bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple micronutrient deficiencies persist during early childhood in Mongolia
Authors: Lander Rebecca , Enkhjargal Tserennadmid , Bailey Karl , Gibson Rosalind
Published: June 2021

The effectiveness of interventions in Mongolia to combat micronutrient deficiencies in children is uncertain. We determined the prevalence of anemia, iron deficiency anemia (IDA), and deficiencies of Fe, Zn, Se, and vitamin A among young Mongolian children. Anthropometry and non‐fasting morning blood samples were collected from 243 children (126 M, 117 F) aged 6–36 months from Ulaanbaatar and 4 rural capitals for hemoglobin (Hb), serum (s) ferritin, Zn, Se, and retinol assays. Children with α‐1‐glycoprotein>1.2 mg/L (n = 27) indicative of infection were excluded. Of the children 14.5% were stunted and none were wasted. Zn deficiency (sZn<9.9μmol/L) had the highest prevalence (74%). The prevalence of anemia (24%) and iron deficiency anemia (IDA) (16%) were lower, with the oldest children (24–36 mos) at lowest risk. 21% of the children had Fe deficiency, and 33% had vitamin A (sretinol <0.70 μmol/L) deficiency. Serum Se values were low, as noted for South Island NZ children, where soil Se is low. Regional differences (p<0.05) existed for anemia, deficiencies of Se and vitamin A, but not Zn or IDA. Age, location, log serum ferritin, infection, and serum zinc were major predictors of hemoglobin. Of the children, 64% were at risk of two or more coexisting micronutrient deficiencies emphasizing the need for multimicronutrient interventions in Mongolia. Funded by WHO, UNICEF, World Vision Mongolia, University of Otago.
Sulfur Amino Acids Are Necessary for Normal Intestinal Mucosal Growth in Neonatal Piglets
Authors: Bauchart‐Thevret Caroline , Stoll Barbara , Chang Xiaoyan , Cui Liwei , Burrin Douglas
Published: June 2021

Sulfur amino acids (SAAs), methionine and cysteine, play important metabolic and functional role in human health and disease. Gastrointestinal tract is an important site of transmethylation and transsulfuration of methionine and metabolizes ∼20% of the dietary methionine intake (Riedijk et al. PNAS, 2007, 104: 3408). Our aim was to determine intestinal mucosal growth in neonatal piglets (3‐d‐old) enterally‐fed a control diet (n=9) or SAA‐free, elemental, isonitrogenous diet (n=7) for 7 days. After 7 d, both small intestine (jejunum and ileum) mass (−33%; g/kg body weight) and weight gain (−70%; g/kg/d) were decreased in SAA‐free vs. control pigs. Also, protein and DNA mass in jejunum and ileum was also lower in SAA‐free pigs vs. control pigs (−50 to −60%). Histological analysis of jejunum, ileum and colon showed lower (−20 to −40%) cross‐section area, number of villi, villus height and crypt depth in SAA‐free pigs vs. control pigs. Moreover, absolute number of goblet cells and Ki‐67 positive proliferative cells in jejunum and colon were lower in SAA‐free vs. control pigs (−40 to −60%). These results show that SAA deficiency significantly alters intestinal epithelial cell proliferation and survival. We conclude that SAAs are necessary for normal intestinal mucosal growth in piglets.

(Supported by ADREN/SFNEP and Nutricia Research Foundation).
Detection and quantification of endothelial VCAM‐1 expression using 19F magnetic resonance spectroscopy and imaging
Authors: Kaneda Megan M. , Southworth Richard , Zhang Lei , Zhang Huiying , Chen Junjie , Lanza Gregory M. , Wickline Samuel A.
Published: June 2021

The early upregulation of vascular adhesion molecule‐1 (VCAM‐1) by the endothelium in disease states such as inflammation makes VCAM‐1 an attractive target for early detection of disease. The objective of this study was to develop a molecularly targeted perfluorocarbon nanoparticle (PFC‐NP) and quantify its binding to VCAM‐1 using fluorine spectroscopy. We have previously shown that VCAM‐1 expression is increased by the renal endothelium of apolipoprotein E deficient (ApoE −/−) mice fed a high cholesterol diet over control C57BL/6 mice. Liquid perfluorocarbon nanoparticle emulsions (PFC‐NP) targeted to VCAM‐1 were injected via the tail vein into ApoE−/− mice and allowed to circulate for 2h. Ex‐vivo kidney 19F imaging and spectroscopy was performed on an 11.7T Varian scanner. The fluorine signal was significantly higher in ApoE−/− kidneys treated with VCAM‐1 targeted PFC‐NP versus ApoE−/− kidneys injected with non‐targeted PFC‐NP (p < 0.01). Additionally, the kidneys of C57BL/6 control mice treated with VCAM‐1 targeted PFC‐NP had a fluorine signal significantly lower than VCAM‐1 targeted ApoE−/− signal (p < 0.05). These results suggest that changes in VCAM‐1 expression can be detected and quantified using 19F spectroscopy and that this technique may provide a non‐invasive method for early detection of inflammation. This work was supported by NIH grant #HL073646
Oxidative Stress Increases RGS2 Expression which Suppresses Increased cAMP Accumulation in Response to CRF Activation in CATH.a Cells
Authors: Salim Samina , Eikenburg Douglas , Dessauer Carmen W , Hite Brian
Published: June 2021

Regulator of G‐protein Signaling Protein (RGS)‐2 has been suggested as a modulator of anxiety and dysregulation of oxidative stress is implicated in the development of anxiety. In the present study, we report that hydrogen peroxide‐induced oxidative stress increases RGS2 expression and increases nuclear translocation of RGS2 in CATH.a cells. CATH.a cells express CRF1 receptors and RGS2 and are a model of locus coeruleus neurons. It is well known that stress causes excessive CRF1 receptor activation, increased cAMP production and increased locus coeruleus firing. We have observed that the cAMP response to CRF1 receptor activation is reduced by chronic hydrogen peroxide treatment in CATH.a cells. This reduced cAMP response may be the result of direct inhibition of adenylyl cyclase by RGS2 in response to oxidative stress. In addition, expression of antioxidant proteins including superoxide dismutase and heme oxygenase are also increased in response to increased oxidative stress. In conclusion, we hypothesize that the hydrogen peroxide treatment increases RGS2 expression as part of a compensatory mechanism to protect against oxidative stress. This protection is via two mechanisms; 1) increasing the expression of antioxidant proteins, and 2) direct inhibition of adenylyl cyclase to reduce cAMP accumulation caused by excessive CRF1 receptor activation. (Supported by NARSAD and NIH grant #GM060419).
Role of eicosanoids, NO and PDE5 in the changes in vascular reactivity to acetylcholine (Ach) and phenylephrine (PE) in rats with diabetes mellitus (DM)
Authors: Sabra Ramzi , Bassila Jean‐Claude , Ghali‐Ghoul Rana , Tahseldar‐Roumieh Rima
Published: June 2021

We previously showed that aortic rings from rats with 2‐weeks DM had an enhanced relaxant response to Ach or DMPPO (PDE5 inhibitor) and a lower contractile response to PE compared with controls. We used indomethacin (INDO) and L‐NAME and measured aortic PDE5 activity to explore the role of eicosanoids, NO and PDE 5 in these changes in streptozotocin‐induced DM. INDO enhanced relaxation by Ach in the control (C) group but had no influence in the DM group, and the response to Ach became similar in the 2 groups. L‐NAME abolished Ach‐induced relaxation in both groups, but C rings had a significant contractile response, not seen in DM rings. When both L‐NAME and INDO were used, the responses to Ach became identical. PE induced contraction was depressed in DM rings. L‐NAME enhanced PE‐induced contraction in both groups but did not eliminate the difference. INDO reduced the response to PE in C but not DM rings, and the difference between them was abolished. The relaxant response to DMPPO was greater in DM rings. INDO reduced the response to DMPPO in DM and slightly enhanced it in C, thus abolishing the difference between them. L‐NAME also abolished the difference but not the vasodilation in response to DMPPO. There was no change in total cGMP hydrolysis or PDE5 activity but an increased total cAMP hydrolysis and PDE 4 activity in DM aorta. The results suggest that there is a reduced production of a constrictor prostanoid in DM that causes greater relaxation to Ach and lower contraction to PE. DMPPO acts by mechanisms other than PDE5 inhibition and accumulation of cGMP; a possible effect on PDE4 may explain its interaction with INDO.
Protective Effects of NOS and/or sGC Inhibitors Combined with Antioxidant in LPS‐Induced Organ Dysfunction
Authors: Doursout Marie‐Francoise , Liang Yang Yan , Sharina Iraida , Sharin Vladislav G. , Murad Ferid
Published: June 2021

Overproduction of nitric oxide (NO) has been described in the pathophysiology of septic shock. Among LPS‐induced proinflammatory metabolites, reactive oxygen species (ROS) are considered to play a crucial pathogenetic role in the tissue injury that can lead to organ failure. Our study was designed to counteract the effects of LPS‐induced hypotension using N‐methyl‐L‐arginine (L‐NMA), an inhibitor of NO production and/or ODQ, a specific inhibitor of sGC activity in the presence and absence of N‐acetylcysteine (NAC) in LPS‐treated rats. Group 1 (n=6) received LPS alone (20 mg/kg IV), whereas Group 2 (n=12) received LPS+L‐NMA (20 mg/kg IV), Group 3 (n=12) received LPS+ODQ, and Group 4 (n=12) received LPS + L‐NMA/ODQ in the presence and absence of NAC. Cardiovascular parameters, NO production, cellular antioxidant capacity and nitrotyrosine formation, as a molecular marker of ROS/RNS were recorded for 24 hrs. Our preliminary data show that LPS induces a cardiac depression associated with iNOS expression leading to increases in nitrate production and nitrotyrosine formation. Combined treatment of L‐NMA/ODQ abolished LPS‐induced hypotension, whereas NAC significantly decreased nitrotyrosine formation. These results indicate that LPS‐detrimental cardiovascular effects can be attenuated by early treatment, e.g. combined NOS and sGC inhibition in the presence of antioxidant.
In vivo administration of KCNQ channel modulators influences blood pressure and mesenteric vascular resistance in rats
Authors: Mackie Alexander R , Formeister Eric J , Tiniakov Ruslan , Scrogin Karie E , Byron Kenneth L
Published: June 2021

Recent work from our laboratory has shown that KCNQ channels are expressed in vascular smooth muscle cells where they contribute to vasopressin‐induced Ca2+ signaling. Suppression of KCNQ currents by vasopressin or pharmacological agents leads to membrane depolarization and activation of L‐type Ca2+ channels. To determine whether inhibition or activation of vascular KCNQ channels influences mesenteric vascular resistance (MVR) and mean arterial pressure (MAP) in vivo, adult Sprague‐Dawley rats were anesthetized with thiobutabarbital (125 mg/kg) and instrumented with femoral venous and arterial catheters for drug administration and arterial pressure determination. A perivascular blood flow probe was placed around the superior mesenteric artery. The KCNQ channel activator (flupirtine) or the inhibitor (linopirdine) was then administered in increasing concentrations (0.01 – 3.0 mg/kg i.v.). Flupirtine produced significant dose‐dependent decreases in MAP and MVR. Flupirtine (3.0 mg/kg) caused a 28.9 ± 1.2 mmHg decrease in MAP and a 33.5% decrease in MVR. In contrast, linopirdine produced dose‐dependent increases in MAP and MVR. A maximally effective concentration of linopirdine (1.0 mg/kg) produced a 9.9 ± 1.3 mmHg increase in MAP and a 19.2% increase in MVR. Flupirtine (3.0 mg/kg) also caused a modest, but significant decrease in HR of 26.3 ± 3.7 beat/min. Our results indicate that KCNQ channels contribute to tonic modulation of mesenteric vascular resistance and blood pressure. This work was supported by the NHLBI (R01 HL070670 to KLB) and the AHA (0715618Z to ARM).
Culturing conditions conducive to the differentiation of hESCs along a hepatic lineage
Authors: Zamule Stephanie Mary , Weyant Denise M , Omiecinski Curtis J
Published: June 2021

The limited availability of hepatic tissue suitable for the treatment of liver disease and for research makes the generation of hepatic‐like cells from alternative sources an attractive proposition. Although a number of studies have demonstrated that human embryonic stem cells (hESCs) are capable of differentiating into hepatic precursors, the methodology has yet to be fully elucidated. To this end, we cultured hESCs for 10 days on collagen substrata in our primary hepatocyte maintenance media comprised of William's E media supplemented with 25 nM dexamethasone, 10 nM insulin, 5 ng/ml selenium, 5 μg/ml transferrin, and 1% linoleic acid. Using quantitative RT‐PCR assays, we found that hESCs cultured under these conditions exhibited a 2‐fold decrease in mRNA levels of the stem cell pluripotency marker, Oct4, a ~30,000‐fold increase in levels of the endodermal marker, α‐fetoprotein, and a ~475‐fold increase in the hepatic marker, albumin. Further, levels of the constitutive androstane receptor, CAR, were increased ~530‐fold, to levels comparable to those in primary cultures of human hepatocytes. These results define a novel, straightforward methodology that may be useful in facilitating hESC differentiation along the hepatic lineage within a relatively short period of time and provide a framework for subsequent studies seeking to develop a renewable supply of human hepatocytes for research applications.

Supported by NIH grant, GM66411.
Genome‐wide expression profiling of a rat acute volume overload model identifies a major inflammatory response associated with extracellular matrix homeostasis disorder
Authors: Chen Yuanwen , Zheng Junying , Pat Betty , Cain Laura , Powell Pamela , Husain Ahsan , Dell’Italia Louis J
Published: June 2021

In acute volume overload (VO) produced by aorto‐caval fistula (ACF) in the rat, a substantial loss of interstitial collagen was observed within 12 hours. Prevention of acute collagen loss attenuated progressive chamber dilation. To investigate the transcriptional changes which may mediate acute collagen loss and latter adverse chamber dilation, we performed a genome‐wide transcriptional profiling of left ventricles from rats subjected to 1 day ACF or sham surgery. In the ACF group, a 44% decrease of collagen was observed. 112 transcripts were up‐regulated and 37 were down‐regulated (fold>=1.5 with P<0.05). About 15% transcripts were selected and verified by real‐time quantitative RT‐PCR and/or immunoblot. Ingenuity Pathways Analysis revealed that the major functions of the altered genes are leukocyte migration and adhesion(MCP‐1, CD14, CCN5, etc) , immune response and inflammatory disease(IL‐1 receptor antagonist, activin A, colony stimulating factor 1, osteoprotegerin, etc ), interleukin and chemokine signaling (CD16, CD32, chemokine(c‐c) motif ligand 7, 12, 13, 11, etc), as well as connective tissue disorder(CD200 receptor 1, MMP‐19, fibronectin 1, etc). Greater than 50% of genes with 2‐fold or higher change are involved in inflammation. These results support an important role of inflammation in mediating the interstitial collagen loss and LV dilation in the acute VO of ACF. Support: HL54816
HNE contributes to macrophage foam cell formation through increased expression of SRA in ApoE‐deficient mice
Authors: Kim Chi Dae , Yun Mi Ran , Im Dong Soon , Lee Seong Jin , Seo Kyo Won , Woo Joong Won , Bae Sun Sik
Published: June 2021

To investigate the role of HNE in atherogenesis, we determined the role of HNE in oxLDL uptake by macrophages and the underlying mechanism implicated in HNE‐induced expression of scavenger receptors (SR). ApoE‐deficient mice fed a high‐fat diet exhibited multiple plaque lesions in the aorta including aortic sinus. These lesions were accompanied by increased accumulation of HNE adducts in the enhanced Mac‐2 stained area. In an in vitro study, HNE exposure to J774A.1 macrophages led to increased expression of class A SR (SR‐A) and CD36 at the protein level with a concomitant increase in endocytic uptake of oxLDL. In contrast to CD36 protein expression, which was associated with an increase in CD36 mRNA expression, the HNE‐enhanced SR‐A protein expression was neither accompanied by its mRNA expression nor affected by actinomycin D, a transcription inhibitor. However, the HNE‐induced SR‐A protein expression was effectively attenuated by translation inhibitors such as cycloheximide and rapamycin. Moreover, HNE enhanced the incorporation rates of 35S‐Met/Cys into SR‐A, representing increased de novo synthesis of SR‐A in mouse macrophages. These data indicate that accumulated HNE in the plaque lesions contributes to macrophage foam cell formation through increased SR‐A synthesis at the level of mRNA translation.
Mast cell mediators increase endothelial cell MMP‐2 production in a β‐catenin‐dependent manner
Authors: Doyle Jennifer Lynn , Haas Tara L
Published: June 2021

In skeletal muscle, mast cells reside in close proximity to capillaries and may be activated in response to muscle stretch. Angiogenesis may be initiated by factors released from mast cells including vascular endothelial growth factor (VEGF) and histamine. Both VEGF and histamine increase endothelial permeability, and as our results show, regulate matrix metalloproteinase (MMP)‐2 production and induce nuclear translocation of β‐catenin. Our objective is to determine how VEGF and histamine increase MMP‐2 production in endothelial cells isolated from the fat pad of rats. We have found that histamine acts through its H2 receptor as stimulation with the H2 receptor agonist dimaprit induced a significant increase in MMP‐2 mRNA levels and promoter activity (4.1±0.5 vs control, p< 0.05, n=3, 1.4±0.1 vs control, p<0.05, n=3). To determine if β‐catenin mediates transcription of MMP‐2, rat endothelial cells were transduced with Ad‐ICAT or Ad‐Bgal prior to growth factor stimulation. Transduction of ICAT blocked the VEGF induced increase in MMP‐2 mRNA levels (1.8±0.04 VEGF vs 1.0 for Bgal, p<0.05 n=3, 0.95±0.1 ICAT + VEGF vs Bgal, p<0.05 n=3). These results indicate a role for β‐catenin in the induction of endothelial cell MMP‐2 expression following VEGF and histamine stimulation. Funding by NSERC.
Shear stress‐dependent suppression of endothelial angiopoietin‐2 is mediated via transcription factor FOXO1
Authors: Pries Axel Radlach , Chlench Sven , DaSilva‐Azevedo Luis , Hoffmann Christian , Hohberg Margret , Baum Oliver , Bongrazio Mauro , Zakrzewicz Andreas
Published: June 2021

While vascular endothelial cells are normally exposed to shear stress, there is no flow and consequently reduced shear stress in capillary sprouts. To explore, if this lack in mechano‐stimulation alters the endothelial phenotype, we analysed the influence of shear stress on the activity of transcription factor FOXO1 and the expression of its target gene angiopoietin‐2 (ang2), an antagonist of the endothelial tyrosine kinase receptor tie2.

Endothelial cells were exposed to shear stress using a cone‐and‐plate system with or without inhibition of PI3K (using LY294,002) or FOXO1 (using siRNA) and analysed by real time RT‐PCR, Western blots, immunoprecipitation, and fluorescence microscopy.

Shear stress (6°dyn/cm2) increased phosphorylation of Akt and FOXO1 in a PI3K‐dependent manner and FOXO1 was translocated out of the nucleus. In addition, expression of FOXO1 on the mRNA‐ and protein level was strongly reduced by shear stress. Suppression of FOXO1 by shear stress was accompanied by a decrease of ang2‐mRNA and ‐protein, which was similarly be achieved by siRNA against FOXO1. Following flow stop, ang2‐mRNA expression increased within two hours to steady state values under static conditions.

So, in capillary sprouts, lacking shear stress activates endothelial FOXO1 by which ang2 is rapidly induced. Thus, the tie2‐system will be inactivated during capillary sprouting, which may turn the vessel wall more susceptible to additional angiogenic factors. By contrast, the onset of flow will activate the tie2‐system and thereby contribute to stabilize the vessel wall.
cAMP‐dependent Rac 1 inactivation is associated with LPS‐induced endothelial barrier breakdown
Authors: Schlegel Nicolas , Baumer Yvonne , Drenckhahn Detlev , Waschke Jens
Published: June 2021

Lipopolysaccharide (LPS)‐induced breakdown of endothelial barrier functions contributes largely to the pathogenesis of sepsis. However, the underlying mechanisms are still unclear. In the present study, incubation of human dermal microvascular endothelial cells with LPS led to a breakdown of endothelial barrier functions within 2–2.5h as revealed by measurements of FITC‐dextran flux and of transendothelial electrical resistance. This was associated with the formation of intercellular gaps, stress fibers and fragmentation of VE‐cadherin immunostaining. Moreover, claudin 5 immunostaining at cell borders was reduced after LPS‐treatment. Activity of small GTPase Rho A, which has been suggested to mediate the LPS‐induced breakdown of the endothelial barrier was not increased after 2h. In contrast, activity of Rac 1, which is known to be important for maintenance of endothelial barrier functions, was significantly reduced to 65 ± 9% after 2h. All LPS‐induced changes of endothelial cells were blocked by a Forskolin/Rolipram‐mediated increase of cAMP. Consistently, LPS led to a significant decrease of intracellular cAMP‐levels to 41 ± 2% after 1h and to 46 ± 8% after 2h. In summary, we suggest that LPS disrupts endothelial barrier functions by decreasing intracellular cAMP‐levels which in turn leads to an inactivation of Rac 1‐activity. Moreover, this mechanism appears to be involved in claudin 5 regulation.
Impact of accumulated oxygen depletion (AOD) on measurements of oxygen tension (PO2) using phosphorescence quenching microscopy (PQM)
Authors: Golub Aleksander Sergeevich , Pittman Roland Nathan
Published: June 2021

PQM is a powerful method to measure PO2 in microvessels. However, its application to stationary tissue and fluids has never been substantiated. Multiple flash excitations applied in PQM to the same sampled volume produce AOD and distort the measured PO2. The magnitude of the AOD is determined by the radius of the excitation area R and rate of excitation light pulses F. We have analyzed the performance of two types of instruments in order to explain reported paradoxical PO2 results and optimize PQM design. The combination of a large R and high F reduces PO2 in perivascular tissue to 64 % of the actual value, creating a large transmural PO2 drop previously interpreted as the result of a large O2 consumption by the vessel wall. The AOD also occurs in the smallest vessels due to their low blood velocity V. An instrument with a large R and F has a high critical velocity Vcr. When V in a vessel is low (V/Vcr < 0.1), PO2 is strongly underestimated. This artifact is responsible in part for the reported longitudinal PO2 gradients in arterioles, PO2 rise in venules and low PO2's in lymphatic microvessels. The PQM technique combining a small R with a low F prevents the AOD in flowing blood and lymph. Artifact‐free PO2 measurements in stationary tissues and fluids were achieved with the scanning excitation principle that excludes multiple excitation of the same sampled volume. Support: AHA‐0655449U and NIH‐HL18292.
Relationship between rate of change of DAF‐2T fluorescence and nitric oxide (NO) concentration in vitro
Authors: Carvalho Helena , Pittman Roland N
Published: June 2021

DAF‐2 has been used for measurements of NO in vitro and in vivo, by virtue of its reaction with an auto‐oxidation product of NO to form the strongly fluorescent product DAF‐2T. Previously, we reported that the rate of change of DAF‐2T fluorescence was proportional to [NO]2, as predicted by the reaction kinetics of NO and DAF‐2 in the presence of O2. In the present study we demonstrated the influence of light intensity that promoted photobleaching and the importance of [O2] on the reaction between DAF‐2 and NO. The generation of DAF‐2T was evaluated in mixtures of 10 μM DAF‐2 with different concentrations of NO (range 100–1200 nM) made by mixing appropriate proportions of PBS equilibrated with room air with PBS equilibrated with 800 ppm NO/balance N2 (1200 nM NO); this procedure produced mixtures with varied PO2. Sequences of fluorescence images (at 10‐sec intervals over 300 sec) were acquired with a digital camera. Light intensities normally used in microscopic measurements induced strong photobleaching when the solutions were illuminated at intervals of 1‐10 sec. To avoid photobleaching, the incident light was attenuated using a 10% transmission neutral density filter. The non‐linear relationship between DAF‐2T fluorescence and [NO] was adequately described by the chemical kinetic model when the PO2 of the mixture was taken into account. These results are being used to interpret DAF‐2T fluorescence in terms of [NO] in vitro. Supported by NIH grants HL18292 and HL 79087.
MODULATION OF MESENCHYMAL STEM CELL (MSC) PARATHYROID HORMONE‐RELATED PROTEIN (PTHrP)/PEROXISOME PROLIFERATOR‐ACTIVATED RECEPTOR GAMMA (PPARγ) SIGNALING FOR LIPOFIBROBLAST(AIF)DIFFERENTIATION
Authors: Guo Pinzheng , Cruz Emmanuel , Wang Ying , Sakurai Reiko , Cerny Laura , Torday John S , Rehan Virender
Published: June 2021

Lung PTHrP/PPARγ epithelial‐mesenchymal interactions result in differentiation of alveolar AIFs critical for injury repair. We attempted MSC differentiation into AIFs via PTHrP/PPARγ signaling as a strategy for stem cell therapy for chronic lung disease (CLD). MSCs derived from Sprague Dawley rat bone marrow (BM) were induced (α‐MEM+10% FBS, 10 μg/ml insulin, 1 μM dexamethasone, and 0.5 mM 3‐isobutyl‐1‐methylxanthine) for 11 days. Oil Red O (ORO) staining demonstrated adipocytic induction. Expression of the key markers of AIF differentiation [PPARγ and α‐smooth muscle actin (αSMA)] was determined by RT‐PCR and Western blot. Isolated MSCs expressed characteristic markers‐ 99% CD45 (−), 60% CD73 (+), and 91% CD90 (+). Small, spindly MSCs became large round adipocyte‐like cells, with lipid droplets (ORO+) in >50% of cells, a 10‐fold increase in PPARγ expression, a marker of adipocytic differentiation, and minimal expression of α‐SMA, a smooth muscle cell marker. Treatment with 500nM PTHrP and/or 1nM Rosiglitazone, a PPARγ agonist, increased the number of ORO‐staining cells significantly, with larger, more numerous lipid droplets and a significant decrease in αSMA. PTHrP and PPARγ drive MSCs to adipocytic cells. Since AIFs are essential for lung development and injury/repair, our data suggest exciting potential for stem cell‐based therapy for CLD. Funding‐NIH HL75405 & HL55268 & TRDRP 15IT‐0250 & 14RT‐0013.
Surfactant Secretagogues Increase Phosphorylation of Annexin A7 in Type II Cells
Authors: Chander Avinash , Chen Xiao‐Liang , Callender Delon
Published: June 2021

Membrane fusion between lamellar bodies and plasma membrane is an obligatory distal step in lung surfactant secretion in alveolar type II (T2) cells. We have previously postulated that annexin A7 (A7) can facilitate such membrane fusion in vitro and can increase surfactant secretion in permeabilized T2 cells. We now report that surfactant secretagogues increase in vivo phosphorylation of A7 in T2 cells that can possibly facilitate stimulated secretion of lung surfactant. Isolated T2 cells were cultured overnight and treated with 80 nM phorbol myristate acetate (PMA) for varying periods. The cells were extracted in lysis buffer and A7 was immunoprecipitated with anti‐annexin A7 antibodies that were raised against recombinant rat A7. Western blot analysis of immunoprecipitated proteins with anti‐ A7, anti‐phosphoserine and anti‐phosphothreonine antibodies showed that PMA‐treatment increased A7 phosphorylation in T2 cells in a time‐dependent manner at both serine and threonine residues. The phosphorylation reached maximum within 5–10 min and was elevated during the study period (30min). Treatment of T2 cells for 10 min with other secretagogues, ATP, terbutaline or calcium ionophore A23187 also showed increased phosphorylation of A7 suggesting that it was related to increased surfactant secretion. In vitro phosphorylation of recombinant A7 with a protein kinase C preparation showed increased membrane fusion activity of annexin A7. These observations suggest that secretagogue activation of protein kinase causes phosphorylation of A7 for increased membrane fusion activity to facilitate increased surfactant secretion.
Effects of Central Sodium On Epithelial Sodium Channels (ENaC) Expression In Rat Brain
Authors: Wang Hong‐Wei , Amin Md Shahrier , Whitman Stewart C. , Tuana Balwant S. , Leenen Frans HH
Published: June 2021

Mineralocorticoid receptors (MR) and ENaC mediate pressor responses to increases of CSF [Na+]. We evaluated MR‐dependent effects of central sodium on ENaC subunits (α, β ,γ) expression as well as its endogenous regulator, Sgk1. Male Wistar rats were infused icv for 2 weeks with regular or Na+‐rich artificial CSF (aCSF) with or without spironolactone. Brain areas studied included ependyma of the anteroventral third ventricle, supraoptic nucleus (SON), paraventricular nucleus (PVN) and subfornical organ (SFO). In ependyma, Na+‐rich aCSF increased expression of the α and β subunits, primarily in the basolateral membrane. Spironolactone did not prevent these changes. In SON, Na+ increased expression of mRNA for α and γ subunits without affecting α protein levels, and increased Sgk1 mRNA levels although a decrease in phosphorylated Sgk1 protein (p‐Sgk1) was noted. Spironolactone prevented the increases in mRNA encoding ENaC but not Sgk1. In PVN, Na+ decreased the α mRNA but not protein levels, and significantly decreased p‐Sgk1 protein with no change in Sgk1 mRNA. In SFO, Na+ only significantly increased γ mRNA. Spironolactone did not prevent the changes in ENaC mRNA in PVN or SFO. Regulation of ENaC and Sgk1 expression in the brain in response to Na+ appears quite region specific. Spironolactone prevents only some of the increases in ENaC subunit expression implying that, in addition to aldosterone, other regulators are involved.
Regulation of human organic cation transporter 3 (hOCT3) by new interacting partners
Authors: Ciarimboli Giuliano , Albiker Christian , Brast Sabine , Humberg Julia , Schlatter Eberhard
Published: June 2021

hOCT3 is a transmembran protein with broad tissue distribution involved in the transport of monoamine substrates and other organic cations across the cell membrane. Since interactions of hOCT3 with other proteins are important for its function and regulation, we identified new interaction partners of hOCT3 with a membrane‐based Split‐Ubiquitin‐Yeast‐Two‐Hybrid (YTH) technique, which, in contrast to classical YTH systems, allows to detect interactions taking place directly at cell membranes. Using this system, we could identify PDZK1 interacting protein 1, growth hormone inducible transmembrane protein, insulin‐like growth factor binding protein 5, the tetraspanin CD63 and the dual‐specifity kinase DYRK1A as interaction partners of hOCT3. We have further focused on DYRK1A and CD63. These proteins affected the function of hOCT3. Inhibition of DYRK1A activity with epigallocatechingallate (0.6 μM) resulted in a significant decrease in hOCT3 transport rate of the fluorescent organic cation 4‐4‐dimethylamino‐styryl‐N‐methylpyridinium (ASP) (−25±3%, n=16), suggesting an activating effect of DYRK1A on hOCT3. Cotransfection of hOCT3 with CD63 in HEK‐cells decreased their ASP‐uptake (−28±4%,n=12), suggesting a negative effect of CD63 on hOCT3 transport, probably mediated by CD63‐accompanied endocytosis of transporters from the membrane.
Production of an AQP2‐Enriched Clonal mpkCCD Cell Line
Authors: Yu Ming‐Jiun , McCoy J. Philip , Shen Rong‐Fong , Knepper Mark A.
Published: June 2021

The collecting duct cell line mpkCCD (developed by A. Vandewalle) expresses vasopressin‐regulated water channel AQP2 endogenously, but at low levels relative to native inner medullary collecting ducts (IMCDs). To obtain an optimized model for study of AQP2 regulation, we sub‐cloned a line that expresses AQP2 at high levels (mpkCCD‐BY11) from the original mpkCCD cell line. AQP2 expression in these cells was found to be induced to levels similar to the native IMCD cells when grown on membrane filter and exposed to 0.1 nM synthetic vasopressin analog dDAVP at the basolateral medium. Semiquantitative immunoblotting revealed 5.0‐fold enrichment of AQP2 protein relative to the original cells, while AQP2 mRNA was increased 1.7‐fold. Immunoblotting of the mpkCCD‐BY11 cells showed dose‐ and time‐dependent increases in AQP2 abundance in response to dDAVP. Immunoblotting with phospho‐specific antibodies showed increased phosphorylation at Ser‐256 and Ser‐269 of AQP2 in response to dDAVP, similar to the native IMCD cells. When exposed to dDAVP, AQP2 was found at the apical plasma membrane by confocal fluorescence microscopy. Upon withdrawal of dDAVP, AQP2 internalized into intracellular vesicles in 4 hours. Re‐applying dDAVP caused AQP2 to redistribute to the apical plasma membrane in 0.5 hour. Thus, the mpkCCD‐BY11 cells recapitulated vasopressin‐induced AQP2 trafficking as seen in the native IMCD principal cells.
Li fluxes reveal presence of Na‐Cl Cotransport (NCC) in Human Lens Epithelial Cells (LECs)
Authors: Adragna Norma Cristina , Chimote Ameet A. , Lauf Peter K.
Published: June 2021

Na‐K‐2Cl cotransport (NKCC) moves RbCl/KCl+NaCl/LiCl and, in FHL124 LECs, is not inhibited by hypotonicity. Here, we studied Li fluxes through NKCC in hyposmotic high K media and Rb fluxes in hyposmotic high Na to understand this effect. Li and Rb uptake were measured by atomic absorption spectrophotometry to determine LiKCC or NRbCC through bumetanide‐sensitive (BS) and Cl‐dependent fluxes in Na/K or Na‐free/K‐free (N‐methyl‐D‐glucamine) and Cl or Cl‐free (sulfamate or nitrate) media ± ouabain (O) or [O+B], ± thiazides, at varying Rb, Li or Cl molar fractions (MF). Main findings are: Li influx in isosmotic (300 mOsM) K was 1/3 of Rb influx in Na, and was mainly mediated by a Cl‐dependent Li flux (LiKCC). In 200 mOsM high K, LiKCC was abolished, whereas in high Na, NRbCC remained active. LiKCC and BS‐Li influx (BS‐LiKCC) showed bell‐shape curves for 0.1–1 Li MF, maxing at ∼0.6 MF. BS‐LiKCC was ¼ of LiKCC. The difference, i.e. the B‐insensitive/Cl‐dependent‐Li influx, saturated with Li and Cl MFs. Kms for Li were 11 with and 7–8 mM without external K, respectively; and ∼40 mM for Cl. Neither furosemide (<100 μM) nor thiazide derivatives inhibited LiCC. RTPCR, Western blots and immunochemistry revealed NCC RNA and protein, with NCC‐specific antibodies, besides the expected NKCC1. Thus, a K‐independent/Cl‐dependent Li flux (LiCC) revealed the presence of NCC in FHL124 LECs.
Probiotics Stimulate DRA Expression in Human Intestinal Caco‐2 Cells
Authors: Saksena Seema , Borthakur Alip , Tyagi Sangeeta , Esmaili Ali , Gill Ravinder K , Singla Amika , Alrefai Waddah A , Ramaswamy Krishnamurthy , Dudeja Pradeep K
Published: June 2021

DRA (Down Regulated in Adenoma) plays a major role in apical Cl−/OH− (HCO3−) exchange process in the human intestine. Dysregulation of DRA has been implicated in diarrheal conditions, such as congenital chloride diarrhea and enteropathogenic infections. However, little is known about the regulation of DRA by probiotics, currently used as an alternative treatment for diarrhea. Therefore, the present studies were undertaken to determine the regulation of DRA expression and function by Lactobacilli in human intestinal Caco2 cells. Culture supernatant (CS) from an overnight grown Lactobacilli culture of ~1.5X107 CFU/ml was obtained and used at 1:50 dilution. L. rhamnosus or L. acidophilus CS treatment of Caco2 cells for 24h significantly increased (~2 fold) the relative abundance of DRA mRNA. To determine whether Lactobacilli alter the rate of DRA gene transcription, DRA promoter activity was assessed. Parallel to the increased mRNA expression, luciferase reporter gene assays showed ~2.5 fold increase in DRA promoter activity. Both L. rhamnosus or L. acidophilus stimulated Cl−/OH− exchange activity as measured by DIDS‐sensitive 36Cl− uptake (~ 50 %, P<0.05). These findings suggest that the soluble factors from Lactobacilli stimulate DRA expression and Cl− absorption and may aid in the development of novel therapeutic interventions for treatment of diarrheal disorders. (Supported by NIDDK and Dept of Veteran Affairs).
Acid‐Sensing Ion Channel 2 modulates the current properties and neuropeptide potentiation of proton‐gated currents
Authors: Sherwood Thomas Walworth , Askwith Candice C
Published: June 2021

Acid‐sensing ion channels (ASICs) are proton‐gated channels required for normal behavior and sensory transduction. ASICs may also play an important role in injuries where prolonged acidosis occurs including stroke and inflammation. The specific biophysical properties of ASIC channels are dependent on their subunit composition and the presence of modulatory compounds such as RFamide neuropeptides. ASIC2 subunits regulate ASIC1a current via the formation of heteromeric channels, but the specific properties modulated by these subunits are not entirely understood. In this study, we examined the biophysical properties of ASIC1/2 heteromeric channels expressed in Xenopus oocytes. We determined that ASIC response to RFamide neuropeptides is dramatically altered by the presence of ASIC2 subunits. Surprisingly, we found that steady‐state desensitization of both heteromeric and ASIC2a homomeric channels was inhibited by RFamide peptides. Furthermore, heteromeric channels were responsive to a larger number of peptides compared to ASIC1a homomultimers. Our data suggest that in addition to affecting primary current properties, the ASIC2 subunits may also confer enhanced peptide sensitivity to proton‐gated currents in vivo, which may be important for the physiological and pathological roles of ASICs.
Dynamic behavior of lasp‐1 and dynamin‐2 regulated by epidermal growth factor and forskolin
Authors: Okamoto Curtis T. , Rajapaksa Thejani , Chew Catherine S.
Published: June 2021

Lasp‐1 is a nebulin family actin‐binding phosphoprotein and is expressed in a variety of epithelial cells. In gastric parietal cells, lasp‐1 is localized to the apical and basolateral membrane, sites enriched in F‐actin, and it is serine‐phosphorylated upon histamine stimulation. Affinity columns with lasp‐1 as bait bind to dynamin‐2 (dyn‐2) and clathrin from parietal cell lysates, suggesting that lasp‐1 may play a role in endocytosis. We have expressed a fusion protein of enhanced Green Fluorescent Protein and lasp‐1 (eGFP‐lasp‐1) in HeLa cells to characterize the dynamic behavior of lasp‐1 and its role in endocytosis by live‐cell imaging. Imaging of eGFP‐lasp‐1 revealed that it is targeted to filopodia and lamellipodia in HeLa cells, especially after cells have been incubated with epidermal growth factor. Lasp‐1‐containing structures resembling actin comet tails are also observed, often in spatiotemporal proximity to structures resembling large endocytotic vesicles or macropinosomes. Incubation of cells with forskolin results in a significantly lower level of cortical membrane activity of eGFP‐lasp‐1. However, in cells co‐expressing untagged lasp‐1 and eGFP‐dyn‐2, forskolin treatment results in recruitment of eGFP‐dyn‐2 to these large vesicles. These data suggest that lasp‐1, possibly together with dyn‐2, may regulate the formation or transport of these large membrane vesicles.
Uptake of Zn2+ across the basolateral membrane of the gastric parietal cell is regulated by [Ca2+]i
Authors: Liu JingJing , Mathew Jeff , Kohler Jonathan , Blass Amy , Soybel David
Published: June 2021

Aims: Our recent work has suggested that basolateral to apical movement of Zn2+ across the gastric parietal cell (PC) is matched to its secretory state. In this study, we tested the hypothesis that changes in [Ca2+]i may regulate uptake of Zn2+ across the basolateral membrane.

Methods: Isolated rabbit gastric glands were loaded with a Zn2+‐sensitive fluorescent dye, fluozin‐3. Cytoplasm loading of Zn2+ in PCs was monitored during a 15 min exposure to solutions containing a “free” [Zn2+] of 10μM. To lower [Ca2+]i, glands were pre‐treated with Ca2+‐free Ringer's (containing 0.3mM EGTA) and thapsigaragin (TG, 2μM), which irreversibly depletes intracellular stores of Ca2+ and opens store‐operated channels (SOCs) that permit Ca2+ to escape from the cytoplasm. To raise [Ca2+]i, glands were pre‐treated with Ca2+‐Ringer's and TG, which permits Ca2+ loading of the cytoplasm through the SOCs.

Results and Conclusions: When exposed briefly to Ringer's without TG (n= 21), [Zn2+]i increased 157% ± 2.4 above baseline, indicating PCs have a capacity for Zn2+ uptake. Exposure to Ca2+‐free Ringer's containing TG abolished this response (3.75%± 0.2, n= 42). When exposed to Ca2+‐containing Ringer's and TG, [Zn2+]i increased 60% ± 0.6 (n=25), indicating that extracellular Ca2+ partially restores Zn2+ loading and that uptake of Zn2+ across the basolateral membrane of the PC is regulated by [Ca2+]i.
Rosiglitazone Protects Against Renal Damage in Ovariectomized Female Dahl Rats
Authors: Sartori‐Valinotti Julio C. , Yanes Licy L. , Greenhaw Bradley , Iliescu Radu , Reckelhoff Jane F.
Published: June 2021

Estradiol (E) has been shown to be protective against renal injury in aging female Dahl Salt‐sensitive rats (DS). New evidence suggests that the peroxisome proliferator activator receptor gamma agonist, rosiglitazone (RO), confers renal protection in humans and animal models of various diseases. We tested the hypothesis that RO protects against renal damage in female DS during salt‐induced rise in blood pressure (BP). Methods: Intact (Int) and ovariectomized (Ovx) female DS (10 weeks; n=15/group) received low salt diet (LS: 0.3% NaCl) or LS+RO (100mg/kg diet) for 1 week and then challenged for 1 week to high salt (HS: 8% NaCl) or HS+RO. BP was recorded by telemetry. Plasma estradiol, urinary protein and renal mass were assessed on HS. Results: On HS diet BP increased in Int and Ovx rats and RO blunted the rise in BP only in Ovx. RO also decreased proteinuria and prevented kidney enlargement only in Ovx. These data show for the first time that RO prevents renal damage in ovx DS on HS diet and provides support for its use as a therapeutic tool for treatment of renal injury associated with salt‐sensitive hypertension in postmenopausal women (supported by NIH HL 66072, AHA 0725561B)
Testosterone‐Induced Vasorelaxation of Rat Mesenteric Microvasculature is Neuronal Nitric Oxide Synthase‐Dependent But Androgen Receptor‐Independent
Authors: White Richard E. , Sellers Minga M. , Stallone John N.
Published: June 2021

Recent studies reveal that testosterone (TES) produces rapid vasorelaxation of mesenteric arterioles at physiological concentrations; however, little is known about TES mechanisms in vascular smooth muscle (VSM). Therefore, acute mechanisms of TES were examined in endothelium‐intact rings of mesenteric arterioles (MA, 200 μm, dia.) prepared from male Sprague‐Dawley (SD) and Testicular‐feminized (Tfm; androgen receptor‐deficient) rats (14–18 wks age) for isometric tension using Halpern‐Mulvany myography. MA were precontracted with phenylephrine (20 μM) and a concen.‐response to TES was obtained (0.1–300 nM). Data are means+S.E (N = 3–13 rats). TES produced vasorelaxation at physiological concentrations in SD (50 ± 7%, EC50 = 2.3 ± 0.5 nM) and in Tfm (38 ± 5%, EC50 = 1.5 ± 0.5 nM). Pretreatment of Tfm MA with the neuronal NOS inhibitor LNPA (10 μM) nearly abolished TES effects (5.5 ± 3.8%). In SD VSM cells, TES activated the fluorescent nitric oxide (NO) indicator DAF‐2DA, while LNPA (10 μM) eliminated this effect of TES. These data suggest that: 1) TES exerts rapid vasorelaxing effects on VSM primarily through activation of nNOS and nitric oxide production; 2) TES‐induced vasorelaxation is a direct, nongenomic effect on VSM, independent of the cytosolic androgen receptor. (NIH: HL‐080402)
Cytosolic Calcium‐Dependence of Elevated cAMP in Polycystic Kidney Disease
Authors: Chu Wen‐Feng , Komlosi Peter , Haycraft Courtney J. , Zhang Zhi‐Ren , Bell Phillip Darwin
Published: June 2021

In polycystic kidney disease there is altered cell signaling, accelerated proliferation, and cyst formation. Previously, we found increased apical Ca2+ permeability resulting in chronic elevated sub‐apical [Ca2+]i in immortalized cell lines derived from the collecting duct of Tg737orpk mice (denoted cilia (−) cells versus the control cell line denoted cilia (+) cells). We hypothesize that an elevated intracellular [Ca2+]i in cilia (−) cells may lead to an increased production of cAMP by activating Ca2+‐sensitive adenylate cyclases. To test this hypothesis we measured intracellular cAMP levels in both cilia (−) and cilia (+) cells. Cells were cultured on permeable supports for 24–96 hours prior to [cAMP] measurement. Under resting conditions, cAMP levels were ~2.5‐fold higher in cilia (−) cells compared in cilia (+) cells. Pretreatment of both cell types with 10 μM BAPTA‐AM, a Ca2+ chelator, abolished the difference in cAMP levels between cilia (+) and cilia (−) cells. Our data suggest that there is elevated cAMP levels in cilia (−) cells compared to cilia (+) cells, which may be the consequence of Ca2+‐dependent adenylate cyclase activity in polycystic kidney disease.
Shear stress increases NO production by thick ascending limb cells
Authors: Garvin Jeffrey L. , Hong Nancy J.
Published: June 2021

Luminal flow induces NO production in the thick ascending limb (TAL) by activating the enzyme that produces NO, NOS3. Shear stress, cellular stretch and transmural pressure all change when flow increases. Shear stress stimulates NO production in endothelial cells and inner medullary collecting ducts. However, it is unclear whether flow‐induced NO production in TALs is mediated by increases in shear stress, stretch or pressure. We hypothesized that shear stress acts as a mechanical stimulus for NO production in the TAL. We subjected primary cultures of rat TAL cells to shear stresses of 0.02 (low shear stress; LSS) and 0.55 dyne/cm2 (high shear stress; HSS) and measured intracellular NO using the fluorescent dye DAF‐2 DA. The rate of increase in dye fluorescence is indicative of NO production. TAL cells under LSS produced NO at a rate of 55 ± 10 AU/s. NO production increased to 315 ± 93 AU/s at HSS (p < 0.03, n = 7). Pre‐treatment with the NOS inhibitor L‐NAME (5 mM) inhibited the shear stress‐induced increase in NO production (44 ± 24 vs. 48 ± 12 AU/s for LSS and HSS, respectively; n = 6). The PI3‐kinase inhibitor wortmannin (150 nM) also blocked NO production stimulated by increased shear stress (26 ± 9 AU/s for LSS vs.27 ± 8 AU/s for HSS; n = 5). We conclude that increases in shear stress mediate flow‐induced NO production by NOS via the PI3‐kinase pathway. Changes in flow may play an important role in regulation of TAL function by NO.

Research support: NIH Grants HL 28982 and HL 70985
Preservation of renal function in the obese Zucker rat by antioxidant diet
Authors: Inman Sharon Ruth , Jackson Edwin , Oshogwemoh Olusayo , Slyvka Yuriy , Nowak Felicia V
Published: June 2021

Type 2 diabetes accounts for 90–95% of all diabetes mellitus and is the worldwide leading cause of end‐stage renal disease (ESRD). The obese Zucker rat model of Type 2 diabetes was used to determine the gender differences of ESRD and effect of administration of an antioxidant (AO) diet on the progression of ESRD. Male and female obese Zucker rats (fa/fa) were studied at 13 and 20 weeks of age on regular (REG) and AO diets.Disease progression was determined by blood glucose, mean arterial pressure (MAP), and glomerular filtration rate (GFR). The AO diet had effects on GFR, MAP and blood glucose in female rats. GFR in 20 week old females on the AO diet was higher than in rats on the REG diet (0.346 ± 0.098 vs 0.053± 0.031 ml/min, p=0.028). This difference was not observed in males. MAP in females at 20 weeks was lower on the AO diet than on the REG diet (121.5±5.8 vs 146.2±1.7 mm Hg, p=0.02). The data also suggest that the AO diet lowers blood glucose in 13 week female rats (371.3±81.1 vs 688.3±31.9 mg/dl, p=0.005). A sex difference was also observed. MAP was higher in males at 20 weeks than in females, on both AO and REG diets.

Conclusion: An AO diet preserves several important parameters in obese Zucker rats, including GFR, MAP and blood glucose. The effect was more significant in females.

Supported by NIH R15 DK073066 to SRI and FVN