Mitophagy-dependent necroptosis contributes to the pathogenesis of COPD

Abstract

Mitochondria/cytosol fractionation in vitro. Mitochondria and cytosol were fractionated using the Mitochondria/Cytosol Fractionation Kit according to the manufacturer's protocol (Enzo Life Sciences).Cytotoxicity assays. Cytotoxicity was assessed by measuring the release of LDH into the media (LDH-Cytotoxicity Colorimetric Assay Kit II; BioVision) according to the manufacturer's protocol.Flow cytometry. To discriminate live and dead cells, cells were simultaneously stained with green fluorescent calcein-AM to indicate intracellular esterase activity and red fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity using the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes). To assess the functional mitochondrial pool, cells were stained for 20 minutes at 37°C with 100 nM TMRE (Abcam), followed by CSE treatment. mtROS was measured in cells by MitoSOX (Invitrogen) staining (2.5 μM for 10 minutes at 37°C). Data were acquired with aIn vivo CS and chemical treatments. Age-matched mice (6-12 weeks old) were exposed to RA or CS in whole-body exposure chambers as described (5) Human lung bronchial epithelial Beas-2B cells were purchased from ATCC and maintained in DMEM containing 10% FBS and gentamicin (100 μg/ml). The primary alveolar epithelial cells of mouse lung were obtained as previously described and used for experiments before passage (70, 71). CSE was prepared and added to culture media as previously described (5, 6). , and Drp1 in human lung homogenate samples from control subjects and COPD patients. β-Actin served as the standard. PINK1, RIP3, and Drp1 expression was assessed by densitometry of immunoblots. Band intensities were normalized to β-actin. n = 9 samples/group. Representative immunohistochemical study (original magnification, ×200) for PINK1 (B) or RIP3 (C) in human lung sections from never-smokers (n = 3 patients, 5 images/patient) or COPD patients (n = 6 patients, 5 images/patient). Scale bar: 100 μm. Outlined areas are shown enlarged at right (scale bar: 20 μm). (D) Immunofluorescence staining (original magnification, ×40) for PINK1 (green), RIP3 (red), and nuclear (blue) in human lung tissue from never-smokers (n = 2 patients, 3 images/patient) and COPD patients (n = 2 patients, 3 images/patient). Scale bar: 50 μm. Yellow-outlined areas are shown enlarged in bottom panels (scale bar: 10 μm). Data represent the mean ± SEM (A). **P < 0.01 by unpaired, 2-tailed Student's t test (A). The Journal of Clinical Investigation R e s e a R c h a R t i c l e4 0 0 1

PARK2-mediated mitophagy is involved in regulation of HBEC senescence in COPD pathogenesis

“…[18][19][20][21] Programmed cell death (PCD) has been widely implicated in COPD pathogenesis and the involvement of autophagy, including mitophagy, has been reported in not only apoptosis but also programmed necrosis, necroptosis. 32,33 The central purpose of both PCD and cell senescence is eliminating damaged cells for tissue regeneration, indicating that the extent of cell damage may be critical for determination of cell fate. 34 In contrast to our finding of a protective role for mitophagy in cell senescence induced by lower Figure 4 (See previous page).…”

“…Open bar is no treatment, filled bar is CSE-treated. *P < 0.05, **P < 0.001. concentration of CSE (1.0%), a recent paper showing involvement of PINK1-mediated mitophagy in necroptosis used higher concentration of CSE (20%), 32 raising the following possibilities. First, excessive mitophagy activation by lethal stress may induce PCD, suggesting that mitophagy can lead to either PCD or senescence depending on the amount of damage.…”

“…Second, PINK1 has been shown to induce not only mitophagy but also mitochondrial depolarization during CSE exposure, indicating that PINK1-mediated mitochondrial damage has a dominant role in the setting of higher concentrations of CSE, resulting in PCD. 32 Mitochondrial division inhibitor-1 (Mdivi-1), which was used as a mitophagy inhibitor for necroptosis reduction, 32 has also been known to attenuate mitochondrial damage via inhibition of mitochondrial fission and mitochondrial outer membrane permeabilization (MOMP), suggesting that both mitophagy and mitochondrial integrity have a key regulatory role in determining cell fate in response to CSE exposure. Although we have demonstrated the involvement of insufficient mitophagymediated ROS production in regulation of CSE-induced HBEC senescence, a causal link between mitochondrial ROS and cellular senescence remains controversial.…”

SESN2/sestrin2 suppresses sepsis by inducing mitophagy and inhibiting NLRP3 activation in macrophages

“…55 mt-mKeima was used as a sensitive and quantitative assessment of mitophagy as described. 56 BMDMs were infected with mtmKeima retroviral particles for 2 d just before use, primed with LPS for 12 h followed by ATP treatment, fixed with 4% paraformaldehyde for 15 min at room temperature, and analyzed by confocal microscopy using 2 excitation filters (438 nm and 550 nm) and a 610LP emission filter at 800 x magnification. Ratio (550 ex :438 ex ) images of mt-mKeima were created and analyzed using MetaMorph software.…”

Wybrane aspekty opieki pielęgniarskiej w opiece nad pacjentem w podeszłym wieku z przewlekłą obturacyjną chorobą płuc - studium przypadku

“…Patogeneza tego schorzenia jest wieloczynnikowa [6]. Odnosi się ona zarówno do aspektów genetycznych, jak i środowiskowych i obejmuje zapalenie (przewlekłe zapalenie oskrzeli), utratę powierzchni pęcherzyków płucnych oraz zniszczenie oskrzelików oddechowych [7]. Schorzenie to w znaczący sposób obniża jakość życia chorych.…”