bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of TRPV4 Channels Improves Cardiac Function and Remodeling Following Myocardial Infarction and Transverse Aortic Constriction
Authors: Adapala Ravi K , Luther Daniel J , Ohanyan Vahagn A , Luli Jordan , Thoppil Roslin , Paruchuri Sailaja , Chilian William M , Meszaros J. Gary , Thodeti Charles K
Published: June 2021

Differentiation of cardiac fibroblasts (CF) into hypersecretory and contractile myofibroblasts during cardiac remodeling leads to increased extracellular matrix (ECM) deposition and contributes to diastolic dysfunction and eventual heart failure. Recently, our work has demonstrated that a mechanosenstive ion channel, TRPV4, is involved in mediating CF differentiation into myofibroblasts, by integrating mechanical and soluble signals in vitro. In the present study we investigated the physiological significance of TRPV4 during in vivo cardiac remodeling. Wild type (WT) and TRPV4 null mice were subjected to either myocardial infarction (MI) by ligating the left anterior descending (LAD) artery or pressure overload by transverse aortic constriction (TAC). Cardiac function was analyzed by 2D and PW Doppler echocardiography while cardiac fibrosis was measured using picrosirius red staining. We found that while survival rates are unchanged in MI animals, TRPV4 null mice showed improved survival rate compared WT in mice subjected to TAC. In addition, ejection fraction was preserved in TRPV4 null mice exposed to both MI and TAC surgeries compared to WT mice and fibrosis was attenuated in the TRPV4 null hearts as well. Our findings thus suggest that the absence of TRPV4 channels improves cardiac function and remodeling following myocardial injury.
Both collagen and elastin matrices are remodeled in the failing ovine atria – a role for elastin‐degrading enzymes in atrial structural remodeling
Authors: Horn Margaux A , Graham Helen K , Borland Samantha J , Clarke Jessica D , Dibb Katharine M , Trafford Andrew W
Published: June 2021

Left atrial (LA) remodeling is a feature of the full spectrum of heart failure (HF) and LA structural remodeling (fibrosis) will contribute to diminished ventricular contractile function and risk of atrial fibrillation. Whilst the fibrotic potential of the LA in HF has been documented, the mechanism by which this occurs, and the role of other matrix proteins is less well understood.

HF was induced in female sheep by rapid ventricular pacing. LA dilatation was present in symptomatic animals as measured by echocardiography. Paraffin embedded tissue sections from the left auricle were stained with picro‐sirius red or Millers stain for quantification of collagen and elastin, respectively. Microscopy revealed both elastic, and collagenous components and in HF, collagen:elastin ratio was increased. Gelatinase activity was measured by zymography and protein levels of the elastin‐degrading MMP ‐3, TIMPs 1–4 and secreted protein acidic and rich in cysteine (SPARC) were measured by immunoblotting. MMP‐2 activity, MMP‐3 and SPARC proteins were increased after rapid pacing, whereas levels TIMP‐1 were decreased.

Both collagen and elastin networks are altered in the ovine LA in HF which is likely to contribute towards a stiffer, less compliant phenotype. Changes to different MMP profiles in failing atria may provide a mechanism for this atrial extracellular matrix remodeling.

Work was funded by the BHF.
BCAT2 regulates BCAA metabolic fate during adipogenesis
Authors: Noriega Lilia G , Estrada Isabela , Tenorio Miriam R , Torres Nimbe , Tovar Armando R.
Published: June 2021

Adipogenesis is a controlled differentiation process regulated by a network of transcription factors that include C/EBPβ and PPARγ. PPARγ binds to the vicinity of >;5000 genes through the different stages of differentiation (Nielsen et al, Genes Dev 2008(22):2953–2967). An analysis of these genes revealed that one of them is the mitochondrial branched‐chain aminotransferase (BCAT2) gene. BCAT2 catalyze the first enzymatic step of the branched chain amino acids (BCAA) catabolism. However, the role of BCTA2 during adipogenesis is barely known. Thus, we have evaluated BCAT2 expression and the oxidation, incorporation to lipids or proteins of the three BCAA, leucine, isoleucine and valine, during adipogenesis. Undifferentiated 3T3‐L1 did not express BCAT2 but both its mRNA and protein levels were gradually increased after induction of differentiation, reaching their maximum level at day 12. Regarding BCAA metabolic fate, BCAA were incorporated into protein when BCAT2 was not present, and were mainly oxidized and incorporated into lipids as BCAT2 increased during the differentiation process. Interestingly, leucine was mainly oxidized in adipocytes from lean mice and this was decreased in adipocytes from obese mice. These data demonstrate that BCAT2 regulates the metabolic fate of BCAA during adipogenesis and establish a process that is altered during obesity. Supported by CONACYT‐155949 to LGN.
Leucine + phenylalanine modify the glucose and insulin response to glucose
Authors: Nuttall Frank Quentin , Iverson Jennifer F , Gannon Mary Carol
Published: June 2021

We have shown that phenylalanine (Phe) or leucine (Leu), when ingested with glucose, reduced the glucose response. Leu increased, whereas Phe had little effect on the insulin response. We then were interested in whether the effects would be additive if Phe and Leu were ingested together with glucose. On 4 separate days, 8 healthy subjects were studied. Test meals were given at 0800. Subjects received water, or 25 g glucose, or Leu+Phe (1 mmol/kg fat free body mass each) ± 25 g glucose. Blood glucose, insulin and glucagon were measured frequently thereafter for 2.5 hrs. Net areas under the curve were calculated. The insulin area response increased by 77% (p<0.05) and the glucose area response decreased by 66% (p<0.05) after ingestion of glucose+Leu+Phe compared to ingestion of glucose alone. The insulin response to Leu+Phe was additive compared to previous results obtained when the individual amino acids (AA) were ingested with glucose. In contrast, the decrease in glucose response to Leu+Phe+glucose was less than additive compared to the individual AA ingested with glucose. Blood glucose was unchanged when Leu or Phe, alone or in combination, were ingested without glucose. Thus, ingestion of Leu+Phe with glucose produced an additive increase in insulin area response but a less than additive reduction in glucose area response in healthy subjects.
Connexin 40 is necessary for recovery of ischemic hindlimb perfusion ‐ inflammation and gender considerations
Authors: Burt Janis M , Querin Lauren L , Oulton Jeremy R , Fang Jennifer S
Published: June 2021

Recovery of tissue perfusion in the ischemic hindlimb is reduced, inflammatory response increased, and survival of distal limb tissue compromised in Cx40‐deficient (40KO) compared to wild‐type (WT) mice. Distal limb perfusion is significantly less in 40KO vs. WT mice despite comparable vascular density suggesting compromised temporal regulation of blood flow distribution in 40KO mice. Preliminary data suggested females of both genotypes were at greater risk than males for poor ischemic outcome. Using two models of surgically induced hindlimb ischemia, one preserving gracilis collaterals the other not (mild vs. severe surgery, respectively), we find that this dysregulated flow distribution, compromised arteriogenesis and persistent inflammation contribute to poor post‐surgery outcome in 40KO mice. The persistence (at 14 days) of activated macrophages in the 40KO ischemic limb is preceded by an earlier onset (8 vs. 12h) invasion of neutrophils, both consistent with the pro‐inflammatory phenotype of 40KO mice. Using the VCD model of induced ovarian failure to examine the role of estrogen in ischemic limb recovery, we find that recovery is further compromised in menopausal 40KO mice but improved in menopausal WT mice. Our data indicate that connexin 40 is necessary for recovery of ischemic limb perfusion and may act in synergy with estrogenregulated properties of the vasculature. Support: NIH R01HL058732
FoxO induced inhibition of secreted molecule signalling pathways: role in promoting angiostasis in the ischemic muscle
Authors: Roudier Emilie Annie , Milkiewicz Malgorzata , Haas Tara L
Published: June 2021

We showed previously that FoxO1 promotes an angiostatic environment within of the vasculature of the ischemic muscle by upregulating of the extracellular matrix (ECM) protein thrombospondin‐1. Here, we aim to identify FoxO targets involved in ECM signalling. BMP and activin membrane‐bound inhibitor (BAMBI) and soluble frizzled‐related protein 1 (sFRP1) are respective inhibitors of the signals of 2 secreted molecules TGF‐β and Wnt. Primary cultures of microvascular endothelial cells (MEC) of mice having the 3 FoxO genes flanked with LoxP sites (FoxOL/L) were transduced with adeno‐Cre recombinase to delete FoxO genes. This increased BAMBI mRNA levels by 7 fold, and decreased sFRP1 expression by 2.8 fold. Conversely, over‐expression of constitutively active FoxO1 in MECs lowered BAMBI mRNA by 70% and increased sFRP1 mRNA levels by 110%. Within murine ischemic muscle (4 days post femoral artery ligation), the levels of sFRP1 were increased (p=0.055) and BAMBI expression was reduced significantly (p=0.03). Ischemia‐induced alterations in sFRP1 and BAMBI expression were abolished in mice with EC‐directed deletion of FoxO (ΔFoxO). Our data indicate that sFRP1 and BAMBI are regulated reciprocally by FoxO1. The FoxO‐dependent regulation of Wnt and TGF‐β signalling pathways may contribute to establish an angiostatic microenvironment within ischemic muscle.
Post‐treatment with the heat shock protein 90 (hsp90) inhibitor, 17‐AAG, reduces pulmonary inflammation, hyper‐permeability and airway dysfunction associated with LPS‐induced acute lung injury (ALI) in mice.
Authors: Dimitropoulou Christiana , Joshi Atul , Barabutis Nektarios , Shaw Mary , Patel Vijay , Catravas John D
Published: June 2021

Pre‐treatment with the heat shock protein 90 (hsp90) inhibitor, 17‐ AAG, reduces the magnitude of LPS‐induced ALI in mice. We now tested the hypothesis that post‐treatment with 17‐AAG would also reduce the magnitude of LPS‐induced ALI. 24h after the intratracheal instillation of vehicle or LPS (1.5mg/kg), mice received either vehicle or 17‐AAG (i.p. 10mg/kg). 48h later bronchoalveolar lavage fluid (BALF) was collected, and the number of cells and protein concentration were estimated as indicators of inflammation and permeability, respectively. In separate groups, lung capillary permeability was estimated by the Evans’ Blue Dye (EBD) method. LPS alone increased BALF cellularity (452±50 ×103cells/ml vs 33±2 ×103cells/ml in vehicle) and BALF protein concentration (204±5% from vehicle). 17‐AAG post‐treatment reduced the LPS‐induced BALF cellularity (140±32 ×103cells/ml) and protein concentration (119±18% from vehicle). Furthermore, LPS produced a 193±18% increase in EBD extravasation which was also significantly reduced to 135±27% in mice post‐treated with 17‐AAG. In additional mice, pulmonary function tests were performed. LPS caused a nearly 100% increase in airway resistance that was completely prevented by post‐treatment with 17‐AAG. These data strongly suggest important reparative actions of hsp90 inhibitors when administered after the LPS insult.
Effect of combined aerobic and resistance exercise training on circulating progenitor cells in sedentary men
Authors: Lin Chin‐Pu , Wang Jong‐Shyan
Published: June 2021

Circulating progenitor cells (CPCs) may improve hemodynamic function by enhancing neovasculogenesis. This study investigated whether combined aerobic and resistance exercise training affected cardiac and muscular hemodynamics by modulating CPCs. Twelve healthy sedentary males were trained on a bicycle ergometer (60% VO2max for 30 minutes) for 3 days/week and an isokinetic dynamometer (alternating 60°–180°/s for 6 to 10 sessions) for 2 days/week for 6 weeks. A noninvasive bioreactance device was used to measure cardiac hemodynamics, and near‐infrared spectroscopy was applied to assess perfusion/oxygenation in vastus lateralis. The levels of various CPCs were detected by three‐color flow cytometry. The results showed that combined aerobic and resistance exercise training significantly increased work‐rate and VO2 at maximal performance, which was accompanied by the increases in cardiac output and perfusion/oxygenation of vastus lateralis during exercise. This training regimen simultaneously increased CD34+/KDR+, CD34+/KDR+/CD117+, CD34+/KDR+/CD133+, or CD34+/KDR+/CD31+ cells, as well as, decreased these CPCs with exposure of phosphatidylserine in blood. Hence, we conclude that combined aerobic and resistance exercise training promotes mobilization and suppresses apoptosis of CPCs, which is associated with the improvements in cardiac and muscular hemodynamic functions.
Enhanced spontaneous cage activity induced by continuous low intensity spinal cord epidural stimulation in complete spinal cord transected adult rats
Authors: Gad Parag , Choe Jaehoon , Rath Mrinal , Shah Prithvi , Gerasimenko Yury , Zhong Hui , Roy Roland R , Edgerton V Reggie
Published: June 2021

Epidural stimulation (ES) of the spinal cord (SC) has been shown to have great potential for improving motor function after a spinal cord injury. Subthreshold levels of ES are used so as to not induce but to enable these motor functions to be initiated and controlled by proprioception from the hindlimbs (HL). Three completely paralyzed humans were implanted with a commercially available electrode array. In these three subjects ES in combination with the proproceptive input and weeks of stand training resulted in full weight‐bearing standing. Given the importance of training and ES, we hypothesized that in paralyzed rats, ES during movements in the cage would greatly increase the of activation of locomotor circuits, resulting in improvements in motor function. The spinal rats were very lethargic in their cages with little movement. Without ES the rat's body remained close to the cage floor. When the rats used their forelimbs (FLs) to move, the HLs dragged behind with little or no the flexor activity. In contrast when stimulated the rats were highly active. With ES the HLs showed alternating flexion and extension resulting in step‐like movements when the forelimbs were used to move. With ES, the rats often would stand using the sides of the cages as support. These data provide evidence that ES, in combination with proproceptive input, can modulate the neural networks such that motor tasks are likely to be practiced spontaneously.
Endothelium dependent vasodilation in young, obese adults: contribution of NOS
Authors: Lee Peltonen Garrett , Johansson Rebecca E , Harrell John W , Limberg Jacqueline K , Crain Meghan K. , Sebranek Joshua J , Walker Benjamin J , Marlowe Eldridge W , Schrage William G
Published: June 2021

Endothelium dependent vasodilation (EDD) is impaired in older, obese adults. However, it remains unknown whether this impairment exists in young, obese adults. We hypothesized that young, obese adults would exhibit attenuated EDD and altered nitric oxide (NO) production. In 10 young, lean (age: 27±2 yrs; BMI: 23±1 kg/m2) and 7 young, obese (23±2 yrs; 38±4 kg/m2) adults forearm EDD was assessed with infusion of acetylcholine (ACH) into the brachial artery. Contribution of NO to EDD was determined by simultaneous infusion of LNMMA and ACH. Smooth muscle responses to NO were tested with nitroprusside (NTP). Forearm blood flow (FBF) was measured with Doppler ultrasound and normalized for mean arterial pressure (MAP) by calculating forearm vascular conductance (FVC =FBF/MAP). ACH infusion increased FVC (p<0.01). FVC was greater in obese versus lean adults expressed as either an absolute value (207 vs. 130 ml min−1 mmHG−1; p<0.01) or change from baseline (165 vs. 90; p<0.01). NOS inhibition decreased ACH mediated EDD (p=0.02) equally in lean and obese subjects. FBF responses to NTP were similar between groups. In conclusion, our data suggest that young obese adults exhibit greater EDD, but this is not due to altered NOS function.

Support: NIH R01 HL105820.
Non‐Uniform Age‐Related Decrements in Regional Sweating and Skin Blood Flow
Authors: Smith Caroline J. , Alexander Lacy M. , Kenney W. Larry
Published: June 2021

Aging is associated with attenuated thermoregulatory effector function which varies regionally over the body. Our aim was to examine age‐related alterations in the relation between regional sweating (RSR) and skin blood flow (SkBF) in response to thermal and pharmacological stimuli. Four microdialysis (MD) fibers were placed in the ventral forearm, abdomen, thigh, and lower back of 8 healthy older (O, 64 ± 2 yrs) and 9 young (Y, 23 ± 2 yrs) men and women matched for anthropometric characteristics and VO2max. RSR and SkBF (laser‐Doppler flux; LDF) were measured over each MD site during whole body heating (WBH; Δ1°C oral temperature) and acetylcholine dose response protocols (ACh 1×10−7–0.1M). During WBH RSRs were attenuated (p<0.001) and onset thresholds were delayed at all sites (p<0.05) in the O. SkBF was also attenuated with age (p<0.001) with few differences in onset (Y vs. O back p<0.05). Within groups, RSR (p<0.001) but not SkBF varied among sites. Cholinergic sensitivity was similar between groups for RSR but varied for SkBF (arm 1.0–10mM P<0.05; abdomen p<0.05). RSR did not differ among sites but SkBF varied in the O (p<0.05). Together these data suggest regional age‐related decrements in heat‐activated sweat gland function but not cholinergic sensitivity. Site‐specific difference in sweating tend to disappear with aging.

Supported by NIH RO1‐ AG007004‐22 (W.L.K.) and ACSM Foundation Research Endowment 2012 (C.J.S).
Characterization of the cutaneous blood flow‐local temperature response through its entire range
Authors: Bruning Rebecca S , Alexander Lacy M. , Kenney W. Larry
Published: June 2021

Thermal provocation of the skin circulation has been used to assess resistance vessel function, yet the response to dose‐dependent changes in local skin temperature (Tsk) has not been examined across the range from full vasoconstriction (VC) to full vasodilation (VD). Skin blood flow responses were assessed in 6 young healthy subjects (3 men, 3 women) using laser Doppler flowmetry (LDF) during two different heating and cooling protocols. 3 sites on the ventral forearm were randomly assigned to cooling (34°C–17°C), heating (31°C–41°C), or thermoneutral. R was a continuous ramp (0.6°C•min−1); S used stepwise heating/cooling at 0.33°C•min−1. Cutaneous vascular conductance (CVC) was calculated as LDF/MAP. Both protocols elicited highly reproducible CVC‐Tsk function curves, whether expressed as absolute or normalized CVC. There were no significant differences in thresholds for VD (R: 36.0 ± 0.6°C, S: 36.6 ± 0.7°C, p=0.37) and VC (R: 30.9 ± 0.8°C, S: 32.3 ± 0.8°C, p=0.12), the max (R: 2.15 ± 0.26, S: 2.23 ± 0.20, p=0.81), min (R: 0.04 ± 0.01, S: 0.04 ± 0.01, p=0.85), or range (R: 2.08 ± 0.29, S: 2.17 ± 0.19, p=0.79) of CVC between protocols. Only R elicited both an axon reflex and cold‐induced VD. Standardized thermal provocation elicits highly reproducible changes in skin blood flow and may provide a means to noninvasively assess changes in resistance vessel function across the entire response range in health and disease.
A novel look at KIR channels and potassium in human skin
Authors: Francisco Michael A , Fujii Naoto , Minson Christopher T , Brunt Vienna E
Published: June 2021

Tetraethylammonium (TEA) abolishes a large portion of the vasodilation observed with both cutaneous thermal and reactive hyperemia by blocking calcium‐sensitive potassium (KCa) channels. Some studies have shown effects of TEA on other potassium channels such as inward‐rectifying potassium (KIR) channels, which are activated by increases in extracellular K+ and blocked by barium (Ba2+). Our primary goal was to test the specificity of TEA to KCa channels by ensuring TEA has no effect on KIR‐mediated vasodilation to KCl. In doing so, we also observed novel insights into the roles of KIR channels and K+, which have not been studied in skin. The following drugs were infused via microdialysis: 1) Control; 2) 50mM TEA; 3) 100μM barium chloride (BaCl2; KIR channel blocker to verify the effects of KCl). KCl was infused for 2 min at each site at 1mM, 10mM, and 100mM doses. Dilation was characterized as Δ peak from baseline, and presented as % maximal cutaneous vascular conductance (CVC). Dilation at the control site (1mM: 10.3±4.8%, 10mM: 10.2±4.2%,100mM: 11.9±4.4%) indicated 1mM KCl is sufficient to achieve maximal activation of KIR channels, which is only ~ 10% of max. All dilation to KCl can be blocked with 100μM BaCl2 (1mM KCl: 4.0±2.1%, 10mM KCl: 0.8±0.7%). Furthermore, TEA had no effect on KCl‐mediated dilation (1mM KCl: 10.8±4.6%, 10mM: 11.9±5.6%), confirming that TEA is specific to KCa channels.

Supported by NIH Grant HL081671
Reduced contribution of NOS and CO to beta adrenergic vasodilation in obesity
Authors: Crain Meghan Kathleen , Johansson Rebecca E , Peltonen Garrett L , Harrell John W , Schrage William G , Limberg Jacqueline K , Sebranek Joshua J , Walker Benjamin J , Eldridge Marlowe W
Published: June 2021

Research indicates older obese (OB) individuals (>;55 yrs) exhibit impaired endothelial dependent dilation (EDD). However, there is limited information on the effects of obesity on EDD in younger adults (< 40 yrs). We hypothesized that young OB adults would exhibit decreased β‐adrenergic mediated EDD and demonstrate a lower contribution of nitric oxide synthase (NOS) and cyclooxygenase (COX) to vasodilation compared to lean controls. Forearm blood flow (FBF) was measured with Doppler Ultrasound during infusion of Isoproteronol (ISO), into a brachial artery catheter in 7 OB adults (23±2yrs, BMI=38±4) and 11 lean adults (27±2yrs, BMI=23±1). Forearm Vascular Conductance (FVC=FBF/BP) was calculated to normalize for blood pressure. ISO infusion was repeated after combined inhibition of NOS and COX, with L‐NMMA and Ketorolac, respectively. ISO increased FVC similarly in both groups. Combined NOS‐COX inhibition decreased FVC in both groups (p<0.01), but the decrease tended (p=.06) to be larger in lean (−30 ml/min/mmHg) than the OB group (−11 ml/min/mmHg). EDD to ISO is similar in lean and OB groups, however lean adults rely more on NOS and COX for vasodilation than the OB adults. These data suggest young OB adults maintain β‐adrenergic EDD by activating mechanisms largely independent of NOS and COX. Support NIH R01 HL105820.
Rett Syndrome mutation MeCP2 T158A affects hypoxic ventilatory response in mice
Authors: Bissonnette John M , Knopp Sharon J , Zhou Zhaolan
Published: June 2021

Rett syndrome (RTT), whose phenotype includes respiratory disorders, is caused by loss‐of‐function mutations in methyl‐CpG‐binding protein 2 (MeCP2). Mouse models of the syndrome that have been generated by deletion of Mecp2 have an altered ventilatory response to acute hypoxia (HVR) characterized by a minute ventilation (VE) that exceeds wild type (WT) and that persists in the early recovery period. Such large deletions, however, account for only ~10% of cases. Here we determine if mice with a common single point mutation retain this respiratory pattern. Knockin mice that carry a RTT‐associated threonine to alanine mutation at residue 158 (T158A) show RTT‐like behavioral phenotypes similar to Mecp2‐null mice (Nature Neuroscience 15:274,2011). Experiments were performed in heterozygous T158A female mice at 9 months of age. Baseline respiratory frequency, tidal volume and minute ventilation in T158A animals was similar to WT. Compared to WT littermates relative VE of T158A at 1 min of 8% O2 was twice as large. WT mice showed the expected post‐hypoxia frequency decline (224±9.5 to 203±12 bpm) while T158A had a sustained tachypnea (203±10 to 219±14.5). HVR in mice with a T158A mutation is similar to that with a null mutation of MeCP2.

Support: IRSF, RSRT, NewLife Foundation (JMB); NIH, IRSF (ZZ)
Role of vascular dysfunction in prostate tumor perfusion during exercise
Authors: McCullough Danielle J. , Siemann Dietmar W , Behnke Bradley J
Published: June 2021

During exercise, blood flow to inactive tissues is reduced through changes in vascular tone elicited largely by the autonomic nervous system. However, in tumors the reduced innervation of feed arteries and possibly blunted adrenergic vasoconstriction may lead to an increased tumor perfusion during exercise. We used control (CON; n=16) and prostate tumor bearing (TB; n=14) Copenhagen rats to test the hypothesis that α‐adrenergic (via norepinephrine; NE) and myogenic vasoconstrictor responses are diminished in the prostatic tumor feed arteries (PTA), resulting in an increased tumor perfusion during exercise. Dunning R‐3327 MatLyLu tumor cells (104) were injected into the ventral prostates of rats. PTA and prostate arteries (CON) were studied in vitro and exposed to cumulative additions of NE (10−9to 10−4M) and increases in intraluminal pressure (myogenic response; 0 to 135 cmH2O). In vivo quantification of perfused tumor vessels during rest and acute exercise (treadmill; 5 min) was determined using Hoechst‐33342 staining. Both α‐adrenergic (7 ± 6% vs. 88 ± 2%, p<0.05) and myogenic vasoconstriction were blunted in PTA versus CON, while the number of PTA perfused increased ~113% with exercise. These data suggest that dysfunction in the PTA results in an inability to augment vascular resistance during exercise, resulting in an enhanced tumor perfusion.

Supp.: NIH (AG‐31317) & Florida Biomed. Res. Prog. (1BN‐02).
Alterations in the Coagulation and Fibrinolytic Systems following an Ultra‐marathon
Authors: Kupchak Brian Richard , Volk Brittanie M , Kunces Lauren , Phinney Lauren , Hoffman Martin D , Phinney Stephan D , Volek Jeff S
Published: June 2021

The aim of this study was to examine coagulation and fibrinolytic responses in runners competing in the Western States Endurance Run (WSER, June 23 to 24, 2012). The WSER is a 160‐km trail foot race through the Sierra Nevada Mountains that involves 15,540 ft of climb and 22,970 ft of descent. We examined 12 men and 4 women (mean±SE, age 44.6±3.0 y), who completed the race (24.64±1.07 hrs). Blood samples were collected the morning before the race, immediately post‐race, and 1 (D1) and 2 (D2) days post‐race (corresponding to 51 – 54 hr, and 75 – 78 hr from the start of the race, respectively). Muscle damage was assessed by serum creatine kinase (CK) and myoglobin. Hypercoagulable state was characterized by prothrombin fragment 1+2 (PTF 1+2) and thrombin‐antithrombin complex. Fibrinolytic state was assessed by plasminogen activator inhibitor antigen (PAI‐1 Ag), tissue plasminogen activator antigen (tPA Ag), and D‐Dimer. Significant increases were measured immediate post‐race for thrombin generation markers, PTF 1+2 and TAT; markers of fibrinolysis, tPA Ag, PAI‐1 Ag, and D‐Dimer; muscle damage markers, CK and myoglobin. Most markers returned to baseline levels at D1, except for PTF 1+2 and D‐Dimer. Muscle damage markers, CK and myoglobin; also continued to be elevated at D1 and D2 time points. In spite of these increases, no clinical evidence of vascular occlusion was observed following the race. In conclusion, the WSER produced extensive muscle damage and activated both the coagulation and fibrinolytic systems. Moreover, these systems are in balance thus limiting the potential of a thrombotic episode.
Is Sympathetic Restraint of Skeletal Muscle Blood Flow Present During Exercise?
Authors: Barrett‐O'Keefe Zachary , Ives Stephen J. , Trinity Joel D. , Witman Melissa A.H. , Rossman Matthew J. , Groot H. Jonathan , Morgan David E. , Gmelch Benjamin , Richardson Russell S. , Wray David Walter
Published: June 2021

To better define the role of sympathetic vasoconstriction in the regulation of exercising skeletal muscle blood flow, five healthy men (24 ± 1 yrs) performed knee‐extensor exercise at 0, 5, 10, and 15 watts during intra‐arterial infusion of phenylephrine (PE, alpha‐1 agonist), phentolamine (PHEN, non‐selective alpha adrenergic antagonist), and saline. Arterial blood pressure, leg blood flow (Doppler ultrasound), and leg vascular conductance were determined continuously. During exercise, PE‐mediated reductions in leg blood flow (−27 ± 11, −22 ± 9, −8 ± 8, −13 ± 9% at 0, 5, 10, and 15W, respectively) and vascular conductance (−27 ± 11, −23 ± 9, −9 ± 8, −13 ± 9%) were blunted in an intensity‐dependent manner. In contrast, PHEN administration provoked a 30–40% increase in both leg blood flow (32 ± 8, 40 ± 12, 42 ± 12, 35 ± 9%) and leg vascular conductance (38 ± 10, 45 ± 14, 44 ± 12, 36 ± 9%) that persisted during all exercise intensities. Arterial blood pressure remained unchanged throughout all drug infusions. Together, these data support the concept of “functional sympatholysis” during exercise, but indicate a more prominent role for endogenous, alpha receptor‐mediated restraint of skeletal muscle blood flow than has been reported previously. Research support from NIH PO1 HL‐091830, VA RR&D E6910R, and AHA 0835209N.
Adenosine receptor inhibition attenuates cutaneous vasoconstriction during whole‐body cooling
Authors: Kenny Glen P , McGinn Ryan , Swift Brendan , Gagnon Daniel
Published: June 2021

It has been suggested that adenosine may modulate cutaneous vasoconstriction. Therefore, we examined if adenosine receptor inhibition would attenuate decreases in cutaneous vascular conductance (CVC) during cold stress. Two microdialysis probes were inserted into the dermis layer of the forearm in eight subjects (4 females, 4 males). The probes were infused continuously with Ringer's lactate (CON) or theophylline (THEO). Skin blood flow was measured continuously (laser‐Doppler) to calculate CVC (% of max). Following 45 min of infusion to ensure complete blockade of adenosine receptors, subjects were exposed to two 20 min periods of whole‐body cooling (via water‐perfused suit), each separated by 45 min of heat stress. Decreases in CVC did not differ between CON and THEO sites during the first cooling period (p=0.218). CVC increased to similar levels during the heat stress period (p=0.820). During the second cooling period, CVC was modeled using nonlinear regression, which revealed that the curves differed between sites (F(4,168) = 7.712, p<0.01). Minimum values of CVC were significantly greater at THEO (38 ± 10%) compared to CON (19 ± 13%, p<0.01). These results suggest that adenosine receptor inhibition significantly attenuates cutaneous vasoconstriction during whole‐body cold stress, but only following cutaneous vasodilation induced via passive heating. Supported by NSERC grant RGPIN‐298159–2009 (held by GP Kenny).
Carbonic anhydrase activity underlies spike frequency adaptation in avian intrapulmonary chemoreceptors (IPC)
Authors: Lee Sheridan Krystal , Lonjaret Jean‐Guillaume , Hempleman Steven C
Published: June 2021

IPC are vagal respiratory afferents that sense intrapulmonary CO2 and control breathing in birds. Most IPC show spike frequency adaptation (SFA) which helps encode phasic and tonic information about CO2 changes within each breath. Carbonic anhydrase (CA) is required for CO2 transduction, and we hypothesized that rapid CA‐catalyzed CO2 hydration and dehydration underlies SFA in IPC. Small doses of CA inhibitor acetazolamide (AZ) were used to test if phasic IPC CO2 responses (SFA) could be inhibited independently of tonic CO2 responses. Single cell IPC spike trains were studied in seven pentobarbital‐anesthetized mallards (Anas platyrhynchos). SFA was decreased 23.0% and 66.2% (p<0.05) at 0.5 and 2.0 mg/kg AZ, respectively, while tonic IPC responses were not affected until AZ dosages ≥5 mg/kg. Results suggest that SFA in IPC is due to rapid intracellular CA catalysis of CO2 hydration resulting in a kinetic overshoot of intracellular pH with abrupt changes in intrapulmonary CO2. We present a model of linked differential equations that explores this hypothesis. Support: NIH R15 HL087269–02 and R25 GM056931–15.
Intermittent hypercapnia‐induced GABAergic plasticity sufficiently enhances ventilatory CO2/pH chemosensitivity to overcome serotonergic dysfunction
Authors: Mosher Bryan Patrick , Taylor Barbara E. , Harris Michael B.
Published: June 2021

Serotonergic dysfunction compromises ventilatory CO2/pH chemosensitivity and is thought to enhance vulnerability to Sudden Infant Death Syndrome (SIDS). We have shown raphé contributions to central chemosensitivity involving both serotonin (5‐HT) and γ‐aminobutyric acid (GABA) mediated mechanisms. We test the hypothesis that exposure to mild intermittent hypercapnia (IHc) during postnatal development will induce long‐lasting respiratory plasticity, due in part to strengthening of GABA mechanisms. Rat pups were exposed to daily IHc (9 consecutive cycles; 5 min 5% CO2 + 10 min air; constant air control) for 5 days beginning at postnatal day P12. We subsequently assessed CO2/pH chemosensitivity using an unanesthetized juvenile rat in situ perfused decerebrate brainstem preparation. Chemosensitivity to a 4% arterial CO2 challenge was tested at P21–35, P36–50 and P51–65. 5‐HT or combined 5‐HT and GABA dysfunction were induced pharmacologically with the 5‐HT2A antagonist ketanserin alone or combined with GABAA and GABAB antagonists bicuculline and saclofen. Sensitivity was sufficiently enhanced by IHc to overcome 5‐HT dysfunction in all groups indicating long‐lasting plasticity. Sensitivity was abolished with combined 5‐HT and GABA antagonism, indicating that GABA mechanisms contributed to plasticity. Supported by NIH 2U54NS041069–06A1 and 5P20RR016466.
Hypercapnic acidosis (HA) increases glutamatergic but decreases GABAergic input to chemosensitive neurons from the caudal nucleus tractus solitarius (cNTS) from neonatal rats
Authors: Li Ke‐Yong , Putnam Robert W.
Published: June 2021

We examined the modulation of synaptic input by HA in cNTS neurons. Whole‐cell patch clamp was used to measure the postsynaptic currents in brainstem slices from neonatal rats. The frequency of spontaneous excitatory postsynaptic currents (sEPSC) was increased from 1 to 1.6 Hz (p=0.02; n=11) by HA (15% CO2) and from 1.3 to 2 Hz (n=7; p=0.03) by HA (10% CO2) in chemosensitive cNTS neurons. Neither 10% nor 15% CO2 had an effect on the amplitude of sEPSCs (10–15 pA). In the presence of tetrodotoxin, HA (15% CO2) resulted in an increase in the frequency of miniature excitatory postsynaptic currents (mEPSC) from 0.4 to 0.6 Hz (n=4; p=0.006) but did not affect mEPSC amplitude (19 pA). In contrast, HA (15% CO2) resulted in a significant decrease in the frequency of spontaneous inhibitory postsynaptic currents (sIPSC) from 0.12 to 0.04 Hz (p=0.03; n=5), with no change in amplitude (6–8 pA). In nonchemosensitive cNTS neurons, HA (10% CO2) had no effect on sEPSC frequency (2.2 Hz) or amplitude (10 pA). The GABA‐A receptor agonist muscimol (3 μM) completely inhibited the firing rate of chemosensitive neurons in slices from neonatal rats aged P1‐P3, suggesting that GABA is inhibitory from birth in cNTS neurons. In conclusion, HA enhances presynaptic release of glutamate while reducing GABA release showing that the chemosensitive response of cNTS neurons is modulated by changes in synaptic input. Supported by WSU Foundation Grant.
Erythropoietin increases the hypoxic sensitivity of the neural respiratory control system in newborn mice
Authors: Soliz Jorge
Published: June 2021

We demonstrated previously that cerebral erythropoietin (Epo) enhances the ventilatory response under conditions of reduced oxygen availavility (hypoxia).. Specificaly, by using adult mice, we showed that Epo activates the central respiratory network under conditions of acute and chronic hypoxia. However, the impact of Epo on the respiratory control system of newborn has not yet been investigated. In this study, we hypothesized that cerebral Epo crucially affects the neural respiratory system during the postnatal development of mice. To investigate the central hypoxic sensitivity, we used the in vitro (en bloc brainstem‐spinal cord preparations ) electrophysiological technique in animals at postnatal day 4. When perfused with hypoxic artificial cerebrospinal fluid (5% CO2 in N2), Epo‐incubated brainstems showed increased time‐ and dose‐dependent phrenic activity. Moreover, this effect was dramatically reversed when bainstems were incubated with soluble Epo receptor (the Epo endogenous antagonist). To assess the effect of Epo on ventilatory response to hypoxia (RVH) in vivo, we applied the whole body plethysmography technique in mice at postnatal days P7, P10, P15 and P21. Once exposed to acute hypoxia (12%, 10% and 6% O2), Epo‐treated animals at all tested ages (i.p injection of 100U Epo 12h before experimentation) showed higher ventilatory response than corresponding controls animals. These results imply that cerebral Epo plays a crucial role in the neural development of respiratory system, and suggest that Epo in brain has potential clinical implications in newborn with respiratory dysfunctions, especially in those born preterm.
A fetal mouse lung slice model for molecular live cell imaging of the pulmonary neuroepithelial body microenvironment
Authors: Schnorbusch Kathy , Brouns Inge , Lembrechts Robrecht , Pintelon Isabel , Timmermans Jean‐Pierre , Adriaensen Dirk
Published: June 2021

The prominent presence of differentiated neuroepithelial bodies (NEBs) from early embryonic development on, with the highest density in the perinatal period, reflects important prenatal functions and involvement in the transition from fetal to neonatal life. We therefore adapted our established ex vivo live cell imaging lung slice model to allow functional studies in fetal mouse lungs.

Using Fluo‐4, changes in [Ca2+]i could be monitored, revealing that ATP causes a [Ca2+]i rise in fetal airway epithelial cells. Additional identification of prenatal NEBs with the vital dye 4‐Di‐2‐ASP, showed that extracellular high K+ evoked a reversible [Ca2+]i increase in fetal NEB cells. Fixed lung slices of prenatal mice revealed immunostaining for CGRP in NEBs, and a P2X3 ATP receptor‐expressing population of NEB‐associated nerve terminals, highlighting the possibility to directly link physiology to neurochemistry in this model. Since slices of fetal GAD67‐GFP knock‐in mice show selective GFP fluorescence in NEB cells, use of these animals will definitely improve physiological studies of NEBs.

The presented fetal mouse lung slice models offer excellent possibilities for molecular live cell imaging to further unravel the functional significance of the NEB microenvironment during the pre‐ and perinatal period.

Grants: IWT (SB 81162); FWO (G.0589.11), Hercules Foundation (AUAH‐09–001); UA (GOA BOF 2007; KP BOF 2011)
Angiopoietin like‐2 knock‐down improves the lipid profile of high‐fat diet‐fed mice and maintains vascular endothelial function
Authors: Yu Carol , Thorin Eric
Published: June 2021

Angiopoietin like‐2 (angptl2), a member of the angiopoietin‐like protein family, can contribute to insulin resistance in obesity (Tabata 2009). However, angptl2 involvement in lipid profiling and vascular endothelial function remain largely unknown.

Objective

An angptl2 knock‐down (KD) mouse was used to study the role of angptl2 in lipid handling and endothelial function in the femoral artery.

Methods/results

3 month‐old wild‐type (WT) and KD mice were fed a regular diet (RD) or high‐fat diet (HFD) until 6 months of age. WT and KD mice displayed similar lipid profiles under the RD. While total cholesterol and high‐density lipoprotein (HDL) levels increased similarly in WT and KD mice after a HFD, low‐density lipoprotein (LDL) levels increased 2.7‐fold in WT and only 1.9‐fold in KD so that total cholesterol‐to‐HDL and LDL‐to‐HDL ratios increased significantly (P<0.05) only in WT but not in KD following HFD. Under a RD, KD mice displayed improved endothelial function as measured by the EC50 of the acetylcholine‐induced dose‐response curve. Although this difference in sensitivity between the two strains was lost after a HFD, contribution by nitric oxide (NO) was maintained in KD but not in WT.

Conclusion

Angptl2 knock‐down in mice may improve lipid handling when challenged with a HFD, and lead to maintenance of endothelial function, specifically NO contribution to vasorelaxation. Supported by CIHR 14496 and NSERC.
Compartmentalization of redox processes, Nox4 and endoplasmic reticulum chaperones within lipid droplets in vascular smooth muscle cells
Authors: Abrahao Thalita Balsamo
Published: June 2021

Lipid droplets (LD) are more than just sites for neutral lipid storage. Present in most cell types, LD display a complex proteomic profile suggestive of signaling functions. NADPH oxidase‐driven redox signaling involves association with protein disulfide isomerase (PDI), an endoplasmic reticulum (ER) redox chaperone that appears in LD proteomes. Knowing that LD are present in aorta tunica media, our objective is to investigate if LDs are dynamic compartments for NADPH oxidase transport/location in vascular smooth muscle cells (VSMC). Known stimulators of NADPH oxidases, such as angiotensin II, promoted changes in LD size, dispersion and number. Subcellular fractionation showed that LD fraction carries PDI and ER chaperones. Nox4 is detectable in LD fraction by western blot and confocal microscopy. Contrarily, the known Nox4 regulators Poldip2 and p22phox were not detectably associated with LD. VSMC knockout for Nox4 or with pharmacological inhibition of Nox4 display more lipid droplets than control, consistent with an anti‐lipogenic effect of Nox4. Thus, LD lipogenesis/lipolysis seems to be regulated by Nox4. Conversely, siRNA PDI and SOD/catalase treatment promoted decrease in number of LD and oxidized lipids were detected within LD. We conclude that LD are relevant to redox signaling processes and take part in Nox4 (patho)biology and ER‐associated events. Support: FAPESP, CNPq
Systemic oxidative stress in older adults: Do peripheral blood mononuclear cells contribute?
Authors: Schanzle Jennifer C , Deo Shekhar H , Fadel Paul J
Published: June 2021

Although it is well known that systemic oxidative stress increases with age, the sources and mechanisms contributing to aging induced increase in oxidative stress remain unclear. Recent findings in animals indicate that peripheral blood mononuclear cells (PBMCs) are a major source of systemic oxidative stress via stimulation of the angiotensin II type 1 receptor (AT1R)‐NADPH oxidase pathway. Thus, to better understand this pathway and its potential contribution to increased oxidative stress with age, PBMCs were isolated from whole blood in healthy recreationally active older (61 ± 2 years, n=5) and younger men (25 ± 2 years, n=7). Intracellular superoxide production was measured using dihydroethidium fluorescence and NADPH oxidase subunits (gp91phox, p22phox, and p67phox) and superoxide dismutase 1 and 2 (SOD1 and SOD2) mRNA expression was measured using real time RT‐PCR. Interestingly, intracellular superoxide production was similar between groups (2 ± 0.4 older vs. 2 ± 0.3 young, Relative Fluorescence Units; P>;0.05). Likewise, gp91phox and p22phox were not different between groups, whereas p67phox was actually significantly lower in older subjects. Aging did not influence SOD2 mRNA expression, while SOD1 expression was lower in older subjects. These preliminary findings suggest that PBMCs may not be a primary source of systemic oxidative stress in healthy older individuals.

Supported by NIH Grant R01 HL093167
Correlation between cardiovascular autonomic control dysfunction and oxidative stress in an experimental model of hypertensive and menopause submitted to fructose overload
Authors: Conti Filipe Fernandes , Brito Janaina O. , Palma Renata K. , Bernardes Nathalia , Sanches Iris C. , Irigoyen Maria C. , De Angelis Kátia
Published: June 2021

The aim of this study was to verify the effects of fructose overload in an experimental model of hypertensive and menopause on cardiovascular autonomic control and cardiac oxidative stress. Female SHR rats were divided into (n=8/group): hypertensive (H), hypertensive ovariectomized (HO) and hypertensive ovariectomized submitted to fructose overload (100g/L in drinking water) (FHO). Arterial pressure (AP) signals were directly recorded. Cardiovascular autonomic modulation was evaluated by spectral analysis. Oxidative stress was evaluated by chemiluminescence (CL), total reactive antioxidant potential (TRAP) and redox balance of glutathione (GSH/GSSG) determinations. AP was higher in FHO group (174±4 vs. H: 146±4 HO: 164±5 mmHg) than others groups. FHO group showed reduced vagal tonus (5±3 vs. H: 23±4; HO: 35±7 bpm) and increased sympathetic tonus (91±18 vs. H: 56±11; HO: 60±3 bpm) when compared to other groups. CL was higher in FHO group than other groups. GSH/GSSG was lower in FOH group (8.94±0.80 umol/g) than HO group (13.0±1.4 umol/g). Negative correlation was obtained between VAR‐SAP and TRAP (r: −0.71, p≤0.05) and a positive correlation between RMSSD index and GSH/GSSG (r: 0.68, p≤0.05). In conclusion, fructose overload induced impairment in cardiovascular autonomic control associated with increased oxidative stress in hypertensive rats submitted to ovarian hormones deprivation.
Resolvins play a role in the resolution of acute lung injury
Authors: Cox Ruan R , Phillips Oluwakemi , Fukumoto Jutaro , Fukumoto Itsuko , Parthasarathy Prasanna Tamarapu , Lagishetty Venu , Luong Tran , Remsen Natalie , Lockey Richard , Kolliputi Narasaiah
Published: June 2021

Patients that receive oxygen therapy are at risk for developing a debilitating respiratory syndrome known as hyperoxic acute lung injury (HALI). Resolution of this syndrome stems from rectifying the imbalance that exists in favor of pro‐inflammatory over anti‐inflammatory cytokines. Resolution phase interaction products (resolvins), pro‐resolution lipid mediators, have been shown to reduce the inflammation seen in acute injuries; however the ability to confer beneficial effects in HALI is not yet known. Thus, we hypothesized that resolvins decrease the pathophysiology seen in HALI. To test this, C57BL/6 mice were treated with resolvins following hyperoxia exposure. Alveolar epithelial cells (A549) were also pre‐treated with resolvins prior to exposure to pro‐inflammatory cytokines IL‐1β or TNF□. In‐vivo results revealed that resolvin treatment, following hyperoxia, resulted in improved lung tissue histology, coupled with decreased barrier dysfunction and neutrophil infiltration. A549 cells treated with resolvins demonstrated reduced adhesion molecule expression and pro‐inflammatory cytokine secretion in comparison to cytokine treated controls. Our results also showed a suppression of MAP kinase and NFκB signaling in resolvins treated cells. Taken together, these results provide evidence that resolvins could be an effective treatment for acute injury syndromes such as HALI.
Oxidative stress and ER stress induce Zidovudine (AZT)‐mediated hepatic lipid accumulation
Authors: Banerjee Atrayee , Abdelmegeed Mohamed A , Jang Sehwan , Song Byoung Joon
Published: June 2021

Zidovudine (AZT) was the first drug approved by the U.S. Food and Drug Administration for treating AIDS patients. The clinical effectiveness of AZT is constrained due to its various adverse side effects including hepatotoxicity. However, the mechanism of hepatic lipid accumulation in AZT‐treated individuals is unknown. We hypothesized that AZT‐induced oxidative stress alters the mitochondrial fat oxidation pathway, leading to fat accumulation. Our study showed significant increase in lipid accumulation, inflammation and toxicity in female mice exposed to AZT for 10 days. Markers of oxidative stress such as CYP4A levels, protein oxidation, nitration, glycation and lipid peroxidation were significantly higher in the AZT‐treated mice as compared to the controls. Further, the levels of ER stress marker proteins like GRP78, p‐PERK, and p‐ELF2α were significantly elevated in AZT‐treated mice. In addition, significantly increased levels of nuclear SREBP‐1c and acetyl‐CoA carboxylase along with significant decrease in the levels of phospho‐AMP kinase and PPARα, a key transcription factor for fatty acid oxidation, were observed in AZT‐treated group. Collectively, these data indicate that oxidative stress and ER stress play a critical role in lipid accumulation and hepatotoxicity in AZT‐treated animals. [Support: Intramural Research Program of NIAAA].