Pharmacologic inhibition of S-nitrosoglutathione reductase protects against experimental asthma in BALB/c mice through attenuation of both bronchoconstriction and inflammation

BACKGROUND: S-nitrosoglutathione (GSNO) serves as a reservoir for nitric oxide (NO) and thus is a key homeostatic regulator of airway smooth muscle tone and inflammation. Decreased levels of GSNO in the lungs of asthmatics have been attributed to increased GSNO catabolism via GSNO reductase (GSNOR) leading to loss of GSNO- and NO- mediated bronchodilatory and anti-inflammatory actions. GSNOR inhibition with the novel small molecule, N6022, was explored as a therapeutic approach in an experimental model of asthma. METHODS: Female BALB/c mice were sensitized and subsequently challenged with ovalbumin (OVA). Efficacy was determined by measuring both airway hyper-responsiveness (AHR) upon methacholine (MCh) challenge using whole body plethysmography and pulmonary eosinophilia by quantifying the numbers of these cells in the bronchoalveolar lavage fluid (BALF). Several other potential biomarkers of GSNOR inhibition were measured including levels of nitrite, cyclic guanosine monophosphate (cGMP), and inflammatory cytokines, as well as DNA binding activity of nuclear factor kappa B (NFκB). The dose response, onset of action, and duration of action of a single intravenous dose of N6022 given from 30 min to 48 h prior to MCh challenge were determined and compared to effects in mice not sensitized to OVA. The direct effect of N6022 on airway smooth muscle tone also was assessed in isolated rat tracheal rings. RESULTS: N6022 attenuated AHR (ED(50) of 0.015 ± 0.002 mg/kg; Mean ± SEM) and eosinophilia. Effects were observed from 30 min to 48 h after treatment and were comparable to those achieved with three inhaled doses of ipratropium plus albuterol used as the positive control. N6022 increased BALF nitrite and plasma cGMP, while restoring BALF and plasma inflammatory markers toward baseline values. N6022 treatment also attenuated the OVA-induced increase in NFκB activation. In rat tracheal rings, N6022 decreased contractile responses to MCh. CONCLUSIONS: The significant bronchodilatory and anti-inflammatory actions of N6022 in the airways are consistent with restoration of GSNO levels through GSNOR inhibition. GSNOR inhibition may offer a therapeutic approach for the treatment of asthma and other inflammatory lung diseases. N6022 is currently being evaluated in clinical trials for the treatment of inflammatory lung disease

ADH IB expression, but not ADH III, is decreased in human lung cancer.

Endogenous S-nitrosothiols, including S-nitrosoglutathione (GSNO), mediate nitric oxide (NO)-based signaling, inflammatory responses, and smooth muscle function. Reduced GSNO levels have been implicated in several respiratory diseases, and inhibition of GSNO reductase, (GSNOR) the primary enzyme that metabolizes GSNO, represents a novel approach to treating inflammatory lung diseases. Recently, an association between decreased GSNOR expression and human lung cancer risk was proposed in part based on immunohistochemical staining using a polyclonal GSNOR antibody. GSNOR is an isozyme of the alcohol dehydrogenase (ADH) family, and we demonstrate that the antibody used in those studies cross reacts substantially with other ADH proteins and may not be an appropriate reagent. We evaluated human lung cancer tissue arrays using monoclonal antibodies highly specific for human GSNOR with minimal cross reactivity to other ADH proteins. We verified the presence of GSNOR in ≥85% of specimens examined, and extensive analysis of these samples demonstrated no difference in GSNOR protein expression between cancerous and normal lung tissues. Additionally, GSNOR and other ADH mRNA levels were evaluated quantitatively in lung cancer cDNA arrays by qPCR. Consistent with our immunohistochemical findings, GSNOR mRNA levels were not changed in lung cancer tissues, however the expression levels of other ADH genes were decreased. ADH IB mRNA levels were reduced (>10-fold) in 65% of the lung cancer cDNA specimens. We conclude that the previously reported results showed an incorrect association of GSNOR and human lung cancer risk, and a decrease in ADH IB, rather than GSNOR, correlates with human lung cancer

Quantitation of GSNOR positive lung cancer tissue sections.

<p>Four identical sets of 120 lung tissue cores (12 normal, 108 cancer) were stained with the GSNOR antibodies indicated below. The stained tissue cores were graded as positive or negative for GSNOR expression by three reviewers blinded to antibody used (SCM, GJR, JPR). The percent of tumor and normal lung cores graded as positive for GSNOR expression are shown below.</p

GSNOR and ADH gene expression in lung cancer cDNA arrays.

<p>GSNOR (gene name <i>ADH5</i>), ADH IB (<i>ADHIB</i>), ADH II (<i>ADH4</i>), and ADH IV (<i>ADH7</i>) mRNA levels were evaluated by qPCR in human lung cancer cDNA arrays (Origene, #HLRT504, lot #0411, Rockville, MD) and normalized to β-actin levels. Arrays contained 23 matched pairs of normal and cancerous lung tissue. Tumor expression relative to the matched normal sample was calculated using the ΔΔCt method. Relative quantities <1 represent decreased expression in the tumor sample. Lung cancer specimens included Stage IA (n = 4), IB (n = 4), IIA (n = 2), IIB (n = 8), IIIA (n = 3), and IIIB (n = 2) with each sample paired with adjacent normal tissue. No correlation between GSNOR mRNA levels and lung cancer was observed, while expression of ADH IB was strongly reduced in lung cancer samples.</p

GSNOR is present in normal lung tissue.

<p>Normal human lung tissue microarrays were stained with N30-C3 monoclonal GSNOR antibody (1 µg/mL) followed by DAB detection. Bronchial epithelial cells, alveolar macrophages, and type 2 pneumocytes in alveoli are strongly stained.</p

Polyclonal GSNOR antibodies react with other ADHs.

<p>22.5 ng purified recombinant proteins were separated by SDS-PAGE, and immunoblots were performed to determine antibody reactivity to GSNOR, ADH IA, ADH IB, ADH II, and ADH IV. With the exception of ADH IA, the purified recombinant proteins were generated with a fusion protein tag during cloning, and the tag was cleaved off during purification as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052995#s2" target="_blank">Materials and Methods</a>. The molecular weights of the fusion proteins are approximately 50–51 kDa, and the final, purified proteins are 39–41 kDa. Human GSNOR consistently migrates faster than the calculated molecular weight. As seen by for the presence of both 50 and 40 kDa bands for ADH II, the majority of the protein in this preparation still contains the fusion protein tag. However, the purified GSNOR protein used for immunization was confirmed to be full length, and free of additional tag sequence as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052995#s2" target="_blank">Materials and Methods</a>. A) Commercially available rabbit polyclonal GSNOR antibody (Proteintech #11051-1-AP). B) In-house polyclonal antibody generated by immunization of rats with purified, recombinant, full length human GSNOR protein at Biomodels (Watertown, MA) for N30 Pharmaceuticals.</p

GSNOR is found in human lung cancer tissues stained with monoclonal GSNOR antibodies.

<p>Normal human lung cancer tissue microarrays were stained with monoclonal GSNOR antibodies (N30-C3, N30-F6, N30-G11) or a commercially available polyclonal GSNOR antibody (11051-1-AP) followed by DAB detection. Various human lung cancer tissues are strongly stained by all three GSNOR monoclonal antibodies, but staining is less prominent when the non-specific polyclonal GSNOR antibody is used.</p