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.

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. @ERSpublications Cell cycle-dependent regulation of ACE-2 by a JNK-mediated transcriptional mechanism explains ACE-2 downregulation in IPF

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.idiopathic pulmonary fibrosis; BRICHOS domain mutations; ADAM17/ TACE; substituted peptide receptor antagonists APOPTOSIS OF ALVEOLAR EPITHELIAL cells (AECs) is increasingly being recognized as a critical event that initiates and propagates fibrosis in the lung parenchyma (20). The concept that AEC death was a critical determinant of fibrosis vs. normal repair was first proposed many years ago on the basis of two-hit toxicological experiments that demonstrated that fibrogenesis could be induced by experimental delay of epithelial repair after lung injury (1, 6), regardless of the presence or absence of inflammation. More recent evidence in support of this theory was found in the ability of caspase inhibitors (8, 25) or genetic deletion of apoptosis signaling molecules (2) to block fibrogenesis subsequent to lung injuries aimed at the epithelium (5).Understanding the regulation of AEC apoptosis is therefore critical to understanding the pathogenesis of lung fibrosis. Earlier work from this laboratory found that endogenous (24, 26) or xenobiotic inducers of AEC apoptosis (11) activate the autocrine synthesis of angiotensin (ANG)II from its precursor angiotensinogen (AGT) as well as its subsequent enzymatic processing to the mature peptide ANGII, all by the AEC itself. Further work demonstrated that the autocrine production of ANGII by AEC...

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 down-regulation. 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 β-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 increased in cells allowed to recover in 21% O2. αSMA N-Cadherin and β-Catenin proteins were significantly decreased in both groups. 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 β-catenin were also significantly decreased under these conditions.

A 23-year-old woman was referred to the allergy and immunology clinic for recurrent abdominal, cutaneous and joint swelling and pain with a history of mucosal infections since childhood. Her history and clinical findings were suggestive of two rare and complex disorders, hereditary angioedema (HAE) and Ehlers-Danlos syndrome (EDS). Her recurrent episodes of abdominal and joint pain were initially misattributed to more common diagnoses such as esophagitis, depression and chronic pain syndrome. However, the coexistence of HAE and EDS likely contributed to a delay in diagnoses as the combination of these two rare but overlapping disorders is less understood by physicians. She had persistently low levels of C4 and C1-esterase inhibitor (C1-INH) with low to low-normal C1-esterase function, normal C1Q and no C1Q antibodies. In the setting of recurrent abdominal pain with cutaneous swelling, this supported the diagnosis of HAE type I. The increase in joint extensibility with recurrent shoulder subluxations since childhood was a manifestation of EDS. Although no known genetic mutations were identified for EDS, her diagnosis was confirmed by a geneticist based on her clinical phenotype. Before the diagnosis of HAE and EDS, our patient had at least 100 visits/year to the emergency department/hospitalisations for these recurrent symptoms. After starting on C1-INH replacement therapy, the frequency has decreased 10-fold. She also noted a 70% improvement in her quality of life. Familiarity with these rare disorders will assist healthcare providers in recognising HAE and EDS and include them as part of their differential diagnoses. Early diagnosis is important for a patient’s well-being as both these chronic disorders have been associated with poor quality of life. Additionally, proper diagnoses will reduce healthcare costs by preventing unnecessary procedures due to misdiagnoses. Proper treatment will help to decrease hospitalisations and avoidance of life-threatening consequences (such as asphyxiation from fatal laryngeal attacks of HAE and rupture of aneurysms in EDS).

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For many patients, end-of-life care (EOLC) wishes are unknown and are generally only brought up during healthcare crises. During such healthcare episodes, loved ones are often distraught, and as such, can find it difficult to focus on details surrounding the event. The best place for these discussions may be in non-acute settings including primary care clinics. The purpose of this study was to examine how well a sample of patients (N = 177) in three Michigan-based primary care teaching clinics thought they and their loved ones were prepared in terms of having their EOLC wishes known. METHODS METHODS Prospective data were collected from three Internal Medicine teaching clinics in the Metro-Detroit area through an anonymous written survey addressing EOLC issues in a 16-item cross-sectional study. Respondents were adult patients at one of three participating primary care clinics. Perceived preparedness for EOLC was measured by: 1) possibly having had a prior EOLC discussion with a healthcare provider 2) having created an Advance Directive, such as a Durable Power of Attorney (DPOA) or Living Will with medical decision preferences, 3) reported preferences for quality versus quantity of life, and 4) preferences for CPR and other specific life-sustaining interventions. RESULTS RESULTS In this sample, 77 (43.5%) of 177 respondents had discussed their EOLC wishes with a provider. Regarding Advance Directives, 63/177 (35.6%) had established a DPOA, and 59/ 177 (22%) had made a Living Will. The majority of respondents preferred quality over quantity of life. The most difficult EOLC questions included the decision for CPR, tracheostomy, and PEG tube placement. CONCLUSIONS CONCLUSIONS Based on these results, EOLC discussions probably occur infrequently in the primary care or other healthcare settings. Most survey responses indicated that sample patients were unprepared concerning the details of future EOLC decisions. Engagement of such discussions should be a part of routine visits in the primary care clinic and should be re-visited when there is a change in a patient's health. Further larger-scale studies using validated surveys are required in this vitally important area of practice.

STUDY OBJECTIVEOur prior work showed that ER stress‐induced apoptosis of AECs is regulated by the autocrine ANGII/ANG1‐7 system. MG132 or surfactant protein C (SP‐C) BRICHOS domain mutation G100S induced apoptosis in human AECs by activating cathepsin D and by decreasing the protective angiotensin‐converting enzyme 2 (ACE‐2). This study tested the hypothesis that ER stress‐induced apoptosis of human AECs might be mediated by UPR pathways that in turn modulate the autocrine ANGII/ANG1‐7 system of these cells.METHODSA549 cells were challenged with the proteasome inhibitor MG132 or the SP‐C mutant G100S. UPR markers and ANG system components were measured by Western blotting. Apoptosis was measured by caspase activation and nuclear fragmentation.RESULTSMG132‐ or G100S mutation‐induced ER stress activated the UPR pathways (phospho PERK, phospho eIF2α, ATF4, phospho IRE1, spliced XBP1, ATF6), which led to an increase of cathepsin D and a decrease of anti‐apoptotic ACE‐2. Blockade of UPR pathways with UPR chaperone 4‐PBA or siRNAs (siATF6, siIRE1, siPERK) prevented both the changes in cathepsin D and ACE‐2 and the AEC apoptosis caused by MG132 or G100S.CONCLUSIONSThese data show that ER stress induces apoptosis in human AECs through UPR‐mediated pathways that in turn modulate the autocrine ANGII/ANG1‐7 system. They also demonstrate that ER stress‐induced apoptosis of human AECs can be prevented by inhibition of UPR‐specific pathways.