doi:10.3181/0712-RM-337
© 2008 Society for Experimental Biology and Medicine
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Sagi Sarada1,
Patir Himadri,
Chitaranjan Mishra,
Pradhan Geetali,
Mastoori Sai Ram and
Govindan Ilavazhagan
Department of Experimental Biology, Defence Institute of Physiology and Allied Sciences, Timarpur, Delhi, India
1 Department of Experimental Biology, Defense Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi-54, India. E-mail: saradasks{at}gmail.com
Abstract |
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TOP Abstract Introduction Materials and Methods Results Discussion Conclusion References |
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Hypoxia is well known to increase the free radical generation in the body, leading to oxidative stress. In the present study, we have determined whether the increased oxidative stress further upregulates the nuclear transcription factor (NFkB) in the development of pulmonary edema. The rats were exposed to hypobaric hypoxia at 7620 m (280 mm Hg) for different durations, that is, 3 hrs, 6 hrs, 12 hrs, and 24 hrs at 25 ± 1°C. The results revealed that exposure of animals to hypobaric hypoxia led to a significant increase in vascular leakage, with time up to 6 hrs (256.38 ± 61 rfu/g) as compared with control (143.63 ± 60.1 rfu/g). There was a significant increase in reactive oxygen species, lipid peroxidation, and superoxide dismutase levels, with a concurrent decrease in lung glutathione peroxidase activity. There was 13-fold increase in the expression of NFkB level in nuclear fraction of lung homogenates of hypoxic animals over control rats. The DNA binding activity of NFkB was found to be increased significantly (P < 0.001) in the lungs of rats exposed to hypoxia as compared with control. Further, we observed a significant increase in proinflammatory cytokines such as IL-1, IL-6, and TNF- with concomitant upregulation ofcell adhesion molecules such as ICAM-I, VCAM-I, and P-selectinin the lung of rats exposed to hypoxia as compared with control.Interestingly, pretreatment of animals with curcumin (NFkB blocker)attenuated hypoxia-induced vascular leakage in lungs with concomitantreduction of NFkB levels. The present study therefore revealsthe possible involvement of NFkB in the development of pulmonaryedema.
Keywords: HAPE, NFkB, oxidative stress, proinflammatory cytokines
Introduction |
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TOP Abstract Introduction Materials and Methods Results Discussion Conclusion References |
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High-altitude pulmonary edema (HAPE) is a non-cardiac, acute, and potentially lethal pulmonary disorder. The rapid ascent to altitudes above 2450 m can lead to high-altitude pulmonary edema (5, 23) in nonacclimatized individuals. The altitude, speed, and mode of ascent are the most important determinants for the occurrence of HAPE. This illness usually occurs 2 to 3 days after acute exposure to altitudes above 2500 to 3000 m. It is characterized by increased pulmonary arterial pressure, vasoconstriction with elevated vascular permeability, and hypoxemia (22). The incidence of subclinical HAPE may be almost 70% in individuals exposed to an altitude of 4500 m. However, a recent report suggests that HAPE may occur in individuals performing heavy exercise at altitudes as low as 2,400 m (19). In spiteof intensive research, the pathophysiology of HAPE is yet tobe elucidated.
Clinical investigations reported that HAPE-susceptible people show a patchy peripheral distribution of edema in lungs with normal wedge pressure (7). On the other hand, some studies reported that increased pulmonary artery pressure with precapillary vasoconstriction leads to edema formation in HAPE patients (7, 24). The pathogenesis of HAPE is attributed to hydrostatic mechanisms leading to capillary leak in pulmonary edema (24). Urano et al. (44) have reported that exposure to severe hypoxia resulted into uneven blood flow distribution followed by increased vascular permeability. Similar results were also reported by Hopkins et al. (23). However, HAPE-susceptible subjects showed a significantly higher increase of pulmonary artery systolic pressure (PASP) during hypoxia at rest compared with controls (16), and considered that PASP measurements at rest during hypoxia or exercise in normoxia are most feasible for the identification of HAPE-susceptible subjects. On the other hand, several studies have reported that the inflammation process plays an important role in the pathogenesis of HAPE (6, 26).
It has been reported earlier that high-altitude exposure results in increased reactive oxygen species (ROS) generation, leading to enhanced oxidative damage to lipids, proteins, and DNA (42). However, there is no direct evidence on the role of ROS in causing acute mountain sickness (AMS), HAPE, and high-altitude cerebral edema (HACE). Numerous oxidative stress–sensitive transcription factors such as nuclear factor kB (NFkB) and activator protein 1 (AP-1) can mediate an inflammatory response caused by oxidative stress by inducing gene transcription of cytokines such as IL-1, IL-6, TNF-, and adhesion molecules ICAM-1 (intracellular cell adhesion molecule) and VCAM-1 (vascular cell adhesion molecule) (21). Furthermore, Carol and Brain (10) reported that the celladhesion molecules like ICAM-I and VCAM-I genes contain a NFkBsite on the promoter region and are known to be regulated byNFkB. However, no direct evidence on the role of inflammatorycytokines and cell adhesion molecules in HAPE is known.
We were particularly interested in exploring the molecular mechanisminvolved in hypoxia-induced pulmonary edema. Therefore, thepresent study was designed to determine whether hypobaric hypoxia-inducedoxidative stress leads to activation of NFkB (in HAPE) in thelungs of rats. The study was also proposed to determine thelevels of proinflammatory cytokines and cell adhesion moleculesin the lungs of rats exposed to hypoxia. Our hypothesis in thisstudy is that oxidative stress and NFkB signaling would certainlycontribute to hypoxia-induced pulmonary vascular leakage. Further,we reasoned that if this hypothesis were true, then treatmentwith NFkB blocker (curcumin) during hypoxia would result inreduced transvascular leakage in the lungs of rats.
Materials and Methods |
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TOP Abstract Introduction Materials and Methods Results Discussion Conclusion References |
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Experimental Design.
Animals.
The experiments were carried out using male Sprague-Dawley rats, weighing 150–200 g. Rats were maintained at 25 ± 1°C with 12:12-hr light to dark cycles each and given food and water ad libitum. This study had the approval of the Institute’sEthic Committee and followed the guidelines of UniversitiesFederation for Animal Welfare (UFAW) guidelines for animal research.
Hypoxic Exposure.
The experiment was conducted in two phases, that is, phase Iand phase II.
Phase I experiment.
A total of 60 rats were used in the phase I experiment; ratswere divided into five groups of 12 rats each. Group 1 servedas control or normoxia (0 hrs); Group 2 was exposed to hypoxiafor a 3-hr duration; Group 3 was exposed to hypoxia for a 6-hrduration; Group 4 was exposed to hypoxia for a 12-hr duration;and Group 5 was exposed to hypoxia for a 24-hr duration.
Phase II experiment.
In phase II, the NFkB blockade study was carried out using curcuminin 48 rats, which were divided into four groups of 12 rats each.Group 1 served as control or normoxia (0 hrs) receiving onlyvehicle; Group 2 (hypoxia) received only vehicle and was exposedto hypoxia for 6 hrs; Group 3 was supplemented with curcumin50 mg/ kg body weight (BW); and Group 4 (hypoxia + curcumin)was supplemented orally with curcumin 50 mg/kg BW and was exposedto hypoxic stress for 6 hrs.
The rats were exposed to a simulated altitude of 7620 m (25,000ft) in a hypobaric chamber (Decibel Instruments India Limited,Delhi, India) for different periods of time, namely 3, 6, 12,and 24 hrs. The temperature of the hypobaric chamber was maintainedat 25 ± 1°C with an air flow rate of 4 liter/hr andbarometric pressure of 280 mm Hg. The partial pressure of arterialoxygen in control rats was found to be 95 ± 2 mm Hg,and in hypobaric rats it was found to be 38 ± 2 mm Hg,indicating that the rats were exposed to reduced levels of partialpressure of oxygen in the hypobaric chamber. The animals wereprovided with adequate quantities of food and water during exposureto hypoxia. We have exposed the rats to hypobaric hypoxia (280mm Hg) because the smaller animals have higher capillary densityin tissues, making them more resistant to hypoxia than humansare.
Sample Preparation and Measurement of Oxidative Stress.
After hypoxic exposure, animals were sacrificed and the lungs were perfused with cold phosphate-buffered saline (PBS). The lung tissue was collected and then washed with cold saline (0.9% NaCl) and a 10% homogenate in 0.154 M KCl was prepared at 4°Cfor estimating various biochemical parameters.
Determination of Biochemical Parameters.
The production of free radicals (ROS) in the lung homogenate was determined time dependently, that is, at 0 hrs, 1.5 hrs (90 mins), 3 hrs, 6 hrs, 12 hrs, and 24 hrs by using DCFH-DA (2,7,dichlorofluorescein diacetate), and the fluorescence was measured by spectrofluorometer (Varian, Walnut Creek, CA) with an excitation at 485 nm and emission at 530 nm (13). Malondialdehyde (MDA) levels were also determined time dependently, that is, at 0 hrs, 1.5 hrs (90 mins), 3 hrs, 6 hrs, 12 hrs, and 24 hrs in the lungs (34) by 2-thiobarbuturic acid assay and measuring the absorbance at 532 nm using 1,1,3,3-tetra-ethoxy propane as standard. The reduced glutathione (GSH) was determined in the lungs by the method of Kum-Talt and Tan (27) using dithionitro-benzoic acid (DTNB) reagent and measuring the absorbance at 412 nm. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) levels in lungs were measured using commercial kit (Randox, Crumlin, UK) as per manufacturer’s instructions. The protein concentration was estimated by the method of Lowry et al. (28).
Determination of Pulmonary Edema.
Determination of vascular permeability.
The vascular permeability of lungs was determined following the method of Baba et al. (3), with some modifications. In brief,half an hour before the completion of hypoxic exposure, therats were taken out of the hypobaric chamber, and 200 µlof sodium fluorescein (5 mg/kg BW in PBS; Sigma Chemical Co.,St. Louis, MO) was injected through the tail vein. Later therats were placed back in the hypoxia chamber and exposed againto hypoxia for additional 30 mins. Later the animals were takenout of the hypoxia chamber, anesthetized, and perfused withPBS through the left heart ventricle to remove the fluorescenttracer from the vascular bed. The lungs were removed, washedwith cold saline, and divided into two equal parts. One partof the lung was kept in 3% formamide for about 18 hrs at roomtemperature (RT). Later, the tissues were centrifuged for 10mins at 3,000 rpm, and the fluorescence in the supernatant wasmeasured using a spectrofluorometer (Varian) with 485 nm excitationand emissions at 530 nm. The other part of the lung was weighedand kept in an oven at 80°C for 72 hrs. Later, the dry tissueswere collected and weighed again to determine the dry weight.The results were presented as relative fluorescence units pergram (rfu/g) dry weight.
Determination of lung water content.
To quantify the lung water content in lungs from both normoxic and hypoxic animals, the wet weight of the lungs was determined immediately after removal. The samples were rinsed with the cold PBS and dried at 80°C for 72 hrs, and the edema index was expressed as wet-to-dry weight ratio (W/D ratio) (46).
Protein Expression Studies.
Sample preparation.
After hypoxic exposure, the animals were sacrificed using ketamine hydrochloride (80 mg/kg) and xylaxine (20 mg/ kg) anesthesia and the lungs were perfused with cold PBS. The whole lung was removed and washed with cold saline and then homogenized in a buffer containing 0.01 M Tris HCl, pH 7.6, 0.1 M NaCl, 0.1 mM dithiothreitol, 0.001 M EDTA, 100 µg/ml phenylmethylsulfonylfluoride (PMSF) and 10 µl/ml protease inhibitor cocktail(Sigma). The contents were centrifuged at 3000 rpm for 15 minsat 4°C, and the supernatant was collected and stored at–80°C.
The proteins (50 µg) were separated on 10% sodium dodecylsulphate-polyacryl–amide gel electrophoresis (Bio-Rad, Hercules, CA) and electroblotted onto nitrocellulose membranes (Millipore, Billerica, MA). The membranes were blocked with 3% bovine serum albumin (BSA) for 2 hrs and thoroughly washed with PBST (phosphate-buffered saline with 0.1 % Tween-80) and were probed with primary antibodies in 1:2000 dilution (IL-I, IL-6, TNF-, ICAM-I, VCAM–I andP-selectin; Santa Cruz Biotechnology, Santa Cruz, CA) for 2hrs. The membranes were washed and incubated with secondaryantibodies conjugated with horseradish peroxidase (1:50,000;Santa Cruz Biotechnology) for 1 hr at RT. Later, the membraneswere thoroughly washed with PBST (five to six times), and thebands were developed on x-ray film (Kodak, Rochester, NY) usingchemiluminescent peroxidase substrate (Sigma). The optical densityof bands was quantified using Labworks software (UVP Bio-imagingSystems, Upland, CA).
Nuclear and Cytoplasmic Fraction.
Oxidative stress is known to modulate the redox-sensitive transcriptionalproteins. For determining NFkB, nuclear and cytoplasmic fractionswere isolated from lung homogenate using a commercial kit (Bio-Vision,Mountain View, CA) according to the manufacturer’s instructions.The NFkB protein levels in both nuclear and cytoplasmic fractionswere determined by Western blotting as described above.
Transcription Factor (NFkB) Activation Studies.
Electrophoretic mobility shift assay (EMSA).
The EMSA for NFkB was carried out using a commercial kit (Pierce, Rockford, IL). The binding mixture (25 µl) containing 10 µg protein of nuclear extract and 1 µg of poly dI-dC were incubated in a Tris-EDTA buffer (10 mM TrisHCL, pH 7.4, 50 mM NaCl, 50 mM KCl, 1 mM MgCl2, 1 mM EDTA, 5 mM DTT) on ice for 15 mins. Later 10 ng of biotinylated double-stranded NFkB probe (NFkB oligonucleotide probe supplied by Operon [Cologne, Germany], the sequence being NFkB, F 5′-AGT TGA GGG GAC TTT CCC AGG C-3′, NFB R 5′-GCC TGG GAA AGT CCC CTC AAC T-3′, NFB mutant F 5’AGT TGA GGC GAC TTT CCC AGG C 3′, NFB mutant R 5’GCCTGG GAA AGT CGC CTC AAC T3′) (the underlining indicates a DNA-bindingsite for NFkB) was added and incubated at RT for 30 mins. ThenDNA protein complexes were separated on native 6% polyacrylamideDNA retardation gel and electroblotted onto positively chargednylon membranes. Biotinylated DNA/ protein complexes were detectedwith peroxidase-conjugated streptavidin and chemiluminescentsubstrate kit (Pierce).
NFkB Blockade Study.
The rats were administered with curcumin (50 mg/kg BW) orally1 hr before the hypoxic exposure (6 hrs). Determination of pulmonaryedema and NFkB protein expression were carried out as describedabove.
Statistical Analysis.
Statistical analysis was performed using SPSS for Windows (15.0) software (SPSS Inc., Chicago, IL). Comparisons between five experimental groups for various time points and also curcumin-treated groups were made by using one-way ANOVA with Student-Newman-Keuls test for multiple comparisons between groups. Whereas, comparisons between normoxia-exposed (0 hrs) and hypoxia-exposed (6 hrs) animals were made using Student’s t test. Differences were considered statistically significant for P < 0.05. Resultsare expressed as mean ± SD.
Results |
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TOP Abstract Introduction Materials and Methods Results Discussion Conclusion References |
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Determination of Pulmonary Edema.
Vascular Permeability.
The effect of different periods of hypoxic exposure on developmentof pulmonary edema is shown in Figure 1. Exposure to hypoxiaresulted in a significant increase in lung wet/dry weight ratiorelative to control animals. The maximum lung water contentwas obtained at 6 hrs and 12 hrs of hypoxic exposure (3-foldincrease in both the hours of exposures). Further increase inexposure to 24 hrs resulted in a marked decrease in wet/dryratio (Fig. 1).
Alternatively, the vascular leakage was determined by measuringthe relative fluorescence intensity in the lungs of rats exposedto hypoxia. There was a significant increase in relative fluorescenceintensity (40%) in the lungs of rats exposed to 7620 m for 3hrs as compared with control animals. Maximum increase in relativefluorescence intensity (60%) was observed in animals exposedto 6 hrs of hypoxia. However, further increase in exposure time(i.e., 12 hrs and 24 hrs) resulted in a considerable fall invascular permeability (Fig. 2). Because maximum vascular leakagewas obtained at 6 hrs of hypoxic exposure, further experimentswere conducted by exposing the rats to hypoxic stress for 6hrs.
ROS, Lipid Peroxidation, and Antioxidant Levels.
A gradual but significant increase in ROS generation (as revealedby increase in DCF fluorescence) was observed in lungs of ratsexposed to hypoxia with time. To determine whether increasedROS levels led to increased membrane peroxidation, we determinedthe malondialdehyde (a volatile carbonyl) levels, an importantbiomarker released from the oxidation and decomposition of PUFA(polyunsa-turated fatty acids). The results revealed a markedincrease in MDA levels in the lungs of rats exposed to hypoxiaup to 6 hrs relative to control values (Table 1). Further increasein hypoxic exposure resulted in marginal decrease in MDA levels.
Because the maximum increase in pulmonary vascular leakage wasobserved during 6 hrs of hypoxic exposure, all other parameterswere determined by exposing the animals for 6 hrs only.
Antioxidant Levels.
A marginal but nonsignificant increase in GSH levels was observedin the lungs of hypoxia exposed rats related to control animals.However, exposure to hypoxia resulted in to an appreciable fallin GPx levels while the SOD remained higher as compared withcontrol animals (Table 2).
NFkB Levels.
There was a nearly 13-fold increase in NFkB levels (nuclearfraction) in the lungs of hypoxia-exposed animals as comparedwith that of control animals. However, the cytoplasmic levelsof NFkB remained more or less similar (Fig. 3). To further confirmwhether increased translocation of NFkB also results in increasedDNA-binding activity, gel shift assays were performed usinga highly specific biotinylated-oligonucleotide probe. The resultsrevealed a significant increase in NFkB DNA-binding activityin the lungs of rats exposed to hypoxia over control animals(Fig. 4).
Proinflammatory and Cell Adhesion Molecules.
To confirm whether the enhanced levels of NFkB lead to upregulation of inflammatory molecules, which are known to be regulated by it, we measured the expressions of IL-1, IL-6, and TNF- levels in the lungs of animals during hypoxic exposure by immunoblotting. In control animals, IL-1 was not detectable; however, its level was increased appreciably upon exposure to hypobaric hypoxia (Fig. 5a). A significant increase in IL-6 and TNF- levels also were noticed in the lungs of hypoxic rats. The fold increase in IL-6 and TNF- was 7 and 4.5 times, respectively, relativeto control levels (Fig. 5b, c).
We also determined the expression profiles of cell adhesionmolecules such as ICAM-I, VCAM-I and selectins (P-selectin)in the lungs of control and hypoxic rats. ICAM-I was virtuallyundetectable in the lung of control rats; however, upon exposureto hypoxia, a significant increase in its level was observed(Fig. 6a). At the same time, VCAM-1 levels increased by about3-fold in the lungs of rats exposed to hypoxia over controlanimals (Fig. 6b). Similarly, exposure of rats to hypoxia alsoresulted in to a significant increase in lung P-selectin levelsalthough in control animals P-selectin was not detectable (Fig.6c).
NFkB Blockade by Curcumin.
To understand the involvement of NFkB in the development ofpulmonary vascular leakage, the animals were administered curcumin(50 mg/kg BW) orally 1 hr before hypoxic exposure. Interestingly,a significant reduction in water content (45.6%) and transvascularleakage (60%) was noted in curcumin +hypoxia group when comparedwith the hypoxic group (Fig. 7a, b). However, the normoxia groupadministered with curcumin did not show any significant differencein vascular leakage compared with control. Similarly, as expected,curcumin administration markedly inhibited hypoxia-induced NFkBlevels by about four times (Fig. 8a, b) compared with the hypoxicgroup.
Discussion |
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TOP Abstract Introduction Materials and Methods Results Discussion Conclusion References |
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The present study was undertaken to determine the association of oxidative stress and activation of NFkB in causing vascular leakage during exposure to hypobaric hypoxia. An animal model (rat) was used, where rats were exposed to simulated hypobaric hypoxia for different durations, that is, 3 hrs, 6 hrs, 12 hrs, and 24 hrs at 7620 m at 25 ± 1°C. The results showed that exposure of animals to hypoxia for 6 hrs led to increased vascular leakage as evidenced by increased water content and fluorescein leakage in lungs as compared with control animals. Further, there was a significant increase in ROS generation and lipid peroxidation, decreased antioxidative enzymes, enhanced expression of NFkB, and higher levels of proinflammatory cytokines and cell adhesion molecules in the lungs of rats exposed to hypoxia compared with normoxic animals. It was also observed that the curcumin administration 1 hr prior to hypoxic exposure significantly (P < 0.01) reduced the transvascular leakagein the lungs of rats by down-regulating the activation of NFkBcompared with the hypoxia-exposed rats. In this limited study,we speculated the possible role of oxidative stress and NFkBin causing the vascular leakage. Further, using the NFkB blockercurcumin, we showed the involvement of NFkB for the first timein the cause of pulmonary edema. These findings provide cluesfor developing new therapeutic drugs against HAPE. The schematicrepresentation of hypoxia-induced trans-vascular leakage inthe present study will be sequentially discussed in the ensuingparagraphs (Fig. 9).
HAPE is associated with an increase in leakage of both fluid and proteins in the alveolar space, with enhanced arterial pressure followed by pulmonary vasoconstriction. It is well known that HAPE occurs on ascent to altitudes above 2450 m (22) while its incidence decreases with lowering altitudes. In our studies, we have used rat as an animal model because of the following reasons: (1) the physiologic mechanisms of its acclimatization to HA are very much similar to that of humans (17); (2) smaller animals have higher capillary density and shorter diffusion distance compared with larger species (37); and (3) hypoxia can lead to pulmonary edema in some species (especially in the rat) as occurs in the development of HAPE in humans, and hypoxia may also exaggerate the effects of a prior inflammatory insult into the lung (12, 25). Therefore, in the present study, we exposed the rats to a simulated altitude of 7620 m at 25 ± 1°C for different durations, that is, 3 hrs, 6 hrs, 12 hrs, and 24 hrs in order to find out the optimum time for appearance of vascular leakage in the lungs. We found that maximum lung wet/dry weight ratio was obtained in rats exposed to 6 hrs to 12 hrs duration. Further exposure resulted into a marginal decrease in wet/dry weight ratio. This could be attributed to considerable water loss caused by severe hyperventilation during exposure to hypoxia. To confirm further, we measured the transvascular leakage (common cause of edema) by direct method using a fluorescent probe, sodium fluorescein. Sodium fluorescein is a fluorescence tracer that can be measured easily in lungs to exactly quantify dye extravasation and therefore vascular permeability. We found that the fluid accumulation, as revealed by increase in fluorescein content in the lungs of rats, began at 3 hrs of hypoxic exposure and reached the peak at 6 hrs of exposure. In this regard our results are in consistence with the earlier studies (9).
Because exposure of animals to hypobaric hypoxia for 6 hrs resulted in a maximum fluid leakage in lungs, we studied the role of oxidative stress, that is, ROS in the development of HAPE. The increased MDA levels (P < 0.001) in lung tissue indicated increased free-radical production. Hypoxia is known to increase plasma and tissue MDA levels at high altitudes and even in in vitro conditions (36, 47), and our results are in accordance with earlier studies (33). To cope with the oxidative stress, a marginal but insignificant increase in GSH levels was noticed in the the lungs of rats exposed to hypoxia as compared with control animals. Further, there was a significant increase in SOD levels in the lungs of rats exposed to hypoxia as compared with control animals. It is speculated that the increased SOD levels will attenuate the superoxide radicals (O2–), which are produced during exposure to hypoxia. Recently, it has been shown that ROS are involved and may even play a causative role in the AMS, HAPE, and HACE (4, 8). However, the mechanism through which ROS contribute to the pathophysiology of organ injury to the lung remains incompletely understood. It is not yet completely known whether pulmonary edema is an inflammatory disease or not. Studies on human subjects showed elevated RBC counts and serum proteins in bronchioalveolar lavage (BAL) fluid of HAPE subjects within a day of ascent to 4559 m. However, there was no significant difference in proinflammatory mediators, that is, IL-1, IL-6, TNF-, IL-8, thromboxane, PEG2, and leuko-triene B4 in the BAL fluid of HAPE subjects (40). The main drawback of this study is that the levels of inflammatory cytokines were measured in subjects at the beginning and at end of the study, but no afford was made to measure these cytokines before the onset of HAPE. Therefore, it is possible that these inflammatory cytokines would have disappeared after causing the inflammation. This would possibly explain the fact that production of inflammatory mediators might precede the onset of pulmonary edema. Interestingly, in the present study, we found that NFkB levels are elevated at 6 hrs of hypoxic exposure (starting of increased vascular leakage). Because NfKB directly regulates proinflammatory cytokine production, we speculate that production of inflammatory cytokines also precedes the vascular leakage, and once their action is over, they disappear. However, studies on BAL fluid of mountaineers with advanced HAPE at Mount McKinley (38) and also hospitalized HAPE patients in Japan (26) showed elevated levels of cytokines and increased granulocytes. This shows that in advanced cases of HAPE, inflammation may occur and contribute to enhanced pulmonary capillary permeability. Earlier, Ono et al. (35) showed thatthe rats primed with endotoxin developed pulmonary edema. Further,the BAL fluid of endotoxin-primed and hypoxia-exposed rats containeda greater number of white blood cells and a higher concentrationof proteins than that of endotoxin-primed normoxic rats. Thisindicates the involvement of inflammatory components in theoccurrence of pulmonary edema. The present study is focusedmainly on the role of oxidative stress (hypoxia) driven–inflammationmediated by NFkB in causing vascular leakage in the lungs.
It is reported recently that oxidant stress increases the vascular endothelial permeability and expression of redox-sensitive transcription factor NFkB (29). In the present study too, we found an appreciable (nearly 13-fold) increase in NFkB levels in the nuclear extract of the lungs of animals exposed to hypoxia over control animals. NFkB is normally found in its inactive form in the cytosol as the heterodimer p50/P65 unbound to its inhibitory unit IkB. Upon appropriate cell stimulation, IkB and IkBβ are rapidly phosphorylated on specific amino-terminal serine, signaling for ubiquitination and degradation by the 26 S proteosome. This results in the exposure of a nuclear localization sequence and DNA-binding domains, allowing the NFkB to enter the nucleus and stimulate the transcription of target genes. The DNA-binding studies in the present study have shown an increased NFkB-binding (nearly 3-fold) activity in the lungs of hypoxia-exposed rats compared with control. NFkB and IKB are present in the nucleusin low concentrations even in nonactivated cells, and the signalingmolecules of NFkB shuttle between the cytoplasm and the nucleus.This could be the reason for the presence of NFkB in the cytoplasmicfraction of the lung in the present study. To the best of ourknowledge, we report for the first time the involvement of NFkBin the development of pulmonary vascular leakage.
Earlier it was reported that altered NFkB can mediate an inflammatory cytokine response and also induce gene transcription of adhesion molecules, that is, ICAM-I, VCAM-1, etc. (21). Therefore, we sought to determine whether increased NFkB levels lead to enhanced inflammatory cytokines. Interestingly, we found a significant increase in IL-1 and IL-6 levels in the lungs of rats exposed to hypoxia over control animals. Further, there was a significant increase in TNF- levels during hypoxia exposure. TNF- is a polypeptide that influences endothelial cell function by promoting neutrophil adherence to vascular endothelium (20). TNF- has been shown to inhibit the release of endothelium-derived relaxing factor (EDRF) in isolated carotid artery rings, and therefore causes endothelial dysfunction besides increasing reactive oxygen species levels (15). Deruelle et al. (18) showed that the increased level of TNF- is responsible for development of right ventricularhypertrophy (RVH) in rats exposed to 10% oxygen for 3 weeks.
The cytokines such as TNF- and IL-1 cause the appearance of ICAM-I and VCAM-I and the selectins such as P-selectin and E-selectin in the capillary endothelial cells and permit lymphocytes and monocytes to adhere and move into inflamed tissues. In the present study, there was a concomitant upregulation of cell adhesion molecules such as ICAM-I, VCAM-I, and P-selectin besides proinflammatory cytokines in the lungs of rats exposed to the hypoxia over control animals. It has been shown earlier that O2 deprivation (a common cause of hypoxia) leads to upregulation of genes involved in inflammation. Carol and Brain (10) reported that the cell adhesionmolecules like ICAM-I and VCAM-I genes contain NFkB site onthe promoter region and are known to be regulated by NFkB. Wetherefore hypothesize that the observed increase in these proinflammatoryand cell adhesion molecules could be attributed to enhancedNFkB levels in the lungs of hypoxic rats.
Recent reports revealed that curcumin, a yellow pigment and a major component of turmeric (Curcumin longa L.), has the ability to downregulate the NFkB activation, which has been linked to a number of inflammatory diseases (1, 14, 45). Therefore, NFkB blockade studies were carried out to know whether this strategy would inhibit hypoxia-induced fluid accumulation in the lungs. It was evident from our present data that curcumin administration significantly attenuated vascular leakage in the lungs of rats exposed to hypobaric hypoxia and maintained their values similar to that of normoxic animals. Further, curcumin significantly inhibited hypoxia-induced NFkB expression and maintained its levels similar to that of control levels. Curcumin from C. longa has been demonstrated as an anti-inflammatory agent in in vivo animal models (2). Curcumin is reported to scavenge free radicals, inhibit lipid peroxidation, protect the SH group of GSH, and activate glutathione-S-transferase as well as inhibit nitrite radical-induced oxidation of hemoglobin and prostaglandin biosynthesis (32, 43). However, recent studies revealed that dexamethasone—a corticosteroid recommended as prophy-lactic drug to prevent AMS and HACE—has been shown to inhibit NFkB (11) and leads to a reduction in systemic pulmonary artery pressure in HAPE subjects (30, 39). Further, nifedipine is nowadays recommended as prophy-lactic drug of choice against HAPE if progressive high-altitude acclimatization is not possible (31). Nifedipine is effective in inhibiting NFkB activation and thereby contributing to decreased inflammation followed by increased endothelial function in the coronary circulation (41). However, in the presentstudy, the ability of curcumin to prevent hypoxia-induced vascularpermeability is not linked to its antioxidant activity, as administrationof antioxidants such as vitamin C or vitamin E have little effecton hypoxia-induced vascular leakage in lungs (data not shown).Further in the present study, there is no linear relationshipbetween ROS generation and vascular leakage. ROS continued toaccumulate even after 24 hrs while maximum lung water contentwas observed between 6 hrs to 12 hrs. However, increased NFkBactivity was directly associated with pulmonary edema. Thisshows that ROS are not solely responsible for occurrence ofvascular leakage although they might be contributing to occurrenceof vascular leakage, probably through upregulation of NFkB.This also possibly explains the inability of antioxidant vitaminsto prevent hypoxia-induced pulmonary vascular leakage. Therefore,the observed reduction in fluid efflux in our study might becaused by the anti-inflammatory activity of curcumin. We thereforehypothesize that exposure of animals to hypoxia activates NFkB,which in turn results in upregulation of inflammatory mediators,finally leading to pulmonary vascular leakage. Further research,including a generation of overexpression/knockout studies ofNFkB and micro-array analysis of rat lungs, will help us tounravel the exact molecular mechanism involved in hypoxia-inducedtrans-vascular leakage.
Conclusion |
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TOP Abstract Introduction Materials and Methods Results Discussion Conclusion References |
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Taken together, our results revealed that exposure of rats to hypoxia increased the oxidative stress in the animals as evidenced by enhanced ROS, increased MDA levels, and marginal elevation in endogenous antioxidants such as SOD and GSH levels in the lungs of rats. This increased oxidative stress might have activated NFkB and its translocation into the nucleus, leading to upregulation of the proinflammatory cytokines (IL-1, IL-6, and TNF-) andcell adhesion molecules (ICAM-1, VCAM-I, and P-selectin). Thisin turn might be responsible for causing vascular leakage inrats exposed to hypoxia. Administration of curcumin significantlyinhibited hypoxia-induced vascular leakage and NFkB levels inlungs. These findings, therefore, suggest that oxidative stress–drivenincreases in lung NFkB content can contribute to the formationof pulmonary edema (seen in this model). Because NFkB playsa significant role in inflammation, the present study opensa new area for developing better therapeutic strategies forthe prevention of HAPE.
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Received for publication December 14, 2008.
Accepted for publication May 13, 2008.
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