The renin–angiotensin system (RAS) is a critical component in regulating multiple tissue and organ functions, such as those of the cardiovascular system, kidney, lung, and liver, specifically by maintaining homeostasis of blood pressure, electrolyte balance, and inflammatory responses (1, 2). Conventionally, the RAS was viewed as a systemic regulatory mechanism, but findings from the last few decades have revealed that the RAS also plays a critical role locally in various organs and tissues. Normally, the RAS is constitutively active to execute biological functions and it is regulated by an array of stimuli to maintain physiological hemostasis. Altered activation of the RAS is attributed to the pathogenesis of many diseases such as hypertension, myocardial infarction, heart failure, diabetes, and inflammatory lung disease (3–5).
Renin, a protease that is generated predominantly in the kidneys, cleaves angiotensinogen to generate angiotensin I (Ang I). Subsequently, angiotensin-converting enzyme, a protease critically involved in regulating the RAS, cleaves Ang I to produce Ang II. Ang II is a key effector of the RAS and exerts biological functions through the two Ang II receptors: Ang II receptor type 1 (AT1R) and Ang II receptor type 2 (AT2R) (6–8). Ang II can be hydrolyzed by various angiotensinases, such as angiotensin-converting enzyme 2 (ACE2), and neprilysin to generate Ang1–7, Ang III, Ang IV, and Ang A (9), which can bind to their respective receptors to act as an agonist or an antagonist for ANG II receptors to mediate physiological processes.
In supporting a notion that RAS is a multicrine system, several new factors of the RAS have been uncovered. One of the novel members of the RAS is angiotensin-converting enzyme 2 (ACE2), a terminal carboxypeptidase and a type I trans membrane glycoprotein, which was the first homolog of human angiotensin converting enzyme cloned in 2000 (10). Importantly, ACE2 has emerged as a potent negative regulator of the RAS, and the imbalanced activity of ACE/ACE2 systemically and/or locally has been proposed to be an important contributor in many disease pathogeneses including inflammatory lung disease (11).
ACE2 gene contains 18 exons, which code for an 805 amino acid type I transmembrane glycoprotein. In mammals, ACE2 is quite conserved at the protein level in that all mammals display a 70% or higher identity of the protein (12). Although ACE2 maps to the X chromosome, sex-related differential gene and protein expression has not been consistently observed. For instance, reported by Xie et al. (13), there was no sex-related difference of ACE2 protein level in young-adult and middle-aged rat lung; however, a significantly higher ACE2 content was detected in old female rat lung than in counterpart male. Chen et al. observed no sex effect in sheep renal ACE2 mRNA during development. But they further investigated that the regulation of ACE2 is more complex and the effect of estrogen on ACE2 is different depending upon the physiological or pathophysiological status of the subject (14).
Systemically and locally, RAS does not function alone; it has been well recognized to cross talk with other systems while pursing its biological functionality. In the setting of inflammation, the cross talk between RAS and the Kinin–Kallikrein system (KKS) needs to be highlighted in that it has been well delineated that among the systems that are activated during inflammation, the KKS plays a prominent role. The KKS–RAS systems have long been thought to participate in several biological and pathological processes and interact at various levels (15). This concept has been validated not only by their functional interaction, but also by the identification of a structural network (16). Therefore, it is appropriate to consider the two highly regulated proteolytic systems, involved in various physiological and pathological processes, a biological network. In addition, des-Arg9-bradykinin (DABK), a bioactive metabolite of bradykinin, is a substrate of ACE2 in biochemical setting and if DABK is proven to be a biological substrate of ACE2, which will provide an additional factor to the already complicated network (Fig. 1).
The current review will focus on recent studies of pulmonary ACE2 biology, its roles in inflammatory lung disease pathogenesis, and possible underlying mechanisms. Finally, we will discuss our perspectives on pulmonary ACE2 as a potential therapeutic target for inflammatory lung disease.
Studies of ACE2 mRNA distribution and enzymatic activity in rodent and human tissues confirmed the hypothesis of complete local tissue-related RAS activity (17, 18). Although highest ACE2 mRNA expression levels were detected in the intestinal epithelium, pulmonary ACE2 expression and function have been given extensive attention in recent years due to the findings that ACE2 serves as the receptor for the Severe Acute Respiratory Syndrome Corona Virus (19) and its beneficial role in acute lung injury (20).
Rodent pulmonary ACE2 expression, as that in other tissues, is developmentally regulated (21, 22), even though the ontogeny of mRNA levels and protein abundance has been conversely detected. ACE2 mRNA levels were observed to be the highest on E14 and lowest when mice reached adulthood, whereas the protein abundances were detected adversely (22). This observation is, in part, a reflection of a supply/demand balance at the time when the ACE2 was detected. Interestingly, ACE2 mRNA expression is not only developmentally regulated, but also impacted by aging. A study of ACE2 gene expression revealed that in young adult rat lung, ACE2 mRNA levels are almost 2 to 3-fold higher than that in middle aged or old rat lung (13).
In the lung, the ACE2 protein colocalizes with cholesterol and sphingolipid-rich lipid raft microdomains in the plasma membrane (23, 24) and its expression level is positively correlated to the state of airway epithelial differentiation (11, 25). Interestingly, ACE2, as its homolog ACE, is released from the surface of epithelia into airway surface liquid (26) via cleavage by TACE (ADAM17) and other sheddases (27, 28). This soluble ACE2 (sACE2) is catalytically active in ASL. Release of sACE2 from airway epithelial cells is dynamic, occurring both constitutively and in response to stimuli. Previous research indicated that a single mutation in human ACE2 gene at residue 584 prevented ACE2 shedding from the cell membrane (27). In vitro studies on non-sheddable ACE2 cell surface expression have demonstrated impaired enzymatic activity, suggesting that releasing ACE2 from the cell membrane is a critical step in catalyzing substrates and implying that attenuation of ACE2 shedding might contribute to disease pathogenesis in which ACE2 activity is crucial in disease initiation and progression.
ACE2 and acute lung injury (ALI)
Many detrimental factors can cause ALI such as viral infection (SARS-CoV, H1N1, H5N1 influenza viruses) (29, 30), bacterial pneumonia, sepsis, remote lung injury from pancreatitis, trauma or inflammatory gastrointestinal diseases, and some noninfectious/inflammatory incidences such as gastric aspiration (31, 32). Compelling evidence in recent years has demonstrated a pivotal role of ACE2 in the pathogenesis of acute lung injury (33, 34). A clear example of a role that ACE2 plays in the pathogenesis of ALI is SARS. In this special case, ACE2 is a multifaceted player. On the one hand, it is the receptor for SARS-CoV and our previous studies linked the susceptibility of human airway epithelia to SARS-CoV infection with the state of cell differentiation and ACE2 expression (11, 25). We and other groups have demonstrated that ACE2 is predominantly localized on the apical surface of well-differentiated airway epithelia, especially ciliated cells. Since a virus must dock and enter the cells before it can replicate, surface localization of ACE2 and the state of cell differentiation may have strong impact on SARS-CoV disease and other pathologies triggered by acute lung injury. On the other hand, once SARS-CoV binds to its receptor, the abundance on the cell surface, mRNA expression, and the enzymatic activity of ACE2 are significantly reduced. These effects are, in part, due to enhanced shedding/internalizing processes and other unknown mechanisms (35, 36). The work by Kuba et al. in a mouse acute lung injury model induced by SARS-CoV spike protein, which mimicked the active SARS-CoV-mediated lung injury, also suggested that the spike protein binds to ACE2 and subsequently down regulated ACE2 protein expression and resulted in worsened acid aspiration pneumonia. They also demonstrated that the mechanism underlying this exacerbated pneumonia by lacking of ACE2 in the lung is that Ang II levels were elevated and vascular permeability was enhanced. So they proposed that ACE2 and other components of the renin–angiotensin system might play a pivotal role in the pathogenesis and progression of acute lung failure from a variety of cues (37).
Imai et al. discovered in murine ARDS models of acid aspiration and sepsis that the lack of ACE2 expression in the lung resulted in attenuated vascular permeability, enhanced lung edema, neutrophil infiltration, and further deteriorated lung function. More interestingly, catalytically active but not mutant, catalytically inactive recombinant ACE2 protein alleviates the symptoms of acute lung injury in wild-type mice, as well as in ACE2 knockout mice, suggesting functional ACE2 protects the lung from acute injury (20). The above observation was further evidenced in a recent report of large animals in an endotoxin infusion-induced ARDS model. In this study, active ACE2 protein, supplied through i.v., significantly improved the outcome of respiratory failure by its ability to increase the oxygen levels by almost 40% in pigs (38).
As mentioned earlier, our group and others confirmed a soluble form of ACE2 in the lung. In response to stimuli, many membrane proteins undergo either shedding or internalization as a negative feedback to control the biological and pathological processes. Enhanced pulmonary ACE2 shedding has been observed in several in vitro and in vivo models. Haga et al. reported that the S protein from SARS-CoV, once binds to ACE2, induces ACE2 shedding by further activating cellular Adam17 (TACE). Ours own study indicated that bacterial endotoxin, LPS, also induces ACE2 shedding. The results seem to suggest a negative feedback on SARS-CoV infection and other infectious and inflammatory status. Indeed, our study demonstrated that soluble ACE2 does not facilitate SARS-CoV S protein pseudotyped FIV transduction in vitro and the pseudotyped FIV transduces cells coexpressing full length of human ACE2 and ectodomain of human ACE2 poorly, a observation that strongly supported the notion that shed ACE2 serves as a soluble receptor for SARS-CoV and prohibits the viral entry of the cells. However, Haga et al. discovered in their model that shedding of ACE2 enhanced SARS-CoV and S protein pseudotyped FIV transduction and exacerbated viral infection. The result highlighted the importance of Adam17 in the processes of SARS-CoV entry of the cells because our in vitro model does not involve the action of Adam 17 to generate soluble ACE2. The role of soluble ACE2 in other infectious and inflammatory lung disease has not been extensively investigated due to the lack of animal models, in which only soluble ACE2 or membrane-bound ACE2 expressed.
Taken together, these studies suggested that the attenuation of ACE2 catalytic function perturbs the pulmonary renin–angiotensin system (RAS), resulting in enhanced inflammation and vascular permeability. Thus, ACE2 may serve as a novel therapeutic reagent for acute lung injury.
Ang II/AT1R axis, Ang1-7/MAS1R axis, and role of ACE2 in the pathogenesis of ALI
Although it is an adopted view that ACE2 plays a protective role in ALI and a lack of functional ACE2 in the lung might contribute to the pathogenesis of ALI, the underlying mechanisms remain incompletely understood. Angiotensin II (Ang II) is the key player in RAS functioning and the predominant substrate of ACE2 in current understanding. There is a great deal of research which aims to dissect the mechanisms as to how ACE2 participated in the initiation and progression of ALI has been focused on ACE2 enzymatic activity, its substrate Ang II and its catalytic product Ang1–7.
Ang II exerts multiple biological actions by stimulating its two specific receptors, Ang II receptor subtype 1 (AT1 R) and subtype 2 (AT2 R). In general, AT1R and AT2R mediate the adverse effects upon Ang II stimulation; however, other studies have also indicated that Ang II stimulates expression of proinflammatory mediators such as interleukin-8/Cytokine-induced Neutrophil Chemoattractant -3 and interleukin-6 via both receptor subtypes (39). Substantial evidence indicates that Ang II triggers inflammatory process in multiple tissues and cells mainly through the activation of AT1R. Once AT1R is activated, it activates transcription factors NF-kB and activating protein 1 (AP-1) (40) to induce cytokine expression, apoptosis, vasoconstriction, fibroproliferation, the retention of Na+, and the enhancement of lung injury. The endogenous Ang II inhibits alveolar fluid clearance (AFC) and dysregulates ENaC expression via AT1R, which contribute to alveolar filling and pulmonary edema in LPS-induced ALI. Plasma and lung Ang II levels were dramatically increased in a time-dependent manner in models of rat ALI (41). These results are consistent with studies in which the AT1R antagonist, losartan, prevented ALI-induced interstitial edema and inflammatory cell infiltration. The mechanism of losartan against ALI/ARDS cannot be attributed to an antihypertensive effect since the blood pressures of rats in all experimental groups did not differ. It is highly likely that losartan has its anti-ALI/ARDS effect through inhibition of the activity of Ang II and AT1 receptor signaling. Recently, Yang et al. (42) demonstrated that lack of ACE2 in the lung exacerbates H7N9 virus-induced acute lung injury by failing to inactivate Ang II/AT1R pathway. Yu et al. (43) also reported that ACE2 could reduce vascular permeability in acute lung injury by antagonizing VEGFa in vascular endothelia. The observation is in agreement with the report by Ji et al. (44), in which they indicated that ACE2, by inhibiting Ang II cascade, could suppress the ALI-induced pulmonary vascular endothelial apoptosis, thus protect lung from injury.
Given the fact that Ang II has a very short half-life, about 15 min in local tissue compartments (45), most in vitro and in vivo experiments that focus on investigating the roles of Ang II in inflammatory process have to undergo long term or continuous perfusion or stimulation. Thus, these model systems may inadvertently therefore be studying unexpected physiological responses that are induced not by Ang II per se. For instance, several signal transduction pathways have been proposed to be involved in Ang II/ AT1R cascades under the same condition in the same cells (46); some studies suggested that toll-like receptor 4 (TLR4) activation up-regulates AT1R expression but an LPS-induced decrease in AT1R expression was observed by other investigators in whole rat and in human mesangial cells (47, 48). Imai et al. (20) elucidated that ACE2-deficient mice displayed drastic symptoms of ARDS, and the detrimental phenotype could be altered by concomitant ablation of ACE gene expression or supplementation of AT1 receptor antagonists. Nonetheless, one of the consequences of impaired ACE2 activity in the lung is reduced production of Ang1–7, a bioactive metabolite of Ang II by ACE2. Similar to AT1R antagonists, Ang1–7, binding to its receptor namely Mas1R, promotes an array of biological responses to counteract Ang II mediated processes such as apoptosis, angiogenesis, vasoconstriction, and inflammation in many tissues and organs including the lung (31, 49). The half-life of Ang1-7 in circulation is approximately 10 s and circulating levels of Ang1-7 are reported to be 20 pg/mL (50). Although the mechanisms by which Ang1-7 exert their effects in these systems are still largely unknown, studies by Rowe et al. (51) suggested that, due to low affinity, the effects of Ang1-7 were unlikely to be mediated by signaling via the angiotensin receptors. Receptor-binding studies later demonstrated that Ang1–7 could bind to the G-protein coupled receptor Mas1R. Only until 2007 has the ACE2/ Ang1–7/ Mas1R pathway developed as a potential target for the regulation of blood pressure control (52). Further research in Mas-deficient mice identified Ang1–7 as the endogenous ligand for this receptor. Moreover, blockade of Mas1 receptor by D-Ala7-Ang1–7 (A-779), the selective antagonist of Mas receptors, completely inhibited the Ang1–7-induced physiological processes while AVE 0991, the agonist of Ang1–7 receptors, mimicked the actions of Ang1–7 (53). Interestingly, despite that Ang1–7 has relatively low affinity for the AT2R, it may also elicit certain biological effects via this receptor. For instance, in stable cell lines that express either the AT1R or AT2R, Ang1–7 was found to bind the AT2R with higher affinity than the AT1R (54). The Ang1–7- AT2R interaction has also been observed in vivo(55). Several studies have demonstrated the anti-inflammatory effects of the angiotensin receptor 1 blockers (ARBs) Telmisartan and Olmesartan (56). Other investigators reported that ARBs induce ACE2, Ang1–7, and Mas expression in line with reduction of pro-inflammatory cytokines and induction of IL-10, an anti-inflammatory cytokine. These anti-inflammatory effects of ARBs were associated with the down-regulation of multiple signaling pathway-related proteins such as PI3K, phospho- Akt, phospho-p38 MAPK, phospho-JNK, phospho-ERK, and phospho-MAPK-2 (57) (Fig. 2).
ACE2 and chronic inflammatory lung diseases
Although direct evidence that ACE2 plays a role in chronic inflammatory lung disease pathogenesis is limited, yet, its potential roles in the diseases have been suggested. Especially its effects on Ang II/ AT1R and Ang1–7/Mas1R axes make ACE2 a plausible target in preventing and treating chronic inflammatory lung diseases.
ACE2 and pulmonary fibrosis
Excessive inflammatory cell influx and pro-inflammatory cytokine release in the acute phase of inflammation have been proposed to be one of the initial steps of tissue fibrosis that leads to the chronic phase of inflammation. ACE2 modulates neutrophil infiltration in the lung by inhibiting Ang II/AT1R axis, thus potentiates injured lungs prone to fibrotic. TGF-β1 is the most potent profibrotic cytokine and acts downstream of Ang II in vascular smooth muscle, myofibroblasts, and macrophages (58, 59). However, positive feedback of TGF-β on Ang II expression has also been reported (60). Recent studies have evaluated the role of ACE2/ Ang1–7 /Mas axis in modifying expression of TGF-β1 and key components of the downstream pathway. A similar observation was made in a study that examined intratracheal administration of lentiviruses expressing Ang1–7 in lungs of rats given bleomycin (61). The report is supportive of the notion that regulation of TGF-β synthesis contributes significantly to the anti-fibrogenic effects of the ACE2/ Ang1–7 /Mas axis. Evidence is emerging to indicate that ACE2/ Ang1–7/Mas axis might block key signaling profibrogenic events initiated by Ang II, endothelins, and other profibrogenic molecules (57, 62). Proliferation is also a critical step for the fibrotic process. Studies demonstrated that ACE2/ Ang1–7/Mas axis activation inhibits the growth of human lung cells via reduction in serum-stimulated phosphorylation of ERK1 and ERK2 (63, 64).
ROS generation plays an important role in lung fibrosis and nicotinamide adenine dinucleotide phosphate oxidases (NOXs) have been proposed to be the leading sources of ROS in the fibrotic lung (65, 66). ROS could also activate one of small GTPase, RhoA, to regulate cell migration and α-collagen I secretion.
Substantial evidence indicates that Ang II, by binding to the AT1 receptor, induces NOX-dependent generation of superoxide and initiates extra cellular matrix deposition. Thus, Ang II is an important profibrotic mediator to induce normal lung fibroblast migration and collagen synthesis (67). In contrary, ACE2, by converting Ang II to Ang1–7, counter the Ang II-induced lung fibrosis. It has been reported that treatment with Ang1–7 or ACE2 improved lung fibrosis and that ACE2 depletion exacerbated collagen deposition in mice (68). Recently, Meng et al. (69) reported that ACE2 overexpression prevented Ang II-induced lung fibroblast migration and BLM-induced lung fibrosis by inhibiting the NOX4/ROS/RhoA/Rock pathway.
ACE2 and COPD
Chronic inflammation of the central and peripheral airways has been recognized as a hallmark of chronic obstructive pulmonary disease (COPD), which encompass the accumulation of inflammatory cells and the release of proinflammatory cytokines (70, 71). The effects of the RAS on COPD have also been highlighted by the actions of Ang II. Clinical research data has demonstrated a 5 to 6-fold increase in the AT1R/AT2R ratio in regions of marked fibrosis surrounding bronchioles, which correlated with a reduction in FEV1 (72). Ang II stimulates the release of multiple cytokines, especially, alveolar macrophage-derived MCP-1, which has been shown to activate mast cells in response to acute alveolar hypoxia, thus triggering systemic inflammation (73). Indeed, blockage of Ang II reduces cytokine production. In addition, Ang II mediated ROS generation, mitochondrial dysfunction and impaired redox signaling have been observed in COPD (74, 75). Direct evidence regarding the role of ACE2 in COPD is limited currently. Tian et al. recently studied the effects of ACE2 over expression on lung function and inflammatory responses, as well as oxidative stress in a COPD rat model. They showed that ACE2 mRNA expression in COPD rat lung significantly decreased compared with that in wild-type animals (74). Over expressing ACE2 in rat trachea significantly improved the lung function and pathological manifestations of COPD, indicating that down regulation of ACE2 and imbalance of ACE/ACE2 are involved in the pathogenesis and progression of COPD.
ACE2 and pulmonary hypertension
Despite many years of intensive research, the precise mechanism of pulmonary hypertension (PH) remained unknown. However, chronic inflammation and deregulation of endothelial nitric oxide synthase (eNOS) with enhanced oxidative stress have been proposed to be the major causes of PH (76, 77).
The RAS has long been suggested to play a role in the pathogenesis of PH, and prominent research of such kind also focused on the effects of Ang II. Several lines of study have indicated that Ang II promotes the development of PH by inducing vasoconstriction, the proliferation of endothelial cells, and cell/tissue inflammation (78, 79). It is conceivable that ACE2 has been hypothesized to play a protective role in the progression of PH. In supporting this notion, several studies demonstrated that the exogenous expression ACE2 or Ang1–7 blocks experimental PH by suppressing Ang II-induced inflammation and oxidative stress (80, 81).
Right ventricle (RV) remodeling characterized by myocyte hypertrophy and fibrosis has been observed in PH patients at late stages of the disease (82, 83). Zisman et al. (84) revealed that ACE2 activity and Ang1–7 content are higher in PH patients, suggesting a compensatory response of the two to protect the heart. Others also reported the protective role of ACE2 in modulating RV remodeling and heart failure (85, 86).
Additional mechanisms by which ACE2 plays a protective role in PH have also been studied. For instance, studies have indicated that attenuated ACE2 activity is associated with hyper proliferation and enhanced migration of pulmonary smooth muscle cells (87). In vivo investigations have also shown that overexpression of ACE2 in the lung inhibited pulmonary blood vessel wall thickening and muscularization of blood vessel (88). Thus, an ACE2 activator to be used in treating PH patients and preventing progression of the disease has been proposed (89, 90).
ACE2 and asthma
Bronchial hyperresponsiveness, an enhanced bronchoconstrictor response to inhaled stimuli, is a common and core feature of asthma. Asthma is characterized predominantly by eosinophilic inflammation and inflammation involving type 2 helper T (Th2) lymphocytes. Patients with asthma may also exhibit neutrophilic inflammation if the asthma is severe and late-onset or chronic infections is involved (91, 92). Exhaled nitric oxide has been described as a marker of asthmatic airway inflammation (93). Additionally, reactive oxygen species (ROS) production may play an important role in the pathogenesis and progression of asthma (94).
The role of RAS in the pathogenesis of asthma has also been focused on angiotensin-converting enzyme and the effector Ang II. As of now, there is no direct evidence supporting a role of ACE2 in the pathogenesis of asthma; nonetheless, its anti-inflammatory nature and ability to inactivate Ang II/ATR1 axis and activate Ang1–7/Mas1R axis, the two counteractive systems strongly related to the asthmatic lung disease, ensured possible role of ACE2.
Previous studies indicated that AT1 receptor activation by Ang II played a critical role in bronchoconstriction (95). A report also suggested that Ang II can potentiate endothelin-1-induced contraction of bovine bronchial smooth muscle (96). AT1 receptor antagonist was proven to prevent eosinophil accumulation and antigen-induced airway hyper-responsiveness in guinea pigs (97) and to regulate airway remodeling process in asthmatic rat model (98).
Accumulating experimental results indicated that Ang1–7, an ACE2-generated metabolite of Ang II, through its anti-inflammatory effect, can attenuate allergic asthma (99, 100). In an ovalbumin-challenged mouse model of allergic asthma, Ang1–7 altered ovalbumin-induced increases in total cell counts, eosinophils, lymphocytes, and neutrophils. Ang1–7 also decreased the ovalbumin-induced perivascular and peribronchial inflammation, fibrosis and goblet cell hyper/metaplasia. Mas-1 receptor antagonist A779 can abolish the effect of Ang1–7, suggesting Ang1–7/Mas1R axis is involved in the process (100). A similar study reported by Magalhaes et al. also concluded that Ang1–7 exerted beneficial attenuation of three major features of chronic asthma: lung inflammation, airway remodeling, and hyperresponsiveness. Their results further supported an important protective role of Ang1–7 in allergic asthma (99).
ACE2 and pulmonary stem/progenitor cells
Mesoderm-derived mesenchymal stem cells (MSCs) are pluripotent stem cells with a great capacity for self-renewal while maintaining their multipotency. Substantial evidence is emerging that MSCs are involved in inflammation suppression, anti-oxidative stress, and anti-apoptotic processes critical for lung repair and regeneration (101, 102). Lung repair and regeneration is complex and conditional. Basal cells, Clara cells, type-II penumocytes, and the putative pulmonary stem/progenitor cells are proposed to contribute to lung repair in response to lung injury (103, 104). These cells are characterized as CD34+ Sca-1+ CD45− PE-CAM−, localized at the bronchoalveolar duct junction of adult lungs and are termed bronchoalveolar stem cells (BASCs) (105). In addition, the POU-homeodomain transcription factor Oct-4 is expressed in the cells at the bronchoalveolar junction of the neonatal lung (106). Interestingly, Chen et al. showed that the stem/progenitor cell markers CD34 and Oct-4, but not markers for cytokeratin or surfactant, are expressed in SARS-CoV-infected lung cells, which are known to express ACE2, the receptor for SARS-CoV. These proposed lung stem/progenitor cells have also been identified in some non-SARS individuals and are capable of being infected by SARS-CoV ex vivo(107). It is hypothesized that infection of these stem/progenitor cells by SARS-CoV may contribute to the loss of lung repair capacity and thus lead to respiratory failure. This observation raised a very important mechanistic question: what is the role of ACE2 in these putative stem/progenitor cells in the lung? Answers to this question are limited thus far. Liu et al. (32) reported recently that ACE2 facilitates human umbilical cord mesenchymal stem cells to heal ischemia-reperfusion-induced lung injury, suggesting a possibility that ACE2 could confer MSCs proliferation or differentiation and/or both in the process. Similar effects of ACE2 on endothelial progenitor cells were observed as well (108) and subsequent investigations demonstrated that AC2 improves endothelial progenitor cell function via regulating eNOS and Nox pathways (109).
Ang II has long been shown to have dual effects on embryonic stem cells and other stem/progenitor cells (110, 111). It promotes proliferation/differentiation of the cells, defined as an angiogenic effect, by modulating RhoA and MAPK signalling (112), but it also mediates apoptotic processes of the cells, termed an inflammatory effect, via NFκb-AP-1- MAPK signalling (113, 114). It is tempting to speculate that it is the balance of the two effects of Ang II that regulates, in part, the stem/progenitor cell function in tissues or cells where the RAS plays an important role in regeneration and renewal. Conceivably, ACE2 plays a critical role in this process. In this regard, further investigation of the roles and mechanisms of ACE2 in regulating/modulating stem/progenitor cells in the lung under native and injurious condition is needed (Table 1).
CONCLUSIONS AND PERSPECTIVES
The critical roles of the RAS, especially ACE2, in airway inflammation and lung injury have been evidenced by an array of basic and clinical research. ACE2 participates in all phases of inflammatory lung disease, including induction of lung inflammation, progression of inflammatory lung disease, and the repair and regeneration of injured lung. The involvement of ACE2 in these processes relies on both its enzymatic activity and non-catalytic functionality in the lung, although a majority of the research has focused on its altered activity, as metalloproteinase, during inflammatory lung disease. Current thinking indicates that ACE2, by removing a single residue of Ang II at its C-terminus, generates Ang1–7. Moreover, it is thought that the attenuation of ACE2 activity or imbalance of the ACE/ACE2 ratio will have a phenomenal impact on Ang II/AT1R and Ang1–7/Mas axes resulting in impaired regulation of inflammatory processes in the lung and other tissues/organs. However, Ang II is not the sole target of ACE2 as a metalloproteinase, and Ang II/AT1R, Ang1–7/Mas axes are not the only effector system of ACE2 enzymatic activity. As suggested by Penninger's group, it is possible that substrates of ACE2 other than Ang II might play an equal or even more important role in the pathogenesis of inflammatory lung disease (20, 37). The notion highlights the importance of finding new biological substrates of ACE2 in the lung to better understand ACE2 biology.
Another potential area of research that deserves more attention is the role of ACE2 in remote lung injury. For instance, a few groups have demonstrated that inflammatory intestinal/colon diseases such as colitis and necrotizing enterocolitis could remotely induce inflammatory lung disease/injury manifested by neutrophil infiltration, pro-inflammatory cytokine release and oxidative stress (115, 116). It is well known that the GI tract, especially the small intestine, is the most abundant ACE2 expression site and studies have shown that ACE2 expression and activity are attenuated in inflammatory colitis (117, 118). It is possible that the impaired local ACE2 in intestine during the course of inflammation has an impact on lung inflammation remotely. Another example of remote organ damage is traumatic lung injury. It has been discovered that intestinal epithelial TLR4 activation is required to induce lung injury in a mouse traumatic model (119). It is also possible that trauma, via the activation of intestinal epithelial TLR4, impairs intestinal ACE2 activity and in turn facilitates excessive neutrophil influx in the lung, which subsequently induces lung injury.
Although it is still debatable as to which cells serve as stem/progenitor cells in the lung, the beneficial effects of stem/progenitor cells in repairing the damage from inflammatory lung disease have been reported. The mechanism underlying how stem/progenitor cells repair lung damaged by inflammation is still far from clear. Interestingly, ACE2 is not only expressed on the cell membrane and released to extracellular compartments. Several lines of study indicated that ACE2 is expressed within nuclei (120, 121). The role of nuclear ACE2 is not fully understood, but limited studies have demonstrated that exogenous ACE2 could facilitate MSCs proliferation and differentiation and participate in healing injured lung from inflammatory lung disease (32, 122). So it is not unreasonable to hypothesize that nuclear ACE2, via an unknown mechanism, affects gene transcription, editing, and repair; thus, modulating cell proliferation/differentiation and impacting the healing processes of lungs injured by inflammation.
It is the beneficial effects of ACE2 that make it a popular idea that recombinant active ACE2 can be used as therapeutic reagent to prevent and treat inflammatory lung injury. In fact, a few clinical trials are under way based upon this idea. There is no doubt about the beneficial effects of supplementing ACE2 to individuals who suffer from inflammatory lung disease, but some concerns remain. For stance, Takahashi et al. (123) reported the detection of an ACE2 autoantibody from patients with connective tissue pathologies, including pulmonary hypertension and persistent digital ischemia, and the antibody significantly reduced ACE2 activity in serum. So far, there is no report yet about recombinant ACE2-induced antibody generation within the same species; however, the potential of such exogenous ACE2-induced antibody and associated pathologies is still a possibility. Thus, the usage of ACE2 activator in preventing and treating inflammatory lung disease might be a better approach instead. Currently, several small-molecule ACE2 activators have been identified (89, 124), so it is feasible to select a potent ACE2 activator with the least side effects for clinical practice. Moreover, angioedema, which occurs as an episodic, adverse effect to ACE inhibitor, presents the emergency physicians with some of the most challenging and distressing airway emergencies encountered in common clinical practice (125). Although the rate of angioedema is low, it could be life threatening. ACE2 primarily functions as an ACE inhibitor but it further promotes Ang1–7/Mas axis activity, to which ACE inhibitor is unable. We do not know if the latter activity of ACE2 would mosaic the potential ACE inhibitor-like effect of angioedema. Obviously, the possibility needs to be closely watched in animal models and in people who undergo ACE2 clinical trial.
It has been over a decade since the discovery of ACE2. It is still a long way to go to fully understand ACE2 biology and the RAS in general. Nevertheless, the potential for ACE2 as a therapeutic target in preventing and treating inflammatory lung disease is evident.
The author is tremendously grateful to Dr David J Hackam for his unequivocal and generous supports, especially for his efforts in reading and editing this article critically. The author also thanks Ms Ana M. Niño for her scientific review and editing.
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