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

Effective administration of flavopiridol in advanced-stage chronic lymphocytic leukemia (CLL) is often associated with early biochemical evidence of tumor cell lysis. Previous work using other cell types showed that flavopiridol impacts mitochondria, and in CLL cells flavopiridol down-regulates the mitochondrial protein Mcl-1. We therefore investigated mitochondrial structure and function in flavopiridol-treated CLL patient cells and in the lymphoblastic cell line 697 using concentrations and times at which tumor lysis is observed in treated patients. Mitochondrial membrane depolarization was detected in flavopiridol-treated CLL cells by 6 hours, well before the onset of cell death. Flavopiridol-induced mitochondrial depolarization was not blocked by caspase inhibitors or by the calcium chelator EGTA, but was reduced by Bcl-2 overexpression. Intracellular calcium mobilization was noted at early time points using fluorescence microscopy. Furthermore, electron paramagnetic resonance oximetry showed a gradual but significant reduction in cellular oxygen consumption rate by 6 hours, corresponding with ultrastructural mitochondrial damage detected by electron microscopy. IntroductionFlavopiridol is a semisynthetic flavone (N-methylpiperidinyl chlorophenyl flavone) that is considered to act broadly as a cyclindependent kinase (CDK) inhibitor. 1 However, the in vivo mechanism of action of flavopiridol is not well understood, and may involve actions other than or inclusive of CDK inhibition. Our group recently demonstrated significant clinical efficacy of flavopiridol in patients with refractory chronic lymphocytic leukemia (CLL) using a novel schedule of administration. 2 Approximately 50% of CLL patients who receive flavopiridol using this schedule exhibit biochemical signs of tumor lysis (elevated potassium and phosphate levels, reduced calcium levels) occurring as early as 4.5 hours after treatment initiation. In limited cases with highly elevated peripheral white blood cell counts, this tumor lysis can be severe enough to require dialysis. This observation suggests that flavopiridol, when effectively administered, induces very rapid cell death that is atypical of classical apoptosis. However, more work is needed to understand this process and to better predict which patients may experience severe tumor lysis.Multiple previous studies in different cell types have incriminated mitochondrial mechanisms in flavopiridol-induced apoptosis, although conditions and time points under which this is noted vary widely and are often reported in combination with other agents. [3][4][5][6][7][8][9][10][11] Several reports showed that flavopiridol induces mitochondrial membrane disruption and release of cytochrome c in the U937 human monoblastic leukemia cell line and that these effects were potentiated by phorbol myristate acetate (PMA). 4,9,10 This process in U937 cells was noted to be partially caspase independent, as a general caspase inhibitor blocked flavopiridol-induced loss of mitochondrial membrane potential (⌬⌿ m ) but ...

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...

Synthesis and characterization of an inert perchlorotriphenylmethyl triester radical, PTM-TE, are reported. PTM-TE was prepared by a facile 3-step synthesis using Friedal-Crafts reaction of tetrachlorobenzene with chloroform followed by ethoxycarbonylation and subsequent oxidation. PTM-TE is paramagnetic, and detected as a single sharp peak by EPR spectroscopy. In solution, the EPR linewidth of PTM-TE is highly sensitive to the dissolved oxygen content, thus enabling accurate measurement of oxygen concentration (oximetry). In addition, the radical also shows high reactivity to superoxide. The ester radical has the potential for use as a high-sensitive probe for determination of oxygen concentration and superoxide in biological systems. KeywordsPerchlorotriphenylmethyl radical; perchlorotriphenylmethyl triester; superoxide; free radicals; EPR; oximetry; spectrophotometry In 1900, Moses Gomberg reported the existence of a stable, trivalent organic free radical which challenged the prevailing belief that carbon could only have four chemical bonds. 1 As this report provided new possibilities to carbon chemistry, research aimed at understanding and developing these radicals continued to thrive and permeated through various fields of science including medicine and industrial applications. Most notably, Gomberg's work paved the way for future research on using persistent trityl radicals in clinical applications. [2][3][4] However, it is known that some radicals and diradicals are short-lived and very reactive -the half-life of triphenylmethyl radicals in aerated solution may be as low as a fraction of a second. Studies have also shown that these reactive radicals become stable and chemically inert upon perchlorination. [5][6][7][8] In light of this endeavor, highly chlorinated mono-, di-, and triarylmethanes were developed and since then they have become the most valuable chemical precursors of inert free radicals with estimated half-lives in the order of Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript 100 years. 6 Together, these studies have been regarded as both promising and significant. 5 As a result, the synthesis of perchlorinated trityl radical has become an immense undertaking with promising implications.The role of reactive oxygen species (ROS) such as superoxide, hydroxyl, and alkylperoxyl radicals has been linked to a variety of pathophysiological processes. 9 Since, oxidative stress leads to an increased production of ROS, especially superoxide, accurate determinatio...