bluuebunny/crossref_metadata_2025 huggingface dataset:
SNPs Outside Response Elements Impact Aryl Hydrocarbon Receptor (AHR) Binding and Gene Regulation: Genome‐wide SNP‐dependent Transcriptional Regulation
Authors: Neavin Drew , Liu Duan , Li Hu , Wang Liewei , Weinshilboum Richard
Published: June 2021

BACKGROUND

Most functionally‐significant single nucleotide polymorphisms (SNPs) are outside of open reading frames. Mechanisms by which these SNPs contribute to gene regulation are poorly understood. Aryl hydrocarbon receptor (AHR) is a nuclear receptor that regulates drug metabolism, xenobiotic response and immune response. Our previous data indicate that SNPs outside response elements can impact transcription factor binding and gene regulation. We hypothesized that SNPs near but outside AHR response elements (AhREs) might impact AHR binding and gene regulation.

METHODS

We have collected dense SNP‐genotypes across 300 lymphoblastoid cell lines (LCLs) – a powerful tool for generating and testing pharmacogenomic hypotheses. Eight LCLs were used to generate hypotheses about AHR SNP‐dependent transcription utilizing RNA‐ and ChIP‐seq, which were tested in additional LCLs using qRT‐PCR and ChIP after AHR agonist or antagonist treatment.

RESULTS

69 genes were highly significantly differentially expressed after agonist or antagonist treatment and 80% of those genes had an AhRE within 50kb. Five SNP‐gene pairs have been functionally validated in additional LCLs (Table 1).

CONCLUSION

SNPs that are outside of AhREs can alter AHR binding and gene transcription. This is a general phenomenon that may have large‐scale influence on gene transcriptional regulation and pharmacogenomics.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of KRas‐Mediated Signaling by G‐Protein Coupled Receptors in Pancreatic Cancer
Authors: Feigin Michael E.
Published: June 2021

Pancreatic ductal adenocarcinoma is a highly lethal malignancy with few effective treatment options. We used computational approaches to identify new drug targets within this heterogeneous patient population, focusing on the G‐protein coupled receptors (GPCRs). GPCRs transduce extracellular signals from a variety of ligands through the plasma membrane, resulting in the modulation of intracellular signaling pathways and regulation of many physiological processes. This wide array of functions has resulted in the extensive utilization of GPCR targeted therapeutics, accounting for 30–50% of all currently used drugs. However, drugs targeting GPCRs are rarely utilized in cancer treatment, despite evidence that GPCRs mediate many aspects of tumorigenesis. Genomic analyses have uncovered GPCR mutations, copy number alterations and gene expression and methylation changes in a wide variety of cancers. Determining the biological implication of these genomic alterations will allow utilization of GPCR targeted therapeutics in those patients with GPCR‐driven tumors. Our preliminary evidence has uncovered the orphan GPCR GPRC5A as a critical regulator of the progression from early stage PanIN lesions to PDA.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect and mechanism of several polyphenols on inflammation related cancers
Authors: LI Xuejun
Published: June 2021

Inflammation plays a key role in the development and progression of cancers. Inhibition of inflammation will prevent carcinogenesis. Cancer and inflammation are linked by two pathways. The extrinsic pathway can be activated by inflammation or infection and the activation of the intrinsic pathway can be caused by oncogene activation. The two pathways co‐activate the transcription factors. The neutrophils are important part of the pathways. Previously, we had found that curcumin upregulated α1‐AT in cancer tissue, which is an endogenous inhibitor of neutrophil elastase (NE). Neutrophils and cancer cells are similar in character.

Firstly, we used the C57 mouse bearing lung cancer model to observe the effect of EGCG on cancer. EGCG could prolong the survival time of cancer mice, reduce cancer volume. At a lower dose of EGCG showed a significantly reduction on the number of metastases in lung tissue and reduced the MPO activity as well. EGCG reversed the migration of NE‐induced A549 cells, and inhibited the activity of NE in a dose‐dependent manner. Using 2D‐MS, we examined the differential protein profiling of EGCG in mouse cancer tissue and detected 12 differential proteins. Signal pathway enrichment analysis by ToppCluster indicated that the PI3K‐Akt‐mTOR signal pathway had the best network correlation. And the IRS‐1, NE substrate and 14‐3‐3e in the network had higher score than other proteins. EGCG up‐regulated α1‐AT, IRS‐1, inhibited PI3K/Akt. Molecular docking and SPR results revealed that EGCG could dosedependently bind to NE. In addition, we had found that curcumin antagonized α1‐AT mRNA levels and up‐regulates the activity of the α1‐AT promoter, indicating that the expression of α1‐AT is regulated at the transcriptional level. Moreover, molecular docking results showed that curcumin also dose‐dependently bound to NE. Our study shows that EGCG and curcumin may have a certain inhibitory effect on inflammation‐related cancers, and the mechanism is related to their up‐regulation on α1‐AT and their inhibition on NE, the both may be the potential drug target for cancer prevention and treatment.

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 Effects of Nitric Oxide Synthase Inhibitors on Mitochondrial Respiration in Isolated Mouse Brain Mitochondria
Authors: Sperling Jared A. , Sakamuri Siva SVP. , Sure Venkata N. , Dholakia Monica H. , Peterson Nicholas R. , Satou Ryousuke , Katakam Prasad VG.
Published: June 2021

Objective

Nitric oxide synthase (NOS) plays an important role in intracellular and intercellular signaling pathways. Experimental studies in mitochondria from various tissues, including the brain, have shown the possibility of the presence of a novel mitochondrial variant of NOS (mtNOS) located within the mitochondrial matrix. We hypothesized that administration of NOS inhibitors significantly affects the mitochondrial respiration in isolated brain mitochondria.

Methods

Mitochondria were isolated from the mice aged 8–12 weeks old and plated in 24‐well microplates for oxygen consumption rate (OCR) readings in the Seahorse XFe24 Analyzer. Mitochondria were incubated with inhibitors of nNOS (ARL‐17477 and N‐ω‐propyl‐L‐arginine), eNOS (N5‐(1‐iminoethyl)‐L‐ornithine), or a non‐specific NOS inhibitor (L‐NG‐Nitroarginine methyl ester) for 30 minutes prior to the mitochondrial stress test in the XFe24 Analyzer and compared to an untreated control group.

Results

Pharmacological inhibition of nNOS in isolated brain mitochondria reduced basal respiration and ATP production. Treatment with nNOS‐specific and non‐specific NOS inhibitors showed reduced basal OCR compared to control mitochondria. Control OCR values of 90.76±1.89 pmol/min were significantly higher than treatment with L‐NAME (74.17±2.12 pmol/min), NPA (77.92±2.83 pmol/min) and ARL (76.60±2.85 pmol/min). eNOS‐specific inhibitor treatment showed no significant change in mitochondrial respiration. ATP production as a bioenergetics parameter represents the portion of basal oxygen consumption that was used to drive ATP synthesis. Isolated mitochondria treated with non‐specific inhibitor L‐NAME and nNOS‐specific ARL‐17477 showed a significant decrease in ATP production. Isolated mitochondria treated with NPA show a slight trend towards decreased ATP production, but the statistical analysis did not show significance. Treatment with NIO demonstrated no significant change in ATP production.

Conclusions

Pharmacological inhibition of NOS isoforms affects brain mitochondrial respiration. Given the significant effects demonstrated by both nNOS‐specific inhibitors and L‐NAME, it may be postulated that mtNOS may be a new isoform of NOS sharing specific properties of the traditional three isoforms.

Support or Funding Information

American Heart Association (PVG: 14SDG20490359 and VNS: 16PRE31450006); National Institute of Health: National Institute of Neurological Disorders and Stroke and National Institute of General Medical Sciences (PVK: R01NS094834); and National Institute of Diabetes and Digestive and Kidney Diseases (RS: DK107694).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of ET‐1‐ and non‐ET‐1‐mediated endothelium‐dependent vasoconstriction in endotoxemic hypotensive shock
Authors: TSENG TZU‐LING , Chen MEI‐FANG , LIU CHIN‐HUNG , Lee Tony JF.
Published: June 2021

Effective managements of refractory hypotension to vasoconstrictors in severe sepsis are limited. A new strategy to ameliorate endotoxemic hypotension by inducing endothelium‐dependent constriction of endotoxemic large arteries was examined. In lipopolysaccharide (LPS)‐induced endotoxemic rats, oroxylin‐A (OroA, 15 mg/kg, iv) administered 6 hours after LPS‐challenge promptly reversed/normalized diminished mean arterial pressure and heart rate. OroA (1–10 μM), which did not constrict isolated normal arteries, repeatedly induced exclusive endothelium‐dependent sustained‐constriction without tachyphylaxis of isolated endotoxemic aortic and tail arteries. OroA‐induced constriction of mesenteric arteries with intact‐endothelium (EC+) was blocked by endothelin‐1 (ET‐1)‐receptor antagonists which, did not affect that of tail or aortic arteries, suggesting ET‐1‐mediated and non‐ET‐1‐substance(s)‐mediated vasoconstrictions, respectively. In parallel, repeated applications of ET‐1 to mesenteric arteries lead to significant tachyphylaxis, OroA, however, induced repeated constriction of tail arteries without tachyphylaxis, Moreover, OroA significantly decreased LPS‐induced expression of ET‐1 mRNA and proteins in tail arteries, while OroA increased those in the mesenteric arteries, suggesting the heterogeneities of the endothelial cells. OroA‐induced ET‐1‐mediated and non‐ET‐1‐substance(s)‐mediated constrictions of all isolated arteries (EC+) were blocked exclusively by ROCK inhibitors. OroA reversed LPS‐induced suppression of RhoA‐activities and enhanced ROCK‐phosphorylation only in arteries with endothelium. Activated muscle ROCK‐activities in mesenteric arteries (EC+) but not those in tail or aortic arteries (EC+) were blocked by ET‐1‐receptor antagonists. Moreover, OroA‐post‐treatment suppressed, via inhibiting NF‐κB, inducible‐NOS expression and circulating NO. LPS induces region‐dependent expression of endothelial ET‐1‐ and/or non‐ET‐1‐vasoconstrictors. Both ET‐1 and non‐ET‐1 were released specifically by OroA‐activated muscle RhoA/ROCK‐pathway, causing prompt‐and‐sustained vasoconstriction that partly is due to OroA‐induced decrease of circulating NO. Simultaneous preservation of endothelial functions, induction of endothelium‐dependent constriction of large arteries, and suppression of systemic inflammation, as shown by OroA, offer new strategies for acute management of endotoxemic‐hypotensive shock.

Support or Funding Information

supported by MOST & Tzu Chi Foundation

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 Sphingosine‐1‐Phosphate on Endothelial Barrier Function Following Hypoxic Injury and Hemorrhagic Shock
Authors: Alves Natascha Guimaraes , Yuan Sarah , Breslin Jerome William
Published: June 2021

Our lab has previously demonstrated that S1P reduces hemorrhagic shock and resuscitation (HSR)‐induced microvascular hyperpermeability. HSR can cause poor perfusion and hypoxia in the gut, leading to endothelial apoptosis and injury to the endothelial surface layer (glycocalyx). In the current study our objective was to investigate the extent to which S1P can prevent apoptotic signaling, shedding of the (glycocalyx), and elevated microvascular permeability in response to HSR in vivo or hypoxia in a cultured endothelial cell model. For our in vivo studies, we used a rat model of HSR, in which S1P was administered in the intravenous resuscitation fluid. The mesenteric microvasculature was dissected and protein was extracted for the assessment of pro‐apoptotic proteins by western blot. We also performed immunofluorescence of the mesenteric microvasculature to evaluate paracellular junction protein interaction. As an indicator of glycocalyx health, we used an intravenous injection of FITC‐labeled lectin and measured the amount of lectin bound to the microvascular endothelium. For the in vitro studies, we placed cultured primary endothelial cells in a low‐oxygen environment and applied S1P during the hypoxic challenge. We tested whether S1P has the potential to protect barrier function of endothelial cells grown on Transwell membranes (0.4 μm pores) from hypoxia‐induced hyperpermeability by determining permeability to FITC‐albumin. We have found that HSR causes an increase in pro‐apoptotic protein BAK in vivo, but S1P fails to blunt this increase. However, S1P seems to decrease levels of cytosolic cytochrome c compared to HSR‐treated rats. Furthermore, we found that the amount of lectin bound to the mesenteric microvasculature is higher in HSR animals treated with S1P compared to HSR only. In our in vitro studies, we found that S1P prevents hypoxia‐induced permeability in different types of endothelial cells, characterized by a decrease in endothelial permeability. Taken together, our results suggest that S1P has the potential to protect microvascular barrier dysfunction caused by HSR by preventing glycocalyx shedding. However, how S1P reduces the activation of apoptotic cascade following HSR remains elusive.

Support or Funding Information

Supported by NIH grants R01HL098215 and R01GM120774.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selective disruption of IGF‐1 signaling in astrocytes impairs neurovascular coupling in mice: implications for cerebromicrovascular aging
Authors: Tarantini Stefano , Fulop Gabor A. , Yabluchanskiy Andriy , Kiss Tamas , Sonntag William E. , Csiszar Anna , Ungvari Zoltan
Published: June 2021

Insulin‐like growth factor 1 (IGF‐1) exerts multifaceted protective effects on the cerebral microcirculation and age‐related reduction of circulating IGF‐1 levels is known to promote cognitive decline. Astrocyte‐mediated neurovascular coupling (NVC), which adjusts cerebral blood flow to increased energy requirements of activated neurons, plays a critical role in the maintenance of normal neuronal function. Although previous studies demonstrate that circulating IGF‐1 deficiency significantly impairs NVC, the role of astrocyte‐specific effects of IGF‐1 in regulation of NVC is not completely understood. On the basis of previous findings we hypothesized that disruption of IGF‐1 signaling specifically in astrocytes impairs NVC responses mediated by epoxyeicosatrienoic acids (EETs). To test this hypothesis, we assessed changes in local cerebral blood flow elicited by contralateral whisker stimulation in mice with astrocyte‐specific knock‐down of IGF‐1 receptor 1 (Igfr1f/fGfap‐Cre), and respective controls using laser speckle contrast imaging. We found that NVC responses were significantly impaired in Igfr1f/fGfap‐Cre mice (4±1%) as compared to that in control mice (10±1%). In control mice NVC responses were significantly decreased by pharmacological inhibition of EETs synthesis (MS‐PPOH) (4±1%). In contrast, NVC responses in Igfr1f/fGfap‐Cre were unaffected by MS‐PPOH. Collectively, our findings support the concept that normal astrocyte function is essential for the adequate NVC responses and provide evidence that decreased IGF‐1 input to astrocytes results in impaired EET mediation of NVC responses, which may contribute to cognitive decline associated with age‐related IGF‐1 deficiency.

Support or Funding Information

This work was supported by grants from the American Heart Association, the National Center for Complementary and Alternative Medicine, and the National Institute on Aging

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Cerebral Vascular Dysfunction on Alzheimer‐Like Cognitive Deficits in Diabetic T2DN rats
Authors: Fan Fan , Wang Shaoxun , He Xiaochen , Travis Olivia , Do Nga , Amaral Lorena , Cornelius Denise , LaMarca Birdie
Published: June 2021

Diabetes mellitus is the leading risk factor for cerebrovascular disease and vascular cognitive impairment. However, the underlying mechanisms remain to be determined. The present study examines whether cerebrovascular function is impaired in diabetic T2DN rat, and whether it induces inflammation and neurodegeneration that contribute to the development of Alzheimer‐like cognitive deficits. We found that cerebral blood flow rose by 137 ± 15% and 36 ± 5%, respectively, in T2DN and SD rats when MAP was increased from 100 to 180 mmHg. Levels of infiltrated T cells (CD3+CD4+) and macrophages (CD68+) were significantly higher in the brains of T2DN (506.6 ± 26.2 and 60.5 ± 13.0 cells/g, respectively) than SD rats (45.6 ± 18.5 and 13.0 ± 1.38 cells/g, respectively). Levels of IL‐1 β and IL‐2, and the expression of Amyloid β 42 (Aβ) and p‐tau (S416) was significantly higher in the brains of T2DN vs. SD rats. The T2DN rats exhibited neurodegeneration in the hippocampus and cortex. Elderly T2DN rats exhibited learning and short and long term memory disabilities. Latency of escape were longer in an eight‐arm water maze test in T2DN rats (2‐hour: T2DN 96 ± 12 vs. SD 13 ± 3 seconds; 24‐hour: T2DN 105 ± 15 vs. SD 8 ± 2 seconds), and they spent less time in the target arm 48 hours after removal of target platform (T2DN 3.4 ± 2.6 vs. SD 45.0 ± 1.7%). These results indicate that diabetes is associated with cerebral vascular dysfunction that induces inflammation and neurodegeneration and may all contribute to Alzheimer‐like cognitive deficits in T2DN rats.

Support or Funding Information

This study was supported by grants AG050049 (FF), P20GM104357 ( FF ) from the National Institutes of Health; 16GRNT31200036 (FF) from the American Heart Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Obesity in aging exacerbates neuroinflammation and alters eicosanoid profiles in the mouse hippocampus: potential role in impaired synaptic plasticity and cognitive decline
Authors: Kiss Tamas , Valcarcel‐Ares Marta Noa , Yabluchanskiy Andriy , Tarantini Stefano , Deak Ferenc , Ungvari Zoltan , Csiszar Anna
Published: June 2021

There is strong evidence that obesity has deleterious effects on cognitive function of older adults. Previous preclinical studies demonstrate that obesity in aging is associated with a heightened state of systemic inflammation, which exacerbates blood brain barrier disruption, promoting neuroinflammation and oxidative stress. To test the hypothesis that synergistic effects of obesity and aging on inflammatory processes exert deleterious effects on hippocampal function, young and aged C57BL/6 mice were rendered obese by chronic feeding of a high fat diet followed by measurement of long‐term potentiation (LTP) in acute hippocampal slices, assessment of changes in hippocampal expression of genes relevant for synaptic function and determination of synaptic spine density. Because there is increasing evidence that altered production of lipid mediators modulate LTP, neuroinflammation and neurovascular coupling responses, the effects of obesity on hippocampal levels of relevant eicosanoid mediators were also assessed. We found that aging exacerbates obesity‐induced microglia activation, which is associated with deficits in hippocampal‐dependent learning and memory tests, impaired LTP, decreased synaptic density and dysregulation of genes involved in regulation of synaptic plasticity. Obesity in aging also resulted in an altered hippocampal eicosanoid profile, including decreases in vasodilator and pro‐LTP epoxy‐eicosatrienoic acids (EETs). Collectively, our results taken together with previous findings suggest that obesity in aging promotes hippocampal inflammation, which in turn may contribute to synaptic dysfunction and cognitive impairment.

Support or Funding Information

This work was supported by grants from the American Heart Association, the National Center for Complementary and Alternative Medicine, and the National Institute on Aging

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Partial loss of Ca2+‐Activated K+ channel (KCa) in Rat Cerebral Arterial Muscle in Insulin‐dependent diabetes mellitus
Authors: Gebremedhin Debebe , Harder David Rae
Published: June 2021

Cerebral arteries depolarize and constrict upon pressurization. This is one of the intrinsic mechanisms underlying autoregulation of cerebral blood flow (CBF). The Ca2+‐activated K+ channel (KCa) channel in arterial muscle is an important target through which cerebral arterial response to changes in endogenous or environmental stimulatory factors are sensed. The level of open state probability (NPo) of KCa channels determines the degree of myogenic tone and cytosolic Ca2+ concentration. In the present study we report impaired pressure‐dependent constriction of cerebral arteries and diminished density and NPo of KCa channel currents associated with reduced expression of KCa channel α‐ and β‐subunit proteins in cerebral arteries of genetically insulin‐dependent diabetic rats. In this study we also found that the expression of the CYP 4A ω‐hydroxylase protein and the production of 20‐HETE was significantly increased in the type 1 diabetic cerebral arteries compared to that in the non‐diabetic cerebral arteries. The observed changes in KCa channel function and expression of CYP 4A ω‐hydroxylase and production level of 20‐HETE could represent an important pathologic mechanisms that could underlie abnormalities in cerebral arterial reactivity in insulin‐dependent diabetes mellitus. It is concluded that the diminished KCa channel function, increased production of 20‐HETE and altered pressure‐dependent cerebral arterial constriction in insulin‐dependent diabetes mellitus may lead to impaired autoregulation of CBF that could contribute to increased risk of stroke in this disease condition.

Support or Funding Information

NIH RO1 HL0338833

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prefrontal and muscle oxygenation responses to supraventricular tachycardia
Authors: Ishii Kei , Machino Takeshi , Yamagami Fumi , Nogami Akihiko , Aonuma Kazutaka , Tsurushima Hideo , Hatori Yasuhiro , Gwak Jongseong , Takahashi Naoki , Komine Hidehiko , Kitazaki Satoshi , Akamatsu Motoyuki
Published: June 2021

Supraventricular tachycardia (SVT) causes hypotension and cerebral hypoperfusion leading to syncope at times. The cerebral and peripheral vascular systems probably operate to maintain cerebral oxygenation against SVT‐induced hypoperfusion. However, time courses of cerebral and peripheral oxygenation responses to SVT has not been fully understood. The aim of this study was to examine the time course and relationship between cerebral and peripheral oxygenation responses and SVT‐induced hypotension. Prefrontal and forearm tissue oxygen index (TOI) and normalized tissue hemoglobin index (nTHI) were collected by near‐infrared spectroscopy (NIRS) during electrophysiological study (EPS) in 14 patients (55 ± 4 years old, 9 men) with SVT. AP decreased during 2–20 s of SVT. The prefrontal TOI and nTHI began to decrease (P < 0.05) transiently at 6 s of SVT and then returned to the baseline level from 14 s. The initial decrease of prefrontal TOI appears to occur when hypotension was over approximately 20 mmHg. In contrast, the forearm TOI and nTHI decreased continuously from 11 s (P < 0.05). The AP and prefrontal NIRS responses suggest that SVT‐induced great hypotension (>20 mmHg) caused the transient reduction of cerebral oxygenation and blood volume, which may be followed by a recovering process owing to cerebral vasodilatation. The continuous reductions of forearm TOI and nTHI during SVT suggest restriction of muscle blood flow by vasoconstriction, which may contribute to redistribution of the blood to the brain.

Support or Funding Information

This research is supported by Automotive and Medical Concert Consortium (AMECC).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mental Stress Increases Cerebral Perfusion, More So During Hypercapnia
Authors: Shoemaker Leena , Wilson Luke , Lucas Sam , Machado Liana , Cotter James
Published: June 2021

Acute mental stress has been shown to cause peripheral vascular dysfunction, but its effect on cerebrovasculature is unclear. Thus, the aim of this study was to examine cerebrovascular and hemodynamic responses to mental stress. Sixteen healthy participants (aged 23±4 y, 5 female) completed seated conditions of baseline, followed by low and high mental stress. Stress conditions were mental arithmetic at two levels of difficulty (i.e., counting backwards from 1000 and subtracting 13 from a frequently changing 4‐digit number), which successfully manipulated reported stress levels (p<0.001 between conditions). During each condition, end‐tidal CO2 pressure (PETCO2) was clamped at normocapnia and two elevated levels (p<0.001): 37±3, 41±1 and 46±1 mm Hg. Middle cerebral artery blood velocity (MCAv) was measured using transcranial Doppler ultrasound, and right prefrontal cortex hemodynamics measured using near‐infrared spectroscopy (NIRS). NIRS provided variables of oxygenated hemoglobin (HbO2), deoxygenated hemoglobin (HHb), total hemoglobin (tHb), and depth‐resolved measures of tissue oxygenation (total oxygenation index, TOI). Blood pressure was measured beat‐by‐beat using finger photoplethysmography, to calculate mean arterial pressure (MAP). Stroke volume and cardiac output (Q̇) were derived with Modelflow. Cerebrovascular reactivity to CO2 (CVRCO2, ΔMCAv/ΔPETCO2), cerebrovascular conductance (CVC, MCAv/MAP), and total peripheral resistance (TPR, MAP/Q̇) were calculated. Effects of mental stress and CO2 were assessed using 2‐way repeated measures ANOVA. The acute mental stress increased MCAv, and more so at higher PETCO2 (interaction: p=0.015). No other physiological variable showed interactive responses (p>0.069). Compared to baseline, low stress increased HR, MAP, TOI, HbO2, and decreased HHb (all p<0.003), but these variables were not altered thereafter with high stress (p>0.05; low vs. high stress). In contrast, CVRCO2 was elevated during high stress only (p=0.017 vs. baseline; p=0.706 for low stress vs. baseline). Finally, neither stress nor CO2 significantly influenced TPR (p≥0.241). In conclusion, mental stress increased MCAv and this was potentiated during hypercapnia. Increased cerebral perfusion during mental stress was mediated by cardiac‐driven increases in MAP. Hypercapnic‐induced blunting of cerebral autoregulation indicates that there may be a greater risk of cerebral overperfusion (e.g., hemorrhagic stroke) as a consequence of mental stress when innate cerebral autoregulation processes are impaired (e.g. early morning).

Support or Funding Information

Internal postgraduate research fund ‐ The School of Physical Education, Sport and Exercise Sciences

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Blood Pressure in Wild‐type and Ts65Dn Mice at 6 Months of Age
Authors: DeRuisseau Lara R. , Buchholz Haley , Purdy Rebecca , Receno Candace , Cunningham Caitlin
Published: June 2021

Down syndrome (Ds) results from triplication of the 21st chromosome, and is estimated to occur 1 in every 700 births in the United States. Many people with Ds exhibit cardiovascular challenges including pulmonary hypertension and lower resting blood pressure. We tested the hypothesis that Ts65Dn mice, a model of Ds, would exhibit lower blood pressure compared to wild‐type mice. The CODA volume‐pressure tail cuff system by Kent Scientific (Torrington, CT) was used to quantify systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) in 6–month wild‐type (n=9) and Ts65Dn (n=7) male mice. Two trial days were administered for each mouse that included 15 inflation/deflation cycles of the cuff on each day. The trial was considered successful if ≥7 of the 15 cycles were accepted by the software. The mean of the 7–15 cycles was calculated for each mouse (DBP, SBP and MAP separated). If the 2nd trial mean differed by >10 mmHg for SBP the mouse was excluded from the analysis. Data are shown as wild‐type vs. Ts65Dn; MEAN ± SD; * = p<0.05. Ts65Dn mice displayed a reduced SBP (104 ± 9 vs. *96 ± 4 mmHg). DBP (77 ± 9 vs. 69 ± 5 mmHg) and MAP (85 ± 9 vs. 78 ± 4 mmHg) were not different in this early analysis. These data suggest that Ts65Dn mice may be an appropriate model to use in understanding cardiovascular function in Ds. Further investigation is warranted to confirm these findings across the lifespan of wild‐type and Ts65Dn mice.

Support or Funding Information

Funded by 1 R15 HD076379‐01A1 (LRD), HKB supported by the Ignite fellowship and Le Moyne College Student Research Committee, CNR supported by 1 R15 HD076379‐01A1S1.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hemodynamic Hyper‐reactivity to Acute Stress in Individuals Reporting Adversity during Childhood: Role of Endothelin‐1
Authors: Tucker Matthew A. , Looney Jacob , Blackburn Marsha , Pollock Jennifer S. , Pollock David M. , Harris Ryan A.
Published: June 2021

Individuals exposed to adverse childhood experiences (ACE) have an increased risk of developing cardiovascular disease versus those with no ACE. Moreover, these individuals also exhibit an exaggerated blood pressure response to an acute sympathetic stressor; a response that is associated with an increased risk of developing hypertension and shown to be mediated by endothelin‐1 (ET‐1) in animal models. The role of ET‐1 as a modulator of the hemodynamic response to acute stress in humans with high ACE, however, is unknown.

Purpose

To compare hemodynamic and plasma ET‐1 responses to a cold pressor test (CPT) in individuals who reported high ACE versus matched controls with no reported ACE.

Methods

14 participants volunteered for this investigation; 7 of which reported an ACE score ≥4 via questionnaire (HI‐ACE; 5 men, 2 women) and 7 control participants (CON) who were matched for age, ethnicity, sex, and body composition. All participants underwent a CPT protocol which consisted of a 5 min baseline followed by immersion of the right hand up to the wrist in ice water (~2°C) for 3 min. Hemodynamic parameters including total peripheral resistance index (TPRI), cardiac index (CI), and systolic/diastolic blood pressure (SBP/DBP) were measured throughout the protocol using beat‐to‐beat monitoring of finger arterial pressure (Finapres NOVA). Plasma concentrations of ET‐1 were determined at baseline and during the second minute of the CPT.

Results

There were no differences (all p>0.05) between groups in age, ethnicity, sex, body composition, or fasting blood glucose. Similarly, there were no differences (all p>0.05) in baseline values of TPRI (0.39 ± 0.31 vs. 0.43 ± 0.25 mmHg/ml/m2), CI (3.5 ± 0.8 vs. 3.5 ± 0.6 L/min/m2), SBP (134 ± 14 vs. 133 ± 12 mmHg), or DBP (83 ± 10 vs. 85 ± 11 mmHg) between HI‐ACE and CON. During CPT, a significantly greater increase in SBP (48 ± 20 vs. 29 ± 10 mmHg; p=0.041) and DBP (35 ± 15 vs. 19 ± 5 mmHg; p=0.028) occurred in HI‐ACE compared to CON. While concentrations of ET‐1 at baseline were similar between HI‐ACE and CON (1.28 ± 0.21 vs. 1.22 ± 0.33 pg/ml, respectively), the ET‐1 response during CPT tended (p=0.098) to be higher in HI‐ACE versus CON (Δ1.12 ± 0.03 vs. Δ0.50 ± 0.37 pg/ml).

Conclusion

These data suggest that greater plasma concentrations of ET‐1 accompany the exaggerated hemodynamic response to a CPT in individuals who reported high ACE. Future studies are warranted to further elucidate the role of ET‐1 as a mediator of hemodynamic responses to acute stress in individuals with high ACE.

Support or Funding Information

Supported in part by NIH/NHLBI P01HL06999.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Responses in the Splenic CD4+ T Cell Proteome Following Ang II‐Induced Hypertension in VCD‐Treated Menopausal Mice
Authors: Husband Nathaniel A. , Moffett Caitlin , Romero‐Aleshire Melissa J. , Uhlorn Joshua A. , Uhrlaub Jennifer , Barker Natalie , Nunez Franchesca , Nikolich‐Zugich Janko , Langlais Paul , Brooks Heddwen L.
Published: June 2021

Cycling female mice are protected against Angiotensin II (Ang II) hypertension, and inducing ovarian failure (menopause) eliminates this protection. T lymphocytes are required for the development of Ang II hypertension in male mice, however premenopausal females are resistant to T cell‐induced hypertension. Following menopause (loss of estrogen) resistance is lost, allowing activation of T cell‐induced hypertension in postmenopausal females. The purpose of this study was to identify, in vivo, T cell‐specific proteome responses to Ang II, before and after menopause. 10‐week‐old C57BL/6 female mice received i.p. 4‐vinylcyclohexene diepoxide (VCD) injections for 20 consecutive days to induce ovarian failure (VCD menopause model). Cyclicity was monitored daily via vaginal cytology. Once in menopause, Ang II was infused (800 ng/kg/min) for 14 days, which has been shown to cause an increase in SBP of 25 mmHg. Ang II in VCD‐treated menopausal females (VCD/AngII) resulted in significantly decreased heart rates versus controls (Control 682 ± 5.6 vs. VCD/AngII 592.5 ± 28.5, p < 0.05). Splenic CD4+ T cells were purified via negative immune‐magnetic selection and CD4+ purity was measured via flow cytometry (>90% purity). Proteomic analysis was performed on control, pre‐menopausal/Ang II and VCD/AngII mice (n=4 per group). Protein lysates from the CD4+ T cells were separated by SDS‐PAGE and subjected to in‐gel tryptic digestion followed by tandem mass spectrometry analysis, resulting in 7,123 proteins identified across the entire experiment. Quantitative proteomics were performed via label‐free quantification using Progenesis software‐based extracted ion abundance. Of the 5,857 proteins identified with two or more unique peptides, 474 of the proteins exhibited significant abundance differences between control, Ang II and VCD/Ang II groups as assessed by One‐Way ANOVA statistical analysis (p ≤ 0.05). Gene Ontology (GO) enrichment analysis of these 474 proteins identified 159 GO biological pathways that were significantly overrepresented (p ≤ 0.05). Pathways associated with increased inflammation and reduced inhibition of inflammatory cytokines were among those identified. Using IHC we show that renal macrophage infiltration is significantly increased in VCD/Ang II mice compared to Ang II and control groups. Overall, our data suggests that following Ang II infusion, splenic T cells are differently impacted by the loss of estrogen, leading to an increased pro‐hypertensive cytokine milieu compared to pre‐menopausal Ang II infused mice.

Support or Funding Information

R01HL131834 (HLB)Sarver Heart Center Endowment for Women's Health Research (HLB)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of the RNA binding protein HuR reduces cardiac cell death following ischemia/reperfusion injury
Authors: Slone Samuel , Anthony Sarah R. , Lanzillotta Lindsey , Nieman Michelle L. , Green Lisa C. , Lorenz John N. , Tranter Michael
Published: June 2021

Despite medical advances, ischemic cardiac injury (ischemia/reperfusion (I/R) injury or myocardial infarction) remains a leading cause of morbidity and huge economic burden in the United States. It is known that the extent of total damage to the heart following I/R injury is directly linked to the percentage of affected individual cardiac myocytes that undergo apoptotic cell death. RNA binding proteins are becoming recognized as potential mediators of cardiac physiology and pathology, but the role of HuR, an RNA binding protein highly expressed in myocytes, in acute cardiac I/R injury is unknown. It is widely accepted that after an ischemic injury, reperfusion, the return of blood flow and therefore oxygen, introduces reactive oxygen species (ROS) that further activates downstream pro‐apoptotic pathways. We have previously shown HuR to be activated downstream of p38 MAPK in cardiac myocytes; since p38 MAPK is known to be downstream of ROS, we postulated that HuR is a potential mediator of apoptotic gene expression during cardiac I/R injury.

To address the functional role of HuR in I/R, we utilized an inducible cardiomyocyte‐specific HuR deletion mouse (iCM‐HuR−/−) and subjected these mice to 30 minutes of LAD (left anterior descending artery) ligation followed by 24 hours of reperfusion. Analysis of infarct size showed that iCM‐HuR−/− mice had a significantly smaller infarct compared to control mice (41% infarct/risk in iCM‐HuR−/− vs. 51% infarct/risk in control, N=3, P<0.05). To identify an underlying mechanism for this cardioprotective effect, we performed simulated I/R (simI/R) in vitro on neonatal rat ventricular myocytes (NRVMs) by subjecting them to 6 hrs of simulated ischemia (glucose deprivation and hypoxia at ≤1% O2) followed by 24 hrs of reperfusion (reoxygenation and reintroduction of glucose). Similar to our in vivo results, siRNA‐mediated knockdown or pharmacological inhibition of HuR significantly reduced simI/R‐induced cell death and caspase‐3 activity. We also observed an increase in expression of key apoptotic genes (Bcl‐2, Bad, and Bax) in NRVMs following simI/R, and ongoing work will identify the specific role of HuR in mediating expression of these genes.

In conclusion, our results suggest that ablation of HuR results in a significant reduction in infarct size following I/R injury through a reduction in caspase 3‐dependent apoptotic cell death.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endoplasmic Reticulum Stress Mediated Inflammation may involve in Doxorubicin‐induced Cardiomyopathy
Authors: Bagchi Ashim K. , Vashisht Vandana , Akolkar Gauri , Singal Pawan K.
Published: June 2021

Many subcellular signaling pathways have been proposed to explain myocardial dysfunction triggered by doxorubicin (Dox). Although endoplasmic reticulum (ER) stress has been suggested to be an important factor in myocardium dysfunction, its role in Dox‐induced cardiomyopathy (DIC) has not been fully understood. Here, we show that ER stress promotes inflammation through iNOS‐regulated Toll‐like receptor 2 (TLR 2) activation in DIC. Cardiomyopathy in male Wistar rats was produced by repeat six equal injections of Dox (each 2.5 mg/kg ip) over three weeks for a cumulative dose of 15 mg/kg body wt. Controls were given only saline. Hearts from these animals were used two weeks after the last treatment. ER stress in the DIC hearts was indicated by an upregulation of binding immunoglobulin proteins (Bip). Furthermore, there was an increased unfolded protein response and dimerization of Bip with ATF6. This increase in ER stress in DIC promoted iNOS activity. The latter was associated with increased expression of TLR2 and TRAF2 together with increased NFκB105/50 expression. This in‐turn promoted pro‐inflammatory cytokines TNF‐ α, and IL‐1α in the myocardium. Overall these data indicate that inflammation, mediated by ER stress, via a direct interaction of Bip and ATF6, upregulates iNOS as well as pro‐inflammatory cytokine production in DIC. Manipulation of this complex signaling pathway may be of therapeutic benefit to prevent cardiomyopathy due to Dox.

Support or Funding Information

Supported by CIHR and Research Manitoba

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High glucose induces Glucose transporter‐2 (GLUT2), but not Na‐glucose cotransporter‐1 (SGLT1) mediated glucose absorption
Authors: Walton Nerissa , Rhodes Ryon Sun , Rajendran Vazhaikkurichi M. , Coon Steven D.
Published: June 2021

Background

Glucose dependent insulinotropic peptide (GIP) is a hormone that has a cyclic pathway in the stimulation of glucose transport. GIP increases transport of glucose, which in turn stimulates GIP secretion. This cycle increases the jejunal absorption of glucose, which stimulates insulin release. High glucose results in excess GIP secretion that causes unusually high glucose absorption and high blood glucose that stimulates insulin release that causes insulin insensitivity. Presence of high blood glucose and insulin lead to obesity and Type‐2 diabetes. Although SGLT1 is the primary apical glucose absorptive process, GLUT2 has been localized on the intestinal apical membranes of obese human and mouse models. However, signaling mechanisms of how GIP regulates glucose transporters in the presence of normal versus high levels of extracellular glucose concentrations is not known.

Hypothesis

Extracellular glucose concentration that alters cellular GIP level would regulate glucose transporters on to the apical membrane of differentiated cells.

Methods

Intestinal epithelial crypt cell‐6 (IEC6 cells) where grown 5‐days post confluent on transwell snapwells using standard DMEM media supplemented with 5% FBS and either 4.5 or 0.45 g/liter of glucose for high and low glucose, respectively. Mucosal to serosal (m‐s) glucose fluxes were measured across IEC6 cell layers that were mounted under voltage clamp conditions in Ussing chambers. Short circuit currents were measured to distinguish the SGLT1 (i.e. electrogenic) and GLUT2 (i.e. electroneutral) mediated glucose absorption. Apical membranes were isolated from IEC6 cell monolayers using a biotinylation technique. Western blot analyses were performed using SGLT1 and GLUT2 antibodies.

Results

Glucose absorption is substantially higher in IEC6 cells that were grown in high glucose medium compared to that grown in low glucose medium. In contrast to increased rate of glucose absorption, Isc is not significantly altered as an indication that high glucose increased GLUT2 mediated, but not SGLT1 mediated glucose absorption in IEC6 cells. Western blot analyses revealed that GLUT2, but not SGLT1 specific protein expression is increased on the apical membrane of IEC6 cells grown in the high glucose medium.

Conclusions

High extracellular glucose environment stimulates GLUT2, but not SGLT1 expression and GLUT2 mediated glucose absorption in intestinal epithelial cells.

Speculation

We speculate that GLUT2 might be regulated similarly in in the villus cells of mouse intestine.

Support or Funding Information

IMBRE grant #G110‐18‐W6619

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 Protective Role of TRPC6 Knockout in the Progression of Diabetic Nephropathy
Authors: Spires Denisha , Ilatovskaya Daria V. , Levchenko Vladislav , Palygin Oleg , Staruschenko Alexander
Published: June 2021

Diabetic nephropathy (DN) is a chronic kidney pathology that leads to end stage renal disease (ESRD). It was previously found that a gain‐of‐function mutation in the Transient Receptor Potential Canonical 6 (TRPC6) channel, a non‐selective cation channel that is highly permeable for calcium ions, causes Focal Segmental Glomerulosclerosis (FSG) that is characterized with podocyte loss. Previous studies from our laboratory indicate that there is an association between the development of DN and over‐activation of the TRPC6 channel. We have demonstrated that there is an increase in TRPC6 expression and activity in the streptozotocin (STZ)‐induced diabetic Dahl Salt‐sensitive (SS) rat, an established model of type 1 DN. We have created the TRPC6 knock‐out on a SS rat background (SSTRPC6−/−), and test the hypothesis that the deletion of TRPC6 will protect podocytes and glomeruli during the development of DN. Four groups of animals – control SS, SSTRPC6−/−, and STZ‐treated SS and SSTRPC6−/− – were utilized in this study. Diabetes development was monitored for 11 weeks after STZ injection with periodic weight, glucose, and urinary output measurements. Glomerular filtration rate (GFR) was measured and tissue collections were made at the end of the protocol. Freshly isolated glomeruli of perfused kidneys were used from all four groups for either electron microscopy or confocal fluorescence microscopy to assess the foot process effacement and basal level of intracellular calcium ([Ca2+]i), respectively. Histochemical analysis of whole kidney slices was done to assess renal injury. There was a decrease in urinary output (49±29 vs 73±39 mL/day/100g BW) and a trend for blunted increase in albuminuria (97±34 vs 112±19 mg/day x 100g BW) in the STZ‐treated SSTRPC6−/− rats compared to the STZ‐SS animals. There was no significant difference in GFR between either of the 4 experimental groups; however there does appear to be a trend for hyperfiltration in the STZ‐treated animals. Importantly, pathological increases in basal [Ca2+]i levels and foot process damage of podocytes during the development of DN was attenuated in the STZ‐treated SSTRPC6−/− animals. Overall, our data indicates that inhibiting the TRPC6 channel may have renoprotective effects and the potential to lead to the development of new pharmacological tools to treat or prevent the progression of DN and kidney damage.

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