bluuebunny/crossref_metadata_2025 huggingface dataset:
The PLA2 activity is required for Angiotensin II‐mediated Endothelial NADPH oxidase
Authors: NGUYEN SU DUY , CHATTERJEE SHAMPA , FISHER ARON B
Published: June 2021

Peroxiredoxin 6 (Prdx6), an antioxidant enzyme with both GSH peroxidase and phospholipase A2 (PLA2) activities, plays a protective function against oxidative stress. Unexpectedly, we have found that ROS generation by NADPH oxidase activation is markedly diminished in cells lacking Prdx6. We thus hypothesized that Prdx6 is a necessary component of the NADPH oxidase complex. ROS production upon Angiotensin II‐induced NADPH oxidase activation was determined in isolated lungs and pulmonary microvascular endothelial cells (PMVEC) using ROS‐specific fluorophores, dihydrodichlorofluorescein diacetate, dihydroethidium, and amplex red. Ang II‐treated lungs and PMVEC from WT mice but not from Prdx6−/− mice showed a significant increase in ROS formation that was attenuated by MJ33, a selective inhibitor of PLA2 activity of Prdx6. Lysophosphatidylcholine treatment enhanced ROS generation in both wild‐type and Prdx6−/− cells, an effect that was abolished by the NADPH oxidase inhibitor, apocynin. Transfection of Prdx6‐null PMVEC with construct only expressing Prdx6 PLA2 activity, but not those expressing only Prdx6 peroxidase activity, partially rescued the ROS response. These results indicate that Prdx6 participates in NADPH oxidase activation and this mechanism is mediated through phospholipase A2 activity of Prdx6.
THE INHIBITOR OF PROTEIN ARGININE METHYLTRANSFERASES, AMI‐1 IS A POTENT SCAVENGER OF NOX‐DERIVED SUPEROXIDE
Authors: Chen Feng , Fulton David
Published: June 2021

The methylation of proteins is an important post‐translational mechanism that has been established to influence the activity of nuclear and nucleic acid binding proteins. Much less is known about the importance of protein methylation in the regulation of cytosolic proteins. Increased methylation of proteins is observed in cardiovascular disease and occurs in conjunction with elevated production of reactive oxygen species. However, the nature of the relationship between reactive oxygen species and protein methylation is poorly understood. Therefore, the goal of the current study was to determine whether protein methylation influences the catalytic activity of the NADPH oxidases which are a family of enzymes responsible for the generation of superoxide. We found that the selective inhibitor of protein arginine methyltransferases, AMI‐1 was a potent antagonist of Nox‐derived superoxide production. However, structurally and mechanistically dissimilar inhibitors of protein methylation and co‐expression of the arginine methyltransferase, PRMT1 did not influence Nox activity. Rather, the effect of AMI‐1 was rapidly reversible and could be demonstrated in an assay using chemically synthesized superoxide. We conclude that protein methylation does not regulate the activity of NADPH‐oxidases and that AMI‐1 is a potent antioxidant with a greater potency than Tiron and Tempol.
Colocalization of the novel plasminogen receptor, Plg‐RKT, with the epithelial sodium channel (ENaC)
Authors: Chen Kevin W , Bai Hongdong , Kiosses William B , Krajewski Stan , Miles Lindsey A , Parmer Robert J
Published: June 2021

Binding of plasminogen (Plg) to cells via specific receptors markedly enhances Plg activation, and localizes the proteolytic activity of plasmin (P) on cell surfaces. Exposure of kidney tubular cells to P results in cleavage and activation of ENaC leading to increased Na+ uptake, and Na+ retention in nephrotic syndrome in which Plg is present in urine. We recently isolated a novel transmembrane Plg receptor, Plg‐RKT, from macrophages. Here, we examined kidney cells for the presence and subcellular localization of Plg‐RKT as a potential component of the mechanism in which P processes ENaC. Human kidney sections were stained with specific monoclonal anti‐Plg‐RKT. Strong staining was observed in distal tubular epithelium (intensity=200–256) > proximal tubular cells (intensity=140–199) ≫ glomeruli (intensity=80–139). In western blotting of MDCK cells and M1 mouse collecting duct cells Plg‐RKT was highly expressed in membrane fractions, but was not detected in cytoplasm. Using confocal microscopy, Plg‐RKT was immunodetected on the apical surface, an orientation allowing for interaction with urinary Plg. Furthermore, in merged images, Plg‐RKT colocalized with both ENaC and the urokinase receptor (uPAR). The extent of colocalization of Plg‐RKT with ENaC was 98±1% and 89±3% in MDCK and M1 cells, respectively, and with uPAR was 96±1% in both cell types. Thus, Plg‐RKT, uPAR and ENaC are present in very close proximity on the kidney tubular cell surface in an orientation to promote Plg activation and ENaC processing.
CHRONIC FOOTSHOCK STRESS CAUSES ENDURING CHANGES IN CIRCADIAN RHYTHMS, WITHOUT PROVOKING HYPERTENSION IN RATS
Authors: Nalivaiko Eugene , Beard Daniel , Bondarenko Evgeny , Beig Mirza I , Jobling Phil , Walker Frederick R , Day Trevor AP , Bobrovskaya Larisa
Published: June 2021

We determined whether chronic stress causes enduring hypertension and, if so, whether this is reflected in biochemical markers of sympathetic activity. Adult male Wistar rats were subjected to six weeks of footshocks (FS+, n=5), with constant monitoring of AP and HR by telemetry; another group served as controls (FS−, n=5). Chronic stress did not alter daily average values of AP or HR, mean AP being 122±1 and 116±2 mmHg for FS+ and FS−, respectively, post‐stress, vs. 120±3 and 119±3 mmHg, respectively, pre‐stress. Chronic stress failed to alter cardiac susceptibility to arrhythmias, and did not alter AT1 receptor expression or tyrosine hydroxylase activity in the stellate ganglia or the adrenal gland. However, chronic stress dramatically altered circadian rhythms, significantly disrupting the normal dark phase rises in AP and HR. These changes were already detectable after the first week of stress, and lasted for at least one week post‐stress. We conclude that chronic footshock stress does not provoke sustained changes in daily average values of AP or HR, or in biochemical markers of sympathetic activity, but causes enduring disruption of circadian rhythms.
Alterations to the Liver RAS in Antenatal Induced MLPD Systemic Hypertension
Authors: Stewart John Michael , Goyal Ravi , Longo Lawrence
Published: June 2021

Objective

It has been well established that systemic hypertension can be result of fetal programming by antenatal maternal low protein diet (MLPD). Furthermore, as apposed to the ‘classical’ systemic renin‐angiotensinogen system (RAS) several studies have suggested that many tissues including lungs, pancreas, kidney, brain, and liver have their own ‘alternative’ RAS. In mice, we tested the hypothesis that hypertensive mice resulting from antenatal MLPD show changes in the liver specific RAS pathway.

Methods

Mice dams were fed a specified isocaloric low protein diet from 7 days precoitus maintained thoughout gestation. We isolated DNA, mRNA, and protein from livers of offspring female and male mice exposed to 50% MLPD and 33% MLPD at 3 weeks and 33 weeks of age. We examined eight RAS genes with qPCR and dot blot analysis at the transcript and protein level.

Results

AGT transcript levels were altered in MLPD mice while there was no significant change in protein. Female AGT transcripts were increased at 3 weeks of age but not 33 weeks and protein levels were not significantly different from control.

Conclusions

In response to an antenatal maternal low protein diet AGT transcript levels change while protein levels do not. These changes in liver RAS, may help elucidate mechanisms responsible for fetal programming of disease, especially increased blood pressure.
A Dual Role of Cyclic GMP in Cardiac Survival Signaling
Authors: Lee SungRyul , McIntosh Rachel , Shen Xinagjun , Chanoit Guillaume , Zvara David A , Xu Zhelong
Published: June 2021

Cyclic guanosine 3′5′‐monophosphate (cGMP) plays an important role in cardioprotection against ischemia/reperfusion injury through activation of protein kinase G (PKG). The purpose of this study was to determine the impacts of cGMP on cardiac survival signaling pathways. We found that cGMP prevents the mitochondrial permeability transition pore (mPTP) opening by inactivating glycogen synthase kinase 3â (GSK‐3â) via protein kinase G (PKG) in cardiac H9c2 cells. Although Akt is well known as an important upstream regulator of GSK‐3â and its activation leads to inactivation of GSK‐3â, we found that cGMP negatively regulated Akt activity. We further found that the negative regulatory effect of cGMP on Akt activity is mediated by the protein phosphatase PP2A but not by PKG. While GSK‐3â and Akt are critical for acute cardioprotection, an excessive activation of Akt or GSK‐3â inactivation can also result in cardiac hypertrophy. Therefore, the negative effect of cGMP on Akt activity may serve as an important mechanism leading to prevention of hypertrophy. Taken together, cGMP not only inactivates GSK‐3â through PKG (this leads to acute cardioprotection) but also negatively regulates Akt activity (this may lead to prevention of hypertrophy and heart failure) in cardiac cells. We propose that cGMP is a versatile signal with a dual beneficial role in cardiac survival.

This study was supported by NIHR01HL08336
Carboxybutyrylated glucosamine inhibits LPS‐induced iNOS expression in RAW 264.7 macrophages via increased protein O‐GlcNAc levels
Authors: Zou Luyun , Zou Chenhang , Wang Pengfei , Wang Yun , Marchase Richard B , Chatham John C
Published: June 2021

We have shown that increasing levels of O‐linked‐N‐acetylglucosamine (O‐GlcNAc) on nucleocytoplasmic proteins, with glucosamine (GlcN) attenuated the inflammatory response of cells treated with lipopolysaccharides (LPS). Others reported that carboxybutyrated glucosamine (CGlcN) attenuated LPS induced inflammatory response in macrophage cell line (RAW264.7). We hypothesized, therefore, that the anti‐inflammatory effect of CGlcN was mediated via an increase in O‐GlcNAc levels. We found that CGlcN increased O‐GlcNAc levels in RAW cells in a dose dependent manner and that at 24 hrs the effect of CGlcN was greater than GlcN. Treatment with 0.1μg/ml LPS significantly decreased RAW O‐GlcNAc levels, which was prevented by both GlcN and CGlcN. LPS treatment significantly increased iNOS expression compared to untreated cells and this was attenuated by both GlcN and CGlcN; however CGlcN appeared to have a greater effect on attenuating iNOS expression that GlcN. We also tested other compounds based on variants of CGlcN; however, none of these compounds had any effect on RAW O‐GlcNAc levels or the response to LPS treatment. These data suggest that CGlcN inhibits LPS induced iNOS expression via increased O‐GlcNAcylation and that this effect appears to be more pronounced than GlcN; however, the mechanism by which this occurs remains to be determined. Grants: NIH HL067464/HL079364.
Effects of hypertonic saline solution in inflammatory response in sepsis
Authors: Theobaldo Mariana Cardillo , Soriano Francisco Garcia
Published: June 2021

Sepsis is a systemic inflammatory response to infection and a major cause of mortality. The hypertonic saline solution (HSS) has been showed to improve hemodynamic and inflammatory response. We studied the hypertonic solution effects in sepsis through cecal ligation and puncture (CLP) in mice Balb‐C, divided in 3 groups: CLP without treatment (C); CLP treated with saline solution 0,9% ‐ 34mg/Kg (SS); CLP treated with HSS 7,5% ‐ 4ml/Kg (SH); and a 4° group to indicate the basal values without both CLP and treatment; both treatments (HSS and saline) were applied 30 min after CLP. The lung was collected after 6, 12 e 24 h to analyze cytokines levels by ELISA and nitric oxide (NO) by Griess method. The group SH showed higher survival rates (60%) when compared to SS (46,6%) and C (33,3%). The SS group (27,3 ± 3,05 pg/ml) presented increased TNF‐α level in 12 h when compared to group C (14,07 ± 1,68 pg/ml) and SH (19,66±3,19 pg/ml). After 24 h the SH (6,2 ± 3,5 pg/ml) decreased TNF‐α when compared to C (73,40± 49,52 pg/ml); IL‐10 level in 24 h was increased in C (CLP ‐ 3,12 ± 0,46 pg/ml) and SS (2,9 ± 0,9 pg/ml) in relation to SH (0,59 ± 0,34 pg/ml). NO concentration was lower in SH (2,26 ± 1,34 pg/ml) compared with C (10,96 ± 2,2 pg/ml) after 24 h. The HSS protects the animal against sepsis, but saline solution doesn't. Our results suggest that the volume replacement of both treatments modulate pro and anti‐inflammatory cytokines and NO. CAPES.FAPESP.
Delayed resuscitation with physostigmine increases end organ damage in intoxicated rats
Authors: Sulzer Jesse , Molina Patricia
Published: June 2021

Acute alcohol intoxication (AAI) increases injury risk and complicates the clinical course of trauma victims. We have shown that AAI impairs hemorrhagic shock (HS)‐induced neuroendocrine activation and restoration of mean arterial blood pressure (MABP) during fluid resuscitation (FR) with Ringer's lactate. These outcomes are improved with central cholinesterase inhibition with physostigmine (PHYS). We examined whether delayed FR+PHYS alters the intervention's effectiveness and safety. Conscious and chronically catheterized male Sprague Dawley rats received alcohol (2.5 g/kg bolus+ 0.3 g/kg/h × 15 h) infusion or isovolemic dextrose (DEX) prior to HS (40 mmHg × 60 min) followed by immediate or delayed (60 min) FR +/− PHYS (100 ug/kg). Immediate FR improved MABP in DEX‐HS animals. This response was delayed by AAI. Delayed FR did not improve MABP in DEX‐ or AAI‐HS animals. Immediate and delayed FR+PHYS effectively increased MABP in DEX‐ and AAI‐HS animals. No differences were noted in markers of organ dysfunction (ALT, AST, BUN, creatinine) in DEX‐HS with respect to time of FR+PHYS. However, delayed FR+PHYS of AAI‐HS animals exacerbated the rise in all markers of organ dysfunction compared to time‐matched FR‐vehicle treated animals. These findings suggest that AAI increases susceptibility to tissue injury in response to FR+PHYS following prolonged hypotension. DOD‐PR‐054196, BoR‐110350057A, NIAAA‐7577.
Fluid Resuscitation Physiological Responses in Hemorrhaged Rats
Authors: Doursout Marie‐Francoise Joelle , Philip Asher , Liang Yang Yan , Deng Xiyun , Conyers Jodie , Holcomb John
Published: June 2021

Hemorrhagic hypotension leads to a well‐characterized sequence of physiological events. This includes decreases in cardiac output and blood pressure, the release of vasoconstrictors, hyporeactivity and vascular decompensation leading to the production of reactive oxygen species (ROS) and nitrogen oxide (peroxynitrite). ROS caused injuries to the vascular endothelium resulting in endothelial dysfunction leading to multiple organ failure in hemorrhagic shock (HS). Our study assessed the production of nitrate, ROS and peroxynitrite in hemorrhaged rats with and without resuscitation. Rats were hemorrhaged at a rate of 2ml/kg over 10 min until mean arterial blood pressure dropped to 30 mmHg. One hour following HS rats were resuscitated with either Fresh Frozen Plasma (FFP; Day 0/Day 5) or Lactated Ringer (LR). Additional rats included HS alone and sham control. Our data demonstrated that FFP Day 0 increased nitrate production, supporting a vasodilatation in vascular beds, as compared to FFP Day 5; LR and HS alone. Our data shown that Xanthine Oxidase, an enzyme that generates ROS, was predominant in HS alone and in LR‐resuscitated rats whereas rats resuscitated with FFP Day 0 decreased ROS production. Finally, resuscitation with FFP at Day 0 as compared to FFP Day 5 and LR decreased nitrotyrosine production. Our results indicate that FFP Day 0 improved resuscitation as compared to FFP Day 5 and LR in rats subjected to HS.
Adenosine A1 Receptor Elicits Coronary In‐stent Stenosis in Metabolic Syndrome
Authors: Long Xin , Alloosh Mouhamad , Kitsongsermthon Jutarat , He Yuanzu , Park Kinam , Sturek Michael
Published: June 2021

Adenosine is widely thought to elicit coronary vasodilation and attenuate smooth muscle proliferation, thereby providing cardioprotection. We cloned the porcine adenosine A1 receptor (A1R) subtype and found that it paradoxically stimulated proliferation of cultured coronary smooth muscle cells by extracellular signal‐regulated protein kinases 1 and 2 (ERK1/2) signaling pathways, thus suggesting A1R dysregulation could play a role in coronary in‐stent restenosis. We utilized the Ossabaw swine model of metabolic syndrome (MetS) to test the hypothesis that A1R up‐regulation and A1R‐ERK1/2 signaling play a role in the development of coronary in‐stent stenosis in MetS. Intracoronary stent deployment followed by different durations of recovery from stenting showed A1R upregulation occurred before maximal amount of in‐stent stenosis. Swine were fed standard chow (Lean) or excess calorie atherogenic diet for over 20 weeks, which elicited MetS characteristics. We observed reduced coronary in‐stent stenosis by TAXUS (paclitaxel‐eluting) stents in MetS. Selective A1R antagonism with DPCPX‐eluting stents decreased coronary ERK1/2 activation and decreased in‐stent stenosis comparable to TAXUS. Conclusion: A1R upregulation and A1R‐ERK1/2 activation contributes to coronary in‐stent stenosis in vivo in the setting of MetS. Support: NIH RR013223, HL062552, Purina TestDiet to MS; HL078715 to KP; AHA to XL.

Grant Funding Source: NIH
Dyslipidemia in metabolic syndrome is necessary to elicit severe coronary artery disease and non‐alcoholic steatohepatitis
Authors: Neeb Zachary P , Alloosh Mouhamad , Saxena Romil , Chalasani Naga , Sturek Michael
Published: June 2021

Risk of coronary artery disease (CAD) is increased in patients with metabolic syndrome (MetS) and non‐alcoholic steatohepatitis (NASH); however, the relative importance of each component of MetS (i.e. obesity, insulin resistance, glucose intolerance, dyslipidemia, hypertension) is unknown. We hypothesized that occlusive and complex CAD and NASH are primarily driven by dyslipidemia within the MetS milieu and that all other components of MetS combined are not sufficient. Ossabaw swine were fed standard chow (Lean; N=16), chow with fructose (FMetS; N=9), trans‐fat/cholesterol/fructose (TMetS; N=14), or mixed‐source fat/cholesterol/fructose (MMetS; N=6) for 22 weeks. FMetS developed normolipidemic MetS. TMetS, and to a greater extent MMetS, developed MetS with the dyslipidemia component. Lean demonstrated mild CAD and no NASH. Although FMetS developed MetS CAD and NASH were virtually non‐existent. CAD and liver disease were directly related to dyslipidemia in MetS, with TMetS showing fatty/fibrous atheroma and MMetS progressing to stenotic, diffuse, and complex CAD and NASH. Total monounsaturated/polyunsaturated fatty acid ratio and fatty acid metabolism enzyme indices were associated with CAD and NASH, however LDL gram‐years most reliably predicted CAD and NASH. Thus, dyslipidemia was a necessary component of MetS for eliciting severe CAD and NASH in swine that superbly mimic human pathologies.
Trefoil factor‐3 is down regulated while CYP24a1 is increased in the ageing rat kidney
Authors: Hultström Michael , Iversen Bjarne M.
Published: June 2021

The large scale expressional changes in ageing kidneys are not well described. In the present experiment we compared the genome wide expression of mRNAs in both kidneys from five 90 week old rats against five 10 week old rats.

The expression from left and right kidneys were compared to determine if the samples should be used as independent samples or as replicates. No significant differences in gene expression were found between left and right kidneys. They showed a homogeneity almost comparable to technical replicates.

Pooling results from left and right kidneys produced 20 genes showing a larger than two‐fold change. The most down regulated gene was Trefoil factor‐3, which has been shown to be lower in ageing kidneys previously. The most upregulated gene was cytochrome P450‐24a1, known to hydroxylate Vitamin D3.

In conclusion, the left and right kidney cortex show very similar expression patterns, making them very useful for paired experiments but not appropriate as independent samples. Vitamin D3 metabolism is likely changed in the ageing kidney as a result of increased CYP24a1 expression.
Left ventricle transcriptome in aldosterone‐mediated cardiac injury
Authors: Romero Damian G , Price Jana E
Published: June 2021

Primary hyperaldosteronism patients develop cardiac injury and dysfunction. In animal experimental models, excess aldosterone (ALDO) only causes cardiac injury in the presence of high salt (HS) intake. To study which molecular mechanisms are involved ALDO/HS‐mediated cardiac injury and its dependence on HS intake, we studied cardiac whole genome gene expression (GE). Male Sprague Dawley rats (n=6 per group) were uninephrectomized, and treated with ALDO (0.75 μg/h delivered by miniosmotic pumps), 0.9 % NaCl in the drinking water or a combination of both of them for 2 days, or 1, 2, 4 or 8 wks. Whole genome GE was performed using Illumina Beadarrays and GE was analyzed using GeneSpring software. ALDO/HS caused severe left ventricle GE changes after only 2 wks of treatment. Although either ALDO or HS alone changed GE (209 and 289 genes, respectively) the combination synergistically modified the expression of 1046 genes (493 UP and 553 DOWN). Bioinformatics analysis indicate that the Gene Ontology set of inflammatory response genes (n=17) and genes with the Sp1 transcription factor binding site in their promoter are significantly enriched. Similar GE results were observed after 4 and 8 wks of treatment. In summary, we report a left ventricle time series GE analysis in an animal experimental model of ALDO‐mediated cardiac injury highlighting many new genes involved in this pathological condition.
Alleles of a S.LEW congenic rat spanning 320.6kb further increases the BP of the hypertensive Dahl S rat: Evidence for Rffl and/or two miRNAs as potential QTL effectors
Authors: Gopalakrishnan Kathirvel , Thangavel Jayakumar , Kumarasamy Sivarajan , Yerga‐Woolwine Shane , Farms Phyllis , Morgan Eric , Kalinoski Andrea , Liu Lijun , Joe Bina
Published: June 2021

Individuals with normal blood pressure (BP) may possess susceptibility to the development of high BP, but the expression of these alleles is masked by the existence of other alleles conferring resistance to high BP. This phenomenon is exhibited in a congenic model of the hypertensive Dahl (S) rat with a <320.6Kb segment from the normotensive LEW rat. The BP of this congenic strain was higher than that of the S rat. In this report the genetic element causing this increase in BP was fine mapped to a region with a single gene Rffl with in 42.496 Kb. There were no coding sequence variants within Rffl, but significant cardiac differential expression was observed. Interestingly higher intrinsic cardiac hypertrophy was also evident in the congenic rats compared with S. Possibilities for the observed differential expression of Rffl were explored with a comprehensive genomic sequence miRNA analysis and transcriptome analysis of the critical region combined with functional analysis of Rffl. The data obtained through this study 1) identified a total of 171 variants within the critical region; 2) identified that 13 out of 110 predicted rat miRNAs within the critical region contained variants; 3) detected multiple gene networks perturbed between S and congenic rats; and 4) provided preliminary evidence for the functional significance of Rffl as a QTL effector.
Evidence that prenatal steroid exposure produces oxidative stress in the kidney in male sheep
Authors: Bi Jianli , Contag Stephen , Chappel Mark , Rose James C
Published: June 2021

Prenatal glucocorticoid administration reduces renal function in adult offspring. But little is known about the effect of prenatal steroid exposure on oxidative stress in kidney.

The objective of this study was to assess responses of a marker for oxidative stress in the kidney to infusion of angiotensin (ANG) peptides in adult male sheep exposed to steroid prenatally.

1–1.5 year old rams exposed to either Betamethasone (B) (n=6) or Vehicle (V) (n=6) at 80–81 days gestation were given intra renal ANG II or ANG 1–7 infusions(1 ng/kg/min) either alone or with an AT 1 R blocker (Candesartan; 0.3 mg/kg), AT 2 R blocker (PD 123319 10ng/kg/min) or Mas receptor blocker (D‐Alaangiotensin 1–7, D‐ALA, 10ng/kg/min) for 3 hours. Urinary 8‐isoprostane(8‐PGF) excretion was measured by EIA kit. ANOVA was used for data analysis.

ANG II increased 8‐PGF excretion in B (from1.9±0.1 to 2.8±0.5ng/kg/h) but not in V sheep(p<0.05,F=6.1). Candesartan blocked the response to Ang II but PD didn't. ANG 1–7 infusion decreased the excretion of 8‐PGF in both groups (P<0.05,F=6.6). This response was blocked by D‐ALA.

The results suggest that prenatal B exposure promotes an oxidative stress response in kidney. Blockade of the response by candesartan but not by PD suggests that the increased 8‐PGF excretion is mediated through AT1R activation. The decreased 8‐PGF excretion during ANG 1–7 infusion indicates ANG 1–7 might attenuate oxidative damage in B sheep. Supported by HD‐47584 and HD‐17644
RENAL VASOCONSTRICTION AND NATRIURESIS INDUCED BY TUMOR NECROSIS FACTOR (TNF)‐α ARE MEDIATED BY TNF RECEPTOR TYPE 1
Authors: Majid Dewan S. A. , Castillo Alexander
Published: June 2021

Infusion of TNF‐α exerts renal vasoconstriction as well as natriuresis in mice. To define the receptor subtypes involved in these renal vascular and tubular actions of TNF‐α, experiments were conducted to assess the responses to human recombinant TNF‐α (0.33ng/min/g BW; i.v. infusion for 75 min) in gene knockout mice for either TNF receptor type 1 (TNFR1KO; n=6) or type 2 (TNFR2KO; n=6) and compared these results with those obtained in corresponding wild type (WT; C57BL6; n=6) mice. Renal blood flow (RBF) and glomerular filtration rate (GFR) were determined by PAH and inulin clearances respectively. As reported previously, TNF‐α infusion in WT mice resulted in decreases of 20±5% (P<0.03) in RBF, 40±2% (P<0.003) in GFR as well as increases in 83±27% (0.05>P>0.01) in urine flow (V) and 363±104% (P<0.05) in sodium excretion (UNaV). These responses are completely absent in TNFR1KO mice. TNF‐α infusion in TNFR1KO mice resulted in non‐significant changes in renal parameters (RBF; 8±22%; GFR, −18±14%; V, 15±16%; UNaV, −47±12%). However, TNF‐α infusion in TNFR2KO mice showed similar changes in renal parameters as observed those in the WT mice. The responses in TNFR2KO mice are as follows: RBF; −22±10 % (0.05>P>0.01); GFR, −36±7% (P<0.002); V, 91±19% (P<0.007); UNaV, 327±70% (P<0.01). These data demonstrate that TNF receptor type 1, not the type 2, is involved in mediating acute renal vascular and tubular actions of TNF‐α.
The RNA Binding Complex Translin‐Trax Mediates Pro‐Excitatory Activity in Neurons
Authors: Cuadra Adolfo Ernesto , Li Zhi , Sumners Colin , Baraban Jay M , Raizada Mohan K
Published: June 2021

In animal models of neurogenic hypertension the Rostral Ventrolateral Medulla (RVLM), of the brain, displays increased excitatory activity that is linked to elevated sympathetic tone and blood pressure. Analysis of gene expression in the RVLM of normotensive Wistar Kyoto (WKY) and Spontaneous Hypertensive Rat (SHR) revealed reduction of the protein Translin (TSN) in SHR. TSN and its partner Trax (TRX) form a complex that is thought to mediate dendritic trafficking of mRNA. To examine the functional effect of this complex, we dialyzed purified TSN‐TRX into cultured brain neurons from either WKY or SHR and measured its effect on the rate of action potential firing (APF). Basal APF rate was 1.4 Hz in WKY neurons and after 20 min control treated neurons showed a similar rate of 1.3 Hz. In contrast, TSN‐TRX treatment mediated a significant increase in APF rate to 1.9 Hz. Tsn alone had no effect in WKY neurons and SHR neurons showed no response to TSN‐TRX. Pretreatment with the RNA translation inhibitor, cycloheximide, abolished the excitatory effect of Tsn‐TRX in WKY neurons. Our results suggest that the excitatory effect of TSN‐TRX in WKY neurons is translation‐dependent. We hypothesize that homeostatic feedback mechanisms in SHR neurons lead to downregulation of TSN levels and the resistance of these cells to the excitatory effects of the TSN‐TRX complex. This work was supported by funding from NHLBI, NIDA of the NIH and NARSAD.
GABA, glutamate and taurine in the paraventricular nucleus (PVN) during the onset of renal wrap hypertension
Authors: Northcott Carrie A , Burnett Robert , MacDonald Molly , Haywood Joseph R , Parsell Dawn
Published: June 2021

The PVN is involved in the development of renal wrap hypertension. We hypothesized that there is increased glutamate and reduced GABA neurotransmitter levels in the PVN during the onset of renal wrap hypertension (Day 7). PVN targeted microdialysis cannulae were stereotaxically placed unilaterally in sham and renal wrap rats one week prior to conscious microdialysis perfusion with artificial cerebrospinal fluid (aCSF) at a rate of 1ul/min for retrieval of perfusate for HPLC detection of GABA, glutamate and taurine. In addition to baseline, aCSF with the GABA uptake inhibitor, nipecotic acid (40 nM), and nipecotic acid (40 nM) plus veratradine (300 uM) were added to the perfusate. The rats were also instrumented with arterial femoral catheters for mean arterial blood pressure (MAP) and heart rate measures. MAP was significantly higher in renal wrap rats compared to sham [100 ± 8 vs. 128 ± 3 mm Hg (n=5–8); p≤0.05]. There were no differences in either PVN GABA or taurine levels. However, PVN glutamate levels were significantly greater in the perfusate throughout all treatments from renal wrap rats compared to sham (baseline: 46.87 ± 9.46 vs. 110.33 ± 17.72 ng/ml; nipecotic acid: 84.91 ± 20.76 vs. 196.97 ± 40.86 ng/ml; nipecotic acid + veratradine: 77.24 ± 17.46 vs. 236.72 ± 90.97 ng/ml). In conclusion there is enhanced basal glutamate as well as altered glutamate handling in the PVN during the onset of renal wrap hypertension.
Short and Long term Peroxide Removal Modulates Relaxing and Contracting Mechanisms Influencing the Response of Bovine Pulmonary Arteries to Hypoxia
Authors: Ahmad Mansoor , Kelly Melissa R. , Kandhi Sharath , Arudra Sri Krishna Chaitanya , Wolin Michael S.
Published: June 2021

Endothelium removed bovine pulmonary arteries (BPA) contract in the presence of hypoxia due to removal of a basal relaxation mediated by superoxide derived peroxide. Previous studies from our lab have shown that short term removal of peroxide with 0.1mM ebselen (30 minutes before hypoxia) results in increased force development to KCl and an attenuation of hypoxic pulmonary vasoconstriction (HPV) accompanied with decreased 5μM lucigenin and horseradish peroxidase‐luminol detectable superoxide and peroxide levels respectively. Inhibition of the potential source of superoxide Nox‐4 with siRNA treatment results in inhibition of HPV without affecting force development to KCl. Prolonged exposure of BPA to ebselen (beginning 45 minutes before exposing rings to passive tension) resulted in effects similar to siRNA treatment where HPV was attenuated without affecting KCl contraction. Thus short term removal of peroxide results in rapid attenuation of a relaxing mechanism promoting increased KCl force development and attenuating HPV. Prolonged removal of peroxide results in HPV attenuation without affecting KCl force, suggesting that peroxide also maintains a force enhancing mechanism which is not rapidly reversed by hypoxia.

Supported by NIH grants HL31069, HL43023 and HL66331.
Suppressor of Cytokine Signaling‐1 Inhibits Hyperoxic Induced Inflammasome Activation
Authors: Kolliputi Narasaiah , Shaik Rahamthulla , Waxman Aaron B
Published: June 2021

The inflammasome is a newly discovered molecular platform required for the caspase‐1 activation and maturation of IL‐1β. IL‐1β is the most active cytokine in acute lung injury patients. Recently it has been reported that reactive oxygen species (ROS) are required for purinergic P2X7 receptor (P2X7R)‐mediated NALP3 inflammasome activation. Our previous results suggest that suppressor of cytokine signaling‐1 (SOCS‐1) functions as negative regulator in ROS‐induced apoptotic responses by inducing ASK‐1 degradation. Thus, we hypothesized that the toxic effects of hyperoxia could be mediated by ASK‐1‐induced P2X7 mediated inflammasome and that the protective effects of SOCS‐1 could be due, at least in part, to its ability to suppress inflammasome response. To test this hypothesis we administered SOCS–1 adenovirus (Ad‐SOCS‐1) into the lung and exposed mice to 100%O2. Ad‐GFP infected mice were used as controls. Inflammasome complex formation was assessed by immunoprecipitation and western blot. Ad‐SOCS‐1 mice lived significantly longer in hyperoxia when compared to Ad‐GFP. After 72 hours of hyperoxia, inflammasome was activated and associated with enhanced caspase‐1and IL‐1 β cleavage in the Ad‐GFP mice but inflammasome assembly was abolished in the Ad‐SOCS‐1 mice with significant decrease in caspase‐1and IL‐1 β cleavage. These findings suggest that SOCS‐1 over‐expressing mice are protected from hyperoxia‐induced inflammation, which is associated with inactivation of inflammasome, thus demonstrating a critical role for SOCS‐1 in inflammasome‐mediated inflammation. (This work was funded by T32‐HL007874).
PPARγ regulates hypoxia‐induced Nox4 expression in human pulmonary artery smooth muscle cells through NF‐κB
Authors: Lu Xianghuai , Murphy Tammy , Nanes Mark , Hart C. Michael
Published: June 2021

The Nox4 NADPH oxidase, which is highly expressed in vascular wall cells, is selectively upregulated in the lungs of human subjects and experimental animals with pulmonary hypertension. We demonstrated that rosiglitazone, a synthetic PPARγ agonist, attenuated hypoxia‐induced pulmonary hypertension, vascular remodeling, Nox4 induction and reactive oxygen species generation in the mouse lung. The current study examined PPARγ‐regulated, hypoxia‐induced Nox4 expression in human pulmonary artery smooth muscle cells (HPASMC). Exposure to 1% oxygen for 72 hours increased Nox4 gene expression and HPASMC H2O2 production, both of which were reduced by treatment with rosiglitazone or with siRNA to Nox4. Rosiglitazone also attenuated hypoxia‐induced HPASMC proliferation and the activity of a Nox4 promoter luciferase reporter. Chromatin immunoprecipitation assays demonstrated that hypoxic‐induced binding of the NF‐κB subunits, p65 and C‐rel, to the Nox4 promoter was attenuated by rosiglitazone. The role of NF‐κB in Nox4 regulation was further supported by demonstrating that overexpression of p65 stimulated Nox4 promoter activity whereas siRNA to p50 or p65 attenuated hypoxic‐stimulated Nox4 promoter activity. These results provide novel evidence for NF‐κB‐mediated stimulation of Nox4 expression in HPASMC that can be negatively regulated by PPARγ.
Recruitment of Inflammatory cells to the Lung is Dependent upon Platelet Activating Factor production in Smokers
Authors: McHowat Jane , Young Dawn M
Published: June 2021

To recruit inflammatory cells to the airways, cells must move from the circulation across the endothelial and epithelial cell barriers. Platelet‐activating factor (PAF) is involved in transendothelial migration in several vascular beds. We hypothesize that an increase in PAF production and cell recruitment may exacerbate inflammatory disease in smokers. We incubated human lung microvascular endothelial cells (HMVEC‐L) with cigarette smoke extract (CSE). CSE (20 μg/ml) significantly inhibited PAF‐acetylhydrolase (PAF‐AH, hydrolyzes and inactivates PAF) activity after 12 hours of CSE incubation (2.1 ± 0.1 to 0.7 ± 0.1 nmol/mg protein/min, n=6, p<0.01). Inhibition of PAF‐AH resulted in increased PAF production (538 ± 60 to 1580 ± 241 dpm, n=6, p<0.01) and PMN adherence (14 ± 3 to 47 ± 3%, n=4, p<0.01). Pretreatment of PMN with the PAF receptor antagonist CV3988 (10μM for 10 min) resulted in complete inhibition of PMN adherence (0.4 ± 0.1, n=4, p<0.01). In the lung, PMN accumulation in the small airways represents one of the initial steps in inflammation and emphysema. In conclusion, these studies have shown that PAF likely plays a central role in the recruitment of inflammatory cells to the small airways of cigarette smokers via inactivation of PAF‐AH and may exacerbate or initiate inflammatory lung disease.
Effect of GSK1016790A, a potent TRPV4 agonist, on lung endothelial permeability
Authors: Jian Ming‐Yuan , Thorneloe Kevine , Townsley Mary I.
Published: June 2021

Activation of the vanilloid transient receptor potential channel TRPV4 with 4α‐phorbol‐12,13‐didecanoate (4αPDD) causes Ca2+ entry‐dependent lung injury in rat and mouse lung. However, 4αPDD has no impact on pulmonary arterial pressure despite TRPV4 expression in pulmonary vascular smooth muscle. In this study, we compared the effect of GSK1016790A, a potent and selective TRPV4 agonist, with that elicited by 4αPDD. Lungs isolated from wild type or TRPV4−/− mice were perfused with Earle's buffer/4% albumin at constant flow. The filtration coefficient (Kf, an index of endothelial permeability) and hemodynamics were measured before and after GSK1016790A or 4αPDD treatment. At 3nM (n=4), GSK1016790A had little impact on permeability. However, Kf increased 5.5‐ and 10.6‐fold with 10nM (n=6) and 30nM (n=5) doses, respectively. The permeability response to GSK1016790A was accompanied a transient increase in pulmonary arterial pressure. In contrast, 4αPDD (10μM, n=4) increased Kf 3.2‐fold without a concomitant pressor response. Neither 30nM GSK1016790A (n=4) nor 10μM 4αPDD (n=4) had impact on permeability or perfusion pressure in lungs from TRPV4−/− mice. In summary, GSK1016790A is a much more potent stimulus than 4αPDD for TRPV4‐mediated acute lung injury and pressor responses in isolated mouse lung. Supported by GlaxoSmithKline and HL066299.
S‐nitrosylation of Caveolin‐1 Cys156 Regulates Oligomer Stability and Caveolae Trafficking
Authors: Bakhshi Farnaz , Chen Zhenlong , Husain Yawer , Shajahan Ayesha , Liu Guoquan , Malik Asrar B. , Minshall Richard D.
Published: June 2021

Caveolin‐1 (Cav‐1) is required for caveolae formation but it also binds to signaling proteins such as endothelial nitric oxide synthase (eNOS), which generally negatively regulates their activity. Interestingly, activation of caveolae trafficking is associated with an increase in NO production. Here, we test the hypothesis that a NO feedback mechanism directly modifies the assembly and mobility of caveolae. We determined whether eNOS‐derived NO affects Cav‐1 oligomer stability by S‐nitrosylation (SNO) of Cys156 and thereby regulates vesicular trafficking. Endothelial cells stimulated with TNFα for up to 4 hr were lysed and used to measure Cav‐1 SNO by biotin‐switch assay as well as Cav‐1 oligomer stability in non‐reduced SDS PAGE gels. TNFα increased NO production and Cav‐1 SNO and also decreased Cav‐1 oligomerization, both of which were blocked by L‐NAME. We next generated Cav‐1−/− cells expressing GFP‐tagged wild type Cav‐1 or Cys156Ser‐Cav‐1 mutant to determine the role of Cys156 in caveolin oligomerization and vesicle trafficking as characterized by immunoblot analysis and live‐cell spinning disc confocal imaging. These studies revealed that Cys156Ser‐Cav‐1 mutant‐labeled caveolae were less mobile and that oligomers were less stable compared to WT Cav‐1. Thus, S‐nitrosylation of Cav‐1 may be an important regulatory mechanism of caveolae trafficking in endothelial cells. Supported by R01 HL071626, P01 HL060678, and T32 HL007829.
Reactive oxygen species induce endothelial tissue factor expression in lungs
Authors: Emin Memet T. , Bhattacharya Sunita
Published: June 2021

To determine the interaction of coagulant and inflammatory pathways in acid‐induced lung injury (AALI), we addressed the role of reactive oxygen species (ROS) in the endothelial induction of the coagulation initiator, tissue factor (TF). To establish AALI, we intra‐tracheally instilled HCl (pH1.2, 2 ml/kg) in anesthetized mice. After 2 h, through the pulmonary artery we sequentially injected chilled buffer and a mixture of chilled collagenase (1000 units/ml) and trypsin (3ml). We collected the left atrial effluent containing ~4×104cells. To label with fluorophore‐conjugated mAbs to TF and the endothelial marker, vWf, we fixed and permeabilized one group of cells with paraformaldehyde and triton‐x. In a second group, we labeled surface proteins with the mAbs in unfixed cells. We determined endothelial fluorescence by flow cytometry. In control lungs, vWf and TF expressions in treated endothelial cells were similar in wild type (WT) and oxidant‐incompetent gp91phox−/− mice (n=3). However in lungs of AALI mice, surface endothelial vWf and TF expressions were markedly blunted in gp91phox−/− as compared with WT mice (p<0.05, n=6). These findings indicate that AALI causes surface endothelial expression of the key initiator of coagulation, TF through a ROS‐dependent mechanism. We propose that in AALI ROS‐induced establishment of a procoagulant endothelial milieu might aggravate lung injury (Support: HL36024).
In utero administration of GABA promotes fetal lung development
Authors: Chintagari Narendranath Reddy , Wang Yang , Xi Dong , Liu Lin
Published: June 2021

Fetal lung secretes chloride‐rich fluid during glandular stages of development. The fluid accumulation is critical for lung distension and normal development. The multisubunit γ‐amino butyric acid receptors (GABAR) mainly act by mediating chloride ion fluxes. Our previous studies revealed the differential expression of several GABAR subunits in fetal lung epithelial cells. We therefore investigated the role of GABAR in fetal lung development in vivo. Timed‐pregnant rats of 18 day gestation underwent an in utero surgery for administration of GABAR modulators into the fetuses. The fetuses were isolated on day 21 and analyzed for changes in fetal lung development. Fetuses injected with GABA (500 μM) had a significantly higher body weight (4732.36 ± 90.80 mg) and lung weight (160.54 ± 4.02 mg) when compared to those of saline (control) injected fetuses (4441.83 ± 81.93 mg and 131.58 ± 4.57 mg). The increase in fetal lung weight was effectively blocked by GABA‐A receptor antagonist, bicuculline. GABA‐injected fetal lungs had 30% higher number of saccules when compared to the control. Bicuculline blocked the increase in saccule number. In conclusion, our results indicate that GABA receptors accelerate fetal lung development.
The magnitude of the hypoxic ventilatory response and ventilatory long‐term facilitation are reduced during sleep when compared to wakefulness
Authors: Gerst D. G. , Syed Z. , Yokhana S. S. , Lin H. S. , Mateika J. H.
Published: June 2021

Purpose

To determine the impact of arousal state on the magnitude of the hypoxic ventilatory response (HVR − Δ VE/Δ SaO2) and ventilatory long‐term facilitation (vLTF).

Methods

5 healthy male subjects were exposed to IH on two occasions. The IH protocol consisted of 12‐2 minute episodes of hypoxia (PETO2 – 50 mmHg) in the presence of PETCO2 levels sustained 3 mmHg above baseline. Each episode was followed by a 2 minute normoxic recovery period with the exception of the last recovery period which was 30 minutes in duration. One IH trial was completed during wakefulness, with the other completed during sleep.

Results

The HVR was greater during wakefulness compared to sleep in all participants (0.8 ± 0.2 vs. 0.5 ± 0.1 L/min/% SaO2, p = 0.06). Moreover, minute ventilation was greater during recovery compared to baseline, during both wakefulness and sleep (Wakefulness – 22.5 ± 1.8 vs. 18.6 ± 1.5 L/min, p = 0.03; Sleep – 15.2 ± 1.8 vs. 14.3 ± 1.8 L/min, p = 0.02). However, the magnitude of vLTF when standardized to baseline was significantly greater during wakefulness compared to sleep (1.21 ± 0.07 vs. 1.07 ± 0.02, p = 0.05).

Conclusions

Exposure to IH elicits vLTF during both wakefulness and sleep. However, the magnitude of the HVR and vLTF is significantly reduced during sleep compared to wakefulness.

Funding: VA‐Merit Award
Electrocortical activity response to intermittent transient tracheal occlusion
Authors: Hotchkiss Mark T , Hooker Jodi , Adams Sherry , Pate Kathryn , Davenport Paul W
Published: June 2021

Respiratory loads elicit mechanosensation in conscious humans and animals. Intermittent transient tracheal occlusion (ITTO) produces an observable change in the attentive state of rats. We hypothesized that ITTO would produce a significant frequency power shift in electrocorticogram (ECoG) activity. The relative power of ECoG frequency ranges associated with relaxed wakefulness and sleep were expected to decrease: Delta (0.8–4.5 Hz), Theta (4.75–6.75 Hz), and Alpha (7.00–12.50 Hz). An increase in relative power was expected for frequency ranges associated with attentive wakefulness and perception: Beta (12.75–35 Hz), Gamma (35.25–100.00 Hz), and UHF (100.25 Hz and up). Rats were instrumented with electrodes on the surface of the cortex and an inflatable occluder was sutured around the trachea. Following post‐surgical recovery, the tracheal cuff was inflated to obstruct 3–10 breaths, and then deflated for a minimum of 15 breaths. The obstruction cycle was repeated for 15–20 minutes once each day for 10 days. Results of this study demonstrated the expected changes in ECoG frequency power associated with increased arousal. These results support the use of this model to study airway obstruction‐related arousal in unanesthetized rats.
Behavioral and physiological changes associated with chronic tracheal obstructions (TO) in conscious rats
Authors: Pate Kathryn M , Scheuer Deborah A , Davenport Paul W
Published: June 2021

Our laboratory uses a cuff implanted around the trachea to elicit respiratory TO in conscious rats. We hypothesized that repeated TO in conscious rats will elicit load compensation, blood pressure (BP) and stress responses. All animals were instrumented with a BP probe, diaphragm electromyography electrodes and tracheal cuff. After recovery, experimental animals (Exp) received cuff inflations, 3–8 seconds per inflation, over the course of a 20 minute time period. Control animals (Con) were placed in the testing chamber for the same amount of time with no TO. This was repeated once a day for 10 days. All animals were tested on an elevated plus maze (EPM) for anxiety on the day before obstructions began and on day 10. Animals were sacrificed on the 10th day. Blood was collected for measurement of corticosterone (CORT) and adrenals weighed. Baseline physiological measurements and EPM were compared within animals for the first and last day and between groups. In Exp, TO response was compared for the first and last day. In Exp, TO elicited load compensation and increased BP. Adrenal weight/body ratio was greater in Exp than Con (p = 0.02), and Exp animals spent more time on the closed arms of EPM post‐TO conditioning compared to Con and pre‐TO. The results suggest that TO conditioning elicits load compensation, hypertensive, and anxiety stress responses.