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Am J Physiol Lung Cell Mol Physiol 298: L117-L125, 2010. First published November 6, 2009; doi: 10.1152/ajplung.00218.2009.--Mechanical ventilation may lead to an impairment of the endogenous surfactant system, which is one of the mechanisms by which this intervention contributes to the progression of acute lung injury. The most extensively studied mechanism of surfactant dysfunction is serum protein inhibition. However, recent studies indicate that hydrophobic components of surfactant may also contribute. It was hypothesized that elevated levels of cholesterol significantly contribute to surfactant dysfunction in ventilation-induced lung injury. Sprague-Dawley rats (n = 30) were randomized to either high-tidal volume or low-tidal volume ventilation and monitored for 2 h. Subsequently, the lungs were lavaged, surfactant was isolated, and the biophysical properties of this isolated surfactant were analyzed on a captive bubble surfactometer with and without the removal of cholesterol using methyl-[beta]-cyclodextrin. The results showed lower oxygenation values in the high-tidal volume group during the last 30 rain of ventilation compared with the low-tidal volume group. Surfactant obtained from the high-tidal volume animals had a significant impairment in function compared with material from the low-tidal volume group. Removal of cholesterol from the high-tidal volume group improved the ability of the surfactant to reduce the surface tension to low values. Subsequent reconstitution of high-cholesterol values led to an impairment in surface activity. It is concluded that increased levels of cholesterol associated with endogenous surfactant represent a major contributor to the inhibition of surfactant function in ventilation-induced lung injury. mechanical ventilation; surface tension

Nucleotides are released from cells in response to mechanical stimuli and signal in an autocrine/ paracrine manner through cell surface P2 receptors. [P2rx7.sup.-/-] mice exhibit diminished appositional growth of long bones and impaired responses to mechanical loading. We find that calvarial sutures are wider in [P2rx7.sup.-/-] mice. Functional P2X7 receptors are expressed on osteoblasts in situ and in vitro. Activation of P2X7 receptors by exogenous nucleotides stimulates expression of osteoblast markers and enhances mineralization in cultures of rat calvarial cells. Moreover, osteogenesis is sup pressed in calvarial cell cultures from [P2rx7.sup.-/-] mice compared with the wild type. P2X7 receptors couple to production of the potent lipid mediators lysophosphatidic acid (LPA) and prostaglandin [E.sub.2]. Either an LPA receptor antagonist or cyclooxygenase (COX) inhibitors abolish the stimulatory effects of P2X7 receptor activation on osteogenesis. We conclude that P2X7 receptors enhance osteoblast function through a cell-autonomous mechanism. Furthermore, a novel signaling axis links P2X7 receptors to production of LPA and COX metabolites, which in turn stimulate osteogenesis.

Lung injury due to mechanical ventilation is associated with an impairment of endogenous surfactant. It is unknown whether this impairment is a consequence of or an active contributor to the development and progression of lung injury. To investigate this issue, the present study addressed three questions: Do alterations to surfactant precede physiological lung dysfunction during mechanical ventilation? Which components are responsible for surfactant's biophysical dysfunction? Does exogenous surfactant supplementation offer a physiological benefit in ventilation-induced lung injury? Adult rats were exposed to either a low-stretch [tidal volume ([V.sub.T]) = 8 ml/kg, positive end-expiratory pressure (PEEP) = 5 cm[H.sub.2]0, respiratory rate (RR) = 54-56 breaths/rain (bpm), fractional inspired oxygen ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.]) = 1.0] or high-stretch ([V.sub.T] = 30 ml/kg, PEEP = 0 cm[H.sub.2]O, RR = 14-16 bpm, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII.] = 1.0) ventilation strategy and monitored for either 1 or 2 h. Subsequently, animals were lavaged and the composition and function of surfactant was analyzed. Separate groups of animals received exogenous surfactant after 1 h of high-stretch ventilation and were monitored for an additional 2 h. High stretch induced a significant decrease in blood oxygenation after 2 h of ventilation. Alterations in surfactant pool sizes and activity were observed at 1 h of high-stretch ventilation and progressed over time. The functional impairment of surfactant appeared to be caused by alterations to the hydrophobic components of surfactant. Exogenous surfactant treatment after a period of high-stretch ventilation mitigated subsequent physiological lung dysfunction. Together, these results suggest that alterations of surfactant are a consequence of the ventilation strategy that impair the biophysical activity of this material and thereby contribute directly to lung dysfunction over time. mechanical ventilation; biophysical activity; ventilation-induced lung injury

Exposing bovine lipid extract surfactant (BLES), a clinical surfactant to reactive oxygen species (ROS) alters surfactant protein B (SP-B). ROS treatment of BLES clinical surfactant results in delayed absorption and an impairment of the ability to attain low surface tensions during film compression.

Pulmonary surfactant, a complex mixture of lipids and proteins, lowers the surface tension in terminal air spaces and is crucial for lung function. Within an animal species, surfactant composition can be influenced by development, disease, respiratory rate, and/or body temperature. Here, we analyzed the composition of surfactant in three heterothermic mammals (dunnart, bat, squirrel), displaying different torpor patterns, to determine: 1) whether increases in surfactant cholesterol (Chol) and phospholipid (PL) saturation occur during long-term torpor in squirrels, as in bats and dunnarts; 2) whether surfactant proteins change during torpor; and 3) whether PL molecular species (molsp) composition is altered. In addition, we analyzed the molsp composition of a further nine mammals (including placental/marsupial and hetero-/homeothermic contrasts) to determine whether phylogeny or thermal behavior determines molsp composition in mammals. We discovered that like bats and dunnarts, surfactant Chol increases during torpor in squirrels. However, changes in PL saturation during torpor may not be universal. Torpor was accompanied by a decrease in surfactant protein A in dunnarts and squirrels, but not in bats, whereas surfactant protein B did not change in any species. Phosphatidylcholine (PC)16:0/16:0 is highly variable between mammals and is not the major PL in the wombat, dunnart, shrew, or Tasmanian devil. An inverse relationship exists between PC16:0/16:0 and two of the major fluidizing components, PC16:0/16:1 and PC16:0/14:0. The PL molsp profile of an animal species is not determined by phylogeny or thermal behavior. We conclude that there is no single PL molsp composition that functions optimally in all mammals; rather, surfactant from each animal is unique and tailored to the biology of that animal. lung; temperature; surfactant proteins; electrospray ionization mass spectrometry; cholesterol

C-reactive protein (CRP) and surfactant protein A (SP-A) are phosphatidylcholine (PC) binding proteins that function in the innate host defense system. We examined the effects of CRP and SP-A on the surface activity of bovine lipid extract surfactant (BLES), a clinically applied modified natural surfactant. CRP inhibited BLES adsorption to form a surface-active film and the film's ability to lower surface tension ([gamma]) to low values near 0 mN/m during surface area reduction. The inhibitory effects of CRP were reversed by phosphorylcholine, a water-soluble CRP ligand. SP-A enhanced BLES adsorption and its ability to lower [gamma] to low values. Small amounts of SP-A blocked the inhibitory effects of CRP. Electron microscopy showed CRP has little effect on the lipid structure of BLES. SP-A altered BLES multilamellar vesicular structure by generating large, loose bilayer structures that were separated by a fuzzy amorphous material, likely SP-A. These studies indicate that although SP-A and CRP both bind PC, there is a difference in the manner in which they interact with surface films. acute respiratory distress syndrome; captive bubble tensiometer; electron microscopy; surface tension; surfactant biophysical impairment

The lung contains two distinct forms of phosphatidic acid phosphatase (PAP). PAP1 is a cytosolic enzyme that is activated through fatty acid-induced translocation to the endoplasmic reticulum, where it converts phosphatidic acid (PA) to diacylglycerol (DAG) for the biosynthesis of phospholipids and neutral lipids. PAP1 is [Mg.sup.2+] dependent and sulfhydryl reagent sensitive. PAP2 is a six-transmembrane-domain integral protein localized to the plasma membrane. Because PAP2 degrades sphingosine-1-phosphate (S1P) and ceramide-1-phosphate in addition to PA and lyso-PA, it has been renamed lipid phosphate phosphohydrolase (LPP). LPP is [Mg.sup.2+] independent and sulfhydryl reagent insensitive. This review describes LPP isoforms found in the lung and their location in signaling platforms (rafts/caveolae). Pulmonary LPPs likely function in the phospholipase D pathway, thereby controlling surfactant secretion. Through lowering the levels of lyso-PA and S1P, which serve as agonists for endothelial differentiation gene receptors, LPPs regulate cell division, differentiation, apoptosis, and mobility. LPP activity could also influence transdifferentiation of alveolar type II to type I cells. It is considered likely that these lipid phosphohydrolases have critical roles in lung morphogenesis and in acute lung injury and repair. epidermal growth factor receptors; caveolae; lysophospholipids; phospholipase D; surfactant secretion

Acute lung injury and lung transplantation influence in vitro subtype conversion of pulmonary surfactant. Am J Physiol Lung Cell Mol Physiol 282: L67-L74, 2002.--The effects of surfactant treatment on surfactant subtype conversion after lung injury were examined. Dogs were subjected to hyperventilation for 8 h with or without surfactant treatment. Lungs were stored for 17 h, and the right lung was transplanted and reperfused for 6 h. Conversion of large aggregate (LA) surfactant to small aggregates was investigated using in vitro surface area cycling. LA from transplanted lungs (Transplant-LA) from the nontreated group converted more rapidly than Transplant-LA from the treated group. Transplant-LA from both groups converted more rapidly than LA from normal lungs. Calculations based on [[sup.3]H]dipalmitoylphosphatidylcholine in the administered surfactant [bovine lipid extract surfactant (BLES)] showed that the endogenous component of Transplant-LA converted more rapidly than the exogenous component. This indicates exogenous BLES did not equilibrate completely with endogenous surfactant. LA from hyperventilated, stored donor right lungs and from the recipients' native lungs from the nontreated group converted more rapidly than corresponding LA in the BLES-treated group. Similar relative conversions were observed with exogenous components from all lungs. Relative conversion of endogenous component from Transplant-LA was more rapid than that from LA from donor's stored right lung or from the recipient's native right lung. Low levels of phenylmethylsulfonyl fluoride inhibited conversion of Transplant-LA to a greater extent than normal LA. LA from all experimental groups had similar protein levels. These studies show acute lung injury, transplant, ischemia-reperfusion, and surfactant treatment have major effects on surfactant subtype integrity. carboxyesterase; convertase; dipalmitoylphosphatidylcholine; ischemia-reperfusion; surfactant subtype conversion

Nanjundan, Meera, and Fred Possmayer. Molecular cloning and expression of pulmonary lipid phosphate phosphohydrolases. Am J Physiol Lung Cell Mol Physiol 281: L1484-L1493, 2001.--Pulmonary lipid phosphate phosphohydrolase (LPP) was shown previously to hydrolyze phosphatidic acid and lysophosphatidic acid in purified rat lung plasma membranes. To better investigate the nature of pulmonary LPP isoforms and their role in the lung, LPPs were cloned by RT-PCR from both adult rat lung and type II cell RNA. The RT-PCR generated LPP1 (849 bp), up to three LPP1 variants, and LPP3 (936 bp) cDNAs. The three LPP1 variants include LPP1a (852 bp) and two novel isoforms, LPP1b (697 bp) and LPP1c (1004 bp). The pulmonary LPP1 and LPP3 isoforms are essentially identical to the previously cloned rat liver and intestinal LPPs, respectively, and the LPP1a isoform has 80% sequence identity to the human homolog. The LPP2 isoform was not detected in lung by RT-PCR. Northern analyses revealed that the mRNAs for LPP1 and LPP3 increase in fetal rat lung in late gestation to day 1 after birth. These mRNAs decrease somewhat during the neonatal period but increase slightly during postnatal development. Expression of LPP1, LPP1a, and LPP3 cDNAs in HEK 293 cells established that they encode functional LPP. In contrast, the novel isoforms LPP1b and LPP1c contain frameshifts that would result in premature termination, producing putative catalytically inactive polypeptides of 30 and 76 amino acids, respectively. Further investigation of the LPP1b isoform revealed that it was present across a variety of tissues, although at lower levels than LPP1/1a. Transient mammalian expression of LPP1b failed to increase phosphatidate phosphohydrolase activity in HEK 293 cells. lung growth; novel isoforms; rat lung; type II cells; tissue expression; overexpression; surfactant secretion

Rat surfactant protein A (SP-A) trimers in association with phospholipid monolayers have been analyzed to determine their three-dimensional structure. The glycosylation site of SP-A was at the side of each subunit, which would suggest that the covalently linked carbohydrate moiety likely is in the spaces between the adjacent globular domains, which would not sterically interfere with ligand binding.

A series of transmission-electron-microscopic studies were conducted using purified surfactant protein-A and lipid vesicles made in vitro as well as native surfactant from bovine lung to elucidate the interaction of SP-A and various lipids commonly found in tubular myelin (TM). Dipalmitoylphosphatidylcholine-egg phosphatidylcholine bilayers formed corrugations, folds, and predominantly laticelike structures. These results offer evidence that SPs and lipid composition play crucial roles in the formation of the lipid membranes of TM and TM-like structures.

Research was conducted to examine the hypothesis that chronic placental insufficiency resulting in fetal growth restriction (FGR) leads to an increase in fetal lung surfactant-related protein (SP) gene expression. A further objective was to determine if the potential increase in surfactant apoprotein gene expression with FGR was associated with changes in fetal cortisol levels in late gestation. Results indicate that FGR is related to changes in fetal lung SP.

A study was conducted to describe the mechanisms of pulmonary surfactant protein A (SP-A) as an immune suppressive agent. SP-A was found to inhibit human T lymphocyte proliferation in a dose dependent manner and that the effect is not mediated by the carbohydrate recognition domain of SP-A. Results implied a potential role for SP-A in inhibiting lymphocyte responses to exogenous stimuli, as well as support the hypothesis that SP-A contributes to the inhibition of in vivo T cell proliferation and that the effect helps maintain the hyporesponsive state of pulmonary leukocytes.

Palmitoyled or depalmitoyled bovine surfactant-associated protein C (SP-C) were analyzed by utilizing captive bubble surfactometer to determine the effects of palmitoylation on SP-C function and structure. Captive bubble surfactometer analysis indicated the importance of palmitoylation on the full activity of SP-C as a pulmonary surfactant. Furthermore, palmitoylation enhanced SP-C lipid absorption compared to deplamitoyled SP-C.

The study explored the inherent structural and functional determinants within the amino acid sequence of mature surfactant protein SP-B, porcine SP-B. It concluded that SP-B-like polypeptides lacing disulfides could still be potentially good mimics of native disulfide-linked SP-B.

Surfactant protein interactions with neutral and acidic phospholipid films. Am J Physiol Lung Cell Mol Physiol 281: L231-L242, 2001.--The captive bubble tensiometer was employed to study interactions of phospholipid (PL) mixtures of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) at 50 [micro]g/ml with physiological levels of the surfactant protein (SP) A SP-B, and SP-C alone and in combination at 37 [degrees] C. All surfactant proteins enhanced lipid adsorption to equilibrium surface tension ([Gamma]), with SP-C being most effective. Kinetics were consistent with the presence of two adsorption phases. Under the conditions employed, SP-A did not affect the rate of film formation in the presence of SP-B or SP-C. Little difference in [[Gamma].sub.min], was observed between the acidic POPG and the neutral POPC systems with SP-B or SP-C with and without SPA. However, [[Gamma].sub.max] was lower with the acidic POPG system during dynamic, but not during quasi-static, cycling. Considerably lower compression ratios were required to generate low [[Gamma].sub.min] values with SP-B than SP-C. DPPC-POPG-SP-B was superior to the neutral POPC-SP-B system. Although SP-A had little effect on film formation with SP-B, surface activity during compression was enhanced with both PL systems. In the presence of SP-C, lower compression ratios were required with the acidic system, and with this mixture, SP-A addition adversely affected surface activity. The results suggest specific interactions between SP-B and phosphatidylglycerol, and between SP-B and SP-A. These observations are consistent with the presence of a surface-associated surfactant reservoir which is involved in generating low [Gamma] during film compression and lipid respreading during film expansion. acute lung injury; acute respiratory distress syndrome; captive bubble tensiometer; dipalmitoylphosphatidylcholine; lipid monolayer; phosphatidylglycerol; phosphatidylcholine; respiratory distress syndrome; surface area cycling; surface tension; synergism

Braems, Geert A., Li-Juan Yao, Kevin Inchley, Anne Brickenden, Victor K. M. Han, Allen Grolla, John R. G. Challis, and Fred Possmayer. Ovine surfactant protein cDNAs: use in studies on fetal lung growth and maturation after prolonged hypoxemia. Am J Physiol Lung Cell Mol Physiol 278: L754-L764, 2000.--cDNAs for ovine surfactant-associated protein (SP) A, SP-B, and SP-C have been cloned and shown to possess strong similarity to cDNAs for surfactant apoproteins in other species. These reagents were employed to examine the effect of fetal hypoxia on the induction of surfactant apoprotein expression in the fetal lamb. Postnatal lung function is dependent on adequate growth and maturation during fetal development. Insulin-like growth factor (IGF) I and IGF-II, which are present in all fetal tissues studied, possess potent mitogenic and proliferative actions, and their effects can be modulated by IGF-specific binding proteins (IGFBPs). Hypoxia can lead to increases in circulating cortisol and catecholamines that can influence lung maturation. Therefore, the effects of mild hypoxia in chronically catheterized fetal lambs at gestational days 126-130 and 134-136 (term 145 days) on the expression of pulmonary surfactant apoproteins and IGFBPs were examined. Mild hypoxia for 48 h resulted in an increase in plasma cortisol that was more pronounced at later gestation, and in these animals, there was a twofold increase in SP-A mRNA. SP-B mRNA levels also increased twofold, but this was not significant. SP-C mRNA was not altered. No significant changes in apoprotein mRNA were observed with the younger fetuses. However, these younger animals selectively exhibited reduced IGFBP-5 mRNA levels. IGF-I mRNA was also reduced at 126-130 days, although this conclusion is tentative due to low abundance. IGF-II levels were not affected at either gestational age. We conclude that these data suggest that mild prolonged fetal hypoxia produces alterations that could affect fetal cellular differentiation early in gestation and can induce changes consistent with lung maturation closer to term. complementary deoxyribonucleic acid; cloning; fetal lamb; pulmonary surfactant; insulin-like growth factors; insulin-like growth factor binding protein-5; messenger ribonucleic acid; glucocorticoids; differentiation; respiratory distress syndrome

Nag, Kaushik, James G. Munro, Kevin Inchley, Samuel Schurch, Nils O. Petersen, and Fred Possmayer. SP-B refining of pulmonary surfactant phospholipid films. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L1179-L1189, 1999.--Pulmonary surfactant stabilizes the alveoli by lining the air-fluid interface with films that reduce surface tension to near 0 mN/m ([[Gamma].sub.min]). Surfactant protein B (SP-B) enhances the surface activity of surfactant phospholipids. A captive bubble tensiometer (CBT) was used to study the properties of adsorbed films of dipalmitoylphosphatidylcholine (DPPC) with acidic 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) or neutral 1-palmitoyl-2oleoyl-sn-glycerol-3-phosphocholine with (7:3) and without 1% dimeric SP-B. SP-B enhanced the adsorption rate of DPPC-containing neutral or acidic lipid suspensions (1 mg/ ml) to a similar extent. Quasi-static cycling of these films revealed that SP-B significantly decreased the film area reduction required to reach [[Gamma].sub.min] for the acidic but not for the neutral system. The results obtained with DPPC-phosphatidylglycerol (PG)-SP-B were consistent with selective DPPC adsorption into the surface monolayer during film formation. Film area reduction required to reach [[Gamma].sub.min] with this system (with and without calcium) approached that of pure DPPC, suggesting selective DPPC insertion and PG squeeze-out. Dynamic cycling of such films showed that larger film area reductions were required to reach [[Gamma].sub.min] for the neutral than for acidic system, even after 20 cycles. Fluorescence microscopy of solvent-spread DPPC-POPG-SP-B planar films revealed highly condensed structures at ~25 mN/m, although no specific PG phase-segregated structures could be identified. The study suggests that specific interactions of SP-B with acidic phospholipids of surfactant may be involved in the generation and maintenance of DPPC-rich films in the alveoli. captive bubble tensiometer; surface tension; dipalmitoylphosphatidylcholine; phosphatidylglycerol; lipid-protein interactions; surfactant protein B; respiratory distress syndrome; squeeze-out; selective adsorption

Tan, Rosemarie C., Machiko Ikegami, Alan H. Jobe, Li Yuan Yao, Fred Possmayer, and Philip L. Ballard. Developmental and glucocorticoid regulation of surfactant protein mRNAs in preterm lambs. Am. J. Physiol. 277 (Lung Cell. Mol. Physiol. 21): L1142-L1148, 1999.--Glucocorticoid treatment increases content of surfactant protein (SP) A and SP-B in lung tissue and lavage fluid of preterm lambs. To investigate this process, we determined the ontogeny and glucocorticoid induction of SP mRNAs. In separate treatment protocols, each with its own controls, sheep were injected with betamethasone 15 h, 48 h, or weekly for 1-4 doses before preterm delivery. Using ovine SP cDNAs, we found an increase equal to or more than threefold in basal levels of all three SP mRNAs between 125 days and term. After betamethasone treatment, SP-B and SP-C mRNA levels increased by 15 h and all SP mRNAs were elevated after 24 h ([is greater than or equal to] 2-fold); mRNA levels in fetuses delivered 1-3 wk after betamethasone were not different from control. We conclude that in vivo betamethasone rapidly induces a coordinated increase in SP mRNAs, which is fully reversible within 7 days despite repetitive doses of betamethasone. Similar increases in mRNA and protein contents for SP-A and SP-B suggest that glucocorticoid regulation of these SPs in vivo is primarily pretranslational. betamethasone; ontogeny

Transmission electron microscopy is used to describe how filaments of purified surfactant protein-A or SP-A, the major SP, from cattle interact with the surfaces of lipid vesicles made in vitro. Findings demonstrate that the surface environment created on lipid membranes can potentially stabilize spontaneous protein-protein interactions. Such protein-lipid arrangements form structures that are similar in appearance to those found in native pulmonary surfactant.