Modulation of lung inflammation by vessel dilator in a mouse model of allergic asthma

Abstract

Background: Atrial natriuretic peptide (ANP) and its receptor, NPRA, have been extensively studied in terms of cardiovascular effects. We have found that the ANP-NPRA signaling pathway is also involved in airway allergic inflammation and asthma. ANP, a C-terminal peptide (amino acid 99-126) of pro-atrial natriuretic factor (proANF) and a recombinant peptide, NP73-102 (amino acid 73-102 of proANF) have been reported to induce bronchoprotective effects in a mouse model of allergic asthma. In this report, we evaluated the effects of vessel dilator (VD), another N-terminal natriuretic peptide covering amino acids 31-67 of proANF, on acute lung inflammation in a mouse model of allergic asthma.

Methods: A549 cells were transfected with pVD or the pVAX1 control plasmid and cells were collected 24 hrs after transfection to analyze the effect of VD on inactivation of the extracellular-signal regulated receptor kinase (ERK1/2) through western blot. Luciferase assay, western blot and RT-PCR were also performed to analyze the effect of VD on NPRA expression. For determination of VD's attenuation of lung inflammation, BALB/c mice were sensitized and challenged with ovalbumin and then treated intranasally with chitosan nanoparticles containing pVD. Parameters of airway inflammation, such as airway hyperreactivity, proinflammatory cytokine levels, eosinophil recruitment and lung histopathology were compared with control mice receiving nanoparticles containing pVAX1 control plasmid.

Results: pVD nanoparticles inactivated ERK1/2 and downregulated NPRA expression in vitro, and intranasal treatment with pVD nanoparticles protected mice from airway inflammation.

Conclusion: VD's modulation of airway inflammation may result from its inactivation of ERK1/2 and downregulation of NPRA expression. Chitosan nanoparticles containing pVD may be therapeutically effective in preventing allergic airway inflammation.

Figure 1

Experimental schedule of sensitization and induction of allergic airway response. Chicken ovalbumin was used to sensitize and challenge mice (n = 6–8 per group).

Figure 2

pVD inactivates ERK1/2 and downregulates NPRA expression. (A) A549 cells were transfected with pVD, pKP2 or pVAX1 control plasmids. Cells were collected 24 hrs after transfection. Expression of ERK1/2 and phospho-ERK1/2 was detected by western blot. (B) HEK293 cells grown on 96-well plates were cotransfected with 0.5 μg of pNPRA-Luc and 1 μg pVAX1 or pVD. Cells were lysed 48 hrs later and luciferase activity was measured in the lysates (p < 0.01). (C) Effect of VD on NPRA expression in vitro. HEK-GCA cells were transfected with pVAX1, pVD or pMut. NPRA expression was detected by western blot. Non-transfected cells were used as control. (D). Effect of VD on NPRA expression in vivo. NPRA mRNA expression was detected by RT-PCR in the lungs of mice intranasally treated with chitosan nanoparticles containing 20 μg of pVAX1 (n = 4), pVD (n = 4) or pMut (n = 4). Mice from the naïve group (n = 4) served as mock controls. All experiments were repeated, and the results of a representative experiment are shown.

Figure 3

VD prevents airway hyperresponsiveness in the mouse model. Pulmonary resistance was measured using the forced oscillation technique. Mice from each group were treated with methacholine at increasing concentrations. Actual maximum resistance is displayed for each group. Mice given pVD chitosan nanoparticles had significantly lower resistance than those from the OVA control group or the group receiving pVAX1 control nanoparticles (p < 0.05).

Figure 4

VD attenuates lung inflammation in BALB/c mice. (A) Mice were sensitized and challenged with OVA and then given nanoparticles containing pVD or control pVAX1 plasmids. Mice were sacrificed 48 hrs after the final treatment, and BAL fluids were collected for differential cell counts. Values are reported as mean ± SEM. Treatment with pVD significantly reduced eosinophil recruitment to the lungs compared to pVAX1 control (p < 0.05). Mac, macrophages; Eos, eosinophils; Neu, neutrophils; Lym, lymphocytes. (B) Lung sections from mice treated with VD nanoparticles also showed a substantial decrease in lung inflammation, goblet cell hyperplasia and infiltration of inflammatory cells compared to the non-OVA-challenged group or the group treated with pVAX1. All experiments were repeated and the results of a representative experiment are shown.

Figure 5

VD reduces proinflammatory cytokines in lung homogenates. Lungs from each group were collected and homogenized. Supernatants of the homogenates were used to measure proinflammatory cytokines with the mouse Th1/Th2 cytokine CBA kit. Significant reduction of IL-4, IL-5, IL-13 and IFN-γ were observed in the pVD nanoparticle-treated group compared to the OVA and pVAX1 nanoparticle-treated group (p < 0.05). All experiments were repeated at least once and the results of a representative experiment are shown.