bluuebunny/crossref_metadata_2025 huggingface dataset:
Inducible nitric oxide synthase inhibition attenuates lung injury and decreases NADPH oxidase expression and activation during ischemia‐reperfusion in the ventilated rabbit lung
Authors: Roberts Andrew M , Akabua Ewoenam A , Lominadze David , Gozal Evelyne
Published: June 2021

Lung ischemia‐reperfusion (IR) causes arteriolar constriction and platelet adhesion in correlation with inducible nitric oxide synthase (iNOS) expression/activity and reactive nitrogen species (RNS). While iNOS was elevated during reperfusion, RNS were only elevated in lungs that underwent ischemia. To determine oxidative stress, and if attenuation of vascular dysfunction by iNOS inhibition could be due to decreased NADPH oxidase stimulation, we assessed p47 phox translocation and vascular leakage. In anesthetized rabbits with open chest, we ventilated the lungs with an oxygen mixture and examined subpleural microvessels and alveoli in the right lung during brief periods when the lungs were statically inflated (10 cm H2O), IR was induced by occluding the right pulmonary artery for 2 h. Fluorescently labeled albumin was injected via the right atrium to assess vascular leakage. Oxidative stress was assessed by translocation of the cytosolic NADPH oxidase subunit, p47 phox, to the membrane fraction by immunoblotting. Vascular leakage and translocation of p47 phox were greater in reperfused ventilated lungs than in contralateral or control lungs, and were attenuated by pretreatment with an iNOS activity inhibitor (1400W). Our data suggest that stimulation of NADPH oxidase is an important component in acute lung injury in conjunction with iNOS overexpression. Supported by ALA of KY and NIH grant # HL080394.
Embolization with 10, 15 and 50 micrometer spheres recruits intrapulmonary shunt vessels in isolated rat lungs
Authors: Bates Melissa , Watson Kal , Eldridge Marlowe
Published: June 2021

We hypothesized that intrapulmonary shunt vessels (IPAVs) are present in the rat, located in the lung apex, and are recruited by increased regional blood flow. We used an isolated lung preparation, perfused with 5% albumin via the pulmonary artery at 20 cmH2O and ventilated between 5 and 15 cmH2O. We progressively embolized the lung with fluorescent spheres to redirect flow and isolate IPAVs. We injected 1 million 50μm spheres into the pulmonary artery (N=1) and counted 114 spheres in the left atrial effluent. Next, we progressively injected 15μm spheres (N=3) in 250,000 sphere aliquots. Initially very few spheres traversed the lung (5±2). With progressive embolization there existed a threshold at which a larger number of spheres appeared in the effluent (48±9). Using 10μm spheres (N=2), we found 48 and 233 spheres in the effluent after the first injection. Again, with progressive embolization there was a threshold at which a larger number of spheres appeared in the effluent (365 and 603). Once embolization decreased flow >50%, the lungs were perfused with Evans blue‐labeled albumin, dried, sectioned, and imaged with a confocal microscope. We found patent vessels limited mainly to the apex and pleural surface and have identified a few vessels with the potential to be IPAVs. These findings suggest that IPAVs are recruited with progressive embolization and the majority of the IPAVs appear to be located in the apex.
Effects of a lower level of control O2, hyperoxia and hypercapnia on solitary complex neurons in medullary tissue slices
Authors: Matott Michael P. , Putnam Robert W. , Dean Jay B.
Published: June 2021

Studies of neuronal O2‐/CO2‐chemosensitivity in the brainstem often employ tissue slices maintained in medium equilibrated with 95%O2, which we now know produces hyperoxia, increased production of reactive species, and oxidative stress. Accordingly, the objectives of this study were to determine if solitary complex (SC) neurons in medullary slices, incubated in 40%O2, exhibit spontaneous, stable activity and are responsive to hyperoxia and hypercapnia. Medullary slices (300‐400µm, P3‐P21) were harvested in 95%O2 + 5%CO2 (2‐4°C) and immediately transferred to 40%O2 + 5%CO2 (22‐24°C). Whole‐cell recordings (~37°C) of 69 neurons show a mean resting membrane potential of ‐42mV with spontaneous and evoked action potentials that overshoot 0mV. Whole cell recordings were made within 2‐7.5hr post‐slicing. To date, 25 neurons were tested using hyperoxia (95% O2 + 5% CO2), hypercapnia (40% O2 + 10%CO2) and hyperoxic hypercapnia (90%O2 + 10%CO2). The majority of neurons (13) were insensitive to hyperoxia/hypercapnia. Eight neurons were stimulated by hyperoxia and five by hypercapnia. Of these, four were stimulated by hyperoxic hypercapnia. Two neurons were inhibited by hypercapnia and one was inhibited by hyperoxia. We conclude that medullary slices can be maintained in 40%O2 for many hours for studies of CO2/O2‐chemosensitivity at normobaric pressure. (ONR N000140710890, NIH R01 HL 56683‐09)
Chronic hyperoxia alters the early and late phases of the hypoxic ventilatory response in neonatal rats
Authors: Bavis Ryan W , Young Kristen M , Barry Kevin J , Boller Matthew R , Kim Eugene , Klein Peter M , Ovrutsky Alida R , Rampersad Donna A
Published: June 2021

Perinatal hyperoxia causes long‐lasting attenuation of the hypoxic ventilatory response (HVR) in rats, but the timing of these ventilatory changes has not been investigated during the hyperoxic exposure. Rats were raised in room air (control) or 60% O2 from birth until studied at 4, 6‐7, and 13‐14 days of age. Ventilation was measured by head‐body plethysmography in 21% O2 and in 12% O2. Normoxic ventilation was significantly reduced at P4 and P6‐7 in hyperoxia‐treated rats, which may be partially explained by lower metabolic rates. Control rats exhibited a biphasic HVR at P4 and P6‐7; the secondary decline diminished with advancing age and was no longer observed by P13‐14. Hyperoxia‐treated rats exhibited a sustained increase in ventilation at all ages, however, suggesting that hyperoxia hastens postnatal maturation of the central components of the HVR. The early (carotid body‐mediated) phase of the HVR tended to be greater in hyperoxia‐treated rats relative to controls at P4 (P=0.09 at minute 1) but decreased as the hyperoxic exposure progressed (i.e., significantly reduced at P6‐7 and P13‐14 vs. controls). Thus, it appears that short exposures to chronic hyperoxia initially enhance the HVR but that this is gradually overcome by impaired carotid body function. Supported by NIH grant R15 HL‐083972.
Differences between two inbred rat strains in number of neurons expressing K+ ion channels in the medullary raphe nucleus (MRN)
Authors: Riley Desire , Neumueller Suzanne , Bonis Josh , Dwinell Melinda , Qian Baogang , Forster Hubert
Published: June 2021

Male (M) and female (F) Dahl salt sensitive (SS) and Brown Norway (BN) inbred rat strains increase pulmonary ventilation by 183 and 37% respectively when exposed to 7% inspired CO2. Since K+ ion channels are hypothesized determinants of ventilatory CO2 sensitivity, we presently determined whether there is a difference between these two rat strains in number of neurons expressing K+ channels in the chemosensitive MRN. Rats were sacrificed, and the brains were perfused, extracted, cyrosectioned at 25μm into 4 series, and alternate sections were stained for Nissl and Task‐1, Kv1.4, or Kir2.3 K+ channel antibodies. From 0‐2mm rostral to obex, the stained neurons in the MRN were microscopically visualized and counted. We found more Nissl stained MRN neurons in BN than in SS rats in both M (6%, P<0.001) and F (8.2%, P<0.001) rats. However, for Task‐1, Kv1.4, and Kir2.3, there were 12, 13.8, and 13.9% respectively more (P<0.001) immunostained MRN neurons in SS than in BN rats. Additionally, F BN rats had 5.3 (P=0.014), 7.3 (P=0.040), and 7.6% (P=0.005) more Task‐1, Kv1.4, and Kir2.3 respectively stained MRN neurons than in F SS rats. We conclude that the number of neurons expressing K+ channels in the MRN is genetically determined. Supported by NIH 25739, HL066579, and the Department of Veterans Affairs.
Hypercapnia inhibits both transient and sustained potassium currents in chemosensitive neurons from neonatal rat locus coeruleus (LC)
Authors: Li Ke‐Yong , Putnam Robert W.
Published: June 2021

Increased ventilation is stimulated by an increase in CO2/H+, which is detected by peripheral chemoreceptors and central chemosensitive neurons. We studied CO2/H+‐sensitive K+ channels in rat LC neurons. We hypothesize that multiple ion channels are involved in the neuronal response to changes in CO2/H+, with K+ channels being an important component mediating this response. In this study, we used whole cell voltage clamp to test the effect of 15% CO2 on both transient and sustained K+ currents of LC neurons from neonatal rat brain slices. Neurons were clamped at ‐80mV. Voltage‐dependent K+ currents were evoked by various depolarizing pulses (300 ms duration) in 10 mV increments from ‐100mV to 50mV in the presence of TTX (1 μm) and Cd2+ (200 μm) to block Na+ and Ca2+ channels, respectively. Transient K+ currents were inhibited by 4‐amino pyridine (4‐AP; 5mM) and sustained K+ currents were inhibited by tetraethylammonium (TEA; 20mM). Five minutes after perfusion with aCSF equilibrated with 15% CO2, the conductances of both transient and sustained K+ channels were decreased by 28 ± 9.5% (n=3) and 19 ± 9.6% (n=3), respectively. These results support the hypothesis that hypercapnia increases the firing frequency of LC neurons through depolarization due to inhibition of multiple K+ channels, including transient and sustained K+ conductances.

Supported by NIH Grant R01‐HL56683‐11.
Pressure‐diameter behavior of airways measured from tantalum bronchograms in isolated mouse lungs
Authors: Lai‐Fook Stephen J. , Houtz Pamela K.
Published: June 2021

In a previous study, conscious mice in response to methacholine aerosol reached a limit in airway constriction that occurred near zero airway elastic recoil, based on an analysis using elastic and active properties of large dog airways (J. Appl. Physiol. 105: 933‐941, 2008). We wondered whether mice have airway elastic properties similar to dogs. Balb/c mice (n = 5, 23‐27 g) were anesthetized and tantalum dust (5 micron) was insufflated via a 22‐G catheter into the lungs using a 0.2 ml air injection to produce a well‐defined bronchogram. Postmortem, the chest was opened and the lungs were inflated to 25 cmH2O transpulmonary pressure (Ptp) then deflated in steps to 10, 5, 0, ‐5 and ‐10 cmH2O. Tantalum bronchograms were taken at each Ptp. Airway diameters (8‐10) located between 1 and 8 airway generations were measured from the bronchograms of each mouse. Airway diameter decreased from a maximum (Dmax) at 25 cmH2O Ptp to 87, 79, 60, 50 and 47 %Dmax at 10, 5, 0, ‐5 and ‐10 cmH2O Ptp, respectively. The pressure‐diameter (PD) behavior was similar for both large (Dmax of 0.75 ± 0.11 (SD) mm) and small (Dmax of 0.32 ± 0.026 mm) airways. The measured PD behavior of mice was similar to that reported for dog airways (J. Appl. Physiol. 44: 859‐868, 1978). Supported by NIH grant HL 36597.
Na+/Ca2+ Exchange and Airway Smooth Muscle Inflammation
Authors: Venkatachalem Sathish , Thompson Michael , Pabelick Christina , Prakash Y S , Sieck Gary C
Published: June 2021

Airway inflammation leads to increased intracellular Ca2+ ([Ca2+]i) in airway smooth muscle (ASM). There is now growing evidence that the bidrectional plasma membrane Na+/Ca2+ exchange (NCX) may contribute to ASM [Ca2+]i regulation. However, the roles of NCX‐mediated Ca2+ influx vs. efflux, or the role of NCX in airway inflammation are largely unknown. In the present study, we examined NCX expression and function in human ASM exposed to the pro‐inflammatory cytokines TNFα and IL‐13. Western blot analysis of human ASM tissue showed significant expression of the NCX1 isoform, with substantial increase in expression following overnight cytokine exposure. In fura‐2 loaded human ASM cells, NCX‐mediated Ca2+ influx as well as efflux (measured as slopes of changes in [Ca2+]i) was elicited using techniques previously described for cardiac muscle. Contribution of NCX was verified by measuring [Na+]i using the fluorescent Na+ indicator SBFI. NCX‐mediated influx was verified by demonstrating prevention of rising [Ca2+]i or falling [Na+]i in the presence of the NCX inhibitor KBR7943. In second set of studies, NCX siRNA transfection of human ASM substantially blunted efflux and influx mode of NCX (siRNA effect verified by Western blot). Exposure to TNF‐α as well as IL‐13 significantly enhanced influx mode of NCX. Although both cytokines also increased efflux mode of NCX, these effects were relatively smaller. These data suggest that NCX may be one mechanism contributing to inflammation‐induced enhancement of [Ca2+]i.

Supported by NIH grants HL74309, RR024150, and the Flight Attendants Medical Research Institute (FAMRI). Additional funding from the Department of Anesthesiology, Mayo Clinic.
Regulation of IK by PI3 Kinase/Akt Pathway in Cardiac Myocytes
Authors: Alvin Zikiar , Zhao Aiqiu , Duan Rong , Laurence Graham G. , Teos Leyla , Coleman Bernell R , Haddad Georges E.
Published: June 2021

Abnormal regulation of the repolarization process in cardiac myocytes is related to the induction of certain arrhythmias and ventricular dysfunction. Regulation of the repolarizing delayed outward rectifier current (IK) can be modulated by inotropic and inotrophic hormones, such as IGF‐1. The aim of this work was to assess the regulation of IK by the cell‐survival pathway, IGF‐1/PI3 K/Akt, in freshly isolated neonatal rat ventricular myocytes, using patch‐clamp technique in whole‐cell configuration. Cells were stimulated from holding potential of ‐40mV, by 500msec, 10‐mV step voltages to +40mV. IGF‐1 reduced the functional density of IK from 4.1 ± 0.5 pA/pF to 2.6 ± 0.3 pA/pF. IGF‐1 effect was eliminated by the PI3 Kinase inhibitor, LY294002 (3μM). Adenoviral transfection of cardiocytes with a constitutively activated PI3 K, BD110, mimicked the IGF‐1 effect on IK (2.5 ± 0.4 pA/pF), which was abrogated by LY 294002. Similar to BD110, transfection of the cardiac myocytes with wild‐type Akt reduced IK (2.4 ± 0.3 pA/pF), which was completely reversed by LY 294002. We conclude that IK is negatively regulated by IGF‐1 through downstream activation of PI3 Kinase and Akt. Such a mechanism could play an anti‐arrhythmic role during cardiac events that are associated with the activation of the PI3 K/Akt signaling, such as cardiac hypertrophy.
Novel catheter guidance technique to the site of origin of the arrhythmia
Authors: Lee Kichang , Lv Wener , Ter‐Ovanesyan Evgeny , Voysey Graham E. , Armoundas Antonis A. , Cohen Richard J.
Published: June 2021

The purpose of this study was to evaluate the accuracy of our catheter guidance system using Single Equivalent Moving Dipole (SEMD) method to guide the tip of the cardiac ablation catheter to the site of origin of the arrhythmia. 3 different patterns of 8 bipolar electrodes were sutured on the right ventricular myocardium of 3 swine through a standard thoracotomy. Electrode cables passed subcutaneously to a ventral interscapular position were exteriorized, fixed by stay sutures, and the chest closed in layers. The animals' heart rate, pulse oximetry, femoral artery blood pressure, and body temperature were continuously monitored. As the epicardial electrodes connected to a stimulator delivered pacing spikes simulating focal ventricular tachycardia at a rate of 120 bpm, the BioSemi ActiveTwo system at a frequency of 1Khz with a symmetrically patterned array of 64 electrodes placed on the chest measured body surface potentials. A paralytic drug was used to reduce error in cardiac mapping by minimizing muscular activity and allowing for full respirator control of breathing. Both simulation and real catheter's movements were well detected (R=0.95) and the final localization error of a simulation catheter to a pre‐defined target was less than 1 cm in imaginary space, which is equal to 0.4 cm in real space. These data support the validity of SEMD method for localizing the origin of arrhythmia in swine model.
T Wave Alternans Observed on Intracardiac Bipolar Electrograms Predicts Ischemic Ventricular Tachycardia Due to Focal and Reentrant Mechanisms
Authors: Chaudhary Ashok K. , Ely Debra R. , Dove Edwin , Martins James B.
Published: June 2021

Recently T Wave Alternans (TWA) measured on intracardiac electrograms has been shown to predict ventricular tachycardia (VT) in animal models and in patients with implantable defibrillators. We measured intracardiac bipolar electrograms (IBE) recorded in 3‐D from ischemic and normal zones in the acutely ischemic heart to predict location and mechanism of VT.

Methods

18 alpha chloralose anesthetized dogs were studied with 3‐D mapping after coronary artery occlusion (CAO); infarct size (IS) was measured as % of all IBE recorded which had voltage drop >45%. Mapping localized the 3‐D origin of spontaneous VT. TWA was measured on the body surface (TWA‐surf) as well as on IBE (from up to 62 anterior septal transmural sites measured by visual estimate) filtered from 3‐1300 Hz (TWA‐IBE).

Results

TWA‐surf correlated with IS in the first 30 minutes of CAO; thereafter it did not. TWA‐surf correlated with the number of IBE with TWA found in normal and ischemic sites in both epicardium and endocardium. Compared to hearts without VT the density of TWA on IBE sites was greater in hearts with VT (4.8 ± 1.7 SE vs 19.5 ± 2.6, p<0.05) Spontaneous VT tended to be focal endocardial (56%) had TWA‐IBE at the site of origin in 81%. However epicardial reentry had only 54% sites, p<0.05 vs focal, with TWA‐IBE.

Conclusion

TWA‐surf correlates well with TWA‐IBE. TWA‐IBE is a marker of VT regardless of mechanism. Density of TWA predicts VT.
Adenosine antagonist 8‐SPT decreases rate of microvessel growth in mouse left ventricular angiogenesis assay in vitro
Authors: Adair Thomas H. , Horton William B.
Published: June 2021

Adenosine is thought to play a pivotal role in the regulation of angiogenesis. We tested the hypothesis that an adenosine receptor antagonist, 8‐(p‐Sulfophenyl) theophylline (8‐SPT), can decrease angiogenesis. Two in vitro angiogenesis assays were used: rat aortic ring and mouse left ventricle (LV). Explants were embedded in collagen and incubated in various O2 environments for 14 days. Aortic rings sprouted microvessels (MVs) in serum‐free media (EBM, Lonza) when incubated in room air (peak increase, 80‐90 MVs on days 6‐7): 2% O2 inhibited MV growth on days 4‐10 by 40‐50%, 12% O2 had no effect, and 5% O2 caused a 20‐30% increase in MVs on days 6‐10, compared to room air controls. 8‐SPT (5x10‐5 M; 3x week) had no effect on MV growth in aortic rings incubated in either room air or 5% O2. LV explants sprouted MVs in EBM/10% FBS media when incubated in 2% O2 (peak increase: 237±17, day 8); MVs did not grow in room air. 8‐SPT decreased the rate of angiogenesis in mouse LV from 81±14 MVs/day (control, day 6) to 31±4 MVs/day (8‐SPT, day 6) in cultures incubated in 2% O2. 8‐SPT also decreased (p<0.01) the peak number of MVs from 237±17 (control, day 8) to 161±17 (8‐SPT, day 10). The results support the hypothesis that adenosine plays an important role in mediating hypoxia‐induced angiogenesis in mouse left ventricle, but does not play a role in mediating MV growth in rat aorta in vitro. (Supported by HL‐51971)
Benzamil decreases microvessel growth in rat aortic rings in vitro
Authors: Adair Thomas H. , Drummond Heather A.
Published: June 2021

Epithelial Na+ Channel (ENaC) and Acid Sensing Ion Channel (ASIC) proteins are thought to be required for mechano‐dependent responses, such as migration of vascular smooth muscle cells. We tested the hypothesis that ENaC/ASIC proteins are required for angiogenesis, a response dependent on vascular/endothelial cell migration. Aortic rings from 78 g Fisher rats were embedded in rat tail collagen and incubated at 37° C in 20% O2/5% CO2 for 8 days. Cultures were fed daily with serum‐free media (EBM, Lonza) containing benzamil (10 μM); control cultures received media containing an equal volume of vehicle. In control cultures (n=23), the number of microvessels (MVs) reached a peak value of 81±6 (mean±SE) on day 5, and then decreased progressively for the next 3 days as shown. Cultures treated with benzamil (n=18) showed an early peak in MV growth on day 4 (58±7) followed by MV regression on days 5‐8. Inhibition of ENaC/ASIC activity with 10 μM benzamil decreased MV number by 32±9% on day 5 (p<0.05), 32±6% on day 6 (p<0.05), and 45±4% on day 7 (p<0.01). The results are consistent with the hypothesis that ENaC/ASIC proteins are required for normal MV growth or maintenance of MVs in the rat aortic ring assay. (Supported by HL‐51971)
Effect of PDIII inhibitor (Cilostazol) on ischemic angiogenesis in diabetic mice
Authors: Otagiri Aoi , Matsushita Shonosuke , Sakakibara Yuzuru
Published: June 2021

Purpose

Cilostazol (Cil) is a Class A drug for peripheral arterial disease (PAD) and used worldwide. Since diabetes mellitus (DM) is a main exacerbating factor of PAD, the effect of Cil on ischemic angiogenesis is investigated to clarify its beneficial effect on DM in regard to cellular and local components.

Methods

Male mice (n=18) were divided into three groups; Control (C), DM (D), and DM? (L). DM was induced by streptozocin. Cil was administered by oral intake. Femoral artery was ligated in all mice. 2 months after operation, mice were evaluated by (1) capillary density of soleus muscle, (2) Laser Doppler flow meter of posterior tibial artery, (3) number of endothelial progenitor cells (EPCs) by FCM, (4) analysis of immunohistochemistry (IHC) by eNOS antibody.

Results

Capillary density was decreased in DM compared to Control, however, DM? recovered to control level (C 15.1 7.6, D 9.6 4.1, L 14.1 3.5 per field; X400, p<0.05 C vs D, p<0.005 D vs L). Laser Doppler flow was as follows: (C 849%, D 6413%, L 63 6%, p<0.05 C vs D, p<0.001 C vs L). There was no difference in the number of EPCs among three groups (C 84, D 82, L 75 per µl). Mice with Cilostazol increased eNOS expression than DM by semiquantitative analysis (p<0.001).

Conclusion

Cilostazol enhanced angiogenesis in diabetic ischemic limb. It is not due to the involvement of bone‐marrow derived EPCs, but may due to enhanced eNOS expression.
Interferon γ (IFN‐γ) Augments Angiotensinogen (AGT) and Angiotensin Converting Enzyme (ACE) Expression in Rat Mesangial Cells (MCs)
Authors: Satou Ryousuke , Urushihara Maki , Miyata Kayoko , Ohashi Naro , Saito Toshie , Gonzalez‐Villalobos Romer A , Katsurada Akemi , Acres Omar W , Navar L Gabriel , Kobori Hiroyuki
Published: June 2021

Intrarenal angiotensin II accelerates renal injury accompanied with augmentation of T cell infiltration and IFN‐γ. IFN‐γ increases monocyte chemoattractant protein‐1 (MCP‐1) through STAT1 activation in MCs. However, the contribution of IFN‐γ to expression of renin‐angiotensin system (RAS) components in MCs has not been established. This study was performed to test the hypothesis that IFN‐γ augments AGT and ACE expression in MCs. Primary cultured MCs were treated with 0‐20 ng/ml IFN‐γ for 24 h. Activation of STAT1 and MCP‐1, AGT and ACE expression were evaluated by real‐time RT‐PCR and western blot analysis. IFN‐γ induced phosphorylation, translocalization of STAT1 to the nucleus, and MCP‐1 expression (8.99±0.77, relative ratio). Two ng/ml IFN‐γ increased AGT expression (1.54±0.07 in mRNA, 1.56±0.15 in protein, relative ratio). Moreover, 20 ng/ml IFN‐γ increased ACE expression (9.46±0.78 in mRNA, 2.65±0.60 in protein, relative ratio). These results indicate that IFN‐γ activates not only a proinflammatory cytokine but also RAS in MCs, which may lead to the development of glomerular damage. NIDDK/NIH (R01DK072408), NCRR/NIH (P20RR017659), NHLBI/NIH (R01HL026371)
High salt diet during pregnancy: effects on the renin ‐angiotensin‐aldosterone system in adult male offspring of Wistar rats
Authors: Ramos Débora Rothstein , Costa Nauilo L. , Oliveira Ivone B. , Lopes Karen L. , Heimann Joel C. , Furukawa Luzia NS.
Published: June 2021

Objective

To study the effect of high salt intake during pregnancy on the renin‐angiotensin system, COX‐2 and nNOS in adult male offspring of Wistar rats.

Methods and Results

Female Wistar rats were fed with normal salt ‐ NSD (1.3%), high salt 1 ‐ HSD1 (4.0%) or high salt 2 HSD2 (8.0% NaCl) diet during pregnancy. Dams after delivery and offspring after weaning were fed NSD.

Table (mean ± SEM): Parameters analyzed in 12 week‐old offspring. * p <0.05 vs. NR

Conclusion

High sodium intake during pregnancy induces changes in renal renin‐angiotensin‐aldosterone system, COX‐2 and nNOS in male offspring of Wistar rats.

Financial Support

FAPESP
Growth Hormone‐Releasing Peptide‐2 reduces oxidative stress in ApoE ‐/‐ mice but does not reduce atherosclerosis
Authors: Titterington Jane S. , Sukhanov Sergiy , Vaughn Charlotte , Delafontaine Patrick
Published: June 2021

Growth Hormone‐Releasing Peptide‐2 (GHRP‐2) is a synthetic peptide that increases circulating Growth Hormone and Insulin‐Like Growth Factor‐1 (IGF‐1) levels. It also binds to CD36, a scavenger receptor for oxidized low‐density lipoprotein (OxLDL), and may prevent cellular uptake of this proatherogenic complex. To determine its potential antiatherogenic effects, GHRP‐2 (20 µg BID) was administered subcutaneously to ApoE ‐/‐ mice for 12 weeks. GHRP‐2 increased circulating IGF‐1 1.2‐1.6 fold and decreased circulating interferon‐γ (IFN‐γ) by 66%. While GHRP‐2 did not alter atherosclerotic plaque area, it decreased aortic production of superoxide as assessed by DHE staining. GHRP‐2 decreased aortic gene expression of 15‐lipoxygenase by 92% and reduced the aortic expression of IFN‐γ, Cu/Zn superoxide dismutase, macrophage migration inhibitory factor, and IGF‐1 receptor (IGF‐1R). In cultured aortic smooth muscle cells, GHRP‐2 prevented the OxLDL‐induced generation of peroxides, downregulation of IGF‐1R, and apoptosis. In macrophages, GHRP‐2 reduced lipid accumulation with OxLDL exposure. In summary, GHRP‐2 exerts antioxidant effects in vivo and in vitro but does not reduce plaque burden. The lack of antiatherogenic effect may be due to the inability of GHRP‐2 to prevent OxLDL‐induced downregulation of IGF‐1R in vivo, thereby blunting the effect of increased IGF‐1. Funding: NIH HL070241
TRP channels provide the depolarisation initiating vasomotion and vessel tone in cerebral resistance arteries
Authors: Wolfle Stephanie Elisabeth , Navarro‐Gonzalez Manuel Francisco , Beech David J , Hill Caryl E
Published: June 2021

Coordinated oscillations in diameter occur spontaneously in cerebral vessels and depend on the opening of voltage dependent calcium channels. We investigated the mechanism that provides the initial depolarisation required for vasomotion. Branches of juvenile rat basilar arteries were studied by measurement of diameter, calcium and membrane potential. Removal of extracellular calcium ([Ca2+]o) abolished vasomotion, relaxed arteries (125% resting vessel diameter, RVD), reduced intracellular Ca2+ ([Ca2+]I; 71%), but paradoxically depolarized cells (‐40mV to ‐27mV). Reduction of [Na+]o, from 140mM to 100mM, produced comparable effects, except that vessels hyperpolarized (‐52mV), even in the absence of [Ca2+]o. TRP channel blockers, SKF96365 and gadolinium, also relaxed vessels (111% RVD), abolished vasomotion, decreased [Ca2+]i (87%) and hyperpolarized (‐57mV) vessels. Depolarisation of SKF96365‐treated vessels with 40mM KCl reinstated vasomotion. Quantitative PCR revealed TRPC1 to be the predominantly expressed TRPC subtype and incubation with a function blocking TRPC1 antibody delayed the onset of vasomotion. We conclude that TRPC1 channels provide the initial depolarising current that activates voltage dependent calcium channels to provide vessel tone and initiate vasomotion. Selective TRP channel inhibition could provide a novel target for the treatment of cerebral vasospasm.
Physiological Consequences of eNOS Subcellular Targeting
Authors: Qian Jin , Zhang Qian , Fulton David
Published: June 2021

Endothelium‐derived NO influences vascular function via two distinct mechanisms, activation of the NO‐soluble guanynyl cyclase (sGC)‐cGMP signaling pathway or through S‐nitrosylation of proteins with reactive thiols. The importance of eNOS subcellular localization to these pathways is not yet known. Two major proteins that are nitrosylated in endothelial cells are N‐ethylmaleimide sensitive factor (NSF), which controls the release of Von Willebrand Factor (vWF) and IκB kinase, which phosphorylates IκB and coordinates inflammatory signaling through the NF‐κB pathway. vWF production was greatly attenuated in eNOS "knockdown" HAEC re‐expressing PM eNOS as compared to Golgi localized eNOS. PM eNOS released more NO than Golgi eNOS and induced greater S‐nitrosylation of NSF. Further, reconstitution of HAEC with Golgi or PM‐targeted Ca2+/CaM‐insensitive eNOS constructs, which produces equal amounts of NO regardless of location, resulted in equivalent inhibition of vWF secretion. In contrast, NO released from both the Golgi and PM eNOS did not affect NF‐κB‐dependent increases in ICAM expression in response to TNFα. Titration of NO levels with the NO donor, DETA‐NONOate, revealed that much higher levels of NO are required to suppress NF‐κB signaling. These results suggest that the nitrosylation of proteins is more dependent on the overall amount of NO produced rather than the location of synthesis.
An experimental and theoretical study of DAF‐FM activation by NO: Toward calibration of an NO‐sensitive fluorescent dye
Authors: Namin Shabnam M. , Joshi Mahesh S. , Bautista Carolina , Nofallah Sara , Tsoukias Nikolaos M.
Published: June 2021

The quantitative measurement of NO in vitro has been hampered by the lack of a method that can detect nanomolar levels with spatial and temporal resolution and without interference from other reaction byproducts. 4,5 diaminofluorescein (DAF‐FM) is a sensitive NO fluorescence probe used widely for qualitative but not quantitative assessment of cellular NO production. With the aid of a mathematical model of NO/DAF‐FM reaction kinetics, we have conducted experimental studies to calibrate the fluorescence signal and estimate NO generation in different systems. Kinetic analysis suggests the following dependence of DAF‐FM triazole (DAF‐T) generation rate:

DAF‐T fluorescence was measured in a series of experiments with varying DAF‐FM and NO donor concentrations (spermine/NO). Experiments showed that the slope of fluorescent intensity is proportional to [NO]2 and exhibits a Monod type dependence on DAF‐FM concentration, thus validating the proposed kinetic mechanism. A calibration procedure was formed and applied in a cell culture system. Human umbilical vein endothelial cells (HUVEC) were stimulated with hemodynamic and agonist stimuli and the release rate of NO was assessed.

[Supported by AHA grant NSDG043506N and NIH grant HL095101]
The hepatic microvascular system in health and disease
Authors: McCuskey Robert S.
Published: June 2021

The hepatic microvascular system includes portal venules and hepatic arterioles, the sinusoids, and central and hepatic venules. The sinusoids are unique exchange vessels lined by: (a) fenestrated endothelial cells (SEC) lacking a basal lamina which have endocytotic and immune functions; (b) phagocytic Kupffer cells (KC) which are sources of mediators for host defense as well as hepatic injury; and (c) stellate cells which are contractile pericytes containing fat droplets that store Vitamin A and produce collagen when activated. Stellate and SEC play a major role in regulating the diameters of sinusoids and the distribution of blood flow in individual sinusoids, lobules or segments of lobules. The SEC also are a sensitive and early target for hepatic injury. The responses of the hepatic microcirculation to toxicants are of two basic types: (a) an inflammatory response involving paracrine activation of SEC by mediators released from adjacent KC following stimulation by toxicants leading to the upregulation of adhesion molecules and the subsequent adhesion of leukocytes to the SEC as well as swelling of the SEC both of which restrict sinusoidal blood flow; and (b) direct injury of the SEC resulting in loss of fenestrae, formation of gaps and penetration of the sinusoidal lining by blood cells. Subsequently, the sinusoid may collapse or disintegrate reducing blood flow. Supported by NIH AA12436, AG02582, and HD26013.
Identification of differentially‐expressed miRNAs in the subfornical organ (SFO) of the mouse brain in a slow pressor angiotensin (Ang)‐II infusion model of hypertension
Authors: Guruju mallikarjun Rao , Peterson Jeffrey R , Infanger David W , Braga Valdir A , Zhou Yi , Sharma Ram V , Davisson Robin L
Published: June 2021

Hypertension caused by systemic Ang‐II infusion is mediated by central neurogenic mechanisms. Circulating Ang‐II interfaces with central sites lacking a blood brain barrier, e.g. SFO, which may respond to chronic Ang‐II signaling by altering gene expression. Given recent evidence suggesting that altered miRNA expression is involved in cardiovascular disease, we hypothesized that miRNA expression in the SFO is altered following slow‐pressor Ang‐II infusion. Mice were implanted subcutaneously with osmominipumps for systemic delivery of saline or Ang‐II (600ng*kg‐1*min‐1) for 2wks. Microarray analyses for miRNA expression performed on RNA from SFO punches (5 mice/sample, n=3) from Ang‐II or saline‐infused mice revealed that 5 miRNAs were differentially expressed (p <0.05). MiRNAs mmu‐miR‐133b, mmu‐miR‐24‐2* and mmu‐mir‐499 were down‐regulated, whereas, mmu‐miR‐712 and mmu‐miR‐193b were up‐regulated Sequence analysis revealed interactions of these miRNAs with many candidate genes involved in neuronal activation. For example, mmu‐miR‐712 interacts with CaMKIIa, glutathione peroxidase 3, and neural growth regulator 1, whereas mmu‐miR‐133b interacts with AP1 family members Tcfap2a and Tcfap2d. Our findings suggest that differential expression of miRNAs following Ang‐II infusion may contribute to changes in gene expression in the SFO that could lead to the development of hypertension.
Courses in modeling transport and metabolism in cardiovascular and respiratory systems
Authors: Raymond Gary M. , Butterworth Erik A. , Bassingthwaighte James B.
Published: June 2021

The National Simulation Resource, Department of Bioengineering, University of Washington, Seattle, will give two courses this summer in modeling transport and metabolism in cardiovascular and respiratory systems. The courses are designed for clinical and research scientists interested in quantitative analysis of biological systems. The focus is on approaches to data analysis, from the use of mathematical models for precision in hypothesis formulation to experimental design and evaluation by fitting models to data. Participants are encouraged to bring data and models for group discussion. The participants will make extensive use of computer models from www.physiome.org/Models and will develop new models using standard tools including JSim, a simulation system for building and analyzing quantitative models primarily in physiology and biomedicine. JSim runs on PC, Mac, and Linux platforms and is free and downloadable from www.physiome.org/jsim/download/index.html.

Course 1, June 14‐19, 2009, emphasizes biophysics and physiology of transport and metabolism in these systems. Course 2, August 23‐28, emphasizes the translation of basic science models to clinical situations. (Supported by NIH T15 HL088516.)
Teaching undergraduate introductory biology in a workshop format results in high student retention and academic performance
Authors: Johnson Kathryn Mercedes Stettler , Fass Marion Field
Published: June 2021

Structured around the theme of heart disease and diabetes, Human Biology, an introductory biology course in an undergraduate liberal arts environment, teaches enhanced critical thinking skills associated with the biological sciences. We have implemented a workshop format focused around hands‐on and discussion based learning. Using assigned text readings as background information, classes focus on the integration of group work, discussion, laboratory activities, and lecture. Student demographic included majors and non‐majors. Classes held in a specifically designed classroom meet for three‐110 min periods per week and are limited to 24 students. Two sections taught in the fall semester of 2008 had a student retention rate of greater than 90% and an average grade of 85 ± 10% at the midterm. Additional evaluation, using Student Assessment of their Learning Gains (SALG), of the workshop format is currently underway. Parameters of this evaluation include student perceived: 1) critical thinking skills, 2) knowledge of science content, and 3) ability to make real world applications, and 4) desire to take additional science work.
Implementation of inquiry‐based physiology laboratories in undergraduate majors and non‐majors courses
Authors: Casotti Giovanni , Rieser‐Danner Loretta , Knabb Maureen
Published: June 2021

Inquiry‐based curricula were introduced to Human Anatomy and Physiology (non‐majors) Human Physiology (Pre‐medical majors) and Comparative Vertebrate Physiology (Biology majors). Our aims are to increase understanding of physiological principles, increase understanding of the scientific approach and stimulate creative and critical thinking. Students design and implement their own experiments, collect and analyze their data and presented their work verbally to the rest of the class. We evaluated the curricula changes by lab exams and oral presentations in non‐majors, and oral presentations, a lab notebook, lab reports and an independent project for the majors. Pre‐ and Post course surveys were given in each class. Non‐majors identified traditional labs as being most useful in better understanding of physiological concepts and majors inquiry labs. All students identified inquiry labs as being better able help them to understand the scientific approach and creative and critical thinking skills. Exam questions show high grades except those pertaining to understanding physiological principles that did not meet expectations. Scores on lab notebooks and lab reports improving significantly as the semester progressed. Scores for independent projects were above 90% for all groups. Post course surveys showed improvement in student confidence in all 3 aims. Supported by NSF CCLI 0509161.
Computerized Dog Lab for Teaching Cardiovascular Physiology to First Year Medical Students
Authors: Cornish Kurtis G , Zucker Irving H
Published: June 2021

Purpose

To provide the students with an experience similar to that provided through animal labs but without using animals.

Methods

Dogs were chronically instrumented to demonstrate cardiovascular function and reflexes. This included aortic, left atrial and left ventricular catheters (LV), aortic and coronary flow probes, inferior vena cava, coronary artery and aortic occluders and in some instances LV dimension crystals. This allowed demonstration of the cardiac cycle and the direct and reflex responses to various interventions in conscious animals. The interventions included increasing and decreasing preload and afterload with drugs or vascular occluders, injecting an alpha or beta agonist, nitroglycerine or veratradine into the left atria. Reactive hyperemia was demonstrated with coronary occlusion.

Results

Data were obtained over a period of years, negating the need for animals in student labs. The data were organized in a html format allowing the playback of avi files or figures showing the response to the intervention. For more advanced analysis the Chart 5 program (ADInstruments) was used.

Conclusion

This lab has repeatedly been very highly evaluated by the students. They appreciate observing the integration of the cardiovascular system without using animals for their "educational use." This lab has replace much of the experience previously obtained with live animal labs.
An enhancer region of the ALK1 gene resides in its intron 2
Authors: Suzuki Rumi , Yonenaga Yoshikuni , Lan Fei , Seki Tsugio
Published: June 2021

Expression of activin receptor‐like kinase 1 (ALK1) gene is undetectable in most of the organs in adults; however, it is specifically induced in arteries feeding ischemic lesions. This gene, with its unique expression pattern, might be exploited to aid in the delivery of a therapeutic peptide to downstream ischemic tissues. Previously, we generated a shortened 4.8 kb Alk1PIB (Promoter‐Intron 2B) regulatory fragment that recapitulates the endogenous mouse ALK1 regulatory activity. In order to elucidate the regulatory mechanisms further, we utilized evolutionarily conserved region (ECR) analysis to identify highly conserved regulatory regions in this study. An alignment of ALK1 gene sequences revealed a 152 bp sequence conserved among 11 mammals within the intron 2B region. To test the regulatory function of this 152‐bp coreECR sequence, recombinant adenoviruses expressing fluorescent reporter were created. Compared to an Alk1PIB virus, the coreECR‐deleted Alk1PIB(d1) virus showed significantly reduced reporter gene expression in cultured arterial endothelial cells, indicating an enhancer function of this sequence. This approach should help us further engineer the ALK1 regulatory region to create short and strong fragment that may be utilized in future gene therapy of various disorders including malignant tumors and ischemic heart diseases. This work was supported by the American Heart Association.
Potential role of ATF2 and MEF2C in shear stress‐mediated VEGF production
Authors: Gee Eric , Fudalewski Tomasz , Haas Tara
Published: June 2021

Increased capillary shear stress in skeletal muscle causes a form of angiogenesis termed luminal splitting. An increased number of capillaries are observed at 7 days of chronic shear stress that is reliant on p38 signaling. Others have shown that VEGF is essential for luminal splitting. We hypothesize that VEGF production is p38 dependent and is increased by p38 dependent transcription factors Creb, ATF2 and MEF2C in response to shear stress. Male Sprague‐Dawley rats were administered prazosin (50 mg/L drinking water) to increase shear stress, and the extensor digitorum longus was extracted at 1, 2, 4 or 7 days. By Western blot, p38 was activated only at 2 days. Skeletal muscle endothelial cells were sheared (12 dynes/cm2) with or without 10 μM SB203580 (p38 inhibitor), then lysed and analyzed by RT‐PCR or Western blot. In vitro, shear stress induced VEGF mRNA and protein elevation at 2 and 6 hours, respectively. These increases were abolished by SB203580. Shear stress increased both ATF2 phosphorylation and MEF2C production at 2 hours, which were abrogated by SB203580. Creb phosphorylation was unchanged by shear stress. Our data show that shear stress‐mediated p38 activation is required for VEGF production. ATF2 phosphorylation and MEF2C production are increased by shear stress, but further investigation is necessary to determine if they mediate p38‐induced VEGF production. Supported by HSF and NSERC.

Grant Funding Source

The Heart and Stroke Foundation
Genetic regulation of the flow‐induced carotid compartment remodeling in mice
Authors: Korshunov Vyacheslav A. , Berk Bradford C.
Published: June 2021

Subclinical atherosclerosis is measured by intima‐media thickening (IMT) in human carotid arteries and strongly predicts cardiovascular risks. Human studies showed that carotid IMT is determined by genetic factors. Recently we identified several quantitative trait loci (QTL) that influence intima formation in response to low blood flow in a mouse genetic cross (C3HeB/FeJxSJL/J, N2=134). We hypothesized that different genomic regions control artery compartments of the remodeled carotid. In particular, we investigated 4 carotid compartment traits: intima+media, media ratio of the left‐to‐right (L/R), external elastic lamina (EEL) ratio L/R, and adventitia ratio L/R. Using genome‐wide scan we identified three highly significant QTLs: Imm1 (intima+media modifier 1 locus) on chromosome 2 (chr2; LOD=6.8), Imm2 on Chr11 (LOD=5.1), and Eelm1 (EEL modifier 1 locus) on chr2 (LOD=5.6). There were five significant QTLs: Imm3 on Chr18 (LOD=3.0), Mrm1 (media ratio L/R modifier 1; LOD=3.9) and Arm1 (adventitia ratio L/R modifier 1; LOD=3.3) on chr2; Mrm2 (LOD=2.9) and Arm2 (LOD=3.9) on chr11. Interval mapping showed that significant intima loci co‐localized with carotid remodeling traits on chr2 and chr11. However, the most significant loci for intima+media, media and EEL traits were localized on chr2, while the most significant adventitia trait locus was on chr11. In summary, for the first time we mapped artery compartment QTLs that regulate carotid remodeling in response to changes in blood flow. These results are important for our understanding of the genetic mechanisms that contribute to carotid IMT and atherosclerosis in humans. This study was supported by NIH HL‐62826 (PI: B.C. Berk).
Keratin hydrogel increases survival time in femoral artery injury model via potential platelet adhesion mechanism
Authors: Rouse Jillian , Burnett Luke , Orebaugh Clinton , Stahle Mary , Hantgan Roy , Van Dyke Mark
Published: June 2021

Keratin hydrogel is shown to be hemostatic in a porcine lethal extremity hemorrhage model compared to gauze and HemCon control groups with the presumptive mechanism of action being platelet adhesion and aggregation. Results showed that keratin hydrogels reduced shock index and increased survival time in comparison to both HemCon and gauze. Experiments to elucidate the mechanism of keratin hemostasis showed that platelets readily adhered to hydrogel films and that adherent cells contained numerous pseudopods, indicative of activation. Studies employing integrin function‐blocking antibodies reduced platelet adhesion. Collectively, these studies indicate that keratin promotes hemostasis via platelet adhesion by an integrin‐mediated process. Keratin hydrogels were shown to promote hemostasis and increase survival time in a lethal hemorrhage model with platelet adhesion and activation as the likely basis for the hemostatic property of keratin biomaterials.

This work was supported by the US Army, SNS Nanofiber Technologies, American Heart Association, and Mid‐Atlantic Affiliate Grant‐in‐Aid.
Absence of mimecan causes medial damage associated with atherosclerotic lesions in apoE‐deficient mice
Authors: Fernández Borja , Fernández María Carmen , Moncayo Javier , Durán Ana Carmen , Such Miguel , Sans‐Coma Valentín
Published: June 2021

Mimecan is an abundant proteoglycan of the arterial wall; its expression is regulated during atherosclerosis in man and rabbit. The function of mimecan in the vascular system and atherosclerosis remains unknown. In order to assess the effect of mimecan deficiency in atherosclerosis, we compared normal and atherosclerotic arteries of 16 weeks old mimecan mutant (mime−/−), C57BL/6 (wt), apoE mutant (apoE−/−) and apoE‐mime double mutant (apoE−/−mime−/−) mice. Six animals per group were used. Histochemistry and immnunofluorescence were performed in the aortic valve, ascending aorta and aortic arch. The arteries from mime−/− mice showed a normal histological structure compared to that of wt mice. In apoE−/− and apoE−/−mime−/− mice, atherosclerotic lesions composed of foam cells and leukocytes, some of them with fibrous tissue, were present in the aortic arch and valve sinuses. In 3 apoE−/−mime−/− mice, there were severe alterations in the aortic and sinus walls associated with the lesions, including aneurism, medial degeneration, inflammation, leukocyte extravasation through the media, and accumulation in the pericardial space. We conclude that mimecan is an important factor for atherosclerosis progression that may affect leukocyte and medial reactivity. This study was supported by grants from Ministerior de Educación y Ciencia, Spain (SAF2006‐01548), and Spanish Society of Cardiology.