Positive correlation of airway resistance and serum asymmetric dimethylarginine (ADMA) in bronchial asthma patients lacking evidence for systemic inflammation

Bronchial asthma (hereinafter referred to as asthma) has high socioeconomic impact stemming from premature morbidity, poor quality of life, significant healthcare utilization and loss of work productivity [1, 2]. The key pathomechanistic properties of asthma are the presence of chronic inflammation in the lower respiratory tract and consequent airflow limitation manifested as dyspnea (predominantly in the form of recurrent bouts) [3, 4]. Contribution of the nitric oxide (NO) pathway to the evolution of inflammation in asthma has long been proposed, nonetheless the net effect of altered NO homeostasis in the inflammatory state characteristic of asthma is yet to be elucidated.

The source of controversy is that, similar to other tissues like vasculature [5, 6], NO may confer both protective and detrimental effects that depends on the activity of different NO synthase (NOS) isoforms, on the affected tissue compartment and on some underlying conditions [7]. Of the three NOS isoforms (each expressed in the lung), endothelial NOS (eNOS) and neuronal NOS (nNOS) are Ca-calmodulin dependent constitutive enzymes that liberate low (ranging from femto- to picomolar) concentrations of NO within seconds of receptor activation. The eNOS, apart from vasculature, is chiefly localized in the bronchial epithelium and type II alveolar cells [8], and NO released by this isoform leads to broncho- and vasodilation. The nNOS is chiefly located in peripheral nerves innervating bronchial smooth muscle and submucosal secretory glands. Density of innervation decreases from trachea to smaller bronchi conferring reduced NO-mediated neural bronchodilation in smaller airways [9]. The third isoform is the inducible NOS (iNOS) that, albeit continuously expressed in lung epithelial cells [10], is chiefly present upon its induction by pro-inflammatory cytokines. These latter molecules activate the nuclear transcription factor NF-κB that leads to iNOS expression and thereby sustained high release of NO (in nanomolar concentration) over the course of hours to days [8]. Based on the results of preclinical and clinical studies it seems that iNOS may be produced by alveolar type II epithelial cells, lung fibroblasts, airway and vascular smooth muscle cells, endothelial cells, mast cells and neutrophils [8, 11], and its expression is inhibited by glucocorticoids [12]. Under conditions of inflammation, NO, synthetized by iNOS, and superoxide anion, formed by activated macrophages and neutrophils, react to form peroxynitrite and subsequent inflammatory cell recruitment, airway constriction and remodeling [13]. Change in the level of all three isoforms in distinct compartments has been described in asthma [7]. In asthma patients, increased expression of iNOS was described in airway epithelium [7, 14, 15], while decline in activity of constitutive isoforms was also observed [16]. Furthermore, l-arginine or tetrahydrobiopterin depletion may cause uncoupling of all three NOS isoforms, switching the enzyme’s function to produce superoxide instead of NO [11, 17, 18].

Asymmetric dimethylarginine (ADMA), a metabolite of protein turnover throughout the body, is considered as a significant factor in NO homeostasis that may interfere with several processes related to the evolution of inflammatory airway diseases. ADMA is viewed as an endogenous competitive inhibitor of NOS showing higher selectivity for the constitutive isoforms [11]. In addition, animal studies showed that ADMA is a natural uncoupler of all the three NOS isoforms leading to increased superoxide formation and oxidative as well as nitrosative stress [19]. ADMA may also compete with l-arginine for intracellular transport, thus it may limit l-arginine’s availability as a substrate for NOS [20] and thereby contribute to intracellular l-arginine depletion. Furthermore, exogenous ADMA per se was shown to cause airway hyperresponsiveness, to increase collagen formation and to induce reversible fibrosis in mice [19]. This latter effect may be due to ADMA’s ability to enhance the activity of arginase [20]. While lung (more specifically: airway epithelium) has been shown to be a major source of ADMA [11, 20], conflicting reports have emerged related to the systemic ADMA concentration in asthma [21] with reports describing both higher [22] and normal/low-normal [23] serum levels (Fig. 1).

Starting from the above-mentioned findings, we set out to elucidate whether ADMA is a risk or protective factor in the inflammatory state characteristic of asthma, by assessing the relationship of ADMA with lung function parameters descriptive of airflow limitation.

The main finding of the current study is that serum ADMA shows significant positive correlation with airway limitation characterized by R_aw in adult asthma patients receiving asthma controller therapy (including, for most patients, an inhaled corticosteroid). This relationship remained significant even after adjusting for potential confounders. Furthermore, this positive association was more pronounced when the analysis was restricted to patients attaining a high level of asthma control. Upon evaluating the inflammatory status of our patient cohort, we found that general markers of inflammation (CRP, procalcitonin and fibrinogen) are in the normal range and show no significant difference with respect to airflow limitation (R_aw). This lack of evidence for systemic inflammation could be due to the fact that these patients all received control-based asthma therapy at the time of our investigations (94% of them (n = 146) were on inhaled corticosteroid therapy).

These findings have several implications regarding l-arginine-NO homeostasis including the interaction between ADMA and distinct NOS isoforms. Prior reports demonstrated that use of even low-dose corticosteroids is able to inhibit the activation of NF-κB and the subsequent iNOS synthesis [12]. Moreover, glucocorticoids can decrease systemic inflammatory signals needed for inducing iNOS transcription [8]. Considering these effects together with the observation that ADMA seems to preferentially inhibit constitutive NO synthases [38], we postulate that, in our cohort (mainly in the well-controlled stratum), iNOS is not or minimally induced and thus its inhibition by ADMA is marginal. This means that the potential beneficial effect of ADMA conferred by inhibition of iNOS may be insignificant. Therefore, we suggest that, in our sample, inhibition of NOS is only evident with respect to the constitutive isoforms. As such, elevated levels of ADMA may elicit deleterious effects by decreasing NO produced by the constitutive NOS isoforms. Furthermore, ADMA, by uncoupling NOS, could lead to increased reactive oxygen and nitrogen species formation in airway epithelial cells [38]. Starting from the observation that ADMA enhanced arginase activity leading to collagen production [20], a reversible fibrosis provoked by ADMA (Fig. 1) may also contribute to the increase of R_aw. This concept is further supported by the weaker association seen between R_aw and serum ADMA levels (indicated by the lower value of the regression coefficient and lack of statistical significance). Given the fact that presence of wheezing is possibly indicative of airway inflammation (being present regardless of the medication used), it may be presumed that these inflammatory processes induce iNOS. It would follow that inhibition of iNOS by ADMA in these patients (e.g. in the not well-controlled stratum) hence would counteract the deleterious effects exerted by iNOS and would manifest in a lower increase of R_aw.

The fact that significant difference was only evidenced for dynamic lung function parameters when patients were compared with respect to their level of symptom control (Table 2) may be due to fact that flow parameters (as well as the Tiffeneau index) compile information regarding airway resistance and respiratory effort. The latter is influenced by elastic recoil of the lung, respiratory muscle strength and stiffness of the chest. It is highly probable that those patients who experienced wheezing over the past 4 weeks are in a poorer general physical condition, further reflected by the significantly and clinically meaningful differences in the Symptoms, Activity and Impacts component scores as well as the totals score for SGRQ. So, it seems probable that the significant difference seen with respect to flow parameters is due to the difference in general physical state and consequent muscle strength of these two groups. This difference is absent in objective measures of airflow limitation, e.g. R_aw and G_aw as these measures are not influenced by respiratory effort.

Previously we have proposed that, in the range of normal concentrations (0.35–1.0 μmol/L), ADMA confers protection against atherosclerosis [6], a lesion where iNOS is induced, by causing a more pronounced inhibition on iNOS due to the lower EC₅₀ value of ADMA for iNOS than that for eNOS [39, 40]. Although, in the present study, serum ADMA levels were also in the normal range, this beneficial effect of ADMA was not seen possibly due to the inhibitory effect inhaled corticosteroids have on airway iNOS expression (which makes inhibition of iNOS by ADMA insignificant in asthma patients receiving corticosteroid therapy).

In general, few reports are available regarding the systemic level of ADMA in pediatric and adult asthma patients. Our ADMA values are comparable with results of others. In a recent study, systemic ADMA levels of 0.37 μmol/L (IQR: 0.29, 0.59) and 0.48 μmol/L (IQR: 0.35, 0.7) were reported for adult patients suffering from early- and late-onset asthma, respectively [22]. Upon assessment of children with asthma, a group found serum ADMA levels corresponding to 0.53 μmol/L (CI: 0.47, 0.6) [41], while yet another group reported mean plasma ADMA concentration of 0.58 ± 0.05 μmol/L [23]. Others described considerably higher circulating levels of ADMA in children suffering from asthma (0.92 ± 0.20 μmol/L), however this showed no significant difference when compared to healthy controls also included in that study (0.91 ± 0.23 μmol/L, p = 0.88) [42].

Our finding that the final model describing the relationship between R_aw and ADMA includes FEF25–75% % pred (Table 3) could be interpreted in view of the well-established notion that asthma compromises the function of the small airways [43]. This is especially true for airways with internal diameter lower than 2 mm [44]. In fact, small airways were implicated to be one of the chief sites of airflow limitation [45]. In line with these observations, we have found that FEF25–75% % pred shows a strong negative correlation with R_aw implying the contribution of small airways to airflow limitation (accordingly, G_aw, the reciprocal of R_aw, exhibits a significant positive correlation with FEF25–75% % pred). The fact that this strong correlation proved to be significant irrespective of the level of asthma control achieved is interesting if one considers that targeting distal airways by means of an inhalational therapy remains a challenge [43]. Our finding that inflammation of small airways (reflected by decreased FEF25–75% % pred) parallels that of larger airways (indicated by increased R_aw) emphasizes that special attention must be paid to optimal delivery of inhalational medication in daily clinical practice to allow for sufficient relief of airflow limitation stemming from the distal airways.

The third set of parameters that contribute significantly to the explanation of the relationship between R_aw and ADMA is related to the health impairment reported by asthma patients of our cohort. In agreement with prior studies [46], we have found a significant negative correlation between the Total score of SGRQ and FEV1% pred. Furthermore, we found that Activity score shows significant positive correlation with R_aw, indicating that airway limitation is associated with loss of quality of life conferred by disturbance of physical activity. The negative association seen between the Impacts score and R_aw in our cohort may be due to the correction of overrepresentation of the influence of Activity score due to the fact that it is included in the Total score as well (so, it might be an inherent feature of SGRQ). The clinically meaningful difference of the three components as well as the Total score compared between groups of lower and higher R_aw values (Table 1) further emphasizes the deleterious effect of airway limitation on the quality of life of patients.

A limitation of the present study is the lack of direct evidence for the activity of distinct NOS isoforms (as specimens related to the different compartments). Thus, we only presume a suppressed iNOS activity starting from the absence of elevated inflammatory markers in the serum that makes the explanation of our findings somewhat speculative. Further limitation of the study relates to the lack of characterization of oxidative and nitrosative stress by means of determining stable end products (nitrite and nitrate). In addition, it must be acknowledged that Raw was not expressed as percent predicted (described by [47]) due to technical limitations, hence this measure was not adjusted for weight and height. Nevertheless, height and BMI identified as significant regressors for Raw and/or (log)ADMA (by the simple linear regression (Table 3)) were included in the initial multiple model. However, both of these parameters were omitted due to their lack of significant contribution to the final model. FEF25–75% % pred should be interpreted with caution because of difficulties with reproducibility previously reported by others [36]. Finally, it should be mentioned that our analysis does not address the timing of the measurements in relationship to the administration of bronchodilators or other medications, thus although patients were examined while being on maintenance therapy, timing of the medication consumption may induce a variability.

To the best of our knowledge, this is the first study in which the relationship was investigated between serum ADMA level and R_aw, an objective functional parameter characteristic of airflow limitation in asthma. The strong positive correlation between ADMA and R_aw in a multiple linear regression model may indicate that ADMA contributes to the development of bronchoconstriction in asthma patients receiving control-based asthma therapy. This is further emphasized by the fact that this correlation became even stronger when the analysis was limited to the stratum of well-controlled patients. Based on the correlation between ADMA and R_aw, we shed light on the potential importance of ADMA in the pathomechanism of asthma. In addition, our results further demonstrate that R_aw is a valuable parameter in the assessment of airflow limitation in asthma patients.