SHOCK, Vol. 43, No. 4, pp. 395Y404, 2015
ANGIOTENSIN-CONVERTING ENZYME INHIBITION ATTENUATES LIPOPOLYSACCHARIDE-INDUCED LUNG INJURY BY REGULATING THE BALANCE BETWEEN ANGIOTENSIN-CONVERTING ENZYME AND ANGIOTENSIN-CONVERTING ENZYME 2 AND INHIBITING MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION Yingchuan Li, Zhen Zeng, Yachun Li, Weifeng Huang, Ming Zhou, Xiaoli Zhang, and Wei Jiang Department of Anesthesiology, The Sixth People’s Hospital Affiliated to Shanghai Jiaotong University, Shanghai, China Received 3 Sep 2014; first review completed 19 Sep 2014; accepted in final form 7 Nov 2014 ABSTRACT—Activation of the renin-angiotensin system (angiotensin-converting enzyme [ACE]/angiotensin II [Ang II] and angiotensin-converting enzyme 2 [ACE2]/Ang-1Y7) has been implicated in the pathophysiology of inflammatory response and acute lung injury (ALI). Previous studies have shown that the ACE inhibitor captopril (Cap) may be a potent therapeutic drug for ALI. However, the mechanisms of its protective effects on ALI are still largely unknown. In this study, we evaluated the effects of Cap on preventing lipopolysaccharide (LPS)Yinduced lung injury and further investigated the underlying mechanisms of these protective effects. Rats were intraperitoneally pretreated with Cap (50 mg/kg) 30 min prior to an intravenous administration of LPS (7.5 mg/kg). Furthermore, following a 30-min pretreatment with Cap (10j5 mol/mL) or combined with the ACE2 inhibitor MLN4760 (10j7 mol/mL), rat pulmonary microvascular endothelial cells (PMVECs) were stimulated with LPS (1 mg/mL). Captopril pretreatment significantly attenuated LPS-induced pathophysiological changes in the lung, inhibited secretion of tumor necrosis factor ! and interleukin 6, reduced the ratio of Ang II to Ang-1Y7, and reversed the increased ratio of ACE to ACE2, which was remarkably decreased from 7.07 (LPS only) to 1.71 (LPS + Cap). The protective effects of Cap on ALI were also confirmed by in vitro studies, in which Cap suppressed LPS-induced secretion of proinflammatory cytokines and modulated the expression levels of ACE and ACE2. After Cap pretreatment, the ratio of ACE to ACE2 expression was remarkably decreased from 5.18 (LPS alone) to 1.52 (LPS + Cap). Furthermore, Cap given before LPS administration led to inhibition of p38 mitogen-activated protein kinase (MAPK), ERK (extracellular signal-regulated kinase) 1/2, and JNK (c-Jun N-terminal kinase) phosphorylation in PMVECs, whereas MLN4760 abolished the protective effects of Cap on LPS-induced secretion of proinflammatory cytokines and abolished Cap-induced blockade of p38MAPK, ERK1/2, and JNK phosphorylation. Our findings reveal that Cap exerts protective effects on LPS-induced lung injury and the cytotoxicity of PMVECs, and these effects may, at least in part, regulate the balance of ACE and ACE2 expression and inhibit the activation of MAPKs. KEYWORDS—Acute lung injury, lipopolysaccharide, angiotensin-converting enzyme, captopril, mitogen-activated protein kinases ABBREVIATIONS—RAS V renin-angiotensin system; ACE V angiotensin-converting enzyme; ACE2 V angiotensinconverting enzyme 2; ACEI V angiotensin-converting enzyme inhibitor; ALI V acute lung injury; ARDS V acute respiratory distress syndrome; LPS V lipopolysaccharide; IL-6 V interleukin 6; TNF-! V tumor necrosis factor !; PMVECs V pulmonary microvascular endothelial cells; Ang I V angiotensin I; Ang II V angiotensin II; AT-1 V Ang II type 1 receptor; SARS V severe acute respiratory syndrome; BALF V bronchoalveolar lavage fluid; Cap V captopril; MAPKs V mitogen-activated protein kinases; ERK V extracellular signal-regulated kinase; JNK V c-Jun N-terminal kinase
INTRODUCTION
public health issue as each year more than 1 million patients suffer from ARDS (1). Acute respiratory distress syndrome is commonly associated with systemic problems such as severe infections, sepsis, major trauma or burns, and acute pancreatitis, leading to indirect damage to the lung. Among all these contributing factors, sepsis is one of the most common causes of ARDS. It has been reported that approximately 25% of ARDS cases are induced by severe sepsis (2, 3). Although the therapeutic technology has been greatly improved in the past two decades, ARDS still remains an important cause of death in intensive care units, with a mortality rate of 40% to 60%, particularly among elderly patients with sepsis and a prior comorbidity (4, 5). At present, the available methods for ARDS treatments include supportive therapy, such as lung protective mechanical ventilation and lung recruitment with a high level of positive end-expiratory pressure. Specific and effective pharmacological therapy for ARDS that can attenuate
Acute respiratory distress syndrome (ARDS) is a critical illness syndrome characterized by acute respiratory insufficiency and severe hypoxemia. It has become a worldwide
Address reprint requests to Wei Jiang, PhD, Department of Anesthesiology, The Sixth People_s Hospital Affiliated to Shanghai Jiaotong University, No. 600, Yishan Rd, Xuhui District, Shanghai 200233, China. E-mail: [email protected]. This work was supported by the National Natural Science Foundation of China (no. 81272145). Authors_ Contributions: Yingchuan L. carried out the experiments and drafted the manuscript. Z.Z. carried out the immunoassays. Yachun L. and W.H. performed histological analysis. M.Z. and X.Z. participated in the design of the study and performed the statistical analysis. W.J. conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript. The authors declare that they have no competing interests. DOI: 10.1097/SHK.0000000000000302 Copyright Ó 2015 by the Shock Society
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lung injury and improve lung repair has not been clinically applied (6, 7). Recently, the role of the renin-angiotensin system (RAS) in ARDS has been well investigated in both clinical and experimental studies (8, 9). Angiotensin-converting enzyme (ACE), a key enzyme of the RAS, converts angiotensin I (Ang I) by cleavage to angiotensin II (Ang II). Angiotensin II, in terms of binding to the Ang II type 1 receptor (AT-1), induces activation of an intracellular signaling cascade, leading to upregulation of proinflammatory genes and a subsequent inflammatory response (10). In an animal study, the Ang II level of the lung was increased in response to lipopolysaccharide (LPS)Yinduced lung injury, but the increased Ang II ultimately exacerbated lung endothelium injury. Nevertheless, these Ang IIYassociated changes can be eliminated by selective blockage of AT-1 (11). In addition, the level of ACE has been evaluated in bronchoalveolar lavage fluid (BALF) of ARDS patients, particularly those with infectious causes of lung injury (12). The number of ARDS patients with the DD genotype and greater ACE activity is elevated, and this mutation is significantly associated with mortality; conversely, the ACE I/I allele has been found to be correlated with an increased survival rate (13, 14). Moreover, treatment to block the Ang II receptor or inhibit ACE activity had a similar inhibitory effect on the inflammatory response, and lung injury occurred during mechanical ventilation (15). Angiotensin-converting enzyme 2 (ACE2) is a homolog of ACE and shares a similar sequence in the catalytic domains with ACE (16). However, ACE2 and ACE appear to act on different catalytic substrates and generate different enzymatic products. Angiotensin-converting enzyme converts Ang I into Ang II, whereas ACE2 cleaves a single residue from Ang II to yield Ang-1Y7 (17). Angiotensin-converting enzyme 2 has been identified as a key receptor in severe acute respiratory syndrome (SARS) caused by SARS-CoV infections (18). Angiotensin-converting enzyme 2Yknockout mice with ARDS had more severe lung injury than did wild-type mice (19). Treatment with an injection of catalytically active recombinant ACE2 protein effectively improved the symptoms and attenuated arterial hypoxemia in a piglet model of LPS-induced lung injury (20). Angiotensin-converting enzyme inhibitor (ACEI) has been widely used as a clinically useful treatment for hypertension and cardiac disease for a long time. Angiotensin-converting enzyme inhibitor is also considered as a specific anti-inflammatory therapeutic agent that can attenuate either lung injury caused by LPS or ventilator-induced lung injury (15, 21Y23). In addition, an imbalance of ACE/ACE2 expression was found in BALF collected from ventilated LPS-exposed animals, in which ACE activity was enhanced, but ACE2 activity was reduced (24). However, it is still not clear whether the protective effect of ACEI in lung injury correlates with the balance of pulmonary ACE and ACE2 expression in ARDS; moreover, the mechanisms of this protective effect are still largely unknown. Therefore, in the present study, we tested the hypothesis that the ACEI captopril (Cap) has direct protective effects in experimental models of ARDS by regulating the balance of pulmonary ACE and ACE2 expression and inhibiting mitogen-activated protein kinase (MAPK) activation.
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METHODS Drugs Lipopolysaccharide(Escherichia coli, O127:B8) and Cap were purchased from Sigma (St Louis, Mo). The ACE2 inhibitor MLN-4760 was a product from Millennium Pharmaceuticals (Boston, Mass). The ACE rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, Calif). The ACE2 rabbit monoclonal antibody was purchased from Abcam (Cambridge, Mass). Rabbit anti-p38MAPK, antiYphospho-p38MAPK, antiY extracellular signal-regulated kinase 1/2 (ERK1/2), antiYphospho-ERK1/2, antiYc-Jun N-terminal kinase (JNK), and antiYphospho-JNK were all purchased from Cell Signaling Technology (Danvers, Mass). All enzymelinked immunosorbent assay (ELISA) kits for Ang II, Ang-1Y7, tumor necrosis factor ! (TNF-!), and IL-6 were products of BioSource International (Carlsbad, Calif).
In vivo studies Animals—All experiments were performed according to the protocol approved by the Ethics Committee of Animal Research at the College of Medicine, Shanghai Jiaotong University (Shanghai, China). A total of 48 male Sprague-Dawley rats weighing 200 to 250 g were obtained from the Laboratory Animal Science Department of Fudan University (Shanghai, China). Animals had access to food and drinking water ad libitum. Experimental protocols—The animal model of LPS-induced acute lung injury (ALI) was developed with some modifications as described by Hagiwara et al. (21). Briefly, the animals were randomly divided into the following groups (n = 12 for each group): (a) control group: receiving intraperitoneal administration of 0.9% NaCl solution 30 min prior to an injection of 0.9% NaCl solution into the tail vein; (b) Cap group: receiving intraperitoneal administration of Cap (50 mg/kg) 30 min prior to an intravenous injection of 0.9% NaCl solution into the tail vein; (c) LPS group: receiving intraperitoneal administration of 0.9% NaCl solution 30 min prior to intravenous injection of LPS (7.5 mg/kg); and (d) LPS + Cap group: receiving intraperitoneal administration of Cap (50 mg/kg) 30 min prior to an intravenous injection of LPS (7.5 mg/kg) into the tail vein. Eight hours after LPS administration, animals were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg) and killed by exsanguinations. Blood samples were centrifuged (3,200g at 4-C for 15 min), and collected serum was stored in aliquots at j80-C. Meanwhile, the lung was dissected for immunostaining. All experimental animals were breathing spontaneously. Lung wet-to-dry weight ratio—To assess tissue edema, the weight of rat lungs (six lungs per group) was measured, followed by a drying step of the lungs in an oven at 80-C for 48 h until the weight of samples became constant. Then, the lung wet-to-dry weight ratio was calculated. Histopathologic and immunohistochemical analysis—To determine the lung injury score, the left lung was fixed immediately in 4% paraformaldehyde, followed by serial dehydration in graded alcohol solutions, then embedded in paraffin, and cut into 4-2m-thick sections. The tissue sections of samples were stained with hematoxylin-eosin for microscopic observation. The degree of lung injury was evaluated by a double-blind examination by a pathologist and was semiquantitatively scored according to Murakami and colleagues_ (25) technique, by which 10 fields of lung parenchyma were graded on a scale of 0 to 4 (0, absent, appears normal; 1, light; 2, moderate; 3, strong; 4, intense) for edema, inflammation, and hemorrhage. A mean score for each of the parameters was then calculated. The final lung injury score was obtained by averaging the score from the animals within each group. The lung tissue sections were pretreated with heat-induced antigen retrieval for optimal immunostaining and then probed with polyclonal rabbit anti-ACE antibody (dilution 1:100) and monoclonal rabbit anti-ACE2 antibody (dilution 1:200). Finally, the sections were incubated with peroxidase-conjugated goatYantiYrabbit immunoglobulin G and visualized by the streptavidinbiotin-peroxidase complex system using a SABC kit (Boster, Wuhan, China). As negative controls, the primary antibodies were replaced with irrelevant immunoglobulins. Western blot—Tissue specimens (approximately 100 mg each) were obtained from the right lung and stored at j80-C. The frozen lung tissue was homogenized in cold RIPA lysis buffer. The prepared lysates were placed on ice for 20 min and then centrifuged at 14,000g for 10 min at 4-C. The supernatant fraction was collected for quantification of protein concentration, and equal amounts (50 2g) of protein extracts were then separated using sodium dodecyl sulfateYpolyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes by using a semidry blot system (Bio-Rad Laboratories, Mississauga, Ontario, Canada). The membranes were blocked with 5% (wt/vol) skim milk in Tris-buffered saline and 0.1% Tween-20 (TBST) for 2 h at room temperature, then incubated with either 1:1,000 rabbit anti-ACE polyclonal antibody or 1:500 rabbit anti-ACE2 monoclonal
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FIG. 1. Effect of Cap treatment on lung injury in LPS-exposed rats. The histological changes of lung injury were examined by hematoxylin-eosin staining of lung sections from animals in the control (A: original magnification 20), Cap (B: original magnification 20), LPS (C: original magnification 20), and LPS + Cap (D: original magnification 20) groups (n = 6), respectively.
antibody at 4-C overnight. After washing three times with TBST, the membranes were incubated with horseradish peroxidaseYconjugated secondary antibody (1:5,000) for 2 h at room temperature. Hybridizing signals were visualized by using chemiluminescence reagents. Densitometric values were quantified using an image-analysis software program (Quantity One software; Bio-Rad Laboratories), and the signal of ACE or ACE2 was normalized to "-actin. Measurement of secreted cytokines, Ang II, and Ang-1Y7—The serum levels of interleukin 6 (IL-6), TNF-!, Ang II, and Ang-1Y7 were assayed using an ELISA. The 96-well plates were coated with monoclonal antibody specific to IL-6, TNF-!, Ang II, and Ang-1Y7 (BioSource International), respectively. The measurement of these inflammatory mediators was performed according to the procedure described by the manufacturer. The plate was read at an absorbance of 450 nm using a microplate reader (Bio-Rad Laboratories). The serum samples were tested twice each.
medium was collected at 8 h after exposure to LPS, and the supernatant was used for measurements. Measurement of secreted cytokines Ang II and Ang 1Y7—The concentrations of IL-6, TNF-!, Ang II, and Ang-1Y7 secretion in the culture supernatant were measured using the ELISA sandwich method. The procedure was similar to that used to measure the serum levels of cytokines and Ang II in the in vivo study. Western blot—Pulmonary microvascular endothelial cells were digested with 0.25% trypsin-EDTA. The harvested cells were washed with ice-cold phosphate-buffered saline and then resuspended in 0.3 mL of ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% Triton X-100; 1 mM phenylmethylsulfonyl fluoride; 0.02% sodium azide; and 1 g/mL aprotinin). After 30 min on ice, the cell lysates were centrifuged at 12,000g for 10 min at 4-C to collect the supernatant for the Western blot assay. The procedure for the Western blot assays to detect ACE, ACE2 expression, and the phosphorylation of p38MAPK, ERK1/2, and JNK in the cell lysates was similar to that described for the tissue samples in the in vivo study.
In vitro studies Cell culture—Isolation and culture of pulmonary microvascular endothelial cells (PMVECs) were performed according to a modified method as described by Cheng et al. (26). Each experimental animal was killed after receiving anesthesia with an intraperitoneal injection of pentobarbital sodium. Then, a thoracotomy was performed, in which the lung circulatory system was perfused with an injection of 50 mL of ice-cold phosphate-buffered saline via the right ventricle, and then the lungs were removed and washed with 30 mL of ice-cold serum-free Dulbecco modified Eagle medium. The pleura was carefully cut away and discarded, from which a 1-mm-thick section was cut from the outer edge of the remaining lung tissue and trimmed into small pieces. These pieces of lung tissue were then inserted into the glass pellets (Costar, Cambridge, Mass) and rinsed in endothelial cell medium (Sciencell Research, Carlsbad, Calif) containing 20% fetal calf serum, 100 U/mL penicillin/ streptomycin, and 2.5 g/mL amphotericin B. The lung tissues in the glass pellets were removed after incubation for 60 h at 37-C (Thermo, Waltham, Mass) in humidified air containing 5% CO2. Thereafter, the cell culture medium in the glass pellets was replaced every 3 days. As the growing cells reached 80% to 90% confluence, the cells were split with 0.25% trypsin-EDTA (Gibco, Grand Island, NY) and then subcultured at a 1:2 split ratio. The cultured cells were identified as endothelial cells by their cobblestone morphology as well as immunostaining with antiYhuman factor VIIIYrelated antigen, CD31 (Boster), and FITC-BSI (Santa Cruz Biotechnology). The third to fifth generations of cultured cells were used for the following experiment. Cell viability assay—The viability of PMVECs was determined by using CCK-8 kits (Dojido Molecular Technologies, Japan), according to the manufacturer_s instructions. Prepared PMVECs were plated in 96-well microplates and incubated with serum-free Dulbecco modified Eagle medium for 24 h. Then, the cells were pretreated or untreated with 10j5 M Cap for 30 min prior to exposure to 1 mg/mL LPS for 8 h. To examine cell viability, 10 2L of CCK-8 reagent was added into each well of the 96-well plate. After incubation for 4 h, the absorbance of the plate was read at 492 nm using a microplate reader. Experimental groups—The treatments were divided into four groups, including control group, without any drug treatment; Cap group, treated with Cap (10j5 mol/mL) only; LPS group, stimulated with LPS (1 mg/mL) alone; and LPS + Cap group, pretreated with Cap (10j5 mol/mL) for 30 min prior to LPS stimulation (1 mg/mL); and LPS + Cap + MLN4760 group, pretreated with Cap (10j5 mol/mL) and ACE2 inhibitor MLN4760 (10j7 mol/mL) for 30 min prior to LPS stimulation (1 mg/mL). The therapeutic effects of Cap were also detected in two extra groups, including (a) the LPS + Cap (after) group, which was treated with Cap (10j5 mol/mL) after LPS stimulation (1 mg/mL) for 30 min; and (b) LPS + Cap + MLN4760 (after) group, which was treated with Cap (10j5 mol/mL) and the ACE2 inhibitor MLN4760 (10j7 mol/mL) after LPS stimulation (1 mg/mL) for 30 min. The cell culture
Statistical analysis All data are presented as mean T SD. To determine the difference between two independent groups, data were analyzed by using one-way analysis of variance and Duncan multiple-range test with SPSS 13.0 software (SPSS Inc, Chicago, Ill). P G 0.05 was accepted as statistically significant.
RESULTS In vivo studies
Effect of Cap on lung injury—Signs of lung injury in the lung tissues from the LPS group were observed, including a marked increase in inflammatory cell infiltration, edema, and hemorrhage in the interstitium and alveolus (Fig. 1B). But, LPS-induced lung edema, hemorrhage, and inflammatory cell infiltration were significantly attenuated by pretreatment with Cap (Fig. 1D). Similarly, the lung injury scores were compatible with the histological changes in the same treatment group. The results
FIG. 2. Lung injury scores of the different treatment groups. The lung injury scores indicating histological changes, including edema, inflammation, and hemorrhage, were calculated in the following groups: control (black bars), Cap (gray bars), LPS (white bars), and LPS + Cap (hatched bars). All data are expressed as mean T SD (n = 6). *P G 0.05 versus the control group, #P G 0.05 versus the LPS group.
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FIG. 3. Effect of Cap on the lung wet-to-dry weight ratio in LPSexposed rats. A comparison was performed among the different treatment groups: control (black bar), Cap (gray bar), LPS (white bar), and LPS + Cap (hatched bar). All data are expressed as mean T SD (n = 6). *P G 0.05 versus the control group, #P G 0.05 versus the LPS group.
showed that the score was significantly greater in the LPS group than that in the control group. Pretreatment with Cap in the LPS + Cap group markedly reduced the lung injury scores in comparison with the LPS group (Fig. 2). In addition, the wet-to-dry weight ratio of the lungs in the LPS + Cap group was significantly less than that in the LPS group (Fig. 3). Effect of Cap on the serum levels of Ang II, Ang-1Y7, IL-6, and TNF-!—In this study, we found that the serum levels of Ang II, IL-6, and TNF-! in the LPS group were significantly greater than those in the control group. Meanwhile, pretreatment with Cap significantly suppressed the secreted Ang II, IL-6, and TNF-!, compared with the serum levels of
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those factors between the Cap + LPS group and the LPS group (Fig. 4, A, D, and E). The level of Ang-1Y7 in the LPS group was also markedly higher than that of the control group, whereas it was also significantly suppressed by pretreatment with Cap (Fig. 4B). However, the ratio of Ang II to Ang-1Y7 was obviously decreased from 1.64 in the LPS group to 0.99 in the LPS + Cap group (Fig. 4C). Effect of Cap on the expression of pulmonary ACE and ACE2—In the lung tissues from the LPS group, the immunohistochemical (IHC) staining analysis results showed that there was a remarkable increase in ACE-positive staining but a decrease in ACE2-positive staining, as compared with the control group (ACE comparison between Fig. 5C and Fig. 5A; ACE2 comparison between Fig. 6C and Fig. 6A). In contrast, Cap treatment significantly suppressed the ACE expression but enhanced ACE2 expression in the LPS + Cap group, compared with the LPS group (ACE: comparison between Fig.5D and Fig. 5C; ACE2: comparison between Fig. 6D and Fig. 6C). Consistent with the IHC staining analysis results, densitometric Western blotting data showed that ACE expression was significantly increased in lung tissues from the LPS group, but the ACE2 level was decreased in the same tissue sample, whereas in lung tissue samples from the LPS + Cap group, the ACE level was dramatically decreased, but ACE2 expression was enhanced (Fig. 7, A and B). In addition, the expression level of pulmonary ACE2 was also notably increased in the Cap group as compared with the control group (P G 0.05, Fig. 7B). The expression of pulmonary ACE in the LPS-exposed animals was increased by 1.43-fold, as compared with the unexposed
FIG. 4. Effect of Cap on the serum levels of Ang II, Ang-1Y7, IL-6, and TNF-! in LPS-exposed rats. The serum levels of (A) Ang II and (B) Ang-1Y7, (C) the ratio of Ang II to Ang-1Y7, and serum levels of (D) TNF-! and (E) IL-6 were measured in the different treatment groups: control (black bar), Cap (gray bar), LPS (white bar), and LPS + Cap (hatched bar). All data are expressed as mean T SD (n = 6). *P G 0.05 as compared with the control group, #P G 0.05 as compared with the LPS group.
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FIG. 5. Effect of Cap on the expression of pulmonary ACE in LPS-exposed rats. The expression of ACE in lung tissues was examined by IHC staining methods. The lung specimens were obtained from the control group (A), Cap group (B), LPS group (C), and LPS + Cap group (D), n = 6 in each group. The results were observed under a microscope (original magnifications 20, 40 inset).
ones, but the expression of pulmonary ACE2 in the same animal was decreased by 1.55-fold, leading to a change of the ACE/ ACE2 ratio from 3.17 to 7.07, whereas the ratio of ACE to ACE2 was greatly reduced with Cap pretreatment prior to LPS stimulation, showing a change in the ratio from 7.07 in the LPS group to 1.71 in the LPS + Cap group. In vitro studies
Effect of Cap and ACE2 inhibitor on LPS-induced cytotoxicity—As shown in Figure 8, pretreatment with Cap (10j5 mol/mL) significantly attenuated LPS-induced cytotoxicity in PMVECs, but this protective effect of Cap was abolished by the ACE2 inhibitor MLN-4760. On the other hand, Cap treatment after LPS exposure slightly improved the cell viability, but the change was not statistically significant compared with the LPS group. Effect of Cap and ACE2 inhibitor on secreted cytokines, Ang II, and Ang-1Y7—There was a significant increase in the levels of Ang II and Ang-1Y7 in the supernatant of cultured PMVECs that were exposed to LPS. Lipopolysaccharide-induced Ang II and Ang-1Y7 secretion was significantly inhibited by pretreatment with Cap. The ratio of Ang II to Ang-1Y7 was markedly decreased from 12.67 in the LPS group to 5.42 in the LPS + Cap group. Pretreatment with MLN-4760 obviously abolished the inhibitory effect of Cap on LPS-induced Ang II secretion, but the level of Ang-1Y7 was more obviously decreased in the Cap + MLN-4760+LPS group, as compared with the LPS + Cap group. The ratio of Ang II to Ang-1Y7 was markedly increased from 5.42 in the LPS + Cap group to 27.77 in the LPS + Cap + MLN4760 group (Fig. 9, AYC). Similarly, the IL-6 and TNF-! levels in the supernatant were also elevated after exposure to LPS, but LPS-induced secretion of IL-6 and TNF-! was significantly inhibited by pretreatment with Cap.
Pretreatment with MLN-4760 obviously abolished the inhibitory effect of Cap on LPS-induced IL-6 and TNF-! secretion (Fig. 9, D and E). In addition, Cap treatment after LPS exposure exerted similar effects on the secretion of cytokines, Ang II, and Ang-1Y7 as pretreatment with Cap. MLN-4760 administration also caused a remarkable increase in the Ang II/Ang-1Y7 ratio from 5.74 (LPS + Cap [after]) to 26.56 (LPS + Cap + MLN4760 [after]) (Fig. 9). Effect of Cap on the expression of ACE and ACE2 in PMVECs—The Western blot results showed that after LPS stimulation, the expression level of ACE was significantly increased in PMVECs, but pretreatment with Cap effectively abolished LPS-induced ACE expression. In contrast, the expression of ACE2 in PMVECs exposed to LPS was significantly decreased. Thus, Cap pretreatment could restore the expression level of ACE2 (Fig. 10, A and B). Furthermore, a single treatment with Cap also caused a significant decrease in the expression level of ACE, as compared with the control group (Fig. 10A). Our results revealed a 1.18-fold increase in the ACE expression in LPS-exposed cells, compared with the unexposed control, whereas in the same exposed cells, ACE2 expression was decreased by 2.12-fold, leading to a change of the ACE/ACE2 ratio from 2.39 (control) to 5.18 (LPS group). Conversely, pretreatment with Cap markedly changed the ACE/ACE2 ratio from 5.18 (LPS group) to 1.52 (LPS + Cap group). Effects of Cap and ACE2 inhibitor on the phosphorylation of p38MAPK, ERK1/2, and JNK—The Western blot analysis showed that the phosphorylation levels of ERK1/2, p38MAPK, and JNK in normal PMVECs that were exposed to LPS for 8 h (LPS group) were significantly higher than those in the untreated control group. Compared with the LPS group, pretreatment with Cap 30 min before LPS exposure could
FIG. 6. Effect of Cap on the expression of pulmonary ACE2 in LPS-exposed rats. The expression of ACE2 in lung tissues was examined by IHC staining methods. The lung specimens were obtained from the control group (A), Cap group (B), LPS group (C), and LPS + Cap group (D), n = 6 in each group. The results were observed under a microscope (original magnifications 20, 40 inset).
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FIG. 7. Evaluation of the expression levels of pulmonary ACE (A) and ACE2 (B) in the different treatment groups. The expression levels of pulmonary ACE (A) and ACE2 (B) were examined by Western blot assays of the following groups: control (black bar), Cap (gray bar), LPS (white bar), and LPS + Cap (hatched bar). All data are expressed as mean T SD (n = 6). *P G 0.05 as compared with the control group, #P G 0.05 as compared with the LPS group.
suppress the phosphorylation of p38MAPK, ERK1/2, and JNK. In contrast, compared with the LPS + Cap group, combined pretreatment with MLN-4760 and Cap (inhibition of both ACE and ACE2) 30 min before exposure to LPS (LPS + Cap + MLN-4760 group) obviously reversed the Cap-mediated blockade of p38MAPK, ERK1/2, and JNK activation (Fig. 11, AYC). DISCUSSION We found that in a rat model of LPS-induced lung injury, ACE expression in lung tissue was remarkably increased, but ACE2 expression was decreased, with high levels of cytokines and Ang II in the serum, whereas pretreatment with Cap could abolish LPS-mediated aberrant expression of ACE and ACE2 in lung tissue, decrease lung injury scores, and attenuate the inflammatory response, as reflected by reduced serum levels of cytokines and Ang II. Similar results were also found in the in vitro experiments. Captopril significantly attenuated LPSinduced cytotoxicity in PMVECs, which correlated with its inhibitory effects on LPS-mediated aberrant expression of ACE and ACE2 as well as the secretion of cytokines and Ang II. Meanwhile, ACE2 inhibitor administration abolished the protective effects of Cap on LPS-induced secretion of proinflammatory cytokines and cell cytotoxicity. These data indicate that an imbalance of ACE and ACE2 expression plays a pivotal role in LPS-induced lung injury. Sepsis is the major clinical pathogenic cause of ARDS in intensive care units (27). Endotoxin, i.e., bacterial LPS, released from gram-negative bacterial cell walls, is considered as an important eliciting factor for the development of sepsis. In animal studies, LPS stimulation can mimic the main pathological features of ARDS in human patients (28). Consistent with previous studies (21), we found that the LPS-induced lung injury was associated with increased levels of TNF-! and IL-6 in a rat model. Similarly, in the in vitro experiments, LPS stimulation could promote elevated secretion of cytokines in PMVECs that had emerging damage caused by LPS. According to these
observations, it was speculated that PMVECs might be the target cells in LPS-induced lung injury. Angiotensin II is essential for proinflammation via a mechanism involving the release of cytokines, chemokines, adhesion molecules, growth factors, and reactive oxygen species via AT-1 receptors (10, 29). Several reports also have implicated the active role of Ang II in LPS-induced lung inflammation and injury through its binding to the AT-1 receptor of the lung microvascular endothelium (11, 21). Treatment with an AT-1 inhibitor prevents sepsis-induced ALI and attenuates the secretion of TNF-!, IL-6, and IL-1 (30). In agreement with other studies (21, 22), our study demonstrated that the level of Ang II was substantially increased in LPS-induced lung injury, with elevated TNF-! and IL-6 levels. Secreted cytokines
FIG. 8. Effects of Cap and ACE2 inhibitor on LPS-induced cytotoxicity. Cell viability of PMVECs was evaluated as the spectrophotometric value obtained from the microplate reader for the different treatment groups: control (black bar), Cap (gray bar), LPS (white bar), LPS + Cap (hatched bar), LPS + Cap + MLN-4760 (dotted bar), LPS + Cap (after) (checkered bar), and LPS + Cap + MLN-4760 (after) (vertical lines bar). All data are expressed as mean T SD (n = 6). *P G 0.05 as compared with the control group, #P G 0.05 as compared with the LPS group.
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FIG. 9. Effects of Cap and ACE2 inhibitor on secreted cytokines, Ang II, and Ang-1Y7 in the supernatant of cultured PMVECs. The measurement for levels of (A) Ang II and (B) Ang-1Y7, (C) the ratio of Ang II to Ang-1Y7, and levels of (D) TNF-! and (E) IL-6 was performed in the following groups: control (black bar), Cap (gray bar), LPS (white bar), LPS + Cap (hatched bar), LPS + Cap + MLN-4760 (dotted bar), LPS + Cap (after) (checkered bar), and LPS + Cap + MLN4760 (after) (vertical lines bar). All data are expressed as mean T SD (n = 6). *P G 0.05 as compared with the control group, #P G 0.05 as compared with the LPS group.
FIG. 10. Effect of Cap on the expression of ACE and ACE2 in LPS-treated PMVECs. The expression levels of ACE (A) and ACE2 (B) were determined by using Western blot analysis in the following groups: control (black bar), Cap (gray bar), LPS (white bar), and LPS + Cap (hatched bar). All data are expressed as mean T SD (n = 6). *P G 0.05 as compared with the control group, #P G 0.05 as compared with the LPS group.
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FIG. 11. Effects of Cap and ACE2 inhibitor on the phosphorylation of p38MAPK, ERK1/2, and JNK in LPS-treated PMVECs. The expression levels of p38MAPK (A), ERK1/2 (B), and JNK (C) were determined by Western blot analysis in the following groups: control (black bar), Cap (gray bar), LPS (white bar), LPS + Cap (hatched bar), and LPS + Cap + MLN-4760 (dotted bar). All data are expressed as mean T SD (n = 6). *P G 0.05 as compared with the control group, #P G 0.05 as compared with the LPS group.
related to the early phase of the inflammatory response have been associated with the development of lung injury (31, 32). As a primary inflammatory mediator, TNF-! also activates other cytokines in sepsis. In terms of the importance of cytokines in sepsis, some studies have suggested that the levels of IL-6 measured at 6 h after the initiation of sepsis can accurately predict the risk of death in sepsis patients (33). In recent reports, elevated levels of ACE and Ang II are correlated with various types of ARDS, implicating the pivotal role of ACE in the pathogenesis of ARDS (15, 19). In agreement with other studies (21), we demonstrated that pretreatment with the ACE inhibitor Cap markedly reduced lung injury scores in LPS-induced lung injury. The administration of Cap 30 min prior to LPS injection could attenuate the lung
inflammatory process, suppress secretion of TNF-! and IL-6, and decrease serum levels of Ang II. Furthermore, Cap pretreatment significantly abolished the LPS-induced aberrant expression of ACE in lung tissues. Overall, our findings implicated that the inhibition of ACE might be through blockage of Ang II signals to reduce the inflammatory responses. Angiotensin-converting enzyme 2 acts as a negative regulator of the RAS by cleaving a single residue from Ang II to generate Ang-1Y7 (34). Angiotensin-converting enzyme 2 was also identified as a key receptor for coronavirus infections responsible for SARS and as a potential protective factor in lung diseases. The report by Imai et al. (19) demonstrated that exacerbated lung injury was found in ACE2-knockout mice as compared with that in wild-type mice using three different
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ACE/ACE2 REBALANCE IN LPS-INDUCED LUNG INJURY
types of ARDS models. Loss of ACE2 in mice resulted in enhanced vascular permeability, increased lung edema, neutrophil accumulation, and worsened lung function (19). Depletion of the ACE2 gene in mice aggravated lung injury and the subsequent severity of lung fibrosis in a model of bleomycininduced experimental lung injury. Conversely, recombinant ACE2 protected mice from bleomycin-induced fibrosis (35). Moreover, emerging evidence also indicates that the unbalanced status of Ang II/Ang-1Y7 is associated with the development of ARDS, and a change in the ratio of Ang II to Ang-1Y7 is closely related to the ratio of ACE2 to ACE. In an animal experiment, it has been shown that the imbalanced expression of ACE and ACE2 in lung tissue accompanied by changes in the ratio of Ang II to Ang-1Y7 is closely related to ALI caused by limb ischemia-reperfusion in mice (36). In the BALF of ventilated animals exposed to LPS, the ACE2 activity is reduced, whereas the ACE activity is enhanced, and these changes are correlated with enhanced BALF levels of Ang II and reduced levels of Ang-1Y7 (24). Our study also revealed that an imbalance of ACE/ACE2 expression was associated with LPS-induced lung injury in vivo and in vitro. Meanwhile, in vivo, pretreatment with Cap abolished LPSinduced aberrant expression of ACE and ACE2, converting the ratio of pulmonary ACE to ACE2 from 7.07 (LPS) to 3.17 (LPS + Cap) and decreasing the ratio of Ang II to Ang-1Y7 from 1.64 (LPS) to 0.99 (LPS + Cap). Our results from in vitro experiments also showed that Cap pretreatment prior to LPS stimulation in PMVECs could reverse the LPS-induced balance change of ACE/ACE2 expression by converting the ACE/ ACE2 ratio from 5.18 (LPS) to 1.52 (LPS + Cap), while reducing the Ang II/Ang-1Y7 ratio from 12.67 (LPS) to 5.42 (LPS + Cap). Furthermore, the ACE2 inhibitor MLN-4760 abolished the protective effects of Cap on LPS-induced cell apoptosis and inflammatory response, and it remarkably reversed the ratio of Ang II to Ang-1Y7 from 5.42 (LPS + Cap) to 27.77 (LPS + Cap + MLN-4760). In addition, we also revealed that Cap treatment after LPS exposure exerted slight therapeutic effects on cell cytotoxicity as well as suppressed cytokine secretion and reduced the Ang II/Ang-1Y7 ratio from 12.67 (LPS) to 5.74 (LPS + Cap [after]). Taken together, our findings suggest that the protective effects of ACE inhibition on LPS-induced lung injury might be through mechanisms related to regulation of local RAS activity, suppression of inflammatory response, and rebalance of the expression levels of ACE and ACE2, accompanied by changes of the ratio of Ang II to Ang-1Y7. In addition, an interesting phenomenon was found in our in vivo experiments, i.e., the single treatment with Cap induced a notable increase in ACE2 expression, as compared with the control group. It has been reported that the binding of ACE inhibitor to ACE leads to phosphorylation of specific serine residues in the tail of ACE, affecting ACE expression and phosphorylation of the important molecules involved in intracellular signaling pathways, such as JNK, CK2, and c-Jun (37, 38). In our in vitro study, we found that LPS stimulation induced higher phosphorylation of ERK1/2, p38MAPK, and JNK, whereas pretreatment with Cap 30 min before LPS exposure suppressed the phosphorylation of these three MAPKs.
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The ACE2 inhibitor MLN-4760 attenuated Cap-mediated blockade of p38MAPK, ERK1/2, and JNK activation. In fact, inhibition of p38MAPK phosphorylation and activity protects against pulmonary infiltration of leukocytes as well as lung edema (39). Furthermore, ACE2 regulates the balance of Ang II/Ang-1Y7 to inhibit apoptosis of alveolar epithelial cells through blockade of JNK phosphorylation (40). Pretreatment with Ang-1Y7 prevents Ang IIYinduced lung fibroblast apoptotic resistance by inhibiting the MAPK/nuclear factor .B pathway (41). In conclusion, Cap, an ACE inhibitor, protects the lung from injury in an LPS-exposed rat model and reduces LPS-induced cytotoxicity of cultured PMVECs. Furthermore, Cap suppresses LPS-induced cytokine secretion. These effects are associated with the regulation of RAS activity in local lung tissues, partially through rebalancing the ratio of ACE to ACE2 expression and inhibiting activation of the MAPK pathway. Although the effects of Cap administered after the onset of ARDS caused by sepsis have not been fully investigated in the present study, our findings identified the rebalance of ACE/ACE2 as a potent new therapeutic target for the treatment of this disease. However, further investigations are needed to elucidate the mechanisms underlying the regulation of pulmonary ACE2 expression in LPS-induced lung injury. REFERENCES 1. Herridge MS, Angus DC: Acute lung injuryVaffecting many lives. N Engl J Med 353:1736Y1738, 2005. 2. Goss CH, Brower RG, Hudson LD, Rubenfeld GD; ARDS Network: Incidence of acute lung injury in the United States. Crit Care Med 31:1607Y1611, 2003. 3. Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, Stern EJ, Hudson LD: Incidence and outcomes of acute lung injury. N Engl J Med 353:1685Y1693, 2005. 4. Phua J, Badia JR, Adhikari NK, Friedrich JO, Fowler RA, Singh JM, Scales DC, Stather DR, Li A, Jones A, et al.: Has mortality from acute respiratory distress syndrome decreased over time?: A systematic review. Am J Respir Crit Care Med 179:220Y227, 2009. 5. Mikkelsen ME, Shah CV, Meyer NJ, Gaieski DF, Lyon S, Miltiades AN, Goyal M, Fuchs BD, Bellamy SL, Christie JD: The epidemiology of acute respiratory distress syndrome in patients presenting to the emergency department with severe sepsis. Shock 40:375Y381, 2013. 6. The Acute Respiratory Distress Syndrome Network: Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med 342:1301Y1308, 2000. 7. Wheeler AP, Bernard GR: Acute lung injury and the acute respiratory distress syndrome: a clinical review. Lancet 369:1553Y1564, 2007. ¨ zlu¨ F, Tuli A, O ¨ zcan K, Yıldızdaz HY: Polymorphism of 8. Satar M, Tazkın E, O the angiotensin-converting enzyme gene and angiotensin-converting enzyme activity in transient tachypnea of neonate and respiratory distress syndrome. J Matern Fetal Neonatal Med 25:1712Y1715, 2012. 9. Marshall RP, McAnulty RJ, Laurent GJ: Angiotensin II is mitogenic for human lung fibroblasts via activation of the type 1 receptor. Am J Respir Crit Care Med 161:1999Y2004, 2000. 10. Suzuki Y, Ruiz-Ortega M, Lorenzo O, Ruperez M, Esteban V, Egido J: Inflammation and angiotensin II. Int J Biochem Cell Biol 35:881Y900, 2003. 11. Zhang H, Sun GY: LPS induces permeability injury in lung microvascular endothelium via AT(1) receptor. Arch Biochem Biophys 441:75Y83, 2005. 12. Idell S, Kueppers F, Lippmann M, Rosen H, Niederman M, Fein A: Angiotensin converting enzyme in bronchoalveolar lavage in ARDS. Chest 91:52Y56, 1987. 13. Marshall RP, Webb S, Bellingan GJ, Montgomery HE, Chaudhari B, McAnulty RJ, Humphries SE, Hill MR, Laurent GJ: Angiotensin converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome. Am J Respir Crit Care Med 166:646Y650, 2002. 14. Jerng JS, Yu CJ, Wang HC, Chen KY, Cheng SL, Yang PC: Polymorphism of the angiotensin-converting enzyme gene affects the outcome of acute respiratory distress syndrome. Crit Care Med 34:1001Y1006, 2006.
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ANGIOTENSIN-CONVERTING ENZYME INHIBITION ATTENUATES LIPOPOLYSACCHARIDE-INDUCED LUNG INJURY BY REGULATING THE BALANCE BETWEEN ANGIOTENSIN-CONVERTING ENZYME AND ANGIOTENSIN-CONVERTING ENZYME 2 AND INHIBITING MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION Yingchuan Li, Zhen Zeng, Yachun Li, Weifeng Huang, Ming Zhou, Xiaoli Zhang, and Wei Jiang Department of Anesthesiology, The Sixth People’s Hospital Affiliated to Shanghai Jiaotong University, Shanghai, China Received 3 Sep 2014; first review completed 19 Sep 2014; accepted in final form 7 Nov 2014 ABSTRACT—Activation of the renin-angiotensin system (angiotensin-converting enzyme [ACE]/angiotensin II [Ang II] and angiotensin-converting enzyme 2 [ACE2]/Ang-1Y7) has been implicated in the pathophysiology of inflammatory response and acute lung injury (ALI). Previous studies have shown that the ACE inhibitor captopril (Cap) may be a potent therapeutic drug for ALI. However, the mechanisms of its protective effects on ALI are still largely unknown. In this study, we evaluated the effects of Cap on preventing lipopolysaccharide (LPS)Yinduced lung injury and further investigated the underlying mechanisms of these protective effects. Rats were intraperitoneally pretreated with Cap (50 mg/kg) 30 min prior to an intravenous administration of LPS (7.5 mg/kg). Furthermore, following a 30-min pretreatment with Cap (10j5 mol/mL) or combined with the ACE2 inhibitor MLN4760 (10j7 mol/mL), rat pulmonary microvascular endothelial cells (PMVECs) were stimulated with LPS (1 mg/mL). Captopril pretreatment significantly attenuated LPS-induced pathophysiological changes in the lung, inhibited secretion of tumor necrosis factor ! and interleukin 6, reduced the ratio of Ang II to Ang-1Y7, and reversed the increased ratio of ACE to ACE2, which was remarkably decreased from 7.07 (LPS only) to 1.71 (LPS + Cap). The protective effects of Cap on ALI were also confirmed by in vitro studies, in which Cap suppressed LPS-induced secretion of proinflammatory cytokines and modulated the expression levels of ACE and ACE2. After Cap pretreatment, the ratio of ACE to ACE2 expression was remarkably decreased from 5.18 (LPS alone) to 1.52 (LPS + Cap). Furthermore, Cap given before LPS administration led to inhibition of p38 mitogen-activated protein kinase (MAPK), ERK (extracellular signal-regulated kinase) 1/2, and JNK (c-Jun N-terminal kinase) phosphorylation in PMVECs, whereas MLN4760 abolished the protective effects of Cap on LPS-induced secretion of proinflammatory cytokines and abolished Cap-induced blockade of p38MAPK, ERK1/2, and JNK phosphorylation. Our findings reveal that Cap exerts protective effects on LPS-induced lung injury and the cytotoxicity of PMVECs, and these effects may, at least in part, regulate the balance of ACE and ACE2 expression and inhibit the activation of MAPKs. KEYWORDS—Acute lung injury, lipopolysaccharide, angiotensin-converting enzyme, captopril, mitogen-activated protein kinases ABBREVIATIONS—RAS V renin-angiotensin system; ACE V angiotensin-converting enzyme; ACE2 V angiotensinconverting enzyme 2; ACEI V angiotensin-converting enzyme inhibitor; ALI V acute lung injury; ARDS V acute respiratory distress syndrome; LPS V lipopolysaccharide; IL-6 V interleukin 6; TNF-! V tumor necrosis factor !; PMVECs V pulmonary microvascular endothelial cells; Ang I V angiotensin I; Ang II V angiotensin II; AT-1 V Ang II type 1 receptor; SARS V severe acute respiratory syndrome; BALF V bronchoalveolar lavage fluid; Cap V captopril; MAPKs V mitogen-activated protein kinases; ERK V extracellular signal-regulated kinase; JNK V c-Jun N-terminal kinase
INTRODUCTION
public health issue as each year more than 1 million patients suffer from ARDS (1). Acute respiratory distress syndrome is commonly associated with systemic problems such as severe infections, sepsis, major trauma or burns, and acute pancreatitis, leading to indirect damage to the lung. Among all these contributing factors, sepsis is one of the most common causes of ARDS. It has been reported that approximately 25% of ARDS cases are induced by severe sepsis (2, 3). Although the therapeutic technology has been greatly improved in the past two decades, ARDS still remains an important cause of death in intensive care units, with a mortality rate of 40% to 60%, particularly among elderly patients with sepsis and a prior comorbidity (4, 5). At present, the available methods for ARDS treatments include supportive therapy, such as lung protective mechanical ventilation and lung recruitment with a high level of positive end-expiratory pressure. Specific and effective pharmacological therapy for ARDS that can attenuate
Acute respiratory distress syndrome (ARDS) is a critical illness syndrome characterized by acute respiratory insufficiency and severe hypoxemia. It has become a worldwide
Address reprint requests to Wei Jiang, PhD, Department of Anesthesiology, The Sixth People_s Hospital Affiliated to Shanghai Jiaotong University, No. 600, Yishan Rd, Xuhui District, Shanghai 200233, China. E-mail: [email protected]. This work was supported by the National Natural Science Foundation of China (no. 81272145). Authors_ Contributions: Yingchuan L. carried out the experiments and drafted the manuscript. Z.Z. carried out the immunoassays. Yachun L. and W.H. performed histological analysis. M.Z. and X.Z. participated in the design of the study and performed the statistical analysis. W.J. conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript. The authors declare that they have no competing interests. DOI: 10.1097/SHK.0000000000000302 Copyright Ó 2015 by the Shock Society
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lung injury and improve lung repair has not been clinically applied (6, 7). Recently, the role of the renin-angiotensin system (RAS) in ARDS has been well investigated in both clinical and experimental studies (8, 9). Angiotensin-converting enzyme (ACE), a key enzyme of the RAS, converts angiotensin I (Ang I) by cleavage to angiotensin II (Ang II). Angiotensin II, in terms of binding to the Ang II type 1 receptor (AT-1), induces activation of an intracellular signaling cascade, leading to upregulation of proinflammatory genes and a subsequent inflammatory response (10). In an animal study, the Ang II level of the lung was increased in response to lipopolysaccharide (LPS)Yinduced lung injury, but the increased Ang II ultimately exacerbated lung endothelium injury. Nevertheless, these Ang IIYassociated changes can be eliminated by selective blockage of AT-1 (11). In addition, the level of ACE has been evaluated in bronchoalveolar lavage fluid (BALF) of ARDS patients, particularly those with infectious causes of lung injury (12). The number of ARDS patients with the DD genotype and greater ACE activity is elevated, and this mutation is significantly associated with mortality; conversely, the ACE I/I allele has been found to be correlated with an increased survival rate (13, 14). Moreover, treatment to block the Ang II receptor or inhibit ACE activity had a similar inhibitory effect on the inflammatory response, and lung injury occurred during mechanical ventilation (15). Angiotensin-converting enzyme 2 (ACE2) is a homolog of ACE and shares a similar sequence in the catalytic domains with ACE (16). However, ACE2 and ACE appear to act on different catalytic substrates and generate different enzymatic products. Angiotensin-converting enzyme converts Ang I into Ang II, whereas ACE2 cleaves a single residue from Ang II to yield Ang-1Y7 (17). Angiotensin-converting enzyme 2 has been identified as a key receptor in severe acute respiratory syndrome (SARS) caused by SARS-CoV infections (18). Angiotensin-converting enzyme 2Yknockout mice with ARDS had more severe lung injury than did wild-type mice (19). Treatment with an injection of catalytically active recombinant ACE2 protein effectively improved the symptoms and attenuated arterial hypoxemia in a piglet model of LPS-induced lung injury (20). Angiotensin-converting enzyme inhibitor (ACEI) has been widely used as a clinically useful treatment for hypertension and cardiac disease for a long time. Angiotensin-converting enzyme inhibitor is also considered as a specific anti-inflammatory therapeutic agent that can attenuate either lung injury caused by LPS or ventilator-induced lung injury (15, 21Y23). In addition, an imbalance of ACE/ACE2 expression was found in BALF collected from ventilated LPS-exposed animals, in which ACE activity was enhanced, but ACE2 activity was reduced (24). However, it is still not clear whether the protective effect of ACEI in lung injury correlates with the balance of pulmonary ACE and ACE2 expression in ARDS; moreover, the mechanisms of this protective effect are still largely unknown. Therefore, in the present study, we tested the hypothesis that the ACEI captopril (Cap) has direct protective effects in experimental models of ARDS by regulating the balance of pulmonary ACE and ACE2 expression and inhibiting mitogen-activated protein kinase (MAPK) activation.
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METHODS Drugs Lipopolysaccharide(Escherichia coli, O127:B8) and Cap were purchased from Sigma (St Louis, Mo). The ACE2 inhibitor MLN-4760 was a product from Millennium Pharmaceuticals (Boston, Mass). The ACE rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, Calif). The ACE2 rabbit monoclonal antibody was purchased from Abcam (Cambridge, Mass). Rabbit anti-p38MAPK, antiYphospho-p38MAPK, antiY extracellular signal-regulated kinase 1/2 (ERK1/2), antiYphospho-ERK1/2, antiYc-Jun N-terminal kinase (JNK), and antiYphospho-JNK were all purchased from Cell Signaling Technology (Danvers, Mass). All enzymelinked immunosorbent assay (ELISA) kits for Ang II, Ang-1Y7, tumor necrosis factor ! (TNF-!), and IL-6 were products of BioSource International (Carlsbad, Calif).
In vivo studies Animals—All experiments were performed according to the protocol approved by the Ethics Committee of Animal Research at the College of Medicine, Shanghai Jiaotong University (Shanghai, China). A total of 48 male Sprague-Dawley rats weighing 200 to 250 g were obtained from the Laboratory Animal Science Department of Fudan University (Shanghai, China). Animals had access to food and drinking water ad libitum. Experimental protocols—The animal model of LPS-induced acute lung injury (ALI) was developed with some modifications as described by Hagiwara et al. (21). Briefly, the animals were randomly divided into the following groups (n = 12 for each group): (a) control group: receiving intraperitoneal administration of 0.9% NaCl solution 30 min prior to an injection of 0.9% NaCl solution into the tail vein; (b) Cap group: receiving intraperitoneal administration of Cap (50 mg/kg) 30 min prior to an intravenous injection of 0.9% NaCl solution into the tail vein; (c) LPS group: receiving intraperitoneal administration of 0.9% NaCl solution 30 min prior to intravenous injection of LPS (7.5 mg/kg); and (d) LPS + Cap group: receiving intraperitoneal administration of Cap (50 mg/kg) 30 min prior to an intravenous injection of LPS (7.5 mg/kg) into the tail vein. Eight hours after LPS administration, animals were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg) and killed by exsanguinations. Blood samples were centrifuged (3,200g at 4-C for 15 min), and collected serum was stored in aliquots at j80-C. Meanwhile, the lung was dissected for immunostaining. All experimental animals were breathing spontaneously. Lung wet-to-dry weight ratio—To assess tissue edema, the weight of rat lungs (six lungs per group) was measured, followed by a drying step of the lungs in an oven at 80-C for 48 h until the weight of samples became constant. Then, the lung wet-to-dry weight ratio was calculated. Histopathologic and immunohistochemical analysis—To determine the lung injury score, the left lung was fixed immediately in 4% paraformaldehyde, followed by serial dehydration in graded alcohol solutions, then embedded in paraffin, and cut into 4-2m-thick sections. The tissue sections of samples were stained with hematoxylin-eosin for microscopic observation. The degree of lung injury was evaluated by a double-blind examination by a pathologist and was semiquantitatively scored according to Murakami and colleagues_ (25) technique, by which 10 fields of lung parenchyma were graded on a scale of 0 to 4 (0, absent, appears normal; 1, light; 2, moderate; 3, strong; 4, intense) for edema, inflammation, and hemorrhage. A mean score for each of the parameters was then calculated. The final lung injury score was obtained by averaging the score from the animals within each group. The lung tissue sections were pretreated with heat-induced antigen retrieval for optimal immunostaining and then probed with polyclonal rabbit anti-ACE antibody (dilution 1:100) and monoclonal rabbit anti-ACE2 antibody (dilution 1:200). Finally, the sections were incubated with peroxidase-conjugated goatYantiYrabbit immunoglobulin G and visualized by the streptavidinbiotin-peroxidase complex system using a SABC kit (Boster, Wuhan, China). As negative controls, the primary antibodies were replaced with irrelevant immunoglobulins. Western blot—Tissue specimens (approximately 100 mg each) were obtained from the right lung and stored at j80-C. The frozen lung tissue was homogenized in cold RIPA lysis buffer. The prepared lysates were placed on ice for 20 min and then centrifuged at 14,000g for 10 min at 4-C. The supernatant fraction was collected for quantification of protein concentration, and equal amounts (50 2g) of protein extracts were then separated using sodium dodecyl sulfateYpolyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes by using a semidry blot system (Bio-Rad Laboratories, Mississauga, Ontario, Canada). The membranes were blocked with 5% (wt/vol) skim milk in Tris-buffered saline and 0.1% Tween-20 (TBST) for 2 h at room temperature, then incubated with either 1:1,000 rabbit anti-ACE polyclonal antibody or 1:500 rabbit anti-ACE2 monoclonal
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FIG. 1. Effect of Cap treatment on lung injury in LPS-exposed rats. The histological changes of lung injury were examined by hematoxylin-eosin staining of lung sections from animals in the control (A: original magnification 20), Cap (B: original magnification 20), LPS (C: original magnification 20), and LPS + Cap (D: original magnification 20) groups (n = 6), respectively.
antibody at 4-C overnight. After washing three times with TBST, the membranes were incubated with horseradish peroxidaseYconjugated secondary antibody (1:5,000) for 2 h at room temperature. Hybridizing signals were visualized by using chemiluminescence reagents. Densitometric values were quantified using an image-analysis software program (Quantity One software; Bio-Rad Laboratories), and the signal of ACE or ACE2 was normalized to "-actin. Measurement of secreted cytokines, Ang II, and Ang-1Y7—The serum levels of interleukin 6 (IL-6), TNF-!, Ang II, and Ang-1Y7 were assayed using an ELISA. The 96-well plates were coated with monoclonal antibody specific to IL-6, TNF-!, Ang II, and Ang-1Y7 (BioSource International), respectively. The measurement of these inflammatory mediators was performed according to the procedure described by the manufacturer. The plate was read at an absorbance of 450 nm using a microplate reader (Bio-Rad Laboratories). The serum samples were tested twice each.
medium was collected at 8 h after exposure to LPS, and the supernatant was used for measurements. Measurement of secreted cytokines Ang II and Ang 1Y7—The concentrations of IL-6, TNF-!, Ang II, and Ang-1Y7 secretion in the culture supernatant were measured using the ELISA sandwich method. The procedure was similar to that used to measure the serum levels of cytokines and Ang II in the in vivo study. Western blot—Pulmonary microvascular endothelial cells were digested with 0.25% trypsin-EDTA. The harvested cells were washed with ice-cold phosphate-buffered saline and then resuspended in 0.3 mL of ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 1% Triton X-100; 1 mM phenylmethylsulfonyl fluoride; 0.02% sodium azide; and 1 g/mL aprotinin). After 30 min on ice, the cell lysates were centrifuged at 12,000g for 10 min at 4-C to collect the supernatant for the Western blot assay. The procedure for the Western blot assays to detect ACE, ACE2 expression, and the phosphorylation of p38MAPK, ERK1/2, and JNK in the cell lysates was similar to that described for the tissue samples in the in vivo study.
In vitro studies Cell culture—Isolation and culture of pulmonary microvascular endothelial cells (PMVECs) were performed according to a modified method as described by Cheng et al. (26). Each experimental animal was killed after receiving anesthesia with an intraperitoneal injection of pentobarbital sodium. Then, a thoracotomy was performed, in which the lung circulatory system was perfused with an injection of 50 mL of ice-cold phosphate-buffered saline via the right ventricle, and then the lungs were removed and washed with 30 mL of ice-cold serum-free Dulbecco modified Eagle medium. The pleura was carefully cut away and discarded, from which a 1-mm-thick section was cut from the outer edge of the remaining lung tissue and trimmed into small pieces. These pieces of lung tissue were then inserted into the glass pellets (Costar, Cambridge, Mass) and rinsed in endothelial cell medium (Sciencell Research, Carlsbad, Calif) containing 20% fetal calf serum, 100 U/mL penicillin/ streptomycin, and 2.5 g/mL amphotericin B. The lung tissues in the glass pellets were removed after incubation for 60 h at 37-C (Thermo, Waltham, Mass) in humidified air containing 5% CO2. Thereafter, the cell culture medium in the glass pellets was replaced every 3 days. As the growing cells reached 80% to 90% confluence, the cells were split with 0.25% trypsin-EDTA (Gibco, Grand Island, NY) and then subcultured at a 1:2 split ratio. The cultured cells were identified as endothelial cells by their cobblestone morphology as well as immunostaining with antiYhuman factor VIIIYrelated antigen, CD31 (Boster), and FITC-BSI (Santa Cruz Biotechnology). The third to fifth generations of cultured cells were used for the following experiment. Cell viability assay—The viability of PMVECs was determined by using CCK-8 kits (Dojido Molecular Technologies, Japan), according to the manufacturer_s instructions. Prepared PMVECs were plated in 96-well microplates and incubated with serum-free Dulbecco modified Eagle medium for 24 h. Then, the cells were pretreated or untreated with 10j5 M Cap for 30 min prior to exposure to 1 mg/mL LPS for 8 h. To examine cell viability, 10 2L of CCK-8 reagent was added into each well of the 96-well plate. After incubation for 4 h, the absorbance of the plate was read at 492 nm using a microplate reader. Experimental groups—The treatments were divided into four groups, including control group, without any drug treatment; Cap group, treated with Cap (10j5 mol/mL) only; LPS group, stimulated with LPS (1 mg/mL) alone; and LPS + Cap group, pretreated with Cap (10j5 mol/mL) for 30 min prior to LPS stimulation (1 mg/mL); and LPS + Cap + MLN4760 group, pretreated with Cap (10j5 mol/mL) and ACE2 inhibitor MLN4760 (10j7 mol/mL) for 30 min prior to LPS stimulation (1 mg/mL). The therapeutic effects of Cap were also detected in two extra groups, including (a) the LPS + Cap (after) group, which was treated with Cap (10j5 mol/mL) after LPS stimulation (1 mg/mL) for 30 min; and (b) LPS + Cap + MLN4760 (after) group, which was treated with Cap (10j5 mol/mL) and the ACE2 inhibitor MLN4760 (10j7 mol/mL) after LPS stimulation (1 mg/mL) for 30 min. The cell culture
Statistical analysis All data are presented as mean T SD. To determine the difference between two independent groups, data were analyzed by using one-way analysis of variance and Duncan multiple-range test with SPSS 13.0 software (SPSS Inc, Chicago, Ill). P G 0.05 was accepted as statistically significant.
RESULTS In vivo studies
Effect of Cap on lung injury—Signs of lung injury in the lung tissues from the LPS group were observed, including a marked increase in inflammatory cell infiltration, edema, and hemorrhage in the interstitium and alveolus (Fig. 1B). But, LPS-induced lung edema, hemorrhage, and inflammatory cell infiltration were significantly attenuated by pretreatment with Cap (Fig. 1D). Similarly, the lung injury scores were compatible with the histological changes in the same treatment group. The results
FIG. 2. Lung injury scores of the different treatment groups. The lung injury scores indicating histological changes, including edema, inflammation, and hemorrhage, were calculated in the following groups: control (black bars), Cap (gray bars), LPS (white bars), and LPS + Cap (hatched bars). All data are expressed as mean T SD (n = 6). *P G 0.05 versus the control group, #P G 0.05 versus the LPS group.
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FIG. 3. Effect of Cap on the lung wet-to-dry weight ratio in LPSexposed rats. A comparison was performed among the different treatment groups: control (black bar), Cap (gray bar), LPS (white bar), and LPS + Cap (hatched bar). All data are expressed as mean T SD (n = 6). *P G 0.05 versus the control group, #P G 0.05 versus the LPS group.
showed that the score was significantly greater in the LPS group than that in the control group. Pretreatment with Cap in the LPS + Cap group markedly reduced the lung injury scores in comparison with the LPS group (Fig. 2). In addition, the wet-to-dry weight ratio of the lungs in the LPS + Cap group was significantly less than that in the LPS group (Fig. 3). Effect of Cap on the serum levels of Ang II, Ang-1Y7, IL-6, and TNF-!—In this study, we found that the serum levels of Ang II, IL-6, and TNF-! in the LPS group were significantly greater than those in the control group. Meanwhile, pretreatment with Cap significantly suppressed the secreted Ang II, IL-6, and TNF-!, compared with the serum levels of
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those factors between the Cap + LPS group and the LPS group (Fig. 4, A, D, and E). The level of Ang-1Y7 in the LPS group was also markedly higher than that of the control group, whereas it was also significantly suppressed by pretreatment with Cap (Fig. 4B). However, the ratio of Ang II to Ang-1Y7 was obviously decreased from 1.64 in the LPS group to 0.99 in the LPS + Cap group (Fig. 4C). Effect of Cap on the expression of pulmonary ACE and ACE2—In the lung tissues from the LPS group, the immunohistochemical (IHC) staining analysis results showed that there was a remarkable increase in ACE-positive staining but a decrease in ACE2-positive staining, as compared with the control group (ACE comparison between Fig. 5C and Fig. 5A; ACE2 comparison between Fig. 6C and Fig. 6A). In contrast, Cap treatment significantly suppressed the ACE expression but enhanced ACE2 expression in the LPS + Cap group, compared with the LPS group (ACE: comparison between Fig.5D and Fig. 5C; ACE2: comparison between Fig. 6D and Fig. 6C). Consistent with the IHC staining analysis results, densitometric Western blotting data showed that ACE expression was significantly increased in lung tissues from the LPS group, but the ACE2 level was decreased in the same tissue sample, whereas in lung tissue samples from the LPS + Cap group, the ACE level was dramatically decreased, but ACE2 expression was enhanced (Fig. 7, A and B). In addition, the expression level of pulmonary ACE2 was also notably increased in the Cap group as compared with the control group (P G 0.05, Fig. 7B). The expression of pulmonary ACE in the LPS-exposed animals was increased by 1.43-fold, as compared with the unexposed
FIG. 4. Effect of Cap on the serum levels of Ang II, Ang-1Y7, IL-6, and TNF-! in LPS-exposed rats. The serum levels of (A) Ang II and (B) Ang-1Y7, (C) the ratio of Ang II to Ang-1Y7, and serum levels of (D) TNF-! and (E) IL-6 were measured in the different treatment groups: control (black bar), Cap (gray bar), LPS (white bar), and LPS + Cap (hatched bar). All data are expressed as mean T SD (n = 6). *P G 0.05 as compared with the control group, #P G 0.05 as compared with the LPS group.
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FIG. 5. Effect of Cap on the expression of pulmonary ACE in LPS-exposed rats. The expression of ACE in lung tissues was examined by IHC staining methods. The lung specimens were obtained from the control group (A), Cap group (B), LPS group (C), and LPS + Cap group (D), n = 6 in each group. The results were observed under a microscope (original magnifications 20, 40 inset).
ones, but the expression of pulmonary ACE2 in the same animal was decreased by 1.55-fold, leading to a change of the ACE/ ACE2 ratio from 3.17 to 7.07, whereas the ratio of ACE to ACE2 was greatly reduced with Cap pretreatment prior to LPS stimulation, showing a change in the ratio from 7.07 in the LPS group to 1.71 in the LPS + Cap group. In vitro studies
Effect of Cap and ACE2 inhibitor on LPS-induced cytotoxicity—As shown in Figure 8, pretreatment with Cap (10j5 mol/mL) significantly attenuated LPS-induced cytotoxicity in PMVECs, but this protective effect of Cap was abolished by the ACE2 inhibitor MLN-4760. On the other hand, Cap treatment after LPS exposure slightly improved the cell viability, but the change was not statistically significant compared with the LPS group. Effect of Cap and ACE2 inhibitor on secreted cytokines, Ang II, and Ang-1Y7—There was a significant increase in the levels of Ang II and Ang-1Y7 in the supernatant of cultured PMVECs that were exposed to LPS. Lipopolysaccharide-induced Ang II and Ang-1Y7 secretion was significantly inhibited by pretreatment with Cap. The ratio of Ang II to Ang-1Y7 was markedly decreased from 12.67 in the LPS group to 5.42 in the LPS + Cap group. Pretreatment with MLN-4760 obviously abolished the inhibitory effect of Cap on LPS-induced Ang II secretion, but the level of Ang-1Y7 was more obviously decreased in the Cap + MLN-4760+LPS group, as compared with the LPS + Cap group. The ratio of Ang II to Ang-1Y7 was markedly increased from 5.42 in the LPS + Cap group to 27.77 in the LPS + Cap + MLN4760 group (Fig. 9, AYC). Similarly, the IL-6 and TNF-! levels in the supernatant were also elevated after exposure to LPS, but LPS-induced secretion of IL-6 and TNF-! was significantly inhibited by pretreatment with Cap.
Pretreatment with MLN-4760 obviously abolished the inhibitory effect of Cap on LPS-induced IL-6 and TNF-! secretion (Fig. 9, D and E). In addition, Cap treatment after LPS exposure exerted similar effects on the secretion of cytokines, Ang II, and Ang-1Y7 as pretreatment with Cap. MLN-4760 administration also caused a remarkable increase in the Ang II/Ang-1Y7 ratio from 5.74 (LPS + Cap [after]) to 26.56 (LPS + Cap + MLN4760 [after]) (Fig. 9). Effect of Cap on the expression of ACE and ACE2 in PMVECs—The Western blot results showed that after LPS stimulation, the expression level of ACE was significantly increased in PMVECs, but pretreatment with Cap effectively abolished LPS-induced ACE expression. In contrast, the expression of ACE2 in PMVECs exposed to LPS was significantly decreased. Thus, Cap pretreatment could restore the expression level of ACE2 (Fig. 10, A and B). Furthermore, a single treatment with Cap also caused a significant decrease in the expression level of ACE, as compared with the control group (Fig. 10A). Our results revealed a 1.18-fold increase in the ACE expression in LPS-exposed cells, compared with the unexposed control, whereas in the same exposed cells, ACE2 expression was decreased by 2.12-fold, leading to a change of the ACE/ACE2 ratio from 2.39 (control) to 5.18 (LPS group). Conversely, pretreatment with Cap markedly changed the ACE/ACE2 ratio from 5.18 (LPS group) to 1.52 (LPS + Cap group). Effects of Cap and ACE2 inhibitor on the phosphorylation of p38MAPK, ERK1/2, and JNK—The Western blot analysis showed that the phosphorylation levels of ERK1/2, p38MAPK, and JNK in normal PMVECs that were exposed to LPS for 8 h (LPS group) were significantly higher than those in the untreated control group. Compared with the LPS group, pretreatment with Cap 30 min before LPS exposure could
FIG. 6. Effect of Cap on the expression of pulmonary ACE2 in LPS-exposed rats. The expression of ACE2 in lung tissues was examined by IHC staining methods. The lung specimens were obtained from the control group (A), Cap group (B), LPS group (C), and LPS + Cap group (D), n = 6 in each group. The results were observed under a microscope (original magnifications 20, 40 inset).
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FIG. 7. Evaluation of the expression levels of pulmonary ACE (A) and ACE2 (B) in the different treatment groups. The expression levels of pulmonary ACE (A) and ACE2 (B) were examined by Western blot assays of the following groups: control (black bar), Cap (gray bar), LPS (white bar), and LPS + Cap (hatched bar). All data are expressed as mean T SD (n = 6). *P G 0.05 as compared with the control group, #P G 0.05 as compared with the LPS group.
suppress the phosphorylation of p38MAPK, ERK1/2, and JNK. In contrast, compared with the LPS + Cap group, combined pretreatment with MLN-4760 and Cap (inhibition of both ACE and ACE2) 30 min before exposure to LPS (LPS + Cap + MLN-4760 group) obviously reversed the Cap-mediated blockade of p38MAPK, ERK1/2, and JNK activation (Fig. 11, AYC). DISCUSSION We found that in a rat model of LPS-induced lung injury, ACE expression in lung tissue was remarkably increased, but ACE2 expression was decreased, with high levels of cytokines and Ang II in the serum, whereas pretreatment with Cap could abolish LPS-mediated aberrant expression of ACE and ACE2 in lung tissue, decrease lung injury scores, and attenuate the inflammatory response, as reflected by reduced serum levels of cytokines and Ang II. Similar results were also found in the in vitro experiments. Captopril significantly attenuated LPSinduced cytotoxicity in PMVECs, which correlated with its inhibitory effects on LPS-mediated aberrant expression of ACE and ACE2 as well as the secretion of cytokines and Ang II. Meanwhile, ACE2 inhibitor administration abolished the protective effects of Cap on LPS-induced secretion of proinflammatory cytokines and cell cytotoxicity. These data indicate that an imbalance of ACE and ACE2 expression plays a pivotal role in LPS-induced lung injury. Sepsis is the major clinical pathogenic cause of ARDS in intensive care units (27). Endotoxin, i.e., bacterial LPS, released from gram-negative bacterial cell walls, is considered as an important eliciting factor for the development of sepsis. In animal studies, LPS stimulation can mimic the main pathological features of ARDS in human patients (28). Consistent with previous studies (21), we found that the LPS-induced lung injury was associated with increased levels of TNF-! and IL-6 in a rat model. Similarly, in the in vitro experiments, LPS stimulation could promote elevated secretion of cytokines in PMVECs that had emerging damage caused by LPS. According to these
observations, it was speculated that PMVECs might be the target cells in LPS-induced lung injury. Angiotensin II is essential for proinflammation via a mechanism involving the release of cytokines, chemokines, adhesion molecules, growth factors, and reactive oxygen species via AT-1 receptors (10, 29). Several reports also have implicated the active role of Ang II in LPS-induced lung inflammation and injury through its binding to the AT-1 receptor of the lung microvascular endothelium (11, 21). Treatment with an AT-1 inhibitor prevents sepsis-induced ALI and attenuates the secretion of TNF-!, IL-6, and IL-1 (30). In agreement with other studies (21, 22), our study demonstrated that the level of Ang II was substantially increased in LPS-induced lung injury, with elevated TNF-! and IL-6 levels. Secreted cytokines
FIG. 8. Effects of Cap and ACE2 inhibitor on LPS-induced cytotoxicity. Cell viability of PMVECs was evaluated as the spectrophotometric value obtained from the microplate reader for the different treatment groups: control (black bar), Cap (gray bar), LPS (white bar), LPS + Cap (hatched bar), LPS + Cap + MLN-4760 (dotted bar), LPS + Cap (after) (checkered bar), and LPS + Cap + MLN-4760 (after) (vertical lines bar). All data are expressed as mean T SD (n = 6). *P G 0.05 as compared with the control group, #P G 0.05 as compared with the LPS group.
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FIG. 9. Effects of Cap and ACE2 inhibitor on secreted cytokines, Ang II, and Ang-1Y7 in the supernatant of cultured PMVECs. The measurement for levels of (A) Ang II and (B) Ang-1Y7, (C) the ratio of Ang II to Ang-1Y7, and levels of (D) TNF-! and (E) IL-6 was performed in the following groups: control (black bar), Cap (gray bar), LPS (white bar), LPS + Cap (hatched bar), LPS + Cap + MLN-4760 (dotted bar), LPS + Cap (after) (checkered bar), and LPS + Cap + MLN4760 (after) (vertical lines bar). All data are expressed as mean T SD (n = 6). *P G 0.05 as compared with the control group, #P G 0.05 as compared with the LPS group.
FIG. 10. Effect of Cap on the expression of ACE and ACE2 in LPS-treated PMVECs. The expression levels of ACE (A) and ACE2 (B) were determined by using Western blot analysis in the following groups: control (black bar), Cap (gray bar), LPS (white bar), and LPS + Cap (hatched bar). All data are expressed as mean T SD (n = 6). *P G 0.05 as compared with the control group, #P G 0.05 as compared with the LPS group.
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FIG. 11. Effects of Cap and ACE2 inhibitor on the phosphorylation of p38MAPK, ERK1/2, and JNK in LPS-treated PMVECs. The expression levels of p38MAPK (A), ERK1/2 (B), and JNK (C) were determined by Western blot analysis in the following groups: control (black bar), Cap (gray bar), LPS (white bar), LPS + Cap (hatched bar), and LPS + Cap + MLN-4760 (dotted bar). All data are expressed as mean T SD (n = 6). *P G 0.05 as compared with the control group, #P G 0.05 as compared with the LPS group.
related to the early phase of the inflammatory response have been associated with the development of lung injury (31, 32). As a primary inflammatory mediator, TNF-! also activates other cytokines in sepsis. In terms of the importance of cytokines in sepsis, some studies have suggested that the levels of IL-6 measured at 6 h after the initiation of sepsis can accurately predict the risk of death in sepsis patients (33). In recent reports, elevated levels of ACE and Ang II are correlated with various types of ARDS, implicating the pivotal role of ACE in the pathogenesis of ARDS (15, 19). In agreement with other studies (21), we demonstrated that pretreatment with the ACE inhibitor Cap markedly reduced lung injury scores in LPS-induced lung injury. The administration of Cap 30 min prior to LPS injection could attenuate the lung
inflammatory process, suppress secretion of TNF-! and IL-6, and decrease serum levels of Ang II. Furthermore, Cap pretreatment significantly abolished the LPS-induced aberrant expression of ACE in lung tissues. Overall, our findings implicated that the inhibition of ACE might be through blockage of Ang II signals to reduce the inflammatory responses. Angiotensin-converting enzyme 2 acts as a negative regulator of the RAS by cleaving a single residue from Ang II to generate Ang-1Y7 (34). Angiotensin-converting enzyme 2 was also identified as a key receptor for coronavirus infections responsible for SARS and as a potential protective factor in lung diseases. The report by Imai et al. (19) demonstrated that exacerbated lung injury was found in ACE2-knockout mice as compared with that in wild-type mice using three different
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types of ARDS models. Loss of ACE2 in mice resulted in enhanced vascular permeability, increased lung edema, neutrophil accumulation, and worsened lung function (19). Depletion of the ACE2 gene in mice aggravated lung injury and the subsequent severity of lung fibrosis in a model of bleomycininduced experimental lung injury. Conversely, recombinant ACE2 protected mice from bleomycin-induced fibrosis (35). Moreover, emerging evidence also indicates that the unbalanced status of Ang II/Ang-1Y7 is associated with the development of ARDS, and a change in the ratio of Ang II to Ang-1Y7 is closely related to the ratio of ACE2 to ACE. In an animal experiment, it has been shown that the imbalanced expression of ACE and ACE2 in lung tissue accompanied by changes in the ratio of Ang II to Ang-1Y7 is closely related to ALI caused by limb ischemia-reperfusion in mice (36). In the BALF of ventilated animals exposed to LPS, the ACE2 activity is reduced, whereas the ACE activity is enhanced, and these changes are correlated with enhanced BALF levels of Ang II and reduced levels of Ang-1Y7 (24). Our study also revealed that an imbalance of ACE/ACE2 expression was associated with LPS-induced lung injury in vivo and in vitro. Meanwhile, in vivo, pretreatment with Cap abolished LPSinduced aberrant expression of ACE and ACE2, converting the ratio of pulmonary ACE to ACE2 from 7.07 (LPS) to 3.17 (LPS + Cap) and decreasing the ratio of Ang II to Ang-1Y7 from 1.64 (LPS) to 0.99 (LPS + Cap). Our results from in vitro experiments also showed that Cap pretreatment prior to LPS stimulation in PMVECs could reverse the LPS-induced balance change of ACE/ACE2 expression by converting the ACE/ ACE2 ratio from 5.18 (LPS) to 1.52 (LPS + Cap), while reducing the Ang II/Ang-1Y7 ratio from 12.67 (LPS) to 5.42 (LPS + Cap). Furthermore, the ACE2 inhibitor MLN-4760 abolished the protective effects of Cap on LPS-induced cell apoptosis and inflammatory response, and it remarkably reversed the ratio of Ang II to Ang-1Y7 from 5.42 (LPS + Cap) to 27.77 (LPS + Cap + MLN-4760). In addition, we also revealed that Cap treatment after LPS exposure exerted slight therapeutic effects on cell cytotoxicity as well as suppressed cytokine secretion and reduced the Ang II/Ang-1Y7 ratio from 12.67 (LPS) to 5.74 (LPS + Cap [after]). Taken together, our findings suggest that the protective effects of ACE inhibition on LPS-induced lung injury might be through mechanisms related to regulation of local RAS activity, suppression of inflammatory response, and rebalance of the expression levels of ACE and ACE2, accompanied by changes of the ratio of Ang II to Ang-1Y7. In addition, an interesting phenomenon was found in our in vivo experiments, i.e., the single treatment with Cap induced a notable increase in ACE2 expression, as compared with the control group. It has been reported that the binding of ACE inhibitor to ACE leads to phosphorylation of specific serine residues in the tail of ACE, affecting ACE expression and phosphorylation of the important molecules involved in intracellular signaling pathways, such as JNK, CK2, and c-Jun (37, 38). In our in vitro study, we found that LPS stimulation induced higher phosphorylation of ERK1/2, p38MAPK, and JNK, whereas pretreatment with Cap 30 min before LPS exposure suppressed the phosphorylation of these three MAPKs.
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The ACE2 inhibitor MLN-4760 attenuated Cap-mediated blockade of p38MAPK, ERK1/2, and JNK activation. In fact, inhibition of p38MAPK phosphorylation and activity protects against pulmonary infiltration of leukocytes as well as lung edema (39). Furthermore, ACE2 regulates the balance of Ang II/Ang-1Y7 to inhibit apoptosis of alveolar epithelial cells through blockade of JNK phosphorylation (40). Pretreatment with Ang-1Y7 prevents Ang IIYinduced lung fibroblast apoptotic resistance by inhibiting the MAPK/nuclear factor .B pathway (41). In conclusion, Cap, an ACE inhibitor, protects the lung from injury in an LPS-exposed rat model and reduces LPS-induced cytotoxicity of cultured PMVECs. Furthermore, Cap suppresses LPS-induced cytokine secretion. These effects are associated with the regulation of RAS activity in local lung tissues, partially through rebalancing the ratio of ACE to ACE2 expression and inhibiting activation of the MAPK pathway. Although the effects of Cap administered after the onset of ARDS caused by sepsis have not been fully investigated in the present study, our findings identified the rebalance of ACE/ACE2 as a potent new therapeutic target for the treatment of this disease. However, further investigations are needed to elucidate the mechanisms underlying the regulation of pulmonary ACE2 expression in LPS-induced lung injury. REFERENCES 1. Herridge MS, Angus DC: Acute lung injuryVaffecting many lives. N Engl J Med 353:1736Y1738, 2005. 2. Goss CH, Brower RG, Hudson LD, Rubenfeld GD; ARDS Network: Incidence of acute lung injury in the United States. Crit Care Med 31:1607Y1611, 2003. 3. Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, Stern EJ, Hudson LD: Incidence and outcomes of acute lung injury. N Engl J Med 353:1685Y1693, 2005. 4. Phua J, Badia JR, Adhikari NK, Friedrich JO, Fowler RA, Singh JM, Scales DC, Stather DR, Li A, Jones A, et al.: Has mortality from acute respiratory distress syndrome decreased over time?: A systematic review. Am J Respir Crit Care Med 179:220Y227, 2009. 5. Mikkelsen ME, Shah CV, Meyer NJ, Gaieski DF, Lyon S, Miltiades AN, Goyal M, Fuchs BD, Bellamy SL, Christie JD: The epidemiology of acute respiratory distress syndrome in patients presenting to the emergency department with severe sepsis. Shock 40:375Y381, 2013. 6. The Acute Respiratory Distress Syndrome Network: Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med 342:1301Y1308, 2000. 7. Wheeler AP, Bernard GR: Acute lung injury and the acute respiratory distress syndrome: a clinical review. Lancet 369:1553Y1564, 2007. ¨ zlu¨ F, Tuli A, O ¨ zcan K, Yıldızdaz HY: Polymorphism of 8. Satar M, Tazkın E, O the angiotensin-converting enzyme gene and angiotensin-converting enzyme activity in transient tachypnea of neonate and respiratory distress syndrome. J Matern Fetal Neonatal Med 25:1712Y1715, 2012. 9. Marshall RP, McAnulty RJ, Laurent GJ: Angiotensin II is mitogenic for human lung fibroblasts via activation of the type 1 receptor. Am J Respir Crit Care Med 161:1999Y2004, 2000. 10. Suzuki Y, Ruiz-Ortega M, Lorenzo O, Ruperez M, Esteban V, Egido J: Inflammation and angiotensin II. Int J Biochem Cell Biol 35:881Y900, 2003. 11. Zhang H, Sun GY: LPS induces permeability injury in lung microvascular endothelium via AT(1) receptor. Arch Biochem Biophys 441:75Y83, 2005. 12. Idell S, Kueppers F, Lippmann M, Rosen H, Niederman M, Fein A: Angiotensin converting enzyme in bronchoalveolar lavage in ARDS. Chest 91:52Y56, 1987. 13. Marshall RP, Webb S, Bellingan GJ, Montgomery HE, Chaudhari B, McAnulty RJ, Humphries SE, Hill MR, Laurent GJ: Angiotensin converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome. Am J Respir Crit Care Med 166:646Y650, 2002. 14. Jerng JS, Yu CJ, Wang HC, Chen KY, Cheng SL, Yang PC: Polymorphism of the angiotensin-converting enzyme gene affects the outcome of acute respiratory distress syndrome. Crit Care Med 34:1001Y1006, 2006.
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