bluuebunny/crossref_metadata_2025 huggingface dataset:
ACE2 Inhibits Angiotensin‐II Mediated NADPH Oxidase Activation In The Central Nervous System
Authors: Xia Huijing , Suda Sonia , Xu Ping , Chhabra Kavaljit , Lazartigues Eric
Published: June 2021

ACE2 plays a protective role in the central regulation of autonomic function by reducing oxidative stress. To assess the mechanisms involved, ACE2 KO and non transgenic (NT) mice (n=6) were infused with AngII (osmotic pump: 600 ng/kg/min sc) and infected with a hACE2 or control (eGFP) adenovirus (2×106 pfu, 200 nL) in the PVN 3d post infusion. After 10d of infusion, NADPH oxidase (NOX) and superoxide dismutase (SOD) activity (U/mg), and SOD 1 and 2 protein expression (Immunofluorescence, arbitrary units) were assessed in the brainstem. NOX (2.8 ±0.1 vs. 1.0 ±0.02) and SOD activities (1.4 ±0.1 vs. 1.0 ±0.05; P<0.05) were significantly increased in KO mice (P<0.05 vs. NT), as well as SOD1 (2.5 ±0.6 vs. 1.0 ±0.04) and SOD2 (1.7 ±0.1 vs. 1.0 ±0.05) expression (P<0.05 vs. NT). Post AngII, NOX activity was increased in both KO (3.1 ±0.2) and NT (2.1 ±0.2) and remained higher in the KO (P<0.05 vs. NT), while SOD1 and 2 expression and activity were only increased in the NT (2.3±0.6; 1.6±0.1; 1.2±0.1; respectively, P<0.05 vs. baseline). ACE2 over‐expression reduced NOX activity in the KO without changing SOD expression or activity. These data suggest that ACE2 gene deletion promotes AngII‐mediated oxidative stress, and ACE2 compensation in the PVN decreases oxidative stress in the brainstem by inhibiting NOX activation, thus contributing to the improvement of autonomic function in this model. (HL093178 and APS Postdoctoral Fellowship)
INVOLVEMENT OF THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS (PVN) IN CARDIOVASCULAR AND AUTONOMIC MODULATION DURING LPS ENDOTOXEMIA IN AWAKE RATS
Authors: Martins‐Pinge Marli Cardoso , de Abreu Samantha Bagolan , Pinge‐Filho Phileno
Published: June 2021

During sepsis, areas of the central nervous system are activated and have a role in physiological adaptations to immunity challenge. The aim of this study was to evaluate the involvement of the paraventricular nucleus of the hypothalamus (PVN) on mean arterial pressure (MAP), heart rate (HR) and spectral analysis of HR variability (HRV) after administration of lipopolysaccharide (LPS) in conscious rats. Male Wistar rats were anesthetized for implantation of guide cannulae to the PVN. After 5 days the rats were anesthetized for chronic catheterization of the femoral artery and vein. 24 hours later it was performed in bilateral PVN of conscious rats, microinjection of 100 nl of saline or muscimol followed by LPS (iv). Cardiovascular parameters were analyzed by 2 hours. Saline microinjection did not modify the baseline parameters and after LPS was observed hypotension and tachycardia (Δ MAP= −35 ± 9 mmHg, Δ HR = 31 ± 14 bpm). Muscimol microinjection caused less decrease in MAP and a higher increase in HR (ΔMAP=−1±9 mmHg, ΔHR=122±31 bpm). The HRV was abolished during sepsis (Basal = 58.25 ± 15.95; Sepsis = 4.67 ± 1.61) and was not different for arterial pressure. Muscimol did not alter HRV in sepsis, but decreased the variability of BP (Saline = 20.04 ± 3.11; Muscimol = 10.93 ± 2.06). Our data suggest an involvement of PVN in cardiovascular and autonomic modulation during the initial phase of sepsis by LPS.
Chronic increases in dorsal hindbrain (DHB) corticosterone (Cort) enhance the blood pressure response to restraint stress without changing peripheral Cort
Authors: Daubert Daisy L. , Dong Ying , Scheuer Deborah A.
Published: June 2021

Chronic increases in dorsal hindbrain (DHB) corticosterone (Cort) enhance the blood pressure response to restraint stress without changing peripheral Cort. We tested the hypothesis that blockade of peripheral vasopressin V1 receptors (AVPX) would normalize the blood pressure response to restraint in DHB Cort‐treated rats. DHB 10% Cort or Sham pellets were implanted in male Sprague‐Dawley rats. Blood pressure was measured by radiotelemetry during a control period, 60 min of stress and 30 min post‐restraint on days 6 and 13. A vasopressin antagonist (AVPX, i.v.) was administered on one day, and saline was administered on the other day. Neither DHB Cort nor AVPX altered baseline arterial pressure, which averaged between 97±3 and 101±3 mmHg per group. DHB Cort significantly increased the arterial pressure response to stress and recovery in saline experiments (total integrated increase 1071±59 and 1628±267 mmHg in Sham‐ and Cort‐treated rats respectively, P<0.05). AVPX normalized the arterial pressure response only during the second 30 min of stress in Cort‐treated rats (566±86 and 340±68 mmHg in saline‐ and AVPX‐treated Cort rats respectively, P<0.05), but had no significant effect in Sham‐treated rats. (356±45 and 300±71 mmHg in saline‐ and AVPX‐treated Sham rats respectively). The data suggest that Cort acts in the DHB to recruit vasopressin secretion in response to restraint stress. Funded by NIH grant #HL 076807.
HYPOTHALAMIC REGIONS ACTIVATED DURING VOLUNTARY EXERCISE AND AIRPUFF STARTLE
Authors: Dampney Roger , Lam Alex , Horiuchi Jouji , McDowall Lachlan , Furlong Teri , Iiigaya Kamon , Polson Jaimie
Published: June 2021

Both exercise and acute psychological stress are associated with stereotyped cardiovascular and respiratory responses. In this study we used the method of Fos expression to compare the pattern of neuronal activation in the hypothalamus of rats after either voluntary exercise or a mild acute psychological stress (air puff startle). In the exercise group, rats (n=5) had free access to a running wheel. Two hours after the period in which the running activity was maximal (4–6 am), rats were euthanized with an overdose of sodium pentobarbitone, and the brains removed and processed to identify Fos‐positive neurons. Control rats (n=4) were housed without the running wheel. Another group of rats (n=4) were subjected to air puff startle and euthanized 2 hours later. After voluntary exercise there was a marked increase in Fos expression in the hypothalamic perifornical area compared with the dorsomedial hypothalamus (P < 0.05), whereas after air puff startle the reverse was the case (P < 0.05). A high proportion (> 60%) of orexin‐containing neurons in the perifornical area expressed Fos after voluntary exercise. The results suggest that the central mechanisms generating cardiorespiratory responses to stress and exercise may, at least at the level of the hypothalamus, be quite different.
Altered balance of excitatory/inhibitory synaptic inputs in supraoptic (SON) and paraventricular (PVN) neurosecretory neurons in heart failure rats.
Authors: Potapenko Evgeniy , Biancardi Vinicia Campana , Stern Javier E
Published: June 2021

Neurohumoral hyperactivation, including enhanced vasopressin (VP) release, is a hallmark of heart failure (HF). VP release from neurosecretory neurons in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei is tightly controlled by their electrical activity, which in turn depends on a balanced glutamate (GLU) excitatory and GABA inhibitory synaptic inputs. Here, we investigated if changes in the GABA/GLU balance in SON neurons may constitute an underlying mechanism in VP overactivation in HF. GABA and GLU IPSCs/EPSCs were simultaneously recorded in SON neurons from sham and coronary ligated HF rats (n= 17 and 27, respectively). Our data indicate an enhanced excitatory synaptic strength (~60% longer lasting and ~52% larger potency EPSCs), concomitant with a blunted inhibitory synaptic strength (~ 35% faster and ~38% reduced potency IPSCs) in neurons from HF rats. The GABA/GLU strength ratio within each cell was decreased in HF rats (~50%, P<0.01). Blockade of GABA inputs with 15 μM gabazine prolonged EPSCs duration by ~20%, in sham but not in HF rats. These results support that a shift in the GABA/GLU balance towards an excitatory predominance may contribute to enhanced VP release in HF. Supported by NIH HL68725.
Altered Blood Brain Barrier Integrity in Hypertensive Rats: Role of Angiotensin II.
Authors: Son Sook Jin , Ahmadi Sahra , Filosa Jessica A. , Stern Javier E.
Published: June 2021

The blood brain barrier (BBB) acts as a physical barrier and a transport mechanism in the CNS, restricting access of systemic harmful chemicals while allowing entrance of essential nutrients. Growing evidence supports compromised BBB integrity under pathological conditions. Here, we aimed to assess if BBB integrity is altered in hypertensive rats. WKY and SHR rats at two developmental stages (7–8 & 11–13 week old) received intracarotid infusions of two fluorescently labeled dyes, RHO‐dextran 70kD and FITC‐dextran 10kD (28 mg/kg.bw, 30 sec). Dyes were allowed to circulate for 20min before the brains were removed, fixed, and processed for confocal imaging. BBB integrity was assessed based on the degree of dye leakage into the extravascular CNS space of hypothalamic and brainstem nuclei. RHO‐70 (larger dye) was largely confined within microvessels, and no differences in extravascular leakage were detected between WKY and SHRs (P>0.5). Conversely, extravascular FITC‐10 mean fluorescence intensity significantly increased in the PVN, NTS and RVLM of SHR compared to WKY rats at both stages (20–200% increase). These changes were largely prevented in SHR rats treated with Losartan (0.2mg/ml, 7 weeks). Our results supports the notion of a disrupted BBB within key CNS areas involved in cardiovascular control in hypertensive rats, and further suggests an AngII‐dependent mechanism. Supported by AHA 0640092N.
Prevention of Mycobacterium avium subsp. paratuberculosis (MAP) infection in BALB/c mice by feeding probiotic Lactobacillus acidophilus NP‐51®
Authors: Osman Mohamed A , Stabel Judy R , Hostetter Jesse M , Nettleton Daniel S , Beitz Donald C
Published: June 2021

The objective of this study was to examine effects of feeding Lactobacillus acidophilus strain NP51 to mice challenged with Mycobacterium avium subspecies paratuberculosis (MAP). Mice were randomized to ten treatment groups; sentinels, control, heat‐killed MAP, viable MAP, heat‐killed NP51, viable NP51, heat‐killed NP51 plus heat‐killed MAP, heat‐killed NP51 plus viable‐MAP, viable‐NP51 plus heat‐killed MAP, viable‐NP51 plus viable‐MAP. Mice were fed 1 × 106 CFU of NP51· mice−1· day−1. On day 45, mice were challenged with 1 × 108 CFU of MAP intraperitonealy. We hypothesized that feeding NP51would increase Th‐1 responses and decrease progression of Johne's disease in mice. Ten mice from each group were euthanized on days 45, 90, 135 and 180. Supernatante from in vitro splenocyte culture was examined for IFN‐γ production. Feeding Heat‐killed NP51 to heat‐killed MAP‐ or viable MAP‐infected mice increased interferon (IFN)‐γ by 1.4‐ and 4‐fold (44.32 and 127.14 vs. 31.69 pg/mL, respectively) over that of heat‐killed MAP‐infected control. Similarly, feeding viable‐NP51 to heat‐killed MAP‐ or viable MAP‐infected mice increased IFN‐γ by 3‐ and 4‐ fold (98.6 and 130.68 vs. 31.69, respectively) over that of heat‐killed MAP‐infected control. These data suggest that feeding NP51 can simulate IFN‐γ production and prevent progression of Johne's disease in mice.

Funding source: Nutrition Physiology Company, Guymon, OK.

Grant Funding Source: Nutrition Physiology Company
GLUTAMATE SUPPRESSES HELICOBACTER PYLORI‐INDUCED GASTRIC ATROPHY ‐MECHANISMS OF CHIEF CELL PROTECTION
Authors: Nakamura Eiji , Hasumura Mai , Gabriel Ana San , Tashima Kimihito , Amagase Kikuko , Uneyama Hisayuki , Takeuchi Koji , Torii Kunio
Published: June 2021

Luminal free glutamate (Glu) induces gastroduodenal mucus secretion, suggesting the protective roles of dietary free Glu during digestion process. We have previously reported that Glu supplemented in the diet reduces Helicobacter pylori (HP)‐induced gastric mucosal damages in gerbils. Surprisingly, gastric atrophy (i.e. the loss of pepsinogen‐secreting chief cells and acid‐secreting parietal cells) was also reduced by Glu supplementation. To clarify the mechanisms how Glu reduces gastric atrophy against HP, we investigated the direct protective role of Glu on the cell viability of cultured chief cells against ammonia, a known HP toxin. NH4Cl (an ammonia donor) significantly reduced cell viability. Glu, glutamine, asparagine and aspartate all protected cultured chief cells against NH4Cl. Pharmacological analysis revealed that Glu synthetase inhibitors, L‐methionine sulfoximine and L‐2‐amino adipic acid protected chief cells against NH4Cl‐induced cell death. In contrast, glutathione synthetase inhibitor, L‐buthionine sulfoximine had no effect on Glu‐induced chief cell protection against NH4Cl. These results suggest that Glu reduces HP‐induced gastric atrophy partly via glutamine synthetase‐mediated pathway. Thus, we propose that free Glu in the diet would contribute to the protection from gastric atrophy against HP in daily life.
Abnormal Reactivity of Afferent Neuronal Pathway from the Heart to the Brain in Heart Failure Patients
Authors: Suzuki Hideaki , Hirose Masanori , Wakayama Yuji , Watanabe Satoshi , Miura Shoko , Fukudo Shin , Shimokawa Hiroaki
Published: June 2021

Background

The involvement of afferent neuronal pathway from the heart to the brain in heart failure (HF) remains unknown.

Methods and Results

We recorded cardiac cerebral evoked potentials (CEPs) induced by ventricular (VVI) pacing in 21 patients with or without HF [64.5±2.6 yrs (SEM), 16 males and 5 females] within a week after pacemaker implantation or cardiac resynchronized therapy. Electroencephalogram (EEG) was simultaneously recorded from the vertex (Cz, international 10–20 system) during the VVI pacing of control stimulation (1.5 V) and an intense stimulation [6–8 (7.21±0.15) V] at 70–100 (80.5±1.9) bpm for 2 min. We averaged 100 EEG waves triggered by the VVI pacing to obtain CEPs. Cardiac CEPs were obtained by subtracting CEPs with control stimulation from those with intense stimulation. In Stage B/C HF patients (n=7 each), cardiac CEPs were reproducibly identified with first negative (N1) peak at 67–93 msec, first positive (P1) peak at 128–147 msec and second negative (N2) peak at 180–194 msec after the stimuli, whereas no such clear CEPs were detected in the remaining 7 patients without organic heart disease and HF.

Conclusions

These results suggest that the abnormal reactivity of cardiac afferent neurons from the heart to the brain is associated with development of HF.
Hyaluronic acid attenuates expression of HIF‐1 alpha and iNOS in experimental rheumatoid arthritis
Authors: Chang Pei‐Lin , Hsieh Yueh‐Ling , Tu Pin‐Wen , Chou Li‐Wei , Ou Hsiu‐Chung
Published: June 2021

Rheumatoid arthritis (RA) is known to be associated with hypoxia‐related joint diseases. The role of hyaluronic acid (HA) on management of RA and its possible anti‐hypoxic abilities are not well known. The purpose of this study is to assess the effects of intra‐articular injection of HA on expression of hypoxia‐inducible factor‐1 alpha (HIF‐1α) and inducible nitric oxide synthase (iNOS) in adjuvant‐induced arthritis rats. Adult SD rats with complete Freund's adjuvant‐induced arthritis (CIA) were subjected to short‐term (day 1) or long‐term (days 1, 5, 10) intra‐articular HA treatments. Buffer‐treated CIA rats served as arthritis controls. Effects of HA in CIA animals were determined by von Frey filament test, joint circumference assessment and HIF‐1α and iNOS immunohistochemistry. The extent and grade of joint swelling and pain behavior in the long‐term HA injection rats were less severe than those of short‐term HA injection and control rats. HA significantly suppressed expressions of HIF‐1α and iNOS in cartilage and synovium after long‐term HA injection. Results indicated that presence of hypoxia in inflamed joints of this CIA model is suppressed by HA dose‐dependent manner. Therefore, down‐regulation of HIF‐1α and iNOS might be important mechanisms of intra‐articular injection of HA in the treatment of RA. The study is supported partially by a grant from the China Medical University (CMU98‐S‐10) of Taiwan.
Hypoxia induced periventricular white matter damage in the developing brain
Authors: Kaur Charanjit , Tay Samuel SW
Published: June 2021

Normal development and maturation of the brain are affected by hypoxia. The white matter peripheral to the lateral ventricles, called periventricular white matter (PWM), is selectively vulnerable to hypoxic damage in premature infants. PWM damage (PWMD) contributes significantly to neonatal mortality and long‐term neurodevelopmental deficits such as cerebral palsy, impaired vision, hearing impairments and mental retardation. This study investigated the hypoxic damage to the PWM in the neonatal brain and the potential of melatonin in ameliorating the damage. PWM of 1‐day old rats was examined at 3 hours to 7days after hypoxic exposure. Vascular endothelial growth factor (VEGF), nitric oxide (NO), glutathione (GSH) and malondialdehyde (MDA) content in the PWM was examined following the hypoxic exposure. VEGF, NO and MDA concentration was increased whereas that of GSH was reduced in the PWM after the hypoxic exposure. Along with the above, degenerating axons, apoptotic and necrotic cells and vacuolation of capillary endothelial cells was observed. The neighbouring ependymal and choroid plexus in the lateral ventricles also appeared to undergo structural alteration. Increased vascular permeability in the PWM of hypoxic rats was evidenced by the leakage of rhodamine isothiocyanate (RhIC) which was taken up by the amoeboid microglial cells. Hypoxic rats treated with melatonin showed decreased VEGF, NO and MDA concentration, increased GSH content and reduced RhIC leakage in the PWM. The ultrastructure of axons, endothelial, ependymal and choroid plexus cells appeared relatively normal in the hypoxic animals treated with melatonin. The results of this study suggest that melatonin has the potential to protect the developing white matter against hypoxic damage and its protective effect was possibly due to its antioxidant properties.
Reactive Oxygen Species‐Mediated Activation of NFAT Leads to Pulmonary Hypertension
Authors: Bosc Laura Gonzalez , Nitta Carlos , Friedman Jessica
Published: June 2021

Reactive oxygen species (ROS) are known to be elevated in pulmonary hypertension. ROS has been shown to activate the Ca+2‐calcineurin‐NFAT pathway. We have shown that NFATc3 is activated in pulmonary arteries (PA) from chronic hypoxia (CH) mice. Also, NFATc3 is required for CH‐induced pulmonary arterial hypertension (PAH). We hypothesized that elevated ROS levels increase NFAT activity leading to PAH in superoxide dismutase 1 −/− mice (SOD1). To test this hypothesis, we backcrossed NFAT‐luciferase reporter mice with SOD1 +/− until NFAT‐luciferase/SOD1 +/+ and −/− were generated. Right ventricular systolic pressure (RVSP) and right ventricle/total heart weight (% RV/T) were measured in four month old mice. Intra PA were isolated to measure luciferase activity which reflects NFAT activation. Lung sections were stained with dihydroethidium to determine ROS levels. Results: NFAT activity is significantly elevated in PA from SOD1 −/− vs. SOD1 +/+ mice (0.9±0.1 vs. 0.5±0.1 AU/μg protein, 7, p<0.02). RVSP is also significantly elevated in SOD1 −/− (31.4±3.6 vs. 22.2±2.2 mmHg, 5, p<0.05) although RV hypertrophy was not observed. ROS levels were significantly elevated in PA from SOD1 −/−. These results suggest that increased ROS levels in the SOD1 −/− mice lead to NFAT‐mediated PAH. Future studies will address this possibility by inhibiting NFAT in the SOD1 −/− to reverse the elevated RVSP. Supported by NIH R01HL088151.
Antenatal Maternal Hypoxic Stress: Epigenetic Adaptations in Fetal Lung Renin‐Angiotensin System
Authors: Goyal Ravi , Leitzke Arthur , Goyal Dipali , Gheorghe Ciprian P. , Longo Lawrence D.
Published: June 2021

Antenatal maternal hypoxia (AMH) can lead to intra‐uterine growth restriction (IUGR), as well as idiopathic pulmonary hypertension of newborn and adult. This may be a consequence of alterations in the local pulmonary renin‐angiotensin system (RAS). Little is known of these adaptations, however. Thus, we tested the hypothesis that antenatal maternal hypoxia is associated with fetal programming of the pulmonary renin‐angiotensin system. In FVB/NJ mice, we studied mRNA and protein expression, as well as promoter DNA methylation and miRNA levels in response to 48 hours hypoxia (10.5% O2) at 15.5 day post coitum (DPC). In response to AMH, the pulmonary mRNA levels of angiotensin converting enzyme (ACE) 1.2, ACE‐2 and angiotensin II type 1b (AT‐1b) receptors were increased significantly, as compared to controls (N=4). In hypoxia fetal lungs, protein levels of renin and ACE‐2 also were increased significantly, whereas ACE‐1 protein expression was reduced. In hypoxic fetal lungs, we also observed reduced expression of the miRNAs: mmu‐mir −199b, −27b, −200b, and −468 that putatively increase the translation of renin, ACE‐1, ACE‐2, and AT‐1 receptors, respectively. Promoter methylation of ACE did not change with AMH. We conclude that AMH leads to changes in expression of pulmonary RAS of fetal mice. This may have significant implications for the regulation of pulmonary vascular contractility in the lung.
In utero hypoxia augments hypoxic pulmonary vasoconstriction in newborn lamb
Authors: Wilson Sean M , Papamatheakis Demosthenes G , Terry Michael R , Merritt Travis , Merrill‐Henry Jeanette , Schroeder Hobe , Longo Lawrence D , Blood Arlin B
Published: June 2021

Chronic hypoxia (CH) is a risk factor in the development of pulmonary hypertension (PH) and recent evidence shows in utero hypoxic stress alters pulmonary structure and function in fetal lambs. Yet, it is unknown whether gestational CH causes pulmonary hypertension in newborn lambs. Reports also indicate Rho‐kinase pathways hold therapeutic promise for the treatment of pulmonary hypertension. Thus, we tested the hypotheses that in utero CH would increase pulmonary arterial pressure (PAP) and that Rho‐kinase inhibition would reduce PH caused by hypoxia. These hypotheses were tested on 12 to 17 day old lambs born at low altitude (LA) or following 100+ days of in utero and postnatal high altitude exposure (HA) at 3801 m. Pulmonary and systemic arterial pressures were measured on anesthetized and ventilated lambs and myography performed on isolated pulmonary arteries (PA). HA did not change baseline PAP, however it significantly augmented hypoxic pulmonary vasoconstriction (HPV). Rho‐kinase inhibition with 2 mg/kg fasudil (intravenous) reduced PAP and blunted HPV in both LA and HA lambs. Rho‐kinase inhibition with 10 μM fasudil or Y27632 also attenuated serotonin mediated PA contractility. Augmentation of HPV in HA lambs and alleviation by fasudil supports the relevance of Rho‐kinase pathways in the treatment of PH in newborns. NIH P01HD031226, R01HD003807 (LDL), R01HL95973 (AB)
Role of Glucose‐6‐Phosphate Dehydrogenase (G6PD) in Chronic Hypoxia‐induced Pulmonary Hypertension
Authors: Chettimada Sukrutha , Oka Masahiko , McMurtry Ivan F , Gupte Sachin A
Published: June 2021

One of the major causes of pulmonary hypertension (PH) is alveolar hypoxia. A reduction in Po2 causes hypoxic pulmonary vasoconstriction (HPV). While HPV is beneficial by matching the ventilation‐to‐perfusion ratio, chronic generalized alveolar hypoxia, such as occurs in chronic mountain sickness, COPD and sleep apnea results in sustained PH. We have shown that G6PD promotes HPV. We, therefore, hypothesized that G6PD could play a role in development of chronic hypoxia (CH)‐induced PH. To investigate our hypothesis, we exposed rats to 3 weeks of hypoxia (~10% O2). Hypoxic rats had elevated PA pressure (111%), RV hypertrophy (40%) and showed a marked increase in lung NADPH levels (150%), as compared to normoxic rats. G6PD is the major source of NADPH in cells, and as expected lung G6PD activity and protein levels were higher (36% and 10% respectively) in CH versus control animals. Further, we investigated the location of increased G6PD in lungs of PH rats by IHC, and found that PASMC expressed higher G6PD. We then exposed cultured PASMC to hypoxia (3% O2) or CoCl2 (100 uM), which mimics hypoxic conditions, for 1 to 72 hrs and measured the protein expression, and activity of G6PD and NADPH. Activity and protein levels of G6PD was increased (P<0.05) between 1 and 8 hrs of hypoxia and CoCl2 treatment as compared to untreated PASMCs. In conclusion, these results suggest that increased G6PD expression and activation in PASMC plays an important role in promoting HPV and development of PH. [Support by NIH grant HL085352]
Contribution of mitochondrial reactive oxygen species to increased pulmonary vasoconstrictor reactivity following intermittent hypoxia
Authors: Snow Jessica , Kanagy Nancy , Walker Benjimen , Resta Thomas
Published: June 2021

Reactive oxygen species (ROS) have been implicated in mediating intermittent hypoxia (IH)‐induced pulmonary hypertension and associated increases in pulmonary vasoconstrictor reactivity to endothelin‐1 (ET‐1). Based on evidence that acute hypoxia increases mitochondrial ROS generation to elicit pulmonary arterial constriction, we hypothesized that IH enhances pulmonary vasoconstrictor reactivity to ET‐1 via production of mitochondrial ROS. To test this hypothesis, we measured vasoconstrictor responses to ET‐1 in the presence of the mitochondrial ROS scavenger MITO‐CP (0.5 μM) or the complex I inhibitor rotenone (10 μM) in endothelium‐disrupted, pressurized pulmonary arteries (~130 μm inner diameter) from IH (3 min cycles of 5% O2, 5% CO2/air flush, 7 hr/day, 4 wk) and Sham (air/air cycling for equal duration) rats. Effects of IH on basal and ET‐1‐induced O2− generation were also assessed in pressurized, dihydroethidium (DHE)‐loaded arteries by fluorescence microscopy. Both MITO‐CP and rotenone attenuated ET‐1‐mediated vasoconstriction in IH arteries and normalized responses between groups. Furthermore, IH increased both basal and ET‐1‐stimulated DHE fluorescence in isolated pulmonary arteries. We conclude that mitochondrial ROS contribute to augmented pulmonary vasoconstrictor reactivity to ET‐1 following IH.
Neprilysin Peptidase‐Independent Complex Formation In Pulmonary Artery Smooth Muscle Cells
Authors: Wick Marilee J. , Miller York E. , Dempsey Edward C.
Published: June 2021

Pulmonary hypertension (PHTN) results from vasoconstriction and remodeling of pulmonary arteries (PA), including accumulation and migration of smooth muscle cells (SMCs). We found that the activity/expression of the transmembrane peptidase neprilysin (NEP) is decreased in chronic hypoxic PHTN, and that chronically hypoxic NEP null mice have increased pulmonary vascular remodeling. We hypothesize that, besides peptidase effects, the peptidase‐independent mechanisms of NEP, mediated through coupling of NEP to intracellular proteins, are important in mediating the effects of NEP in PA SMCs.

Silver‐stained gels of C57BL/6 wt mouse PA SMCs, co‐immunoprecipitated with an antibody to NEP, show bands of 40, 55, 65, 75, 90 and 150 kD. Western analyses (n=2–5) show that NEP forms complexes with the MAPK ERK1/2 and the protein kinase Lyn. Human PA SMCs show similar results. Future studies will investigate the growth‐ and migratory‐inhibitory properties of these complexes, as well as characterize NEP complexes with other proteins. These findings indicate that peptidase‐independent mechanisms may contribute to the mechanisms of action of NEP and may aid in understanding NEP's involvement in preventing/reversing pulmonary vascular remodeling, and in designing treatment and prevention strategies for PHTN.

Research Support: NHLBI RO1 HL‐078929‐01 and PPG HL‐014985, UCD Dept. of Medicine, and the VA.
Protein kinase G deficiency induces pulmonary hypertension in mice and humans
Authors: Zhao You‐Yang , Mirza Muhammad K , Sun Sophie , Wasiukanis Kristen , Vogel Stephen M , Hofmann Franz , Zhao Yidan
Published: June 2021

Protein Kinase G (PKG) is a critical mediator of nitric oxide signaling which regulates cardiovascular homeostasis. It has been shown that PKG‐I−/− mice exhibit impaired nitric oxide/cGMP‐dependent vasorelaxation and systemic hypertension. However, it remains unknown whether PKG‐I deficiency induces pulmonary hypertension. Here we characterize the hypertensive pulmonary phenotype in PKG‐I−/− mice and delineate the underlying molecular basis. We observed significant increases in RVSP and muscularization of distal pulmonary vessels in PKG‐I−/− mice. Microangiography revealed impaired integrity of the pulmonary vasculature in PKG‐I−/− mice. Mechanistically, phosphorylation of RhoA Ser188 was significantly decreased resulting in Rho kinase activation in PKG‐I−/− lung tissues. Intra‐tracheal administration of fasudil, a Rho kinase inhibitor reversed the hypertensive pulmonary phenotype in PKG‐I−/− mice. The clinical relevance of the data was indicated by the observation that PKG‐I protein levels were drastically decreased in lung tissues from patients with severe pulmonary fibrosis. Together, these data show that PKG‐I deficiency induces pulmonary hypertension through Rho kinase activation. Inhibition of Rho kinase may represent an effective approach for the treatment of pulmonary hypertension in patients. This work was supported by NIH grant R01 HL 085462 to YYZ.
Hyperosmolarity stimulates heat shock protein 40 and 70 interaction with UT‐A1 urea transporter
Authors: Chen Guangping , Feng Xiuyang , Frohlich Otto , Yang Yuan , Klein Janet D , Sands Jeff M
Published: June 2021

Hyperosmolarity increases urea transporter UT‐A1 activity in the kidney inner medulla. Osmotic stress induces heat shock protein (HSP) expression. When using a 126‐aa fragment of the intracellular N‐terminus of UT‐A1 as bait in a yeast two‐hybrid assay, we identified heat shock protein 40 (HSP40) as a UT‐A1 interacting protein. HSP40 functions to regulate complex formation between HSP70 and client proteins. HSP70 has been shown to regulate water channel (AQP2), K+ channel, and CFTR activity. Increasing osmolarity (300 to 800 mOsm) by addition of NaCl in 0.5% FCS DMEM for 30 min increased both UT‐A1 and HSP40 expression in native IMCD suspension. We examined the direct interaction of UT‐A1 and heat shock proteins in HEK293 cells co‐transfected with UT‐A1, FLAG‐HSP40 and/or FLAG‐HSP70. Hyperosmolarity (500 mOsm for 30 min) enhanced HSP70 binding to UT‐A1. Functionally, co‐injection of UT‐A1 with HSP40 or HSP70 in oocytes increased UT‐A1 activity but it did not reach statistical significance. However, co‐injection of UT‐A1 with both HSP40 and HSP70 significantly increased UT‐A1 urea transport activity. In our study, we found that HSP40 is associated with UT‐A1 and regulates UT‐A1 urea transport activity in collaboration with HSP70.
Sex steroids and renal protein excretion in mice: the importance of testosterone
Authors: Rouch Al , White Nevin , Burke Josh , Gadd Nicole , Kudo Lucia
Published: June 2021

Sex steroids are involved in the renoprotection in females. Our purpose was to determine the effect of estrogen and testosterone on protein excretion (PE, mg/day) in female and male mice. Two studies were conducted: one with normal and ovariectomized (ovx) females and one with normal and castrated (cast) males (3 groups each study, n=4). Female groups were normal, ovx+vehicle, and ovx+17β‐estradiol (sc injection, 5μg/day). Pellets were implanted in males. Normal and cast groups were given placebo (P) pellets and another cast group was given 35mg pellets of testosterone proprionate (T). Mice (25–30 days old) were placed in metabolic cages for 25 days and consumed 40% casein. In females, no differences in PE occurred between the three groups. In males, average PE was: normal+P = 18.1 ± 2.9*, cast+P = 5.1 ± 1.0, and cast+T = 20.9 ± 3.27* (*− p<.0001 to cast+P). On day 25, kidney weight‐to‐body weight (kw/bw) did not differ in females. In males, kw/bw differed in the profile: cast+T > normal+P > cast+P, and that for cast+P did not differ from females. Results suggest that the absence of testosterone rather than the presence of estrogen accounts for the renoprotection in females.
Peripheral vs central administration of NaCl differently modulates vasopressin secretion through the regulation of PVN Gαq sub‐unit proteins
Authors: Kapusta Daniel R , Wainford Richard D
Published: June 2021

Hypothesis

PVN Gαq sub‐unit protein gated pathways, which are stimulatory on AVP secretion, are endogenously down‐regulated to prevent excess AVP secretion following peripheral, but not central, administration of NaCl.

Methods

Sprague‐Dawley rats were maintained on 0.4% or 8% NaCl chow, or were infused intracerebroventricularly (i.c.v.) with aCSF or 0.8M NaCl aCSF for 21‐days (N=5/gp). PVN tissue was examined for Gαz & Gαq protein expression (normalized to GAPDH) via immunoblotting. Plasma AVP levels were determined via ELISA. In rats treated chronically as described above, N/OFQ (peptide inhibitor of AVP) was injected i.c.v. (5.5 nmol) and urine output was collected for 90‐min (10‐min periods). Separate rats infused with 0.8M NaCl (21‐days) were pre‐treated i.c.v. with a Gαq oligodeoxynucleotide (ODN; 25 μg) 24‐h prior to plasma AVP and PVN Gαz/q protein measurement.

Results

0.4% NaCl diet
8% NaCl diet
aCSF i.c.v. infusion
0.8M NaCl aCSF i.c.v. infusion
0.8M NaCl aCSF i.c.v. infusion + Gαq ODN

PVN Gαq Protein levels [ODU]
0.99±0.07
0.12±0.05*
1.16±0.06
1.09±0.08
0.22±0.04#

PVN Gαz Protein levels [ODU]
2.24±0.13
2.34±0.12
2.01±0.12
2.12±0.13
2.08±0.11

AVP [pg/ml]
1.39±0.07
1.38±0.14
1.50±0.17
2.12±0.17#
0.99±0.06#

Peak Diuresis to N/OFQ [μl/min]
199±16
191±11
177±23
54±12#
T.B.D.

*, # Sig diff vs 0.4% NaCl or aCSF gp;

Conclusion

In response to a peripheral salt load, which does not elevate CSF NaCl levels, the down‐regulation of PVN Gαq proteins is an endogenous counter regulatory mechanism to prevent salt‐induced increases in plasma AVP levels and water retention. When CSF NaCl is increased, this counter‐regulatory mechanism fails, leading to increased circulating AVP and an impaired diuretic ability. AHA 2250585, DK43337, HL071212, AHA 0855293E, P20 RR018766
Hypertension in response to agonistic autoantibodies to the angiotensin II type I receptor (AT1‐AA): role of reactive oxygen species (ROS)
Authors: LaMarca Babbette , Parrish Marc , Weimer Abram , Arany Marrietta , Cockrell Kathy , Dechend Ralf
Published: June 2021

AT1‐AA and ROS are implicated in hypertension in preeclamptic women. Administration of purified rat AT1‐AA significantly increases mean arterial pressure (MAP) and placental ROS in normal pregnant rats. The objective of this study was to determine the role of ROS in mediating AT1‐AA induced hypertension during pregnancy.

To achieve this goal, MAP and ROS were analyzed in AT1‐AA induced hypertensive pregnant rats in the presence and absence of a superoxide dismutase mimetic, Tempol. Rat AT1‐AA (1:50) was infused intraperitoneally via osmotic minipumps and Tempol (30mg/kg/day)was delivered in drinking water of pregnant rats beginning on day 12 of gestation. On day 19 MAP was analyzed and placentas were collected for ROS analysis via lucigenin chemiluminescence technique.

MAP was 101 + 2 in NP rats and 116 + 2 mmHg AT1‐AA rats (p=0.002). Placental basal and NADPH oxidase stimulated ROS was 29 +/− 6 and 92 +/− 10 RLUs in NP rats. These levels increased to 159 +/− 29 (P<0.01) and 287 +/− 60 RLUs (P<0.001) in AT1‐AA rats. MAP in AT1‐AA + Tempol rats was 109 +/−3 vs 109+/−2 mmHg in Tempol treated controls. Administration of Tempol decreased ROS in AT1‐AA treated rats (121+/−13; 262 +/−21 RLUs) compared to Tempol treated controls (69 +/−24; 141 +/− 33 RLUs)

AT1‐AA induced hypertension is associated with placental placenta oxidative stress and is attenuated with administration of an SOD mimetic
Timing of placental ischemia is important in the development of an angiogenic imbalance during pregnancy
Authors: Soldner Emma Louise Bahe , Johnson Britt K , Gilbert Jeffrey S
Published: June 2021

Although recent studies indicate an angiogenic imbalance between factors such as vascular endothelial growth factor (VEGF) and soluble fms‐like tyrosine kinase‐1 (sFlt‐1) is important in the pathophysiology of preeclampsia, it is recognized this is not characteristic of all preeclamptic pregnancies. To this end, we hypothesized that chronic reductions in uteroplacental perfusion (RUPP) for five days during either mid (12–17 days) or late (14–19 days) gestation would have disparate effects on plasma sFlt‐1 and VEGF levels. Chronic RUPP was achieved by placement of silver clips on the abdominal aorta and ovarian arteries on either gestational day (GD) 12 or 14. Arterial pressure (AP) was increased in RUPP vs. normal pregnant (NP) (113±3 vs. 100±1 mmHg ; p <0.05) and (126±2 vs. 100±4 mmHg; p <0.05) on both GD 17 and 19, respectively. Circulating sFlt‐1 was increased (1290±374 vs. 350±27 pg/mL; p<0.05) and VEGF was decreased (665±49 vs. 961±101 pg/mL; p<0.05) in the RUPP compared to NP dams on GD 19 but not GD 17 group. Fetal and placental weights were decreased in RUPP compared to NP dams on GD 19 but not GD 17 (p < 0.05). The present data indicate that RUPP has different effects on angiogenic balance depending on the onset of placental ischemia. These findings suggest that the timing of ischemic insult during gestation may be an important factor in determining the mechanism by which hypertension occurs during preeclampsia.
Dahl salt‐sensitive rats on a high‐fat diet develop hypertension and enhanced constriction to angiotensin II without changing endothelial‐dependent vasorelaxation
Authors: Spradley Frank T , Pollock David M , Pollock Jennifer S
Published: June 2021

Experiments were designed to test the hypothesis that genetic salt‐sensitivity is associated with hypertension and vascular dysfunction under conditions of a high‐fat diet. We established a breeding colony of Dahl salt‐sensitive rats at the Medical College of Georgia (SS/Mcg) that are genotypically similar to SS/Mcw rats. Mean arterial pressure (MAP) was determined by telemetry in SS/Mcg rats on a high‐fat, normal‐salt diet (HF; 59% calories from fat; 0.4% NaCl) and compared to SS.BN13 genetic controls. SS/Mcg rats were hypertensive following 4 wk of HF (152 ± 5 mmHg; n=5) compared to SS/Mcg on a normal diet (ND; 15% calories from fat; 0.4% NaCl) (133 ± 6 mmHg; p<0.05; n=4). MAP in SS.BN13 rats was unchanged by HF (n=4). Vasorelaxation to acetylcholine and nitroprusside as well as vasoconstriction to KCl and phenylephrine were similar in aortic rings from HF and ND SS/Mcg and SS.BN13. In contrast, vasoconstriction in response to Ang II was enhanced in aortic rings from HF SS/Mcg compared to HF SS.BN13 (% maximum constriction: 30 ± 4 (n=5) vs. 2 ± 1 (n=6); p<0.05). However, SS/Mcg and SS.BN13 on ND had similar Ang II‐induced constriction (% maximum constriction: 11 ± 4 (n=5) vs. 13 ± 5 (n=6), respectively; p>0.05). In conclusion, a salt‐sensitive genotype predicts the MAP and Ang II vasoconstrictor response following a short‐term HF diet without promoting endothelial dysfunction. Supported by NIH: HL69999 and HL076146.
Focal acidification of the caudal ventrolateral medulla (Loeschcke's area) increases ventilation during wakefulness but not in sleep
Authors: Silva Glauber SF , Li Aihua , Nattie Eugene E
Published: June 2021

The classic central chemoreceptors were originally described as the regions bilaterally located on the surface of the rostral and caudal ventrolateral medulla (rVLM and cVLM). The cVLM, just lateral to the pyramidal tract between the roots of the hypoglossal nerve, also known as Loeschcke's area, was excited by focal application of H+ and subsequently has not received much of attention in the past decade. Salgueiro et al. recently showed that some of the neurons in the cVLM increased firing rate in response to focal acidification in anaesthetized rats (2003). Here we hypothesize that in conscious rat, focal acidification of the cVLM region would increase ventilation in a sleep‐awake state dependent manner. In unanesthetized rats, normal (5%) or high (25%) CO2 equilibrated aCSF was unilaterally dialyzed into the cVLM region and ventilation was recorded using a whole body plethysmograph. In the responsive group (n=8), focal acidification with high CO2 significantly increased ventilation by 17% in wakefulness due to both VT and frequency but not in sleep comparing with the baseline. In the non‐responsive group (n=7), focal acidification did not affect VE, f and VT either in wakefulness or sleep. The focal acidification data suggest that the cVLM, the area that is partially overlapping Loeschcke's area, participates in the central chemoreception in an arousal state dependent manner. (HL‐28066)
Global knockout of AMP‐activated protein kinase alpha subunits attenuates the hypoxic ventilatory response in mice
Authors: Wyatt Christopher N , Jordan Heidi L , Barr Barbara L , Viollet Benoit , Peers Chris , Hardie D. Grahame , Evans A. Mark
Published: June 2021

The AMP‐activated protein kinase (AMPK) is a heterotrimer composed of catalytic α and regulatory βγ subunits. AMPK monitors the AMP:ATP ratio as an index of metabolic stress and, when activated, adjusts cell metabolism to maintain ATP supply. Previously we have proposed that AMPK may also underpin hypoxia‐response coupling in the carotid bodies, which mediate the entire ventilatory response of the body to acute hypoxia. We therefore sought to determine whether or not AMPK was sufficient and necessary for this process by global knockout of the catalytic alpha subunits in mice.

AMPKα1 and AMPKα2 knockout mice (European Mutant Mouse Archive) were bred onto a C57BL/6: 129/X1 background to obtain knockout (−/−) animals. Breathing during normoxia (21% O2) and the hypoxic ventilatory response to 8% O2 (3 min) were monitored using a Respiromax plethysmography system (Columbus Instruments). Minute ventilation at rest was lower in AMPKα1−/− (1.7 ± 0.1 ml/g/min) and AMPKα2−/− (1.6 ± 0.1 ml/g/min) mice when compared to controls (2.3 ± 0.1 ml/g/min, P < 0.0004). Most significantly, the increase in minute ventilation triggered by hypoxia was attenuated in AMPKα2−/− mice (3.8 ± 2.8%) when compared with controls (19.0 ± 4.1%, P < 0.005). These data provide further support for the new hypothesis that AMPK regulates oxygen and thereby energy supply at the whole body level.

Funded by the American Heart Association and the Wellcome Trust
Chronic sustained hypoxia reduces neuronal excitability in 2nd‐order neurons of peripheral chemoreceptors in the NTS
Authors: Zhang Weirong , Mifflin Steve
Published: June 2021

Chronic sustained hypoxia (CSH) induces alterations in neural control of cardio‐respiratory function. Here we studied CSH effect on the excitability of NTS neurons receiving direct inputs from peripheral chemoreceptors. Whole‐cell recordings were performed in NTS neurons in brainstem slices. Second‐order neurons were identified following DiA labeling of carotid bodies. After exposure to 10% O2 for 7 d, current injection evoked fewer action potentials (APs) in CSH than normoxic (NORM) cells. Currents of 50, 100, 150 pA elicited 5±1, 10±1, 16±1 APs in CSH cells (n=13), 7±1, 16±1, 22±1 APs in NORM cells (n=14, p<0.001). There was no significant difference in intrinsic membrane properties between groups, including input resistance (NORM 1.0±0.1 GΩ vs CSH 1.1±0.1 GΩ, p>0.05). A‐type K+ currents potently regulate neuronal excitability. Although there was no difference in activation and inactivation of A‐currents in NTS neurons, peak steady‐state currents were greater in CSH (n=6) than NORM cells (n=5, p<0.05). An increased A‐current may contribute to decreased excitability. This could be one adaptive mechanism in response to we previously reported CSH‐induced enhancement of glutamatergic synaptic transmission in the NTS. Current results further reveal the complexity of CSH‐induced alteration of neural function in peripheral chemoreflex pathways. This work is supported by AHA BGIA 0865269F and NIH HL88052.
Developmental regulation of glucosensing in rat adrenomedullary chromaffin cells
Authors: Livermore Simon J. , Piskuric Nikol A. , Buttigieg Josef A. , Nurse Colin A.
Published: June 2021

During birth, asphyxial stimuli such as hypoxia and acid hypercapnia trigger the release of catecholamines (CAT) from adrenomedullary chromaffin cells (AMCs), which protects the heart and prepares the lungs of the neonate for air breathing. These direct chemosensing properties are present in neonatal AMCs but are lost by ~2 weeks postnatally, as these cells become innervated by the splanchnic nerve. Asphyxia also leads to glucose deprivation though it is unknown whether or not neonatal AMCs are also directly sensitive to low glucose (hypoglycemia). Recently, we demonstrated that 0 glucose induced membrane depolarization and a rise in intracellular Ca2+ in an immortalized, fetal‐derived chromaffin cell line. In the present study, we tested the hypothesis that primary neonatal rat AMCs are sensitive to hypoglycemia and that this property is developmentally regulated. Using fura‐2 Ca2+ imaging and carbon fiber amperometry we found that hypoglycemia induced a rise in intracellular Ca2+ and quantal vesicular CAT secretion in most neonatal AMCs. Furthermore, these responses were markedly reduced or absent in 2‐week old juvenile AMCs. Though the molecular mechanisms remain to be elucidated, this study suggests that the neonatal rat adrenal medulla may act as a glucosensor, perhaps similar to that recently proposed for the carotid body. This work was supported by the Heart and Stroke Foundation of Ontario and NSERC.
Modulation of Acute Hypoxia on Canonical Transient Receptor Potential Channel 3
Authors: Yang Qin , Huang Jun‐Hao , Yao Xiao‐Qiang , He Guo‐Wei
Published: June 2021

Canonical transient receptor potential channel 3 (TRPC3) is involved in intracellular Ca2+ ([Ca2+]i) regulation therefore is important in cell function. Whether this channel is affected by ischemia/hypoxia has been poorly studied. We investigated the response of the TRPC3 channel to acute hypoxia. Human embryonic kidney cells (HEK293 cells) were transiently overexpressed with TRPC3 gene and exposed to either normoxia or acute hypoxia (10 min, PO2<10 mmHg). Ionic currents were measured by patch‐clamp and protein expression was determined by western blot. Application of 1‐oleyl‐2‐acetyl‐sn‐glycerol (OAG, 100 μM) evoked significant cation currents in TRPC3‐overexpressing HEK293 cells (3.1±0.4 vs. 1.9±0.3 pA/pF, p<0.01) but not in wild‐type cells (2.0±0.4 vs. 1.9±0.4 pA/pF, p>0.05). Acute exposure to hypoxia enhanced the increase of current induced by OAG (5.7±0.9 vs. 3.0±0.4 pA/pF in normoxia, p<0.05). With the presence of specific anti‐TRPC3 antibody, OAG failed to induce current change in both normoxia (1.9±0.2 vs. 2.1±0.1 pA/pF) and hypoxia‐exposed cells (2.0±0.4 vs. 2.4±0.4 pA/pF) (p>0.05). Protein expression of TRPC3 was not altered. Acute hypoxia enhances the electrophysiological activity of the TRPC3 channel. This may be a mechanism involved in [Ca2+]i dysregulation under hypoxic/ischemic conditions.

Supported by Hong Kong GRF grants CUHK4651/07M & 4789/09M and CUHK direct grant 2041457.