Pleural Effusion in COVID-19 Pneumonia: Clinical and Prognostic Implications-An Observational, Retrospective Study.

BACKGROUND: COVID-19 presents with a wide spectrum of clinical and radiological manifestations, including pleural effusion. The prevalence and prognostic impact of pleural effusion are still not entirely clear. PATIENTS AND METHODS: This is a retrospective, single-center study including a population of consecutive patients admitted to the University Hospital of Cisanello (Pisa) from March 2020 to January 2021 with a positive SARS-CoV-2 nasopharyngeal swab and SARS-CoV-2-related pneumonia. The patients were divided into two populations based on the presence (n = 150) or absence (n = 515) of pleural effusion on chest CT scan, excluding patients with pre-existing pleural effusion. We collected laboratory data (hemoglobin, leukocytes, platelets, C-reactive protein, procalcitonin), worst PaO2/FiO2 ratio as an index of respiratory gas exchange impairment, the extent of interstitial involvement related to SARS-CoV-2 pneumonia and data on intensity of care, length of stay and outcome (discharge or death). RESULTS: The prevalence of pleural effusion was 23%. Patients with pleural effusion showed worse gas exchange (p < 0.001), longer average hospital stay (p < 0.001), need for more health care resources (p < 0.001) and higher mortality (p < 0.001) compared to patients without pleural effusion. By multivariate analysis, pleural effusion was found to be an independent negative prognostic factor compared with other variables such as increased C-reactive protein, greater extent of pneumonia and older age. Pleural effusion was present at the first CT scan in most patients (68%). CONCLUSIONS: Pleural effusion associated with SARS-CoV-2 pneumonia is a relatively frequent finding that is confirmed to be a negative prognostic factor. Identifying early prognostic factors in an endemic-prone disease such as COVID-19 is necessary to optimize its clinical management. Further clinical studies aimed at better characterizing pleural effusion in these patients will be appropriate in order to clarify its pathogenetic role.

Gemcitabine: An Alternative Treatment for Oxaliplatin-Resistant Colorectal Cancer.

Resistance to treatments is one of the leading causes of cancer therapy failure. Oxaliplatin is a standard chemotherapy used to treat metastatic colorectal cancer. However, its efficacy is greatly reduced by the development of resistances. In a previous study, we deciphered the mechanisms leading to oxaliplatin resistance and highlighted the roles played by ROS production and the p38 MAPK pathway in this phenomenon. In this report, we studied the effects of different chemotherapy molecules on our oxaliplatin-resistant cells to identify alternative treatments. Among all the studied molecules, gemcitabine was the only one to present a major cytotoxic effect on oxaliplatin-resistant cancer cells both in vivo and in vitro. However, the combination of oxaliplatin and gemcitabine did not present any major interest. Indeed, the study of combination efficiency using Chou and Talalay's method showed no synergy between oxaliplatin and gemcitabine. Using PamGene technology to decipher gemcitabine's effects on oxaliplatin-resistant cells, we were able to show that gemcitabine counteracts chemoresistance by strongly inhibiting the Akt and src/p38 MAPK pathways, leading to apoptosis induction and cell death. In view of these results, gemcitabine could be an interesting alternative therapy for patients with colorectal cancer not responding to oxaliplatin-based protocols such as FOLFOX.

Tau Protein as Therapeutic Target for Cancer? Focus on Glioblastoma.

Despite being extensively studied for several decades, the microtubule-associated protein Tau has not finished revealing its secrets. For long, Tau has been known for its ability to promote microtubule assembly. A less known feature of Tau is its capability to bind to cancer-related protein kinases, suggesting a possible role of Tau in modulating microtubule-independent cellular pathways that are associated with oncogenesis. With the intention of finding new therapeutic targets for cancer, it appears essential to examine the interaction of Tau with these kinases and their consequences. This review aims at collecting the literature data supporting the relationship between Tau and cancer with a particular focus on glioblastoma tumors in which the pathological significance of Tau remains largely unexplored. We will first treat this subject from a mechanistic point of view showing the pivotal role of Tau in oncogenic processes. Then, we will discuss the involvement of Tau in dysregulating critical pathways in glioblastoma. Finally, we will outline promising strategies to target Tau protein for the therapy of glioblastoma.

Tau Regulates Glioblastoma Progression, 3D Cell Organization, Growth and Migration via the PI3K-AKT Axis.

The Microtubule-Associated Protein Tau is expressed in several cancers, including low-grade gliomas and glioblastomas. We have previously shown that Tau is crucial for the 2D motility of several glioblastoma cell lines, including U87-MG cells. Using an RNA interference (shRNA), we tested if Tau contributed to glioblastoma in vivo tumorigenicity and analyzed its function in a 3D model of multicellular spheroids (MCS). Tau depletion significantly increased median mouse survival in an orthotopic glioblastoma xenograft model. This was accompanied by the inhibition of MCS growth and cell evasion, as well as decreased MCS compactness, implying N-cadherin mislocalization. Intracellular Signaling Array analysis revealed a defective activation of the PI3K/AKT pathway in Tau-depleted cells. Such a defect in PI3K/AKT signaling was responsible for reduced MCS growth and cell evasion, as demonstrated by the inhibition of the pathway in control MCS using LY294002 or Perifosine, which did not significantly affect Tau-depleted MCS. Finally, analysis of the glioblastoma TCGA dataset showed a positive correlation between the amount of phosphorylated Akt-Ser473 and the expression of MAPT RNA encoding Tau, underlining the relevance of our findings in glioblastoma disease. We suggest a role for Tau in glioblastoma by controlling 3D cell organization and functions via the PI3K/AKT signaling axis.

Leptin Promotes Prostate Cancer Proliferation and Migration by Stimulating STAT3 Pathway.

To better understand the link between obesity and prostate cancer (PC) aggressiveness, we investigate the role of leptin, an obesity associated adipokine, and its receptor (Ob-R) in PC cells migration. The migration assay (Wound-healing) was used to study the leptin impact on DU-145 and PC3 cells lines. STAT3 activation was performed by Western Blot. E-cadherin expression was studied using fluorescence microscopy and Ob-R expression in PC and benign prostatic Hyperplasia (BPH) biopsies was assessed by RT-PCR. In this study we demonstrate that high dose of leptin promotes PC cells migration and EMT transition via the stimulation of STAT3 pathway. In addition, we report that although Ob-R mRNA is expressed by ADK and BPH resections biopsies, significant higher levels were observed for ADK patients. Finally, we found a positive association between Ob-R mRNA expression and worse PC prognosis. A better understanding of the molecular processes of leptin signaling is crucial for identifying appropriate approaches for treatment of obesity-related PC patients.

Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas.

Protein disulfide isomerases including PDIA1 are implicated in cancer progression, but underlying mechanisms are unclear. PDIA1 is known to support vascular Nox1 NADPH oxidase expression/activation. Since deregulated reactive oxygen species (ROS) production underlies tumor growth, we proposed that PDIA1 is an upstream regulator of tumor-associated ROS. We focused on colorectal cancer (CRC) with distinct KRas activation levels. Analysis of RNAseq databanks and direct validation indicated enhanced PDIA1 expression in CRC with constitutive high (HCT116) vs. moderate (HKE3) and basal (Caco2) Ras activity. PDIA1 supported Nox1-dependent superoxide production in CRC; however, we first reported a dual effect correlated with Ras-level activity: in Caco2 and HKE3 cells, loss-of-function experiments indicate that PDIA1 sustains Nox1-dependent superoxide production, while in HCT116 cells PDIA1 restricted superoxide production, a behavior associated with increased Rac1 expression/activity. Transfection of Rac1G12V active mutant into HKE3 cells induced PDIA1 to become restrictive of Nox1-dependent superoxide, while in HCT116 cells treated with Rac1 inhibitor, PDIA1 became supportive of superoxide. PDIA1 silencing promoted diminished cell proliferation and migration in HKE3, not detectable in HCT116 cells. Screening of cell signaling routes affected by PDIA1 silencing highlighted GSK3ß and Stat3. Also, E-cadherin expression after PDIA1 silencing was decreased in HCT116, consistent with PDIA1 support of epithelial-mesenchymal transition. Thus, Ras overactivation switches the pattern of PDIA1-dependent Rac1/Nox1 regulation, so that Ras-induced PDIA1 bypass can directly activate Rac1. PDIA1 may be a crucial regulator of redox-dependent adaptive processes related to cancer progression.

Tau regulates the microtubule-dependent migration of glioblastoma cells via the Rho-ROCK signaling pathway.

The pathological significance of Tau (encoded by MAPT) in mechanisms driving cell migration in glioblastoma is unclear. By using an shRNA approach to deplete microtubule-stabilizing Tau in U87 cells, we determined its impact on cytoskeletal coordination during migration. We demonstrated here that the motility of these Tau-knockdown cells (shTau cells) was significantly (36%) lower than that of control cells. The shTau cells displayed a slightly changed motility in the presence of nocodazole, which inhibits microtubule formation. Such reduced motility of shTau cells was characterized by a 28% lower number of microtubule bundles at the non-adhesive edges of the tails. In accordance with Tau-stabilized microtubules being required for cell movement, measurements of the front, body and rear section displacements of cells showed inefficient tail retraction in shTau cells. The tail retraction was restored by treatment with Y27632, an inhibitor of Rho-ROCK signaling. Moreover, we clearly identified that shTau cells displayed relocation of the active phosphorylated form of p190-RhoGAP (also known as ARHGAP35), which inhibits Rho-ROCK signaling, and focal adhesion kinase (FAK, also known as PTK2) in cell bodies. In conclusion, our findings indicate that Tau governs the remodeling of microtubule and actin networks for the retraction of the tail of cells, which is necessary for effective migration.

Synthesis and biological evaluation of 4 arylcoumarin analogues as tubulin-targeting antitumor agents.

The synthesis of twenty-six 4-arylcoumarin analogues of combretastatin A-4 (CA-4) led to the identification of two new compounds (25 and 26) with strong cytotoxic activity. Both compounds had a high cytotoxic effect on a CA-4-resistant colon adenocarcinoma cell line (HT29D4). The compounds affected cell cycle progression characterized by a mitotic block. The activity of these compounds against microtubules both in vitro and in cells was examined and both compounds were found to potently inhibit in vitro microtubule formation via a sub-stoichiometric mode like CA-4. By immunofluorescence, it was observed that both compounds induced strong microtubule network disruption. Our results provide a strong experimental basis to develop new potent anti-tubulin molecules targeting CA-4-resistant cancer cells.

Peptide screen identifies a new NADPH oxidase inhibitor: impact on cell migration and invasion.

The NADPH oxidase proteins catalyse the formation of superoxide anion which act as signalling molecules in physiological and pathological processes. Nox1-dependent NADPH oxidase is expressed in heart, lung, colon, blood vessels and brain. Different strategies involving Nox1 inhibition based on diphenylene iodonium derivatives are currently tested for colorectal cancer therapy. Here, after peptides screening on Nox1-dependent NADPH oxidase assay in HT-29 cells, we identify a peptide (referred to as NF02), cell-active, that potently block Nox1-dependent reactive oxygen species generation. Study of DEPMPO adduct formation by electron paramagnetic resonance showed that NF02 has no superoxide scavenging activity and no impact on cellular reactive oxygen species-producing enzymes such xanthine oxidase. NF02 was not cytotoxic, inhibited reactive oxygen species production of reconstituted Nox1/Noxo1/Noxa1 complex in HEK293 and did not decrease Nox2 dependent cellular NADPH oxidase reactive oxygen species production. Finally, NF02 inhibited cell migration and invasion of colorectal cancer cells which is consistent with the described impact of Nox1 inhibitors on cell migration. NF02 peptide is a new NADPH oxidase inhibitor specific for Nox1 over Nox2 and xanthine oxidase which might represent a useful Nox1 tool with potential therapeutic insights.

Rutin inhibits proliferation, attenuates superoxide production and decreases adhesion and migration of human cancerous cells.

Lung and colorectal cancer are the principal causes of death in the world. Rutin, an active flavonoid compound, is known for possessing a wide range of biological activities. In this study, we examined the effect of rutin on the viability, superoxide anion production, adhesion and migration of human lung (A549) and colon (HT29 and Caco-2) cancer cell lines. In order to control the harmlessness of the tested concentrations of rutin, the viability of cancer cell lines was assessed using a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. ROS generation was measured by lucigenin chemiluminescence detecting superoxide ions. To investigate the effect of rutin on the behavior of human lung and colon cancer cell lines, we performed adhesion assays, using various purified extracellular matrix (ECM) proteins. Finally, in vitro cell migration assays were explored using modified Boyden chambers. The viability of cancerous cells was inhibited by rutin. It also significantly attenuated the superoxide production in HT29 cells. In addition, rutin affected adhesion and migration of A549 and HT29 cell. These findings indicate that rutin, a natural molecule, might have potential as anticancer agent against lung and colorectal carcinogenesis.

ß-eudesmol, a sesquiterpene from Teucrium ramosissimum, inhibits superoxide production, proliferation, adhesion and migration of human tumor cell.

Reactive oxygen species are well-known mediators of various biological responses. Recently, new homologues of the catalytic subunit of NADPH oxidase have been discovered in non phagocytic cells. These new homologues (Nox1-Nox5) produce low levels of superoxides compared to the phagocytic homologue Nox2/gp91phox. In this study we examined the effect of ß-eudesmol, a sesquiterpenoid alcohol isolated from Teucrium ramosissimum leaves, on proliferation, superoxide anion production, adhesion and migration of human lung (A549) and colon (HT29 and Caco-2) cancer cell lines. Proliferation of tumor cells was inhibited by ß-eudesmol. It also significantly inhibited superoxide production in A549 cells. Furthermore, ß-eudesmol inhibited adhesion and migration of A549 and HT29 cell. These results demonstrate that ß-eudesmol may be a novel anticancer agent for the treatment of lung and colon cancer by different ways: by inhibition of superoxide production or by blocking proliferation, adhesion and migration.

Epothilone B inhibits migration of glioblastoma cells by inducing microtubule catastrophes and affecting EB1 accumulation at microtubule plus ends.

Invasion of normal brain tissue by tumor cells is a major contributing factor to the recurrence of glioblastoma and its resistance to therapy. Here, we have assessed the efficacy of the microtubule (MT) targeting agent Epothilone B (patupilone) on glioblastoma cell migration, a prerequisite for invasive tumor cell behavior. At non-cytotoxic concentrations, patupilone inhibited glioblastoma cell movement, as shown by transwell cell migration, random motility and spheroid assays. This anti-migratory effect was associated with a reduced accumulation of EB1 and other MT plus end tracking proteins at MT ends and with the induction of MT catastrophes, while the MT growth rate and other MT dynamic instability parameters remained unaltered. An increase in MT catastrophes led to the reduction of the number of MTs reaching the leading edge. Analysis of the effect of patupilone on MT dynamics in a reconstituted in vitro system demonstrated that the induction of MT catastrophes and an alteration of EB1 accumulation at MT plus end are intrinsic properties of patupilone activity. We have thus demonstrated that patupilone antagonizes glioblastoma cell migration by a novel mechanism, which is distinct from suppression of MT dynamic instability. Taken together, our results suggest that EB proteins may represent a new potential target for anti-cancer therapy in highly invasive tumors.

Bcl-2 overexpression in type II epithelial cells does not prevent hyperoxia-induced acute lung injury in mice.

Bcl-2 is an anti-apoptotic molecule preventing oxidative stress damage and cell death. We have previously shown that Bcl-2 is able to prevent hyperoxia-induced cell death when overexpressed in a murine fibrosarcoma cell line L929. We hypothesized that its specific overexpression in pulmonary epithelial type II cells could prevent hyperoxia-induced lung injury by protecting the epithelial side of the alveolo-capillary barrier. In the present work, we first showed that in vitro Bcl-2 can rescue murine pulmonary epithelial cells (MLE12) from oxygen-induced cell apoptosis, as shown by analysis of LDH release, annexin V/propidium staining, and caspase-3 activity. We then generated transgenic mice overexpressing specifically Bcl-2 in lung epithelial type II cells under surfactant protein C (SP-C) promoter (Tg-Bcl-2) and exposed them to hyperoxia. Bcl-2 did not hinder hyperoxia-induced mitochondria and DNA oxidative damage of type II cell in vivo. Accordingly, lung damage was identical in both Tg-Bcl-2 and littermate mice strains, as measured by lung weight, bronchoalveolar lavage, and protein content. Nevertheless, we observed a significant lower number of TUNEL-positive cells in type II cells isolated from Tg-Bcl-2 mice exposed to hyperoxia compared with cells isolated from littermate mice. In summary, these results show that although Bcl-2 overexpression is able to prevent hyperoxia-induced cell death at single cell level in vitro and ex vivo, it is not sufficient to prevent cell death of parenchymal cells and to protect the lung from acute damage in mice.

NADPH oxidase-1 plays a crucial role in hyperoxia-induced acute lung injury in mice.

RATIONALE: Hyperoxia-induced acute lung injury has been used for many years as a model of oxidative stress mimicking clinical acute lung injury and the acute respiratory distress syndrome. Excess quantities of reactive oxygen species (ROS) are responsible for oxidative stress-induced lung injury. ROS are produced by mitochondrial chain transport, but also by NADPH oxidase (NOX) family members. Although NOX1 and NOX2 are expressed in the lungs, their precise function has not been determined until now. OBJECTIVES: To determine whether NOX1 and NOX2 contribute in vivo to hyperoxia-induced acute lung injury. METHODS: Wild-type and NOX1- and NOX2-deficient mice, as well as primary lung epithelial and endothelial cells, were exposed to room air or 100% O(2) for 72 hours. MEASUREMENTS AND MAIN RESULTS: Lung injury was significantly prevented in NOX1-deficient mice, but not in NOX2-deficient mice. Hyperoxia-dependent ROS production was strongly reduced in lung sections, in isolated epithelial type II cells, and lung endothelial cells from NOX1-deficient mice. Concomitantly, lung cell death in situ and in primary cells was markedly decreased in NOX1-deficient mice. In wild-type mice, hyperoxia led to phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), two mitogen-activated protein kinases involved in cell death signaling, and to caspase-3 activation. In NOX1-deficient mice, JNK phosphorylation was blunted, and ERK phosphorylation and caspase-3 activation were decreased. CONCLUSIONS: NOX1 is an important contributor to ROS production and cell death of the alveolocapillary barrier during hyperoxia and is an upstream actor in oxidative stress-induced acute lung injury involving JNK and ERK pathways in mice.

Antiangiogenic vinflunine affects EB1 localization and microtubule targeting to adhesion sites.

The motile behavior of endothelial cells is a crucial event for neoangiogenesis. We previously showed that noncytotoxic concentrations of vinflunine inhibit capillary-like tube formation on Matrigel and endothelial cell migration with a concomitant increase in interphase microtubule dynamic instability. In this article, we further investigated the effects of vinflunine on migration and cytoskeleton interaction dynamics in HMEC-1 endothelial cells. We confirmed that vinflunine, at low and noncytotoxic concentrations (0.01-1 nmol/L), inhibited endothelial cell random motility by 54%. This effect was associated with a decrease in the percentage of stable microtubules and in the mean duration of pauses for dynamic ones. Moreover, we found that vinflunine altered adhesion site targeting by microtubules and suppressed the microtubule (+) end pause that occurs at adhesion sites during cell migration (from 151 +/- 20 seconds in control cells to 38 +/- 7 seconds in vinflunine-treated cells, P < 0.001). This effect was associated with the inhibition of adhesion site dynamics and the formation of long-lived stress fibers. Importantly, we found that vinflunine altered EB1 localization at microtubule (+) ends. These results highlight a new mechanism of action of vinflunine, which act by disrupting the mutual control between microtubule and adhesion site dynamics and strengthen the role of +TIPs proteins such as EB1 as key regulators of endothelial cell motility.

Poly(ADP-ribose) polymerase-1 (PARP-1) controls lung cell proliferation and repair after hyperoxia-induced lung damage.

Oxygen-based therapies expose lung to elevated levels of ROS and induce lung cell damage and inflammation. Injured cells are replaced through increased proliferation and differentiation of epithelial cells and fibroblasts. Failure to modulate these processes leads to excessive cell proliferation, collagen deposition, fibrosis, and chronic lung disease. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated in response to DNA damage and participates in DNA repair, genomic integrity, and cell death. In this study, we evaluated the role of PARP-1 in lung repair during recovery after acute hyperoxia exposure. We exposed PARP-1 -/- and wild-type mice for 64 h to 100% hyperoxia and let them recover in air for 5-21 days. PARP-1-deficient mice exhibited significantly higher lung cell hyperplasia and proliferation than PARP-1 +/+ animals after 5 and 10 days of recovery. This was accompanied by an increased inflammatory response in PARP-1 -/- compared with wild-type animals, characterized by neutrophil infiltration and increased IL-6 levels in bronchoalveolar lavages. These lesions were reversible, since the extent of the hyperplastic regions was reduced after 21 days of recovery and did not result in fibrosis. In vitro, lung primary fibroblasts derived from PARP-1 -/- mice showed a higher proliferative response than PARP-1 +/+ cells during air recovery after hyperoxia-induced growth arrest. Altogether, these results reveal an essential role of PARP-1 in the control of cell repair and tissue remodeling after hyperoxia-induced lung injury.

Bcl-2 protects against hyperoxia-induced apoptosis through inhibition of the mitochondria-dependent pathway.

Bcl-2 is an antiapoptotic molecule that prevents oxidative stress damage and cell death. We investigated the possible protective mechanisms mediated by Bcl-2 during hyperoxia-induced cell death in L929 cells. In these cells, hyperoxia promoted apoptosis without DNA fragmentation. Overexpression of Bcl-2 significantly protected cells from oxygen-induced apoptosis, as shown by measurement of lactate dehydrogenase release, quantification of apoptotic nuclei, and detection of Annexin-V-positive cells. Bcl-2 partially prevented mitochondrial damage and interfered with the mitochondrial proapoptotic signaling pathway: it reduced Bax translocation to mitochondria, decreased the release of cytochrome c, and inhibited caspase 3 activation. However, treatment with the caspase inhibitor Z-VAD.fmk failed to rescue the cells from death, indicating that protection provided by Bcl-2 was due not only to caspase inhibition. Bcl-2 also prevented the release of mitochondrial apoptotic inducing factor, a mediator of caspase-independent apoptosis, correlating with the absence of oligonucleosomal DNA fragmentation. In addition, Bcl-2-overexpressing cells showed significantly higher intracellular amounts of glutathione after 72 h of oxygen exposure. In conclusion, our results demonstrate that the overexpression of Bcl-2 is able to prevent hyperoxia-induced cell death, by affecting mitochondria-dependent apoptotic pathways and increasing intracellular antioxidant compounds.

Long-term cultures of polarized airway epithelial cells from patients with cystic fibrosis.

The poor ability of respiratory epithelial cells to proliferate and differentiate in vitro into a pseudostratified mucociliated epithelium limits the general use of primary airway epithelial cell (AEC) cultures generated from patients with rare diseases, such as cystic fibrosis (CF). Here, we describe a procedure to amplify AEC isolated from nasal polyps and generate long-term cultures of the respiratory epithelium. AEC were seeded onto microporous permeable supports that carried on their undersurface a preformed feeder layer of primary human airway fibroblasts. The use of fibroblast feeder layers strongly stimulated the proliferation of epithelial cells, allowing the expansion of the cell pool with successive passages. AEC at increasing passage were seeded onto supports undercoated with airway fibroblasts and exposed to air. Either freshly isolated or amplified AEC could differentiate into a pseudostratified mucociliated epithelium for at least 10 mo. Thus, CF epithelia cultures showed elevated Na+ transport, drastic hyperabsorption of surface liquid, and absence of cAMP-induced Cl- secretion as compared with non-CF cultures. They were also characterized by thick apical secretion that hampered the movement of cell surface debris by cilia. However, CF respiratory epithelia did not show increased production of mucins or IL-8. The method described here is now routinely used in our laboratory to establish long-term cultures of well differentiated respiratory epithelia from human airway biopsies.

Poly(ADP-ribose)polymerase activation mediates lung epithelial cell death in vitro but is not essential in hyperoxia-induced lung injury.

Hyperoxia induces extensive DNA damage and lung cell death by apoptotic and nonapoptotic pathways. We analyzed the regulation of Poly(ADP-ribose)polymerase-1 (PARP-1), a nuclear enzyme activated by DNA damage, and its relation to cell death during hyperoxia in vitro and in vivo. In lung epithelial-derived A549 cells, which are known to die by necrosis when exposed to oxygen, a minimal amount of PARP-1 was cleaved, correlating with the absence of active caspase-3. Conversely, in primary lung fibroblasts, which die mainly by apoptosis, the complete cleavage of PARP-1 was concomitant to the induction of active caspase-3, as assessed by Western blot and caspase activity. Blockade of caspase activity by Z-VAD reduced the amount of cleaved PARP-1 in fibroblasts. Hyperoxia induced PARP activity in both cell types, as revealed by poly-ADP-ribose accumulation. In A549 cells, the final outcome of necrosis was dependent on PARP activity because it was prevented by the PARP inhibitor 3-aminobenzamide. In contrast, apoptosis of lung fibroblasts was not sensitive to 3-aminobenzamide and was not affected by PARP-1 deletion. In vivo, despite evidence of PARP activation in hyperoxia-exposed mouse lungs, absence of PARP-1 did not change the extent of lung damage, arguing for redundant oxidative stress-induced cell death pathways.

Mitochondrial cytochrome c release is a key event in hyperoxia-induced lung injury: protection by cyclosporin A.

Hyperoxia is known to induce extensive alveolar cell death by still poorly defined mechanisms. In this study, the mitochondria-dependent cell death pathway was explored during hyperoxia-induced lung injury in mice. We observed a progressive release of cytochrome c from the mitochondria into the cytosol of alveolar cells. This release was accompanied by the translocation of the proapoptotic protein Bax from cytosol to mitochondria without detectable activation of caspase-3. As cytochrome c release can be induced by mitochondrial membrane alteration and permeability transition (MPT), mice were treated with cyclosporin A, which specifically inhibits MPT. Cyclosporin A treatment prevented mitochondrial release of cytochrome c during hyperoxia and concomitantly preserved mitochondria from extensive swelling and crista disorganization, as assessed by electron microscopy analysis of alveolar epithelial cells. These morphological and biochemical observations correlated with decreased lung tissue damage, as evaluated by morphological score and lung weight. In conclusion, mitochondrial damage and cytochrome c release are important linked events in hyperoxia-induced lung injury and can be efficiently blocked by cyclosporin A.