Inflamm. Res. DOI 10.1007/s00011-014-0733-5
Inflammation Research
ORIGINAL RESEARCH PAPER
Inducible nitric oxide synthase inhibition reverses pulmonary arterial dysfunction in lung transplantation Jing-xiang Wu • Hong-wei Zhu • Xu Chen • Jiong-lin Wei Xiao-feng Zhang • Mei-ying Xu
•
Received: 30 August 2013 / Revised: 30 March 2014 / Accepted: 31 March 2014 Ó Springer Basel 2014
Abstract Background Ischemia–reperfusion injury (IRI) after lung transplantation remains a significant cause of morbidity and mortality. Lung IRI induces nitric oxide synthesis (iNOS) and reactive nitrogen species, decreasing nitric oxide bioavailability. We hypothesized that ischemiainduced iNOS intensifies with reperfusion and contributes to IRI-induced pulmonary arterial regulatory dysfunction, which may lead to early graft failure and cause pulmonary edema. The aim of this study was to determine whether ischemia–reperfusion alters inducible and endothelial nitric oxide synthase expression, potentially affecting pulmonary perfusion. We further evaluated the role of iNOS in posttransplantation pulmonary arterial disorder. Methods We randomized 32 Sprague–Dawley rats into two groups. The control group was given a sham operation whilst the experimental group received orthotropic lung transplants with a modified three-cuff technique. Changes in lung iNOS, and endothelial nitric oxide synthase expression were measured after lung transplantation by enzyme-linked immunosorbent assay (ELISA). Vasoconstriction in response to exogenous phenylephrine and vasodilation in response to exogenous acetylcholine of pulmonary arterial rings were measured in vitro as a measure of vascular dysfunction. To elucidate the roles of iNOS in regulating vascular function, an iNOS activity
inhibitor (N6-(1-iminoethyl)-L-lysine, L-NIL) was used to treat isolated arterial rings. In order to test whether iNOS inhibition has a therapeutic effect, we further used L-NIL to pre-treat transplanted lungs and then measured posttransplantation arterial responses. Results Lung transplantation caused upregulation of iNOS expression. This was also accompanied by suppression of both vasoconstriction and vasodilation of arterial rings from transplanted lungs. Removal of endothelium did not interfere with the contraction of pulmonary arterial rings from transplanted lungs. In contrast, iNOS inhibition rescued the vasoconstriction response to exogenous phenylephrine of pulmonary arterial rings from transplanted lungs. In addition, lung transplantation led to suppression of PaO2/FiO2 ratio, increased intrapulmonary shunt (Qs/ Qt), and increase of lung wet to dry ratio (W/D), malondialdehyde and myeloperoxidase levels, all of which were reversed upon iNOS inhibition. Furthermore, inhibition of iNOS significantly rescued vascular function and alleviated edema and inflammatory cell infiltration in the transplanted lung. Conclusions Our data suggest that lung transplantation causes upregulation of iNOS expression, and pulmonary vascular dysfunction. iNOS inhibition reverses the posttransplantational pulmonary vascular dysfunction. Keywords Nitric oxide synthase Ischemia–reperfusion injury Rat lung transplantation Pulmonary arterial rings
Responsible Editor: John Di Battista. J. Wu H. Zhu X. Chen J. Wei X. Zhang M. Xu (&) Department of Anesthesiology, Shanghai Chest Hospital, Shanghai Jiaotong University, 241 West Huaihai Road, Shanghai 200030, People’s Republic of China e-mail: [email protected]
Introduction Lung transplantation is the ultimate therapeutic option for end-stage malignant pulmonary diseases. However, the
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1-year survival rate is only 80 % [1]. Ischemia–reperfusion injury (IRI) is a significant factor contributing to primary graft failure in lung transplantation and increases the risk of bronchiolitis obliterans syndrome after lung transplantation [2]. Ischemia–reperfusion injury causes damage to the pulmonary vascular endothelium, resulting in decreased alveolar perfusion, transplant exhaustion and graft failure [3, 4]. Despite extensive efforts to interpret the observed vascular damage, mechanisms contributing to this damage remain unclear. Nitric oxide (NO) plays a crucial role in the regulation of post-transplantational pulmonary function [5]. Two different nitric oxide synthases (NOS) are involved in regulation of pulmonary vascular function. Endothelial NOS (eNOS), mostly distributed in vascular endothelium, produces low levels of NO (picomoles) in a calciumdependent manner [6] to maintain vascular dilation and permeability. In contrast, inducible NOS (iNOS) is expressed by inflammatory cells such as neutrophils and macrophages, and is now known to be widely distributed in the airway epithelium and vascular smooth muscles [7]. iNOS is calcium-independent, and can produce a large, continuous flux of NO that is 1,000-fold higher than that of eNOS, lasting for hours to days in response to conditions of stress and inflammation [8, 9]. It is thought that eNOS is cell-protective, while iNOS has cytotoxic functions [10]. Numerous studies have been performed to study the association between NOS and post-transplantational pulmonary function. A significant increase in iNOS and a decrease in eNOS mRNA was found after reperfusion during rat lung transplantation [9]. Increased iNOS and decreased eNOS activity may contribute to ischemia– reperfusion-induced pulmonary vasoconstriction [11]. Overproduction of NO via iNOS and oxidative stress may lead to reactive oxygen and nitrogen species formation and vascular dysfunction during acute lung injury [12]. A recent study has shown that hepatic ischemia–reperfusion leads to lung injury by promoting pulmonary iNOS expression [13]. However, it remains unclear whether there is a causal relationship between iNOS upregulation and post-transplantational pulmonary vascular dysfunction. Furthermore, it is not known whether iNOS inhibition has any therapeutic role in post-transplantational pulmonary vascular dysfunction. Accordingly, the current study combines the in vivo orthotropic lung transplantation model and the ex vivo technique of isolated pulmonary arterial rings and an iNOS-selective inhibitor (N6-(1-iminoethyl)-L-lysine, L-NIL) [14, 15] to study the role of iNOS in lung transplant-related pulmonary vascular dysfunction during ischemia–reperfusion injury.
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Materials and methods Animals and operation process Studies were performed in accordance with the Guidelines for Animal Experiments of Shanghai Jiaotong University, PR China. Male Sprague–Dawley rats (340 ± 5 g) were housed with free access to food and water under a natural day/night cycle and acclimated for 1 week before any experimental procedures. The rat orthotropic left lung transplantation was carried out by using the modified three-cuff technique [16]. Briefly, donor rats were anesthetized by intraperitoneal injection of ketamine chloride (100 mg/kg). Rats were intubated and ventilated with 100 % oxygen, tidal volume of 10 mL/kg and a respiratory rate of 70 breaths/min. After heparinization (1,000 U/kg intravenously), a median sternotomy was performed, the lungs were flushed through the main pulmonary artery with 20 mL of cold low-potassium dextran solution (LPD; Perfadex, Vitrolife, Gothenburg, Sweden), the pulmonary artery, vein and main stem bronchus were ligated and tied off, and the inflated left lung was placed into LPD solution at 4 °C for 3 h. Recipient rats were anesthetized, intubated, and mechanically ventilated at the same parameters as for the donor rats. A left thoracotomy was performed through the fourth intercostal space. The hilum of the left lung was dissected, and the pulmonary artery, pulmonary vein and main bronchus were clamped with microvascular clamps. After incising the pulmonary artery and vein, the donor lung was anastomosed with the corresponding recipient structures. The thorax was closed and the mechanical ventilation was maintained, and 30 mg/kg ketamine were added according to the occurrence of limb movements and corneal reflexes to prevent the rats from regaining consciousness before they were sacrificed by overdose of chloral hydrate after 2 h of reperfusion [17]. For ex vivo experiments, the left pulmonary artery was isolated and cut into three cross sections for vasoconstriction, vasodilation and endothelial denudation assays. The left lung tissue was also cut into two pieces. One piece was used to measure lung wet to dry weight ratio (W/D). The other piece was kept at -80 °C for future malondialdehyde (MDA), myeloperoxidase (MPO), iNOS and eNOS measurements. For in vivo experiments, some of the transplanted rats were injected with 1 mL (3 mg/kg) N6(1-iminoethyl)-L-lysine (L-NIL, an iNOS activity inhibitor) solution diluted with 0.9 % saline solution by tail vein injection 5 min before the reperfusion and were sacrificed after 2 h of reperfusion. Left lung tissues were isolated and analyzed as mentioned above.
Inducible nitric oxide synthase inhibition
In vitro vascular preparation
Assessment of lung function
Arterial rings were prepared as previously described [18]. Briefly, following sacrifice of the rats, hearts and lungs were immediately removed and placed in a petri dish containing 4 °C Krebs–Henseleit (KH) solution (in mM/L: 118.40 NaCl; 5.01 KCl; 1.20 KH2PO4; 2.5 CaCl2; 1.2 MgCl2; 10.1 glucose; 25 NaHCO3) saturated by a gas mixture (95 % O2, 5 % CO2). The pulmonary arterial tree was rapidly dissected from the lung parenchyma with small scissors and forceps. The left pulmonary artery was carefully separated from pulmonary artery branches under the microscope. Peripheral adipose and connective tissues were removed. The left pulmonary artery was cut into three cross sections, each ring measuring 3–4 mm. Two retained an intact endothelium whilst the third was subjected to removal of the endothelium by friction. Vascular rings were suspended by stainless steel hooks attached to the tension sensor and mounted in thermostatically controlled 37 °C Krebs buffer (pH = 7.4, 10 mL) and gassed with 95 % O2 and 5 % CO2. The mechanical activity was recorded isotonically under a resting load of 1 g. The mechanical signals were processed by bridge amplifiers and were recorded by the MPA2000 multi-channel physiological recorder system (Alcott Technology Co. Ltd, Shanghai China). The arterial segments were viable at least for 4 h under the experimental conditions. The KH solution was renewed every 20 min. The arterial rings were equilibrated in their baths for 1 h under a resting load of 1 g. The endothelium denudation was performed by gently and systematically rubbing the intimal surface of the everted cylindrical segment using moistened cotton wool fixed on a thin wooden stick. Successful removal of endothelial cells from the rings was confirmed by the inability of acetylcholine to induce relaxation [18, 19]. At the beginning of each experiment, the endothelial integrity was examined by pre-contracting the isolated pulmonary artery with a submaximal concentration (EC80) of phenylephrine hydrochloride (Phe) (0.1 lM) and testing the ability of acetylcholine (Ach) (1 lM) to relax the arterial strips [20]; this effect is absent in endothelium denuded preparations [21]. Ach-induced relaxation of the arterial rings by [ 80 % or \ 20 % of the Phe-induced pre-contraction was taken as indicative of structurally intact or damaged endothelium, respectively [22]. After three washes during the next 30 min, the concentration–response relationship for Ach (0.001–100 lM) and Phe (0.0001–10 lM) was determined by cumulative exposure to increasing drug concentrations at 3 min intervals. Some of the vascular rings were pre-incubated with the selective iNOS inhibitor, L-NIL, (10 lmol/L) for 10 min to analyze the role of iNOS in vascular constriction and dilatory function.
Transplant graft function was assessed by measuring amount of blood shunted/total blood flow (Qs/Qt) levels and partial arterial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2) ratio in blood taken from the pulmonary vein at the completion of the 2-h reperfusion period (n = 6 for each group). Qs/Qt = (CcO2 - CaO2)/ (CcO2 - CvO2) (CcO2, oxygen content of pulmonary capillary blood; CaO2, oxygen content of arterial blood; CvO2, oxygen content of mixed venous blood) [23]. Qs/Qt and partial pressures of oxygen were measured with a blood-gas analyzer (Radiometer ABL 800, Copenhagen, Denmark) to report PaO2, FiO2 and Qs/Qt values. Wet-todry lung tissue weight ratios were calculated at the end of the 2-h reperfusion period as a measurement of lung edema (n = 6 for each group). The left lung was dissected with a sharp blade. All surface liquid was removed using filter paper. Lung tissue was weighed and placed in an oven at 80 °C for 48 h. Then, the dried lung portion was reweighed, and the ratio of the lung weight before and after drying was calculated. Determination of MDA, MPO, iNOS and eNOS levels in lung tissue As an index of membrane lipid peroxidation, levels of MDA were determined as previously described [24]. Lung tissues were homogenized (100 mg/mL) in 10 volumes of 1.15 % KCl solution containing 0.85 % NaCl and then centrifuged at 1,500 g for 15 min. Next, 200 lL of lung tissue homogenate was added to a reaction mixture consisting of 1.5 mL 0.8 % thiobarbituric acid, 200 lL 8.1 % sodium dodecyl sulfate, 1.5 mL 20 % acetic acid (adjusted to pH 3.5 with NaOH) and 600 lL distilled H2O. The mixture was then heated at 95 °C for 40 min. After cooling to room temperature, the samples were centrifuged (10,0009g, 10 min) and supernatant absorbance was measured at 532 nm, using 1,1,3,3-tetramethoxypropane as an external standard. The level of lipid peroxide was expressed as nmol MDA/mg protein (Bradford assay) (n = 6 for each group). Activity of MPO, an enzyme present in neutrophils, was used as a marker of neutrophil infiltration. MPO activity was determined in lung as described before [25]. Lung tissues (100 mg of tissue) were homogenized in 2 mL of 20 mM potassium phosphate buffer (pH 7.4). After centrifugation (15,0009g, 20 min), the pellet was resuspended in 2 mL of 50 mM potassium phosphate buffer (pH 6.0) containing 0.5 % hexadecyltrimethylammonium bromide and sonicated for 30 s. After being heated at 60 °C for 2 h, the samples were centrifuged (10,0009g, 10 min). The supernatant (25 lL) was added to 725 lL of 50 mM phosphate buffer (pH 6.0) containing
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Fig. 1 Decreased pulmonary arterial ring constriction in response to phenylephrine and dilation in response to Ach after lung transplantation. a Comparison of pulmonary artery constriction response to phenylephrine between lung transplantation group (lung Tx) and sham group (sham). Both groups were treated with different concentrations of phenylephrine (0.0001–10 lM) and then the concentration–response relationship of vasoconstriction was recorded. b Comparison of pulmonary artery dilation in response to Ach
(0.001–100 lM) between lung transplantation group and sham group. Dilation is shown as reduction of constriction. The dilation values are expressed as a percentage of the phenylephrine-induced pre-constriction level recorded immediately before Ach administration. (Relaxation % = (present constriction-pre-constriction)/pre-constriction 9 100 %. The values represent mean ± SEM, n = 6. **P \ 0.01 vs. sham, repeated measures ANOVA
0.167 mg/mL o-dianisidine and 5 9 10-4 % hydrogen peroxide. MPO activity was measured spectrophotometrically as the change in absorbance at 460 nm at 37 °C, using a Spectramax microplate reader (Molecular Devices, Sunnyvale, CA, USA). Results were expressed as units of MPO activity per gram wet tissue (1 U defined as change in absorbance of 1 nm/min) (n = 6 for each group). The levels of eNOS and iNOS were measured by enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions (n = 6 each group).
decreased dilation in response to Ach (Fig. 1b). The maximum contractile effect for Phe, a a-adrenergic agonist, was significantly decreased in pulmonary artery rings from transplanted rats after 2 h of in vivo reperfusion compared to shams (Emax = 2.11 ± 0.38 vs. 4.15 ± 0.39 mN/mm, n = 6, P \ 0.01, respectively). The dilation of pulmonary artery rings from transplanted lungs to Ach (0.001–100 lM) was significantly decreased as compared to the sham group (Fig. 1b). Transplantation leads to increased iNOS, MPO and MDA levels
Statistical analysis SPSS11.0 statistical software was used to analyze the results. The data are expressed as mean ± SD. Repeated measures of analysis of variance (ANOVA), calculated using the general linear model, were used to compare the concentration response curves obtained in pulmonary arteries. The other results were analyzed using one-way ANOVA followed by the Student–Newman–Keuls test. All statistical analyses were considered significant when P \ 0.05.
Results Lung transplantation leads to diminished pulmonary arterial reactivity Following lung transplantation, arterial ring constriction decreased in response to Phe (Fig. 1a), and there was
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Because NOS plays a crucial role in post-transplantational pulmonary function regulation, we measured the levels of iNOS and eNOS in sham-treated and transplanted lungs. We found that lung transplantation caused upregulation of iNOS and downregulation of eNOS levels (P \ 0.05, n = 6) (Table 1). In accordance with the iNOS and eNOS changes, the activity of MPO was significantly increased after transplantation, (P \ 0.05, n = 6) (Table 1). MDA, another marker for oxidative stress [26], was also significantly increased after lung transplantation (P \ 0.05, n = 6) (Table 1). iNOS inhibition but not endothelium-denuded arterial rings affected the post-transplantational pulmonary arterial dysfunction To elucidate whether decreased post-transplantational pulmonary vasoconstriction was endothelium-dependent, we removed the arterial endothelium. However, endothelial
Inducible nitric oxide synthase inhibition Table 1 Changes in iNOS, eNOS, MPO and MDA levels (x ± SD) Groups
iNOS
eNOS
MPO
MDA
Sham
1.02 ± 0.36
0.71 ± 0.07
19.05 ± 5.03
17.01 ± 2.82
LungTx
2.63 ± 0.61a
0.19 ± 0.02a
39.36 ± 6.81a
26.36 ± 2.73a
b
b
b
18.69 ± 2.79b
LungTx ? L-NIL
0.93 ± 0.18
0.52 ± 0.11
13.16 ± 1.39
Levels of MDA, iNOS, eNOS and activity of MPO in different groups following transplantation. Level of MDA was expressed as nmol MDA/mg protein. The levels of eNOS and iNOS were presented as ng/ml. Activity of MPO was presented as units of MPO activity per gram wet tissue. n = 6 in each group iNOS inducible nitric oxide synthase, eNOS endothelial nitric oxide synthase, MPO myeloperoxidase, MDA malondialdehyde, LungTx lung transplantation, L-NIL N6-(1-iminoethyl)-L-lysine a
P \ 0.05 compared with sham group,
b
P \ 0.05 compared with LungTx group
removal only moderately enhanced Phe-induced vasoconstriction of arterial rings from sham-operated lungs (P \ 0.05, n = 6) (Fig. 2a). On the contrary, constriction of arterial rings from transplanted lungs to Phe was not affected (Fig. 2b). Since Ach produced an endotheliumdependent relaxation of the sustained contraction to Phe, it cannot induce endothelium-dependent relaxation of the endothelium-removed pulmonary arterial rings. We then tested whether iNOS affected pulmonary function in transplanted lungs by inhibiting iNOS using its inhibitor L-NIL. We found that L-NIL could effectively inhibit post-transplantational levels of iNOS, MDA, as well as MPO activity (Table 1). L-NIL had no effect on vasoconstriction of arterial rings from sham-operated lungs in response to Phe (Fig. 2c). However, iNOS inhibition significantly rescued vasoconstriction ability from transplanted lungs (P \ 0.01, n = 6) (Fig. 2d). This suggests that iNOS contributes to the constriction of the rings from transplanted lungs to Phe. Furthermore, we found that pre-treatment of lung with L-NIL protected the dilatory response to Ach in the rings from transplanted lungs (Fig. 3). iNOS inhibition reverses pulmonary function defects after lung transplantation Finally, we tested whether iNOS inhibition could benefit post-transplantational pulmonary function. Compared with the sham-operated rats, the transplanted rats developed significant pulmonary dysfunction as evidenced by decreased PaO2/FiO2 ratio (lung transplant group (LTx) vs. sham, 280.0 ± 8.7 vs. 406.3 ± 10.9, P \ 0.01), increased intrapulmonary shunt (Qs/Qt, LTx vs. sham, 26.3 ± 0.7 vs. 8.7 ± 0.2, P \ 0.01) and lung edema (W/D weight ratio, LTx vs. sham, 5.62 ± 0.1 vs. 4.4 ± 0.1, P \ 0.01). However, L-NIL treatment significantly improved the pulmonary function in transplanted lungs by improving gas exchange (PaO2/FiO2 ratio 360 0.0 ± 13.6, Qs/Qt 20.5 ± 1.4) and reducing lung (W/D 4.9 ± 0.1) edema (Fig. 4a–c).
Discussion The pathogenesis of lung ischemia–reperfusion is characterized by two major processes. First, the increased capillary permeability results in pulmonary edema and impaired gas exchange due to tissue fluid leakage [27]. Second, the pulmonary microvasculature constricts abnormally and the relaxation of pulmonary artery is impaired [28], which causes vascular dysfunction. These two factors cause lung ventilation-perfusion ratio mismatch, hypoxemia, and acute respiratory distress syndrome in the clinic [29]. Previous study has found that cold ischemia and reperfusion may produce different patterns of pulmonary vasomotor dysfunction and contribute to increased pulmonary vascular resistance in the transplanted lung [30]. Endothelium-dependent pulmonary arterial relaxation was significantly impaired by reperfusion, and pulmonary neutrophil sequestration could be the main determinant of ischemia–reperfusion-induced vascular dysfunction in lung transplantation [4]. In the present study, we found that lung transplantation led to significantly decreased pulmonary arterial ring vasoconstriction and decreased dilation. Consistent with previous discoveries [9, 11], lung transplantation also caused increased levels of iNOS, MDA, enhanced MPO activity and decreased levels of eNOS, which indicated increased oxidative stress, inflammatory cell infiltration, and pulmonary capillary permeability, suggesting increased post-transplantational infiltration of iNOS-producing neutrophils. We further showed that endothelial removal did not affect Phe-induced vasoconstriction of pulmonary arterial rings from transplanted lungs, although it moderately enhanced it in the sham group. The difference in endothelial dependency between transplantation and sham groups suggests that the endothelium may already be injured or depressed in transplanted lungs [31]. Recently, it has been described that NO may modulate its own production due to cross-talk between the eNOS and iNOS isoforms [32]. iNOS could inhibit eNOS production and suppress the function of
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Fig. 2 iNOS inhibition but not endothelium-removal affects the posttransplantational pulmonary vascular dysfunction. Pulmonary arterial rings from the sham group and lung transplantation group were treated with different concentrations of phenylephrine (0.0001–10 lM) and then the concentration–response relationship of vasoconstriction was recorded. The endothelial vasoconstriction responses in the sham group (a) and the lung transplant group (b) after removal of endothelium are shown. E- without
endothelium, E? intact endothelium. In thelower panel, the vascular rings were pre-incubated with the selective iNOS inhibitor (L-NIL, 10 lmol/L) for 10 min to analyze the role of iNOS in vasoconstriction in response to Phe. L-NIL did not affect the constriction to Phe in the rings from sham rats (c). iNOS inhibition rescued vasoconstriction to Phe in pulmonary rings from transplanted lungs (d). The values represent mean ± SEM, n = 6. *P \ 0.05, **P \ 0.01 vs. sham, repeated measures ANOVA
endothelial cells [32], and inhibition of iNOS activity could significantly reverse dysfunctional endothelial cells by the upregulation of eNOS [31]. Consistent with these effects, we discovered that lung transplantation caused increased production of iNOS and decreased levels of eNOS. These findings suggested that iNOS-derived NO repressed the vascular contraction by relaxing vascular smooth muscle but not by vascular endothelium, since removal of the endothelium had no effect, although in normal condition, the pulmonary vascular tone is modulated by eNOSderived NO in an endothelium-dependent way. In addition, iNOS contributed to the post-transplantational pulmonary vascular dysfunction. iNOS inhibition using L-NIL abrogated these vascular defects and decreased W/D ratio, MDA, and MPO levels in transplanted lung, demonstrating that elevated iNOS plays a pivotal role in ischemia– reperfusion-induced lung injuries following transplantation. Therefore, the augmented expression of iNOS may be an underlying mechanism of the pulmonary artery hyporesponsiveness to vasoconstrictors and Ach-related
relaxation. Furthermore, iNOS inhibition may serve as a strategy to increase the success of lung transplantation. We only used pulmonary arterial rings in our study for vascular dysfunction. Vessels from different segments or even vessels with different diameters respond differently to vasoconstricting or dilating factors [33–36]. It has been shown that pulmonary microvessels are more sensitive to various vasoconstrictors compared with pulmonary arteries, including endothelin-1 and thromboxane A2 analogue U46619 [37, 38]. Although we used phenylephrine hydrochloride instead of endothelin-1 and Thromboxane A2 analogue, it’s still reasonable to speculate that responses of pulmonary arteries and microvessels are different. In our study, we have directly shown the role of iNOS in the pulmonary arterial response. We measured the W/D ratio changes in response to iNOS inhibition, which indirectly reflect the pulmonary microvessels responses. Our data suggest that both pulmonary arterial dysfunction and the W/D ratios are regulated by iNOS inhibition, suggesting that iNOS regulates both pulmonary arteries and microvessels.
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Fig. 3 iNOS inhibition restored vasodilation to Ach in transplanted arterial rings. The vascular rings were pre-incubated with the selective iNOS inhibitor (L-NIL, 10 lmol/L) for 10 min to analyze the role of iNOS in vascular dilatory function. The concentration–response relationship for Ach (0.001–100 lM) was determined by cumulative exposure to increasing drug concentrations at 3 min intervals. Vessels of lung transplant group were compared with the sham group. The values represent mean ± SEM, n = 6. **P \ 0.01 vs. sham, repeated measures ANOVA
To study vascular relaxation, we used phenylephrine hydrochloride (Phe) pre-contracted vessels, which are commonly used to measure vascular constriction. The major limitation with this method is that it is a1-adrenergic receptor dependent. Other factors, including endothelin-1 and thromboxane A2 analogue, can induce vascular constriction [39–45], while prostacyclin and histamine can induce relaxation of pulmonary microvessels [46]. These factors could also be induced after lung transplantation. In addition to using pre-contacted vessels, another method to measure arterial relaxation response is to directly measure
Fig. 4 NOS inhibition by L-NIL reverses lung functions in transplanted rats. iNOS inhibitor L-NIL (3 mg/kg) was administrated by tail vein injection 5 min before the reperfusion of experimental orthotropic lung transplantation. Transplant graft function was assessed by measuring PaO2/FiO2 (partial arterial pressure of oxygen/fraction of inspired oxygen) ratio (a), Qs/Qt (amount of blood
the dynamic pulmonary vessels dilation (the changes in internal vessel diameter and changes in blood velocity) using in vivo videomicroscopy. This method, however, is beyond the scope of this study. Nitric oxide plays a central role in the regulation of pulmonary circulation under physiological and pathophysiological circumstances [6, 47]. NO can be protective or injurious, depending on its site of release, concentration, and local conditions [10, 47–49]. NO exerts many of its physiological effects by activation of guanylyl cyclase [7]. Under normal physiological conditions, NO is primarily generated by the eNOS pathway to reduce vascular tone and inhibit leukocyte and platelet exudation [48]. Activated eNOS binds to heat shock protein 90 (Hsp90) to form a protein kinase complex Akt-eNOS-Hsp90 [50–52] that improves eNOS activity by increasing eNOS affinity for calmodulin [53]. NO derived from eNOS binds to this complex to activate guanylyl cyclase and to modify its function in a calcium-dependent manner; this is the key step in vasodilation induced by endothelium-derived NO [32]. NO can also be generated by iNOS after stimulation by oxidative stress, toxins and inflammatory mediators such as tumor necrosis factor and cytokines [32]. iNOSgenerated NO then induces pathophysiological changes via cytotoxic effects and exacerbation of tissue damage [12, 48, 54, 55]. The availability of other reactive intermediates with which NO may interact also determines its toxicity [56]. Because of its radical character, NO reacts with superoxide radicals (O2-) at a near diffusion-limited rate, which in turn produces another highly reactive species, peroxynitrite (ONOO-) [57–60]. Peroxynitrite causes oxidative/nitrative modification to biomolecules, thus modulating physiological and pathophysiological processes [61, 62]. In addition, peroxynitrite can be converted to
shunted/total blood flow) ratio (b) and W/D (wet-to-dry lung tissue) weight ratio (c) levels at the completion of the 2-h reperfusion period. Data are means ± SEM of six rats for each group. **P \ 0.01 vs. sham group, **P \ 0.01 vs. lung transplant (LT) group, one-way ANOVA followed by the Student–Newman–Keuls test
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highly reactive hydroxyl-like radicals [63], which inhibit the formation of Akt-eNOS-Hsp90 complex and thus block vascular endothelial relaxation [53]. Enhanced iNOS activity in the vessel wall inhibits eNOS activity and reduces NO bioavailability in endothelial cells, which causes vascular dysfunction as manifested by decreased endothelium-dependent relaxation to acetylcholine [11]. Our results demonstrated that lung transplantation caused suppression of Ach-induced arterial relaxation (Fig. 1b), indicating vascular endothelial dysfunction in transplanted lungs. Reduced eNOS was detected in transplanted lungs (Table 1). This is consistent with the idea that decreased eNOS is associated with vascular endothelial dysfunction. Pre-treatment with the selective iNOS inhibitor L-NIL restored the relaxation ability of transplanted lung arteries to control levels. iNOS inhibition also restored the levels of eNOS. These results suggest that the decline in endothelial eNOS activity may be caused by the high level of iNOS in transplanted lungs. iNOS inhibition restored the levels of eNOS, which possibly rescued vascular endothelial function. The contractile response to Phe in isolated pulmonary arteries was significantly suppressed after lung transplantation. However, vasoconstriction defects were not affected by removal of vascular endothelium (Fig. 2a, b). We found that high levels of post-transplantational iNOS were associated with reduced pulmonary vasoconstriction. We further showed that iNOS inhibition using L-NIL restored pulmonary vasoconstrictive reaction in vitro (Fig. 2d). Collectively, these data support the hypothesis that iNOSderived NO represses vascular contraction by relaxation of smooth muscle cells but not endothelium. Smooth muscle cells and possibly peroxynitrite may be responsible for reduced pulmonary arterial constriction. Exposure of vascular tissue to peroxynitrite can result in a prolonged relaxation mediated through a glutathione-dependent generation of NO [64]. Firstly, in tissue bath, superoxide can react with NO to generate perocynitrite [65]. At physiological conditions, this production of peroxynitrite can exert multiple biological activities [66]. This could explain the source of perocynitrite and the possible effect for reduced arterial constriction. Secondly, iNOS could inhibit eNOS production and suppress the function of endothelial cells [11, 32] and inhibition of iNOS activity could significantly reverse dysfunctional endothelial cells by the upregulation of eNOS [67]. In summary, we found that lung transplantation led to defects in vasoconstriction and vasodilation reactivity of the pulmonary artery. This was associated with increased iNOS levels. Pre-treatment with the selective iNOS inhibitor L-NIL not only restored vascular function of the transplanted lung, but also reversed transplant-related lung ischemia–reperfusion injury and improved pulmonary
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function. Therefore, inhibition of iNOS may serve as a potential strategy to protect the lung from ischemia– reperfusion injury after lung transplantation. Acknowledgments The authors thank Dr. Wei-gang Guo, Dr. Qiang Tan and Yi-qingYang for their advice and helpful suggestions. This work was supported by Shanghai Natural Science Foundation No. 12ZR1428700, ‘‘1050’’ Foundation for the Talents by Shanghai Chest Hospital and Shanghai Joint development project for Municipal hospitals (SHDC12010222). Dr. Wu and Zhu contributed equally to this work.
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Inflammation Research
ORIGINAL RESEARCH PAPER
Inducible nitric oxide synthase inhibition reverses pulmonary arterial dysfunction in lung transplantation Jing-xiang Wu • Hong-wei Zhu • Xu Chen • Jiong-lin Wei Xiao-feng Zhang • Mei-ying Xu
•
Received: 30 August 2013 / Revised: 30 March 2014 / Accepted: 31 March 2014 Ó Springer Basel 2014
Abstract Background Ischemia–reperfusion injury (IRI) after lung transplantation remains a significant cause of morbidity and mortality. Lung IRI induces nitric oxide synthesis (iNOS) and reactive nitrogen species, decreasing nitric oxide bioavailability. We hypothesized that ischemiainduced iNOS intensifies with reperfusion and contributes to IRI-induced pulmonary arterial regulatory dysfunction, which may lead to early graft failure and cause pulmonary edema. The aim of this study was to determine whether ischemia–reperfusion alters inducible and endothelial nitric oxide synthase expression, potentially affecting pulmonary perfusion. We further evaluated the role of iNOS in posttransplantation pulmonary arterial disorder. Methods We randomized 32 Sprague–Dawley rats into two groups. The control group was given a sham operation whilst the experimental group received orthotropic lung transplants with a modified three-cuff technique. Changes in lung iNOS, and endothelial nitric oxide synthase expression were measured after lung transplantation by enzyme-linked immunosorbent assay (ELISA). Vasoconstriction in response to exogenous phenylephrine and vasodilation in response to exogenous acetylcholine of pulmonary arterial rings were measured in vitro as a measure of vascular dysfunction. To elucidate the roles of iNOS in regulating vascular function, an iNOS activity
inhibitor (N6-(1-iminoethyl)-L-lysine, L-NIL) was used to treat isolated arterial rings. In order to test whether iNOS inhibition has a therapeutic effect, we further used L-NIL to pre-treat transplanted lungs and then measured posttransplantation arterial responses. Results Lung transplantation caused upregulation of iNOS expression. This was also accompanied by suppression of both vasoconstriction and vasodilation of arterial rings from transplanted lungs. Removal of endothelium did not interfere with the contraction of pulmonary arterial rings from transplanted lungs. In contrast, iNOS inhibition rescued the vasoconstriction response to exogenous phenylephrine of pulmonary arterial rings from transplanted lungs. In addition, lung transplantation led to suppression of PaO2/FiO2 ratio, increased intrapulmonary shunt (Qs/ Qt), and increase of lung wet to dry ratio (W/D), malondialdehyde and myeloperoxidase levels, all of which were reversed upon iNOS inhibition. Furthermore, inhibition of iNOS significantly rescued vascular function and alleviated edema and inflammatory cell infiltration in the transplanted lung. Conclusions Our data suggest that lung transplantation causes upregulation of iNOS expression, and pulmonary vascular dysfunction. iNOS inhibition reverses the posttransplantational pulmonary vascular dysfunction. Keywords Nitric oxide synthase Ischemia–reperfusion injury Rat lung transplantation Pulmonary arterial rings
Responsible Editor: John Di Battista. J. Wu H. Zhu X. Chen J. Wei X. Zhang M. Xu (&) Department of Anesthesiology, Shanghai Chest Hospital, Shanghai Jiaotong University, 241 West Huaihai Road, Shanghai 200030, People’s Republic of China e-mail: [email protected]
Introduction Lung transplantation is the ultimate therapeutic option for end-stage malignant pulmonary diseases. However, the
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1-year survival rate is only 80 % [1]. Ischemia–reperfusion injury (IRI) is a significant factor contributing to primary graft failure in lung transplantation and increases the risk of bronchiolitis obliterans syndrome after lung transplantation [2]. Ischemia–reperfusion injury causes damage to the pulmonary vascular endothelium, resulting in decreased alveolar perfusion, transplant exhaustion and graft failure [3, 4]. Despite extensive efforts to interpret the observed vascular damage, mechanisms contributing to this damage remain unclear. Nitric oxide (NO) plays a crucial role in the regulation of post-transplantational pulmonary function [5]. Two different nitric oxide synthases (NOS) are involved in regulation of pulmonary vascular function. Endothelial NOS (eNOS), mostly distributed in vascular endothelium, produces low levels of NO (picomoles) in a calciumdependent manner [6] to maintain vascular dilation and permeability. In contrast, inducible NOS (iNOS) is expressed by inflammatory cells such as neutrophils and macrophages, and is now known to be widely distributed in the airway epithelium and vascular smooth muscles [7]. iNOS is calcium-independent, and can produce a large, continuous flux of NO that is 1,000-fold higher than that of eNOS, lasting for hours to days in response to conditions of stress and inflammation [8, 9]. It is thought that eNOS is cell-protective, while iNOS has cytotoxic functions [10]. Numerous studies have been performed to study the association between NOS and post-transplantational pulmonary function. A significant increase in iNOS and a decrease in eNOS mRNA was found after reperfusion during rat lung transplantation [9]. Increased iNOS and decreased eNOS activity may contribute to ischemia– reperfusion-induced pulmonary vasoconstriction [11]. Overproduction of NO via iNOS and oxidative stress may lead to reactive oxygen and nitrogen species formation and vascular dysfunction during acute lung injury [12]. A recent study has shown that hepatic ischemia–reperfusion leads to lung injury by promoting pulmonary iNOS expression [13]. However, it remains unclear whether there is a causal relationship between iNOS upregulation and post-transplantational pulmonary vascular dysfunction. Furthermore, it is not known whether iNOS inhibition has any therapeutic role in post-transplantational pulmonary vascular dysfunction. Accordingly, the current study combines the in vivo orthotropic lung transplantation model and the ex vivo technique of isolated pulmonary arterial rings and an iNOS-selective inhibitor (N6-(1-iminoethyl)-L-lysine, L-NIL) [14, 15] to study the role of iNOS in lung transplant-related pulmonary vascular dysfunction during ischemia–reperfusion injury.
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Materials and methods Animals and operation process Studies were performed in accordance with the Guidelines for Animal Experiments of Shanghai Jiaotong University, PR China. Male Sprague–Dawley rats (340 ± 5 g) were housed with free access to food and water under a natural day/night cycle and acclimated for 1 week before any experimental procedures. The rat orthotropic left lung transplantation was carried out by using the modified three-cuff technique [16]. Briefly, donor rats were anesthetized by intraperitoneal injection of ketamine chloride (100 mg/kg). Rats were intubated and ventilated with 100 % oxygen, tidal volume of 10 mL/kg and a respiratory rate of 70 breaths/min. After heparinization (1,000 U/kg intravenously), a median sternotomy was performed, the lungs were flushed through the main pulmonary artery with 20 mL of cold low-potassium dextran solution (LPD; Perfadex, Vitrolife, Gothenburg, Sweden), the pulmonary artery, vein and main stem bronchus were ligated and tied off, and the inflated left lung was placed into LPD solution at 4 °C for 3 h. Recipient rats were anesthetized, intubated, and mechanically ventilated at the same parameters as for the donor rats. A left thoracotomy was performed through the fourth intercostal space. The hilum of the left lung was dissected, and the pulmonary artery, pulmonary vein and main bronchus were clamped with microvascular clamps. After incising the pulmonary artery and vein, the donor lung was anastomosed with the corresponding recipient structures. The thorax was closed and the mechanical ventilation was maintained, and 30 mg/kg ketamine were added according to the occurrence of limb movements and corneal reflexes to prevent the rats from regaining consciousness before they were sacrificed by overdose of chloral hydrate after 2 h of reperfusion [17]. For ex vivo experiments, the left pulmonary artery was isolated and cut into three cross sections for vasoconstriction, vasodilation and endothelial denudation assays. The left lung tissue was also cut into two pieces. One piece was used to measure lung wet to dry weight ratio (W/D). The other piece was kept at -80 °C for future malondialdehyde (MDA), myeloperoxidase (MPO), iNOS and eNOS measurements. For in vivo experiments, some of the transplanted rats were injected with 1 mL (3 mg/kg) N6(1-iminoethyl)-L-lysine (L-NIL, an iNOS activity inhibitor) solution diluted with 0.9 % saline solution by tail vein injection 5 min before the reperfusion and were sacrificed after 2 h of reperfusion. Left lung tissues were isolated and analyzed as mentioned above.
Inducible nitric oxide synthase inhibition
In vitro vascular preparation
Assessment of lung function
Arterial rings were prepared as previously described [18]. Briefly, following sacrifice of the rats, hearts and lungs were immediately removed and placed in a petri dish containing 4 °C Krebs–Henseleit (KH) solution (in mM/L: 118.40 NaCl; 5.01 KCl; 1.20 KH2PO4; 2.5 CaCl2; 1.2 MgCl2; 10.1 glucose; 25 NaHCO3) saturated by a gas mixture (95 % O2, 5 % CO2). The pulmonary arterial tree was rapidly dissected from the lung parenchyma with small scissors and forceps. The left pulmonary artery was carefully separated from pulmonary artery branches under the microscope. Peripheral adipose and connective tissues were removed. The left pulmonary artery was cut into three cross sections, each ring measuring 3–4 mm. Two retained an intact endothelium whilst the third was subjected to removal of the endothelium by friction. Vascular rings were suspended by stainless steel hooks attached to the tension sensor and mounted in thermostatically controlled 37 °C Krebs buffer (pH = 7.4, 10 mL) and gassed with 95 % O2 and 5 % CO2. The mechanical activity was recorded isotonically under a resting load of 1 g. The mechanical signals were processed by bridge amplifiers and were recorded by the MPA2000 multi-channel physiological recorder system (Alcott Technology Co. Ltd, Shanghai China). The arterial segments were viable at least for 4 h under the experimental conditions. The KH solution was renewed every 20 min. The arterial rings were equilibrated in their baths for 1 h under a resting load of 1 g. The endothelium denudation was performed by gently and systematically rubbing the intimal surface of the everted cylindrical segment using moistened cotton wool fixed on a thin wooden stick. Successful removal of endothelial cells from the rings was confirmed by the inability of acetylcholine to induce relaxation [18, 19]. At the beginning of each experiment, the endothelial integrity was examined by pre-contracting the isolated pulmonary artery with a submaximal concentration (EC80) of phenylephrine hydrochloride (Phe) (0.1 lM) and testing the ability of acetylcholine (Ach) (1 lM) to relax the arterial strips [20]; this effect is absent in endothelium denuded preparations [21]. Ach-induced relaxation of the arterial rings by [ 80 % or \ 20 % of the Phe-induced pre-contraction was taken as indicative of structurally intact or damaged endothelium, respectively [22]. After three washes during the next 30 min, the concentration–response relationship for Ach (0.001–100 lM) and Phe (0.0001–10 lM) was determined by cumulative exposure to increasing drug concentrations at 3 min intervals. Some of the vascular rings were pre-incubated with the selective iNOS inhibitor, L-NIL, (10 lmol/L) for 10 min to analyze the role of iNOS in vascular constriction and dilatory function.
Transplant graft function was assessed by measuring amount of blood shunted/total blood flow (Qs/Qt) levels and partial arterial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2) ratio in blood taken from the pulmonary vein at the completion of the 2-h reperfusion period (n = 6 for each group). Qs/Qt = (CcO2 - CaO2)/ (CcO2 - CvO2) (CcO2, oxygen content of pulmonary capillary blood; CaO2, oxygen content of arterial blood; CvO2, oxygen content of mixed venous blood) [23]. Qs/Qt and partial pressures of oxygen were measured with a blood-gas analyzer (Radiometer ABL 800, Copenhagen, Denmark) to report PaO2, FiO2 and Qs/Qt values. Wet-todry lung tissue weight ratios were calculated at the end of the 2-h reperfusion period as a measurement of lung edema (n = 6 for each group). The left lung was dissected with a sharp blade. All surface liquid was removed using filter paper. Lung tissue was weighed and placed in an oven at 80 °C for 48 h. Then, the dried lung portion was reweighed, and the ratio of the lung weight before and after drying was calculated. Determination of MDA, MPO, iNOS and eNOS levels in lung tissue As an index of membrane lipid peroxidation, levels of MDA were determined as previously described [24]. Lung tissues were homogenized (100 mg/mL) in 10 volumes of 1.15 % KCl solution containing 0.85 % NaCl and then centrifuged at 1,500 g for 15 min. Next, 200 lL of lung tissue homogenate was added to a reaction mixture consisting of 1.5 mL 0.8 % thiobarbituric acid, 200 lL 8.1 % sodium dodecyl sulfate, 1.5 mL 20 % acetic acid (adjusted to pH 3.5 with NaOH) and 600 lL distilled H2O. The mixture was then heated at 95 °C for 40 min. After cooling to room temperature, the samples were centrifuged (10,0009g, 10 min) and supernatant absorbance was measured at 532 nm, using 1,1,3,3-tetramethoxypropane as an external standard. The level of lipid peroxide was expressed as nmol MDA/mg protein (Bradford assay) (n = 6 for each group). Activity of MPO, an enzyme present in neutrophils, was used as a marker of neutrophil infiltration. MPO activity was determined in lung as described before [25]. Lung tissues (100 mg of tissue) were homogenized in 2 mL of 20 mM potassium phosphate buffer (pH 7.4). After centrifugation (15,0009g, 20 min), the pellet was resuspended in 2 mL of 50 mM potassium phosphate buffer (pH 6.0) containing 0.5 % hexadecyltrimethylammonium bromide and sonicated for 30 s. After being heated at 60 °C for 2 h, the samples were centrifuged (10,0009g, 10 min). The supernatant (25 lL) was added to 725 lL of 50 mM phosphate buffer (pH 6.0) containing
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Fig. 1 Decreased pulmonary arterial ring constriction in response to phenylephrine and dilation in response to Ach after lung transplantation. a Comparison of pulmonary artery constriction response to phenylephrine between lung transplantation group (lung Tx) and sham group (sham). Both groups were treated with different concentrations of phenylephrine (0.0001–10 lM) and then the concentration–response relationship of vasoconstriction was recorded. b Comparison of pulmonary artery dilation in response to Ach
(0.001–100 lM) between lung transplantation group and sham group. Dilation is shown as reduction of constriction. The dilation values are expressed as a percentage of the phenylephrine-induced pre-constriction level recorded immediately before Ach administration. (Relaxation % = (present constriction-pre-constriction)/pre-constriction 9 100 %. The values represent mean ± SEM, n = 6. **P \ 0.01 vs. sham, repeated measures ANOVA
0.167 mg/mL o-dianisidine and 5 9 10-4 % hydrogen peroxide. MPO activity was measured spectrophotometrically as the change in absorbance at 460 nm at 37 °C, using a Spectramax microplate reader (Molecular Devices, Sunnyvale, CA, USA). Results were expressed as units of MPO activity per gram wet tissue (1 U defined as change in absorbance of 1 nm/min) (n = 6 for each group). The levels of eNOS and iNOS were measured by enzymelinked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions (n = 6 each group).
decreased dilation in response to Ach (Fig. 1b). The maximum contractile effect for Phe, a a-adrenergic agonist, was significantly decreased in pulmonary artery rings from transplanted rats after 2 h of in vivo reperfusion compared to shams (Emax = 2.11 ± 0.38 vs. 4.15 ± 0.39 mN/mm, n = 6, P \ 0.01, respectively). The dilation of pulmonary artery rings from transplanted lungs to Ach (0.001–100 lM) was significantly decreased as compared to the sham group (Fig. 1b). Transplantation leads to increased iNOS, MPO and MDA levels
Statistical analysis SPSS11.0 statistical software was used to analyze the results. The data are expressed as mean ± SD. Repeated measures of analysis of variance (ANOVA), calculated using the general linear model, were used to compare the concentration response curves obtained in pulmonary arteries. The other results were analyzed using one-way ANOVA followed by the Student–Newman–Keuls test. All statistical analyses were considered significant when P \ 0.05.
Results Lung transplantation leads to diminished pulmonary arterial reactivity Following lung transplantation, arterial ring constriction decreased in response to Phe (Fig. 1a), and there was
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Because NOS plays a crucial role in post-transplantational pulmonary function regulation, we measured the levels of iNOS and eNOS in sham-treated and transplanted lungs. We found that lung transplantation caused upregulation of iNOS and downregulation of eNOS levels (P \ 0.05, n = 6) (Table 1). In accordance with the iNOS and eNOS changes, the activity of MPO was significantly increased after transplantation, (P \ 0.05, n = 6) (Table 1). MDA, another marker for oxidative stress [26], was also significantly increased after lung transplantation (P \ 0.05, n = 6) (Table 1). iNOS inhibition but not endothelium-denuded arterial rings affected the post-transplantational pulmonary arterial dysfunction To elucidate whether decreased post-transplantational pulmonary vasoconstriction was endothelium-dependent, we removed the arterial endothelium. However, endothelial
Inducible nitric oxide synthase inhibition Table 1 Changes in iNOS, eNOS, MPO and MDA levels (x ± SD) Groups
iNOS
eNOS
MPO
MDA
Sham
1.02 ± 0.36
0.71 ± 0.07
19.05 ± 5.03
17.01 ± 2.82
LungTx
2.63 ± 0.61a
0.19 ± 0.02a
39.36 ± 6.81a
26.36 ± 2.73a
b
b
b
18.69 ± 2.79b
LungTx ? L-NIL
0.93 ± 0.18
0.52 ± 0.11
13.16 ± 1.39
Levels of MDA, iNOS, eNOS and activity of MPO in different groups following transplantation. Level of MDA was expressed as nmol MDA/mg protein. The levels of eNOS and iNOS were presented as ng/ml. Activity of MPO was presented as units of MPO activity per gram wet tissue. n = 6 in each group iNOS inducible nitric oxide synthase, eNOS endothelial nitric oxide synthase, MPO myeloperoxidase, MDA malondialdehyde, LungTx lung transplantation, L-NIL N6-(1-iminoethyl)-L-lysine a
P \ 0.05 compared with sham group,
b
P \ 0.05 compared with LungTx group
removal only moderately enhanced Phe-induced vasoconstriction of arterial rings from sham-operated lungs (P \ 0.05, n = 6) (Fig. 2a). On the contrary, constriction of arterial rings from transplanted lungs to Phe was not affected (Fig. 2b). Since Ach produced an endotheliumdependent relaxation of the sustained contraction to Phe, it cannot induce endothelium-dependent relaxation of the endothelium-removed pulmonary arterial rings. We then tested whether iNOS affected pulmonary function in transplanted lungs by inhibiting iNOS using its inhibitor L-NIL. We found that L-NIL could effectively inhibit post-transplantational levels of iNOS, MDA, as well as MPO activity (Table 1). L-NIL had no effect on vasoconstriction of arterial rings from sham-operated lungs in response to Phe (Fig. 2c). However, iNOS inhibition significantly rescued vasoconstriction ability from transplanted lungs (P \ 0.01, n = 6) (Fig. 2d). This suggests that iNOS contributes to the constriction of the rings from transplanted lungs to Phe. Furthermore, we found that pre-treatment of lung with L-NIL protected the dilatory response to Ach in the rings from transplanted lungs (Fig. 3). iNOS inhibition reverses pulmonary function defects after lung transplantation Finally, we tested whether iNOS inhibition could benefit post-transplantational pulmonary function. Compared with the sham-operated rats, the transplanted rats developed significant pulmonary dysfunction as evidenced by decreased PaO2/FiO2 ratio (lung transplant group (LTx) vs. sham, 280.0 ± 8.7 vs. 406.3 ± 10.9, P \ 0.01), increased intrapulmonary shunt (Qs/Qt, LTx vs. sham, 26.3 ± 0.7 vs. 8.7 ± 0.2, P \ 0.01) and lung edema (W/D weight ratio, LTx vs. sham, 5.62 ± 0.1 vs. 4.4 ± 0.1, P \ 0.01). However, L-NIL treatment significantly improved the pulmonary function in transplanted lungs by improving gas exchange (PaO2/FiO2 ratio 360 0.0 ± 13.6, Qs/Qt 20.5 ± 1.4) and reducing lung (W/D 4.9 ± 0.1) edema (Fig. 4a–c).
Discussion The pathogenesis of lung ischemia–reperfusion is characterized by two major processes. First, the increased capillary permeability results in pulmonary edema and impaired gas exchange due to tissue fluid leakage [27]. Second, the pulmonary microvasculature constricts abnormally and the relaxation of pulmonary artery is impaired [28], which causes vascular dysfunction. These two factors cause lung ventilation-perfusion ratio mismatch, hypoxemia, and acute respiratory distress syndrome in the clinic [29]. Previous study has found that cold ischemia and reperfusion may produce different patterns of pulmonary vasomotor dysfunction and contribute to increased pulmonary vascular resistance in the transplanted lung [30]. Endothelium-dependent pulmonary arterial relaxation was significantly impaired by reperfusion, and pulmonary neutrophil sequestration could be the main determinant of ischemia–reperfusion-induced vascular dysfunction in lung transplantation [4]. In the present study, we found that lung transplantation led to significantly decreased pulmonary arterial ring vasoconstriction and decreased dilation. Consistent with previous discoveries [9, 11], lung transplantation also caused increased levels of iNOS, MDA, enhanced MPO activity and decreased levels of eNOS, which indicated increased oxidative stress, inflammatory cell infiltration, and pulmonary capillary permeability, suggesting increased post-transplantational infiltration of iNOS-producing neutrophils. We further showed that endothelial removal did not affect Phe-induced vasoconstriction of pulmonary arterial rings from transplanted lungs, although it moderately enhanced it in the sham group. The difference in endothelial dependency between transplantation and sham groups suggests that the endothelium may already be injured or depressed in transplanted lungs [31]. Recently, it has been described that NO may modulate its own production due to cross-talk between the eNOS and iNOS isoforms [32]. iNOS could inhibit eNOS production and suppress the function of
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Fig. 2 iNOS inhibition but not endothelium-removal affects the posttransplantational pulmonary vascular dysfunction. Pulmonary arterial rings from the sham group and lung transplantation group were treated with different concentrations of phenylephrine (0.0001–10 lM) and then the concentration–response relationship of vasoconstriction was recorded. The endothelial vasoconstriction responses in the sham group (a) and the lung transplant group (b) after removal of endothelium are shown. E- without
endothelium, E? intact endothelium. In thelower panel, the vascular rings were pre-incubated with the selective iNOS inhibitor (L-NIL, 10 lmol/L) for 10 min to analyze the role of iNOS in vasoconstriction in response to Phe. L-NIL did not affect the constriction to Phe in the rings from sham rats (c). iNOS inhibition rescued vasoconstriction to Phe in pulmonary rings from transplanted lungs (d). The values represent mean ± SEM, n = 6. *P \ 0.05, **P \ 0.01 vs. sham, repeated measures ANOVA
endothelial cells [32], and inhibition of iNOS activity could significantly reverse dysfunctional endothelial cells by the upregulation of eNOS [31]. Consistent with these effects, we discovered that lung transplantation caused increased production of iNOS and decreased levels of eNOS. These findings suggested that iNOS-derived NO repressed the vascular contraction by relaxing vascular smooth muscle but not by vascular endothelium, since removal of the endothelium had no effect, although in normal condition, the pulmonary vascular tone is modulated by eNOSderived NO in an endothelium-dependent way. In addition, iNOS contributed to the post-transplantational pulmonary vascular dysfunction. iNOS inhibition using L-NIL abrogated these vascular defects and decreased W/D ratio, MDA, and MPO levels in transplanted lung, demonstrating that elevated iNOS plays a pivotal role in ischemia– reperfusion-induced lung injuries following transplantation. Therefore, the augmented expression of iNOS may be an underlying mechanism of the pulmonary artery hyporesponsiveness to vasoconstrictors and Ach-related
relaxation. Furthermore, iNOS inhibition may serve as a strategy to increase the success of lung transplantation. We only used pulmonary arterial rings in our study for vascular dysfunction. Vessels from different segments or even vessels with different diameters respond differently to vasoconstricting or dilating factors [33–36]. It has been shown that pulmonary microvessels are more sensitive to various vasoconstrictors compared with pulmonary arteries, including endothelin-1 and thromboxane A2 analogue U46619 [37, 38]. Although we used phenylephrine hydrochloride instead of endothelin-1 and Thromboxane A2 analogue, it’s still reasonable to speculate that responses of pulmonary arteries and microvessels are different. In our study, we have directly shown the role of iNOS in the pulmonary arterial response. We measured the W/D ratio changes in response to iNOS inhibition, which indirectly reflect the pulmonary microvessels responses. Our data suggest that both pulmonary arterial dysfunction and the W/D ratios are regulated by iNOS inhibition, suggesting that iNOS regulates both pulmonary arteries and microvessels.
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Inducible nitric oxide synthase inhibition
Fig. 3 iNOS inhibition restored vasodilation to Ach in transplanted arterial rings. The vascular rings were pre-incubated with the selective iNOS inhibitor (L-NIL, 10 lmol/L) for 10 min to analyze the role of iNOS in vascular dilatory function. The concentration–response relationship for Ach (0.001–100 lM) was determined by cumulative exposure to increasing drug concentrations at 3 min intervals. Vessels of lung transplant group were compared with the sham group. The values represent mean ± SEM, n = 6. **P \ 0.01 vs. sham, repeated measures ANOVA
To study vascular relaxation, we used phenylephrine hydrochloride (Phe) pre-contracted vessels, which are commonly used to measure vascular constriction. The major limitation with this method is that it is a1-adrenergic receptor dependent. Other factors, including endothelin-1 and thromboxane A2 analogue, can induce vascular constriction [39–45], while prostacyclin and histamine can induce relaxation of pulmonary microvessels [46]. These factors could also be induced after lung transplantation. In addition to using pre-contacted vessels, another method to measure arterial relaxation response is to directly measure
Fig. 4 NOS inhibition by L-NIL reverses lung functions in transplanted rats. iNOS inhibitor L-NIL (3 mg/kg) was administrated by tail vein injection 5 min before the reperfusion of experimental orthotropic lung transplantation. Transplant graft function was assessed by measuring PaO2/FiO2 (partial arterial pressure of oxygen/fraction of inspired oxygen) ratio (a), Qs/Qt (amount of blood
the dynamic pulmonary vessels dilation (the changes in internal vessel diameter and changes in blood velocity) using in vivo videomicroscopy. This method, however, is beyond the scope of this study. Nitric oxide plays a central role in the regulation of pulmonary circulation under physiological and pathophysiological circumstances [6, 47]. NO can be protective or injurious, depending on its site of release, concentration, and local conditions [10, 47–49]. NO exerts many of its physiological effects by activation of guanylyl cyclase [7]. Under normal physiological conditions, NO is primarily generated by the eNOS pathway to reduce vascular tone and inhibit leukocyte and platelet exudation [48]. Activated eNOS binds to heat shock protein 90 (Hsp90) to form a protein kinase complex Akt-eNOS-Hsp90 [50–52] that improves eNOS activity by increasing eNOS affinity for calmodulin [53]. NO derived from eNOS binds to this complex to activate guanylyl cyclase and to modify its function in a calcium-dependent manner; this is the key step in vasodilation induced by endothelium-derived NO [32]. NO can also be generated by iNOS after stimulation by oxidative stress, toxins and inflammatory mediators such as tumor necrosis factor and cytokines [32]. iNOSgenerated NO then induces pathophysiological changes via cytotoxic effects and exacerbation of tissue damage [12, 48, 54, 55]. The availability of other reactive intermediates with which NO may interact also determines its toxicity [56]. Because of its radical character, NO reacts with superoxide radicals (O2-) at a near diffusion-limited rate, which in turn produces another highly reactive species, peroxynitrite (ONOO-) [57–60]. Peroxynitrite causes oxidative/nitrative modification to biomolecules, thus modulating physiological and pathophysiological processes [61, 62]. In addition, peroxynitrite can be converted to
shunted/total blood flow) ratio (b) and W/D (wet-to-dry lung tissue) weight ratio (c) levels at the completion of the 2-h reperfusion period. Data are means ± SEM of six rats for each group. **P \ 0.01 vs. sham group, **P \ 0.01 vs. lung transplant (LT) group, one-way ANOVA followed by the Student–Newman–Keuls test
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highly reactive hydroxyl-like radicals [63], which inhibit the formation of Akt-eNOS-Hsp90 complex and thus block vascular endothelial relaxation [53]. Enhanced iNOS activity in the vessel wall inhibits eNOS activity and reduces NO bioavailability in endothelial cells, which causes vascular dysfunction as manifested by decreased endothelium-dependent relaxation to acetylcholine [11]. Our results demonstrated that lung transplantation caused suppression of Ach-induced arterial relaxation (Fig. 1b), indicating vascular endothelial dysfunction in transplanted lungs. Reduced eNOS was detected in transplanted lungs (Table 1). This is consistent with the idea that decreased eNOS is associated with vascular endothelial dysfunction. Pre-treatment with the selective iNOS inhibitor L-NIL restored the relaxation ability of transplanted lung arteries to control levels. iNOS inhibition also restored the levels of eNOS. These results suggest that the decline in endothelial eNOS activity may be caused by the high level of iNOS in transplanted lungs. iNOS inhibition restored the levels of eNOS, which possibly rescued vascular endothelial function. The contractile response to Phe in isolated pulmonary arteries was significantly suppressed after lung transplantation. However, vasoconstriction defects were not affected by removal of vascular endothelium (Fig. 2a, b). We found that high levels of post-transplantational iNOS were associated with reduced pulmonary vasoconstriction. We further showed that iNOS inhibition using L-NIL restored pulmonary vasoconstrictive reaction in vitro (Fig. 2d). Collectively, these data support the hypothesis that iNOSderived NO represses vascular contraction by relaxation of smooth muscle cells but not endothelium. Smooth muscle cells and possibly peroxynitrite may be responsible for reduced pulmonary arterial constriction. Exposure of vascular tissue to peroxynitrite can result in a prolonged relaxation mediated through a glutathione-dependent generation of NO [64]. Firstly, in tissue bath, superoxide can react with NO to generate perocynitrite [65]. At physiological conditions, this production of peroxynitrite can exert multiple biological activities [66]. This could explain the source of perocynitrite and the possible effect for reduced arterial constriction. Secondly, iNOS could inhibit eNOS production and suppress the function of endothelial cells [11, 32] and inhibition of iNOS activity could significantly reverse dysfunctional endothelial cells by the upregulation of eNOS [67]. In summary, we found that lung transplantation led to defects in vasoconstriction and vasodilation reactivity of the pulmonary artery. This was associated with increased iNOS levels. Pre-treatment with the selective iNOS inhibitor L-NIL not only restored vascular function of the transplanted lung, but also reversed transplant-related lung ischemia–reperfusion injury and improved pulmonary
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function. Therefore, inhibition of iNOS may serve as a potential strategy to protect the lung from ischemia– reperfusion injury after lung transplantation. Acknowledgments The authors thank Dr. Wei-gang Guo, Dr. Qiang Tan and Yi-qingYang for their advice and helpful suggestions. This work was supported by Shanghai Natural Science Foundation No. 12ZR1428700, ‘‘1050’’ Foundation for the Talents by Shanghai Chest Hospital and Shanghai Joint development project for Municipal hospitals (SHDC12010222). Dr. Wu and Zhu contributed equally to this work.
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