scholarly journals Nanotoxicologic Effects of PLGA Nanoparticles Formulated with a Cell-Penetrating Peptide: Searching for a Safe pDNA Delivery System for the Lungs

Pharmaceutics ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 12 ◽  
Author(s):  
Larissa Gomes dos Reis ◽  
Wing-Hin Lee ◽  
Maree Svolos ◽  
Lyn Margaret Moir ◽  
Rima Jaber ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Membrane Integrity ◽  
A549 Cells ◽  
Intracellular Delivery ◽  
Plga Nanoparticles ◽  
Cell Penetrating Peptides ◽  
Lung Epithelial Cells ◽  
Mitochondrial Activity ◽  
Lung Epithelial ◽  
Cell Penetrating

The use of cell-penetrating peptides (CPPs) in combination with nanoparticles (NPs) shows great potential for intracellular delivery of DNA. Currently, its application is limited due to the potential toxicity and unknown long-term side effects. In this study NPs prepared using a biodegradable polymer, poly(lactic–co–glycolic acid (PLGA) in association with a CPP, was assessed on two lung epithelial cell lines (adenocarcinomic human alveolar basal epithelial cells (A549) and normal bronchial epithelial cells (Beas-2B cells)). Addition of CPP was essential for intracellular internalization. No effects were observed on the mitochondrial activity and membrane integrity. Cells exposed to the NPs–DNA–CPP showed low inflammatory response, low levels of apoptosis and no activation of caspase-3. Increase in necrotic cells (between 10%–15%) after 24 h of incubation and increase in autophagy, induced by NPs–DNA–CPP, are likely to be related to the lysosomal escape mechanism. Although oxidative stress is one of the main toxic mechanisms of NPs, NPs–DNA–CPP showed decreased reactive oxygen species (ROS) production on Beas-2B cells, with potential antioxidant effect of CPP and no effect on A549 cells. This NP system appears to be safe for intracellular delivery of plasmid DNA to the lung epithelial cells. Further investigations should be conducted in other lung-related systems to better understand its potential effects on the lungs.

Ionizing radiation enhances matrix metalloproteinase-2 production in human lung epithelial cells

2001 ◽  
Vol 280 (1) ◽  
pp. L30-L38 ◽  
Author(s):  
Jun Araya ◽  
Muneharu Maruyama ◽  
Kazuhiko Sassa ◽  
Tadashi Fujita ◽  
Ryuji Hayashi ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Ionizing Radiation ◽  
Basement Membrane ◽  
Lung Injury ◽  
Human Lung ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Lung Epithelial ◽  
Radiation Induced ◽  
Human Lung Epithelial Cells

Radiation pneumonitis is a major complication of radiation therapy. However, the detailed cellular mechanisms have not been clearly defined. Based on the recognition that basement membrane disruption occurs in acute lung injury and that matrix metalloproteinase (MMP)-2 can degrade type IV collagen, one of the major components of the basement membrane, we hypothesized that ionizing radiation would modulate MMP-2 production in human lung epithelial cells. To evaluate this, the modulation of MMP-2 with irradiation was investigated in normal human bronchial epithelial cells as well as in A549 cells. We measured the activity of MMP-2 in the conditioned medium with zymography and the MMP-2 mRNA level with RT-PCR. Both of these cells constitutively expressed 72-kDa gelatinolytic activity, corresponding to MMP-2, and exposure to radiation increased this activity. Consistent with the data of zymography, ionizing radiation increased the level of MMP-2 mRNA. This radiation-induced increase in MMP-2 expression was mediated via p53 because the p53 antisense oligonucleotide abolished the increase in MMP-2 activity as well as the accumulation of p53 after irradiation in A549 cells. These results indicate that MMP-2 expression by human lung epithelial cells is involved in radiation-induced lung injury.

scholarly journals TRIM28 regulates SARS-CoV-2 cell entry by targeting ACE2

2020 ◽  
Author(s):  
Yinfang Wang ◽  
Yingzhe Fan ◽  
Yitong Huang ◽  
Tao Du ◽  
Zongjun Liu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Nk Cells ◽  
Interleukin 2 ◽  
A549 Cells ◽  
Endogenous Retrovirus ◽  
Alveolar Epithelial Cells ◽  
Cell Entry ◽  
Lung Epithelial Cells ◽  
Lung Epithelial ◽  
Ifn Γ

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), it binds to angiotensin-converting enzyme 2 (ACE2) to enter into human cells. The expression level of ACE2 potentially determine the susceptibility and severity of COVID-19, it is thus of importance to understand the regulatory mechanism of ACE2 expression. Tripartite motif containing 28 (TRIM28) is known to be involved in multiple processes including antiviral restriction, endogenous retrovirus latency and immune response, it is recently reported to be co-expressed with SARS-CoV-2 receptor in type II pneumocytes; however, the roles of TRIM28 in ACE2 expression and SARS-CoV-2 cell entry remain unclear. This study showed that knockdown of TRIM28 induces ACE2 expression and increases pseudotyped SARS-CoV-2 cell entry of A549 cells and primary pulmonary alveolar epithelial cells (PAEpiCs). In a co-culture model of NK cells and lung epithelial cells, our results demonstrated that NK cells inhibit TRIM28 and promote ACE2 expression in lung epithelial cells, which was partially reversed by depletion of interleukin-2 and blocking of granzyme B in the co-culture medium. Furthermore, TRIM28 knockdown enhanced interferon-γ (IFN-γ)-induced ACE2 expression through a mechanism involving upregulating IFN-γ receptor 2 (IFNGR2) in both A549 and PAEpiCs. Importantly, the upregulated ACE2 induced by TRIM28 knockdown and co-culture of NK cells was partially reversed by dexamethasone in A549 cells but not PAEpiCs. Our study identified TRIM28 as a novel regulator of ACE2 expression and SARS-CoV-2 cell entry.

Dual mechanism forMycobacterium tuberculosiscytotoxicity on lung epithelial cells

2012 ◽  
Vol 58 (7) ◽  
pp. 909-916 ◽  
Author(s):  
Jorge Castro-Garza ◽  
W. Edward Swords ◽  
Russell K. Karls ◽  
Frederick D. Quinn
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Neutralizing Antibodies ◽  
Cytotoxic Effect ◽  
Alveolar Epithelial Cell ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Lung Epithelial

Mycobacterium tuberculosis strains CDC1551 and Erdman were used to assess cytotoxicity in infected A549 human alveolar epithelial cell monolayers. Strain CDC1551 was found to induce qualitatively greater disruption of A549 monolayers than was strain Erdman, although total intracellular and cell-associated bacterial growth rates over the course of the infections were not significantly different. Cell-free culture supernatants from human monocytic cells infected with either of the 2 M. tuberculosis strains produced a cytotoxic effect on A549 cells, correlating with the amount of tumor necrosis factor alpha (TNF-α) released by the infected monocytes. The addition of TNF-α-neutralizing antibodies to the supernatants from infected monocyte cultures did prevent the induction of a cytotoxic effect on A549 cells overlaid with this mixture but did not prevent the death of epithelial cells when added prior to infection with M. tuberculosis bacilli. Thus, these data agree with previous observations that lung epithelial cells infected with M. tuberculosis bacilli are rapidly killed in vitro. In addition, the data indicate that some of the observed epithelial cell killing may be collateral damage; the result of TNF-α released from M. tuberculosis-infected monocytes.

scholarly journals Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells

2004 ◽  
Vol 78 (15) ◽  
pp. 8146-8158 ◽  
Author(s):  
Santanu Bose ◽  
Mausumi Basu ◽  
Amiya K. Banerjee
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Human Lung ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Parainfluenza Virus ◽  
Lung Epithelial ◽  
Human Parainfluenza Virus ◽  
Type 3 ◽  
Human Lung Epithelial Cells

ABSTRACT Human parainfluenza virus type 3 (HPIV-3) is an airborne pathogen that infects human lung epithelial cells from the apical (luminal) plasma membrane domain. In the present study, we have identified cell surface-expressed nucleolin as a cellular cofactor required for the efficient cellular entry of HPIV-3 into human lung epithelial A549 cells. Nucleolin was enriched on the apical cell surface domain of A549 cells, and HPIV-3 interacted with nucleolin during entry. The importance of nucleolin during HPIV-3 replication was borne out by the observation that HPIV-3 replication was significantly inhibited following (i) pretreatment of cells with antinucleolin antibodies and (ii) preincubation of HPIV-3 with purified nucleolin prior to its addition to the cells. Moreover, HPIV-3 cellular internalization and attachment assays performed in the presence of antinucleolin antibodies and purified nucleolin revealed the requirement of nucleolin during HPIV-3 internalization but not during attachment. Thus, these results suggest that nucleolin expressed on the surfaces of human lung epithelial A549 cells plays an important role during HPIV-3 cellular entry.

Induction of proinflammatory cytokines in human lung epithelial cells during Rhodococcus equi infection

2013 ◽  
Vol 62 (8) ◽  
pp. 1144-1152 ◽  
Author(s):  
Sara Remuzgo-Martínez ◽  
Lilian Pilares-Ortega ◽  
Lorena Álvarez-Rodríguez ◽  
Maitane Aranzamendi-Zaldunbide ◽  
Daniel Padilla ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Lung ◽  
A549 Cells ◽  
Rhodococcus Equi ◽  
Airway Epithelial Cells ◽  
Lung Epithelial Cells ◽  
Initial Contact ◽  
Structural Level ◽  
Lung Epithelial ◽  
Human Lung Epithelial Cells

Rhodococcus equi is an opportunistic human pathogen associated with immunosuppressed people. While the interaction of R. equi with macrophages has been comprehensively studied, little is known about its interactions with non-phagocytic cells. Here, we characterized the entry process of this bacterium into human lung epithelial cells. The invasion is inhibited by nocodazole and wortmannin, suggesting that the phosphatidylinositol 3-kinase pathway and microtubule cytoskeleton are important for invasion. Pre-incubation of R. equi with a rabbit anti-R. equi polyclonal antiserum resulted in a dramatic reduction in invasion. Also, the invasion process as studied by immunofluorescence and scanning electron microscopy indicates that R. equi make initial contact with the microvilli of the A549 cells, and at the structural level, the entry process was observed to occur via a zipper-like mechanism. Infected lung epithelial cells upregulate the expression of cytokines IL-8 and IL-6 upon infection. The production of these pro-inflammatory cytokines was significantly enhanced in culture supernatants from cells infected with non-mucoid plasmid-less strains when compared with cells infected with mucoid strains. These results demonstrate that human airway epithelial cells produce pro-inflammatory mediators against R. equi isolates.

scholarly journals Proliferation of Lung Epithelial Cells Is Regulated by the Mechanisms of Autophagy Upon Exposure of Soots

2021 ◽  
Vol 9 ◽  
Author(s):  
Rituraj Niranjan ◽  
Kaushal Prasad Mishra ◽  
Sachchida Nand Tripathi ◽  
Ashwani Kumar Thakur
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Epithelial Cells ◽  
Antioxidant Properties ◽  
A549 Cells ◽  
Beclin 1 ◽  
Lung Epithelial Cells ◽  
Akt Inhibitor ◽  
Fullerene Soot ◽  
Lung Epithelial

BackgroundSoots are known to cause many diseases in humans, but their underlying mechanisms of toxicity are still not known. Here, we report that soots induce cell proliferation of lung epithelial cells via modulating autophagy pathways.ResultsFullerene soot and diesel exhaust particles (DEP) induced cell proliferation of lung epithelial, A549 cells via distinct autophagic mechanisms and did not cause cell death. Exposure of fullerene soot protected the cell death of A549 cells, caused by hydrogen peroxide, and inhibited LPS-induced autophagy. Fullerene soot co-localized with the autophagic proteins and inhibited starvation-induced autophagy (downregulated ATG-5, beclin-1, p62, and LC3 expressions) independent of its antioxidant properties. Similarly, it decreased the expression profile of autophagic genes and upregulated the proliferation-responsive gene, Ki-67, in mice. We observed that expressions of fullerene soot-responsive genes (Beclin-1, ATG-5, and p62) were reverted by Akt Inhibitor X, indicating an important role of the Akt pathway. At an elemental level, we found that elemental carbon of fullerene soot may be converted into organic carbon, as measured by OCEC, which may point fullerene soot as a source of carbon. On the other hand, DEP upregulated the expressions of autophagy genes. Akt Inhibitor X did not attenuate DEP-induced cell proliferation and autophagic response. However, an autophagic inhibitor, chloroquine, and significantly inhibited DEP-induced cell proliferation.ConclusionIt can be said that distinct autophagic mechanisms are operational in cell proliferation of lung epithelial cells due to soots, which may be responsible for different diseases. Understanding the mechanism of these pathways provides some important targets, which can be utilized for the development of future therapeutics.

scholarly journals Detection of Viral RNA Fragments in Human iPSC-Cardiomyocytes following Treatment with Extracellular Vesicles from SARS-CoV-2 Coding-Sequence-Overexpressing Lung Epithelial Cells

2020 ◽  
Author(s):  
Youjeong Kwon ◽  
Sarath Babu Nukala ◽  
Shubhi Srivastava ◽  
Hiroe Miyamoto ◽  
Nur Izzah Ismail ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Extracellular Vesicles ◽  
A549 Cells ◽  
Induced Pluripotent Stem Cell ◽  
Conclusive Evidence ◽  
Lung Epithelial Cells ◽  
Viral Rna ◽  
Lung Epithelial ◽  
Viral Genes ◽  
Novel Coronavirus

ABSTRACTThe novel coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into a worldwide pandemic. Early data suggest that the prevalence and severity of COVID-19 appear to be higher among patients with underlying cardiovascular risk factors. Despite the expression of angiotensin-converting enzyme 2 (ACE2), a functional receptor for SARS-CoV-2 infection, in cardiomyocytes, there has been no conclusive evidence of direct viral infection although the presence of inflammation and viral genome within the hearts of COVID-19 patients have been reported. Here we transduced A549 lung epithelial cells with lentivirus overexpressing selected genes of the SARS-CoV-2. We then isolated extracellular vesicles (EVs) from the supernatant of A549 cells and detected the presence of viral RNA within the purified EVs. Importantly, we observed that human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were able to actively uptake these EVs and viral genes were subsequently detected in the cardiomyocytes. Accordingly, uptake of EVs containing viral genes led to an upregulation of inflammation-related genes in hiPSC-CMs. Thus, our findings indicate that SARS-CoV-2 RNA-containing EVs represent an indirect route of viral RNA entry into cardiomyocytes.

scholarly journals Necrosis of Lung Epithelial Cells during Infection withMycobacterium tuberculosis Is Preceded by Cell Permeation

2000 ◽  
Vol 68 (11) ◽  
pp. 6300-6310 ◽  
Author(s):  
Karen M. Dobos ◽  
Ellen A. Spotts ◽  
Frederick D. Quinn ◽  
C. Harold King
Keyword(s):  
Epithelial Cells ◽  
A549 Cell ◽  
Cell Monolayer ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Lung Epithelial ◽  
Infected Cells ◽  
Cellular Necrosis ◽  
Ldh Release

ABSTRACT Mycobacterium tuberculosis establishes infection, progresses towards disease, and is transmitted from the alveolus of the lung. However, the role of the alveolar epithelium in any of these pathogenic processes of tuberculosis is unclear. In this study, lung epithelial cells (A549) were used as a model in which to examine cytotoxicity during infection with either virulent or avirulent mycobacteria in order to further establish the role of the lung epithelium during tuberculosis. Infection of A549 cells with M. tuberculosis strains Erdman and CDC1551 demonstrated significant cell monolayer clearing, whereas infection with eitherMycobacterium bovis BCG or Mycobacterium smegmatis LR222 did not. Clearing of M. tuberculosis-infected A549 cells correlated to necrosis, not apoptosis. Treatment of M. tuberculosis-infected A549 cells with streptomycin, but not cycloheximide, demonstrated a significant reduction in the necrosis of A549 cell monolayers. This mycobacterium-induced A549 necrosis did not correlate to higher levels of intracellular or extracellular growth by the mycobacteria during infection. Staining of infected cells with propidium iodide demonstrated that M. tuberculosis induced increased permeation of A549 cell membranes within 24 h postinfection. Quantitation of lactate dehydrogenase (LDH) release from infected cells further demonstrated that cell permeation was specific to M. tuberculosis infection and correlated to A549 cellular necrosis. Inactivated M. tuberculosis or its subcellular fractions did not result in A549 necrosis or LDH release. These studies demonstrate that lung epithelial cell cytotoxicity is specific to infection by virulent mycobacteria and is caused by cellular necrosis. This necrosis is not a direct correlate of mycobacterial growth or of the expression of host cell factors, but is preceded by permeation of the A549 cell membrane and requires infection with live bacilli.

scholarly journals Cytosolic phospholipase A2α activation induced by S1P is mediated by the S1P3 receptor in lung epithelial cells

2008 ◽  
Vol 295 (2) ◽  
pp. L326-L335 ◽  
Author(s):  
Li-Yuan Chen ◽  
Grzegorz Woszczek ◽  
Sahrudaya Nagineni ◽  
Carolea Logun ◽  
James H. Shelhamer
Keyword(s):  
Epithelial Cells ◽  
Rho Kinase ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Calcium Flux ◽  
Lipid Mediator ◽  
Dependent Manner ◽  
Cytosolic Phospholipase ◽  
Lung Epithelial ◽  
Sphingosine 1 Phosphate

Cytosolic phospholipase A2α (cPLA2α) activation is a regulatory step in the control of arachidonic acid (AA) liberation for eicosanoid formation. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator involved in the regulation of many important proinflammatory processes and has been found in the airways of asthmatic subjects. We investigated the mechanism of S1P-induced AA release and determined the involvement of cPLA2α in these events in A549 human lung epithelial cells. S1P induced AA release rapidly within 5 min in a dose- and time-dependent manner. S1P-induced AA release was inhibited by the cPLA2α inhibitors methyl arachidonyl fluorophosphonate (MAFP) and pyrrolidine derivative, by small interfering RNA-mediated downregulation of cPLA2α, and by inhibition of S1P-induced calcium flux, suggesting a significant role of cPLA2α in S1P-mediated AA release. Knockdown of the S1P3 receptor, the major S1P receptor expressed on A549 cells, inhibited S1P-induced calcium flux and AA release. The S1P-induced calcium flux and AA release was associated with sphingosine kinase 1 (Sphk1) expression and activity. Furthermore, Rho-associated kinase, downstream of S1P3, was crucial for S1P-induced cPLA2α activation. Our data suggest that S1P acting through S1P3, calcium flux, and Rho kinase activates cPLA2α and releases AA in lung epithelial cells. An understanding of S1P-induced cPLA2α activation mechanisms in epithelial cells may provide potential targets to control inflammatory processes in the lung.

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