PC‐3 cell exosomes, too, indicating that aggressive prostate cancer exhibit higher levels of adhesion or cohesion proteins. We also show that MCAM (CD146) was strongly associated to PC‐3 mobility in vitro. These data provided that MCAM should be useful as a new candidate biomarker for prostate cancer progression.

Support or Funding Information

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) (OGM4391711)

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