In Vitro Functional and Structural Characterization of A Synthetic Clinical Pulmonary Surfactant with Enhanced Resistance to Inhibition.

CHF5633 is a novel synthetic clinical pulmonary surfactant preparation composed by two phospholipid species, dipalmitoyl phosphatidylcholine (DPPC) and palmitoyloleoyl phosphatidylglycerol (POPG), and synthetic analogues of the hydrophobic surfactant proteins SP-B and SP-C. In this study, the interfacial properties of CHF5633 in the absence and in the presence of inhibitory serum proteins have been assessed in comparison with a native surfactant purified from porcine lungs and with poractant alpha, a widely used clinical surfactant preparation. The study of the spreading properties of CHF5633 in a Wilhelmy balance, its ability to adsorb and accumulate at air-liquid interfaces as revealed by a multiwell fluorescence assay, and its dynamic behavior under breathing-like compression-expansion cycling in a Captive Bubble Surfactometer (CBS), all revealed that CHF5633 exhibits a good behavior to reduce and sustain surface tensions to values below 5 mN/m. CHF5633 shows somehow slower initial interfacial adsorption than native surfactant or poractant alpha, but a better resistance to inhibition by serum proteins than the animal-derived clinical surfactant, comparable to that of the full native surfactant complex. Interfacial CHF5633 films formed in a Langmuir-Blodgett balance coupled with epifluorescence microscopy revealed similar propensity to segregate condensed lipid domains under compression than films made by native porcine surfactant or poractant alpha. This ability of CHF5633 to segregate condensed lipid phases can be related with a marked thermotropic transition from ordered to disordered membrane phases as exhibited by differential scanning calorimetry (DSC) of CHF5633 suspensions, occurring at similar temperatures but with higher associated enthalpy than that shown by poractant alpha. The good interfacial behavior of CHF5633 tested under physiologically meaningful conditions in vitro and its higher resistance to inactivation by serum proteins, together with its standardized and well-defined composition, makes it a particularly useful therapeutic preparation to be applied in situations associated with lung inflammation and edema, alone or in combined strategies to exploit surfactant-facilitated drug delivery.

Exposure to diethylhexyl phthalate (DEHP) and monoethylhexyl phthalate (MEHP) promotes the loss of alveolar epithelial phenotype of A549 cells.

Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer that is metabolized to mono(2-ethylhexyl) phthalate (MEHP). Inhalation is an important exposure route for both phthalates, and their effects on lungs include inflammation, alteration of postnatal maturation (alveolarization), enlarged airspaces and cell differentiation changes, suggesting that alveolar epithelial cells-2 (AEC) are targets of phthalates. This study evaluated the cell progression, epithelial and mesenchymal markers, including surfactant secretion in A549 cells (AEC) that were exposed to DEHP (1-100 µM) or MEHP (1-50 µM) for 24-72 h. The results showed an increased cell proliferation at all concentrations of each phthalate at 24 and 48 h. Cell migration showed a concentration-dependent increase at 24 and 48 h of exposure to either phthalate and enlarged structures were seen. Decreased levels of both surfactants (SP-B/SP-C) were observed after the exposure to either phthalate at 48 h, and of SP-C positive cells exposed to MEHP, suggesting a loss of the epithelial phenotype. While a decrease in the epithelial marker E-cadherin and an increase in the mesenchymal marker fibronectin were observed following exposure to either phthalate. Our results showed that DEHP and MEHP altered the structure and migration of A549 cells and promoted the loss of the epithelial phenotype.

Comparative study of the effects of PM1-induced oxidative stress on autophagy and surfactant protein B and C expressions in lung alveolar type II epithelial MLE-12 cells.

BACKGROUND: There is a strong link between smaller air pollution particles and a range of serious health conditions. Thus, there is a need for understanding the impacts of airborne fine particulate matter (PM) with an aerodynamic diameter of <1µm (PM1) on lung alveolar epithelial cells. In the present study, mouse lung epithelial type II cell MLE-12 cells were used to examine the intracellular oxidative responses and the surfactant protein expressions after exposure to various concentrations of PM1 collected from an urban site and a steel-factory site (referred as uPM1 and sPM1 hereafter, respectively). METHODS: Physicochemical characterization of PM1 was performed by using scanning electron microscopy and transmission electron microscopy. Cytotoxicity and autophagy induced by PM1 were assessed by using comprehensive approaches after MLE-12 cells were exposed to different concentrations of PM1 for various times. Expression of surfactant proteins B and C in MLE-12 cells was determined by Western blotting. RESULTS: All of the tested PM1 induced cytotoxicity evidenced by significant decrease of cell viability and increase of lactate dehydrogenase (LDH) release in a time- and concentration-dependent manner in the exposed cells compared with the unexposed cells. A similar pattern of increase of intercellular reactive oxygen species (ROS) generation and decrease of superoxide dismutase (SOD) and catalase (CAT) activities was also observed. PM1-induced autophagy was evidenced by an increase in microtubule-associated protein light chain-3 (LC3) puncta, accumulation of LC3II, and increased levels of beclin1. Data from Western blotting showed significant decrease of surfactant protein B and C expressions. Relatively high concentrations of transition metals, including Fe, Cu and Mn, may be responsible for the higher toxicity of sPM1 compared with uPM1. Moreover, pretreatment with N-acetylcysteine (NAC) or Chelex (a metal chelating agent, which removes a large suite of metals from PM1) prevented the increase of PM1-inudced ROS generation and autophagy, and down-regulated the expression of surfactant proteins B and C. CONCLUSION: PM1, particularly PM1 with high concentrations of transition metals, such as Fe, Cu and Mn, induces oxidative damage and autophagy, as well as inhibits surfactant protein B and C expressions in lung alveolar type II epithelial cells. GENERAL SIGNIFICANCE: This study will help to understand the mechanism underlying the toxicological effects of PM1 in lung alveolar type II epithelial cells. This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andrew J. Ghio and Weidong Wu.

A small key unlocks a heavy door: The essential function of the small hydrophobic proteins SP-B and SP-C to trigger adsorption of pulmonary surfactant lamellar bodies.

The molecular basis involving adsorption of pulmonary surfactant at the respiratory air-liquid interface and the specific roles of the surfactant proteins SP-B and SP-C in this process have not been completely resolved. The reasons might be found in the largely unknown structural assembly in which surfactant lipids and proteins are released from alveolar type II cells, and the difficulties to sample, manipulate and visualize the adsorption of these micron-sized particles at an air-liquid interface under appropriate physiological conditions. Here, we introduce several approaches to overcome these problems. First, by immunofluorescence we could demonstrate the presence of SP-B and SP-C on the surface of exocytosed surfactant particles. Second, by sampling the released particles and probing their adsorptive capacity we could demonstrate a remarkably high rate of interfacial adsorption, whose rate and extent was dramatically affected by treatment with antibodies against SP-B and SP-C. The effect of both antibodies was additive and specific. Third, direct microscopy of an inverted air-liquid interface revealed that the blocking effect is due to a stabilization of the released particles when contacting the air-liquid interface, precluding their transformation and the formation of surface films. We conclude that SP-B and SP-C are acting as essential, preformed molecular keys in the initial stages of surfactant unpacking and surface film formation. We further propose that surfactant activation might be transduced by a conformational change of the surfactant proteins upon contact with surface forces acting on the air-liquid interface.

In silico interaction of methyl isocyanate with immune protein responsible for Mycobacterium tuberculosis infection using molecular docking.

This article reports in silico analysis of methyl isocyanate (MIC) on different key immune proteins against Mycobacterium tuberculosis. The analysis shows that MIC is released in the Bhopal gas tragedy in 1984, which is highly toxic and extremely hazardous to human health. In this study, we have selected immune proteins to perform molecular docking with the help of Autodock 4.0. Results show that the CD40 ligand and alpha5beta1 integrin have higher inhibition compared to plasminogen activator urokinase, human glutathione synthetase, mitogen-activated protein kinase (P38 MAPK 14), surfactant protein-B, -D (SP-D), and pulmonary SP-D. MIC interacted with His-125, Try-146 residue of CD40 ligand and Ala-149, and Arg-152 residue of alpha5beta1 integrin and affects the proteins functioning by binding on their active sites. These inhibitory conformations were energetically and statistically favored and supported the evidence from wet laboratory experiments reported in the literature. We can conclude that MIC directly or indirectly affects these proteins, which shows that survivals of the disaster suffer from the diseases like tuberculosis infection and lung cancer.

Biophysical inhibition of pulmonary surfactant function by polymeric nanoparticles: role of surfactant protein B and C.

The current study investigated the mechanisms involved in the process of biophysical inhibition of pulmonary surfactant by polymeric nanoparticles (NP). The minimal surface tension of diverse synthetic surfactants was monitored in the presence of bare and surface-decorated (i.e. poloxamer 407) sub-100 nm poly(lactide) NP. Moreover, the influence of NP on surfactant composition (i.e. surfactant protein (SP) content) was studied. Dose-elevations of SP advanced the biophysical activity of the tested surfactant preparation. Surfactant-associated protein C supplemented phospholipid mixtures (PLM-C) were shown to be more susceptible to biophysical inactivation by bare NP than phospholipid mixture supplemented with surfactant protein B (PLM-B) and PLM-B/C. Surfactant function was hindered owing to a drastic depletion of the SP content upon contact with bare NP. By contrast, surface-modified NP were capable of circumventing unwanted surfactant inhibition. Surfactant constitution influences the extent of biophysical inhibition by polymeric NP. Steric shielding of the NP surface minimizes unwanted NP-surfactant interactions, which represents an option for the development of surfactant-compatible nanomedicines.

Mutation of a lysine residue in a homeodomain generates dominant negative thyroid transcription factor 1.

Thyroid transcription factor 1 (TTF-1) is a 42 kDa homeodomain (HD) containing the tissue-specific transcription factor of Nkx2 family members (also termed TEBP and Nkx2.1). TTF-1 is an essential transcription factor required for lung development and lung-specific gene expression. Transgenic mice carrying TTF-1 DNA-binding site mutations completely abolish expression of the human surfactant protein B (hSP-B) 1.5 kb lacZ reporter gene in the lung in vivo. Acetylation of transcription factors by nuclear receptor coactivators is an important mechanism for gene regulation. TTF-1 is acetylated by nuclear receptor coactivators including the activator of the thyroid and retinoic acid receptor, CREB-binding protein, and steroid receptor coactivator 1 (SRC-1) in cell transfection and immunoprecipitation studies. A glutathionine transferase pull-down assay shows TTF-1 direct interaction with the SRC-1 histone acetyltransferase domain. Site-specific mutagenesis identifies that the lysine residue at position 182 in the TTF-1 HD is acetylated in respiratory epithelial cells. Mutation at this acetylation site shows a dominant negative effect on SP-B gene transcription. The study supports a concept that acetylation is an important mechanism for TTF-1 activity.