Stuart Horowitz

Mitogen-activated protein kinase pathway mediates hyperoxia-induced apoptosis in cultured macrophage cells

We have previously demonstrated that the lungs of mice can exhibit increased programmed cell death or apoptosis after hyperoxic exposure in vivo. In this report, we show that hyperoxic exposure in vitro can also induce apoptosis in cultured murine macrophage cells (RAW 264.7) as assessed by DNA-laddering, terminal deoxynucleotidyltransferase dUTP nick end-labeling, and nucleosomal assays. To further delineate the signaling pathway of hyperoxia-induced apoptosis in RAW 264.7 macrophages, we first show that hyperoxia can activate the mitogen-activated protein kinase (MAPK) pathway, the extracellular signal-regulated kinases (ERKs) p42/p44, in a time-dependent manner as assessed by increased phosphorylation of ERK1/ERK2 by Western blot analyses. Neither the c-Jun NH(2)-terminal kinase/stress-activated protein kinase nor the p38 MAPK was activated by hyperoxia in these cells. Chemical or genetic inhibition of the ERK p42/p44 MAPK pathway by PD-98059, a selective inhibitor of MAPK kinase, and dominant negative mutants of ERK, respectively, attenuated hyperoxia-induced apoptosis as assessed by DNA laddering and nucleosomal ELISAs. Taken together, our data suggest that hyperoxia can induce apoptosis in cultured murine macrophages and that the MAPK pathway mediates hyperoxia-induced apoptosis.We have previously demonstrated that the lungs of mice can exhibit increased programmed cell death or apoptosis after hyperoxic exposure in vivo. In this report, we show that hyperoxic exposure in vitro can also induce apoptosis in cultured murine macrophage cells (RAW 264.7) as assessed by DNA-laddering, terminal deoxynucleotidyltransferase dUTP nick end-labeling, and nucleosomal assays. To further delineate the signaling pathway of hyperoxia-induced apoptosis in RAW 264.7 macrophages, we first show that hyperoxia can activate the mitogen-activated protein kinase (MAPK) pathway, the extracellular signal-regulated kinases (ERKs) p42/p44, in a time-dependent manner as assessed by increased phosphorylation of ERK1/ERK2 by Western blot analyses. Neither the c-Jun NH2-terminal kinase/stress-activated protein kinase nor the p38 MAPK was activated by hyperoxia in these cells. Chemical or genetic inhibition of the ERK p42/p44 MAPK pathway by PD-98059, a selective inhibitor of MAPK kinase, and dominant negative mutants of ERK, respectively, attenuated hyperoxia-induced apoptosis as assessed by DNA laddering and nucleosomal ELISAs. Taken together, our data suggest that hyperoxia can induce apoptosis in cultured murine macrophages and that the MAPK pathway mediates hyperoxia-induced apoptosis.

Retinal vascular endothelial growth factor (VEGF) mRNA expression is altered in relation to neovascularization in oxygen induced retinopathy

The temporal and spatial expression of vascular endothelial cell growth factor (VEGF) mRNA was studied in normal developing cat retina, and in oxygen induced retinopathy. Unexposed control and oxygen-exposed animals (80 h of 80% oxygen from day 3, n = 16) were studied at 1, 2, 4, and 6 weeks after birth. India ink injected retinal flat mounts were used to study vessel progression, and in situ hybridizations using retinal cross sections were used to assess VEGF mRNA accumulation. In controls, as the retina matured, VEGF mRNA hybridization was evident in the ganglion cell layer in a scattered line of distinct cells prior to the ingrowth of vessels, involved the most cells in regions just peripheral to invading vessels and persisted in a fewer positive cells, widely spaced in the vascularized retinas of control, six week animals. In the inner nuclear layer, hybridization initially appeared diffusely and later became localized to a narrow portion of that layer and persisted there. In animals with oxygen induced retinopathy, a substantial increase in hybridization was observed in both the ganglion cell and inner nuclear layers of the avascular retina anterior to the advancing neovascularization. VEGF hybridization decreased abruptly to background levels in both layers at the point were neovascularization met avascular retina. By six weeks, when the neovascularization reached the ora, there was a return of VEGF mRNA in the inner nuclear layer which was similar to normal control expression. A low level of unchanging expression was also observed in the retinal pigment epithelium in both groups at all ages. These results indicate that VEGF mRNA abundance is regulated during retinal vascularization and is increased in relation to oxygen induced neovascularization, suggesting that VEGF may play an important role in both normal retinal vessel development and in the pathophysiology of retinopathy of prematurity.

Hyperoxia Inhibits Oxidant-induced Apoptosis in Lung Epithelial Cells

It has previously been shown that hyperoxia induces nonapoptotic cell death in cultured lung epithelial cells, whereas hydrogen peroxide (H2O2) and paraquat cause apoptosis. To test whether pathways leading to oxidative apoptosis in epithelial cells are sensitive to molecular O2, A549 cells were exposed to 95% O2 prior to exposure to lethal concentrations of H2O2. The extent of H2O2-induced apoptosis was significantly reduced in cells preexposed to hyperoxia compared with room-air controls. Preexposure of the hyperoxia-resistant HeLa-80 cell line to 80% O2 also inhibited oxidant-induced apoptosis, suggesting that this inhibition is not due to O2toxicity. Because hyperoxia generates reactive oxygen species and activates the redox-sensitive transcription factor nuclear factor κB (NF-κB), the role of antioxidant enzymes and NF-κB were examined in this inhibitory process. The onset of inhibition appeared to be directly related to the degradation of IκB and subsequent activation of NF-κB (either by hyperoxia or TNF-α), whereas no significant up-regulation of endogenous antioxidant enzyme activities was found. In addition, suppression of NF-κB activities by transfecting A549 cells with a dominant-negative mutant construct of IκB significantly augmented the extent of H2O2-induced apoptosis. These data suggest that hyperoxia inhibits oxidant-induced apoptosis and that this inhibition is mediated by NF-κB.

Pulmonary apoptosis in aged and oxygen-tolerant rats exposed to hyperoxia.

Accumulating evidence demonstrates that genotoxic and oxidant stress can induce programmed cell death or apoptosis in cultured cells. However, little is known about whether oxidative stress resulting from the deleterious effects of hyperoxia can induce apoptosis in vivo and even less is known regarding the functional significance of apoptosis in vivo in response to hyperoxia. Using hyperoxia as a model of oxidant-induced lung injury in the rat, we show that hyperoxic stress results in marked apoptotic signals in the lung. Lung tissue sections obtained from rats exposed to hyperoxia exhibit increased apoptosis in a time-dependent manner by terminal transferase dUTP nick end labeling assays. To examine whether hyperoxia-induced apoptosis in the lung correlated with the extent of lung injury or tolerance (adaptation) to hyperoxia, we investigated the pattern of apoptosis with a rat model of age-dependent tolerance to hyperoxia. We show that apoptosis is associated with increased survival of aged rats to hyperoxia and with decreased levels of lung injury as measured by the volume of pleural effusion, wet-to-dry lung weight, and myeloperoxidase content in aged rats compared with young rats after hyperoxia. We also examined this relationship in an alternate model of tolerance to hyperoxia. Lipopolysaccharide (LPS)-treated young rats not only demonstrated tolerance to hyperoxia but also exhibited a significantly lower apoptotic index compared with saline-treated rats after hyperoxia. To further separate the effects of aging and tolerance, we show that aged rats pretreated with LPS did not exhibit a significant level of tolerance against hyperoxia. Furthermore, similar to the hyperoxia-tolerant LPS-pretreated young rats, the nontolerant LPS-pretreated aged rats also exhibited a significantly reduced apoptotic index compared with aged rats exposed to hyperoxia alone. Taken together, our data suggest that hyperoxia-induced apoptosis in vivo can be modulated by both aging and tolerance effects. We conclude that there is no overall relationship between apoptosis and tolerance.

Synergistic cytotoxicity from nitric oxide and hyperoxia in cultured lung cells

Exogenous nitric oxide (NO) is being tested clinically for the treatment of pulmonary hypertension in infants and children. In most cases, these patients receive simultaneous oxygen (O2) therapy. However, little is known about the combined toxicity of NO + hyperoxia. To test this potential toxicity, human alveolar epithelial cells (A549 cells) and human lung microvascular endothelial lung cells were cultured in room air (control), hyperoxia (95% O2), NO (derived from chemical donors), or combined hyperoxia + NO. Control cells grew normally over a 6-day study period. In contrast, cell death from hyperoxia was evident after 4-5 days, whereas cells neither died nor divided in NO alone. However, cells exposed to both NO and hyperoxia began to die on day 2 and died rapidly thereafter. This cytotoxic effect was clearly synergistic, and cell death did not occur via apoptosis. As an indicator of peroxynitrite formation, nitrotyrosine-containing proteins were assayed using anti-nitrotyrosine antibodies. Two protein bands, at molecular masses of 25 and 35 kDa, were found to be increased in A549 cells exposed to NO or NO + hyperoxia. These results indicate that combined NO + hyperoxia has a synergistic cytotoxic effect on alveolar epithelial and lung vascular endothelial cells in culture.Exogenous nitric oxide (NO) is being tested clinically for the treatment of pulmonary hypertension in infants and children. In most cases, these patients receive simultaneous oxygen (O2) therapy. However, little is known about the combined toxicity of NO+hyperoxia. To test this potential toxicity, human alveolar epithelial cells (A549 cells) and human lung microvascular endothelial lung cells were cultured in room air (control), hyperoxia (95% O2), NO (derived from chemical donors), or combined hyperoxia+ NO. Control cells grew normally over a 6-day study period. In contrast, cell death from hyperoxia was evident after 4-5 days, whereas cells neither died nor divided in NO alone. However, cells exposed to both NO and hyperoxia began to die on day 2 and died rapidly thereafter. This cytotoxic effect was clearly synergistic, and cell death did not occur via apoptosis. As an indicator of peroxynitrite formation, nitrotyrosine-containing proteins were assayed using anti-nitrotyrosine antibodies. Two protein bands, at molecular masses of 25 and 35 kDa, were found to be increased in A549 cells exposed to NO or NO+hyperoxia. These results indicate that combined NO+hyperoxia has a synergistic cytotoxic effect on alveolar epithelial and lung vascular endothelial cells in culture.

Intracellular uptake of recombinant superoxide dismutase after intratracheal administration

We have previously demonstrated that recombinant human copper-zinc superoxide dismutase (rhCu,ZnSOD) is rapidly incorporated into cells of airways, respiratory bronchioles, and alveoli after intratracheal administration. The present study examines whether this cellular uptake is specific for rhCu,ZnSOD or whether other proteins are similarly incorporated into lung cells. Twenty-two newborn piglets (2-3 days old, 1.2-2.0 kg) were intubated and mechanically ventilated. Eight piglets received fluorescently labeled recombinant human manganese superoxide dismutase (rhMnSOD), six received fluorescently labeled albumin, two received free (unbound) fluorescent label intratracheally, and two piglets served as untreated controls. To determine whether endogenous surfactant was important in the process of intracellular uptake, four additional piglets were made surfactant deficient by repeated bronchoalveolar lavage and then given rhCu,ZnSOD intratracheally. All animals were killed after 30-60 min. Lung sections were examined blindly by laser confocal microscopy. Similar to our previous observations with rhCu,ZnSOD, intracellular uptake of rhMnSOD and albumin was noted throughout the lung. The free label did not localize intracellularly. The uptake of proteins did not appear to be affected by surfactant deficiency. rhMnSOD administration was associated with a greater than twofold increase in lung MnSOD activity. Data suggest that the cellular uptake of antioxidants and other proteins in the lung may reflect a nonspecific host defense system for clearing proteins from the lumen of airways and alveoli.

Interleukin-1 expression during hyperoxic lung injury in the mouse.

An important component of the pathophysiologic response to hyperoxia (O2) is pulmonary inflammation, although the roles of specific inflammatory mediators during pulmonary O2 toxicity are not completely known. Interleukin-1 (IL-1) is an early inflammatory mediator and is sufficient to elicit many of the responses associated with acute injury. The IL-1 family comprises two bioactive proteins, IL-1alpha and IL-1beta, and their natural antagonist IL-1ra. Here we report studies of IL-1 regulation during hyperoxic lung injury in the adult mouse. When assayed by Northern blot, increases in IL-1beta mRNA were seen after 2 days of hyperoxia. In contrast, IL-1alpha mRNA was barely detectable before 4 days of hyperoxia. To further understand the cellular origin of IL-1beta expression in lungs, in situ hybridization and immunohistochemical analyses were performed. IL-1beta mRNA or protein was not detected in the lungs of unexposed animals. At 3 days, we observed the accumulation of IL-1beta transcripts in pulmonary interstitial macrophages and in a subset of neutrophils, and immunodetectable IL-1beta protein was co-localized in adjacent sections. At 4 days of exposure, IL-1beta transcripts were widespread in lung tissue, but many areas rich in IL-1beta mRNA were devoid of immunodetectable IL-1beta. However, it is not known whether increased synthesis of IL-1beta or the uncoupling of IL-1beta protein and mRNA accumulation has a role in pathophysiology of pulmonary O2 toxicity.

Cell-specific expression of fibronectin in adult and developing rabbit lung

Fibronectin (FN), a glycoprotein component of the extracellular matrix, plays a role in tissue morphogenesis and tissue-specific differentiation through its effects on cell adhesion, cell shape, and cytoskeletal organization. Immunohistochemistry has been used to show that during lung development FN deposition changes, yet the cell-specific sites of pulmonary FN synthesis have not been determined. Because cellular FN synthesis is reflected by FN mRNA abundance, we performed in situ hybridizations to identify pulmonary tissue with the capacity to synthesize FN. Both in situ mRNA hybridization and immunohistochemical staining were performed on tissue sections from lungs of adults and late gestation fetal and neonatal rabbits. In adults, FN transcripts and immunostaining were clearly seen in endothelial cells, smooth muscle cells, and chondrocytes. During lung development, FN transcripts were virtually ubiquitous except in airway epithelium. There was a gradual decrease in FN mRNA abundance with advancing fetal age, but low levels of FN mRNA persisted in neonatal and adult lungs. In contrast, parenchymal immunostaining increased throughout fetal development and remained elevated in the newborn. FN immunostaining was lower in adult lung. In all tissues examined, airway epithelial cells contained no FN transcripts above background. However, immunostaining was detected in airway basement membrane zones and on luminal surfaces of some epithelial cells. The lack of FN transcripts in airway epithelial cells suggests that FN synthesis does not normally occur in this cell type and that its associated FN immunostaining is from another source. The colocalization of FN mRNA and protein in pulmonary endothelial cells, smooth muscle cells, and chondrocytes in adults strongly suggests that these cells are sites of FN synthesis.

Cell-specific expression of fibronectin and EIIIA and EIIIB splice variants after oxygen injury

Cellular fibronectin (cFN) expression is characteristic of injured tissues. Unlike plasma FN, cFN mRNA often contains the EIIIA or EIIIB domains. We examined the lung cell-specific expression of total cFN mRNA and the EIIIA and EIIIB splice variants in rabbits after acute oxygen injury. By in situ hybridization, control lung had low cFN mRNA. After exposure to > 95% oxygen, mRNAs for total cFN and EIIIA were noted primarily in alveolar macrophages and large-vessel endothelial cells. By 3-5 days recovery, cFN and EIIIA mRNA abundance was increased in alveolar septal cells (i.e., alveolar epithelial, interstitial, or endothelial cells) and in some large-vessel endothelial cells but was low in bronchial epithelial cells. During recovery, EIIIB mRNA was low in alveolar septal cells but was noted mainly in chondrocytes. Immunostaining for EIIIA increased during recovery, paralleling the in situ hybridizations. Because FN may modulate alveolar type II cell phenotype, we investigated type II cell cFN mRNA expression in vivo. During recovery, neither isolated type II cells nor cells with surfactant protein C mRNA in vivo contained FN mRNA. In summary, these data suggest that cFN with the EIIIA domain has a role in alveolar cell recovery from oxygen injury and that type II cells do not express cFN during recovery.

DISTINCT PATTERNS OF APOPTOSIS DURING TOTAL LIQUID VENTILATION (TLV) VS. CONVENTIONAL VENTILATION (CV). |[dagger]| 1516

Mechanical ventilation with high pressures and supraphysiologic concentrations of oxygen is often needed to treat critically ill infants, but it can be quite toxic to the lung. This toxicity can lead to cell death by two distinct modes: apoptosis or necrosis. Apoptosis is often a scheduled and physiologically regulated event, while necrosis is the result of unscheduled, acute injury (often accompanied by inflammation). We have previously shown that in vivo, hyperoxia can cause significant cell death in the lung by apoptosis. TLV is a technique that may be associated with less lung injury and better physiological outcomes compared to CV. To examine whether TLV causes less lung injury and apoptosis compared to CV, we studied 2 groups of premature lambs delivered at 110 and 120 days gestation. Animals were ventilated with 100% O2 and TLV (Liquivent, Alliance Pharm., Hoechst-Marion-Roussel) or CV from birth for up to 4h. Unventilated, gestational matched controls were also studied. Standard physiologic and histologic parameters were analyzed. We also utilized the in situ TUNEL assay, which labels 3′-OH ends of DNA cut by endonucleases that are activated during apoptosis, to evaluate the amount of apoptosis occurring in the lung. Similar to previous reports, the TLV group had better gas exchange and less histologic evidence of lung injury compared to animals receiving CV. Approximately 5% of lung cells were apoptotic in both experimental groups, while gestational matched controls showed virtually no apoptosis. However, the pattern of apoptosis seen with both modes of ventilation was distinctly different. CV was associated with the presence of apoptotic cells within the lumen of immature air spaces, often with these cells completely occluding the lumen. In contrast, during TLV apoptosis was limited to a small number of epithelial cells lining the airspaces, with only a few cells found within the lumen. Although the specific cell types have not yet been identified, these observations suggest that the severity of lung injury might be related to a variety of factors including cell specific apoptosis, rather than apoptosis per se. Supported by a research grant from the March of Dimes(1-FY96-0752).