bluuebunny/crossref_metadata_2025 huggingface dataset:
Thioredoxin‐Interacting Protein Mediates Hcys‐induced NLRP3 Inflammasome Activation in Mouse Podocytes
Authors: Abais Justine , Boini Krishna M. , Xia Min , Li Pin‐Lan
Published: June 2021

Our recent studies have demonstrated that reactive oxygen species (ROS) derived from NADPH oxidase activates NLRP3 inflammasomes causing homocysteine (Hcys)‐induced podocyte and glomerular injury. However, the precise mechanism regarding how ROS activates the inflammasome is still unknown. The current study explored whether thioredoxin‐interacting protein (TXNIP) mediates Hcys‐induced NLRP3 inflammasome activation in cultured podocytes. TXNIP, the regulatory inhibitor of the antioxidant thioredoxin (TRX), is thought to dissociate from TRX in response to elevated levels of oxidative stress to bind to inflammasome protein NLRP3 and activate the inflammasome complex. It was found that TXNIP protein expression was decreased by Hcys treatment in a concentration‐dependent manner. However, TRX protein expression increased upon Hcys stimulation. At an inflammasome‐activating dose of 40 μM Hcys, TXNIP protein expression decreased by 60.4% and TRX increased 2.0‐folds versus the control. However, co‐immunoprecipitation demonstrated that Hcys increased TXNIP binding to NLRP3 by 1.5‐folds over the control, which was attenuated by TXNIP siRNA. Confocal microscopy showed that inhibition of TXNIP also prevented Hcys‐induced NLRP3 inflammasome formation by reducing colocalization of NLRP3 with ASC or caspase‐1. Hcys‐induced NLRP3 inflammasome activation was also attenuated by TXNIP siRNA, as shown by a 45.3% inhibition of caspase‐1 activity and 39.2% reduction of IL‐1β production. In addition, TXNIP inhibition protected podocytes from injury as demonstrated by normal expression levels of podocyte markers like podocin and desmin. From these results it is concluded that TXNIP binding to inflammasome protein NLRP3 is a key signaling mechanism for Hcys‐induced formation and activation of the NLRP3 inflammasome, thereby inducing podocyte injury (supported by NIH grants DK54927 and 1F31AG043289).
Effect of the Epac activator 8‐pCPT‐2′‐O‐Me‐cAMP (8‐pCPT) on the expression pattern of VE‐cadherin (VE‐cad) & connexins (Cxs) in Human Coronary Artery Endothelial Cells (HCAECs)
Authors: Quinn Rachael Ellen , Roberts Owain , Kamishima Tomoko , Dart Caroline , Quayle John
Published: June 2021

Homocellular junctions between endothelial cells include adherens junctions, formed by cadherins, & gap junctions, formed by Cxs. Dynamic regulation of these junctions is necessary to maintain the endothelial barrier. We examined the effect of Ca2+ & 8‐pCPT, a selective activator of exchange protein directly activated by cAMP (Epac), on the expression pattern of VE‐cad & Cxs in HCAECs. VE‐cad, Cx37, 40 & 43 were identified in HCAECs by immunocytochemistry. VE‐cad was identified in parts of the cell membrane, without overlapping with Cx37, 40 or 43. Application of 8‐pCPT produced more continuous VE‐cad membrane staining that overlapped with Cx37. Disruption of VE‐cad interactions using VE‐cad antibody changed HCAEC morphology from flattened & continuous to small dispersed colonies. Removal of Ca2+ from medium resulted in rounded cells isolated from each other. Following Ca2+ restoration, a normal pattern of VE‐cad membrane staining returned, followed by return of membrane Cx staining. Addition of VE‐cad antibody blocked this recovery. Epac activation promotes localisation of VE‐cad to the membrane & adherens junction formation. Redistribution of VE‐cad has a subsequent effect on Cx localisation. Adherens junction formation in HCAECs therefore precedes & promotes Cx interactions at cell junctions. Funded by the University of Liverpool
In vitro effects of organophosphorus pesticide malathion on the reactivity of rat aorta
Authors: Fagundes Agnes Afrodite Sumarelli Albuquerque , Carvalho Marco Túlio Menezes , Ferreira Luciana Garros , Silveira Ana Paula Cassiano , de Nadai Tales Rubens , Capellini Verena Kise , Evora Paulo Roberto Barbosa , Celotto Andrea Carla
Published: June 2021

In vitro studies have demonstrated organophosphate‐mediated vasodilator effects, suggesting that organophosphates may have peripheral activity independent of acetylcholinesterase inhibition. To evaluate the effect of the organophosphate malathion, on the reactivity of isolated rat aorta as well as to explore the mechanisms involved in this response, including the role of nitric oxide (NO) and prostacyclin (PGI2). The study protocol included two methods: in vitro vascular reactivity and flow cytometry (using DAF‐FM DA dye). In rat thoracic aorta segments pre‐constricted with phenylephrine (Phe), malathion induced vasodilation in arteries with endothelium. Acetylcholine‐mediated vasodilatation was not affected when incubate with malathion. Malathion‐mediated vasodilation was blocked by L‐NAME a non‐specific nitric oxide synthase inhibitor and/or indomethacin, an unspecific cyclooxygenase inhibitor. In thoracic aortas rings, with endothelium, Phe promoted dose‐dependent contraction, which was reduced by malathion. In rings with endothelium incubated with malathion and the two blockers, the Phe contraction, was 100% restored. The role of NO was confirmed using flow cytometry. Malathion causes endothelium‐dependent relaxation mediated synergistically by NO (cGMP pathway) and PGI2 (cAMP pathway) that would be a preventive mechanism against spasm and thrombosis. Research support FAEPA.
Evaluation of the iHealth Blood Pressure Monitoring System
Authors: Midriff Whitney , Latman Neal S.
Published: June 2021

Self‐monitoring of blood pressure (SMBP) at home has several significant advantages compared to blood pressure (BP) monitoring by a clinician in a clinical setting. The purpose of this study was to evaluate the accuracy of a SMBP system, the iHealth, and determine the effectiveness of wirelessly transferring the results via the internet to another site, such as a clinician's office. The iHealth accuracy was determined on 91 subjects, age range 18 to 83, with a systolic BP range of 97 to 171 and diastolic range of 46 to 140 mmHg. The BP of each subject was measured 3 times with the iHealth test device alternating with 3 measurements with the reference instrument, the MABIS SmartSpeed Plus. Accuracy was determined by the ANSI/AAMI SP10:2002/A1:2003 standard. The systolic and diastolic mean difference was −1.9 (S.D.=7.6) and −1.0 (S.D.=4.2) mmHg. Therefore, in this study, the iHealth satisfied the ANSI/AAMI BP accuracy standard for systolic and diastolic of +/− 5 mmHg or less and S.D.=8 or less. The iHealth accurately transmitted the BP data via the attached iPad in 100% of the 150 attempts. In conclusion, the iHealth BP monitor with attached iPad appeared to accurately measure BP and reliably transmit the information over the wireless internet connection in this study.
Treatment of cerebral small vessel disease with relaxin
Authors: Chan Siu‐Lung , Cipolla Marilyn
Published: June 2021

Cerebral small vessel disease (SVD) associated with hypertension is a common cause of vascular cognitive impairment thought to be due to inward remodeling of brain parenchymal arterioles (PA) and white matter hypoperfusion. Here, we examined structural remodeling of PA in spontaneous hypertensive (SHR) and Wistar‐Kyoto rats (WKY; n=8/group) and if treatment with relaxin (4 μg/hr, osmotic pump for 2 wks), a peptide hormone shown to selectively enlarge PA, could reverse structural remodeling in SHR. PA were dissected from the brain parenchyma and studied pressurized in an arteriograph for passive structural measurements. PA from SHR had significantly smaller inner and outer diameters than WKY (49±3 vs. 58±3 μm and 62±3 vs. 71±3 μm at 60 mmHg; p<0.01) that was reversed by relaxin treatment (56±4 and 67±4 μm; p<0.01 vs. SHR). Relaxin treatment of SHR also increased PA distensibility (34±3 vs. 10±2 % in SHR at 60 mmHg; p<0.01) without affecting blood pressure (MAP with and without relaxin in SHR: 140±5 vs.138±2 mmHg). In summary, chronic hypertension caused inward remodeling in PA that was reversed by relaxin treatment. The effect of relaxin on PA diameter and distensibility may improve perfusion to the white matter in chronic hypertension and thus may improve cerebral SVD. This study is supported by NIH NS045940 and HL095488.
Age‐related cerebrovascular endothelial dysfunction is associated with cognitive impairment in C57BL/6 mice
Authors: Asano Shinichi , Seals Douglas R , Zigler Melanie C , Justice Jamie N , Weber Stewart A , Yoshizawa Mutsuko , Sindler Amy L
Published: June 2021

Mild cognitive impairment (MCI) is an age‐related disorder that contributes to a reduced quality of life in older adults. However, the underlying mechanisms contributing to MCI with aging are incompletely understood. We tested the hypothesis that old C57 male mice demonstrate cerebrovascular endothelial dysfunction associated with MCI. Endothelium‐dependent dilation (EDD) (ex vivo dilation of mid cerebral and posterior communication arteries to acetylcholine [ACh]) was impaired in old (O: 22–26 mo, n= 17) vs. young (Y: 3–6 mo, n=11) mice (51.2 ± 5.9 % vs. 72.4 ± 5.4 %, p<0.05), and this was associated with reduced L‐NAME‐sensitive dilation (O: 16.1 ± 9.1 % vs. Y: 49.5 ± 9.5 %, p<0.05). Basal NO inhibition by L‐NAME had no effect on phenylephrine‐induced vasoconstriction in O (Con. 23.1 ± 2.8 % vs. L‐NAME 31.1 ± 4.1 %), but increased constriction in Y (Con 19.9 ± 3.1 % vs. L‐NAME 38.0 ± 3.9 %, p<0.05). O (n = 7) had impaired spatial learning during the Morris water maze test (p<0.05) and this was unrelated to differences in vision. In the probe trial, O had lower platform crossing numbers than Y (n =8) (O: 2.7 ± 0.5 vs. Y: 5.1 ± 0.7, p<0.05), suggesting O had impaired spatial memory compared to Y. These results indicate that cerebrovascular endothelial dysfunction, as reflected by reduced basal NO production and NO‐dependent EDD, develops with aging and is associated with MCI in C57 mice.

Work supported by NIH AG013038 and AG000279.
Intermittent oxygen inhalation increase maximal workload and maximal heart rate in elite athletes
Authors: Lindholm Peter , Boberg Olle , Frånberg Oskar , Larsson Åke , Gullstrand Lennart
Published: June 2021

OBJECTIVE

Recent studies on pulmonary shunt during exercise suggest that oxygen per se could close shunts and thus increase arterial PO2. We hypothesized that a small increase in intrapulmonary PO2 (5–10kPa) would suffice to get performance enhancing effect of oxygen. We also hypothesized that intermittent addition of 10–20l/min of oxygen to inspired air could yield this increase without using premixed gas in athletes ventilating 150–250 l/min.

Methods

7 athletes were tested on an ergometer cycle in a blinded cross‐over design on two separate occasions. Tests were done on two sub‐maximal workloads (160 and 240W) and thereafter a VO2max test increasing workload by 22.5W/min until exhaustion. Measurements included VO2max(air), heart rate, SaO2. O2 or Air was added intermittently to the inspired air with one exercise protocol ending with air and the other with oxygen. O2 was added at 10–20 liters/minute by an apparatus developed by Oxelerate™, Sweden.

RESULTS

Max workloads were 437(58)W (Mean(SD)) with air and 453(57)W with O2, p<0,001. Maximal heart rate were 188 (12) beats/min with air and 192(12) with O2 (p<0,001). VO2max(air) were 5,18(0,76)L/min. End‐tidal FO2 was 21,23(0,52)% at maximal workload with O2 addition.

CONCLUSIONS

These data suggest that intermittent addition of oxygen to the inspired air is enough to increase work performance and maximal heart rate in highly trained athletes.
Acute Immune Responses to Prolonged Exercise in Environmental Extremes
Authors: Hassan Emad Shaban , Tabba Hani D
Published: June 2021

This study is to investigate the effect of prolonged exercise with and without a thermal clamp on leukocyte cell, stress hormones, and cytokine responses. Fifteen healthy male volunteers ( means ± SD: age 22 ± 3yr; mass 75.8 ± 3.2kg; maximal oxygen uptake 55 ± 7ml/min/kg) randomly completed four chamber trials of 1 hour each, in different environment and separated by 7 days. Trials were: 1. exercise induced heating (EX‐Heating: temperature/humidity, 38°C/50%); 2. exercise with a thermal clamp (EX‐Cooling: temperature/humidity, 18°C/ 50%); 3. passive heating (PA‐H: temperature/ humidity, 38°C/ 50%); 4. passive cooling (PA‐C: temperature/ humidity, 18°C/50%). EX‐H and EX‐C were composed of 1h treadmill runs at 80% Individual anaerobic threshold (IAT). Blood samples were collected at pre‐post, and 1h post environments exposure. Compared to EX‐H, exercise‐induced increases in core temperature, heart rate, cortisol, human growth hormone, Interleukin‐6, leukocyte and monocyte counts were significantly (p≤0.01) more pronounced after EX‐C. Our results suggest that the additional impact of elevated ambient temperatures on stress responses to endurance exercise in trained subjects seems to affect primarily the hormonal systems and resulting changes in leukocyte number, monocyte and interleukine‐6.

This study was supported by the King Saud University
Effects of Long Term Exercise on Age‐related changes in Cardiac Fibrosis and Cell Death Profiles
Authors: Akins Tiffany , Barton Greg , Hoffman Marie , Aiken Judd , Diffee Gary
Published: June 2021

Aging results in changes to cardiac structure and function but the mechanisms underlying these effects are not well understood. Exercise training appears to have some positive effects on age related functional changes, but the effects of exercise on age‐related structural changes have not been well explored. We characterized cardiac remodeling in response to aging and exercise in male FBN rats, starting with rats age 24 mo and continuing until age 36 mo. Rats were divided into 3 groups; high intensity (H), and moderate intensity (M) treadmill exercise, and sedentary (S). M and H animals trained at 13 m/min for 30 min/day, with the H group at 5% incline. At the end of training, hearts were excised, embedded and sectioned. Sections were stained with H&E and Masson's Trichrome. Cardiac sections were analyzed via immunohistochemistry for autophagy (using an anti‐Becilin antibody) and necrosis (using an anti‐C5b9 antibody). Fibrosis deposition significantly increased with age, but was decreased by exercise training. The number of cells positive for C5b9 and Becilin were significantly higher in aged hearts, but this number was decreased with exercise training. These results show that aging increases fibrosis deposition and markers for autophagy and necrosis. Prolonged endurance exercise training into advanced ages appears to minimize a number of these age‐related changes.

Supported by: NIH AG030423
The Effects of Exercise Training (ET) on Cardiac Arrhythmias and Left Ventricular (LV) Connexin 43 (Cx43) Expression in Aged and Young Rats
Authors: Jepson Amanda J , Garnett Emily R , Davenport Ashley N , Firkins Rachel M , Kadow Zachary A , Henry Matthew K , Moffitt Julia A
Published: June 2021

Reduced LV expression of the myocardial gap junction protein Cx43 is associated with increased arrhythmogenesis. The occurrence of cardiac arrhythmias increases with age although mechanisms are unknown. We hypothesized that aging would result in increased cardiac arrhythmias and decreased LV Cx43 expression while ET would reverse these effects in young (4–6 mo) and aged (24–25 mo) F344 rats. Rats underwent 10–11 weeks of treadmill ET or sedentary handling. Subcutaneous electrocardiographic leads were implanted after the young sedentary (YS n=9), young exercise (YEx n=7), aged sedentary (AS n=6), and aged exercise (AEx n=6) protocols to allow for data acquisition via the Actiwave telemetry system. The arrhythmic index (AR) was calculated using a modified scoring system during baseline (BL), sympathoexcitation (isoproterenol (ISO), 0.15mg/kg, s.c.) and brief restraint (BR). The total AR (BL+ISO+BR) was significantly higher in aged vs young rats (p<0.05) while ET showed a trend toward reversing this effect (p=0.065). Western blot analysis (YS=3, YEx=3, AS=3, AEx=2) revealed reduced Cx43 expression in aged vs young rats while exercise partially reversed this effect in AEx rats. These data support the hypothesis that ET provides a protective benefit against cardiac arrhythmias in young and aged rats. Future studies will further investigate the role of Cx43 in mediating this response.
Exercise Training Restores Muscle Mechano and Metaboreflex Sensitivity in Heart Failure Patients
Authors: Antunes‐Correa Ligia M , Nobre Thais S , Groehs Raphaela V , Alves Maria Janieire NN , Rondon Maria Urbana PB , Mady Charles , Almeida Dirceu R , Oliveira Patricia , Lima Marta F , Mathias Wilson , Brum Patricia C , Rossoni Luciana V , Oliveira Edilamar M , Middlekauff Holly R , Negrao Carlos Eduardo
Published: June 2021

Increased sympathetic muscle mechanoreflex sensitivity and attenuated sympathetic muscle metaboreflex sensitivity have been described in heart failure (HF) patients. We tested the hypothesis that exercise training (ET) would improve sympathetic mechano and metaboreflex sensitivity in HF patients. 24 consecutive, randomized, HF patients, Functional Class II‐III NYHA, EF≤40% were divided into two groups: Exercise‐trained (n=12, 55±2 years) and untrained (n=12, 54±2 years). 10 normal controls (NC) were also studied. Muscle sympathetic nerve activity (MSNA) was directly recorded from the peroneal nerve. Mechanoreceptors were activated by passive exercise. Metaboreceptors were activated by post‐exercise circulatory arrest. ET consisted of three 60‐minutes exercise sessions per week for 4 months. ET significantly reduced MSNA in HF patients (34 vs. 40 bursts/min, P<0.05). ET significantly reduced MSNA responses to passive exercise (Δ = 2 vs. 5 bursts/min, P<0.05) and increased MNSA responses during post‐exercise circulatory arrest (Δ = 5 vs. −1 bursts/min, P<0.0 5). These changes were so dramatic that the difference between HF patients and NC were no longer observed. No changes in untrained HF patients were found. In conclusion, ET restores sympathetic muscle mechano and metaboreflex sensitivity in HF patients, which may contribute to the reduction in MSNA and clinical outcomes in these patients.
Exercise regulates obesity‐induced chemerin expression in a tissue‐dependent manner
Authors: Keslacy Stefan , Gladstone Miriam , Kim Yuho
Published: June 2021

Chemerin has been involved in obesity‐induced inflammation and insulin resistance and is preconized as a new biomarker for diabetes. We previously showed that exercise modulates systemic chemerin, but the underlying mechanism is still unknown.

Purpose

To determine the source of the exercise‐induced changes in obesity‐related chemerin expression.

Methods

44 mice (DIO C57Bl/6J) were separated into 2 cohorts: high fat diet (HFD) and normal diet (ND). Following a 12 week‐feeding period, diabetes‐relevant tissues (adipose, muscle, pancreas and liver) have been harvested. Chemerin gene expression and protein levels were assessed using pcr and western blot respectively.

Results

HFD resulted in enhanced chemerin gene expression in fat and muscle (P<0.005) as well as its receptor ChemR (P<0.05), but not in pancreas. Following exercise chemerin gene expression in fat did not change in ND but increased in HFD (P<0.05). We did not observe changes in chemerin or its receptor in muscle and pancreas following exercise. Our preliminary data suggested that exercise modulates chemerin protein expression in adipose tissue.

Conclusion

Our data suggest that the effect of exercise on obesity‐increased chemerin expression could be explained by adipose tissue. While chemerin gene expression increased in muscle with obesity, the effect of exercise seemed specific to adipose tissue. Study supported by Syracuse University.
Effect of aerobic training on the capillary and arteriolar remodeling in heart and skeletal muscle of hypertensive rats
Authors: Alves Tatiana Pereira , El Chaar Laiali Jurdi , Batista Alvaro Martins , Barboza Tatiane Evelyn , Lima Cintia Taniguti , Michelini Lisete Compagno , Lacchini Silvia
Published: June 2021

We evaluated if aerobic training improves blood pressure and if determine capillary and arteriolar remodeling in heart and skeletal muscle of hypertensive rats.

We used SHR and WKY male rats submitted or not to aerobic training (AT, 1hr, 5 times per week, in treadmill). Rats were trained for 1, 2, 4, 8 and 12 weeks or kept sedentary for 0 and 12 weeks (n=8/group). At the end of each time, artery was catheterized for mean arterial pressure (MAP) and heart rate (HR) measurements. Heart, soleus and temporalis skeletal muscles were harvested for histologic evaluation, with capillary density and arteriolar wall/lumen ratio measurements.

Both SHR and WKY improved physical performance after 12 weeks of AT (S0:1.02±0.3 vs 1.62±0.4, T12:1.92±0.4 vs 2.40±0.4km/h, WKY vs. SHR). AT induced a rest bradycardia in WKY (326±4 to 294±3bpm), and in SHR (385±7 to 357±2bpm). Only SHR reduced MAP after AT (174±2 to 164±1mmHg). AT decreased arteriolar wall/lumen ratio in SHR in the second week of training in soleus (−0.23±0.03μm), and in the fourth week in heart (−0.06±0,02 μm) and temporalis (−0.09±0.02μm). AT also induced fast (week 1) capillary increase in the heart of WKY (+31.1%) and SHR (+202.8%), and in soleus of WKY (+22%) and SHR (+110%).

The data indicates that AT induced beneficial vascular remodeling after hemodynamic adjusts, improving arteriolar wall/lumen ratio and capillary density, more pronounced in hypertensive rats.
Difference in bone metabolism beetwen experienced SCUBA divers and controls
Authors: Bozic Josko , Kurir Tina Ticinovic , Novak Anela , Supe‐Domic Daniela , Salamunic Ilza , Jurin Zdravka , Bilopavlovic Nada , Valic Maja , Valic Zoran
Published: June 2021

The bones of divers are subjected to stress from water pressure, from the forces generated when their muscles resist water pressure, and from weightlessness. Several previously published studies have shown conflicting results. The aim of this study was to determine bone mineral density (BMD) and biochemical markers of bone turnover in experienced SCUBA divers. In this study we included 10 experienced SCUBA divers with a mean age of 47±2.4 years (mean±SEM). The average number of diving years was 26.1 with 381 diving hours per year. The control group included 10 healthy man who were matched by age, weight, and height. The BMD of lumbar spine and hip regions was assessed by dual energy x‐ray absorptiometry and biochemical markers of bone turnover were measured. The BMD value for total lumbar spine in divers was 1.252±0.031 vs. 1.136±0.041 g/cm2 in control group and for total hip was 1.172±0.047 vs. 1.102±0.041 g/cm2 in control group. T scores were 1.54±0.28 vs. 0.42±0.37 and 1.05±0.27 vs. 0.45±0.28 for lumbar and hip regions, respectively. There were no significant differences in bone turnover markers between groups, except in sRANKL concentration (in divers group 1322±466 vs. 2316±1046 pmol/L in control group. In conclusion, our results show that middle aged, experienced divers have increased BMD and decreased sRANKL levels.

This study was supported by Croatian Ministry of Science, Education and Sports.
Physical Exercise Increases Bone Mineral Density of Osteopenic Diabetic Rats
Authors: Sumida Doris Hissako , de Melo Yamamoto Aline Pedro , Astolphi Rafael Dias , Louzada Mario Jefferson Quirino
Published: June 2021

This study evaluated the changes caused by the absence of load in Bone Mineral Density (BMD) of diabetic rats (D) and the effectiveness of exercise on bone tissue. 64 male rats (5 days old) were divided into: control (C) and D (Streptozotocin i.p.). Sixty days later, the first BMD of the left tibia was measured by DEXA. Then, these groups were subdivided into 4 groups: C, Osteopenic control (OC), D and Osteopenic diabetic (OD). OC and OD were suspended by their tails for 21 days. Afterwards, other densitometric analyses (DA) were performed. Then, some of them were subjected to exercises (for 12 weeks) and the rats were subdivided again into 8 groups: 1) Exercised OC (EOC); 2) Sedentary OC (SOC); 3) Exercised Control (EC); 4) Sedentary Control (SC); 5) Exercised OD (EOD); 6) Sedentary OD (SOD); 7) Exercised Diabetic (ED); 8) Sedentary Diabetic (SD). After 12 weeks, the third DA was performed. In the first analysis (before suspension), there was a difference (p<0.05) in BMD between C and D. In the second evaluation (after suspension), an exacerbated reduction in BMD in OD compared to C, OC and D occurred (p<0.05). After exercising for 12 weeks, there was an increase in BMD in ED and EOD regarding SD and SOD (p<0.05). We concluded that there was a decrease in DMO in D compared to C group. Furthermore, the suspension period increased the loss of bone mass of OD and exercise improved BMD in diabetic rats. FAPESP 2010/14755–5.
Deterioration of mouse femur microstructure by heavy cigarette smoking and the effect of smoking cessation
Authors: Chan Ruby Lok Yi , Cho Chi Hin
Published: June 2021

Smoking is a risk factor of osteoporosis. To investigate the dose effect of smoking, mice were subjected to ambient air, 2% or 4% cigarette smoke exposure for 14 weeks. To evaluate the effect of smoking cessation, ambient air or 4% cigarette smoke were given to mice for 20 weeks, while the smoking cessation group stopped smoking in the last 6 weeks. Urine and serum bone turnover markers; as well as femora and tibias mineral content, histology and microstructure were assessed.

Deoxypyridinoline (DPD), minerals excretion in urine and serum alkaline phosphatase (ALP) activity increased after heavy smoking. Bone weight and mineral contents had no difference in all groups. Relative bone volume and trabecular thickness were lower with elevation in structure model index after 4% smoking. Smoking cessation returned body weight to control level. Serum ALP activity decreased after smoking cessation. Urine DPD level in smoking groups were significantly higher. Less intact trabecular structure in distal femur and reduced femur growth plate thickness were found in smoking groups.

To conclude, chronic heavy smoking increases bone turnover and deteriorates bone microstructure in mice. Smoking cessation does not reverse the ill effects of chronic heavy smoking. The study was supported by research funds from the Jockey Club Centre for Care and Control and the Direct Grant, the Chinese University of Hong Kong.
Exercise does not increase skeletal muscle FNDC5 protein or mRNA in pigs
Authors: Laughlin M. Harold , Company Joseph M , Booth Frank W , Padilla Jaume , Jenkins Nathan T , Sacks Harold S , Fain John N
Published: June 2021

Endurance exercise training (EX) is reported to increase FNDC5 expression in skeletal muscle (SkM) of mice (Nature 481:463, 2012). We tested the hypothesis that EX increases FNDC5 expression in pig SkM. Castrated male Rapacz hypercholesterolemic (FH) and normal Yucatan miniature swine (Y) were divided into EX and unexercised (SED) groups. After 16–20 weeks pigs were anesthetized and tissue samples collected and FNDC5 gene expression measured by RT‐PCR. Citrate synthase activity (CS) (index of EX‐induced adaptation) was increased in SkM samples of both FH and Y EX. FNDC5 mRNA appeared 64% less deltoid and 25% less in long head of triceps brachii (LHT) SkM of FH EX pigs. EX did not appear to affect FNDC5 mRNA expression in fat or heart muscle. FNDC5 mRNA expression in subcutaneous fat was comparable to that in the SkM. EX enhanced PGC1α mRNA levels which were much higher in the SkM samples than in fat samples. The changes in PGC1α mRNA levels were not correlated with FNDC5 mRNA. Despite the increase in CS activity in SkM, there was no difference in FNDC5 protein content (Western blot) between SED and EX SkM of Y pigs. We conclude that EX does not increase FNDC5 mRNA or protein in pig SkM.
The effect of Insulin Like Growth Factor I on matrix synthesis in engineered human tendon tissue
Authors: Herchenhan Andreas , Schjerling Peter , Bayer Monika Lucia , Kjaer Michael
Published: June 2021

Growth factors are essential during tissue formation and maintenance. In tendon tissue, insulin like growth factor I (IGF‐I) plays a key role in cell response to mechanical loading by increasing the production of extracellular matrix proteins. However, its role in formation and regeneration of human tendon is unknown. We hypothesized that IGF‐I improves neoformation of human tendon and particularly collagen synthesis. To study this, we engineered tendon‐like tissue from adult human tendon cells and exposed the tissue to medium with or without IGF‐I, respectively, supplemented with either 0.5% or 10% fetal bovine serum (FBS). The cells increased collagen type I, scleraxis and tenomodulin gene expression, and the total collagen protein content rose with supplementation of IGF‐I (p<.05). This increase can be partially explained by a higher collagen fibril diameter in the presence of IGF‐I (p<.05). It is intriguing that 10% FBS had an inhibitory effect on tendon matrix formation compared to 0.5% FBS and suggests that serum proteins inhibit matrix synthesis. These results demonstrate that IGF‐I enhances collagen production in human tendon‐like tissue and implicates potential treatment options following tendon injuries. The suppressive effect of highly concentrated FBS illustrates the challenge in subjecting cells to high dosages of various growth factors.

(Financial Support: Nordea Fondation, Danish Medical Research Council)
Prolactin as an osmoregulatory hormone in zebrafish: transcriptional regulation of a Na+/Cl− cotransporter (ncc) in the gill
Authors: Breves Jason , Serizier Sandy , Goffin Vincent , McCormick Stephen , Karlstrom Rolf
Published: June 2021

Prolactin (PRL) is a well‐known regulator of ion and water transport within osmoregulatory tissues across vertebrate species, yet how PRL acts on some of its targets remains poorly understood. Along with the genetic and experimental accessibility of the zebrafish (Danio rerio) model, the high capacity for ion‐uptake exhibited by this freshwater teleost positions them as a powerful model for studies aimed at elucidating how the endocrine system governs effectors of ion transport. Here, we show that ionocytes in the gill may directly respond to systemic PRL to regulate ion uptake. Ion‐poor conditions led to increases in the expression of PRL receptor (prlra), Na+/Cl− cotransporter (ncc; slc12a10.2), Na+/H+ exchanger (nhe3b), and epithelial Ca2+ channel (ecac) transcripts within the gill. Intraperitoneal injection of ovine PRL (oPRL) increased ncc and prlra transcripts, but did not affect nhe3b or ecac. Consistent with direct action, the addition of oPRL to cultured gill filaments stimulated ncc in a concentration‐dependent manner, an effect blocked by a pure human PRL receptor antagonist (Δ1–9‐G129R‐hPRL). These results suggest that endocrine PRL signaling through PRL receptors regulates ncc expression, thereby suggesting PRL may be a key hormonal regulator of Cl− uptake mechanisms in zebrafish gill. Supported by NIH [T32‐MH020051–07 and F32‐DK095575].
Hypoxia up‐regulates hemoglobin expression in cultured human retinal pigment epithelium (RPE)
Authors: Mangini Nancy J , Fetea Teodora , Schaab Tara , Kennedy Brian G.
Published: June 2021

RPE is a transporting epithelial monolayer situated between sensory retina and its choroidal blood supply. The retina is among the most metabolically active tissues, with photoreceptors exhibiting the highest O2 demand. 100% of O2 delivered to photoreceptors comes across the RPE; even small decrements in oxygenation lead to measurable changes in vision. Recent studies suggest hemoglobin in RPE facilitates O2 delivery. The present study examined the effect of hypoxia on hemoglobin expression in RPE. Proteomic analysis revealed the presence of hemoglobin chains in cultured human RPE and demonstrated up‐regulation in response to a treatment that altered pH. Hemoglobin antibodies were used for western blotting and immunocytochemistry to compare RPE hemoglobin expression under control‐and hypoxic conditions. Our studies demonstrated the presence of hemoglobin alpha‐, beta‐and delta chains in RPE. Immunocytochemistry revealed increased staining and a marked subcellular redistribution in response to hypoxic conditions. The effect was most pronounced for hemoglobin delta. Hemoglobin is expressed in cultured RPE and expression is sensitive to oxygen levels. Supported by IUSM‐NW Research Funds
Attenuation of the hypercapnic ventilatory response in the Brown Norway (BN) rat occurs prior to postnatal (P) day 26 and does not appear to be influenced by gender
Authors: Puissant Madeleine M , Hodges Matthew
Published: June 2021

Adult (<49 days of age) inbred Brown Norway (BN) rats exhibit a blunted hypercapnic ventilatory response compared to inbred Dahl salt sensitive (SS) and outbred Sprague Dawley (SD) strains (Hodges et al., 2002), despite developing high CO2 sensitivity from post‐natal (P) day P12–14 until at least P20–21 (Davis et al., 2006). Thus, we hypothesized that CO2 sensitivity is attenuated between P30–P49 in the BN, but not the SS rat strain, and that the identification of a time window and/or potential gender difference may provide insights into the cause. The hypercapnic ventilatory response (FICO2=0.07) of male (n=6) and female (n=6) BN, and male SS (n=6) rats was measured with flow‐through plethysmography from P26–47, and expressed as a percentage of room air breathing (VE; % control). VE increased during hypercapnia in both male and female BN rats to (151%) at the earliest age studied (P26–27), and was similar throughout the remainder of the ages studied with one exception. At P30–31 there was a modest increase in the hypercapnic ventilatory response in male BN rats, but it was also highly variable. Additional comparisons to the SS strain await further analysis. We conclude from these data that the apparent attenuation of CO2 sensitivity in BN rats occurs prior to P26, and does not appear to be dependent upon gender. Supported by NIH HL097033.
Novel Mechanism of Na/K Pump Inhibition by Chelerythrine (CET), a PKC Inhibitor, Uncovers Potential Early Signal Transducers of Apoptosis.
Authors: Lauf Peter K , Heiny Judith , Alter Gerald M , Meller Jarek , Brown Thomas L , Adragna Norma C
Published: June 2021

In human lens epithelial cells (HLECs), CET abolishes Na/K pump (NKP) activity, concomitantly inhibits K fluxes via the Na‐K‐2Cl isoform 1 and stimulates K channels (Lauf et al. Cell Physiol Biochem 2012). Since in HLECs neither ATP, nor phosphorylation of the α1 subunit or FXYD2 proteins were altered, nor ouabain binding to pig renal Na/K ATPase, an alternate direct action of CET on NKP was explored. CET is a BH3‐mimetic for proapoptotic Bcl‐2 proteins interacting with their BH1 domains. Thus we hypothesized that the NKP might also possess BH1 domains to which a) the BH3‐mimetic CET and b) BH3‐domain type Bcl‐2 proteins would bind and hence modify NKP activity crucial for cellular homeostasis. Indeed, MAFT sequence alignments between Bcl‐2 and BclXl revealed the presence of at least one BH1‐like domain within the NKP potentially interacting with CET or Bcl‐2 BH3 proteins. This site consists of some 13 amino acids (aa) within the N‐terminal 100 aa of the actuator (A) within the NKP (and other P‐type pumps). We propose that CET inhibits the NKP by conformational changes of its A domain thus disrupting the α1 subunit catalytic activity. Consequently, by interacting with the putative NKP CET‐binding BH1‐like site, Bcl‐2 BH3 domain proteins may be intermediary cytosolic sensors of normal NKP function thus constituting important signal transducers in the initial phases of apoptosis.
Targeting CIKS expression or CIKS/IL‐17RA interaction inhibits IL‐17A‐induced cardiomyocyte death by reversing JNK activation and DUSP1/MKP‐1 inhibition
Authors: Valente Anthony J. , Yoshida Tadashi , Morris Gilbert F. , Somanna Naveen , Siebenlist Ulrich , Chandrasekar Bysani
Published: June 2021

Myocardial ischemic injury is characterized by cardiomyocyte (CaM) death. Here we investigated whether IL‐17‐induced cardiomyocyte death is CIKS (connection to IKK and SAPK/JNK )‐dependent, and determined the underlying molecular mechanisms. IL‐17A induced significant death of CaM, an effect blocked by anti‐IL‐17A or IL‐17RA Ab. CIKS knockdown or a CC′ loop decoy peptide that blocks IL‐17RA/CIKS physical interaction abolished the IL‐17A‐induced death response. Further, IL‐17A induced CIKS/TRAF6‐mediated ASK1‐dependent and ASK‐1‐independent JNK, c‐Jun, and AP‐1 activation. In contrast, IL‐17A markedly attenuated the JNK phosphatase DUSP1/MKP‐1 via miR‐101 induction. Notably, forced expression of MKP‐1 or mutant JNK1 reversed IL‐17A‐induced CaM death. IL‐17A activated both intrinsic (Bax, cytochrome c release, caspase‐9) and extrinsic (Bad, Bim, Bid, Fas‐L) pro‐apoptotic signaling pathways, and attenuated the pro‐survival Bcl‐2 and Bcl‐xL expression. These effects were reversed by CIKS knockdown or JNK inhibition. These results indicate that IL‐17A shifts the balance between pro‐survival and pro‐apoptotic signaling pathways in favor of cell death via CIKS‐dependent JNK activation, and suggest that CIKS is a critical intermediate and potential therapeutic target in myocardial injury and inflammation.
The choroid plexus regulation of cerebrospinal fluid pH
Authors: Christensen Henriette Lajgaard , Damkier Helle Hasager , Brown Dennis , Praetorius Jeppe
Published: June 2021

The choroid plexus epithelium (CPE) lines the ventricular spaces of the brain and secretes the majority of the cerebrospinal fluid (CSF). Changes in blood pCO2 are efficiently transmitted to the CSF, which lack protein buffers. Nevertheless, the CSF pH fluctuates significantly less than would be predicted from the pCO2 changes. This ascertains a proper respiratory center response.

Despite the clear clinical importance, strikingly little is known about the molecular mechanisms behind CSF pH control. The aim of this study is to determine whether the CPE is involved in CSF pH regulation.

As a first step, we wished to identify the types of putative pH, HCO3− or CO2 sensor proteins and luminal acid‐base extruders expressed in the CPE. RT‐PCR on mouse choroid plexus revealed mRNA encoding the sensor proteins TDAG8, OGR1, Gpr4, Ptprg, and sAC. We have also demonstrated mRNA expression of the putative luminal acid‐base extruders NHE1 and NBCe2, as well as the A, B2, C1, C2, d1 and d2 subunits of the vacuolar H+‐ATPase (V‐ATPase). NHE1 and NBCe2 are known luminal proteins in the CPE, while preliminary immunolocalization of the V‐ATPase suggests a mainly vesicular distribution.

In conclusion, the CPE expresses a molecular machinery that may well confer both acid‐base sensing and potent transport capacity to regulate CSF pH within suitable limits.
The electroneutral Na+HCO3− cotransporter NBCn1 plays an essential role in duodenal acid/base balance and colonic mucus layer build‐up in anaestetised mice
Authors: Seidler Ursula Elisabeth , Singh Anurag Kumar , Xia Weiliang , Riederer Brigitte , Juric Marina , Li Junhua , Zheng Wen , Xiao Fang , Bachmann Oliver , Praetorius Jeppe , Aalkjaer Christian
Published: June 2021

Background

Duodenal mucosa needs efficient defence strategies against gastric acidity, while colonic mucosa counteracts damage by pathogens through the build‐up of an adherent mucus layer.

Aim

This study delineates the significance of the electroneutral Na+HCO3− cotransporter NBCn1 (Slc4a7) for duodenal defence against acid and for colonic mucus release.

Methods and Results

NBCn1 was localised to the basolateral membrane of duodenal villous enterocytes and of colonic crypt cells. Duodenal villous enterocyte pHi was studied before and during a luminal acid load by two‐photon microscopy in exteriorised, vascularly perfused, pH indicator‐loaded duodenum of anaesthetised mice. Acid‐induced HCO3− secretion was measured in vivo by single‐pass perfusion and pH‐stat titration. After a luminal acid load, NBCn1‐deficient duodenocytes were unable to rapidly recover from intracellular acidification and could not adequately respond with a protective HCO3− secretory response. Colonic mucus layer build‐up was delayed, and a decreased thickness of the adherent mucus layer was observed, suggesting that basolateral HCO3− uptake is essential for optimal colonic mucus release.

Conclusions

The electroneutral Na+HCO3− cotransporter NBCn1 is essential for intestinal mucosal protective functions, such as pHi‐recovery and HCO3− secretion after an acid load in the duodenum, as well as mucus secretion in the colon.
Visualization of dysferlin‐vesicle dynamics reveals a role for microtubules and kinesin‐motors in dysferlin‐vesicle transport and fusion in skeletal muscle cells
Authors: McDade Joel Ryan , Michele Daniel
Published: June 2021

Muscle cells reseal the plasma membrane following wounding, and this process is disrupted in cells lacking dysferlin. Dysferlin‐vesicles accumulate at membrane lesions by an unknown mechanism, and the contribution of dysferlin‐vesicle fusion to resealing is unclear. We used live‐cell imaging of dysferlin‐eGFP expressing L6 myotubes to examine the role of microtubules (MTs) and kinesin motors (KIF5) in dysferlin‐vesicle transport and damage‐induced fusion. Dysferlin‐vesicles colabel with KIF5 and are distributed along microtubules throughout the cytoplasm. Long‐range movement of dysferlin‐vesicles is significantly reduced following pharmacological disruption of MTs, indicating that MTs are required for dysferlin vesicle transport in muscle cells. We demonstrate for the first time that wounding induces fusion of dysferlin‐vesicles to form extremely large cytoplasmic vesicles. Disruption of MTs or expression of dominant‐negative KIF5 inhibited large vesicle formation following wounding, indicating that MT‐based transport of dysferlin‐vesicles is critical for dysferlin‐vesicle fusion. Our data suggest that dysferlin‐vesicles undergo vesicle‐vesicle fusion following wounding, and identify a novel role for MTs and KIF5 in regulating dysferlin‐vesicle function in skeletal muscle cells. This research is supported by the American Heart Association Pre‐doctoral Fellowship
Effects of lipopolysaccharides on placental oxidative stress and angiogenic factors: fetal repercussion
Authors: Vieira‐Filho Leucio Duarte , Farias Juliane Silva , Barros Nelson Góes , Ribeiro Natalie Emanulle , Cabral Edjair Vicente , Paixão Ana Durce Oliveira
Published: June 2021

Disturbances during intrauterine development are linked to fetal growth retardation and programming of diseases at adult life. Placental oxidative stress and placental impaired angiogenesis may be underlying mechanisms. It was investigated whether maternal inflammation, induced by lipopolysaccharides (LPS) elevates oxidative stress and affects vascular endothelial growth factor (VEGF) in placenta of rats. LPS was administered in pregnant Wistar rats at days 13, 15, 17 and 19 of pregnancy, accompanied or not by α‐tocopherol treatment during all pregnancy. At 20th day of gestation, fetal and placental weights were not affected by LPS, however placental and fetal hepatic lipid peroxidation was increased, as well as, placental and maternal hepatic reduced glutathione (GSH) levels were lowered. Moreover, LPS treatment increased VEGF and VEGF receptor type‐1 (VEGFR‐1) expression. Maternal α‐tocopherol treatment prevented lipid peroxidation changes induced by LPS, albeit it did not recover GSH levels or changed expressions of VEGF and VEGFR‐1. Thus, although LPS increases oxidative stress, it may increase placental angiogenesis. CAPES, CNPq and FACEPE.
Corticotrophin‐releasing hormone up regulated IL‐6, IL‐8 and MCP‐1 output in primary human myometrial smooth muscle cells via CRHR1 by activating the PKA pathway
Authors: Xu chen , You xingji , Ni xin
Published: June 2021

CRH has been implicated to be involved in controlling the length of pregnancy and initiating the onset of parturition in human. Given that parturition is an inflammatory process, we hypothesized that CRH regulates the inflammation responses by controlling the production of cytokines and chemokines in human myometrium, thereby leading to parturition. Primary myometrial cells were isolated and cultured from lower segmental myometrial tissues obtained from pregnant women at term. CRH treatment caused an increase in IL‐6, IL‐8 and MCP‐1 production in myometrial cells. These effects were reversed by CRHR1 antagonist but not by CRHR2 antagonist. Knockdown of CRHR1 resulted in a loss of CRH effect. CRH stimulated active Gαs protein and resulted in an increase in cAMP production in myometrial cells. CRH‐induced production of IL‐6, IL‐8 and MCP‐1 was blocked by either adenylate cyclase (AC) inhibitor SQ22536 or PKA inhibitor H89. In conclusion, CRH acts on CRHR1 to stimulate the production of cytokine and chemokine in myometrial cells. These effects are dependent on AC/PKA signaling pathway. Our data suggest that CRH can directly stimulate myometrial cells to secret chemokines and cytokines, which could recruit leukocytes to the uterus and activation of inflammation response.
Rb influx in K‐Cl cotransporter 3 (KCC3)‐transfected human embryonic kidney 293 (HEK293) cells and effect of external Na
Authors: Ravilla Nagendra , Lauf Peter K. , Kahle Kristopher T. , Adragna Norma C.
Published: June 2021

K‐Cl cotransport (KCC) is encoded by 4 genes (SLC12A4–7) with at least four protein isoforms (KCC1‐KCC4) and electroneutrally extrudes K and Cl under physiological conditions. Major functions of KCC involve cell volume and Cl regulation and thus abnormalities in its activity are associated with numerous pathological conditions. The purpose of this study is to characterize the functional properties of KCC3 in a human cell line. Rb, a K congener, was used to study KCC ± ouabain or bumetanide, inhibitors of other K transport mechanisms i.e. Na/K pump and Na‐K‐2Cl cotransport (NKCC), respectively, ± Cl (sulfamate replacement) ± Na (N‐methyl‐D‐glucamine replacement) in KCC3 transfected HEK293 cells. Rb uptake through KCC was linear up to 10 min whereas Rb uptake through NKCC was not linear even at the earliest time point tested (2.5 min). NKCC was 3 fold >; KCC in Na whereas KCC was about 6 fold >; NKCC in Na‐free medium. NKCC was the largest component of the total Rb flux in Na (Na/K pump 25 %, NKCC 63 %, and KCC 11 %). These findings indicate that KCC3‐transfected HEK293 cells behave like non‐transfected cells from various tissues for KCC, NKCC and Na/K pump activity both in Na or Nafree medium under baseline conditions, thus constituting a promising mammalian experimental model to study the specific role of KCC3 in cell volume regulation and in different signaling pathways. Supported by WSU Foundation and NIH R25
The K‐Cl cotransporter KCC3 is a catalytic switch of cellular K and volume homoestasis
Authors: Adragna Norma C. , Ravilla Nagendra , Lauf Peter K. , Kahle Kristopher T.
Published: June 2021

The SLC12A cation cotransporters NKCC1 and KCCs play a crucial role in volume homeostasis, with KCC3 as key mediator of regulatory volume decrease. Dephosphorylation of human KCC3 at T991 & T1048 by protein phosphatase 1is triggered by cell swelling, but its physiological effects have not been systematically explored. By creating isogenic human epithelial cell lines with inducible expression of wild type KCC3 (WT) or a constitutively‐active double alanine KCC3 mutant mimicking the dephosphorylated KCC3 (AA), and measuring KCC3 activity with Rb, a K congener, and cell K (Ki) ± transport inhibitors, we gain insight into the role of KCC3 in cell K and volume homeostasis. In isotonic medium, WT is phosphorylated and of low activity, as assessed by phospho‐specific antibodies against T991 & T1048 and Rb flux. In contrast, induction of AA resulted in a 10 fold greater furosemide‐sensitive (FS) KCC3 activity. In induced AA but not WT cells, Ki loss was >; 90 % in Na and ~ 70 % in Na‐free media, time‐ and external K and Rb concentrations‐dependent and could not be prevented by various Cl, K and Ca channel blockers. In contrast to NKCC and KCC, the induced Ki loss was insensitive to F in the preincubation and flux. Addition of F during induction of AA significantly reduced Ki loss in sulfamate with concomitant inhibition of KCC. Thus, a constitutively active KCC3 promotes an equal FS Ki loss and Rb influx, which may impact certain pathologies. Supported by WSU Foundation and NIH R25.