Lung fluid absorption is induced in preterm guinea pigs ventilated with low tidal volumes.
Time Factors; Animals; Gestational Age; Guinea Pigs; *Premature Birth; Phosphorylation; Enzyme Activation; Permeability; Extracellular Signal-Regulated MAP Kinases/metabolism; Intubation; Epithelial Sodium Channels/metabolism; Absorption; Sodium-Potassium-Exchanging ATPase/metabolism; Fetal Organ Maturity; Epinephrine/blood; Extravascular Lung Water/*metabolism; *Tidal Volume; Albumins/administration & dosage/*metabolism; Hydrocortisone/blood; Lung/embryology/*metabolism/physiopathology; MAP Kinase Kinase Kinases/metabolism; Ventilator-Induced Lung Injury/metabolism/physiopathology/*prevention & control; Newborn; Intratracheal; Artificial/adverse effects/*methods; Respiration
The objective of this study was to determine if low tidal volume (V(t)) ventilation was beneficial when ventilating preterm fetuses. The authors ventilated preterm guinea pig fetuses at gestation day (GD) 67, 3 days before birth, newborn, and 10-day-old (PD10) guinea pigs with low V(t) (6 mL/kg body weight [bw]) and compared them to age-matched fetuses/animals ventilated with higher potentially injurious V(t) (12 mL/kg bw). Lung fluid absorption was measured after intratracheal instillation of 5% albumin in 0.9% NaCl. Low V(t) ventilation stimulated lung fluid absorption when compared to higher V(t) in all groups. The increased lung fluid absorption in low V(t)-ventilated fetuses was associated with increased alpha epithelial Na channel (alphaEnaC) mRNA. However, alphaENaC and betaENaC protein was unchanged over the 1-hour study. Because stretch induces mitogen-activated protein (MAP) kinase expression and MAP kinases may affect lung fluid absorption, the authors investigated if MAP kinase (MAPK) expression was affected by V(t). Extracellular signal-regulated kinase (ERK) and MAPK/ERK kinase (MEK) were phosphorylated in the higher V(t)-ventilated guinea pig fetuses. This suggested that a reduced activation of MAP kinases might explain the increased lung fluid absorption in the low V(t)-ventilated fetuses. Thus these data suggest that low V(t) ventilation increases fetal lung fluid absorption and thus may be preferential to use clinically.
Koshy Shyny; Beard LaMonta L; Kuzenko Stephanie R; Li Tianbo; Folkesson Hans G
Experimental lung research
2011
2011-02
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.3109/01902148.2010.514024" target="_blank" rel="noreferrer noopener">10.3109/01902148.2010.514024</a>
RNA interference for CFTR attenuates lung fluid absorption at birth in rats.
*RNA Interference; Animals; Biological Transport/drug effects; Cells; Cultured; Cystic Fibrosis Transmembrane Conductance Regulator/*genetics/*metabolism; Epithelial Sodium Channels/genetics/metabolism; Female; Gene Expression Regulation/drug effects; Lung/cytology/drug effects/*metabolism; Messenger/metabolism; Newborn; Pregnancy; Rats; Respiratory Mucosa/cytology/drug effects/metabolism; RNA; Small Interfering/*pharmacology; Sprague-Dawley; Water/*metabolism
BACKGROUND: Small interfering RNA (siRNA) against alphaENaC (alpha-subunit of the epithelial Na channel) and CFTR (cystic fibrosis transmembrane conductance regulator) was used to explore ENaC and CTFR function in newborn rat lungs. METHODS: Twenty-four hours after trans-thoracic intrapulmonary (ttip) injection of siRNA-generating plasmid DNA (pSi-0, pSi-4, or pSi-C2), we measured CFTR and ENaC expression, extravascular lung water, and mortality. RESULTS: alphaENaC and CFTR mRNA and protein decreased by approximately 80% and approximately 85%, respectively, following alphaENaC and CFTR silencing. Extravascular lung water and mortality increased after alphaENaC and CFTR-silencing. In pSi-C2-transfected isolated DLE cells there were attenuated CFTR mRNA and protein. In pSi-4-transfected DLE cells alphaENaC mRNA and protein were both reduced. Interestingly, CFTR-silencing also reduced alphaENaC mRNA and protein. alphaENaC silencing, on the other hand, only slightly reduced CFTR mRNA and protein. CONCLUSION: Thus, ENaC and CFTR are both involved in the fluid secretion to absorption conversion around at birth.
Li Tianbo; Koshy Shyny; Folkesson Hans G
Respiratory research
2008
2008-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1186/1465-9921-9-55" target="_blank" rel="noreferrer noopener">10.1186/1465-9921-9-55</a>
Stimulation of MAP kinase pathways after maternal IL-1beta exposure induces fetal lung fluid absorption in guinea pigs.
Absorption/drug effects; Animal/*metabolism; Animals; Body Fluids/*metabolism; Extracellular Signal-Regulated MAP Kinases/metabolism; Female; Fetus/metabolism; Gestational Age; Guinea Pigs; Hydrocortisone/*metabolism; Injections; Interleukin-1beta/administration & dosage/*pharmacology; Lung/*embryology; Mitogen-Activated Protein Kinase Kinases/metabolism; Mitogen-Activated Protein Kinases/*metabolism; Pregnancy; Rats; Recombinant Proteins/administration & dosage/pharmacology; Subcutaneous
BACKGROUND: We tested the hypothesis that maternal interleukin-1beta (IL-1beta) pretreatment and induction of fetal cortisol synthesis activates MAP kinases and thereby affects lung fluid absorption in preterm guinea pigs. METHODS: IL-1beta was administered subcutaneously daily to timed-pregnant guinea pigs for three days. Fetuses were obtained by abdominal hysterotomy and instilled with isosmolar 5% albumin into the lungs and lung fluid movement was measured over 1 h by mass balance. MAP kinase expression was measured by western blot. RESULTS: Lung fluid absorption was induced at 61 days (D) gestation and stimulated at 68D gestation by IL-1beta. Maternal IL-1beta pretreatment upregulated ERK and upstream MEK expression at both 61 and 68D gestation, albeit being much more pronounced at 61D gestation. U0126 instillation completely blocked IL-1beta-induced lung fluid absorption as well as IL-1beta-induced/stimulated ERK expression. Cortisol synthesis inhibition by metyrapone attenuated ERK expression and lung fluid absorption in IL-1beta-pretreated fetal lungs. JNK expression after maternal
Bhattacharjee Reshma; Li Tianbo; Koshy Shyny; Beard LaMonta L; Sharma Kapil; Carter Ethan P; Garat Chrystelle; Folkesson Hans G
Respiratory research
2007
2007-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1186/1465-9921-8-27" target="_blank" rel="noreferrer noopener">10.1186/1465-9921-8-27</a>
Alveolar epithelial ion and fluid transport: recent progress.
Animals; Biological Transport; Body Fluids/*metabolism; Epithelium/*metabolism; Humans; Lung/cytology/*metabolism/pathology; Pulmonary Alveoli/*cytology; Sodium Channels/metabolism
Folkesson Hans G; Matthay Michael A
American journal of respiratory cell and molecular biology
2006
2006-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1165/rcmb.2006-0080SF" target="_blank" rel="noreferrer noopener">10.1165/rcmb.2006-0080SF</a>
Beta-adrenoceptor stimulation of alveolar fluid clearance is increased in rats with heart failure.
Adrenergic; Animals; beta/*metabolism; Body Fluids/*metabolism; Epithelial Cells/drug effects/metabolism/pathology; Gene Expression Regulation/drug effects; Heart Failure/blood/diagnostic imaging/*metabolism/*pathology; Hormones/blood; Hyperplasia; Ion Channels/genetics/metabolism; Male; Messenger/genetics/metabolism; Myocardial Infarction/blood/diagnostic imaging/pathology; Pulmonary Alveoli/drug effects/*pathology; Rats; Receptors; RNA; Sprague-Dawley; Terbutaline/pharmacology; Ultrasonography
The alveolar epithelium plays a critical role in resolving pulmonary edema. We thus hypothesized that its function might be upregulated in rats with heart failure, a condition that severely challenges the lung's ability to maintain fluid balance. Heart failure was induced by left coronary artery ligation. Echocardiographic and cardiovascular hemodynamics confirmed its development at 16 wk postligation. At that time, alveolar fluid clearance was measured by an increase in protein concentration over 1 h of a 5% albumin solution instilled into the lungs. Baseline alveolar fluid clearance was similar in heart failure and age-matched control rats. Terbutaline was added to the instillate to determine whether heart failure rats responded to beta-adrenoceptor stimulation. Alveolar fluid clearance in heart failure rats was increased by 194% after terbutaline stimulation compared with a 153% increase by terbutaline in control rats. To determine the mechanisms responsible for this accelerated alveolar fluid clearance, we measured ion transporter expression (ENaC, Na-K- ATPase, CFTR). No significant upregulation was observed for these ion transporters in the heart failure rats. Lung morphology showed significant alveolar epithelial type II cell hyperplasia in heart failure rats. Thus, alveolar epithelial type II cell hyperplasia is the likely explanation for the increased terbutaline-stimulated alveolar fluid clearance in heart failure rats. These data provide evidence for previously unrecognized mechanisms that can protect against or hasten resolution of alveolar edema in heart failure.
Maron Michael B; Luther Daniel J; Pilati Charles F; Ohanyan Vahagn; Li Tianbo; Koshy Shyny; Horne Walter I; Meszaros J Gary; Walro Jon M; Folkesson Hans G
American journal of physiology. Lung cellular and molecular physiology
2009
2009-09
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajplung.90629.2008" target="_blank" rel="noreferrer noopener">10.1152/ajplung.90629.2008</a>
IL-1beta-induced cortisol stimulates lung fluid absorption in fetal guinea pigs via SGK-mediated Nedd4-2 inhibition.
Absorption/drug effects; Animals; Base Sequence; Body Fluids/metabolism; DNA/genetics; Endosomal Sorting Complexes Required for Transport; Epithelial Sodium Channels/metabolism; Female; Guinea Pigs; Hydrocortisone/*biosynthesis/blood/physiology; Immediate-Early Proteins/genetics/*metabolism; Interleukin-1beta/*pharmacology; Lung/*drug effects/*physiology; Molecular Sequence Data; Nedd4 Ubiquitin Protein Ligases; Pregnancy; Protein-Serine-Threonine Kinases/genetics/*metabolism; Recombinant Proteins/pharmacology; Ubiquitin-Protein Ligases/*antagonists & inhibitors/genetics
We tested the hypothesis that interleukin (IL)-1beta-induced cortisol synthesis stimulates distal lung fluid absorption in fetal guinea pigs via induction of serum- and glucocorticoid-regulated kinase (SGK) and inhibition of neural precursor cell expressed, developmentally downregulated protein 4-2 (Nedd4-2).
Li Tianbo; Koshy Shyny; Folkesson Hans G
American journal of physiology. Lung cellular and molecular physiology
2009
2009-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajplung.90506.2008" target="_blank" rel="noreferrer noopener">10.1152/ajplung.90506.2008</a>
The adenosine 2A receptor agonist GW328267C improves lung function after acute lung injury in rats.
Acute Lung Injury/*drug therapy/metabolism/pathology; Adenosine A2A/*chemistry; Adenosine/*analogs & derivatives/therapeutic use; Amiloride/pharmacology; Animals; Biological Transport; Bronchoalveolar Lavage; Cystic Fibrosis Transmembrane Conductance Regulator/metabolism; Endotoxemia/*drug therapy/metabolism/microbiology; Epithelial Sodium Channel Blockers; Epithelial Sodium Channels/metabolism; Escherichia coli; Escherichia coli Infections/drug therapy/metabolism/microbiology; Immunoblotting; Male; Pneumonia/*drug therapy/metabolism/microbiology; Pulmonary Alveoli/cytology/*drug effects/metabolism; Pulmonary Edema/*drug therapy/metabolism/pathology; Rats; Receptor; Respiratory Physiological Phenomena; Sodium Channel Blockers/pharmacology; Sprague-Dawley; Triazoles/*therapeutic use
There is a significant unmet need for treatments of patients with acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS). The primary mechanism that leads to resolution of alveolar and pulmonary edema is active vectorial Na(+) and Cl(-) transport across the alveolar epithelium. Several studies have suggested a role for adenosine receptors in regulating this fluid transport in the lung. Furthermore, these studies point to the A(2A) subtype of adenosine receptor (A(2A)R) as playing a role to enhance fluid transport, suggesting that activation of the A(2A)R may enhance alveolar fluid clearance (AFC). The current studies test the potential therapeutic value of the A(2A)R agonist GW328267C to accelerate resolution of alveolar edema and ALI/ARDS in rats. GW328267C, at concentrations of 10(-5) M to 10(-3) M, instilled into the airspaces, increased AFC in control animals. GW328267C did not increase AFC beyond that produced by maximal beta-adrenergic stimulation. The effect of GW328267C was inhibited by amiloride but was not affected by cystic fibrosis transmembrane conductance regulator inhibition. The drug was tested in three models of ALI, HCl instillation 1 h, LPS instillation 16 h, and live Escherichia coli instillation 2 h before GW328267C instillation. After either type of injury, GW328267C (10(-4) M) decreased pulmonary edema formation and restored AFC, measured 1 h after GW328267C instillation. These findings show that GW328267C has beneficial effects in experimental models of ALI and may be a useful agent for treating patients with ALI or prophylactically to prevent ALI.
Folkesson Hans G; Kuzenko Stephanie R; Lipson David A; Matthay Michael A; Simmons Mark A
American journal of physiology. Lung cellular and molecular physiology
2012
2012-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajplung.00395.2011" target="_blank" rel="noreferrer noopener">10.1152/ajplung.00395.2011</a>
Prolonged isoproterenol infusion impairs the ability of beta(2)-agonists to increase alveolar liquid clearance.
Adrenergic; Adrenergic beta-Agonists/*pharmacology; Animals; beta/metabolism; Down-Regulation/drug effects; Drug Interactions; Epithelial Cells/metabolism; Extravascular Lung Water/*metabolism; Isoproterenol/*pharmacology; Male; Pulmonary Alveoli/cytology/*metabolism; Pulmonary Circulation/physiology; Pulmonary Edema/metabolism; Rats; Receptors; Respiratory Mucosa/cytology/*metabolism; Sprague-Dawley; Terbutaline/pharmacology
We determined if prolonged isoproterenol (Iso) infusion in rats impaired the ability of the beta(2)-adrenergic agonist terbutaline to increase alveolar liquid clearance (ALC). We infused rats with Iso (at rates of 4, 40, or 400 microg.kg(-1).h(-1)) or vehicle (0.001 N HCl) for 48 h using subcutaneously implanted miniosmotic pumps. After this time, the rats were anesthetized, and ALC was determined (by mass-balance after instillation of Ringer lactate containing albumin into the lungs) under baseline conditions and after terbutaline administration. Baseline and terbutaline-stimulated ALC in vehicle-infused rats averaged, respectively, 19.6 +/- 1.2% (SE) and 44.7 +/- 1.5%/h. The ability of terbutaline to increase ALC was eliminated at 400 microg.kg(-1).h(-1)Iso, inhibited by 26% at 40 microg.kg(-1).h(-1) Iso, and was not affected by 4 microg.kg(-1).h(-1) Iso. beta-adrenergic receptor (betaAR) density of freshly isolated alveolar epithelial type II (ATII) cells from Iso-infused rats was reduced by the 40 and 400 microg.kg(-1).h(-1) infusion rates. These data demonstrate that prolonged exposure to beta-agonists can impair the ability of beta(2)-agonists to stimulate ALC and produce ATII cell betaAR downregulation.
Morgan Eric E; Hodnichak Cheryl M; Stader Sonya M; Maender Kay C; Boja John W; Folkesson Hans G; Maron Michael B
American journal of physiology. Lung cellular and molecular physiology
2002
2002-04
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajplung.00381.2001" target="_blank" rel="noreferrer noopener">10.1152/ajplung.00381.2001</a>
Postreceptor defects in alveolar epithelial beta-adrenergic signaling after prolonged isoproterenol infusion.
8-Bromo Cyclic Adenosine Monophosphate/pharmacology; Adenylyl Cyclases/metabolism; Adrenergic; Adrenergic beta-Agonists/*pharmacology; Animals; beta/*metabolism; Colforsin/pharmacology; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases/metabolism; Cyclic AMP/metabolism; Extravascular Lung Water/metabolism; Isoproterenol/*pharmacology; Male; Pulmonary Alveoli/*drug effects/*metabolism; Pulmonary Edema/metabolism; Rats; Receptors; Respiratory Mucosa/drug effects/metabolism; Signal Transduction/drug effects; Sprague-Dawley
We previously found that prolonged isoproterenol (Iso) infusion in rats impaired the ability of beta-adrenoceptor (beta-AR) agonists to increase alveolar liquid clearance (ALC). Here, we determined if postreceptor defects in beta-AR signaling contribute to this impairment. Iso was infused using subcutaneous miniosmotic pumps (4, 40, or 400 microg. kg-1. h-1) in rats for 48 h. At this time, forskolin-stimulated ALC was measured by mass balance. Forskolin-stimulated ALC [33.4 +/- 2.1%/h (mean +/- SE) in vehicle-infused rats] was reduced by 25 and 38%, respectively, after the 40 and 400 microg. kg-1. h-1 Iso infusions. The ability of forskolin to increase cAMP was reduced by 70% in alveolar type II (ATII) cells isolated from rats infused with 400 microg. kg-1. h-1 Iso. Additionally, the ability of the stable cAMP analog 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Sp-isomer, to increase ALC (48.7 +/- 3.0% in vehicle-infused rats) was reduced by 25 and 51%, respectively, after the 40 and 400 microg. kg-1. h-1 infusions. Finally, the ability of cAMP to increase protein kinase A activity was eliminated in ATII cells isolated from rats infused with Iso at 400 microg. kg-1. h-1. These data demonstrate that prolonged beta-AR agonist exposure can impair alveolar epithelial beta-AR signaling downstream of the beta-AR.
Morgan Eric E; Stader Sonya M; Hodnichak Cheryl M; Mavrich Kate E; Folkesson Hans G; Maron Michael B
American journal of physiology. Lung cellular and molecular physiology
2003
2003-09
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajplung.00339.2002" target="_blank" rel="noreferrer noopener">10.1152/ajplung.00339.2002</a>
Oxytocin-induced labor augments IL-1beta-stimulated lung fluid absorption in fetal guinea pig lungs.
*Labor; Absorption/drug effects; Amiloride/administration & dosage; Animals; Enzyme Activation/drug effects; Enzyme Inhibitors/administration & dosage; Epinephrine/metabolism; Extravascular Lung Water/*metabolism; Female; Fetal Organ Maturity/drug effects; Fetus/metabolism; Guinea Pigs; Hydrocortisone/metabolism; Induced; Interleukin-1/*administration & dosage/metabolism; Ion Transport/drug effects; Lung/*embryology; Maternal-Fetal Exchange/drug effects; Metyrapone/administration & dosage; Oxytocics/*administration & dosage; Oxytocin/*administration & dosage; Pregnancy; Propranolol/administration & dosage; Sodium Channel Blockers/administration & dosage; Sodium-Potassium-Chloride Symporters/metabolism; Vasodilator Agents/administration & dosage
We tested the hypothesis that oxytocin-induced labor augmented
Nair Prem K; Li Tianbo; Bhattacharjee Reshma; Ye Xin; Folkesson Hans G
American journal of physiology. Lung cellular and molecular physiology
2005
2005-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajplung.00256.2004" target="_blank" rel="noreferrer noopener">10.1152/ajplung.00256.2004</a>
IL-1beta stimulates alveolar fluid absorption in fetal guinea pig lungs via the hypothalamus-pituitary-adrenal gland axis.
Adrenocorticotropic Hormone/blood; Animals; Epinephrine/blood; Epithelial Sodium Channels; Extravascular Lung Water/*metabolism; Female; Guinea Pigs; Hydrocortisone/blood; Hypothalamo-Hypophyseal System/*physiology; Interleukin-1/blood/*pharmacology; Norepinephrine/blood; Pituitary-Adrenal System/*physiology; Pregnancy; Pulmonary Alveoli/*drug effects/embryology/*metabolism; Sodium Channels/metabolism; Sodium-Potassium-Exchanging ATPase/metabolism
We tested the hypothesis that interleukin (IL)-1beta-induced cortisol synthesis stimulates alveolar fluid clearance in preterm fetuses. IL-1beta was administered subcutaneously daily to timed-pregnant guinea pigs for 3 days with and without simultaneous cortisol synthesis inhibition by metyrapone. Fetuses were obtained by abdominal hysterotomy at 61 and 68 days gestation and instilled with isosmolar 5% albumin in the lungs, and alveolar fluid movement was measured over 1 h from the change in alveolar protein concentration. Alveolar fluid clearance was induced at 61 days gestation and stimulated at 68 days gestation by IL-1beta, which both were attenuated by cortisol synthesis inhibition. Plasma ACTH and cortisol concentrations were increased by IL-1beta at both gestational ages, and metyrapone reduced cortisol concentrations. IL-1beta was mostly low or undetectable in both fetal and maternal blood. Prenatal alveolar fluid clearance, when present as well as IL-1beta induced, was always propranolol and amiloride sensitive, suggesting that beta-adrenoceptor stimulation and amiloride-sensitive Na+ channels were critical for fluid absorption. IL-1beta increased lung beta-adrenoceptor density at gestation day 61, and cortisol synthesis inhibition attenuated the increased beta-adrenoceptor density. Epithelial Na+ channel and Na+-K+-ATPase subunit expressions were both increased by IL-1beta and attenuated by cortisol synthesis inhibition. These results may explain why babies delivered preterm after intrauterine inflammation have a reduced risk of developing severe respiratory distress.
Ye Xin; Acharya Reshma; Herbert Jonathan B; Hamilton Sarah E; Folkesson Hans G
American journal of physiology. Lung cellular and molecular physiology
2004
2004-04
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajplung.00214.2003" target="_blank" rel="noreferrer noopener">10.1152/ajplung.00214.2003</a>
RNA interference for alpha-ENaC inhibits rat lung fluid absorption in vivo.
Absorption/drug effects; Adrenergic beta-Agonists/pharmacology; Amiloride/pharmacology; Animals; Body Fluids/*metabolism; Capillary Permeability; Dose-Response Relationship; Drug; Endothelium; Epithelial Cells/metabolism; Epithelial Sodium Channels; Epithelium/metabolism; Inhibitory Concentration 50; Lung/enzymology/*metabolism; Male; Messenger/antagonists & inhibitors/metabolism; Permeability; Plasmids/administration & dosage/pharmacology; Rats; RNA; Small Interfering/genetics/pharmacology; Sodium Channels/drug effects/genetics/metabolism/*physiology; Sprague-Dawley; Terbutaline/pharmacology; Tissue Distribution; Vascular/metabolism
We used siRNA against the alpha-ENaC (epithelial Na channel) subunit to investigate ENaC involvement in lung fluid absorption in rats by the impermeable tracer technique during baseline and after beta-adrenoceptor stimulation by terbutaline. Terbutaline stimulation of lung fluid absorption increased fluid absorption by 165% in pSi-0-pretreated rat lungs (irrelevant siRNA-generating plasmid). Terbutaline failed to increase lung fluid absorption in rats given the specific alpha-ENaC siRNA-generating plasmid (pSi-4). pSi-4 pretreatment reduced baseline lung fluid absorption by approximately 30%. alpha-ENaC was undetectable in pSi-4-pretreated lungs, regardless of condition but was normal in pSi-0-pretreated lungs. We carried out a dose-response analysis where rats were given 0-200 microg/kg body wt pSi-4, and alpha-ENaC mRNA and protein expressions were analyzed. To reach IC(50) for alpha-ENaC mRNA expression, 32 microg/kg body wt pSi-4 was needed, and to reach IC(50) for alpha-ENaC protein expression, 59 microg/kg body wt pSi-4 was needed. We tested for lung tissue specificity and found no changes in beta-ENaC expression, at either mRNA or protein level, as well as no changes in alpha(1)-Na-K-ATPase protein expression. We isolated alveolar epithelial type II cells 24 h after in vivo pSi-4 pretreatment. In these cells, alpha-ENaC mRNA was undetectable, demonstrating that alveolar epithelial ENaC expression was attenuated after intratracheal alpha-ENaC siRNA-generating plasmid DNA instillation. We tested for organ specificity and found no changes in kidney alpha- and beta-ENaC mRNA and protein expression. Thus we provide conclusive evidence that beta-adrenoceptor stimulation of lung fluid absorption is critically ENaC dependent, whereas baseline lung fluid absorption seemed less ENaC dependent.
Li Tianbo; Folkesson Hans G
American journal of physiology. Lung cellular and molecular physiology
2006
2006-04
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajplung.00205.2005" target="_blank" rel="noreferrer noopener">10.1152/ajplung.00205.2005</a>
Distal air space epithelial fluid clearance in near-term rat fetuses is fast and requires endogenous catecholamines.
Adrenergic beta-Antagonists/pharmacology; Aging/metabolism; Animals; Body Fluids/*metabolism; Body Water/metabolism; Catecholamines/*physiology; Embryonic and Fetal Development; Epinephrine/blood; Epithelium/metabolism; Female; Fetus/drug effects/metabolism; Gestational Age; Lung/drug effects/*embryology/metabolism; Male; Newborn/metabolism; Propranolol/pharmacology; Rats; Sprague-Dawley; Time Factors
Knowledge about the conversion of the epithelium in the distal air spaces of the lung from secretion to absorption is imperative to the understanding of postnatal lung development; little such information is available in rats. Distal air space fluid clearance was therefore measured in 21- to 22-day gestation rat fetuses and newborn (40 h) rats. Distal air space fluid clearance was measured from the increase in (131)I-albumin concentration in an isosmolar, physiological solution instilled into the developing lungs. There was no net fluid movement across the distal air space epithelium in the lungs of 21-day gestation fetuses. Twenty-four hours later, distal air space fluid was cleared at a rapid rate in the 22-day gestation fetuses. Within the first 40 h after birth, the rate rapidly declined to adult levels. The high distal air space fluid clearance at 22 days gestation and at 40 h after birth was mediated by beta-adrenergic receptors as demonstrated by elevated plasma epinephrine levels and inhibition by propranolol. Interestingly, the elevated distal air space fluid clearance in the 22-day gestation fetuses was only minimally amiloride sensitive; however, amiloride sensitivity increased over the first 40 h after birth. In conclusion, these studies demonstrate that 1) rapid rates of net alveolar fluid clearance occur late in gestation in the rat and 2) this clearance is driven by elevations of endogenous epinephrine.
Folkesson Hans G; Matthay Michael A; Chapin Cheryl J; Porta Nicolas F M; Kitterman Joseph A
American journal of physiology. Lung cellular and molecular physiology
2002
2002-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajplung.00183.2001" target="_blank" rel="noreferrer noopener">10.1152/ajplung.00183.2001</a>
Involvement of {alpha}ENaC and Nedd4-2 in the conversion from lung fluid secretion to fluid absorption at birth in the rat as assayed by RNA interference analysis.
*RNA Interference; Absorption; Animals; Body Fluids/*metabolism; Death; Endosomal Sorting Complexes Required for Transport; Epithelial Sodium Channels/genetics/*physiology; Extravascular Lung Water/metabolism; Gene Silencing; Lung/*metabolism; Nedd4 Ubiquitin Protein Ligases; Newborn/*metabolism; Rats; RNA; Small Interfering/metabolism; Sodium-Potassium-Exchanging ATPase/metabolism; Sprague-Dawley; Tissue Distribution; Ubiquitin-Protein Ligases/genetics/metabolism/*physiology
To explore interactions between the epithelial Na channel (ENaC) and neural precursor expressed, developmentally downregulated protein 4-2 (Nedd4-2) at the conversion of the rat lung from fluid secretion to absorption at birth, we used small-interfering RNA (siRNA) against alphaENaC and Nedd4-2. siRNA-generating plasmid DNA (pDNA) was administered via trans-thoracic intrapulmonary (ttip) injection 24 h before ENaC and Nedd4-2 expression, extravascular lung water, and mortality were measured. alphaENaC mRNA and protein were specifically reduced by approximately 65% after pSi-4 injection. Nedd4-2 mRNA and protein were reduced by approximately 60% after pSi-N1 injection. Interestingly, alphaENaC and betaENaC mRNA and protein expression were increased after Nedd4-2 silencing. Extravascular lung water was significantly increased after alphaENaC silencing and reduced after Nedd4-2 silencing. alphaENaC silencing resulted in a fourfold increase in newborn mortality, whereas silencing Nedd4-2 did not affect mortality. We also isolated distal lung epithelial (DLE) cells after in vivo alphaENaC or Nedd4-2 silencing and measured alphaENaC or Nedd4-2 expression in freshly isolated DLE cells. In these DLE cells, there were attenuated alphaENaC or Nedd4-2 mRNA and protein, thus demonstrating that alphaENaC and Nedd4-2 silencing occurred in alveolar epithelial cells after ttip injection. We also looked for pDNA by PCR to determine pDNA presence in the lungs and found strong evidence for pDNA presence in both lungs. Thus we provide evidence that ENaC and Nedd4-2 are involved in the transition from lung fluid secretion to fluid absorption near term and at birth.
Li Tianbo; Koshy Shyny; Folkesson Hans G
American journal of physiology. Lung cellular and molecular physiology
2007
2007-10
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajplung.00151.2007" target="_blank" rel="noreferrer noopener">10.1152/ajplung.00151.2007</a>
PKA delivery to the distal lung air spaces increases alveolar liquid clearance after isoproterenol-induced alveolar epithelial PKA desensitization.
Adrenergic beta-Agonists/*pharmacology; Animals; Body Fluids/physiology; Cyclic AMP-Dependent Protein Kinases/*administration & dosage; Desensitization; Extravascular Lung Water/metabolism; Immunologic; Isoproterenol/*pharmacology; Male; Pulmonary Alveoli/cytology/*drug effects/*metabolism; Pulmonary Circulation/physiology; Pulmonary Edema/metabolism; Rats; Respiratory Mucosa/cytology/drug effects/metabolism; Sprague-Dawley
Isoproterenol (Iso) infusion for 48 h in rats decreases the ability of beta-adrenoceptor (beta-AR) agonists to increase alveolar liquid clearance (ALC). An impairment in protein kinase A (PKA) function appears to be critical in producing the desensitized ALC response. To test this hypothesis, we used a novel protein delivery reagent (Chariot, Active Motif) to deliver either the PKA catalytic subunit or the PKA holoenzyme to the distal lung epithelium of Iso-infused rats (400 microg.kg(-1).h(-1), 48 h). After this infusion, ALC was measured by mass balance over 2 h. ALC in Iso-infused rats was 27.9% (SD 5.8) of instilled volume absorbed. Delivery of the catalytic PKA subunit to Iso-infused rats increased ALC to 47.7% (SD 8.9) (P \textless 0.05). ALC in Iso-infused rats delivered the inactive PKA holoenzyme [29.6% (SD 2.5)] was not increased above baseline values. Subsequent holoenzyme activation by intravenous infusion of the stable cAMP analog Sp-8-Bromo-cAMPS increased ALC to 41.7% (SD 8.8) (P \textless 0.05). Immunohistochemical localization of Chariot-delivered PKA revealed staining in the alveolar and distal airway epithelium. These data indicate that protein delivery reagents can be used to rapidly deliver biologically active proteins to the distal lung epithelium and that PKA desensitization may be an important rate-limiting event in the development of Iso-induced desensitization of the alveolar epithelial beta-AR signaling pathway.
Maron Michael B; Folkesson Hans G; Stader Sonya M; Walro Jon M
American journal of physiology. Lung cellular and molecular physiology
2005
2005-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajplung.00134.2004" target="_blank" rel="noreferrer noopener">10.1152/ajplung.00134.2004</a>
Congenital diaphragmatic hernia prevents absorption of distal air space fluid in late-gestation rat fetuses.
*Gestational Age; *Hernias; Absorption; Amiloride/pharmacology; Animals; Body Fluids/*metabolism; Congenital; Diaphragmatic; Diaphragmatic/*metabolism; Epinephrine/metabolism; Epithelial Sodium Channels; Female; Fetal Organ Maturity; Fetus/*metabolism; Hernia; Male; Newborn; Phenyl Ethers/administration & dosage; Pregnancy; Propranolol/pharmacology; Rats; Respiratory System Abnormalities; Sodium Channels/metabolism; Sodium-Potassium-Exchanging ATPase/metabolism; Sprague-Dawley
We hypothesized that congenital diaphragmatic hernia (CDH) may decrease distal air space fluid absorption due to immaturity of alveolar epithelial cells from a loss of the normal epithelial Na+ transport, as assessed by amiloride and epithelial Na+ channel (ENaC) and Na-K-ATPase expression, as well as failure to respond to endogenous epinephrine as assessed by propranolol. Timed-pregnant dams were gavage fed 100 mg of nitrofen at 9.5-day gestation to induce CDH in the fetuses, and distal air space fluid absorption experiments were carried out on
Folkesson Hans G; Chapin Cheryl J; Beard LaMonta L; Ertsey Robert; Matthay Michael A; Kitterman Joseph A
American journal of physiology. Lung cellular and molecular physiology
2006
2006-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajplung.00124.2005" target="_blank" rel="noreferrer noopener">10.1152/ajplung.00124.2005</a>
Impaired alveolar liquid clearance after 48-h isoproterenol infusion spontaneously recovers by 96 h of continuous infusion.
*Pulmonary Alveoli/drug effects/metabolism; Adrenergic; Adrenergic beta-Antagonists/pharmacology; Animals; beta-2/metabolism; Bronchodilator Agents/administration & dosage/*pharmacology; Isoproterenol/administration & dosage/*pharmacology; Lung/drug effects/metabolism; Male; Propranolol/pharmacology; Rats; Receptors; Sprague-Dawley; Terbutaline/pharmacology; Time Factors
We previously demonstrated that 48-h isoproterenol (Iso) infusion in rats impaired the ability of beta-adrenoceptor (beta-AR) agonists to increase alveolar liquid clearance (ALC). In this study, we determined whether this impairment persisted over longer time periods by infusing 400 mug.kg(-1).h(-1) Iso by osmotic minipump for 24-144 h (n = 6-7/group). ALC in control rats was 19.0 +/- 2.4 (SD)% of instilled volume absorbed per hour. In Iso-infused rats, ALC was elevated at 24 h (34.9 +/- 2.4%) and decreased at 48 h (15.2 +/- 4.4%) and had recovered to 24 h values at 96 h (37.3 +/- 3.8%) and 144 h (35.2 +/- 3.3%). Plasma Iso concentrations remained elevated at all Iso infusion times. Peripheral lung beta(2)-AR expression exhibited a parallel time course, with a reduction in expression observed at 48 h, followed by an increase to 24 h values at 96 and 144 h. Propranolol prevented the increase in ALC observed at 96 and 144 h, indicating that the recovery in ALC was mediated by a recovery of beta-AR function and beta-AR signaling. ALC at 96 and 144 h could not be further increased by terbutaline, indicating that ALC was maximally stimulated. These data indicate that recovery of beta-AR-stimulated ALC can occur in the continued presence of Iso and is mediated by a recovery of the ability of the distal lung epithelium to respond to beta-AR stimulation.
Maron Michael B; Folkesson Hans G; Stader Sonya M; Hodnichak Cheryl M
American journal of physiology. Lung cellular and molecular physiology
2006
2006-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajplung.00022.2006" target="_blank" rel="noreferrer noopener">10.1152/ajplung.00022.2006</a>
Type VI collagen induces cardiac myofibroblast differentiation: implications for postinfarction remodeling.
Angiotensin II/pharmacology; Animals; Cell Differentiation/*drug effects; Cell Proliferation/drug effects; Cells; Collagen Type I/physiology; Collagen Type III/physiology; Collagen Type VI/*physiology; Coronary Vessels/pathology; Cultured; Extracellular Matrix/*physiology; Fibroblasts/*cytology; Fibrosis/etiology; Ligation; Male; Myocardial Infarction/physiopathology; Rats; Sprague-Dawley; Ventricular Remodeling/*physiology
Cardiac fibroblast (CF) proliferation and differentiation into hypersecretory myofibroblasts can lead to excessive extracellular matrix (ECM) production and cardiac fibrosis. In turn, the ECM produced can potentially activate CFs via distinct feedback mechanisms. To assess how specific ECM components influence CF activation, isolated CFs were plated on specific collagen substrates (type I, III, and VI collagens) before functional assays were carried out. The type VI collagen substrate potently induced myofibroblast differentiation but had little effect on CF proliferation. Conversely, the type I and III collagen substrates did not affect differentiation but caused significant induction of proliferation (type I, 240.7 +/- 10.3%, and type III, 271.7 +/- 21.8% of basal). Type I collagen activated ERK1/2, whereas type III collagen did not. Treatment of CFs with angiotensin II, a potent mitogen of CFs, enhanced the growth observed on types I and III collagen but not on the type VI collagen substrate. Using an in vivo model of myocardial infarction (MI), we measured changes in type VI collagen expression and myofibroblast differentiation after post-MI remodeling. Concurrent elevations in type VI collagen and myofibroblast content were evident in the infarcted myocardium 20-wk post-MI. Overall, types I and III collagen stimulate CF proliferation, whereas type VI collagen plays a potentially novel role in cardiac remodeling through facilitation of myofibroblast differentiation.
Naugle Jennifer E; Olson Erik R; Zhang Xiaojin; Mase Sharon E; Pilati Charles F; Maron Michael B; Folkesson Hans G; Horne Walter I; Doane Kathleen J; Meszaros J Gary
American journal of physiology. Heart and circulatory physiology
2006
2006-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1152/ajpheart.00321.2005" target="_blank" rel="noreferrer noopener">10.1152/ajpheart.00321.2005</a>
A noninflammatory interleukin-1beta fragment stimulates fetal lung fluid absorption in guinea pigs.
Absorption; Adrenergic; Adrenocorticotropic Hormone/blood; Animals; beta/analysis; Body Fluids/drug effects/metabolism; Epithelial Sodium Channels/genetics; Female; Fetus/drug effects; Guinea Pigs; Hydrocortisone/biosynthesis; Interleukin-1beta/*pharmacology; Lung/*drug effects/metabolism; Messenger/analysis; Oxytocin/pharmacology; Peptide Fragments/*pharmacology; Pregnancy; Receptors; RNA
We have previously demonstrated that full-length interleukin (IL)-1beta can induce and stimulate lung fluid absorption in near-term guinea pig fetuses via stimulation of fetal cortisol synthesis and release. To develop a potentially clinically useful drug, we tested the hypothesis that maternal administration of a noninflammatory IL-1beta-fragment (IL-1beta(Fr)) induced cortisol synthesis and stimulated lung fluid absorption in preterm fetuses. IL-1beta(Fr) was administered s.c. daily to timed-pregnant guinea pigs for 3 days with and without simultaneous cortisol synthesis inhibition by metyrapone. Fetuses were obtained by abdominal hysterotomy at 61 and 68 days gestation and instilled with isosmolar 5% albumin into the lungs, and lung fluid absorption was measured over 1 h by mass balance. Lung fluid absorption was induced at 61 days and stimulated at 68 days gestation by IL-1beta(Fr), which both were attenuated by cortisol synthesis inhibition. Moreover, induction of labor by oxytocin stimulated lung fluid absorption at 61 days but had no stimulatory effect at 68 days gestation when given with the IL-1beta(Fr). Plasma adrenocorticotropin and cortisol concentrations were increased by IL-1beta(Fr) at 61 days gestation and remained high but unstimulated by IL-1beta(Fr) at 68 days gestation, and metyrapone always reduced cortisol concentrations. Prenatal lung fluid absorption, when present as well as IL-1beta(Fr)-induced, was always propranolol- and amiloride-sensitive, suggesting that beta-adrenoceptor stimulation and the epithelial Na(+) channel (ENaC) were critical for the induced/stimulated lung fluid absorption. ENaC expression was increased by IL-1beta(Fr) and attenuated by cortisol synthesis inhibition. Thus, our results suggest a potential clinical use of IL-1beta(Fr) therapeutically to induce lung fluid absorption in fetuses at risk of preterm delivery.
Li Tianbo; Varadarajulu Shilpa; Beard LaMonta L; Yun June; Folkesson Hans G
The Journal of pharmacology and experimental therapeutics
2007
2007-02
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1124/jpet.106.111369" target="_blank" rel="noreferrer noopener">10.1124/jpet.106.111369</a>
Anthocyanin-rich black currant (Ribes nigrum L.) extract affords chemoprevention against diethylnitrosamine-induced hepatocellular carcinogenesis in rats.
Animals; Anthocyanins/*therapeutic use; Anticarcinogenic Agents/therapeutic use; Apoptosis/drug effects; bcl-2-Associated X Protein/metabolism; Cell Proliferation/drug effects; Chemoprevention; Diethylnitrosamine; Down-Regulation; Experimental/metabolism/pathology; Liver Neoplasms; Liver Neoplasms/chemically induced/*prevention & control; Liver/pathology; Male; Phenobarbital; Plant Extracts/*therapeutic use; Proliferating Cell Nuclear Antigen/metabolism; Proto-Oncogene Proteins c-bcl-2/metabolism; Rats; Ribes/chemistry; Sprague-Dawley; Up-Regulation
Anthocyanins are known to possess potent anticarcinogenic properties against several cancers thus demonstrating potential for cancer prevention. Black currant (Ribes nigrum L., Grossulariaceae) fruits have a high anthocyanin content. This "superfruit" is known to possess various pharmacological effects including alleviation of chronic oxidative stress and inflammation. In contrast to a large volume of literature on the health benefits of black currant, limited evidence on antitumor effects of black currant exists with virtually no data on the prevention of experimental carcinogenesis. In the current study, we have investigated the chemopreventive effects of an anthocyanin-rich black currant skin extract (BCSE) utilizing our well-characterized model of rat liver carcinogenesis. Initiation of hepatocarcinogenesis was done by intraperitoneal injection of diethylnitrosamine (DENA) followed by promotion with phenobarbital. The rats were exposed to dietary BCSE for 4 weeks prior to initiation, and the treatment was continued for 22 consecutive weeks. BCSE dose-dependently decreased the incidence, total number, multiplicity, size and volume of preneoplastic hepatic nodules. The antihepatocarcinogenic effect of BCSE was confirmed by histopathological examination of liver sections. Immunohistochemical analysis of proliferating cell nuclear antigen and DNA fragmentation revealed BCSE-mediated inhibition of abnormal cell proliferation and induction of apoptosis in
Bishayee Anupam; Mbimba Thomas; Thoppil Roslin J; Haznagy-Radnai Erzsebet; Sipos Peter; Darvesh Altaf S; Folkesson Hans G; Hohmann Judit
The Journal of nutritional biochemistry
2011
2011-11
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.jnutbio.2010.09.001" target="_blank" rel="noreferrer noopener">10.1016/j.jnutbio.2010.09.001</a>
Stimulation of distal airspace fluid clearance in guinea pigs involves bumetanide-sensitive ion transport.
*Ion Transport; Absorption; Adrenergic; Adrenergic beta-Antagonists/pharmacology; Animals; beta/physiology; Body Fluids/*metabolism; Bumetanide/*pharmacology; Diuretics/*pharmacology; Epinephrine/blood; Epithelium; Fetus/metabolism; Gestational Age; Guinea Pigs; Lung/*embryology/metabolism; Newborn; Propranolol/pharmacology; Receptors
OBJECTIVE: This study was undertaken to test the hypothesis that beta-adrenoceptor stimulation of fetal lung fluid absorption in near-term guinea pig fetuses involves bumetanide-sensitive ion transport. STUDY DESIGN: Fetuses were obtained from timed-pregnant guinea pigs at 61 to 69 days' gestation with and without oxytocin-induced preterm labor. The fetuses were placed on continuous positive airway pressure oxygenation, and an isosmolar 5% albumin solution was instilled into the lungs. Distal airspace fluid clearance was measured over 1 hour from the increase in distal airspace protein concentration as fluid was reabsorbed with and without the Cl(-) transport inhibitor bumetanide. RESULTS: Fetal lungs began to absorb distal airspace fluid at 64 to 66 days' gestation, and at birth, distal airspace fluid clearance rapidly quadrupled. Labor induction by oxytocin stimulated distal airspace fluid clearance. Distal airspace fluid clearance, when present, was sensitive to propranolol-inhibition and depended on beta-adrenoceptor stimulation. Fluid secretion at 61 days' gestation was reduced by bumetanide instillation. Bumetanide addition was only inhibitory when distal airspace fluid clearance was propranolol-sensitive. CONCLUSION: Beta-adrenoceptor stimulation from endogenous fetal epinephrine increased fetal distal airspace fluid clearance and involved bumetanide-sensitive ion transport.
Ye Xin; Norlin Andreas; Folkesson Hans G
American journal of obstetrics and gynecology
2004
2004-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.ajog.2003.09.074" target="_blank" rel="noreferrer noopener">10.1016/j.ajog.2003.09.074</a>
Fetal lung epithelial ion channels relocate in the cell membrane during late gestation.
Animals; Blotting; Caveolin 1/*metabolism; Cell Membrane/*metabolism; Cl transport; Cystic Fibrosis Transmembrane Conductance Regulator/*metabolism; distal lung fluid absorption; Epithelial Sodium Channels/*metabolism; Epithelium/embryology/*metabolism; Female; fetal lung development; Fetus/*metabolism; Gestational Age; Guinea Pigs; IL-1beta; Immunoprecipitation; Interleukin-1beta/metabolism; Ion Channels/metabolism; Lung/embryology/*metabolism; Na transport; Western
Near the end of gestation, the direction of ion and fluid flow across the alveolar epithelium rapidly changes from secretion to absorption. Thus, the relative cell membrane location of epithelial Na channels (ENaCs) and cystic fibrosis transmembrane regulator (CFTR) Cl channels during late fetal lung development and after maternal interleukin-1beta (IL-1beta) pretreatment was the focus of our study. Western blot analysis after sucrose gradient separation of caveolin-1-(Cav-1)-rich membrane regions (CRR) and Cav-1-poor membrane (non-CRR) revealed primary CRR ENaC localization at gestation day (GD) 61 in guinea pigs. Correlating with the natural induction of distal lung fluid absorption, ENaC appeared in the non-CRR cell membrane regions at GD68. Conversely, CFTR was present in the non-CRR cell membrane regions at GD61 and in the CRRs at GD68.
Beard LaMonta L; Li Tianbo; Hu Yang; Folkesson Hans G
Anatomical record (Hoboken, N.J. : 2007)
2011
2011-09
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1002/ar.21363" target="_blank" rel="noreferrer noopener">10.1002/ar.21363</a>