bluuebunny/crossref_metadata_2025 huggingface dataset:
Pulmonary Vascular Pressures and Ventilatory Control during Exercise in Heart Failure with Preserved or Reduced Ejection Fraction
Authors: Van Iterson Erik H. , Snyder Eric M. , Olson Thomas P.
Published: November 2023

Introduction

A disproportionate increase in ventilation relative to carbon dioxide output (VE/VCO2) slope is a strong prognostic indicator in heart failure with reduced (HFrEF) or preserved ejection fraction (HFpEF) patients. Similarly, secondary pulmonary hypertension also poses a profound mortality risk in these patients. Exaggerated ventilation and pulmonary pressures appear to parallel one another during exercise in HF. However, an underlying pathophysiologic mechanism relating these two outcomes remains incompletely understood. Thus, the aim of this study was to determine associations between anatomical and physiologic metrics of ventilatory control and pulmonary pressures during exercise in HFrEF and HFpEF.

Methods

32 HFpEF and 33 HFrEF (EF: 62±2 vs 22±1%; male: 17 and 31; age: 70±2 and 55±2 yrs; BMI: 34±1 and 28±1 kg/m2; weight: 98±4 and 86±3 (all P<0.05), respectively, participated in cardiopulmonary exercise testing during right heart catheterization. Measures of pulmonary pressures included: systolic (PASP); diastolic (PADP); mean (mPAP); wedge (PCWP); pulse (PP); transpulmonary gradient (TPG). Partial pressure of arterial oxygen (PaO2) and carbon dioxide (PaCO2) were measured via radial arterial catheterization. Ventilation was measured continuously using a breath by breath metabolic measurement system. We calculated dead space to tidal volume [Vd/Vt=(VE×PaCO2)‐(863×VCO2)/(VE×PaCO2)] and Alveolar‐arterial oxygen gradient (A‐a)=PAO2‐PaO2; VE/VCO2 slope=rest to peak exercise.

Results

At rest, pulmonary pressures did not differ between groups (P>0.05); whereas Vd/Vt was lower in HFpEF vs HFrEF (0.42±0.02 vs 0.48±0.01, respectively, P=0.03). At peak exercise, HFpEF had higher PCWP (34.8±1.2 vs 30.2±1.5 mm Hg, P=0.02) and PADP (35.4±1.3 vs 29.8±1.6 mm Hg, P=0.02), but lower Vd/Vt (0.32±0.02 vs 0.38±0.01, P=0.03) and VE/VCO2 slope (35.3±1.6 vs 43.5±2.0, P<0.01) vs HFrEF. At peak in HFpEF, Vd/Vt was related to PASP (r=0.40, P=0.03), PP (r=0.44, P=0.01), and TPG (r=0.53, P<0.01), and A‐a was related to PASP (r=0.43, P=0.01), PP (r=0.44, P=0.01), and TPG (r=0.46, P<0.01). At peak in HFrEF, Vd/Vt was related to PADP (r=0.41, P=0.02), and A‐a was related to PASP (r=0.43, P<0.01), PADP (r=0.42, P=0.01), and mPAP (r=0.44, P<0.01). There were no significant relationships between measures of pulmonary pressures and PaCO2. Remaining correlations between pulmonary pressures and anatomical or physiologic metrics of ventilatory control were negligible in both groups (P>0.05).

Conclusion

These data suggest pulmonary pressures play a role in dead space ventilation during exercise in HF. Moreover, as suggested by the A‐a gradient, augmented pulmonary pressures may contribute to an exacerbation of ventilation/perfusion mismatch during exercise in both HFrEF and HFpEF patients.

Support or Funding Information

None
CO2 Sensitivity of the Ventilatory Response to Muscle Metaboreflex Stimulation in Humans
Authors: White Michael , AlGaith Jassim , Balanos George
Published: November 2023

The role of skeletal muscle metaboreceptive afferents in human cardiorespiratory control is isolated by using a period of post exercise circulatory occlusion (PECO). Previously we showed that the ventilatory response to a period of PECO is augmented in humans during concurrent hypercapnia, which elevated end tidal pCO2 by ~10mmHg above resting. In addition we found that this augmentation did not depend on peripheral chemoreflex involvement or local muscle hypercapnia. This suggested an interaction between central chemoreflex and muscle metaboreflex activation. We now aim to further examine this interaction by determining the lower level of end tidal pCO2 elevation required to cause the sustained elevation of ventilation during PECO.

With local Ethical Committee approval, young healthy subjects performed 4 trials in which end tidal pCO2 was elevated by 1, 3, 7 or 10 mmHg above resting levels. In each trial subjects were seated in an isometric dynamometer designed to measure ankle plantar flexor force. Rest for 2 minutes in room air was followed by 15 minutes of exposure to one of the four levels of hypercapnia. During this hypercapnia phase, 5 minutes further rest was followed by 2 minutes of sustained isometric calf muscle contraction, at 50% of the previously determined maximal voluntary strength. Just prior to cessation of exercise, a cuff around the upper leg was inflated rapidly to 200mmHg. This remained inflated for 3 more minutes before deflation and a further 5 minutes of exposure to hypercapnia. Beat to beat blood pressure (Finapres) and heart rate (ECG) were measured throughout as was breath‐by‐breath ventilation (VE) and end tidal PCO2 using a computer controlled dynamic end tidal forcing system.

PECO maintained mean blood pressure significantly above baseline by ~15 mmHg in all trials, indicating a consistent and well controlled level of muscle chemoreflex activation. However, ventilation during PECO was only sustained above baseline levels when end tidal pCO2 was maintained at or above 3 mmHg.

These results suggest that metaboreflex activation plus this threshold level of end tidal CO2 elevation is required to cause the ventilatory response to PECO. This further supports the idea of a synergistic interaction between muscle afferent activation and central chemoreflex stimulation, resulting in additional respiratory neurone excitation and increased ventilation in man.
Opioid‐induced suppression of augmented breaths (‘sighs’) persists during extended morphine administration at sub‐analgesic dosage
Authors: Donkin Joshua D , Grzesek Kimberly , Gulick Cody J , Bernardo Magalie , Nickels Cooper R , Bell Harold James
Published: November 2023

Our previous work has demonstrated that acute morphine administration causes powerful suppression of augmented breaths (ABs) at the low‐to‐mid dosage range for analgesia in adult rats. The present study was performed to determine the extent to which this suppression of ABs persists in the course of prolonged morphine administration. We studied breathing non‐invasively in adult rats in 2 separate experiments: Exp 1: animals were monitored immediately before, and repeatedly following s.c. bolus injections of morphine, 4 mg/kg loading dose and maintenance doses of 2 mg/kg given each hour for 8 consecutive hours (n=8). Exp 2: Animals were monitored immediately before and again repeatedly across 10 consecutive days of morphine treatment self‐administered via oral dosing in drinking water (n=7). Appropriate sham/control experiments were performed using saline injections and normal drinking water. In Exp 1, morphine injection caused a potent suppression of ABs, observed immediately upon first injection (6.0 ± 2.9 vs 0.5 ± 0.5 ABs/15 min, pre vs. post), and persisting throughout the 8 hours of study (P<0.001). This occurred despite there being no depression of minute ventilation. The prevalence of ABs in the sham injection condition remained unchanged across the 8 hours of study. In Exp 2, morphine concentration in drinking water was titrated up to 0.4 mg/mL across days 1 to 5, and remained constant at 0.4 mg/mL through day 10. This concentration was chosen so as to avoid taste aversion and drinking abstinence commonly seen when attempting to use higher concentrations of morphine. Animals self‐administered an average of 33.1 ± 6.9 mg/day (1.4 mg/kg/hr) on day 7 and 36.6 ± 3.4 mg/kg/day (1.5 mg/kg/hr) on day 10. This relatively low dosage was sub‐analgesic in efficacy as confirmed by lack of prolongation of tail flick latency. Despite the low dosage, continuously administered morphine caused significantly fewer ABs to be expressed on day 10 of morphine treatment (3.3 ± 1.5 ABs/15 min) compared to either pre‐treatment (7.0 ± 1.8) or day 10 of the control (6.3 ± 1.8) condition. No depression of minute ventilation was observed across the 10 days of treatment. These results confirm that the opioid‐induced suppression of ABs occurs rapidly and persists across extended treatment of at least 10 days. Moreover, this side effect occurs even in the sub‐analgesic dose range in the absence of any depression of minute ventilation.

Support or Funding Information

This work was supported by research funds provided by the Central Michigan University College of Medicine, and an Early Career Grant awarded by the Office of Research and Sponsored Programs at Central Michigan University.
Activation of Neurons in the Intermediate and Medial Nucleus of Tractus Solitarius by Multiple Afferent Stimuli
Authors: Tsai Hsiu‐Wen , Davenport Paul W
Published: November 2023

Nucleus of tractus solitaries (NTS) and subjacent reticular formation has been demonstrated to receive central suprapontine inputs and peripheral respiratory afferent information from facial (VII), glossopharyngeal (IX) and vagus (X) nerves for the regulation of breathing pattern. It has been reported that electrical stimulation of phrenic nerve and application of respiratory occlusion in both anesthetized and conscious rats elicited c‐fos expression in the intermediate and medial NTS (imNTS). In the present study, we electrically stimulated media nerve in anesthetized rats and vagus nerve in anesthetized guinea pigs, and applied methacholine to induce bronchoconstriction in both anesthetized rats and guinea pigs. Depending on the specific stimulus, cFOS expression was found in the rostral, central, intermediate/medial, caudal and ventrolateral regions of the NTS. The imNTS was the only subdivision of the NTS that had cFOS expression elicited by all stimulus modalities. These results support the hypothesis of a brainstem sensory integration of multiple afferent modalities within an intrinsic neural network in the NTS and the imNTS as a candidate subdivision for afferent convergence and integration.
Adaptive Mechanisms Governing Tracheobronchial Cough: In Vivo Experiments and Model Simulations
Authors: Musselwhite M. Nicholas , Pitts Teresa E. , Morris Kendall F. , Shen Tabitha Y. , Davenport Paul W. , Bolser Donald C.
Published: November 2023

Repetitive coughing can be suppressed in a dose‐dependent manner in response to administration of cough suppressant drugs in anesthetized cats. Cough suppression by these drugs manifests as a reduction in cough number and expiratory motor drive. Cough phase durations and inspiratory motor drive are unaffected by these drugs in the cat. The mechanism by which antitussive drugs exert these effects is unknown. We speculated that depression of synaptic excitability at the first central synapse of cough receptor afferents could contribute to this effect. To test this hypothesis, we conducted simulations using a well‐established computational model of the peripheral afferents and the central circuit that is proposed to account for cough and breathing. Modulation of the conductance of the first central synapse and then the firing probability of cough receptors predicted inverse relationships between these parameters, and cough number and expiratory motor drive. However, these simulations also predicted that reductions in the magnitudes of these parameters would result in longer cough cycle times, with a greater impact of synaptic conductance on this relationship. These predictions are not consistent with the known action of antitussive drugs on the cough motor pattern. Similarities to the results of these simulations were observed between coordinated in vivo experiments in anesthetized spontaneously breathing cats. A repetitive cough protocol was conducted in which naïve animals were challenged with a sequence of trials in which the intrathoracic trachea was mechanically stimulated to elicit coughing. Cough number and expiratory motor drive decreased by approximately 50% over the course of 15–20 consecutive trials. Total cough cycle duration increased by 46% (p<0.02) accounting for the decreased number of coughs during the protocol. Significant prolongations of cough TI, E1, and E2 were also observed. The data support: (i) significant adaptive processes of naïve animals to a sequential cough stimulation protocol, (ii) a different phenotype of this adaptive process than that associated with cough suppressants, (iii) potential mechanisms for this adaptive process that could include reduced excitation of both peripheral afferents and their first synapse with a larger contribution of the central component to the adaptation.

Support or Funding Information

Supported by HL 103415, HL 111215, HL 109025
Frequency Dependent Inhibitory Mechanisms Controlling the Laryngeal Adductor Response (LAR)
Authors: Shen Tabitha Y. , Gestreau Christian , Musselwhite M. Nicholas , Rose Melanie J. , Pitts Teresa E. , Morris Kendall F. , Lindsey Bruce G. , Davenport Paul W. , Bolser Donald C.
Published: November 2023

Electrical stimulation of the superior laryngeal nerve (SLN) has been used to induce behaviors with airway protective components, such as the LAR. The LAR has both short (R1) and long latency (R2) peaks in response to stimulation, but the neuronal pathways that produce and modulate the LAR are not well understood. It has been reported that there exists a frequency‐dependent suppression of R2 for SLN stimulation frequencies above 2.5 Hz. The R1 can follow frequencies of electrical stimulation of the SLN approaching 200 Hz, suggesting a high fidelity of brainstem neuronal pathways, but to our knowledge there is no report on whether R1 exhibits frequency‐dependent depression. We hypothesized that the R1 would exhibit frequency‐dependent depression consistent with the existence of modulatory short latency inhibitory mechanisms. To test this hypothesis, we monitored EMG activity from the thyroarytenoid (TA) muscle and electrically stimulated the SLN unilaterally with a range of frequencies between 1 and 40 Hz. All frequencies employed induced both repetitive swallowing and LAR. The R1 peak of the LAR was time‐locked to each stimulus pulse (peak R1 latencies were 13.7±0.5 ms (5–10 Hz) and 14.2±0.6 ms (35–40 Hz)), and the R2 peak was not observed. Lower frequency stimulations (5–10 Hz) of the SLN elicited significantly higher LAR amplitudes (52.8 ±2.9 % max) than higher frequency stimulations (35–40 Hz; 36.8±3.2 % max). In other experiments, analysis of neuronal responses recorded in the ventral respiratory column revealed evidence of stimulus‐induced synchrony as well as disruption of concurrent functional relationships among respiratory‐modulated neurons. These observations suggest that more complex regulatory mechanisms exist in addition to frequency‐dependent depression of the LAR.

Support or Funding Information

Supported by NIH HL 103415, HL 111215, HL109025.
Effects of Two‐Hit Lipopolysaccharide (LPS) Administration on Basal Inspiratory Motor Activity in Spontaneously Breathing Adult Male C57BL/6 Mice
Authors: Wadolowski Robert M , Ahmad Mohammed S , Wang Minqi , Solomon Irene C
Published: November 2023

Ongoing studies designed to induce peripheral inflammation, and its accompanying neuroinflammation, have begun to examine various aspects of neural control of breathing. These studies have implemented either a systemic (ip) or intratracheal (IT) administration protocol using the bacterial endotoxin lipopolysaccharide (LPS) to produce models of sepsis and acute lung injury/acute respiratory distress syndrome (ALI/ARDS). In chronic lung disease, there is often a combination of systemic inflammation with an airway/lung‐mediated exacerbation; thus, a combined ip‐IT LPS administration protocol could better reflect chronic (lung/airway) disease states. Here, we implemented a ‘two‐hit’ LPS administration protocol in which systemic LPS (ip, 3 mg/kg) was administered ~24 hr prior to an IT LPS (0.5 mg/kg) injection, and examined the ventilatory behavior in both unanesthetized (recorded using whole body plethysmography) and urethane‐anesthetized (recorded as EMG activities from diaphragm (EMGDia) and genioglossus (EMGGG) muscles) spontaneously breathing adult male C57BL/6 mice at 2–6 hr post ‘two‐hit’ LPS administration. A two‐hit protocol using ip‐IT saline injections served as a control. We found that following ip‐IT LPS administration, (1) breathing frequency was initially increased (within ~2 hr post injections) after which it markedly declined by as much as ~50% of the pre‐LPS breathing rate, (2) inspiratory burst durations were minimally altered but expiratory durations were increased and highly variable, (3) the expiratory flow profile was altered to a pattern consistent with an obstructive disease phenotype, (4) a progressive alteration in breathing pattern that included periodic robust discharges in EMGGG as compared to EMGDia and periods of dysrhythmic breathing emerged, (5) the shape of the EMG bursts was altered with the peak in both EMGDia and EMGGG bursts being shifted to an earlier time point, and (6) the duration and location of EMGDia and EMGGG spectral activities were differentially affected, including an enhancement of MFO spectral power in a subset of mice. These findings indicate that ‘two‐hit’ administration of LPS not only markedly alters basal ventilatory behaviors, including inspiratory‐phase synchronization, but also may differentially affect the central neural mechanisms controlling diaphragm versus genioglossus muscle activities. Moreover, our data support using a ‘two‐hit’ administration approach as viable option for producing (chronic) lung disease and its accompanying neuroinflammation.
Influence of Two‐Hit Lipopolysaccharide (LPS) Administration on Acute Hypoxic Ventilatory Response (HVR) in Urethane‐Anesthetized Spontaneously Breathing Adult Male C57BL/6 Mice
Authors: Ahmad Mohammed S , Wadolowski Robert M , Wang Minqi , Solomon Irene C
Published: November 2023

In chronic lung/airway disease, there is often a combination of systemic inflammation with an airway/lung‐mediated exacerbation. Under these conditions, patients may experience bouts of acute hypoxia; however, to date, there is limited information regarding the influence of this inflammation paradigm, including its accompanying neuroinflammation, on the acute hypoxic ventilatory response (HVR). To begin to address this issue, we used a ‘two‐hit’ lipopolysaccharide (LPS) administration protocol in which systemic LPS (ip, 3 mg/kg) was administered ~24 hr prior to an IT LPS (0.5 mg/kg) injection in spontaneously breathing adult male C57BL/6 mice, and at 2–6 hr post ‘two‐hit’ LPS administration, we recorded EMG activities from diaphragm (EMGDia) and genioglossus (EMGGG) muscles under baseline conditions (40% O2 in a balance of N2), during an acute bout of hypoxia (HVR, 10% O2 for 90 s), and during recovery from hypoxia. A similar recording protocol was used in control experiments in which mice received two‐hit ip‐IT saline injections. We found that ip‐IT LPS administration not only altered basal inspiratory motor output, but markedly altered the acute HVR. Specifically, ‘two‐hit’ LPS administration blunted the hypoxia‐induced amplitude response, such that amplitude increased by ≤10% (versus 30–50% increase in control mice), and produced an exaggerated frequency response, such that frequency increased by ≥30% (versus~15–20% increase in control mice). In addition, in LPS treated mice in which a dysrhythmic breathing pattern was observed, hypoxia either worsened the dysrhythmia or improved it to yield a more normal (but higher frequency) rhythm. Finally, ‘two‐hit’ LPS administration slightly altered the EMG burst pattern and attenuated the reduction in inspiratory burst complexity (as reflected by a decrease in the approximate entropy (ApEn) value) when compared to changes observed in control mice. During recovery from hypoxia, the inspiratory motor activity in both LPS treated and control mice returned toward baseline levels. These findings indicate that ‘two‐hit’ administration of LPS significantly alters the acute HVR, and further support the use of a ‘two‐hit’ administration approach for studying the role of lung/airway inflammation and its accompanying neuroinflammation in central respiratory control, including the chemical drive to breath.
Differential Effects of Systemic versus Intratracheal Lipopolysaccharide (LPS) Administration on Inspiratory Motor Output in Anesthetized Spontaneously Breathing Sprague‐Dawley Rat
Authors: Wang Minqi , Wadolowski Robert M , Ahmad Mohammed S , Solomon Irene C
Published: November 2023

Both systemic (ip) and intratracheal (IT) administration of the bacterial endotoxin lipopolysaccharide (LPS) have been used independently to elicit neuroinflammation. While both methods are reported to induce time‐dependent changes in expression levels of pro‐inflammatory cytokines in the CNS, their effects on various aspects of neural control of breathing are only beginning to be appreciated. Here, we characterize the effects of LPS delivered systemically (3 mg/kg, ip) or IT (0.5 mg/kg) in urethane‐anesthetized spontaneously breathing Sprague‐Dawley rats on basal inspiratory motor output (recorded as EMG activities from diaphragm (EMGDia) and genioglossus (EMGGG) muscles), inspiratory‐phase synchronization (evaluated as time‐varying spectral activity), and the acute hypoxic ventilatory response (HVR, 10% O2 for 90 s) at 2–4 hr post administration. We found that both ip and IT LPS altered basal inspiratory motor activity and the HVR; however, the effects were not identical. While both ip and IT LPS increased breathing frequency, the changes in both burst duration (TI) and the time between bursts (TE) were highly variable following ip LPS. In addition, while both ip and IT LPS blunted the HVR, the hypoxia‐induced increase in breathing frequency was greater for ip LPS than for IT LPS. Finally, while both ip and IT LPS altered inspiratory‐phase spectral activities, ip LPS evoked a marked increase in MFO power while IT LPS shifted spectral activities to earlier in the inspiratory burst. These findings indicate that while both methods of administration alter ventilatory activity, the resulting ventilatory patterns appear to be markedly different. We suggest that differences in the extent of ip versus IT LPS‐induced neuroinflammatory changes in respiratory‐related brainstem and spinal cord regions mediate, at least in part, these differences in inspiratory motor behavior.
Ventilatory Acclimatization to Hypoxia Requires Early Microglia Activation
Authors: Stokes Jennifer Ann , Arbogast Tara Elizabeth , Powell Frank L
Published: November 2023

Ventilatory acclimatization to hypoxia (VAH) is the time‐dependent increase in ventilation, which persists upon return to normoxia, and involves plasticity in central nervous system (CNS) respiratory centers and peripheral chemoreceptors. Plasticity within the CNS increases ventilatory motor output for a given chemoreceptor input and the mechanisms of such plasticity in the CNS are unknown. It is clear from the literature that glial cells influence neuronal transmission and central respiratory control. Previously we showed a role for microglia in VAH (Stokes JA et al., 2015), where pharmacological blockade of microglia by minocycline inhibited VAH during a 7‐day chronic sustained hypoxia (CSH) exposure (10% O2). Building upon this work, we investigated the effect of timing of microglia inhibition on VAH. In this study rats were exposed to 7 days of CSH (10% O2) and administered minocycline (45mg/kg i.p.) in one of two regimes: 1) Early minocycline – minocycline days 0–3 of hypoxia exposure and saline days 4–7; 2) Late minocycline – saline days 0–3 of hypoxia and minocycline days 4–7. Ventilation was recorded using whole‐body plethysmography on day 4 (before treatment change) and on day 7, after final hypoxic day. Early minocycline administration initially blocked VAH on day 4, but after saline administration during the final days of hypoxia VAH was observed on day 7. Late minocycline administration was not able to reverse the VAH, which was observed on day 4 with early saline administration. These results show that microglial activation is required for the initial plasticity involved in acclimatization but once acclimatization is established, blocking microglia activation does not reverse the adaptation. To investigate possible mechanisms, we used RT‐PCR to assess cytokine production in the nucleus tractus solitarius (NTS) following initial hypoxia exposure, as activated microglia release cytokines which activate neighboring glial cells and subsequently neurons. After 24 hours of hypoxia, Il‐6 and TNF‐α mRNA expression increased in the NTS and this increase was blocked by minocycline. Taken together these results support a model for microglia activation and release of cytokines early in sustained hypoxia that induce neural plasticity in VAH, which is generally assumed to be a healthy response to hypoxia.

Support or Funding Information

NIH RO1 HL‐081823 (FLP) and T32 5T32HL098062‐04 (JAS)
Lipopolysaccharide‐induced IL‐6 and TNF‐alpha expression in neonatal rat nucleus tractus solitarii (NTS) in vivo
Authors: Johnson Rhaya , Murray Samuel , Wilson Christopher
Published: November 2023

Systemic infection is a multi‐billion dollar healthcare burden in the United States. Pre‐term infants are at high risk for respiratory tract infection which can occur due to intrauterine inflammation (choriamnionitis), during normal vaginal birth, or before innate immunity is fully developed. Airway infection results in immune up‐regulation, including production and release of the early pro‐inflammatory cytokines interleukin‐1‐beta (IL‐1beta), interleukin‐6 (IL‐6) and tumor necrosis factor‐alpha (TNFa). Our laboratory has previously shown that LPS injected into the trachea of neonatal rats causes rapid increases in IL‐1beta expression in the autonomic brainstem regions responsible for the control of breathing including: nucleus tractus solitarii (NTS) and hypoglossal motonucleus (XII). These changes occur within a few hours and are correlated with changes in breathing pattern. Here we test the hypothesis that IL‐6 and TNFa are up‐regulated in NTS and XII in response to low doses of LPS (0.5 mg/kg) instilled in the trachea of neonatal rats (postnatal days 10 to 12) within two hours post treatment. We used immunohistochemistry and unbiased stereology to quantify changes in IL‐6 and TNFa in NTS and XII. Our preliminary results show that IL‐6 increased significantly (32%, p < 0.05) when compared to sham‐operated animals and this increase could be arrested by vagal nerve stimulation (25 KHz, 30 min). TNFa also showed increased expression when compared to sham animals. Our results show that cytokine production occurs extremely rapidly in the brainstem after LPS exposure in the periphery suggesting rapid transduction of the inflammatory signal.
An Evaluation of the Cytotoxic and Pro‐inflammatory Effects of Various E‐cigarette Liquid Flavors on Monocytes and Macrophages
Authors: Stanley Corshe D. , Clapp Phillip W. , Jones Shannon Z. , Jaspers Ilona
Published: November 2023

The usage of electronic cigarettes is becoming very popular in today's society. Although the popularity of these products is increasing, they are not currently regulated by the Food and Drug Administration and the long term health effects are still unknown. The objective of the present study was to identify the potential cytotoxic and inflammatory effects of various flavors of E‐cigarette liquids in THP‐1 monocytes and alveolar macrophages. THP‐1 cells and alveolar macrophages were used as experimental models because these cells are important components of our immune system and first line of defense against infections, especially in the lung. Alveolar macrophages are the most predominant resident immune cell in the healthy human lung. Macrophages and other immune cells use cytokines to communicate with other components of the immune system and to signal the onset of infections. In this project we challenged macrophages/THP‐1 cells with seven different flavors of e‐cigarette liquids. The cells were harvested 24 hours post‐challenge and examined for the secretion of IL‐6 and IL‐8 as markers of inflammation and immune responses. We also calculated cell viability following challenge, using trypan blue exclusion. Our hypothesis was that E‐cigarette liquids are pro‐inflammatory and potentially cytotoxic and thus potentially adversely affecting the ability of these cells to function properly. Our data suggest that of the seven E‐cigarette liquids tested, two induced significant cytotoxicity in THP‐1 monocytes, while one of the flavors had no effect on cytokine secretion or cell viability. It is important to note that the two most cytotoxic liquids contained the cinnamon‐flavoring chemical cinnamaldehyde. These results suggest that, individually, E‐cigarette liquids may contribute to inflammatory and cytotoxic responses.

Support or Funding Information

UNC TCORS grant (5‐33779)
Compromised Lung Mechanics in Cigarette Smoke Exposed Mice with Inhibited Pulmonary VEGF Expression
Authors: Lee Jasmine H. , Fine Janelle , Gutierrez Alma , Witte Tyler , Wong Fiona , Bigby Timothy , Breen Ellen C.
Published: November 2023

Cigarette smoke (CS) exposure is well known to lead to progressive lung destruction that is characterized by enlarged airspaces and a decrease in lung elastic recoil. We hypothesized that VEGF expression by airway cells may protect against the early onset of excessive lung damage. Adult VEGFLoxP mice (5 months of age) were exposed to daily periods (5 days/week) of “nose‐only” CS or remained in room air (RA) for 4 months. Half of the mice in each exposure group were instilled one month earlier with AAV‐Cre to delete the VEGF gene in airway cells or a control virus (AAV‐LacZ). An additional group of VEGF promoter/luciferase reporter mice were exposed to CS to monitor VEGF transcriptional activation by in vivo optical imaging. Static lung mechanics were characterized from pressure‐volume curves and oxygen saturation levels were measured in awake mice breathing 21% O2 or 10% O2 by a pulse oximeter. VEGF transcriptional activity in the lungs peaked at 7 days and returned towards baseline level by 14 days (Day 0, 1.14e7 ± 2.4e6 relative light units (RLU); Day 1, 3.46e7 ± 9.5e6 RLU, Day 7, 7.40e7 ± 1.9e7 RLU, Day 14, 3.10e7 ± 5.4e6 RLU, p=0.055). There was a progressive left shift and increased hysteresis of the P‐V relationship in mice from the RA‐LacZ < CS‐LacZ < RA‐Cre < CS‐Cre groups. At an average airway pressure of 20 cmH2O, lung volumes were 0.9 ml RA‐LacZ group, 1.0 ml CS‐LacZ group, 1.1 ml RA‐Cre group, and 1.2 ml CS‐Cre group. O2 saturation levels were not different among groups when breathing RA. In mice challenged with 10% O2, the oxygen saturation levels were lowest in the CS‐Cre group (RA‐LacZ, 71.0 ± 1.8%; CS‐LacZ, 75.2 ± 4.9%; RA‐Cre, 70.4 ± 3.9%; CS‐Cre, 62.6 ± 3.5%, p<0.05). These data suggest that airway epithelial VEGF is vital for maintaining lung function and when deleted from the airway, obstructive physiology develops. This pattern is further exacerbated by exposure to chronic cigarette smoke.

Support or Funding Information

Department of Veterans Affairs
Heparan Sulfate is Shed into the Alveolus after Intratracheal Lung Injury and Facilitates Alveolar Epithelial Growth Factor Signaling and Lung Repair
Authors: Haeger Sarah , Yang Yimu , Zemans Rachel , Suflita Matt , Zhang Fuming , Linhardt Robert , Tuder Rubin , Schmidt Eric
Published: November 2023

Heparan sulfate proteoglycans (HSPGs) are proteins covalently bound to heparan sulfate (HS), a linear highly‐sulfated polysaccharide, and are present in both the extracellular matrix and on cell surfaces. During tissue injury, HSPGs are thought to be shed from the injured cell surface; for example, the protein component of the cell membrane‐bound HSPG, syndecan‐1, has been shown to be shed from the respiratory epithelial cell surface into the airway during naphthalene lung injury. However, it remains unknown whether heparan sulfate (HS) is also shed into the airspace during intratracheal (IT) lung injury. Additionally, as HS is known to function as a scaffolding molecule for numerous growth factor ligands and receptors, facilitating ligand‐receptor binding and enhancing signaling, it is important to determine if HS is shed into the airspace during lung injury, as the shed HS may affect growth factor signaling and lung repair.

To determine if HS is shed into the airspace during lung injury, we intratracheally instilled lipopolysaccharide (LPS) in mice and performed mass spectrometry for HS in bronchoalveolar (BAL) fluid collected from mice days 2, 4, 6, and 8 after IT LPS instillation. We detected increased BAL HS on days 2 (2.3 fold) and 4 (3.2 fold) after IT LPS instillation in comparison to animals instilled with PBS as control. To determine if the airspace HS enhances lung epithelial growth factor signaling and repair, we tested if exogenous HS enhances ERK signaling in alveolar type II (ATII) epithelial cells, progenitor cells that re‐epithelialize the alveolus after injury. We treated MLE‐12 cells (a mouse ATII cell line) with 0.5μg/ml HS and determined that, while HS inhibits ERK signaling in naïve MLE‐12 cells, HS permits ERK signaling in MLE‐12 cells that are enzymatically devoid of cell‐surface HS. This finding suggests that airspace HS may facilitate growth factor signaling in ATII cells that have shed their cell‐surface HS during injury. Finally, to determine if supplementation with exogenous HS enhances lung repair in vivo, we treated animals with intravenous HS on day 4 after IT LPS instillation and measured outcomes of lung injury. Twenty‐four hours after supplementation, HS reduced BAL total protein by 30%, indicating that exogenous HS supplementation may enhance alveolar epithelial barrier function and lung repair.

Support or Funding Information

NHLBI RO1 HL125371
Resident Lung Macrophages Are Sparsely Amongst Alveoli
Authors: Li Li , Gusarova Galina A , Bhattacharya Jahar
Published: November 2023

Lung macrophages are major determinants of pro‐ and anti‐inflammatory mechanisms underlying multiple lung diseases, including Acute Lung Injury (ALI). Our recent findings in optically imaged mouse lungs indicate that resident macrophages in alveoli adhere to the alveolar epithelium (Nature 2014). We called these cells sessile alveolar macrophages (SAMs) to distinguish them from non‐adherent macrophages that can be recovered in the bronchoalveolar lavage. Here we report new studies in which we determined distribution of SAMs by lung histology. We anesthetized mice, then exsanguinated them by cardiac puncture (n=3). Through a tracheal cannula we instilled tissue embedding compound (OCT, R&D Systems) diluted 1:1 in PBS to inflate the lungs at 25 cmH2O. After 20 min, we isolated the lungs, froze the lungs in dry ice, then stored the lungs overnight at −80°C. We obtained tissue blocks that we sectioned in a tissue microtome to obtain 8 micron sections at 100–200 microns below the pleural surface. For immunohistochemistry, we incubated the sections at 4°C overnight with primary antibodies against the lung macrophage markers, CD11c and Siglec‐F, and the primary antibody against the alveolar wall marker, connexin 43 (Cx43). Then after washing the sections, we further exposed them to secondary antibodies tagged with different fluorescent markers. We viewed the sections by fluorescence microscopy. We identified alveolar walls as Cx43 expressing cells (green), and SAMs as cells co‐expressing CD11c (cyan) and Siglec‐F (red). In each lung, we counted the number of SAMs in 100 alveoli in 7–8 imaging fields. Normalized for a single imaging field, our counts indicated that 5±2 SAMs were present in 14±5 alveoli, indicating that SAMs were distributed as approximately 1 per 3 alveoli. We conclude, SAMs are sparsely distributed in the parenchyma, suggesting that SAMs employ communicative strategies for pathogen detection in macrophage‐free alveoli (Support: HL57556).

Support or Funding Information

Support: HL57556
Exacerbated Headache‐Related Pain in the Single Prolonged Stress Model of Post‐traumatic Stress Disorder
Authors: Standifer Kelly M. , Simpson‐Durand Cindy , Zhang Yong
Published: November 2023

Post‐traumatic stress disorder (PTSD) frequently shares co‐morbidity with chronic pain and negatively impacts the course of treatment for both disorders. Single‐prolonged stress (SPS), an established animal model for PTSD, models many of the symptoms of PTSD reported in humans including exaggerated negative feedback of the HPA axis, hypocortisolism, enhanced fear and anxiety responses and cognitive impairment. We and others reported that SPS induces long‐lasting mechanical and thermal allodynia and visceral and inflammatory hypersensitivity. Chronic headache pain is one of the most commonly reported co‐morbid pain conditions by PTSD patients and one of the most resistant to effective treatment, yet this has never been modeled in an animal model of PTSD. We modified the chronic headache pain model recently developed by Pradhan and colleagues (2014) to investigate the contribution of SPS to sodium nitroprusside (SNP)‐induced hyperalgesia. SPS and control Sprague Dawley rats were randomly divided into SNP or vehicle‐treated groups. SNP (2 mg/kg, i.p.) was injected every second day from day 7 to 15 after initiation of SPS. Rat paw withdrawal threshold (PWT) to von Frey stimuli and tail flick latencies (TFL) dramatically decreased as early as 7 days after SPS and lasted until at least day 21. Basal PWT and TFL, as assessed prior to each SNP administration, also significantly decreased during the SNP treatment period. The lower pain thresholds recovered 7 days following the final SNP injection in SNP group, but not in SPS/SNP group. Nociceptin/orphanin FQ (N/OFQ) plays an important role in maintenance of hyperalgesia and allodynia following SPS. Elevated N/OFQ levels observed in cerebrospinal fluid (CSF) of SPS rats were even higher in CSF of the SPS/SNP group. Real‐time PCR revealed that glial fibrillary acidic protein (GFAP) and N/OFQ peptide (NOP) receptor mRNA expression increased in dorsal root ganglia (DRG) 21 days after SPS exposure; mRNA increases in the SPS/SNP group was more pronounced than SPS or SNP alone. PreproN/OFQ mRNA expression decreased in both SPS and SPS/SNP rats compared to control, but no difference was observed between the two groups. SNP treatment alone did not alter GFAP, NOP receptor or N/OFQ mRNA levels in DRG. Significant interaction between SNP and SPS was noted following immunoblot analysis of TNF alpha protein expression in DRG and NOP receptor expression in trigeminal ganglia. Our results indicate that chronic SNP‐induced nociceptive hypersensitivity (headache pain) is exaggerated in rats exposed to PTSD, and that N/OFQ may play an important role in the interaction between SPS and SNP.

The animal protocol was approved by the OUHSC IACUC committee; studies conformed to the FASEB Statement of Principles for the use of animals in research and education.

Support or Funding Information

University of Oklahoma Health Sciences Center Vice President for Research and Presbyterian Health Foundation Bridge Grant Programs.
Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis
Authors: Tsvetanova Nikoleta , von Zastrow Mark
Published: November 2023

G protein‐coupled receptors (GPCRs) comprise the largest family of signaling receptors and a very important class of therapeutic targets. GPCRs are well known to signal via cyclic AMP (cAMP) production at the plasma membrane, but it is now clear that various receptors also signal after internalization. This endosome‐derived signal is already known to modify the temporal profile of downstream responsiveness but it remains unknown, particularly for signals transduced by diffusible second messengers, if there is any functional significance to the spatial segregation of initiation sites. Using the beta2‐adrenoceptor (β2‐AR) as a model, we show that endocytosis is required for the full repertoire of downstream cAMP‐dependent transcriptional control. Next, we describe an orthogonal optogenetic approach to definitively establish that the location of cAMP production is indeed the critical variable determining the transcriptional response. Further, we demonstrate that the endocytosis‐dependent transcriptional signal is highly sensitive and saturable, effectively limiting the downstream cellular response to a uniform level. These properties allow the endosome to function as a ‘noise filter’, reducing variability of the downstream response and thus enhancing reliability of cognate ligand detection. In summary, our findings reveal a discrete principle for achieving cellular signaling specificity, based on endosome‐mediated spatial encoding of intracellular second messenger production and ‘location aware’ downstream transcriptional control.

Support or Funding Information

National Institute on Drug Abuse of the NIH; American Heart Association Postdoctoral Fellowship
Ubiquitin plays an atypical role in GPCR‐induced p38 MAP kinase activation on endosomes
Authors: Grimsey Neil , Narala Rachan , Aguilar Berenice , Rada Cara , Smith Thomas , Soohoo Amanda , Puthenveedu manojkumar , Nizet Victor , Trejo JoAnn J
Published: November 2023

Protease‐activated receptor 1 (PAR1) is a G protein–coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen‐activated protein kinase signaling. The mechanisms that govern PAR1‐induced p38 activation remain unclear. Here, we define an atypical ubiquitin‐dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63‐linked ubiquitination is mediated by the NEDD4‐2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor‐β–activated protein kinase‐1 binding protein‐2 (TAB2). The ubiquitin‐binding domain of TAB2 was essential for recruitment to PAR1‐containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y1 purinergic GPCR also stimulated p38 activation via NEDD4‐2–mediated ubiquitination and TAB1–TAB2. TAB1–TAB2‐dependent p38 activation was critical for PAR1‐promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1‐induced vascular leakage in vivo. These studies define an atypical ubiquitin mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption.
Methamphetamine‐Induced Ubiquitination and Trafficking of Dopamine Transporters
Authors: Hinshaw Tyler , Sun Haiguo , Leussen Deborah , Chen Rong
Published: November 2023

Introduction

The dopamine transporter (DAT) is the primary terminator of synaptic dopamine transmission by reuptake of dopamine, and is a major target for psychostimulants including cocaine and methamphetamine. DAT undergoes constitutive and stimulated endocytosis and postendocytic trafficking. It has been shown that psychostimulants cause aberrant trafficking of DAT although the mechanisms remain elusive. Posttranslational modification of transporters by ubiquitin conjugation has recently emerged as a major regulatory mechanism of internalization and intracellular sorting of transporters and receptors. However, it is unknown whether psychostimulants alter ubiquitination of DAT. Moreover, recent evidence indicates that membrane rafts play an important role in DAT trafficking and activity. Thus, the purpose of this study is to investigate the consequence of neurotoxic treatment of methamphetamine on DAT ubiquitination and localization in lipid rafts.

Methods

Male Sprague Dawley rats were treated with methamphetamine (7.5 mg/kg, IP, every 2 hrs for a total of 4 injections) or saline. This treatment regimen is known to induce hyperthermia which produces neurotoxicity. To prevent the effect of hyperthermia on DAT function, rats were cooled following each methamphetamine injection. Animals were sacrificed two or ten days after the treatment. Striatal tissues were immunoprecipitated with specific DAT antibody and immunoblotted with ubiquitin antibody. The localizations of striatal DAT in lipid rafts were examined using discontinuous sucrose gradient.

Results

Our data showed that neurotoxic doses of methamphetamine significantly enhanced DAT ubiquitination only two days after the treatment, suggesting a transient effect of methamphetamine on DAT posttranslational modification. The heightened DAT ubiquitination was not observed after 10 days, which indicate DAT ubiquitination is reversible. We also examined the changes of DAT localization in lipid rafts and non‐lipid rafts. Methamphetamine‐induced alterations in DAT ubiquitination and translocation in lipid rafts may be associated with impaired DAT activity.

Support or Funding Information

This project is supported by the ASPET Zannoni Summer Undergraduate Research Fellow (SURF) Individual Awards and NIH DA006634.
Regulation of α2‐Adrenergic Receptor Cell Surface Transport by Spliced GGA Variants
Authors: Zhang Maoxiang , Huang Wei , Davis Jason , Wu Guangyu
Published: November 2023

Golgi‐localized, γ‐adaptin ear domain homology, ADP ribosylation factor‐binding proteins (GGAs) have been well characterized to function as adaptor proteins for clathrin‐coated vesicles and are involved in the sorting of cargo proteins at the trans‐Golgi network (TGN) into the endosomal/lysosomal pathway. We have recently reported that GGAs are involved in the anterograde transport of α2B‐adrenergic receptor (α2B‐AR), a prototypic G protein‐coupled receptor. Here we have determined the role of several highly expressed GGA variants in the cell surface targeting of α2B‐AR and many other cargos. We demonstrated that the naturally occurring truncated form of GGA1 (GGA1t) lacking the N‐terminal domain had normal intracellular localization and formed homodimers and heterodimers with other GGAs. Interestingly, overexpression of GGA1t inhibited the cell surface export of α2B‐AR and arrested the receptor in the Golgi compartment. We also found that, in marked contrast to its wild‐type counterpart, GGA1t was unable to bind to the cargo α2B‐AR and to recruit clathrin onto the TGN. In addition, enhanced GGA1t expression also blocked the trafficking of CI‐MPR, a well‐known cargo that transports via the GGA‐mediated pathway. These data provide evidence implicating that the truncated form of GGA1 behaviors as a dominant‐negative regulator for the cell surface export of α2B‐AR which is mediated via multiple mechanisms.