Sham Lungs Research Articles

Abstract 361: The Augmentation of Oxygen-sensitive Generation of Oxygen Radicals in Mitochondria and the Attenuation of Hyperoxia-induced Oxidative Stress After Cardiac Arrest

Introduction: Hyperoxia can induce oxidative stress resulting in organ injuries after cardiac arrest (CA). Mitochondrial reactive oxygen species (ROS) are key to understanding its mechanism, however the augmentation of oxygen-sensitive ROS generation caused by CA ( Fig 1 ) has not been well described. Methods: Rats were assigned into: 1) sham with normoxia, 30% O2; 2) sham with hyperoxia, 100% O2; 3) CA with normoxia; and 4) CA with hyperoxia (n=15 for each group). CA was induced by 10-minute asphyxia and CPR was delivered. Neurological deficit score (NDS), histological lung injury score (LIS), survival time were obtained from the CA groups. Carbonyl protein as an oxidative stress indicator was measured at 2 hrs after resuscitation. H2O2 generation from isolated mitochondria were measured ex vivo at a normoxic and hyperoxic condition set up with nitrogen and air saturated medium. Results: Between the CA groups, the normoxia group had a higher 48hr-survival rate (77%), lower NDS (359±140), and lower LIS (4.3±2.9) compared to those (28%, 452±85, 14±2, respectively) of the hyperoxia group (p<0.01, p<0.05, and p<0.01, respectively). In the brain, lung, and kidney, CA augmented the hyperoxia-induced increase in carbonyl protein (absolute change: sham brain, 0.18 nmol/mg protein vs. CA brain, 0.61 nmol/mg protein; sham lung, 0.29 nmol/mg protein vs. CA lung, 0.72 nmol/mg protein; sham kidney, 0.04 nmol/mg protein vs. CA kidney, 0.55 nmol/mg protein, respectively). In the brain and kidney isolated mitochondria, CA augmented the hyperoxia-induced increase in H2O2 (absolute change: sham brain, 18 pmol/mg protein vs. CA brain, 60 pmol/mg protein; sham kidney, 16 pmol/mg protein vs. CA kidney, 45 pmol/mg protein, respectively). Conclusions: The normoxic therapy may attenuate organ damage and improve survival of CA. The reduction of augmented mitochondrial ROS generation, which is oxygen-sensitive, would be an important mechanism and therapeutic target of post-CA care.

2058 miRNA manipulation to improve CFTR correction in cystic fibrosis

OBJECTIVES/SPECIFIC AIMS: CFTR is the mutant protein that causes cystic fibrosis (CF), a fatal respiratory diseases affecting 1 in 3500 children. CFTR modulators are small molecules that directly address mutant CFTR function. Improving correction of the F508del CFTR mutation (affecting 90% of CF patients) is one of the most pressing unmet needs in CF. Currently available F508del therapeutics only marginally improve CF, In vitro, we have identified a miRNA that impairs utility of CFTR directed therapies. miR-145 is upregulated by TGF-β (a genetic modifier of CF lung disease) with a direct binding site on the 3’-untranslated region of CFTR mRNA. Binding of miR-145 to CFTR destabilizes mRNA transcript and impedes protein translation. Overexpression of miR-145 abolishes benefit of F508del CFTR correction. Antagonists to miR-145 block TGF-β suppression of CFTR function and augment response to CFTR correction. This project evaluate in vivo impact of TGF-beta and miRNA manipulation on CFTR functional readouts including nasal potential difference (NPD) and short circuit current (Isc) across tracheal explants in addition to standard biochemical measures. METHODS/STUDY POPULATION: Wild-type Sprague-Dawley rats were inoculated with an adenoviral vector containing bioactive TGF-beta or sham at 1×109 pfu/animal placed in the left nares. Seven days post-inoculation, functional, and biochemical measures were conducted. NPD was measured with a microelectrode placed in the left nare and grounded the tail. The nare was sequentially perfused with standard Ringer’s solution, amiloride (to block the ENaC sodium channel), low chloride Ringer’s (to stimulate chloride efflux), forskolin (to open the CFTR channel) and CFTRinh-172 (to block the CFTR channel. Tracheal explants were harvested, microdissected, and placed on modified Ussing chambers. RESULTS/ANTICIPATED RESULTS: We have inoculated WT rats with bioactive TGF-β Versus sham delivered by intranasal inoculation of an adenoviral vector. Functional readout of CFTR function is by Isc across tracheal epithelia and NPD. Lung homogenates are analyzed for TGF-β signaling, miRNA expression, and CFTR transcripts. Both tracheal explants and NPD indicate TGF-β stimulation diminishes CFTR function in vivo. In tracheal explants, TGF-β exposure diminishes CFTR response to forskolin-stimulation by 75%. Loss of current after CFTR inhibition (CFTRinh-172) is halved. By nasal PD, TGF-β inoculation similarly halves the bioelectric response to low chloride and forskolin stimulation. Evaluation by qPCR reveals a strong increase in TGF-β signaling demarcated by PAI-1, prompting a reduction in CFTR mRNA. miR-145 is expressed highly in rat pulmonary tissue, but no change in overall miR-145 levels was detected between TGF-β and sham exposed rats. This finding reflects what we have observed in human lungs, with a localized increased miR-145 expression in CF epithelia, but similarly high levels of miR-145 in both CF and non-CF whole lung homogenates. Although expressed at lower levels than miR-145, we did find increased expression in TGF-β relevant miR-101, miR-494, and miR-144 that have a predicted binding site on rat 3’-UTR in TGF-β exposed Versus sham lungs. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data indicate the relevance of TGF-β stimulation to suppress CFTR synthesis and function in vivo. Future work will evaluate whether these additional miRNA with CFTR binding sites may mediate TGF-β suppression of CFTR in the rat model, and the utility of miRNA manipulation to augment F508del CFTR correction.

Effect of the oestrogen receptor antagonist fulvestrant on the cirrhotic rat lung.

It has been postulated that cirrhosis-related lung vasodilatation and the subsequent hepatopulmonary syndrome are partly explained by an increased estradiol level through an enhanced endothelial formation of nitric oxide (NO). In this study, we assessed whether the oestrogen receptor antagonist fulvestrant (F) improves cirrhosis-related lung abnormalities. Cirrhosis was induced in rats by chronic bile duct ligation (CBDL). Four groups were studied: CBDL, CBDL+F, sham, and sham+F. Histological, immunohistochemical, and Western blot analyses were performed on lung samples. In the lung, the endothelial NO synthase and the nitrotyrosine protein expressions were increased in CBDL as compared to sham rats. Both parameters were significantly reduced by fulvestrant in the CBDL rats. Surprisingly, the level of pVASP (an indirect marker of NO formation and action) was decreased in CBDL rats, and fulvestrant had no effect on this parameter. The level of the vascular endothelial growth factor, the diameter of small lung vessels, and the number of macrophages were increased in CBDL lungs in comparison with sham lungs, and these parameters were unaffected by fulvestrant treatment. In conclusion, fulvestrant may not be relevant to improve lung abnormalities in cirrhosis because NO may not be biologically active and because key events contributing to the lung abnormalities are not affected by fulvestrant.

Ultrastructural Pathology of Rat Lung Injury Induced by Ischemic Acute Kidney Injury

Ischemic acute kidney injury (AKI) is a common complication during inpatient hospitalization, and often induces acute lung injury (ALI). A lot of studies have concentrated on the relevance between AKI and ALI, but the underlying mechanisms of AKI- associated ALI have remained unclear until now. One reason is that evidence of the ultrastructural pathology of AKI-associated ALI has been scarce and needed to be accumulated. The aims of present study are to observe ultrastructural changes, and to reveal leukocyte trafficking of ALI induced by ischemic AKI in rats. For this purpose light microscopy (LM) and electron microscopy (EM), as well as morphometric analysis, were employed in present study. LM observations revealed distinct regions of collapsed alveoli, hemorrhage in alveoli, and interstitial edema in AKI-induced ALI. EM examinations provided facts that alveolar epithelial cells, including type I and type II cells, were necrotic, and endothelia cells undergoing apoptosis as well as interstitial cells undergoing necroptosis were noted in AKI lungs. In addition, shrinkage and decreased or disappeared lamellar bodies were evident in alveolar type II cell of AKI rat lungs. Leukocyte numerical density on area (NA) in AKI lungs was significantly more than that in sham lungs. Based on the morphological criteria from EM examinations and morphometric analysis, a conclusion was that necrosis, including necroptosis, and apoptosis were involved in damaged lung induced by AKI. And inflammation also contributed to acute lung injury of rats with AKI.

Alveolar macrophage activation is a key initiation signal for acute lung ischemia-reperfusion injury

Lung ischemia-reperfusion (I/R) injury is a biphasic inflammatory process. Previous studies indicate that the later phase is neutrophil-dependent and that alveolar macrophages (AMs) likely contribute to the acute phase of lung I/R injury. However, the mechanism is unclear. AMs become activated and produce various cytokines and chemokines in many inflammatory responses, including transplantation. We hypothesize that AMs respond to I/R by producing key cytokines and chemokines and that depletion of AMs would reduce cytokine/chemokine expression and lung injury after I/R. To test this, using a buffer-perfused, isolated mouse lung model, we studied the impact of AM depletion by liposome-clodronate on I/R-induced lung dysfunction/injury and expression of cytokines/chemokines. I/R caused a significant increase in pulmonary artery pressure, wet-to-dry weight ratio, vascular permeability, tumor necrosis factor (TNF)-alpha, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2 expression, as well as decreased pulmonary compliance, when compared with sham lungs. After AM depletion, the changes in each of these parameters between I/R and sham groups were significantly attenuated. Thus AM depletion protects the lungs from I/R-induced dysfunction and injury and significantly reduces cytokine/chemokine production. Protein expression of TNF-alpha and MCP-1 are positively correlated to I/R-induced lung injury, and AMs are a major producer/initiator of TNF-alpha, MCP-1, and MIP-2. We conclude that AMs are an essential player in the initiation of acute lung I/R injury.

Modulation of hypoxic pulmonary vasoconstriction is time and nitric oxide dependent in a peritonitis model of sepsis.

This study assessed modulation of hypoxic pulmonary vasoconstriction (HPV) in isolated perfused rat lungs during sepsis induced by cecal ligation and perforation (CLP) at different times and its relationship to nitric oxide synthases (NOS). Prospective controlled trial in a university research laboratory. 102 male Sprague-Dawley rats. Groups 1-3 received sham laparotomy 6 h before lung isolation: group 1, only laparotomy; group 2, concurrently L- N6-(1-iminoethyl)-lysine (L-NIL, 3 mg/kg); group 3, concurrently N(Omega)-nitro-L-arginine methylester (L-NAME, 5 mg/kg). Groups 4-6 received CLP 6 h before lung isolation: group 4, only CLP; group 5, concurrently L-NIL; group 6, concurrently L-NAME. The same experiments were carried out with sham and CLP treatment for 24 h (groups 7-12). Exhaled NO from rats' lungs was measured after anesthesia and tracheostomy. After the pulmonary circuit was isolated and perfused, angiotensin II (0.1 microg) was injected into the inflow tract. The lungs were ventilated with the hypoxic mixture (HPV, 3% O2) for 10 min and then again with the normoxic mixture (21% O2) for an equal period. Changes in perfusion pressure were measured. Endothelial (eNOS) and inducible NOS (iNOS) expression of the lungs was determined. Treatment with L-NAME but not L-NIL increased HPV in sham lungs. HPV was unaltered after CLP 6 h and decreased after CLP 24 h compared to sham. In CLP animals eNOS protein expression was reduced whereas iNOS expression was increased compared to sham animals. Exhaled NO, reflecting NOS activity was twice as high in the CLP 24 h group than in the CLP 6 h group. In the CLP sepsis model modulation of HPV was time-dependent. In addition, vasoconstriction to hypoxic stimuli was dependent on NOS activity.

ADAR1 is involved in the development of microvascular lung injury.

Deamination of adenosine on pre-mRNA to inosine is a recently discovered process of posttranscription modification of pre-mRNA, termed A-to-I RNA editing, which results in the production of proteins not inherent in the genome. The present study aimed to identify a role for A-to-I RNA editing in the development of microvascular lung injury. To that end, the pulmonary expression and activity of the RNA editase ADAR1 were evaluated in a mouse model of endotoxin (15 mg/kg IP)-induced microvascular lung injury (n=5) as well as in cultured alveolar macrophages stimulated with endotoxin, live bacteria, or interferon. ADAR1 expression and activity were identified in sham lungs that were upregulated in lungs from endotoxin-treated mice (at 2 hours). Expression was localized to polymorphonuclear and monocytic cells. These events preceded the development of pulmonary edema and leukocyte accumulation in lung tissue and followed the local production of interferon-gamma, a known inducer of ADAR1 in other cell systems. ADAR1 was found to be upregulated in alveolar macrophages (MH-S cells) stimulated with endotoxin (1 to 100 microg/mL), live Escherichia coli (5x10(7) colony-forming units), or interferon-gamma (1000 U/mL). Taken together, these data suggest that ADAR1 may play a role in the pathogenesis of microvascular lung injury possibly through induction by interferon.

Inhibition of K(Ca) channels restores blunted hypoxic pulmonary vasoconstriction in rats with cirrhosis.

Rats with liver cirrhosis exhibit the hepatopulmonary syndrome composed of blunted hypoxic pulmonary vasoconstriction and arterial hypoxemia. The purpose of this study was to investigate the roles of nitric oxide (NO) and endothelin-1 (ET-1) in the blunted hypoxic pressor response (HPR) in rats with common bile duct ligation (CBDL). Lungs from CBDL rats exhibited markedly blunted HPR, increased endothelial NO synthase (NOS) protein expression, and decreased ET-1 mRNA and peptide expression. The blunted HPR was not reversed by sequential NOS and soluble guanylyl cyclase inhibition by nitro-L-arginine and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), respectively, or by NOS inhibition combined with ET-1 addition. The blunted HPR was not due to a generalized inability to vasoconstrict because perfusion pressure was equally elevated by increased perfusate KCl in CBDL and sham lungs. After KCl vasoconstriction, HPR was potentiated and did not differ between CBDL and sham lungs. Blunted HPR was also completely restored in CBDL lungs treated with nitro-L-arginine, ODQ, and the Ca(2+)-activated K(+) channel blockers apamin and charybdotoxin. These results indicate that although CBDL-induced liver cirrhosis is associated with increased NO and decreased ET-1 in the lung, the blunted HPR is a result of additional factors and appears to involve Ca(2+)-activated K(+) channel activation.

PULMONARY VASCULAR REMODELING DISTAL TO PULMONARY ARTERY LIGATION IS ACCOMPANIED BY UPREGULATION OF ENDOTHELIN RECEPTORS AND NITRIC OXIDE SYNTHASE

There is increasing evidence that the pathogenesis and progression of many forms of pulmonary vasculopathy are related to abnormalities in endothelial mediators, including endothelin-1 (ET-1) and nitric oxide (NO). Using a rat model of chronic unilateral pulmonary artery ligation, we investigated the role of ET-1 and NO in postobstructive pulmonary vasculopathy (POPV). Eight months after a left thoracotomy with either left main pulmonary artery ligation (ligated group) or no ligation (sham group), rat lungs, including those contralateral to the ligation (hyperperfused group), were fixed and mounted for histologic sectioning. Morphometric measurements were carried out by computer-assisted image analysis and immunohistochemical staining was performed using specific antibodies against ET-1, ETA, and EBB receptors, and endothelial NO synthase (eNOS). Compared to sham lungs, the ligated lungs showed (1) an increase in muscular, adventitial, and intimal thickness of pulmonary artery; (2) increase in external diameter of the bronchial artery (39.8+/-2.2 mum vs.16.8+/-0.9 mum in sham group; P <. 005) and number of bronchial arteries per bronchiole (3.21+/-mu0.26vs.1.86+/-mu0.21 in sham group; P <. 001); and (3) increase in the intensity of eNOS and ETA, B receptor immunoreactivity. No morphometric or immunohistochemical differences were observed between the hyperperfused and sham lungs. These ndings suggest that increased synthesis of endothelial NO may serve as a compensatory mediator in ET-1 mediated vascular remodeling.

Decreased pulmonary nitric oxide synthase activity in the rat model of congenital diaphragmatic hernia

Because nitric oxide (NO) dilates vascular smooth muscle cells a deficiency of endogenous pulmonary nitric oxide production by nitric oxide synthase (NOS) has been suggested to be involved in the pathophysiology of pulmonary hypertension in congenital diaphragmatic hernia (CDH). Our aim was to determine whether experimentally induced CDH in rats results in a decrease in the synthesis of NO in the lungs. Adult Sprague-Dawley rats were fed 300 mg/kg of nitrofen at 10.5 days' gestation. CDH, control, and sham (dosed with nitrofen, but without CDH) lungs were homogenized at full term (22 days' gestation) for measurement of NOS activity using the 14C-l-arginine to 14C-l-citrulline conversion assay. Western blot analysis with anti-endothelial cell NOS (EC-NOS) monoclonal antibody (mAb) was performed, and NOS expression was measured by densitometry. NOS activity was highest in the pulmonary parenchyma of control rat lungs (0.420 ± .020 fmol/min/mg lung; n = 11), intermediate in sham lungs (0.370 ± 0.010 fmol/min/mg lung; n = 14), and lowest in CDH lungs (0.300 ± 0.04 fmol/min/mg lung; n = 12). NOS activity in the CDH and sham lungs was significantly lower than that of control lungs (P < .05). There was no difference in pulmonary NOS activity between sham and CDH lungs. NOS protein expression by Western blot analysis paralleled the observation for NOS activity in all groups, with the highest concentrations in controls, intermediate expression in sham lungs, and lowest expression in CDH lungs. Both NOS expression and NOS activity are significantly decreased in CDH rat lungs. Pulmonary hypertension in this model may be attributable to a deficiency of endogenous NO. This is the first reported study to suggest that decreased NOS activity may result in pulmonary hypertension in CDH.