bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Chlorogenic Acid on Reactive Oxygen Species and Neutrophil Extracellular Trap Formation
Authors: Foley Anna , Abouelkheir Mahmoud , Petreaca Melissa
Published: June 2021

Innate immune cells such as neutrophils respond to signals at sites of infection or tissue damage by moving to the affected area and eliminating microbes by producing reactive oxygen species (ROS), very reactive molecules that damage and kill microbes, and neutrophil extracellular traps (NETs), expelled DNA chromatin and histones that form net‐like structures with sticky surfaces that can trap and kill microbes. Although ROS and NET formation are needed to prevent life‐threatening infections, excessive inflammation can lead to host tissue damage and dysfunction, and, indeed, does so in many types of diseases. Chlorogenic acid (CGA), a dietary polyphenol found in coffee, has antioxidant and potentially anti‐inflammatory effects. The possible anti‐inflammatory impacts of CGA have important medical implications due to the widespread, global availability of coffee, even in areas with reduced access to affordable and accessible pharmaceuticals. We tested the impacts of CGA on PMA‐induced ROS and NET formation in neutrophil‐like HL‐60 cells. We found that CGA functions as an antioxidant that significantly decreases PMA‐induced ROS production cells in these cells without affecting the production of superoxide or the formation of NETs. This suggests that CGA may function as an antioxidant in vivo and thereby reduce inflammation by decreasing the tissue‐damaging functions of ROS in inflamed tissues. We are currently investigating this idea further by elucidating the impact of chlorogenic acid on Lipopolysaccharide (LPS)‐induced ROS production by human polymorphonuclear neutrophils (PMN) to determine whether CGA maintains similar effects in a more physiological system.

Support or Funding Information

DePauw University and the Science Research Fellows program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Aquaporin 5 and Carbonic Anhydrase II on the Diffusion of CO2 in Xenopus Oocytes
Authors: Wang Dengke , Boron Walter F
Published: June 2021

Transport of gases across the plasma membrane is not only dependent on the permeability, but also the concentration gradient. Carbon dioxide (CO2) is one of the most important physiological gases in the human body, but undergoes slow hydration/dehydration reactions. As a gas channel, human (h) AQP5 can accelerate the diffusion of CO2 by increasing membrane permeability to CO2. Carbonic anhydrases (CAs) also can accelerate the transport of CO2 across the membrane by maximizing the CO2 gradient via the consumption/production of CO2. Here, by measuring the maximal extracellular‐surface pH increase (DpHS) caused by the introduction of extracellular CO2/HCO3− at constant extracellular pH (pHo), we obtain a semiquantitative index of the CO2 influx into Xenopus oocytes. On Day 1, we injected oocytes with the cRNA encoding hAQP5 (or H2O as a control). On Day 4, we injected purified bovine CA II protein in Tris buffer (or Tris buffer as a control). In agreement with previous reports, both hAQP5 and CA II individually increase DpHS vs. H2O/Tris‐injected control oocytes. However, the effect of CA II alone in raising DpHS even at very high CA II concentrations (1000ng/oocyte), is not as effective as AQP5 alone. Most likely, the native CO2 permeability of Xenopus oocytes is so low that it limits transmembrane CO2 diffusion. Interestingly, injection of CA II—even at CA II levels so low that they barely increase DpHS in oocytes not expressing AQP5—into oocytes expressing AQP5 causes DpHS to increase substantially vs. AQP5 oocytes without CA II. Our data suggest that when membrane CO2 permeability is low, the effects of AQP5 and CA on CO2 fluxes are supra‐additive.

Support or Funding Information

NIH U01‐GM111251, N00014‐15‐1‐2060, N00014‐16‐1‐2535

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lymph nodes harbor compartments of substantial acidity
Authors: Swietach Pawel , Wu Hao , Estrella Veronica , Enriquez‐Navas Pedro , Abrahams Dominique , Ibrahim‐Hashim Arig , Reshetnyak Yana , Luddy Kimberly , Damaghi Mehdi , Pilon‐Thomas Shari , Gillies Robert J
Published: June 2021

The effector function of T cells (e.g. secretion of cytokines) is profoundly inhibited by low extracellular pH (pHe), such as that encountered in solid tumors. Because the effect of acid on T cells is so robust, we hypothesized that it plays a role in normal physiological function. A plausible location where this acid signaling could take place physiologically is in lymph nodes (LNs), where T cells become activated, but must refrain from secreting inflammatory cytokines to avoid damaging the host organ. While LNs are known to be hypoxic1, measurements of their pH are lacking. We show, using four different approaches, that LNs have acidic compartments, reporting pHe in the range 6.2–6.8. LN pHe was interrogated using microelectrodes2, chemical exchange saturation transfer (CEST) MRI3, imaging with pH‐low inserting peptide (pHLIP)4, and intravital imaging of dextran‐conjugated cSNARF1, a pH sensitive fluorescent dye delivered by tail vein or footpad injection5. Furthermore, intravital imaging and pHLIP‐immunostaining showed that this acidity was restricted to subcortical regions, known to contain antigen‐presenting dendritic cells and lymphocytes. LN acidity was not washed‐away with NaHCO3 administered orally, suggesting that it is unlikely to arise from diffusion‐limited retention of acid. Our findings suggest that an acidic milieu in LNs is involved in the fundamental process of developing adaptive immunity.

Support or Funding Information

Funding: PS: Fulbright Scholarship (US Department of State) RJG: Grant from NCI (R01CA077571)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of High Environmental CO2 Levels on Atlantic Salmon Post‐Smolts
Authors: Gharbi Naouel , Mota Vasco , Terjesen Bendik Fyhn , Kolarevic Jelena , Ytteborg Elisabeth , Ebbesson L.O.E , Handeland Sigurd O , Nilsen Tom Ole
Published: June 2021

Atlantic salmon has demonstrated the capacity to physiologically acclimate when exposed to increased pCO2 levels in aquaculture flow‐through systems. Less is known about tolerance to increased pCO2 in Recirculation Aquaculture System (RAS). In the present study, Atlantic salmon post‐smolt in a RAS were kept at 12 ppt (12°C) and exposed to six different pCO2 levels ranging from <5 mg/L (PCO2<1.83 mm Hg), 12 mg/L (PCO2=4.40 mm Hg), 19 mg/L (PCO2=6.94 mm Hg), 26 mg/L (PCO2=9.50 mm Hg), 33 mg/L (PCO2=12.06 mm Hg) and 40 mg/L (PCO2=14.61 mm Hg). After 12 week treatments fish were transferred to full strength seawater with the lowest tested pCO2 level. We found that post‐smolts demonstrated a classic transient physiological compensatory response to cope with elevated pCO2, despite a clear and persistent decrease in growth rate when reared above 19 mg/L CO2. After twelve weeks of variable pCO2 exposures post‐smolt were challenged with rapid changes from high (40 mg/L) to low (5 mg/L) pCO2 levels and vice‐versa. Here we also will present physiological compensatory responses 1 and 24 hours and up to 6 days following rapid changes in pCO2 exposure.

Support or Funding Information

Center for Research Based Innovation, “CtrlAqua” Project Number 237856/030

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How Does Temperature Affect Primary Barrier Functions in Atlantic Salmon (Salmo Salar L.) Post‐Smolts?
Authors: Tang Patrik , Nilsen Tom Ole , Gharbi Naouel , Osberg Camilla , Handeland Sigurd , Ebbesson Lars , Käkelä Reijo , Tronci Valentina , Pedrosa Cindy , Balseiro Pablo , Stefansson Sigurd
Published: June 2021

Environmental temperature has a considerate impact over near all aspects of physiology in both wild and cultured salmonids. In addition to climate change, insight regarding the effects of temperature over physiology is crucial for the aquaculture sector, as temperature differences during the seaward transfer of fish from (semi‐) closed containment systems to open sea pens may be great and ultimately dependent on the prevailing season. For example, transferring of fish will likely occur towards higher temperatures during the summer months and lower temperatures during winter months. Further, open‐pen rearing of post‐smolt salmonids occur throughout the year at varying seasonal temperatures. Due to these temperature differences, it is therefore important to know how temperature influences tissue characteristics and modulates physiological functioning and adaptation, which processes maybe challenged during stress, thereby contributing to the stress response and overall robustness of the fish. Here we present how differences in temperature, ranging from 4°C to 18°C, affects Atlantic salmon (Salmo salar L.) post‐smolt primary barrier composition and functioning of osmoregulation in seawater. Our results suggest that primary barrier characteristics change with shifting temperatures, resulting in new physiological set points to match osmoregulatory demand in those conditions. Further, we elucidate how prior acclimation history affects the capacity of these barrier systems to respond when confronted by an additional acute stress. In this study, we investigated key changes in gill and skin phospholipid (PL) and fatty acid (FA) compositions utilizing electrospray ionization‐tandem mass spectrometry (ESI‐MS/MS) and gas chromatography (GC‐FID), in concert with qPCR expression profile changes of vital genes involved in osmoregulation, thereby aiming to understand how these correlate to changing patterns in primary barrier performance at different temperatures.

Support or Funding Information

Center for Research Based Innovation, “CtrlAqua” Project Number 237856/030

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ablation of PHOX2B‐Derived Astrocytes Results in Neuronal Dystrophy‐Like Neuropathology in the RTN
Authors: de Melo e Silva Talita , Czeisler Catherine M , Fair Summer R , Alzate‐Correa Diego , Takakura Ana C T , Moreira Thiago S , Otero Jose J
Published: June 2021

The transcription factor Phox2b is a regulator of the autonomic nervous system development and is especially expressed by respiratory circuits involved in chemosensory integration, as neurons of solitary tract nucleus (NTS) that relay information from peripheral chemoreceptors to the ventral respiratory column, and by central chemoreceptors located in the retrotrapezoid nucleus (RTN). In addition to neurons, recent studies show that astrocytes from ventral medullary surface also detect PCO2 increase, and contribute to respiratory drive. Since PHOX2B biased progenitor cells generate astrocytes that have a developmental connection with RTN neurons, the aim of this study is determine the extent to which selective ablation of these astrocytes affect synapse integrity in the retrotrapezoid nucleus. We utilized the astrocyte ablation technique described by Tsai et al., 2012. In this model, the astrocyte‐specific ALDH1L1 promoter induces GFP expression in the absence of cre recombinase, and in the presence of cre recombinase expresses diphtheria toxin A (DTA). We interbred ALDH1L1loxp‐GFP‐STOP‐loxp‐DTA mice to PHOX2Bcre mice. Transmission electron photomicrographs were captured from the RTN/VLM area and morphometric analysis was performed using FIJI. We noted that axon terminals in the mutants (PHOX2Bcre, ALDH1L1loxp‐GFP‐STOP‐loxp‐DTA) were on average larger in area and perimeter. Furthermore, we noted an increase in the percentage of axon terminals showing autophagic vacuoles/phagosomes. The proportion of axon terminals with other organelles, including mitochondria and dense core neurosecretory vesicles, was not different between groups. In summary, PHOX2B‐derived, astrocyte ablated mice show synaptic pathology characterized by dysmorphic neuroaxonal morphology, suggesting an underlying defect in the afferent input into the RTN. These data suggest that PHOX2B‐derived astrocytes may play a role in regulating the synaptic integrity of neuronal inputs into RTN neurons. Other experiments are being performed in order to elucidate if the cell body of these neurons is located in the commissural NTS, and also, if PHOX2b derived astrocytes depletion can imply local regulation of presynaptic autophagy, which can lead to neurodegeneration and breathing impair.

Support or Funding Information

FAPESP and NIH

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of the Cerebellum on the Control of Tracheobronchial Cough in Spontaneously Breathing Cats
Authors: Musselwhite M Nicholas , Shen Tabitha Y , Rose Melanie J , Reed Mitchell D , Poliacek Ivan , Pitts Teresa , Bolser Donald C
Published: June 2021

Prior work exploring the role of the cerebellum in cough has been limited to paralyzed preparations. Xu et al. (1997) found in paralyzed, artificially ventilated, and decerebrated cats that after cerebellectomy cough number and expiratory magnitude was reduced with no change in phrenic bursting. However, we hypothesized that the cerebellum would have a different effect on coughing in unparalyzed animals than what was observed by Xu et al. (1997) due to significant behavior‐specific feedback. Cough was induced by mechanical stimulation of the tracheobronchial airways in anesthetized, spontaneously breathing adult cats (n=6). Electromyograms (EMGs) were recorded from upper airway, chest wall and abdominal respiratory muscles. Cough trials were performed before and after a total cerebellectomy. Post cerebellectomy, we observed a decrease in respiratory rate (23±2 to 18±2 breaths/min, p = 0.02) as a result of an increase in the duration of the expiratory phase of breathing (1.5±0.2 to 2.3±0.3 seconds, p = 0.03) and increase in total cycle time (2.7±0.3 to 3.5±0.3 seconds, p = 0.04). Cerebellectomy had no effect on cough number, frequency, esophageal pressure, or bout duration. Post cerebellectomy, there was a significant increase in thoracic inspiratory EMG magnitudes during cough. When normalized to the maximal control effort, on average diaphragm EMG increased from 67±6% to 81±9% (p = 0.04). The left parasternal EMG increased from 70±5% to 98±8% (p = 0.04), and the right parasternal EMG increased from 71± 4% to 93±5% (p = 0.01). The average thyroarytenoid EMG amplitude increased post cerebellectomy (66±6% to 118±26%, p = 0.03), with no change observed in the posterior cricoarytenoid EMG. No significant changes were observed in cough phase durations. Changes in cough EMG magnitudes suggest that the cerebellum modulates cough motor drive, but not cough phase timing or number of behaviors in response to mechanical stimuli in this model. These results suggest an important role of the cerebellum and behavior‐related sensory feedback in the regulation of coughing.

Support or Funding Information

Supported by NIH SPARC OT2OD023854‐01 and HL131716.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence for stretch feedback in the thyroarytenoid muscle and potential implications in spasmodic dysphonia
Authors: Reed Mitchell , Huff Alyssa , Iceman Kimberly , Mian Seema , Howland Dena , Pitts Teresa
Published: June 2021

Muscle position feedback, via receptors for stretch (muscle spindles) and force (golgi tendon organs), is critical for the muscular coordination and proprioception that is essential for most movements. Muscle spindles are innervated by gamma motoneurons that modulate their sensitivity to stretch. Recent reports suggest that 5‐HT1D receptors can modulate gamma motoneurons. Adduction of the vocal folds in the larynx is regulated by the thyroarytenoid muscle, which is active during breathing, cough, swallow, and vocalization. We tested the hypothesis that activation of gamma motoneurons via 5‐HT1D agonists would modulate stretch‐dependent thyroarytenoid activity. Muscle activity (electromyography, EMG) of both the left and right thyroarytenoid muscles was monitored in freely‐breathing sodium pentobarbital anesthetized Sprague Dawley rats. The thyroid and cricoid cartilages were mechanically stretched in an inferior‐superior direction before and after the addition of the 5‐HT1D agonist L‐694,247. Stretching elicited EMG activity from the thyroarytenoid muscle, which increased with the addition of the agonist. Similarly, phasic EMG activity observed during breathing increased with sustained stretching, and further increased with addition of the agonist. These results provide evidence that the previously reported “spindle‐like” structures act in a stretch feedback manner, and modulation of receptors known to exist on gamma motoneurons can amplify this feedback. Identifying and understanding stretch reception in muscles of the larynx may be pivotal to determining the mechanisms of voice disorders including muscle tension dysphonia and spasmodic dysphonia.

Support or Funding Information

This work was supported by NIH grants HL 111215, HL 103415 and OT20D001983, the Craig F. Neilson Foundation Pilot Research Grant 546714, Veterans Affairs Rehabilitation, Research and Development RCSB92495 (DRH), Kentucky Spinal Cord and Head Injury Research Trust, Rebecca F. Hammond Endowment (DRH), and the Commonwealth of Kentucky Challenge for Excellence.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optogenetic stimulation of the pre‐Bötzinger Complex and the control of breathing in a mouse model of Parkinson's disease
Authors: Santos Luiz Marcelo Oliveira , Baertsch Nathan A , Moreira Thiago Santos , Takakura Ana Carolina , Ramirez Jan‐Marino A
Published: June 2021

Parkinson's disease (PD) is a neurodegenerative disorder well known for its progressive loss of dopaminergic neurons in the substantia nigra compacta (SNpc). Much less known, yet clinically very important, are the detrimental effects on breathing associated with this disease. Studies in the 6‐hydroxydopamine (6‐OHDA) rat model of PD revealed reductions of respiratory frequency (fR) and NK1r‐immunoreactivity in the pre‐Bötzinger complex (preBötC), a medullary region critical for generating inspiration. To unravel mechanisms that underlie breathing disturbances in PD, we used a transgenic approach to specifically label or stimulate different excitatory (Dbx1‐ERT2Cre, Vglut2‐Cre) and inhibitory (Vgat‐Cre) neural populations in the preBötC. Homozygous Cre mice were bred with homozygous Ai6 (ZsGreen) or Ai32 (ChR2) mice. Adult offspring then received bilateral injections of vehicle or 6‐OHDA (10 μg/μl) into the striatum (CPu) to induce PD; and Ai32 mice also received preBötC fiber optic cannulation. 10–12 days later, whole body plethysmography was used to measure ventilation at rest and during optogenetic photo‐stimulation of the preBötC in unanaesthetized and unrestrained mice. As expected, fR at rest was reduced by 24.9% in PD mice (154.6 ± 6.0 vs. vehicle: 205.9 ± 3.7 bpm, p<0.05). Dbx1 photo‐stimulation during the inspiratory phase did not affect the rhythm in control animals, but increased fR by 17% in PD animals. Similarly, Vglut2 photo‐stimulation in control animals did not change the rhythm, but increased fR by 30% in PD animals. No changes were elicited by photo‐stimulation during expiration. Photo‐stimulation of preBötC inhibitory Vgat neurons during inspiration increased fR by 21%, but only in vehicle‐injected mice; no changes were observed in PD mice. Histology confirmed that 6‐OHDA injections reduced the number of catecholaminergic neurons in the SNpc by 69% (150.4 ± 4.9 vs. vehicle: 494.7 ± 14.5 cells). Surprisingly, the number of Dbx1 and Vglut2 neurons in the preBötC and the adjacent ventral respiratory column (VRC) were also reduced by 47.6 and 17.3%, respectively (p<0.05), whereas the number of Vgat neurons did not change. In conclusion, our study reveals that the 6‐OHDA model of PD is associated with a specific reduction in the number of excitatory neurons within the preBötC, which includes rhythmogenic Dbx1 neurons. Photo‐stimulating specifically these neurons can restore breathing in this PD mouse model.

Support or Funding Information

NIH and FAPESP.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cigarette smoke and Pseudomonas aeruginosa synergistically cause ARDS via impairment of mitophagic flux and NLRP3 inflammasome activation
Authors: Lu Qing , White Alexis , Wang Zhengke , Chambers Eboni , Ayala Alfred , Rounds Sharon , Wang Xing
Published: June 2021

Itis increasingly recognized that cigarette smoke (CS) increases the incidenceand mortality associated with acute respiratory distress syndrome (ARDS), however, the underlying mechanism(s) for this is unknown. Pseudomonas aeruginosa is a frequent pathogen causing pneumonia, which is the most prevalent cause of ARDS. Whether CS increases prevalence of ARDS in individuals with P. aeruginosa infection is unknown. We have previously shown that CS increases severity of acute lung injury in mice challenged by lipopolysaccharide. In this study, we demonstrated that exposure of mice to CS either briefly (6h) or sub‐acutely (2weeks) suppressed lung bacterial clearance and exacerbated P. aeruginosa (PA103)‐induced lung edema, inflammation and mortality. CS and PA103 also synergistically increased lung microvascular endothelial cell permeability in vitro. To investigate the mechanisms of CS priming for lung injury, we used the clinically relevant double‐hit mouse model of ARDS induced by CS priming, followed by PA103 infection. We found that CS increased mitochondrial fission and mitochondrial oxidativestress and inhibition of mitochondrial fission and oxidative stress prevented CS‐induced increase in endothelial permeability. CS pre‐exposure did not affect PA103‐induced increase in steady‐state mitophagy, but impaired mitophagic fluxin alveolar macrophages and lung endothelial cells; an effect associated with accumulation of fragmented/swollen mitochondria and double‐membraned mitophagosomes in the lungs and NLRP3 inflammasome activation in alveolar macrophages. We further found that inhibition of NLRP3 inflammasome signaling attenuated CS/PA103 double‐hit‐induced lung inflammation and lung edema and improved bacterial clearance, as well as overall animal survival. Our results suggest that CS/PA103 double‐hit injury causes ARDS via enhancement of mitochondrial fission and impairment of mitophagic flux and resultant NLRP3 inflammasome activation. Suppression of mitochondrial fission, improvement of mitophagic flux and inhibition of the inflammasome may be novel targeted therapeutic strategies for smokers with ARDS.

Support or Funding Information

COBREP20GM103652 (Rounds, Lu), R01 HL130230 (Lu), VA Merit Review (Rounds)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Influenza virus NS1 protein upregulates epithelial sodium channel expression and function by inhibiting ubiquitin ligase Nedd4‐2
Authors: Lazrak Ahmed , Lazrak Samuel , Liu Evan , Yu Zhihong , Matalon Sadis
Published: June 2021

Influenza is a contagious respiratory virus that infects epithelial cells throughout the respiratory tract. The viral infection is facilitated by the attachment of the viral hemagglutinin to the sialic acid residues on the host cell membrane. Once the virus is inside the host cell, the acidic endosomal environment facilitates the release of the viral ribonucleoproteins, which are, subsequently, transported to the host cell nucleus for replication. The mechanism by which replicating influenza viruses affect the function of ion channels in the lung epithelia is not well understood. We have studied the effects of the matrix protein 2 (M2) on the Epithelial Sodium Channel (ENaC) and the Cystic Fibrosis Transmembrane Conductance regulator1,2. However, the effects of other viral proteins is still unknown. In this study we explored the mechanisms by which the Non‐Structural protein 1 (NS1) regulates the expression and the function of ENaC heterologously expressed in Xenopus Laevis oocytes. in‐vivo, NS1 acts by, mainly, inhibiting the host immune response. Herein, we found NS1 to act by a dual mechanism when heterologously expressed with ENaC in Xenopus Laevis oocytes. 1) We observe an inhibition of ENaC expression and function (−5.64±2.7 μA to −1.14±0.71 μA; p<0.001) when both cRNAs, ENaC's and NS1's, were consecutively injected the same day, NS1 acted by inhibiting the replication and proteins synthesis, a known effect of NS1 on host proteins and RNAs. 2) NS1 acted by enhancing ENaC expression and function when its cRNA was injected 24 hours after ENaC cRNA was injected, a situation simulating the infection of an epithelail cell by a flu virus. NS1 increased the expression of ENaC proteins at the surface of the oocytes, at least, three times the control values, measured by western blot. The increased ENaC proteins at the membrane of the oocyte resulted in a 3 fold increase of recorded amiloride sensitive sodium currents (from −6.62±0.67 μA to −17.15±0.62 μA; p<0.001) measured by the double voltage clamp technique. In conclusion, NS1 acted by increasing the number of active ENaC at the surface of oocytes. NS1 may have acted by inhibiting Nedd4‐2, the ubiquitin ligase, responsible th regulation of ENaC expression at the cell surface. Nedd4‐2 acts by ubiquitinilating and marking ENaC proteins for removal by endocytosis, internalization, and degradation in the proteasome or lysosome.

Support or Funding Information

5U01ES026458 and 1U01ES02769701

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lung Hydrogel Implantation System to Study the Pathogenesis of Pulmonary Hypertension
Authors: Mammoto Akiko , Mammoto Tadanori
Published: June 2021

Remodeling of distal pulmonary arterioles (PAs) associated with marked accumulation of pulmonary artery smooth muscle cells (PASMCs) represents one of the major pathologic features of pulmonary hypertension (PH). The levels of a transcription factor, Twist1 are upregulated in the lungs of patients with pulmonary arterial hypertension (PAH). However, the mechanism by which endothelial Twist1 stimulates SMC accumulation to distal PAs in PH remains unclear. Here we have found that Twist1 overexpression increases the expression of platelet‐derived growth factor (PDGFB) in cultured human pulmonary arterial endothelial cells (HPAECs). Conditioned media collected from Twist1 overexpressing HPAECs induce SMC migration and growth. Knockdown of Twist1 in ECs attenuates hypoxia‐induced accumulation of alpha‐smooth muscle actin (SMA)‐positive cells to the PAs in mice. To further study the mechanism by which endothelial Twist1 controls vascular remodeling and SMC accumulation in the lung, we implant fibrin gel supplemented with fluorescently labeled HPAECs on the mouse lung. Supplemented HPAECs form vascular structures and hypoxia treatment stimulates accumulation of alpha‐SMA–positive cells to blood vessels and upregulates expression of PDGFB in the gel. Twist1 overexpression in supplemented HPAECs or hypoxia treatment also induces endothelial‐to‐mesenchymal transition (EndMT) in the implanted gel, which also partly contributes to the pathogenesis of PH. Endothelial Twist1 plays a key role in SMC accumulation to PAs through PDGFB signaling and the mouse lung gel implantation system would be a good model system to study the pathogenesis of PH and other lung diseases.

Support or Funding Information

AHA18TPA34170129, MCW Research Affair Committee New Investigator Award

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Responses of Caudal NTS Neurons to Acute Tissue Hypoxia
Authors: Paundralingga Obed , Wang Lei , Mifflin Steve Wayne
Published: June 2021

The neurons of caudal nucleus tractus solitarii (nTS) initiate an integrated response to hypoxia by virtue of its afferent inputs and efferent projections. Furthermore, these neurons are also subjected to tissue hypoxia. In this study we investigated the effects of acute tissue hypoxia on intracellular Ca2+ ([Ca2+]i) levels in neurons from rat caudal nTS. Adult male Sprague‐Dawley rats were anesthetized with isoflurane and the hindbrain removed and cut into 250 μm thick sections. The NTS slices were then incubated for 45 min with 10 μM Fura‐2AM and 30 μL of F127 at room temperature and then washed for 20 min in artificial cerebrospinal fluid (aCSF) bubbled with 95% O2/5%CO2. A single slice was transferred to the recording chamber on an upright epifluorescent microscope and superfused with normal aCSF at a rate of 2.5 ml/min. Acute hypoxia was established by exposing hindbrain slices containing nTS to aCSF bubbled with 95% N2/5% CO2. The slice was superfused with either 20 μM nifedipine, 10 μM ruthenium red, or 10 μM ryanodine dissolved in aCSF. Cell viability was confirmed by 63 mM KCl. Fluorescence of Fura‐2AM was excited by epi‐illumination with light provided by a 75 W Xenon lamp band‐pass filtered alternatively at 340 or 380 nm. Emission light pass through a barrier filter (510 nm). Pairs of 340 and 380 nm images were acquired at intervals of 5 s and analyzed off‐line with NIS‐Elements AR 3.2 software. All images were captured with a charge‐coupled device (CCD) camera. A total of 20 sections were examined from 9 rats and an average of 4 cells/slice were analyzed in each section. Five minutes of hypoxic aCSF triggered a 41.37±20.95 % reversible increase in 340/380 ratio normalized to KCl relative to baseline with a latency of 56.9 s ± 25.1 s. This increase was due to calcium influx from extracellular compartment as removing calcium from extracellular bathing solution abolished the response. Nifedipine reduced the increase in [Ca2+]i during hypoxia by 14.62±11.88% (n=25 cells, p<0.05). Neither ryanodine nor ruthenium red altered the [Ca2+]i response to hypoxia. These findings suggest that the elevated [Ca2+]i as a response to acute tissue hypoxia in caudal NTS is dependent, at least in part, upon influx through L‐type voltage gated calcium channel. Other types of Ca channels, ROS generation, and mitochondrial release could also contribute to the response and are being investigated. Hypoxia‐induced elevations in intracellular Ca are likely to alter caudal NTS neuronal function under hypoxic conditions.

Support or Funding Information

Supported by HL088052

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
H2S synthesis inhibitor prevents hypoxia‐evoked periodic breathing in spontaneous hypertensive rats
Authors: Zhang Xiuli , Peng Ying‐Jie , Nanduri Jayasri , Muzzio Miguel , McCormick David L , Prabhakar Nanduri R
Published: June 2021

An augmented carotid body (CB) response to hypoxia has been implicated as one of the mechanism for evoking periodic breathing (PB). Spontaneous hypertensive (SH) rats exhibit CB hypersensitivity to hypoxia; this hypersensitivity is mediated by H2S produced by cystathionine‐g‐lyase (CSE; Peng et al. PNAS, 2017). Using whole‐body plethysmography, we demonstrate that acute hypoxia evokes PB in unsedated 2–4 month old SH rats; PB was characterized by episodes of hyperventilation and hypoventilation (decreased tidal volume and respiratory rate). The magnitude of PB in SH rats was linked to the severity of hypoxia: exposure to 12% O2 elicited 40 ± 3 episodes/hr of PB, while more severe hypoxia (8–10% O2) elicited 114 ± 4 episodes/hr of PB. Oral administration of the CSE inhibitor, L‐propargylglycine (L‐PAG) prevented CB hypersensitivity to hypoxia and stabilized breathing in dose‐related fashion. L‐PAG is rapidly absorbed after oral dosing (Tmax = 0.25 to 0.5 hr) and oral bioavailability is 100%. These data demonstrate that CSE inhibition provides a viable mechanism for prevention of breathing instability arising from CB hypersensitivity to hypoxia

Support or Funding Information

(Supported by NHLBI grant UH3HL‐123610).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhanced Carotid Body Chemosensory Discharge is Essential for the Hypertension Induced by Chronic Intermittent Hypoxia
Authors: Dias Ana Carolina , Arias Paulina , Andrade David , Del Rio Rodrigo , Iturriaga Rodrigo
Published: June 2021

Chronic intermittent hypoxia (CIH), the main feature of obstructive sleep apnea, enhances carotid body (CB) discharges, leading to sympathetic overflow and hypertension. CIH produces oxidative stress (ROS) in the CB, which contributes to enhance the CB drive to the brainstem and hypothalamic nuclei related to the cardiorespiratory control, such as the nucleus of the tractus solitary (NTS), the rostral ventrolateral medulla (RVLM) and the paraventricular nucleus (PVN). We hypothesized whether the enhanced CB chemosensory drive may increase ROS levels in central cardiovascular nuclei during CIH‐induced hypertension. Accordingly, we studied the effects of CB ablation on ROS formation in the NTS, PVN and RVLM of hypertensive CIH‐rats. Male Sprague‐Dawley rats (200 g) were exposed to CIH (5–6% inspired O2 for 20s, followed by room air for 280s, 12 times/h, 8 h/day), for 28 days. Arterial blood pressure (BP) was measured by radio‐telemetry (DSI, USA). Following 21 days of CIH exposure, rats were anesthetized with isofluorane in 2% O2, and the CBs were cryogenically destroyed bilaterally using a fine‐tipped forcep cooled in liquid N2. Then, rats were maintained in CIH conditions for one more week. Another group of rats were maintained in CIH or Sham conditions for 28 days. At the end of the experiments, all animals were euthanized (pentobarbital 120mg/kg i.p.) and brains were removed and frozen at −20°C. Coronal sections of the NTS, RVLM and PVN were cut and incubated for 30 min in the dark with the ROS sensitive fluorescent probe dihydroethidium (DHE 10μM, Invitrogen USA). DHE fluorescence was measured using epifluorescence microscopy (Olympus Optical, Japan) at an excitation wavelength of 543 nm. Data were analyzed by one‐way ANOVA followed by Bonferroni's test. Compared to Sham rats, CIH‐rats showed a significant increase in mean BP (□ 10 mmHg, p<0.05). The CIH‐induced hypertension was abolished by the ablation of the CBs (CBA). ROS formation was markedly reduced by CBA in the NTS, RVLM and PVN of CIH‐exposed rats (CIH vs. CIH‐CBA: NTS: 6.2±1.5 vs. 1.5±02 au, RVLM: 4.9± 1.5 vs. 2.1±1.6 au, PVN: 5.4±1.8 vs. 1.6±05 au, p <0.05, n= 5–6 rats). Results support the evidence for an essential role of the CBs in the development and maintenance of the CIH‐induced hypertension and oxidative stress present in the NTS, RVLM and PVN

Support or Funding Information

FONDECYT 1150040

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of NLRP3 inflammasome in regulating the progression of prostate cancer
Authors: Wu Hsin‐En , Lai Yi‐Xin , Li Chia‐Yang
Published: June 2021

Chronic inflammation is a hallmark of cancers and plays a critical role in modulating tumor progression and metastasis. Inflammasomes are a group of cytosolic protein complexes, composed of an NOD‐like protein (NLR), the adaptor apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), and caspase‐1, which are responsible for the cleavage of pro‐IL‐1β and pro‐IL‐18 proteins into mature and biologically active IL‐1β and IL‐18, respectively. IL‐1β levels in tumor tissues were previously connected to poor prognosis for cancer patients. However, it remains unclear the function of inflammasomes in tumor growth and metastasis. The aim of this study is to examine whether the activation of NLRP3 inflammasome regulates the progression of prostate cancer. First, we screened the ability of NLRP3 inflammasome activation in prostate cells due to cancer heterogeneity. Prostate cell lines were primed with LPS, followed by treated with ATP or nigericin to induce NLRP3 inflammasome activation. Our results showed that the secretion of IL‐1β was significantly increased in PC3 (human prostate cancer cell line), BPH‐1 (human benign prostate hyperplasia epithelial cell line) and RWPE‐1 (human prostate epithelial cell line) cells. Second, we tested the effect of hypoxic condition in regulating the activation of NLRP3 inflammasome. Our results showed that hypoxia also induced the secretion of IL‐1β in PC3, BPH‐1 and RWPE‐1 cells. Third, we found that NLRP3 inflammasome activation involved in regulating tumor growth and metastasis. Our results indicated that the activation of NLRP3 inflammation plays an important role in modulating the progression of prostate cancer. Therefore, targeting NLRP3 inflammasome could be a strategy for prostate cancer therapy.

Support or Funding Information

This study was supported by the Ministry of Science and Technology, Taiwan, R.O.C. (Grant No. MOST 107‐2320‐B‐037‐019).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effect of gonadotropin releasing hormone (GnRH) antagonism on feeding behavior in female rats
Authors: Izumi Ayana , Hirano Ayaka , Iida Kurumi , Nouchi Mizuki , Fujimaki Saori , Morimoto Keiko , Takamata Akira
Published: June 2021

Estrogen replacement in ovariectomized rats reduces food intake specifically during the light phase, suggesting that estrogen exerts an anorectic action by modifying circadian feeding rhythm. Ovariectomy is known to elevate levels of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) via the negative feedback system of the hypothalamic‐pituitary‐gonadal (HPG) axis. However, the contribution of GnRH and/or LH to the hypophagia induced by ovariectomy remains unknown. To elucidate the role of GnRH and/or LH in the feeding regulation, we examined the effect of GnRH antagonism on feeding behavior in female rats. Female Wistar rats were injected subcutaneously with either 1mg/kg degarelix, a GnRH antagonist, (De group) or 5% mannitol (Veh group), and were provided free access to water and a standard rodent chow. We found that daily food intake and body weight gain were significantly greater in the De group than the Veh group. Food intake during the light phase was not different between the groups, while food intake during the dark phase was greater in the De group than the Veh group. These results suggest that estrogen regulates daily energy intake, and GnRH and/or LH possibly modulate diurnal pattern of feeding behavior.

Support or Funding Information

JPSP KAKENHI #15K12355 and 24500975 to Akira Takamata

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gene expression profile and cytokine analysis in experimental autoimmune encephalomyelitis (EAE) in AVP deficient rats
Authors: Quintanar‐Stephano Andres , Viñuela‐Berni Verónica , Macías‐Segura Noe , Valdez‐Urias Fernando , Kovacs Kalman
Published: June 2021

Introduction

Human Multiple Sclerosis (MS) is a medically important disease. MS and its experimental autoimmune encephalomyelitis (EAE) paradigm are central nervous system demyelinizing diseases in which the immune system is directly involved. The pursuit for new treatments for MS still as an important goal for research. The use of the EAE animals to study the immune mechanisms that mediate the disease still being the best strategy to deep into the immune‐neuroendocrine interactions during the pathogenesis. Previously we have demonstrated that neurointermediate pituitary lobectomy (NIL) decreases humoral and cell‐mediated immune responses and that AVP is directly involved. Nevertheless, the role of AVP has not been entirely clarified.

Objective

To investigate the mechanism by which AVP deficiency (by NIL) and desmopressin (DP, a synthetic analog of AVP) affects the development of the clinical signs, the immune response and gene expression profile in the EAE.

Methods

Female Lewis rats were divided into intact control (IC), EAE immunized, NIL and NIL treated with desmopressin (NIL+DP). Except the IC, the remaining groups were immunized for EAE. The EAE was induced by encephalitogen subcutaneous injection. Animals were sacrificed at the peak of the disease (15 days after immunization). EAE clinical signs, cytokine serum profiles (IL‐17A, IL‐4, IL‐2, IL‐10, IL‐6, TNF‐α and INF‐γ) (flow cytometry) and spleen gene expression profile (microarray) were evaluated. DP treatment started 3 days before immunization.

Results

As compared with the EAE group, NIL animals developed just a mild clinical signs of EAE and significant decreased IFN‐γ and IL‐17 levels. NIL+DP treated animals restored its susceptibility to EAE clinical signs, whereas no‐significant differences on cytokine levels occurred. Significant differences in gene expression profile among groups were evident; in the NIL group the expression pattern of the TGF‐β pathway was down‐regulated as compared with the EAE group, whereas in the NIL+DP group the same genes were up‐regulated.

Conclusions

Findings add novel data indicating that directly AVP plays a crucial role in regulating the immune responses and suggests that AVP receptor blockers may be used for the MS treatment and possibly autoimmune diseases and other inflammatory processes.

Support or Funding Information

Supported by UAA‐PIFF14‐1 and CONACYT‐221262 (AQS). México. VVB: CONACT Doctoral scholarship. NMS: CONACYT post doctoral Fellow. México.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Decreased Amplitude of the Photopic Negative Response (PhNR) in the Ins2Akita Mouse Model of Type 1 Diabetes Mellitus
Authors: Fortepiani Lourdes Alarcon , Le Dung , Akimov Nikolay P , Sohn Jeong‐Hyeon , Rentería René C
Published: June 2021

Diabetic retinopathy (DR) is a common, severe complication of diabetes mellitus. Current clinical detection of DR is based primarily on visible retinal vascular damage, and earlier detection of impending DR might allow earlier treatment to protect function. Recent studies in both animal models and humans with diabetes have shown that degradation of retinal ganglion cell (RGC) function occurs at an early stage, before visible vascular complications and before significant RGC loss. The photopic negative response (PhNR), obtained from full‐field electroretinogram (ERG) with photopic stimuli is thought to arise directly from RGC activity. Here, we hypothesized that PhNR amplitudes are reduced in a type 1 diabetic mouse model, the Ins2Akita mouse, which develops hyperglycemia by 5 weeks of age and visual function and ocular blood flow deficits early in the disease progression, before overtly visible vascular damage. Diabetic (male heterozygotes) and control (male WT littermates) mice were grouped according to age: young, adult, and old (5–6, 9–11, and 12–14 months of age, respectively). ERG recording was performed with 1.62 log cd/m2 white flashes over a constant 40 cd.s/m2 green background (Diagnosys Espion and ColorDome ganzfeld system) using gold ring contact lens electrodes (LKC Techniologies) under isoflurane anesthesia. Following 1 hour adaptation to the green background illumination, PhNR, a‐wave, and b‐wave amplitudes and implicit times were measured in response to 25 10‐msec flashes.

The mean PhNR amplitude of the adult diabetic mice was 36% of their age‐matched controls (p < 0.01). For the old diabetic group, the mean PhNR amplitude was 49% of their age‐matched controls (p < 0.05). However, no significant difference was found in the young group. A‐wave amplitudes were decreased in the diabetic adult group, to a value only 41% of their age‐matched controls (p < 0.05). B‐wave amplitudes were decreased in the diabetic old group, to 35% of their age‐matched controls (p < 0.05). However, no significant difference was found among groups for PhNR, a‐wave, or b‐wave implicit times. Our results suggest that PhNR amplitude is altered relatively early in the Ins2Akita mouse model of diabetes. This suggests PhNR measurements could be used to detect diabetic retinopathy at earlier stages than the current clinical standard.

Support or Funding Information

NIH R01 EY023290 and UIWRSO Faculty Development Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characteristics of the Cortical Silent Period in Individuals with and without Spinal Cord Injury
Authors: Sfreddo Hannah J , Wu Yu‐Kuang , Maher Matthew T , Limonta James , Levine Jonah M , Wecht Jaclyn , Harel Noam Y
Published: June 2021

Objective

To increase our knowledge of corticospinal excitability changes in persons with spinal cord injury (SCI) by comparing the cortical silent periods (CSPs) of persons with chronic, incomplete cervical SCI to the CSPs of able‐bodied (AB) volunteers.

Methods

The CSP is a corticospinal inhibitory response (1). It appears as gap in electromyographic (EMG) activity of a voluntarily contracting muscle due to transcranial magnetic stimulation (TMS) delivered to the motor cortex (see Figure A). We measured TMS‐induced CSPs in 5 participants with chronic, incomplete cervical SCI and 5 AB volunteers as an exploratory outcome of a larger study assessing the effects of non‐invasive stimulation in persons with and without SCI. EMG data was collected from the abductor pollicis brevis muscle of the dominant hand in each subject. We recorded a total of 139 CSPs in the SCI participants and 176 in the AB participants. Further analysis of 4 more SCI subjects and 10 more AB subjects is ongoing.

Results

The preliminary results suggest that the average CSP duration of the SCI group (89 ± 34 ms) was less than that of the AB group (116 ± 30 ms). While statistically insignificant, (p = 0.230; Figure B), our results corroborate previous findings of shortened CSPs in persons with SCI (2). Moreover, the proportion of CSPs with at least one “interruption,” visualized as a momentary spike in EMG activity lasting 5–10 ms (see Figure C), was higher in the SCI group (14.48% of CSPs) than in the AB group (2.56% of CSPs; ns; Figure D).

Conclusions

Our preliminary findings, of shorter CSP duration and higher frequency of CSP interruptions in subjects with SCI, may imply that cortical inhibitory pathways are damaged or less excitable in persons with SCI. CSP parameters may be valuable outcome measures in the study of SCI.

Support or Funding Information

New York Spinal Cord Injury Research Board (DOH01‐CARTID‐2015‐00037)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.