bluuebunny/crossref_metadata_2025 huggingface dataset:
Real time measurement of hydrogen peroxide (H2O2) or nitric oxide (NO) in femoral ischemia/reperfusion (I/R): Effects of protein kinase C (PKC) epsilon activator (ε+) or inhibitor (ε‐) combined with tetrahydrobiopterin (BH4) or dihydrobiopterin (BH2)
Authors: Zambrano Christopher John , Krass Samuel , Chen Qian , Perkins Kerry‐Anne , Rueter Brian , Young Lindon H.
Published: June 2021

Endothelial NO synthase (eNOS) uncoupling is a source of superoxide and H2O2 during I/R when the cofactor BH4 is oxidized to BH2. We previously showed that BH2 increases H2O2/ decreases NO release whereas BH4 reverses this effect in I/R. Moreover, PKC ε+ regulates eNOS to increase H2O2/ decrease NO release whereas PKC ε‐ reverses this effect in I/R. Therefore, the effects of combining BH4 or BH2 with PKC ε+ or ε‐ on NO or H2O2 release were measured to evaluate the mechanisms of eNOS uncoupling during I/R in real‐time. NO or H2O2 microsensors (100 µm diameter) were inserted into both femoral veins in the anesthetized rat, one is subjected to I/R, and the other served as a sham. The femoral artery and vein on one side was clamped for 20 min and released for 45 min to induce I/R. H2O2 release increased in the I/R compared to the sham vein in the control group (n=7, P<0.01). The combination of PKC ε+ (0.9 mg/kg)/BH2 (2mg/kg) (n=5, P<0.05) significantly increased H2O2 release compared to control suggesting facilitation of eNOS uncoupling in I/R increases H2O2. By contrast, PKC ε‐ (0.4 mg/kg)/BH2 (2 mg/kg) (n=5, P<0.05) or PKC ε+ (0.9 mg/kg)/BH4 (0.8 mg/kg) (n=6, P<0.01) significantly reduced H2O2 release compared to control suggesting that attenuation of eNOS uncoupling reduces oxidative stress during reperfusion. This study was supported by NHLBI Grant 2R15HL‐076235‐02A1 and Center for Chronic Disorders of Aging at PCOM.
The effects of an antioxidant cocktail on apoptotic signaling in the soleus muscles of aged hindlimb suspended rats
Authors: Nichols Cody E , Jackson Janna R , Regal Steve M , Alway Stephen E
Published: June 2021

Hindlimb suspension is a technique used in rats to simulate non‐weight bearing situations. In using this technique the hindlimbs atrophy. It is known that slow twitch muscles atrophy severely under hindlimb suspension. It has also been shown that oxidative stress could be a possible cause of apoptosis, and apoptosis is a cause of atrophy. This experiment was conducted to see if by using an antioxidant cocktail the amount of apoptosis and atrophy could be decreased. This was accomplished by orally gavaging the rats with compound: seven days before suspension and while being suspended for fourteen days. The rats were then sacrificed and the right soleus was removed in vivo. Many apoptotic markers were then found using a western blotting technique. After running an ANOVA on the data obtained by a caspase assay, it was shown that the suspension had an effect on caspase 3 activity and supplementation had an effect on caspase 9 and caspase 3 activity. (Supported by NIH Grant 5P20RR016477 to the West Virginia IDeA Network for Biomedical Research Excellence)
Effects of allopurinol on plasma uric acid concentrations and leukocyte oxidative activity in broilers
Authors: Carro Maria Dolores , Falkenstein Elizabeth , VanDyke Knox , Klandorf Hillar
Published: June 2021

Allopurinol (AL) is a xanthine oxidase inhibitor that has been recently proposed as a therapy to reduce oxidative stress in human tissues. The effects of xanthine oxidase on oxidative stress are complex, since it forms uric acid (a potent antioxidant) but also catalyzes the formation of reactive oxygen species (ROS). This study was designed to investigate the effects of AL on plasma uric acid (PUA) concentrations and oxidative activity in broilers. Fifteen 5 wk‐old male chickens were divided into 3 groups, and each was randomly assigned to one of the following treatments: CON (control), AL25 (25 mg AL/kg body weight (BW)) and AL50 (50 mg AL/kg BM). Birds were weighed twice weekly for 4 wks, feed intake was measured every two days, and leukocyte oxidative activity (LOA) and PUA levels were determined weekly. Feeding AL decreased feed intake in both groups (P < 0.01), and by wk 4 reduced (P < 0.005) BW to 86.9 and 73.2% of that in CON group for AL25 and AL50 birds, respectively. As expected, AL reduced (P < 0.001) PUA at each wk, mean values being 41.8 and 36.3% of those in CON for AL25 and AL50, respectively. There was no effect (P > 0.05) of AL on LOA over the first 3 wk, but at wk 4 LOA in AL25 birds was 4.6 times greater (P = 0.008) than that in CON. The general lack of effect of AL on LOA would indicate a decrease in xanthine oxidative activity associated with a reduction in ROS production and growth rate.

Support M.D. Carro had a Spanish MICIN scholarship
Lipid‐raft redox signaling platform and apoptosis of podocytes upon homocysteine stimulation
Authors: Zhang Chun , Hu Jun‐Jun , Bao Jun‐Xiang , Brimson Christopher , Li Ning‐Jun , Li Pin‐Lan
Published: June 2021

Lipid raft (LR) redox signaling platform associated with NADPH oxidase (NOX) was reported to mediate the actions of death receptor activation in different cells. It is interesting to know whether this LR redox signaling platform is also involved in podocytes injury during hyperhomocysteinemia. The present study first characterized the presence of the NOX membrane subunit‐gp91phox and it LR clusters in podocytes, and then tested whether this redox signaling platform contributes to homocysteine (Hcys)‐induced podocytes apoptosis. It was found that Hcys markedly increased the expression of gp91phox and stimulated NOX‐dependent superoxide (O2.−) production in a concentration‐dependent manner, as measured by electronic spin resonance (ESR). Using confocal microscopy, gp91phox was found to aggregate in LR clusters upon Hcys stimulation, which was inhibited by lipid‐raft disruptors, methyl‐β ‐cyclodextrin (MCD) and filipin. Functionally, increased O2.− production associated with these LR‐gp91phox platform was also blocked by MCD or filipin. Flow cytometry showed that Hcys induced podocytes apoptosis, which could be attenuated by gp91phox siRNA or LR disruptors. Our results indicate that the formation of gp91phox‐associated LR redox signaling platform importantly contributes to podocytes injury during exposure to high Hcys levels (supported by NIH grants DK54927, HL075316, and HL57244).
Sphingosine 1‐phosphate (S1P)‐induced autophagy plays a protective role in human prostate PC‐3 cells
Authors: Chang Chi‐Lun , Chen Chiung‐Nien , Lee Hsinyu , Huang Wei‐Pang
Published: June 2021

Sphingosine 1‐phosphate (S1P) is a platelet‐enriched lysophospholipid (LPL) which regulates various cellular functions through binding and activating a specific family of G protein‐coupled S1P receptors. In general, S1P promotes cell proliferation among cancer cells. Interestingly, our previous studies suggested that S1P inhibited proliferation in PC‐3 cells independent of apoptosis. Moreover, autophagy, a sub‐cellular membranes trafficking process which leads to the degradation and recycling of cytoplasm under starvation stress, was also elevated upon S1P stimulus through S1P5 activation. In the present study, we intend to investigate the relationship between cell death and autophagy induced by S1P. By using a potent autophagy inhibitor, 3‐methyladenine (3‐MA), we observed that inhibition of S1P‐induced autophagy by 3‐MA leads to a significant decrease of cell numbers as compared to S1P treatment. These results indicate that the autophagy activity induced by S1P treatment plays a protective role in PC‐3 cells.
TRPC1 contribute to hypoxic pulmonary hypertension and right heart hypertrophy: evidence from TRPC1 knockout mice
Authors: Yang Xiao‐Ru , Cao Yuan‐Ning , Birnbaumer Lutz , Sham James S.K.
Published: June 2021

Prolonged exposure to aveolar hypoxia causes pulmonary hypertension (PAH) and right heart hypertrophy. Hypoxic PAH is associated with alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). We have previously shown that the store‐operated TRPC1 and receptor‐operated TRPC6 channels are upregulated in PASMCs of chronic hypoxic rats; and the enhanced store‐operated Ca2+ entry is responsible for the elevated [Ca2+]i and vascular tone in pulmonary arteries (PAs). In this study, we used trpc1−/− and trpc6−/− mice to examine whether these channels are required for the development of hypoxic PAH. Real‐time RT‐PCR showed that TRPC1, TRPC2, TRPC3, TRPC4 and TRPC6 channels were expressed in wild‐type mouse PASM. There was no significant shift in the expression of other TRPC subtypes in PAs, except the absence of TRPC1 and TRPC6 transcripts in the trpc1−/− and trpc6−/− mice, respectively. In PA and aorta of trpc1−/− mice, store‐operated Ca2+ entry induced by cyclopiazonic acid was slightly reduced, suggesting that TRPC1 and other channels were involved. Phenylephrine and serotonin induced vasoconstriction were unaltered in trpc1−/− PAs and aorta. However, when trpc1−/− mice were exposed to 4 weeks of hypoxia (10% or 8.5% O2), right ventricular systolic pressure (RVSP) and right heart mass ratio (RV/(LV+S)) was significantly attenuated. There was no significant difference in RVSP and RV/(LV+S) between chronic hypoxic trpc6−/− and wild‐type mice. These results, for the first time, suggest that TRPC1 might play a significant role in the development of hypoxic PAH and right heart hypertrophy.
Study of central CO2 chemosensitivity using a combined Goldman–Hodgkin‐Katz (GHK) and Hodgkin‐Huxley (HH) type of model
Authors: Cordovez Juan M , Clausen Chris , Moore Leon , Solomon Irene
Published: June 2021

Our previous work has focused on identifying putative mechanisms for explaining the differences in intracellular pH (pHi) regulation in CO2 chemosensitive versus non‐chemosensitive neurons using a mathematical model. The model incorporates the kinetics of the Na+, K+‐ ATPase, Na+ ‐ H+ exchanger (NHE), anion exchanger (AE), and passive diffusion of ions, as described by the Goldman‐Hodgkin‐Katz (GHK) equation. Other studies using mathematical models to investigate central CO2 chemoreception have focused on identifying ion channels responsible for the firing responses of putative CO2 chemosensitive neurons. Parameters for this kind of model are generally based on invertebrate studies, and the kinetics of acid/base transporters are often simulated by a mathematical expression that fits experimental data rather than underlying chemical kinetics. To overcome this limitation, we have coupled our mathematical model with a Hodgkin‐Huxley (HH) type of model in order to combine the Na+ and K+ voltage‐gating mechanisms with the kinetics of transmembrane transporters and passive diffusion of ions. This approach links CO2 signaling with neuronal firing and membrane potential responses. To evaluate the new GHK‐HH coupled model, we compare its behavior to that of a simple HH model and its ability to generate limit cycle oscillations (e.g., trains of actions potentials). Supported by NS04532
Impact of chronic intermittent hypoxia on ventilatory long term facilitation and the hypoxic ventilatory response in participants with sleep apnea
Authors: Gerst D. G. , Lee D. S. , Badr M. S. , Qureshi T. , Mateika J. H.
Published: June 2021

Purpose

To determine whether ventilatory long‐term facilitation (vLTF) and the hypoxic ventilatory response (HVR ‐ ? VE/? SaO2) are enhanced after exposure to acute and chronic intermittent hypoxia (AIH and CIH).

Methods

7 male subjects with mild/moderate sleep apnea were exposed to AIH daily for a period of 8 days. The AIH protocol consisted of 12‐4 minute episodes of hypoxia (PETO2 ‐ 50 mmHg) in the presence of PETCO2 levels sustained 3 mmHg above baseline. Each episode was followed by a 4 minute normoxic recovery period with the exception of the last recovery period which was 30 minutes in duration. Before and after exposure to AIH participants were exposed to a single 4 min episode of normocapnic hypoxia (PETO2 ‐ 50 mmHg).

Results

On day 1, minute ventilation (VE) and the HVR were greater after AIH compared to baseline (VE ‐ 18.6 ± 1.1 vs. 14.4 ± 1.3 L/min, p = 0.001; HVR ‐ 1.1 ± 0.1 vs. 0.8 ± 0.1 L/min/% SaO2, p = 0.02). Similar relationships also existed on Day 8 (VE ‐ 21.1 ± 1.0 vs. 15.8 ± 1.3 L/min, p = 0.005; HVR ‐ 1.3 ± 0.2 vs. 0.8 ± 0.2 L/min/% SaO2, p = 0.04). Moreover, increases in VE and the HVR observed after AIH on Day 8 were greater than on Day 1 (Day 8 vs. Day 1: VE ‐ 21.1 ± 1.0 vs. 18.6 ± 1.1 L/min, p = 0.005; HVR ‐ 1.3 ± 0.2 vs. 1.1 ± 0.1 L/min/% SaO2, p = 0.04).

Conclusions

Exposure to AIH elicits vLTF and enhances the HVR in participants with sleep apnea. These phenomena are further potentiated following exposure to CIH.

Funding

NIH (HL‐085537)
Estrogen reduces AHR, stimulates NO release and inhibits ROS production in murine asthmatic airways
Authors: Dimitropoulou Christiana , Intapad Suttira , Snead Connie , Wiseman Dean A , Black Stephen M , Catravas John D
Published: June 2021

We have reported that estrogen (E2) reduces inflammation and airway hyper‐responsiveness (AHR) in a murine model of allergic asthma. To study the underlying mechanisms, female BALB/c ovariectomized mice were implanted with sc pellets releasing either vehicle or E2 and subjected to a 25‐day regimen of ovalbumin (OVA) sensitization. Acute (30min) exposure of tracheal rings isolated from vehicle‐treated, OVA‐sensitized mice (asthmatic) to 100nM E2 reduced the constrictor effects of carbachol. Western blot analysis revealed increased pNOS/NOS ratio in asthmatic lung homogenates exposed to E2 (100nM) for 30min, compared to vehicle‐exposed homogenates. Similarly, in tracheal smooth muscle cells harvested from asthmatic mice (SMC), nitric oxide (NO) production increased after 30min exposure to E2 (100nM); this was reversed by the NOS1 inhibitor 7‐NINA. 30min E2 exposure reduced superoxide production in SMC. To investigate the chronic effects of E2, Rac1 and p47phox expression was analyzed by Western blotting in lung homogenates from asthmatic mice treated with vehicle or E2. E2‐treated asthmatic mice exhibited lower lung expression of both Rac1 and p47phox, suggesting inhibition of NAD(P)H oxidase. These data suggest that E2, acutely or chronically, inhibits AHR, stimulates NOS1 activation and NO release and reduces NAD(P)H oxidase function and superoxide production in a mouse model of allergic asthma.
Roles of pulmonary collectins in host defense against Legionella pneumophila infection
Authors: Ariki Shigeru , Sawada Kaku , Yamazoe Masami , Nishitani Chiaki , Shimizu Takeyuki , Takahashi Motoko , Yokota Shin‐ichi , Fujii Nobuhiro , Takahashi Hiroki , Kuroki Yoshio
Published: June 2021

Legionella pneumophila (L. pneumophila) is one of the serotypes of Legionella, and causes serious and life‐threatening pneumonia. Surfactant proteins A and D (SP‐A and SP‐D) are members of a collectin group of the C‐type lectin family and are believed to play pivotal roles in innate immunity of the lung. The purposes of this study were to determine the direct interactions between pulmonary collectins and L. pneumophila, and to investigate the roles of collectins against L. pneumophila infections. Pulmonary collectins bind to L. pneumophila in a Ca2+dependent manner. Lipopolysaccharides isolated from L. pneumophila significantly but partially inhibited the binding of the collectins to L. pneumophila, suggesting that LPS is one of the ligands for the collectins. In addition, pulmonary collectins inhibited the growth of L. pneumophila in a Ca2+dependent manner in culture media. Pulmonary collectins also attenuated the pore‐forming activity and the intracellular growth of L. pneumophila in infected THP‐1 cells. This study provides the evidence that pulmonary collectins play important roles in L. pneumophila infections.

This work was supported in part by a Grant‐in Aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan.
Comparative effects of Bevacizumab and Ranibizumab on endothelial cells. Implications in age‐related macular degeneration
Authors: Costa Raquel , Pirraco Ana , Falcão Manuel , Carneiro Ângela , Rocha Ana , Soares Raquel
Published: June 2021

Angiogenesis, is required for choroidal neovascularisation (CNV), one of the principal causes of blindness in age‐related macular degeneration (AMD). Recently, anti‐VEGF molecules like Bevacizumab and Ranibizumab, have been used intravitreally to control CNV. The aim of this study was to evaluate the effects of these agents in human microvascular endothelial cells (HMECs).

Cell cultures were established and treated during 24 h using a range of concentrations that include the clinical dose (0,25 mg/ mL for Bevacizumab and 0,125 mg/ mL for Ranibizumab). Controls were incubated with the excipient.

Cell cytotoxicity (MTS assay) was not present at any concentration. Apoptosis (TUNEL assay) was significantly increased and cell proliferation (BrdU incorporation assay) was effectively reduced. Even more, these treatments resulted in a significant decrease in cell migration (injury and double‐chamber assays) and cord structures formation (matrigel assay). These findings were accompanied by a decrease in VEGF receptor expression (Western Blotting) and in secreted VEGF (ELISA assay) upon antibodies incubations.

Our findings show that these compounds are able to affect several processes in HMEC. Given the lower price and prolonged half‐life of Bevacizumab, this agent is probably more advantageous in the prevention of visual acuity loss, which is increased in ocular pathologies, such as AMD.

Supported by Under‐graduation project by University of Porto (IRIC IPG 81/07); FCT PTDC/EME‐PME/70155/2006
Role of Mitochondrial Production of Reactive Oxygen Species on Angiotensin II Dependent Skeletal Muscle Angiogenesis.
Authors: Stodola Timothy , Joseph Joy , Kalyanaraman Balaraman , Greene Andrew
Published: June 2021

Reactive oxygen species (ROS) play an essential role in physiological angiogenesis and are known to be produced in the mitochondria of working skeletal muscles. The goal of this study was to determine how dose‐dependent targeting of different sources of ROS could affect angiogenesis. To accomplish this we used TEMPOL, a general ROS scavenger, and Mito‐TEMPOL, a TEMPOL variant that has been demonstrated to accumulate in mitochondria. Seven week‐old Sprague‐Dawley rats were placed on 4% NaCl diet to suppress the Renin‐Angiotensin System. Electrical stimulators were implanted to intermittently stimulate the peroneal nerve in one hind leg, contracting the Tibialis Anterior (TA) and Extensor Digitorum Longus (EDL) muscles for 8 hours/day for 7 days while they were infused with ANGII at a subpressor dose. The rats were treated with TEMPOL (26.0 to 540.3 µmoles*kg−1*day−1) or Mito‐TEMPOL (1.0 to 35.3 µmoles*kg−1*day−1) in drinking water. Control rats had an 18.6% increase in microvessel density in the stimulated TA. Doses up to 118.9 of TEMPOL and 7.6 of Mito‐TEMPOL also had a significant increase in vessel density. Doses higher than 142.5 of TEMPOL and 11.6 of Mito‐TEMPOL suppressed the increase in vessel density. These data reinforce the importance of ROS in physiological angiogenesis and suggest that the source of ROS in ANGII and ES dependent angiogenesis is of mitochondrial origin. Supported by NIH HL 29587.
High salt intake induces left ventricular interstitial fibrosis by a blood pressure and angiotensin II type 1 receptor‐independent mechanism
Authors: Ferreira Daniele Nunes , Katayama Isis A. , Oliveira Ivone B. , Rosa Kaleizu T. , Coelho Michella S. , Heimann Joel C.
Published: June 2021

Objective

To evaluate the mechanisms of left (LV) and right (RV) ventricular hypertrophy induced by salt overload.

Methods

18‐week‐old Wistar rats were fed normal (NS: 1.3% NaCl), high 1 (HS1 4%) or high 2 (HS2 8%) salt diet since weaning. Starting on the 7th week of age, two HS2 subgroups received hydralazine or losartan (HZ 15 and LOS 20 mg/kg/day). Tail cuff blood pressure (BP), myocyte transverse diameter (TD), interstitial fibrosis (IF) and angiotensin II type 1 (AT1) receptor and collagen type I (COL 1) protein expression were measured.

Results (mean±SEM ‐ p<0.05, n=7‐8/group)

At 18th week of age, BP (mmHg) was higher on HS1 (146±2) and HS2 (147±1) than on NS (121±2). BP on HS2+HZ (123±1) and HS2+LOS (120±2) did not differ from NS. TD in the LV was higher on HS1 (14±0.2), HS2 (16±0.5) and HS2+HZ (15±0.2) than on NS (12±0.4) and HS2+LOS (12±0.1). TD in the RV was higher on HS2 (15±0.5) and HS2+HZ (15±0.5) than on NS (12±0.3), HS1 (13±0.5) and HS2+LOS (11±0.1). IF and COL 1 (AU) in the LV and RV were higher on HS1, HS2 and HS2+LOS than on NS and HS2+HZ (COL 1 LV: HS1 1.04±0.05, HS2 1.10±0.01, HS2+HZ 0.91±0.04, HS2+LOS 1.02±0.02 and NS 0.87±0.02). AT1 (AU) in the LV and RV was higher on HS2, HS2+HZ than NS, HS1 and HS2+LOS (AT1 LV: HS2 1.1±0.02, HS2+HZ 1.1±0.01, HS2+LOS 0.9±0.02 and NS 0.9±0.08).

Conclusions

HS leads to LV hypertrophy by a BP independent effect. It can be speculated that the effect of HS on TD and IF is due to different mechanisms.

FAPESP
Vascular smooth muscle cells limit endothelial control of vasoreactivity via ROS production through myoendothelial communications
Authors: Billaud Marie , Marthan Roger , Savineau Jean‐Pierre , Guibert Christelle
Published: June 2021

Although cross‐talk between endothelial (EC) and smooth muscle cells (SMC) through myoendothelial gap junctions play a major role in the vasoreactivity, the transduction pathways remain unclear. Here we tested the hypothesis of the role of ROS and gap junctions in this cross‐talk.

Connexin (Cx)‐ mimetic peptide targeted against Cx 37 and Cx 43 (37,43Gap27) reduced (1) the contractile and calcium responses to serotonin (5‐HT) simultaneously recorded in small intrapulmonary arteries and (2) abolished the diffusion, in the SMC, of carboxyfluorescein‐AM loaded in EC. Quantitative RT‐PCR, Western Blot analyses and immunofluorescent labeling showed the presence of Cx 37, 40 and 43. Superoxyde dismutase and catalase which destroy superoxyde anion and H2O2 respectively also decreased the contractile and calcium responses to 5‐HT. Both Cx‐ and ROS‐mediated effects on the responses to 5‐HT were reversed by L‐NAME and/or endothelium removal thus reflecting a cross‐talk between SMC and EC through ROS and gap junctions. Electronic paramagnetic resonance demonstrated that superoxyde anion production by 5‐HT originated from the SMC only. Moreover, 5‐HT decreased cyclic GMP content in isolated arteries.

These data support a model where superoxyde anion produced by 5‐HT in the SMC will pass through myoendothelial gap junctions to reduce endothelial NO‐dependent control of vasoreactivity.
Cerebrovascular actions of insulin
Authors: Katakam Prasad V.G. , Domoki Ferenc , Lenti Laura , Gaspar Tamas , Snipes James A , Busija David W
Published: June 2021

Insulin resistance is a major risk factor for cerebrovascular and neurodegenerative diseases; however, cerebrovascular actions of insulin have not been studied. Therefore, we evaluated the vascular actions of insulin in isolated rat cerebral arteries (CAs). Diameter measurements of CAs showed a biphasic response to insulin; an initial constriction was followed by vasodilation. Endothelial denudation or large conductance calcium‐activated K+ channels (KCa) inhibition resulted in vasoconstriction to insulin. Inhibition of small/intermediate conductance KCa or inward‐rectifier or voltage gated or ATP dependent K+ channels reduced insulin induced vasodilation. Inhibition of nitric oxide (NO) synthase or cytochrome P450 reduced vasodilation while inhibition of cyclooxygenase enhanced vasodilation to insulin. Inhibition of endothelin type A receptors enhanced the vasodilation while type B blockade diminished vasodilation to insulin. Inhibition of reactive oxygen species (ROS) production or scavenging the ROS abolished the vasoconstriction to insulin. Fluorescence studies of cultured rat cerebral microvascular endothelial cells showed elevation of intracellular calcium and enhanced generation of NO and ROS in response to insulin. Thus, insulin induced vasodilation was dependent K+ channels and endothelial factors while vasoconstriction was mediated by ROS, prostanoids and endothelin.
Exploiting the cellular actions of SKCa and IKCa channels to manipulate endothelial function and vascular tone
Authors: Braun Andrew P , Sheng Jianzhong , Ella Srikanth , Davis Michael J , Hill Michael A
Published: June 2021

Growing evidence indicates that small‐ and intermediate‐conductance, Ca2+‐activated K+ channels (SKCa and IKCa channels, respectively) play pivotal roles in the cellular mechanisms contributing to stimulated vasorelaxation (i.e. nitric oxide (NO) synthesis, actions of EDHF). Using single cell patch clamp and fluorimetry, along with arteriolar pressure myography, we have obtained novel results demonstrating that activators of SKCa and IKCa channels (i.e. NS309 and DCEBIO, 0.01 ‐ 1 μM) enhance a number of cellular processes associated with stimulated vasorelaxation (i.e. cytosolic Ca2+ elevations, membrane hyperpolarization and NO synthesis). These observed effects of NS309 and DCEBIO were largely prevented by the SKCa and IKCa channel blockers apamin and TRAM‐34 in combination, but not when applied separately. In rat cremaster arterioles pressurized to 70 mmHg, NS309 and DCEBIO reduced myogenic tone and further augmented ACh‐induced vasodilation. The eNOS inhibitor L‐NAME (0.1 mM) decreased ACh‐evoked inhibition of myogenic tone, but did not affect the dilatory responses to NS309 and DCEBIO. The observed inhibition of NS309/DCEBIO action following endothelial denudation confirmed that these agents acted predominantly via the vascular endothelium. Finally, we will discuss initial efforts to discern the contribution of STIM1 to SKCa and IKCa activity and endothelial vasodilatory processes.
Effects of methylarginines on superoxide production from uncoupled eNOS.
Authors: Forbes Scott Preston , Pope Arthur J. , Druhan Lawrence J. , Karrupiah Kanchana , Cardounel Arturo J.
Published: June 2021

The endogenous methylarginines, asymmetric dimethylarginine (ADMA) and NG‐monomethyl‐L‐arginine (L‐NMMA) regulate nitric oxide (NO) production from endothelial NO synthase (eNOS). Under conditions of tetrahydrobiopterin (BH4) depletion eNOS generates ▸O2‐, however, the effects of methylarginines on eNOS‐derived ▸O2‐ generation are poorly understood. Therefore, using electron paramagnetic resonance spin trapping techniques we measured the dose‐dependent effects of ADMA and L‐NMMA on ▸O2‐ production from eNOS under conditions of BH4 depletion. In the absence of BH4, ADMA dose‐dependently increased NOS‐derived ▸O2‐ generation, with a maximal increase of 151 % at 100 uM. L‐NMMA also dose‐dependently increased NOS‐derived ▸O2‐, but to a lesser extent, demonstrating a 102 % increase at 100 uM L‐NMMA. Moreover, methylarginines also increased eNOS derived ROS from endothelial cells. Measurements of NADPH consumption from eNOS demonstrated that binding of methylarginines increased the rate of NADPH oxidation. Spectrophotometric studies demonstrate that the binding of ADMA and L‐NMMA shifts the eNOS heme to the high‐spin state, enabling enhanced electron transfer from the reductase to the oxygenase site. These results demonstrate that the methylarginines can profoundly shift the balance of NO and ▸O2‐ generation from eNOS and play an important role in endothelial dysfucntion.
Tissue Glutathione Mediates the Conversion of Nitrite to Nitric Oxide in the Vascular Wall to Facilitate Vasodilation
Authors: Alzawahra Wael Fares , Jourd'heuil Frances , Jourd'Heuil David
Published: June 2021

Nitrite (NO2‐) is emerging as an important mediator of vasodilation during tissue hypoxia. Recent studies have indicated that the vascular wall can directly convert nitrite back to NO and stimulates vasodilation, the mechanism for which remains unclear. The goal of the present study was to test the specific hypothesis that tissue associated glutathione is required for nitrite‐dependent hypoxic‐vasodilation. Using chemiluminescent and electrochemical detection methods, we found that incubation of nitrite with glutathione (GSH) resulted in the formation of namolar concentrations of nitric oxide (NO) at neutral pH. This effect was potentiated upon deoxygenation of the solution, indicating that molecular oxygen inhibited GSH‐dependent NO generation from nitrite. Also determined was the photosensitivity of NO production by nitrite and GSH. Nitrite alone did not yield any significant amounts of NO, unless pH was decreased. Precontracted rat aortic segments relaxed upon addition of incremental amounts of nitrite and the sensitivity of the vessel rings to nitrite was increased at low oxygen tension and in the absence of light. Inhibition of glutamate‐cysteine ligase, the rate limiting enzyme for GSH synthesis, by L‐buthionine‐S,R‐sulfoximine resulted in profound changes in the response of rat aortic rings to nitrite. Overall, these studies provide insights into the mechanism by which nitrite regulates vasodilation.
The relationship between cardiovascular collapse in Methicillin‐resistant Staphylococcus aureus‐induced sepsis and excessive expression of reactive nitrogen and oxidative species
Authors: Jonkam Collette , Zhu Yong , Traber Daniel , Bansal Kamna , Rehberg Sebastian , Sousse Linda , Traber Lillian , Enkhbaatar Perenlei
Published: June 2021

MRSA Sepsis remains a serious problem. Increased microvascular permeability is associated with high mortality in septic patients. Currently, we investigated the relationship between microvascular hyperpermeability and tissue expression of oxidative and reactive nitrogen species in MRSA sepsis.

METHODS

Sheep were surgically prepared and randomized to the groups: Control (non injured, n=6), and MRSA (injured, n=8). Injury consisted of smoke insufflation and instillation of 2‐5 x 10^11 CFU of live MRSA into the lungs. All animals were resuscitated with Lactated Ringer's solution.

RESULTS

Cardiovascular variables were stable in control animals. MRSA septic animals developed hypotensive and hyperdynamic sepsis, severe microvascular hyperpermeability (significantly increased flank lymph flow and permeability index, decreased plasma protein and oncotic pressure, and increased net fluid balance) compared to the control. The MRSA group also showed significantly higher plasma nitrate/nitrite levels, heart tissue inducible, endothelial, and neuronal nitric oxide synthase mRNA expressions, 3‐nitrothyrosine, and poly‐ADP ribose protein expressions compared to the control.

CONCLUSION

The data provides evidence that cardiovascular collapse in MRSA sepsis is mediated by excessive formation of reactive radicals and subsequent microvascular hyperpermeability to fluid and to protein.
A Simple and Intelligent, Web‐Based Dietary Assessment Tool
Authors: Buzzell Paul R. , Power Mariya , Pintauro Stephen J.
Published: June 2021

In person dietary assessment methods, such as the 24 hour recall method, are expensive and require the assistance of a highly trained interviewer. This often limits their use to relatively small and perhaps only local populations. We developed a Web‐based dietary assessment tool that is simple enough to be used by children and that is capable of generating results similar to that obtained from an in‐person 24 hour recall. In addition, it can be easily incorporated into a Website for online food diary or journal use. The tool is designed for use by young adolescents, but it can easily be adapted for use by adults. Its intuitive interface, featuring a talking "healthy food buddy," is self‐explanatory and allows easy navigation through its screens. The user is guided and prompted through all steps of diet entry and analysis. In addition, the program is designed to be "intelligent." That is, it will "learn" what foods in the USDA database are most often matched to a particular food entry term or phrase. The more the program is used, the better it will perform at quickly matching food entry phrases to the correct database choices. These real‐time enhancements to the web application, based upon usage of the application, occur transparently to the end‐user. We are currently in the process of validating this program against the in‐person 24‐hour recall method in a group of middle school students.
Successful implementation of a multi‐site graduate physiology course
Authors: Pawelczyk James A , Lang Charles
Published: June 2021

Penn State's Interdisciplinary Graduate Program in Physiology, initiated in 1966, includes students and faculty divided between the University Park (UP) and College of Medicine (CoM) campuses (150 km separation). By 2001 the increased availability of technology at both campuses afforded the opportunity to combine graduate course offerings in mammalian physiology.

Method

We established a set of best practices and course principles to govern this team taught course, utilizing course directors at each site, a common textbook, equal number of lectures originating from each site and established technology solutions (videoconferencing, file sharing, and a common course website). Over a six‐year period, we compared test scores for 156 students in the areas of membrane biophysics, autonomic, muscle, cardiovascular, respiratory and renal physiology with two‐way ANOVA, using the instructor and student campus of origin (CoM or UP) as factors.

Results

There was no significant interaction between the site of instructor and the site of the student (F=0.58, P=0.617). Thus, performance with a distance learning (remote instructor) format did not differ from a traditional learning (on‐site instructor) format.

Conclusion

Using technology and defining best practices a priori allowed us to revise graduate physiology education in a manner that had no negative effects on student learning. (Supported in part by NIIH T32 GM 08619)
Mast cells increase in number following muscle overload but their activation is not critical to the angiogenic process
Authors: Doyle Jennifer L , Haas Tara L
Published: June 2021

In skeletal muscle, mast cells reside in close proximity to capillaries and release factors capable of initiating angiogenesis. Overload (OL) of skeletal muscle induces angiogenesis as evidenced by increased capillary to muscle fibre ratio (C:F). We determined if mast cells play a role in the angiogenic response to muscle OL. OL of the extensor digitorum longus (EDL) was induced in male rats through extirpation of the tibialis anterior muscle. EDL was removed after 1, 2, 4 and 7 days of OL and stained with toluidine blue to visualize mast cells. Mast cell number in OL muscle did not differ from that seen in sham animals at the 1, 2 and 4 day timepoints but was significantly increased following 7 days OL. To determine if mast cells are critical to the angiogenic response, animals received Cromolyn (Cr) (160mg/kg), an inhibitor of mast cell degranulation, by i.p. injection pre‐surgery and again one day post surgery. 14 days OL and 14 days OL + Cr significantly increased C:F compared to sham + vehicle (2.64±0.13 and 2.48±0.10 vs. 1.69±0.06). Matrix metalloproteinase‐2 protein was assessed as it is crucial to OL‐induced angiogenesis. Levels were elevated after 7 days OL and 7 days OL + Cr compared to sham + vehicle (1.64±0.20 and 1.49±0.17 vs. 1.11±0.16). Our results indicate that EDL mast cell number increases after 7 days OL. However, mast cell activation is not critical to the angiogenic response. Funding by NSERC and OGS.

Grant Funding Source

NSERC
The changing landscape of anatomical education: one opinion on how we might increase the value of our stock
Authors: Haines Duane E.
Published: June 2021

Advances in medicine over the last 25 years have significantly improved the treatment of patients and, perhaps unwittingly, presented unique opportunities to the medical educator. While these advances were the product of medical exigency they have given medical educators unique innovations to embrace and use to their own advantage. The real question is, are we really integrating clinical concepts, information, or content in our courses?

MRI and CT are unique and powerful educational tools that function in parallel with clinical terminology. During the time period in which these methods have become commonplace, there have been repeated efforts to reduce or diminish course content in the anatomical sciences. Have we, as medical educators, allowed this decease in emphasis to occur by our failure to embrace the very tools and concepts that show the absolute essential value of anatomy in medical education?

There are several examples where MRI, CT, or clinical terminology, can be used to significant advantage in the basic science setting. First, CT and MRI show anatomy in vivo; such images should be an integral, if not dominate, part of gross anatomy. Second, when teaching cross‐sectional anatomy, "laterality" must be taken into account. A cross‐section in an "anatomical" orientation that does not match a corresponding MRI or CT is intrinsically flawed. Third, brainstem "anatomy" should be taught to match the "anatomy" in MRI; otherwise clinical correlations are difficult to make and time consuming to explain. Fourth, there are numerous examples where a clinical term can be used to designate an anatomical structure within its proper context.
Exercise induces Hsp70 in smooth muscle cells of the cerebral vasculature
Authors: Van Aarsen Marcus J , Noble Earl G
Published: June 2021

Exercise (EX) is a complex physiological stressor of the cardiovascular system. EX is known to increase expression of the cytoprotective inducible isoform of the 70 kDa heat shock protein (Hsp70) in blood vessels of working muscle. The objective of this study was to determine whether EX induces a similar increase in Hsp70 content in the cerebral vasculature. Sprague‐Dawley rats (n=10) were randomly assigned to either an EX (60 min treadmill running, 30 m/min, 5 consecutive days) or control group. Brain was harvested 24 hours following the final EX bout and sectioned for immunohistological localization of Hsp70, vascular endothelium and smooth muscle. Results showed increased Hsp70 expression in the cerebral vasculature post‐EX versus controls. Post‐EX, Hsp70 was always observed in the vascular smooth muscle of large cerebral vessels adjacent to the midbrain, and was also infrequently present in the vascular endothelium of these vessels. No Hsp70 was detected in neural regions of the brain. Although unclear, increased expression of Hsp70 may be a response to the change in characteristics of blood and blood flow that occur due to EX, or due to increased autonomic outflow to the vascular smooth muscle. Regardless, the neuroprotective nature of EX may be in part due to the protection of cerebral blood vessels by Hsp70.

Supported by NSERC #8170‐05

Grant Funding Source

Internal
Characterization of Myogenic Factors in Normal Rat Mesenteric Veins
Authors: Enouri Saad Salama , Johnson Ron
Published: June 2021

Vascular tone is maintained by the sympathetics, other neurohormones and myogenic factors. Myogenic factors have been characterized in arteries; however limited studies have been completed in veins. The objective of this study was to evaluate the contribution of myogenic factors to vascular tone in a large capacitance bed, the mesenteric veins, of normal male rats. Pressure myography was used to study isolated cannulated third order mesenteric venules (2‐12 mmHg) and arterioles (20‐140 mmHg). Arterioles showed myogenic contractions to pressure increases beginning at 60 mmHg, while venules showed evidence of myogenic tone (resistance to stretch) but not myogenic contractions. The contribution of endothelial dilators (N‐nitro‐L‐argentine, 100 µM) to net tone with increasing luminal pressure was minimal in both vessel types. The L‐type calcium channel blocker, Nifedipine (1 µM), abolished myogenic contributions to net tone in arterioles while only partially reducing responses in venules. The protein kinase C inhibitor (PKC), Chelerythrine (2.5 µM), also abolished myogenic contributions to net tone in arterioles; however, responses in venules were only substantially reduced. These findings suggest that in mesenteric venules (1) myogenic factors contribute to net venous tone and (2) myogenic tone is due in part to extracellular calcium and PKC activation and not altered by endothelial nitric oxide release.

Grant Funding Source

NSERC
Variant Distribution of the Right Coronary Artery at the Crux of the Heart (Anatomical and Multislice CT Imaging Study)
Authors: El‐Maasarany Shirley Hilal , Aboul‐Enein Fatma Adel
Published: June 2021

Background

In case of dominance of the right coronary artery RCA (80%‐90%), the later reaches the crux to give the posterior interventricular artery (posterior descending artery PDA) , the atrio‐ventricular nodal artery AVNA and continues to give postero‐lateral branches that supply the inferior surface of the left ventricle.

Aim

The aim of the present work was to study the course, relations and distribution of the right coronary artery at the crux of the heart and its variations.

Material and methods

Twenty embalmed human hearts showing no gross pathological lesions in the walls of the chambers were used. The crux of the heart was dissected to study the arteries in this region, their origin, relations and their destinations.

Multi‐slice Computed Tomography MSCT of twenty healthy individual, with no obstructed lesions were studied retrospectively.

Results

At the crux the RCA forms a loop upwards which gives the PDA of a considerable size and smaller AVNA. Double and triple PDAs were seen in two specimens. AVNA, in the majority of specimens, took origin from the loop of the RCA. It coursed upwards and forwards in the inferior septal space ISS to reach the AVN. Variant origin of the AVNA was seen in three of the specimens, where it took origin from the PDA or from its first septal perforator. Double AVNA was encountered in 4 of the 20 specimens. In MSCT the AVNA was recognized by its vertical course off the distal RCA.

Conclusions

These variant anatomical findings should be considered during various interventional and surgical procedures encountering the crux of the heart.
Dynamic Analysis of Blood Vessel Formation in Transgenic Quail Embryos
Authors: Sato Yuki , Huss David , Poynter Greg , Fraser Scott E , Lansford Rusty
Published: June 2021

Blood vessels help to establish the circulatory system essential for gas, nutrient, and waste exchange. Blood vessels also play a vital role in formation of the nervous system and visceral organs during development. Although numerous molecules required for blood vessel formation have been identified, a detailed understanding of normal blood vessel formation and direct effects of the gene defects is lacking due to the difficulty of imaging living embryos. To take advantage of the easy accessibility to the developing embryos, we generated Tie1.H2B‐EYFP transgenic quail that express H2B‐eYFP in endothelial cells. The Tie1.H2B‐EYFP transgenic quail enable us to image nucleus of every blood vessel endothelial cell in living embryo by EYFP signals under the confocal laser microscopes. We are studying aorta formation to better understand various types of dynamic morphogenetic events including cell migration/sprouting, cell division, mesenchymal‐to‐epithelial transition, and rearrangement of luminal structure. We dynamically track individual endothelial cells to determine their individual behavior and their collective morphometric changes during the dorsal aorta formation. By combining dynamic imaging with DNA electroporation technique, we are trying to visualize the functional role of Shh signaling for the endothelial cell in angiogenesis and cardiogenesis.

Grant Funding Source

NIH, Center of Excellence in Genomic Science (CEGS), Japan Society for Promotion of Science (JSPS)
The osteopetrosis seen in the op rat is not the result of a mutation within the coding region of the Mcpt7 gene.
Authors: Dobbins David E.
Published: June 2021

The spontaneously occurring mutation in op rats produces an animal with severe osteopetrosis. Production of affected F2 animals with recombinant events has localized this mutation to a 0.51 cM portion of rat chromosome 10. Lying within the genetic critical region for op is the Mcpt7 gene. Mcpt7 (mast cell protease 7, known as TPSD1 in humans) is a serine protease precursor that is highly expressed in human bone marrow. In this study, we tested if this gene could be responsible for the osteopetrosis seen in the op rat. M‐13 tailed primer sets were designed from rat genomic sequence to cover the 5 exons of the rat Mcpt7 gene. These primer sets were amplified by PCR against genomic DNA from affected mutant animals and the two parental strains, BN and LEW. Mutational analysis of the Mcpt7sequences from the three strains failed to reveal significant differences between the affected mutant animals and the two parental strains although single nucleotide polymorphisms between BN and the other two strains were identified in exons 1,3,4 and 5. These data indicate that the osteopetrosis seen in the op rat is not the result of a mutation within the coding region of the Mcpt7 gene. Analysis of additional candidate genes will be required to identify the causative mutation in this animal model of failed bone homeostasis. Elucidation of the op mutation will improve our understanding of the genetic control of osteoclast function and may indicate novel therapeutic paradigms in osteopetrosis, osteoporosis and rheumatoid arthritis where failed bone homeostasis is an instigating or exacerbating component of the disease process.
Protocol to assess growth plate kinetics during long‐term in vivo multiphoton imaging using BrdU as a physiological marker
Authors: Liu Wenhua , Williams Rebecca M. , Farnum Cornelia E.
Published: June 2021

With the ability to collect optical images deep into biological tissues, multiphoton microscopy (MPM) is a powerful tool to study growth plate kinetics in real time, but the act of imaging introduces variables with the potential to alter chondrocytic kinetics. This study is to develop a procedure to assess chondrocytic viability during in vivo MPM imaging on the proximal tibial growth plate of 4‐wk‐old mice. The BrdU labeling index is used as a physiological marker to assess imaging variables, following IACUC procedures. Isoflurane anesthesia was used at 1L/min (no ventilation) to maintain breathing at ~1.5/sec. Fluorescein was injected IP for a vascular concentration of ~1mM. After 2 hrs, a second dye injection was given with ~200 ul of isotonic fluids to avoid hydropenia. Body temperature was maintained at ~350C. The surgical limb was bathed in warm Ringer's. BrdU was given IP 0.5 hr before euthanasia. Fixation and plastic embedment followed, with analysis of BrdU LI by standard protocols. Maintenance of core and limb temperature, hydration, reinjection of the dye after 2 hrs, and limiting imaging to 3.5 hrs proved to be key to successful long‐term imaging. This standard protocol for BrdU‐based analysis of proliferative capacity will allow comparative assessment of multiple vital dyes for monitoring chondrocytic dynamics during multi‐hour MPM imaging. Supported by NIH‐R01AR052003.

Grant Funding Source

NIH