Modulation of lung inflammation by vessel dilator in a mouse model of allergic asthma
© Wang et al. 2009
Received: 03 October 2008
Accepted: 17 July 2009
Published: 17 July 2009
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.
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.
pVD nanoparticles inactivated ERK1/2 and downregulated NPRA expression in vitro, and intranasal treatment with pVD nanoparticles protected mice from airway inflammation.
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.
Asthma is a complex disease, characterized by reversible airway obstruction, airway hyperresponsiveness and chronic airway inflammation. According to the Third National Health Nutrition Examination Survey, about 54% of the US population is allergic to one or more allergens, and over the last two decades, the rates of asthma have increased worldwide . Current pharmacologic treatments for asthma include bronchodilating beta2-agonists and antiinflammatory glucocorticosteroids. These agents act only on symptoms and do not target the main cause of the disease, the generation of pathogenic Th2 cells [2–5]. Hence, there is a continued search for novel agents against allergy and asthma.
The family of natriuretic hormone peptides has broad physiologic effects including vasodilation, cardiovascular homeostasis, sodium excretion and inhibition of aldosterone secretion. There have been several reports demonstrating involvement of the atrial natriuretic peptide (ANP) signaling pathway in immunity in the heart and lung . The natriuretic peptide prohormone is a polypeptide of 126 amino acids that gives rise to four peptides: long acting natriuretic peptide (LANP, amino acids 1–30), vessel dilator (VD, amino acids 31–67), kaliuretic peptide (KP, amino acids 79–98) and atrial natriuretic peptide (ANP, amino acids 99–126) . In contrast to ANP, the N-terminal proANP peptides (LANP, VD, KP) are slowly metabolized and their plasma concentration is higher than ANP consistent with their important role in electrolyte balance and regulation of vascular tone. ANP and its principal receptor, NPRA, have been extensively studied in terms of cardiovascular effects . ANP signals primarily through NPRA by increasing cGMP and activating cGMP-dependent protein kinase (PKG). Activated PKG turns on ion transporters and transcription factors, which together affect cell growth and proliferation, and inflammation . NPRA is widely expressed in the lung and has been associated with allergic inflammation and asthma [9–11].
We have reported that both ANP and NP73-102 showed bronchoprotective effects [12, 13]. Expression of NP73-102 induced constitutive nitric oxide production and decreased activation of a number of transcription factors including nuclear factor kappa B in human epithelial cells . However, there is no report of the functions of the N-terminal proANP peptides including LANP, VD and KP in modulating lung inflammation. In this report we show that overexpression of VD attenuates airway inflammation in a mouse model of allergic asthma. The effects of VD on airway inflammation may result from its inactivation of ERK1/2 and downregulation of NPRA expression.
BALB/c mice were purchased from Harlan Sprague Dawley, Inc. and maintained under specific pathogen-free conditions within the vivarium at Louisiana State University Health Sciences Center (New Orleans, LA) or at the University of South Florida (Tampa, FL). Sentinel mice within each colony were monitored and were negative for specific known mouse pathogens. All animal protocols were prepared in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and approved by the Institutional Animal Care and Use Committee at Louisiana State University Health Sciences Center or at University of South Florida.
Preparation of pVD chitosan nanoparticles
The cDNAs encoding VD were cloned between the EcoRI and XhoI sites of the mammalian expression vector pVAX1 (Invitrogene, CA) using standard molecular biology procedures. Similarly, we also constructed a plasmid, pMut, which expresses a mutated VD peptide with the reversed amino acid sequence of VD. Stocks of pVD, pMut and pVAX1 plasmids were prepared using Qiagene endotoxin-free Gigaprep kits (Qiagen, CA). We have developed a nanoparticle delivery system utilizing the polysaccharide chitosan that allows intranasal administration of peptides, plasmids, and drugs . The nanoparticles protect the natriuretic peptide expression plasmids from nuclease degradation and improve delivery to cells. Complex coacervation of the DNA with chitosan (33 kDa, with 90% deacetylation, obtained from TaeHoon Bio (Korea) at a chitosan:DNA weight ratio of 1:3) was achieved by vortexing for 2 min at room temperature. Coacervates were used immediately after preparation or stored at 4°C.
Analysis of ERK1/2 expression and NPRA in pVD-transfected cells by Western blot
A549 human alveolar carcinoma epithelial cells (ATCC, Manassas, VA) were grown in 6-well plates and transfected with 1 μg of pVD or pVAX1 using Fugene 6 under manufacture's instruction (Roche, NJ). To extract whole-cell protein, cells were harvested 48 hrs after transfection and resuspended in lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 0.5% NP-40, 0.1 mM phenylmethylsulfonyl fluoride, 2.5 μg/ml leupeptin, 0.5 mM NaF, and 0.1 mM sodium vanadate. Fifty μg of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10% polyacrylamide gel and then transferred onto nitrocellulose membranes. Western blotting was performed using primary antibodies against extracellular signal-regulated kinase (ERK)1/2 according to the manufacturer's instructions (Cell Signaling Technology, Beverly, MA). For analysis of the effect of VD on NPRA expression in vitro, HEK-GCA (human embryonic kidney cells stably transfected with the natriuretic peptide receptor, GC-A, which is the same as NPRA) cells grown in 6-well plates were transfected with 1 μg of pVAX1, pVD or pMut. Differential expression of NPRA was detected by western blot using primary antibody against NPRA (Santa Cruz Biotechnology, CA).
Luciferase assay to analyze the VD effect on NPRA promoter activity
Human embryonic kidney cells (HEK293; ATCC, Manassas, VA) were grown on 6-well plates and co-transfected with 1 μg of pVD plus 0.5 μg of pNPRA-Luc(-1575) which contains the 1575-bp fragment of NPRA promoter inserted upstream of the luciferase gene. In the control wells, cells were co-transfected with 1 μg of empty vector, pVAX1, and 0.5 μg pNPRA-Luc (-1575). Forty-eight hrs later, cells were washed with PBS, scraped off and suspended in 200 μl of luciferase assay lysis buffer (Promega, Madison, WI). Cell suspensions were kept on ice for 15 min and then vortexed for 15 sec before centrifugation for 1 min at 13,200 rpm. The supernatant was removed and aliquots were stored at -80°C. After protein concentration measurement, equal amounts of total protein from each transfection assayed for luciferase according to manufacturer's instructions (Pierce Biotechnology Inc., Rockford, IL).
RT-PCR detection of NPRA expression in the lung
For analysis of the effect of VD on NPRA expression in vivo, three groups of mice (n = 4) were intranasally treated with 50 μl of chitosan nanoparticles containing 20 μg of pVAX1, pVD or pMut. Mice were sacrificed 48 hrs after nanoparticle treatment and lungs were collected. Approximately 100 mg of lung tissue from each mouse was treated with RNAlater (Invitrogen, CA), and total cellular RNA was extracted using Trizol reagent (Invitrogen, CA). RNA from each mouse was reverse transcribed and analyzed for NPRA by RT-PCR by using the following primers: NPRA-forward: 5'-cctgagtacttggaattcctgaagc-3'; NPRA-reverse, 5'-gttccacatccgctgagtgatgtt-3'. Mouse β-actin was used as housekeeping gene.
Sensitization and induction of allergic airway response with OVA
Measurement of airway responsiveness to methacholine
Pulmonary resistance was measured using the forced oscillation technique as previously described . Two sets of experiments that each included mice from all four experimental groups were performed on different days. Anesthetized animals were mechanically ventilated with a tidal volume of 10 ml/kg and a frequency of 2.5 Hz using a computer-controlled piston ventilator (Flexivent, SCIREQ; Montreal, Canada). Responses were determined in response to increasing concentrations of aerosolized methacholine (MeCh, at 0, 6.25, 12.5, and 25 mg/ml in isotonic saline). The single compartment model was used to determine airway resistance values and peak values obtained after each MeCh challenge were plotted .
Modulation of lung inflammation by VD
To test the effects of VD on airway inflammation, a separate experiment was performed. BALB/c mice were divided into four groups (n = 8 per group). One group served as naïve control with no OVA sensitization and challenge while the second group received OVA sensitization and OVA challenge on days 18, 19, 20 and 21. Animals in the third group got OVA sensitization, OVA challenge and i.n. treatment with VD NPs on day 18, 19, 20 and 21. The last group was OVA sensitized and challenged, but treated with control NPs containing pVAX1. All mice were sacrificed on day 23 to collect bronchoalveolar lavage (BAL) fluid. Lungs were rinsed with intratracheal injections of PBS, perfused with 10% neutral buffered formalin, then removed, paraffin-embedded, sectioned at 20 μm and stained with hematoxylin and eosin (H & E). Lung homogenates for cytokine measurement were also prepared.
For differential cell enumeration, BAL fluid was centrifuged at 1,200 rpm for 5 min and the cell pellet was suspended in 200 μl of PBS and counted using a hemocytometer. The cell suspensions were centrifuged onto glass slides using a cytospin centrifuge at 1,000 rpm for 5 min at room temperature. Cytocentrifuged cells were air dried and stained with a modified Wright's stain (Leukostat, Fisher Scientific, Atlanta, GA) which allows differential counting of monocytes and lymphocytes. At least 300 cells per sample were counted by direct microscopic observation. For evaluation of proinflammatory cytokines, the levels of IL-2, IL-4, IL-5, IL-13, IFN-γ and TNFα in lung homogenates were measured using a mouse Th1/Th2 Cytokine CBA kit following the manufacturer's instruction (BD Bioscience, CA).
All experiments were repeated at least once. The data are expressed as means ± SEM (standard error of the mean) and differences are considered significant at p < 0.05. Comparisons were done using the 2-tailed Student's t test or 2-way ANOVA with Bonferroni post-test.
VD prevented ERK1/2 activation in A549 cells
VD down-regulated NPRA expression
We have reported that NPRA plays a role in airway inflammation. Knockout of NPRA in mice resulted in less lung inflammation . Inhibition of NPRA by small inferfering RNA against NPRA attenuated lung inflammation in a mouse model of asthma . There is a feedback regulation of the circulating concentration of natriuretic peptides such that ANP decreases kaliuretic peptide and vice versa . Although there is no direct evidence that VD interacts with NPRA, we investigated the effect of VD on NPRA expression. By luciferase assay, it was found that VD significantly decreased NPRA promoter activity up to 99% (p < 0.01, Fig. 1B). Downregulation of NPRA expression was also observed in HEK-GCA cells transfected with pVD (Fig. 2C) and by RT-PCR in the lungs of mice treated i.n. with pVD NPs (Fig. 2D) compared to pVXA1 and pMut controls. Further molecular mechanism studies are needed to demonstrate whether VD directly binds to the NPRA promoter or affects NPRA transcription though additional cellular factors.
VD reduced airway hyperresponsiveness
VD treatment attenuated eosinophilia and lung pathology
VD treatment reduced TH2 inflammatory cytokines IL-4, IL-5 and IL-13
Here we demonstrate that intranasal treatment with pVD NPs decreases lung inflammation and protects against allergen-induced airway hyperresponsiveness. No VD-specific receptor has been identified, and the mechanism of how VD reduces airway inflammation is unknown. Here we show that VD inactivates ERK1/2 in A549 lung epithelial carcinoma cells, suggesting that VD may achieve its effect by interfering with the ERK1/2 signaling pathway [6, 22, 19–23].
We also tested whether VD attenuates lung inflammation through its interference with the ANP-NPRA signaling pathway. It has been reported that there is a feedback regulation of the circulating concentration of the N-terminal natriuretic peptide and C-terminal natriuretic peptide such that ANP decreases KP and vice versa . We hypothesized that VD may behave like KP and that overexpression of VD may decrease the level of ANP and its receptor NPRA. We demonstrated that VD reduced NPRA promoter activity in a luciferase assay. Downregulation of NPRA expression was also confirmed both in vitro and in vivo by western blot and RT-PCR. Therefore, the observed attenuation of airway inflammation by VD is consistent with our previous report that NPRA-deficient mice or mice treated with siRNA for NPRA have less eosinophilia and lower levels of Th2-like cytokines compared to wild type mice [10, 11].
Since no signal peptide sequence was placed in front of the VD ORF when pVD was constructed in our investigation, expressed VD remains inside the transfected cells (primarily lung epithelial cells). This differs from the normal biology of VD in which cleavage of the prohormone into N-terminal and C-terminal fragments occurs outside the cell. However, the intracellular expression of VD in lung cells may help us to meet our goal of developing a safe anti-inflammatory drug targeting the respiratory system. Because VD is a cardiovascular hormone, overexpression and circulation of VD may cause side effects. We will test the expression of a secreted form of VD in the future and it will be interesting to compare those results to the current data. Irrespective of the mechanism, the finding that ANP-NPRA is involved in the inflammatory immune response to allergens opens new avenues of research into the pathogenesis of allergic disease and asthma.
The current study demonstrates that vessel dilator, VD, inactivates ERK1/2 and down-regulates NPRA expression. Inhibition of ANP-NPRA and ERK1/2 signaling pathways by VD affords bronchoprotection and anti-inflammatory activity; therefore, chitosan nanoparticles containing VD may be therapeutically effective in preventing allergic airway inflammation.
This study is supported by NIH grant RO1 (5HL71101A2), VA Merit Review and Career Scientist Awards and Mabel and Ellsworth Simmons Professorship to SSM and the Joy McCann Culverhouse Endowment to the University of South Florida Division of Allergy and Immunology and by R01 (ES015050) to SAC.
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