American Journal of Respiratory Cell and Molecular Biology

Accumulating evidence has demonstrated that up-regulation of the angiotensin (Ang)-converting enzyme (ACE)/AngII/AngII type 1 receptor (AT1R) axis aggravates pulmonary fibrosis. The recently discovered ACE2/Ang-(1-7)/Mas axis, which counteracts the activity of the ACE/AngII/AT1R axis, has been shown to protect against pulmonary fibrosis. However, the mechanisms by which ACE2 and Ang-(1-7) attenuate pulmonary fibrosis remain unclear. We hypothesized that up-regulation of the ACE2/Ang-(1-7)/Mas axis protects against bleomycin (BLM)-induced pulmonary fibrosis by inhibiting the mitogen-activated protein kinase (MAPK)/NF-κB pathway. In vivo, Ang-(1-7) was continuously infused into Wistar rats that had received BLM or AngII. In vitro, human fetal lung-1 cells were pretreated with compounds that block the activities of AT1R, Mas (A-779), and MAPKs before exposure to AngII or Ang-(1-7). The human fetal lung-1 cells were infected with lentivirus-mediated ACE2 before exposure to AngII. In vivo, Ang-(1-7) prevented BLM-induced lung fibrosis and AngII-induced lung inflammation by inhibiting the MAPK phosphorylation and NF-κB signaling cascades. However, exogenous Ang-(1-7) alone clearly promoted lung inflammation. In vitro, Ang-(1-7) and lentivirus-mediated ACE2 inhibited the AngII-induced MAPK/NF-κB pathway, thereby attenuating inflammation and α-collagen I production, which could be reversed by the Mas inhibitor, A-779. Ang-(1-7) inhibited AngII-induced lung fibroblast apoptotic resistance via inhibition of the MAPK/NF-κB pathway and activation of the BCL-2-associated X protein/caspase-dependent mitochondrial apoptotic pathway. Ang-(1-7) alone markedly stimulated extracellular signal–regulated protein kinase 1/2 phosphorylation and the NF-κB cascade. Up-regulation of the ACE2/Ang-(1-7)/Mas axis protected against pulmonary fibrosis by inhibiting the MAPK/NF-κB pathway. However, close attention should be paid to the proinflammatory effects of Ang-(1-7).

The antifibrotic effects of angiotensin-(1-7) make the heptapeptide a candidate for a therapeutic target in humans with pulmonary fibrosis.

Pulmonary fibrosis is a progressive and fatal lung disease characterized by chronic inflammation, the migration and proliferation of fibroblasts, the accumulation of the extracellular matrix (ECM), and remodeling of the lung parenchyma (1, 2). Currently, there are no effective antifibrotic therapies for pulmonary fibrosis (3).

A growing body of evidence indicates that angiotensin (Ang) II, a bioactive peptide of the renin–Ang system (RAS), plays a key role in the initiation and the maintenance of lung fibrosis (4). Ang-converting enzyme (ACE) inhibitors (5) and AngII type 1 receptor (AT1R) blockers (6) have been used for the treatment of pulmonary fibrosis in animal models and humans. Consequently, down-regulation of the ACE/AngII/AT1R axis may be a viable therapeutic strategy for pulmonary fibrosis.

The recent discovery of the ACE2/Ang-(1-7)/Mas axis offers an alternative approach for counter-regulating the ACE/AngII/AT1R axis to produce beneficial effects (4, 7). The heptapeptide Ang-(1-7), an enzymatic product of ACE2 (8), has been shown to counteract the detrimental effects of AngII in the liver (9), heart (10), and kidneys (11). Recently, Ang-(1-7) and ACE2 have been shown to prevent bleomycin (BLM)-induced pulmonary fibrosis (4). Hence, the ACE2/Ang-(1-7)/Mas axis may potentially offer a novel therapeutic strategy for pulmonary fibrosis. However, the exact molecular mechanism by which the ACE2/Ang-(1-7)/Mas axis protects against pulmonary fibrosis remains unclear.

NF-κB, which can be activated by mitogen-activated protein kinases (MAPKs) (12), is responsible for the transcription of inflammatory factors and profibrotic cytokines, which promote an inflammatory response and fibrosis (13, 14). Moreover, as a cell survival pathway, the activation of NF-κB signaling results in the apoptotic resistance of lung fibroblasts, which facilitates the accumulation of lung fibroblasts (15), all of which contribute equally to pulmonary fibrogenesis. AngII has been reported to activate fibroblasts and promote collagen accumulation by activating MAPKs (16) and NF-κB (17). However, Ang-(1-7) inhibited the MAPK and NF-κB pathways in the liver (18) and in a murine model of asthma (19). The loss of ACE2 enhanced NF-κB–driven renal inflammation in a mouse model of obstructive nephropathy (20). Whether the ACE2/Ang-(1-7)/Mas axis inhibits the MAPK and NF-κB pathways stimulated by AngII in lung fibroblasts has not yet been investigated. We hypothesized that the ACE2/Ang-(1-7)/Mas axis protects against pulmonary fibrosis by inhibiting the MAPK/NF-κB pathway.

Hence, our study aimed to investigate the mechanism by which the ACE2/Ang-(1-7)/Mas axis protects against pulmonary fibrosis in vivo and in vitro. We found that Ang-(1-7) and lentivirus-mediated ACE2 (lenti-ACE2) protected against BLM- and AngII-induced inflammation and ECM accumulation by inhibiting the MAPK/NF-κB pathway. Ang-(1-7) inhibited AngII-induced apoptotic resistance of lung fibroblasts by inhibiting the MAPK/NF-κB pathway and activating the BCL-2-associated X protein (bax)/caspase–dependent mitochondrial apoptotic pathway.

Materials

AngII, Ang-(1-7), A-779 (a selective Mas inhibitor), SB203580 (a p38 MAPK inhibitor), PD98059 (a specific extracellular signal–regulated protein kinase [ERK] 1/2 inhibitor), SP600125 (a Jun N-terminal kinase [JNK]/MAPK inhibitor), and BAY117082 (an IκK inhibitor) were purchased from Sigma-Aldrich (St. Louis, MO). Irbesartan (an AT1R blocker) was kindly provided by Merck and Co. (Darmstadt, Germany). BLM was purchased from Nippon Kayaku (Tokyo, Japan). Alzet osmotic pumps (models 2004 and 2ML4) were purchased from Durect Corporation (Cupertino, CA). The other reagents are described subsequently here.

Animals

Male Wistar rats weighting 200–300 g were provided by the Central Animal Care Facility of Southern Medical University (Permission No. SCXK 2009-015). The animals were housed in a controlled environment (12 h light/12 h dark at 22–24°C), and received food and water ad libitum. All of the animals received humane care in compliance with the Chinese Animal Protection Act, which is in accordance with the National Research Council criteria.

Animal Treatment Regimens

We established two animal models. In the first model, 24 male Wistar rats were randomly divided into four groups of six rats each: a control group, a BLM treatment group; a BLM plus Ang-(1-7) treatment group; and a BLM plus AngII treatment group. All of the rats received a single intratracheal instillation of 200 μl of sterile saline while under pentobarbital anesthesia. The three BLM groups received sterile saline that contained 5 mg/kg of BLM sulfate. While the animals were under anesthesia, micro-osmotic pumps were implanted subcutaneously to enable 28 days of continuous infusion with AngII or Ang-(1-7) at a rate of 25 μg/kg/h. The animals in the control group and the BLM treatment groups received a constant subcutaneous infusion of saline.

In the second study model, 24 male Wistar rats were randomly divided into four groups: a control group; an Ang-(1-7) treatment group; an AngII treatment group; and an AngII plus Ang-(1-7) treatment group. Micro-osmotic pumps were implanted subcutaneously in these animals to enable 28 days of infusion with AngII and/or Ang-(1-7) at a rate of 25 μg/kg/h. The control animals each received a constant subcutaneous infusion of saline.

Cell Culture

Human fetal lung (HFL)-1 cells (fibroblasts) were obtained from the American Type Culture Collection (Manassas, VA). Pulmonary primary fibroblasts were isolated from rat lungs as previously described (21). The cells were cultured in α-Dulbecco’s modified Eagle medium (GIBCO BRL, Gaithersburg, MD) with antibiotics and 15% FBS. Next, the cells were preincubated for 1 hour with irbesartan (10−5 mol/L), A-779 (10−5 mol/L), PD98059 (10−5 mol/L), SB203580 (10−5 mol/L), SP600125 (10−5 mol/L), or BAY117082 (10−5 mol/L) before being exposed to AngII (10−7 mol/L) or Ang-(1-7) (10−7 mol/L) for 1 hour.

Statistical Analysis

All of the data are expressed as means (± SEM). The data were analyzed using an ANOVA with least significant difference for multiple comparisons. The differences were considered significant at P values less than 0.05. All of the data were analyzed using SPSS 13.0 (SPSS, Inc., Chicago, IL).

Complementary Assays

The use of hematoxylin and eosin, Masson’s trichrome, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining, the hydroxyproline assay, immunohistochemistry, immunofluorescence, quantitative real-time RT-PCR, the production of ACE2 lentiviral particles, and Western blot analysis are described in the online supplement.

Effects of Ang-(1-7) or AngII on BLM-Induced Pulmonary Fibrosis

The animals in the BLM group exhibited higher Ashcroft scores (22) compared with the control animals. The BLM-treated animals presented characteristic histological changes in lung tissue, including areas of inflammatory infiltration, thickening of the alveolar walls, increased interstitial collagen deposition, and a fibroblastic appearance. Chronic infusion with Ang-(1-7) resulted in a significantly lower Ashcroft score, indicating a protective effect of Ang-(1-7) against lung fibrosis. However, treatment with AngII significantly aggravated BLM-induced fibrosis, resulting in a higher Ashcroft score for the BLM plus AngII treatment group than for the BLM group (Figures 1A and 1C). Along similar lines, an increase in lung collagen accumulation (assessed by measuring the collagen area and lung hydroxyproline levels) was observed in the BLM group; this increase was significantly reduced by Ang-(1-7) treatment and exacerbated by AngII treatment (Figures 1D and 1E). Plaminogen activator inhibitor-1 (PAI-1) massage RNA (mRNA) levels and connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and α-collagen I protein levels increased in the lungs of the BLM group; the increases were significantly reduced by Ang-(1-7) treatment and augmented by AngII treatment (Figures 1F–1H; see also Figure E2 in the online supplement). Moreover, lung fibroblast apoptosis in vivo was confirmed using immunofluorescence with an FITC-conjugated terminal deoxynucleotidyl transferase dUTP nick end labeling reaction mixture in combination with CY3 NHS ester–conjugated anti-Vimentin antibody (Figure E1).

Exogenous Ang-(1-7) Regulated the Balance between the ACE/AngII/AT1R Axis and the ACE2/Ang-(1-7)-Mas Axis

The locally based RAS is involved in the development of lung fibrosis, and the ACE2/Ang-(1-7)/Mas axis counteracts the profibrotic effects of the ACE/AngII/AT1R axis. Hence, the balance of these two axes determines the development of pulmonary fibrosis. To evaluate the influences of Ang-(1-7) and AngII on the balance of these two axes in BLM-induced pulmonary fibrosis, the lung protein levels of ACE2 and ACE were assessed using immunohistochemistry (Figures 2A–2C) and Western blot (Figure 2D); the lung mRNA levels of components of the RAS, including ACE2, Mas, ACE, and AT1R, were measured using quantitative real-time RT-PCR (Figures 2E–2H). The results show that BLM treatment resulted in an obvious up-regulation of the ACE protein level and increases of approximately 21-fold and 32-fold in the ACE and AT1R mRNA levels, respectively. However, BLM treatment augmented the ACE2 protein level and increased the ACE2 and Mas mRNA levels roughly 1.7-fold and 7-fold, respectively, compared with the control group. Thus, BLM treatment shifted the balance toward the ACE/AngII/AT1R axis. Strikingly, Ang-(1-7) promoted the ACE2 and Mas levels and decreased the ACE and AT1R levels compared with the BLM group, regulating the balance from the ACE/AngII/AT1R axis toward the ACE2/Ang-(1-7)/Mas axis. In contrast, AngII shifted the balance toward the ACE/AngII/AT1R axis (Figure 2).

Constant Infusion with Exogenous Ang-(1-7) Inhibited the MAPK/NF-κB Pathway

It is well known that the MAPKs, including ERK1/2, p38, JNK, and NF-κB, are crucial for lung fibrogenesis. Increased phosphorylation of MAPKs can stimulate NF-κB activation; increased nuclear translocation of activated NF-κB initiates a cascade of responses, including abundant expression of proinflammatory factors, adhesion molecules, and cytokines that have been shown to be involved in the pathology of lung fibrosis. We determined the effects of administering Ang-(1-7) or AngII on the MAPK/NF-κB pathway induced by BLM. The results of these experiments revealed that exogenous Ang-(1-7) treatment inhibits the BLM-induced phosphorylation of ERK1/2, p38 MAPK, and JNK (Figure 3A), and inhibits the activation of the NF-κB cascade (Figures 3B–3D) in lung tissue. In contrast, the coadministration of AngII and BLM resulted in marked increases in the activation of the MAPK/NF-κB pathway (Figures 3A–3D).

Dual Effects of Exogenous Ang-(1-7) on the MAPK/NF-κB Pathway and Collagen Deposition in Lung Tissue

To determine whether Ang-(1-7) has a dual effect on the MAPK/NF-κB pathway in lung tissue, we performed a second experiment. The morphological results show higher scores for both the AngII and the Ang-(1-7) treatment groups compared with the control group, suggesting that the administration of Ang-(1-7) alone initiated lung inflammation and ECM accumulation. However, the coadministration of AngII and Ang-(1-7) resulted in a significantly lower score than administering AngII alone, demonstrating that Ang-(1-7) has a protective effect against lung injury, induced by AngII (Figures 4A–4D). Both Ang-(1-7) and AngII significantly increased the levels of ACE, AT1R, and Mas mRNA; thus, both up-regulated the two axes of the local RAS. Interestingly, the coadministration of Ang-(1-7) and AngII led to obviously higher ACE2 and Mas mRNA levels and markedly lower ACE and AT1R mRNA levels than the administration of AngII alone, suggesting that Ang-(1-7) facilitates the regulation of the balance of the two axes from the ACE/AngII/AT1R axis toward the ACE2/Ang-(1-7)/Mas axis (Figure 4E). Finally, both Ang-(1-7) and AngII significantly augmented the phosphorylation of ERK1/2, p38 MAPK, and JNK (Figure 4G), in addition to augmenting the activation of the NF-κB cascade (Figures 4F and 4H) and the protein levels of CTGF,α-SMA, and collagen (Figure 4I) in lung tissue. In contrast, the coadministration of Ang-(1-7) and AngII markedly attenuated the activation of the MAPK/NF-κB pathway and the deposition of collagen compared with the AngII treatment group (Figures 4F–4I).

Effects of Ang-(1-7) on the Phosphorylation of MAPKs in HFL-1 Cells

As demonstrated previously here, Ang-(1-7) plays a dual role in the phosphorylation of MAPKs in lung tissue. To determine whether Ang-(1-7) has similar effects in HFL-1 cells, we performed a third experiment. As shown in Figures 5A–5B, the Ang-(1-7) treatment alone stimulated the phosphorylation of ERK1/2 in HFL-1 cells in a dose- and time-dependent manner. However, Ang-(1-7) markedly blunted the phosphorylation of JNK, and did not affect the phosphorylation of p38. The Ang-(1-7) treatment also significantly decreased the phosphorylation of MAPKs stimulated by AngII; this effect could be reversed by A-779, a Mas antagonist (Figure 5C).

Ang-(1-7) Promoted Apoptosis and Suppressed AngII-Induced Collagen Production via Mas in Lung Fibroblasts

It is well known that the apoptosis resistance of lung fibroblasts facilitates the accumulation of fibroblasts and contributes to pulmonary fibrosis. To assess the effects of Ang-(1-7) or AngII on fibroblastic apoptosis, flow cytometry was performed (Figures 6A and 6B). The rate of early apoptosis of primary lung fibroblasts treated with AngII was lower (0.03%) than that in the control group (0.1%). In contrast, treatment with Ang-(1-7) alone markedly enhanced the rate of early apoptosis (14.1%) compared with the AngII-treated group and the control group. Moreover, the inhibitory effect of AngII on the early apoptosis rate could be reversed by Ang-(1-7), PD98059, SB203580, SP600125, and BAY117082. In addition, A-779 clearly abolished the effect of Ang-(1-7).

Next, we investigated the concrete mechanism. On the one hand, AngII inhibited the bax/caspase–dependent mitochondrial apoptotic pathway by up-regulating the B-cell lymphoma-2 (bcl-2) protein level and down-regulating the bax and caspase 3 protein levels, resulting in a decrease in lung fibroblast apoptosis (Figure 6C). On the other hand, AngII stimulated NF-κB signaling (Figure 5D), a cell survival pathway, leading to apoptosis resistance in lung fibroblasts. Ang-(1-7) markedly inhibited the AngII-induced MAPK/NF-κB pathway (Figure 5D) and activated the bax/caspase–dependent mitochondrial apoptotic pathway suppressed by AngII (Figure 6C), resulting in a reversal of the decrease in apoptosis induced by AngII (Figure 6A). Interestingly, Ang-(1-7) alone either stimulated NF-κB signaling (Figure 5D) or activated the bax/caspase–dependent mitochondrial apoptotic pathway in lung fibroblasts (Figure 6C), resulting in an increase in apoptosis.

Our study also found that AngII treatment resulted in significant increases in the mRNA levels of transforming growth factor (TGF)-β and PAI-1 (Figure 6E) and the protein levels of CTGF, α-SMA, and α-collagen I (Figure 6D) compared with those in the control group. However, the effects of AngII were suppressed by Ang-(1-7), MAPK blockers (Figures 6D and 6E), and the inhibitor of nuclear factor kappa-B kinase (IKK) inhibitor, BAY117082 (Figures E4B and E4C). The effects of Ang-(1-7) were negated by A-779 (Figures 6D and 6E). These results demonstrate that AngII protects lung fibroblasts against apoptosis and promotes the deposition of ECM, which can be inhibited by Ang-(1-7).

Lenti-ACE2 Inhibited the MAPK/NF-κB Pathway and the α-Collagen I Level Stimulated by AngII in HFL-1 Cells

To determine whether ACE2 protects against lung fibrosis in vitro, HFL-1 cells were transfected with lenti-ACE2. The overexpression of ACE2 protein in the transfected cells was verified with a Western blot assay (Figure 7A). We found that lenti-ACE2 attenuated the increase in the phosphorylation of MAPKs (Figure 7B), the activation of the NF-κB cascade (Figures 7C and 7D), and the increase in the CTGF, α-SMA, and α-collagen I protein levels (Figure 7E) induced by AngII; these effects of lenti-ACE2 could be reversed by A-779. The results indicate that ACE2, by cleaving AngII onto Ang-(1-7), inhibits the AngII-induced MAPK/NF-κB pathway and the synthesis of α-collagen I via Mas.

This study reached four novel conclusions with respect to the antifibrotic effect of the ACE2/Ang-(1-7)/Mas axis on pulmonary fibrosis. First, Ang-(1-7) regulates the balance of the RAS from the ACE/AngII/AT1R axis toward the ACE2/Ang-(1-7)/Mas axis. Second, Ang-(1-7) and lenti-ACE2 protect against BLM- or AngII-induced inflammation and ECM accumulation by inhibiting the MAPK/NF-κB pathway. Third, Ang-(1-7) inhibits the AngII-induced apoptotic resistance of lung fibroblasts by inhibiting the MAPK/NF-κB pathway and activating the mitochondrial apoptotic pathway. Fourth, Ang-(1-7) alone initiated the proinflammatory response in vivo and in vitro.

Research has found that the ACE/AngII/AT1R axis is up-regulated in BLM-induced lung fibrosis (4), resulting in increases in the ACE level, local AngII production, and the AT1R level, all of which contribute to the initiation and progression of lung fibrosis (23). Consistent with these reports, we observed that BLM enhanced the ACE and AT1R levels in the lungs. Constant infusion with exogenous AngII not only significantly aggravated lung fibrosis in BLM-treated rats, but also shifted the balance toward the ACE/AngII/AT1R axis. On the other hand, constant infusion with exogenous Ang-(1-7) down-regulated the ACE/AngII/AT1R axis by decreasing the mRNA or protein levels of ACE and AT1R, which led to the amelioration of lung fibrosis in BLM-treated rats. Interestingly, we found that the levels of both Mas and ACE2 were higher in fibrotic lungs than in normal lungs, which was not consistent with previous reports on lung fibrosis (4, 24). The inconsistent results may be due to the differences in the dosage and the duration of time used in our animal model and other investigators’ studies. In our study, BLM (5 mg/kg) -induced lung fibrosis lasted for 4 weeks. In a study by Li and colleagues (24), however, BLM (1 mg/kg) -induced lung fibrosis only lasted for 2 weeks. The differences in the dosage and the duration of animal model studies may affect the ACE2 protein levels. We suppose that, in the early stages of a BLM-induced lung injury (2 wk), the protective protein, ACE2, was inhibited; after that period, ACE2 gradually increased to protect against the injury. However, this explanation could be refuted by the finding that ACE2 is severely down-regulated in humans with idiopathic pulmonary fibrosis (24). Hence, the dynamics of ACE2 in fibrotic tissue deserve further study.

Although the ACE2/Ang-(1-7)/Mas axis ameliorated lung fibrosis in rats, the molecular mechanism remains unclear. Therefore, we performed additional experiments to determine whether the ACE2/Ang-(1-7)/Max axis attenuated lung fibrosis by inhibiting the MAPK/NF-κB pathway stimulated by AngII. The MAPK family (ERK 1/2, p38 kinase, and JNK) and NF-κB regulate cellular growth and inflammation, leading to lung fibrosis (13, 25, 26). Consistent with these reports, we found that the MAPKs and the NF-κB cascade were activated by BLM or AngII in vivo or in vitro. The study by Bancroft and colleagues (27) and our in vitro data show that inhibiting MAPKs suppressed the AngII-stimulated NF-κB cascade. Furthermore, the increased levels of TGF-β, CTGF, α-SMA, and α-collagen I induced by AngII could be inhibited by MAPK inhibitors and an IκK blocker. These data highlight the important role of the MAPK/NF-κB pathway in the pathogenesis of AngII-mediated lung fibrosis. However, in BLM- or AngII-treated rats and in lung fibroblasts, Ang-(1-7) prevented the phosphorylation of MAPKs and the NF-κB cascade; these results align with the results from other cell systems of lung (2830). Similarly, we also observed that the overexpression of ACE2 alleviated the proinflammatory and profibrotic effects of AngII. A-779, a Mas antagonist, was able to reverse the suppressive effects of ACE2, suggesting that ACE2 exerted its effect by generating Ang-(1-7) (8). These results indicate that the ACE2/Ang-(1-7)/Mas axis inhibited the MAPK/NF-κB pathway induced by AngII, leading to the attenuation of inflammation and collagen secretion.

A large number of reports have demonstrated that increased apoptosis of alveolar epithelial cells and decreased apoptosis of fibroblasts play important roles in the pathogenesis of pulmonary fibrosis (2, 31). Previous study has shown that apoptosis of alveolar epithelial cells was induced by AngII through the mitochondrial pathway (29). In contrast, we found that AngII up-regulated the bcl-2 protein level and down-regulated bax and caspase 3 protein levels, resulting in a decrease in lung fibroblast apoptosis. As a cell survival pathway, activation of the NF-κB pathway promotes the apoptotic resistance of fibroblasts (15). Consistent with this, we found that inhibition of the MAPK/NF-κB pathway could reverse down-regulation of bax and caspase 3 protein levels and the decreased apoptosis of lung fibroblasts induced by AngII. These results indicate that AngII promoted pulmonary fibrosis by inhibiting lung fibroblast apoptosis through activation of the MAPK/NF-κB pathway and inhibition of the bax/caspase–dependent mitochondrial apoptotic pathway. On the other hand, Studies by Uhal and colleagues (29, 32) showed that Ang-(1-7) could inhibit AngII- and endoplasmic reticulum stress–induced epithelial apoptosis. However, we found that Ang-(1-7) markedly promoted fibroblast apoptosis and activated the bax/caspase–dependent mitochondrial apoptotic pathway inhibited by AngII. The proapoptotic effects of Ang-(1-7) were reversed by pretreatment with A-779. Hence, we supposed that, besides inhibiting epithelial apoptosis, Ang-(1-7) also ameliorated lung fibrosis by suppressing the inflammation and the apoptotic resistance of lung fibroblasts via inhibition of the AngII-induced MAPK/NF-κB pathway.

Many studies on the biological properties of Ang-(1-7) have led to widespread consensus that Ang-(1-7) acts as a counter-regulatory peptide in the RAS, often opposing the inflammatory response and the proliferative actions of AngII. However, the effects of Ang-(1-7) are variable: inflammation and growth-stimulatory pathways are activated in some cases (3335). In agreement with these studies, the present study shows that constant infusion of Ang-(1-7) in rats significantly stimulated the MAPK/NF-κB pathway and increased collagen deposition in the lungs. Furthermore, Ang-(1-7) alone stimulated ERK1/2 phosphorylation in a time- and dose-dependent manner, but it blunted the phosphorylation of JNK in HFL-1 cells. Interestingly, although Ang-(1-7) alone stimulated NF-κB signaling (a cell survival pathway), it also promoted the apoptosis of primary lung fibroblasts by activating the bax/caspase–dependent mitochondrial apoptotic pathway. Therefore, in comparison with the NF-κB cascade, bax/caspase–dependent mitochondrial apoptotic signaling may be a main apoptosis-regulating pathway for Ang-(1-7).

How can Ang-(1-7) exert both protective and deleterious effects? To some extent, the variable responses to Ang-(1-7) can be explained by the state of local RAS activation. In the presence of high glucose levels, Ang-(1-7) attenuated the epithelial–mesenchymal transition and TGF-β1 production in rat kidney epithelial cells (36). In contrast, in the absence of RAS activation or high glucose levels, Ang-(1-7) induced epithelial–mesenchymal transition and increased TGF-β1 and CTGF production in rat kidney epithelial cells (37). In accordance with these results, we found that Ang-(1-7) attenuated inflammation and the accumulation of collagen in BLM- and AngII-treated animals, whereas Ang-(1-7) alone significantly induced an inflammatory response and collagen deposition. A plausible interpretation of these results is that, when the ACE/AngII/AT1R axis is activated by BLM or AngII, exogenous Ang-(1-7) not only significantly down-regulates the ACE/AngII/AT1R axis, directly attenuating lung fibrosis, but also up-regulates the ACE2/Ang-(1-7)/Mas axis, thereby facilitating the hetero-oligomerization of Mas (a physiological antagonist of AT1R) with the AT1R and interfering with AngII action (38). In contrast, in the absence of ACE/AngII/AT1R axis activation, Ang-(1-7) exerts proliferative and proinflammatory effects by interacting with the AT1R (39, 40). These results raise the possibility that the dual effects of Ang-(1-7) observed in the present study were determined by the state of activation of the ACE/AngII/AT1R axis. Nevertheless, in human aortic endothelial cells (41), Ang-(1-7) alone inhibited MAPK phosphorylation, reflecting the functional diversity of the heptapeptide in different cell types. The exact molecular mechanism of Ang-(1-7)/Mas signaling in different cells deserves further examination.

In summary, our study demonstrate that exogenous Ang-(1-7) and ACE2 overexpression protect against BLM- or AngII-induced pulmonary fibrosis by down-regulating the MAPK/NF-κB pathway. However, constant infusion of Ang-(1-7) paradoxically initiates an inflammatory response in the lungs. The antifibrotic effects of Ang-(1-7) noted here make the heptapeptide a strong candidate for a therapeutic target in humans with pulmonary fibrosis. Further studies to identify the precise mechanism of its action are needed to fully understand its role and to determine the therapeutic possibilities.

The authors thank Prof. Ji-Man He for his critical review of this manuscript.

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Correspondence and requests for reprints should be addressed to Xu Li, Ph.D., Department of Emergency Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China. E-mail:

This work was supported by National Science Foundation of China research grant 30900659.

Author Contributions: Conception and design, X.L., S.X.-C.; analysis and interpretation, Y.M., C.-H.Y., W. Li, T.L., W. Luo, S.H., P.-S.W.; drafting the manuscript for important intellectual content, Y.M., C.-H.Y.

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2012-0451OC on October 29, 2013

Author disclosures are available with the text of this article at www.atsjournals.org.

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