Terminal Deoxyribonucleotidyl Transferase-mediated Deoxyuridine Research Articles

Role of Microglia Autophagy in Microglia Activation After Traumatic Brain Injury

We evaluated the role of microglia autophagy in microglia activation after traumatic brain injury (TBI) in rats. TBI was induced by a fluid percussion TBI device. All rats were killed 24 hours after TBI. The ipsilateral hippocampus in all rats was analyzed with hematoxylin-eosin staining. Immunohistochemistry and Western blotting of ionized calcium-binding adapter molecule 1 was used to determine changes in microglia activation. Double staining of microtubule-associated protein light chain 3, Beclin-1, and ionized calcium-binding adapter molecule 1 was used to assess changes of microglia autophagy. Enzyme-linked immunosorbent assay of tumor necrosis factor-α and interleukin-1β was used to evaluate changes in inflammatory responses. Terminal deoxyribonucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick-end labeling staining was used to determine cell death in the ipsilateral hippocampus. At 24 hours after TBI, microglial cells became activated, and the autophagy inhibitor 3-methyladenine (3-MA) further promoted microglia activation. Protein light chain 3- and Beclin-1-positive microglial cells were increased after TBI, whereas 3-MA decreased the number of positive microglial cells, increasing the expression of tumor necrosis factor-α and interleukin-1β; terminal deoxyribonucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick-end labeling staining demonstrated that 3-MA could increase the number of terminal deoxyribonucleotidyl transferase-mediated deoxyuridine 5'-triphosphate nick-end labeling-positive cells (16.83±0.83 vs. 11 ± 0.82, P < 0.001). Our data demonstrated that TBI induced microglia activation and microglia autophagy. Inhibition of microglia autophagy with 3-MA increased microglia activation and neural apoptosis. These findings indicate that targeting microglia autophagy may be a therapeutic strategy for TBI.

Protective effect of polydatin against burn-induced lung injury in rats.

Polydatin (PD) has anti-inflammatory and anti-apoptotic effects in ischemic-reperfusion injury. Moreover, inflammatory responses and apoptosis play a role in the development of burn-induced lung injuries. Based on these findings, in this study we investigated the hypothesis that PD can ameliorate lung injury induced by extensive burns via reduction of inflammation and apoptosis. Rats were subjected to 30% total body surface area burn injury followed by resuscitation. The treatment group received 45 mg/kg PD, and the burn group received the same amount of normal saline solution. No burn injury was inflicted in the sham group. Microvascular permeability, interstitial edema, neutrophil recruitment, and histopathological changes were detected by measuring Evans blue concentration, wet-to-dry lung weight ratio (W/D), myeloperoxidase (MPO) activity, and hematoxylin and eosin staining, respectively. To investigate the mechanism of action of PD, enzyme-linked immunosorbent assay, cell counting, terminal deoxyribonucleotidyl transferase-mediated deoxyuridine 5-triphosphate-digoxigenin nick end labeling (TUNEL) staining, fluorometric assay, and Western blot were used for assessing levels of inflammatory cytokines (tumor necrosis factor alpha, interleukin [IL]-1β, and IL-6), total number of cells, and concentration of polymorphonuclear leukocytes (PMNs) in bronchoalveolar lavage fluid (BALF), the number of apoptotic cells, caspase-3 activity, and apoptosis-related proteins including Bax and Bcl-xl, respectively. Burn-injury rats exhibited significant lung injury characterized by the deterioration of histopathological characteristics, pulmonary microvascular hyperpermeability, and a high W/D, which were attenuated by PD (P = .007 for permeability, P = .004 for W/D). PD inhibited the burn-induced inflammatory response, as evidenced by the down-regulation of lung MPO activity (P = .008), total number of cells, PMN concentration in BALF, and the local and systemic levels of the pro-inflammatory cytokines examined. Moreover, PD treatment dramatically prevented burn-induced pulmonary cell apoptosis in lungs, as reflected by the decrease in the number of TUNEL-positive cells (P = .002) and changes in Bax, Bcl-xl, and caspase-3 activity (P = .03). PD ameliorates burn-induced lung injury via its anti-inflammatory and anti-apoptotic effects, and PD treatment may therefore serve as a potential therapeutic target for the treatment of critical burn injuries.

Activation of Peroxisome Proliferator–activated Receptor β/δ Attenuates Acute Ischemic Stroke on Middle Cerebral Ischemia Occlusion in Rats

Peroxisome proliferator-activated receptor (PPAR)-β/δ is a transcription factor that belongs to the nuclear hormone receptor family. There is little information about the effects of the immediate administration of specific ligands of PPAR-β/δ (GW0742) in animal models of acute ischemic stroke. Using a rat model of middle cerebral ischemia occlusion (MCAO) invivo, we have investigated the effect of pretreatment with GW0742 before MCAO. The neuroprotective effect of GW0742 against acute ischemic stroke was evaluated by the neurologic deficit score (NDS), dry-wet weight, and 2,3,5-triphenyltetrazolium chloride staining. The levels of interleukin (IL)-1β, nuclear factor (NF)-κB, and tumor necrosis factor (TNF)-α were detected by an enzyme-linked immunosorbent assay. The expressions of inducible nitric oxide synthase (iNOS), Bax, and Bcl-2 were detected by Western blot. The apoptotic cells were counted by in situ terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay. The pretreatment with GW0742 significantly increased the expression of Bcl-2, and significantly decreased in the volume of infarction, NDS, edema, expressions of IL-1β, NF-κB, TNFα, and Bax, contents of iNOS and the apoptotic cells in infarct cerebral hemisphere compared with rats in the vehicle group at 24hours after MCAO. The study suggests the neuroprotective effect of the PPAR-β/δ ligand GW0742 in acute ischemic stroke by a mechanism that may involve its anti-inflammatory and antiapoptotic action.

A major role for Tau in neuronal DNA and RNA protection in vivo under physiological and hyperthermic conditions

Nucleic acid protection is a substantial challenge for neurons, which are continuously exposed to oxidative stress in the brain. Neurons require powerful mechanisms to protect DNA and RNA integrity and ensure their functionality and longevity. Beside its well known role in microtubule dynamics, we recently discovered that Tau is also a key nuclear player in the protection of neuronal genomic DNA integrity under reactive oxygen species (ROS)-inducing heat stress (HS) conditions in primary neuronal cultures. In this report, we analyzed the capacity of Tau to protect neuronal DNA integrity in vivo in adult mice under physiological and HS conditions. We designed an in vivo mouse model of hyperthermia/HS to induce a transient increase in ROS production in the brain. Comet and Terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assays demonstrated that Tau protected genomic DNA in adult cortical and hippocampal neurons in vivo under physiological conditions in wild-type (WT) and Tau-deficient (KO-Tau) mice. HS increased DNA breaks in KO-Tau neurons. Notably, KO-Tau hippocampal neurons in the CA1 subfield restored DNA integrity after HS more weakly than the dentate gyrus (DG) neurons. The formation of phosphorylated histone H2AX foci, a double-strand break marker, was observed in KO-Tau neurons only after HS, indicating that Tau deletion did not trigger similar DNA damage under physiological or HS conditions. Moreover, genomic DNA and cytoplasmic and nuclear RNA integrity were altered under HS in hippocampal neurons exhibiting Tau deficiency, which suggests that Tau also modulates RNA metabolism. Our results suggest that Tau alterations lead to a loss of its nucleic acid safeguarding functions and participate in the accumulation of DNA and RNA oxidative damage observed in the Alzheimer’s disease (AD) brain.

Antioxidant and antiapoptotic properties of chlorogenic acid on human umbilical vein endothelial cells

Chlorogenic acid (CGA) is one of the phenols isolated from Lonicera japonica Thumb. This study was designed to investigate whether CGA prevents human umbilical vein endothelial cells (HUVECs) from hydrogen peroxide (H2O2)–induced damage. The cell viability was examined by methylthiazolyl tetrazolium (MTT) assay. Malondialdehyde (MDA) level was measured using the thiobarbituric acid method. Reactive oxygen species (ROS) levels were determined using the 2',7'-dichlorofluorescin-diacetate (DCFH-DA) assay. The antioxidant enzymes activities, including glutathione peroxidase (GPx) and superoxide dismutase (SOD) were examined photometrically. Total antioxidant capacity (T-AOC) was measured using the ferric reducing activity of plasma (FRAP) assay. Apoptotic cells were detected by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL), annexin-V binding and by assessment of caspase-3. Protein and mRNA expression of Bax and Bcl-2 were determined by western blot and reverse transcription-polymerase chain reaction (RT-PCR). In the process of H2O2-induced damage, the expression of Bcl-2 was decreased and the expression of Bax was increased. The antioxidant enzymes activities and T-AOC levels were decreased. The content of MDA and the level of ROS were increased. Pretreatment with CGA significantly reduced oxidative stress by increased T-AOC levels and antioxidant enzymes activities, decreased ROS levels and lipid peroxidation. CGA significantly reduced apoptosis by up-regulation the expression of Bcl-2 and down-regulation the expression of Bax. Taken together, these results suggest that CGA exhibits antioxidant and antiapoptotic properties to prevent H2O2-induced cytotoxicity in HUVECs. Key words: Chlorogenic acid, human umbilical vein endothelial cells (HUVECs), hydrogen peroxide, oxidative stress, apoptosis.

Neuroprotective Capabilities of Tanshinone IIA against Cerebral Ischemia/Reperfusion Injury via Anti-apoptotic Pathway in Rats

Danshen, derived from the dried root or rhizome of Salviae miltiorrhizae BGE., has Tanshinone IIA (TSA) as one of its active ingredients. Recent reports have shown that TSA can inhibit the apoptosis induced by serum withdrawal or ethanol in cultured PC12 cells. However, whether TSA has any neuroprotective effect remains unknown. In this study, we investigated the effects of TSA on cerebral apoptosis induced by middle cerebral artery occlusion (MCAO) in which cerebral ischemia had been induced 2 h earlier. Twenty-four hours after reperfusion, the rats were assessed for infarct volume etc. Intraperitoneal administration of 25 and 40 mg/kg TSA 10 min after MCAO significantly diminished infarct volume and brain water content and improved neurological deficits in a dose-dependent manner. The 25 mg/kg dosage was more effective. Treatment with 25 mg/kg TSA significantly improved symptoms and reduce infarct volume at different points in time, of which 10 min after MCAO was the most significant. Nissl-staining and HE-staining of the 25 mg/kg TSA group were more appreciable in terms of improvement relative to the vehicle group in the infarct core. TSA of dosage 25 mg/kg significantly decreased the expression of cleaved caspase-3 protein and increased the expression of B-cell lymphoma 2 (bcl-2) protein in the ischemic cortex. Fewer terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labeling (TUNEL)-positive cells were found in the penumbra of the treated group, but they were significantly more common in the vehicle group. We here conclude that the neuroprotective effects of TSA against focal cerebral ischemic/reperfusion injury are likely to be related to the attenuation of apoptosis.

Morphology of the developing muscularis externa in the mouse esophagus

Muscularis externa of mouse esophagus is composed of two skeletal muscle layers in the adult. But less attention is paid to the histogenesis of the muscularis externa of the esophagus, and controversies still exist about the developmental process and the spatio-temporal expression characteristics of muscle-specific proteins during the development of esophageal muscularis externa. To further probe into the developmental pattern of muscularis externa of the mouse esophagus and the expression characteristics of different muscle-specific proteins, immunohistochemical and terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick-end labeling apoptotic staining methods are used to investigate the expression patterns of different muscle-specific proteins and to elucidate the relationship of these protein expressions with the development of muscularis externa of the mouse esophagus. Thus, an understanding of the developing esophageal muscularis externa may be important for developing therapeutic strategies for the treatment of human esophagus diseases. Serial sections of mouse embryos from embryonic day (ED) 12 to ED18, and full-length esophagi from postnatal first to 5th day were stained with monoclonal antibodies against α-smooth muscle actin (α-SMA), α-sarcomerical actin (α-SCA), desmin, and monoclonal anti-skeletal myosin (MHC), while apoptosis was determined using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling assay. The expression of α-SMA was started at ED12. During the development of ED14-ED15, α-SMA positive cells were seen extending from the walls of left three, four, and six arch arteries toward the dorsal wall of esophagus. Stronger expression of α-SCA and desmin could be detected at ED14 and ED15, expression intensity in caudal segment and inner layer was stained stronger than that of cranial segment and outer layer, but after ED16, strong expression of α-SCA and desmin was found in the outer layer of muscularis externa. Expression of MHC was first detected in the outer layer of cranial segment of muscularis externa at ED17. At ED18, MHC had extended to the level of thyroid gland, staining intensity in the outer layer and cranial segment was stronger than that of inner layer and caudal segment. One to five days after birth, the thickness of the esophageal muscle layer was obviously increased. Most of the muscle cells in the cranial segment of esophagus showed strong expression of α-SCA and clear cross striations at higher magnification. With progression toward the caudal segment, expression intensity of α-SCA became weaker, but the expression intensity of desmin was the same at different levels of esophagus. The muscle fibers were arranged densely with high expression of MHC in the cranial segment. During the development of esophageal muscularis externa, few apoptotic cells were observed. α-SMA, α-SCA, desmin, and MHC show different expression patterns. The differentiation of outer layer of esophageal muscularis externa is quicker than that of inner layer, and the caudal segment is quicker than that of the cranial segment. Besides, apoptosis may not participate in the development of esophageal muscularis externa. The smooth muscle cells from arch arteries may participate in the development of esophageal muscularis externa.

Catalpol inhibits apoptosis in hydrogen peroxide-induced endothelium by activating the PI3K/Akt signaling pathway and modulating expression of Bcl-2 and Bax

Catalpol, an iridoid glucoside found in the root of Rehmannia glutinosa Libosch, has been demonstrated to reduce apoptosis in neuronal cell lines. Recent data suggests that catalpol also exerts anti-apoptotic effects on other cell types. The aim of the present study was to investigate whether catalpol protects against hydrogen peroxide (H 2O 2) induced apoptosis in human umbilical vein endothelial cells (HUVECs). Apoptotic cells were detected by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling, Annexin V-fluorescein isothiocyanate binding assay and by assessment of caspase-3 activity. The level of intracellular reactive oxygen species was quantified by 2′, 7′-dichlorofluorescein diacetate assay. Expression of Akt, Bad, Bcl-2 and Bax mRNA and protein was determined by real-time semiquantitative reverse transcription-polymerase chain reaction and Western blotting. Apoptosis in HUVECs was associated with increased Bax, decreased Bcl-2 activity and inactivated phosphorylation of Akt and Bad after 24 h of H 2O 2 exposure. Pre-treatment of HUVECs with catalpol significantly reduced H 2O 2-induced intracellular reactive oxygen species release. Catalpol not only increased the expression of Bcl-2, while decreasing Bax expression, but also induced Akt activation and Bad phosphorylation, and ultimately reduced H 2O 2-induced apoptosis. The protective effects of catalpol were partially inhibited by the phosphatidylinositol 3-kinase (PI3K) antagonist wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). Taken together, these results suggest that pre-treatment of HUVECs with catalpol can block H 2O 2-induced apoptosis, and that the underlying mechanism involves reactive oxygen species scavenging, activation of the PI3K/Akt–Bad signaling pathway and increased Bcl-2 and decreased Bax expression.

Endothelial cell apoptosis in severe drug-induced bullous eruptions

Toxic epidermal necrolysis (TEN) and Stevens-Johnson syndrome (SJS) are characterized by extensive keratinocyte apoptosis mediated by cytotoxic proteins. Similar features have been found in another severe dysimmune syndrome, allogeneic acute graft-versus-host disease, where endothelial cell apoptosis has been recently characterized. To determine whether endothelial cell apoptosis occurs in dermal vessels of TEN and SJS, and whether it is linked to expression of cytotoxic proteins. Skin biopsies of eight patients with severe drug-induced bullous eruptions (four TEN, four SJS), eight with drug-induced urticaria and eight healthy controls were compared. Blood vessel damage was studied by electron microscopy and quantified by CD31 immunostaining. Apoptotic cells, characterized by electron microscopy, were quantified on terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling assay. Immunohistochemistry was also used to characterize and quantify inflammatory cells and granzyme B, tumour necrosis factor (TNF)-alpha and Fas ligand (FasL) expression. Endothelial cell apoptosis was observed in all TEN and SJS cases: it occurred in 85% of the vessel sections. It occurred in one case of drug-induced urticaria, in 5% of vessel sections, but not in healthy controls. Numbers of CD68+ macrophages and CD8+ T lymphocytes were significantly higher in TEN and SJS compared with both other groups; granzyme B and TNF-alpha but not FasL were expressed. Characterization of endothelial cell apoptosis in TEN and SJS is important to assess a factor worsening skin damage, with possible extension to other organs. It may also be useful for the development of novel therapeutic strategies.

An ethanolic extract of leaves of Piper betle (Paan) Linn mediates its antileishmanial activity via apoptosis

An unprecedented increase in the incidence of unresponsiveness to antimonial compounds has highlighted the urgent need to develop new antileishmanial agents. The leaves of Piper betle (locally known as Paan) have long been in use in the Indian indigenous system of medicine for its antimicrobial properties but its antileishmanial potential has not been studied. Accordingly, an ethanolic extract of leaves of Piper betle (PB) was tested for its antileishmanial activity that was evidenced in both promastigotes and amastigotes, with IC50 values of 9.8 and 5.45 microg/ml, respectively; importantly, it was accompanied by a safety index of >12-fold. This leishmanicidal activity of PB was mediated via apoptosis as evidenced by morphological changes, loss of mitochondrial membrane potential, in situ labeling of DNA fragments by terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling, and cell-cycle arrest at the sub-G0/G1 phase. Taken together, the data indicate that PB has promising antileishmanial activity that is mediated via programmed cell death and, accordingly, merits consideration and further investigation as a therapeutic option for the treatment of leishmaniasis.

Identification and quantification of apoptosis in the kidney using morphology, biochemical and molecular markers

Renal cell apoptosis is important in both physiological conditions such as normal renal development and pathological processes affecting the glomerular, vascular or tubulointerstitial compartments. Apoptosis may result in the detrimental loss of cells following many renal diseases or damaging changes, with significant loss of function. In contrast, apoptosis may control and limit inflammatory processes in both the acute and chronic phases of renal disease. Investigators interested in the presence of apoptotic cells in different forms of renal disease and development need methods to accurately determine the level of apoptosis within the kidney. Apoptosis is a gene-driven mode of cell death that may be identified by distinct morphological features, endonuclease-initiated DNA degradation, and by the involvement of specific apoptosis-regulating proteins. Many research papers that analyse the presence of apoptosis use the in situ terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay that detects DNA strand breaks in situ in tissue sections. Localization of activated caspase-3 is now seen as an alternative to TUNEL. This review will discuss some methods of identifying apoptosis in the kidney, using both morphological and biochemical or molecular characteristics, and also discuss some of the pitfalls of entire reliance on biochemical means of apoptotic cell identification without some morphological checks and balances. Although there are some caveats to the methods for identifying apoptotic cells in renal disease, those investigators who take the time to undertake such analysis often gain insightful data that provide explanations for the disease or condition being studied.

Insulin-Like Growth Factor-I Is an Important Antiapoptotic Factor for Rat Leydig Cells during Postnatal Development

The present investigation examines the influence of IGF-I and the role of IGF-I receptor (IGF-IR) in the apoptosis/survival of Leydig cells. Immunohistochemical analysis of the rat testis at different ages revealed that the level of the phosphorylated IGF-IR increases from birth to d 20 of postnatal life, remaining high in the adult testis. Western blotting revealed that this level is higher in Leydig cells isolated from 40-d-old than from 10- or 60-d-old rats. Application of the terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay revealed that IGF-I decreases the level of apoptosis in Leydig cells at all stages of development, and the selective inhibitor of IGF-IR, picropodophyllin, blocks this antiapoptotic effect. The mechanism underlying the antiapoptotic action of IGF-I involves the phosphatidylinositol 3-kinase/Akt pathway, and in immature Leydig cells, this growth factor enhances the expression of Bcl-2 and cellular inhibitor of apoptosis proteins 2, while preventing activation of caspase-3 by cleavage. Furthermore, IGF-II and high concentrations of insulin also evoke phosphorylation of IGF-IR and, like IGF-I, enhance the expression of the steroidogenic acute regulatory protein by Leydig cells. Inhibition of IGF-IR by picropodophyllin decreases the survival of Leydig cells, both in the presence and absence of IGF-I, demonstrating that signaling via the IGF-IR plays an important role in Leydig cell survival.

Activation of the maternally preset program of apoptosis by microinjection of 5-aza-2'-deoxycytidine and 5-methyl-2'-deoxycytidine-5'-triphosphate in Xenopus laevis embryos.

The present study examines the effects on embryogenesis of microinjecting Xenopus laevis fertilized eggs with 5-aza-2'-deoxycytidine (5-Aza-CdR), which induces hypomethylation of DNA, and 5-methyl-2'- deoxycytidine-5'-triphosphate (5-methyl-dCTP), which induces hypermethylation of DNA. Embryos injected with either one of these analogs cleaved normally until the mid-blastula stage, but underwent massive cell dissociation and stopped development at the early gastrula stage. Dissociated cells that appeared here were positive by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end-labeling and contained fragmented nuclei with condensed chromatin. The DNA from these cells formed a "ladder" on electrophoresis. Furthermore, the induction of cell dissociation by 5-Aza-CdR and 5-methyl-dCTP was postponed by 2-3 h by co-injection of Bcl-2 mRNA and the normal metabolite (CdR and dCTP, respectively). Using a specific antibody against 5-methyl-cytosine, we confirmed that 5-Aza-CdR induces hypomethylation, whereas 5-methyl-dCTP induces hypermethylation in X. laevis embryos before the onset of cell dissociation. Incorporation of radioactive precursors revealed that synthesis of DNA, and also RNA, is inhibited significantly in both 5-Aza-CdR-injected and 5-methyl-dCTP-injected embryos. These results show that 5-Aza-CdR and 5-methyl-dCTP are incorporated into DNA and induce apoptosis, probably through alteration of DNA methylation coupled with inhibition of DNA replication and/or transcription.

Apoptosis in neonatal murine lung exposed to hyperoxia.

Exposure to high concentrations of oxygen in the neonatal period may impair lung growth and is a major contributing factor to the development of bronchopulmonary dysplasia. Cell death from hyperoxic injury may occur through either an apoptotic or nonapoptotic pathway, and we were interested in determining the type of cell death that occurs in the lung of neonatal mice exposed to hyperoxia. We found increased levels of Bax messenger RNA, a gene associated with apoptosis, in the lungs of neonatal mice born and raised in 92% hyperoxia. We next determined the extent of apoptosis taking place in the lungs of neonatal mice exposed to hyperoxia using terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling in 3.5-, 4.5-, and 5.5-d-old neonatal lung. The number of apoptotic cells in peripheral lung was significantly higher in the 3.5-, 4.5-, and 5.5-d-old mice treated with oxygen compared with that in the room-air control mice. Further, the number of apoptotic cells in the lung increased with longer exposure duration. In murine lung bronchus cells exposed to hyperoxia, growth arrest occurred after 48 h of oxygen exposure. Using annexin V binding, necrotic cell death was found to be the major form of cell death in these cells after 72 h of hyperoxic exposure. We conclude that 92% hyperoxia causes significant lung injury in neonatal mice exposed to hyperoxia, and that the number of apoptotic cells in the lung increases the longer the duration of exposure. The increase in apoptosis from hyperoxic exposure during a critical period of lung development may be an important factor in the impaired lung growth and remodeling that occur in animals exposed to high oxygen concentrations. Finally, it appears that hyperoxic injured cells in neonatal lung undergo both apoptotic and nonapoptotic cell death.

The cyclin-dependent kinase inhibitor p21 protects the lung from oxidative stress.

The lung is a major target tissue for oxidative stress, including hyperoxia used to relieve tissue hypoxia. Unfortunately, severe hyperoxia damages DNA, inhibits proliferation, and kills cells, resulting in morbidity and mortality. Although hyperoxia induces the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1) (p21), their role in pulmonary injury remains unknown. Using p53- and p21-deficient mice we demonstrate that hyperoxia induces p21 in the absence of p53, suggesting that previous conclusions that p53 does not modify hyperoxic lung injury cannot be extrapolated to p21. In fact, mean survival of p21-deficient mice decreased by 40% and was associated with terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining of alveolar debris, indicative of DNA fragmentation and cell death. Ultrastructural analyses revealed that alveolar endothelial and type I epithelial cells died rapidly by necrosis. Although hyperoxia decreased DNA replication in p21-wild-type lungs, it had no effect on replication in p21-deficient lungs. Our findings suggest that p21 protects the lung from oxidative stress, in part, by inhibiting DNA replication and thereby allowing additional time to repair damaged DNA. Our findings have implications for patients suffering from the toxic effects of supplemental oxygen therapies.

Pseudomonas aeruginosa induction of apoptosis in respiratory epithelial cells: analysis of the effects of cystic fibrosis transmembrane conductance regulator dysfunction and bacterial virulence factors.

Airway epithelial cells can respond to infection by activating several signaling pathways. We examined the induction of apoptosis in response to Pseudomonas aeruginosa PAO1 in normal cells and several cystic fibrosis (CF) and corrected cell lines. Epithelial cells in monolayers with tight junctions, confirmed by apical ZO-1 staining demonstrated by confocal microscopy, were entirely resistant to PAO1-induced apoptosis. In contrast, cell lines such as 9HTEo(-) cells that do not form tight junctions were susceptible, with 50% of the population apoptotic after 6 h of exposure to PAO1. CF transmembrane conductance regulator (CFTR) dysfunction caused by different mechanisms (trafficking mutations, overexpression of the regulatory domain or antisense constructs) did not alter rates of apoptosis, nor were differences apparent in terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling detection of apoptotic airway cells from PAO1 infected cftr -/- or control mice. Bacterial expression of specific adhesins, complete lipopolysaccharide, and a functional type III secretion system were all necessary to evoke apoptosis even in susceptible epithelial cells. Unlike other mucosal surfaces, the airway epithelium is highly resistant to apoptosis, and this response is activated only when the appropriate epithelial conditions are present as well as fully virulent P. aeruginosa capable of coordinately expressing both adhesins and cytotoxins.