bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of posture on left ventricular long- and short-axis shortening
Authors: Sundblad Patrik , Wranne Bengt
Published: March 2015

End-diastolic volume and left ventricular stroke volume are increased in the supine compared with upright position, but the contribution of long-axis (LAS) and short-axis shortening (SAS) to these changes with change in posture has not been established. We examined long- and short-axis motion and dimensions with echocardiography in 10 healthy subjects in the upright and supine position. Long-axis length at end diastole was almost identical, whereas the diastolic short-axis diameter was increased in the supine position. At end systole, there was a decreased long-axis length and increased short-axis length in the supine vs. upright position. Both LAS and SAS were enhanced in supine vs. upright positions [LAS: 9.3 ± 2.2 vs. 15.1 ± 3.1 mm ( P < 0.001); SAS: 12.7 ± 3.2 vs. 16.3 ± 2.8 mm ( P < 0.001)], presumably via Starling mechanisms. LAS increased more in the lateral part of the mitral annulus than in the septal part [7.7 ± 2.6 vs. 4.0 ± 2.8 mm ( P < 0.006)], which implies that the more spherical form, in the supine position, induces more stretch at the lateral free wall than in the ventricular septum. These findings support the notion that Starling mechanisms affect systolic LAS.
Aging-induced decrease in the PPAR-α level in hearts is improved by exercise training
Authors: Iemitsu Motoyuki , Miyauchi Takashi , Maeda Seiji , Tanabe Takumi , Takanashi Masakatsu , Irukayama-Tomobe Yoko , Sakai Satoshi , Ohmori Hajime , Matsuda Mitsuo , Yamaguchi Iwao
Published: March 2015

Peroxisome proliferator-activated receptor (PPAR)-α, a transcriptional activator, regulates genes of fatty acid (FA) metabolic enzymes. To study the contribution of PPAR-α to exercise training-induced improvement of FA metabolic capacity in the aged heart, we investigated whether PPAR-α signaling and expression of its target genes in the aged heart are affected by exercise training. We used hearts of sedentary young rat (4 mo old), sedentary aged rat (23 mo old), and swim-trained aged rat (23 mo old, training for 8 wk). The mRNA and protein expression of PPAR-α in the heart was significantly lower in the sedentary aged rats compared with the sedentary young rats and was significantly higher in the swim-trained aged rats compared with the sedentary aged rats. The activity of PPAR-α DNA binding to the transcriptional regulating region on the FA metabolic enzyme genes, the mRNA expression of 3-hydroxyacyl CoA dehydrogenase (HAD) and carnitine palmitoyl transferase-I, which are PPAR-α target genes, and the enzyme activity of HAD in the heart altered in association with changes of the myocardial PPAR-α mRNA and protein levels. These findings suggest that exercise training improves aging-induced downregulation in myocardial PPAR-α-mediated molecular system, thereby contributing to the improvement of the FA metabolic enzyme activity in the trained-aged hearts.
Effects of varying impulse number on cotransmitter contributions to sympathetic vasoconstriction in rat tail artery
Authors: Bradley Eamonn , Law Andrea , Bell David , Johnson Christopher D.
Published: March 2015

We examined the contributions of the cotransmitters norepinephrine (NE), ATP, and neuropeptide Y (NPY) to sympathetically evoked vasoconstriction in the rat tail artery in isolated vascular rings by using 1–100 stimulation impulses at 20 Hz. Phentolamine (2 μM), the α-adrenoceptor antagonist, markedly reduced responses to all stimuli, although responses to lower impulse numbers were reduced less than responses to longer trains. The purinergic receptor antagonist suramin (100 μM) reduced all responses, but to a much greater extent with few impulse trains. Responses were further reduced or abolished by addition of the second antagonist. Any remaining responses were abolished by the NPY-Y1 receptor antagonist BIBP-3226 (75 nM). NPY had a direct agonist action and potentiated sympathetically mediated responses. NPY (75 nM) potentiated responses and BIBP-3226 decreased responses to 2- and 20-impulse trains. Both affected responses from 2 impulses to >20 impulses, but there was no preferential effect on purinergic contributions to responses because neurally released NPY potentiated both “pure” NE and ATP responses equally. We conclude that all three cotransmitters contribute significantly to vascular responses and their contribution varies markedly with impulse numbers. There is considerable synergy between cotransmitters, especially with lower impulse numbers where NPY contributions are greater than expected.
Mechanism of glutamate stimulation of CO production in cerebral microvessels
Authors: Leffler Charles W. , Balabanova Liliya , Fedinec Alexander L. , Waters Christopher M. , Parfenova Helena
Published: March 2015

Dilation of piglet pial arterioles to glutamate involves carbon monoxide (CO) produced from heme by heme oxygenase-2 (HO-2). Piglet cerebral microvessels and endothelial and smooth muscle cells grown on microcarrier beads were used to address the hypothesis that glutamate increases endothelial CO production by increasing HO-2 catalytic activity. CO was measured by gas chromatography/mass spectrometry. Glutamate increased CO production from endogenous heme by cerebral microvessels, endothelial cells, and smooth muscle cells. Glutamate increased the conversion of exogenous heme to CO. Protein tyrosine kinase inhibition blocked glutamate stimulation of CO production. Inhibition of protein tyrosine phosphatases stimulated CO production. Conversely, neither phorbol myristate acetate nor H-7 changed glutamate stimulation of CO production. The mechanism of HO-2 stimulation by glutamate appears to be independent of cytosolic Ca, because stimulation of CO production by glutamate was the same in Careplete medium, Ca-free medium with ionomycin, and Careplete medium with ionomycin. Therefore, glutamate appears to increase HO-2 catalytic activity in cerebral microvessels via a tyrosine kinase mediated pathway.
Weight loss improves neurovascular and muscle metaboreflex control in obesity
Authors: Trombetta Ivani Credidio , Batalha Luciana T. , Rondon Maria Urbana P. B. , Laterza Mateus Camaroti , Kuniyoshi Fátima H. S. , Gowdak Márcia M. G. , Barretto Antonio Carlos P. , Halpern Alfredo , Villares Sandra M. F. , Negrão Carlos Eduardo
Published: March 2015

We studied the effects of a hypocaloric diet (D, n = 24, age: 32.2 ± 1.4 yr, body mass index: 34.7 ± 0.5 kg/m2) and a hypocaloric diet associated with exercise training (D + T, n = 25, age: 32.3 ± 1.3 yr, body mass index: 32.9 ± 0.4 kg/m2) on muscle metaboreflex control, muscle sympathetic nerve activity (MSNA, microneurography), blood pressure, and forearm blood flow (plethysmography) levels during handgrip exercise at 10% and 30% of maximal voluntary contraction in normotensive obese women. An additional 10 women matched by age and body mass index were studied as a nonadherent group. D or D + T significantly decreased body mass index. D or D + T significantly decreased resting MSNA (bursts/100 heartbeats). The absolute levels of MSNA were significantly lower throughout 10% and 30% exercise after D or D + T, although no change was found in the magnitude of response of MSNA. D + T, but not D, significantly increased resting forearm vascular conductance. D + T significantly increased the magnitude of the response of forearm vascular conductance during 30% exercise. D or D + T significantly increased MSNA levels during posthandgrip circulatory arrest when muscle metaboreflex is isolated. In conclusion, weight loss improves muscle metaboreflex control in obese women. Weight loss reduces MSNA, which seems to be centrally mediated. Weight loss by D + T increases forearm vascular conductance at rest and during exercise in obese individuals.
Blood monocyte concentration is critical for enhancement of collateral artery growth
Authors: Heil Matthias , Ziegelhoeffer Tibor , Pipp Frederic , Kostin Sawa , Martin Sandra , Clauss Matthias , Schaper Wolfgang
Published: March 2015

0.1152/ajpheart. 01098.2001.—Arteriogenesis has been associated with the presence of monocytes/macrophages within the collateral vessel wall. We tested the hypothesis that arteriogenesis is functionally linked to the concentration of circulating blood monocytes. Monocyte concentrations in peripheral blood were manipulated by single injections of the antimetabolite 5-fluorouracil (5-FU), resulting in a marked rebound effect in New Zealand White rabbits. Collateral artery growth was assessed by the use of a model of acute femoral artery ligation. Seven days after ligation, collateral conductance and the number of visible collateral arteries were increased in the rebound group. This increase was accompanied by an increased monocyte accumulation as demonstrated by immunohistology in the thigh 3 days after surgery. In a second animal model (129S2/SvHsd mice), 5-FU treatment caused a remarkable decrease in blood monocyte numbers at day 4, followed by a rebound effect at day 12. Foot blood flow, assessed by laser-Doppler imaging before and at various time points after surgery, increased from day 7 through day 21 in mice from the rebound group. In contrast, ligation during the phase of monocyte depletion resulted in a reduction of blood flow reconstitution. This inhibition could be reversed by an injection of isolated monocytes. In conclusion, we have demonstrated a functional link between the monocyte concentration in the peripheral blood and the enhancement of arteriogenesis.
Nonreentrant focal activations in pulmonary veins in canine model of sustained atrial fibrillation
Authors: Zhou Shengmei , Chang Che-Ming , Wu Tsu-Juey , Miyauchi Yasushi , Okuyama Yuji , Park Angela M. , Hamabe Akira , Omichi Chikaya , Hayashi Hideki , Brodsky Lauren A. , Mandel William J. , Ting Chih-Tai , Fishbein Michael C. , Karagueuzian Hrayr S. , Chen Peng-Sheng
Published: March 2015

Repetitive rapid activities are present in the pulmonary veins (PVs) in dogs with pacing-induced sustained atrial fibrillation (AF). The mechanisms are unclear. We induced sustained (>48 h) AF by rapidly pacing the left atrium (LA) in six dogs. High-density computerized mapping was done in the PVs and atria. Results show repetitive focal activations in all dogs and in 12 of 18 mapped PVs. Activation originated from the middle of the PV and then propagated to the LA and distal PV with conduction blocks. The right atrium (RA) was usually activated by a single large wavefront. Mean AF cycle length in the PVs (left superior, 82 ± 6 ms; left inferior, 83 ± 6 ms; right inferior, 83 ± 4 ms) and LA posterior wall (87 ± 5 ms) were significantly ( P < 0.05) shorter than those in the LA anterior wall (92 ± 4 ms) and RA (107 ± 5 ms). PVs in normal dogs did not have focal activations during induced AF. No reentrant wavefronts were demonstrated in the PVs. We conclude that nonreentrant focal activations are present in the PVs in a canine model of pacing-induced sustained AF.
Chronic effects of angiotensin-converting enzyme inhibition on kinin receptor binding sites in the rat spinal cord
Authors: Ongali Brice , Buck Hudson de Sousa , Cloutier Frank , Legault Francine , Regoli Domenico , Lambert Chantal , Thibault Gaétan , Couture Réjean
Published: March 2015

With the use of in vitro receptor autoradiography, this study aims at determining whether the higher level of kinin B2 receptor density in the spinal cord of the spontaneously hypertensive rat (SHR) is secondary to arterial hypertension and whether chronic treatment with angiotensin I-converting enzyme inhibitors (ACEI) can regulate neuronal B1 and B2 receptors. SHR received, from the age of 4 wk, one of the two ACEI (lisinopril or zofenopril, 10 mg · kg−1 · day−1) or for comparison, the selective AT1 antagonist (losartan, 20 mg · kg−1 · day−1) in their drinking water for a period of 4, 12, and 20 wk. Age-matched untreated SHR and Wistar-Kyoto rats (WKY) were used as controls. B2 receptor binding sites in most laminae were higher in SHR than in WKY from the age of 8 to 24 wk. Whereas B1receptor binding sites were significantly present in young SHR and WKY, they were barely detectable in adult rats. ACEI (16 and 24 wk) and AT1 antagonist (24 wk) enhanced the number of B2 without changing B1 receptor binding sites. However, at 8 wk the three treatments significantly increased B1 and decreased B2 receptors in lamina I. It is concluded that 1) the higher density of B2receptors in the spinal cord of SHR is not due to hypertension, 2) kinin receptors are regulated differently by ACEI in neuronal and vascular tissues, and 3) aging may have a profound impact on levels of B1 and B2receptors in the rat spinal cord.
Muscle pump function during locomotion: mechanical coupling of stride frequency and muscle blood flow
Authors: Sheriff Don D.
Published: March 2015

We imposed opposing oscillations in treadmill speed and grade on nine rats to test for direct mechanical coupling between stride frequency and hindlimb blood flow. Resting hindlimb blood flow was 15.5 ± 1.7 ml/min. For 90 s at 7.5 m/min, rats alternated walking at −10° for 10 s and +10° for 10 s. This elicited oscillations in hindlimb blood flow having an amplitude of 4.1 ± 0.5 ml/min (18% of mean flow) with a delay presumably due to metabolic vasodilation. Similar oscillations in speed (5.5–9.5 m/min) elicited oscillations in hindlimb blood flow (amplitude 3.4 ± 0.5 ml/min, 15% of mean flow) with less of a delay, possibly due to changes in vasodilation and muscle pump function. We then simultaneously imposed these speed and grade oscillations out of phase (slow uphill, fast downhill). The rationale was that the oscillations in vasodilation evoked by the opposing oscillations in speed and grade would cancel each other, thereby testing the degree to which stride frequency affects hindlimb blood flow directly (i.e., muscle pumping). Opposing oscillations in speed and grade evoked oscillations in hindlimb blood flow having an amplitude of 3.3 ± 0.6 ml/min (16% of mean flow) with no delay and directly in phase with the changes in speed and stride frequency. The finding that hindlimb blood flow changes directly with speed (when vasodilation caused by changes in speed and grade oppose each other) indicates that there is a direct coupling of stride frequency and hindlimb blood flow (i.e., muscle pumping).
Expression levels strongly affect ligand-induced sequestration of B2bradykinin receptors in transfected cells
Authors: Faussner Alexander , Bauer Alexandra , Kalatskaya Irina , Jochum Marianne , Fritz Hans
Published: March 2015

Transfection of cells with expression vectors is one of the most important tools used to assess the effects of receptor mutations on ligand-induced receptor sequestration. Most transfection methods give rise to transiently or stably transfected clones with a wide range of receptor expression levels that may also depend on the mutations made. It is, therefore, important to determine how the regulation of the receptors depends on their numbers per cell. In Chinese hamster ovary (CHO) and human embryonic kidney (HEK)-293 cells expressing high levels of B2kinin receptors, we observed poor sequestration indicated by <20% reduction in cell surface receptor number after 10 min of stimulation with 1 μM bradykinin (BK) compared with >70% in low-expressing cells. Whereas the rate of [3H]BK internalization (internalized [3H]BK in percentage of total bound [3H]BK) in low-expressing cells was independent of the ligand-concentration used, in high-expressing cells a strong rate decrease was observed with higher (>1 nM) concentrations. Lower ligand concentrations, however, led to internalization rates identical to those obtained in low-expressing cells. Transiently transfected HEK and COS-7 cells showed results similiar to those of stably high-expressing cells. Our results demonstrate the difficulty in determining the internalization pattern of (mutated) B2kinin receptors, and possibly of G protein-coupled receptors in general, using a sequestration assay in high-expressing cells or transiently transfected cells with high numbers of receptors per transfected cell. However, the receptor (mutant)-specific internalization rate can be measured, provided that the ligand concentrations used are below a threshold at which the internalization rate is still independent of the ligand concentration.
Increased calcium intake reduces plasma cholesterol and improves vasorelaxation in experimental renal failure
Authors: Jolma Pasi , Kööbi Peeter , Kalliovalkama Jarkko , Kähönen Mika , Fan Meng , Saha Heikki , Helin Heikki , Lehtimäki Terho , Pörsti Ilkka
Published: March 2015

Chronic renal failure (CRF) is associated with abnormal lipid metabolism and high prevalence of vascular complications. Calcium salts are commonly used in CRF as phosphate binders. Increased calcium intake may also lower plasma cholesterol and beneficially influence vascular tone. Therefore, we investigated the influence of increasing dietary calcium from 0.3% to 3.0% for 8 wk after 5/6 nephrectomy (NTX) on plasma cholesterol and mesenteric resistance vessel tone in male Sprague-Dawley rats. The groups were Sham, Sham-Calcium, NTX, and NTX-Calcium ( n = 10–11). Blood pressure was modestly elevated after NTX, whereas the plasma creatinine, urea nitrogen, phosphate, and parathyroid hormone levels were clearly increased. The high-calcium diet suppressed plasma phosphate and parathyroid hormone but was without effect on blood pressure. The NTX resulted in 1.6-fold elevation in plasma total cholesterol and 40% reduction in high density-to-low density lipoprotein ratio (HDL/LDL). However, the lipid profile in NTX rats on the high-calcium diet did not differ from sham-operated controls. The endothelium-mediated relaxations induced by acetylcholine were impaired in NTX rats, whereas the response was normalized by a high-calcium diet. No differences in vasorelaxations by the endothelium-independent vasodilator nitroprusside were detected. In conclusion, improved vasorelaxation after a high-calcium diet could be due to reduced plasma total cholesterol and ameliorated HDL/LDL ratio, although decreased plasma phosphate and parathyroid hormone may also play a significant role in the vascular effects of increased calcium intake.
Transmural recording of monophasic action potentials
Authors: Zhou Xiaohong , Huang Jian , Ideker Raymond E.
Published: March 2015

To investigate the possibility of transmural recording of repolarization through the ventricular wall, KCl monophasic action potential (MAP) electrodes positioned along plunge needles were developed and tested. The MAP electrode consists of a silver wire surrounded by agarose gel containing KCl, which slowly eluted into the adjacent tissue to depolarize it. In six dogs, a plunge needle containing three KCl MAP electrodes was inserted into the left ventricle to simultaneously record from the subepicardium, midwall, and subendocardium. In six pigs, eight plunge needles containing three KCl MAP electrodes and two plunge needles containing similar electrodes except for the absence of KCl were inserted into the ventricles. In three guinea pig papillary muscles, a KCl electrode was used to record MAPs along with two microelectrodes for recording transmembrane potentials. Transmural MAP recordings could be made for >1 h in dogs and >2 h in pigs with a significant decrease in MAP amplitude over time but without a significant change in MAP duration. With the electrodes without KCl in pigs, the injury potentials subsided in <30 min. When the pacing rate was changed to alter the action potential duration and refractory period in dogs, the MAP duration correlated with the local effective refractory period ( r = 0.94). The time course of the MAP duration recorded with a KCl MAP electrode in guinea pig papillary muscles corresponded well with that of the transmembrane potential recorded with an adjacent microelectrode. It is possible to record transmural repolarization of the ventricles with KCl MAP electrodes on plunge needles. The MAP is caused by the KCl rather than being a nonspecific injury potential.
Calcium channels contribute to the decrease in blood pressure of pregnant rats
Authors: Simaan May , Cadorette Chanterelle , Poterek Matthieu , St-Louis Jean , Brochu Michèle
Published: March 2015

Pregnancy is associated with hemodynamic changes such as reduced vascular resistance and blood pressure. We reported that, during late pregnancy, the activity of voltage-dependent calcium channels (VDCC) is altered in the adrenal cortex and vascular smooth muscle. These observations suggested that the late pregnancy-induced decrease in blood pressure is linked to diminished VDCC function. We attempted to prevent pregnancy-induced reduced blood pressure with a calcium channel activator (CGP 28392) in pregnant rats and to mimic it by administration of a calcium channel blocker (nifedipine) to nonpregnant rats. Treatment was given from the 15th day of gestation for 7 days. The systolic blood pressure of CGP 28392-treated pregnant rats rose transiently for 2 days and then declined toward values of nontreated pregnant controls, although remaining higher. However, nonpregnant rats maintained their high arterial pressure throughout CGP 28392 treatment. Nifedipine lowered the blood pressure in nonpregnant rats to values of nontreated term-pregnant controls. Both agents did not affect body weight, water or food intake, plasma renin activity, and plasma aldosterone or corticosterone levels. Nifedipine and CGP 28392 treatment of nonpregnant and pregnant animals, respectively, did not modify the response of aortic rings to KCl. These results show that VDCC activation caused hypertension, which modified the extent of the decrease in blood pressure at the end of pregnancy.
Induction of oxidative stress and disintegrin metalloproteinase in human heart end-stage failure
Authors: Hunt Matthew J. , Aru Giorgio M. , Hayden Melvin R. , Moore Charles K. , Hoit Brian D. , Tyagi Suresh C.
Published: March 2015

Collagen degradation is required for the creation of new integrin binding sites necessary for cell survival. However, a complete separation between the matrix and the cell leads to apoptosis, dilatation, and failure. Previous studies have demonstrated increased metalloproteinase activity in the failing myocardium. To test the hypothesis that disintegrin metalloproteinase (DMP) is induced in human heart end-stage failure, left ventricle tissue from ischemic cardiomyopathic (ICM, n = 10) and dilated cardiomyopathic (DCM, n = 10) human hearts were obtained at the time of orthotopic cardiac transplant. Normal ( n = 5) tissue specimens were obtained from unused hearts. The levels of reduced oxygen species (ROS) were 12 ± 2, 25 ± 3, and 16 ± 2 nmol (means ± SE, P < 0.005) in normal, ICM, and DCM, respectively, by spectrofluorometry. The percent levels of endothelial cells were 100 ± 15, 35 ± 19, and 55 ± 11 in normal, ICM, and DCM, respectively, by CD31 labeling. The levels of nitrotyrosine by Western analysis were significantly increased, and endothelial nitric oxide (NO) by the Griess method was decreased in ICM and DCM compared with normal tissue. The synthesis and degradation of β1-integrin and connexin 43 were significantly increased in ICM and DCM compared with normal hearts by Western analysis. Levels of DMP were increased, and levels of cardiac inhibitor of metalloproteinase (CIMP) were decreased. Aggrecanase activity of DMP was significantly increased in ICM and DCM hearts compared with normal. These results suggest that the occurrence of cardiomyopathy is significantly confounded by the increase in ROS, nitrotyrosine, and DMP activity. This increase is associated with decreased NO, endothelial cell density, and CIMP. In vitro, treatment of CIMP abrogated the DMP activity. The treatment with CIMP may prevent degradation of integrin and connexin and ameliorate heart failure.
PI3K is required for proliferation and migration of human pulmonary vascular smooth muscle cells
Authors: Goncharova Elena A. , Ammit Alaina J. , Irani Carla , Carroll Richard G. , Eszterhas Andrew J. , Panettieri Reynold A. , Krymskaya Vera P.
Published: March 2015

Human vascular smooth muscle cell proliferation and migration contribute to vascular remodeling in pulmonary hypertension and atherosclerosis. The precise mechanisms that regulate structural remodeling of the vessel wall remain unknown. This study tests the hypothesis that phosphatidylinositol 3-kinase (PI3K) activation is both necessary and sufficient to mediate human pulmonary vascular smooth muscle (PVSM) cell proliferation and migration. Microinjection of human PVSM cells with a dominant-negative class IA PI3K inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis by 65% ( P < 0.001; χ2analysis) compared with cells microinjected with control plasmid, whereas microinjection of cells with a constitutively active class IA PI3K (p110*-CA) was sufficient to induce DNA synthesis (mitotic index of p110*-CA-microinjected cells was 15% vs. 3% in control cells; P < 0.01). Transfection of PVSM cells with p110*-CA was also sufficient to promote human PVSM cell migration. In parallel experiments, stimulation of human PVSM cells with PDGF induced PI3K-dependent activation of Akt, p70 S6 kinase, and ribosomal protein S6 but not mitogen-activated protein kinase. PDGF-induced proliferation and migration was inhibited by LY-294002. These results demonstrate that PI3K signaling is both necessary and sufficient to mediate human PVSM cell proliferation and migration and suggest that the activation of PI3K may play an important role in vascular remodeling.
SP-B deficiency causes respiratory failure in adult mice
Authors: Melton Kristin R. , Nesslein Lori L. , Ikegami Machiko , Tichelaar Jay W. , Clark Jean C. , Whitsett Jeffrey A. , Weaver Timothy E.
Published: March 2015

Targeted deletion of the surfactant protein (SP)-B locus in mice causes lethal neonatal respiratory distress. To assess the importance of SP-B for postnatal lung function, compound transgenic mice were generated in which the mouse SP-B cDNA was conditionally expressed under control of exogenous doxycycline in SP-B-/-mice. Doxycycline-regulated expression of SP-B fully corrected lung function in compound SP-B-/-mice and protected mice from respiratory failure at birth. Withdrawal of doxycycline from adult compound SP-B-/-mice resulted in decreased alveolar content of SP-B, causing respiratory failure when SP-B concentration was reduced to <25% of normal levels. Decreased SP-B was associated with low alveolar content of phosphatidylglycerol, accumulation of misprocessed SP-C proprotein in the air spaces, increased protein content in bronchoalveolar lavage fluid, and altered surfactant activity in vitro. Consistent with surfactant dysfunction, hysteresis, maximal tidal volumes, and end expiratory volumes were decreased. Reduction of alveolar SP-B content causes surfactant dysfunction and respiratory failure, indicating that SP-B is required for postnatal lung function.
C/EBP transcription factors in the lung epithelium
Authors: Cassel Tobias N. , Nord Magnus
Published: March 2015

During recent years, the biological roles of CCAAT/enhancer binding proteins (C/EBPs) in the lung have started to be uncovered. C/EBPs form a family within the basic region-leucine zipper class of transcription factors. In the lung epithelium C/EBPα, -β, and -δ are expressed. Lung-specific target genes for these transcription factors include the surfactant proteins A and D, the Clara cell secretory protein, and the P450 enzyme CYP2B1. As more information is gathered, a picture is emerging in which C/EBPα has a role in regulating proliferation as well as differentiation-dependent gene expression, whereas C/EBPβ and -δ, in addition to a partly overlapping role in regulating expression of differentiation markers, also seem to be involved in responses to injury and hormones.
Guanylyl cyclases, nitric oxide, natriuretic peptides, and airway smooth muscle function
Authors: Hamad Ahmed M. , Clayton Andrew , Islam Baharul , Knox Alan J.
Published: March 2015

Airway smooth muscle (ASM) plays an important role in asthma pathophysiology through its contractile and proliferative functions. The cyclic nucleotides adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP) are second messengers capable of mediating the effects of a variety of drugs and hormones. There is a large body of evidence to support the hypothesis that cAMP is a mediator of the ASM's relaxant effects of drugs, such as β2-adrenoceptor agonists, in human airways. Although most attention has been paid to this second messenger and the signal transduction pathways it activates, recent evidence suggests that cGMP is also an important second messenger in ASM with important relaxant and antiproliferative effects. Here, we review the regulation and function of cGMP in ASM and discuss the implications for asthma pathophysiology and therapeutics. Recent studies suggest that activators of soluble and particulate guanylyl cyclases, such as nitric oxide donors and natriuretic peptides, have both relaxant and antiproliferative effects that are mediated through cGMP-dependent and cGMP-independent pathways. Abnormalities in these pathways may contribute to asthma pathophysiology, and therapeutic manipulation may complement the effects of β2-adrenoceptor agonists.
Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein
Authors: Roper Jason M. , Staversky Rhonda J. , Finkelstein Jacob N. , Keng Peter C. , O'Reilly Michael A.
Published: March 2015

The unique morphology and cell-specific expression of surfactant genes have been used to identify and isolate alveolar type II epithelial cells. Because these attributes can change during lung injury, a novel method was developed for detecting and isolating mouse type II cells on the basis of transgenic expression of enhanced green fluorescence protein (EGFP). A line of transgenic mice was created in which EGFP was targeted to type II cells under control of the human surfactant protein (SP)-C promoter. Green fluorescent cells that colocalized by immunostaining with endogenous pro-SP-C were scattered throughout the parenchyma. EGFP was not detected in Clara cell secretory protein-expressing airway epithelial cells or other nonlung tissues. Pro-SP-C immunostaining diminished in lungs exposed to hyperoxia, consistent with decreased expression and secretion of intracellular precursor protein. In contrast, type II cells could still be identified by their intrinsic green fluorescence, because EGFP is not secreted. Type II cells could also be purified from single-cell suspensions of lung homogenates using fluorescence-activated cell sorting. Less than 1% of presorted cells exhibited green fluorescence compared with >95% of the sorted population. As expected for type II cells, ultrastructural analysis revealed that the sorted cells contained numerous lamellar bodies. SP-A, SP-B, and SP-C mRNAs were detected in the sorted population, but T1α and CD31 (platelet endothelial cell adhesion molecule) were not, indicating enrichment of type II epithelial cells. This method will be invaluable for detecting and isolating mouse type II cells under a variety of experimental conditions.
Lung alveoli: endogenous programmed destruction and regeneration
Authors: De Carlo Massaro Gloria , Radaeva Svetlana , Clerch Linda Biadasz , Massaro Donald
Published: March 2015

Mammalian alveoli, complex architectural and cellular units with dimensions that are linked to the organism's O2consumption (V˙o2), are thought to be destroyed only by disease and not to spontaneously regenerate. Calorie restriction of adult mammals lowersV˙o2, and ad libitum refeeding returnsV˙o2to pre-calorie-restriction values. We took advantage of these relationships and tested the hypothesis in adult mice that calorie restriction (two-thirds reduction for 2 wk) followed by ad libitum refeeding (3 wk) would cause alveolar destruction and regeneration, respectively. Calorie restriction diminished alveolar number 55% and alveolar surface area 25%. Refeeding fully reversed these changes. Neither manipulation altered lung volume. Within 72 h, calorie restriction increased alveolar wall cell apoptosis and diminished lung DNA (∼20%). By 72 h of refeeding, alveolar wall cell replication increased and lung DNA rose to amounts in mice that were never calorie restricted. We conclude that adult mice have endogenous programs to destroy and regenerate alveoli, thereby raising the danger of inappropriate activation but the possibility of therapeutic induction, if similar programs exist in humans.
Receptors and signaling pathway underlying relaxations to isoprostanes in canine and porcine airway smooth muscle
Authors: Catalli Adriana , Zhang Dawei , Janssen Luke J.
Published: March 2015

Using muscle bath techniques, we examined the inhibitory activities of several E- and F-ring isoprostanes in canine and porcine airway smooth muscle. 8-Isoprostaglandin E1and 8-isoprostaglandin E2(8-iso PGE2) reversed cholinergic tone in a concentration-dependent manner, whereas the F-ring isoprostanes were ineffective. Desensitization with 8-iso-PGE2and PGE2implicated isoprostane activity at the PGE2receptor (EP). We found that the inhibitory E-ring isoprostane responses were significantly augmented by rolipram (a type IV phosphodiesterase inhibitor), while 1 H-[1,2,4]-oxadiazolo[4,3- a]quinoxalin-1-one (a guanylate cyclase inhibitor) had no effect, suggesting a role for cAMP in isoprostane-mediated relaxations. 8-Iso-PGE2did not reverse KCl tone, suggesting that voltage-dependent Ca2+influx and myosin light chain kinase are not suppressed by isoprostanes. Patch-clamp studies showed marked suppression of K+currents by 8-iso-PGE2. We conclude that E-ring isoprostanes exert PGE2receptor-directed, cAMP-dependent relaxations in canine and porcine airway smooth muscle. This activity is not dependent on K+channel activation or the direct inhibition of voltage-operated Ca2+influx or myosin light chain kinase.
An endothelin receptor antagonist, SB-217242, inhibits airway hyperresponsiveness in allergic mice
Authors: Henry Peter J. , Mann Tracy S. , D'Aprile Angela C. , Self Glenn J. , Goldie Roy G.
Published: March 2015

Within the airways, endothelin-1 (ET-1) can exert a range of prominent effects, including airway smooth muscle contraction, bronchial obstruction, airway wall edema, and airway remodeling. ET-1 also possesses proinflammatory properties and contributes to the late-phase response in allergic airways. However, there is no direct evidence for the contribution of endogenous ET-1 to airway hyperresponsiveness in allergic airways. Allergic inflammation induced in mice by sensitization and challenge with the house dust mite allergen Der P1 was associated with elevated levels of ET-1 within the lung, increased numbers of eosinophils within bronchoalveolar lavage fluid and tissue sections, and development of airway hyperresponsiveness to methacholine ( P < 0.05, n = 6 mice per group). Treatment of allergic mice with an endothelin receptor antagonist, SB-217242 (30 mg · kg−1· day−1), during allergen challenge markedly inhibited airway eosinophilia (bronchoalveolar lavage fluid and tissue) and development of airway hyperresponsiveness. These findings provide direct evidence for a mediator role for ET-1 in development of airway hyperresponsiveness and airway eosinophilia in Der P1-sensitized mice after antigen challenge.
Surfactant protein A is a required mediator of keratinocyte growth factor after experimental marrow transplantation
Authors: Haddad Imad Y. , Milla Carlos , Yang Shuxia , Panoskaltsis-Mortari Angela , Hawgood Samuel , Lacey David L. , Blazar Bruce R.
Published: March 2015

We reported an association between the ability of recombinant human keratinocyte growth factor (rHuKGF) to upregulate the expression of surfactant protein A (SP-A) and to downregulate pulmonary inflammation that occurs after allogeneic bone marrow transplantation (BMT). To establish a causal relationship, rHuKGF (5 mg/kg) was administered subcutaneously for three consecutive days before irradiation to SP-A-sufficient and -deficient [SP-A(+/+) and SP-A(-/-), respectively] mice given inflammation-inducing allogeneic spleen T cells at the time of BMT. In contrast with SP-A(+/+) mice, rHuKGF failed to suppress the high levels of TNF-α, IFN-γ, and nitric oxide contained in bronchoalveolar lavage fluids collected on day 7 after BMT from SP-A(-/-) mice. Early post-BMT weight loss was attenuated by rHuKGF in both SP-A(+/+) and SP-A(-/-) recipients. In the absence of supportive respiratory care, however, SP-A deficiency eventually abolished the ability of rHuKGF to prevent weight loss and to improve survival monitored for 1 mo after allogeneic BMT. In further experiments, the addition of cyclophosphamide (which is known to cause severe injury to the alveolar epithelium in donor T cell-recipient mice) to the conditioning regimen prevented rHuKGF-induced upregulation of SP-A and suppression of lung inflammation in both SP-A(+/+) and SP-A(-/-) mice. We conclude that endogenous baseline SP-A levels and optimal upregulation of SP-A are required for the anti-inflammatory protective effects of KGF after allogeneic transplantation.
Contribution of the KCachannel to membrane potential and O2sensitivity is decreased in an ovine PPHN model
Authors: Olschewski Andrea , Hong Zhigang , Linden Bradley C. , Porter Valerie A. , Weir E. Kenneth , Cornfield David N.
Published: March 2015

Ca2+-sensitive K+(KCa) channels play an important role in mediating perinatal pulmonary vasodilation. We hypothesized that lung KCachannel function may be decreased in persistent pulmonary hypertension of the newborn (PPHN). To test this hypothesis, pulmonary artery smooth muscle cells (PASMC) were isolated from fetal lambs with severe pulmonary hypertension induced by ligation of the ductus arteriosus in fetal lambs at 125–128 days gestation. Fetal lambs were killed after pulmonary hypertension had been maintained for at least 7 days. Age-matched, sham-operated animals were used as controls. PASMC K+currents and membrane potentials were recorded using amphotericin B-perforated patch-clamp techniques. The increase in whole cell current normally seen in response to normoxia was decreased (333.9 ± 63.6% in control vs. 133.1 ± 16.0% in hypertensive fetuses). The contribution of the KCachannel to the whole cell current was diminished in hypertensive, compared with control, fetal PASMC. In PASMC from hypertensive fetuses, a change from hypoxia to normoxia caused no change in membrane potential compared with a −14.6 ± 2.8 mV decrease in membrane potential in PASMC from control animals. In PASMC from animals with pulmonary hypertension, 4-aminopyridine (4-AP) caused a larger depolarization than iberiotoxin, whereas in PASMC from control animals, iberiotoxin caused a larger depolarization than 4-AP. These data confirm the hypothesis that the contribution of the KCachannel to membrane potential and O2sensitivity is decreased in an ovine model of PPHN, and this may contribute to the abnormal perinatal pulmonary vasoreactivity associated with PPHN.
Involvement of protein tyrosine kinase in Toll-like receptor 4-mediated NF-κB activation in human peripheral blood monocytes
Authors: Chen Ling-Yu , Zuraw Bruce L. , Zhao Ming , Liu Fu-Tong , Huang Shuang , Pan Zhixing K.
Published: March 2015

Bacterial lipopolysaccharide (LPS) is a powerful activator of the innate immune system. Exposure to LPS induces an inflammatory reaction in the lung mediated primarily by human blood monocytes and alveolar macrophages, which release an array of inflammatory chemokines and cytokines including IL-8, TNF-α, IL-1β, and IL-6. The signaling mechanisms utilized by LPS to stimulate the release of cytokines and chemokines are still incompletely understood. Pretreatment with the protein tyrosine kinase-specific inhibitors genistein and herbimycin A effectively blocked LPS-induced NF-κB activation as well as IL-8 gene expression in human peripheral blood monocytes. However, when genistein was added 2 min after the addition of LPS, no inhibition was observed. Utilizing a coimmunoprecipitation assay, we further showed that LPS-stimulated tyrosine phosphorylation of Toll-like receptor 4 (TLR4) may be involved in downstream signaling events induced by LPS. These findings provide evidence that LPS-induced NF-κB activation and IL-8 gene expression use a signaling pathway requiring protein tyrosine kinase and that such regulation may occur through tyrosine phosphorylation of TLR4.
IL-13 and IL-4 promote TARC release in human airway smooth muscle cells: role of IL-4 receptor genotype
Authors: Faffe Débora S. , Whitehead Timothy , Moore Paul E. , Baraldo Simonetta , Flynt Lesley , Bourgeois Kerri , Panettieri Reynold A. , Shore Stephanie A.
Published: March 2015

The chemokine thymus- and activation-regulated chemokine (TARC) induces selective migration of Th2, but not Th1, lymphocytes and is upregulated in the airways of asthmatic patients. The purpose of this study was to determine whether human airway smooth muscle (HASM) cells produce TARC. Neither IL-4, IL-13, IL-1β, IFN-γ, nor TNF-α alone stimulated TARC release into the supernatant of cultured HASM cells. However, both IL-4 and IL-13 increased TARC protein and mRNA expression when administered in combination with TNF-α but not IL-1β or IFN-γ. Macrophage-derived chemokine was not expressed under any of these conditions. TARC release induced by TNF-α + IL-13 or TNF-α + IL-4 was inhibited by the β-agonist isoproterenol and by other agents that activate protein kinase A, but not by dexamethasone. To determine whether polymorphisms of the IL-4Rα have an impact on the ability of IL-13 or IL-4 to induce TARC release, HASM cells from multiple donors were genotyped for the Ile50Val, Ser478Pro, and Gln551Arg polymorphisms of the IL-4Rα. Our data indicate that cells expressing the Val50/Pro478/Arg551 haplotype had significantly greater IL-13- or IL-4-induced TARC release than cells with other IL-4Rα genotypes. These data indicate that Th2 cytokines enhance TARC expression in HASM cells in an IL-4Rα genotype-dependent fashion and suggest that airway smooth muscle cells participate in a positive feedback loop that promotes the recruitment of Th2 cells into asthmatic airways.
Intratracheal gene transfer of decorin reduces subpleural fibroproliferation induced by bleomycin
Authors: Shimizukawa Minoru , Ebina Masahito , Narumi Ko , Kikuchi Toshiaki , Munakata Hiroshi , Nukiwa Toshihiro
Published: March 2015

Decorin, a small leucin-rich proteoglycan, is a negative regulator of transforming growth factor-β, but the antifibrotic effect of decorin gene transfer has not been examined in a mouse model of usual interstitial pneumonia (UIP). We constructed a replication-defective recombinant adenovirus harboring human decorin gene (AdCMV.DC) and administered 1 × l09plaque-forming units of AdCMV.DC intratracheally or intravenously to C57BL/6 mice with intraperitoneal injection of bleomycin, which induces a subpleural fibroproliferation, mimicking UIP, by day 28. Only intratracheal administration of AdCMV.DC increased decorin mRNA expression in the lung and decreased the hydroxyproline content augmented in bleomycin-induced pulmonary fibrosis (1.13 ± 0.02 to 0.96 ± 0.02, P = 0.006). In contrast, intravenous administration of AdCMV.DC increased the decorin expression only in the liver, but not in the lung, and without reducing lung fibrosis. These results indicate that adenoviral decorin gene transfer is effective only by direct administration to fibrosing lungs.
LPS-induced lung injury in neonatal rats: changes in gelatinase activities and consequences on lung growth
Authors: Franco Marie-Laure , Waszak Paul , Banalec Gaëlle , Levame Micheline , Lafuma Chantal , Harf Alain , Delacourt Christophe
Published: March 2015

Postnatal lung growth disorders may involve imbalance between metalloproteinases and their inhibitors. Inflammatory cell 92-kDa gelatinase overactivity has been reported in adults with lung injury but has not been looked for in neonates. We compared gelatinase activity in neonatal and adult rats and evaluated postnatal lung growth after lipopolysaccharide (LPS)-induced lung injury. Significant intra-alveolar inflammatory cell recruitment occurred in adults and neonates; cell counts increased 16-fold in adults and 2.7-fold in neonates. Total 92-kDa gelatinase activity was increased in neonates and adults and was significantly correlated to inflammatory cell counts. For a given cell count, 92-kDa gelatinase increased more in neonates than in adults. Morphometric neonatal lung analysis showed that LPS-injured lungs had decreases in absolute values of lung volume ( P < 0.03), alveolar surface ( P < 0.004), and air space volume ( P< 0.03). Doxycycline, a nonspecific metalloproteinase inhibitor, partly inhibited LPS-induced 92-kDa gelatinase overactivity but did not improve LPS-induced alveolar growth disorders. LPS-mediated lung injury in neonatal rats induced both gelatinase B overactivity and alveolar growth disorders, although no causal link between these two effects was demonstrated.
TNF-α disruption of lung endothelial integrity: reduced integrin mediated adhesion to fibronectin
Authors: Rotundo Robert F. , Curtis Theresa M. , Shah Melissa D. , Gao Baochong , Mastrangelo Anthony , LaFlamme Susan E. , Saba Thomas M.
Published: March 2015

Tumor necrosis factor-α (TNF-α) causes an increase in transendothelial protein permeability of confluent monolayers of calf pulmonary artery endothelial (CPAE) cells, and the addition of plasma fibronectin (pFn) to the culture medium can attenuate this increase in permeability. We determined if reduced integrin function had a role in decreased endothelial cell adhesion to immobilized Fn after exposure of the endothelial monolayers to TNF-α. TNF-α also causes a reorganization of the subendothelial Fn rich matrix and a significant loss in RGD-dependent adhesion of TNF-α treated CPAE cells to pFn coated surfaces. However, flow cytometry revealed no decrease in α5β1or total β1integrin expression on the surface of the CPAE cells after TNF-α. Reduced CPAE adhesion to immobilized Fn was, in part, due to a loss of β1-integrin function since the β1-integrin blocking antibody mAb 13 significantly ( P < 0.05) prevented the adhesion of normal control CPAE cells but did not further reduce the adhesion of TNF-α-treated cells. In addition, antibodies which activate β1integrins restored ( P < 0.05) adhesion of TNF-α-treated cells to immobilized pFn but did not alter the adhesion of control cells. Despite reduced ability to adhere to immobilized Fn, TNF-α-treated CPAE monolayers demonstrated increased binding and incorporation of fluid-phase pFn into the subendothelial extracellular matrix (ECM) as measured by the analysis of the deoxycholate (DOC) detergent insoluble pool of125I-Fn in the cell layer. In contrast to the RGD-mediated adhesion of CPAE cells to matrix Fn, the increased binding of soluble pFn after TNF-α was not inhibited by RGD peptides or mAb 13. Thus reduced integrin-dependent adhesion of the CPAE cells to matrix Fn as well as disruption of the Fn matrix may contribute to the increased protein permeability of previously confluent endothelial monolayer after TNF-α. In addition, increased ability for the monolayer to incorporate fluid-phase Fn into the ECM after TNF-α via a non-β1- integrin dependent mechanism may be a compensatory response to stabilize the Fn matrix and the endothelial barrier.