bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypoxia increases endothelial cell reactive oxygen species production and reduces thioredoxin 2 levels
Authors: Adesina Sherry Eniola , Porter Kristi M , Hart C. Michael , Sutliff Roy L
Published: June 2021

Pulmonary hypertension (PH) is characterized by pulmonary artery remodeling and increased vascular resistance. Current evidence suggests that reactive oxygen species (ROS) contribute to PH pathogenesis, though the role of mitochondria‐derived ROS in PH remains controversial. Antioxidants, such as mitochondrial thioredoxin 2 (Trx2), may attenuate deleterious ROS effects. The effects of hypoxia on Trx2 expression are unknown. We hypothesize that hypoxia alters endothelial expression of Trx2 to contribute to endothelial dysfunction in PH. Human pulmonary artery endothelial cells (HPAEC) were cultured in normoxia (21% O2) or hypoxia (1% O2) for 72 hours. Cytosolic ROS were measured by Amplex red and DCF staining. Mitochondrial ROS was visualized using MitoSOX. Western blot analysis and quantitative real‐time PCR were employed to assess Trx2 expression. Results indicate that hypoxia exposure increases H2O2 production measured by Amplex red and DCF staining. Hypoxia also increases superoxide detection in mitochondria by MitoSOX staining. Hypoxia decreased Trx2 expression. These studies suggest hypoxia‐induced decreases in Trx2 levels may exacerbate hypoxia‐induced ROS generation that contributes to alterations in pulmonary vascular endothelial function and PH. Funding: T32 ES 012870 Toxicology Training Grant, Research Service of the Atlanta VAMC, and NIH grants DK074519/HL102167 (CMH and RLS).
Prediction Models for Assessment of Abdominal Adiposity
Authors: Freeland-Graves Jeanne H , Lee Jane J , Yao Ming , Xu Bugao
Published: June 2021

Abdominal visceral adiposity increases risk for diabetic/cardiovascular diseases. This study developed prediction equations for visceral and subcutaneous adiposity via 3‐ dimensional stereovision body imaging (SBI). 121 adults were measured for anthropometrics via SBI and magnetic resonance imaging (MRI). Abdominal fat volumes were evaluated by MRI umbilicus scans. Adjusted measurements for stereovision (SV) umbilicus and hip circumferences, central obesity depth (COD), and frontal widest girth of abdominal area (WG) were measured via SBI. Prediction models were developed by stepwise multiple linear regression, with body measurements and demographics as independent predictors; visceral/subcutaneous adiposity was dependent. Strongest predictors were: subcutaneous fat (mm2), − 439.35 + 7.45 SV umbilicus circumferences (cm) − 40.09 gender − 1.71 age (yr) (R2=0.88, p<0.001); visceral fat (mm2), 90.93 + 11.90 COD (cm) – 4.30 WG (cm) +0.51 age (yr) −1.90 SV hip circumference (cm) + 1.30 SV umbilicus circumference (cm) (R2=0.72, p<0.001). Inclusion of SV significantly improved R2. In contrast, visceral fat with only anthropometrics and demographics reduced the R2 to 0.57. Subcutaneous adiposity prediction did not improve with SBI. Thus, SBI is an effective technique to create visceral adiposity prediction equations that can be applied to assess obesity in clinical settings. NIH 3R21DK081206‐02S1.
Toll‐like receptor 2 is elevated in rat corpus cavernosum in response to nitric oxide deficiency
Authors: Davis Jason , Nunes Kenia , Webb R. Clinton
Published: June 2021

The prevalence of erectile dysfunction (ED) is significantly higher among men with comorbid diseases, such as cardiovascular disease and or diabetes mellitus. One common underlying vascular condition that has been shown in both diseases when associated with ED is endothelial dysfunction, a process in which nitric oxide (NO) bioavailability, is reduced. Thus, our objective was to reveal that in the early stages of ED, an innate immune reaction is generated in response to endothelial dysfunction. Therefore, our hypothesis is that toll‐like receptor 2 (TLR2), a key player in innate immunity, is increased in the corpus cavernosum following treatment with N (G)‐nitro‐L‐arginine methyl ester (L‐NAME), an endothelium nitric oxide synthase inhibitor, that mimics endothelial dysfunction. Corpus cavernosum tissues from male Sprague‐Dawley (SD) rats were incubated with L‐NAME (100 uM) for 6, 12 or 24 hours, respectively. Western blot analysis demonstrated that, TLR2 expression is augmented in a time dependent manner, (6hrs:0.007±0.002, 12hrs:0.015±0.008, 24hrs:0.066±0.022), in response to L‐NAME. These results suggest a novel role of TLR2 activation in the causal path leading to ED.
Early myocardial contractility depression in female endotoxemic rats
Authors: Goncalves Ronald Paiva Moreno , da Silva Santos J. Eduardo , Guarido Karla Lorena , Assreuy Jamil
Published: June 2021

Although several in vitro studies have addressed heart function in sepsis, this subject has been scarcely investigated in vivo. Using a pressure‐volume (PV) conductance catheter, we evaluated the cardiac function in situ in Wistar rats injected 6 or 24 h before with E. coli lipopolysaccharide (LPS, 10 mg/kg, i.p.). The rats were anesthetized with xylazine (2 mg/kg) and isoflurane (1–3%), plus lidocaine on surgical sites. The catheter was inserted into the left ventricle (LV) through the carotid artery. Compared to control, stroke work (SW) and LV relaxation time constant Tau were reduced by 57% and 63% in LPS 6 h, and by 36% and 57% in LPS 24 h group, respectively. LV mean pressure was decreased ~ 20 mmHg both 6 and 24 h after LPS (control: 48.9 ± 7 mmHg; p < 0.05). Interestingly, the mean arterial pressure was reduced from 88.7 ± 7.3 in control to 63.2 ± 3.2 mmHg in LPS 24 h, but it was unchanged in LPS 6 h. Heart rate did not change in LPS 24 h, but was increased from 253 ± 21 to 314 ± 13 bpm in control and LPS 6 h groups, respectively. Our study suggests that heart function is impaired in early stages of sepsis, even before the well‐described hypotension, and that this dysfunction may be hidden by compensatory mechanisms.

Research support: CNPq and FAPESC (1366/2010‐8).
Hemoperitonium Increases Carbon Monoxide and Reduces Platelet Aggregation in Trauma Patients
Authors: JOHNSON ROBERT A , JOHNSON FRUZSINA K , DURANTE WILLIAM , STEWART RONALD M
Published: June 2021

Hemorrhage into body cavities is a common complication from blunt trauma. While it used to be standard practice to clear such a hemoperitoneum, more recently they been left untreated due to various concerns with patient stability.

Objective

Determine if hemoperitoneum elevates carbon monoxide production and impairs platelet aggregation in trauma patients.

Methods

Male patients with verified hemoperitoneum were enrolled along with non‐hemoperitoneum head injury controls. Three days after admission, whole patient carbon monoxide excretion was measured via solid‐phase gas chromatography. A blood sample was drawn to evaluate collagen‐ and thrombin‐induced aggregation.

Results

Three days after injury, patients with hemoperitoneum displayed higher carbon monoxide excretion (0.54±0.10 vs 0.96±0.11 μmol/Kg per hr, control vs hemoperitoneum, P<0.05), and carboxyhemoglobin levels 24% higher than the non‐hemoperitoneum controls. Patients with hemoperitoneum also displayed decreased amplitude of both collagen and thrombin activated platelet aggregation (−23 and − 34%, respectively).

Conclusions

Abdominal blood drives endogenous carbon monoxide formation and interferes with hemostatic processes, so inhibitors of carbon monoxide formation may potentially serve as a therapeutic to stabilize affected patients. Supported by grants Department of Army #W81XWH‐07‐1‐0717 (RAJ and RMS), AHA 0865241F (FKJ) and NIH HL064577 (RAJ).
Variations in the density of blood during hemorrhage
Authors: Nunez Fiesky A. , Kislukin V. , Thurmalla N. , Krivitski N. , Callahan M. F. , Smith T. L.
Published: June 2021

It is known that hematocrit of the whole body (Htb) and venous or systemic hematocrit (Ht1) are different. The cell factor(ratio of whole body to venous Hct) F_c= Htb/Ht1. is around 0.85. Thus one can expect a drop in blood density during blood loss. Since the delivery of O2 (DO2) is proportional to cardiac output x Ht1; the variation of F_c reflects both the redistribution of (a) blood volume toward central circulation, to increase venous return and (b) RBC toward central circulation for the partially restoration of Ht1, the variation/stability of F_c could be a mechanism for compensation of variations of DO2.

The aim of this study is to present a method of estimation of Htb/Ht1 during blood loss and to analyze variation of Htb/Ht1 during this challenge.

Male Sprague‐Dawley rats (n=55) were hemorrhaged in 3–4 steps with 7 ml/kg body weight withdrawal of blood at each step. An arterio‐venous extracorporeal catheter (priming volume 0.8ml) was connected the carotid artery and jugular vein. Clamp on transit time sensors, TS450 flowmeter and an automatic algorithm (Transonic Systems Inc.) were used to calculate the cardiac output and blood volumes such as central blood volume and actively circulating blood volume.

Results

The drop in blood density is proportional to F_cell and inversely proportional to the circulating blood volume. A linear drop in density during first and second withdrawals indicates the constancy of F_cell. After completion of the blood withdrawal the Htb/Ht1 was on average, slightly elevated (7% +/−6%), possibly due to the starting of refilling of intravascular volume.
Tissue‐specific expression of angiotensin‐converting enzyme 2 (ACE2) from two promoter regions is unaffected by elevated levels of renin and angiotensinogen
Authors: Nguyen Van Kim , Pedersen Kim Brint , Xia Huijing , Lazartigues Eric
Published: June 2021

Angiotensin‐Converting Enzyme type 2 (ACE2) counteracts Renin‐Angiotensin System (RAS) over‐activity and has been proposed to protect against the development of cardiovascular morbidity. ACE2 is transcribed from a proximal and distal promoter. ACE2 gene regulation is furthermore dependent on tissue type. Hypothesis: tissue‐specific regulation of the ACE2 gene in response to RAS over‐activity is due to tissue‐specific promoter usage. We developed real‐time qRT‐PCR assays for a proximal promoter transcript (PPT) and a distal promoter transcript (DPT). We analyzed brain, kidney, lung, and heart RNA from wild‐type mice and hypertensive transgenic mice with overactive RAS due to expression of human renin and angiotensinogen. Surprisingly, the genotype did not affect the ACE2 transcript levels. However, the tissues differed in promoter usage: the PPT is significantly (p < 0.001) more abundant than the DPT in the kidney, brain, and heart (>20‐fold), whereas the DPT is significantly more abundant than the PPT in the lung (49‐fold). Among tissues, the kidney had the highest concentration of the PPT whereas the lung had the highest concentration of the DPT. Our work illustrates that tissue‐specific promoter usage should be taken into account when developing new therapies based on modulating ACE2 expression. Support: NIH/NHLBI HL093148, NCRR P20RR016456, and Louisiana Board of Regents Support Fund.
Effects of Commonly Used Chinese Herbal Medicines and Electroacupuncture on Experimental Myocardial Infarct Size
Authors: Dow Joan Stephanie , Painovich Jeannette , Hale Sharon L , Longhurst John C , Tjen-A-Looi Stephanie , Kloner Robert A
Published: June 2021

Background

We hypothesized that herbal therapy or electroacupuncture (EA) would reduce infarct size and ischemia‐reperfusion induced cardiac arrhythmias.

Methods

An anesthetized rat model of proximal left coronary artery occlusion (30 min) and reperfusion (180 min) was used. Infarct size (IS; triphenyltetrazolium chloride) and ischemic risk zone (blue dye technique) were assessed. In Protocol 1 rats received either saline, Dan Shen (DS) or DS+Jiangxiang (JX), starting 30 minutes before coronary occlusion. In Protocol 2, rats were either untreated, received static needle placement or EA at the PC5 and 6 acupuncture points of the forearm starting 5 minutes before occlusion.

Results

Neither treatment, herbal or EA, reduced IS expressed as a fraction of the ischemic risk zone or altered hemodynamics. Protocol 1: 0.43±0.06 in controls, 0.39±0.05 in DS and 0.42±0.04 in JX; Protocol 2: control (0.38±0.04), static needle (0.47±0.4) and EA (0.32±0.05) (p=NS). In a separate study EA failed to reduce ventricular tachycardia and VPB's. Results may be related to the absence of hemodynamic effects, a major factor underlying the reduction of experimental myocardial ischemia during EA.

Conclusion

Our studies do not suggest a cardioprotective effect of DS or JX or EA in experimental myocardial ischemia/reperfusion, in the absence of any significant changes in myocardial oxygen demand.
Low‐intensity interval exercise training normalizes coronary blood flow‐O2 demand coupling and improves cardiac efficiency in miniature swine with LV hypertrophy
Authors: Emter Craig A
Published: June 2021

We have previously reported low‐intensity interval exercise training prevents coronary vascular dysfunction in miniature swine with LV hypertrophy. The purpose of this study was to determine how changes in coronary vascular function impact metabolic and left ventricular (LV) function in aortic‐banded (AB) sedentary (AB‐S, n=5), AB exercise trained (AB‐TR, n=5) and control sedentary (CON, n=4) male Yucatan miniature swine. Pressure‐volume loops, myocardial oxygen consumption (MVO2), and coronary blood flow (CBF) were measured simultaneously in vivo under varying afterloads by increasing MAP to 80, 90, 100, and 120 mmHg under anesthesia. Baseline MVO2 (p=0.09) and heart rate across all pressure loads (p<0.05) were significantly elevated in AB‐S animals. For a given rate‐pressure product, CBF was decreased in AB‐S animals compared to AB‐TR and CON groups (p=0.057). Consequently, O2 extraction was increased and venous O2 pressure and saturation reduced for any given MVO2 in AB‐S animals. O2 extraction and venous O2 content were the same in both AB‐TR and CON animals. Lactate metabolism was unaltered in all groups. LV stroke work (SW) was elevated in both AB‐S and AB‐TR groups, but cardiac efficiency (SW:MVO2) was increased only in AB‐TR animals (p<0.05). In conclusion, low‐intensity interval exercise normalizes coronary blood flow‐O2 demand coupling and improves LV efficiency in AB mini‐swine. NIH HL101332
Aerobic Exercise Training Partially Reverses Inward Hypertrophic Coronary Arteriole Remodeling in Type 2 Diabetic db/db Mice
Authors: Trask Aaron J. , Delbin Maria A. , Katz Paige S. , Zanesco Angelina , Lucchesi Pamela A.
Published: June 2021

The aim of this study was to determine whether aerobic exercise training improves impaired coronary arteriole remodeling in type 2 diabetic db/db mice. Eight wk old male mice were divided into: control sedentary (Db/db), diabetic sedentary (db/db), and diabetic trained (db/db‐TR). Exercise training (ET) was performed on a mouse treadmill for 5 days/wk, 60 min/day, at a speed of 9–10 m/min, 0% grade without electric stimulation for 8 wks. Coronary arterioles were mounted on a pressure myograph to measure wall thickness and diameters over a range of pressures (0–125 mmHg). Coronary arterioles isolated from db/db mice exhibited decreased luminal diameter (Db/db: 119 ± 6 μm vs. db/db: 102 ± 5 μm p < 0.05) with thicker microvascular walls (Db/db: 7.0 ± 0.2 μm vs. db/db: 9.1 ± 0.6 μm p < 0.01), which significantly increased the wall:lumen ratio (Db/db: 6.2 ± 0.4 vs. db/db: 8.8 ± 0.6, p < 0.01). Compared to db/db mice, coronary arterioles isolated from db/db‐ TR mice had similar internal diameter (109 ± 6 μm, p > 0.05) and wall thickness (7.5 ± 0.5 μm, p = 0.14), while wall:lumen ratio (6.8 ± 0.2 μm, p < 0.05) and growth index (db/db: 22.8 vs. db/db‐TR: 5.8, % of Db/db) were reduced. External diameters were not different among groups. These data suggest that aerobic exercise training can mitigate inward hypertrophic remodeling observed in coronary arterioles of type 2 diabetic mice.

Support: NIH T32HL098039 to AJT; NIH HL056046 and NCH to PAL.
Evidence of the regulatory potential of human skeletal muscle feed arteries
Authors: Ives Stephen James , Andtbacka Robert Hans Ingemar , Noyes R. Dirk , Donato Anthony , Park Song Young , Gifford Jayson , Lesniewski Lisa , McCullagh Joseph , Richardson Russell S
Published: June 2021

Prior work has documented the regulatory role of skeletal muscle feed arteries in rodent models, however it is unknown if human skeletal muscle feed arteries display such functionality. Feed arteries (888±188 μm ID) from 6 humans (53±3 yrs) were studied using pressure myography. Vessel function was assessed using potassium chloride (KCl), phenylephrine (PE), acetylcholine (ACh), and sodium nitroprusside (SNP) dose response curves (DRCs) to characterize non‐receptor and receptor‐mediated vasoconstriction as well as endothelium‐dependent and independent vasodilation, respectively. Vascular resistance was calculated using internal diameter changes due to pharmacological stimulation and Poiseuilles’ law (R= 8nL/πr4). Vessel function protocols revealed robust vasoconstriction in response to PE and KCl (37 ± 6; 43 ± 19 % vasoconstriction, respectively), but also achieved significant vasodilation with ACh and SNP (97 ± 11; 117 ± 24 % vasodilation, respectively). Using the maximal PE and KCl‐induced vasoconstriction, vascular resistance was calculated to increase by 304 and 392%, respectively. These findings provide evidence that human skeletal muscle feed arteries are capable of generating significant changes in vascular resistance, and thus likely play a role in blood flow regulation. Support by NIH PO1 HL‐091830.
Acute exercise increases prostate tumor blood flow in rats
Authors: McCullough Danielle J , Stabley John N. , Davis Robert T. , Siemann Dietmar W. , Behnke Bradley J.
Published: June 2021

During exercise there is a redistribution of blood flow within the body based upon the functional vascular compliance of a given tissue. Given the altered vasculature morphology within tumors (e.g., less smooth muscle), there may be a profound increase in tumor blood flow during exercise. We utilized vehicle control (CON; n=5) or tumor bearing (TB; n=6) Copenhagen rats to test whether prostate tumor perfusion increases during moderate intensity exercise. Dunning R‐3327 MatLyLu tumor cells were injected (104cells) into the ventral lobe of the prostate of TB animals and blood flow experiments performed 21 days later. Blood flow and vascular conductance to the tumor and prostate tissue was measured using radiolabelled microspheres at rest and during exercise (treadmill; 5 min @ 15 m/min, 10° incline). There was a marked increase in tumor blood flow during exercise (rest, 17 ± 2; exercise, 52 ± 5 ml/min/100 g; p<0.001). Tumor vascular conductance increased by ~ 270% from rest to exercise. Blood flow to the surrounding prostate tissue (TB) or the prostate (CON) did not change with exercise. This is the first investigation to demonstrate that tumor perfusion is significantly increased with exercise. The precise mechanisms for the increase perfusion (e.g., blunted vasoconstriction), and whether increase in blood flow during exercise affect tumor growth, remains to be determined. Support: NIH (AG‐31317); FBRP (1BN‐02).
Treatment of male mice with estrogen for the study of contractile dysfunction
Authors: Kararigas Georgios , Bito Virginie , Nguyen Ba Tiep , Jarry Hubertus , Sipido Karin R , Regitz-Zagrosek Vera
Published: June 2021

In older and/or obese men, estrogen (E2) levels rise significantly, which have been associated with increased incidence of cardiovascular disease. In particular, high E2 levels are a significant predictor of poor prognosis and higher mortality in men with heart failure. We developed the present model to study contractile dysfunction in males and to verify the causal relationship between high E2 levels and impaired contractility. We injected i.p. middle‐aged male mice with 0.2 mg/kg E2 and after five hours we isolated cardiomyocytes (CMs). E2 led to reduced CM contractility, which was characterized by increased expression of the myosin regulatory light chain (MRLC) interacting protein (MYLIP) E3 ubiquitin ligase. We found that MRLC is an in vivo substrate of MYLIP leading to increased MRLC ubiquitination. Hence, MRLC levels decreased in E2‐treated CMs. In addition, three months of oral E2 treatment in male mice led to structural remodeling resulting in a hypertrophic response of the heart. Our model is the first to show that E2 has direct deleterious effects in the male heart. We believe it will be useful to unravel the underlying pathways and to identify novel therapeutic targets for heart failure.
Physical training modulates adiposity and protects mice from obesity and glucose intolerance
Authors: Higa Talita Sayuri , Spinola Acauã Vida , Fonseca-Alaniz Miriam Helena , Evangelista Fabiana Sant'Anna
Published: June 2021

Obesity increases the risk of glucose intolerance (GI) and diabetes. Physical training (PT) has been recommended to prevention and treatment of obesity and GI. We investigated the role of adipose tissue to prevents GI by PT. Male C57BL/6J mice were assigned into chow‐fed controls (C, n=10), cafeteria diet (CAF, n=10), chow‐fed trained (TR, n=13), and cafeteria diet plus trained (CAF‐TR, n=12). PT was performed simultaneously with diet and consisted of 8‐wk running session of 60 min at 60% of maximal speed, 5 days/wk. After PT, TR and CAF‐TR increased exercise tolerance and showed lower BW compared to C and CAF (p<0,05). Retroperitoneal and periepydidimal fat pads were higher in CAF than C and TR (p<0,05), and lower in TR than CAF‐TR (p<0,05). Adipocyte diameter, BW gain and daily food intake increased only in CAF (p<0,05), which were counteracted by PT in CAF‐TR. Resting oxygen uptake and insulin did not change. Before PT fasting glycemia and intraperitoneal glucose tolerance test response were similar among groups, however after PT the CAF group showed hyperglycemia compared to C and TR (p<0,05) and glucose intolerance compared to others groups (p<0,05). Lipolytic activity increased in CAF (5607,2±723,6 nmol/106cels/h) compared to C and TR (2167,2±601,6 and 2549,5±398,6 nmol/106cels/h, respectively, p<0,05). In conclusion, PT prevents GI and this response is associated with reduced adipose tissue and BW.
ROLE OF ESTROGEN THERAPY ASSOCIATED WITH PHYSICAL TRAINING ON OXIDATIVE STRESS PROFILE IN OVARIECTOMIZED RATS
Authors: de Brito Sebastião , de Oliveira Brito Janaina , Bernardes Nathalia , Dias Danielle , Llesuy Susana , Irigoyen Maria-Cláudia , De Angelis Kátia
Published: June 2021

The aim of study was to verify the effects of 17â estradiol therapy (ET) associated or not with physical training (PT) on cardiac, renal and muscular oxidative stress in ovariectomized (OVX) rats. Female Wistar rats were divided into: control (SC), sedentary OVX (SO), sedentary OVX+ET (SOE) and trained OVX+ET (TOE) (n=8 each group). Estradiol pellets were implanted and PT was performed on a treadmill (8wks). Oxidative stress was evaluated by chemiluminescence (CL), thiobarbituric acid reactive substances (TBARS), redox balance of glutathione (GSH/GSSG), catalase (CAT), superoxide dismutase (SOD) and gluthatione peroxidase (GPx) determinations. Oxidative stress was higher in SO (CL, GSH/GSSG: heart and TBARS: kidney and muscle) than in SC group. The SOE and TOE showed reduction compared to SO group in: cardiac CL (SOE:60%; TOE:61%), renal CL (SOE:45%; TOE:55%) and TBARS (SOE:20%; TOE:27%), and muscle TBARS (SOE:84%; TOE:84%). The cardiac GSH/GSSG was higher in SOE and TOE in relation to SO group (SOE: 14.85 ± 2.18; TOE: 16.20 ± 1.76 vs. SO: 4.59 ± 0.82 umol/g). The SOE and TOE showed increased renal CAT, cardiac and renal GPx compared to SO group, attenuating OVX alterations. In conclusion, long‐term ET, associated or not with PT, improved oxidative stress profile in OVX‐rats, suggesting a positive role of these therapies association after menopause.

Financial support: 2009/06865‐8; 2007/57595‐5
Endurance exercise training enhances regional femoral and tibial bone blood flow during exercise
Authors: Stabley John N. , Davis Robert T. , Moningka Natasha C. , Behnke Bradley J. , Delp Michael D.
Published: June 2021

The principal nutrient artery to the femur demonstrates an increase in endothelium‐dependent, nitric oxide mediated vasodilation in rats after endurance exercise training. The present study sought to determine whether exercise training alters hindlimb bone blood flow distribution at rest and during exercise. Sprague‐Dawley rats were exercise trained (ET, 6–8 mo. old, n=10) with treadmill (TM) exercise at 15 m/min, 15° incline for 60 min/d over a 10–12 wk period. Sedentary (SED, n=10) control animals were acclimated to TM exercise for 5 min/d during the week preceding their blood flow measurements using the microsphere method. During exercise, ET rats demonstrated increases in blood flow to the femoral diaphysis (rest: 27 ± 8, TM: 56 ± 10 ml/min/100 g, p<0.05), femoral diaphyseal marrow (rest: 92 ± 13, TM: 154 ± 29 ml/min/100 g, p<0.05), distal tibial metaphysis (rest: 43 ± 13, TM: 108 ± 22 ml/min/100 g, p<0.05), and tibial diaphysis (rest: 37 ± 14, TM: 68 ± 12 ml/min/100 g, p<0.05). The tibial diaphyseal marrow was the only hindlimb bone region to elevate flow during exercise (rest: 92 ± 7, TM: 202 ± 21 ml/min/100 g, p<0.05) in SED animals. The more generalized elevations in bone perfusion during exercise in ET rats may serve to augment medullary fluid pressures and bone interstitial fluid flow, thus benefiting bone integrity. Supported by NASA NNX09AP06G and NIH AG‐31317.
Effect of Exercise on the Mitochondrial Profile of Testicular Tumors in Aged Fischer 344 Rats
Authors: Welter Ashleigh E , Nguyen Linda M-D , Behnke Bradley J , Dominguez James M , Adhihetty Peter J
Published: June 2021

Tumors exhibit an altered mitochondrial metabolic profile and suppressed mitochondrial‐mediated apoptosis. Exercise improves mitochondrial function and reduces susceptibility to mitochondrial‐mediated apoptosis in a variety of tissues but its effect on aged tumorigenic testes is unexplored. Thus, we investigated whether key mitochondrial signaling markers, mitochondrial content, and/or apoptotic factors differed in, 1) young control testes (YC; 6m; Fischer 344; n=4 group), 2) young exercised‐trained testes (YE; 6m; 10 weeks treadmill training) 3) old control non‐tumorigenic testes (OC; 22–24m; Fischer 344), 4) old tumorigenic testes (OT), 5) old tumorigenic exercise‐trained (OT‐E) testes. OT and OT‐E testes mass were similar but significantly larger (1.5‐ fold) than YC, YE and OC. Mitochondrial content was significantly elevated by 70–80% in both OT and OT‐E testes when compared to YC, YE, and OC, and similar trends were evident in upstream mitochondrial biogenesis signaling markers LKB1, AMPK, p38 and PGC‐1á. Bax:Bcl‐2 ratio, an apoptotic index, was significantly reduced by 70–80% in both OT and OT‐E when compared to YC, YE, and OC. Our findings indicate old tumorigenic testes exhibit enhanced mitochondrial biogenesis signaling and mitochondrial content but have reduced mitochondrial apoptotic susceptibility when compared to young healthy testes and this was unaffected by exercise.
Skeletal muscle OGT and OGA expression: associations with hormone replacement therapy, physical activity and muscle area in postmenopausal women
Authors: Toivonen Minna Heini Maria , Pöllänen E , Ahtiainen M , Suominen H , Taaffe D , Cheng S , Takala T , Kujala U , Tammi M , Sipilä S , Kovanen V
Published: June 2021

In skeletal muscle, prolonged increase in O‐GlcNAc transferase (OGT) mediated protein O‐linked N‐acetylglucosaminylation (O‐GlcNAcylation) is associated with age and muscle atrophy. The current study investigated the effects of estrogen based hormone replacement therapy (HRT) and power training (PT) on OGT and O‐GlcNAcase (OGA; enzyme releasing O‐GlcNAc moiety from proteins) gene expression in postmenopausal women and the association of OGT and OGA gene expression with muscle crosssectional area (CSA). Twenty seven 50–57‐year‐old women from a yearlong randomized placebo‐controlled trial formed the three study groups (HRT, n=10; PT, n=8; control, CO, n=9). Following the one year, the ratio of OGT to OGA, reflecting O‐GlcNAcylation capacity, increased 20.0% in the CO group, while in the PT (15.2%) and HRT (−3.9%) group the change was not significant (HRT vs. CO: p=0.004 and PT vs. CO: p=0.54). OGT/OGA correlated negatively with muscle CSA (r= −0.61, p=0.001). Our results suggest that an increase in O‐GlcNAcylation capacity is associated with a decrease in muscle CSA, and that HRT and possibly PT influence muscle OGT and OGA expression, which may be one mechanism by which HRT and PT prevent age‐related loss of muscle mass. Supported by the Academy of Finland, Finnish Ministry of Education and Culture and Finnish Cultural Foundation.
The effects of varying caffeine levels in a carbohydrate‐containing sports drink on the metabolism and performance of triathletes
Authors: Borgard Chris patrick , OˈHara Christie , LaGuire Tiev C , Reaves Scott K
Published: June 2021

The objectives of the study included the development of a sports drink using a novel combination of carbohydrates, caffeine, electrolytes and antioxidants and to test its effects on endurance performance using a randomized, double‐blind design that included two test formulas with different levels of caffeine and a placebo control beverage (P). The two test beverages were 6% carbohydrate (CHO) and only varied in their caffeine content. The low caffeine formula (CHO/LC) contained 10.6 mg caffeine per 100 ml beverage and the high caffeine formula (CHO/HC) contained twice that amount (21.2 mg/100 mL). Ten male collegiate triathletes were tested once with each beverage with a one week washout period. Each test included a 20‐minute swim followed immediately by a 70‐minute cycling event and then a graded treadmill run to exhaustion of approximately 25 minutes. Beverages were provided 30 minutes prior to the swim and at three other points during exercise. Respiratory exchange ratio (RER) data suggested similar substrate use in the CHO/LC and CHO/HC and each had higher RERs compared to P. Times to exhaustion and average VO2max at the end of the run for CHO/LC and CHO/HC were both greater than P but CHO/LC vs. CHO/HC were not significantly different. Overall, substrate use was altered and performance enhanced in CHO/LC and CHO/HC compared to P but the additional caffeine in the CHO/HC group did not further improve performance.
Aerobic Interval Training Ameliorates Exertional Dyspnea by Improving the Ventilatory‐Hemodynamic Efficiency in Patients with Systolic Heart Failure
Authors: HUANG SHU-CHUN , Wang Jong-Shyan
Published: June 2021

This study investigated how aerobic interval training (AIT) affects dyspnea during daily activities by modulating ventilatory‐hemodynamic efficiency in patients with systolic heart failure (SHF, left ventricular ejection fraction<40%). Sixty patients with SHF were randomized to divided intoAIT (n=30) and general healthcare (GHC, n=30) groups. The AIT subjects performed the supervised hospital‐based training(3‐minute intervals at 40% and 80%VO2peak) for30 minutes/day, 3 days/week for 12 weeks; and the GHC subjects only engaged in general home‐based health care. Following the intervention, the AIT group exhibited higher VO2peak, oxygen uptake efficiency slope (OUES), and lowerVE‐VCO2 slope than the GHC group. Additionally, AIT, but not GHC, increased cardiac output (CO)and reduced systemic vascular resistance (SVR)at the relative workload during the graded exercise test. This regimen simultaneously reduced total and exertional dyspnea‐related scores (MLHF‐ED) in Minnesota Living with Heart Failure Questionnaire. Moreover, reduced MLHF‐ED score were associated with increased OUES and CO and decreased VE‐VCO2 slope and SVR. Hence, we conclude that AIT ameliorates exertionaldyspnea by improving the ventilatory‐hemodynamic efficiency in patients with SHF.
Role of bone marrow‐derived mesenchymal stem cells in recovery following cervical spinal hemisection
Authors: Mantilla Carlos B , Zhan Wen-Zhi , Restrepo Laura , Arias Alejandra , Sieck Gary C
Published: June 2021

Following spinal cord hemisection at C2 (C2SH), removal of descending excitatory drive to phrenic motoneurons results in ipsilateral diaphragm muscle (DIAm) paralysis. Over time, there is partial recovery of rhythmic phrenic activity post‐C2SH. Intrathecal delivery of brain‐derived neurotrophic factor (BDNF) enhances this functional recovery, with 100% of rats displaying rhythmic phrenic activity at 14 days (14D) post‐C2SH; but in humans intrathecal BDNF is associated with adverse effects that limit application. Stem cell transplantation may facilitate a targeted BDNF delivery and minimize side effects. In adult rats, bilateral DIAm EMG activity was chronically monitored before and after C2SH to assess recovery. Wild type, GFP‐expressing bone‐marrow derived mesenchymal stem cells (MSC‐wt) or BDNF‐GFP expressing MSC (MSC‐BDNF) were injected at C2. Seventy % of MSC‐BDNF treated rats displayed functional recovery at 7D and 100% at 14D post‐C2SH. Functional recovery in BDNF‐wt treated C2SH rats was not different from untreated C2SH controls (~10% at 7D and ~40% at 14D). Cellular incorporation of MSC into the spinal cord was evident by GFP immunoreactivity in both MSC groups. We conclude that targeted MSC‐based delivery of BDNF enhances functional recovery of DIAm respiratory activity following an upper cervical spinal cord injury.

Supported by NIH grant HL096750 and Mayo Clinic.
Gap‐junction coupling in a computational model of the hypoglossal motor nucleus
Authors: Memelli Heraldo , Horn Kyle G. , Wittie Larry D. , Solomon Irene C.
Published: June 2021

Hypoglossal motoneurons (HMs) innervate the tongue muscles and play an important role in the maintenance of upper airway patency. Various studies have focused on investigating the biophysical properties of individual HMs, and recent work from our laboratory has focused on developing a series of mathematical HM models that accurately simulate the spiking behaviors and electrophysiological properties recorded experimentally in single HMs. As part of our HM modeling efforts, we have developed a computational model of a large‐scale network of HMs that simulates the entire hypoglossal motor nucleus (nXII) of the rat (approximately 1700 neurons). The HMs are connected with each other through gap junctions, and we analyze different aspects of their coupling mechanisms. Among other results, we noticed the emergence of different firing groups in a rather homogenous population of HMs. We also examined the network with less homogenous HMs composed of slight differences in their biophysical properties, and created a series of networks with different connectivity topologies. Finally, we analyzed the synchrony of our (nXII) model via numerous computer simulations. Supported by HL63175 and NS049310
Differential expression of candidate genes associated with voluntary physical activity levels in inbred mice
Authors: Dawes Michelle , Ferguson David , Lightfoot Tim
Published: June 2021

This study investigated the levels of gene expression for eight potential candidate genes speculated to play a role in voluntary physical activity: Leptin Receptor (Lepr), Actinin alpha 2 (Actn), Myostatin (Mstn), Calsequestrin1 (Casq1), Dopamine Receptor 1 (Drd1), Nescient helix loop helix 2 (Nhlh2), and Glucose Transporter member 4 (Slc2a4) in soleus and nucleus accumbens of high active (C57L/J, n=3) and low active (C3H/HeJ, n=4) inbred mice as well as high (n=16) and low active (n=18) F2 offspring. All animal procedures were conducted in conformance with the FASEB Statement of Principles for the use of animals in research and education. The F2 cohort was used as a control to remove gene expression due to the homozygosity of the inbred animals. mRNA was isolated and quantitative real‐time PCR was performed. Gene expression was calculated as a ratio of the cross point (ct) of the target gene to RN18S (control gene). Mstn (p=0.02) and Casq1 (p=0.04) had higher expression in the soleus of low active animals. Mstn was also found to be differentially expressed in soleus between the high and low active F2 animals (p=.045), as well as between the progenitors (p=.03), when the groups were analyzed separately. While other genes may also regulate activity, none appear to function by differences in transcriptional product as do Mstn and Casq1 in this study. This work is supported by Texas A&M University.
65‐gene based risk score predicts recurrence and survival in hepatocellular carcinoma
Authors: Kim Soo Mi , Lee Ju-Seog
Published: June 2021

We aimed to develop an easy‐to‐use risk score with a limited number of genes that can predict the prognosis of hepatocellular carcinoma (HCC) patients. In 139 patients, we identified 65 genes whose expression patterns were significantly associated with overall survival and recurrence in two independent HCC cohorts. The risk score was developed by using Cox coefficient values for the 65 genes from the training set. The risk score was a highly significant predictor of both overall survival and recurrence in the test cohort. The risk score also predicted the prognosis of 65‐base invasion. High‐risk HCC was characterized by activation of IGFR (P = 2.2 × 10–4) and AKT (P = 0.003), whereas low‐risk disease was associated with active mutations of CTNNB1 (P= 0.05). Our new risk score can identify two clinically distinct HCC subtypes in a simple and highly reproducible manner across multiple data sets.
Caveolin‐1 regulation of mouse lung epithelial progenitor cell differentiation and morphogenesis.
Authors: Wynia Brian Paul , Chernaya Olga , Shinin Vasily , Minshall Richard D.
Published: June 2021

Caveolin‐1 (Cav‐1) is an intracellular scaffolding protein that regulates cell signaling and caveolae formation. We previously observed morphological and patterning defects in Cav‐1−/− mouse lungs and thus hypothesized that Cav‐1 may play an important role in the regulation of lung epithelial cell differentiation and morphogenesis. Distal lung epithelial progenitor cells (LEPCs) isolated from wild‐type (WT) and Cav‐1−/− mouse lungs expressed generic epithelial markers E‐cadherin and pan‐cytokeratin, but were negative for differentiation markers Clara Cell Secretary Protein (CCSP) and Aquaporin‐5 (AQP5). Cav‐1−/− LEPC colonies grown on plastic showed disrupted E‐cadherin and ß‐catenin junctional staining after induction of differentiation by addition of high calcium. qRT‐PCR and Western blot analysis of WT and Cav‐1−/− cells cultured in matrigel showed induction of CCSP and AQP5 in WT cells only. Also, epithelial cell cyst formation was observed in both WT and Cav‐1−/− cells, however cyst diameter was significantly greater for Cav‐1−/− cysts. From these studies, we speculate that defective differentiation and cell‐cell communication in the absence of Cav‐1 leads to dysfunctional distal lung epithelial cell morphogenesis.
cAMP regulates polarization and apoptosis during mammary epithelial acini formation in vitro
Authors: Nedvetsky Pavel I. , Kwon Sang-Ho , Debnath Jayanta , Mostov Keith E.
Published: June 2021

Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three‐dimensional (3D) culture models utilizing MCF10A mammary epithelial cells, hollow acini form due to integrin‐dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking ECM contact. Here, we report that cAMP‐dependent protein kinase A (PKA) promotes hollow acini formation via two mechanisms. First, cAMP accelerates the polarization of outer cells due to ERK/MAPK inhibition. Second, cAMP, promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed resulting in profound luminal filling. cAMP‐dependent apoptosis is accompanied by a posttrascriptional increase in the proapoptotic protein BIM, which is at least partially ERK‐independent. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cell cultures both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus.

This study was supported by NIH grants to K.E.M (R01 DK074398 and P01 AI53194), S.K. (DK082115), and J.D (RO1 CA126792). P.I.N. was supported by a fellowship from the German Research Society (DFG; Ne‐897/1‐1).
Liver x receptors regulate human organic anion transporter 1 in renal proximal tubule
Authors: Kittayaruksakul Suticha , Soodvilai Sunhapas , Asavapanumas Nithi , Chatsudthipong Varanuj
Published: June 2021

Liver x receptors (LXRs) play important role in the regulation of cholesterol and glucose homeostasis by regulating several transporters. In this study, we investigated the effects of LXRs on OAT1‐mediated organic anion transport in renal S2 cells expressing hOAT1 and mouse kidney. Exposure the S2 cells to LXRs agonists (TO901317 and GW3965) and their endogenous ligand (22(R)‐hydroxycholesterol) decreased OAT1‐mediated [14C]‐PAH uptake. This inhibition was abolished by co‐incubation with 22(S)‐hydroxycholesterol, a LXRs antagonist. Kinetic analysis revealed that LXRs activation decreased the maximum rate of PAH transport (Jmax) but had no effect on the affinity of the transporter (Kt). Data from western blot analysis showed the decrease in hOAT1 protein expression following LXRs activation. Similarly, we found the inhibition of [14C] PAH uptake in renal cortical slices as well as a decreased mOAT1 protein expression in mouse kidney. Our findings indicated for the first time that OAT1 function was downregulated by LXRs activation in renal proximal tubule.

This work is supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program.
Real‐time visualization of calcium oxalate crystals in Drosophila ‐ a gut and renal model of kidney stones
Authors: Bondeson Daniel P , Hirata Taku , Cabrero Pablo , Ritman Erik L , Dow Julian AT , Romero Michael F
Published: June 2021

Background

Kidney stones (nephrolithiasis) are an expensive and painful health problem with a complex etiology with ~70% of stones composed of calcium oxalate (CaOx). The Slc26 gene family contains several proteins that transport oxalate in the gut and kidney. Slc26a6 and its homologs exchange Cl− for oxalate (ox2−), as well as SO4 2−, HCO3 − and formate. Slc26a6−/− mice have hyperoxaluria and CaOx stones illustrating a physiologic collaboration between the gut and kidney. We developed a Drosophila genetic model for CaOx stones (dPrestin, Slc26a5/6, manipulation) allowing rapid gut, renal and whole‐body physiological assessment (min/h v. mo/y).

Methods

(Tissue) First, Malpighian tubules (MT, renal tubule) were dissected from wild type and dPrestin knockdown mutants and soaked in ox2− solutions. CaOx stones were visualized by DIC microscopy. Second, we generated Drosophila expressing eYFP in the MT, MT+gut or whole body. eYFP is a halide (Cl−) sensor, allowing quantification of Cl−/ox2− exchange by native dPrestin. (Whole body) Wild type and dPrestin knockdown flies were fed ox2− enriched food, and flies imaged (CaOx quantified) by micro computed tomography (MicroCT).

Results

Exposing dPrestin‐knockdowns to bath ox2− elicited almost no CaOx crystals. However, exposing wt‐MT to ox2− elicited many CaOx crystals in 20 min. Increased CaOx crystal formation was also coupled with an eYFP intensity increase, matching dPrestin Cl−/ox2− exchange characteristics. MicroCT showed more CaOx crystals in flies that fed on ox2− longer.

Conclusions

Our observations indicate that Drosophila and dPrestin manipulation can be used for rapid, detailed studies of gut, renal and whole organism oxalate metabolism & CaOx stone formation. [DK83007]
Isolation and characterisation of lipid rafts containing the kidney‐specific Na(+)K(+)2Cl(−) cotransporter
Authors: Dathe Christin , Daigeler Anna , Jankowski Vera , Kaminski Henriette Josephine , Jankowski Joachim , Fähling Michael , Bachmann Sebastian , Paliege Alexander
Published: June 2021

Transport activity of the thick ascending limb (TAL) Na(+)K(+)2Cl(−) cotransporter (NKCC2) critically depends on its association with lipid raft membrane microdomains (LR). Aim of the present study was to characterize the protein composition of NKCC2‐containing LR. To this end we isolated detergent‐resistant LR from rat renal outer medullary tissue samples using a discontinuous sucrose gradient and ultracentrifugation (floating assay). NKCC2‐containing LR were purified from total LR by immunoprecipitation. Protein content of the precipitates was analyzed by western blot and mass spectrometry (MS). Floating assay confirmed accumulation of NKCC2 in LR‐enriched fractions. Analysis of the immunoprecipitate demonstrated the presence of NKCC2 and, among others, the lipid raft proteins flotillin‐1 and annexin A2. In conclusion, we have successfully established a protocol for the isolation of NKCC2‐containing LR from kidney samples. Further analysis of the protein content of these LR may provide insight into the regulation of NKCC2 function.

Support: DFG FOR667
Intestinal epithelial barrier dysfunction induces IL‐17 production by γδ intraepithelial lymphocytes: A novel mechanism of mucosal tolerance
Authors: Edelblum Karen L , Turner Jerrold R
Published: June 2021

Myosin light chain kinase (MLCK) is essential for pathophysiological epithelial tight junction regulation. Transgenic (Tg) mice with intestinal epithelial‐specific expression of constitutively‐active MLCK (CA‐MLCK) demonstrate increased tight junction permeability and mucosal Th1 activation, but do not develop spontaneous disease. Therefore, we hypothesized that chronic luminal antigen exposure induces tolerance, which in turn prevents immune‐mediated disease in CA‐MLCK Tg mice. To identify underlying mechanisms, colonic intraepithelial and lamina propria T cell functions were assessed. CA‐MLCK expression increased lamina propria CD4+ and CD8+, but not Foxp3+, T cell numbers, suggesting that tolerance was not driven by Foxp3+ T regulatory cells. Further, cytokine production after ex vivo stimulation was similar in lamina propria CD8+ T cells, although slight increases in CD4+T cell IL‐17 production were noted. In contrast, IL‐17 production by intraepithelial γδ TCR+, but not αβ TCR+, T cells was markedly increased in CA‐MLCK mice. Antibiotic treatment normalized IL‐17 production in CA‐MLCK Tg mice, suggesting that the epithelial barrier regulates microbial activation of Th17 responses. These data suggest that limited Th17 activation may dampen Th1 responses and thereby induce tolerance that prevents disease in the setting of chronic epithelial barrier dysregulation. Funding: AGA and NIH.
Hibernation alters the gut microbial community in ground squirrels
Authors: Carey Hannah V , Walters William A. , Knight Rob
Published: June 2021

Diet is a major factor that influences the structure of gut microbiota. Hibernating mammals that fast for several months each winter provide an extreme example of natural dietary change that can impact the microbiome. We explored this relationship using 16S rRNA pyrosequencing of DNA extracted from cecal contents of 13‐lined ground squirrel litters over one year. In all seasons dominant phyla were Bacteroidetes and Firmicutes. Principal component analysis of UniFrac distances showed that samples clustered strongly by season, with summer communities distinct from winter, and spring (2 wk after feeding resumed) intermediate. For hibernators, time since start of torpor (i.e., length of fast) influenced community diversity but torpor/arousal state did not. Bacterial taxa that distinguished hibernator microbiotas from summer included greater representation of mucin degraders (i.e., Verrucomicrobiaceae and Bacteroidales), reduced occurrence of Clostridiales and the absence of Lactobacilliaceae. The diversity of maternal microbiotas had little influence on seasonal clustering of their young. The results from this natural system underscore the profound impact of host diet on the structure of gut microbial communities. Supported by funds from UW‐Madison School of Veterinary Medicine (HVC), the Bill and Melinda Gates Foundation (RK) and the Howard Hughes Medical Institute (RK).