bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects Of 5‐HT2A And 5‐HT7 Receptors In The RTN Region On Breathing During Eupnea And Hypercapnia
Authors: Muere Clarissa , Hodges Matthew
Published: November 2023

Raphé‐derived neuromodulators, such as serotonin (5‐HT), increase the excitability of many neurons, including Phox2b‐expressing retrotrapezoid nucleus (RTN) neurons. Here, we sought to test the hypothesis that unilateral blockade of 5‐HT receptors in the RTN region will decrease ventilation at rest and during hypercapnia. We chronically implanted guide cannulae targeting the RTN in Sprague‐Dawley (SD, n=2) and Dahl salt‐sensitive (SS, n=2) rats. After >7 days recovery, we inserted a probe for reverse microdialysis of mock cerebrospinal fluid (mCSF) and serotonergic receptor antagonists, while measuring breathing in room air and in 7% CO2 in unrestrained, conscious animals. Preliminary results show that dialysis of the 5‐HT2A receptor antagonist, MDL 11,939 (20 μM), consistently reduced eupneic (−19 ± 2%) and CO2 breathing (−29 ± 5%) compared with control conditions (dialysis of mCSF alone) in SS rats. Furthermore, dialysis of MDL 11,939 consistently reduced eupneic breathing (−37 ± 16%) and had mixed effects on CO2 breathing (−20 ± 25%) in SD rats. Dialysis of the 5‐HT7 receptor antagonist, SB258719 (100 μM), reduced eupneic breathing (−11.2 to −43%) and enhanced the CO2 chemoreflex (+65 to +248%) in one rat from each strain. Combined dialysis of MDL 11,939 and SB258719 in these same rats, however, reduced eupneic (−34 ± 8%) and CO2 breathing (−26 ± 10%) compared with control conditions. Thus, unilateral blockade of 5‐HT2A receptors, 5‐HT7 receptors, or both consistently decreases eupneic ventilation, but has mixed effects on the CO2 chemoreflex. These data suggest that 5‐HT2A and 5‐HT7 receptors in the RTN may modulate breathing at rest and during hypercapnia in vivo.

Support or Funding Information

This study was supported by National Institutes of Health grant HL122358.
Orexin 1 Receptors Are necessary for Phase‐Specific Enhancement of The Hypercapnic Ventilatory Response in Female Rats Subjected to Neonatal Stress
Authors: Lopes Luana Tenorio , Gulemetova Roumiana , Drolet Guy , Bretzner Frédéric , Kinkead Richard
Published: November 2023

Panic disorder (PD) is an anxiety disorder that affects ~3 times more women than men. Respiratory manifestations of PD appear after puberty and include an increased sensitivity to CO2. This observation suggests that ovarian function contributes to the onset of PD. In humans and rodents, exposure to stress during early life alters brain development and is considerate a risk factor for PD. Using Neonatal Maternal Separation (NMS) as a stressor, we showed that NMS augments the ventilatory response to CO2 in adult female rats but these effects are not present in pre‐pubertal rats. Based on these observations we first tested the hypothesis that cyclic hormone fluctuations influence chemosensitivity. In addition to their involvement in fear and vigilance responses, orexin neurons (ORX) are CO2 sensitive. Based on these properties, ORX has been linked to PD. Thus, we also tested the hypothesis that ORX receptors are necessary for stress‐related enhancement of the CO2 response.

Pups subjected to NMS were placed in an incubator for 3 h/day from postnatal day 3 to 12. Control pups remained undisturbed. Female rats were reared until adulthood (P60–70), and the ventilatory response to CO2 was measured by whole‐body plethysmography (FICO2 = 0.05, for 10 min). OX1 antagonist (SB334867; 15mg/kg) or vehicle (ip) was injected 45 to 60 minutes before start the recordings. After the experiment, the oestrus cycle was verified by vaginal smear.

In controls, the ventilatory response to CO2 did not change across the estrus cycle. The hypercapnic ventilatory response of NMS females was phase‐dependent with the greatest responsiveness observed during proestrus. Pharmacological inactivation of OX1 receptors had not effect on the hypercapnic response of controls, but reduces the respiratory response to CO2 in NMS females, the most important effects were observed during proestrus and metaestrus phases.

Based on the phase‐dependent increase in NMS females, we conclude that stress interferes with the influence that sex‐hormones exert on respiratory control. These effects may take place in the hypothalamic region that contain ORX neurons since activation of OX1 receptors are necessary for stress‐related enhancement of the hypercapnic ventilatory response. These results provide new insight into the pathophysiology of PD.

Support or Funding Information

Canadian Institutes of Health Research and CNPq ‐ Brazil.
Chronic Exposure to Elevated Ambient CO2 Influences the Orexinergic System in the Lateral Hypothalamus and Dorsocaudal Medulla
Authors: Sherwood Jacob William , Beard Richard S , Landon Carol S , Dean Jay B
Published: November 2023

Central chemoreceptors (CCRs) of the caudal solitary complex (cSC) are activated by hypercapnic acidosis (HA). CCRs also receive input from orexinergic projections originating in the lateral hypothalamus (LH). Indeed both HA and the neuropeptides orexin A&B stimulate ventilation and gastric acid secretion (Dean 2011 Resp. Physiol. & Neurobiol. 175:189–209). Acute exposure to severe hypercapnia (Wang et al. 2013 Respirology 18:102–107) decreases orexins and increases orexin‐1 receptor (OX1) expression in the hypothalamus of mice while OX1 expression in the brain stem remains unchanged. We hypothesized that chronic CO2 retention (e.g., disabled submarines, underventilated workspaces, hypoventilation syndromes) causes changes in the orexinergic system. Given orexins’ roles in arousal, feeding, energy consumption, and respiration‐among others, this change could impair health and well being in CO2 retainers. Male Sprague‐Dawley rats (~250–300g) were placed in a Biospherix chronic CO2 environmental chamber (66”W×20”D×20”H) equipped with two controllers capable of regulating ambient CO2 at 0.1–4.0% CO2 in air. Rats were placed in the box for 1–4 weeks and fed ad libitum. Chamber humidity and temperature were recorded using the HOBO data logger and ambient CO2 was recorded using Lab Scribe (iWorx). Prior to harvesting brains, rats were transcardially perfused with tomato lectin before perfusion with fixative. Coronal sections representing the LH and cSC were stained with anti‐OX1 and GFAP antibodies and examined using confocal microscopy. Rats not perfused with fixative had hypothalamus and brainstem removed for qPCR analysis. To date, we have tested 6 rats in 2% CO2 and 16 rats in 1% CO2 in air for 1–4 weeks. Confocal micrographs of the LH and cSC show a marked increase in neurons expressing OX1 in both regions as early as one week at 2% CO2. Interestingly, we also saw a sensitization of astrocytes to orexin‐A indicated by an induction of OX1 expression. Additionally, analysis of qPCR data show marked changes in both neuropeptide and receptor expression in both regions that trend toward normalization at 4 weeks. In conclusion, our findings indicate that chronic exposure to hypercapnia changes expression of both orexins and their receptors in the LH and cSC. These changes presumably modify systems activated by orexins. This includes respiration during sleep and wakefulness; gastric acid secretion and feeding; and even astrocyte activation (Support provided by the ONR Undersea Medicine Program).
Role of central serotonin in sleep architecture and arousal in infant rat pups
Authors: Young Jacob , Hodges Mathew , Cummings Kevin
Published: November 2023

Sudden Infant Death Syndrome (SIDS) occurs during sleep and is the leading cause of infant death in the post‐neonatal period. SIDS cases have more active sleep (AS), a sleep state associated with apnea and cardiovascular instabilities. It is also thought that SIDS involves a failure to arouse from an apneic event during sleep. Defects within the brainstem serotonin (5‐hydroxytryptamine, 5‐HT) system (including a loss of 5‐HT content) are associated with SIDS, and a causal role 5‐HT deficiency is suggested by recent studies using rodents deficient in central 5‐HT. However, how a specific loss of central 5‐HT in neonatal life affects sleep, respiratory control across sleep states and arousal from sleep has not been investigated. We hypothesized that 5‐HT deficiency increases the amount of active sleep (AS), compromises breathing mostly during quiet sleep (QS) when 5‐HT neurons still fire, and blunts the arousal response to hypercapnia. We monitored sleep and breathing over ~2hrs in two groups of unanaesthetized, 2 week‐old rat pups deficient in tryptophan hydroxylase‐2 (TPH2−/−) and wild‐type controls (WT). One group (n=6 WT, 5 TPH2−/−) was kept in normoxic conditions and the other group (n=10 WT, 8 TPH2−/−) was given intermittent challenges of 5% CO2 at the beginning of episodes of AS and QS until arousal. Sleep state was determined using nuchal electromyography and behavioral observation. Breathing was monitored using whole body plethysmography. We measured the duration of AS and QS episodes (i.e. latency to arousal) in normocapnia and hypercapnia, and in normocapnia measured respiratory variables in each sleep state: frequency (fB), tidal volume (VT), ventilation (VE) and the co‐efficient of variation of the respiratory period (CV%). While the duration of QS episodes was not influenced by 5‐HT deficiency, TPH2−/− pups experienced episodes of AS that were ~2‐times longer than those of controls (~190 vs 90 sec; p=0.003). In AS, hypercapnia had no effect on the latency to arousal of either WT or TPH2−/− pups. In QS, hypercapnia reduced the arousal latencies of WT pups (from ~108 sec to 72 sec), but had no influence on the latencies of TPH2−/− pups (state × gas × genotype: p=0.02). 5‐HT deficiency significantly reduced fB and VE in both sleep states (p<0.001 for both). Breathing stability was significantly compromised by 5‐HT deficiency, but only in AS (genotype × state: p<0.001). We conclude that at an age close to infancy, central 5‐HT deficiency increases the duration of AS episodes, destabilizes breathing during AS, and delays arousal from hypercapnia during QS. These findings lend support the hypothesis that reduced 5‐HT signaling alters sleep architecture and breathing in a way that increases SIDS risk.

Support or Funding Information

Funding for this research was provided by the American Physiological Society Undergraduate Student Research Fellowship Program (to J.Y.) and by an American Heart Association Scientist Development Grant (14SDG18560022; PI: KJC).
Role of central serotonin (5‐HT) in sleep architecture and the control of breathing during sleep in adult rats
Authors: Magnusson Jennifer , Li Aihua , Hodges Mathew , Cummings Kevin
Published: November 2023

5‐HT neurons have a relatively high firing frequency during wakefulness (QW), a reduced firing rate during quiet sleep (SWS), and are essentially quiescent during rapid eye movement sleep (REMS). Studies have shown that mice lacking tryptophan hydroxylase 2 (TPH2), the rate limiting enzyme in central 5‐HT synthesis, have a reduced arterial blood pressure (BP) and breathing frequency (fB). However, the specific role of brainstem 5‐HT in sleep architecture and cardiorespiratory regulation across sleep states is unclear. We hypothesized that, relative to control littermates, TPH2‐deficient adult rats (TPH2−/−) would have increased sleep, and that during QW and QS they would have reduced arterial blood pressure (BP), fB and ventilation (VE) and a destabilized respiratory pattern relative to controls. To test these hypotheses, we used electroencephalography and electromyography to monitor the sleep state of adult rats deficient in tryptophan hydroxylase 2 (TPH2−/−; n=17) and littermate controls (TPH2+/+; n=13). Femoral arterial catheters were used to measure BP and heart rate (HR), and whole body plethysmography was used to measure respiratory variables, including fB, tidal volume (VT), ventilation (VE), and metabolic rate (VO2). We also measured the coefficient of variability in breathing (CV%) as an index of respiratory stability. TPH2−/− rats experienced episodes of REMS and SWS that, on average, were ~110s and ~40s longer than controls, respectively (p<0.0001 and p=0.028, respectively). 5‐HT deficiency had no effect on BP or HR in any sleep‐state. A loss of central 5‐HT reduced breathing fB in REM sleep (10 bpm; p<0.001), but not in QW or QS. TPH2−/− rats had a significantly increased VT compared to controls (~0.0015ml/g ; p=0.013) but, contrary to our hypothesis, genotype had no significant effect on VE or VE/VO2 in any state. However, across all states female TPH2−/− rats displayed a significantly destabilized breathing pattern compared to female TPH2+/+ (p=0.003). In summary, our data suggest that central 5‐HT signaling regulates the duration of REM sleep as well as the respiratory rhythm and pattern during REM sleep.

Support or Funding Information

Funding for this research was provided by an American Heart Association Scientist Development Grant (14SDG18560022; PI: KJC).
Caffeine improves survival in autoresuscitation‐impaired serotonin deficient rats
Authors: Lee Stella , Nattie Eugene
Published: November 2023

Our previous investigation demonstrated that an existing underlying vulnerability in rat neonates (i.e. brainstem 5‐HT deficiency) in combination with a stressor (i.e. pre‐ and early post‐natal nicotine exposure) exacerbates autoresuscitation failure from an anoxic event at a critical developmental period (P10 rats). During this critical period, we hypothesize that caffeine, a respiratory stimulant, will improve successful autoresuscitation rates in the 5‐HT deficient, nicotine exposed P10 neonates and reduce mortality. Preliminary data demonstrate that an intraperitoneal injection of 10mg kg−1 caffeine prior to anoxic challenge improves autoresuscitation rates (survival rate approximately 40%) in the 5‐HT deficient, nicotine exposed P10 rats in comparison to the rats who received IP saline vehicle injections (survival rate of 25%). Prior investigation suggests that eupnea and heart rate recovery following an anoxic event (which elicits an apnea accompanied by a bradycardia) is significantly delayed in 5‐HT deficient rats treated with nicotine, making them more susceptible to failure of autoresuscitation (p<0.05). Presence of caffeine is likely facilitating successful autoresuscitation by shortening apnea duration and promoting an early onset of gasping post‐apnea for a faster eupnea and heart rate recovery.

Support or Funding Information

Research is supported by the NIH Program Project Grant HD036379 (NICHD, PI, Dymecki, S. M.; Project PI, Nattie, E. E.). The authors would also like to acknowledge the generous support of the Tenney Fund and the Ryan Fellowship (awarded to S. Lee).
New Theory of Holistic Integrative Physiology and Medicine IV: Fetus Does Not Breathing and How We Start It After Birth
Authors: Sun Xing‐Guo
Published: November 2023

Objective

The fetus does not have any true breathing. After birth, we have to start breathing and breathing is our vital sign for life before we expired. Just the short period before and after birth we should have the same system and same mechanism of breathing control. However, the knowledge of traditional respiratory systemic physiology could not explain these clearly. Based on new constructed HOLISTIC INTEGRATIVE PHYSIOLOGY AND MEDICINE, we explained how we control our breathing after birth (see II abstract), but we did not talk about those before birth and short period after birth.

Methods

Now we plan to use the different arterial blood gas signals (trinity of PO2, PCO2 and [H+]) at different time period to discuss mechanisms of why fetus no breathing and how we start our first breathing after birth.

Results

Why fetus does not need breathing? Mother is normal breathing with mean PaO2=~100mmHg, PaCO2=~40mmHg and pHa=~7.40 of up‐down waveform in artery as described in II abstract. The fetus obtains oxygen and nutrients from the mother through the placenta and the umbilical cord. When the uterus arterial blood goes through the placenta capillary, the magnitude of waveform in umbilical vein decreases, and then the blood pass through PFO from RA into LA was diluted more than 10 times by venous return blood, so that mean PfO2=~30mmHg, PfCO2=~45mmHg and pHf=~7.38 in fetus artery without up‐down waveform. Even if they are very low O2, high CO2 and [H+]; the stable mean levels of them do not change the stimulation via peripheral chemical receptors and nerve net. So fetus does not have any true breathing.

How we start our first breathing after birth? After birth, newborn's cell is metabolic and circulation is running, so that before breathing its arterial blood gases will go to much lower O2 and higher CO2 and [H+], progressively. The newborn mean are PaO2=6–30mmHg, PaCO2=42–66mmHg and pHa=7.18–7.38, before first breathe. While the PaO2, PaCO2 and pHa reach some threshold, the stimulation via peripheral chemical receptors and nerve net to start our first breathing. The mind is meaning that “we are air hunger, do we breathe or die? After our first breathing started, we breathing by arterial blood gases up‐down signals switching ins‐ and expiration via peripheral chemical receptors and nerve net; and maintaining stability via central chemical receptors and nerve net (see II abstract).

Conclusion

We can explain the mechanisms of stable arterial blood gases (none waveform) in fetus does not need any true breathing, and after birth, the decreasing of O2 and increasing of CO2 and [H+] will start our first breathing via chemical receptors and nerve net. Based on it, we also explain some existing questions in physiology of respiration and circulation: How we set of FRC and why we close our PFO after birth etc. in different abstracts.

Support or Funding Information

CNSF‐Medical Science Program (81470204); NCCVD Fuwai Hospital Start Program (2012‐YJR02)
Hypoxia Reduces HUWE1 by Downregulating PPARγ in Mouse Lungs and Human Pulmonary Artery Endothelial Cells
Authors: Kang Bum‐Yong , Kleinhenz Jennifer M , Murphy Tamara C , Sutliff Roy L , Hart C Michael
Published: November 2023

Rationale

Pulmonary hypertension (PH) causes significant morbidity and mortality. Hypoxia is an important mediator in the pathogenesis of PH. We recently reported that the peroxisome proliferator‐activated receptor gamma (PPARγ) ligand, rosiglitazone (RSG), attenuates hypoxia‐induced: a) increases in nuclear factor kappa‐B p65 (NF‐κB p65) activation and b) decreases in lung microRNA‐98 (miR‐98) levels.

Objective

We hypothesized that reductions in PPARγ reduce levels of a homology to E6‐AP C terminus E3 ubiquitin ligase (HUWE1), an E3 ubiquitin ligase and host gene of miR‐98, leading to increased NF‐κB p65 expression in hypoxia‐induced PH.

Methods and Results

Male C57BL/6 mice were exposed to hypoxia (10% O2) or normoxia (21% O2) for 3 weeks. Hypoxia reduced HUWE1 levels in mouse lungs. In contrast, in mice treated with RSG (10 mg/kg/d by gavage) for 10 days, RSG attenuated reductions in HUWE1 in mouse lung. In vitro, human pulmonary artery endothelial cells (HPAECs) were exposed to hypoxia (1% O2) or normoxia (21% O2) for 72 hours. Hypoxia reduced HUWE1 expression. These alterations were attenuated by treatment with RSG (10 μM) during the last 24 hours of hypoxia exposure. Loss of PPARγ function reduced HUWE1 levels and gain of PPARγ function increased HUWE1 levels in vivo and in vitro. Furthermore, while knockdown of NF‐κB p65 does not regulate HUWE1 levels, the depletion of HUWE1 increases NF‐κB p65 levels. These novel findings are consistent with the postulate that PPARγ increases HUWE1 to induce ubiquitination of p65 and termination of NF‐κB signaling.

Conclusion

Collectively, these findings suggest that PPARγ attenuates hypoxia‐induced reductions in HUWE1 levels by inhibiting activation of hypoxia‐responsive transcription factors, such as NF‐κB p65. Targeting PPARγ represents a novel therapeutic strategy to inhibit NF‐κB p65 that contributes to PH pathogenesis.

Support or Funding Information

Supported by Atlanta VA Merit Review 1I01BX001910, NIH HL 102167 (RLS and CMH), AHA‐SDG 13SDG14150004 (BYK).
Chronic Hypoxia Elevates Basal Tone in Neonatal Pulmonary Hypertension through PKCβ and Reactive Oxygen Species Signaling
Authors: Sheak Joshua R , Jernigan Nikki L , Walker Benjimen R , Resta Thomas C
Published: November 2023

Vasoconstriction contributes to chronic hypoxia (CH)‐induced pulmonary hypertension in neonates. However, little is known regarding mechanisms of pulmonary vasoconstriction in this setting. PKCβ is a conventional PKC isoform that contributes to a variety of cardiovascular disorders associated with oxidative stress. We hypothesized that neonatal CH mediates spontaneous pulmonary arterial tone through PKCβ and reactive oxygen species (ROS) signaling. To test this hypothesis, we measured baseline pulmonary vascular resistance (PVR) in the presence and absence of PKCβ inhibition with LY‐333,531 (10 nM) or ROS scavenging with the superoxide dismutase mimetic, TEMPOL (1 mM) using salt‐perfused lungs (in situ) from 2‐wk‐old control and CH (2 wk at 0.5 atm) rats. Microvascular pressure was determined by double occlusion technique, allowing calculation of arterial and venous resistances. As indices of pulmonary hypertension, we assessed peak right ventricular systolic pressure (RVSP) and right heart remodeling. Neonatal CH rats displayed elevated RVSP compared to controls (p<0.05), assessed by direct cardiac puncture in anesthetized animals. Furthermore, CH increased Fulton's index [right ventricle (RV)/left ventricle plus septum weight], indicative of RV hypertrophy. Lungs from animals in each group were pre‐treated with the NO synthase inhibitor, Nω‐nitro‐L‐arginine (L‐NNA; 300 μM) or vehicle to evaluate the contribution of endogenous NO to PVR. The contribution of active tone to total and segmental baseline PVR was assessed by examining responses to exogenous NO (spermine NONOate, 100 μM). In the absence of L‐NNA, CH resulted in significant increases in total and arterial baseline vascular resistance (p<0.05), but had no effect on venous resistance. L‐NNA exaggerated CH‐dependent increases in total and arterial vascular resistance (p<0.05 vs. vehicle), but had no significant effect in control lungs. Consistent with our hypothesis, either PKCβ inhibition or ROS scavenging reduced total and arterial baseline tone in lungs from CH neonates (p<0.05 vs. vehicle), while having no significant effect in controls. We conclude that PKCb and ROS contribute to spontaneous pulmonary arterial constriction in neonatal CH rats. Furthermore, this response to CH is limited by endogenous NO.
Pressurized Pulmonary Arteries from the TWIK‐2 Knockout Mouse Exhibit Increased Vascular Reactivity
Authors: Kitagawa Melanie G , Durgan David J , Reynolds Julia , Bryan Robert M , Pandit Lavannya M
Published: November 2023

TWIK‐2 is a member of the two‐pore domain potassium channel family. C57/BL6‐SV129 mice lacking functional TWIK‐2 channels develop pulmonary hypertension at 20 but not 10 weeks of age. We hypothesize that pulmonary arteries from TWIK‐2 KO mice show perturbations in vascular reactivity when compared to wild‐type (WT) mice.

Right ventricular systolic pressure, a surrogate for pulmonary artery (PA) pressure, was measured in mice using isoflurane anesthesia. Right main PAs were harvested from 20 to 22‐week‐old WT and TWIK‐2 knockout (KO) mice and mounted between two glass micropipets. Each PA was bathed in Krebs buffer gassed with 20% O2 and 5%CO2 to achieve a pH of 7.4. Development of spontaneous tone was assessed by comparing PA diameters at pressures of 5, 15, and 30mmHg in 3 mM Ca+2 Krebs buffer to that in Ca+2‐free buffer. Constrictor responses were compared in PAs from WT and KO mice when phenylephrine (PE) was added to the bath at log increments from 10−10 M to 10−5 M. Following the constriction with PE, each PA was exposed to 10−5M acetylcholine (Ach) abluminally to measure endothelial‐mediated vasodilation. The data was analyzed using a repeated measure two‐way ANOVA or Mann‐Whitney Rank Sum test as appropriate.

PA pressures were 22 ± 3 and 34 ± 1 mmHg in WT and KO mice respectively (p=0.01). In the presence of Ca+2, PA diameters were similar between genotype (898 ± 40 and 936 ± 40 μm for WT and KO respectively at 5 mmHg) and increased linearly 21 ± 3 μm / mmHg (R2 = 0.90) at 15 and 30 mmHg. In Ca+2‐free buffer, the diameters were not increased compared to that in the presence of Ca+2 at the three pressures studied. Thus, the right main branch of the PA does not develop spontaneous tone and dilates passively with increases in pressure. Concentration response curves to PE showed a similar maximal constrictor response; however, there was a significant interaction between genotype and PE concentration (p=0.013) and the EC50, the concentration for half of the maximal response, was 42‐fold less in KO (1.68 X 10 −10 M) compared to WT (7.03 X 10−9 M, p=0.029). Endothelial‐mediated dilations to acetylcholine (~11% of maximal diameter) were similar between genotype. The increased sensitivity to PE indicates the potential for an enhanced constrictor response in PAs from KO mice. This enhanced constrictor response in PAs from TWIK‐2 KO mice may have a role in the development of pulmonary hypertension.

Support or Funding Information

Veterans Affairs Career Development Award Program (L.M. Pandit)
Reduced Membrane Cholesterol Following Chronic Hypoxia Limits Depolarization‐induced Ca2+ Entry In Pulmonary Arterial Endothelial Cells
Authors: Zhang Bojun , Naik Jay , Jernigan Nikki , Resta Thomas , Walker Benjimen
Published: November 2023

Chronic hypoxia (CH)‐induced pulmonary hypertension is associated with diminished production of endothelium‐derived Ca2+‐dependent vasodilators such as nitric oxide. Interestingly, ATP‐induced endothelial Ca2+ entry as well as membrane cholesterol (Chol) are decreased in pulmonary arteries from rats exposed to CH (4 wk, PB = 380 torr) compared to normoxic control rats. Furthermore, Chol supplementation restores ATP‐induced Ca2+ entry to the level of controls, suggesting that reduced membrane Chol following CH limits the activity of ion channels required for agonist‐mediated Ca2+ entry. Depolarization‐induced Ca2+ entry is a major component of the response to ATP and is also decreased after CH. However, the effect of Chol on this component of Ca2+ entry is unknown. We hypothesized that depolarization‐induced entry also requires membrane Chol, and that CH attenuates this response by decreasing membrane Chol. To test this hypothesis, we administered Chol (2 mM) or epicholesterol (EpiCh; epimeric form of cholesterol, 2 mM) to acutely isolated pulmonary arterial endothelial cells (PAEC) from control and CH rats to either supplement or replace native Chol. The efficacy of membrane Chol manipulation was confirmed by filipin staining in isolated PAECs. Ca2+ influx in response to a depolarizing stimulus of KCl (60 mM) was assessed by Mn2+‐quenching of fura‐2 fluorescence. Whereas KCl‐induced Ca2+ entry was observed in cells from control rats, exposure to CH nearly abolished this response. EpiCh treatment greatly reduced KCl‐induced Ca2+ influx in PAECs from control rats (p<0.05 vs. vehicle) without further effect in those from CH animals. Chol treatment, however, restored depolarization‐induced Ca2+ entry in PAECs from CH rats (p<0.05 vs. vehicle). Ca2+ influx in response to KCl was inhibited (p<0.05 vs. vehicle) by the T‐type Ca2+ channel antagonist, mibefradil (10 μM), but not by the L‐type Ca2+ channel inhibitor, diltiazem (50 μM). We conclude that membrane Chol is required for depolarization‐induced Ca2+ entry through T‐type channels in PAECs and that reduced Ca2+ entry after CH may be due to loss of this key regulator.

Support or Funding Information

AHA Southwest Affiliate Award #15GRNT21080001
G Protein‐Coupled Receptor (GPCR) Expression in Pulmonary Artery Smooth Muscle Cells of Human and Experimental Models of Pulmonary Arterial Hypertension
Authors: Aroonsakool Nakon , Sriram Krishna , Michkov Alexander V. , Insel Paul A.
Published: November 2023

Pulmonary Arterial Hypertension (PAH) occurs as a primary (idiopathic) disease (IPAH) and secondary to various conditions. Both types are characterized by increased pulmonary vascular resistance and remodeling of the pulmonary artery, which derives in part from the proliferation of pulmonary artery smooth muscle cells (PASMCs), and can lead to right ventricular (RV) heart failure. Current treatments for PAH are inadequate, especially in terms of impact on mortality; thus, new therapies are needed. Animal models that mimic features of human PAH have been used to assess pathophysiology and evaluate treatments for PAH and include the exposure of rodents to chronic hypoxia (in the absence or presence of the VEGFR inhibitor SU5416) or treatment of rats with monocrotaline. Our studies are testing the hypothesis that previously unrecognized G protein‐coupled receptors (GPCRs) may contribute to PAH pathophysiology and may be new therapeutic targets. To begin to test this hypothesis, we used TaqMan® GPCR arrays as an unbiased approach to identify and quantify GPCRs in PASMCs of humans, rats and mice, including from patients with PAH and experimental PAH animal models. Development of PAH and RV hypertrophy in the animal models was confirmed by immunostaining/histology and quantified using the Fulton index (the [right ventricle]/[left ventricle + septum] weight). We found that PASMCs express >100 GPCRs and that the GPCR expression profiles of mouse and rat PASMCs are similar to that of human PASMCs. Moreover, we find numerous changes (of at least 2‐fold) in GPCR expression of PASMCs from PAH patients compared to control PASMCs. GPCRs with altered expression include receptors that link to G proteins whose signaling effectors (e.g., cAMP, Ca++, etc.) are predicted to regulate PASMC proliferation and tone. These results show that PASMCs express many more GPCRs than previously known and that the GPCR expression profile differs in PAH‐PASMCs. GPCRs with altered expression may contribute to altered function in PAH and thus may be novel therapeutic targets for this disease.

Support or Funding Information

Supported by DOD AWARD: No. W81XWH‐14‐1‐0372
Calcineurin ‐NFAT Signaling Molecules Regulate Proliferation, Migration and Apoptosis in PASMCs of Pulmonary Hypertensive Rats induced by Monocrotaline
Authors: Lin Mo‐Jun , He Rui‐Lan , Liu Xiao‐Ru , Gui Long‐Xin , Wang Rui‐Xing , Mu Yun‐Ping , Sham James S.K.
Published: November 2023

Pulmonary arterial hypertension (PAH) is characterized by increased resting intracellular free Ca2+ concentration of pulmonary arterial smooth muscle cells (PASMCs), angiogenesis and vascular remodeling. Previous studies of PAH show that canonical transient receptor potential (TRPC) genes are upregulated and store‐operated Ca2+ entry is augmented in PASMCs of monocrotaline (MCT)‐treated rats. While increased intracellular Ca2+ can further activate calcineurin (CaN) and make nuclear factor of activated T cells (NFAT) dephosphorylation and nuclear translocation. Since CaN and NFAT may functionally regulate the transcription of a variety of cytokines, we sought to examine the effects of CaN‐NFAT signaling molecules on proliferation, apoptosis and migration in PASMCs of control and MCT‐induced rats. Rats developed severe PAH and right ventricular hypertrophy within three weeks after single intraperitoneal injection of MCT at a dose of 60 mg/kg. MCT treatment can upregulated mRNA and protein expression of STIM1/2‐ORAI1/2‐CaNBβ‐NFATc2/3/4 on PAs, as well as developed proliferation and migration and decreased apoptosis on PASMCs. Pretreatment with Cyclosporine A (CsA) inhibited proliferation and migration on PASMCs of control (CON) and MCT rats, in a dose dependence manner (15, 30 60μM), but inhibited apoptosis only on high concentration (60μM). Proliferation, migration and apoptosis resistance of PASMCs in CON and MCT rats were obviously inhibit by siRNA of NFATc2 and NFATc4. Pretreatment with siRNA of NFATc3 were no effect to proliferation, migration and apoptosis resistance of PASMCs in CON rats, but can obviously suppressed proliferation and migration, promoted apoptosis of PASMCs in MCT rats. Our results hence show that MCT treatment caused significant alterations in the expression of STIM1/2‐ORAI1/2‐CaNβ‐NFATc2/3/4 signaling molecules. NFATc2/4 are essential factor for PASMCs growth, however, NFATc2/34 may contribute to pulmonary vascular remodeling in PAH induced by MCT.

Support or Funding Information

This work is supported by NSFC31171104, NSFC 31371165, NSFC 31571179 and FJMU09ZD010
HDAC6‐Hsp90 interplay in Pulmonary Arterial Hypertension
Authors: Chabot Sophie , Boucherat Olivier , Ruffenach Grégoire , Breuils‐Bonnet Sandra , Tremblay Eve , Provencher Steeve , Bonnet Sébastien
Published: November 2023

Background

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by the elevation of mean pulmonary arterial pressure. Pulmonary arterial smooth muscle cells (PASMCs) from PAH patients exhibit a cancer‐like metabolic‐dependent pro‐proliferative, pro‐migratory and anti‐apoptotic phenotype leading to development of progressive pulmonary artery remodeling. The histone deacetylase 6 (HDAC6) is mainly cytoplasmic and involved primarily in “non‐histone” functions. HDAC6 interacts and deacetylates various proteins such as α‐tubulin and Hsp90 promoting proto‐oncogene activation such as Survivin (implicated in PAH) to carry out cancerous functions. We hypothesize that HDAC6/Hsp90 axis is up‐regulated in PAH and contributes to the development of the cancer‐like phenotype through in part Survivin stabilization.

Method/Results

Using a translational molecule‐cell‐organ‐animal and multidisciplinary approach, we demonstrated that both HDAC6 and HSP90 are up‐regulated (immunofluorescence and immunoblot; p<0.01) in the lungs and PASMCs isolated from PAH patients (n=5) compared to age‐matched controls (n=5). HDAC6 inhibition via a pharmacological (Tubastatin A) or molecular (siHDAC6) approach dose dependently increased α‐tubulin acetylation and decreased PAH‐PASMC migration (wound healing assay; p<0.05), resistance to apoptosis (Annexin‐V; p<0.05) and proliferation (Ki67; p<0.01). Increased apoptosis following Tubastatin A treatment was associated with mitochondrial membrane depolarization (TMRM) of PAH‐PASMCs and inhibition of the oxygen consumption rate in a concentration‐dependent manner (Seahorse XFe 24 system). All these effects were associated with a down‐regulation of Survivin. Similarly, inhibition of Hsp90 (AT13387 or siHsp90) reverses the cancer‐like phenotype and this effect was associated with a down‐regulation of HDAC6, suggesting that HDAC6 up‐regulation in PAH is Hsp90‐mediated. In vivo, HDAC6 inhibition in monocrotaline‐induced PAH decreases both mean pulmonary artery pressure (right heart catheterization, p<0.01) and right ventricular hypertrophy (Fulton index).

Conclusion

We provide evidence that Hsp90 and HDAC6 are specifically up‐regulated in human PAH and contributes to the proliferative, migratory and anti‐apoptotic phenotype seen in PAH‐PASMCs. As HDAC6 inhibitors have been tested in cancer, this offers a short‐term new therapeutic avenue for PAH patients.

Support or Funding Information

Canada Research Chairs and CIHR grants to S. Bonnet and S. Provencher supported this work
JNK2 Regulates Vascular Remodeling in Pulmonary Hypertension
Authors: Das Mita , Zawada W. Michael , West James , Stenmark Kurt
Published: November 2023

Structural alterations of pulmonary arterial (PA) wall are the key pathological features of pulmonary hypertension (PH). Although such vascular abnormalities correlate with heightened phosphorylation of c‐Jun N‐terminal kinases 1/2 (JNK1/2) in rat models of PH, contribution of each individual JNK isoform to the pathophysiology of PH is unknown. Hence, we hypothesized that activation of either one, or both JNK isoforms regulates PA wall thickening in PH. Comparing JNK1/2 activation in lungs of healthy individuals vs lungs of PH patients showed robust JNK1/2 phosphorylation (an index of JNK activation) accompanied by remarkable vascular thickening only in the disease‐affected lungs. In PH patients’ lungs, increased JNK1/2 phosphorylation paralleled marked reduction in MAP kinase phosphatase 1 (a key JNK dephosphorylator) expression. Association of JNK1/2 activation with vascular wall thickening was then confirmed in a well‐established neonatal calf model of hypoxia‐induced PH. To ascertain the role of each JNK isoform in PH pathophysiology, wild‐type (WT), JNK1 null (JNK1−/−), and JNK2 null (JNK2−/−) mice were exposed for six weeks to either normoxia (ambient air) for control or chronic hypoxia (10% O2) to develop PH. Hypoxia stimulated an increase in JNK1/2 phosphorylation that associated with the development of PH like pathology i.e. excessive accumulation of extracellular matrix, small vessel (20–100 μm diameter) muscularization with medial wall thickening, and up‐regulation of small vessel density in WT mouse lungs. Similar to the hypoxia‐exposed WT lungs, increase in deposition of extracellular matrix and structural remodeling of the vascular wall were also detected in hypoxic JNK1−/− lungs. In contrast, JNK2−/− mice lacked such hypoxia‐induced up‐regulation of matrix accumulation as well as vascular abnormalities in the lung. However, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) were increased to a similar magnitude by hypoxia among all three genotypes. Our findings suggest that JNK2 regulates hypoxia‐induced PA structural thickening and that selectively modulating JNK2 actions might quell vascular abnormalities in PH.

Support or Funding Information

National Institutes of Health HL64917
Ubiquitin Proteasome System in Pulmonary Hypertension
Authors: Wade Brandy E , Hart C Michael , Sutliff Roy L
Published: November 2023

Pulmonary Hypertension (PH) is characterized by a sustained increase in pulmonary arterial pressure. Increased pulmonary vascular pressure and resistance result in right ventricle (RV) hypertrophy and can ultimately lead to RV failure and death. The mechanisms leading to vascular cell proliferation and vascular remodeling in PH are incompletely defined. Current therapeutic approaches that stimulate vasodilation inadequately address the cellular proliferation and vascular remodeling that underlies the pathogenesis of PH. To more directly address mechanisms of cell proliferation in PH, this project focuses on the ubiquitin proteasome system (UPS) which plays an important role incellular homeostasis by regulating protein stability. Current evidence demonstrates changes in UPS activity in PH; however, the pathways and proteins influenced by these changes are poorly defined. Mice exposed to normoxic or hypoxic conditions for 3 weeks were used to identify proteins with altered ubiquitination in response to hypoxia exposure. Lungs were collected, and the PTMScan® Ubiquitin Remnant Motif (K‐ɛ‐GG) Kit was used to precipitate ubiquitinated proteins for analysis by mass spectrometry (MS). 245 proteins were identified with a ±2 fold change. In silico analysis identified several proteins that promote cell proliferation. Tax1bp1, Hspa8, Hspb1, Fhl1, Gnb1 interact with important cellular proliferation pathways and exhibit decreased ubiquitination in lungs from mice exposed to hypoxia, suggesting an increase in their stability in hypoxia. These results using MS and in silico analysis suggest that hypoxia‐induced changes in the UPS may impact proteins involved in pathways that regulate cellular proliferation by increasing or decreasing their stability.

Support or Funding Information

This work was supported by NIH RO1 HL 102167 and the T32 Training Program in Academic Pulmonary Medicine (T32HL076118).
Sex Differences in Sepsis‐Induced Nitric Oxide and EDHF Signaling Pathways in Rat Pulmonary Artery
Authors: Kuntamallappanavar Guruprasad , Leo M. Dennis , Subramani Jaganathan , Mishra Santosh
Published: November 2023

Sepsis still remains a major cause of hospital infection related deaths in the US. Pulmonary manifestation of sepsis is characterized by pulmonary hypertension, pulmonary edema and refractory hypoxemia. Pulmonary hypertension is associated with abnormal regulation of vascular tone in sepsis. Evidence exists that males are more susceptible to sepsis than females. The objective of present study was to elucidate the influence of gender on endothelial function, particularly with reference to nitric oxide (NO) and endothelium‐derived hyperpolarizing factor (EDHF) signaling pathways in pulmonary artery of septic rats. Sepsis was induced in rats by cecal ligation and puncture (CLP). To determine the effect of sepsis‐induced alteration in the NO and EDHF signaling pathways in both sexes, arterial relaxation was measured using isometric tension experiments. Results demonstrate that in control animals the potency but not the amplitude of the response to the vasoconstrictor, phenylephrine, was significantly greater in the pulmonary arteries from female than male rats. Sepsis caused a significant reduction in the efficacy of phenylephrine in pulmonary arteries from either sex. In controls, acetylcholine (ACh) was more potent in dilating pulmonary arteries from female than male rats and sepsis identically inhibited ACh‐induced relaxations in both male and female animals. Sepsis completely abolished the EDHF response in pulmonary arterial rings from both sexes. Pre‐treatment with L‐NAME (eNOS inhibitor) and indomethacin (prostacyclin inhibitor) significantly increased the basal tone of pulmonary arterial rings from female rats and was significantly reduced in sepsis. However, L‐NAME and indomethacin did not alter the basal tone of pulmonary artery from both control and septic male rats. Real time PCR analysis of mRNA expression revealed that sepsis decreased IKCa, SKCa1, and SKCa2 mRNA expression levels in female rats and IKCa and SKCa2 mRNA expression levels in male rats. SKCa1 SKCa1 mRNA expression levels did not change in male rats while sepsis had no effect on SKCa3 mRNA expression levels in both sexes. In conclusion, the results of the present study suggest that sepsis‐induced alteration in the reactivity of pulmonary artery is gender dependent. This can be attributed to a decrease in NO but predominantly EDHF‐mediated vasorelaxation resulting from a significant decrease in expression of endothelium localized potassium channels.
Enabling a Viable Pipeline of NHLBI Extramural Investigators in Fiscally Challenging Times
Authors: Maric‐Bilkan Christine , Charette Marc , Oh Young , Schum Paula , Gao Yunling , McDonald Cheryl , Reid Diane , Robinson Valerie , Stanley Dennis , Fuentes Kelin , Olive Michelle , Eblen Matthew , Pearson Katrina , Tolunay Eser , Galis Zorina
Published: November 2023

A major goal of the National Heart, Lung, and Blood Institute (NHLBI) is to maintain a viable and robust pipeline of extramural investigators. We have examined data on NIH funding trends among research project grant (RPG) awardees using economic and demographic data between fiscal year 1998 and fiscal year 2014. We have divided the investigators who received RPG awards during this period into three age groups: “early‐stage” (24–40 years), “mid‐career” (41–55 years) and ”established” (56 years and greater). We observed that there has been a low level, but progressively steady shift, in the distribution of NHLBI RPG awardees. The cause of this shift appears to be due, in part, to a parallel shift in the demographics of NHLBI RPG applicants. The combined result of these shifts has implications for the overall distribution of NHLBI RPG direct cost dollars. The magnitude of these effects is significantly increased during periods of flat or slightly declining levels of RPG funding. The three main drivers of these trends, 1) the shifting demographics of NHLBI RPG applicants, 2) the changing distribution of RPG direct cost dollars, and 3) the adverse effects of prolonged periods of level or slightly declining research funding, will be presented in detail along with suggested proposals that are intended to ensure the NHLBI will maintain its steady pipeline of qualified investigators now and for the foreseeable future.

Support or Funding Information

None
Tactical post hoc Exam Key Review
Authors: Price Joseph A.
Published: November 2023

The immediate task in reviewing course exam (typically one best answer multiple choice) results is to make any key revisions in order to release the grades on that exam in an often controversial, emotionally debated, inconsistent, and too often misguided process. Here I show a tactical approach to post hoc exam review that will deal with poorly performing test items, and/or a too low class average, and do so in a manner fair to the students as a class. The specific method shown in the flow chart is grounded on and extension of established educational and psychometrics literature and principles, and in a novel way takes a seemingly complicated subjective process and organizes decisions into an easy to follow but consistent, effective solution, based on specific predetermined criteria while preserving instructor's options. This psychometrics driven method is based on the answers to two questions: “What portion of the class should get an item correct?”, and “How many students is it acceptable to fail?”, and on an implementation of a philosophy of education. It is particularly useful for newer medical school faculty and course directors.

Support or Funding Information

No extramural support.
An Active Learning Activity Utilizing a Headline News Story to Engage Students, Enhance Interest, and Improve In‐class Learning of Cell Cycle Regulation
Authors: Dirks‐Naylor Amie J
Published: November 2023

An active learning activity was utilized to engage students and enhance in‐class learning of cell cycle regulation in a Pharm.D. integrated biological sciences course. The aim of the study was to determine the effectiveness and perception of the in‐class activity. Following completion of lecture on the topic of cell cycle regulation, students completed a 10‐question multiple choice quiz before and after engaging in the activity. The activity involved reading of a headline news article published by ScienceDaily.com entitled, “One Gene Lost Equals One limb Regained”. The name of the gene was deleted from the article and, thus, the end goal of the activity was to determine the gene of interest by the description in the story. The activity included compiling a list of all potential gene candidates before sufficient information was given to identify the gene of interest (p53). A survey was completed to determine student perceptions of the activity. Quiz scores improved by an average of 20% after the activity (40.1 ± 1.95 vs 59.9 ± 2.14; p< 0.0001; N= 96). Students enjoyed the activity, found the news article interesting, and believed that the activity improved their understanding of cell cycle regulation. The majority of students agreed that the in‐class activity peaked their interest for learning the subject matter and also agreed that if they understand a concept during class, they are more likely to want to study that concept outside of class. In conclusion, the activity improved in‐class understanding and enhanced interest in cell cycle regulation.

Support or Funding Information

None