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The mechanisms contributing to hypoxia in lung contusion remain unclear and not temporally associated with the peak onset of acute inflammation.
We investigated the role of oxidative stress in alteration of pulmonary arterial (PA) reactivity following LC. Additionally, the role of antioxidants in reversing this process was examined.
PaO2 and PA reactivity were measured in rats subjected to bilateral LC. Rings were pretreated with a NO synthase (NOS) inhibitor, L-nitro arginine (LNA 10−3 M) or PEG-superoxide dismutase (SOD) and PEG-catalase (CAT) or both (LNA+SOD/CAT). Rings were constricted with norepinephrine (NE) and relaxed with an NOS agonist (A23187) or NO donor (SNAP). Immunochemical and mass spectrometric quantification for nitrotyrosine were performed.
Rats were hypoxemic at 4h post-contusion compared to controls, but recovered by 24h (PaO2/FiO2 ratio: baseline- 443±28, 4h-288±46 and 24h-417±23). PA constriction to NOS inhibition and relaxation to A23187 were impaired 4h after LC. PA relaxation to SNAP was decreased at 4h and 24h after LC. These alterations in PA reactivity were reversed by SOD/CAT pretreatment. SOD1 and 2 mRNA was up-regulated and soluble guanylyl cyclase (sGC) mRNA was down-regulated 24h after LC. IHC and mass spectrometry revealed that levels of 3-nitrotyrosine were increased markedly at 4h following LC consistent with superoxide generation and formation of peroxynitrite.
Collectively, this data suggests that consumption of NO due to excess superoxide resulting in peroxynitrite formation leads to diminished vascular reactivity following LC.
Lung contusion (LC) is a common consequence of blunt chest trauma (1) and is an independent risk factor for acute respiratory distress syndrome (ARDS) and ventilator associated pneumonia (VAP). The pathogenesis of hypoxia, a pathognomonic feature of LC, is not well understood. There are also conflicting reports on the degree of correlation between the severity of hypoxemia and the volume of LC (2, 3). Previous studies performed in our laboratory indicate that the peak onset of inflammatory response is at 24h, a time point at which there is almost a complete reversal of hypoxia. (4). Previous studies have postulated that the acute pulmonary dysfunction caused by contusion can be secondary to release of blood and plasma into the alveoli resulting in ventilation and perfusion mismatch and increased pulmonary vascular resistance (5). Additionally, some studies have reported that the alterations in pulmonary vascular reactivity associated with lung contusion may increase pulmonary vascular resistance and lead to right ventricular strain (6). However these results have not been completely substantiated in small animal models of LC. Moreover, the pulmonary vascular reactivity in animal models of LC has not been studied in detail.
We have described a rat model of isolated bilateral LC induced by blunt chest trauma (7). In this model, hypoxemia was found to be profound between 4h–8h after contusion and resolved by 24–48h. Nitric oxide (NO) is an important determinant of pulmonary vascular reactivity(8, 9). Vascular superoxide anions react with NO to form toxic peroxynitrite (10). The main determinant of bioavailability of endogenous NO is the local concentration of superoxide anions and activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (11).
We hypothesized that LC would result in generalized alteration in pulmonary arterial reactivity in all areas of the lung. We further hypothesized that changes in pulmonary vaso-reactivity following LC would be secondary to alterations in the NO-superoxide pathway. The overall goals were to identify factors responsible for hypoxemia including changes in pulmonary vascular reactivity and understand the role of antioxidants and NO as potential therapeutic options in LC. Two time points were chosen based on our prior studies; 4h (significant period of hypoxemia that extends to up to 8 hr) and 24h (representing the period of recovery from hypoxemia) after LC. We sought to evaluate contraction to norepinephrine (NE) and relaxation to nitric oxide synthase (NOS) agonists and NO donors in rings from third generation pulmonary arteries (PA) in control and rats following LC. A subset of PAs was pretreated with NOS inhibitors and superoxide scavengers (SOD + catalase) to study their impact on the NO-superoxide pathway. Changes in mRNA expression of the NO-Endothelin pathway were measured by RT-PCR. Our analyses, as discussed below, revealed that NO pathways are well preserved and the reversal of hypoxemia at 24h was associated with increased levels of SOD 1 and 2. Additionally 3-nitrotyrosine (a marker for peroxynitrite formation) levels in the pulmonary vasculature as determined by fluorescent immunohistochemistry were found to be elevated, particularly at 4 h. These data demonstrate that consumption of NO due to excess superoxide resulting in peroxynitrite formation leads to diminished vascular reactivity following LC
Adult male Long-Evans male rats (250–300 g body wt., Harlan Sprague-Dawley, Indianapolis, IN) were utilized in this study. All procedures performed were approved by the Institutional Animal Care and Use Committee at the University of Buffalo, SUNY, and the University of Michigan, and complied with State, Federal, and National Institutes of Health regulations.
LC was induced in 38 halothane-anesthetized rats by dropping a hollow aluminum cylindrical weight (300g) from a height of 80cm onto the chest as described earlier (7). The key part of the instrument is the presence of a protective plastic shield that protects the mediastinal structures and selectively injures the lungs bilaterally (7). Others and we have identified it as a significantly useful model to study pulmonary contusion. In the model the force used to injure the rodent was maximal and non-lethal (we strove to achieve a reproducible model with less than 10% mortality). Use of a higher potential energy was associated with 30% mortality and hence we chose the energy equivalence of 2.2 joules for the experiments (7). This protocol therefore used an impact energy of 2.2 J and is associated with low mortality (2/38 rats). Twelve rats that did not undergo contusion were used as controls. Control rats and contused rats (4h, n=13 and 24h, n=23 after contusion), were anesthetized with 2% halothane and 98% oxygen. Arterial blood gas samples were obtained from the abdominal aorta and the rats were sacrificed.
After the induction of LC and in uninjured controls, the heart and lungs were removed en bloc. The lungs were examined for areas of contusion. Third-generation pulmonary arteries (inner diameters of ~ 0.5 mm) were dissected from areas of the lungs without macroscopically visible injury, isolated and cut into rings as described previously (12, 13). Rings were suspended in water-jacketed chambers filled with aerated (21% O2 – 6% CO2, remainder N2) modified Krebs-Ringer solution (in mM: 118 sodium chloride, 4.7 potassium chloride, 2.5 calcium chloride, 1.2 magnesium sulfate, 1.2 potassium biphosphate, 25.5 sodium bicarbonate and 5.6 glucose). A continuous recording of isometric force generation was obtained by tying each vessel ring to a force displacement transducer (model UC2, Statham Instruments, Hato Rey, PR) that was connected to a recorder (Gould Instrument Systems, Valley View, OH). After the arterial rings were mounted, they were allowed to equilibrate for 20 min in the bathing solution (PO2 – 140 to 150 mmHg). A micrometer was used to stretch the tissues repeatedly in small increments over the following 45 min until resting tone remained stable at a passive tension of 0.6 g. Preliminary experiments and literature search (14, 15) determined that this procedure provided optimal length for generation of active tone to exogenous norepinephrine. Wet tissue weights were obtained at the end of each experiment, and contraction responses were normalized to tissue weight.
The following pharmacological agents were used (Table 1): DL-propranolol, norepinephrine hydrochloride (NE), calcium ionophore A 23187 and nitric oxide donor S-nitrosyl amino penicillamine (SNAP), nitric oxide synthase antagonist, Nitro-L-arginine (LNA), phosphodiesterase 5 (PDE5) inhibitor, E 4021, superoxide dismutase (SOD) linked to polyethylene glycol (PEG-SOD) and catalase linked to polyethylene glycol (PEG-catalase). E4021 was manufactured by Eisai Tsukuba Research Laboratory was a gift from the Animal Health Trust (Newmarket, Suffolk, UK). All other drugs were obtained from Sigma-Aldrich. SNAP was dissolved first in a small quantity of DMSO and then diluted in distilled water. E4021 was dissolved in 0.01N sodium hydroxide and LNA was dissolved in warmed Kreb’s solution by sonication. All other drugs were dissolved in distilled water. DMSO and dilute sodium hydroxide, at the concentrations used in these experiments, did not alter the preexisting tone of the pulmonary arteries.
Isolated pulmonary arteries were pretreated with propranolol (10−6 M) to block β adrenergic receptors and LNA to block endogenous nitric oxide synthesis. Some additional vessels were pretreated with PEG-SOD (75 units/ml) and PEG-catalase (1200 units/ml) to scavenge superoxide anions and to evaluate the role of reactive oxygen species in LC. Both these agents were added simultaneously to avoid significant accumulation of H2O2 in the tissue bath as prolonged exposure to H2O2 constricts rat pulmonary arteries(16). The arteries were first constricted with increasing doses of NE (10−8 to 3 × 10−6 M). Constriction responses were recorded as grams of force and were normalized for wet arterial ring weight (expressed as grams of force/grams weight). Two rings were studied from each animal and the mean contraction from these rings was used for analysis. Additional arterial rings were used for relaxation studies: these vessels were constricted with an EC50 concentration of NE. Some of these vessels were pretreated with LNA or PEG-SOD and PEG-catalase or both. These arterial rings were then relaxed with A23187 (10−6 M), SNAP or E4021 (10−8 to 10−5 M). These experiments were done in a dark room as LNA is sensitive to light.
The quantitative real time PCR methods were performed as described previously(17, 18). Briefly, frozen, isolated PA tissue was ground on liquid nitrogen, total RNA was isolated and prepared to cDNA. Real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad) with the iCycler iQ real-time PCR detection system (Bio-Rad), with 40 cycles of real-time data collection at 95°C for 20 s and 59.6°C for 1 min, followed by melt-curve analysis to verify the presence of a single product. Specific primers for NO-Endothelin pathway were obtained from SA Biosciences® Valencia, CA. PCR product size was verified by agarose gel electrophoresis, and all samples were analyzed in duplicate. For each reaction, negative controls containing reaction mix and primers without cDNA were performed to verify that primers and reaction mixtures were free of template contamination. Relative NOS3 (endothelial NOS), sGC (soluble guanylate cyclase – target enzyme for NO) and PDE 5 amounts were normalized to 18S expression using the cycle threshold (CT) method. Data are fold values relative to control rats.
Lung sections were prepared and stained as previously described (17). Briefly, the rat lung tissues sections were fixed in 10% (w/v) PBS-buffered formaldehyde and 7μm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeated with 0.1% (w/v) Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the sections in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with biotin and avidin, respectively. Sections were incubated overnight with anti-nitrotyrosine rabbit polyclonal antibody (1:500 in PBS, v/v). Sections were washed with PBS and incubated with secondary antibody. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG and avidin-biotin peroxidase complex (DBA). To confirm that the immunoreaction for the nitrotyrosine was specific, some sections were also incubated with the primary antibody (anti-nitrotyrosine) in the presence of excess nitrotyrosine (10 mM).
Lung sections were prepared and stained as previously described (17). Quickly, the right, middle lobe of the lung was removed, and OCT compound (Tissue–Tek, Torrance CA) was pushed gently into the deflated lobe and allowed to solidify on ice for 15–20 min. Blocks were prepared and cut into 7μm sections that were mounted onto charged slides for staining and stored at −80°C. Frozen lung sections were subsequently fixed with acetone and exposed to 5 μM dihydroethidium (DHE; Molecular Probes/Invitrogen) in PBS. Slides were incubated in a light-protected humidified chamber at 37°C for 30 min. Ethidium-stained slices were observed by fluorescence microscopy with excitation at 518 nm and emission at 605 nm. Fluorescent images were captured using a Nikon Eclipse Ti Microscope with NIS elements AR imaging software (Nikon Inc, NY)
Plasma protein-bound oxidized tyrosine marker, nitrotyrosine was measured by HPLC/MS by triple quadruple tandem MS as described previously(19). Briefly, the tissue was homogenized and the protein pellet was isolated. Isotopically labeled internal standards were added, and samples were hydrolyzed with 4N methane sulfonic acid at 110°C for 24 h under argon, Quantification of oxidized amino acids were performed using isotope dilution electrospray ionization MS as described previously(19).
All data are expressed as mean ± SEM, with ‘n’ representing the number of animals studied. Statistical comparisons of the curves were performed with repeated measures ANOVA. Student-Newman-Keuls post-hoc testing was used as needed to compare multiple groups. Statistical analysis was performed with Stat View software (Abacus Concepts, Berkley CA), and GraphPad Prism5.01 (20). *P < 0.05 compared to corresponding control animals.
Thirty-eight rats underwent LC under halothane anesthesia. Two rats died soon after contusion. Surviving rats recovered in room air. Four hours after contusion, arterial blood gas was obtained by directed aortic puncture before sacrifice by exsanguination under halothane anesthesia after administration of 100% oxygen for about 5 min in 14 rats. Similar blood gas samples were obtained from the rats 24h after contusion. The results of the blood gas analysis are shown in Table 2. Rats were significantly hypoxemic at 4h following LC but showed recovery by 24h. There was no significant difference in PaCO2values between the three groups.
Constriction to increasing concentrations of NE in different groups of rats (control, 4h post -contusion, and 24h post-contusion) are shown in Fig. 1-A, B and C. Fig. 1-D shows constriction to the highest dose of NE (0.3 μM) based on the four treatment groups (no pretreatment, pretreatment with PEG-SOD and PEG-catalase, pretreatment with NOS inhibitor, LNA and pretreatment with PEG-SOD, PEG-catalase and LNA). Treatment with a high dose of potassium chloride resulted in PA constriction in control PA (484 ± 120g/g). There was no significant change in constrictor response to potassium chloride after contusion ((560 ± 39 g/g after 4h and 592±98 g/g after 24h of LC). Pretreatment with SOD/CAT or LNA did not significantly alter response to potassium chloride.
Pulmonary arteries constricted well in response to norepinephrine in a concentration dependent manner in control rats. Pretreatment with PEG-SOD and PEG-catalase did not significantly alter the contraction response to norepinephrine (suggesting that oxidative stress did not play a role in constriction response to NE). Inhibition of NOS with LNA significantly increased the contraction response to NE (reflective of normal endogenous baseline NOS activity). Addition of PEG-SOD and PEG-catalase prior to pretreatment with LNA did not alter the response compared to LNA alone. The concentration-response curve to NE without any pretreatment of the 4h post-contusion rats did not differ significantly from control rats (Fig. 1B and D). Pretreatment with PEG-SOD and PEG-catalase significantly reduced the contraction response (Fig. 1B, suggesting oxidative stress contributing to constriction). Inhibition of NOS with LNA markedly enhanced the contraction to NE (reflective of enhanced endogenous NOS activity four hours after contusion). Addition of PEG-SOD and PEG-catalase prior to pretreatment with LNA reversed the increased contraction observed with LNA alone. Concentration-response curves to NE without any pretreatment from pulmonary arteries isolated from 24h post-contusion rats did not differ significantly from controls (Fig. 1C and D). Pretreatment with PEG-SOD and PEG-catalase did not significantly alter the constriction response (suggesting that oxidative stress was resolved or there was a compensatory increase in anti-oxidant activity). NOS inhibition with LNA markedly enhanced constriction to NE. Pretreatment with PEG-SOD and PEG-catalase, prior to addition of LNA, did not alter constriction to NE compared to LNA alone.
Subsequently maximal constriction to a high concentration of potassium chloride was evaluated. Potassium chloride elicits electromechanical contraction of vascular smooth muscle by causing depolarization resulting in an influx of ionic calcium into the cytoplasm. There was no statistically significant difference in potassium chloride constriction among all the groups Pretreatment with LNA significantly enhanced constriction to KCl 4h after contusion again suggesting enhanced endogenous NOS activity during this period.
Following pretreatment with NE, relaxation to a single dose of 10−6M of A23187 was analyzed. This response studies the ability of the endothelial NOS (NOS 3) to produce NO, bioavailability of NO and the effectiveness of the NO–soluble guanylate cyclase (sGC) pathway. The relaxation to A23187 was impaired following LC both at 4h and 24h post -contusion (Fig. 2A). Pretreatment with PEG-SOD and PEG-catalase did not alter the relaxation to A23187 in control PA. In sharp contrast, it completely reversed the impaired response following LC (Fig. 2B). Pulmonary arteries isolated from control rats relaxed in a concentration dependent manner to SNAP (Fig. 3A). Pretreatment with either LNA or PEG-SOD and PEG-catalase or their combination did not alter the relaxation response to SNAP in control pulmonary arteries.
Relaxation to SNAP was significantly impaired after contusion (Fig. 3D). Interestingly, scavenging superoxide anions with PEG-SOD and PEG-catalase enhanced relaxation to SNAP (Fig. 3B) in pulmonary arteries isolated from rats four hours after contusion. Inhibiting NOS by pretreatment with LNA also significantly enhanced SNAP mediated relaxation in this group (Fig. 3B). Pretreatment with LNA and PEG-SOD and PEG-catalase resulted in a significantly better relaxation response compared to PEG-SOD and PEG-catalase alone 4 h after contusion. Relaxation to SNAP continued to be impaired 24h after LC despite scavenging superoxide anions (Fig. 3D). Pretreatment with PEG-SOD and PEG-catalase did not enhance relaxation to SNAP in this group. In contrast, NOS inhibition with LNA enhanced relaxation to SNAP in pulmonary arteries isolated from rats 24h after LC. Pulmonary arteries pretreated with LNA with PEG- SOD and PEG-catalase relaxed significantly better to SNAP compared to arteries pretreated with PEG-SOD and PEG-catalase alone (Fig. 3C).
To determine the role of cyclic GMP mediated relaxation induced by NO, the role of phosphodiesterase 5 enzyme was investigated. Inhibition of phosphodiesterase 5 enzymes by the specific inhibitor, E4021 resulted in concentration-dependent relaxation of PA isolated from rats (80 ± 7% relaxation with 10−6 M) similar to our previous studies in lambs (21, 22). There was no significant difference in relaxation between control and post-contusion PA (87 ±11%).
Messenger RNA expression of endothelial NOS (NOS3) was not significantly altered following LC. There was a significant decrease in soluble guanylate cyclase (sGC) mRNA expression following contusion reaching significance 24h after LC. There was no change in the expression of phosphodiesterase-5 enzyme (Fig. 4A). The mRNA expression of cytosolic Cu-Zn- SOD (SOD 1), mitochondrial MnSOD (SOD 2), extracellular SOD (SOD 3) and catalase were analyzed in pulmonary arteries. LC increased expression of SOD 1 and SOD 2 mRNA 24h after contusion (Fig. 4B). There were no differences in the mRNA levels of Endothelin in the various groups when compared with uninjured controls (data not shown).
The lung sections stained positive for superoxide anions with dihydroethidium (DHE) staining. The pulmonary arteries from post-contusion lung samples stained with similar fluorescent intensity to DHE compared to control lungs without contusion (Fig. 5).
3-Nitrotyrosine is used as a marker of peroxynitrite formation secondary to interaction between superoxide anions and nitric oxide (Fig. 6). The intensity of peroxynitrite staining was low in pulmonary arteries from control lungs (Fig. 6C). There was a significant increase in fluorescent intensity at 4h and 24h following LC compared to uninjured rats (florescent intensity of 11.1 ± 4.6 at 4hr, 9.0± 2.2 compared with 0.9 ±0.19, p<0.05 compared to uninjured controls) (Fig. 6D and E).
We investigated whether oxidative stress is increased with LC and in order to determine the source of the excess oxidant in the lung tissue performed quantification of oxidized amino acids using isotope dilution electrospray ionization MS as described previously(19). Lung tissue nitrotyrosine levels were markedly increased ~2 fold in LC vs. controls (p < 0.001) (Figure 7).
In this study, abnormalities of vaso-reactivity and alterations in the nitric oxide – superoxide pathway in pulmonary arteries isolated from segments of the lung without any evidence of visible contusion, were observed. Rats were hypoxemic at 4h post-contusion compared to controls, but recovered significantly at 24h after contusion. Constriction to LNA+NE was higher and relaxation to A23187 was impaired 4h after contusion compared to controls. These alterations were reversed by pretreatment of the PA with SOD/CAT. Relaxation to SNAP was impaired 4h and 24h after contusion and pretreatment with SOD/CAT improved these responses. Evaluation of the pulmonary arteries revealed that SOD1 and 2 mRNA were up-regulated and sGC mRNA was down-regulated 24h after contusion. There was no difference in superoxide anion staining in pulmonary arteries with LC. IHC staining for 3-nitrotyrosine markedly increased following LC and these markers including dityrosine were confirmed by MS/MS. Taken together we conclude that peroxynitrite formation is responsible for altered pulmonary arterial activity following LC.
Nitric oxide (NO) released by the endothelial nitric oxide synthase (eNOS or NOS3) is an important relaxing factor and plays a role in maintaining basal tone of pulmonary arteries. NO can react with superoxide anions, forming a highly potent oxidant, peroxynitrite. The local bioavailability of NO in a tissue is determined by the local concentration of superoxide anions and by the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase which scavenge superoxide and prevent formation of peroxynitrite. Constriction responses observed in these vessels are a result of a balance between vasoconstrictor and dilators. Pulmonary arteries isolated from control rats without LC constricted well to norepinephrine. Inhibition of NOS by LNA removed the vasodilator effects of endogenous NO resulting in significant enhancement of constriction to NE (Figure 1A). In contrast, antioxidant enzymes, PEG-SOD and PEG-catalase did not alter constriction to NE. This suggests that the local concentration of reactive oxygen species (ROS) such as superoxide anions is low in control rat lungs resulting in higher bioavailability of NO. This is illustrated by a robust relaxation response to both endogenous NO (released as a result of stimulation of eNOS by A23187, figure 2) and exogenous NO (released by a direct donor, SNAP, Figure 3A). Relaxation to A23187 and SNAP were not altered by pretreatment with PEG-SOD and PEG-catalase.
Four hours after sustaining LC, rats were hypoxemic (Table 2). Despite a significant drop in PaO2, PaCO2 was only marginally increased. Constriction response to norepinephrine in the absence of any pretreatments did not differ significantly from control rats (figure 1B and D). Inhibition of NOS resulted in a marked increase in contraction response to norepinephrine suggesting that the NO pathway was active under basal conditions. In fact, eNOS mRNA expression was up-regulated in pulmonary arteries 4h after LC compared to controls (Figure 4A), although this difference did not reach statistical significance (p = 0.08). This led to the possibility of NO avidly binding to superoxide anions resulting in peroxynitrite formation contributing to pulmonary vasoconstriction and hypoxemia. There was a marked (12-fold) increase in pulmonary arterial 3-nitrotyrosine levels 4h after contusion compared to controls (Figure 6 and and7).7). Pretreatment of pulmonary arteries with PEG-SOD and PEG-catalase to scavenge superoxide anions resulted in a significant reduction in contractile response to norepinephrine (Figure 1B). Pretreatment with antioxidant enzymes also reversed the increase in contractility seen in response to NOS inhibition. There was no difference in contraction to KCl suggesting that the contractile apparatus in the smooth muscle was not altered by LC.
NOS agonist, A23187 mediated relaxation was decreased following LC (Figure 2A) but improved to levels seen in control rats following pretreatment of pulmonary arteries with PEG-SOD and PEG-catalase. Relaxation to SNAP was significantly impaired 4h after LC, compared to controls (Figure 3D). There was a tendency towards increased NOS3 mRNA and a decrease in sGC mRNA compared to controls (Figure 4A). Scavenging superoxide anions by adding PEG-SOD and PEG-catalase to the vessel bath resulted in increased relaxation to SNAP in pulmonary arteries isolated from rats 4h after LC (Figure 3B). These results indicate this decrease in response to exogenous NO could be secondary to reduced production of NO by NOS, or increased scavenging of NO by superoxide anions, or changes in the activity of target enzymes (soluble guanylate cyclase – sGC and phosphodiesterase 5). Inhibition of NOS resulted in enhanced relaxation to SNAP suggesting that uncoupled NOS could be a potential source of superoxide anions after LC (Figure 8). Such uncoupling of NOS has been reported in other models of pulmonary hypertension (8). There was no difference in relaxation response to E4021, a phosphodiesterase inhibitor and there was no significant difference in PDE5 mRNA levels in pulmonary arteries after LC (Figures 4A). These findings suggest that ROS such as superoxide anions and peroxynitrite increase significantly after LC and scavenging ROS by antioxidant enzymes reduces contractility and improves vaso-relaxation(Figure 8).
Twenty-four hours after LC, rats recovered from hypoxemia (Table 2). The contractile response to norepinephrine following NOS inhibition with LNA decreased and was similar to controls. Pretreatment with PEG-SOD and PEG-catalase did not significantly alter the constriction response to norepinephrine either in the presence or absence of NOS inhibition with LNA (Figure 1B). This could be attributed to a compensatory increase in SOD1 and SOD2 mRNA levels 24h after contusion (Figure 4B). Relaxation to A23187 and SNAP were significantly reduced (Figure 2 and and3D)3D) probably secondary to increased ROS and decreased sGC levels (Figure 4A). Interestingly, pretreatment with PEG-SOD and PEG-catalase and inhibition of NOS with LNA significantly improved relaxation to SNAP.
Overall these results suggest that ROS play an important role in the pathogenesis of vasoconstriction and hypoxemia in LC. Increased 3-nitrotyrosine staining suggestive of peroxynitrite formation seen four hours after LC (Figure 6B) coincides with the period of hypoxemia (Table 2). It is very likely that by 24h after LC, compensatory increases in cytosolic (SOD1) and mitochondrial (SOD2) SODs (Figure 4B, ,8)8) contribute to improvement in pulmonary vasoconstriction and oxygenation.
There are several limitations to this study. We did not measure right ventricular or pulmonary arterial pressure following LC in rats. However, there are prior reports of right ventricular strain and pulmonary hypertension in LC (6). We only evaluated mRNA expression of various enzymes in the NO-superoxide pathway. We did not evaluate protein levels and activity because of the small size and limited quantities of third generation pulmonary arterial tissue available for study. Finally, the rats were exposed to 100% oxygen to obtain blood gases prior to sacrifice. This could have resulted in an increase in superoxide anions observed in all tissues.
In conclusion, the data presented in the current manuscript suggest that that consumption of NO due to excess superoxide resulting in peroxynitrite formation leads to diminished vascular reactivity following LC. The results, additionally leads us to conclude that compensatory increases in cytosolic SOD improves arterial reactivity and hypoxia at 24 hours following LC. Taken together it is likely that appropriate antioxidant therapy may be a potential therapeutic option in patients with LC.
The authors gratefully acknowledge the support of National Institutes of Health Grants RO-1 HL-102013 (KR)
This work was supported by National Institutes of Health Grants HL-102013 (KR) and HL094230 (SP). Mass Spectrometry experiments were performed in Molecular Phenotyping Core, Michigan Nutrition and obesity Center (DK 89503).
Author contributions: (Authors MVS and SL contributed equally to this study) Conception and design, M.V.S., S.L and K.R. Performed research, M.V.S., S.L., B.Y., L.C.N., J. D. H., L.Z., S.F.G Analysis and interpretation, M.V.S., S.P, K.R.
None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.