bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional characterization of a novel urate efflux transporter URATv1 (SLC2A9) and its relation to renal hypouricemia.
Authors: Jutabha Promsuk , Anzai Naohiko , Ichida Kimiyoshi , Kimura Toru , Kitamura Kenichiro , Hisatome Ichiro , Endou Hitoshi , Sakurai Hiroyuki
Published: June 2021

Reanl urate handling is clinically important because underexcretion of urate causes hyperuricemia. In 2002, we identified an apical urate‐anion exchanger, URAT1 encoded by SLC22A12. But it is currently not known how urate taken up via URAT1 exits from the tubular cell. In this study, we functionally characterized SLC2A9 gene product GLUT9 in search of urate excreting transporter. GLUT9 expressed Xenopus oocytes transported [14C]urate time‐ and concentration‐dependent manner (Km, 365 µM). Interestingly, we found that its transport was Na+‐independent and enhanced at high concentrations of extracellular potassium, indicating that GLUT9‐mediated urate transport is affected by the changes in membrane potential. GLUT9‐expressing oocytes preloaded with radiolabelled urate showed a time‐dependent efflux of radioactivity in the standard uptake solution Thus, we renamed this novel urate transporter as voltage‐driven urate transporter (URATv1). In vivo role of URATv1 is supported by the fact that a renal hypouricemia patient without any mutations in SLC22A12 was found to have a missense mutation P412R in SLC2A9, which reduced urate transport activity in vitro. Considering its basolateral expression in proximal tubules in human kidney, URATv1 is proposed to be a basolateral exit pathway of urate, which is likely to act in tandem with URAT1 for urate reabsorption.
Sprint interval training increases VO2max and central circulatory capacity in sedentary, overweight women
Authors: Trilk Jennifer L , Singhal Arpit , Bigelman Kevin A , Cureton Kirk J
Published: June 2021

Sprint interval training (SIT) increases muscle oxidative capacity and may increase maximal oxygen uptake (VO2max), but whether central circulatory capacity is increased is unknown. The objective was to examine the effects of SIT on VO2max and its determinants defined by the Fick equation in sedentary, overweight women. Twenty‐eight women with BMI=26 were randomly assigned to SIT (n=14; age=30±7 yr, BMI=36±6 kg/cm2; DXA %fat=48±6; VO2max 22±4 ml/kg min) or control (CON, n=14; age= 31±6 yr, BMI=35±6 kg/cm2; DXA %fat=47±5; VO2max 20±3 ml/kg min) groups. SIT trained 3 d/wk for 4 wk, with 1‐2 d rest between sessions, while CON continued baseline physical activity (exercise=1 day/wk). Training consisted of 4‐7, 30‐s sprints on a stationary bike with 5% body weight as resistance and 4 min active recovery between sprints. Pre‐ and post‐intervention, VO2max and HRmax were measured via cycling graded exercise test, and a 20‐min ride at 50%VO2max was used to estimate cardiac output (Q) and stroke volume (SV) by CO2 rebreathing. Maximal responses were estimated using measured VO2max and HRmax, and assuming SV was maximal at 50%VO2max. Qmax and (a‐v)O2 diffmax were calculated. Increases in SIT were greater than CON (p<0.05) for VO2max (0.245 L/min, 12% vs ‐0.011 L/min, ‐1%), Qmax (1.5 L/min, 10% vs ‐0.9 L/min, ‐6%) and SVmax (10 ml/beat, 11% vs ‐3.2 ml/beat, 3%). Changes by SIT and CON in HRmax (‐2 bpm, 1% vs ‐2 bpm, 1%) and (a‐v)O2diffmax (0.4 ml/100 ml, 3% vs 0.6 ml/100 ml, 5%) were not significant (p>0.05). We conclude that 4 wk of SIT improves VO2max by increasing SVmax and Qmax.
Adenosine receptor blockade accentuates the responses of muscle sympathetic nerve activity to fatiguing exercise
Authors: Cui Jian , Blaha Cheryl , Leuenberger Urs A. , Yoder Jonathan , Sinoway Lawrence I.
Published: June 2021

The role adenosine plays in evoking the exercise pressor reflex in humans remain controversial. Interestingly, animal studies suggest that adenosine can inhibit type 1 vanilloid receptors activity. Thus, we hypothesized that adenosine receptor blockade would accentuate muscle sympathetic nerves activity (MSNA) response to fatiguing exercise in humans. Blood pressure (Finometer), heart rate, and MSNA from peroneal nerve were assessed in 11 healthy young volunteers during fatiguing isometric handgrip, post exercise muscle ischemia (PEMI), and passive muscle stretch during PEMI. The protocol was performed before and after adenosine receptor blockade by local infusion of 40 mg aminophylline in saline via forearm Bier block (regional intravenous anesthesia). In the second experiment, the same amount of saline was infused via the Bier block. After aminophylline, the MSNA and blood pressure responses to fatiguing handgrip, PEMI and passive stretch (all P<0.05) were significantly greater than those in control condition. Saline Bier block had no similar effects on the MSNA and blood pressure responses. These data verified our hypothesis, and suggest that adenosine may act to inhibit MSNA response in exercise pressor reflex. Supported by NIH P01 HL077670, M01 RR010732 and AHA 0635245 N.
Activation of Transient Receptor Potential Vanilloid‐1 (TRPV1) Channels in the Rostral Ventrolateral Medulla (RVLM) Produces Renal Sympathoinhibition
Authors: Adams Julye M , McCarthy John J , Derbenev Andrei , Smith Bret , Stocker Sean D
Published: June 2021

The RVLM plays an important role in the regulation of sympathetic nerve activity (SNA) and arterial blood pressure (ABP). The TRPV1 channel is sensitive to temperature, chemical and mechanical stimuli, but recent evidence suggests that TRPV1 channels also modulate neurotransmission. The present study examined whether activation of TRPV1 channels in the RVLM altered SNA and ABP. In urethane‐chloralose anesthetized rats, unilateral injection (60nL) of the TRPV1 agonist capsaicin into the RVLM dose‐dependently decreased renal SNA (0.5nmol: ‐9±1%; 1.0nmol: ‐44±6%; n>4) and mean ABP (0.5nmol: ‐4±1; 1 nmol: ‐15±2 mmHg; n>4). The sympathoinhibitory response to 1nmol capsaicin was eliminated by pretreatment with 600pmol of the TRPV1 antagonist capsazepine (renal SNA: ‐1±1%; mean ABP: 0±0 mmHg, n=6). Blockade of GABA receptors by pretreatment with bicuculline (60pmol) and CGP35348 (400pmol) did not prevent, but rather enhanced the sympathoinhibitory response to 1nmol capsaicin (renal SNA: ‐58±1%; mean ABP: ‐51±3; n=3). Blockade of ionotropic glutamate receptors by injection of kynurenic acid (3nmol) did not alter the capsaicin‐induced decrease in renal SNA (‐42±9%) or mean ABP (‐13±3mmHg, n=4). The present findings suggest that activation of TRPV1 channels in the RVLM inhibit renal SNA and ABP independently of GABAergic or glutamatergic neurotransmission. Supported by NIH HL090826, AHA 0630202N & 0625260B
Smooth muscle BK channels oppose sympathetic nerve‐evoked constrictions in mesenteric resistance arteries
Authors: Krishnamoorthy Gayathri , Heppner Thomas , Nelson Mark T.
Published: June 2021

Large conductance, calcium‐ and voltage‐sensitive potassium (BK) channels have been shown to oppose constrictions to changes in intravascular pressure ("myogenic tone"). The role of BK channels in the modulation of sympathetic tone is not known. Here, we propose that smooth muscle BK channels oppose nerve‐evoked constrictions in resistance arteries of the mesenteric circulation. Sympathetic nerves on third order pressurized (60 mm Hg) arteries were stimulated by short (1s) and long (50s) electrical pulse trains. Diameter changes were recorded using edge‐detection software in the presence and absence of BK channel blockers. The blocker of BK channels, paxilline (1 μM), increased nerve‐induced constrictions by about 1.6‐fold for short and long stimulation pulses. In addition, paxilline increased the duration of the evoked constrictions (4.55 min ± 0.17 sem in control vs. 5.6 min ± 0.19 sem in paxilline). The effect of paxilline on neurotransmission was determined by measuring junctional calcium transients (jCaTs), which reflect the release of ATP from nerve varicosities. Paxilline also increased constrictions to exogenous application of phenylephrine, an α‐adrenergic agonist, by about 1.8‐fold. These results indicate that BK channels act in a negative feedback fashion to oppose nerve‐induced constrictions and regulate the sympathetic tone of resistance arteries of the mesenteric circulation. These studies highlight the importance of the BK channel in opposing sympathetic tone.
Cerebral ischemic preconditioning protects neurons from apoptosis via decoy receptors
Authors: Panneerselvam Mathivadhani , Kidd Michael W , Head Brian P , Davis Daniel P , Roth David M , Patel Piyush , Patel Hemal H
Published: June 2021

Cerebral ischemia induces cytokine secretion resulting in neuronal damage. One such factor is TNF related apoptotic‐inducing ligand (TRAIL). Ischemic preconditioning (IPC) is a phenomenon where brief episodes of ischemia protect the brain from lethal injury. Decoy receptors, a subset of TRAIL receptors containing ligand binding but no signaling capacity, act as a sink to compete for TRAIL inhibiting apoptosis. We hypothesize that IPC protects neurons against TRAIL‐induced neuronal death by increasing expression of decoy receptors. Neurons were isolated from 1‐day‐old rat pubs and subjected to TRAIL‐induced apoptosis with and without IPC. IPC was induced by oxygen‐glucose deprivation for 20 min one day prior to TRAIL treatment. Cell death and apoptotic protein expression was assessed 24 h after TRAIL treatment. Trypan blue inclusion assay showed that IPC protects neuron form TRAIL‐induced death. Western blot analysis showed IPC increased expression of decoy receptors as well as decreased pro‐apoptotic (cleaved caspase‐3, CAD and Bad) and increased anti‐apoptotic (Bcl‐2) protein expression 24 h after TRAIL treatment. These data suggest that the protective effects of IPC may result from altered expression of decoy receptors that efficiently compete to limit pro‐apoptotic signaling in neurons. Thus, decoy receptors represent a novel target to limit stroke‐induced neuronal injury.
Bone marrow stromal cells isolated from the infarcted region of the postischemic brain secrete neuroprotective and angiogenic factors
Authors: Yilmaz Gokhan , Erkuran Yilmaz Cigdem , Alexander J. Steven , Stokes Karen Y. , Granger D. Neil
Published: June 2021

Although the therapeutic potential of bone marrow stromal cells (BMSC) has been demonstrated in stroke, there is limited information about the neuroprotective factors secreted by BMSC in the infarcted brain. The objective of this study was to compare the expression of genes for extracellular (EXC) factors produced by BMSC infiltrating brain tissue vs their naïve counterparts. Ischemic stroke was induced in C57BL/6 mice by middle cerebral artery occlusion for 1 h, followed by 24 h reperfusion. BMSC were isolated from H‐2Kb‐tsA58 mice (cells can grow at 33°C), and were administered (i.v.) at 24 h of reperfusion. Infarcted hemispheres were cultured at 33°C for BMSC isolation. Microarray analysis and RT‐PCR were performed to compare gene expression patterns between the BMSC populations in 9 mice. Z‐scoring revealed a robust change in the extracellular region of analyzed cells. Pair‐wise analysis detected 163 extracellular factor genes that were upregulated (≥ 2 fold, P<0.05) between naïve and infiltrated BMSC. Fibroblast growth factors, bone morphogenetic protein, angiopoietins, neural and vascular growth factors were among the expressed genes detected. Our findings indicate that extracellular factors secreted by BMSC infiltrating the postischemic brain may explain the beneficial therapeutic effects of stem cell treatment and reveal potential therapeutic agents for treatment of ischemic stroke. (HL26411)
DOCA‐salt hypertension promotes leukocyte (but not platelet) adhesion in cerebral venules
Authors: Rodrigues Stephen Fernandes , Granger Daniel Neil
Published: June 2021

Although hypertension is a major risk factor for the development of ischemic stroke, the mechanisms underlying the influence of increased blood pressure on stroke risk remain poorly understood. Since both platelets and inflammatory cells have been implicated in the pathogenesis of ischemic stroke, we examined whether DOCA‐salt induced hypertension alters the behavior of platelets and leukocytes in cerebral venules in male mice. A 50 mg DOCA pellet was implanted via s.c. in uninephrectomized C57Bl/6 mice placed on 1 % saline for a period of 3 weeks, which produced a significant increase in blood pressure (from 103±2 to 127±2 mmHg) as well as cardiac and renal hypertrophy (relative to controls). Platelets isolated from matched donor mice were labeled ex vivo with CSFE while rhodamine was administered i.v. for in vivo labeling of leukocytes. Intravital microscopic observation of blood cell trafficking in pial venules revealed a large increase in the number of adherent leukocytes in DOCA‐salt mice (558±58 per mm2 vs 183±39 per mm2). However, platelet adhesion within venules was unaffected by DOCA‐salt (12±7 per mm2 vs 4±4 per mm2). These findings indicate that DOCA‐salt induced hypertension induced a pro‐inflammatory, but not a pro‐thrombogenic, phenotype in the cerebral microvasculature. The accumulation of inflammatory cells in the brain during hypertension may predispose this tissue to ischemic tissue injury. (Supported by HL26441).
Impaired Behavioral Performance after Prolonged Moderate Hypobaric Hypoxic Exposure in Mice
Authors: Xu Kui , Sun Xiaoyan , Tsipis Constantinos P , LaManna Joseph C
Published: June 2021

Hypobaric hypoxia can be used to simulate the conditions of high altitude to study adaptive responses. The brain is completely reliant on oxygen for oxidative energy metabolism, and even mild decrease in ambient O2 could result in altered function. In this study we investigated specific motor and cognitive performance in mice adapting to moderate hypoxia. Mice (C57BL/6) were exposed to the sea level equivalent of 8% O2 for 3 weeks in hypobaric chambers at a pressure of 300 mmHg (0.4 ATM); littermate normoxic controls were kept in the same location. Motor function was tested using the Rotarod and the inclined‐screen tests. Cognitive function was measured using an object recognition test and a fear conditioning test. Capillary density was estimated from parietal cortical sections. After 2 weeks of hypoxia, the motor performance was declined as represented by decreased latency in the Rotarod test and increased latency in the inclined screen test; the hypoxic mice also performed poorly in the fear conditioning and object recognition tests. Similar to the previous mouse and rat studies, the hypoxic mice went through a decrease in body weight, increases in hematocrit (%, 80 ± 7 vs. 45 ± 1) and an increase in capillary density (N/mm2, 721 ± 63 vs. 533 ± 69) compared to the normoxic controls. Motor and cognitive functions were impaired in the simulated high‐altitude condition despite successful vascular adaptation.
Distribution of ferritin chains in noncataractous and cataractous canine lenses
Authors: Goralska Malgorzata , Nagar Steven , Fleisher Lloyd N. , McGahan M. Christine
Published: June 2021

Ferritin consists of 24 subunits of light (19 kDa) and heavy (21 kDa) chains each with roles in storing iron and protecting cells from iron induced oxidative damage. Changes in subunits can alter iron storage in ferritin. Nuclear cataractogenesis is associated with age and oxidative damage. We compared ferritin chains from fiber cells of normal and cataractous canine lenses separated into six layers. The ferritin chains from each layer were analyzed by Western. The iron binding of assembled ferritin was determined by 59Fe labeling. The chains in lens fiber cells differed from the standard canine ferritin H (21 kDa) and L‐chain (19 kDa). The L‐chain was larger (30 kDa) and there were two modified ferritin H‐chains (12 and 29 kDa). The 30 kDa L‐chain was present in the cortex of all lenses. The 12 kDa H‐chain was found in cortex and outer nuclei of both lens types. The 29 kDa H‐chain was present only in nuclei of cataractous lenses. 59Fe labeling showed that assembled ferritin from cataractous lenses bound more iron than ferritin from normal lenses. All altered ferritin chains in lens fibers were unevenly distributed throughout the lens. The characteristics and distribution of H‐chain in cataractous lenses were different from those in normal lenses. The higher content of differentially modified H‐chain in the nucleus and higher affinity for iron of ferritin in cataractous lenses may be a part of cell defense against oxidative damage.
Aging alters the mitochondrial stress response
Authors: Haak Jodie , Kregel Kevin
Published: June 2021

The mitochondrial stress response involves a set of stress proteins known as heat shock proteins (Hsps) that repair and restrict protein damage in the mitochondria. Specifically, Hsp60, Hsp10, and mitochondrial Hsp70 (mtHsp70) are members of the mitochondrial stress protein arsenal. While it has been established that aging impairs the cell stress response, the effect of age on mitochondrial stress proteins remains unknown. To examine the effect of age on the mitochondrial stress response, young (6 mo) and old (24 mo) Fischer 344 rats were heat stressed (41.0°C for 30 min) on two consecutive days, and their liver mitochondria were isolated at 2 and 24 h after the second stress. Immunoblotting revealed that mitochondria exhibit altered stress protein expression after heat stress. The differences in protein levels were most notable in Hsp60, the main heat‐inducible mitochondrial stress protein. Hsp60 protein levels increased significantly 2 h after heating in young mitochondria and remained elevated 24 h after heating. In old mitochondria, a delayed and blunted elevation in Hsp60 levels was observed as compared to young. Additionally, mtHsp70 and Hsp10 protein levels increased solely in young and not old liver mitochondria in response to stress. These data indicate that aging causes mitochondria to lose their ability to respond properly to physiological stress. (Supported by NIH AG12350)
Hydrogen peroxide increases cerebral arterial KCa channel opening through activation of Akt signaling pathway
Authors: Gebremedhin Debebe , Maia Terashvili , Harder David R
Published: June 2021

The oxidant hydrogen peroxide (H2O2) is not only an inducer of cellular damage, but can also play a role as a modulator of vascular function. Exogenous application of H2O2 (3‐300 µM) resulted in a concentration‐dependent increase in NPo of KCa single‐channel currents recorded from cell attached patches at a patch potential of +40 mV, whereas this oxidant did not change the NPo of KCa single‐channel current when recorded from excised inside‐out membrane patches of cerebral arterial muscle cells (CAMCs). The membrane permeant organic peroxide tert‐butyl hydroperoxide (t‐BOOH) (30 µM) also induced similar increase in NPo of KCa single‐channel currents in the cell‐attached patches but had no effect on NPo of the single‐channel KCa currents recorded from excised inside‐out patches at a the same patch potential. In an attempt to elucidate possible signaling mechanism mediating the effects of these oxidants on KCa channel currents in CAMCs, we examined the impact of inhibition of the phospoinositide 3‐kinase‐Akt (PI3K‐Akt) pathway on the stimulatory actions of both H2O2 and t‐BOOH on KCa channel current. Pretreatment of CAMCs with two structurally different PI3K‐Akt pathway inhibitors wortmannin (10 nM) and LY294002 (5 µM) significantly attenuated the H2O2‐ or t‐BOOH‐induced increase in NPo of single‐channel KCa currents in CAMCs. On the other hand, stimulation of cerebral arterial muscle with either H2O2 or t‐BOOH for 10 minutes induced activation of Akt as determined by western blot analysis. These findings suggest that activation of the Akt signaling pathway contributes to the H2O2‐or t‐BOOH‐induced enhancement of KCa channel currents in cerebral arterial muscle cells.
Chronic hypoxia decreases response to central chemoreceptor stimulation in the nucleus tractus solitarius (NTS)
Authors: Wilkinson Katherine A. , Nichols Nicole L. , Putnam Robert W. , Powell Frank L.
Published: June 2021

One hallmark of acclimatization to chronic hypoxia (CH) is hyperventilation and a lower arterial carbon dioxide (CO2) level. These effects persist upon return to normoxia, suggesting a change in the regulation of CO2 during CH. We tested the hypothesis that plasticity in one central chemoreceptor site, the NTS, contributes to altered CO2 regulation in CH. In urethane‐anesthetized rats, we stimulated chemoreceptors in the NTS by a unilateral microinjection of acetazolamide (ACZ, 1 nl of 0.3 μM), to produce focal acidosis. The product of rectified, integrated phrenic nerve amplitude and frequency (Phr) was measured 30 min after ACZ in normoxic control (N; n=5) and CH rats (n=5). ACZ increased Phr significantly more in N rats than CH rats (N=263±14%; CH=83±18%; p<0.001). ACZ in adjacent brainstem regions had no effect on Phr. All rats showed the capacity to increase Phr to a 10% inspired CO2 challenge following ACZ, suggesting that the dose of ACZ did not produce a maximal phrenic response. These results indicate plasticity in chemosensitivity in the NTS but they cannot easily explain the lower CO2 set point with CH.

Supported by NIH HL81823 (FLP) and AHA 0615069Y (KAW).
Inhibition of the commissural nucleus of the solitary tract inhibits peripheral but not central chemoreflex in rats
Authors: Moreira Thiago S , Menani Jose V , Takakura Ana C , Colombari Eduardo , de Paula Patricia M
Published: June 2021

A large variety of sensory afferents converges to the nucleus of the solitary tract (NTS) and influences the activity of ventrolateral medullary (VLM) neurons involved in cardiovascular and respiratory regulations. Here, we investigated the effects of inhibition of the commissural NTS (commNTS) on cardiovascular and respiratory responses to chemoreflex activation in conscious and anesthetized male Holtzman rats (280‐300g, n = 7/group). In conscious rats, electrolytic lesions of the commNTS eliminated the increase in mean arterial pressure (MAP) (Δ= +8±4, vs. sham: +45±18 mmHg) and reduced the increase in ventilation (404±43, vs. sham: 773±28 ml/min/kg) produced by activation of peripheral chemoreflex with KCN (40 μg/rat) intravenously (iv). However, electrolytic lesion of the commNTS did not change the increase in ventilation produced by activation of central chemoreceptors with increased levels of CO2 (7%) (1189±32, vs. sham: 1055±53 ml/min/kg). In urethane anesthetized animals, the injection of the GABA‐A agonist muscimol (2 mM ‐ 60 nL) into the commNTS eliminated the increase in MAP (Δ= +6±3, vs. saline: +23±18 mmHg), on phrenic nerve discharge (PND) and on splanchnic sympathetic nerve discharge (sSND). Muscimol into the commNTS did not change the activation of central chemoreceptors with CO2 (7%) on PND and sSND. In conclusion, the integrity of commNTS is important for the cardiorespiratory responses elicited by peripheral but not central chemoreflex activation.
Serotonergic neurotransmission in the locus coeruleus modulates hypercapnic ventilatory response
Authors: Gargaglioni Luciane H , Souza‐Moreno Vanessa , Szawka Raphael E , Alselmo‐Franci Janete A. , Bícego Kenia C
Published: June 2021

We assessed the possible modulation of respiratory response to hypercapnia by serotonin (5‐HT), through 5‐HT1A and 5‐HT2 receptors, in the locus coeruleus (LC). We determined the concentrations of 5‐HT and its metabolite, 5‐hydroxyindole‐3‐acetic acid (5‐HIAA), in the LC after hypercapnic exposure. Pulmonary ventilation was measured before and after microinjection of WAY‐100635 (5‐HT1A antagonist, 5.57 e 55.7 mM in 100nL), Ketanserin (5‐HT2 antagonist, 3.66 e 36.6 Mm in 100nL) or DOI (5‐HT2 agonist, 69.9 mM in 100nL) into the LC, followed by a 60‐min period of 7%CO2 exposure. Hypercapnia increased 5‐HTIAA levels and 5‐HT turnover (5‐HIAA/5‐HT) within the LC. WAY‐100635 intra‐LC decreased the hypercapnic ventilatory response due to a lower tidal volume. Ketanserin increased CO2‐drive to breathing and DOI caused the opposite response, both acting on tidal volume. The current results provide evidence of increased 5‐HT release during hypercapnia in the LC. In addition, 5‐HT presents opposite effects on CO2‐ drive to breathing depending on the receptor that it binds in the LC: whereas activation of the 5‐HT1A receptors stimulates hypercapnic ventilatory response, activation of the 5‐HT2 inhibits it. Supported by FAPESP and CNPq.
Spleen tyrosine kinase mediates angiotensin II‐ and vascular endothelial growth factor‐induced angiogenesis
Authors: Buharalioglu Cuneyt Kemal , Yaghini Fariborz A , Song Chi Young , Ghafoor Hafiz U , Estes Anne M , Malik Kafait U
Published: June 2021

Angiotensin II (AngII) and vascular endothelial growth factor (VEGF) stimulate angiogenesis. A 72 kDa non‐receptor tyrosine kinase, spleen tyrosine kinase (Syk) expressed in endothelial cells has been implicated in their migration and proliferation and in vasculogenesis. Syk also mediates AngII‐induced vascular smooth muscle cell hypertrophy. This study was conducted to determine the contribution of Syk to the angiogenic effect of AngII and VEGF. Angiogenesis was determined in Matrigel by tube formation from endothelial cells EA.hy926, and sprouting of rat aortic rings into microvessels. EA.hy926 cells were allowed to form tubular structures for 16 h and aortic rings to sprout into microvessel for 5 days and then phase contrast images were recorded. Both tube length and numbers were calculated. AngII (10 nM) and VEGF (50 ng/ml) stimulated EA.hy926 cells to organize into tubular network and increased aortic sprouting. The effect of VEGF but not AngII to stimulate tube formation and aortic sprouting was inhibited by VEGFR2 blocker SU1498 (10 µM). Both AngII‐ and VEGF‐induced angiogenesis and Syk phosphorylation, as determined by Western blot analysis, was inhibited by piceatannol (10 µM), a Syk kinase inhibitor. These data suggest that AngII‐induced angiogenesis is independent of VEGFR2 phosphorylation and that both AngII‐ and VEGF‐induced angiogenesis is mediated by Syk.
Role of NO and the NADPH Oxidase in Hyperhomocysteinemia induced Cardiac Metabolic Dysfunction in Pregnancy.
Authors: Song Su , Huang Harer , Ojaimi Caroline , Kaminski Pavel , Zhang Suhua , Wang Ziping , Wolin Michael , Hintze Thomas H
Published: June 2021

Hyperhomocysteinemia (HHcy) causes cardiovascular dysfunction and is associated with many complications during pregnancy related to reduced NO bioactivity. This study assessed the mechanisms of HHcy on the NO‐dependent control of cardiac O2 consumption in pregnant rats. Pregnant rats were fed methionine in drinking water to increase plasma homocysteine (Hcy) (3.0±1.2 to 14.6±8.9 uM). The ability of bradykinin and carbachol to reduce myocardial O2 consumption in vitro was impaired by 69% and 57% in heart. This was proportional to the plasma Hcy concentration (r=0.89). The response was restored by ascorbic acid, tempol and apocynin. Pregnant rats fed L‐NAME also have impaired ability of bradykinin and carbachol to regulate myocardial O2 consumption (54% and 56%). O2− production was increased in heart using lucigenin chemiluminescence. HHcy caused a 51% increase in the p47phox subunit of NADPH oxidase, an increase in Rac‐1 and increased nitrotyrosine (21%). Microarray analysis from heart tissues revealed 853 genes to be differentially regulated (P≤0.05). There were no change in the NADPH oxidase subunits and NOS. However, there were up regulation of some structural genes (Myosin, MMP3) and metabolic genes (Aldehyde DH, pyruvate DH and NADH DH). HHcy increases O2− production by NADPH oxidase and reduces the ability of NO to regulate myocardial O2 consumption in pregnancy. Supported by HL50142.
Nitric oxide stores in coronary blood vessels of dogs with metabolic syndrome
Authors: Manukhina Eugenia B. , Caffrey James L. , Barlow Matthew , Vanin Anatoly F. , Mikoyan Vasak D. , Downey H. Fred
Published: June 2021

Nitric oxide (NO) in vivo forms complexes (NO stores) for transport and intracellular storage of NO. A key function of NO stores is defense against NO toxicity during NO overproduction. Metabolic syndrome (MS) is associated with NO overproduction, which is detrimental for endothelium function. We evaluated NO storage in coronary vessels of 4 dogs with MS induced by a high fat diet and in coronary vessels of 4 control dogs. NO stores were indexed by relaxation of isolated left circumflex artery (LCA) rings, precontracted with a thromboxane A2 mimetic, U46619 (625 nmol/L), to N‐acetylcysteine (NAC, 10−3 M). NAC‐induced relaxation of LCA from dogs with MS was larger than in control (11.1±1.6 vs 3.1±1.4%, respectively, P<0.01). This is consistent with NO overproduction in MS. To estimate capacity of NO stores, LCA was incubated with a NO donor, dinitrosyl iron complex (10−5 M) for 20 min. After washout, LCA was precontracted with U46619, and NAC was added to release NO from the accumulated NO stores. Relaxation of LCA from control dogs was significantly greater than that of LCA from dogs with MS (62.4±7.3 vs. 33.9±5.9%, respectively, P<0.03). In conclusion, our findings are consistent with NO overproduction in MS, and with depressed ability of MS coronary vessels to bind excessive NO into NO stores. Thus, an intrinsic defense of coronary vessels against NO toxicity is impaired in MS. (supported by CRI/Integrative Physiology, UNTHSC)
Utility of the Transthyretin Gene (TTR) Intron One for Elucidating Crocodylian Phylogenetic Relationships
Authors: Willis Raymond E
Published: June 2021

The gene encoding transthyretin (TTR) is an attractive candidate for use in phylogenetic analysis because it is a short, single‐copy nuclear gene with regions that are highly conserved across evolutionarily‐divergent organisms. The TTR gene product belongs to a group of thyroid hormone binding proteins that also includes thyroxine‐binding globulin and albumin. The first intron of the TTR gene is well‐suited for sequencing because it is relatively short and the flanking coding regions have sections that are highly conserved, facilitating primer design across diverse species. To explore the utility of TTR intron 1, I selected a group of closely‐related species for which there was overall agreement about their relationships, yet several ambiguities remained. The extant crocodylians met these criteria. TTR intron 1 (787‐bp) was sequenced in 22 crocodylian species and analyzed using maximum likelihood, maximum parsimony and Bayesian analyses. Molecular analysis of the TTR intron 1 data set indicates that it is highly conserved within the crocodylia. Uncorrected pair‐wise divergence values ranged from less than 0.3% when comparing species within Crocodylus to a high of 6.85 % between members of Crocodylus and the caimans. The detailed comparisons of TTR intron 1 resolved the three expected lineages, Alligatoroidea, Gavialoidea, and Crocodyloidea with high support at most nodes (greater than 0.95 in most major nodes). It offered additional evidence for the utility of synapomorphic indels in elucidating higher‐level phylogenetic relationships. It follows that when used in conjunction with other nuclear and mitochondrial markers, as well as fossil and morphological data sets, TTR intron 1 should be a valuable tool in the investigation of other closely‐related groups.
Clock genes regulate circadian rhythmical change of sglt1 gene expression according to feeding schedule through binding of BMAL1 on the promoter region
Authors: Goda Toshinao , Iwashina Ikumi , Mochizuki Kazuki
Published: June 2021

Sodium‐D‐glucose cotransporter SGLT1 plays an important role in the absorption of glucose in the small intestine. To examine whether the circadian rhythmical expression of the intestinal sglt1 gene is regulated by clock genes, we determined the levels of mRNA and BMAL1 binding on the promoter regions of the sglt1 gene by real‐time RT‐PCR and chromatin immunoprecipitation assay, respectively, in the jejunum of mice receiving free access to diet and those with restricted feeding from 9:00 to 17:00 under light (7:00‐19:00)‐dark (19:00‐7:00) cycle. Jejunal expression of sglt1 gene gradually increased from 7:00, and reached a maximal level at 19:00 in mice with free access to diet. The peak was shifted to 7:00 in mice with restricted feeding schedule. The gene expression of bmal1, a transcription factor driving the central feedback loop of clock genes, displayed a maximal expression at the period when sglt1 gene expression started to increase. The gene expression of per1‐3, the clock genes coding negative regulators of BMAL1, was highest at or soon after the time of maximal expression of sglt1 gene in both groups. The binding of BMAL1 to the promoters of sglt1 and per2 genes was associated with their expressional changes. The results suggest that the circadian expressional change of sglt1 gene in the jejunum of mice which is cued by the feeding schedule is directly regulated by a feedback loop of clock genes.
Generating Flash files of Your Lectures: Good Practice Strategies Helping Students Engage with Their Learning
Authors: Dallemagne Catherine
Published: June 2021

Our students come from diverse backgrounds. They need flexibility in their learning. First year students tend to worry when they miss lectures or part of lectures. Having the lecture as an on line resource allows students to miss a lecture without stressing about it and to be more relaxed in the lecture, knowing that anything they may miss will be available later.

The resource: The Windows based program from Blueberry Software (not Blackberry!) ‐ BB Flashback ‐ allows the simultaneous recording of the computer screen together with the audio, as well as Webcam recording.

Editing capabilities include adding pause buttons, graphics and text to the file before exporting it in a flash file. Any diagrams drawn on the board or shown via visualiser can be photographed and easily incorporated.

The audio from the file can be extracted if required to be posted as podcast. Exporting modes other than Flash are also available, allowing vodcasting if you wish.

What you will need:

‐ the recording software: it can be installed on the lecture hall computer just prior to lecture if needed

‐ a computer: either the ones in lecture halls, especially if fitted with audio recording, or a laptop (I have used audio recording via Bluetooth for mobility).

Feedback from students has been positive and will be presented on the poster.
The Protective Role of T cells against Angiogenic Potential of Pulmonary Endothelial Cells
Authors: Taraseviciene‐Stewart Laima , Slepikas Liudas , Fragoso Miguel , Gerasimovskaya Evgenia
Published: June 2021

Rationale

In contrast to the euthymic rats, chronic hypoxia in the athymic rats induces non‐reversible severe pulmonary hypertension and remodeling. Here we investigate whether T cells play a protective role against the development of pulmonary hypertension by suppressing angioproliferative potential of endothelial cells (EC).

Methods

Rats were exposed to chronic hypoxia for 3 weeks. Pulmonary artery pressures (PAP) and right ventricle hypertrophy (RV/LV+S) were measured. Vascular remodeling was assessed by immunohistochemistry (IHC). The de‐differentiated endothelial cell population (DD‐EC) was obtained by excessive trypsinization of human umbilical vein endothelial cells (HUVEC). Carboxyfluorescein diacetate (CFDA)‐labeled DD‐EC were injected into the euthymic and athymic rats.

Results

Hypoxic exposure of athymic rats resulted in a significant pulmonary vascular remodeling, high PAP (PAP = 42±0.6 versus 33±0.4 mmHg) and right ventricle hypertrophy (RV/LV+S = 0.7±0.08 versus 0.45±0.02). CFDA‐labeled DD‐EC were present in the circulation, but not in the lungs of euthymic rats. In contrast, in the athymic rats, CFDA‐DD‐ECs were found in the lung vascular wall.

Conclusions

T cells play a protective role against hypoxia‐induced pulmonary hypertension and remodeling. In the immune competent animals the angioproliferative DD‐ECs do not reside in the lung, but once they escape immune control (athymic rat), they colonize in the lung vasculature. Our data suggest that T cells play a protective role in suppressing angioproliferative potential of vascular endothelial cells.

Funded by AHA 0735388N and AHA 0665464Z
Heart hypertrophy in response to pressure overload in TSP4 knockout mice
Authors: Frolova Ella , Sopko Nikolai , Penn Marc , Stenina Olga , Plow Edward
Published: June 2021

Thrombospondin‐4 (TSP4) is abundant in the heart in blood vessels, including capillaries and the aorta. TSP4 expression increases in hypertrophic and failing hearts and in response to ischemia. TSP4 KO and WT mice were subjected to transverse aortic constriction (TAC). 2 and 20 weeks later TSP4 KO hearts revealed more extensive hypertrophy and higher HW/BW (heart weight/ body weight) ratio compared to WT mice, and the differences were magnified with age: 6.7(0.2 in WT versus 7.4(0.2 in KO of 23 weeks, 4.7(1 in WT versus 9(3 KO at 55 weeks, and 4.3(0.8 in WT versus 8.5(3 in KO at 82 weeks. The increased hypertrophy in TAC TSP4 KO mice was due to higher deposition of collagen. The fibrosis fraction was calculated as the ratio of Masson's trichrome stained connective tissue area to total myocardial area: WT ‐ 7.4%(2.6, KO ‐ 9.6%(5.9 at 23 weeks 20 weeks after TAC; WT ‐ 3.8%(2, KO ‐ 11.2%(8.4 at 55 weeks, 20 weeks after TAC; and WT ‐ 4.6%(2.1, KO ‐ 16.8%(3 at 82 weeks, 2 weeks after TAC.

TSP4 KO hearts experienced the overload even in normal conditions: the cardiomyocytes in sham operated KO trended to be smaller in size; in hearts of old sham KO mice fibrosis fraction was 2 times larger than in WT mice hearts and non‐operated KO hearts showed 1.8 fold increase in glucose uptake.

We conclude that TSP4 is involved in adaptive responses of the heart to pressure overload, suggesting its important role in tissue remodeling. P50 HL077107; R01 DK067532
Ultrastructure and immunolocalisation of a myofibroblast cell population within the rat mitral valve
Authors: Keane Aisling , Edgar Kevin , Gardiner Tom
Published: June 2021

The aim of this study was to investigate the myofibroblast‐like characteristics of valvular interstitial cells (VICs) in the rat mitral valve and explain the potential significance of this finding. Immunohistochemical (IHC) staining was carried out for the presence of á‐ smooth muscle actin (á‐SMA), filamentous actin (F‐actin), desmin, vimentin and integrin linked kinase (ILK). The results showed that immunolocalisation of desmin and ILK in elongated VICs is similar to that of smooth muscle cells. IHC results also show the presence of desmin, which further indicates a myofibroblast phenotype. Electron microscopy results show evidence of communication between the nervous component of the valve and VICs. This last result in combination with the novel finding of ILK in VICs suggests that these cells may have a mechanosensory function in the valve. Overall the results presented show the valve itself may be more dynamic than previously believed and that VICs may play an active role in valvular mechanics.
Collagenase 3 (MMP13) modulates activation of Transforming Growth Factor b (TGFb) by hypertrophic growth plate chondrocytes
Authors: Averna Tiffany , Lehner Joseph , Lau Shukkwan , Routson Stephanie , Selim Abdulhafez , D'Angelo Marina
Published: June 2021

MMP13 expression is concomitant with onset of chondrocyte hypertrophy in the growth plate and can activate TGFβ produced by chondrocytes. Our previous binding studies indicated significant binding of a 19 amino acid MMP13‐derived peptide with the large latent TGFβ complex. Bioinformatics data indicate the site of interaction within close proximity of a non‐catalytic region in MMP13. We hypothesized that MMP13‐derived peptide could competitively inhibit MMP13 binding leading to reduced TGFβ activation. Serum‐free, primary hypertrophic chondrocyte cultures were treated with 10, 100 and 250nM of the peptide and endogenously activated TGFβ measured via ELISA and immunoblot. At 24 hours, the 250nM dose of peptide significantly induced a 5‐fold decrease in activated TGFβ without any change in the total TGFβ produced and this effect was reduced by 48 hours. These data suggest that the MMP13‐19 peptide can interfere with TGFβ activation without effects on total TGFβ produced. In osteoarthritis, aberrant activation of TGFβ?leads to cartilage degradation and inhibitors of the MMP catalytic domain have been used to stop this degradation, but with detrimental side‐effects. This study describes a method of reversible targeted disruption of MMP13 interaction with the TGFβ large latent complex independent of the catalytic site promising novel therapeutic approaches in the treatment of osteoarthritis. Supported by CCDA

Grant Funding Source

CCDA
Articular Chondroprogenitors as tools for cartilage tissue engineering
Authors: Bishop Joanna Charlotte , Khan Ilyas , Williams Rebecca , Archer Charles
Published: June 2021

The use of stem/progenitor cells to form good quality cartilage matrix has been targeted as a therapeutic goal for cartilage repair treatment. We have characterised a progenitor cell population that resides in the surface layer of neonatal bovine articular cartilage [1]. Here, we assess the progenitor cell's chondrogenic potential.

Cloned chondroprogenitors exhibited exponential growth for the first 20 population doublings (PD), then slower linear growth with evidence of replicative senescence at later passages. Mean telomere lengths of exponentially growing chondroprogenitors were significantly longer than dedifferentiated chondrocytes that had undergone a similar number of PD (P<0.05). Chondroprogenitors also had 2.6‐fold greater telomerase activity and maintained sox9 and Notch‐1 mRNA expression whereas dedifferentiated chondrocytes demonstrated little or no detectable expression. Induced differentiation to cartilage in 3D pellet cultures, demonstrated sox9, Notch‐1, aggrecan and PCNA expression.

Maintenenace of telomerase activity, Notch‐1 and sox9 gene expression distinguish clonal chondroprogenitor cells from dedifferentiated chondrocytes. When placed in chondrogenic medium chondroprogenitors appear to engage in co‐ordinated growth and differentiation in stark contrast to dedifferentiated chondrocytes. UK DBERF
Periodontium derived osteoprogenitors isolated from αSMA‐GFP mice expressed bone and stem cell markers
Authors: SANMIGUEL SYMONE MARQUEZ , Li Haitao , Igwe John , Ferrer Vesna , Aguila Hector , Kalajzic Ivo
Published: June 2021

Purpose

This study was conducted to determine if the αSMA promoter would direct GFP expression in osteoprogenitor cells of dental follicle and periodontal ligament.

Methods

Epifluorescence was evaluated in histological sections of mandibles derived from neonatal and 4‐6 week old mice. Transgene expression was also evaluated in primary cultures derived from dental follicle (mDF) and periodontal ligament (mPDL). Osteogenic differentiation was determined by RNA expression of bone markers, histochemical staining for alkaline phosphatase (ALP) and detection of mineralized nodules by xylenol orange. Flow cytometry (FACS) was used to determine cellular profile of freshly isolated or cultured αSMA positive cells.

Results

αSMA‐GFP expression was observed in the dental follicle, and in apical regions of the root. Strong expression of αSMA‐GFP was observed during early culture stages and diminished as mineralization progressed. Intense ALP activity, presence of mineralized nodules and increased expression of bone sialoprotein and dentin matrix protein‐1 following two weeks of osteogenic induction was observed. FACS analysis revealed that freshly digested mDF cells expressed the mesenchymal markersThy‐1 (40%) and Sca‐1 (8.79%).

Conclusion

αSMA promoter has the potential to identify a population of mesenchymal progenitor cells residing within the dental follicle and periodontal ligament.

Grant Funding Source

NIH GRANTU24DE06495
A platelet‐rich plasma‐like growth factor‐protein mixture inhibits development of the osteogenic phenotype in osteoblastic cell cultures grown on titanium
Authors: Tambasco de Oliveira Paulo , Andrade de Oliva Marcos , Maximiano William Marcatti Amarú , Spinola de Castro Larissa Moreira , Ciancaglini Pietro , Beloti Márcio Mateus , Nanci Antonio , Rosa Adalberto Luiz
Published: June 2021

The efficacy of platelet‐rich plasma (PRP) for enhancing osseointegration of titanium (Ti) implants is still subject of debate. The present study evaluated the effects of a well‐defined PRP‐like mixture containing PDGF‐BB, TGF‐β1, TGF‐β2, albumin, fibronectin, and thrombospondin (GFs + proteins) on the development of the osteogenic phenotype on Ti. Human alveolar bone‐derived cells were subcultured on Ti discs and exposed during the first 7 days to osteogenic medium supplemented with GFs + proteins and to osteogenic medium alone thereafter up to 14 days. Control cultures were exposed to only osteogenic medium. Dose‐response experiments were carried out using rat calvarial cells exposed to GFs + proteins and 1:10 or 1:100 dilutions of the mixture. Treated human‐derived cell cultures exhibited a significantly higher number of cells and of Ki‐67‐positive cells, significantly reduced alkaline phosphatase (ALP) activity, and no Alizarin red staining. Although the 1:10 and 1:100 dilutions of the mixture restored the proliferative activity of rat‐derived cells to control levels and promoted a significant increase in ALP activity compared to GFs + proteins, calcified nodule formation was only observed with the 1:100 dilution. The present results demonstrated that a PRP‐like protein mixture inhibits development of the osteogenic phenotype in both human and rat osteoblast cell cultures grown on Ti. FAPESP and CNPq.
Effects of Infrared Light on Release of NO and SO in Osteoblasts
Authors: Rabe Jacob B , Struve Janine A , Weihrauch Dorothee , Marshall Scott A , Ninomiya James T
Published: June 2021

Hypoxia surrounds bone fractures as a result of interruption of the intact vasculature. Nitric Oxide (NO) and superoxide (SO) are important signaling molecules in bone formation. Our previous studies showed infrared (IR) light increases osteoblast (OB) proliferation. We hypothesize that the application of IR light alters OB mediators after hypoxia (hyp). We tested this by determining the hyp tolerance of OBs. MC3T3‐E1 cells were exposed to different durations of hyp in a 1% oxygen tension chamber (1h, 2h, 4h, 6h, 8h, 10h, 18h). Media was tested for LDH release. OBs were exposed to IR light (830nm, 4J/cm2) following 6h hyp insult. Measurements of NO and SO using DAF‐DA and DHE, respectively, followed. Fluorescence intensity was analyzed using confocal microscopy. Protein expression of HIF1α and VEGF was determined using immunohistochemistry. 4h hyp cells without glucose showed significant differences of LDH release compared to all other groups. Differences peaked at 8 h. NO and SO levels from cells not exposed to NIR light, were increased after hyp as compared to those of control. Intensity levels of cells exposed to IR were increased for both hyp and control conditions, as compared to those not exposed to the light. HIF 1α and VEGF expression were up in hypoxia and reduced after IR exposure. IR light might be clinically relevant in enhancing the rate or extent of fracture healing.