Experimental Lung Research, 41, 241–250, 2015 Copyright © 2015 Informa Healthcare USA, Inc. ISSN: 0190-2148 print / 1521-0499 online DOI: 10.3109/01902148.2013.850125
ORIGINAL ARTICLE
Activated protein C attenuates ischemia-reperfusion-induced acute lung injury Chou-Chin Lan,1,2 Chung-Kan Peng,3,4 Shiu-Feng Huang,5 Kun-Lun Huang,3,4,∗ and Chin-Pyng Wu6,∗
Exp Lung Res Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
1
Division of Pulmonary Medicine, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taipei, Taiwan, Republic of China 2 School of Medicine, Tzu Chi University, Hualien, Taiwan, Republic of China 3 4
Division of Pulmonary Medicine, Tri-Service General Hospital, Taipei, Taiwan, Republic of China Institute of Undersea and Hyperbaric Medicine, National Defense Medical Center, Taipei, Taiwan, Republic of China
5
Department of Pathology, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taipei, Taiwan, Republic of China 6
Department of Critical Care Medicine, Li-Shin Hospital, Tao-Yuan County, Taiwan, Republic of China A B STRA CT Ischemia-reperfusion (IR)-induced acute lung injury (ALI) is implicated in several clinical conditions, such as lung transplantation, acute pulmonary embolism after thrombolytic therapy, re-expansion of collapsed lung from pneumothorax, or pleural effusion, cardiopulmonary bypass, etc. Because mortality remains high despite advanced medical care, prevention and treatment are important clinical issues. Activated protein C (APC) manifests multiple activities with antithrombotic, profibrinolytic, and anti-inflammatory effects. We therefore conducted this study to determine the beneficial effects of APC in IR-induced ALI. IR-induced ALI was conducted in a rat model of isolated-perfused lung in situ. The animals were divided into the control group, IR group, and IR+APC group. There were six adult male Sprague–Dawley rats in each group. The IR caused significant pulmonary microvascular hyperpermeability, pulmonary edema and dysfuction, increased cytokines (tumor necrosis factor (TNF)-α, IL-17, CXCL-1), and neutrophils infiltration in lung tissues. Administration of APC significantly attenuated IR-induced ALI with improving microvascular permeability, pulmonary edema, pulmonary dysfunction, and suppression inflammatory response. The current study demonstrates the beneficial effects of APC in IR-induced ALI. This protective effect is possibly associated with the inhibition of TNF-α, IL-17A, CXCL1, and neutrophils infiltration in lung tissues. However, the current results were obtained in an animal model and it is still necessary to confirm these findings in human subjects. If we can demonstrate the benefits of APC to protect IR lung injury, we can postulate that APC is a potential therapeutic drug for lung preservation. KEYWORDS activated protein C, acute lung injury, ischemia-reperfusion, pro-inflammatory cytokines
IR-induced ALI with alveolar damage and pulmonary edema is an important issue in lung transplantation [2]. Despite advancements in organ preservation and perioperative care, IR-induced ALI remains a significant cause of early mortality and morbidity [2]. IR-induced ALI is also implicated in other clinical conditions, including acute pulmonary embolism after thrombolytic therapy, re-expansion of collapsed lung from pneumothorax or pleural effusion, and cardio-pulmonary bypass [3–5]. Reperfusion injury may also increase inflammation in adult respiratory
INTRODUCTION Exposure of the lungs to periods of ischemia and rapid initiation of reperfusion causes ischemiareperfusion (IR)-induced acute lung injury (ALI)[1]. Received 3 July 2013; accepted 26 September 2013 ∗ These authors contributed equally to this work. Address correspondence to Chin-Pyng Wu, M.D., PhD. Department of Critical Care Medicine, Li-Shin Hospital, #77, Kwang-Tai Road, Ping-jen City, Tao-Yuan County, Taiwan, Republic of China. E-mail: [email protected]
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Exp Lung Res Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
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distress syndrome, wherein perfusion of the pulmonary vasculature is often occluded by vasoconstriction, micro-emboli, or in situ thromboses [6]. Mortality from IR-induced ALI remains high even with advanced medical care [7, 8]. Thus, prevention and treatment of IR-induced ALI are important clinical issues. Activated protein C (APC) is an endogenous anticoagulant generated from protein C by the action of the thrombin–thrombomodulin complex on the endothelial cells [9]. It has an important role in coagulation homeostasis through proteolytic cleavage and inactivation of factors Va and VIIIa [9]. It is known to be critical in regulating microvascular coagulation [9] and its action on microcirculation during endotoxemia has been noted [10]. It also has antiinflammatory properties by blocking tumor necrosis factor (TNF), interleukin (IL)-6, and IL-8, and inhibits neutrophils chemotaxis [11, 12]. Therefore, APC manifests multiple effects with antithrombotic, profibrinolytic, and anti-inflammatory activities. Because of its multiple effects, APC may have a potential role in the treatment of complex medical disorders. Sepsis is one of the most challenging medical conditions. Some previous clinical studies have reported that low levels of circulating protein C in patients with sepsis will lead to higher mortality [13]. Administration of APC in sepsis was used in many clinical trials [14, 15]. However, the effect of APC was controversial in clinical trials of Protein C Evaluation in Severe Sepsis (PROWESS) and PROWESS-SHOCK [15]. Nonetheless, more studies on the role of APC in nonseptic inflammatory conditions should be undertaken. Since APC manifests multiple actions and regulates micro-circulation, the present study posits the hypothesis that APC may be beneficial in IR-induced ALI. This study has been conducted to investigate the effects of APC in IR-induced ALI.
lung in situ in the chest were as previously described [16]. After deep anesthesia, tracheostomy was performed and a cannula was inserted into the trachea. The lungs were ventilated with a humidified gas mixture containing 5% CO2 in air at a frequency of 60 cycle/minute, tidal volume of 3 mL, and positive endexpiratory pressure (PEEP) of 1 cm H2 O. Median sternotomy was performed and heparin (1 U/g of body weight) was injected into right ventricle. An afferent silicon catheter was inserted into the pulmonary artery through right ventricle. An efferent catheter was installed into left atrium via left ventricle to collect the effluent perfusate for re-circulation. Pulmonary venous outflow was diverted via the efferent catheter into the reservoir. Pulmonary trunk and aorta were tied. A third ligature was placed at the atrio-ventricular junction to prevent perfusate from entering into left ventricle. Pulmonary arterial pressure (PAP) and venous pressure (PVP) were measured from side-ports in the afferent and efferent catheters. A peristaltic pump (Model 1203, Harvard Apparatus) was used to perfuse the lungs with recirculated perfusate: blood (1:1) mixed with physiologic salt solution (119 mM NaCl, 4.7 mM KCl, 1.17 mM MgSO4 , 22.6 mM NaHCO3 , 1.18 mM KH2 PO4 , 1.6 mM CaCl2 , 5.5 mM glucose, and 50 mM sucrose). Bovine albumin (4 g/dL) was added to maintain osmolarity of the perfusate. The perfusion rate was kept at 8–10 mL/minute by a roller pump and constant temperature (37◦ C) was maintained by a water bath. The preparation was placed on an electronic balance with the isolated lungs remaining in situ. The digital signals of the electronic balance were converted to analog signals by a digital–analog converter and recorded on a polygraph recorder. Weight changes were precalibrated on the electronic balance before preparation of the experiment. In this isolated lung in situ preparation, the changes in body weight reflected the lung weight changes[17].
MATERIALS AND METHODS Isolation and Perfusion of Lungs
Experimental Protocols and Induction of IR-induced ALI
The National Science Council and Animal Review Committee of the National Defense Medical Center (Taipei, Taiwan) approved the study protocol. Animals in this study were cared for in accordance with the “Guide for the Care and Use of Laboratory Animals” published by the United States National Institutes of Health. Male Sprague–Dawley rats weighing 301.0 ± 31.4 g were anesthetized through intraperitoneal injection of pentobarbital sodium (50 mg/kg). Procedures regarding the preparation of isolated-perfused
The animals were divided into three groups (n = 6 per group): control group (without IR), IR group, and IR with APC (IR+APC) group. The IR-induced ALI was performed with 30 minutes of ischemia by stopping ventilation and perfusion [18]. After ischemia, the lungs were reperfused for 90 minutes and ventilated with 5% CO2 + 95% air. The temperature was monitored and kept at 37◦ C during IR and the whole course of experiment. APC (100 μg/kg) was administered via perfusate after ischemia. Lung weight, airway pressure, PAP, and PVP were continExperimental Lung Research
Activated Protein C in Ischemia-Reperfusion ALI
uous monitored during experiment. Microvascular permeability (Kf) was measured at the baseline and post-IR. All of the rats were further studied for lung histopathology, lung wet/dry weight ratio (W/D), total protein concentration, and cytokine levels in broncho-alveolar lavage fluid (BALF). The difference in pressures (between PAP and PVP) divided by the perfusate flow rate measured pulmonary vascular resistance (PVR) [19]. Lung compliance was calculated by tidal volume divided by pressure difference between peak airway pressure and PEEP[19].
Exp Lung Res Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
Microvascular Permeability The measurement of Kf in isolated lungs was as previously described [16]. Kf as an index of microvascular permeability was calculated from the increase in lung weight produced by an elevation in PVP. During the experiment, PVP was elevated rapidly by 10 cm H2 O for 7 minutes to measure Kf. The hydrostatic challenge elicited a biphasic increase in lung weight with an initial rapid component followed by a slow and steady component. The slow, the steady phase of weight gain as a function of time (W/T) was plotted on a semi-logarithmic paper and then extrapolated to zero time to obtain the initial rate of transcapillary filtration. From this plot, Kf was defined as the y-intercept (gm/minute) divides by PVP (10 cm H2 O) and lung weight, and expressed in whole units of grams per minute per centimeter of H2 O multiplied by 100 g [16].
Total Protein Concentration and Cytokine Levels in BALF At the end of the experiment, the left lungs were lavaged twice with 2.5 mL isotonic saline and the returned fluid (BALF) was collected by free drainage. The BALF was centrifuged at 200×g for 10 minutes, and total protein concentration in the supernatant was determined using bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). TNF-α, C-X-C motif ligand 1 (CXCL1) (R&D Systems Inc., Minneapolis, MN, USA) and IL-17A (eBioscience, San Diego, CA, USA) in BALF were measured using an ELISA kit according to the manufacturer’s protocol.
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for 48 hours [16]. The wet and dry weights were then measured to calculate lung W/D.
Lung Histopathology Part of the right lower lung lobe was taken for histologic examination. The tissues were immersed in 10% formaldehyde fixative for 24 hours, then embedded in paraffin wax and cut into 4–6 μm thick sections using a microtome. Histopathologic examination with hematoxylin and eosin (H&E)-stained was performed to verify the micro-anatomic features of lung injury imposed by IR and to assess the effects of APC. For tissue neutrophils quantification, H&E-stained sections were used to count the number of neutrophils per high power field (HPF). For each slide, neutrophils were counted in a blinded fashion on 10 sequential, nonoverlapping HPFs (magnification, 400×) of lung parenchyma beginning at the periphery of the section [20].
Data Analysis All statistical analyses were performed using the SPSS software 18.0 (SPSS Inc., Chicago, IL, USA). All values were reported as means ± SD (standard deviation). Statistical comparisons among groups were performed using one-way ANOVA and Bonferroni post hoc multiple comparison test. A P < .05 was considered statistically significant.
RESULTS APC Decreased Pulmonary Edema in IR-induced ALI The lung weight was significantly higher in IR group than in control group (P < .001; Figure 1A), whereas the lung weight of IR+APC group was significantly lower than that of IR group (P < .001). Lung W/D was significantly increased after IR (Figure 1B) and significantly decreased in IR+APC group (IR vs. control, P < .001; IR vs. IR+APC, P < .001).
APC Improved Lung Compliance in IR-induced ALI Pulmonary Edema Lung W/D was used as an indicator of pulmonary edema. After the experiment, part of the right middle lobe was weighed and then dried in an oven at 60◦ C C
2015 Informa Healthcare USA, Inc.
The lung compliance was significantly lower in IR group than in control group (P < .001) (Figure 1C), whereas the lung compliance of IR+APC group was significantly higher than that of IR group (P < .001).
Exp Lung Res Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
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FIGURE 1. APC decreased pulmonary edema in I/R-induced ALI. The (A) lung weight was significantly higher in IR group than in control group (P < .001), whereas the lung weight of IR+APC group was significantly lower than that of IR group (P < .001). Lung (B) W/D was significantly increased after IR and decreased in IR+APC group (IR vs. control, P < .001; IR vs. IR+APC, P < .001). (C) The lung compliance was significantly lower after IR and higher in IR+APC group (IR vs. control, P < .001; IR vs. IR+APC, P < .001). (D) The PVR was significantly higher after IR and decreased in IR+APC group (IR vs. control, P = .020; IR vs. IR+APC, P = .038). Note. There was significant difference from the ∗ control (P < .05) and # IR (P < .05) groups. APC, activated protein C; IR, ischemia-reperfusion; ALI, acute lung injury; PVR: pulmonary vascular resistance.
APC Decreased Pulmonary Vascular Resistance in IR-induced ALI The PVR was higher in IR group than in control group (P = .02) (Figure 1D), whereas the PVR of IR+APC group was significantly lower than that of IR group (P = .038).
ference (P = .219). The Kf changes (%) were significantly higher in IR (238.6%±106.9%) than control (7.5%±5.6%) and IR+APC (93.1%±43.4%) groups (IR vs. control, P < .001; IR vs. IR+APC, P = .005) (Figure 2C). When comparing between control and IR+APC groups, the Kf changes were without significant difference (P = .126).
APC Attenuated I/R-induced ALI with Microvascular Hyperpermeability
APC Decreased Total Protein Concentration and Cytokines in BALF
The Kf at baseline was similar between the three groups (IR vs. control, p = 1.0; IR vs. IR+APC, p = 1.0 at basline) (Figure 2A). After IR, the Kf was significantly increased and was attenuated by APC (Figure 2B) (IR vs. control, P < .001; IR vs. IR+APC, P < .012). When comparing between control and IR+APC groups, the Kf was without significant dif-
Total protein concentration in BALF was significantly increased after IR (IR vs. control, P < .001) (Figure 3A). However, APC decreased the total protein concentration after IR (IR vs. IR+APC, P < .001). TNF-α was significantly higher in IR than in control group, and decreased in the IR+APC group (IR vs. control, P = .005; IR vs. IR+APC, P
ORIGINAL ARTICLE
Activated protein C attenuates ischemia-reperfusion-induced acute lung injury Chou-Chin Lan,1,2 Chung-Kan Peng,3,4 Shiu-Feng Huang,5 Kun-Lun Huang,3,4,∗ and Chin-Pyng Wu6,∗
Exp Lung Res Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
1
Division of Pulmonary Medicine, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taipei, Taiwan, Republic of China 2 School of Medicine, Tzu Chi University, Hualien, Taiwan, Republic of China 3 4
Division of Pulmonary Medicine, Tri-Service General Hospital, Taipei, Taiwan, Republic of China Institute of Undersea and Hyperbaric Medicine, National Defense Medical Center, Taipei, Taiwan, Republic of China
5
Department of Pathology, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taipei, Taiwan, Republic of China 6
Department of Critical Care Medicine, Li-Shin Hospital, Tao-Yuan County, Taiwan, Republic of China A B STRA CT Ischemia-reperfusion (IR)-induced acute lung injury (ALI) is implicated in several clinical conditions, such as lung transplantation, acute pulmonary embolism after thrombolytic therapy, re-expansion of collapsed lung from pneumothorax, or pleural effusion, cardiopulmonary bypass, etc. Because mortality remains high despite advanced medical care, prevention and treatment are important clinical issues. Activated protein C (APC) manifests multiple activities with antithrombotic, profibrinolytic, and anti-inflammatory effects. We therefore conducted this study to determine the beneficial effects of APC in IR-induced ALI. IR-induced ALI was conducted in a rat model of isolated-perfused lung in situ. The animals were divided into the control group, IR group, and IR+APC group. There were six adult male Sprague–Dawley rats in each group. The IR caused significant pulmonary microvascular hyperpermeability, pulmonary edema and dysfuction, increased cytokines (tumor necrosis factor (TNF)-α, IL-17, CXCL-1), and neutrophils infiltration in lung tissues. Administration of APC significantly attenuated IR-induced ALI with improving microvascular permeability, pulmonary edema, pulmonary dysfunction, and suppression inflammatory response. The current study demonstrates the beneficial effects of APC in IR-induced ALI. This protective effect is possibly associated with the inhibition of TNF-α, IL-17A, CXCL1, and neutrophils infiltration in lung tissues. However, the current results were obtained in an animal model and it is still necessary to confirm these findings in human subjects. If we can demonstrate the benefits of APC to protect IR lung injury, we can postulate that APC is a potential therapeutic drug for lung preservation. KEYWORDS activated protein C, acute lung injury, ischemia-reperfusion, pro-inflammatory cytokines
IR-induced ALI with alveolar damage and pulmonary edema is an important issue in lung transplantation [2]. Despite advancements in organ preservation and perioperative care, IR-induced ALI remains a significant cause of early mortality and morbidity [2]. IR-induced ALI is also implicated in other clinical conditions, including acute pulmonary embolism after thrombolytic therapy, re-expansion of collapsed lung from pneumothorax or pleural effusion, and cardio-pulmonary bypass [3–5]. Reperfusion injury may also increase inflammation in adult respiratory
INTRODUCTION Exposure of the lungs to periods of ischemia and rapid initiation of reperfusion causes ischemiareperfusion (IR)-induced acute lung injury (ALI)[1]. Received 3 July 2013; accepted 26 September 2013 ∗ These authors contributed equally to this work. Address correspondence to Chin-Pyng Wu, M.D., PhD. Department of Critical Care Medicine, Li-Shin Hospital, #77, Kwang-Tai Road, Ping-jen City, Tao-Yuan County, Taiwan, Republic of China. E-mail: [email protected]
241
Exp Lung Res Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
242
C.-C. Lan et al.
distress syndrome, wherein perfusion of the pulmonary vasculature is often occluded by vasoconstriction, micro-emboli, or in situ thromboses [6]. Mortality from IR-induced ALI remains high even with advanced medical care [7, 8]. Thus, prevention and treatment of IR-induced ALI are important clinical issues. Activated protein C (APC) is an endogenous anticoagulant generated from protein C by the action of the thrombin–thrombomodulin complex on the endothelial cells [9]. It has an important role in coagulation homeostasis through proteolytic cleavage and inactivation of factors Va and VIIIa [9]. It is known to be critical in regulating microvascular coagulation [9] and its action on microcirculation during endotoxemia has been noted [10]. It also has antiinflammatory properties by blocking tumor necrosis factor (TNF), interleukin (IL)-6, and IL-8, and inhibits neutrophils chemotaxis [11, 12]. Therefore, APC manifests multiple effects with antithrombotic, profibrinolytic, and anti-inflammatory activities. Because of its multiple effects, APC may have a potential role in the treatment of complex medical disorders. Sepsis is one of the most challenging medical conditions. Some previous clinical studies have reported that low levels of circulating protein C in patients with sepsis will lead to higher mortality [13]. Administration of APC in sepsis was used in many clinical trials [14, 15]. However, the effect of APC was controversial in clinical trials of Protein C Evaluation in Severe Sepsis (PROWESS) and PROWESS-SHOCK [15]. Nonetheless, more studies on the role of APC in nonseptic inflammatory conditions should be undertaken. Since APC manifests multiple actions and regulates micro-circulation, the present study posits the hypothesis that APC may be beneficial in IR-induced ALI. This study has been conducted to investigate the effects of APC in IR-induced ALI.
lung in situ in the chest were as previously described [16]. After deep anesthesia, tracheostomy was performed and a cannula was inserted into the trachea. The lungs were ventilated with a humidified gas mixture containing 5% CO2 in air at a frequency of 60 cycle/minute, tidal volume of 3 mL, and positive endexpiratory pressure (PEEP) of 1 cm H2 O. Median sternotomy was performed and heparin (1 U/g of body weight) was injected into right ventricle. An afferent silicon catheter was inserted into the pulmonary artery through right ventricle. An efferent catheter was installed into left atrium via left ventricle to collect the effluent perfusate for re-circulation. Pulmonary venous outflow was diverted via the efferent catheter into the reservoir. Pulmonary trunk and aorta were tied. A third ligature was placed at the atrio-ventricular junction to prevent perfusate from entering into left ventricle. Pulmonary arterial pressure (PAP) and venous pressure (PVP) were measured from side-ports in the afferent and efferent catheters. A peristaltic pump (Model 1203, Harvard Apparatus) was used to perfuse the lungs with recirculated perfusate: blood (1:1) mixed with physiologic salt solution (119 mM NaCl, 4.7 mM KCl, 1.17 mM MgSO4 , 22.6 mM NaHCO3 , 1.18 mM KH2 PO4 , 1.6 mM CaCl2 , 5.5 mM glucose, and 50 mM sucrose). Bovine albumin (4 g/dL) was added to maintain osmolarity of the perfusate. The perfusion rate was kept at 8–10 mL/minute by a roller pump and constant temperature (37◦ C) was maintained by a water bath. The preparation was placed on an electronic balance with the isolated lungs remaining in situ. The digital signals of the electronic balance were converted to analog signals by a digital–analog converter and recorded on a polygraph recorder. Weight changes were precalibrated on the electronic balance before preparation of the experiment. In this isolated lung in situ preparation, the changes in body weight reflected the lung weight changes[17].
MATERIALS AND METHODS Isolation and Perfusion of Lungs
Experimental Protocols and Induction of IR-induced ALI
The National Science Council and Animal Review Committee of the National Defense Medical Center (Taipei, Taiwan) approved the study protocol. Animals in this study were cared for in accordance with the “Guide for the Care and Use of Laboratory Animals” published by the United States National Institutes of Health. Male Sprague–Dawley rats weighing 301.0 ± 31.4 g were anesthetized through intraperitoneal injection of pentobarbital sodium (50 mg/kg). Procedures regarding the preparation of isolated-perfused
The animals were divided into three groups (n = 6 per group): control group (without IR), IR group, and IR with APC (IR+APC) group. The IR-induced ALI was performed with 30 minutes of ischemia by stopping ventilation and perfusion [18]. After ischemia, the lungs were reperfused for 90 minutes and ventilated with 5% CO2 + 95% air. The temperature was monitored and kept at 37◦ C during IR and the whole course of experiment. APC (100 μg/kg) was administered via perfusate after ischemia. Lung weight, airway pressure, PAP, and PVP were continExperimental Lung Research
Activated Protein C in Ischemia-Reperfusion ALI
uous monitored during experiment. Microvascular permeability (Kf) was measured at the baseline and post-IR. All of the rats were further studied for lung histopathology, lung wet/dry weight ratio (W/D), total protein concentration, and cytokine levels in broncho-alveolar lavage fluid (BALF). The difference in pressures (between PAP and PVP) divided by the perfusate flow rate measured pulmonary vascular resistance (PVR) [19]. Lung compliance was calculated by tidal volume divided by pressure difference between peak airway pressure and PEEP[19].
Exp Lung Res Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
Microvascular Permeability The measurement of Kf in isolated lungs was as previously described [16]. Kf as an index of microvascular permeability was calculated from the increase in lung weight produced by an elevation in PVP. During the experiment, PVP was elevated rapidly by 10 cm H2 O for 7 minutes to measure Kf. The hydrostatic challenge elicited a biphasic increase in lung weight with an initial rapid component followed by a slow and steady component. The slow, the steady phase of weight gain as a function of time (W/T) was plotted on a semi-logarithmic paper and then extrapolated to zero time to obtain the initial rate of transcapillary filtration. From this plot, Kf was defined as the y-intercept (gm/minute) divides by PVP (10 cm H2 O) and lung weight, and expressed in whole units of grams per minute per centimeter of H2 O multiplied by 100 g [16].
Total Protein Concentration and Cytokine Levels in BALF At the end of the experiment, the left lungs were lavaged twice with 2.5 mL isotonic saline and the returned fluid (BALF) was collected by free drainage. The BALF was centrifuged at 200×g for 10 minutes, and total protein concentration in the supernatant was determined using bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). TNF-α, C-X-C motif ligand 1 (CXCL1) (R&D Systems Inc., Minneapolis, MN, USA) and IL-17A (eBioscience, San Diego, CA, USA) in BALF were measured using an ELISA kit according to the manufacturer’s protocol.
243
for 48 hours [16]. The wet and dry weights were then measured to calculate lung W/D.
Lung Histopathology Part of the right lower lung lobe was taken for histologic examination. The tissues were immersed in 10% formaldehyde fixative for 24 hours, then embedded in paraffin wax and cut into 4–6 μm thick sections using a microtome. Histopathologic examination with hematoxylin and eosin (H&E)-stained was performed to verify the micro-anatomic features of lung injury imposed by IR and to assess the effects of APC. For tissue neutrophils quantification, H&E-stained sections were used to count the number of neutrophils per high power field (HPF). For each slide, neutrophils were counted in a blinded fashion on 10 sequential, nonoverlapping HPFs (magnification, 400×) of lung parenchyma beginning at the periphery of the section [20].
Data Analysis All statistical analyses were performed using the SPSS software 18.0 (SPSS Inc., Chicago, IL, USA). All values were reported as means ± SD (standard deviation). Statistical comparisons among groups were performed using one-way ANOVA and Bonferroni post hoc multiple comparison test. A P < .05 was considered statistically significant.
RESULTS APC Decreased Pulmonary Edema in IR-induced ALI The lung weight was significantly higher in IR group than in control group (P < .001; Figure 1A), whereas the lung weight of IR+APC group was significantly lower than that of IR group (P < .001). Lung W/D was significantly increased after IR (Figure 1B) and significantly decreased in IR+APC group (IR vs. control, P < .001; IR vs. IR+APC, P < .001).
APC Improved Lung Compliance in IR-induced ALI Pulmonary Edema Lung W/D was used as an indicator of pulmonary edema. After the experiment, part of the right middle lobe was weighed and then dried in an oven at 60◦ C C
2015 Informa Healthcare USA, Inc.
The lung compliance was significantly lower in IR group than in control group (P < .001) (Figure 1C), whereas the lung compliance of IR+APC group was significantly higher than that of IR group (P < .001).
Exp Lung Res Downloaded from informahealthcare.com by Nyu Medical Center on 06/18/15 For personal use only.
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C.-C. Lan et al.
FIGURE 1. APC decreased pulmonary edema in I/R-induced ALI. The (A) lung weight was significantly higher in IR group than in control group (P < .001), whereas the lung weight of IR+APC group was significantly lower than that of IR group (P < .001). Lung (B) W/D was significantly increased after IR and decreased in IR+APC group (IR vs. control, P < .001; IR vs. IR+APC, P < .001). (C) The lung compliance was significantly lower after IR and higher in IR+APC group (IR vs. control, P < .001; IR vs. IR+APC, P < .001). (D) The PVR was significantly higher after IR and decreased in IR+APC group (IR vs. control, P = .020; IR vs. IR+APC, P = .038). Note. There was significant difference from the ∗ control (P < .05) and # IR (P < .05) groups. APC, activated protein C; IR, ischemia-reperfusion; ALI, acute lung injury; PVR: pulmonary vascular resistance.
APC Decreased Pulmonary Vascular Resistance in IR-induced ALI The PVR was higher in IR group than in control group (P = .02) (Figure 1D), whereas the PVR of IR+APC group was significantly lower than that of IR group (P = .038).
ference (P = .219). The Kf changes (%) were significantly higher in IR (238.6%±106.9%) than control (7.5%±5.6%) and IR+APC (93.1%±43.4%) groups (IR vs. control, P < .001; IR vs. IR+APC, P = .005) (Figure 2C). When comparing between control and IR+APC groups, the Kf changes were without significant difference (P = .126).
APC Attenuated I/R-induced ALI with Microvascular Hyperpermeability
APC Decreased Total Protein Concentration and Cytokines in BALF
The Kf at baseline was similar between the three groups (IR vs. control, p = 1.0; IR vs. IR+APC, p = 1.0 at basline) (Figure 2A). After IR, the Kf was significantly increased and was attenuated by APC (Figure 2B) (IR vs. control, P < .001; IR vs. IR+APC, P < .012). When comparing between control and IR+APC groups, the Kf was without significant dif-
Total protein concentration in BALF was significantly increased after IR (IR vs. control, P < .001) (Figure 3A). However, APC decreased the total protein concentration after IR (IR vs. IR+APC, P < .001). TNF-α was significantly higher in IR than in control group, and decreased in the IR+APC group (IR vs. control, P = .005; IR vs. IR+APC, P
