bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuroplasticity in the nucleus tractus solitarius precedes development of functional changes in autonomic regulation of arterial pressure in obese Zucker rats
Authors: Fu G. Jeffery , Huber Domitila A. , Schreihofer Ann M.
Published: June 2021

Obese Zucker rats (OZR) develop elevated sympathetic nerve activity (SNA) and arterial pressure (AP) between 7–14 wks of age vs. lean Zucker rats (LZR). Baroreflexes are normal in juvenile OZR (7 wk), but impaired in adult OZR (12–14 wk). In adult OZR baroreceptor afferent nerves respond normally to rises in MAP, but direct stimulation of afferents evokes blunted decreases in SNA and MAP, suggesting changes in the brain impair baroreflexes. We examined if nucleus tractus solitarius (NTS), the brain site receiving baroreceptor inputs, is affected in adult OZR. Stimulation of NTS by microinjections of glutamate evoked comparable decreases in SNA and MAP in juvenile OZR vs. LZR, but reduced responses in adult OZR vs. LZR. However, stimulation the caudal ventrolateral medulla, a target of the NTS, evoked comparable decreases in SNA and MAP in adult OZR and LZR. We examined the time course of changes in NTS by expression of ΔFosB protein as a marker of neuroplasiticity. At 5 wks OZR and LZR had comparable ΔFosB+ neurons (203±30 vs. 195±22). However, by 7 wks NTS of OZR had more ΔFosB+ neurons than LZR (514.5±70 vs. 207±24.5; P = 0.027), which persisted at 12 wks (177±15.38 in OZR and 88±15.76 in LZR; P = 0.02). These data suggest reduced excitability of NTS neurons contributes to blunted baroreflexes in adult OZR. Furthermore, changes in the NTS occur prior to the onset of altered baseline SNA and MAP or NTS function. Funding: NIHHL086759.
Group III/IV muscle afferents impair limb blood flow during exercise in patients with heart failure
Authors: Amann Markus , Venturelli Massimo , Ives Steve , Morgan David , Gmelch Benjamin , Witman Melissa , Groot Jon , Wray David Walter , Stehlik Josef , Richardson Russell
Published: June 2021

Patients with heart failure (HF) are characterized by altered muscle afferent feedback which might account for their hemodynamic and autonomic reflex abnormalities. We investigated the influence of group III/IV muscle afferents on the circulatory response to rhythmic exercise in HF. Nine patients (NYHA class II) performed single leg knee extensor exercise (25/50/80% peak workload) under both control conditions (Ctrl) and with lumbar intrathecal fentanyl impairing μ‐opioid receptor‐sensitive muscle afferents. Cardiac output (Q) and femoral blood flow (QL) were determined, and arterial/venous blood samples were collected at each workload. Fentanyl had no effect at rest. During exercise, Q was 8–13% lower with Fentanyl, secondary to a 13% reduction in stroke volume and a 2–5% reduction in heart rate. Lower norepinephrine spillover during Fentanyl exercise implicated a reduced sympathetic outflow and likely accounted for the 25% increase in leg vascular conductance. As a result of this attenuated sympathoexcitation, QL was, despite a 4% lower blood pressure, 10–14% higher during exercise with Fentanyl. Although leg O2 delivery was higher during exercise with Fentanyl, arteriovenous O2 difference remained unchanged resulting in 10–17% increase in leg O2 consumption (p<0.05). Thus, although group III/IV muscle afferents represent a key determinant of the central hemodynamic response in HF, our findings also reveal that these sensory neurons cause a sympathetically‐mediated impairment of QL during rhythmic exercise in this population.
Acute Effects of Conjugate Equine Estrogen on Postischemic Pial Arteriole Vasodilation and Microvascular Cyclooxygenase Expression in Young and Aged Female Rats
Authors: Zeynalov Emil , Ma Li , Rezvani Niloofar , Littleton‐Kearney Marguerite T
Published: June 2021

Objectives

In these studies we evaluated the effects of acute conjugated equine estrogens (CEE) on postischemic pial vasodilatory capacity of young ovariectomized (OVX) and aged, reproductively senescent (RS) rats. Additionally, we evaluated expression of cyclooxygenase‐1 (COX‐1) in isolated pial arterioles in OVX and RS rats.

Methods

First, OVX and RS female rats were anesthetized; a closed cranial window was placed. Rats were then subjected to 15‐ min reversible forebrain ischemia, followed by 2 hr. reperfusion. At the onset of reperfusion CEE (1 mg/kg, I.V.) or vehicle was administered. Pial artery dilatory responses to Ach were tested before and after ischemia. Pial arteries were isolated and analyzed for COX‐1 expression using western blot techniques.

Results

CEE treatment partially preserved postischemic vasodilatory responses (22 ± 5 % vs. 15 ± 6 %) of OVX rats which coincided with increased microvascular expression of COX‐1 protein (0.07±0.01 vs. 0.21 ± 0.03, COX‐1/Actin). However, CEE failed to restore postischemic vasodilatory responses (21 ± 7 % vs. 1 ± 6 %) of RS rats. Increases in COX‐1 expression were diminished by half in RS rats (0.04 ±0.007 vs.0.11 ± 0.03, COX‐1/Actin).

Conclusion

Increases in postischemic pial arteriole COX‐1 expression after acute CEE treatment is associated with improvements of postischemic pial arteriole vasodilatory capacity in OVX, but not RS rats.
PDGF Receptor Antagonism Prevents the Increase in Urinary Angiotensin II Excretion in Cyp1a1‐Ren2 Transgenic Rats with Angiotensin II‐Dependent Malignant Hypertension
Authors: Milani Carlo J , Howard Catherine G. , Seth Dale M. , Davis Porcha D. , Mitchell Kenneth D.
Published: June 2021

The present study was performed to determine if chronic administration of the PDGF receptor antagonist, imatinib mesylate, prevents the augmentation of urinary ANG II excretion that occurs in Cyp1a1‐Ren2 transgenic rats with ANG II‐dependent malignant hypertension. Male Cyp1a1‐Ren2 rats (n=6/group) were induced to develop malignant hypertension by dietary administration of indole‐3‐carbinol (I3C, 0.3% wt/wt) for 10 days. One group was chronically treated with imatinib mesylate by oral gavage (60 mg/kg/d) starting 3 days before initiating I3C induction and maintained on imatinib for the duration of the study. Systolic blood pressures (SBP) were measured daily by tail‐cuff plethysmography. Rats were placed in metabolic cages for measurement of 24‐hour urine output and urinary protein and ANG II excretion. Dietary I3C resulted in pronounced increases in SBP (152±6 to 209±6 mmHg, P<0.001) and urine output (8.5±0.7 to 60±5 ml/day, P<0.001), marked proteinuria (26.8±3.0 to 72.2±11.8 mg/day, P<0.01), and augmented urinary ANG II excretion (448±12 to 987±119 fmol/hr, P<0.05). Chronic imatinib administration did not prevent the development of hypertension (204±3 vs. 157±8 mmHg, P<0.001) or the increase in urine flow (61.8±5.0 vs. 8.5±0.6 ml/day, P<0.001), but prevented the I3C‐induced increases in proteinuria (21.8±2.2 vs. 21.8±0.8 mg/day) and urinary ANG II excretion (263±30 vs. 492±43 fmol/hr). The present findings demonstrate that PDGF receptor antagonism with imatinib mesylate prevents the marked proteinuria and augmented urinary ANG II excretion independent of changes in blood pressure that occur in Cyp1a1‐Ren2 rats with ANG II‐dependent malignant hypertension.
NADPH oxidase and ETA receptors mediate glomerular reactive oxygen species production in sickle cell nephropathy
Authors: Heimlich Brett , O'Connor Paul M , Meiler Steffen E , Pollock David M
Published: June 2021

Sickle cell nephropathy (SCN) is a form of chronic renal disease characterized in part by extensive glomerular damage; however, the mechanism is unknown. Recent evidence suggests that endothelin (ET) contributes to proteinuria and renal injury. We hypothesized that SCN is associated with increased reactive oxygen species (ROS) production in glomeruli, an effect mediated by ET. We found that sickle mice have increased proteinuria (5.7±1.1 vs. 2.8±0.5 mg/mg creatinine/day n=6) and nephrinuria (2.8±0.6 vs. 0.8±0.3 μg/mg creatinine/day n=3–4) at baseline compared to C57/BL6J controls. In isolated glomeruli, we observed that sickle mice had significantly higher glomerular ROS production compared to controls following stimulation with phorbol myristae acetate (PMA)(26.3±4.8 v. 3.4±0.7 AU*min, respectively, p<0.01). This effect was abrogated in vitro by the NADPH oxidase inhibitor, apocynin (0.8±0.2 AU*min, p<0.001 v. untreated sickle glomeruli). Furthermore, we demonstrated that PMA‐induced ROS was absent following in vivo treatment with the ETA antagonist, ABT‐627 (5mg/kg/day, 7 days) (6.7±3.2 AU*min, p<0.01 v. untreated sickle glomeruli). These data indicate a novel mechanism for glomerular injury in the setting of sickle cell disease and suggests that ETA antagonism could provide a viable treatment strategy to prevent the initiation and progression of SCN.
Inhibition of COX‐1 ameliorates endotoxemia‐induced fall in GFR in mice
Authors: Mederle Katharina , Castrop Hayo , Schweda Frank , Höcherl Klaus
Published: June 2021

In this study, we investigated the impact of COX‐1‐ and COX‐2‐derived prostanoids on lipopolysaccharide (LPS)‐induced decrease in glomerular filtration rate (GFR) in mice.

Injection of LPS (3 mg/kg; i.p.; n=8) decreased GFR and the amount of thrombocytes to about 50% of basal values. Plasma, urine and renocortical tissue levels of TxB2 were increased about 10‐, 7‐ and 2‐fold in response to LPS, respectively. Pretreatment with the COX‐1 inhibitor SC‐560 (20 mg/kg) attenuated the LPS‐induced fall in GFR and in thrombocytes to about 75% of basal levels (n=8; p<0.01). Further, SC‐560 abolished the increase in plasma, urine and renocortical tissue levels of TxB2 in response to LPS. Pretreatment with the COX‐2 inhibitor SC‐236 (10 mg/kg) further enhanced the LPS‐induced fall in GFR to about 40% of basal values (n=6; p<0.05 vs. LPS). SC‐236 did not alter thrombocyte levels nor the LPS‐induced increase in plasma, urine and renocortical tissue levels of TxB2. Pretreatment with the P2Y12 antagonist Clopidogrel (n=6; 50 mg/kg × d; oral) for two days inhibited the LPS‐induced fall in thrombocytes, but did not attenuate the LPS‐induced decrease in GFR.

This study demonstrates that endotoxemia‐induced decrease in GFR is mediated in part by COX‐1 derived prostanoids. Our data further suggest that vascular‐rather than thrombocyte‐derived thromboxane is responsible for the fall in GFR in response to LPS.
Ribosomal Protein L22 (RPL22) accumulates as aggregates in distal renal tubules after aldosterone administration
Authors: Cheema Muhammad Umar , Nielsen Jakob , Poulsen Ebbe Toftgaard , Enghild Jan J , Fenton Robert A , Praetorius Jeppe
Published: June 2021

The mineralocorticoid aldosterone is important in the delevelopment of renal disease. We hypothesized that aldosterone increase protein turnover and challenge the cellular protein degradation systems in renal tubules as a primary event in tubular damage. The cellular accumulation of specific protein aggregates and aggresome formation was assessed in kidneys from rats receiving either 50 μg aldosterone/kg body weight/day for 7 days or vehicle in osmotic minipumps. Aldosterone induced accumulation of 60s ribosomal subunit L22 (RPL22) in protein aggregates specifically in the aldosterone sensitive distal renal tubules. RPL22 showed 80% co‐localization with proteasome 20s, while co‐localization with endosomal and lysosomal markers was neglible. A 1.5‐fold increase in proteasome 20s abundance was observed in distal renal tubules with a concomitant increase in proteasome aggregate count. Finally, aldosterone treatment induced foci of early epithelial to mesenchymal transition in renal cortex as judged by vimentin expression and lymphocyte infiltration. We conclude that aldosterone administration leads to accumulation of non‐classical protein aggregates in the distal renal tubule that are not cleared by autophagy.
Losartan retards the development of Chronic Kidney Disease (CKD) after an Acute Kidney Injury (AKI) episode
Authors: Rodríguez Roxana , Barrera‐Chimal Jonatan , Benítez Kenya , Pérez‐Villalva Rosalba , Gamba Gerardo , Bobadilla Norma A.
Published: June 2021

We studied if preventing AKI with Losartan (Lo) stops CKD development. Fifty rats were divided in: Sham‐operated (S), Lo‐treated group (50 mg/kg) (Lo+S), rats underwent bilateral ischemia of 45 min (I), rats receiving Lo before I (Lo+I), and rats receiving Lo, 3 h after I (I+Lo). All groups were observed 270 days. Proteinuria (Prot), creatinine clearance (CrCl), renal blood flow (RBF), tubulo‐interstitial fibrosis (TIF) and glomerular diameter were measured. In addition, interleukin 6 and monocyte chemo attracting protein mRNA levels, as well as urinary kidney injury molecule and H2O2 were measured. Rats underwent ischemia developed CKD characterized by a progressive increase in Prot and a decrease in CrCl and RBF. Glomerular hypertrophy, extensive TIF, and Kim‐1 up‐regulation were also observed, all these findings were associated with an increase in oxidative stress and with greater inflammation. Losartan treatment avoided the progression of CKD evidenced by CrCl and renal architecture preservation without increased inflammation or oxidative stress. We show in this model of renal injury induced by AKI that angiotensin receptor antagonism reduced CKD progression. However, prophylactic treatment was more effective than after the ischemic insult. The mechanisms activated after AKI episode and the angiotensin II involvement could give us the knowledge for designing therapeutic strategies to prevent CKD.
CD40 ligand contributes to angiotensin‐II induced vascular inflammation, oxidative stress and endothelial dysfunction
Authors: Daub Steffen , Hausding Michael , Jurk Kerstin , Becker Christian , Kroeller‐Schoen Swenja , Oelze Matthias , Schwenk Melanie , Stein Judith , Kerahrodi Jasmin Ghaemi , Kossmann Sabine , Schulz Eberhard , Wenzel Philip , Reske‐Kunz Angelika , Münzel Thomas , Grabbe Stephan , Daiber Andreas
Published: June 2021

Methods and Results

Angiotensin‐II (1mg/kg/d for 7d) was infused in C57/Bl6 (control) and CD40L−/− mice using osmotic minipumps. Vascular function was recorded by isometric tension studies, reactive oxygen species (ROS) were monitored in blood and heart of the mice by enhanced chemiluminescence. ELISA of CD4+ T cell supernatants, Western blot analysis and real‐time RT‐PCR were used to analyze pro‐inflammatory cytokines, NAPDH oxidase subunits, T cell transcription factors and other genes of interest. CD40L−/− mice showed an ameliorated endothelial function and decreased oxidative stress in the heart and blood after angiotensin‐II treatment compared to their wild‐type littermates. Moreover, CD40L−/− mice displayed significantly decreased levels of TH1 cytokines released by splenic CD4+ T cells. This observation was accompanied by lower expression levels of NOX‐2 in aortic tissue, T‐bet in splenic cells, as well as P‐selectin in heart tissue from CD40L−/− mice.

Conclusions

Our results demonstrate that CD40L−/− deficiency improves angiotensin‐II induced vascular inflammation, dysfunction and oxidative stress. These data further support our previous observations [Wenzel et al. Circulation, 2011] that immune cells and inflammatory pathways significantly contribute to angiotensin‐II induced arterial hypertension.

Research support

This work was supported by funds from the Federal Ministry of Education and Research (BMBF 01EO1003) to A.D., S.G., P.W. and T.M; S.K.‐S., E.S., A.D. and T.M. hold grants from the Stiftung Mainzer Herz.
Comparative expression profiling of 50–60 year old male competitive athletes and lean healthy individuals
Authors: Edgett Brittany A , Mukherjee Kamalika , St. Amand Tim , Funk Colin D , Gurd Brendon J
Published: June 2021

The current study examined genetic and metabolic adaptations to aerobic exercise in highly‐trained masters athletes (MA; n = 9; age, 53 ± 3 yrs; BMI, 24 ± 3 kg/m2; VO2peak, 59.1 ± 5.2 ml·kg−1·min−1) and age and BMI matched controls (CON; n = 8; 54 ± 5 yrs; 25 ± 3 kg/m2; 35.9 ± 9.7 ml·kg−1·min−1). All participants performed a 45 minute endurance ride at 60% of their VO2peak followed by cycling at 90% VO2peak to fatigue. Blood was sampled before, immediately after, and 24 hours after exercise. Fasted insulin (MA, 18.1 ± 4.3; CON, 32.6 ± 13.8 pmol/L), HDL (MA, 1.91 ± 0.49; CON 1.28 ± 0.26 mmol/L), and lipid ratio (3.00 ± 0.62; CON 4.16 ± 0.86) were different between groups. MA also demonstrated an augmented insulin response to exercise compared to CON. A genome‐wide DNA microarray analysis of RNA from blood samples revealed that a variety of genes involved in insulin activity, cardiovascular and metabolic functions were significantly different between the two groups. These differences will be confirmed for individual changes in gene expression by quantitative real‐time PCR. These results may lead to new insights into signaling pathways that control the beneficial effects of exercise in older men, and may help to identify surrogate markers for monitoring exercise and training load. This research was supported by NSERC.
The effects of mouth guard usage on neuromuscular activation and muscle performance
Authors: Veicsteinas Arsenio , Limonta Eloisa , Cè Emiliano , Rampichini Susanna , Esposito Fabio
Published: June 2021

To assess the effects of a commercially‐available mouth guard (Brux Ltd.) on neuromuscular activation and muscle performance, 8 participants completed a balanced and randomized study with (S‐MG) and without (Con) a sport mouth guard. Each participant performed two tests with both elbow flexors and knee extensors muscles: a 1‐min contraction at 100% of maximum voluntary contraction (MVC) and an 80% MVC until exhaustion. During 100% MVC, force decay (ΔF%) and root mean square (RMS) of electromyogram (EMG) were calculated. During 80% MVC, the time into force target (t‐target) and force coefficient of variation (CV) were determined. MVC was significantly higher in S‐MG than in Con in both muscle groups (P<0.05). ΔF% was significantly lower in S‐MG than in Con in the knee‐extensors (61.1±1.7% and 68.9±1.3% in S‐MG and Con, respectively; P<0.05) but not in the elbow flexors. At 80% MVC, higher t‐target values were observed in S‐MG compared to Con for the knee‐extensors (18.1±2.5 s and 12.9±2.7 s in S‐MG and Con, respectively; P<0.05), but not for the elbow flexors. CV was significantly lower in S‐MG compared to Con in both muscle groups. These findings indicate that despite neuromuscular activation was not altered, the mouth guard usage increased maximum force production, endurance time and force stability, especially in the lower limb muscles.

The study was partially funded by a grant of the University of Milan.
Endurance exercise and doxorubicin‐induced skeletal muscle dysfunction
Authors: Hydock David Scott , Bredahl Eric , Gibson Noah , Quinn Colin , Schneider Carole , Hayward Reid
Published: June 2021

The purpose of this study was to examine the role that prior endurance training plays in combating the skeletal muscle dysfunction and fatigue associated with the anticancer drug doxorubicin (DOX). Ten‐week old, male Sprague‐Dawley rats were randomly assigned to a treadmill training group (progressive treadmill training protocol 5 d/week for 10 weeks) or a sedentary group (normal cage activity for 10 weeks). Following activity treatments, animals were further randomized to receive DOX (15 mg/kg) or saline as a control. Five days following injections, soleus and extensor digitorum longus (EDL) maximal twitch force and fatigue were analyzed ex vivo. DOX treatment resulted in significant reductions in maximal twitch force in the soleus and EDL, and prior treadmill training attenuated this reduction in the soleus only. Additionally, treadmill training attenuated the increased fatigue associated with DOX in the soleus but not in the EDL. Exercise training provided protection against DOX‐induced muscle dysfunction and fatigue in the slow, oxidative soleus muscle but not in the fast, glycolytic EDL. This suggests that treadmill training's protection against DOX myotoxicity may be specific to type I muscle.
Oxidative stress and induction of non‐cardiogenic pulmonary edema following cerebral hypoxia in a canine model
Authors: Khademi Shadi , Jeckel Kimberly , Frye Melinda A , Irwin David , Schroeder Thies , Miller Benjamin F , Monnet Eric , Hamilton Karyn L
Published: June 2021

Neurogenic pulmonary edema (NPE) is a non‐cardiogenic form of pulmonary edema associated with stroke, traumatic brain injury and seizure. NPE is a public health concern due to high morbidity and mortality, yet the mechanism(s) are unknown.

Hypothesis

Cerebral hypoxia, even in the absence of systemic hypoxia, is sufficient to induce pulmonary edema, oxidative stress and compensatory antioxidant mechanisms.

Methods

Using an updated Moss's model, 9 Walker hounds were randomly assigned to cerebral hypoxemia (SaO2 ~55%) with systemic normoxia (SaO2 ~90%) (CH; n=6), or cerebral and systemic normoxia (SaO2 ~90%) (Sham; n=3). Femoral venous (CH) or arterial (Sham) perfusate was delivered from a cardiopulmonary bypass circuit to the brain. Wet to dry lung weight ratios were assessed as an index of pulmonary edema, lipid peroxidation was assessed as an indicator of oxidative stress, and brain and lung compensatory antioxidants were assessed with immunoblotting.

Results

Lung wet:dry weight was greater in the CH compared to sham. Lipid peroxidation was greater in CH and was accompanied by increased compensatory antioxidants (hemeoxygenase‐1, NAD(P)H quinone oxidoreductase‐1).

Conclusion

Brain hypoxemia, despite systemic normoxia, is associated with lipid peroxidation and compensatory antioxidant enzymes, suggesting oxidative stress could contribute to neurogenic pulmonary edema. DARPA N66001‐10‐C‐2134
Gene expression analysis of healthy adults rapidly transported to high altitude
Authors: Herman Nicole Marie , Grill Diane E , Anderson Paul J , Miller Andrew D , O'Malley Kathy A , Johnson Jacob B , Richert Maile L Ceridon , Johnson Bruce D
Published: June 2021

Objective

To identify novel genes that change expression in healthy adults exposed to hypobaric hypoxia.

Background

The uniform and rapid transfer of adults from McMurdo Station (sea level, SL) in Antarctica to the South Pole (2835m, SP) provides a unique opportunity to study the changes in gene expression related to the body's adaptation to high altitude. We hypothesized that a large number of genes would change expression and that many of these genes would be associated with inflammatory processes.

Methods

Peripheral venous blood was drawn at SL and after 2 d at SP in 98 subjects (age 37±9 y, 70% male). Peripheral blood mononuclear cells (PBMCs) were isolated and microarray analysis of the mRNA isolated from these cells was performed (Affymetrix HG U133 Plus 2 microarray chip). Analysis and the computation of expression fold changes was performed using the Bioconductor package in R. Pathway analysis was performed using MetaCore™.

Results

1118 probe sets with significant p‐values (p<0.05) and fold changes were identified and entered into MetaCore™. Several pathways including oxidative phosphorylation, cytoskeletal remodeling, and platelet aggregation were identified as having significant representation in the restricted data set.

Conclusion

Several pathways indicating significantly increased activity in PBMCs were identified. This is indicative of an increased inflammatory response. NSF B‐179‐M.
Hypoxia and cytokines differentially regulate the expression of the tissue plasminogen activator (tPA) and plasminogen activator inhibitor‐1 (PAI‐1) genes in cultured human mesothelial cells (HMCs): Implications for post‐surgical peritoneal fibrinolytic dysregulation
Authors: Keenan Benjamin J , Currie Brian M , Heydrick Stanley J , Stucchi Arthur F
Published: June 2021

Abdominal surgery induces mesothelial hypoxia and inflammation, both implicated in the dysregulation of the peritoneal fibrinolytic system and intraabdominal adhesion formation. Our objective examined the contribution of each using cultured HMCs. Cells were treated with either a cytokine (C) cocktail (TGFβ+IL‐1β+IFN‐γ), the hypoxia mimetic dimethyloxaloylglycine (D) or both, and the expression of tPA and PAI‐1 mRNA was measured at 1, 3, 6, 12, and 18 hours (n=4/group) by RT‐PCR. C alone and C+D increased PAI‐1 mRNA levels at 6 hr (2.4±0.1 fold; p<0.001 and 2.1±0.17 fold; p<0.001 vs controls) and 12 hr (4.1±0.4 fold; p<0.001 and 4.1±0.7 fold; p<0.001 vs controls) while D alone elevated PAI‐1 mRNA levels at 6 and 12 hr (p<0.05). D increased tPA mRNA levels at 12 hr (2.2±0.16 fold; p<0.001) and 18 hr (3.2±0.7 fold; p<0.001) vs control while C+D paralleled closely at both time points (p<0.001). The tPA/PAI‐1 mRNA ratio, a predictor of fibrinolytic capacity, was reduced 40% on average (p<0.001) at 6 hr in all groups vs control. However, it rose thereafter in D and C+D (2.2±0.4 fold; p<0.001 and 3.8±0.7 fold; p<0.001 vs 6 hr) while C continued to drop at 12 hr. Our results show that cytokines appear to regulate PAI‐1 while hypoxia regulates tPA, suggesting that both contribute to peritoneal fibrinolytic dysregulation and adhesiogenesis. Funded by Cooper‐Tyler Endowment, Department of Surgery, Boston University Medical Center.
Prenatal Inflammation (Amnionitis) Impairs Acute Respiratory Response to Hypoxia
Authors: Zenebe Woineshet , Litvin David , Balan Kannan , Martin Richard , Kc Prabha
Published: June 2021

The brainstem autonomic centers regulating the cardio‐respiratory system in infancy are clearly influenced by perturbations in utero, however, long‐term implications on respiratory centers are unknown. One such common perturbation is inflammation attributed to intra‐ or chorio‐amniotic infection. We hypothesized that prenatal inflammation affects brainstem mechanisms altering long‐term respiratory function. In this study, lipopolysaccharide (LPS, 1 μg/amniotic sac) or normal saline (NS) was microinjected in pregnant Sprague Dawley rats at the 20th day of pregnancy into each amniotic sac and pups were delivered spontaneously. Inflammatory signals in the brainstem as well as hypoxic ventilatory response (HVR) using whole body plethysmography were measured. Our multiplex ELISA and supportive immunohistochemical data showed a highest level of brainstem IL1β and TNF‐α at postnatal day one (p1) which progressively declined (p8 and p15). Physiological data showed baseline respiratory frequency was significantly depressed at p3 (NS=139 ±5 vs LPS=113±5 breaths/min, p<0.05) with no significant difference at p8. HVR to acute hypoxia was significantly depressed at both p8 (NS= 65% ±3 vs LPS=39% ±5 increase above baseline) and p15 (NS= 60 % ±4 vs LPS=27% ±4 increase above baseline). These data suggest that prenatal inflammation alters neural development which may induce long‐term alteration in respiratory response to acute hypoxia. We speculate that suppression of inflammatory signals at an early age may be therapeutic in protecting the respiratory control system.

Supported by: NIH R00HL087620
Investigating the effect of temperature on large aggregate conversion of surfactant from ground squirrels
Authors: Cvijanovic Vanja , McCaig Lynda , Staples Jim , Veldhuizen Ruud
Published: June 2021

During hibernation, ground squirrels’ body temperature is 5°C. Our research focuses on pulmonary surfactant during hibernation. Surfactant is a phospholipid‐rich material which regulates surface tension at the alveolar surface. A change in surface‐area during respiration leads to conversion of the large aggregate form of surfactant to the small aggregate form. Since temperature affects aggregate conversion, we tested the hypothesis that hibernation led to less conversion of large to small aggregates. Lungs of hibernating and summer active ground squirrels were lavaged to obtain large and small aggregates which were measured by a phospholipid‐phosphorous assay. Surface‐area cycling was performed at 37°C and 4°C to determine large aggregate conversion. Total amounts of large aggregate were higher in hibernating squirrels compared to summer active group. The large aggregates from all groups underwent a conversion to a greater extent at 37°C than at 4°C. Changes in large aggregate pool sizes occur during hibernation, in vitro aggregate conversion data suggests this may be as a result of reduced aggregate conversion at low temperatures.

Funding: Canadian Institutes for Health Research.
Improvement to the surface tension reducing capability of human pulmonary surfactant with elevated levels of cholesterol via addition of surfactant protein A
Authors: Hiansen Joshua Qua , Yao Li‐juan , Lewis James , Veldhuizen Ruud
Published: June 2021

Pulmonary surfactant is composed of phospholipids, neutral lipids and proteins. Surfactant's composition is important for its ability to reduce surface tensions within alveoli. Previous studies showed that serum‐protein mediated surfactant dysfunction can be overcome by the addition of surfactant protein A (SP‐A) to the material. Elevated cholesterol levels in surfactant can also cause surfactant dysfunction, but it is unknown if SP‐A can reverse this form of impairment. It was hypothesized that the addition of SP‐A to surfactant with elevated levels of cholesterol will improve the material's ability to reduce surface tension. Human surfactant was depleted of cholesterol via repeated acetone precipitation and reconstituted with 0, 5, 10 or 20% cholesterol with or without 5% SP‐A. The minimum achievable surface tension of the samples was tested on a captive bubble surfactometer. Results showed that surfactant with 10 and 20% cholesterol had significantly higher minimum surface tensions than surfactant with 0 and 5% cholesterol. Addition of SP‐A to samples with 10 and 20% cholesterol resulted in significantly lower minimum surface tension as compared to analogous samples without SP‐A. In conclusion, cholesterol mediated surfactant dysfunction can be mitigated by SP‐A. Funding was provided by Canadian Institutes for Health Research.
AMP‐Kinase (AMPK) activation attenuates LPS‐induced lung microvascular endothelial injury
Authors: Jian Ming‐Yuan , Creighton Judy
Published: June 2021

AMPK is a molecular sensor for detection and mediation of cellular adaptations to vascular disruptive stimuli. We have shown previously that AMPKα1 stimulation by endothelial disruptive stimuli enhances repair of injured capillaries. In this study, we hypothesized that bacterial derived endotoxin lipopolysaccharide‐induced activation of AMPK limits vascular injury in the pulmonary capillary circulation. AMPK activity studies reveal that LPS induces a rapid increase in kinase activity that remains elevated over time. Western blot analysis and PCR indicate expression of the AMPKα1 subunit is enhanced by LPS in pulmonary microvascular cells (PMVECs) in culture. These data were confirmed by immunohistology in the lungs of LPS treated rats. Moreover, inhibition of AMPK increased the permeability and decreased the resistance of LPS treated PMVECs in culture and exacerbated LPS‐induced vascular injury in the isolated rat lung. Taken together these data identify AMPK as a barrier protective mechanism in the LPS‐treated lung. Supported by HL102296 and HL 110803.
Hsp90 inhibition attenuates LPS‐mediated NF‐κB‐dependent gene activation by preventing the de‐acetylation and maintaining the DNA binding of acetyl‐histone H3(K9) in human lung microvascular endothelial cells (HLMVEC)
Authors: Thangjam Gagan Singh , Joshi Atul Dattatray , Barabutis Nektarios , Dimitropoulou Christiana , Shaw Mary Cherian , Patel Vijay , Fulton David , Catravas John D
Published: June 2021

Heat shock protein 90 (hsp90) regulates the stability and function of pro‐inflammatory intermediates, including the inflammatory transcription actor, NF‐κB. NF‐κB dependent gene activation requires the displacement of constitutively acetylated histone‐ H3(Lys9) from NF‐κB responsive promoters by, and subsequent binding of, p65. We studied the role of hsp90 in the regulation of histone‐H3/DNA interaction and its effect on NF‐κB gene activation in HLMVEC. Exposure of HLMVEC to LPS (1 EU/ml for 1h) downregulated acetyl‐H3(Lys9) levels and binding to the NF‐κB‐regulated IKBα promoter. This was associated with increased IKBα mRNA expression, increased p65 DNA binding and NF‐κB reporter gene activation. These steps were blocked by treatment with the hsp90 inhibitor, 17‐AAG (5 μg/ml). Since p38 MAPK also enhances transcription by inducing histone‐H3(Ser10) phosphorylation, we also studied its regulation by hsp90. Both 17‐ AAG and the p38 MAPK inhibitor, SB203580 (10 μM), prevented H3(Ser10) phosphorylation and NF‐κB reporter gene activation by LPS. However, unlike 17‐AAG, SB203580 did not block p65 binding to the IKBα promoter. We conclude that hsp90 inhibition blocks LPS‐mediated NF‐κB gene activation by preventing acetyl‐H3(Lys9) downregulation and thus attenuating p65 binding to relevant DNA elements, via a p38 MAPK‐independent mechanism. (Supported by HL093460).
Iloprost suppresses LPS‐induced lung injury and inflammation via Rac1‐dependent mechanism
Authors: Birukov Konstantin G , Wu Tinghuai , Tian Yufeng , Sarich Nicolene
Published: June 2021

Prostacyclin exhibits protective effects on vascular endothelium and enhances endothelial barrier, as reflected by rapid increase in transendothelial resistance. This study investigated for the first time protective effects of stable prostacyclin analog iloprost using in vitro and in vivo models of lung injury and inflammation induced by bacterial wall lypopolysacharide (LPS). Iloprost attenuated disruption of endothelial monolayer and suppressed activation of inflammatory signaling, including p38 mitogen activated protein kinase, NFkB pathway, Rho signaling, attenuated ICAM1 expression, cytokine release, neutrophil adhesion and migration after LPS challenge. In vivo, iloprost exhibited potent inhibition of LPS‐induced lung injury and vascular leak, as detected by suppression of protein and neutrophil accumulation in bronchoalveolar lavage fluid and reduced myeloperoxidase activation, ICAM‐1 expression, and Evans blue extravasation in the lungs. Inhibition of Rac1 activity using pharmacologic inhibitors or siRNA‐mediated knockdown abolished barrier‐protective and anti‐inflammatory effects of iloprost in vitro and in vivo. We conclude that iloprost‐induced elevation of intracellular cAMP triggers Rac signaling, which attenuates LPS‐induced inflammatory pathways of vascular permeability.

Funding: NHLBI HL87823, HL076259, and HL058064
Phosphorylation of TRPC4 as a regulatory mechanism of Isoc
Authors: Vasauskas Audrey A , Cioffi Donna L
Published: June 2021

Calcium signaling mediates a number of cellular events, and in many non‐excitable cells, store‐operated calcium entry (SOCE) represents the major Ca2+ event for signaling. In SOCE, depletion of ER Ca2+ stores leads to extracellular Ca2+ entry through SOC channels. Pulmonary artery endothelial cells (PAECs) exhibit a Ca2+‐selective SOCE current termed Isoc. Activation of Isoc in PAECs leads to formation of inter‐endothelial cell gaps. TRPC proteins 1 and 4 contribute to Isoc channel architecture. The region encompassing the protein 4.1 binding site and adjacent proline‐rich region on the TRPC4 subunit are essential for Isoc channel activation. Isoc inactivation is phosphorylation‐ and Ca2+‐dependent but the identity of kinases/phosphatases and their targets are still being explored. TRPC4 is a promising target for phosphoregulation of Isoc. Here, we examine the phosphorylation status of TRPC4 following activation of thapsigargin‐induced SOCE in PAECs. Following store depletion, TRPC4 phosphorylation increases as evidenced by western blot. In the presence of a calcineurin phosphatase inhibitor, baseline and thapsigargin‐induced TRPC4 phosphorylation is increased, and thapsigargin‐induced inter‐endothelial gap formation is reduced. These data suggest TRPC4 phosphorylation plays a role in Isoc regulation. Supported by 1F32HL112565–01 and 5R00HL089361
Effects of Na/K‐ATPase on cardiac remodeling and regeneration in partial nephrectomized mice
Authors: Drummond Christopher Anson , Wang Xiaoliang , Shapiro Joseph I , Tian Jiang
Published: June 2021

Na/K‐ATPase plays an important role in uremic cardiomyopathy. Reduction of Na/K‐ATPase attenuates survival signaling in cardiac cells. This study tested the role of Na/K‐ATPase in partial nephrectomized (PNx) Na/K‐ATPase α1 heterozygous mice (α1+/−) and their wild‐type (WT) littermates. PNx was established by ligation of one branch of the left renal artery, followed by removal of the entire decapsulated right kidney one week later. Results demonstrated that body weight was lower by the end of the experiment in α1+/− PNx animals (33.63 ± 2.4g vs 36 ± 1.1g in WT), while cardiac hypertrophy was evident in both PNx groups. In α1+/− mice, the heart/body weight ratio was 5.0 ± 0.38 for PNx, vs 3.8 ± 0.08 for sham, whereas in WT animals the ratio was 4.8 ± 0.28 vs 3.8 ± 0.11. PNx significantly increased blood pressure in both groups. Cardiac fibrosis, as indicated by Sirius Red staining, was increased significantly in both genetic groups versus respective shams. To examine regenerative capability cardiac tissues were probed for the progenitor cell marker c‐Kit. The α1+/− mice had a significantly higher number of c‐Kit positive cells in their heart tissue compared to WT mice. We conclude that PNx induces hypertrophic growth and fibrosis regardless of Na/K‐ATPase content. However, regeneration as indicated by c‐Kit expression seems more active in α1+/− mice. Supported by NIH HL‐105649
Abnormal sodium‐potassium dynamics and Na,K‐ATPase expression in Mel‐290 uveal melanoma cells.
Authors: Delamere Nicholas A. , Shahidullah Mohammad , Turco Elena R.
Published: June 2021

Uveal melanoma is the most common form of malignant intraocular tumor. Metastases are common and the mortality for metastatic uveal melanoma approaches 90 %. Here we report on abnormal sodium‐potassium dynamics in a particular uveal melanoma cell line, Mel‐290. The ratio of sodium concentration to potassium concentration in Mel‐290 cells, determined by atomic absorption spectrometry, was 0.57 compared to 0.1 in a different melanoma cell, OCM‐3, and 0.15 in pig ciliary epithelium. Assuming cell Na+K = 140 mM, Mel‐290 cells have resting sodium concentration of approx. 50 mM which is several fold higher than the concentration in normal cells. Western blot studies showed lower abundance of Na,K‐ATPase alpha 1 subunit protein in Mel‐290 compared to OCM‐3. Differences in Na,K‐ATPase alpha subunit phosphorylation also were observed. In contrast to Na,K‐ATPase, H/K‐ATPase beta subunit and H‐ATPase (v‐ATPase) beta subunit were higher in Mel‐290 compared to OCM‐3. A high Na:K ratio was detected in two other melanoma cell types, suggesting the possible existence of a high sodium sub‐group of melanomas. It remains to be determined whether the high‐sodium phenotype is related to a particular pattern of ATPase expression or regulation. It is noteworthy that high cytoplasmic sodium has been detected in certain liver, brain and breast tumors by MRI and other techniques.
Apoptotic gene expression changes in an IRBP‐deficient mouse model of retinal degeneration at 21–27 days of age
Authors: Donmoyer Christine M , Giles Jessica , Homa Linnea
Published: June 2021

In models of retinal degeneration, damage may occur to photoreceptors or retinal pigment epithelial cells, and cellular damage triggers apoptosis. In mice deficient in interphotoreceptor retinoid‐binding protein (IRBP−/−), approx. 40% of photoreceptors are lost by 30 days of age, resulting in partial blindness. TUNEL staining suggests that apoptosis peaks at postnatal day 25 in the IRBP−/−. We asked which genes involved in apoptosis were responsible for photoreceptor loss. IRBP−/− mice, postnatal day 21 through 27 (P21–P27), were sacrificed, and retinas from n=4 mice were pooled. Total RNA from pooled retinas was isolated and used to synthesize cDNA. mRNA abundance was analyzed by quantitative PCR with SYBR green and primers for apoptotic genes; beta‐actin was used as a reference gene. The age with the lowest relative gene expression (cycle threshold, ΔCt) was used as a control (set to 1), and other ages were compared to it (ΔΔCt). The expression ratio (2ΔΔCt) was then calculated. Pro‐apoptotic genes bax, TRADD, ASK‐1, and bnip3l and anti‐apoptotic gene bcl‐2 were studied. mRNA for ASK‐1, bax and bnip31 were most abundant at P23 in IRBP−/− mice, with bax having 300‐fold greater expression than at P21. Anti‐apoptotic bcl‐2 mRNA was also more abundant at P23 (40‐fold more than P21) in IRBP−/− mice, while TRADD expression was highest at P27. In summary, with highest expression at P23, the three pro‐apoptotic genes may be contributing to the apoptotic signal in IRBP‐deficient retinas. Future work can distinguish the apoptotic pathway involved in photoreceptor loss in this mouse model.
Autophagy is induced by hypertonic stress and is associated with microtubule‐dependent pericentrosomsal clustering of autolysosomes
Authors: Nunes Paula , Ernandez Thomas , Roth Isabelle , Qiao Xiaomu , Feraille Eric , Brown Dennis , Hasler Udo
Published: June 2021

Protein damage is a consequence of hypertonic stress, but whether autophagy contributes to the osmoprotective response is unknown. We show that hypertonicity rapidly induced long‐lived protein degradation, LC3 lipidation and Ptdlns3K‐dependent formation of LC3‐ and ATG12‐positive puncta. Lysosomotropic agents but not nutrient deprivation or rapamycin enhanced LC3 lipidation and ATG12‐positive puncta, indicating that hypertonic stress increases autophagic flux. ATG12 siRNA‐mediated knockdown reduced cell survival by hypertonic stress. We show that hypertonicity induces fast reorganization of microtubule (MT) networks, which is associated with strong MT reorganization at centrosomes. MT remodeling was associated with pericentrosomal clustering of ATG12‐positive autolysosomes that co‐localized with p62 and ubiquitin, indicating that autophagy induced by hypertonic stress is partly selective. Efficient autophagy by hypertonic stress required MT remodeling and was dynein‐dependent as autophagosome clustering was enhanced by paclitaxel‐induced MT stabilization and was reduced by nocodazole‐induced tubulin depolymerization as well as chemical (EHNA) or genetic [dynactin 2 (p50) overexpression] interference of dynein activity. The data document a general and hitherto overlooked mechanism, where autophagy and MT remodeling play prominent roles in the osmoprotective response.
Therapeutic potential of a bi‐functional GLP‐1/Glibenclamide targeted to the pancreatic β‐cell
Authors: Hart Nathaniel , Vagner Josef , Weber Craig , Ananthakrishnan Kameswari , Limesand Sean
Published: June 2021

Glucagon like Peptide 1 (GLP‐1) analogs and sulfonyurea drugs are used clinically to enhance pancreatic β‐cell function and treat diabetes. However, rapid degradation and off‐target effects limit their usefulness. Here we describe an approach for targeting β‐cells by synthesizing a bi‐functional agent composed GLP‐1 attached via a flexible linker to a Sulfonyurea receptor antagonist Glibenclamide (Glb). Microscopy shows that this bivalent ligand (Cy5 tagged) binds to, and is rapidly internalized into βTC3 cells which express both receptors. However, GLP‐1/Glb‐Cy5 did not bind significantly to PANC‐1 cells and exhibited right shifted binding to cells that express only one receptor. Glb increases cell Ca2+ and GLP‐1 elevates cAMP. In βTC3 cells, GLP‐1/Glb elicits a half maximal Ca2+ response at ~10nM, but the magnitude of the maximal Ca2+ response was significantly less than that observed for monomeric Glb (~25% of the Glb increase). Unlike the Ca2+response, cAMP production over a range of concentrations elicited by GLP‐1 and GLP‐1/Glb were essentially equal. Even though Ca2+ signaling is reduced, glucose activated insulin secretion remains potentiated by the GLP‐1/Glb ligand (353 +/− 63% increase). The bivalent ligand binds specifically to both receptors and activates signaling in β‐cells. These data suggest that the GLP‐1/Glb ligand has potential as a β‐cell specific therapeutic. Supported by JDRF
Atypical localization of cytoskeletal and anchoring proteins in the choroid plexus of normal and slc4a10 knockout mice
Authors: Damkier Helle Hasager , Praetorius Jeppe
Published: June 2021

The cellular distribution of membrane proteins in the choroid plexus (CP) is known to differ from other transporting epithelia. The Na,K‐ATPase is atypically located in the luminal membrane and AQP1 is located in both membranes. Genetic deletion of slc4a10, which encodes the key basolateral Na+ loader Ncbe, is known to decrease the abundance of AQP1 and Na, K ATPase and cause a redistribution of ezrin that anchors certain membrane proteins to the cytoskeleton.

The aim of the present study was to determine the distribution of known anchoring proteins and cytoskeletal components in CP of normal and NCBE knockout mice by immunohistochemical analysis.

A known binding partner of AQP1, β‐catenin, was located only near the basolateral membrane and was less abundant in the NCBE knockout mouse. Ankyrin 3, a known Na,K ATPase anchor in CP was localized near the luminal membrane, but was less abundant in the NCBE knockout mouse. Spectrin cytoskeleton was localized close to the luminal membrane and is known to bind to Ankyrin 3. In the NCBE knockout mouse, spectrin labelling was observed in both membrane domains, however, but predominantly basolaterally.

In conclusion, AQP1 in the luminal membrane of CP is not anchored to β‐catenin. Furthermore, the removal of the key Na+ loader, NCBE, causes not only a decrease in abundance of the Na, K ATPase binding partner Ankyrin 3, but also a redistribution of the spectrin‐cytoskeleton.
Activating a voltage‐sensitive 5′ phosphatase (VSP) that decreases phosphatidylinositol 4,5‐bisphosphate (PIP2) inhibits electrogenic Na/bicarbonate cotransporter NBCe1‐B and ‐C variants expressed in Xenopus laevis oocytes
Authors: Thornell Ian M. , Bevensee Mark O.
Published: June 2021

Na+‐coupled bicarbonate cotransporters such as NBCe1 variants A, B, and C regulate intracellular pH and promote solute reabsorption/secretion in epithelia. We previously reported that both PIP2 applied to a membrane patch and PIP2 hydrolysis to IP3 in an oocyte can stimulate NBCe1 activity. In the current 2‐ electrode, voltage‐clamp study, we selectively examined the effect of PIP2 on the activity of rat NBCe1 variants expressed in oocytes using a co‐expressed VSP that dephosphorylates PIP2 to PIP. We monitored the time course of a predominantly NBC‐mediated outward current (INBC) elicited by a 5‐s depolarization from −60 mV to either −40 mV (no VSP activation) or +20 mV (VSP activation). For each depolarization, the initial mean INBCs were similar in oocytes expressing NBCe1‐C ± VSP (n=4). At the end of the −40 mV depolarization, mean INBC had further increased only 0.3 ± 0.6% for NBCe1‐C and decayed only 1.9 ± 0.4% for NBCe1‐ C+VSP. However, at the end of the +20 mV depolarization, mean INBC had decayed 77 ± 7.6% for NBCe1‐C+VSP compared to being stimulated by only 7.7 ± 1.5% for NBCe1‐C. Similar results were obtained with NBCe1‐B. VSP markedly inhibited the activity of NBCe1‐B/C in a strongly voltage‐dependent manner. A pronounced VSP‐induced INBC decay for NBCe1‐C was also seen after ER Ca2+ depletion, and is therefore independent of basal PIP2 hydrolysis. Oocyte repolarization and subsequent VSP deactivation restored NBCe1 currents with a time course consistent with restoration of membrane PIP2. The data are consistent with PIP2 itself being required for the activity of NBCe1‐B/C in an intact cell. NIH NS046653