bluuebunny/crossref_metadata_2025 huggingface dataset:
Intramuscular gene transfer of FGF-2 attenuates endothelial dysfunction and inhibits intimal hyperplasia of vein grafts in poor-runoff limbs of rabbit
Authors: Shoji Tetsuya , Yonemitsu Yoshikazu , Komori Kimihiro , Tanii Mitsugu , Itoh Hiroyuki , Sata Shihoko , Shimokawa Hiroaki , Hasegawa Mamoru , Sueishi Katsuo , Maehara Yoshihiko
Published: March 2015

We previously demonstrated that sustained disturbance of endothelium-dependent vasorelaxation and poor distal runoff in ischemic limbs were critical factors affecting the neointimal development of autologous vein grafts (VGs). Also, we recently showed the superior therapeutic potential of basic fibroblast growth factor (bFGF/FGF-2) boosted by the recombinant Sendai virus (SeV) for severe limb ischemia compared with that of vascular endothelial growth factor. Here, the effect of FGF-2 on neointimal hyperplasia of VGs was examined in a rabbit model of poor-runoff limbs. Two weeks after initial surgery for the induction of poor-runoff, SeV-expressing human FGF-2 (SeV-hFGF2) or that encoding firefly luciferase (109plaque-forming units/head) was injected into the thigh and calf muscle. At that time, the femoral vein was implanted in the femoral artery in an end-to-end manner in some groups. FGF-2 gene-transferred limbs demonstrated significantly increased blood flow assessed not only by laser Doppler flow image but also by ultrasonic transit-time flowmeter (USTF). USTF also showed a significant increase in the blood flow ratio of the deep femoral artery to external iliac artery, indicating that collateral flow was significantly restored in the thigh muscles ( P < 0.01). Reduction of neointimal hyperplasia was also observed in the VGs treated by SeV-hFGF2; these grafts demonstrated significant restoration of endothelium-dependent vasorelaxation. These findings thus extend the indications of therapeutic angiogenesis using SeV-hFGF2 to include not only limb salvage but also prevention of late graft failure.
Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO
Authors: Kirima Kazuyoshi , Tsuchiya Koichiro , Sei Hiroyoshi , Hasegawa Toyoshi , Shikishima Michiyo , Motobayashi Yuki , Morita Kyoji , Yoshizumi Masanori , Tamaki Toshiaki
Published: March 2015

The measurement of hemoglobin-nitric oxide (NO) adduct (HbNO) in whole blood by the electron paramagnetic resonance (EPR) method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and unknown radical species overlap the same magnetic field as that of HbNO. To reveal the EPR spectrum of HbNO, we then introduced the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of sample blood using the software. Rats were treated with Nω-nitro-l-arginine methyl ester (l-NAME; 120 mg · kg–1· day–1) for 1 wk to obtain HbNO-depleted blood. When this method was applied to the analysis of untreated fresh whole blood, the five-coordinate state of HbNO was observed. HbNO concentration in pentobarbital-anesthetized rats was augmented (change in [HbNO] = 1.6–5.5 μM) by infusion of l-arginine (0.2–0.6 g/kg) but not d-arginine. Using this method, we attempted to evaluate the effects of temocapril on HbNO dynamics in an l-NAME-induced rat endothelial dysfunction model. The oral administration of l-NAME for 2 wk induced a serious hypertension, and the HbNO concentration was reduced (change in [HbNO] = 5.7 μM). Coadministration of temocapril dose dependently improved both changes in blood pressure and the systemic HbNO concentration. In this study, we succeeded in measuring the blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril improves abnormalities of NO dynamics in l-NAME-induced endothelial dysfunction rats using the EPR HbNO signal subtraction method.
Pharmacological preconditioning with resveratrol: an insight with iNOS knockout mice
Authors: Imamura Gembu , Bertelli Alberto A. , Bertelli Aldo , Otani Hajime , Maulik Nilanjana , Das Dipak K.
Published: March 2015

Resveratrol, a natural antioxidant and polyphenol found in grapes and wine, has been found to pharmacologically precondition the heart through the upregulation of nitric oxide (NO). To gain further insight of the role of NO in resveratrol preconditioning, mouse hearts devoid of any copies of inhibitory NO synthase (iNOS) (iNOS knockout) and corresponding wild-type hearts were perfused with 10 μM resveratrol for 15 min followed by 25 min of ischemia and 2 h of reperfusion. Control experiments were performed with wild-type and iNOS knockout hearts that were not treated with resveratrol. Resveratrol-treated wild-type mouse hearts displayed significant improvement in postischemic ventricular functional recovery compared with those of nontreated hearts. Both resveratrol-treated and nontreated iNOS knockout mouse hearts resulted in relatively poor recovery in ventricular function compared with wild-type resveratrol-treated hearts. Myocardial infarct size was lower in the resveratrol-treated wild-type mouse hearts compared with other group of hearts. In concert, a number of apoptotic cardiomyocytes was lower in the wild-type mouse hearts treated with resveratrol. Cardioprotective effects of resveratrol was abolished when the wild-type mouse hearts were simultaneously perfused with aminoguanidine, an iNOS inhibitor. Resveratrol induced the expression of iNOS in the wild-type mouse hearts, but not in the iNOS knockout hearts, after only 30 min of reperfusion. Expression of iNOS remained high even after 2 h of reperfusion. Resveratrol-treated wild-type mouse hearts were subjected to a lower amount of oxidative stress as evidenced by reduced amount of malonaldehyde content in these hearts compared with iNOS knockout and untreated hearts. The results of this study demonstrated that resveratrol was unable to precondition iNOS knockout mouse hearts, whereas it could successfully precondition the wild-type mouse hearts, indicating an essential role of iNOS in resveratrol preconditioning of the heart.
Age-related functional effects linked to phosphatase activity in ventricular myocytes
Authors: Grey Elizabeth M. , Chan Chun K. , Chen Yi , Hofmann Polly A.
Published: March 2015

Conflicting reports exist regarding the influence of β-adrenergic stimulation on the maximum velocity of shortening ( Vmax) in ventricular myocytes. This may be due to an unrecognized effect of maturation. In the present study, the effects of β-adrenergic receptor stimulation on myocytes from hearts of juvenile nonbred and young adult retired breeder female rats were compared. Ventricular myocytes from young adults had a β-adrenergic-dependent increase in Vmaxand Ca2+-dependent actomyosin ATPase that was not observed in myocytes from juveniles. Myocytes from young adults had both an increase in β-myosin heavy chain (MHC) and higher basal serine/threonine phosphatase activity compared with juvenile rats. Additional studies established moderate increases in β-MHC induced by hypothyroidism do not confer myocardial β-adrenergic responsiveness, whereas inhibition of the higher phosphatase activity in myocytes from young adults blocks the age-dependent, β-adrenergic-induced increase in cross-bridge cycling rates. We propose that the higher phosphatase activity of myocytes from young adults compared with juveniles allows for a greater functional response of the myocardium to β-adrenergic stimulation.
Melatonin as an effective protector against doxorubicin-induced cardiotoxicity
Authors: Liu Xuwan , Chen Zhongyi , Chua Chu Chang , Ma Yan-Shan , Youngberg George A. , Hamdy Ronald , Chua Balvin H. L.
Published: March 2015

The present study was designed to explore the protective effects of melatonin and its analogs, 6-hydroxymelatonin and 8-methoxy-2-propionamidotetralin, on the survival of doxorubicin-treated mice and on doxorubicin-induced cardiac dysfunction, ultrastructural alterations, and apoptosis in mouse hearts. Whereas 60% of the mice treated with doxorubicin (25 mg/kg ip) died in 5 days, almost all the doxorubicin-treated mice survived when melatonin or 6-hydroxymelatonin (10 mg/l) was administered in their drinking water. Perfusion of mouse hearts with 5 μM doxorubicin for 60 min led to a 50% suppression of heart rate × left ventricular developed pressure and a 50% reduction of coronary flow. Exposure of hearts to 1 μM melatonin or 6-hydroxymelatonin reversed doxorubicin-induced cardiac dysfunction. 8-Methoxy-2-propionamidotetralin had no protective effects on animal survival and on in vitro cardiac function. Infusion of melatonin or 6-hydroxymelatonin (2.5 μg/h) significantly attenuated doxorubicin-induced cardiac dysfunction, ultrastructural alterations, and apoptosis in mouse hearts. Neither melatonin nor 6-hydroxymelatonin compromised the antitumor activity of doxorubicin in cultured PC-3 cells. These results suggest that melatonin protect against doxorubicin-induced cardiotoxicity without interfering with its antitumor effect.
Vascular rarefaction in peripheral skeletal muscle after experimental heart failure
Authors: Nusz David J. , White David C. , Dai Qunsheng , Pippen Anne M. , Thompson Michael A. , Walton G. Brant , Parsa Cyrus J. , Koch Walter J. , Annex Brian H.
Published: March 2015

A decrease in vascular density in peripheral skeletal muscle has been associated with exercise intolerance in humans with congestive heart failure (CHF). The purpose of this study was to determine whether CHF results in a reduction in vascular density in peripheral skeletal muscle. In this established model, CHF was induced by coronary artery ligation in New Zealand White rabbits and sham rabbits that underwent an identical surgical procedure without ligation of the coronary artery. At study termination, rabbits underwent hemodynamic testing and skeletal muscle analysis. The first series of rabbits was divided into sham ( n = 6) and CHF ( n = 6) 21 days postoperatively. Ten CHF rabbits were then examined 3 ( n = 3), 7 ( n = 3), and 14 days ( n = 4) postoperatively. Vascular density in sham tibialis anterior muscle was 347 ± 41 capillaries/mm2or 1.20 ± 0.11 capillaries/muscle fiber. In 21-day CHF rabbits, the capillary density was significantly lower, 236 ± 14 capillaries/mm2or 0.84 ± 0.04 capillaries/muscle fiber (both P < 0.00001 vs. sham); PECAM protein was 2-fold lower ( P < 0.0001) in muscle protein lysates; the fraction of apoptotic cells was greater, 3.8 ± 2.2 vs. 0.69 ± 0.56 ( P < 0.02 vs. sham) with many TdT-mediated dUTP-biotin nick-end labeling-positive endothelial cells; and Bax protein was 2.8-fold greater ( P < 0.0001). By regression analysis, vascular density tended to decrease over time ( r2= 0.572, P < 0.0001). Vascular rarefaction and endothelial apoptosis develop after experimental CHF and may contribute to the skeletal muscle abnormalities in this disease. Modulating vascular density may provide new approaches to treat exercise intolerance in CHF.
Mechanisms of altered vagal control in heart failure: influence of muscarinic receptors and acetylcholinesterase activity
Authors: Dunlap Mark E. , Bibevski Steve , Rosenberry Terrone L. , Ernsberger Paul
Published: March 2015

Parasympathetic control of the heart is attenuated in heart failure (HF). We investigated possible mechanisms and sites of altered vagal control in dogs with HF induced by rapid pacing. Muscarinic blockade reduced the R-R interval by 308 ms in controls but only by 32 ms in HF, indicating low levels of resting vagal tone. Vagomimetic doses of atropine sulfate prolonged the R-R interval by 109 ms in controls and increased standard deviation of the R-R interval by 66 ms but only by 46 and 16 ms, respectively, in HF. Bradycardia elicited by electrical stimulation of the vagus nerve was also attenuated in the HF group. Conversely, muscarinic receptor activation by bethanechol, and indirectly by neostigmine, elicited exaggerated R-R interval responses in HF. To investigate possible mechanisms, we measured muscarinic receptor density (Bmax) and acetylcholinesterase activity in different areas of the heart. In sinoatrial nodes, Bmaxwas increased (230 ± 75% of control) and acetylcholinesterase decreased (80 ± 6% of control) in HF. We conclude that muscarinic receptors are upregulated and acetylcholinesterase is reduced in the sinus node in HF. Therefore, reduced vagal control in HF is most likely due to changes of presynaptic function (ganglionic), because postsynaptic mechanisms augment vagal control in HF.
Myocardial preconditioning factors evoke mesenteric ischemic tolerance via opioid receptors and KATPchannels
Authors: Dickson Eric W. , Tubbs Robert J. , Porcaro William A. , Lee Won Jae , Blehar David J. , Carraway Robert E. , Darling Chad E. , Przyklenk Karin
Published: March 2015

We have shown that a reverse-phase concentrate generated from the effluent of preconditioned (PC) rabbit hearts evokes a cardioprotective effect in virgin acceptor hearts. With the use of a model of sustained (1 h) simulated ischemia in isolated, spontaneously contracting rabbit jejunum, our current aims were to 1) determine whether protective factor(s) released from PC hearts can improve ischemic tolerance in noncardiac tissue; and 2) obtain preliminary insight into the mediator(s) involved in triggering and eliciting this remote protection. Recovery of contractile force following reoxygenation (our index of ischemic tolerance) was enhanced in jejunal segments pretreated with concentrate generated from PC hearts (33 ± 3% of baseline, P < 0.01) versus segments that received no concentrate (21 ± 2%) and segments treated with concentrate from normoxic hearts (16 ± 3%; P < 0.01). Protection achieved with PC concentrate was attenuated by coadministration of naloxone or glibenclamide, thereby implicating the involvement of opioids and ATP-sensitive potassium channels. Moreover, evaluation of purified subfractions of the crude PC concentrate identified a specific bioactive fraction that may participate in triggering the improved jejunal ischemic tolerance.
Characterization and regulation of T-type Ca2+channels in embryonic stem cell-derived cardiomyocytes
Authors: Zhang Ying Ming , Shang Lijuan , Hartzell Criss , Narlow Michael , Cribbs Leanne , Dudley Samuel C.
Published: March 2015

T-type Ca2+channels may play a role in cardiac development. We studied the developmental regulation of the T-type currents ( ICa,T) in cardiomyocytes (CMs) derived from mouse embryonic stem cells (ESCs). ICa,Twas studied in isolated CMs by whole cell patch clamp. Subsequently, CMs were identified by the myosin light chain 2v-driven green fluorescent protein expression, and laser capture microdissection was used to isolate total RNA from groups of cells at various developmental time points. ICa,Tshowed characteristics of Cav3.1, such as resistance to Ni2+block, and a transient increase during development, correlating with measures of spontaneous electrical activity. Real-time RT-PCR showed that Cav3.1 mRNA abundance correlated ( r2= 0.81) with ICa,T. The mRNA copy number was low at 7+4 days (2 copies/cell), increased significantly by 7+10 days (27/cell; P < 0.01), peaked at 7+16 days (174/cell), and declined significantly at 7+27 days (25/cell). These data suggest that ICa,Tis developmentally regulated at the level of mRNA abundance and that this regulation parallels measures of pacemaker activity, suggesting that ICa,Tmight play a role in the spontaneous contractions during CM development.
Cardiac homeostasis is independent of calf venous compliance in subjects with paraplegia
Authors: Wecht Jill M. , De Meersman Ronald E. , Weir Joseph P. , Spungen Ann M. , Bauman William A.
Published: March 2015

The purpose of this study was to examine cardiac hemodynamics during acute head-up tilt (HUT) and calf venous function during acute head-down tilt (HDT) in subjects with paraplegia compared with sedentary nondisabled controls. Nineteen paraplegic males (below T6) and nine age-, height-, and weight-matched control subjects participated. Heart rate, stroke volume, and cardiac output were assessed using the noninvasive acetylene uptake method. Venous vascular function of the calf was assessed using venous occlusion plethysmography. After supine measurements were collected, the table was moved to 10° HDT followed by the three levels of HUT (10, 35, and 75°) in random order. Cardiac hemodynamics were similar between the groups at all positions. Calf circumference was significantly reduced in the paraplegic group compared with the control group ( P < 0.001). Venous capacitance and compliance were significantly reduced in the paraplegic compared with control group at supine and HDT. Neither venous capacitance ( P = 0.37) nor compliance ( P = 0.19) increased from supine with 10° HDT in the paraplegic group. A significant linear relationship was established between supine venous compliance and supine cardiac output in the control group ( r = 0.80, P < 0.02) but not in the paraplegic group. The findings of reduced calf circumference and similar venous capacitance at supine rest and 10° HDT in the paraplegic group imply that structural changes may have limited venous dispensability in individuals with chronic paraplegia. Furthermore, the lack of a relationship between supine venous compliance and supine cardiac output suggests that cardiac homeostasis does not rely on venous compliance in subjects with paraplegia.
Differential modulation of glucose, lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart
Authors: Lloyd Steven , Brocks Charlye , Chatham John C.
Published: March 2015

Despite the fact that lactate and pyruvate are potential substrates for energy production in vivo, our understanding of the control and regulation of carbohydrate metabolism is based principally on studies where glucose is the only available carbohydrate. Therefore, the purpose of this study was to determine the contributions of lactate, pyruvate, and glucose to energy production in the isolated, perfused rat heart over a range of insulin concentrations and after activation of pyruvate dehydrogenase with dichloroacetate (DCA). Hearts were perfused with physiological concentrations of [1-13C]glucose, [U-13C]lactate, [2-13C]pyruvate, and unlabeled palmitate for 45 min. Hearts were freeze clamped, and 13C NMR glutamate isotopomer analysis was performed on tissue extracts. Glucose, lactate, and pyruvate all contributed significantly to myocardial energy production; however, in the absence of insulin, glucose contributed only 25–30% of total pyruvate oxidation. Even under conditions where carbohydrates represented >95% of substrate entering the tricarboxylic acid (TCA) cycle, we found that glucose contributed at most 50–60% of total carbohydrate oxidation. Despite being present at only 0.1 mM, pyruvate contributed between ∼10% and 30% of total acetyl-CoA entry into the TCA cycle. We also found that insulin and DCA not only increased glucose oxidation but also exogenous pyruvate oxidation; however, lactate oxidation was not increased. The differential effects of insulin and DCA on pyruvate and lactate oxidation provide further evidence for compartmentation of cardiac carbohydrate metabolism. These results may have important implications for understanding the mechanisms underlying the beneficial effects of increasing cardiac carbohydrate metabolism.
Epoxygenase-driven angiogenesis in human lung microvascular endothelial cells
Authors: Medhora Meetha , Daniels John , Mundey Kavita , Fisslthaler Beate , Busse Rudi , Jacobs Elizabeth R. , Harder David R.
Published: March 2015

Angiogenesis is one of the most recent physiological functions attributed to products of cytochrome P-450 (CYP450) enymes. To test this at a molecular level in human cells, we used a cloned cDNA for the human endothelial enzyme CYP450 2C9 (CYP2C9) to study growth as well as differentiation of human microvascular endothelial cells from the lung (HMVEC-L). Using adenoviral vectors overexpressing mRNA for CYP2C9, we show that the presence of CYP2C9 doubles thymidine incorporation and stimulates proliferation of primary cultures of endothelial cells compared with Ad5-GFP (control) in 24 h. In addition, there is a significant increase of tube formation in Matrigel after infection of HMVEC-L with Ad5-2C9 than with Ad5-GFP. More interestingly, Ad5-2C9 expressing the antisense product of CYP2C9 (2C9AS) inhibited tube formation compared with both Ad5-GFP as well as the Ad5-2C9 constructs. Finally, we tested the most abundant arachidonic acid metabolite of CYP2C9, 14,15-epoxyeicosatrienoic acid, which induced angiogenesis in vivo when embedded in Matrigel plugs and implanted in adult rats. These data support an important role for CYP2C9 in promoting angiogenesis.
Cardioprotective action of CRF peptide urocortin against simulated ischemia in adult rat cardiomyocytes
Authors: Gordon Jennifer M. , Dusting Gregory J. , Woodman Owen L. , Ritchie Rebecca H.
Published: March 2015

The major objective of this study was to determine whether urocortin, a member of the corticotrophin-releasing factor (CRF) family, protects adult rat cardiomyocytes from ischemia that has been simulated by glucose deprivation and acidosis. When it was present during simulated ischemia, urocortin (0.1 μM) markedly attenuated the cellular injury, which was assessed by increases in creatine kinase and lactate dehydrogenase levels. This effect was comparable with that observed with adenosine (10 μM). The cardioprotective effect of urocortin was markedly attenuated by the protein kinase C inhibitor chelerythrine and by 5-hydroxydecanoate, an inhibitor of ATP-sensitive K+channels. Cardiomyocytes were also protected from injury by pretreatment with urocortin, either by incubation for 5 min with a subsequent 10-min recovery or incubation for 20 min with a 20-h recovery before simulated ischemia. Similar cardioprotective effects were observed with ischemic preconditioning protocols during both immediate and delayed phases. In conclusion, in adult cardiomyocytes, urocortin has immediate and delayed cardioprotective actions that mimic ischemic preconditioning. These actions are mediated via protein kinase C and ATP-sensitive K+channels.
Impaired endothelium-mediated relaxation in isolated cerebral arteries from insulin-resistant rats
Authors: Erdös Benedek , Miller Allison W. , Busija David W.
Published: March 2015

Insulin resistance (IR) impairs vascular responses in peripheral arteries. However, the effects of IR on cerebrovascular control mechanisms are completely unexplored. We examined the vascular function of isolated middle cerebral arteries (MCAs) from fructose-fed IR and control rats. Endothelium-dependent vasodilation elicited by bradykinin (BK) was reduced in IR compared with control MCAs. Maximal dilation to BK (10−6 M) was 38 ± 3% ( n = 13) in control and 19 ± 3% ( n = 10) in IR arteries ( P < 0.01). N ω-nitro-l-arginine methyl ester (l-NAME; 10 μM) decreased responses to BK in control arteries by ∼65% and inhibited the already reduced responses completely in IR MCAs. Indomethacin (10 μM) reduced relaxation to BK in control MCAs by ∼40% but was largely ineffective in IR arteries. Combined l-NAME and indomethacin treatments eliminated the BK-induced dilation in both groups. Similarly to BK, endothelium-mediated and mainly cyclooxygenase (COX)-dependent dilation to calcium ionophore A23187 was reduced in IR arteries compared with controls. In contrast, vascular relaxation to sodium nitroprusside was similar between the IR and control groups. These findings demonstrate that endothelium-dependent dilation in cerebral arteries is impaired in IR primarily because of a defect of the COX-mediated pathways. In contrast, nitric oxide-mediated dilation remains intact in IR arteries.
High-sodium intake prevents pregnancy-induced decrease of blood pressure in the rat
Authors: Beauséjour Annie , Auger Karine , St-Louis Jean , Brochu Michèle
Published: March 2015

Despite an increase of circulatory volume and of renin-angiotensin-aldosterone system (RAAS) activity, pregnancy is paradoxically accompanied by a decrease in blood pressure. We have reported that the decrease in blood pressure was maintained in pregnant rats despite overactivation of RAAS following reduction in sodium intake. The purpose of this study was to evaluate the impact of the opposite condition, e.g., decreased activation of RAAS during pregnancy in the rat. To do so, 0.9% or 1.8% NaCl in drinking water was given to nonpregnant and pregnant Sprague-Dawley rats for 7 days (last week of gestation). Increased sodium intakes (between 10- and 20-fold) produced reduction of plasma renin activity and aldosterone in both nonpregnant and pregnant rats. Systolic blood pressure was not affected in nonpregnant rats. However, in pregnant rats, 0.9% sodium supplement prevented the decreased blood pressure. Moreover, an increase of systolic blood pressure was obtained in pregnant rats receiving 1.8% NaCl. The 0.9% sodium supplement did not affect plasma and fetal parameters. However, 1.8% NaCl supplement has larger effects during gestation as shown by increased plasma sodium concentration, hematocrit level, negative water balance, proteinuria, and intrauterine growth restriction. With both sodium supplements, decreased AT1mRNA levels in the kidney and in the placenta were observed. Our results showed that a high-sodium intake prevents the pregnancy-induced decrease of blood pressure in rats. Nonpregnant rats were able to maintain homeostasis but not the pregnant ones in response to sodium load. Furthermore, pregnant rats on a high-sodium intake (1.8% NaCl) showed some physiological responses that resemble manifestations observed in preeclampsia.
Effects of sustained β-adrenergic stimulation on ionic currents of cultured adult guinea pig cardiomyocytes
Authors: Zhang Li-Ming , Wang Zhiguo , Nattel Stanley
Published: March 2015

Short-term stimulation of β-receptors is known to affect cardiac ion channels; however, the impact of longer-term stimulation on intrinsic channel function is poorly understood. To evaluate this, cultured guinea pig ventricular myocytes were exposed to isoproterenol (10 nM), vehicle, or isoproterenol plus propranolol (1 μM) for 48 h. Sustained exposure to isoproterenol decreased the density of the inward rectifier ( I K1), slow delayed rectifier ( I Ks), and L-type Ca2+( I Ca L) currents, effects that were fully prevented by propranolol. Changes in K+ currents were prevented by the β1-selective antagonist CGP-20712A, unaffected by the β2-antagonist ICI-118,551, and mimicked by the membrane-permeable cAMP analog 8-bromo-cAMP. Isoproterenol did not alter the current-voltage relationship of the K+currents but increased the density of T-type Ca2+ current ( I Ca T) and thereby increased the proportion of the total Ca2+ current at more negative potentials. We conclude that sustained exposure to isoproterenol reduces I K1, I Ks, and I Ca L density and increases the density of I Ca T. The direct ionic current remodeling effects of sustained β-adrenoceptor stimulation resemble changes reported with heart failure and may be important in arrhythmogenic ionic remodeling.
Cardiac myofibroblasts isolated from the site of myocardial infarction express endothelin de novo
Authors: Katwa Laxmansa C.
Published: March 2015

Recently it was demonstrated that treatment with a nonselective endothelin (ET) receptor antagonist significantly reduces myocardial infarct size, which suggests a major role for ET in tissue repair following myocardial infarction (MI). Tissue repair and remodeling found at the site of MI are mainly attributed to myofibroblasts (myoFbs), which are phenotypically transformed fibroblasts that express α-smooth muscle actin. It is unclear whether myoFbs generate ET peptides and consequentially regulate pathophysiological functions de novo through expression of the ET-1 precursor (prepro-ET-1), ET-converting enzyme-1 (ECE-1), a metalloprotease that is required to convert Big ET-1 to ET-1 and ET receptors. To address these intriguing questions, we used cultured myoFbs isolated from 4-wk-old MI scar tissue. In cultured cells, we found: 1) expression of mRNA for ET precursor gene ( ppET1), ECE-1, and ETAand ETBreceptors by semiquantitative RT-PCR; 2) phosphoramidon-sensitive ECE-1 activity, which converts Big ET-1 to biologically active peptide ET-1; 3) expression of ETAand ETBreceptors; 4) elaboration of Big ET-1 and ET-1 peptides in myoFb culture media; and 5) upregulation of type I collagen gene expression and synthesis by ET, which was blocked by bosentan (a nonselective ETA- and ETBreceptor blocker). These studies clearly indicated that myoFbs express and generate ET-1 and receptor-mediated modulation of type I collagen expression by ET-1. Locally generated ET-1 may contribute to tissue repair of the infarcted heart in an autocrine/paracrine manner.
Effects of pre-, peri-, and postmyocardial infarction treatment with omapatrilat in rats: survival, arrhythmias, ventricular function, and remodeling
Authors: Lapointe Nathalie , Nguyen Quang Trinh , Desjardins Jean-François , Marcotte Francois , Pourdjabbar Ali , Moe Gordon , Calderone Angelino , Rouleau Jean-Lucien
Published: March 2015

We showed previously that the vasopeptidase inhibitor (VPI) omapatrilat improves peri-myocardial infarction (MI) survival, but the mechanisms involved and whether these effects are sustained remained to be determined, and are the subject of this study. Rats ( n = 272) received omapatrilat (20 mg · kg-1 · day-1) starting 7 days before MI and continued peri- and post-MI, or no treatment (control). One group of rats had continuous ambulatory ECG and blood pressure recordings started 6 h before MI and continued until 24 h after MI, when survival was evaluated, and the rats were killed, and MI size was evaluated. A second group had left ventricular (LV) remodeling evaluated by echocardiography at 30 days and, at 38 days, had cardiac hemodynamics and morphology done and survival evaluated. Survival 24 h after MI ( n = 255) improved with omapatrilat (60% vs. 46% for control; P = 0.0378). Over the next 37 days, there was no further improvement with omapatrilat but the early benefit was sustained. Omapatrilat reduced MI size 24 h after MI (36 ± 2 vs. 42 ± 2 mm2 for controls; P = 0.034). Omapatrilat reduced ventricular arrhythmia score 1–12 h after MI. Omapatrilat decreased blood pressure, but not during the first 24 h after MI. Omapatrilat reduced LV diastolic and systolic dimensions and LV and right ventricular weights compared with control large MI, indicating a decrease in reactive hypertrophy. Improvement in cardiac remodeling was accompanied by improved cardiac hemodynamics. Thus this study indicates that pre-, peri-, and post-MI treatment with the VPI omapatrilat is beneficial in survival, ventricular arrhythmias, LV remodeling, and cardiac function.
Hypothermia preserves myocardial function and mitochondrial protein gene expression during hypoxia
Authors: Ning Xue-Han , Chen Shi-Han , Xu Cheng-Su , Hyyti Outi M. , Qian Kun , Krueger Julia J. , Portman Michael A.
Published: March 2015

Hypothermia before and/or during no-flow ischemia promotes cardiac functional recovery and maintains mRNA expression for stress proteins and mitochondrial membrane proteins (MMP) during reperfusion. Adaptation and protection may occur through cold-induced change in anaerobic metabolism. Accordingly, the principal objective of this study was to test the hypothesis that hypothermia preserves myocardial function during hypoxia and reoxygenation. Hypoxic conditions in these experiments were created by reducing O2concentration in perfusate, thereby maintaining or elevating coronary flow (CF). Isolated Langendorff-perfused rabbit hearts were subjected to perfusate (Po2= 38 mmHg) with glucose (11.5 mM) and perfusion pressure (90 mmHg). The control (C) group was at 37°C for 30 min before and 45 min during hypoxia, whereas the hypothermia (H) group was at 29.5°C for 30 min before and 45 min during hypoxia. Reoxygenation occurred at 37°C for 45 min for both groups. CF increased during hypoxia. The H group markedly improved functional recovery during reoxygenation, including left ventricular developed pressure (DP), the product of DP and heart rate, dP/d tmax, and O2consumption (MVo2) ( P < 0.05 vs. control). MVo2decreased during hypothermia. Lactate and CO2gradients across the coronary bed were the same in C and H groups during hypoxia, implying similar anaerobic metabolic rates. Hypothermia preserved MMP βF1-ATPase mRNA levels but did not alter adenine nucleotide translocator-1 or heat shock protein-70 mRNA levels. In conclusion, hypothermia preserves cardiac function after hypoxia in the hypoxic high-CF model. Thus hypothermic protection does not occur exclusively through cold-induced alterations in anaerobic metabolism.
Effects of changes in left ventricular contractility on indexes of contractility in mice
Authors: Nemoto Shintaro , DeFreitas Gilberto , Mann Douglas L. , Carabello Blase A.
Published: March 2015

Measurement of left ventricular (LV) function is often overlooked in murine studies, which have been used to analyze the effects of genetic manipulation on cardiac phenotype. The goal of this study was to address the effects of changes in LV contractility on indexes of contractility in mice. LV function was assessed in vivo in closed-chest mice by echocardiography and by LV catheterization using a conductance pressure-volume (P-V) catheter with three different interventions that alter contractility by 1) atrial pacing to increase inotropy by augmentation of the force-frequency relation (modest increment of inotropy), 2) dobutamine to maximize inotropy, and 3) esmolol infusion to decrease contractility. Load-independent parameters derived from P-V relations, such as slope of end-systolic P-V relations (ESPVR) and slope of the first maximal pressure derivative over time (dP/d t max)-end-diastolic volume relation (dP/d t-EDV), and standard echocardiographic parameters were measured. The dP/d t-EDV changed the most among parameters after atrial pacing and dobutamine infusion (percent change, 162.8 ± 95.9% and 271.0 ± 44.0%, respectively). ESPVR was the most affected by a decrease in LV contractility during esmolol infusion (percent change, −49.8 ± 8.3%). However, fractional shortening failed to detect changes in contractility during atrial pacing and esmolol infusion and its percent change was <20%. This study demonstrated that contractile parameters derived from P-V relations change the most during a change in LV contractility and should therefore best detect a small change in contractility in mice. Heart rate has a modest but significant effect on P-V relationship-derived indexes and must be considered in the evaluation of murine cardiac physiology.
Subchronic endotoxin inhalation causes persistent airway disease
Authors: Brass D. M. , Savov J. D. , Gavett S. H. , Haykal-Coates N. , Schwartz D. A.
Published: March 2015

The endotoxin component of organic dusts causes acute reversible airflow obstruction and airway inflammation. To test the hypothesis that endotoxin alone causes airway remodeling, we have compared the response of two inbred mouse strains to subchronic endotoxin exposure. Physiological and biological parameters were evaluated after 1 day, 5 days, or 8 wk of exposure to endotoxin [lipopolysaccharide (LPS)] in endotoxin-sensitive (C3HeB/FeJ) and endotoxin-resistant (C3H/HeJ) mice. After 5 days or 8 wk of LPS exposure, only C3HeB/FeJ had elevated airway hyperreactivity to inhaled methacholine. Only the C3HeB/FeJ mice had significant inflammation of the lower respiratory tract after 1 day, 5 days, or 8 wk of LPS exposure. Stereological measurements of small, medium, and large airways indicated that an 8-wk exposure to LPS resulted in expansion of the submucosal area only in the C3HeB/FeJ mice. Cell proliferation as measured by bromodeoxyuridine incorporation contributed to the expansion of the submucosa and was only significantly elevated in C3HeB/FeJ mice actively exposed to LPS. C3HeB/FeJ mice had significantly elevated levels of interleukin-1β protein in whole lung lavage after 1 day and 5 days of LPS exposure and significantly elevated protein levels of total and active transforming growth factor-β1 in whole lung lavage fluid after 5 days of LPS exposure. Our findings demonstrate that subchronic inhalation of LPS results in the development of persistent airway disease in endotoxin-responsive mice.
Chronic carbon monoxide enhanced IbTx-sensitive currents in rat resistance pulmonary artery smooth muscle cells
Authors: Dubuis Eric , Gautier Mathieu , Melin Alexandre , Rebocho Manuel , Girardin Catherine , Bonnet Pierre , Vandier Christophe
Published: March 2015

Exogenous carbon monoxide (CO) can induce pulmonary vasodilation by acting directly on pulmonary artery (PA) smooth muscle cells. We investigated the contribution of K+ channels to the regulation of resistance PA resting membrane potential on control (PAC) rats and rats exposed to CO for 3 wk at 530 parts/million, labeled as PACO rats. Whole cell patch-clamp experiments revealed that the resting membrane potential of PACO cells was more negative than that of PAC cells. This was associated with a decrease of membrane resistance in PACO cells. Additional analysis showed that outward current density in PACO cells was higher (50% at +60 mV) than in PAC cells. This was linked to an increase of iberiotoxin (IbTx)-sensitive current. Chronic CO hyperpolarized membrane of pressurized PA from −46.9 ± 1.2 to −56.4 ± 2.6 mV. Additionally, IbTx significantly depolarized membrane of smooth muscle cells from PACO arteries but not from PAC arteries. The present study provides initial evidence of an increase of Ca2+-activated K+ current in smooth muscle cells from PA of rats exposed to chronic CO.
Activation of multiple signaling modules is critical in angiotensin IV-induced lung endothelial cell proliferation
Authors: Li Yong D. , Block Edward R. , Patel Jawaharlal M.
Published: March 2015

Signaling events involving angiotensin IV (ANG IV)-mediated pulmonary artery endothelial cell (PAEC) proliferation were examined. ANG IV significantly increased upstream phosphatidylinositide (PI) 3-kinase (PI3K), PI-dependent kinase-1 (PDK-1), extracellular signal-related kinases (ERK1/2), and protein kinase B-α/Akt (PKB-α) activities, as well as downstream p70 ribosomal S6 kinase (p70S6K) activities and/or phosphorylation of these proteins. ANG IV also significantly increased 5-bromo-2′-deoxy-uridine incorporation into newly synthesized DNA in a concentration- and time-dependent manner. Pretreatment of cells with wortmannin and LY-294002, inhibitors of PI3K, or rapamycin, an inhibitor of the mammalian target of rapamycin kinase and p70S6K, diminished the ANG IV-mediated activation of PDK-1 and PKB-α as well as phosphorylation of p70S6K. Although an inhibitor of mitogen-activated protein kinase kinase, PD-98059, but not rapamycin, blocked ANG IV-induced phosphorylation of ERK1/2, both PD-98059 and rapamycin independently caused partial reduction in ANG IV-mediated cell proliferation. However, simultaneous treatment with PD-98059 and rapamycin resulted in total inhibition of ANG IV-induced cell proliferation. These results demonstrate that ANG IV-induced DNA synthesis is regulated in a coordinated fashion involving multiple signaling modules in PAEC.
Modulation of endotoxin-induced NF-κB activation in lung and liver through TNF type 1 and IL-1 receptors
Authors: Koay M. Audrey , Christman John W. , Wudel L. James , Allos Tara , Cheng Dong-Sheng , Chapman William C. , Blackwell Timothy S.
Published: March 2015

We investigated the requirement for tumor necrosis factor-α (TNF-α) and interleukin (IL)-1 receptors in the pathogenesis of the pulmonary and hepatic responses to Escherichia coli lipopolysaccharide (LPS) by studying wild-type mice and mice deficient in TNF type 1 receptor [TNFR1 knockout (KO)] or both TNF type 1 and IL-1 receptors (TNFR1/IL-1R KO). In lung tissue, NF-κB activation was similar among the groups after exposure to aerosolized LPS. After intraperitoneal injection of LPS, NF-κB activation in liver was attenuated in TNFR1 KO mice and further diminished in TNFR1/IL-1R KO mice; however, in lung tissue, no impairment in NF-κB activation was found in TNFR1 KO mice and only a modest decrease was found in TNFR1/IL-1R KO mice. Lung concentrations of KC and macrophage-inflammatory peptide 2 were lower in TNFR1 KO and TNFR1/IL-1R KO mice after aerosolized and intraperitoneal LPS. We conclude that LPS-induced NF-κB activation in liver is mediated through TNF-α- and IL-1 receptor-dependent pathways, but, in the lung, LPS-induced NF-κB activation is largely independent of these receptors.
Pulmonary cytochromeP-450 2J4 is reduced in a rat model of acutePseudomonaspneumonia
Authors: Yaghi Asma , Bradbury J. Alyce , Zeldin Darryl C. , Mehta Sanjay , Bend John R. , McCormack David G.
Published: March 2015

We previously reported that the levels of epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are depressed in microsomes prepared from lungs of rats with acute Pseudomonas pneumonia. We also showed a potential role for cytochrome P-450 (CYP) metabolites of arachidonic acid (AA) in contractile responses of both normal pulmonary arteries and pulmonary arteries from rats with pneumonia. The CYP2J subfamily enzymes (endogenous source of EETs and HETEs) are constitutively expressed in human and rat lungs where they are localized in vascular smooth muscle and endothelium. The purpose of this study was to determine if CYP2J proteins are modified in pneumonia. Pseudomonas organisms were injected via a tracheostomy in the lungs of rats. Later (44 h), lungs were frozen, and microsomes were prepared from pneumonia and control rat lung homogenates. Lung microsomal proteins were then immunoblotted with anti-CYP2B1/2B2, anti-CYP4A, anti-CYP2J9pep2 (which reacts with rat CYP2J3), anti-CYP2J6pep1 (which reacts with rat CYP2J4), anti-CYP2J2pep4, or anti-CYP2J2pep3 (both of which react with all known CYP2J isozymes). Western blotting revealed a prominent 55-kDa band with anti-CYP2J2pep3, anti-CYP2J2pep4, and anti-CYP2J6pep1 (but not anti-CYP2J9pep2) that was reduced in pneumonia compared with control lung microsomes. The CYP2B bands (51-52 kDa) were less prominent and not different between pneumonia and control lungs. CYP4A proteins (20-HETE sources) were not detected in rat lung microsomes. Therefore, rat lung contains a protein with immunological characteristics similar to CYP2J4, and this CYP is reduced after pneumonia. We speculate that CYP2J (but not CYP2B) enzymes and their AA metabolic products (EETs) are involved in the modulation of pulmonary vascular tone in pneumonia in rats.
Differential effects of mechanical ventilatory strategy on lung injury and systemic organ inflammation in mice
Authors: Gurkan Ozlem U. , O'Donnell Christopher , Brower Roy , Ruckdeschel Emily , Becker Patrice M.
Published: March 2015

Patients with acute respiratory distress syndrome are at increased risk for developing multiorgan system dysfunction. The goal of this study was to establish an in vivo murine model to assess the differential effects of ventilation-protective strategies on the development of acute lung injury and systemic organ inflammation. C57B/6 mice were randomized to mechanical ventilation (MV) with conventional, high (17 ml/kg) or protective, low (6 ml/kg) tidal volume (VT) after intratracheal hydrochloric acid or no intervention. Mean arterial pressure was continuously monitored during MV and did not differ between groups. After 4 h, lung injury was assessed by measurement of wet/dry lung weight, lung lavage protein concentration and cell count, and histology. Concentration of IL-6, TNF-α, VEGF, and VEGF receptor-2 (VEGFR2) was measured in lung, liver, kidney, and heart. Results were compared with control, spontaneously breathing mice. Lung injury and altered pulmonary cytokine expression were not detected after MV of healthy mice with low or high VT. Although MV did not significantly alter IL-6 or TNF-α in systemic organs, VEGF concentration significantly increased in liver and kidney. After acid aspiration, mice ventilated with high VT manifested lung injury and increased IL-6 and VEGFR2 in lung, liver, and kidney, whereas VEGF increased only in liver and kidney. MV with low VT after acid aspiration attenuated lung injury, both IL-6 and VEGFR2 expression in lung and systemic organs, and hepatic, but not renal, increased VEGF. Our data suggest that MV strategy has differential effects on systemic inflammatory changes and thus may selectively predispose to systemic organ dysfunction.
ChTX induces oscillatory contraction in guinea pig trachea: role of cyclooxygenase-2 and PGE2
Authors: Yagi Yukihiro , Kuwahara Masayoshi , Tsubone Hirokazu
Published: March 2015

We examined the possible role of cyclooxygenase (COX) in charybdotoxin (ChTX)-induced oscillatory contraction in guinea pig trachea. Involvement of prostaglandin E2(PGE2) in ChTX-induced oscillatory contraction was also investigated. ChTX (100 nM) induced oscillatory contraction in guinea pig trachea. The mean oscillatory frequency induced by ChTX was 10.7 ± 0.8 counts/h. Maximum and minimum tensions within ChTX-induced oscillatory contractions were 68.4 ± 1.8 and 14.3 ± 1.7% compared with K+(72.7 mM) contractions. ChTX-induced oscillatory contraction was completely inhibited by indomethacin, a nonselective COX inhibitor. Valeryl salicylate, a selective COX-1 inhibitor, did not significantly inhibit this contraction, whereas N-(2-cyclohexyloxy-4-nitro-phenyl)-methanesulfonamide, a selective COX-2 inhibitor, abolished this contraction. Exogenously applied arachidonic acid enhanced ChTX-induced oscillatory contraction. SC-51322, a selective PGE receptor subtype EP1antagonist, significantly inhibited ChTX-induced oscillatory contraction. Exogenously applied PGE2induced only a slight phasic contraction in guinea pig trachea, but PGE2induced strong oscillatory contraction after pretreatment with indomethacin and ChTX. Moreover, ChTX time-dependently stimulated PGE2generation. These results suggest that ChTX specifically activates COX-2 and stimulates PGE2production and that ChTX-induced oscillatory contraction in guinea pig trachea is mediated by activation of EP1receptor.
The late asthmatic response is linked with increased surface tension and reduced surfactant protein B in mice
Authors: Haczku Angela , Atochina Elena N. , Tomer Yaniv , Cao Yang , Campbell Colleen , Scanlon Seth T. , Russo Scott J. , Enhorning Goran , Beers Michael F.
Published: March 2015

Pulmonary surfactant dysfunction may significantly contribute to small airway obstruction during the asthmatic response, but neither its exact role nor its regulation is clear. Surfactant function and composition was studied in an Aspergillus fumigatus ( Af)-induced late-phase allergic airway response in sensitized BALB/c mice. The peak of Af-induced airway hyperresponsiveness in sensitized and challenged mice 24 h after allergen provocation coincided with a significant fall in surface activity of the pulmonary surfactant. The underlying changes included time-dependent elaboration of eotaxin and IL-5 followed by eosinophil influx into the airways. The height of airway inflammation and hyperresponsiveness was preceded by release of IL-4 and marked reductions in surfactant protein (SP)-B, a hydrophobic surfactant protein responsible for maintaining low surface tension of the lining fluid of distal air spaces. Furthermore, intratracheal administration of IL-4 significantly inhibited SP-B, indicating a regulatory role of this cytokine in the surfactant biophysical changes. Thus surfactant dysfunction induced by an IL-4-driven SP-B deficiency after allergen provocation may be an important part of the late asthmatic airway response.