Yonsei Med J.  2015 May;56(3):825-831. 10.3349/ymj.2015.56.3.825.

Hypoxia Increases Epithelial Permeability in Human Nasal Epithelia

• Min HJ
• Kim TH
• Yoon JH
• Kim CH

• KMID: 2450360
• DOI: http://doi.org/10.3349/ymj.2015.56.3.825

Abstract

PURPOSE
The nasal mucosa is the first site to encounter pathogens, and it forms continuous barriers to various stimuli. This barrier function is very important in the innate defense mechanism. Additionally, inflammation of the nasal sinus is known to be a hypoxic condition. Here, we studied the effect of hypoxia on barrier function in normal human nasal epithelial (NHNE) cells.
MATERIALS AND METHODS
The expression levels of various junction complex proteins were assessed in hypoxia-stimulated NHNE cells and human nasal mucosal tissues. We performed real-time polymerase chain reaction analysis, western blotting, and immunofluorescence assays to examine differences in the mRNA and protein expression of ZO-1, a tight junction protein, and E-cadherin in NHNE cells. Moreover, we evaluated the trans-epithelial resistance (TER) of NHNE cells under hypoxic conditions to check for changes in permeability. The expression of ZO-1 and E-cadherin was measured in human nasal mucosa samples by western blotting.
RESULTS
Hypoxia time-dependently decreased the expression of ZO-1 and E-cadherin at the gene and protein levels. In addition, hypoxia decreased the TER of NHNE cells, which indicates increased permeability. Human nasal mucosa samples, which are supposed to be hypoxic, showed significantly decreased levels of ZO-1 and E-cadherin expression compared with control.
CONCLUSION
Our results demonstrate that hypoxia altered the expression of junction complex molecules and increased epithelial permeability in human nasal epithelia. This suggests that hypoxia causes barrier dysfunction. Furthermore, it may be associated with innate immune dysfunction after encountering pathogens.

Keyword

Hypoxia; ZO-1; E-cadherin; permeability

MeSH Terms

Anoxia/etiology/*metabolism
Blotting, Western
Cadherins/*analysis/genetics
Epithelium/chemistry/pathology
Humans
Membrane Proteins/*analysis
Nasal Mucosa/*chemistry/pathology/*secretion
Permeability/*radiation effects
RNA, Messenger/genetics/metabolism
Real-Time Polymerase Chain Reaction
Tight Junctions/*metabolism
Zonula Occludens-1 Protein
Cadherins
Membrane Proteins
RNA, Messenger
Zonula Occludens-1 Protein

Figure

• 

  Fig. 1 Hypoxia decreases the levels of ZO-1 and E-cadherin in NHNE cells. (A) NHNE cells were incubated under hypoxic conditions, and Western blotting was performed to assess the expressions of HIF-1α, ZO-1, and E-cadherin at 8 and 16 hours of hypoxia. (B) The relative band intensities of ZO-1 and E-cadherin after 8 hours of hypoxia were calculated and normalized to β-actin. (C) NHNE cells were incubated under hypoxic conditions for 8 hours, and real-time PCR analysis was performed to compare the mRNA expressions of ZO-1 and E-cadherin. The relative gene expression was calculated by normalization to β-actin (n=3, *p<0.05, †p<0.01). HIF, hypoxia-inducible factor; NHNE, normal human nasal epithelial; PCR, polymerase chain reaction.

• 

  Fig. 2 Immunofluorescence analysis of NHNE cells. (A) NHNE cells were incubated under hypoxic conditions for 8 hours, and the immunofluorescence assay was performed with ZO-1 and E-cadherin antibodies. Fluorescence images were taken using a confocal microscope. (B) The mean fluorescence intensity was calculated using the ImageJ program (n=3, *p<0.05). NHNE, normal human nasal epithelial.

• 

  Fig. 3 Hypoxia decreases the TER of NHNE cells. NHNE cells were incubated under hypoxic conditions for 8 hours, and TER values were measured immediately. The mean TER values of control and hypoxia-conditioned cells were 407.5±43.76 and 199.5±41.17, respectively (n=4, *p<0.01). TER, trans-epithelial resistance; NHNE, normal human nasal epithelial.

• 

  Fig. 4 ZO-1 and E-cadherin expressions are decreased in the hypoxia-conditioned human nasal mucosa. (A) The maxillary sinus mucosa was harvested from totally (left) and partially (right) obstructed maxillary sinuses, and Western blotting was performed to compare the expressions of ZO-1 and E-cadherin. (B) The expressions of ZO-1 and E-cadherin were decreased in the totally obstructed maxillary sinus mucosa compared with the partially obstructed maxillary sinus mucosa. (C) The experiments were repeated in ten patients, and the relative band intensities of ZO-1 and E-cadherin were calculated and normalized to β-actin. P, sinus mucosa from the partially obstructed maxillary sinus; T, sinus mucosa from the totally obstructed maxillary sinus (n=10, *p<0.05, †p<0.01).

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