bluuebunny/crossref_metadata_2025 huggingface dataset:
The time course of transcriptional regulation in anoxic fibroblasts: Implications for Carpal Tunnel Syndrome
Authors: Pierce Christine Walker , Tucci Michelle A. , Benghuzzi Hamed
Published: June 2021

Hypoxia is the initial cellular insult observed in carpal tunnel syndrome. While much is known about the disease progression, little is known about its molecular basis. These studies sought to determine the transcriptional regulation in fibroblasts subjected to anoxia with respect to HIF‐1α and TGF‐β1 expression.

Methods

MRHF fibroblasts were cultured under anoxic conditions for up to 72 hours alongside normoxic controls. mRNA levels relative to baseline were determined by real‐time PCR. GAP‐DH was used as the reference standard for normalization and relative quantification of fold‐differences in mRNA expression were determined by the comparative CT method.

Results

Transcriptional up‐regulation of both HIF‐1α and TGF‐β1 were observed in response to anoxia with peak up‐regulation occurring at 15 min of anoxia exposure for both HIF‐1α and TGF‐β1. HIF‐1α showed a 7.3‐fold increase relative to baseline, and this increase was sustained over time. In contrast, TGF‐β1 was increased 3.3‐fold over baseline, but this increase was only transient.

Conclusion

Both HIF‐1α and TGF‐β1 have been shown to regulate the expression of connective tissue growth factor (CTGF). The pronounced and sustained up‐regulation of HIF‐1α observed in these studies suggests that CTGF expression in hypoxic carpal tissues is predominantly under the regulatory influence of HIF‐1α.
The role of Methionine sulfoxide reductase A (MsrA) in the anoxia‐tolerant turtle (Trachemys scripta)
Authors: Bruce Lynsey Erin , Milton Sarah L.
Published: June 2021

The enzyme MsrA repairs oxidized proteins, and may act as a scavenger of reactive oxygen species (ROS), making it a potential therapeutic target for age‐related neurodegenerative diseases. The anoxia‐tolerant turtle offers a unique model to observe the effects of oxidative stress on a system that maintains neuronal function following anoxia and reoxygenation, and that ages without senescence. Using RT‐PCR and western blot, we examined MsrA transcript and protein levels in the turtle during anoxia and recovery; mRNA and protein levels were upregulated 9‐ and 7‐fold over 4 hours of anoxia, with mRNA but not protein levels falling upon reoxygenation. MsrA is present in both the mitochondria and cytosol, with protein levels increasing respectively 3‐ and 4‐fold over 4h anoxia, and remaining 2‐fold higher than basal upon reoxygenation. MsrA was knocked down in neuronally‐enriched cell cultures via RNAi transfection. Propidium iodide staining showed no significant cell death during anoxia, but this increased 7‐fold upon reoxygenation, suggesting a role for MsrA in ROS suppression during reperfusion. This is the first report in any system of MsrA transcript and protein levels being regulated by oxygen levels. By delving into the molecular pathways of this unique model, more may be understood about the function and role of MsrA during oxidative stress and the process of aging. This research is funded by an AFAR grant to SLM.
The Novel Soluble Guanylate Cyclase Activator PPCA Reverses Acute Hypoxia‐induced Pulmonary Hypertension in Dogs
Authors: Wang Tao , McCefferty Gerald P. , Schnackenberg Christine G. , Jucker Beat M , Willette Robert N. , Behm David J.
Published: June 2021

Nitric oxide (NO) plays a critical role in regulating pulmonary vascular tone. Impaired bioavilability of the NO receptor, soluble guanylate cyclase (sGC), and/or response to endogeneous NO has been implicated in the pathogenesis of pulmonary hypertension (PH). We hypothesize that activating sGC in a NO‐independent manner will provide a valuable therapy for patients with PH.

We generated a novel NO‐independent type 2 sGC activator, PPCA, and tested it in a canine model of hypoxia‐induced pulmonary hypertension. Under normoxic conditions PPCA dose‐dependently (3–100 μg/kg, IV) decreased mean arterial pressure (MAP) and pulmonary arterial pressure (PAP) in anesthetized and catheterized beagle dogs (n=5). Following 20 minutes of hypoxia (10% O2), either saline or PPCA (15μg/kg, IV) was administered. PPCA significantly decreased PAP (−25.1±4.1%) vs. vehicle (−7.6±1.4%, P< 0.05), MAP (−16.1± 0.1%) vs. vehicle (−2.7±0.1%, P<0.05), PCWP and LVEDP. In addition, PPCA improved SaO2 and relieved labored breathing as compared to vehicle controls. In contrast, renal blood flow, LV dP/dtmax and cardiac output were not altered by PPCA. Early administration of PPCA (30μg/kg, IV) at 5 minutes after hypoxia completely blocked the hypoxic pulmonary vasoconstriction for over 80 minutes.

These data suggest that activation of sGC by PPCA improves pulmonary hemodynamics and function in hypoxia‐induced pulmonary hypertension.
Epigenetic programming of pulmonary vascular dysfunction in mice
Authors: Rexhaj Emrush , Sartori Claudio , Scherrer Urs
Published: June 2021

Fetal insults predispose to premature cardiovascular disease later in life. In mice, male offspring of restricted diet pregnancy (a model mimicking preeclampsia in humans), display pulmonary vascular dysfunction and transmit this dysfunction to the next generation. The mechanism is unknown. Epigenetic alterations may result in stable changes of gene expression that are transmitted throughout generations.

To test for this possibility, we examined the effects of the histone deacetylase inhibitors Butyrate (2 mg/kg/day, i.p for 2 wks) and trichostatin on pulmonary DNA methylation and vascular function in male offspring of restricted diet pregnancies of the 1st and 2nd generation.

In offspring of restrictive diet pregnancies pulmonary DNA methylation was significantly impaired (P=0.02 vs. controls). Butyrate normalized pulmonary DNA methylation and pulmonary vascular responsiveness to acetylcholine in vitro (P<0.0001 vs. vehicle) and to hypoxia in vivo in offspring of the 1st generation, and prevented transmission of vascular dysfunction to the next generation (P<0.0001, Butyrate vs. vehicle). Trichostatin had similar effects.

We demonstrate for the first time that restrictive diet pregnancy alters pulmonary DNA methylation and pulmonary vascular function by an epigenetic mechanism. We speculate that epigenetic mechanisms may underpin vascular dysfunction in offspring of preeclampsia in humans.
ASIC1 mediates enhanced receptor‐mediated vasoconstriction following chronic hypoxia‐induced pulmonary hypertension
Authors: Jernigan Nikki L , Walker Benjimen R , Resta Thomas C
Published: June 2021

Increases in pulmonary vascular smooth muscle (VSM) store‐operated Ca2+ entry (SOCE) are implicated in augmented basal VSM intracellular Ca2+([Ca2+]i) and enhanced vasoconstriction in chronic hypoxia (CH)‐induced pulmonary hypertensive rats. Our laboratory has recently characterized a unique role for acid sensing ion channel 1 (ASIC1) in regulating SOCE in pulmonary VSM. The goal of the present study was to determine the physiological importance of ASIC1‐mediated SOCE to receptor‐mediated pulmonary vasoconstriction. We hypothesized that enhanced Ca2+ entry through ASIC1 contributes to increased receptor‐mediated vasoconstriction following CH. To test this hypothesis we examined vasoconstrictor and VSM [Ca2+]i to UTP, endothelin‐1, KCl (depolarizing stimulus), and elevated intraluminal pressure (myogenic) in fura‐2‐loaded, endothelium‐disrupted, pressurized small pulmonary arteries from control and CH rats (4 wk at 0.5 atm). Specific inhibition of ASIC1 with psalmotoxin 1 (PcTX1) attenuated vasoconstriction and increases in VSM [Ca2+]i to UTP and endothelin‐1 in arteries from control and CH rats; normalizing the responses between groups. However, PcTX1 had no effect on depolarization‐ or pressure‐induced vasoconstriction and changes in VSM [Ca2+]i. These studies support a novel role of ASIC1 in elevated receptor‐stimulated vasoconstriction following CH which is likely mediated through increased SOCE.
Role of NADPH oxidase in enhanced membrane depolarization‐induced Ca2+‐sensitization in pulmonary vascular smooth muscle following chronic hypoxia
Authors: Norton Charles Elbert , Broughton Brad , Walker Benjimen , Resta Thomas
Published: June 2021

Chronic hypoxia (CH) augments membrane depolarization induced myofilament Ca2+‐sensitization in pulmonary vascular smooth muscle (VSM) through O2− dependent stimulation of RhoA, a response that may contribute to the vasoconstrictor component of pulmonary hypertension (PH). We hypothesized that NADPH oxidase (NOX)‐derived O2− mediates this sensitization. To test our hypothesis, we examined effects of apocynin (30 μM) on vasoreactivity to depolarizing concentrations of KCl in isolated, saline perfused lungs from control and CH (4 wk at 0.5 atm) rats. We additionally measured vasoconstrictor responses to KCl in the presence or absence of the NOX inhibitors apocynin or diphenylene iodonium (10 μM) in endothelium‐disrupted, Ca2+ permeabilized, small pulmonary arteries from each group. Parallel experiments in pressurized arteries used the fluorescent O2− indicator, dihydroethidium, to assess effects of NOX inhibition on depolarization induced O2− production. CH augmented KCl‐dependent vasoconstriction in both isolated lungs and arteries and increased O2− generation as previously reported. Furthermore, both NOX inhibitors prevented these effects of CH, resulting in normalization of responses to the level of controls. We conclude that NOX is a source of O2− responsible for enhanced membrane depolarization‐induced pulmonary VSM Ca2+‐sensitization and vasoconstriction in CH‐induced PH.
In vivo determination of the renal activity of the epithelial sodium channel (ENaC) using benzamil natriuresis in male (M) and female (F) mice infused with angiotensin II (Ang II)
Authors: Li Lijun , Tiwari Swasti , Mulroney Susan , Sandberg Kathryn , Ecelbarger Carolyn M.
Published: June 2021

We recently showed sex differences on immunoblots for ENaC subunits with Ang II infusion. To determine whether these protein changes were associated with altered ENaC activity, in Study 1, M and F mice (MF‐1 strain) were infused with Ang II by osmotic minipump (800 ng/kg·bw/min) or received a sham surgery (n = 5/sex/treatment). After 7 days, mice were anesthetized and infused via a jugular catheter with saline (2% body weight/hour), then saline plus benzamil (ENaC antagonist) for 2‐consecutive, 20‐minute periods. Urine was collected from the catheterized bladder. Period 1 natriuresis (response to the infusion) was not different between groups. Period 2 natriuresis (index of ENaC activity) was increased by Ang II, but only in male mice (% of sex respective control group): 141 (MA) and 104 (FA). In Study 2, control M and F MF‐1 mice were treated intraperitoneally with benzamil @1.4 mg/kg·bw in the conscious state and urine collected for 4 hours in metabolic cages. No sex differences were observed in the control state (mmol Na/kg·bw/4 hours): 2.24 ± 0.27 (M) vs 2.47 ± 0.26 (F); however, when Ang II was infused into the same mice (in the subsequent week) only M mice showed a positive increase (Δ mmol Na/kg·bw/4 hours): +1.70 ± 1.79 (M) vs −1.26 ± 0.31 (F). Overall, Ang II increased natriuretic response to benzamil to a greater extent in the males suggesting greater ENaC activity, which may provide insight on therapies for both sexes.
Blood pressure independent sexual dimorphism in proteinuric response to high salt intake in Sprague‐Dawley rats.
Authors: Byrd Charlene Abadie , Rands Vicky F , Prieto Minolfa C , Navar L Gabriel
Published: June 2021

We examined if changes in blood pressure and proteinuria during chronic salt intake exhibit sex disparities in age‐matched male (♂; N=10) and female (♀; N=10) Sprague‐Dawley (SD) rats subjected to either normal (0.7% NaCl) or high salt (8% NaCl) diets for 2‐weeks. Metabolic studies, systolic blood pressures (SBP) measured by tail‐cuff method and proteinuria in 24‐h urine samples, were performed. Baseline SBP was not different between genders (♂:119.1±3 vs. ♀:119.0±4 mmHg) or after 2‐weeks on HS diet (♂:129.0±5 vs. ♀:125.6±6 mmHg). HS intake induced proteinuria in both sexes; however it was greater in male than in female rats [Day ‐1(♂:5±1 vs. ♀:3±1); Day 9 (♂:36±3 vs. ♀:17±2 mg/day); p<0.01]. By day 13, body weights were higher in ♂ than ♀ rats; however there was no difference with salt load [(male NS: 330±8 versus male HS: 324±7 g); ♀ NS: 217±12 vs. ♀ HS: 226±4 g)]. There were no sex dependent differences in food intake after it factored by body weight (♂ NS: 11±1 g/kg BW; ♂ HS: 12±1 g/kg BW; female NS: 11±2 g/kg BW; female HS: 13±1 g/kg BW; p=no significant). SBP under HS intake did not exhibit sexual dimorphism; however the augmented proteinuria in male rats may reflect a greater predisposition to develop renal injury with high salt consumption than female rats.

Grants from the APS (2009 Frontiers in Physiology Research Host Awardees Program); NIH (P20‐RR‐017659; Tulane‐BIRCWH K12HD043451 award).
Urinary Proteomics Analysis of a High Salt Diet
Authors: Jackson Cynthia Ann , Mangaroo Shantae , Ward Ransom E , Robertson Carla J , Jackson Cynthia Ann
Published: June 2021

The American Heart Association recommends that healthy adults should eat < 2.4g of sodium a day. Currently, the average American eats 9–12 g of salt a day. The kidney is the major organ in the body that is responsible for maintaining sodium and water homeostasis in response to the high salt diet. In order to excrete the excess sodium, there is an increase in the renal transport and regulatory proteins. The purpose of this study was to determine if qualitative alterations occurs in urinary proteins in response to a high salt diet. To achieve this goal a portion of 24‐hr urine samples from C57BL10 mice fed either a normal salt (0.3%NaCl) or a high salt diet (8.0%NaCl) for seven days were used in this study. The urine was treated with protease inhibitors and the total protein concentration was determined by a Bradford assay with BSA as the standard. 5.3 ìg of total protein from each sample were analyzed on a 12% SDS‐PAGE. Preliminary analysis revealed that there are more visible bands in the urine of animals fed a chronic HS diet than in urine of animals fed a NS intake. In addition, some bands were present or absent in the HS urine when compared with the protein bands in the NS urine. Although further evaluation is needed, this data suggest that alterations in urinary protein excretion do occur in response to a chronic HS intake. The analysis of urinary proteins could provide a noninvasive method of evaluating physiological.
V‐ATPase membrane accumulation and activity are stimulated by cAMP in A‐type intercalated cells
Authors: Paunescu Teodor G. , Ljubojevic Marija , Russo Leileata M. , Winter Christian , McLaughlin Margaret M. , Wagner Carsten A. , Breton Sylvie , Brown Dennis
Published: June 2021

Recycling of the vacuolar proton (H+) pumping ATPase (V‐ATPase) between intracellular vesicles and the plasma membrane in H+‐secreting cells is central to acid‐base regulation by the kidney. Collecting duct (CD) A‐type intercalated cells (A‐IC) respond to systemic acidosis by accumulating the V‐ATPase in their apical membrane, which correlates with higher rates of H+ secretion and increased microvillar extension. We show that CPT‐cAMP infused in vivo results in an almost two‐fold increase in apical V‐ATPase accumulation in A‐IC within 15 min, and that these cells develop an extensive array of apical microvilli. The cAMP effect on A‐IC is direct, as shown in vitro in cells isolated from the medulla of transgenic mice expressing EGFP in IC (driven by the V‐ATPase B1‐subunit promoter). 3D‐reconstructions of confocal images showed that cAMP induced a dramatic, time dependent growth of microvilli in isolated IC. These morphological changes were paralleled by increased cAMP‐mediated H+ extrusion rates by A‐IC in isolated outer medullary CD measured using the ratiometric probe BCECF. These results support the idea that cAMP, generated by the soluble adenylyl cyclase (sAC ‐ which is enriched in IC) or other effectors, is part of the signal transduction mechanism for acid‐base sensing and V‐ATPase trafficking in IC. Funding source: NIH DK‐73266, DK‐42956, DK‐38452 and HD‐40793.
Effect of knocking out receptor protein tyrosine phosphatase γ (RPTPγ) in the HCO3‐induced inhibition of HCO3 reabsorption by mouse renal proximal tubules
Authors: Zhou Yuehan
Published: June 2021

The accompanying abstract shows that knocking out RPTPγ renders isolated perfused proximal tubules (PTs) incapable of responding to changes in basolateral (BL) [CO2], consistent with the hypothesis that the “orphaned” RPTPγ is a CO2 sensor. Here I test the hypothesis that RPTPγ is also a HCO3 sensor. I perfused PTs from wild‐type vs RPTPγ‐null mice, using out‐of‐equilibrium to vary [HCO3]BL at fixed [CO2]BL and pHBL. In wild‐type PTs, raising [HCO3]BL from 0 to 22 (“baseline”) to 44 mM (fixed [CO2]BL = 5%, pHBL = 7.4) decreased JHCO3 from 222 ± 12 (n = 14) to 136 ± 7 (n = 40) to 55 ± 12 pmol mm−1 min−1 (“pmm”, n = 14). In RPTPγ‐null PTs, baseline JHCO3 was reduced by 23% to 105 ± 7 pmm (p = 0.002, 1‐way ANOVA), and the responses to 0 mM [HCO3]BL (JHCO3 = 112 ± 6, n = 6) and 44 mM [HCO3]BL (JHCO3 = 100 ± 8, n = 6) were eliminated. Finally, when I added 10−8 M candesartan (non‐peptide AT1 antagonist) to the lumen of RPTPγ‐null PTs, baseline JHCO3 further fell by 29% to the “pedestal” level (JHCO3 75 ± 8 pmm, n = 18, p = 0.012, 1‐way ANOVA). Moreover, the responses to 0 mM [HCO3]BL (JHCO3 = 67 ± 14, n = 4) and 44 mM [HCO3]BL (JHCO3 = 55 ± 6, n = 5) were eliminated. Thus, knocking out RPTPγ eliminates the PT response to changes in [HCO3]BL, consistent with the idea that RPTPγ is a HCO3 sensor, perhaps reporting the ratio [CO2]BL/[HCO3]BL, as part of a signal‐transduction cascade that also includes the apical AT1 receptor.
Sham rehydration contributes to increased Fos staining in the hindbrain after water deprivation in the rat
Authors: Little Joel Turner , Knight W David , Ji Lisa Liping , Gottlieb Helmut , Cunningham J Thomas
Published: June 2021

This experiment tested the role of the oropharyngeal and gastric afferents on Fos staining in the hindbrain of dehydrated and rehydrated rats instrumented with gastric fistulae. Rats were divided into control (CON) or 48 hour water deprivation (WD) treatments. WD rats were further subdivided to include 3 rehydration treatments: 2 hour rehydration with fistulae closed (RHC), fistulae open (RHO), or rehydration with isotonic saline (RHS). An open fistula facilitates sham rehydration while closed fistulae allow for complete ingestion; WD and RHS rats also had open fistulae. WD increased osmolality and only RHC treatment reduced osmolality (CON: 296±2 WD: 307±2 RHC: 278±2 RHO: 305±1 RHS: 308±3). Hindbrains were processed for fos and the nucleus of the solitary tract (NTS) for each rat was examined. Water deprivation modestly increased Fos in the NTS (CON: 7±2; WD: 15±3). Rehydration significantly increased Fos staining in both RHO (37±6) and RHC (35±6) rats compared to CON and WD rats. In RHS rats, Fos staining was only slightly elevated compared to WD rats (23±4) and significantly less than RHC and RHO rats. Fos staining in the NTS associated with rehydration appears to be mediated by visceral afferents activated during sham rehydration and possibly independent of the solute concentration of the solution ingested. Supported by NIH R01 HL62579.
Salt intake sensitization and hypothalamic expression of genes related to the renin‐angiotensin system
Authors: Pereira‐Derderian Daniela T. B. , Chiavegatto Silvana , Menani José V. , De Luca Laurival A.
Published: June 2021

Sodium intake occurs either as a spontaneous or an induced behavior, which is increased by repeated episodes of water deprivation followed by partial rehydration (WD‐PR). In this work, we quantified transcripts related to RAS in the hypothalamus of rats that had a history of WD‐PR. Adult male Holtzman rats (n=6–12/group) had water and 0.3 M NaCl measured daily. The animals were submitted to one or three episodes of WD‐PR, with 7‐day interval among the three episodes. The animals were sacrificed at the end of the 1st or 3rd WD‐PR (dehydrated) or four days after none, 1st, or 3rd WD‐PR (hydrated). The hypothalamus was dissected and level of mRNA for components of the RAS was determined by qRT‐PCR. Dehydrated animals had increased hypothalamic transcripts of angiotensinogen, aminopeptidase N, ANG II receptor type‐1‐associated protein, ANG receptor like‐1 or apelin receptor by 43%, 60%, 36%, and 159%, respectively, in the 3rd versus 1st WD‐PR. There was no difference in the hypothalamic mRNA content of RAS among hydrated animals. The 0.3 M NaCl intake was enhanced in the 3rd compared to the 1st sodium appetite test in a distinct group. These data suggest that enhancement in the induced sodium intake is associated with alterations in gene expression related to RAS in the hypothalamus. Therefore, hypothalamic RAS may play a role in the long‐term changes of sodium appetite. Research supported by CNPq and FAPESP.
Photostimulation of channelrhodopsin‐transfected rostral medullary Phox2b‐expressing neurons activates breathing in unaesthetized rats
Authors: Kanbar Roy , Stornetta Ruth L. , Lewis Stephen J , Guyenet Patrice G
Published: June 2021

The Phox2b‐positive neurons of the RVLM were made to synthesize channelrhodopsin‐2 (ChR2) fused to mCherry (ChR2‐mCherry) by injecting a lentivirus that expresses the transgene under the control of the artificial promoter PRSX8. Labeled neurons consisted of ~62% C1 cells and ~38% retrotrapezoid (RTN) neurons. After 3–4 weeks, we examined the effect of photostimulation (~9 mW; 20 Hz, 10 ms pulses, 30 s) on breathing using plethysmography (10 rats) or diaphragmatic EMG (dEMG) recordings (4 rats). Photostimulation increased dEMG amplitude (19.2±7.4 %) and frequency (104±5 to 125±6 bursts/min, P<0.01). Photostimulation increased all plethysmographic respiratory variables including rate (from 76±3 to 108±5 breaths/min, P<0.0001), tidal volume (from 1.7±0.1 to 2.1±0.1 ml, P<0.001) and peak expiratory flow (from 7.9±0.6 to 9.7±0.9, P<0.05). In sum, the Phox2b‐expressing neurons of the RVLM regulate all aspects of breathing including inspiration and active expiration. We attribute these respiratory effects to the activation of RTN neurons but a small contribution of the C1 cells to these effects is not excluded (NIH support: HL 074011 & 28785).
In Vivo Pharmacological Alterations of Murine Carotid Body Responses to Hypoxia: The Role of BK Channels in Hypoxic Sensitivity
Authors: Pichard Luis , Sgambati Francis , Kostuk Eric , Bashai Pejmon , Fitzgerald Robert , Shirahata Machiko
Published: June 2021

Since the discovery of hypoxic inhibition of large‐conductance calcium‐activated K (BK) channels in the rat carotid body (CB), mechanisms of hypoxic sensitivity have been investigated mostly in vitro. Moreover, little is known about the role that BK channels play in the in vivo CB. The aim of this study is to explore the role that BK channels play in the hypoxic sensitivity of the in vivo murine CB. We have previously found the CB of the DBA/2J mice to have a higher expression of BK channels when compared to the A/J strain. However, BK channel opener(s) can still activate BK channels in the A/J's CB. Subsequently, hypoxia will inhibit activated BK channels, resulting in an increased neural activity. DBA/2J and A/J mice were anesthetized and artificially ventilated. Carotid sinus chemoreceptor neural activity was recorded from the central end just prior to its junction to the glossopharyngeal nerve using a glass suction pipette. Baroreceptor activity was mechanically disrupted. Following a baseline recording, the inspired gases were lowered from hyperoxia to FIO2=0.21, 0.15 and 0.10. Subsequently, we filled the space surrounding the CB with Krebs containing NS1619 to activate BK channels. After 10 minutes, the hypoxic challenge was repeated. We observed decreased baseline neural activity after application of NS1619 in both strains, suggesting opening of BK channels and hyperpolarization of cells. Also, alterations of hypoxic responses in the A/J strain became faster and more significant (although lower than DBA/2J's responses) under NS1619. Our results suggest that BK channels play a role in the hypoxic response of the murine CB in vivo. Support: F31HL096450, HL081345, AHA 09GRNT2080158, HL50712, HL61596.
Hypoxia vs hydrogen sulfide (H2S) acting at the carotid body (CB) and elsewhere systemically
Authors: Fitzgerald Robert Schaefer , Shirahata Machiko , Kostuk Eric
Published: June 2021

In vascular smooth muscle hydrogen sulfide (H2S) mimics the effects of hypoxia (HH). The purpose of this study was to determine if in the cat carotid body (CB) H2S mimicked HH. CBs were harvested from anesthetized cats, cleaned of fat and connective tissue, incubated in a salt solution at 37 C and bubbled with 30%O2/5%CO2 for 20min of recovery. Protocols and euthanizing methods were approved by the University's Animal Care and Use Committee. Set 1 of sectioned CBs (5μm) was incubated with antibody to cystathione‐β‐synthase (CBS; a H2S synthesizing enzyme); set 2, with the antibody for the Kir 6.1 subunit of the KATP channel. Set 3 was challenged with HH (6%O2/5%CO2) followed by a challenge with 100μM Na2S (precursor of H2S) while bubbled with 30%O2/5%CO2. The incubation medium was analyzed for release of two excitatory (in cat and rat) transmitters –ACh and ATP. A positive immunohistochemical signal for CBS and for the Kir 6.1 subunit were detected in the CBs’ transmitter‐containing glomus cells. And whereas HH produced a significant increase in ACh and ATP release compared to control, 100μM Na2S produced a significantly reduced release compared to control. The data suggest that Na2S may open the KATP channels producing hyperpolarization (as it does in vascular smooth muscle) reducing the release of the transmitters. Airway smooth muscle was also relaxed by Na2S in two species. Supported in part by NIH award: HL 050712
Intermittent hypoxia enhances chemical sensitivity of pulmonary C fibers in rats: role of reactive oxygen species
Authors: Shen Mei‐Ya , Luo Ya‐Ling , Lai Ching Jung
Published: June 2021

Obstructive sleep apnea (OSA) is characterized by intermittent hypoxia (IH) during sleep. Clinically, OSA may occur concomitantly with noturnal asthma and cough reflex, it is likely that involves hypersensitivity of pulmonary C fibers (PCFs) during airway inflammation. This study was performed to determine whether the excitabilities of PCFs to chemical and mechanical stimuli are altered by IH‐induced production of reactive oxygen species (ROS). In anesthetized, open‐chest, and artificially ventilated rats, delivered ten episodes (30 s of N2 + 30 s of 21% O2) of IH into the lungs significantly enhanced phrenic apnea to intravenous injection of capsaicin, a selective stimulant of PCFs. Bilateral vagotomy abolished apneic response to capsaicin both before and after IH. Subsequent recording of single‐fiber activity of PCFs (PCFA) showed that IH caused a slight increase in baseline PCFA. Additionally, IH markedly enhanced PCFA to capsaicin injection, and this enhanced response lasted for more than 30 min after termination of IH. In contrast, IH did not change the response of PCFA to lung inflation. The potentiating responses of these PCFs to capsaicin after IH were greatly attenuated by pretreatment with dimethylthiourea, a hydroxyl radical scavenger. These results suggest that IH augments the responses of the C‐fiber afferents to capsaicin, and this potentiating effect of IH is mediated through the action of ROS.
Fentanyl Turns Pulmonary C‐Fiber ‐Mediated Rapid Shallow Breathing into an Apnea in Anesthetized Rats
Authors: Zhang Zhenxiong , Zhang Cancan , Zhou Moxi , Xu Fadi
Published: June 2021

It is generally accepted that rapid shallow breathing (RSB) is primarily mediated by pulmonary C‐fibers (PCFs). A low dose of PCF stimulants induces RSB while a high dose produces an apnea, suggesting that the occurrence of RSB or an apnea depends on the excitatory degree of the PCFs. Because vagal nerves contain opioid receptors and PCF activity is increased by opioids, we asked if opioids would sensitize PCFs to turn the PCF‐mediated RSB into an apnea. Right atrial injection of the selective PCF stimulant phenylbiguanide (PBG) was given before and after intravenous administration of selective μ‐, δ‐, or κ‐receptor agonists (fentanyl, 6 μg/kg; DPDPE, 2 mg/kg; or U‐50488H, 4 mg/kg, respectively) in anesthetized and spontaneously breathing rats. PBG at a low dose (3–5 μg/kg) shortened the expiratory duration (TE) from 0.40 ± 0.04 s to 0.27 ± 0.05 s (P < 0.01), while it produced a long‐lasting apnea (0.52 ± 0.06 vs. 3.40 ± 0.46 s for TE, P < 0.01) after fentanyl. PBG at a high dose (10 μg/kg) also induced an apnea (2.3 ± 0.8 s) that was remarkably prolonged by fentanyl (9.1 ± 1.5 s). These facilitating effects of fentanyl were eliminated by naloxone (5 mg/kg, iv, an opioid receptor antagonist). DPDPE or U‐50488H failed to turn the PBG‐induced RSB into an apnea. This study suggests that opioids can facilitate the PCF‐mediated respiratory response through μ‐ rather than δ‐ or κ‐receptors. (Supported by ALA RT‐83131‐N)
The bullfrog neuroventilatory response to hypercapnia is unaffected by cannabinergic potentiation
Authors: Bajada Nicholas Bradford , Brundage Cord Michael , Taylor Barbara E
Published: June 2021

Cannabinoid type 1 receptors (CB1) are G‐protein coupled receptors widely expressed in the vertebrate CNS. Binding at CB1 autoreceptors on pre‐synaptic neurons reduces GABA release. The hypercapnic ventilatory response (HVR), an increase in ventilatory frequency seen under hypercapnic (high PCO2), may be mediated by a reduction in GABAergic activity. We tested the hypothesis that activation of CB1 autoreceptors suppresses GABAergic inhibition and augments the HVR. Brainstems from juvenile bullfrogs were isolated, and whole‐nerve activity associated with breathing was recorded. Putative breathing was recorded during superfusion with standard artificial cerebrospinal fluid (aCSF) and aCSF containing the non‐selective cannabinoid agonist WIN‐55, 212‐2 (WIN; 100 muM) combined with normocapnic and hypercapnic conditions (1.5 and 5% CO2 respectively; balance O2). WIN significantly increased putative breath frequency. Despite this potentiation there was no significant increase in neuroventilatory frequency during hypercapnia with WIN. These data suggest that, although CB1 receptors may augment neuroventilation, their function does not extend to a modulation of the bullfrog HVR.
Raphe serotonin neurons: characteristics of central chemoreceptors in situ
Authors: Iceman Kimberly Erin , Corcoran Andrea E , Harris Michael B
Published: June 2021

The location and identity of central chemoreceptors is debated, and several different neuronal populations may contribute to central chemosensitivity. Rat raphé serotonin (5HT) neurons, intrinsically sensitive to pH change in vitro, are proposed as chemoreceptors in vivo. Characterization of 5HT neurons as chemoreceptors is refuted, as electrophysiological evidence for chemosensitive 5HT neurons is lacking in vivo. We address this controversy using the unanesthetized juvenile rat in situ perfused decerebrate brainstem preparation. This preparation displays respiratory patterns and reflexes comparable to an intact animal. Through extracellular recording in medullary raphe we demonstrate both chemosensitive and insensitive neurons, including cells with firing patterns expected of 5HT neurons. We also report results of extracellular recording combined with juxtacellular labeling of raphe neurons, identifying neurotransmitter phenotypes of sensitive and insensitive cells. Our results support the presence of chemosensitive 5HT neurons in the raphe in situ. Given the intrinsic chemosensitivity of such neurons in vitro, our data support a role for raphe 5HT neurons as central chemoreceptors in vivo. Supported by NIH 2U54NS041069‐06A1
Essential role of Mecp2 in the regulation of myofibroblast differentiation
Authors: Hu Biao , Gharaee‐Kermani Mehrnaz , Wu Zhe , Phan Sem H.
Published: June 2021

DNA methylation is a key mechanism of gene expression and previous studies suggest it suppresses α‐smooth muscle actin (α‐SMA) gene expression during myofibroblast differentiation. However, the trans‐acting factors that interact with the methylated α‐SMA gene and regulate its expression, have not been identified. Using gel shift and ChIP assays, MeCP2 was shown to bind to the methylated α‐SMA gene. Suppression of MeCP2 gene expression by siRNA or its deficiency in MeCP2 knock out mice led to reduced α‐SMA gene expression in isolated lung fibroblasts. In contrast, transient transfection of MeCP2 expression plasmid into fibroblasts enhanced α‐SMA gene expression. Further studies indicated that, compared to their wild type littermates, MeCP2 deficient mice exhibited significantly decreased alveolar wall thickness, inflammatory cell infiltration, interstitial collagen deposition and myofibroblast differentiation upon endotracheal injection of bleomycin. Thus MeCP2 is essential in myofibroblast differentiation and pulmonary fibrosis.
Novel role of NF‐κB‐dependent miR‐146a induced by diazoxide preconditioning in stem cell survival
Authors: Suzuki Yoko , Kim Ha Won , Haider Husnain Kh , Ashraf Muhammad
Published: June 2021

We have previously reported that preconditioning (PC) of bone marrow‐derived mesenchymal stem cells (MSCs) with diazoxide (DZ) improved their survival via NF‐κB signaling. MicroRNAs (miRs) are critical regulators for a wide variety of biological events including apoptosis and differentiation. However, the role of DZ‐induced miRs in stem cell survival remains unknown. Here we show that miR‐146a is the major miR induced by DZ treatment and plays a pivotal role in MSCs survival through NF‐κB signaling. PC of MSCs with DZ (200 μM) for 3 hrs significantly increased phosphorylation of Akt as well as nuclear NF‐κB translocation, thereby improving MSCs survival as evaluated by lactate dehydrogenase (LDH) release assay (45% decrease). Importantly, miR‐146a expression was increased by DZ treatment and knockdown of miR‐146a abolished DZ‐induced cytoprotective effects. In addition, knockdown of NF‐κB ablated DZ‐induced miR‐146a expression as well as MSCs survival, indicating miR‐146a is a downstream of NF‐κB and plays a key role in stem cell survival. We are in the process of determining the target genes of miR‐146a by using PCR array as well as computational analysis. Together, these data demonstrated that cytoprotection afforded by PC with DZ was regulated by miR‐146a which may be a novel therapeutic target for stem cell transplantation therapy.

Grant supports: NIH R37 HL074272, HL080686, HL087246, HL087288, HL089535
X‐ray, proton and iron ion irradiation all increase adhesiveness of aortic endothelium and may accelerate development of atherosclerosis.
Authors: Kucik Dennis F , Khaled Saman , Gupta Kiran , Wu Xing , Yu Tao , Chang Polly Y
Published: June 2021

Radiation from a variety of sources is associated with increased risk of atherosclerosis. The molecular mechanism for this is not known. With the hypothesis that radiation's pro‐atherogenic effect is due, at least in part, to a direct, pro‐adhesive effect on vascular endothelium, our objective was to determine the adhesive response of cultured primary human aortic endothelial cells (HAECs) to both X‐rays and the high energy radiation likely to be encountered in prolonged space flight.

Vascular EC adhesiveness was assayed using a portable flow‐chamber adhesion assay for X‐rays at UAB and for components of cosmic radiation (iron ions and protons) at Brookhaven National Laboratory. Adhesiveness was scored as the number of un‐irradiated monocytic cells that adhered to HAECs per minute under physiological shear stress. We found that radiation increased HAEC adhesiveness, and that the effect depended on dose, time, and type of radiation. Changes in expression of HAEC adhesion molecules, however, were small and may not account for the pro‐adhesive effect.

We conclude that both X‐rays and cosmic radiation increase HAEC adhesiveness, with the potential to increase risk for atherosclerosis. Current work is aimed at identifying molecular mechanisms. Results will be compared to pro‐atherogenic effects of all three types of radiation in a mouse model.

This work was supported by a grant from NSBRI through NASA NCC 9–58.
Positive Inotropic and Lusitropic Effects of Acetylcysteine on HIV‐Tat Transgenic Mice
Authors: Chen Fangping , Lewis William , Xie Sherry , Finkel Mitchell S.
Published: June 2021

We and others have proposed that the cardiomyopathy identified in patients with HIV infection is at least partially mediated through direct effects of HIV proteins on the heart. Our previous data supporting direct myocardial effects of HIV proteins include anatomical, biochemical and physiologic defects in transgenic mice expressing HIV Tat protein targeted to the myocardium, +/− Tat transgenic mice (TG). TG reveal a relative bradycardia by 3 mos, followed by systolic and diastolic dysfunction and mitochondrial defects at 6 mos of age. We further explored possible mechanisms by which mitochondrial defects could contribute to the hemodynamic dysfunction in this animal model. We previously reported salutary effects of the membrane permeable sulfhydryl donor, N‐acetyl‐L‐cysteine (NAC), on excitation‐contraction coupling in another cardiomyopathy model. Accordingly, we treated cardiac myocytes isolated from 6 mos old TG with NAC and noted significant improvements in baseline fractional shortening (%FS; 5.06 ± 0.7 vs. 9.9±0.7; p=0.02; n=8), contractility (+dl/dt; 118.5±12 vs. 272±27; p=0.03; n=8) and lusitropy (−dl/dt; 229± 17 vs 395± 43; p=0.03; n=8). However, the dose‐response curve for the beta adrenergic agonist, isoproterenol, was not significantly affected by NAC (p=NS; n=8. ANOVA). These findings suggest a possible mechanism linking mitochondrial pathology with myocardial dysfunction in TG.
Protective effect of Cardiotonic Pills on ischemia‐reperfusion induced microcirculatory disturbance and myocardial damage in rats
Authors: Han Jing‐Yan , Zhao Na , Liu Yu‐Ying , Wang Fang , Hu Bai‐He , Sun Kai , Chang Xin , Zhao Xin‐Rong , Liu Lian‐Yi , Wei Xiao‐Hong , Yang Ji‐Ying , Wang Chuan‐She , Guo Zhi‐Xin , Fan Jing‐Yu
Published: June 2021

Cardiotonic Pills ® (CP) is a compound preparation widely used in China for the treatment of cardiovascular disease. However, limited data is available regarding the mechanism of action of CP on myocardial function during ischemia‐reperfusion (I/R) injury. In this study, we examined the effect of CP on I/R‐induced coronary microcirculatory disturbance and myocardial damage. Male SD rats were subjected to left coronary anterior descending branch occlusion for 30 min followed by reperfusion with or without pre‐treatment with CP (0.1, 0.4, or 0.8 g/kg). Coronary blood flow, vascular diameter, velocity of RBC, and albumin leakage were evaluated in vivo. In parallel, neutrophil expression of CD18, malondialdehyde, and myocardial infarction, endothelial expression of ICAM‐1, apoptosis‐related proteins, histological and ultrastructural of myocardial damage were assessed. Pre‐treatment with CP (0.8 g/kg) attenuated the I/R‐induced myocardial microcirculatory disturbance. In addition, the drug significantly ameliorated the I/R‐induced myocardial damage and apoptosis indicated by increased malondialdehyde, infarct size, myocardial ultrastructural changes, terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling‐positive myocardial cells, and expression of Bcl‐2, Bax and caspase‐3 in myocardial tissues. The results provide evidence for the potential role of CP in preventing microcirculatory disturbance and myocardial damage following I/R injury.
Early and late effects of iron deficiency on heart and baroreflex function
Authors: Chew Herbert G , Almaw Bethelhem H , Gandy Kayla C , Oh Wunyong , Tolhurst Matthew J
Published: June 2021

Iron deficiency (ID) induces cardiac and neural alterations, such as cardiac hypertrophy and apoptosis, electron transport dysfunction, and brain mitochondrial disruption. Cardiac norepinephrine (NE) depletion suggests chronically high sympathetic nervous system (SNS) traffic to the heart. Thus, ID would be expected to affect heart function and blood pressure, and our previous experiments have shown baroreflex alterations after 4 weeks of ID.

HYPOTHESES

(1) Chronic high SNS output initially leads to a hyperfunctional heart with higher left ventricular pressure (LVP) and heart rates (HR) at each mean arterial pressure (MAP), and (2) eventual depletion of NE, along with heart apoptosis and debility, is coincident with reduced LVP and HR at all MAP.

METHODS

Rats were fed control (CN) or ID diets for 2 or 4 weeks. Baroreflex experiments were conducted using ramp (before and after atropine) and steady state protocols. Hearts were excised for Langendorff experiments to obtain LVP at set flow rates.

RESULTS

Hematocrit verified ID (p ≤ 0.05); heart mass verified hypertrophy. Baroreflex preliminary data suggests higher HR at all MAP at 2 weeks, but lower HR after 4 weeks; experiments continue at time of writing. Langendorff data indicates higher LVP with ID at 2 weeks, but lower LVP after 4 weeks.

CONCLUSION

Changes with ID are adaptive at 2 weeks but pathological after 4 weeks. Supported by WY INBRE (LC4090).
Biochip Array Profiling of Neuron Specific Enolase, Neutrophil Gelatinase‐associated Lipocalin, Soluble Tumor Necrosis Factor Receptor I, D‐Dimer, Thrombomodulin, and C‐reactive Protein in End Stage Renal Disease
Authors: Davis Rachael , Bansal Vinod , Litinas Evangelos , Rahman Saudur , Hoppensteadt Debra , Vinchhi Dipesh , Fareed Jawed
Published: June 2021

Plasma samples from 39 patients with clinically confirmed end‐stage renal disease (ESRD) were collected prior to a maintenance hemodialysis. A pilot group of ten normal individuals, both male and female, were included as control. Cerebral Array II chips were used in the Randox® system to simultaneously measure Neuron Specific Enolase (NSE), Neutrophil Gelatinase‐associated Lipocalin (NGAL), Soluble Tumor Necrosis Factor Receptor I (TNFRI), D‐Dimer (DD), Thrombomodulin (TM), and C‐reactive protein (CRP). As compared to normals, all markers studied showed an upregulation in ESRD patients. Most notably TNFRI showed a 25 fold increase in patients with ESRD (mean 7.4 ± 2.4, range 3.1 to 13.6) compared to the control (mean 0.3 ± 0.1, range 0.2 to 0.5). TM showed a 6.6 fold increase (mean 7.0 ± 2.5, range 2.5 to 14.1) compared to control (mean 1.1 ± 0.2, range 0.8 to 1.4), and NGAL showed a 5.5 fold increase (mean 1416 ± 203, range 839 to 1729), compared to control (mean 257 ± 74, range 115 to 377). Increases in CRP and DD were also noted up to 2.8 fold. These studies show that some newer markers such as TNFRI, NGAL and NSE are upregulated in ESRD. However, the clinical significance of these markers still needs to be further explored. In addition, this study validates the role of endothelial damage and endogenous thrombotic processes in ESRD as evidenced by the increased levels of thrombomodulin and D‐Dimer.
Sex‐specific adaptation of coronary vasculature to post‐myocardial infarction remodeling in middle‐aged rats
Authors: Dedkov Eduard I , Oak Kunal , Christensen Lance P , Tomanek Robert J
Published: June 2021

Increasing evidence indicates that sex can influence left ventricular (LV) remodeling after myocardial infarction (MI). The current study addressed coronary vasculature adaptations during post‐infarction LV remodeling in 12‐month‐old female rats (MI‐F) and compared the data with previous findings on age‐matched males (MI‐M). MI was induced by left coronary artery ligation and sham‐operated rats served as a control. For a final analysis, 4 weeks after MI, we selected the MI‐F rats with the infarct sizes similar to that in MI‐M rats (67.2% vs. 63.8% of the LV free wall [LVFW], respectively). We found that in the LVFW of MI‐F rats the compensatory angiogenesis, i.e. 22.1% (p≤0.05) increase in capillary to myocyte ratio, had exclusively occurred in endomyocardium where it associated with the 58.7% (p≤0.01) enlargement of myocyte cross‐sectional area. In contrast, MI‐M rats have demonstrated significant compensatory angiogenesis associated with myocyte hypertrophy only in epimyocardium. The intraventricular septum remained unaffected in both groups of MI rats. Although, post‐MI remodeling of LVFW led to a significant increase in arteriolar length density in both MI‐F and MI‐M rats (27.6% vs. 22.4%, respectively [p≤0.05]), only MI‐M rats demonstrated the notable increase (28.1%, p≤0.05) in arteriolar growth in the intraventricular septum. Our data provided the first evidence documenting the existence of sex‐related differences in regional adaptation of the coronary vasculature during 4 weeks of post‐MI remodeling. Supported by NIH RO1‐HL62587.
Novel TIE‐2 phosphatase inhibitors restore angiogenesis in RAS suppressed Sprague Dawley rats
Authors: Stodola Timothy , Greene Andrew
Published: June 2021

The TIE‐2 receptor binds with Angiopoietins (Ang‐1), and activation triggers signaling pathways associated with endothelial cell migration, proliferation, survival, permeability, sprouting and tube formation. Previous results showed novel HPTPβ inhibitors could selectively inhibit dephosphorylation of the TIE‐2 receptor. The goal of this study was to determine if these drugs could restore angiogenesis in Renin‐Angiotensin System (RAS) suppressed Sprague Dawley (SD) rats. Seven week old SD rats were placed on 4% NaCl diet to suppress RAS. Electrical stimulators were implanted to intermittently stimulate the peroneal nerve in one hind leg, contracting the Tibialis Anterior (TA) and Extensor Digitorum Longus (EDL) muscles for 8 hours/day for 7 days while they were injected with AKB‐9328 (15 mg/kg) or AKB‐9778 (5, 7.5, 10, 15, 25 mg/kg) s.c. b.i.d. High salt animals receiving vehicle had a 0.9% increase in vessel density, while low salt controls had a 13.6% increase in vessel density. Animals with doses at and above 10 mg/kg of either drug showed significant increases in vessel density, while doses of 5 and 7.5 mg/kg did not show significant increases in vessel density. These data show inhibition of TIE‐2 receptor dephosphorylation can restore physiological angiogenesis in high salt inhibited rats. Supported by Akebia Theraputics, Inc, project AG‐08‐001 and AG‐09‐001.
Prolonged treadmill walking attenuates ischemic pressor response and baroreflex control in Yucatan pigs with femoral artery occlusion
Authors: Yang Hsiao Tung , Terjung Ronald L , Bowles Douglas K , Laughlin Harold M
Published: June 2021

Exercise‐induced hypertension can be extensive in intermittent claudicants (Bakke et al., 2007), likely due to exaggerated sympathetic activation, in part, from peripheral muscle afferents. To study whether exercise training alleviates this ischemic pressor response, Yucatan pigs (35 to 40 kg) received femoral artery occlusion. One wk later, pigs either ran on a treadmill (12 wk, 5 d/wk, at 3.1 mi/hr 0% grade) until fatigue (~30–60 min) (Tr: n=9), or limited to cage activity (Sed: n=8). A stress test by running @1 to 5 mile/h; Aortic blood pressure (BP), heart rate (HR) and collateral blood flow (BF) were monitored at each speed. Baroreflex function was assessed by adenosine (Aden) or Phenylephrine (PE) i.v. BP was 158±2.8 and 140±3.3 at wk 3, and 135±5.8 and 122±4.1 mmHg at 12 wks in Sed and Tr groups (p<0.05), respectively. BFs was ~24% higher in Tr as compared to Sed animals (122±25.3 ml/min; p>0.05). The slope for HR increase to Aden‐induced BP decline was 0.30±0.11 in Tr and 1.6±0.43 in Sed group (p<0.01); whereas, HR decreases to BP increases by PE were not different between groups. Our data showed that exercise training can reduce the exercise‐pressor response, coincident with a modified baroreflex control and possibly related to a modified peripheral muscle afferent input. These findings imply that physical training can temper the exercise‐triggered hypertension in claudicants.

Supported by NIH P01HL 52490