Lung Endothelial Cell Line Research Articles

Fucoidans inhibited tau interaction and cellular uptake

Tau spreading in Alzheimer's disease is mediated by cell surface heparan sulfate (HS). As a class of sulfated polysaccharides, fucoidans might compete with HS to bind tau, resulting in the cessation of tau spreading. The structural determinants of fucoidans for competition with HS binding to tau are not well understood. Sixty previously prepared fucoidans/glycans with different structural determinants were used to determine their binding abilities to tau using SPR and AlphaLISA. Finally, it was found that fucoidans had two fractions (sulfated galactofucan (SJ-I) and sulfated heteropolysaccharide (SJ-GX-3)), which exhibited strong binding abilities than heparin. Tau cellular uptake assays using wild type mouse lung endothelial cell lines were performed. It was shown SJ-I and SJ-GX-3 inhibited tau-cell interaction and tau cellular uptake, suggesting that fucoidans might be good candidates for inhibiting tau spreading. NMR titration mapped fucoidans binding sites, which could provide the theoretical basis for the design of tau spreading inhibitors.

Eco-friendly extraction of Mastocarpus stellatus carrageenan for the synthesis of gold nanoparticles with improved biological activity

Carrageenan was extracted from Mastocarpus stellatus using hot water extraction under atmospheric and pressurized conditions. The influence of heating temperature during a non-isothermal heating profile up to temperatures in the range 70–190 °C was studied to evaluate the extraction yields and properties of the carrageenan fraction. Under the selected conditions (130 °C), extracted carrageenan (CMs) was used for the green synthesis of gold nanoparticles (AuNPs). After the optimization of the reaction conditions, the synthesized gold nanoparticles (Au@CMs) were characterized by UV–Vis spectroscopy, Z potential measurements, electron microscopy, and X-ray diffraction analysis, which confirmed the formation of spherical, polycrystalline, and negatively charged nanoparticles with a mean diameter of 14.3 ± 2.1 nm. The study conducted by scanning transmission electron microscopy, energy dispersive X-ray analysis and mapping confirmed the presence of carrageenan stabilizing AuNPs. Finally, Fourier transformed infrared spectroscopy was performed to analyze the functional groups of CMs involved in the reduction and stabilization of AuNPs. The selective cytotoxicity and the antioxidant activity of the Au@CMs were evaluated in different cell lines and compared to the CMs. Au@CMs showed an improved antioxidant capacity in cells under oxidative stress and the induction of apoptosis in a monocytic cell line, while no antitumor effect was observed in a lung endothelial cell line.

Lung endothelial cells are sensitive to epsilon toxin from Clostridium perfringens

The pore-forming protein epsilon toxin (Etx) from Clostridium perfringens produces acute perivascular edema affecting several organs, especially the brain and lungs. Despite the toxin evident effect on microvasculature and endothelial cells, the underlying molecular and cellular mechanisms remain obscure. Moreover, no Etx-sensitive endothelial cell model has been identified to date. Here, we characterize the mouse lung endothelial cell line 1G11 as an Etx-sensitive cell line and compare it with the well-characterized Etx-sensitive Madin-Darby canine kidney epithelial cell line. Several experimental approaches, including morphological and cytotoxic assays, clearly demonstrate that the 1G11 cell line is highly sensitive to Etx and show the specific binding, oligomerization, and pore-forming activity of the toxin in these cells. Recently, the myelin and lymphocyte (MAL) protein has been postulated as a putative receptor for Etx. Here, we show the presence of Mal mRNA in the 1G11 cell line and the presence of the MAL protein in the endothelium of some mouse lung vessels, supporting the hypothesis that this protein is a key element in the Etx intoxication pathway. The existence of an Etx-sensitive cell line of endothelial origin would help shed light on the cellular and molecular mechanisms underlying Etx-induced edema and its consequences.

Pigment epithelium-derived factor mediates impaired lung vascular development in neonatal hyperoxia.

Bronchopulmonary dysplasia is a chronic lung disease of preterm infants characterized by arrested microvascularization and alveolarization. Studies show the importance of proangiogenic factors for alveolarization, but the importance of antiangiogenic factors is unknown. We proposed that hyperoxia increases the potent angiostatin, pigment epithelium-derived factor (PEDF), in neonatal lungs, inhibiting alveolarization and microvascularization. Wild-type (WT) and PEDF(-/-) mice were exposed to room air (RA) or 0.9 fraction of inspired oxygen from Postnatal Day 5 to 13. PEDF protein was increased in hyperoxic lungs compared with RA-exposed lungs (P < 0.05). In situ hybridization and immunofluorescence identified PEDF production primarily in alveolar epithelium. Hyperoxia reduced alveolarization in WT mice (P < 0.05) but not in PEDF(-/-) mice. WT hyperoxic mice had fewer platelet endothelial cell adhesion molecule (PECAM)-positive cells per alveolus (1.4 ± 0.4) than RA-exposed mice (4.3 ± 0.3; P < 0.05); this reduction was absent in hyperoxic PEDF(-/-) mice. The interactive regulation of lung microvascularization by vascular endothelial growth factor and PEDF was studied in vitro using MFLM-91U cells, a fetal mouse lung endothelial cell line. Vascular endothelial growth factor stimulation of proliferation, migration, and capillary tube formation was inhibited by PEDF. MFLM-91U cells exposed to conditioned medium (CM) from E17 fetal mouse lung type II (T2) cells cultured in 0.9 fraction of inspired oxygen formed fewer capillary tubes than CM from T2 cells cultured in RA (hyperoxia CM, 51 ± 10% of RA CM, P < 0.05), an effect abolished by PEDF antibody. We conclude that PEDF mediates reduced vasculogenesis and alveolarization in neonatal hyperoxia. Bronchopulmonary dysplasia likely results from an altered balance between pro- and antiangiogenic factors.

Lung endothelial cells strengthen, but brain endothelial cells weaken barrier properties of a human alveolar epithelium cell culture model

The blood–air barrier in the lung consists of the alveolar epithelium, the underlying capillary endothelium, their basement membranes and the interstitial space between the cell layers. Little is known about the interactions between the alveolar and the blood compartment. The aim of the present study was to gain first insights into the possible interplay between these two neighbored cell layers. We established an in vitro Transwell model of the alveolar epithelium based on human cell line H441 and investigated the influence of conditioned medium obtained from human lung endothelial cell line HPMEC-ST1.6R on the barrier properties of the H441 layers. As control for tissue specificity H441 layers were exposed to conditioned medium from human brain endothelial cell line hCMEC/D3. Addition of dexamethasone was necessary to obtain stable H441 cell layers. Moreover, dexamethasone increased expression of cell type I markers (caveolin-1, RAGE) and cell type II marker SP-B, whereas decreased the transepithelial electrical resistance (TEER) in a concentration dependent manner. Soluble factors obtained from the lung endothelial cell line increased the barrier significantly proven by TEER values and fluorescein permeability on the functional level and by the differential expression of tight junctional proteins on the molecular level. In contrast to this, soluble factors derived from brain endothelial cells weakened the barrier significantly. In conclusion, soluble factors from lung endothelial cells can strengthen the alveolar epithelium barrier in vitro, which suggests communication between endothelial and epithelial cells regulating the integrity of the blood–air barrier.

Targeted Induction of Lung Endothelial Cell Apoptosis Causes Emphysema-like Changes in the Mouse

Pulmonary gas exchange relies on a rich capillary network, which, together with alveolar epithelial type I and II cells, form alveolar septa, the functional units in the lung. Alveolar capillary endothelial cells are critical in maintaining alveolar structure, because disruption of endothelial cell integrity underlies several lung diseases. Here we show that targeted ablation of lung capillary endothelial cells recapitulates the cellular events involved in cigarette smoke-induced emphysema, one of the most prevalent nonneoplastic lung diseases. Based on phage library screening on an immortalized lung endothelial cell line, we identified a lung endothelial cell-binding peptide, which preferentially homes to lung blood vessels. This peptide fused to a proapoptotic motif specifically induced programmed cell death of lung endothelial cells in vitro as well as targeted apoptosis of the lung microcirculation in vivo. As early as 4 days following peptide administration, mice developed air space enlargement associated with enhanced oxidative stress, influx of macrophages, and up-regulation of ceramide. Given that these are all critical elements of the corresponding human emphysema caused by cigarette smoke, these data provide evidence for a central role for the alveolar endothelial cells in the maintenance of lung structure and of endothelial cell apoptosis in the pathogenesis of emphysema-like changes. Thus, our data enable the generation of a convenient mouse model of human emphysema. Finally, combinatorial screenings on immortalized cells followed by in vivo targeting establishes an experimental framework for discovery and validation of additional ligand-directed pharmacodelivery systems.

A selective small molecule inhibitor of c-Met, PHA-665752, reverses lung premalignancy induced by mutant K-ras.

The c-Met receptor tyrosine kinase has been implicated in cellular transformation induced by mutant Ras, a commonly activated proto-oncogene in non-small cell lung cancer (NSCLC). However, the role of c-Met has not been defined in K-ras-mutant NSCLC, a disease for which no effective targeted therapeutic options currently exist. To acquire a greater understanding of its role, we used genetic and pharmacologic approaches to inhibit c-Met in mice and cultured cells. In Kras(LA1) mice, which develop premalignant lung lesions that progress to multifocal lung adenocarcinomas owing to somatic mutations in K-ras, c-Met was expressed in multiple cell types within premalignant lung lesions, and high concentrations of HGF were detected in bronchoalveolar lavage samples. Short-term treatment with PHA-665752, a c-Met inhibitor, decreased the numbers of premalignant lung lesions and induced apoptosis in tumor cells and vascular endothelial cells within lesions. In cell culture, PHA-665752 induced apoptosis of a lung adenocarcinoma cell line derived from Kras(LA1) mice (LKR-13) and a murine lung endothelial cell line (MEC). c-Met depletion by siRNA transfection induced apoptosis of MECs but not LKR-13 cells. Collectively, these findings suggest that apoptosis was an on-target effect of PHA-665752 in MECs but not in LKR-13 cells. We conclude that PHA-665752 inhibited lung tumorigenesis in Kras(LA1) mice and may provide a novel therapeutic approach to the prevention of K-ras-mutant NSCLC.

Surface Anisotropy Orients Cell Divisions in Contact Guided Cells

The orientation of cell elongation and the plane of cell division were determined in cells growing under isotropic conditions on a plain glass surface or under anisotropic conditions on a scratched glass surface. Four cell lines were analysed, each showing various degrees of contact guidance. Human skin fibroblasts and the skin keratinocyte cell line HaCaT oriented randomly on the smooth isotropic surface of glass, grew and divided randomly. By contrast, on an anisotropic scratched surface these cells showed contact guidance, elongated along scratches, and their planes of division were perpendicular to the long axis of the cell. In these two cell types there was a high degree of correlation between the cell alignment and the plane of cell division, which shows that extracellular factors can influence or even determine the latter. In cell lines in which contact guidance under anisotropic conditions was less evident, viz. the lung endothelial cell line HLMEC and the skin endothelial cell line HSkMEC, the alignment of cell division planes was less ordered. This report concentrates on quantitative phenomenological descriptions of the orientation of cell division as determined by contact guidance.

ERM is expressed by alveolar epithelial cells in adult mouse lung and regulates caveolin‐1 transcription in mouse lung epithelial cell lines

We previously identified an Ets cis-element in the mouse caveolin-1 promoter that is selectively activated in lung epithelial (E10), but not lung endothelial murine lung endothelial cell line (MFLM-4), cell lines and therefore appears important for differential, cell-specific caveolin-1 transcription. In the present study, we demonstrate that immunostaining of adult mouse lung detects the ETS protein Ets-related molecule (ERM PEA3) in distal lung epithelium in alveolar type I and II cells, but not in bronchial epithelium or lung endothelial cells. We tested ERM and polyomavirus enhancer activator 3 (PEA3) for their ability to increase endogenous caveolin-1 transcripts and to activate caveolin-1 promoter fragments containing the -865 Ets cis-element. Chromatin immunoprecipitation (ChIP) assays show that both ERM and PEA3 bind to the caveolin-1 promoter in murine E10, but not MFLM-4, cells. Normalized luciferase activities show that only ERM activates the caveolin-1 promoter in E10 cells, but neither protein enhances promoter activity in MFLM-4 cells. Mutation of the Ets site blocks ERM-mediated promoter activation in E10 cells. Furthermore, overexpression of ERM increases the cellular content of caveolin-1 mRNA and protein, in E10, but not MFLM-4, cells. The effects of PEA3 on the cellular content of endogenous caveolin-1 expression are variable. These results demonstrate that ERM is involved in caveolin-1 regulation in a murine lung epithelial, but not lung endothelial cell line. We conclude that transcriptional regulation of caveolin-1 differs markedly between lung epithelial and endothelial cell lines, perhaps explaining why the onset of caveolin-1 expression differs in epithelial and endothelial cells during lung development.

Lipid Mixing between Lipoplexes and Plasma Lipoproteins Is a Major Barrier for Intravenous Transfection Mediated by Cationic Lipids

It has been previously shown that transfection activity of cationic liposome/DNA lipoplexes delivered systemically is drastically inhibited by lipoproteins (Tandia, B. M., Vandenbranden, M., Wattiez, R., Lakhdar, Z., Ruysschaert, J. M., and Elouahabi, A. (2003) Mol Ther. 8, 264-273). In this work, we have compared the binding/uptake and transfection activities of DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) and diC14-amidine (3-tetradecylamino-N-tert-butyl-N'-tetra-decylpropionamidine)-containing lipoplexes in the presence or absence of purified low density lipoproteins and high density lipoprotein. Binding/uptake of both lipoplexes by the mouse lung endothelial cell line was inhibited to a similar extent in the presence of lipoproteins. In contrast, transfection activity of diC14-amidine-containing lipoplexes was almost completely inhibited (approximately by 95%), whereas approximately 40% transfection activity of DOTAP-containing lipoplexes was preserved in the presence of lipoproteins. Interestingly, the ability of lipoproteins to inhibit the transfection efficiency of lipoplexes was well correlated with their ability to undergo lipid mixing with the cationic lipid bilayer as revealed by fluorescence resonance energy transfer assay. Incubation of lipoplexes with increased doses of lipoproteins resulted in enhanced lipid mixing and reduced transfection activity of the lipoplexes in mouse lung endothelial cells. The role of lipid mixing in transfection was further demonstrated using lipid-mixing inhibitor, lyso-phosphatidylcholine, or activator (dioleoylphosphatidylethanolamine). Incorporation of Lyso-PC into diC14-amidine-containing lipoplexes completely abolished their capacity to undergo lipid mixing with lipoproteins and allowed them to reach a high transfection efficiency in the presence of lipoproteins. On the other hand, the incorporation of dioleoylphosphatidylethanolamine into DOTAP/DNA lipoplex activated lipid mixing with the lipoproteins and was shown to be detrimental toward the transfection activity of these lipoplexes. Taken together, these results indicate that fusion of lipoplexes with lipoproteins is a limiting factor for in vivo transfection.

Transcription of the Caveolin-1 Gene Is Differentially Regulated in Lung Type I Epithelial and Endothelial Cell Lines: A ROLE FOR ETS PROTEINS IN EPITHELIAL CELL EXPRESSION

In the lung, caveolin-1 is expressed in both type I alveolar epithelial and endothelial cells where it is hypothesized to modulate molecular signaling activities and progression of tumorigenesis. Developmentally, caveolin-1alpha is expressed in fetal lung endothelial, but not epithelial, cells; in adult lung, both cell types express caveolin-1alpha. To test the hypothesis that caveolin-1 transcription is differentially regulated in type I and endothelial cells, we characterized the proximal promoter of the mouse caveolin-1 gene in lung cell lines to identify factors that control its cell-specific expression. We show that caveolin-1 expression is regulated by an Ets cis-element in a lung epithelial cell line, but not a lung endothelial cell line, and that three ETS family members, ETS-1, PEA3, and ERM, recognize and bind the Ets site in the epithelial cell line. Based on these findings, we have identified the Ets cis-element as a region that accounts for differential transcriptional regulation of caveolin-1 in lung epithelial and endothelial cells.

Thr 203 of claudin-1, a putative phosphorylation site for MAP kinase, is required to promote the barrier function of tight junctions

Mitogen-activated protein kinase (MAPK) modulates the barrier function of tight junctions. We identified a putative phosphorylation site for MAPK at around Thr 203 (PKPTP) in claudin-1, and determined the biological significance of this site. To this end, using the rat lung endothelial cell line RLE, we generated cells expressing doxycycline (Dox)-inducible wild-type claudin-1 and its mutant with substitution of Thr 203 to Ala, and named them RLE:rtTA:CL1 and RLE:rtTA:CL1T203A, respectively. We herein show, by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin, that functional tight junctions were reconstituted in both cells by Dox-induced expression of claudin-1. Interestingly, the barrier functions of tight junctions were less developed in RLE:rtTA:CL1T203A cells compared with RLE:rtTA:CL1 cells. Consistently, levels of both detergent-insoluble claudin-1 protein and its threonine-phosphorylation after Dox treatment were low in RLE:rtTA:CL1T203A cells compared to RLE:rtTA:CL1 cells. Furthermore, pretreatment with the MAPK inhibitor PD98059 markedly suppressed the barrier function and amount of detergent-insoluble claudin-1 in Dox-exposed RLE:rtTA:CL1 cells, whereas it marginally influenced those in RLE:rtTA:CL1T203A cells. These findings indicate that Thr 203 of claudin-1 is required to enhance the barrier function of claudin-1-based tight junctions, probably via its phosphorylation and subsequent integration into tight junctions.

Use of N-terminal modified poly(L-lysine)-antibody conjugate as a carrier for targeted gene delivery in mouse lung endothelial cells.

A DNA targeted delivery and expression system has been designed based on an N-terminal modified poly(L-lysine) (NPLL)-antibody conjugate, which readily forms a complex with plasmid DNA. Monoclonal antibodies against the cell-surface thrombomodulin conjugated with NPLL were used for targeted delivery of foreign plasmid DNA to an antigen-expressing mouse lung endothelial cell line in vitro and to mouse lungs in vivo. In both cases significant amounts of DNA can be specifically bound to the target cells or tissues. Specific gene expression was observed in the treated mouse lung endothelial cells.

Modulation of adhesion of lymphocytes to murine brain endothelial cells in vitro: relation to class II major histocompatibility complex expression

Adhesion of lymphocytes to mouse brain endothelial cells was studied after treatment of the endothelium with 1000 U/ml gamma interferon (IFN- γ) for 1 h to 2 days. Adhesion was not significantly different from controls after 1 h but at 4 h and thereafter, adhesion increased in a time-related manner. IFN- γ also increased the expression of class II major histocompatibility complex (MHC) and murine intercellular adhesion molecule-1 (ICAM-1) molecules on the endothelial cells. The level of expression of class II MHC molecules was related to the length of exposure to INF- γ. MAb blocking studies suggested that class II molecules were responsible for the INF- γ-induced increase in lymphocyte-endothelial cell adhesion. Transfection of a murine lung endothelial cell line with cDNA for the class II MHC molecule also produced a significant increase in lymphocyte-endothelial cell adhesion, suggesting that the class II MHC molecule may have a role in adhesion which is distinct from antigen presentation.