bluuebunny/crossref_metadata_2025 huggingface dataset:
Female spontaneously hypertensive rats have a compensatory increase in renal regulatory T cells in response to elevated blood pressure (1083.3)
Authors: Tipton Ashlee , Li Beverly , Sullivan Jennifer
Published: June 2021

We recently demonstrated that female SHR have more anti‐inflammatory regulatory T cells (Tregs) in their kidneys than males. The goal of this study was to determine the impact of blood pressure (BP) on Tregs in male (M) and female (F) SHR. We hypothesized that hypertension (HTN) promotes renal T cell infiltration in both sexes, although females will have greater increases in Tregs. SHR were treated with hydrochlorothiazide and reserpine from 6 to 13 wks of age (7‐HCTZ), 11 to 13 wks of age (2‐HCTZ), or vehicle control (N=5‐8) and renal T cells were measured by flow cytometry. 7‐HCTZ attenuated age‐related increases in BP (M: 137±4, F: 132±4 systolic BP mmHg) and 2‐HCTZ reversed established HTN (M: 122±4, F: 116±3 mean arterial BP mmHg; p<0.05) compared to vehicle‐treated SHR (M: 174±4, F: 161±1 mean arterial BP mmHg). Neither 7‐HCTZ nor 2‐HCTZ altered renal CD3+ or CD4+ T cell infiltration in either sex. Both treatments decreased renal Tregs in female SHR (vehicle: 11±0.2 vs 7‐HCTZ: 6±0.4 %CD4+CD3+ cells, p<0.05; vehicle: 9±0.5 vs 2‐HCTZ: 3±0.6, p<0.05); Tregs in males were unaffected. F SHR maintained greater Treg infiltration than males following 7‐HCTZ treatment (males: 7±1 vs females: 6.4±0.4; p<0.05). Treg infiltration was comparable in 2‐HCTZ‐treated M and F SHR (Tregs males: 5±1 vs females: 3.2±0.6, NS). These data suggest that female SHR have a compensatory increase in renal Tregs in response to increases in BP.

Grant Funding Source: Supported by Intramural Funds
A reduction in uterine perfusion pressure induces hypertension during pregnancy in the mouse (1084.5)
Authors: Intapad Suttira , Warrington Junie , Spradley Frank , Palei Ana , Drummond Heather , Ryan Michael , Granger Joey , Alexander Barbara
Published: June 2021

Preeclampsia is a hypertensive disorder of pregnancy and a leading cause of maternal morbidity and mortality; however, the underlying mechanisms are not clear. Reduced uterine perfusion in the pregnant rat induces many of the characteristics of preeclampsia including hypertension and fetal growth restriction. However, the lack of availability of a mouse model prevents harnessing the power of genetically modified mice. Thus, the aim of this study was to develop a mouse model of reduced uterine perfusion pressure (RUPP). Pregnant C57BL/6J mice were subjected to either a sham surgery or a mechanical reduction of the blood flow to the abdominal aorta and both branches of ovarian arteries initiated at day 13 of gestation (E13). Mice were instrumented with a carotid arterial catheter at E17 followed by measurement of mean arterial pressure (MAP) at E18 in the conscious state. MAP at E18 in the RUPP was significantly elevated versus sham (117±4 vs. 97±2 mmHg, P<0.01; respectively). Pup weight from the RUPP were significantly reduced versus sham (0.77±0.02 vs. 0.92±0.03 g, P<0.003; respectively). However, no significant difference in placental weight was noted between RUPP and sham mice (0.07±0.003 vs. 0.08±0.004 g; respectively). Thus, these data indicate that RUPP induces hypertension and fetal growth restriction in the mouse. Importantly, this mouse model of RUPP will be a valuable tool to address novel hypotheses related to the etiology of preeclampsia.
Post‐exercise fate of lactate in the American locust (1101.2)
Authors: Tyler Samantha , Olson Matthew , Kirkton Scott
Published: June 2021

The fate of lactate after exercise varies between animal groups. Mammals oxidize lactic acid in their muscles, fish oxidize lactic acid in their liver, and reptiles use both their muscles and liver to oxidize post‐exercise lactate. Grasshoppers are unique among insects in that they produce lactic acid during repeated jumping. However, it is unclear where the lactate is oxidized. Using the American locust (Schistocerca americana), we measured lactate levels in the jumping muscle at different time points after jumping. Lactate levels were measured using a fluorometric plate assay. Immediately after three minutes of jumping, muscle lactate levels were approximately 23 mmol kg‐1. After fifteen minutes of rest, lactate levels decreased by 40% in the jumping muscle. After one hour of rest, muscle lactate levels had decreased by 70% of their post‐exercise level. After approximately three hours of rest, muscle lactate was similar to pre‐jumping levels. We have previously demonstrated that lactate production correlates with jumping fatigue rate in S. americana grasshoppers. Current work is examining whether hemolymph or fat body lactate levels vary after jumping. A decrease in muscle lactate levels during recovery, combined with a rise in hemolymph and fat body lactate levels would suggest a non‐muscle model for lactate oxidation in grasshoppers.

Grant Funding Source: Supported by Union College Undergraduate Research Grants
Thyroid hormone changes associated with prolonged food deprivation in adult male northern elephant seals (1101.6)
Authors: Lee Debby , Martinez Bridget , Crocker Daniel , Ortiz Rudy
Published: June 2021

During food deprivation in mammals, thyroid hormone (TH) concentrations and deiodinase (DI) content is typically reduced to suppress metabolism. However, in metabolically active elephant seal pups the hypothalamic‐pituitary‐thyroid axis is up‐regulated during prolonged fasting. To better understand the mechanisms regulating the cellular function of TH in a fasting, yet metabolically active mammal, we measured plasma TH concentrations and mRNA expressions of DI type II (DI2) and TH receptor beta‐1 (THrβ‐1) in adult male northern elephant seals (n=8) over 8 weeks of fasting (early vs. late). We hypothesized that natural fasting in male seals decreases TH levels and DI expression. Fasting did not alter the concentrations of plasma reverse T3 and total T4. However, free T4 (0.25 ± 0.03 to 0.78 ± 0.08 ng/dL) and free T3 (0.61 ± 0.06 to 0.83 ± 0.08 pg/mL) increased between early to late fasting suggesting that there is an increased potential for TH‐mediated cellular effects. Conversely, total T3 decreased (63 ± 5 to 50 ± 4 ng/mL) suggesting that DI2 or DI3 may be contributing to the changes in TH metabolism. mRNA expressions of DI2 and THrβ‐1 decreased 87% ± 7 and 93% ± 2, respectively, between early to late fasting suggesting that there is a down‐regulation in cellular TH potential during fasting duration consistent with the effects of fasting in other mammals. When compared to the opposite effects observed in fasting pups, we conclude that elephant seals have evolved age‐dependent changes in fasting‐induced TH dynamics that may reflect differences in TH‐associated metabolic mechanisms.

Grant Funding Source: Supported by NIH/NHLBI 442606‐RO‐29013
Differential effects of clonidine and lisinopril on thermoregulatory and cardiovascular function during chronic exposure to exertional heat stress in rats (1104.30)
Authors: Stauss Harald , Choudhary Navita , Moss Anne , Kregel Kevin
Published: June 2021

The effects of clonidine (C, sympatholytic drug) and lisinopril (L, ACE inhibitor) on thermoregulatory, cardiovascular, and autonomic responses to chronic (5 days, 2 times/day) exertional (ExHS) or non‐exertional (HS) heat stress were assessed in rats. ExHS and HS elicited increases in nocturnal core temperature (Tc) and heart rate (HR) in placebo (P) treated rats. This effect was blunted by C in the ExHS protocol and by both drugs in the HS‐only protocol. Nocturnal blood pressure (BP) increased more during the ExHS than the HS protocol. This hypertensive response to ExHS was blunted by both drugs. In P‐treated rats, baroreflex sensitivity decreased only during the ExHS protocol. L improved this impaired baroreflex function during exposure to ExHS more than C. In P‐treated rats, sympathetic modulation of cardiac and vascular function (low frequency HR and BP variability) was elevated in both protocols. Both drugs reduced this elevated sympathetic modulation of cardiovascular function. In conclusion, C and L elicited beneficial thermoregulatory (Tc), cardiovascular (BP, baroreflex function), and autonomic (cardiac and vascular sympathetic modulation) effects during exposure to HS or ExHS. However, during exposure to ExHS, C appears to be more effective in improving thermoregulation than L, while L may be more effective in improving cardiovascular (baroreflex) function than C.

Grant Funding Source: Supported by DoD/USAMRMC #W81XWH‐10‐2‐0115.
Physiological impact of caloric restriction on the lower critical temperature and body temperature dynamics in mice (1105.2)
Authors: Swoap Steven , Joslin Patrick , Jaramillo Carolina , Shote Shelby
Published: June 2021

The lower critical temperature (TLC) is that ambient temperature (Ta) where metabolic rate (MR) increases linearly with decreasing Ta. We tested the hypothesis that chronic caloric restriction (CR) would elevate TLC. Control male C57Bl/6J mice (n=9) were allowed free access to food and ate 4.2 g/day while CR mice (n=9) were restricted to 2.7 g/day for 8 weeks. Indirect calorimetry demonstrated that CR mice had a higher TLC than control mice (30.3±0.5 vs. 27.7±1.0 °C, respectively), with a lower basal MR (0.45±0.02 vs. 0.66±0.01 mL O2/min), and lower average MR (0.86±0.04 vs. 1.17±0.04 mL O2/min) when compared to controls. In a separate study to examine the impact of CR and Ta on body temperature (Tb), outbred female mice were weaned and separated into one of four groups: 1) housed and given food ad libidum at 20°C, n=37; 2) housed ad lib at 30°C, n=35; 3) CR at 20°C, n=34; and 4) CR at 30°C, n=34. Ad lib feeding and CR lasted ~1 year. CR mice weighed ~55% of their ad lib counterparts for both housing temperatures (20°C and 30°C). A subset (n=7 for each group) of these mice were implanted with Tb telemeters, allowed 2 weeks for recovery, and continued with CR or ad lib. While ad lib mice housed at both 30°C and 20°C had similar minimum Tbs (35.7±0.1 and 35.5±0.2°C, respectively), the CR mice housed at 20°C experienced daily torpor, with a minimum Tb of 24.7±1.1°C, significantly lower than that of CR mice housed at 30°C (32.3±0.3°C). These findings indicate that caloric restriction 1) elevates TLC, and 2) exerts a minimal effect on the Tb of mice housed closed to their TLC, but has a great effect on Tb of mice housed well below their TLC.
Increased expression of caveolin‐1 is associated with upregulation of the RhoA/Rho kinase pathway and smooth muscle contraction in diabetes (1110.11)
Authors: Mahavadi Sunila , Nalli Ancy , Kumar Divya , Bhattacharya Sayak , Zhou Ruizhe , Grider John , Murthy Karnam
Published: June 2021

Gastrointestinal motility disorders are common among type I and type II diabetes and are shown to be associated with depletion of ICC and NOS‐containing neurons. Our recent studies in smooth muscle of diabetic mice and diabetic patients showed that hyperglycemia causes upregulation of RhoA/Rho kinase pathway leading to increase in sustained muscle contraction (tone). Caveolin‐1, an essential protein in the formation of caveolae, positively regulates RhoA/Rho kinase pathway in smooth muscle. Aim. To test the hypothesis that increase in caveolin‐1 expression causes upregulation of the RhoA/Rho kinase pathway in diabetes. Results. mRNA and protein expression of caveolin‐1 was increased in gastric smooth muscle from type 1 (NOD) and type 2 diabetic (db/db) mice and from diabetic patients. Treatment of muscle cells from control mice with 25 mM glucose for 48 h increased expression of caveolin‐1 and augmented ACh‐induced Rho kinase activity. The effect of high glucose on caveolin‐1 expression and Rho kinase activity was reversed by 72 h treatment in 5 mM glucose. Suppression of caveolin‐1 by siRNA attenuated the effect of high glucose on Rho kinase activity, whereas overexpression of caveolin‐1 in control cells augmented ACh‐induced Rho kinase activity. Conclusion. Increased expression of caveolin‐1 in smooth muscle contributes to upregulation of the RhoA/Rho kinase pathway and sustained contraction in diabetes.

Grant Funding Source: DK28300 and DK15564
Circadian clock mediates ER stress signaling in alcoholic fatty liver (1116.12)
Authors: Tsuchiya Hiroyuki , Zhang Yuxia , Lee Sangmin , Smalling Rana , Wang Li
Published: June 2021

[Purpose] This is the first study to investigate how alcohol regulates ER stress and fatty liver through nuclear receptor SHP mediated liver circadian clock machinery.

[Methods] The NIAAA EtOH binge model was established using 8 weeks old, male C57BL/6 (WT) and SHP‐/‐ (SKO) mice (Nat Protoc 2013; 8:627‐637). In brief, the mice were acclimatized to a control liquid diet for 5 days, then fed with 5% Lieber‐DeCarli diet or pair‐fed for 10 days. By day 11, the mice were gavaged maltose (control) or EtOH solutions at zeitgeber time (ZT) 3 (9 am) and sacrificed at ZT12, 18, 0, and 6.

[Results] Metabolomics by GC/MS revealed a global alteration by alcohol in the oscillation of serum intermediate metabolites in pathways of glucose, lipid and amino acid metabolism in SKO mice. The rhythmic expression of core clock genes (Clock, Bmal1, Npas2) and nuclear receptors (Rorα, Rorγ, Rev‐erbα) were disturbed by alcohol. Hepatic lipid metabolic genes and ER stress markers exhibited strong oscillations which were perturbed by alcohol. Chop was a clock controlled gene through the Rorα/Rorγ/Rev‐erbα/SHP regulatory cascade. Liver SREBP‐1c protein cleavage and TG content were drastically induced in control SKO livers, which was markedly decreased by alcohol in both WT and SKO mice.

[Conclusions] SHP mediated clock machinery is significantly disrupted by alcohol, which in turn perturbs ER stress to control AFL.

Grant Funding Source: Supported by AHA and NIDDK
The impact of maternal in utero environment on salt‐induced hypertension in the SS rat (1120.4)
Authors: Geurts Aron , Mattson David , Liu Pengyuan , Skelton Meredith , Kurth Terry , Klotz Jason , Grzybowski Michael , Liang Mingyu , Cowley Allen
Published: June 2021

There is considerable interest in mechanisms by which environmental factors impact disease phenotypes later in life. We previously demonstrated that SS (Dahl‐salt sensitive) male rats fed a grain‐based diet (G) show attenuated salt‐induced blood pressure increase and renal damage than animals fed a casein‐based protein diet, AIN‐76a (A). We hypothesized that differences in dietary‐induced susceptibility may be driven by the in utero environment of pregnant females fed these different diets. Using SS rats fed the casein‐based (SSA), or a grain‐based (SSG) diets for many generations, we performed reciprocal embryo transfers, weaned the offspring to a 0.4% NaCl AIN‐76A diet for 6 weeks, then switched the rats to a 4.0% NaCl AIN‐76A diet for mean arterial pressure (MAP) and urinary albumin excretion (UAE) measurements. Compared to control male F1 generation rats generated by the transfer of SSA embryos into SSA females (SSA>A), F1 males generated by transfer of SSA embryos into SSG female recipients (SSA>G) demonstrated significantly reduced pressure (MAP, 157±6 mmHg vs. 138±4; n=8‐14/group) and renal damage (UAE, 204±16 mg/day vs. 96±13 mg/day; n=12‐18/group) after 21 days of exposure to a 4.0% NaCl diet. Conversely, reciprocal embryo transfer of SSG embryos into SSA recipients (SSG>A) promoted hypertension (MAP, 166±5 mmHg, n=21) and renal disease (UAE, 187±27 mg/day, n=25) in male offspring. These data support the hypothesis that the maternal in utero environment is a significant driving factor in the hypertensive phenotype of the SS rat. Interestingly, following breeding the of the protected SSA>G F1 animals on the A diet, the disease attenuation was lost in the F2 generation (MAP=152±5 mmHg; Ualb=145±25 mg/day, n=12‐13/group), suggesting that; 1) that the protective effect of the G diet is not transgenerational and 2) that severe salt‐induced hypertension disease phentoypes can be restored to SS rats fed the grain‐based diet by switching the breeders to the pro‐hypertensive AIN‐76A diet.
Global DNA methylation and extracellular matrix remodeling in aortic smooth muscle versus endothelial cells in hyperhomocysteinemia (1120.5)
Authors: Narayanan Nithya , Kundu Sourav , Givvimani Srikanth , Pushpakumar Sathnur , Kulkarni Ruhi , Tyagi Suresh
Published: June 2021

Introduction: Homocysteine (Hcy) is a byproduct of DNA methylation. Increased levels of Hcy (HHcy) have been shown to induce smooth muscle cell (SMC) proliferation and inhibit endothelial cell proliferation. It is well known that HHcy causes increased activation of matrix metalloproteinases, a key factor in vascular remodeling. However, the role of global DNA methylation in regulating aortic matrix remodeling is not known.

Hypothesis: In the present study, we hypothesize that global DNA methylation during HHcy regulates the expression of MMPs in mouse aortic SMCs and endothelial cells (MAEC).

Methods: Mouse SMC and MAEC were cultured with 80µM Hcy and 100µM Aza deoxycytidine (AzadC), DNA methyltransferase inhibitor.

Results: Global DNA methylation analysis by ELISA showed that in HHcy, there was a 2 fold and 1.3 fold increase in %5‐mC in SMC and MAECs respectively. During HHcy, there was an increased expression and activity of MMP9 in SMCs, which depreciated after AzadC treatment. Methylation Specific PCR (MS‐PCR) showed increased methylation status of Hcy metabolism genes, CBS and MTHFR in SMCs and MAECs.

Summary: Our results suggest that HHcy causes a greater global hyper‐methylation in aortic SMC than in MAEC. This may suggest that Hcy generated through methylation contributes to vascular remodeling.

Grant Funding Source: NIH
Prolactin influences the excitability of subfornical organ neurons (1129.3)
Authors: Black Emily , Grattan Dave , Ferguson Alastair
Published: June 2021

Prolactin is a hormone that is involved in multiple physiological functions in vertebrates. Prolactin receptor mRNA has been identified in the subfornical organ (SFO) through microarray analysis (Hindmarch et al 2008). The current study was therefore carried out in order to determine the effects of prolactin on the excitability of SFO neurons. We used the whole‐cell patch clamp technique to determine the influence of prolactin on the membrane potential of dissociated SFO neurons. We found that 55% (11 of 20) of neurons responded when treated with prolactin (1 uM and 100nM). Of the cells that responded, 54% (n=6) depolarized with a mean depolarization of 7.7 ± 3.3 mV and 46% (n=5) hyperpolarized with a mean hyperpolarization of ‐8.0 ± 3.6 mV. This study suggests the SFO as a potential central nervous system site at which prolactin may act to influence central autonomic integration.

Grant Funding Source: Canadian Institutes for Health Research
Structural characterization of mixed wt/mutant aquaporin‐2 tetramers using dual‐colored fluorescent subunit counting (1133.1)
Authors: Lussier Yoann , McGuire Hugo , El Tarazi Abdulah , Bissonnette Pierre , Blunck Rikard , Bichet Daniel
Published: June 2021

Mutations in Aquaporin‐2 (AQP2) induce nephrogenic diabetes insipidus, a water reabsorption defect of the kidney. Using co‐immunoprecipitation and functional assays, we have recently shown that (at least) some mild recessive (rec) AQP2 mutants can adequately associate to its wild‐type (wt) counterpart to generate fully functional wt/rec heterotetramers. To better characterize actual subunit distributions within wt/rec AQP2 populations, we have developed a dual‐colored fluorescent subunit counting approach in TIRF condition, in order to identify and quantify individual monomers through bleaching of linked fluorophores. Assays performed on both Xenopus oocyte and transfected cell line have confirmed that mild mutations (K228E, D150E and V24A), similar to wt‐AQP2, adequately oligomerize in tetramer, unlike the severe R187C mutant which only displays monomeric structures. In dual transfections (sfGFP‐wt‐AQP2 + RFP‐rec‐AQP2), we also confirm the presence of mixed wt/rec tetramers for mild mutations, but not with R187C, which remains monomeric. Ultimately, we aim at describing subunit distributions within wt/rec tetramers in order to evaluate the properties, limitations and functional outcome of such heteromeric associations.
Female mice retain more sodium than male mice but maintain lower blood pressure during 30 days on a high salt diet (1135.1)
Authors: Rouch Al , Kudo Lucia , Fan Liming , Hammami Heba , Nadeem Maria , Chatzigiannidis Athena
Published: June 2021

High salt consumption contributes to hypertension. The kidneys must excrete ingested sodium in order to maintain normal blood pressure. Our previous study showed that female mice had lower renal excretion of sodium (Nae) than male mice for a 15‐day period of high salt consumption. The goal of this study was to determine if sex differences in Nae and blood pressure (BP) occur from a 30‐day period of high salt consumption. Intact female and male 12‐week old mice (n=6/group) consumed a 4% salt diet in metabolic cages for 30 days. Nae was determined from daily measurements of urine sodium concentration and urine volume. Sodium intake (Nai) was determined from daily food intake. BP was measured via the tail‐cuff method. Real‐time quantitative PCR was used to measure expression of renal sodium transporters. For the 30‐day period, average daily Nae/Nai (%) was significantly lower in female mice compared to male mice (53.3±2.7 vs 68.1±1.8, respectively, p<0.0001) in addition to the mean BP (mmHg) (78.4 ±1.0 vs 84.9 ±1.2 respectively, <0.0005). Expression of the renal NKCC2 mRNA was over 5‐fold higher in the female kidney. We conclude that female mice retain more sodium and maintain lower BP during high salt consumption compared to male mice. This study indicates that female mice exposed to a high salt diet are protected from hypertension for a period of 30 days despite higher sodium retention.

Grant Funding Source: Supported by OK INBRE 8P20GM103447 Summer Research Program
Impact of uninephrectomy on body L‐arginine homeostasis and blood pressure control (1136.14)
Authors: Pillai Samyuktha , Fingerhut Ralph , Verrey François
Published: June 2021

L‐arginine plays an important role, acting as a precursor for a variety of physiologically important substances including citrulline, urea, ornithine, proline and nitric oxide and the kidney is a major site for its metabolism. Uninephrectomy (UNX) is observed to cause an increase in the size of the remnant kidney and compensation to some degree of the glomerular filtration rate. As very little is known about UNX‐induced effects on blood pressure control and expression levels of transporters and enzymes involved in arginine metabolism, we are testing in mice the hypothesis that renal mass reduction impacts on Arg metabolism and possibly thereby affects blood pressure control. Our results show that in contrast to sham operated controls, mice having undergone UNX display an increased systolic blood pressure (120 ± 2.14 vs. 112 ± 1.97 mmHg by tail cuff measurements, n = 18). This effect was more pronounced in females than males and observed also by telemetry. Plasma levels of asymmetric dimethyl arginine (ADMA), an inhibitor of NOS considered to be a good marker for renal disease, were increased in UNX animals, whereas the level of none of the proteinogenic amino acids was changed significantly. There were also no changes in the mRNA expression levels of Arg transporters and enzymes involved in arginine metabolism. Our observations suggest that UNX affects blood pressure and the effects are less pronounced in males, possibly due to a more important remnant kidney compensatory growth.

Grant Funding Source: Supported by the Swiss NCCR Kidney.ch and the European Community's Seventh Framework Programme
Chromosomal mapping and zygosity check PCR of transgenes in a GFP mouse line (C57BL/6‐Tg(CAG‐EGFP)C14‐Y01‐FM131Osb) determined by genomic walking (1138.13)
Authors: Suzuki Osamu
Published: June 2021

Positional effect of transgenes is important for analyses of transgenic animals. In this study, the transgene insertion site in genomes of a GFP mouse line and primers for zygosity check PCR were determined by genomic walking (Exp Anim 53:103‐111, 2004). Adapter‐ligated, enzyme‐restricted genomic libraries were constructed with genomic DNA from hemizygous GFP mice. The sequence flanking the transgene was determined by sequencing amplicons from PCR of the genomic libraries with transgene‐specific and adaptor primers. The insertion site was located by BLAST search in Ensembl genome database (http://www.ensembl.org) using the genome sequence flanking the transgene. Primer sets for zygosity check were designed based on the transgene and flanking genome sequences. The flanking sequence was mapped on chromosome 11. Three primers were designed for zygosity check (1: CACCGCCGGGTCTCTCACTGTCAGTCT, 2: GACGTCAATGGGCGGGGGTCGTT, and 3: TGGGACCTGATCACGGTAAAACGAGGT). Transgenic and wild alleles could be detected in the genome by PCR with primers 1 and 2, and primer 1 and 3, respectively. The information should be useful for genomic research using the GFP line (C57BL/6‐Tg(CAG‐EGFP)C14‐Y01‐FM131Osb).

Grant Funding Source: Supported by a grant from the Ministry of Health, Labor and Welfare of Japan.
Arginase inhibition prevents vascular endothelial dysfunction in a type 2 diabetes model (1139.13)
Authors: Yao Lin , Xu Zhimin , Toque Haroldo , Bhatta Anil , Xu Xizhen , Huo Yuqing , Caldwell Ruth , Caldwell R
Published: June 2021

Type 2 diabetes is associated with increased levels of reactive oxygen species (ROS) and activation of blood monocytes. Both conditions significantly increase risk of cardiovascular disease, with vascular endothelial dysfunction (VED) recognized as a key event. We previously reported that increased vascular arginase activity (AA) is associated with VED in type 1 diabetic mice. Arginase 1 knockdown or arginase inhibitor (2(S)‐amino‐6‐boronohexanoic acid, ABH) treatment can prevent these effects. We hypothesized that ABH treatment would prevent VED in Type 2 diabetic (db/db obese) mice. To test this, plasma and aorta were collected from 18 wk old lean control (LC) and db/db mice or db/db mice treated with ABH (8 mg/kg/day, drinking water, last 7 weeks). The AA was increased 6.7 fold in plasma and 4.4 fold in aorta of db/db mice vs LC. ABH treatment blocked these increases. The db/db mice exhibited a decrease in maximum vasorelaxation to acetylcholine to 53% vs 76% for LC and a 2.1 fold increase in numbers of circulating, activated monocytes. Both effects were blocked by ABH. Plasma ROS (lipid peroxides) were elevated 2.5 fold in db/db mice and were not altered by ABH treatment. In conclusion, systemic inhibition of arginase prevents type 2 diabetes‐induced VED and blocks the increase in activated monocytes, without altering plasma ROS levels.
TGF‐β1 and HtrA1 interactive pathway in the molecular progression of osteoarthritis (1139.9)
Authors: Andersen Karl , Black Collin , Crepeau Philip , Kartchner Jeff , Mecham David , Robinson MarLeice , Wilhelm Spencer , Chavez Matias Elizabeth , Kooyman David
Published: June 2021

Prior research has indicated that a pathway exists in the molecular progression of Osteoarthritis (OA) involving key biomarkers HtrA1, Ddr‐2 and Mmp‐13. We and others have recently shown, by immunohisotchemical staining, that HtrA1 and TGF‐β1 show a unique, inverse relationship in OA. The objective of this research is to provide supporting data and clarification to this interaction via Enzyme‐Linked Immunosorbent Assay (ELISA) and quantitative Polymerase Chain Reaction (qPCR) analysis. Cartilage samples from the hip and synovial fluid samples form the knee of wild type (WT) mice, treated to induce OA, were assayed for quantitative presence of TGF‐β1 biomarker using ELISA and quantitative mRNA presence using qRTPCR. We observed initially high levels of TGF‐β1 protein that subsequently decreased 50% as OA progressed and became more severe over time. We hypothesize that TGF‐β1, as a cytokine growth factor, is over expressed in early OA resulting in an increased expression of HtrA1, a serine protease, which subsequently impairs TGF‐β1 cell maintenance activity and dooms the cell to apoptosis.
Role of epigenetics and STAT5 in breast cancer resistance protein (BCRP/ABCG2) expression in the lactating mammary gland (1141.3)
Authors: Wu Alex , Dalvi Pooja , Yang Mingdong , Turinsky Andrei , Wang Kelvin , Butcher Darci , Egan Sean , Weksberg Rosanna , Brudno Michael , Harper Patricia , Ito Shinya
Published: June 2021

The multidrug transporter breast cancer resistance protein (ABCG2) is upregulated in the mammary gland during lactation. We have shown that prolactin induces ABCG2 in human breast cancer cells via the recruitment of Signal Transducer and Activator of Transcription‐5 (STAT5) to the human ABCG2 gene. It is unclear if a similar mechanism regulates ABCG2 in vivo. Here we investigated epigenetic and transcriptional mechanisms that may control Abcg2 expression in the lactating mouse mammary gland. Expression profiling of three different Abcg2 mRNA isoforms (E1a, E1b, and E1c) revealed that the E1b isoform is predominately expressed and induced in the lactating mammary gland. Despite this significant induction during lactation, the E1b promoter region in the virgin gland is already hypomethylated and enriched with the open chromatin histone mark H3K4me2. Next we used a forced‐weaning model to stop lactation and to rapidly turn off Stat5 activity in the mammary gland. Within 48h after forced weaning, there was a significant reduction in Abcg2 mRNA expression with a corresponding reduction in Stat5 occupancy at the mouse Abcg2 gene. Taken together, our results suggest that the Abcg2 gene is already poised for expression in the virgin mammary gland and that the binding of Stat5 plays an important role in the regulation of Abcg2 during lactation.

Grant Funding Source: Supported by CIHR.
Ginkgo biloba extract potentiates human vitamin D receptor‐mediated increase in CYP2B6 reporter activity by 1α,25‐dihydroxyvitamin D3 (1141.8)
Authors: Lau Aik Jiang , Chang Thomas
Published: June 2021

Vitamin D receptor (VDR) regulates the expression of a broad array of genes, including those involved in drug biotransformation (e.g. CYP2B6). Ginkgo biloba extract induces CYP2B6, but it is not known whether it activates VDR. Therefore, we characterized the effect of G. biloba extract on human VDR (hVDR) activity. As assessed in a cell‐based dual‐luciferase reporter gene assay, G. biloba extract did not activate hVDR in cells transfected with a hVDR expression plasmid and a CYP2B6‐PBREM/XREM reporter plasmid. In contrast, 1α,25‐dihydroxyvitamin D3, which is a hVDR agonist, increased it by 16‐fold compared to the vehicle‐treated group. The combination of G. biloba extract and 1α,25‐dihydroxyvitamin D3 further increased hVDR activity by another 14‐fold (i.e. 225‐fold increase compared to the vehicle‐treated group). This potentiation effect was evident in multiple lots of G. biloba extract and with other VDR agonists (i.e. lithocholic acid and 9‐cis retinoic acid), and it increased as the concentration of the extract or 1α,25‐dihydroxyvitamin D3 increased. In contrast, the potentiation did not occur when cells were transfected with a plasmid expressing human constitutive androstane receptor, human pregnane X receptor, or mouse VDR. Overall, the findings indicate that G. biloba extract potentiates hVDR‐mediated CYP2B6 reporter activity.

Grant Funding Source: Supported by the Canadian Institutes of Health Research and Michael Smith Fdn. for Health Res.
Mobilization of bone marrow stem/progenitor cells by AMD3100 or G‐CSF is impaired in diabetes (1142.4)
Authors: Vasam Goutham , Bartelmez Stephen , Jarajapu Yagna
Published: June 2021

The success of autologous cell therapies for the treatment of cardiovascular disease in patients with diabetes is dependent on mobilization of bone marrow derived stem/progenitor cells (BMSPCs). We tested the efficacy of bone marrow (BM) mobilizing agents in experimental diabetes. Mice with Streptozotocin‐induced type 1 diabetes were used either at 14 (short‐term, D‐ST) or 22 (long‐term, D‐LT) weeks of diabetes with respective age‐matched controls. BM‐derived stem/progenitor cells (BMSPCs), Sca‐1+ or c‐Kit+ subsets of lineage‐negative cells (LS, LK and LSK), in peripheral blood were enumerated by flow cytometry before and after administration of AMD3100 (5 mg/Kg, s.c.) or G‐CSF (125 mcg/Kg, s.c., twice a day for four days). Basal levels of LK and LSK cells in PB were decreased in both D‐ST and D‐LT compared to age‐matched controls (Ctrl, P<0.005). LS, LK and LSK were increased by AMD3100 or G‐CSF in D‐ST. In D‐ST group, mobilization of BMSPCs by AMD3100 or G‐CSF was similar to that in controls. In D‐LT group mobilization of all three subsets of BMSPCs by AMD3100 or G‐CSF was attenuated compared to the control group (P<0.005 or P<0.001, respectively). The number of SPCs in BM and the in vitro proliferation of BM‐LS cells were decreased in D‐LT (P<0.001 vs control). These results suggest that long‐term diabetes is associated with impaired mobilization of BMSPCs to clinically used mobilizers, decreased proliferative potential and reduced number of resident BMSPCs.

Grant Funding Source: Supported by NIH (P30 GM103332‐01) and AACP‐NIA
Identification of a urinary cyclized metformin‐methylglyoxal metabolite in metformin‐treated diabetes patients (1142.9)
Authors: Kinsky Owen , Anumol Tarun , Hargraves Tiffanie , Snyder Shane , Monks Terrence , Lau Serrine
Published: June 2021

Reactive dicarbonyls such as methylglyoxal (MG) are elevated in type‐2 diabetes mellitus (T2DM) patients and their ability to covalently modify proteins contributes to diabetic complications. The T2DM first‐line drug metformin (MF) significantly reduces diabetes‐related endpoints and mortality more effectively than other glucose‐lowering medications. We have examined whether, in addition to its ability to reduce hepatic gluconeogenesis, MF directly scavenges dicarbonyls as an additional mechanism to reduce T2DM complications. We synthesized a MF/MG cyclized product (183 mw) according to a published method, and characterized the product by ESI‐MS/MS mass spectrometry (MH+, 184 m/z; Agilent 6490) and 13C and 1H NMR (Bruker AVIII‐400). Using an LC‐MS‐based multiple reaction monitoring analysis we measured MF and cyclized metabolite (CM) in human urine with nM sensitivity of detection. The CM was detected in all 30+ MF‐treated T2DM subjects analyzed to date. Quantitation of CM in MF subjects is ongoing, using creatinine or specific gravity normalization. Correlations between CM levels and diabetic complications will be determined. The data reveal that urine from every T2DM patient treated with MF contains this MG scavenging product. The role of the CM in the reduction of diabetic complications warrants further study.

Grant Funding Source: Supported by DK090958, ABRC, ES016652, T32ES007091, P30ES006694.
Andrographolide downregulates pro‐inflammatory cytokines and free radical productions to prevent dopaminergic neuro‐degeneration induced by lipopolysaccharide (1143.16)
Authors: Sani Dahiru , Ramli Noorlela , Kirby Brian , Sumon Shariful , Basri Hamidon , Stanslas Johnson
Published: June 2021

Oxidative stress and inflammation triggers the production of free radicals and toxic pro‐inflammatory cytokines leading to neurodegeneration. Down‐regulation of oxidative stress and inflammatory mediators offers defence against development and progression of the disease. Andrographolide is the most abundant diterpene lactone isolated from the leaves of Andrographis paniculata. Despite the increasing literature studies on the anti‐inflammatory effect of andrographolide, there is still paucity regarding its neuroprotective role as can be ascertained from the search of literature. The present study investigated the potential therapeutic role of andrographolide as a neuroprotective agent via in vitro model of lipopolysaccharide (LPS)‐induced brain injury using microglial (BV2) and dopaminergic (N27) cells. Pre‐treatment of andrographolide at non‐cytotoxic concentration range (0.25‐2.0 µM) of BV2 cells followed by LPS dose‐dependently suppressed nitric oxide production, pro‐inflammatory cytokines (TNF‐α, IL‐6) as well as attenuating intracellular reactive oxygen species (ROS) and thiobarbituric acid reactive substance (TBARS) compared to cells without andrographolide pretreatment. Further, conditioned media (CM) of the pretreatment had diminished toxicity towards dopaminergic neurons as compared to CM without pretreatment.

Grant Funding Source: The Malaysian Ministry of Agriculture through NRGS grant allocation (NH0612D009)
Schizophrenia‐like behaviors in the offspring after maternal stress and prenatal NMDA receptor hypofunction (1144.9)
Authors: Lopez Tenee , Tejada‐Simon Maria
Published: June 2021

Pharmacological animal models have been developed and used to study the neurobiological deficits most commonly associated with schizophrenia. Evidence suggests that NMDA receptor (NMDAR) activity is an integral component in the pathophysiology of the disease, because antagonizing this receptor leads to schizophrenic‐like neurophysiological alterations and behaviors. In this study we sought to determine the influence of maternal NMDAR hypofunction combined with stress in the outcome of schizophrenia‐like behaviors of the offspring. After daily prenatal injections of MK‐801 and induction of stress in female mice, pregnancy was allowed to ensue and the litter was allowed to develop to P21‐P30. Analysis of schizophrenia‐like behaviors was performed on the offspring by measuring cognition as well as anxiety and other related behaviors. Our results suggest that maternal NMDAR dysfunction together with environmental stress impact the neurodevelopmental status of the offspring. These results not only strengthen the view that NMDAR function is involved in normal brain development, but also show that changes induced prenatally can affect the brains of the offspring.
The effects of doxorubicin on the mitochondrial dynamics and mitophagy machinery in varying types of skeletal muscles (1164.1)
Authors: Dirks‐Naylor Amie , Yang Sendra , Kouzi Samir
Published: June 2021

Doxorubicin (DOX) is an effective chemotherapeutic agent, known to cause cardiotoxicity via induction of oxidative stress and apoptosis. Despite toxic effects of DOX, few studies have investigated its effects in skeletal muscles. Multiple studies have examined DOX induced oxidative stress and mitochondrial dysfunction. However, no studies have investigated the effects of Dox on mitochondrial morphology and mitophagy. Thus, the first aim of the study was to determine the effects of DOX on the protein expression of primary regulators of mitochondrial morphology and mitophagy in skeletal muscle. The second aim was to investigate expression differences between three types of skeletal muscles varying in fiber type composition: diaphragm (DIA), extensor digitorum longus (EDL), and soleus (SOL). Male F344 rats were injected IP with 20 mg/kg of DOX or saline. Animals were fasted with no food or water until sacrifice 24 hours later. DOX treatment increased the protein content of DRP1, MFN2, and LC3‐I with no effect on FIS1, OPA1, MFN1, Parkin, PINK1, or p62. These effects were only observed in the DIA. Differences in expression levels between muscle types were present in FIS1, Parkin, LC3‐I, LC3‐II, and P62. In conclusion, DIA is more sensitive to the effects of DOX when compared to the EDL and SOL. Furthermore, the expression of regulators of mitochondria morphology and mitophagy varies among skeletal muscle types.
Ultrasonic bone stimulation increases orthostatic tolerance after 24 hours of bed rest (1171.6)
Authors: Ray Chester , Sauder Charity , Dyckman Damian
Published: June 2021

Our studies indicate that stimulation of the otolith organs by gravitational (head‐down rotation) and non‐gravitational (ultrasonic bone stimulation of mastoid; UBS) activation increases muscle sympathetic activity in humans. These findings suggest that otolith stimulation might assist in maintaining vascular resistance and arterial blood pressure during orthostatic challenges. To test this hypothesis, 16 subjects were placed in head‐down bed rest for 24 hours and then tested twice with an 80° head‐up tilt test once with and without UBS. The order of the two tilt tests was randomized with a maximum test duration of 30 min. Eight subjects were able to complete both tests after bed rest. In the remaining 8 subjects (2 M, 6 F, 26 ± 2 yr, 172.4 ± 3.0 cm, 74.6 ± 5.8 kg) who could not complete the tilt tests, tilt time was significantly greater in 7 of the 8 subjects with UBS compared to without (743 ± 180 vs. 574 ± 158 s, P = 0.002). Mean arterial pressure was 60±6 mmHg at the stoppage of the control tilt test and 90±5 mmHg at the same time point during UBS trial (P = 0.006). However, heart rate was unaffected by UBS during head‐up tilt (95 ± 5 and 93 ± 11 bpm for UBS and control, respectively). These findings indicate that UBS can improve orthostatic tolerance in subjects following bed rest. Similar results might be expected in subjects that experience orthostatic intolerance due to other reasons.

Grant Funding Source: Supported by NIH (HL109952), NSBRI (CA00404)
Transitioning to eupnea during the first hour after birth in term and preterm mice (1177.6)
Authors: Koschnitzky Jenna , Youngquist Tiffany , Smith Charles , Ramirez Jan‐Marino
Published: June 2021

We used lipopolysaccharide (LPS) to induce cytokine‐mediated inflammation and preterm labor. Dams injected with LPS gave birth on G18.5, one day early. Saline injected dams either gave birth at term (G19.5) or had a c‐section (C‐S) on G18.5. Breathing and cardiac function were then assessed <10 and >60 min. after birth. C‐S preterm and term animals had high survival rates (~94%). LPS preterm animals had low survival rates (43%). Survival corresponded with a fast transition from a low frequency, high tidal volume, long duration ('large') breath pattern to a eupneic pattern. Non‐survivors failed to make the transition. Heart rate was directly related to breathing rate and correlated with survival. However, cardiac activity continued after non‐survivors took their last breath indicating that respiratory, not cardiac, failure was the primary cause of death. A subset of animals that did not transition to a eupneic pattern were administered caffeine. Caffeine improved survival outcomes, however, after an anoxic bout these animals had delayed heart and breathing rate recovery times. In conclusion, LPS‐mediated preterm birth increases mortality rates. This is due to a failure to transition from an initial 'large' respiratory pattern to a eupneic pattern and may correspond with an inability to alter network dynamics when transitioning from a low to high oxygen environment.

Grant Funding Source: Supported by Seattle Children's Hospital Inter‐Center Funds
Recovery of respiratory motor function after repeated exposure to intermittent hypoxia in mice genetically depleted of brain serotonin (1178.8)
Authors: Mateika Jason , Richardson Chelsea , Solarewicz Julia , Angoa‐Perez Mariana , Nantwi Kwaku , Kuhn Donald
Published: June 2021

Purpose: Our study was designed to determine if daily exposure to intermittent hypoxia (IH) promotes recovery of respiratory motor function in mice following spinal cord hemisection and to determine if serotonin in the central nervous system is required to induce this process.

Methods: Telemetry transmitters were surgically implanted in 9 wild type mice (Tph2+/+) and 6 tryptophan hydroxylase 2 knockout mice (Tph2‐/‐) to measure electroencephalography, core body temperature and activity. Thereafter, a C2 hemisection was completed. Following recovery mice were exposed to 12 ‐ 4 minute episodes of hypoxia (10% oxygen) for 10 consecutive days while measures of ventilation were monitored via whole body plethysmography.

Results: Following 10 days of exposure to IH, tidal volume (VT) and ventilatory drive (VD) were enhanced compared to baseline measures on the initial day in the Tph2‐/‐ but not the Tph2+/+ mice (VT ‐ 1.25 ± 0.08 vs. 0.97 ± 0.03, p = 0.001; VD ‐ 1.28 ± 0.11 vs. 0.97 ± 0.04, p = 0.001; standardized to baseline on initial day), while breathing frequency was reduced in both groups (0.92 ± 0.04 vs. 0.92 ± 0.04, p = 0.02; standardized to baseline on initial day).

Conclusion: Recovery of respiratory motor function in spinal cord hemisected mice following repeated daily exposure to IH does not require central nervous system serotonin.

Grant Funding Source: Supported by VA award 1IO1 RX000458 (DMK) & 1I21RX001412 (JHM)
Obesity‐induced pancreatic tumor growth is accompanied by the accumulation of myeloid‐derived suppressor cells and altered metabolic mediators (123.6)
Authors: Collins Shawntawnee , Turbitt William , Rogers Connie
Published: June 2021

Obesity increases the risk of several solid tumors, including pancreatic cancer. One mechanism underlying the obesity‐induced increase in tumor incidence may be an obesity‐induced increase in tumor‐derived immunosuppressive factors. Thus, the goal of the current study was to evaluate the emergence and functionality of obesity induced myeloid derived suppressor cells (MDSCs) in our subcutaneous pancreatic tumor model (Panc.02). MDSCs inhibit anti‐tumor immunity, and expand significantly as a result of numerous tumor‐derived inflammatory signals. C57BL/6 mice (n=8/group) were fed either a 30% calorie restricted diet; a 10% kcal from fat diet fed ad libitum; or a 60% kcal from fat diet fed ad libitum to generate lean, control and obese mice, respectively. Mice were injected with 1 X106 Panc.02 cells after 16 weeks on each diet. In this study we observed a progressive accumulation of MDSCs between days 30‐60 post tumor implantation. The accumulation of MDSCs was significantly correlated with body weight (r=0.789, p<0.001); and changes in body weight were positively correlated with insulin (ρ=0.6328; p=0.0027), leptin (ρ =0.699; p<0.001), and monocytes chemoattractant protein‐1 (MCP‐1 (ρ=0.384; p=0.094). Obesity‐induced accumulation of MDSCs may be mediated by obesity‐induced inflammatory and metabolic mediators.

Grant Funding Source: Supported by National Institutes of Health (NIH), National Cancer Institute (NCI)