Developmental delay in hypoxia-induced HO-1 expression predisposes to gut injury.

AIMS: Necrotizing enterocolitis (NEC) is an often fatal disease that affects 5-8% of preterm newborn infants but does not occur in older infants and children. As carbon monoxide (CO) may exert protective effects against NEC, we assessed patterns of intestinal injury and investigated the expression of the CO-producing enzyme heme oxygenase-1 (HO-1) in mature and immature rat guts in response to hypercapnia and reoxygenation (H/R). METHODS: Gut barrier failure (increased permeability for dextran) was assessed in immature (newborn rats) and mature rats (weanling rats) subjected to H/R. Their guts were assayed for apoptosis (caspase-3 activity), expression of inducible NO synthase (iNOS) and HO-1 [quantitative polymerase chain reaction (PCR) and immunoblot]. The role of HO-1 was investigated in experiments involving HO-1 induction by hemin or HO-1 inhibition by tin protoporphyrin IX. RESULTS: In the mature gut, H/R induced the expression of intestinal HO-1 within 48 h, whereas in the immature gut HO-1 up-regulation was delayed by 48 h. Immature, but not mature, rats exhibited gut barrier failure, apoptosis and increased iNOS expression upon H/R. After the induction of HO-1 by hemin, gut barrier failure and apoptosis were abrogated in the immature gut, while the inhibition of HO-1 by tin protoporphyrin IX significantly aggravated gut injury. CONCLUSIONS: These experiments point to an immaturity-dependent lag in HO-1 expression upon H/R in the immature gut and link low HO-1 to gut barrier failure induced by H/R in a non-infectious dam-fed animal model of gut injury.

Early subcutaneous administration of etanercept (Enbrel) prevents from hyperoxia-induced lung injury.

Both hyperoxia-induced proapoptotic sensitization of alveolar type II cells (TII cells) and high-stretch mechanical ventilation induced pulmonary inflammation are tumor necrosis factor alpha (TNFalpha) mediated. Therefore, binding of free TNFalpha should protect from TNFalpha-mediated acute lung injury and ameliorate the subsequently developing chronic lung disease. Here, the authors show that a single subcutaneous pretreatment of rat with etanercept, a recombinant p75 TNF receptor 2 human immunoglobulin G1 (IgG1) construct, inhibits the hyperoxia-induced and TNFalpha-mediated increase in the expression of TNFalpha receptor, the activation of caspase 3 in TII cells, and, as an early indicator of lung injury, the capillary-alveolar leakage and granulocyte number in lung lavage. The authors assume that subcutaneous administration of etanercept might be suitable to prevent acute lung injury and its sequelae induced by hyperoxic ventilation of premature neonates and critically ill patients.

Activation of PPARgamma reverses a defect of surfactant synthesis in mice lacking two types of fatty acid binding protein.

Lung surfactant is a lipid-protein-film covering the inner alveolar surface. We have previously shown that double knock-out (d-ko) mice lacking both the epidermal-type (E-) and the heart-type (H-) fatty acid binding protein (FABP) exhibit a defect of surfactant synthesis in alveolar type II cells that can be corrected by feeding pioglitazone, a drug that activates peroxisome proliferator-activated receptor gamma (PPARgamma). Here, we demonstrate first that healthy surfactant at collapse pressure produces protrusions composed of bilayers but not folds, second that the d-ko effect profoundly perturbs lipid/hydrophobic protein composition, pressure-area isotherm, and structural organisation of the surfactant at nanoscale, parameters that are critical for the normal breathing cycle. In support of these data in vivo measurements of lung function reveal that maximum compliance in d-ko vs. wild-type mice is significantly reduced. Further, we show that the biophysical phenotype can be corrected substantially with pioglitazone. Finally, we show that d-ko alveolar cells up-regulate liver-type (L-) FABP, a member of the FABP family that we have previously shown to interact with PPARgamma. Taken together, these data suggest that PPARgamma agonists could be a tool to repair surfactant damage caused by dysfunctional alveolar lipid metabolism, and provide in vivo support for L-FABP aided signaling.

Formation of reactive oxygen species in lung alveolar cells: effect of vitamin E deficiency.

Reactive oxygen species (ROS) play an important role in the pathogenesis of numerous pulmonary diseases. Various mainly membrane-bound ROS-generating processes exist in alveolar cells. Vitamin E (vit. E) is the most important lipophilic antioxidant. However, the significance of vit. E levels in alveolar cells for the regulation of ROS generation has not been investigated so far. We demonstrated here that feeding rats with vit. E-depleted nourishment for 5 weeks reduced the concentration of vit. E in alveolar type II cell preparations to one-fifth the amount of control animals. This reduction of vit. E levels was associated with an approximately threefold increase in ROS generation in type II pneumocytes, lymphocytes, and macrophages. The contribution of individual processes of ROS formation in control animals differed strongly among these three cell types. However, vit. E deficiency induced predominantly nonmitochondrial ROS formation in alveolar cells. Expression and NAD(P)H-oxidase activity in alveolar type II cell preparations was not affected by vit. E deficiency. Moreover, protein kinase C (PKC) also did not seem to be responsible for vit. E deficiency-induced ROS generation in alveolar cells. Alimentary vit. E supplementation for 2 days corrected the cellular vit. E concentration but failed to normalize ROS generation in alveolar cells. These data let us assume that alimentary vit. E deficiency caused a preferentially nonmitochondria-mediated increase of ROS formation in type II pneumocytes, macrophages, and lymphocytes. However, the short-term supplementation of vit. E does not reverse these effects.

Vitamin E differentially regulates the expression of peroxiredoxin-1 and -6 in alveolar type II cells.

Vitamin E is the primary lipophilic antioxidant in mammals. Lack of vitamin E may lead to an increase of cytotoxic phospholipid-peroxidation products (PL-Ox). However, we could previously show that alimentary vitamin E-depletion in rats did not change the concentrations of dienes, hydroperoxides, and platelet-activating factor-related oxidation products in alveolar type II cells (TII cells). We hypothesized that vitamin E deficiency increases the activity of enzymes involved in the degradation of PL-Ox. Degradation of PL-Ox may be catalyzed by phospholipase A2, PAF-acetylhydrolase, or peroxiredoxins (Prx's). Alimentary vitamin E deficiency in rats increased the expression of Prx-1 at the mRNA and protein levels and the formation of Prx-SO3, but it did not change the expression of Prx-6 or the activity of phospholipase A2 and PAF-acetylhydrolase in TII cells. H2O2-induced oxidative stress in isolated TII cells activated protein kinase Calpha (PKCalpha) and increased the expression of Prx-1 and Prx-6. Inhibition of PKCalpha in isolated TII cells by long-time incubation with PMA inhibited PKCalpha and Prx-1 but not Prx-6. We concluded that the expression of Prx-1 and -6 is selectively regulated in TII cells; PKCalpha regulates the expression of Prx-1 but not Prx-6. Prx-6 expression may be closely linked to lipid peroxidation.

Inhibition of TNFalpha in vivo prevents hyperoxia-mediated activation of caspase 3 in type II cells.

BACKGROUND: The mechanisms during the initial phase of oxygen toxicity leading to pulmonary tissue damage are incompletely known. Increase of tumour necrosis factor alpha (TNFalpha) represents one of the first pulmonary responses to hyperoxia. We hypothesised that, in the initial phase of hyperoxia, TNFalpha activates the caspase cascade in type II pneumocytes (TIIcells). METHODS: Lung sections or freshly isolated TIIcells of control and hyperoxic treated rats (48 hrs) were used for the determination of TNFalpha (ELISA), TNF-receptor 1 (Western blot) and activity of caspases 8, 3, and 9 (colorimetrically). NF-kappaB activation was determined by EMSA, by increase of the p65 subunit in the nuclear fraction, and by immunocytochemistry using a monoclonal anti-NF-kappaB-antibody which selectively stained the activated, nuclear form of NF-kappa B. Apoptotic markers in lung tissue sections (TUNEL) and in TIIcells (cell death detection ELISA, Bax, Bcl-2, mitochondrial membrane potential, and late and early apoptotic cells) were measured using commercially available kits. RESULTS: In vivo, hyperoxia activated NF-kappaB and increased the expression of TNFalpha, TNF-receptor 1 and the activity of caspase 8 and 3 in freshly isolated TIIcells. Intratracheal application of anti-TNFalpha antibodies prevented the increase of TNFRI and of caspase 3 activity. Under hyperoxia, there was neither a significant change of cytosolic cytochrome C or of caspase 9 activity, nor an increase in apoptosis of TIIcells. Hyperoxia-induced activation of caspase 3 gradually decreased over two days of normoxia without increasing apoptosis. Therefore, activation of caspase 3 is a temporary effect in sublethal hyperoxia and did not mark the "point of no return" in TIIcells. CONCLUSION: In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFalpha effect in vivo by anti-TNFalpha antibodies prevents the pro-apoptotic sensitisation of TIIcells.

Naturally derived commercial surfactants differ in composition of surfactant lipids and in surface viscosity.

Pulmonary surfactant biophysical properties are best described by surface tension and surface viscosity. Besides lecithin, surfactant contains a variety of minor lipids, such as plasmalogens, polyunsaturated fatty acid-containing phospholipids (PUFA-PL), and cholesterol. Plasmalogens and cholesterol improve surface properties of lipid mixtures significantly. High PUFA-PL and plasmalogen content in tracheal aspirate of preterm infants reduces the risk of developing chronic lung disease. Different preparations are available for exogenous surfactant substitution; however, little is known about lipid composition and surface viscosity. Thus lipid composition and surface properties (measured by oscillating drop surfactometer) of three commercial surfactant preparations (Alveofact, Curosurf, Survanta) were compared. Lipid composition exhibited strong differences: Survanta had the highest proportion of disaturated PL and total neutral lipids and the lowest proportion of PUFA-PL. Highest plasmalogen and PUFA-PL concentrations were found in Curosurf (3.8 +/- 0.1 vs. 26 +/- 1 mol%) compared with Alveofact (0.9 +/- 0.3 vs. 11 +/- 1) and Survanta (1.5 +/- 0.2 vs. 6 +/- 1). In Survanta samples, viscosity increased >8 x 10(-6) kg/s at surface tension of 30 mN/m. Curosurf showed only slightly increased surface viscosity below surface tensions of 25 mN/m, and viscosity did not reach 5 x 10(-6) kg/s. By adding defined PL to Survanta, we obtained a Curosurf-like lipid mixture (without plasmalogens) that exhibited biophysical properties like Curosurf. Different lipid compositions could explain some of the differences in surface viscosity. Therefore, PL pattern and minor surfactant lipids are important for biophysical activity and should be considered when designing synthetic surfactant preparations.

Phenotype of palmitic acid transport and of signalling in alveolar type II cells from E/H-FABP double-knockout mice: contribution of caveolin-1 and PPARgamma.

Based on the assumption that fatty-acid-binding proteins (FABPs) of the epidermal-type (E-FABP) and heart-type (H-FABP) in murine alveolar type II (TII) cells mediate the synthesis of dipalmitoyl phosphatidylcholine (DPPC), the main surfactant phospholipid, we analysed TII cells isolated from wild-type (wt) and E/H-FABP double-knockout (double-ko) mice. Application of labelled palmitic acid to these cells revealed a drop in uptake, beta-oxidation, and incorporation into neutral lipids and total phosphatidylcholine (PC) of TII cells from double-ko mice. Whereas incorporation of labelled palmitic acid into DPPC remained unchanged, degradation studies demonstrated a substantial shift in DPPC synthesis from de novo to reacylation. In addition, increased expression of mRNAs and proteins of caveolin-1 and PPARgamma, and an increase of the mRNA encoding fatty acid translocase (FAT) was observed in the double-ko phenotype. As caveolin-1 interacted with PPARgamma, we assumed that FAT, caveolin-1, and PPARgamma form a signalling chain for fatty acid or drug. Consequently, PPARgamma-selective pioglitazone was added to the diet of double-ko mice. We found that further activation of PPARgamma could 'heal' the E/H-FABP double-ko effect in these TII cells as transport and utilisation of labelled palmitic acid restored a wt phenocopy. This indicated that E-FABP and/or H-FABP are involved in the mediation of DPPC synthesis in wt TII cells.

Perfluorocarbons are taken up by isolated type II pneumocytes and influence its lipid synthesis and secretion.

OBJECTIVE: Because alveoli fill with perfluorocarbons during liquid ventilation, an uptake of perfluorocarbons by type II pneumocytes can be postulated that might affect synthesis and secretion of pulmonary surfactant. The study was performed to answer the following questions: Do isolated type II pneumocytes take up perfluorocarbons? Do perfluorocarbons affect lipid synthesis of type II cells? Do perfluorocarbons change surfactant secretion of type II pneumocytes? DESIGN: Controlled experiments that used isolated type II pneumocytes. SETTING: Experimental laboratory of a university hospital. SUBJECTS: Male Wistar rats. INTERVENTIONS: To study perfluorocarbon uptake, isolated type II cells were incubated with fluorescence-labeled perfluorocarbons and examined with a laser scanning microscope. The effect of perfluorocarbons on biosynthesis of phospholipids and triglycerides was measured by incubating cells that were pulse-labeled with [H]-palmitic acid for 30 secs, with two different perfluorocarbons (PF 5080 or RM 101) for 10 mins. The effect of perfluorocarbon incubation on lipid secretion was studied by transmission electron microscopy. To quantify secretion, adherent type II pneumocytes (containing radioactively labeled phospholipids) were incubated with perfluorocarbons, and extra- and intracellular radioactivity was measured. MEASUREMENTS AND MAIN RESULTS: We found a significant uptake of labeled perfluorocarbons into lamellar bodies within 10 mins. Both perfluorocarbon species significantly (p <.05) reduced the biosynthesis of phospholipids when compared with control. Perfluorocarbon incubation did not affect mitochondrial activity, tested by MitoTracker staining. Transmission electron microscopy revealed changes that suggest an increased secretion of surfactant by type II cells. Studies with radioactively labeled surfactant revealed a significantly (p <.01) higher amount of extracellular lipids after RM 101 and PF 5080 treatment (RM 101, 17 +/- 7.9%; PF 5080, 9 +/- 1.9%) compared with control (5.3 +/- 1.9%). CONCLUSIONS: Our results suggest that perfluorocarbons are taken up by type II pneumocytes and cause an increased secretion of surfactant, despite a relative reduction in the synthesis of phospholipids.

Vitamin E deficiency reduces surfactant lipid biosynthesis in alveolar type II cells.

Reactive oxygen species play an important role in development of lung injury. Neonates exhibit a high risk of developing acute and/or chronic lung disorder, often associated with surfactant deficiency, and in parallel they show low vitamin E concentration. We investigated whether the vitamin E status of adult rats affects the content of phospholipids (PL) in bronchoalveolar lavage and alveolar type II cells. Phosphatidylcholine (PtdCho) is the dominant and functional most important PL in lung surfactant. Therefore, we determined its formation via de novo synthesis and reacylation of lyso-PtdCho in type II cells. Vitamin E depletion caused a decrease of PL content in bronchoalveolar lavage and type II cells and decreased glycerol-3-phosphate O-acyltransferase (G3P-AT) activity, de novo synthesis of PtdCho, and reacylation of lyso-PtdCho in type II cells. Preincubation of type II cell homogenates with dithiothreitol restored the activity of G3P-AT and de novo synthesis but inhibited reacylation. Reacylation was strongly reduced by chelerythrine-mediated inhibition of protein kinase C. We conclude that antioxidant and PKC-modulating properties of vitamin E regulate de novo synthesis of PtdCho and reacylation of lyso-PtdCho in alveolar type II cells. Vitamin E depletion reduced the two pathways of PL synthesis and caused a decrease of PL content in alveolar surfactant of rats.

Vitamin E as an antioxidant of the lung: mechanisms of vitamin E delivery to alveolar type II cells.

Oxidants play an important role in the development of acute and chronic lung injuries. Alveolar surfactant is the first target of air-borne oxidants. Surfactant contains, besides dipalmitoyl phosphatidylcholine, cholesterol and polyunsaturated phospholipids that play an important functional role. Therefore, vitamin E could be important for protecting surfactant lipids against oxidation and subsequent lung injury. Alveolar type II cells play a central role in synthesis and secretion of surfactant lipids and also supplement the surfactant with vitamin E during intracellular assembly. High density lipoprotein (HDL) is the primary source of vitamin E for type II cells. The uptake of vitamin E by specific lipid transfer is mediated by at least three HDL-specific receptors (scavenger receptor BI, membrane dipeptidase, and HDL-binding protein-2). In addition, cubilin and megalin mediate in a cooperative manner HDL-holoparticle uptake by alveolar type II cells. A temporary vitamin E deficiency induces a reversible change of the expression of pro- and antiinflammatory markers and of markers defining apoptosis, and reduces surfactant lipid synthesis in alveolar type II cells. These metabolic changes of type II cells may prime the lung to develop clinically manifest injury in response to an additional insult, e.g., hyperoxia.

Bioactive oxidized lipids in the plasma of cardiac surgical intensive care patients.

Critical illness is associated with increased oxidative stress that may give rise to the formation of lipid hydroperoxides (LOOH) and various secondary degradation products such as fragmented phosphatidylcholine (FPC) and lipids related to the platelet-activating factor (PAF). Because some oxidized phospholipids are potent proinflammatory agents, we measured the concentration of LOOH, FPC, and PAF-like activity in blood plasma of 36 patients who had undergone cardiac surgery and developed postoperative complications associated with systemic inflammatory response syndrome (SIRS) or multiple organ failure (MOF). These patients were compared to two control groups, namely preoperative patients scheduled for cardiac surgery (n = 13), and postoperative patients without complications (n = 19). Postoperative patents had higher concentrations of LOOH and lower concentrations of FPC than preoperative patients (P < 0.01). However, SIRS and MOF had no significant effect on the concentration of oxidatively modified lipids. This is despite the fact that MOF patients showed evidence of increased lipid peroxidation (7-fold higher ratio of alpha-tocoquinone/alpha-tocopherol compared to control). LOOH correlated positively with the white blood cell count. Postoperative patients had 4-fold higher plasma activities of phospholipase A2 and this activity was further increased in patients with SIRS (P < 0.04). Phospholipase A2 activity correlated negatively with the concentration of FPC. The data suggest that oxidatively modified lipids do not accumulate in patients with SIRS and MOF, perhaps because enhanced peroxidation of lipids is offset by enhanced lipolytic activity.

HDL-holoparticle uptake by alveolar type II cells: effect of vitamin E status.

Alveolar type II cells accumulate vitamin E preferentially from high-density lipoproteins (HDL) and express at least three receptors that are specific for HDL. The expression of these receptors increases in response to vitamin E deficiency. Beside receptors for specific lipid transfer from HDL, cubilin and megalin, several other receptors that mediate HDL-particle uptake were found in the lung. We hypothesize that alveolar type II cells also exhibit the HDL-particle uptake and that this process can be regulated by the vitamin E status. By confocal laser microscopy and flow cytometry we showed that type II cells accumulate protein-labeled HDL-particle. Vitamin E depletion in rats increased HDL-particle uptake in alveolar type II cells and the expression of megalin. The expression of cubilin did not change. Refeeding with vitamin E reversed HDL-particle uptake and megalin expression. Long-time incubation of type II cells with phorbol myristyl acetate (PMA) reduced HDL-holoparticle uptake and megalin expression. We assume that alveolar type II cells exhibit HDL-holoparticle uptake mediated by megalin and cubilin. Megalin represents the regulated element of the megalin/cubilin receptor-cooperation and can be modulated by protein kinase C.

Vitamin E deficiency sensitizes alveolar type II cells for apoptosis.

Pre-term neonates and neonates in general exhibit physiological vitamin E deficiency and are at increased risk for the development of acute lung diseases. Apoptosis is a major cause of acute lung damage in alveolar type II cells. In this paper, we evaluated the hypothesis that vitamin E deficiency predisposes alveolar type II cells to apoptosis. Therefore, we measured markers of apoptosis in alveolar type II cells isolated from control rats, vitamin E deficient rats and deficient rats that were re-fed a vitamin E-enriched diet. Bax and cytosolic cytochrome c increased, and the mitochondrial transmembrane potential and Hsp25 expression was reduced in vitamin E deficiency. Furthermore, increased DNA-fragmentation and numbers of early and late apoptotic cells were seen, but caspases 3 and 8 activities and expression of Fas, Bcl-2, Bcl-x and p53 remained unchanged. Vitamin E depletion did not change the GSH/GSSG ratio and the activities of antioxidant enzymes. Thus, vitamin E deficiency may induce a reversible pro-apoptotic response in lung cells and sensitise them for additional insult. In agreement with this hypothesis, we demonstrate that in vivo hyperoxia alone does not induce apoptosis in type II cells of control rats but reversibly increases DNA-fragmentation and numbers of early apoptotic type II cells in vitamin E-depleted cells.

Cellular cholesterol stimulates acute uptake of palmitate by redistribution of fatty acid translocase in type II pneumocytes.

Cholesterol is an abundant lipid of lung surfactant, where its concentration changes relative to phospholipids in response to certain physiological conditions. We investigated the effect of the cellular cholesterol content on uptake and esterification of palmitic acid, and on cellular distribution of fatty acid translocase (FAT/CD36) in alveolar type II cells. Incubation of type II cells with methyl-beta-cyclodextrin-cholesterol complexes increased the cholesterol content of lamellar bodies. The palmitate uptake of type II cells increased in parallel with the cellular cholesterol content. The content of FAT/CD36 increased in membranes and decreased in cytosol in type II cells. The detergent-insoluble fraction (DIGs), isolated from type II cells, was enriched in FAT/CD36 and caveolin-1 after increasing the cellular cholesterol. The total incorporation of labeled palmitic acid into glycerolipids and cholesterol ester (CE) increased by a factor of about 10 when the amount of unbound (14)C-palmitic acid added to type II cells was increased by a factor of about 1000. Under these conditions, a small but significant increase of the palmitate incorporation into PL occurred. Independent from the amount of added palmitate, palmitate incorporation into triacylglycerol decreased and palmitate incorporation into cholesterol ester increased about 40-65-fold. The beta-oxidation of palmitate significantly decreased. We conclude that alveolar type II cells respond to an increase of the cholesterol level with (i) cellular redistribution of FAT/CD36 into DIGs causing enhanced palmitate uptake and increased cholesterol ester-formation, (ii) storage of cholesterol in lamellar bodies, and (iii) induction of the formation of caveolae-like microdomains in the surface membrane, a structure possibly involved in a lamellar body-independent efflux of free cholesterol via the high-density lipoprotein-specific pathway.

Effect of cholesterol and surfactant protein B on the viscosity of phospholipid mixtures.

Low viscosity of the surface of alveolar fluid is mandatory for undisturbed surfactant function. Based on the known reduction of the viscosity of surfactant-like phospholipid (PL-) mixtures by plasmalogens, the effect of cholesterol and surfactant protein (SP-) B on surface viscosity of these lipid mixtures has been studied. Surface viscosity at the corresponding surface tension was measured with the oscillating drop surfactometer. We found that the viscosity was lowest in cholesterol-, followed by plasmalogen- and SP-B containing samples. Addition of SP-B to a plasmalogen-containing PL-mixture caused a further decrease in viscosity. However, in cholesterol containing mixtures, addition of SP-B led to a significant increase in viscosity, and the effect was reversed by further addition of plasmalogens. We conclude that SP-B, plasmalogens and cholesterol all affect the surface viscosity, thus synergistically regulate monolayer stability. This suggests that they are all needed in vivo for fine tuning of surface properties of pulmonary surfactant.