bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of Bioavailable NO with Enhanced iNOS Expression and Oxidative Stress Increase Pulmonary Arteriolar Tone in Aged Mice
Authors: Drinovac Robert , Jagadapillai Rekha , Gozal Evelyne , Lin Xingyu , Vaishnav Radhika , Friedland Robert P. , Roberts Andrew M.
Published: November 2023

Aging impairs blood vessel function and leads to cardiovascular disease. Vascular dysregulation with aging, caused by endothelial dysfunction in numerous vascular beds, has been reported as a consequence of decreased bioavailable nitric oxide (NO). However, the effect of endothelial dysfunction with aging on pulmonary microcirculation is not well characterized. We hypothesized that aging causes pulmonary microvascular dysfunction by decreasing lung NO bioavailability, resulting in the constriction of pulmonary arterioles. To determine if decreased NO bioavailability in the lung, as occurs in aging, affects pulmonary vascular function, we assessed the effect of aging on pulmonary arteriolar responses in C57BL/6J mice (11–32 months old). Pulmonary microvascular diameters (ranging from 25–54 μm) were examined in the right lungs of anesthetized mice with open chest by intra‐vital microscopy before and after the nitric oxide synthase (NOS) inhibitor, L‐NAME, was administered (0.1mg/g body wt) to produce acute NOS inhibition. Pulmonary arteriolar tone in the older mice (> 2 years 4 months) did not significantly increase (1.4 ± 2.8%, mean ± S.D, n=4) after L‐NAME treatment, suggesting that NO synthesis is decreased in aging mice. In contrast, in the younger mice (≤ 2 years 4 months), pulmonary arteriolar tone increased by 11.5 ± 9.4% (n=4, P < 0.05) after L‐NAME. Expression of inducible NOS (iNOS) and TNF‐α in lung tissue increased with the age of the mice indicating a greater level of oxidative stress and inflammation. These results suggest that, with aging, a loss in the bioavailability of NO and increased oxidative stress could impair the lung's ability to maintain a low microvascular resistance, which could affect alveolar perfusion.
Cutaneous Thermal Injury Increases Vascular Permeability of Rat Mesenteric Venules by a Reactive Oxidant‐Dependent Mechanism
Authors: Pfitzinger Duke M. , Howard James M. , Moncure Michael , Wood John
Published: November 2023

Background

Following major burns, edema develops in systemic organs and contributes to the development of remote organ dysfunction. The underlying mechanisms responsible for microvascular inflammation in this setting are not yet fully known. The purpose of our study was to determine if thermal injury to the skin of rats would increase vascular permeability in the mesenteric microcirculation, and if the mechanism involved generation of reactive oxidants.

Methods

Intravital microscopy was used to examine mesenteric venules of anesthetized rats. FITC‐albumin was used to determine a vascular permeability index (VPI) based on the ratio of extravascular to intravascular fluorescence. Thermal injury was induced by exposing skin of animals to steam for 1 min (~40% of body surface area). Control animals were not exposed to steam. Vehicle or the antioxidant lipoic acid (2 mg/kg bolus, 0.5 mg/kg/h) was administered i.v. to animals after steam injury.

Results

VPI significantly increased after burn (0.39±0.13 at 30 min vs 0.61±0.08 at 4 h, p<0.05, n=5), but not in non‐burned animals (0.08±0.09 at 30 min vs 0.12±0.03 at 4 h, n=5). These values for VPI were significantly different at these times in burned and non‐burned animals (p<0.05). Lipoic acid significantly attenuated VPI in burn animals (0.20±0.06 at 30 min and 0.23±0.05 at 4 h, n=5) compared to vehicle‐treated burn animals (p<0.05).

Conclusions

Thermal injury to the skin of rats increased vascular permeability in the mesenteric microcirculation of rats. Lipoic acid attenuated this increase which supports a role for increased generation of reactive oxidants in this mechanism.

Support or Funding Information

Department of Surgery Research Funds
Decreased Large Conduit Artery Nitric Oxide Bioavailability in Metabolic Syndrome Effects Peripheral Microvessel Reactivity
Authors: Branyan Kayla , DeVallance Evan , Lemaster Kent , Skinner Roy , Brooks Steve , Asano Shinichi , Willimas Justin , Frisbee Jefferson , Chantler Paul
Published: November 2023

One out of every five adults in the U.S. are considered to have multiple components of Metabolic Syndrome (MetS). These components include dyslipidemia, hypertension, insulin resistance, and a chronic pro‐inflammatory state, which significantly increase the risk for poor cardiovascular outcomes. Nitric oxide (NO) bioavailability is decreased in MetS, leading to vascular dysfunction. With this decrease in vascular function, there is an increased risk of poor cardiovascular outcomes. A common animal model used to mimic MetS is the obese Zucker rat (OZR) whereas its lean counterpart is used as a healthy control. The purpose of this study was to determine the extent of impairment in NO bioavailability in large conduit arteries and compare that to the reactivity of the related microvessels, such as the gracilis arteriole. Specifically, a DAF‐FM diacetate assay was used to determine the degree of NO production in the aorta. To assess microvessel reactivity, the gracilis arteriole was isolated, hung in a pressurized microvessel myobath, and was exposed to increasing doses of acetylcholine to measure endothelial function. TEMPOL was also used to determine the role of oxidant stress in modulating reactivity via vasodilation. LZRs had a higher overall NO bioavailability as compared to OZRs (p<0.05). This was reflected in gracilis reactivity where the LZRs had increased gracilis reactivity than OZRs overall (p<0.01). With TEMPOL, percent dilation was doubled in the gracilis of OZRs (p<0.01) but this effect was not seen in the LZRs. These results suggest that oxidant stress causes vascular dysfunction and that a higher NO bioavailability correlates with better microvascular reactivity. When a membrane‐permeable radical scavenger is introduced, reactive oxygen species that are causing the oxidative stress can be decreased to improve reactivity in a MetS model.

Support or Funding Information

AHA

NIH
Effects of Acute Dietary Polyphenols Supplementation on Hemodynamic Responses to Dynamic Exercise in Young Prehypertensive Men
Authors: Choi Hyun‐Min , Kim Kyung‐Ae , Doh Hyung‐Woo , Nho Hosung , Park Seung‐Kook , Yang Seung‐Won , Kim Jong‐Kyung
Published: November 2023

We found previously that exercise may evoke an exaggerated blood pressure (BP) response in prehypertensive individuals due to an increase in peripheral vasoconstriction induced by endothelial dysfunction. It has been reported that dietary polyphenols extracted from grape seed reduces ambulatory blood pressure in hypertensive animals. To examine whether acute dietary polyphenols attenuate BP response to dynamic exercise prehypertensive men. In a double‐blind, randomized, crossover study, 10 prehypertensives (23–27 yrs) consumed polyphenol capsule (PC; 600 mg) or polyphenol‐depleted placebo (PL; 600 mg). The subjects were continuously instrumented to measure heart rate, stroke volume, cardiac output (CO) and mean arterial pressure (MAP) during cycling exercise ranging from low to moderate workloads corresponding to 40% and 60% of VO2peak. Endothelial function is assessed via flow‐mediate dilation (FMD) before and after each supplementation. After PC supplementation, FMD was significantly increased compared to the PL. PC supplementation significantly decreased MAP at rest and across workloads and lowered total peripheral resistance (TPR) [TPR=MAP/(CO×1000)] during exercise compared to the PL. CO was also during exercise. We demonstrated that acute dietary polyphenols supplementation attenuated exercising BP via improved endothelial function in prehypertensive men. This study suggests that such supplements may be useful as a treatment to help reduce both resting and exercising BP in prehypertensive individuals.

Event
Condition
MAP (mmHg)
CO (L/min)
TPR (ml/min/mmHg)

Rest
PC
100±1.3 vs. 98±1.0*
4.2±0.2 vs. 4.5±0.2
24.3±1.1 vs. 22.3±1.3

PL
99±1.1 vs. 98±1.3
4.4±0.1 vs. 4.5±0.2
22.9±0.7 vs. 22.0±0.9

40%
PC
115±1.4 vs. 112±1.6*
8.9±0.5 vs. 9.9±0.6*
13.2±0.8 vs. 11.7±0.9*

PL
111±0.8 vs. 113±0.9
9.2±0.4 vs. 9.3±0.6
12.3±0.6 vs. 12.6±0.9

60%
PC
126±1.6 vs. 123±1.8*
11.7±0.8 vs. 12.8±0.9*
11.2±0.8 vs. 10.0±0.7*

PL
124±1.5 vs. 125±1.8
12.4±0.8 vs. 12.5±1.2
10.5±0.8 vs. 10.9±1.1
Production of Soluble Receptor for Advanced Glycation End‐Products Following Acute Aerobic Exercise is Gender Specific
Authors: Miranda Edwin R , Mey Jacob T , Blackburn Brian K , Farabi Sarah S , Quinn Laurie , Haus Jacob M
Published: November 2023

The Receptor for Advanced Glycation End Products (RAGE) is a transmembrane receptor that initiates a self‐propagating inflammatory cascade and has been implicated in the onset of complications involved with aging, diabetes and neuroinflammation. Soluble RAGE (sRAGE) inhibits this inflammatory signaling by competitively binding to RAGE ligands without stimulating downstream effectors. Evidence from our lab demonstrates chronic aerobic exercise increases the cleaved isoform of sRAGE (sRAGEc). However, the effects of acute aerobic exercise on sRAGEc production have not been comprehensively examined. Furthermore, recent data suggests that estrogen may play a role in exacerbating RAGE signaling and perturbing sRAGE production in diabetic women. Therefore, the primary objective of this study was to investigate changes in plasma sRAGE with acute aerobic exercise in both lean healthy (LH) and obese insulin resistant (OB‐IR) individuals. A secondary objective of the study was to compare exercise responses between men and women. 8 LH participants (4 M, 4 F) and 14 OB‐IR participants (4 M, 10 F) were recruited for the study. VO2max was determined via treadmill test and participants returned to the lab on a separate day following an overnight fast and exercised at 65% VO2max for 30 minutes. Blood samples were collected before and following exercise after participants rested in seated position for 30 minutes. Quantification of plasma sRAGE and endogeonous secretory RAGE were determined via ELISA and sRAGEc was calculated by subtraction. Between‐group comparisons were made via independent T Test and the effect of gender was analyzed via two‐way ANOVA. At baseline the OB‐IR group was older (41 ± 3 y vs. 26 ± 1 y, p < .001), more obese (BMI 35.1 ± 0.9 vs. 22.2 ± 0.9 kg m−2, p < .001) and less aerobically fit (VO2max 27.8 ± 1.8 vs. 50.2 ± 2.9 mL kg−1 min−1, p < .001) compared the LH group. There was no main effect of group (OB‐IR vs. LHC) on change in sRAGE or sRAGEc in response to exercise (ΔsRAGE 20.3 ± 53.2 vs. 13.8 ± 34.4 pg/mL, p = .93), (ΔsRAGEc 28.7 ± 47.1 vs. 14.4 ± 34.8 pg/mL, p = .33). However there was an effect of gender on the response to acute exercise. Males in both groups saw a significantly greater increase in plasma sRAGE (131.49 ± 46.46 vs. −46.94 ± 39.23 pg/mL, p < .05) and plasma sRAGEc (127.73 ± 47.04 vs. −36.08 ± 34.13 pg/mL, p < .05) compared to females. This study is the first to show that young healthy women and obese/insulin resistant women have an impaired ability to increase sRAGE plasma levels with acute aerobic exercise. Recent data has suggested that estrogen can exacerbate RAGE signaling as well as inhibit sRAGE production although the precise mechanism for this interaction is unclear and warrants further investigation.
A Redox Responsive MRI Contrast Agent for the Detection of Oxidative Stress in the Heart
Authors: Schwartz Dean , Yu Meng , Goldsmith Christian R , Beyers Ronald , Cattley Russell C
Published: November 2023

The over‐production of reactive oxygen species (ROS), known as oxidative stress, has been implicated in many mechanisms of tissue injury. The exact role of ROS remains poorly understood, which has motivated efforts to develop imaging techniques capable of differentiating normal from aberrant biological redox activity. A mononuclear Mn(II) complex with the redox‐active ligand N,N′‐bis(2,5‐dihydroxybenzyl)‐N,N′‐bis(2‐pyridinylmethyl)‐1,2‐ethanediamine (H4qtp2) responsive to hydrogen peroxide was synthesized and characterized. The capability of H4qtp2 to serve as a MRI contrast agent was assessed by 3T MR T1 relaxometry. The relationship between the concentration of H4qtp2 and T1 was linear and consistent with the Mn(II)‐H4qtp2 adducts remaining intact in solution. The slope yielded a relaxivity (r1) of 5.27 mM−1 s−1. The r1 of H4qtp2 increased to 7.02 mM−1 s−1 upon the addition of 10 mM hydrogen peroxide. H4qtp2 also behaves as a strong anti‐oxidant, as evidenced by its ability to degrade superoxide and serve as a hydrogen atom donor in vitro. We next assessed the ability of this agent to detect oxidative stress in the heart. For these studies, rats were injected with the anthracycline anticancer drug, doxorubicin (DOX, 15 mg/kg i.p.). The clinical use of DOX is limited due to cumulative dose‐dependent irreversible cardiotoxicity that leads to congestive heart failure. The mechanism of toxicity is widely accepted to result from the excess generation of reactive oxygen species (ROS) and the resulting oxidative stress in cardiac myocytes. DOX treated rats displayed prolonged ECG QT intervals but no indications of oxidative‐stress induced lipid peroxidation on days 3 or 7. There was, however, an increase in the expression of a number of antioxidant genes in the heart. For cardiac MRI, rats were anesthetized with isoflurane and MRI was performed using a Siemens actively‐shielded whole body 7T MRI scanner. Dark‐blood, T1‐weighted Cine was performed to quantify cardiac function. T1‐mapping was performed with an inversion recovery Look‐Locker sequence before and after intravenous infusion of the redox‐responsive MRI agent H4qtp2 (10 mg/kg i.v.). Ejection fraction in control rats was 60.5 +/− 2.5 % and in DOX rats was 50 +/− 2.8% seven days after DOX administration. The T1 (sec) in the myocardium of control rats was similar before and after infusion of H4qtp2. The T1 (sec) decreased by 11.8% in the hearts of DOX‐treated rats whereas there was no change in the T1 in skeletal muscle in these same rats. The data suggest that H4qtp2 can be used to detect oxidative stress in the hearts of doxorubicin treated rats.

Support or Funding Information

Funded in part by Auburn University Research Initiative in Cancer (AURIC).
Endothelial derived hyperpolarization in vivo in hypertensive rats
Authors: Brasen J. Christian , Stannov Søs Urup , Salomonsson Max , Sorensen Charlotte Mehlin
Published: November 2023

Endothelial derived hyperpolarization (EDH) plays a role in regulation of vascular tone, but little is known about its role in renal arteries in vivo. Whether EDH is affected by hypertension or vice versa is also unknown. We have investigated if the magnitude of EDH in renal arteries in vivo is different between normotensive and hypertensive rats and what the difference could be due to. In this study we quantify renal EDH in vivo in normotensive Sprague Dawley (SpD) rats and spontaneous hypertensive rats (SHR). Renal blood flow is measured in sevoflurane anesthetized rats. The ACh‐induced increase in renal blood flow (RBF) is measured before and after inhibition of the nitric oxide synthase and cyclooxygenase with L‐NAME and indomethacin, respectively.

Mean arterial pressure (MAP) is significantly higher in SHR (139 mmHg) compared to SpD (97 mmHg). Infusion of L‐NAME and indomethacin decreased RBF and increased MAP in both strains. We therefore used renal vascular resistance (RVR) to quantify EDH. Following infusion of L‐NAME and indomethacin RVR increased from 14 to 25 mmHg × min/ml in the SpD, whereas RVR increased from 27 to 72 mmHg × min/ml in the SHR. When L‐NAME and indomethacin were infused the decrease in RVR following infusion of ACh was greater in the SHR (from 73 to 67 mmHg × min/ml) compared to the SpD (from 21 to 18 mmHg × min/ml). In the SpD inhibition of the alpha‐1 adrenergic receptor with prazosin had no significant effect on RVR in the presence of L‐NAME and indomethacin. The magnitude of the EDH response was also not significantly different with or without prazosin. In the SHR RVR was not significantly different after prazosin infusion in the presence of L‐NAME and indomethacin. However, the effect of EDH measured as a decrease in RVR was greater in the prazosin treated animals. The vasodilator papaverine did not change EDH in SpD, but in the SHR it was reduced. Infusion of NE or AngII did not change the EDH response the SpD or SHR. These results suggest that alpha adrenoceptor activity might be responsible for the reduced EDH in SHR.
Stimulation of intrarenal endothelin B receptors with Sarafotoxin 6c improves kidney oxygenation in rats with diabetic nephropathy by augmented oxygen availability
Authors: Franzen Stephanie , Palm Fredrik
Published: November 2023

Diabetic nephropathy is the leading cause of end stage renal disease. The damage progression has recently been suggested to involve decreased kidney oxygenation as a result of increased oxygen consumption. Furthermore, endothelin A receptors (ETA) are highly related to kidney damage meanwhile endothelin B receptors (ETB) seem to be more protective towards diabetic nephropathy. Therefore, in this study we examined how acute in vivo intrarenal stimulation of ETB affects the progression of kidney damage in rats with diabetic nephropathy.

Two weeks post induction of hyperglycemia (Streptozotocin 55 mg/kg) rats werestudied for kidney function and oxygen metabolism during acute in vivo procedures. Parameters were collected during a baseline period with intrarenal saline infusion and during an experimental period with Sarafotoxin 6c (S6c) infusion (0.78125 pmol/h).

Hyperglycemic rats had lower kidney oxygenation which increased during S6c infusion. Meanwhile the increased oxygen consumption and glomerular filtration rate was unaffected by the treatment. Renal blood flow, delivered oxygen, urinary sodium excretion and oxidative stress levels increased in both groups during treatment compared to baseline.

Conclusively, stimulating ETB with S6c seems to improve the diminished kidney oxygenation in diabetic nephropathy in the same manner as inhibiting ETA, by improving oxygen availability, although not to the same extent.

Support or Funding Information

All experiments were conducted according to FASEB Statement of Principles. This study was supported by the Swedish Research Council, the Swedish Diabetes Foundation and the Swedish Heart Lung Foundation.
Investigation of MicroRNA Expression Profiles in the Susceptible Population to In‐stent Restenosis
Authors: Chen Chang‐Jui , Wu Chieh‐Hsi , Chun‐Ming Shih , Huang Chun‐Yao , Bi Wei‐Fung , Chan Chao‐Shun , Kao Yung‐Ta , Pan Chun‐Hsu , Chiang Shuo‐Ju , Chiang Kuang‐Hsing , Liu Yun‐Ru , Luo Ji‐Dung
Published: November 2023

In‐stent restenosis (ISR) is a pathological phenomenon in response to vascular injury after balloon angioplasty with stent placement, which occurs in just about 20~30% cases. Because of the lack of ISR‐specific clinical biomarkers, the drug‐eluted stents (DES) were widely abused as preventive interventions at present. A growing number of evidences suggested that abnormal expression profiles of microRNAs (miRNAs) associated with development of numerous diseases. To date, no reports showed that any miRNA candidates have been used to distinguish the population with high‐risk for ISR. Thereby, the purpose of the present study was to identify potential candidates of miRNAs differentially expressed in ISR patients. Based on the correlation network of our preliminary data from the proteiomics analysis of animal disease model and a small‐size SNP array of human samples, the candidate miRNAs with highly potential to involve in ISR development were picked up by using the MetaCore™ software. Thirty participants, including the restenosis group and the non‐restenosis group, were recruited from the Taipei Medical University Hospital, Taiwan. Total miRNA was extracted from blood serum of each participant, and expression profiles of five candidate miRNAs (mir‐142‐5p, mir‐132‐3p, mir‐24‐2‐5p, mir‐424‐5p and mir‐15b‐5p) were analyzed by using SYBR Green‐based real‐time PCR. Our data mentioned that the expression levels of two mRNAs (mir‐424‐5p and mir‐15b‐5p) showed increasing trends but another mRNAs (mir‐142‐5p, mir‐132‐3p and mir‐24‐2‐5p) were decreased in the restenosis group.

Support or Funding Information

Taipei Medical University

Taipei Medical University Hospital
Examination of the Development of Gastrointestinal Brunner's Glands: Stem Cells and Differentiation Factors
Authors: Powell Anne , Parappilly Michael , Lo Yuan‐Hung , Lantz Tyler , Shroyer Noah
Published: November 2023

Gastrointestinal Brunner's Glands are located at the pyloric junction of the stomach and duodenum. They function to secrete sodium barcarbonate to neutralize stomach acid and prevent damage to the intestinal epithelium. To date, a great deal is known about the ways in which Brunner's glands participate in ion transport and production of secreted factors, such as Epidermal Growth Factor Receptor Ligands. It is also known that the Brunner's Glands develop largely postnatally, but nothing is known about the factors that influence their development. Hamartomas of the Brunner's Glands are known to arise in humans, as well as in mice that lack the Epidermal Growth Factor Receptor Negative Regulator Lrig1, so understanding normal Brunner's Gland development may be critical to our understanding of how cancer in this gland could arise. We sought to provide the first significant developmental molecular characterization of this critical gastrointestinal gland. In this study we demonstrate that Brunner's Glands arise from Lrig1‐expressing stem cells, using lineage tracing from Lrig1‐CreERT2/+;R26R‐YFP/+ mice. In addition, we examined mice lacking the transcription factor SAM Pointed Domain Containing ETS Transcription Factor (Spdef) at postnatal days 1–14 and show that Brunner's glad epithelial cells have lost their secretory phenotype and have abnormal differentiation.
Bardet‐Biedl Syndrome (BBS) Proteins in AgRP Neurons Are Required for Energy Homeostasis
Authors: Guo Deng Fu , Rahmouni Kamal
Published: November 2023

BBS is a pleiotropic autosomal recessive human disorder associated with several clinical features including obesity. We previously demonstrated that Bbs1 gene deletion from the nervous system caused obesity in mice highlighting the importance of neuronal BBS proteins for energy homeostasis. Here, we investigated the role of Bbs1 gene in the orexigenic agouti‐related peptide (AgRP) neurons present in the arcuate nucleus of the hypothalamus. We crossed Bbs1fl/fl mice with AgRPCre mice and confirmed Cre recombinase activity in AgRP neurons using td‐Tomato reporter mice. Interestingly, AgRPCre/Bbs1fl/fl mice developed obesity as indicated by the increased body weight (19% vs control littermates) and fat pad masses including brown adipose tissue, inguinal, perirenal and reproduction fats. Moreover, compared to male AgRPCre/Bbs1fl/fl mice, female AgRPCre/Bbs1fl/fl mice displayed profound obesity phenotype. Increased food intake contributed to the development of obesity in AgRPCre/Bbs1fl/fl mice. However, no change in hypothalamic genes regulating food intake (AgRP, proopiomelanocortin and neuropeptide Y) were detected in AgRPCre/Bbs1fl/fl mice relative to controls. The emerging importance of BBS proteins for the trafficking of receptors, particularly G protein‐coupled receptors (GPCR), led us to investigate the contribution of GPCR mistrafficking in AgRP neurons to the obesity phenotype. Using immunohistochemistry, we noticed that in hypothalamic neurons of wild type mice neuropeptide Y2 receptor (NPY2R) localize exclusively to cilia. Strikingly, deletion of the Bbs1 gene from AgRP neurons abolished the ciliary localization of NPY2R in these neurons, but not in other neuronal populations. Adenyl cyclase III, ciliary marker, was not affected by Bbs1 deletion suggesting that ciliary mistrafficking of NPY2R after Bbs1 ablation is specific. In HEK 293 cells, silencing the Bbs1 gene significantly decreased GFP‐tagged NPY2R trafficking to the plasma membrane. These findings demonstrate that BBS1 protein in AgRP neurons is involved in the regulation of energy homeostasis by controlling the trafficking of receptors such as NPY2R.
DREADD (Designer Receptors Exclusively Activated by Designer Drugs)‐induced activation of subfornical organ neurons stimulates thirst and salt appetite
Authors: Nicoleau Marvin H , Nation Haley , Kinsman Brian , Browning Kirsteen , Stocker Sean D
Published: November 2023

Circumventricular organs such as the subfornical organ play a pivotal role in body fluid homeostasis through the ability of these neurons to sense neurohumoral signals and subsequently regulate thirst, salt appetite, neuroendocrine function, and sympathetic outflow. Previous studies have largely relied on lesions or infusion of drug antagonists into a structure that is adjacent to the 3rd ventricle. Recently, a new approach known as DREADD (Designer Receptors Exclusively Activated by Designer Drugs) has been developed to manipulate neuronal activity through virally‐mediated expression of a G‐protein coupled receptor that becomes activated after administration of the exogenous ligand Clozapine N‐Oxide (CNO). The purpose of this study was to investigate whether DREADD expression in SFO neurons could be a useful tool to manipulate body fluid homeostasis. We hypothesized that excitation of SFO neurons using DREADD‐Gq would stimulate thirst and salt appetite. At least 2 week before experiments, male C57/Bl6 mice (12–15 weeks) received an SFO injection of rAAV2‐CaMKII‐HA‐hM3D(Gq)‐IRES‐mCitrine (1×1012 molecular/mL, 50nL). Subsequent injection of CNO (0.25mL, sc) into mice (n=13) with robust expression of the DREADD in SFO (verified immunocytochemically) produced a dose‐dependent increase in water intake: 0.0 mg/kg (0.02±0.01 mL) vs 0.06 mg/kg (0.08±0.03mL, P<0.05) vs 0.1 mg/kg (0.21±0.05mL, P<0.05) vs 1mg/kg (0.32±0.09mL, P<0.05) vs 3 mg/kg (0.32±0.07mL, P<0.05). To test whether DREADD‐induced activation of SFO neurons induces salt appetite, mice (n=13) were given access to distilled water, 0.3M NaCl solution, and 0.01% Na chow for 1 week. Subsequent injection of CNO (3mg/kg, sc) versus vehicle significantly increased the ingestion of water (0.36±0.08 vs 0.05±0.01 mL, respectively; P<0.01) and 0.3M NaCl (0.14±0.04 vs 0.03±0.01mL, respectively; P<0.05). A final set of experiments verified that injection of CNO activated SFO neurons through expression of the early‐intermediate gene Fos. Injection of CNO (3mg/kg, sc) versus vehicle (0.25mL, sc) significantly increased the number of Fos‐positive nuclei in the SFO (159±49 vs 2±1 cells, respectively; n=4/group; P<0.01). Mice with DREADD expression outside of the SFO (n=12) did not increase water intake, salt appetite, or Fos expression after injection of CNO. Collectively, these findings demonstrate that DREADD can be used experimentally to manipulate neuronal activity of SFO neurons and ultimately reveals that direct excitation of SFO neurons stimulates thirst and salt appetite.
Active expiration and sympathetic excitation during hypercapnia require glutamatergic neurotransmission in the retrotrapezoid nucleus
Authors: Lemes Eduardo , Zoccal Daniel
Published: November 2023

Animals exposed to hypercapnia exhibit increases in sympathetic and expiratory motor activities that are depend on, at least in part, the activation of glutamatergic neurons in the retrotrapezoid nucleus (RTN). The generation of active expiratory pattern requires the expiratory neurons of the parafacial respiratory group (pFRG), which are anatomically co‐localized with the RTN neurons. Herein we explored the hypothesis that antagonism of glutamate receptors in the RTN/pFRG prevents the emergence of active expiration and reduces the sympathoexcitation induced by hypercapnia. Microinjections of kynurenic acid (KYN, antagonist of glutamate ionotropic receptor, 100mM, 50 nL) were performed bilaterally in the RTN of arterially‐perfused decerebrated in situ preparations of Holtzman rats (60–70 g) during the exposure to hypercapnia (8% CO2) and the changes in the thoracic sympathetic (tSN), abdominal (AbN) and phrenic nerve (PN) activities were evaluated. Hypercapnia elevated tSN, PN and AbN activities of the in situ preparations (n=7). KYN microinjections in the RTN during hypercapnia reduced the sympathetic‐excitation (ΔtSN: 158±25 vs 80 ± 11%, P<0.05) and greatly attenuated the AbN response (ΔAbN: 41±13 vs 16±3%, P<0.001), but not changed the reflex increase in PN activity. Also, KYN in the RTN during hypercapnia further reduced the inspiratory time (ΔTi: −0.06±0.05 vs −0.13±0.05 s, P<0.05) and the perfusion pressure (ΔPP: −12±1 vs −20±3 mmHg, P<0.05). The results indicate that glutamatergic neurotransmission in the RTN is required for processing of the sympathetic‐excitatory and the generation of active expiratory pattern in response to hypercapnia.

Support or Funding Information

Support by FAPESP (2013/17251‐6), NIH (1R01AT008632‐01).
Acute Effects of Isometric Handgrip Exercise on Blood Pressure and Heart Rate Variability
Authors: Locke Ben
Published: November 2023

Isometric handgrip (IHG) exercise has been shown to elicit post‐exercise hypotension and transient improvements in heart rate variability (HRV) after one session. Typical IHG efforts are held at 30% of maximal voluntary contraction (MVC), with few studies deviating from this intensity. The purpose of this study is to assess the acute effects of different IHG exercise intensities on blood pressure (BP) and HRV. In a crossover study, 20 participants (18–30 years of age) will complete two IHG exercise protocols: (1) four 2‐min isometric contractions at 30% MVC with 2‐min rest periods using the dominant arm, and (2) four 45‐s isometric contractions at 50% MVC with 1‐min rest periods using the dominant arm. BP and HRV will be recorded using a continuous noninvasive arterial pressure (CNAP) monitor and three lead electrocardiography (ECG), respectively, at pre‐exercise, 5‐min post‐exercise, and 15‐min post‐exercise. BP will also be recorded continuously during both exercise protocols to measure BP responses after each IHG contraction. Statistical analysis for BP and HRV will be performed using a 2 × 3 repeated measures ANOVA. Secondary analysis of BP at the end of each isometric contraction will be conducted using a 2 × 5 repeated measures ANOVA. Statistical significance will be set to P < 0.05.
Monomeric cocoa procyanidins enhance functional β‐cell mass
Authors: Rowley Thomas , Bitner Benjamin , Ballard Matthew , Smithson Andrew T. , Neilson Andrew P , TESSEM JEFFERY Sivert
Published: November 2023

Type 2 diabetes (T2D) is increasing at an alarming rate world wide. As T2D progresses individuals have decreased insulin secretion and loss of β‐cells, resulting in decreased functional β‐cell mass. Enhancing functional β‐cell mass could be leveraged as a treatment for T2D. There is much interest regarding cocoa flavanols to prevent and ameliorate T2D. Animals fed a high fat diet supplemented with cocoa procyanidin oligomers demonstrated improved glucose tolerance. These animals, as well as animals fed cocoa procyanidin monomers, presented decreased serum insulin and endotoxin levels. Here we present data looking at the effect of cocoa procyanidin extract, monomers, oligomers and polymers on β‐cell function, survival and proliferation. Our data demonstrates that cocoa monomers significantly enhance glucose stimulated insulin secretion in the 832/13 β‐cell line and primary rat islets. Interestingly, cocoa oligomers and polymers do not induce the same increase in GSIS. Furthermore, cocoa monomers significantly enhance β‐cell proliferation. These data demonstrate that cocoa monomers may enhance functional β‐cell mass, and highlight their potential as a T2D therapeutic intervention.
Improved Insulin‐Mediated Glucose Oxidation in Cultured Human Myotubes Following Roux‐en‐Y Gastric Bypass Surgery
Authors: Zou Kai , Hinkley J. Matthew , Park Sanghee , Zheng Donghai , Dohm G. Lynis , Houmard Joseph A.
Published: November 2023

Roux‐en‐Y gastric bypass (RYGB) leads to remission of type 2 diabetes and markedly improved insulin‐mediated glucose oxidation at the whole body level, as well as in skeletal muscle. However, little is known about whether the improvements occur intrinsically at the level of skeletal muscle. The purpose of this study was to determine whether improved glucose oxidation is intrinsically retained in differentiated myotubes derived from RYGB surgery patients, and if so, the time course of the improvement. Primary human skeletal muscle cells were isolated from muscle biopsies obtained from 6 RYGB patients prior to, 1‐month and 7‐months following surgery (BMI = 50.2 ± 2.0, 43.2 ± 2.8 and 35.7 ± 2.2 kg/m2, respectively) and 6 lean subjects (BMI = 23.4 ± 0.6 kg/m2) and were differentiated to myotubes. Radiolabeled 1‐14C glucose was used to measure complete glucose oxidation (GO) and non‐oxidized glycolysis (NOG) rates in the presence or absence of insulin. Insulin‐stimulated GO rate was significantly elevated to greater extent in myotubes derived from RYGB patients at 1‐month after surgery in comparison to before surgery (7.3 ± 3.4% vs. −2.2 ± 2.3%, respectively, P<0.05). It was further enhanced at 7‐months post‐surgery (13.5 ± 2.1%, P<0.05), which was virtually restored to the level of lean people (15.4 ± 1.4%). On the other hand, NOG production in response to insulin stimulation was not significantly changed in myotubes from RYGB patients at 1‐months post‐surgery in comparison to before surgery (16.0 ± 6.2% vs. 19.5 ± 5.6%), but was largely decreased at 7‐months post‐surgery (−1.3 ± 3.6%, P<0.05), which was almost identical to the lean control group (−1.2 ± 2.5%). These changes lead to the improvement of GO/NOG ratio, an indicator of glucose partitioning, in response to insulin stimulation in myotubes from RYGB patients at both 1‐month (1.8 ± 3.8%, P=0.08) and 7‐months (11.2 ± 1.0%, P<0.05) post‐surgery when compared to before surgery (−10.8 ± 5.4%). Taken together, these data suggest that RYGB surgery can rapidly improve glucose oxidation intrinsically in skeletal muscle as early as 1‐month following surgery and further restore it to the level control level.

Support or Funding Information

National Institute of Health (DK 56112)
Transcriptomic profiling in cardiovascular disease with renal failure in human
Authors: Lee Danielle , Xu Jen , Zhang Emily , Ho Lilun , Kong Tianqing , Lu Tzongshi
Published: November 2023

Background

Chronic kidney disease (CKD) is a major health issue globally; it affects 50 million people worldwide and over 26 million Adults in the United States. Studies indicate that up to 50% of deaths in patients with ens‐stage renal failure are due to cardiovascular disease (CVD) and there is a 10 to 30‐fold higher cardiovascular mortality in CKD patients than in the age‐matched general population, despite adjustment for classical cardiovascular risk factors. To profile the exhaustive signatures in calcifited human endotelials, we performed whole transcriptome analysis to explore novel genes in primary human aortic endothelial cells (HAECs) and human brain microvascular endothelial cells (HBMECs).

Methods

HAECs and HBECs were treated with calcification medium, and total RNA was extracted using the Nucleospin RNA isolation kit (Clontech). cDNA was synthesized and fragmented using SMART‐seq2 and Nextera XT (Illumina), respectively to generate libraries for sequencing on Hi‐seq 2000 (Illumina). Five million single‐ended raw reads from each sample were mapped to the human reference genome consortium GRCh38 by Burrows‐Wheeler Aligner (BWA); the abundancies of each gene, (FPKM, fragment per kilo‐base exon per million mapped reads), were estimated using RNA‐seq by Expectation Maximization (RSEM). Log 2 transformed FPKM were normalized by quantile normalization, which were then subjected to differential expression analysis to calculate significant fold change (Gene‐E, Broad Institute). Gene selections were performed by the combinations of fold‐changes on log 2 ratio, and p value < 0.05. Data were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) and The Database for Annotation, Visualization and Integrated Discovery v6.7 (DAVID, http://david.abcc.ncifcrf.gov/).

Results

Pathway analysis among the top 500 up‐regulated genes in comparing untreated HAECs and HBMECs to those treated with calcification medium revealed that these genes are mainly involved in cell metabolic, proliferation, and apoptosis processes. Among these genes, they were found to play roles in cell cycle regulation, apoptosis, oxidative stress, and stress resistance such as heat shock protein 70 families (HSPA1, HSPA5, and HSP40), NF‐kappa‐B inhibitor alpha, and aryl hydrocarbon receptor nuclear translocator (ARNT) in HIF‐1, NF‐kappa B and c‐AMP signaling pathways. In addition, MT‐RNR1 and cannabinoid receptor 1 are also detected that involved in amount of ATP produced in mitochondria and co‐chaperone of HSP70.

Conclusion

We have identified a group of genes differentially expressed in healthy and calcified endothelial cells. Our data contribute to our understanding of the direct effects in the presence of hyperphosphatemia and hypocalcemia at endothelial cells. This gene set may have pathophysiological significance and may serve to inform rational development of therapeutic strategies to prevent CVD adverse effects in CKD patients.
Extracellular Adenosine Diphosphate Ribose Mobilizes Intracellular Ca2+ via Purinergic‐dependent Ca2+ Pathway in Rat Pulmonary Artery Smooth Muscle Cells
Authors: Huang Chun , Hu Jinxing , Subedi Krishna P. , Lin Amanda H.Y. , Sham James S.K.
Published: November 2023

Adenosine diphosphate ribose (ADPR), a product of β‐NAD+ metabolism by the multifunctional enzyme CD38, is recognized as a novel signaling molecule. The catalytic site of CD38 orients extracellularly or intracellularly, capable of generating ADPR outside and inside the cells. CD38‐dependent pathways have been characterized in pulmonary artery smooth muscle cells (PASMCs); however the physiological function of extracellular ADPR is unclear. Here we sought to characterize the Ca2+ mobilizing and proliferative effects of extracellular ADPR and compared with the ATP‐induced responses in rat PASMCs. ADPR elicited concentration‐dependent increase in [Ca2+]i with a fast transient and a sustained phase in PASMCs. The sustained phase was abolished by Ca2+ removal and inhibited by the non‐selective cation channel blocker SKF‐96365, but was unaffected by TRPM2 antagonists or nifedipine. The IP3‐receptor inhibitor xestospongin C caused a partial inhibition of the peak and the sustained phase, while ryanodine had no effect on the ADPR‐induced Ca response. The purinergic receptor (P2X) antagonist pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulfonate inhibited partially the transient and the sustained Ca2+ response, while the P2(XY) inhibitor suramin and the phospholipase C inhibitor U73122 abolished the sustained Ca2+ influx. The P2Y1 antagonist MRS2179 had no effect on the response. By contrast, ATP and ADP activated Ca2+ response exhibited a high and a low affinity component, and the pharmacological profile of ATP‐induced Ca2+ response was distinctive from that of ADPR. BrdU incorporation assay showed that ADPR caused significant inhibition whereas ATP caused slight stimulation of PASMC proliferation. RT‐PCR analysis found that mRNA of most P2X and P2Y subtypes are expressed in PAs. In conclusion, ADPR and ATP activate Ca2+ responses through different combinations of multiple purinergic receptor subtypes; and extracellular ADPR may exert an autocrine/paracrine action via purinergic receptors on PASMCs.

Support or Funding Information

This work was supported by NIH grants HL071835 and HL075134,, National Natural Science Foundation of China grant NSFC31371165, and Fujian Province “Hundred Experts Award”
How DNA‐Damage Machinery Protects the Genomes of Mouse Oocytes
Authors: da Silva Garcia Rinaldi Vera , Bolcun‐Filas Ewelina , Schimenti John
Published: November 2023

During meiosis hundreds of endogenously induced DNA double stranded breaks (DSB) are made by the SPO11 endonuclease in order to assure homologous chromosome pairing (synapsis) and their proper segregation. Because DSBs are potentially mutagenic, quality control mechanisms are in place to guarantee that they are all repaired, preferentially by interhomolog recombination, otherwise apoptosis is induced. It is currently accepted that this “pachytene checkpoint” senses DSB repair and synapsis by independent mechanisms. However, we have experimental evidence that oocytes defective for either DNA repair or synapsis are both eliminated by a canonical DNA damage response checkpoint pathway (dependent upon signaling of the CHK2 kinase to downstream effectors p53 and p63). During the first meiotic division, mechanisms are in place to inhibit the DNA‐repair machinery from using the sister chromatid as recombination repair donors, making the homologous chromosome the substrate of choice. Whereas this block to sister chromatid repair of DSBs assures interhomolog (IH) recombination, it also prevents the chromosomes that fail to undergo IH repair from repairing residual DSBs. Here, we present evidence consistent with a model whereby asynaptic oocytes accumulate SPO11‐independent DSBs, and are eliminated via CHK2 activation.
Computational Analysis of Next Generation Sequencing Data Predicts Potential Sources of Phenotypic Variation in Human Glucocorticoid Signaling
Authors: Ryan Sean M. , Forst Thomas M. , Martz Flora G. , Murphy Patrick J. M.
Published: November 2023

Genetic differences and the complexity of protein‐protein interactions affecting intracellular hsp90‐mediated glucocorticoid receptor (GR) signaling cause substantial phenotypic variation and impact an individual's response to glucocorticoids. The goal of this study was to identify candidate causal variants and single nucleotide polymorphisms (SNPs) correlated with a previously observed phenotypic variation in in glucocorticoid response. A newly developed computer program was used to analyze whole exome sequencing data from leukocytes of participants with similar demographic profiles yet distinct glucocorticoid response phenotypes. Utilizing user‐defined arguments, identified mutations were filtered to homozygous nonsynonymous SNPs, reducing the total number of candidate casual variants to <2% of the initial total mutation count of 384,889 unique variants. From this number of variants detected among all participants, <500 (<0.15%) were nonsynonymous mutations shared exclusively among those identified as possessing the negative glucocorticoid response phenotype. A subset of these gene mutations were clustered, resulting in translated products with known protein‐protein interactions and identified using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. These data indicate a select group of candidate causal gene variants (<200) could be detected and provide a quantitative approach to reduction in preliminary loci examination for causal variance of glucocorticoid phenotypic response.
Enhanced Sample Preparation for Proteomic Analysis Utilizing Reversible Biotinylation and Polymer Based Protein Engineering
Authors: Lucas Amber , Cummings Chad , Russell Alan , Minden Jonathan
Published: November 2023

Proteome sample preparation for gel‐based and mass spectrometry‐based studies has remained relatively unchanged for many years. The goal of these sample preparation schemes is to remove contaminants such as lipids, DNA, RNA, and carbohydrates from cell or tissue homogenates. The vast majority of current methods rely on protein precipitation followed by centrifugation and resolubilizing the proteins. Unfortunately none of these techniques provide an unbiased sampling of the tentire proteome. Regardless of the method (ammonium acetate precipitation, TCA/acetone precipitation, PEG precipitation, etc.), not all proteins will be precipitated and not all will be resolubilized. We have developed an unbiased method for proteome sample preparation based on protein capture, not precipitation. We recently showed that Biotin‐CDM can be used as a tool to reversibly tag the entire proteome for purification.1 Biotin‐CDM reacts with lysine residues present on the surface of proteins at elevated pH to covalently biotinylate proteins. Biotinylated proteins are bound to Avidin beads and then all non‐protein contaminants are washed away. The biotin tag is then reversed by lowering the pH to release the proteins from the Avidin beads, resulting in pur, unmodified, and unbiased proteome sample. Because an excess of Biotin‐CDM is required to tag all proteins, we utilized polymer based protein engineering to develope a novel method that separates free Biotin‐CDM from biotinylated proteins. Thus, we have developed a method to prepare proteome samples in an unbiased manner that could be used for any organism.
The collection and preservation of urinary proteins using a special diaper
Authors: Qin Weiwei
Published: November 2023

Change is the most fundamental property of biomarker. Homeostatic nature of internal environment limits the amount and extent of changes (biomarkers) in blood, which in turn make the urine a better biomarker source (more changes) than blood, let alone that drawing blood from infants and toddlers is hard and less tolerated. For those patients with immature or no language ability, giving chief complaint is difficult. Thus, using biomarkers in urine can provide valuable clues for diagnosis of diseases especially pediatric ones. Collecting urine from little kids and some adult patients is not as easy as from healthy adults. Here we propose a method using a special diaper to collect urine. Urinary proteins were then eluted and adsorbed onto a piece of nitrocellulose membrane which can be dried and stored in a vacuumed bag. Through this procedure, the protein patterns remain faithfully preserved in SDS‐PAGE. With this simple and economical method, it is possible to collect and preserve urine samples from infants, toddlers and special patients even on large‐scale for prospective studies which are important for biomarker research on related pathophysiological conditions.

Support or Funding Information

National Basic Research Program of China (973 Program)(Nos. 2012CB517605, 2013CB530805).