Targeting ATP12A, a Nongastric Proton Pump α Subunit, for Idiopathic Pulmonary Fibrosis Treatment.

Idiopathic pulmonary fibrosis (IPF) is a pathological condition of unknown etiology that results from injury to the lung and an ensuing fibrotic response that leads to the thickening of the alveolar walls and obliteration of the alveolar space. The pathogenesis is not clear, and there are currently no effective therapies for IPF. Small airway disease and mucus accumulation are prominent features in IPF lungs, similar to cystic fibrosis lung disease. The ATP12A gene encodes the α-subunit of the nongastric H+, K+-ATPase, which functions to acidify the airway surface fluid and impairs mucociliary transport function in patients with cystic fibrosis. It is hypothesized that the ATP12A protein may play a role in the pathogenesis of IPF. The authors' studies demonstrate that ATP12A protein is overexpressed in distal small airways from the lungs of patients with IPF compared with normal human lungs. In addition, overexpression of the ATP12A protein in mouse lungs worsened bleomycin induced experimental pulmonary fibrosis. This was prevented by a potassium competitive proton pump blocker, vonoprazan. These data support the concept that the ATP12A protein plays an important role in the pathogenesis of lung fibrosis. Inhibition of the ATP12A protein has potential as a novel therapeutic strategy in IPF treatment.

Stem-Cell Therapy for Bronchopulmonary Dysplasia (BPD) in Newborns.

Premature newborns are at a higher risk for the development of respiratory distress syndrome (RDS), acute lung injury (ALI) associated with lung inflammation, disruption of alveolar structure, impaired alveolar growth, lung fibrosis, impaired lung angiogenesis, and development of bronchopulmonary dysplasia (BPD) with severe long-term developmental adverse effects. The current therapy for BPD is limited to supportive care including high-oxygen therapy and pharmacotherapy. Recognizing more feasible treatment options to improve lung health and reduce complications associated with BPD is essential for improving the overall quality of life of premature infants. There is a reduction in the resident stem cells in lungs of premature infants with BPD, which strongly suggests a critical role of stem cells in BPD pathogenesis; this warrants the exploration of the potential therapeutic use of stem-cell therapy. Stem-cell-based therapies have shown promise for the treatment of many pathological conditions including acute lung injury and BPD. Mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (EVs) including exosomes are promising and effective therapeutic modalities for the treatment of BPD. Treatment with MSCs and EVs may help to reduce lung inflammation, improve pulmonary architecture, attenuate pulmonary fibrosis, and increase the survival rate.

Regulation of ACE-2 enzyme by hyperoxia in lung epithelial cells by post-translational modification.

BACKGROUND: Bronchopulmonary Dysplasia (BPD) occurs in premature neonates with respiratory distress who require supplemental oxygen in the first days after birth. BPD involves uniform arrest of alveolar development and variable interstitial cellularity and/or fibroproliferation. Previous studies by our lab showed that the enzyme, angiotensin converting enzyme-2 (ACE-2) and its product Ang1-7 exerting action on the receptor Mas oncogene in what is known as ACE-2/Mas axis is protective to lung cells. We also showed that ACE-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic gas lung injury, an effect caused by ACE-2 enzyme shedding mediated by TNF-alpha-converting enzyme (TACE/ADAM17). However, no reports yet exist about the regulation of ACE-2 in the alveolar epithelia in hyperoxic lung injury. OBJECTIVE: In this study we aim to define the effects of hyperoxic lung injury on the protective ACE-2 enzyme in the human lung alveolar epithelial cell line A549. DESIGN/METHODS: Cultured A549 cells were exposed to hyperoxia (95% O2) or normoxia (21% O2) for 3 or 7 days in serum-free nutrient media. Cells were lysed and culture media were collected to test for cellular ACE-2 enzymatic activity and for ACE-2, Mas receptor, TACE/ADAM17, and ubiquitin proteins abundance by immunoblotting. Cells were harvested in Trizol for RNA extraction and ACE-2 qRT-PCR. Whole cell extracts of A549 cell line was used for ACE-2 immunoprecipitation and subsequent ubiquitin immunoblotting. RESULTS: Total ubiquitinated proteins were increased by hyperoxia treatment, while ACE-2 and Mas receptor proteins abundance and ACE-2 enzymatic activity were decreased significantly in A549 cells exposed to hyperoxia relative to the normoxia controls. The percent decrease in ACE-2 activity corresponded with increased time of hyperoxic gas exposure. However, in contrast to our data from lung fibroblasts, no significant change was noted in ACE-2 protein released into the media or in ACE-2 mRNA levels by the hyperoxic treatment. Ubiquitin immunoreactive bands were detectable in the ACE-2 immunoprecipitate. CONCLUSIONS: These data suggest that hyperoxic exposure of the lung epithelial cells decreases the protective enzyme ACE-2 by cell type specific mechanisms independent of shedding by TACE/ADAM17. The data also suggest a regulatory level of ACE-2 downstream of transcription may involve ACE-2 ubiquitination and targeting for degradation.

COVID-19 Vaccines and Thrombosis-Roadblock or Dead-End Street?

Two adenovirus-based vaccines, ChAdOx1 nCoV-19 and Ad26.COV2.S, and two mRNA-based vaccines, BNT162b2 and mRNA.1273, have been approved by the European Medicines Agency (EMA), and are invaluable in preventing and reducing the incidence of coronavirus disease-2019 (COVID-19). Recent reports have pointed to thrombosis with associated thrombocytopenia as an adverse effect occurring at a low frequency in some individuals after vaccination. The causes of such events may be related to SARS-CoV-2 spike protein interactions with different C-type lectin receptors, heparan sulfate proteoglycans (HSPGs) and the CD147 receptor, or to different soluble splice variants of the spike protein, adenovirus vector interactions with the CD46 receptor or platelet factor 4 antibodies. Similar findings have been reported for several viral diseases after vaccine administration. In addition, immunological mechanisms elicited by viral vectors related to cellular delivery could play a relevant role in individuals with certain genetic backgrounds. Although rare, the potential COVID-19 vaccine-induced immune thrombotic thrombocytopenia (VITT) requires immediate validation, especially in risk groups, such as the elderly, chronic smokers, and individuals with pre-existing incidences of thrombocytopenia; and if necessary, a reformulation of existing vaccines.

Oxygen injury in neonates: which is worse? hyperoxia, hypoxia, or alternating hyperoxia/hypoxia.

Premature birth results in an increased risk of respiratory distress and often requires oxygen therapy. While the supplemental oxygen has been implicated as a cause of bronchopulmonary dysplasia (BPD), in clinical practice this supplementation usually only occurs after the patient's oxygen saturation levels have dropped. The effect of hyperoxia on neonates has been extensively studied. However, there is an unanswered fundamental question: which has the most impact-hyperoxia, hypoxia or fluctuating oxygen levels? In this review, we will summarize the reported effect of hypoxia, hyperoxia or a fluctuation of oxygen levels (hypoxia/hyperoxia cycling) in preterm neonates, with special emphasis on the lungs.

Degradation of Lung Protective Angiotensin Converting Enzyme-2 by Meconium in Human Alveolar Epithelial Cells: A Potential Pathogenic Mechanism in Meconium Aspiration Syndrome.

BACKGROUND: Pancreatic digestive enzymes present in meconium might be responsible for meconium-induced lung injury. The local Renin Angiotensin System plays an important role in lung injury and inflammation. Particularly, angiotensin converting enzyme-2 (ACE-2) has been identified as a protective lung enzyme against the insult. ACE-2 converts pro-apoptotic Angiotensin II to anti-apoptotic Angiotensin 1-7. However, the effect of meconium on ACE-2 has never been studied before. OBJECTIVE: To study the effect of meconium on ACE-2, and whether inhibition of proteolytic enzymes present in the meconium reverses its effects on ACE-2. METHODS: Alveolar epithelial A549 cells were exposed to F-12 medium, 2.5% meconium, meconium + a protease inhibitor cocktail (PIc) and PIc alone for 16 h. At the end of incubation, apoptosis was measured with a nuclear fragmentation assay and cell lysates were collected for ACE-2 immunoblotting and enzyme activity. RESULTS: Meconium caused a fourfold increase in apoptotic nuclei (p < 0.001). The pro-apoptotic effect of meconium can be reversed by PIc. Meconium reduced ACE-2 enzyme activity by cleaving ACE-2 into a fragment detected at ~ 37 kDa by immunoblot. PIc prevented the degradation of ACE-2 and restored 50% of ACE-2 activity (p < 0.05). CONCLUSION: These data suggest that meconium causes degradation of lung protective ACE-2 by proteolytic enzymes present in meconium, since the effects of meconium can be reversed by PIc.

Activation of mas restores hyperoxia-induced loss of lung epithelial barrier function through inhibition of apoptosis.

BACKGROUND: Neonatal therapy with a high concentration of oxygen (hyperoxia) is a known cause of bronchopulmonary dysplasia (BPD). BPD is characterized by increased pulmonary permeability and diffuse infiltration of various inflammatory cells. Disruption of the epithelial barrier may lead to altered pulmonary permeability and airways fluid accumulation. Mas receptor is a component of the renin angiotensin system and is the receptor for the protective endogenous peptide angiotensin 1-7. The activation of the Mas receptor was previously shown to have protective pulmonary responses. However, the effect of Mas receptor activation on epithelial barrier integrity has not been tested. OBJECTIVE: To determine the effects of hyperoxia with or without Mas receptor activation on epithelial cell barrier integrity. DESIGN/METHODS: Human epithelial cell line A549 was cultured on transwell polycarbonate porous membrane to confluence and treated with 95% oxygen (hyperoxia) for 72 hours with or without the Mas receptor agonist (AVE0991), or the apoptotic inhibitors Z-VAD-FMK or aurintricarboxylic acid. The cells were then challenged with Rhodamine labeled bovine serum albumin (Rh-BSA) on one side of the membrane. Fluorescent quantitation of Rh-BSA (albumin flux) was performed on the media in the other side of the membrane 3 hours later and was compared with 21% oxygen (Normoxia) control group. A549 cells were also cultured with or without AVE0991 in hyperoxia or normoxia and used for nuclear fragmentation apoptosis assay using propidium iodide staining. RESULTS: Hyperoxia induced an increase in albumin flux that was significantly prevented by AVE0991 treatment and by the apoptosis inhibitors. AVE0991 also significantly decreased the hyperoxia-induced nuclear fragmentation. CONCLUSION: These results suggest that hyperoxia causes a disruption in the epithelial barrier integrity, and that this disruption is inhibited by the Mas receptor agonist AVE0991 through inhibition of epithelial apoptosis. These results reveal a novel potential drug for BPD and pulmonary edema treatment.

The renin angiotensin system in liver and lung: impact and therapeutic potential in organ fibrosis.

Liver and lung fibrosis are two main organ diseases that are of particular importance in both Egypt and the US. Hepatitis C Virus "HCV" infection and idiopathic pulmonary fibrosis (IPF) are fibrotic diseases of the liver and lung respectively. The liver and lung are reported in literature to share many immune/inflammatory responses to damage through the lung-liver axis. Most importantly, HCV was shown to enhance the development of IPF and is considered one of the risk factors for IPF. The renin angiotensin system (RAS) plays a critical role in the fibrogenesis and inflammation damage of many organs including liver and lung. The relatively recently identified component of RAS, angiotensin converting enzyme-2 (ACE-2), has shown a promising therapeutic potential in models of liver and pulmonary fibrosis. This article reviews the role of RAS in organ fibrosis with focus on role of ACE-2 in fibrotic diseases of the liver and the lung.

Local activation of the pulmonary extravascular angiotensin system induces epithelial apoptosis and lung fibrosis.

Previous work suggests that a local extravascular angiotensin system plays an important role in the development of pulmonary fibrosis through stimulation of alveolar epithelial cell (AEC) apoptosis and collagen deposition. To demonstrate a causative role for the local tissue angiotensin (ANG) system in lung fibrosis, we hypothesize that overexpression of the angiotensinogen (AGT) gene or pharmacologic elevation of lung tissue ANG II levels might cause apoptosis of AECs and lung fibrosis. ANGII levels were elevated in rat or mouse lung tissue by intratracheal instillation of either purified ANGII or an adenovirus expressing AGT, or by ubiquitous overexpression of AGT in transgenic mice. Intratracheal instillation of purified ANGII caused significant collagen accumulation in lung tissue, both ex vivo and in vivo. Ubiquitous overexpression of AGT enhanced the profibrotic effect of bleomycin given at suboptimal doses. Intratracheal delivery of an adenoviral vector expressing mouse AGT (Ad-AGT) overexpressed AGT primarily in AECs and caused both apoptosis of AECs and pulmonary fibrosis. The lung collagen accumulation and AEC apoptosis caused by Ad-AGT was blocked by the caspase inhibitor ZVAD-fmk, by the ANG receptor AT1 antagonist Losartan or by the non-selective ANGII receptor antagonist Saralasin. Together, these data support the hypothesis that elevated pulmonary expression of AGT and its conversion to angiotensin II plays a causative role in the development of lung fibrosis through its induction of AEC apoptosis.

Bleomycin-Induced Neonatal Lung Injury Requires the Autocrine Pulmonary Angiotensin System.

BACKGROUND: Previous work from this laboratory demonstrated that apoptosis is regulated by a local angiotensin (ANG) system in alveolar epithelial cells (AECs). Autocrine generation of angiotensin II (ANGII) in response to endogenous or xenobiotic inducers is required for apoptosis in adult rat AECs and in AEC-derived human lung carcinoma cell line A549. Therefore, we hypothesized that a similar mechanism might also be involved in bleomycin (Bleo)-induced murine neonatal lung injury. METHODS: To investigate the local production of angiotensinogen (AGT) and ANGII in neonatal lung injury, lung explants were obtained from C57/BL6 wild type neonatal mice and were treated with Bleo in the presence or absence of an angiotensin converting enzyme (ACE) inhibitor. AGT protein, ANGII levels and caspase-9 were then measured. RESULTS: Exposure to Bleo significantly induced AGT protein (p<0.02), extracellular ANGII levels (p< 0.005) and the active form of caspase-9 (p<0.05) in neonatal lung tissue. Further, Bleo inducetion of both AGT protein and of caspase-9 were prevented by the ACE inhibitor lisinopril. CONCLUSION: These data clearly demonstrate the synthesis of AGT and ANGII in the lungs of neonates in response to Bleo. Furthermore, they suggest that manipulation of the angiotensin system may hold therapeutic potential for neonatal lung injury.

Prior hypoxia prevents downregulation of ACE-2 by hyperoxia in fetal human lung fibroblasts.

UNLABELLED: Purpose/Aim of Study: The renin angiotensin system is involved in experimentally induced lung fibrosis. Angiotensin (ANG)-II is profibrotic. Angiotensin converting enzyme-2 (ACE-2) cleaves ANG-II and is thus protective. ACE-2 has recently been reported to be significantly decreased under hyperoxic conditions. Hyperoxia is linked to Bronchopulmonary Dysplasia and lung fibrosis. Fetal lung cells normally do not undergo fibrotic changes with physiologic hypoxemia. We hypothesized that hypoxia prior to hyperoxic exposure in fetal lung fibroblasts (IMR-90 cell line) might be protective by preventing ACE-2 downregulation. MATERIALS AND METHODS: IMR-90 cells were exposed to hypoxia (1%O2/99%N2) followed by hyperoxia (95%O2/5%CO2) or normoxia (21%O2) in vitro. Cells and culture media were recovered separately for assays of ACE-2, TNF-α-converting enzyme (TACE), αSmooth muscle actin (αSMA)-myofibroblast marker-, N-cadherin, and ß-catenin immunoreactive protein. RESULTS: ACE-2 significantly increased when IMR-90 were hypoxic prior to hyperoxic exposure with no recovery. In contrast to hyperoxia alone, ACE-2 did not decrease when IMR-90 were hypoxic prior to hyperoxic exposure with recovery. TACE/ADAM17 protein and mRNA were significantly decreased under these conditions. αSMA N-cadherin, and ß-catenin proteins were significantly decreased with or without normoxic recovery. CONCLUSIONS: Hypoxia prior to hyperoxic exposure of fetal lung fibroblasts prevented ACE-2 downregulation and decreased ADAM17/TACE protein and mRNA. αSMA, N-cadherin, and ß-catenin were also significantly decreased under these conditions.

Angiotensin-(1-7)/mas inhibits apoptosis in alveolar epithelial cells through upregulation of MAP kinase phosphatase-2.

Earlier work from this laboratory showed that autocrine generation of angiotensin II and c-Jun-NH2-terminal kinase phosphorylation (p-JNK) are both required events in alveolar epithelial cell (AEC) apoptosis. Although earlier data showed that angiotensin-(1-7) [ANG-(1-7)] protects against AEC apoptosis, the pathways by which ANG-(1-7)/mas activation prevent JNK phosphorylation and apoptosis are poorly understood. Therefore, in the current study, it was theorized that ANG-(1-7) activates a mitogen-activated protein kinase phosphatase (MKP) and thereby reduces JNK phosphorylation to inhibit apoptosis and promote cell survival. This hypothesis was evaluated in the human A549 and mouse MLE12 AEC lines and primary cultures of human AECs. Cells were transfected with small-interfering RNAs, antisense oligonucleotides, or inhibitors specific for MKP-2 or mas, and were then assayed for phospho-JNK, caspase-9, loss of mitochondrial membrane potential, and nuclear fragmentation. Silencing of MKP-2 significantly prevented the blockade of all apoptotic markers by ANG-(1-7). Knockdown or blockade of mas receptor by antisense oligonucleotides or by the receptor antagonist A779, respectively, caused significant decreases in MKP-2, and simultaneously increased the apoptotic markers of caspase-9 activation and nuclear fragmentation. These data show that the ANG-(1-7)/mas pathway constitutively prevents JNK phosphorylation and apoptosis of AECs by maintaining activation of the JNK-selective phosphatase MKP-2, and further demonstrate the critical role of the ANG-(1-7) receptor mas in AEC survival.

Hyperoxia downregulates angiotensin-converting enzyme-2 in human fetal lung fibroblasts.

BACKGROUND: Angiotensin (ANG) II is involved in experimental hyperoxia-induced lung fibrosis. Angiotensin-converting enzyme-2 (ACE-2) degrades ANG II and is thus protective, but is downregulated in adult human and experimental lung fibrosis. Hyperoxia is a known cause of chronic fibrotic lung disease in neonates, but the role of ACE-2 in neonatal lung fibrosis is unknown. We hypothesized that ACE-2 in human fetal lung cells might be downregulated by hyperoxic gas. METHODS: Fetal human lung fibroblast IMR90 cells were exposed to hyperoxic (95% O2/5% CO2) or normoxic (21% O2/5% CO2) gas in vitro. Cells and culture media were recovered separately for assays of ACE-2 enzymatic activity, mRNA, and immunoreactive protein. RESULTS: Hyperoxia decreased ACE-2 immunoreactive protein and enzyme activity in IMR90 cells (both P < 0.01), but did not change ACE-2 mRNA. ACE-2 protein was increased in the cell supernatant, suggesting protease-mediated ectodomain shedding. TAPI-2, an inhibitor of TNF-α-converting enzyme (TACE/ADAM17), prevented both the decrease in cellular ACE-2 and the increase in soluble ACE-2 (both P < 0.05). CONCLUSION: These data show that ACE-2 is expressed in fetal human lung fibroblasts but is significantly decreased by hyperoxic gas. They also suggest that hyperoxia decreases ACE-2 through a shedding mechanism mediated by ADAM17/TACE.

Molecular and cellular mechanisms of the inhibitory effects of ACE-2/ANG1-7/Mas axis on lung injury.

An established body of recent literature has demonstrated potent inhibitory effects of the angiotensin converting enzyme-2 (ACE-2)/ANG1-7/ Mas axis on acute lung injury and lung fibrogenesis. One of the mechanisms of this inhibition is the enzymatic action of ACE-2 to degrade its main substrate angiotensin (ANG) II, thereby reducing the injurious and profibrotic activities of this octapeptide. Another, potentially more important mechanism is the production by ACE-2 of the heptapeptide ANG1-7, which inhibits the actions of ANGII through its own receptor Mas, the product of the oncogene of the same name. Very recent efforts to define the molecular and cellular mechanisms of ANG1-7/Mas action have revealed a number of similar, but mechanistically distinct, pathways by which ANG1-7 and Mas act on various lung cell types to inhibit lung injury and fibrosis. In this review we summarize the beneficial actions of the ANG1-7/Mas pathway, specifically on lung cells in non-neoplastic lung injury. We also review the currently known downstream signaling mechanisms of the ANG1-7/Mas pathway in various lung cell types known to be key in acute injury and fibrogenesis.

The Witschi Hypothesis revisited after 35 years: genetic proof from SP-C BRICHOS domain mutations.

Over 35 years ago, Wanda Haschek and Hanspeter Witschi published a theory for the pathogenesis of lung fibrosis that dared to challenge the longstanding view of lung fibrosis as an "inflammatory disease." On the basis of considerable experimental evidence, they proposed that lung fibrosis was initiated and propagated by microfoci of epithelial damage that, if unrepaired, upset the normal epithelial-fibroblast balance to create profibrotic microenvironments, without any obligatory contribution of "inflammatory" cells. Unfortunately, this theory was largely overlooked for many years. In the meantime, the repeated failure of attempts to treat idiopathic pulmonary fibrosis with anti-inflammatory regimens has led some investigators to revive the theory referred to, in decades past, as "The Witschi Hypothesis." This manuscript briefly reviews more recent evidence in support of the "Severity of Epithelial Injury" Hypothesis proposed by Haschek and Witschi. More important, it offers the updated viewpoint that mutations in the BRICHOS domain of surfactant protein C, which cause interstitial lung disease and induce cell death specifically in lung epithelial cells, in effect provide genetic proof that the Witschi Hypothesis is indeed the correct theory to explain the pathogenesis of fibrosis in the lungs.

Angiotensinogen promoter polymorphisms predict low diffusing capacity in U.S. and Spanish IPF cohorts.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in angiotensinogen (AGT) at positions -20 and -6 are associated with increased severity and progression of various fibrotic diseases. Our earlier work demonstrated that the progression of idiopathic pulmonary fibrosis (IPF) was associated with the A-6 allele. This study examined the hypothesis that the homozygous CC genotype at -20 and the AA genotype at -6 would confer worse measures of pulmonary function (measured by pulmonary function tests) in IPF. METHODS: Multiple logistic regression analysis was applied to a NIH Lung Tissue Research Consortium cohort and a Spanish cohort, while also adjusting for covariates to determine the effects of these SNPs on measures of pulmonary function. RESULTS: Analysis demonstrated that the CC genotype at -20 was strongly associated with reduced diffusing capacity in males in both cohorts (p = 0.0028 for LTRC and p = 0.017 for the Spanish cohort). In females, the AA genotype was significantly associated with lower FVC (p = 0.0082) and V alv (p = 0.022). In males, the haplotype CA at -20 and -6 in AGT was also strongly associated with reduced diffusing capacity in both cohorts. CONCLUSIONS: This study is the first to demonstrate an association of AGT polymorphisms (-20A > C and -6G > A) with lower measures of pulmonary function in IPF. It is also the first to relate the effect of gender in lung fibrosis with polymorphisms in AGT.

Abrogation of ER stress-induced apoptosis of alveolar epithelial cells by angiotensin 1-7.

Earlier work showed that apoptosis of alveolar epithelial cells (AECs) in response to endogenous or xenobiotic factors is regulated by autocrine generation of angiotensin (ANG) II and its counterregulatory peptide ANG1-7. Mutations in surfactant protein C (SP-C) induce endoplasmic reticulum (ER) stress and apoptosis in AECs and cause lung fibrosis. This study tested the hypothesis that ER stress-induced apoptosis of AECs might also be regulated by the autocrine ANGII/ANG1-7 system of AECs. ER stress was induced in A549 cells or primary cultures of human AECs with the proteasome inhibitor MG132 or the SP-C BRICHOS domain mutant G100S. ER stress activated the ANGII-generating enzyme cathepsin D and simultaneously decreased the ANGII-degrading enzyme ACE-2, which normally generates the antiapoptotic peptide ANG1-7. TAPI-2, an inhibitor of ADAM17/TACE, significantly reduced both the activation of cathepsin D and the loss of ACE-2. Apoptosis of AECs induced by ER stress was measured by assays of mitochondrial function, JNK activation, caspase activation, and nuclear fragmentation. Apoptosis induced by either MG132 or the SP-C BRICHOS mutant G100S was significantly inhibited by the ANG receptor blocker saralasin and was completely abrogated by ANG1-7. Inhibition by ANG1-7 was blocked by the specific mas antagonist A779. These data show that ER stress-induced apoptosis is mediated by the autocrine ANGII/ANG1-7 system in human AECs and demonstrate effective blockade of SP-C mutation-induced apoptosis by ANG1-7. They also suggest that therapeutic strategies aimed at administering ANG1-7 or stimulating ACE-2 may hold potential for the management of ER stress-induced fibrotic lung disorders.

Cell cycle dependence of ACE-2 explains downregulation in idiopathic pulmonary fibrosis.

Alveolar epithelial type II cells, a major source of angiotensin-converting enzyme (ACE)-2 in the adult lung, are normally quiescent but actively proliferate in lung fibrosis and downregulate this protective enzyme. It was, therefore, hypothesised that ACE-2 expression might be related to cell cycle progression. To test this hypothesis, ACE-2 mRNA levels, protein levels and enzymatic activity were examined in fibrotic human lungs and in the alveolar epithelial cell lines A549 and MLE-12 studied at postconfluent (quiescent) versus subconfluent (proliferating) densities. ACE-2 mRNA, immunoreactive protein and enzymatic activity were all high in quiescent cells, but were severely downregulated or absent in actively proliferating cells. Upregulation of the enzyme in cells that were progressing to quiescence was completely inhibited by the transcription blocker actinomycin D or by SP600125, an inhibitor of c-Jun N-terminal kinase (JNK). In lung biopsy specimens obtained from patients with idiopathic pulmonary fibrosis, immunoreactive enzyme was absent in alveolar epithelia that were positive for proliferation markers, but was robustly expressed in alveolar epithelia devoid of proliferation markers. These data explain the loss of ACE-2 in lung fibrosis and demonstrate cell cycle-dependent regulation of this protective enzyme by a JNK-mediated transcriptional mechanism.

Angiotensin signalling in pulmonary fibrosis.

A large body of evidence demonstrates that angiotensin II and angiotensin receptors are required for the pathogenesis of experimental lung fibrosis. Angiotensin has a number of profibrotic effects on lung parenchymal cells that include the induction of growth factors for mesenchymal cells, extracellular matrix molecules, cytokines and increased motility of lung fibroblasts. Angiotensin is also proapoptotic for lung epithelial cells, and is synthesized by a local system (i.e., entirely within the lung tissue) after lung injury by a variety of agents of both xenobiotic and endogenous origins. Recent evidence shows that the counterregulatory molecule angiotensin 1-7, the product of the enzyme ACE-2, inhibits epithelial cell apoptosis and thus acts as an antifibrotic epithelial survival factor. This manuscript reviews the evidence supporting a role for angiotensin in lung fibrogenesis and discusses the signalling mechanisms underlying its action on lung parenchymal cells important in the pathogenesis of pulmonary fibrosis.

Regulation of alveolar epithelial cell survival by the ACE-2/angiotensin 1-7/Mas axis.

Earlier work from this laboratory demonstrated that apoptosis of alveolar epithelial cells (AECs) requires autocrine generation of angiotensin (ANG) II. More recent studies showed that angiotensin converting enzyme-2 (ACE-2), which degrades ANGII to form ANG1-7, is protective but severely downregulated in human and experimental lung fibrosis. Here it was theorized that ACE-2 and its product ANG1-7 might therefore regulate AEC apoptosis. To evaluate this hypothesis, the AEC cell line MLE-12 and primary cultures of rat AECs were exposed to the profibrotic apoptosis inducers ANGII or bleomycin (Bleo). Markers of apoptosis (caspase-9 or -3 activation and nuclear fragmentation), steady-state ANGII and ANG1-7, and JNK phosphorylation were measured thereafter. In the absence of Bleo, inhibition of ACE-2 by small interfering RNA or by a competitive inhibitor (DX600 peptide) caused a reciprocal increase in autocrine ANGII and corresponding decrease in ANG1-7 in cell culture media (both P < 0.05) and, moreover, induced AEC apoptosis. At baseline (without inhibitor), ANG1-7 in culture media was 10-fold higher than ANGII (P < 0.01). Addition of purified ANGII or bleomycin-induced caspase activation, nuclear fragmentation, and JNK phosphorylation in cultured AECs. However, preincubation with ANG1-7 (0.1 µM) prevented JNK phosphorylation and apoptosis. Moreover, pretreatment with A779, a specific blocker of the ANG1-7 receptor mas, prevented ANG1-7 blockade of JNK phosphorylation, caspase activation, and nuclear fragmentation. These data demonstrate that ACE-2 regulates AEC survival by balancing the proapoptotic ANGII and its antiapoptotic degradation product ANG1-7. They also suggest that ANG1-7 inhibits AEC apoptosis through the ANG1-7 receptor mas.