bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuronal Nitric Oxide Synthase in the Rostral Ventrolateral Medulla of Hindlimb Unloaded Rats
Authors: Zidon Terese M , Fong Angelina Y , Foley C Michael , Mueller Patrick J , Heesch Cheryl M , Hasser Eileen M
Published: June 2021

Prolonged periods of bed rest or spaceflight result in cardiovascular deconditioning and a predisposition for orthostatic intolerance. Hindlimb unloaded (HU) rats are an animal model of deconditioning and exhibit blunted baroreflex sympathoexcitation. Neurons in the rostral ventrolateral medulla (RVLM) are important for baroreflex function. Previous studies have shown that GABAergic inhibition in the RVLM is increased in HU rats. Nitric oxide (NO) is a neuromodulator that increases GABA inhibition and may contribute to enhanced GABAergic inhibition in the RVLM following HU. We hypothesized that 14 days of HU increases the number of neuronal NO synthase (nNOS) and GAD67 positive neurons in the RVLM. The RVLM distribution of nNOS and GAD67 was examined using immunohistochemistry in brainstem sections (30μm) from male Sprague Dawley (300–350g) control (n=5) and HU (n=5) rats. The number of nNOS and GAD67 positive cells was not different between control and HU rats. Interestingly, there was substantial colabeling of RVLM cells with GAD67 and nNOS in both groups. The number of GAD67 positive cells colabeled with nNOS (11 – 13%) and the number of nNOS positively labeled cells colabeled with GAD67 (40–45%) was similar between control and HU rats. Thus, HU does not appear to alter the number or colabeling of nNOS or GAD67 containing neurons in the RVLM. (HL55306)
Angiotensin‐(1‐7) antagonist into the CVLM improves baroreflex bradycardia in renovascular hypertensive rats
Authors: Campagnole‐Santos Maria J , Cangussu Luiza M , Machado Raquel P , Ferreira Patricia M , Silva Marcelo E , Santos Robson AS , Alzamora Andreia C
Published: June 2021

We have shown that Ang‐(1‐7) at the caudal ventrolateral medulla (CVLM) induces a decrease in the baroreflex bradycardia (BRS) in normotensive rats. In this study we evaluated the changes in mean arterial pressure (MAP), heart rate (HR) and BRS induced by CVLM microinjection of the Ang‐(1‐7) antagonist, A779, in normotensive (Sham) and renal hypertensive (2K1C) rats. Blood pressure was recorded by Powerlab system and BRS was estimated by the ratio between changes in HR (as pulse interval) and changes in MAP induced by phenylephrine. CVLM microinjection of A779 produced decreases in MAP in 2K1C (−12 ± 3 mmHg; n=6; Baseline MAP=141 ± 6 mmHg; n=7) in contrast to that observed in Sham (−5 ± 2 mmHg; Baseline MAP= 108 ± 3 mmHg; n=8). No significant changes in HR were observed in Sham or 2K1C. A779 injection significantly increased baroreflex bradycardia in 2K1C (0.44 ± 0.05 ms/mmHg, n=7 vs 0.24 ± 0.03 ms/mmHg, n=7; before). However, A779 did not alter baroreflex bradycardia in Sham rats (0.35 ± 0.04 ms/mmHg, n=7 vs 0.40 ± 0.02 ms/mmHg, n=7; before). These data suggest that increased levels of Ang‐(1‐7), at least in the CVLM, may contribute to the low sensitivity of the baroreflex control of heart rate in renovascular hypertensive rats. Support: CNPq, FAPEMIG, PRONEX, REDE‐TOXIFAR‐UFOP.
Comparison of the cardiovascular responses evoked by chemical activation of the left and right sides of dorsomedial hypothalamus
Authors: Custódio Carlos Henrique Xavier , Campagnole‐Santos Maria José , Fontes Marco Antônio Peliky
Published: June 2021

Activation of dorsomedial hypothalamus (DMH) neurons by microinjections of the GABAA antagonist, bicuculline methiodide (BMI), evokes physiological cardiovascular changes typically seen in experimental emotional stress. We compared the heart rate (HR), renal sympathetic activity (RSNA) and renal blood flow (RBF) responses produced by chemical activation of right (R) and left (L) sides of DMH. Rats were anesthetized (urethane 1.4 g/Kg), prepared for recording of cardiovascular variables and for microinjections. In all animals, RSNA and RBF were recorded on the L side. In protocol 1 (n=5), BMI (40pmol/100nL) was injected first into LDMH and 40 min. later, into the RDMH. Inversely, in the protocol 2 (n=5) BMI was injected first into the RDMH and later into the LDMH. In both cases there were always greater increases in RSNA with reductions in RBF when BMI was injected into the LDMH, i.e., ipsilateral to the side where RSNA and RBF were recorded (average RSNA: L= +50% and R= +26.5%; average RBF: L= −14% and R= −6.5%). In both protocols RDMH activation caused greater tachycardia when compared to LDMH activation (ΔHR Protocol 1: L= 45±3 bpm vs. R= 74±4 bpm; Protocol 2: R= 78±5 bpm vs. L=55±6 bpm; P<0.05). These results indicate that the descending cardiovascular pathways from DMH are predominantly lateralized and suggesting that the right DMH might exert a prominent control on heart rate changes. Support: CNPq, FAPEMIG, CAPES.
Locally released vasopressin increases presympathetic PVN neuronal activity
Authors: Son Sook Jin , Stern Javier E
Published: June 2021

In addition to neurohypophysial secretion, vasopressin (VP) is locally released within the supraoptic and paraventricular (PVN) nuclei, autoregulating VP neuronal activity. Here, we tested the hypothesis that locally produced VP can also influence presympathetic PVN neuronal activity. Patch‐clamp recordings from presympathetic PVN‐RVLM neurons show that focal application of VP (1μM) depolarized, and increased the firing activity of PVN‐RLVM neurons (∼450%, P< 0.01). The effect persisted in the presence of the GABAA and glutamate receptor blockers BIC (20 μM) and KYN (1 mM), respectively. In the voltage‐clamp mode, VP activated an inward current of ∼6–10 pA. VP effects were blocked by the manning compound (1 μM), a V1a receptor antagonist. The expression of V1a receptors in PVN‐RVLM neurons was further confirmed by immunohistochemical studies. Importantly, the V1a receptor antagonist per se induced membrane hyperpolarization (∼4 mV) and reduced the ongoing firing activity of PVN‐RVLM neurons (∼75%, P< 0.02). In summary, our results indicate that locally produced VP tonically excites presympathetic PVN neurons, and suggest that activity‐dependent VP release from magnocellular neurosecretory neurons may constitute an important intercellular signaling pathway in the generation of homeostatic patterns of neuroendocrine and autonomic activities by the PVN. Supported by NIH HL68725.
GABAergic pump cells of solitary tract nucleus innervate retrotrapezoid nucleus chemoreceptors
Authors: Takakura Ana C , Moreira Thiago S , Stornetta Ruth L , Guyenet Patrice G
Published: June 2021

The retrotrapezoid nucleus (RTN) contains chemosensitive neurons that are inhibited by lung inflation via slowly‐adapting pulmonary stretch receptors (SARs). Here we examine whether RTN inhibition by lung inflation could be mediated by a direct projection from SAR second‐order neurons (pump cells). Pump cells (N = 56 neurons, 13 rats) were recorded in the nucleus of solitary tract (NTS) of halothane‐anesthetized rats with intact vagus nerves. Pump cells had discharges that coincided with lung inflation as monitored by the tracheal pressure. Their activity increased when end‐expiratory pressure was raised and stopped instantly when ventilation was interrupted in expiration. Many pump cells could be antidromically activated from RTN (12/36). Nine of those were labeled with biotinamide. Of these 9 cells, eight contained glutamic acid decarboxylase67‐ (GAD67‐) mRNA and seven were found to reside in the lower half of the interstitial subnucleus of NTS (iNTS). Using the retrograde tracer cholera toxin‐B, we confirmed that neurons located in or close to iNTS innervate the RTN (2 rats). Many such neurons contained GAD67‐mRNA and a few contained glycine transporter2 (GLYT2) mRNA. Anterograde tract‐tracing with biotinylated dextranamide (4 rats) applied to iNTS also confirmed that this region innervates RTN via a predominantly GABAergic projection. This work confirms that, in rats many NTS pump cells are located in and around the interstitial subnucleus at area postrema level. We demonstrate that a GABAergic subset of these pump cells innervates the RTN region. We conclude that these inhibitory neurons probably contact RTN chemoreceptors and mediate their inhibition by lung inflation.

Supported by: HL 74011 (PGG) & Capes‐BEX 4402/05‐7 (ACT).
CO2 chemosensitivity and air breathing in the Alaska blackfish
Authors: Hoffman M , Taylor B E
Published: June 2021

Central CO2 chemosensitivity has been reported in primitive and modern air‐breathing fish. Alaska blackfish (Dallia pectoralis) are an arctic air‐breathing fish naturally exposed to variations in water temperature as well as CO2 and O2 content. We tested the hypothesis that Alaska blackfish will exhibit central CO2 chemosensitivity. Mild tissue acidosis was induced in these fish by inhibiting CO2 excretion with acetazolamide (ACZ), a carbonic anhydrase inhibitor. We monitored spontaneous air‐breathing frequency in blackfish acclimatized to a range of temperature before and after treatment with ACZ. Results indicate that ACZ treatment influences spontaneous air‐breathing in a temperature‐dependent manner. These data suggest that central CO2 stimuli can influence air breathing in this species.
Decreased fiber size and mitochondrial content in mouse diaphragm after normobaric hypoxia
Authors: Gamboa Jorge L , Andrade Franciso H
Published: June 2021

Life at high altitude requires that organisms adapt to chronic hypoxia. Increased ventilation is part of the adaptive response to hypoxia. The hindlimb muscles of animals exposed to hypoxia adapt by reducing fiber cross sectional area (FCSA) and mitochondrial capacity. These changes are explained, at least in part, by less physical activity. In contrast to limb muscles, the diaphragm must remain active and increases its workload due to the hypoxia‐induced hyperventilation. Thus, we hypothesized that in the diaphragm hypoxia would not change either FCSA or mitochondrial content. Adult male mice (8wks old) were exposed to normobaric hypoxia (FiO2 = 10%) for 0, 2 or 4 weeks. Then mice were killed and the diaphragm removed for histology and western blot analysis. FCSA was not different after 2 weeks of hypoxia (633±229 sq. um vs. 666±246 sq. um in normoxic controls, mean±standard deviation) but it decreased after 4 weeks (488±213 sq. μm, p<0.001). The content of the mitochondrial complexes IV and V was reduced by 40±7% (p=0.009) and 60±5% (p=0.036), respectively, after 4 weeks of hypoxia. No significant changes were observed after two weeks of hypoxia. These results do not support our initial hypothesis. Instead, they indicate that the diaphragm follows the same adaptive strategy as limb muscles in response to hypoxia, despite its higher workload.
Role of adenosine and nitric oxide in hypoxia‐induced skeletal muscle vasodilation in humans
Authors: Leuenberger Urs A. , Mascarenhas Vernon , Lninton‐Frazier Latoya , Steiner Jill , Hogeman Cynthia , Gray Kristen S.
Published: June 2021

In humans, systemic hypoxia evokes vasodilation in resting skeletal muscle despite significant increases in sympathetic vasoconstrictor nerve activity. This is thought to be due to endogenous vasodilators such as adenosine and nitric oxide (NO). To test whether adenosine and NO act independently, we determined the forearm blood flow responses to systemic hypoxia (FiO2 0.1 for 10 min) during 1) inhibition of adenosine receptors (aminophylline), 2) inhibition of NO‐synthase (NG‐monomethyl‐L‐arginine, L‐NMMA), and 3) combined inhibition of adenosine receptors and NO‐synthase (aminophylline plus L‐NMMA) administered via brachial artery in healthy humans. Forearm blood flow (FBF, venous plethysmography) was compared in the infused (experimental) and opposite (control) forearms. During hypoxia (pO2 35±1 mmHg), FBF in the control forearm increased by ~50%. L‐NMMA alone (n=8) reduced hypoxia‐induced vasodilation by ~37% whereas aminophylline alone (n=9) reduced it by ~50%. Combined blockade with aminophylline plus L‐NMMA (n=8) did not inhibit hypoxia‐induced vasodilation more greatly than either agent alone. These data demonstrate that both adenosine and NO contribute to hypoxia‐induced skeletal muscle vasodilation. The lack of synergy between aminophylline and L‐NMMA suggests that adenosine and NO may be part of the same vasodilator signaling cascade.

(Supported by P01 HL077670 and R01 HL068699)
Gender‐dependent regulation of hypoxic ventilation in mouse and man is mediated by erythropoietin
Authors: Gassmann Max , Soliz Jorge
Published: June 2021

Acclimatization to hypoxic exposure relies on an elevated ventilation and erythropoietic activity. We recently showed that erythropoietin (Epo) links both responses: apart from its well‐known function on erythropoiesis, cerebral and plasma Epo interferes with the central respiratory centers (brainstem) and peripheral chemoreceptors (carotid bodies). Knowing that women cope better than men to reduced oxygen supply (as observed at high altitude), we analyzed the hypoxic ventilatory response in female mice. We used either transgenic mice with elevated Epo levels in brain only (Tg21), or in brain and plasma (Tg6) or wt animals injected or not with rhEpo. Exposure of these animals to moderate and severe acute hypoxia as well as to hyperoxia and injection of domperidone, reveled that the presence of Epo extensively increases the hypoxic ventilatory response in female mice compared to their male siblings. Furthermore, we showed that soluble EpoR (sEpoR) is endogenously synthesized by neural tissue, and abolishes the ventilatory acclimatization to hypoxia when infused in brain. Finally, human volunteers were injected rhEpo and subsequently exposed to 10% oxygen. Compared to men, the hypoxic ventilatory response was significantly increased in women. We conclude that Epo exerts a gender‐dependent effect on hypoxic ventilation most probably involving sexual hormones.
Nitric oxide and zinc contribute to hypoxia induced contractile events in pulmonary endothelial cells
Authors: Bernal Paula Jimena , Watkins Simon Charles , Croix Claudette Marie St
Published: June 2021

We previously reported that acute hypoxia caused a reversible constriction of small non‐muscularized vessels (<100 μM) of the isolated perfused mouse lung (IPL).This led us to study the contractile behavior of isolated endothelial cells using differential interference contrast (DIC) microscopy. We found that rat pulmonary microvascular endothelial cells (RPMVECs) constricted in hypoxia whereas systemic endothelial cells did not. We previously showed that: 1) hypoxia caused NOdependent increases in labile zinc in the IPL that were critically dependent upon the metal binding protein, metallothionein (MT); and 2) hypoxic pulmonary vasoconstriction (HPV) was blunted MT −/− mice or wild‐type mice treated with zinc chelators. We therefore examined a role for NO‐induced zinc release in regulating contraction in RPMVECs, using a NO‐sensitive FRET reporter based on MT and the zinc‐sensitive fluorophore, FluoZin‐3. Hypoxia induced changes in energy transfer consistent with unfolding of MT; accompanied by increases in Zn. Our data suggest that zinc signaling contributes to the effects of hypoxia mediated NO biosynthesis and this pathway may contribute to constriction in lung endothelium.
Ceramide induces inhibition of Kv channels and vasoconstriction in rat pulmonary arteries
Authors: Frazziano Giovanna , Cobeño Laura , Cogolludo Angel , Moreno Laura , Moral Javier , Villamor Eduardo , Perez‐Vizcaino Francisco
Published: June 2021

Voltage‐gated potassium (Kv) channels make a major contribution to the membrane potential of pulmonary artery smooth muscle cells (PASMC). We examined whether ceramide, a sphingolipid‐derived second messenger, inhibits Kv channels and induces pulmonary vasoconstriction. C6‐ceramide and bacterial sphingomyelinase inhibited Kv currents and caused depolarization of freshly isolated PASMC and induced a sustained contractile response in endothelium–denuded pulmonary arteries. C6‐ceramide also increased the production of reactive oxygen species, measured by the fluorescence of dichlorofluorescein. The protein kinase Cζ (PKCζ) pseudosubstrate inhibitor (PKC‐PI) inhibited all the effects elicited by C6‐ceramide. t‐butyl‐hydroperoxide inhibited Kv currents and contracted pulmonary arteries but these effects were not affected by PKCζ‐PI. The NADPH oxidase inhibitor apocynin prevented the C6‐ceramide‐induced Kv current inhibition and vasoconstriction. In contrast to pulmonary arteries, in mesenteric arteries, C6‐ceramide had no effect on either Kv currents or resting tone. The expression of Kv1.5 and PKCζ was significantly higher in pulmonary compared to mesenteric arteries. In conclusion, ceramide induced a pulmonary‐selective inhibition of Kv currents and vasoconstriction via PKCζ and NADPH‐oxidase derived reactive oxygen species
Neutral sphingomyelinase and protein kinase Cζ are involved in hypoxic pulmonary vasoconstriction
Authors: Cogolludo Angel , Moreno Laura , Cobeño Laura , Frazziano Giovanna , Castañeda Javier , Gonzalez Constancio , Villamor Eduardo , Perez‐Vizcaino Francisco
Published: June 2021

Hypoxic pulmonary vasoconstriction (HPV) is a highly conserved physiological mechanism that optimizes blood oxygen saturation by increasing pulmonary vascular resistance in poorly aerated lung regions. We found that hypoxia increased ceramide content measured by immunocytochemistry, phosphorylation of the membrane NADPH oxidase subunit p47phox, reactive oxygen species production measured by either dihydroethidium or dichlorofluorescein fluorescence, inhibited voltage‐gated K+ (Kv) currents, depolarized the membrane and induced a vasoconstrictor response in isolated rat pulmonary arteries (PA). Inhibition of NAPDH oxidase, protein kinase Cζ (PKCζ), neutral sphingomyelinase (nSMase) abrogated the changes in reactive oxygen species, Kv currents and the vasoconstrictor response. nSMase inhibition also inhibited the increase in ceramide and prevented HPV in anesthetized open‐chest rats. The signaling pathway was specific for small PA as compared to mesenteric arteries. nSMase2 mRNA expression was ≈10 fold higher in small PA compared to large PA or mesenteric arteries. In conclusion, nSMase‐derived ceramide production and activation of PKCζ are early and necessary events in the signaling cascade of HPV leading to increased reactive oxygen species production, inhibition of Kv channels, depolarization and vasoconstriction. Supported by SAF2005‐03770, CIBER and Mutua Madrileñ
Four weeks of residential cardiac rehabilitation reduces NT‐proBNP and improves fitness
Authors: Crouse Stephen F. , Berent Robert , vonDuvillard Serge P. , Green John S. , Schmid Peter , Auer Johann , Sinzinger Helmut
Published: June 2021

Cardiac patients (68 women, 298 men, mean aged 63 ± 11 yr) were studied for changes in exercise performance and blood N‐terminal B‐type natriuretic peptide (NT‐proBNP) concentrations, a known marker of heart failure, before and after 25 ± 4 days of residential cardiac rehabilitation. Exercise training consisted of 6 x wk cycle ergometry for 14 – 22 min (mean 17 ± 4 min), and daily walking for 45 min at 60–70% maximal heart rate (HRmax). Nutrition, diet, and pharmacologic prescriptions where initiated when indicated. NT‐proBNP decreased 33% (1061 ± 1406 to 707 ± 826 pg.mL−1) after training (p < 0.0001). NT‐proBNP concentrations were inversely (p < 0.0001) related to pulse pressure at maximal exercise (−0.391), HRmax (−0.267), systolic blood pressure at maximal exercise (−0.402), and VO2max (−0.404). Post‐training maximal ergometry performance (+18%), pulse pressure (+5%) and VO2max (+12%) increased; BMI (−2%), resting heart rate (−8%), and resting diastolic blood pressure (−3%) decreased significantly (p < 0.03 to < 0.0001). These encouraging findings in only 25 days suggest a relatively powerful effect of short‐term exercise training as part of a residential cardiac rehabilitation program to improve myocardial function, and to at least partially ameliorate the symptoms of ventricular systolic and diastolic insufficiency.
Effects of maternal exercise on the fetal heart
Authors: May Linda E , Gustafson Kathleen F , Popescu Anda , Popescu Mihai , Drake William B
Published: June 2021

The hypothesis that exercise exposed fetuses have an improved autonomic tone was tested by measuring fetal heart rate and heart rate variability (HRV) recorded from a biomagnetometer at various gestational ages. Mothers were classified as exercisers if they performed moderate intensity aerobic exercise at least 30 minutes 3 times per week. Mothers in the control category did not partake of a regular exercise regime. After separating maternal and fetal heart rates, fetal R peaks were marked by computer and corrected manually. We found that fetuses exposed to maternal exercise had lower heart rates than non‐exercise exposed healthy fetuses. At each age, the differences between the FHR means of the two groups is statistically significant with p<0.05 using a t‐test with equal variances. The data from 6 uncomplicated pregnancies (3 exercisers and 3 non‐exercisers) have been controlled for fetal gender, fetal activity state, and parity of the mother. As we anticipated, we have found significantly decreased fetal heart rates in exercise exposed fetuses in the same activity state and at the same gestational age compared to non‐exercise exposed fetuses. However, the analysis of short‐ and long‐term variability at 28, 32, and 36 wks gestational age in exercise exposed fetuses relative to non‐exercise exposed fetuses demonstrate no statistically significant differences, which can be presumably accounted for by the small sample size. These results imply an exciting potential benefit of maternal exercise on fetal cardiac autonomic nervous system. These data suggest a prenatal programming effect on the fetal cardiac and autonomic nervous system from maternal physiological events during gestation.
Bicarbonate Regulation of Intracellular cAMP in Pulmonary Endothelial Cells
Authors: Sayner Sarah Louise , Stevens Troy
Published: June 2021

Transmembrane adenylyl cyclases can be stimulated by isoproterenol or forskolin to generate a subplasma membrane cAMP pool in pulmonary endothelial cells. In contrast, toxic bacterial adenylyl cyclases are injected into endothelial cells and require a eukaryotic factor to generate a cytosolic cAMP pool. Recently, a mammalian adenylyl cyclase has been described which lacks transmembrane domains, is insensitive to heterotrimeric G‐protein modulation, and is uniquely activated by bicarbonate and calcium to generate cAMP. We sought to determine whether this bicarbonate‐sensitive adenylyl cyclase is expressed in pulmonary endothelial cells, and whether the enzyme contributes to baseline cAMP concentrations. Using RT‐PCR we demonstrated the endogenous expression of this bicarbonate‐sensitive soluble adenylyl cyclase in pulmonary microvascular and pulmonary artery endothelial cells. Utilizing a bicarbonate free system we determined that activity of this soluble adenylyl cyclase contributes to basal cAMP levels. Further, acetazolamide inhibition of bicarbonate synthesis leads to a dose dependent decrease in basal cAMP levels in pulmonary microvascular endothelial cells. Therefore, bicarbonate stimulation of the soluble adenylyl cyclase contributes to the basal cAMP levels in pulmonary endothelial cells. Supported by HL66299
Calcium phosphate complexation in ISOC channel inactivation
Authors: Chen Hairu , Wu Songwei , Stevens Troy , Cioffi Donna L.
Published: June 2021

The endothelial ISOC channel is a calcium selective store‐operated calcium entry channel comprised of the transient receptor potential canonical (TRPC) proteins 1 and 4. Activation/inactivation properties are important determinants of channel function. ISOC inactivation is broadly described as fast or slow, and we have observed that the endothelial ISOC exhibits a slow inactivation. While it is known that ISOC inactivation is phosphorylation dependent and calcium dependent, it is not known whether the phosphorylation and calcium mediated pathways are part of a common mechanism. Indeed TRPC4 is phosphorylated upon thapsigargin‐induced activation of SOC entry. We hypothesized that free calcium permeating the ISOC channel pore complexes with the phosphate groups located in a proline rich region of TRPC4 to promote channel inactivation. As proof of concept, peptides corresponding to the proline rich region of TRPC4, with and without phosphorylated serine, were generated and isothermal calorimetry studies initiated to determine whether formation of a calcium‐phosphate complex is thermodynamically favorable. A rapid calorimetric transition was detected with phosphorylated peptide upon calcium addition. This observation is consistent with the idea that tight binding occurs between calcium and the phosphate group which is important in ISOC channel inactivation. Supported by HL60024 and 1F32HL086124.
Transendothelial migration of CD133+ hematopoietic progenitor cells isolated from human umbilical cord blood
Authors: Rastogi Prerna , Young Dawn M , White Maureen C , McHowat Jane
Published: June 2021

Transmigration of hematopoietic progenitor cells across the vascular endothelium and subsequent maturation in the tissue helps to maintain the pool of resident mast cells. We determined the potential of CD133+ cells isolated from umbilical cord blood to migrate across an endothelial cell monolayer. CD133+ cells possess mRNA for several adhesion molecules that are critical in the process of neutrophil transmigration. Stem cell factor & its receptor CD117 are required for homing & differentiation of mast cells in the tissue. We detected the presence of CD34, CD117, CD29, & CD18 on the surface of CD133+ cells using flow cytometry. Tryptase stimulation leads to increased endothelial cell platelet activating factor (PAF) production that may facilitate the interaction between the endothelium & the PAF receptor (PTAFR) on circulating CD133+ cells. Treatment with the PTAFR antagonist CV3988 significantly reduced the percentage of transmigrating CD133+ cells across a tryptase stimulated endothelial monolayer. This suggests that CD133+ cell migration depends on the PAF/PTAFR interaction in a manner similar to that involved in neutrophil transmigration. Following transmigration there is the potential for a subset of these cells, in the presence of SCF & IL‐6, to mature into tissue mast cells.

This work was supported by AHA Heartland Affiliate Grant # 0610118Z.
Coupling LCM to gene expression and microproteomics: Novel approaches to molecularly define pediatric coronary artery intimal occlusive lesions
Authors: Juraszek Amy L , Calicchio Monica L , Steen Hanno , Waldon Zachary , Smoot Leslie , Mayer John E , Jr  , Perez‐Atayde Antonio R , Collins Tucker
Published: June 2021

Background‐Cardiac allografts fail due to post‐transplant vasculopathy, a progressive loss of the lumen in coronary arteries. The arterial injury is characterized pathologically by a concentric intimal expansion as a result of the proliferation of smooth muscle cells and the accumulation of extracellular matrix (ECM). The precise pathogenesis of graft arteriosclerosis is unknown.

Methods and Results‐Samples from coronary artery occlusive lesions were captured using LCM. The tissue was then incubated in Liquid Tissue Reagent (Expression Pathology, Gaithersburg, MD), enabling subsequent proteolysis and LC‐MS/MS analysis using a nanoflow HPLC system and a linear ion trap/orbitrap mass spectrometer. Proteins for multiple ECM components were detected in the neointima, including a variety of collagens, fibrillin, laminin, fibronectin and specific proteoglycan core proteins. The findings were validated by immunohistochemical analysis.

Conclusions‐Microproteomic analysis demonstrates multiple ECM components including collagens and specific proteoglycan core proteins in the intimal lesions of post‐transplant vasculopathy. This accumulation of ECM components was present in all lesions, suggesting it plays an important role in their pathogenesis. Further analysis of the ECM in these lesions may provide new insights into the pathobiology of post‐transplant arteriopathy.

NIH/NHLBI 1 P50 HL74734
In vivo caveolin‐3 adenoviral gene delivery restores caveolin‐3 protein expression and caveolae in cardiac myocytes of caveolin‐3 knockout mice
Authors: Kidd Michael William , Tsutsumi Yasuo M , Niesman Ingrid R , Lai N. Chin , Roth David M , Patel Hemal H
Published: June 2021

Caveolae are cholesterol, sphingolipid and caveolin enriched membrane microenvironments that have enriched expression of various signaling molecules. Caveolin‐3 (Cav‐3) knockout mice show no expression of Cav‐3 or formation of caveolae on the surface of cardiac myocytes. As such, these mice have aberrant cardiac function. We sought to determine if Cav‐3 adenoviral gene delivery to Cav‐3 knockout (KO) mice could restore expression of Cav‐3 protein and, more importantly, form caveolae on cardiac myocytes. Adenovirus encoding EGFP (Adv.EGFP) or Cav‐3 (Adv.Cav‐3) was delivered using indirect intracoronary injection in Cav‐3 KO mice. Three days after gene transfer, whole hearts were excised and the presence Cav‐3 protein was assessed. Densitometry of the immunoblot for Cav‐3 showed that Cav‐3 gene transfer resulted in restoration of Cav‐3 protein expression to 30% of wild‐type at three days. No Cav‐3 was detected in the mice injected with EGFP. Seven days after gene transfer, hearts were fixed and electron microscopy was used to assess caveolae formation on cardiac myocytes. Adv.Cav‐3 treated mice had restored caveolae formation, while no caveolae were observed in untreated knockout mice. The results indicate that intracoronary delivery results in efficient transgene expression. Furthermore, Cav‐3 is necessary and sufficient to drive the formation of caveolae in cardiac myocytes. Adv.Cav‐3 may offer a means to modulate caveolae number and cardiac signaling.
Site specific silencing of caveolin acylation causes accumulation of Alexa labeled Cholera Toxin B chain at distinct sites in the lipid raft‐caveolae endosomal system in polarized HepG2 cells
Authors: Everson William V. , Carter Josh , Kentucky Eric J Smart.
Published: June 2021

We have previously shown that silencing of the three distinct acylation sites in caveolin‐1 by substitution of alanine for cysteine at positions 133 (cav 133), 143 (cav 143) and 156 (cav 156) alter the cholesterol transport activity of 2 distinct cytosolic protein complexes. In this study, we examined the effect of these acylation mutants on endocytosis of fluorescent tagged cholera toxin B chain (CT‐B), which binds to the ganglioside GM1 and marks lipid raft/caveolae endocytosis. We stably expressed wild type caveolin (wt cav) or each of the single site acylation mutants in HepG2 cells, a cell line which lacks detectable expression of caveolin. Cells were cultured with Alexa 488 labeled wheat germ agglutinin (WGA), a marker of the constitutive endocytic pathway, and Alexa 555 labeled CT‐B. Ligand uptake was followed by live confocal microscopy at 37 deg or cells were incubated for time points up to 4 hours at 4 deg or 37 deg, fixed and immunostained for caveolin, organelle markers, or annexins (proteins associated with lipid raft/caveolae trafficking). Expression of each of the acylation mutants showed accumulation of CT‐B in distinct intracellular sites compared to cells expressing wt cav. Expression of the mutants also altered the distribution and accumulation of WGA indicating that alteration in caveolae/lipid raft trafficking impacts the constitutive endocytic system. Supported by NIH.
Functional and Molecular Properties of Store‐Operated Ca2+ Entry (SOCE) in Pulmonary Arterial SMCs
Authors: Forrest Abigail Sarah , Angermann Jeff E , Greenwood Iain A , Leblanc Normand
Published: June 2021

SOCE is a prominent Ca2+ entry pathway in rabbit pulmonary arterial smooth muscle (RbPASMC) and it is suggested that Orai and STIM proteins may be the link between intracellular Ca2+ ([Ca2+]i) store emptying and SOCE. Objectives of this study were: 1) to assess whether SOCE is mediated by direct Ca2+ permeation through a channel, or from an indirect Ca2+ influx pathway involving reverse‐mode Na+/Ca2+ exchange (NCX); 2) to explore the molecular basis of SOCE in this tissue. Simultaneous whole‐cell patch clamp and quantitative Fluo‐4 fluorescence techniques were used to voltage clamp RbPA myocytes dialyzed with Na+‐free pipette solution while recording global ([Ca2+]i). In cells incubated with CPA, increasing hyperpolarization from Vh (0mV) led to robust Ca2+ transients. Steps to depolarizing potentials following a hyperpolarization‐evoked Ca2+ transient led to a sharp exponential decline in [Ca2+]i. RT‐PCR analysis of Rb brain and PA cDNA identified gene expression for STIM 1 & 2, and Orai 1 & 2 but not Orai3. Our data indicate that SOCE parallels a Ca2+ gradient‐dependent mechanism which is inconsistent with reverse‐mode NCX. Genes encoding for the basic machinery of SOCE are expressed in PASMC.
TLR5KO mice have dysregulated intestinal gene expression that results in non‐specific resistance to Salmonella‐induced typhoid‐like disease
Authors: Vijay‐Kumar Matam , Aitken Jesse , Kumar Amrita , Neish Andrew , Gewirtz Andrew
Published: June 2021

We recently reported that a subset of TLR5KO mice develop spontaneous colitis. Non‐colitic TLR5KO mice exhibited alterations in basal microflora and colonic gene expression. Here, we examined how non‐colitic TLR5KO mice and WT littermates fared in 2 models of Salmonella challenge. In a model of Salmonella‐induced gastroenteritis, in which antibiotic pre‐treatment ablates commensal microflora, TLR5KO mice exhibited enhanced gut pathology suggesting that TLR5 protects the intestine against this pathogen. In contrast, TLR5KO mice are resistant to the usually‐fatal typhoid‐like illness that results when S. typhimurium disseminates systemically. However, we observed that such resistance was not specific for flagellated S. typhimurium but, rather, TLR5KO mice were also resistant to challenge by flagellin‐deficient S. typhimurium. Pre‐treatment of mice with streptomycin to reduce the level of commensal microbes eliminated such resistance of TLR5KO to S. typhimurium‐induced mortality relative to their WT littermates. TLR5KO mice had elevated basal intestinal expression of genes that mediate innate and adaptive immunity in the gut. Together, these results indicate that resistance of TLR5KO‐mice to Salmonella‐induced typhoid‐like illness results from alterations in their basal phenotype rather than lack of TLR5 ligation during the infection per se.
Biological effects of bacitracin as a therapeutic agent and growth promoting compound
Authors: Lin Jun , Van Hout Amber , Wolff Teddi , Erickson Alan , Francis David
Published: June 2021

Bacitracin is a peptide antibiotic and it has several effects on the microbial population of the intestines that could account for its growth‐promoting ability. Enterotoxigenic E. coli is a major pathogen of weaned pigs and is responsible for significant death loss and retardation in weight gain. We found in in vitro studies that bacitracin at sub‐inhibitory concentrations (390 to 780 μg/ml) preferentially selected for the outgrowth of tetracycline sensitive bacteria from among a population of tetracycline resistant and tetracycline sensitive E. coli and Salmonella. Two E. coli strains were used and showed that bacitracin did select Tetracycline sensitive bacteria (P < 0.05). Nine tetracycline resistant and nine tetracycline sensitive Salmonella strains were pooled in this study. Twenty‐five percent more tetracycline sensitive bacteria were found in LB broth containing bacitracin at the sub‐inhibitory concentration (P < 0.01). These findings suggested that a possible benefit from using bacitracin as a feed additive might be to reduce the Tetrpopulation of pathogens in the intestinal tract. In studies utilizing newly weaned piglets, we established that a feed level concentration of bacitracin in the starter diet did enable piglets to clear a pathogenic bacterium at a faster rate than was the case when piglets were placed on antibiotic‐free feed.

Supported by Alphamra Inc., the South Dakota Agricultural Experiment Station, and the South Dakota Center for Infectious Disease Research and Vaccinology
Bacteria biofilms regulate human cathelicidin transcription in the gut through VDR signaling
Authors: Resta‐Lenert Silvia Cristina , Lee Susan J , Azarenok Serge , Maruggi Marco
Published: June 2021

Antimicrobial cathelicidins, are produced by mucosal cells in innate and adaptive immunity. Crohn's disease (CD) is characterized by low levels of cathelicidins and vit.D receptor (VDR). VDR activation stimulates LL‐37 synthesis. Moreover, certain enteric bacteria are prevalent in the gut of patients with CD. We showed that commensals and probiotics regulate VDR and calcium metabolism in chronic inflammation. We hypothesized that low cathelicidins levels in CD results from inhibition of VDR signaling by enteroadhesive bacteria and that this can be prevented by probiotics. Enteroadhesive E.coli (EADEC from a CD case), and probiotics (S.thermophilus, ST, and L.acidophilus, LA) were grown as biofilms. Caco‐2 and HT29/cl 19A cells were grown as monolayers and treated for 24‐48 hr with/without EADEC and ST/LA. IL‐10 −/− and WT mice were treated with ST/LA for 4 wk in vivo. Cells, and small intestinal (SMI) and colonic tissues (CT) were examined for VDR and cathelicidin (LL‐37 and mCRAMP) by mRNA and protein analysis. A significant reduction of VDR and cathelicidins (3‐ and 5‐fold vs. controls for cells and tissues, respectively) was found in EADEC‐treated cells and in colitic tissues. This was reversed by probiotics. Moreover, a VDR siRNA blocked the effect of probiotics in EADEC‐primed cells. Similarly, lower mCRAMP was observed in ex vivo cultures of SMI and CT treated with an inactive vit. D analog. Our data underscore the role of the gut microbiota in regulating physiologic functions of the host and suggest that complex pathways are activated by probiotic and commensal bacteria. Supported by grants NIH‐DK062096 and UCSD Academic Senate (SRL)
Human Intestinal Ste20‐related Proline/alanine Kinase (SPAK): A Novel Regulator of Intestinal Inflammation
Authors: Yan Yutao , Dalmasso Guillaume , Charrier‐Hisamuddin Laetitia , Nguyen Hang , Sitaraman Shanthi V , Merlin Didier
Published: June 2021

We have recently cloned and characterized a new intestinal inflammation associated ste20‐related protein kinase isoform (SPAK). Here we investigate its express and regulation during inflammatory bowel disease. Real time PCR‐based analysis suggested high expression of SPAK in colonic tissue from IBD patients and DSS mouse colitis compared to normal control colonic tissue. We then isolated the SPAK core promoter. Two transcriptional initiation sites were mapped by primer extension and 5′‐RACE. Direct sequencing of the promoter revealed it a promoter with one NF‐?B and three Sp1 binding sites, but no TATA box. Using EMSA, CHIP, and Luciferase assay, we demonstrated that NF‐?B binding site was essential for stimulated SPAK promoter activity by TNF‐a, whereas Sp1 binding sites were important for the basal promoter activity. Western blots of Caco2‐BBE cells treated with siRNA of Sp1 and NF‐?B demonstrated that knock‐down of NF‐?B but not of Sp1 reduces the SPAK level. By mRNA decay assay, we found that TNF‐a increases SPAK transcripts without affecting its mRNA stability. Microarray analysis demonstrated SPAK can target key inflammatory signaling pathways such as p38, JNK. These findings collectively indicate that the SPAK is expressed during active inflammatory bowel disease in human as well as animal models. TNF‐a is a key regulator of SPAK expression.
Short fasting prevents glucose‐dependent insulinotropic polypeptide (GIP) induced stimulation of intestinal electrolyte secretion
Authors: Flemström Gunnar , Jedstedt Gunilla , Bengtsson Magnus
Published: June 2021

Glucose‐dependent insulinotropic polypeptide (GIP) and glucagon like peptide‐1 (GLP‐1) are important incretins, facilitating release of insulin from pancreatic islets. In addition, these peptides affect satiety and metabolism. Our aim was to study effects of GIP and GLP‐1 on duodenal mucosal bicarbonate secretion in fed and food‐deprived animals. Glucagon like peptide‐2 (GLP‐2) is not an incretin but was tested for comparison. Methods: Lewis x Dark Agouti rats had free access to water and, unless fasted overnight, free access to food. Animals were anesthetized (thiobarbiturate, 120 mg/kg) and a segment of proximal duodenum with intact blood supply cannulated in situ. Mucosal bicarbonate secretion (pH stat) was continuously recorded and peptides were administered to the duodenum by close intra‐arterial infusion. Results: Intra‐arterial infusion of GIP (60‐600 pmol/kg, h) caused dose‐dependent increases in duodenal bicarbonate secretion in fed animals (p<0.01) but not in fasted animals (p>0.05). At a higher dose (6000 pmol/kg, h), GLP‐1 induced a slight increase (p<0.05) in secretion. GLP‐2 did not affect the duodenal secretion. Conclusions: GIP and to a smaller extent GLP‐1, stimulate duodenal bicarbonate secretion in fed but not in overnight fasted rats. Studies of intestinal secretion may require particular evaluation with respect to feeding status.

Supported by the Swedish Research Council
The periodontoduodenal motility reflex in fed rats
Authors: Lorber Mortimer
Published: June 2021

Ten female rats were allowed to feed until pausing. They fed for 7±4 min, taking in 1.52±0.81 g of chow. Under pentobarbital three sequential five min periods of visual observations of the proximal duodenum were made. No instrumentation was used. The baseline began at quiescence 168±129 min after feeding. Simulated mastication involved lowering and raising the jaw until opposing molars contacted transiently at a rate of 3/s for 12s followed by a 3s rest. This was repeated for five min followed by postprocedure observations. Segmentations (mean±SD) at baseline were 19.2±17.5. Procedure values were 38.4±48.5. Postprocedure ones were 85.7±82.7. ANOVA 0.038 and Bonferroni for the first and last periods was P < 0.05. These results reflect that tooth contacts cause the periodontal ligament (PDL) about each dental root to distort as the roots intrude deeper into their sockets. This stimulates PDL mechanoreceptors, Vth nerve afferents, whose axons enter the Gasserian ganglion. The latter's somata course to the NTS where contact is made with dendrites from the dorsal motor nucleus of X. These synapses plus vagal innervation underlie the periodontogastric motility (Lorber Can J Physiol Pharmacol 78:29, 2000) and emptying (Lorber J Dent Res 81:302, 2002) reflexes, and the present one. Personal support.
Mechanical Force Induces G2/M Cell Cycle Arrest in Human Osteosarcoma Cells and Inhibits Their Differentiation
Authors: Chang Shun‐Fu , Chang Cheng Allen , Chiu Jeng‐Jiann , Chien Shu
Published: June 2021

Fluid shear stress plays important roles in proliferation and differentiation of bone cells. We investigated the effects of shear stress on MG63 human osteosarcoma cells. Application of shear stress (12 dynes/cm2) to MG63 cells induced G2/M arrest and inhibited differentiation. Pre‐treatment of MG63 cells with antibodies against alphavbeta3 and beta1 integrins inhibited these shear‐induced responses. Shear stress caused sustained increases in cyclin B and p21CIP1 and decrease in CDK1 expression in MG63 cells. Moreover, shear stress induced sustained activations of Smad1/5 and decrease in runt‐related transcription factor 2 (Runx2) activity in MG63 cells. Transfection of MG63 cells with Smad1/5‐specifc siRNA inhibited these shear‐mediated expression and activation of cell cycle regulators and Runx2. Our results demonstrated that shear stress induces G2/M arrest in MG63 cells and inhibits their differentiation, and that these responses are regulated by alphavbeta3 and beta1 integrins through Smad1/5 and their modulation in Runx2, as well as specific cell cycle regulators. Our findings provide insights into the mechanisms by which shear stress regulates cell cycle progression and differentiation in human osteosarcoma cells.
Anabolic effects of testosterone in bone of gonadectomized male and female rats
Authors: Yarrow Joshua F , Conover Christine F , Conrad Bryan , Zheng Niquan , Wronski Thomas J , Altman Molly K , Franz Sarah E , Borst Stephen E
Published: June 2021

In males (M), testosterone (T) administration augments bone mineral density and maximal bone strength (MBS); however, the direct effects of T‐administration on female (F) bone are less understood. We evaluated the effects of supraphysiological T administration on M and F serum hormone concentrations [T, dihydrotestosterone (DHT), and estradiol (E2)], tibial cancellous bone volume % (CBV%), and femur MBS (3‐point bending test). Fisher SAS rats received Sham surgery + placebo (P), gonadectomy (GNX) + P, or GNX + T (7.0 mg x week‐1) for 28 days. GNX reduced serum T by 99%, DHT by 42–87%, and E2 by 72–82% in both sexes (p<0.05). When compared with Shams, T‐administration resulted in >18‐fold (M) and >100‐fold (F) increases in serum T, 17‐fold (M) and 50‐fold (F) increases in DHT, and a 4‐fold increase in E2 (M) concentrations (p<0.05); while reducing serum E2 by 55% in (F) (p<0.05). T‐administration also reversed the GNX induced reductions in CBV% in M (Sham 4.8±1.0, GNX 1.6±0.5, GNX+T 6.0±0.5; p<0.05) and resulted in a trend towards increased CBV% in F (Sham 9.8±5.7, GNX 3.5±2.6, GNX+T 7.4±3.5; p=0.052), when compared with GNX. Ultimately, GNX+T augmented MBS by ~6–12% in M and ~15–19% in F when compared with Sham and GNX, respectively (p<0.05). Our results indicate that supraphysiological T‐administration directly augments CBV% and MBS after GNX in both M and F rats. Study funded by VA Merit grant (S.E. Borst).
Nuclear Receptor Co‐Regulator Krüppel‐like Factor 9 in Human Endometrial Stromal Cell Differentiation
Authors: Zeng Zhaoyang , Simmen Frank A , Simmen Rosalia CM
Published: June 2021

The biological actions of ligand‐bound estrogen (E) and progesterone (P) receptors are dependent on coregulator partner proteins. We have identified Krüppel‐like Factor 9 (KLF9) as important for E and P actions in endometrial cells. Ablation of KLF9 in mice resulted in subfertility due partly to altered uterine sensitivity to E and P. KLF9 expression at peri‐implantation in mice was highest in endometrial stroma but was undetectable in decidua. To understand the significance of KLF loss with decidual formation, we used an immortalized human endometrial stromal cell line (HESC) responsive to E and P to evaluate the temporal expression of KLF9 during P‐induced decidualization. HESC treated with medium alone or with estradiol + medroxyprogesterone were analyzed for expression of KLF9 and differentiation marker genes by quantitative real‐time PCR. KLF9 transcripts began to rise above control levels at 6d, peaked at 8/9d, and decreased to 6d levels at 12d and 15d of treatment. Expression of the P‐responsive and KLF9‐regulated Hoxa10 gene coincided with that of KLF9 at 6d and 8/9d. By contrast, IGFBP1 transcript levels remained low until a ∼12‐fold increase at 15d, while that of prolactin increased at 8/9d and remained high thereafter. Results suggest that loss of KLF9 expression precedes stromal cell differentiation, suggestive of a preferential role for KLF9 in pre‐decidual stromal proliferation. (NIH‐HD21961)