bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of delta opioid receptor (DOR) responsiveness in vitro by antagonist occupancy of kappa opioid receptor (KOR)
Authors: Berg Kelly A. , Sanchez Teresa A. , Silva Michelle , Rowan Matt P. , Hargreaves Kenneth M. , Clarke William P.
Published: June 2021

Peripheral opioid administration produces analgesia under conditions of tissue injury or inflammation. DOR, expressed on peripheral sensory neurons in culture, are nonfunctional under basal conditions, however, after brief treatment (priming) with inflammatory mediators such as bradykinin (BK) become competent to inhibit adenylyl cyclase (AC) activity and neuropeptide release. DOR have also been shown to function as oligomers with KOR both in vitro and in vivo. Here we report that occupancy of KOR with a selective antagonist, nor‐BNI (3nM), differentially altered the potency of DOR agonists, DADLE and DPDPE, to inhibit PGE2‐stimulated AC in BK‐primed cultures derived from rat trigeminal ganglion (5–6 days in culture). Concentration curves for DPDPE were shifted to the left 10‐fold whereas curves for DADLE were shifted to the right 20‐fold (DPDPE EC50 was 0.24 nM vs 0.06 nM and for DADLE was 0.058 nM vs 1.19 nM (p <0.05) without or with nor‐BNI). In addition, the DOR‐KOR heterodimer‐selective agonist, 6’GNTI, also produced BK‐priming dependent inhibition of PGE2‐stimulated AC. These data suggest that, in vitro, functional interactions occur between DOR and KOR, possibly as a result of oligomerization, in a ligand‐dependent manner. Moreover, we hypothesize that DOR‐KOR interactions may regulate nociceptor function and participate in opioid‐mediated analgesia in inflamed tissue. Supported by a DA 016719.
Pleiotropic properties of minocycline preserve cardiac function during ischemia/reperfusion injury
Authors: Romero‐Perez Diego , Fricovsky Eduardo , Yamazaki Katrina Go , Villarreal Francisco
Published: June 2021

Minocycline, a broad‐spectrum antibiotic, has been reported to exhibit pleiotropic properties that yield protective effects in the setting of cardiac and brain ischemia/reperfusion (I/R) injury. We used in vitro and ex vivo cardiac systems to further explore the mechanisms involved in mediating cardioprotection. In vitro assays indicate minocycline bears comparable levels of antioxidant activity to those of the vitamin E analog Trolox. Recombinant enzyme assays indicate that minocycline acts as a matrix metalloproteinase (MMP) inhibitor with an IC50 of 118μM for MMP‐7 and 134μM for MMP‐9. Minocycline was tested ex vivo to assess recovery of cardiac function in stunned myocardium. Rate pressure product results indicated that 500nM minocycline conferred an average of 50% sustained recovery of contractile function vs. controls. It improves 25% vascular function and also suppresses 85% the release of troponin I vs. controls. We found that cultured cardiac fibroblasts and myocytes display a temperature‐ and time‐dependent active transport mechanism for minocycline uptake. In rats, the concentration of minocycline in normal myocardium is 0.6‐fold higher vs. serum. Furthermore, the concentration in I/R myocardium is 3.5‐fold higher compared to a non‐ischemic portion of the same heart. Altogether these results indicate that the high concentration of minocycline observed inside cardiac cells may allow it to act in a pleiotropic fashion to confer protection from I/R injury. Supported by NIH HL‐43617 and 67922.
Nitric Oxide Scavenging by Environmental Particles and Engineered Nanoparticles: Size and Composition Dependency
Authors: Nurkiewicz Timothy R. , Dreher Kevin L.
Published: June 2021

Inhaled particulate matter (PM) elicits systemic biological responses such as impaired arteriolar dilation and increased venular leukocyte adhesion. One mechanism that may contribute to such effects is NO scavenging by pulmonary translocated particles. The purpose of this study was to determine if PM scavenges NO in vitro, and also determine if PM size or composition influences any such activity. PM was classified as combustion source particles, engineered nanoparticles or nuisance dusts. NO solutions were generated by SNAP and were measured with a NO sensor. A concentration of 1–2.5 μg/ml was used for each particle, and responses were normalized to a bolus SNAP dose, [NO]=841 nm. Among fly ashes, residual oil fly ash was the most potent scavenger, decreasing the signal by 67%. Coal fly ash from Western Kentucky and Montana, as well as Mt. St. Helens ash did not significantly alter NO levels. Diesel exhaust particles attenuated the NO signal by 19%. Among nanoparticles, TiO2 decreased the NO signal by 39%. Single and multi‐walled carbon nanotubes did not produce an effect. These findings suggest that air particulate pollution as well as some engineered nanoparticles have the potential to scavenge NO, and this ability is heavily dependent upon particle size, composition and/or surface properties.

Support: HEI #4730 and NIH RO1 ES015022 to TRN

(This abstract does not necessarily reflect EPA policy)
PACE therapy and its influence on microcirculatory hemodynamics and leukocyte‐endothelial interactions
Authors: Krokowicz Lukasz , Klimczak Alesandra , Duggan William , Grykien Christopher , Mielniczuk Mariusz , Siemionow Maria
Published: June 2021

Purpose: Effect of Pulsed Acoustic Cellular Therapy (PACE) therapy on muscle microcirculatory hemodynamics, neovascularization, leukocyte‐endothelial interactions and muscle flap healing was assessed.

Methods: Cremaster muscles were dissected in 42 Lewis rats, divided into 5 groups: 1) controls (n=10); 2) PACE with 200 or 3) 500 impulses (n=8 each) immediately before dissection; 4) PACE with 200 or 5) 500 impulses (n=8 each) 24h before dissection, followed by microcirculatory hemodynamic recording at 1,2,3,4 h. Immunohistochemistry assessed expression of cell adhesion molecules and proangiogenic factors: VEGF and vWF.

Results: PACE increased diameter of first (A‐1 in group 3) and second order arterioles (A‐2 in groups 2 and 3) (P<0.05). In groups 2, 3 and 4 rolling and sticking leukocytes were significantly decreased compared to controls (P<0.05). 200 impulses of PACE increased number of transmigrating leukocytes compared to 500 PACE impulses and to controls (P<0.05). Leukocyte‐endothelial interaction correlated with upregulation of ELAM‐1, CD15s and VCAM‐1. Synergistic expression of proangiogenic factors was significantly upregulated 24h after 500 impulses of PACE.

Conclusions: PACE therapy increased main arterioles diameters and has direct effect on activation of leukocyte‐endothelial interactions and stimulation of expression of proangiogenic factors at microcirculatory levels
EDHF prevents endothelial dysfunction in response to a high‐fat diet in the Wistar rat
Authors: Azevedo Stefanie M. , Menicucci Steve D. , Naik Jay S.
Published: June 2021

Individuals consuming a high‐fat diet, often display reduced responsiveness to endothelial‐dependant vasodilators such as acetylcholine or bradykinin. Endothelium‐derived hyperpolarizing factor (EDHF) may be an important vasoactive factor in resistance arteries from many vascular beds. However, its importance relative to other endothelial derived relaxing factors such as nitric oxide and prostaglandins is unclear. Although, genetic models of obesity and type II diabetes have demonstrated that EDHF‐mediated vasodilation is impaired; it is unknown if EDHF function is also affected by a high‐fat diet. 3–4 wk old Male Wistar rats were fed either a high‐fat diet (60% lard) or chow for 10–11 wks. Isolated small mesenteric arteries were pre‐constricted with Phenylephrine (PE; 10−9 – 10−5 M) and administered acetylcholine (ACh; 10−8 – 10−6 M) in the presence of either vehicle or Indomethacin (10 μm) and N(ω)‐nitro‐L‐arginine (L‐NNA; 100 μm). Vasoconstrictor responses to PE were also assessed in a separate set of untreated arteries. Blood pressure was determined by tail‐cuff. Body and epididymal fat pad weight were significantly greater in fat‐fed rats compared to chow fed. Mean arterial blood pressure was not different between groups. ACh‐induced dilation in arteries treated with vehicle was similar between chow and fat‐fed rats. Pre‐treating arteries with Indomethacin and L‐NNA blunted ACh‐mediated vasodilation in chow fed rats but did not alter ACh responses in arteries from fat‐fed animals. Reactivity to PE was not different between groups. These results suggest that role for EDHF in ACh‐mediated vasodilation is enhanced in small mesenteric arteries following a high‐fat diet.
Mice heterozygote null for 11βHSD2 do not regulate ENaC activity in response to dietary sodium content
Authors: Bailey Matthew A. , Mullins John J.
Published: June 2021

Mutations in 11β‐hydroxysteroid dehydrogenase type 2 (11βHSD2) cause Apparent Mineralocorticoid Excess (AME), in which the mineralocorticoid receptor (MR) is inappropriately activated by glucocorticoids. Although AME is rare, milder forms may cause salt‐sensitive hypertension. We have assessed blood pressure (BP) and renal function in 11βHSD2 heterozygous null mice, having ~50% reduction in enzyme activity. Mice (n=7) were maintained on either a standard (0.3%) or high (3%) sodium chow for 3 weeks. Renal clearance experiments were used to measure the effect of amiloride (2mg/kg IV) on sodium excretion. On standard chow BP was ~90mmHg in both groups of mice. Increasing dietary sodium increased BP in 11βHSD2+/− mice but not in controls (101±2 vs 90±2mmHg; P<0.01). High salt diet increased fractional excretion of sodium in both groups of animals but the response was significantly blunted in 11βHSD2+/− mice. Whereas sodium loading prompted a down‐regulation of ENaC in control mice, robust amiloride‐sensitive sodium reabsorption was still observed in 11βHSD2+/− mice. Chronic MR blockade rescued both the blood pressure and renal phenotypes. Thus a reduction in 11βHSD2 activity impairs the ability of the kidney to down‐regulate ENaC‐mediated sodium reabsorption in response to sodium loading and this involves MR‐mediated pathways. These data suggest mild‐loss of function of 11βHSD2 may be clinically significant.
FITC localization of renin in rat mesometrial tissue
Authors: Hurley Mary Eder , Quarshie Rudrick , Gibrill Yusif , Critelli Marta , Hurwitz Melissa , Gurita Cristian , Olivieri Marion Pace
Published: June 2021

Renin, a hormone produced primarily by the kidneys in response to low blood pressure, is an immediate downstream gene product of Class I HOX gene regulation in the embryo. Examination of transgenic mice in which renin was linked to gfp reveals renin in renal, adrenal, and gonadal arteries in the embryo, giant trophoblast cells of the placenta, kidney and submandibular gland in both male and female, and uterine artery. In rat, giant trophoblast cells, other cells in fetal and maternal regions of the placenta, and cells of the tunica adventita of uterine artery as well as in the wall of the afferent arteriole of the kidney were labeled with antibody stain. This project examines pregnant and nonpregnant rat mesometrial tissues to identify cells that localize FITC labeled sheep anti‐rat renin antibody. Fluorescent granules were seen in cells resembling fibroblasts throughout the mesometrium and along the walls of small vessels within the mesometrium. Afferent arterioles were labeled and control tissues were not. These results demonstrate that the mesometrium is another site within rat female reproductive tissues in which renin is located, adding support to the hypothesis that extrarenal renin production may play a role in preecclampsia. Work is supported by NSF grant #0132823 and 0726185, and D’Youville College faculty council research funding.
Pathophysiological role of intrarenal Angiotensin II (AngII) in Dahl salt‐sensitive (SS) hypertensive rats
Authors: Mattson David , Lund Hayley
Published: June 2021

Experiments investigated the role of intrarenal AngII in normotensive and salt‐sensitive hypertensive rats. Increasing dietary NaCl from 0.4% to 4.0% in normal Sprague Dawley rats (n=4–7/group) significantly suppressed plasma renin concentration (5.3±1.3 to 2.9±0.7 ng AngI/ml/hr), plasma AngII (11.7±2.2 to 6.2±0.8 pg/ml), and renal tissue AngII (84±12 to 37±6 pg/gram tissue). The ratio of kidney to plasma AngII was unaltered from the 0.4% NaCl value of 7.2±1.0 when sodium intake was increased. In contrast, increasing dietary NaCl from 0.4 to 4.0% in Dahl SS significantly decreased kidney renin concentration (from 1343±247 to 527±142 ng AngI/μg protein/hr) but the ratio of kidney to plasma AngII significantly increased from 5.9±1.1 to 10.8±2.9. Experiments were then performed to evaluate the functional role of the elevated intrarenal AngII in Dahl SS rats fed high salt. Compared to vehicle‐treated SS rats fed 4.0% NaCl, administration of the AT1 receptor antagonist losartan (30 mg/kg/day, oral) to Dahl SS rats led to a significant attenuation of salt‐sensitive hypertension from 163±6 to 137±1 mmHg and albuminuria from 62±12 to 30±5 mg/day. Administration of captopril (15 mg/kg/day, oral) had similar effects on hypertension and albuminuria in Dahl SS fed high salt. These studies indicate that inappropriately elevated intrarenal AngII may participate in the development of Dahl SS hypertension.
Fatty acid dependent regulation of renin transcription by nuclear hormone receptor HNF‐4
Authors: Brinkmeier Thomas , Steege Andreas , Fähling Michael , Kirschner Karin , Martinka Peter , Persson Pontus B. , Scholz Holger , Mrowka Ralf
Published: June 2021

Production and release of renin are pivotal in controlling the Renin‐angiotensin‐system (RAAS), a main long‐term regulator of blood pressure and electrolyte homeostasis. In this context, the regulation of renin transcription by hormonal and nutritional agents is of fundamental interest.

Fatty acid dependent transcription factors LXRalpha and PPARgamma have already been shown to affect renin gene transcription. We focussed our attention on another member of the nuclear hormone receptor family, HNF‐4, which is bound by fatty acyl CoA modulating its transcriptional activity. In co‐transfection experiments, HNF‐4 activated a luciferase reporter construct containing 5797bp upstream the transcriptional start site of the renin gene more than five‐fold. This effect could be completely abolished by the addition of omega3 fatty acids at a concentration of 500μM. Using computational methods, we identified five potential binding sites for HNF‐4 in this sequence. There is first evidence that HNF‐4 binds to these sites depending on the presence of fatty acyl CoAs.

As HNF‐4 is expressed abundantly in proximal tubular cells our results could shed light on the regulation of local proximal tubular RAAS by fatty acids, indicating a new potential role of HNF‐4 in the kidney. Down‐regulation of renin gene expression may contribute to the antihypertensive effect of fish oil omega3 fatty acids.
Renin release is differentially sensitive to clinically used calcineurin inhibitors
Authors: Friis Ulla G , Madsen Kirsten , Skøtt Ole , Hansen Pernille B , Jensen Boye L
Published: June 2021

Renin release from juxtaglomerular cells (JG) is paradoxically inhibited by a rise in [Ca2+]i. We hypothesized that the calmodulin‐dependent phosphatase calcineurin in necessary for the inhibitory effect of [Ca2+]i. The hypothesis was tested in isolated JG‐cells by whole cell patch clamp and by renin release studies. In rat JG‐cells, the calcineurin inhibitor cyclosporine A (CsA, 5 μM) increased membrane capacitance (Cm), an index of cell surface area, in single JG‐cells by 10.7% ± 2.9% (n=5; P<0.05). A structurally distinct calcineurin inhibitor tacrolimus did not change Cm significantly when applied directly to JG‐cells. Similarly, renin release from JG‐cell cultures was stimulated by cyclosporine but not tacrolimus. Binding proteins for both inhibitors were detected in afferent arterioles (cyclophilin and FKBP). The effect on Cm was mimicked by the calmodulin‐inhibitor W‐13 (100 μM) in the patch pipette. When the experiments were repeated with a protein kinase A‐blocker (RpcAMPs (5 μM)) in the patch pipette, CsA and W‐13 still increased Cm significantly (15.8 % ± 4.2 % (n = 4) and 9.3 ± 3% (n = 4), respectively) indicating that the increase in Cm is independent on the cAMP‐PKA pathway. We conclude that calcineurin exerts a tonic suppressor effect on renin release from juxtaglomerular cells and that renin release is differentially sensitive to clinically used calcineurin inhibitors.

This study was supproted by grants from Carlsbergfondet, the Novo Nordisk Foundation and the Danish Heart Foundation.
A new approach to modeling the control of blood pressure
Authors: Ringwood John Vincent , Mangourova Violeta , Power Brendan , VanVliet Bruce , Malpas Simon
Published: June 2021

A variety of integrated models have been developed to account for the control of blood pressure, the most well know being that developed by Guyton and colleagues in the 1960's. However such models are often built using sequential sets of difference equations implemented in computer code. This format can hide the underlying physiological structure and makes certain assumptions about the way in which the underlying differential equations describing the physiology are solved (discretized). We propose the use of a hierarchical block‐diagram simulation environment for physiological models, which can represent physiological structure at varying levels of detail, chosen relative to the application need. Physiological processes are specified in natural differential equation (continuous‐time) form with a varying step length used for the computer solution of the equations, which is important for systems containing both slow and fast dynamics, as physiological systems frequently do. We have modelled how the variety of neural, hormonal and other factors affect blood pressure on a variety of time‐scales from seconds to weeks. The graphical, blockdiagram, representation also facilitates easy recognition of physiological subsystem inputs, outputs and interconnections and allows the model to be easily extended, modified and updated.
Vasomotor changes in controlled and uncontrolled hypertension
Authors: Frances Maria F , Zamir Mair , Usselman Charlotte W. , Goswami Ruma , Masse Nick , Fleischhauer Arlene , Nolan Robert , Shoemaker J. Kevin
Published: June 2021

The relationship between arterial stiffness and cardiovascular disease has been implicated in the development of isolated systolic hypertension. This study tested the hypothesis that hypertension is related to altered vascular mechanics and hence altered hemodynamics in skeletal muscle. The ability to detect such changes was tested using a lumped Windkessel model that incorporates several parameters of vascular control including resistance (R), compliance (C), viscoelasticity (K) and inertance (L). Two groups of middle aged hypertensive individuals were tested, one with normal blood pressure (NBP) following treatment (n=10), and the other with uncontrolled BP (UBP) (n=27). Baseline beat‐by‐beat measures of heart rate (ECG), mean arterial BP (Finometer), and forearm blood flow (FBF; Doppler ultrasound) were collected. Systolic BP, pulse pressure and mean arterial pressure were higher in the UBP group (p<0.01). Furthermore, values of C (0.008 for NBP vs. 0.006 mL/mmHg for UBP; p<0.02) and K (0.161 for NBP vs. 0.233 mmHg/mL/min for the UBP group; p=0.1) indicated that indeed the vessel walls in the UBP group were less compliant and offered more viscous resistance to stretch. Values of R were the same in the two groups. These findings suggest that hypertensive individuals with controlled BP are better able to maintain the function of skeletal muscle vasculature for muscle perfusion.

Supported by the Heart & Stroke Foundation of Canada and the Canadian Stroke Network.
Cardiovascular responses induced by excitation/lesion of caudal paramedial medulla (CPM)
Authors: Mortati Maria Carolina Galli , Seiji Willian Korim , Rosa Daniel Alves , Cravo Sergio L
Published: June 2021

Previous studies suggested that neurons located in the midline and adjacent regions of the caudal medulla participate in cardiovascular regulation. Presently we sought to determine the effects of chemical excitation or lesion of this area. Male Wistar rats were anesthetized, paralized, and artificially ventilated and prepared for mean arterial pressure (MAP), heart rate (HR), and blood flow (BF) recordings. Hindquarter (HQVC) and renal vascular conductance (RVC) were determined by the ratio MAP/BF. Bilateral microinjection of KA (5 Mm, 50 nL) into CPM (1.6 rostral, 0.5 lat and −2.3 mm to the CS) increased MAP (+43±10.3 mmHg), HR (+48±23.6 bpm) and induced renal and hindquarter vasoconstriction (−72±8.3; −75±3.6% of baseline, respectively). Reversely, bilateral microinjection of ibotenic acid (2.5nM, 50nL) induced hypotension (−41±13.3 mmHg) and bradicardia (−75±43.1 bmp) without significant changing RVC or HVC (2±12.8; 33±29.2% of baseline respectively). Forty minutes after IBO microinjection of KA into the same sites overturned these effects, inducing marked hypertension (+38±13.7 mmHg), tachycardia (43±8.3 bpm) and renal and hindlimb vasoconstriction (−74±5.9,−75±6.4% of baseline, respectively). These results suggested that paramedial medulla contains excitatory and inhibitory cardiovascular pathways that may be differentiated by their sensitivity to glutamate agonists.
Glycemic control and muscle metaboreflex‐induced pressor responses in type 2 diabetes patients
Authors: Fisher James P. , Young Colin N. , Alter Adam A. , LeMaster Joseph W. , Fadel Paul J.
Published: June 2021

An exaggerated pressor response to exercise has been reported in type 2 diabetes mellitus (DM) patients. However, the underlying neural mechanisms remain unknown. Also, how alterations in blood pressure (BP) reactivity are influenced by glycemic control is unclear. Therefore, in six type 2 DM patients (43±3 yr) and four age and body weight matched control subjects (39±5 yr) we examined the pressor responses to: 1) isolated activation of metabolically sensitive skeletal muscle afferents (i.e., muscle metaboreflex) during post exercise ischemia following 30% maximum voluntary contraction isometric handgrip (PEI 30%) and 2) a cold pressor test (i.e., non‐exercise stimulus). Pressor responses were related to fasting plasma hemoglobin A1c concentrations (HbA1c), an index for glycemic control. Resting mean BP was not different between groups, however during PEI 30% mean BP was higher in DM patients (103±3 vs. 88±2 mmHg, DM vs. Controls; P<0.05). A positive linear relationship was observed between HbA1c and PEI 30% induced changes in mean BP (R2 = 0.65, P<0.01), but not between HbA1c and cold pressor test induced changes in mean BP (R2 = 0.03, P = 0.69). These preliminary findings suggest that an exaggerated muscle metaboreflex responsiveness is related to impairments in glycemic control and may contribute to augmented increases in blood pressure during exercise in DM patients.
Angiogenic imbalance in a mouse model of pre‐eclampsia, BPH/5
Authors: Woods Ashley K , Hoffmann Darren S , Weydert Christine J , Sharma Ram V , Davisson Robin L
Published: June 2021

Recent clinical studies in pregnant women show an angiogenic imbalance present in a large fraction of women who go on to develop pre‐eclampsia (PE). These angiogenic factors include members of the vascular endothelial growth factor family, VEGF, PlGF, and the soluble form of their receptor: sFlt‐1. Similarily, soluble endoglin, an antagonist in the transforming growth factor β signaling pathway, has been recognized for its antiangiogenic potential. BPH/5 mice exhibit the cardinal symptoms of PE (late gestational hypertension, proteinuria) thus we hypothesized that an angiogenic imbalance would be found in BPH/5 mice. ELISA assays using plasma collected at early (d10.5–12.5), mid (d14.5) and late (d18.5) gestation revealed a decrease in circulating VEGF and PlGF in early gestation in BPH/5 mice (VEGF: 29.0±1.2 pg/ml vs. C57 84.1±15.3 pg/ml, n=3, p<0.05; PlGF:80.08±29.21pg/mL(BPH/5) vs. 418±58.29pg/mL (C57), n=4–5, p<0.05) and an elevated sFlt‐1 late in gestation (BPH/5:21.36±2.48 fold non‐pregnant (NP)state; C57:10.67±0.77 fold NP, n=6–7, p<0.01). Similarly, soluble endoglin levels are increased in BPH/5 mice at early gestation. These support the hypothesis that an angiogenic imbalance may underscore some of the pathological processes and demonstrate that BPH/5 is a good model to study in detail the role of angiogenic imbalance in PE.
Atrial natriuretic peptide inhibits angiotensin II‐induced cell proliferation in fetal sheep cardiomyocytes in vitro
Authors: O'Tierney Perrie Faye , Chattergoon Natasha , Louey Samantha , Giraud George , Thornburg Kent
Published: June 2021

Objectives: The anti‐hypertrophic properties of atrial natriuretic peptide (ANP) in cardiomyocytes have been well‐demonstrated; ANP inhibits the effects of several potent hypertrophic agents (ie. Angiotensin II, endothelin‐1, phenylephrine). During the last 1/3 of gestation the fetal heart grows by both proliferation and hypertrophy. We hypothesized that ANP also has an anti‐proliferative effect on cardiomyocyte (CMC) growth during fetal development.

Methods: Actively dividing cardiomyocytes were isolated from right and left ventricles of late‐gestation fetal sheep. Cultured fetal CMCs were treated with 10uM Bromodeoxyuridine (BrdU) and increasing doses of ANP (3.25, 32.5, 100, 325, 650 nM) with and without 100nM angiotensin II. Following 48 hours of treatment, cells were fixed and stained for myosin and BrdU. BrdU incorporation was used as an index of cell proliferation.

Results: Angiotensin II induced BrdU uptake in fetal CMCs and this was inhibited by ANP in a dose‐dependent manner (P=0.024). Physiological doses of ANP alone have no significant effect on BrdU uptake, though at supraphysiological levels (650nM), ANP increased BrdU uptake by fetal CMCs (P=0.051). No significant differences were seen between ventricles.

Conclusion: Physiological doses of ANP inhibit fetal cardiomyocyte proliferation induced by angiotensin II.

This research was supported by a NICHD (P01HD34430) and a N.L. Tartar Trust research fellowship.
Inositol 1,4,5‐trisphosphate Pathway Mediates Testosterone‐Induced Cardiac Hypertrophy
Authors: Estrada Manuel , Oyarce Cesar , Toyos Marcela , Altamirano Francisco
Published: June 2021

The anabolic hormone testosterone is associated with cardiac hypertrophy, but the signal transduction mechanisms involved are poorly known. In this work, primary cultures of neonatal cardiomyocytes where used to study testosterone effects on intracellular Ca2+ and their relation with cardiac hypertrophy markers: β‐MHC and skeletal α‐actin (SKA). Testosterone (100 nM) induced a rapid increase in intracellular Ca2+ that was blocked by inositol 1,4,5‐trisphosphate (IP3) inhibitors (U73122, 2APB and Xestospongin C) or by overexpression of the Ca2+ buffer protein parvalbumin. Neither nifedipine nor ryanodine affected the testosterone‐induced Ca2+ response. Stimulation with testosterone resulted in an increase of β‐MHC (12h) and SKA (24h) expression. Both in the presence of inhibitors of the IP3 pathway or in cells transfected with parvalbumin, β‐MHC and SKA expression were inhibited. Moreover, testosterone induced an increase in NFAT activation measured as luciferase activity that was inhibited by FK506. NFAT inhibition in turn blocked the hypertrophy markers increase. Cyproterone, an inhibitor of androgen receptor, had no effects on the intracellular Ca2+ or NFAT increase, but attenuated β‐MHC and SKA expression. These results suggest a synergism between pathways involving intracellular signal cascades and the classical genomic actions of androgens for testosterone‐induced cardiac hypertrophy.

Fondecyt 1060077 FONDAP 15010006
Combined Aspirin & Eicosapentaenoic Acid Improve Decreased Acetycholine Vasodilation Mediated by TNF‐ alpha
Authors: Hill‐Pryor Crystal Darlene‐Robin , Tostes Rita C. A. , Webb R. Clinton
Published: June 2021

Aspirin‐triggered lipoxins and similar chemical mediators like the Resolvins (resolution phase interaction products, RvDs) have powerful anti‐inflammatory effects and proresolution properties. RvDs are oxygenated products derived from omega–3 polyunsaturated fatty acids (ω‐3PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Aspirin, by acetylation of cyclooxgenase‐2 (COX‐2), enables the biosynthesis of the epimeric 17R‐series RvDs. The present study was designed to induce these biomolecules in vitro and study their effect on the impaired relaxation caused by TNF‐α in the vascular endothelium. We hypothesize that omega‐3 FA (EPA/DHA) in the presence of aspirin, which acetylates COX‐2 and favors RvDs production, have direct vascular effects, increasing endothelium‐dependent relaxation. After contraction with phenylephrine, vascular responses to acetylcholine (ACH, 10‐9 to 10‐5 M) or sodium nitroprusside (SNP, 10‐9 to 10‐5 M) were evaluated. We conclude that the ω‐3 PUFA DHA, in the presence of COX‐2 inhibition, facilitates endothelium‐mediated vasorelaxation, an effect that possibly is mediated by the 17R‐series RvDs. TNF‐α related decrease in vasorelaxation can be attenuated by the combination of omega‐3 fatty acids and aspirin. This supports the hypothesis that omega‐3 FA derived mediators can reverse the endothelial dysfunction caused by inflammatory cytokines.
Induction of PDE5 attenuates NO mediated vasodilation in resistance arteries under chronic high and low flow
Authors: Zhang Haiying , Lee Hyon Jae , Fisher Steven A.
Published: June 2021

In other studies we have observed the induction of NOS and PDE5 isozymes in the mesenteric artery (MA1) under conditions of sustained high and low flow (HF/LF). In this study we used pharmacological methods to test the role of NO and PDE5 in regulating vascular tone in HF/LF. Alternating pairs of second order MAs are ligated, resulting in chronic LF in the upstream MA1 and chronic HF in the adjacent MA1. Vascular reactivity was studied in a wire myograph system 4 days later, when NOS and PDE5 expression are increased. HF/LF Day4 MA1s were less sensitive than control to the α‐agonist phenylephrine (PE). Pre‐treatment with L‐NAME nearly normalized the sensitivity to PE in HF/LF MA1s. The PDE5 inhibitor Sildenafil at 1 μM without L‐NAME induced more complete relaxation of PE pre‐constricted HF/LF MA1 vs control (HF:76.7±8.0%; LF 90.3±4.7% ; control 46.3±11.1%; P<0.05). This difference could reflect the increased release of NO and/or increased activity of PDE5. To distinguish these, MA1s were pre‐treated with L‐NAME and Sildenafil and dose‐response to DEA/NO measured. The HF/LF MA1s were more sensitive to DEA/NO only in the presence of Sildenafil (EC50: HF 27±4; LF 24±4; Con 45 ± 6 nM; P<0.01). The responses to the PDE1 inhibitor Vinpocetine did not differ. We conclude that induction of PDE5 serves to de‐sensitize the vascular smooth muscle to chronic NO signaling under sustained HF/LF conditions.
VEGF165b is more potent at inhibiting endothelial cell migration than Pegabtanib and is cytoprotective for retinal pigmented epithelial cells
Authors: Magnussen Anette Lieselotte , Churchill Amanda , Harper Steve , Floege Juergen , Bates David
Published: June 2021

Ocular angiogenesis, stimulated by VEGF165 in the retina and choroid, is the principal cause of vision loss in patients with diabetic retinopathy and wet age related macular degeneration. In these conditions there is an excess of VEGF165 over the anti‐angiogenic splice variant VEGF165b. Inhibitors of VEGF such as ranibizumab and pegabtanib are used clinically to treat AMD patients. We have compared the effect of VEGF165b with these anti‐VEGF antibody treatments. Western blotting showed that pegabtanib did not bind VEGF165b, whereas bevacizumab (the antibody from which ranibizumab was derived) bound VEGF165b. Chemotaxis assays of HMVE Cells using a Boyden chamber showed a 2.6 fold decrease of HMVEC migration at 10nM pegabtanib after challenging the cells with 1nM VEGF165 (p<0.01). The same impairment in migration occurred with only 1nM VEGF165b. Treatment of RPE cells with 10mg/ml neutralising antibody to VEGF165b showed a significant increase in cytotoxicity compared to control IgG, measured by lactase dehydrogenase release, indicating that VEGF165b is a survival factor for retinal pigmented epithelial cells. Together these results support the use of VEGF165b as an anti‐angiogenic treatment in ocular neovascularization.
Nitric oxide release from nitric oxide synthase on erythrocytes during hemorrhagic shock
Authors: Chen Kejing , Pittman Roland N. , Popel Aleksander S.
Published: June 2021

A large loss of blood during hemorrhage could result in profound shock, a state of hypotension associated with hemodynamic abnormalities. One of the hypotheses for the hemorrhage‐induced collapse of homeostasis is that the production of nitric oxide (NO), a gas molecule that dilates blood vessels to match O2 delivery to the metabolic activity of local tissue, is significantly impaired because of the reduced blood volume and flow. Recent studies show that erythrocytes express functional endothelial nitric oxide synthase (NOS3), which potentially serves as an intraluminal NO source. We hypothesized that the loss of NOS3 on erythrocytes during hemorrhage contributes to the NO‐deficiency related shock. In this study, we constructed a multi‐cellular computational model that simulates NO production from NOS3 on erythrocytes and its transport around an arteriole. Our simulations show that whether a significant amount of NO from this source can be delivered to vascular smooth muscle is strongly dependent on the existence of a protected mechanism for NO delivery. Our result also predicts that NO delivery from erythrocytes decreases with blood loss during hemorrhage; with a critically low hematocrit (8%), ∼50% of the NO bioactivity from NOS3 on erythrocytes delivered to smooth muscle is lost, compared with the condition of 45% hematocrit. (Supported by NIH grants HL79087 and HL18292).
Direct measurement of periarterial nitric oxide (NO) in resistance vessels of aged WKY rats reveals elevated basal and suppressed flow‐mediated production which is normalized by apocynin.
Authors: Zhou Xiaosun S , Bohlen H Glenn , Unthank Joseph L , Miller Steven J
Published: June 2021

Abnormal regulation of the nitric oxide (NO) system may be responsible for the age‐related impairment in tissue perfusion and collateral growth. Direct, real time, in vivo measurements of NO in aging resistance vessels are not available. This study tested the hypothesis that aging is correlated with reduced basal and flow‐mediated NO production, which can be reversed by anti‐oxidant therapy. Our mesenteric model was used in young (Y) and retired breeder (RB) WKY and RB pre‐treated with apocynin (RB+Apo). NO‐ specific electrodes measured mesenteric periarterial NO simultaneously with arterial flow. After NO baseline measurement, collateral flow was varied from 50–200% of control by placement of microvascular clamps on adjacent arteries. RB had significantly increased baseline NO compared with Y (2053 ± 504 vs. 1059 ± 160 nM, p< 0.05). Periarterial NO levels were altered in proportion to blood flow changes in Y but not in RB rats (Figure 1). In RB+Apo, NO baseline was reduced (760 ± 102 nM) and flow‐mediated NO production restored in RB+Apo (Figure 1). Immunoblotting showed that RB had significantly increased phospho‐eNOS levels compared to either Y or RB+Apo. This suggests that the elevated baseline and suppressed flow‐mediated NO production may result from near maximal activation of eNOS in the RB. eNOS stimulation by reactive oxygen species produced by NADPH oxidase is a mechanism to be explored.

Support: HL42898 and HL20605
Pentoxifylline reduces myocardial infarction by attenuating the inflammatory response of ischemia‐reperfusion injury
Authors: Jiang Rong , Deneve Jeremiah , Eldaif Shady , Granfeldt Asger , Guyton Robert A. , Zhao Zhi‐qing , Vinten‐Johansen Jakob
Published: June 2021

Studies have suggested that Pentoxifylline attenuates contractile dysfunction after ischemia‐reperfusion. This study tested the hypothesis that PTX given specifically at reperfusion (R) reduces infarct size after in vivo coronary artery occlusion‐R by a TNFα‐related mechanism. Methods: 24 male SD rats were divided into three groups of 8 each with 30 min left coronary artery occlusion and followed by 3 hours reperfusion; all drugs/vehicle were administered intraperitoneally 10 minutes before R: 1) Control: saline vehicle; 2) PTX10: 10 mg/kg PTX; PTX50 50mg/kg. Results: There were no hemodynamic differences among the three groups. The area at risk was comparable (C: 35¡À 2%, PTX 10:32¡À2%, PTX 50:33¡À2%). PTX significantly decreased infarct size in a dose‐dependent manner (PTX10 = 48¡À1.9%; PTX50 = 39¡À2.3%) compared to Control (C=56¡À1.4%, p<0.05). Plasma MDA levels increased between baseline and 3 hrs of R but there were no significant group differences. Plasma TNFα levels (pg/mL) were comparable among Control and PTX 50 at baseline (Control: 84¡À71; PTX 50 85¡À95). However, plasma TNFα increased significantly after 1 hour of R in Control (2083¡À513). In contrast, plasma TNFα was significantly reduced in PTX 50 group (758¡À297, P<0.05). Conclusions: PTX given at R decreases infarct size, possibly by a reduction in TNFα generation, but not by a reduction in oxidant‐induced membrane injury.
Differential effects of a green tea derived polyphenol (−)‐epigallocatechin‐3‐gallate on the muscle‐specific decrease in calcium response to acidosis
Authors: Liou Ying‐Ming , Kuo Shih‐Chang
Published: June 2021

(−)‐Epigallocatechin‐3‐gallate (EGCg), a green tea derived polyphenol, has received much attention as a protective agent against cardiovascular diseases. In this study, we determined the effects of EGCg on changes in pH sensitivity of myofilament response to Ca2+ in myofibrils prepared from porcine ventricular myocardium and chicken pectoral muscle. In comparison with skeletal muscle, lowering pH from 7.0 to 6.5 and/or 6.0 EGCg has significantly blunted the acidic effect on the reduction of Ca2+ sensitivity for myofibrillar ATPase activity in cardiac muscle. Studies with recombinant mouse cardiac (cTnC) and chicken fast skeletal troponin C (sTnC) by circular dichroism (CD), intrinsic and extrinsic fluorescence spectroscopy showed that EGCg specifically binds to cTnC with a dissociation constant ~ 3–4 μM, but no effect on sTnC. Presumably binding to the cTnC C‐lobe, EGCg acted to alter the Ca2+‐binding to cTnC and compromised the interference of proton effect on the Ca2+ binding at acidic pH. To demonstrate the isoform‐specific effects on the action of EGCg, the pH‐sensitivity of Ca2+ response was examined in cardiac myofibrils replaced with either sTnC or cTnC and skeletal myofibrils exchanged with whole cardiac Tn complex (cTn). The data obtained suggested that EGCg interactions with the cardiac isoform specific TnC or Tn complex could alter the pH effect on myofilament Ca2+ sensitivity in striated muscle.
TIMP3 regulates p38 MAPK‐dependent myogenesis via TACE
Authors: Li Yi‐Ping , Chen Shuen‐Ei , Liu Huijie , Jin Bingwen
Published: June 2021

Activation of p38 MAPK is a critical step in the onset of myogenesis. However, the upstream signaling mechanism that activates p38 during myogenesis remains undefined. We have recently found that p38 activation in differentiating myoblasts and regenerating muscle is mediated by autocrine TNF‐α release which is the function of TNF‐α converting enzyme (TACE). In the present study, we tested a hypothesis that tissue inhibitor of metallopreoteinase3 (TIMP3), the endogenous inhibitor of TACE, is a physiological suppressor of myogenesis, and is a part of the regulatory mechanism for the activation of myogenesis. We observed a dramatic down‐regulation in TIMP3 level during the first 48 h of C2C12 myoblast differentiation. Overexpression of TIMP3 suppressed TNF‐α release and blocked p38 activation, myogenin and myosin heavy chain expression, and myotube formation. Conversely, addition of recombinant TNF‐α (50 pg/ml) to differentiation medium rescued the impaired myogenesis. Down‐regulation of TIMP3 was also observed in cardiotoxin‐injured mouse soleus muscle; and overexpressed TIMP3 inhibited p38 activation and myogenesis. In addition, the down‐regulation of TIMP3 is accompanied by a decline in TIMP3 mRNA level. These results indicate that TIMP3 is a physiological suppressor of myogenesis and its down‐regulation activates p38‐dependent myogenesis.

Supported by National Institute of Health Grant AR049022
Effect of altered AMP‐activated protein kinase activity and exercise training on muscle PBEF‐1/visfatin levels.
Authors: Brandauer Josef , Koh Ho‐Jin , Jung Michelle M. , Toyoda Taro , An Ding , Yu Haiyan , Fujii Nobuharu , Hirshman Michael F. , Witters Lee A. , Goodyear Laurie J.
Published: June 2021

Pre‐B‐cell enhancing factor 1 (PBEF‐1; also known as visfatin) is a stress‐inducible regulator of intracellular NAD+. Elevated NAD+ levels are associated with increased Sirtuin activity, which can contribute to life span extension in some organisms. Caloric restriction may serve to extend life span via this mechanism. Since AMP‐activated protein kinase (AMPK) serves an important role as a cellular fuel gauge, we investigated the role of AMPK in regulating muscle PBEF‐1 levels. PBEF‐1 was significantly increased (50%) in tibialis anterior muscle from mice expressing an activating mutation of AMPK (γ1R70Q) in skeletal muscle (p<0.01). PBEF‐1 was significantly decreased in the muscles of two models of impaired AMPK activity, muscle‐specific AMPKα2 inactive transgenic mice (13%; P<0.05) and muscle‐specific LKB1 knockout mice (30%; p<0.01). In addition to skeletal muscle, PBEF‐1 was also decreased in cardiac tissue from the AMPKα2 inactive transgenic mice and LKB1 knockouts, and this effect was relatively greater in the heart compared to skeletal muscle. We next investigated the effects of seven weeks of voluntary wheel cage running on PBEF‐1 protein expression in skeletal muscle and heart from rats. Training resulted in 26% increased PBEF‐1 levels in cardiac muscle (p<0.05), but had no effect in triceps muscle despite clearly increased citrate synthase activity in this muscle. In conclusion, AMPK may be an important regulator of the purported anti‐aging protein, PBEF‐1 in skeletal and cardiac muscles. PBEF‐1 levels are significantly elevated after training in cardiac muscle, suggesting a previously unidentified potential aging‐protective role of chronic exercise in the heart.
Thinking outside the BötC (Bötzinger complex): possible brainstem origins of abdominal (AB) expiratory activity in the rat in situ
Authors: Abdala Ana Paula Lima , Rybak Ilya A , Smith Jeffrey C , Paton Julian F.R.
Published: June 2021

We have focused on brainstem regions and mechanisms involved in generating expiratory activity. We recorded phrenic (PN), central vagus (cVN) and AB outflow (T10‐L1) during hypercapnia, following brainstem transections and chemical suppression of the retrotrapezoid nuclei (RTN) & parafacial (PF). In the in situ decerebrate preparation of rat, AB expiratory activity exhibited low amplitude post‐inspiratory (post‐I) discharge. In hypercapnia (7–10% CO2) augmenting late‐expiratory (late‐E) discharge was evoked. Sectioning the rostral pons revealed a 2‐phase rhythm with tonic AB discharge throughout expiration. When the BötC was removed all expiratory activity was abolished and could not be reinstated by hypercapnia (n=7). Suppression of RTN/PF (isoguvacine, 60 nL, 20 mM) attenuated resting post‐I activity in AB (−69±14 %, n=5, P<0.05) and cVN (−68±12 %, n=5, P<0.05), which partially recovered in hypercapnia, and abolished late‐E AB bursts evoked by hypercapnia (n=5). The most effective sites were localised in the caudal half of RTN/PF. We conclude that the integrity of RTN/PF is required for regeneration late‐E AB bursts during hypercapnia. The BötC is essential for generation of expiratory activity for the 2‐phase rhythm. The pons provides essential tonic excitatory drive to post‐I cell populations in the brainstem with RTN/PF contributing to this drive. Support: NIH‐RO1 NS057815.
Effect of cadmium exposure on P‐glycoprotein expression and activity in eastern oysters, Crassostrea virginica
Authors: Ivanina Anna , Sokolova Inna
Published: June 2021

Heavy metal pollution is a worldwide problem, and cadmium (Cd) is one of the most noxious environmental pollutants responsible for episodes of chronic poisoning in humans and posing serious threat to wildlife. One of the possible mechanisms of cell protection against Cd is an increase in expression of P‐glycoprotein (P‐gp) – an ATP‐depending pump which translocates a variety of xenobiotics out of the cell. We studied P‐gp expression and function in control and Cd exposed (50 ìg L‐1 Cd, 30 days) oysters Crassostrea virginica. Expression of P‐gp protein was significantly higher (2–2.5 fold) in cell membranes of Cd‐exposed oysters compared to the controls; no difference in P‐gp expression was observed in mitochondria of control and Cd‐exposed oysters. No difference in P‐gp mRNA expression was found between control and Cd‐exposed oysters indicating regulation at post‐transcriptional level. P‐gp activity was measured using Rhodamine B (RB) efflux assay and showed a 3.5‐7‐fold increase in P‐gP dependent RB extrusion in Cd exposed oysters. Cd exposure resulted in elevated rated of tissue MO2 by 25%. However, P‐gp inhibitors (cyclosporin A and JS‐2190) had no significant effect on tissue oxygen consumption indicating that P‐gp contribution to energy budget is negligible.
Endocrine parameters and ovarian dynamics in Ossabaw miniature swine with metabolic syndrome suggest a model for polycystic ovary syndrome
Authors: Krisher Rebecca L , Lowe Jennifer , Bailey Amanda , Sturek Michael
Published: June 2021

We describe characterization of the Ossabaw pig as a model of polycystic ovary syndrome (PCOS). Gilts (n=17) were fed either a control (lean) or excess kcal atherogenic diet for 22 weeks that elicits metabolic syndrome (MetS). Blood free testosterone and androstenedione were measured by RIA and ovarian activity monitored by ultrasound twice a week. Ovarian follicles were measured individually and area, circumference and diameter recorded. Across the period of the study, androstenedione values did not differ between groups. However, androstenedione was significantly higher in the last third of the study period in MetS gilts, suggesting the onset of hyperandrogenemia. Free testosterone was higher in lean gilts. There were no differences in follicular circumference or diameter between groups. MetS had significantly greater follicular area than lean gilts (MetS = 0.044±0.002, lean = 0.037±0.001), more follicles with an area ≥0.1 cm2 (MetS = 4.98%, lean = 3.54%), more follicles with circumference ≥2 cm (MetS = 1.68%, lean = 0.65%) and more follicles with circumference <0.5 cm (lMetS=16.91%, lean=14.59%), suggesting that MetS gilts produce more small follicles that in turn grow larger than those in lean gilts. We propose that Ossabaw swine with MetS develop reproductive parameters reminiscent of PCOS, and thus may represent a complete animal model of the disease. Support: CBR2‐IUPU, RR013223, HL062552.