RETRACTED ARTICLE: The anti-proliferative and anti-inflammatory response of COPD airway smooth muscle cells to hydrogen sulfide

Hydrogen sulfide (H₂S) which was discovered in human tissues over 15 years ago, has emerged as an important gaseous mediator in several biological processes [1]. H₂S is now considered the third member of a family of gasotransmitters, together with nitric oxide (NO) and carbon monoxide [2]. The bulk of endogenous H₂S synthesis in mammalian tissues appears to be from the pyridoxal-5`-phosphate–dependent enzymes, cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS), and also by 3-mercaptopyruvate sulphur transferase (MPST) [3].

Chronic obstructive pulmonary disease (COPD) is a common, highly debilitating disease of the airways, primarily caused by smoking [4]. Serum H₂S levels are significantly increased in patients with stable COPD as compared to age matched control subjects or those with acute exacerbation of COPD [5]. Serum H₂S levels were negatively correlated with the severity of airway obstruction in patients with stable COPD whereas they were positively correlated with the lung function in all patients with COPD and healthy controls. Patients with acute exacerbations and increased pulmonary artery pressure (PASP) had lower levels of H₂S than those with normal PASP, suggesting a negative relation between H₂S and PASP in COPD exacerbations. Serum H₂S levels are also lower in smokers than non-smokers regardless of their health status (COPD or healthy controls). Furthermore, patients with acute exacerbations, whose serum H₂S levels were decreased, demonstrated greater neutrophil numbers but lower lymphocyte numbers in sputum than patients with stable COPD, suggesting a potential role of H₂S in regulating inflammatory response at different types or stages of COPD.

We have previously demonstrated that mitogen stimulation increases inflammatory mediator release from both ASM IL-6 and CXCL8 release in COPD patients to a greater degree than those from non-smoker subjects [4]. Furthermore, we have shown that H₂S donors inhibit mitogen-induced inflammatory mediator release and proliferation of cells from healthy non-smoking subjects [6]. We therefore set out to determine the effect of H₂S in ASM cells isolated from healthy smokers and patients with COPD. We hypothesized that H₂S may also mediate ASM proliferation, and cytokine release to varying degrees in these diseased cells. We examined the effect of both exogenous and intracellular sources of H₂S in human ASM from 9 donors in each group upon proliferation induced by fetal calf serum (FCS). We used two extracellular H₂S donors; the rapidly releasing H₂S donor, sodium hydrogen sulfide (NaSH), and modelled endogenous H₂S synthesis with the slow H₂S-releasing molecule, GYY4137 [6, 7]. To examine the role of endogenously synthesized H2S, we used an inhibitor of H₂S synthesis (O-(carboxymethyl)-hydroxylamine hemihydrochloride (CHH)) to inhibit CBS [6]. Finally, we also investigated the role of mitogen-activated protein kinase (MAPK) activation in this process.

The presence of H₂S producing enzymes in ASM cells in-situ was determined by immunohistochemical staining of bronchial biopsies from healthy non-smokers, healthy smokers and subjects with COPD. The most intense staining was seen for CSE but no difference in the expression of CSE, CBS or MPST was observed between patient groups (Additional file 1: Figure S1).

For the first time, we demonstrate that both endogenous and exogenous H₂S inhibits human ASM cell proliferation and cytokine release induced by FCS, and that this effect was dependent on the patient. Specifically; proliferation and cytokine release from non-smoker ASM cells returned to basal levels (as previously reported [6]) whereas in smokers, both IL-6 and CXCL8 release were reduced to baseline but proliferation although being significantly reduced did not return to basal levels. In contrast, the effect of H₂S on proliferation and cytokine release from ASM cells isolated from COPD patients was impaired compared to smokers and non-smoker cells. Furthermore, we have shown that endogenous H₂S is produced by the enzymes CBS and MPST, and not by CSE. We found that H₂S differentially inhibited phosphorylation of the MAPKs, ERK-1/2 and p38, according to the patient group and propose that this could be a mechanism by which H₂S inhibits cellular proliferation and cytokine release [4, 16‐19].

ASM proliferation is increased in response to FCS [9, 10, 20] and studies have examined the role of H₂S upon cell proliferation. These have concluded that this gas can induce proliferation [21] or, conversely, inhibit it [6, 22, 23] depending upon the cell type examined. Both the fast-release H₂S donor, NaSH, and the slow-release donor, GYY4137, have been used previously to affect inflammation in both in-vivo and in-vitro models of inflammation, including a mouse models of vascular inflammation and oxidative stress [24], asthma [25], COPD [26], and a rat model of colitis [27]. Our data extends our previous report demonstrating the inhibitory action of H₂S in non-smoker ASM cells [6] and examined its role in smoker and COPD ASM cells. Both NaSH and GYY4137 caused similar inhibitory effects on FCS-induced ASM cell proliferation, IL-6 and CXCL8 release from smokers as well as non-smokers indicating that the rate of release does not modulate the inhibitory effect of H₂S in ASM. We also show for the first time, an effect upon primary ASM cells isolated from patients with COPD. However, the effect of H₂S donors is reduced compared to that seen in cells from smokers and non-smokers which may explain, in part, the increased inflammatory and proliferative status of COPD cells. Indeed, the H₂S enzyme inhibitor CHH had no significant effect on FCS-induced inflammatory protein release from COPD cells in contrast to the effect seen in cells from other subject groups.

We found that, all three H₂S producing enzymes are expressed in ASM cells to a similar extent across the subject groups studied. However, our pharmacological studies suggest that endogenous H₂S production is these cells is most likely to be through the enzymes, CBS, and MPST. In cultured ASM cells, FCS was able to induce CBS and MPST mRNA and protein in cells from non-smokers and smokers but not in COPD cells suggesting that mitogens may induce cells to produce more H₂S. NaSH inhibited both CBS and MPST, likely as a negative-feedback inhibitory mechanism. Currently, CBS appears to be involved in the generation of endogenous H₂S in neural pathways, the brain, vascular tissue, and non-smoker ASM cells [6, 28‐32]. In contrast, CSE is predominantly involved in endogenous H₂S production in rodent smooth muscle and the lung [33‐37], and MPST maintains mitochondrial function [extensively reviewed in [38, 39]]. Clearly cell, species and pathology differences should be taken into consideration when investigating the production of endogenous H₂S.

A role for the ERK-1/2 and p38 MAPKs in regulating ASM cell proliferation and cytokine release is well documented [4, 16‐19] and H₂S has been shown to affect the phosphorylation of these kinases [6, 26, 40‐43]. Hence, we examined the degree of phosphorylation of these kinases in our COPD ASM cells. We noted that FCS induced both ERK-1/2 and p38 MAPK phosphorylation, which was reduced by NaSH in both the non-smoker and smoker ASM cells, but no effect was seen in the COPD cells. Inhibiting these kinases significantly reduced the ASM proliferation and cytokine release and, when they were used before treatment with NaSH, a further decrease in aberrant phenotype was observed, further supporting the possibility that the mechanism of H₂S, at least in part, is via the inhibition of these kinases.

Finally, our data shows that ASM cells of COPD patients indicate an attenuated response to H₂S, as compared to the non-smoker and smoker-groups. But the question remains, why? There are numerous reviews discussing both the importance of H₂S in chronic respiratory diseases [3, 44] and smooth muscle itself [45], however recent studies demonstrate further actions of this gasotransmitter. For example, Fitzgerald et al. demonstrate that H₂S causes the relaxation of human ASM and implicate the role for sarcolemmal KATP channels [46]. In mouse models, Huand et al. indicate that H₂S can induce mouse ASM relaxation by activating BKCa [47], and Castro-Piedras et al. indicate that H₂S causes ASM relaxation by inhibiting Ca⁽²⁺⁾ release through InsP3Rs and consequent reduction of agonist-induced Ca⁽²⁺⁾ oscillations [48]. In other rodent models of lung pathology, endogenous H₂S has been suggested to have a protective role of anti-inflammation and bronchodilation in chronic cigarette smoke-induced pulmonary injury in rats [49], and H₂S provokes tachykinin-mediated neurogenic inflammation that is mediated by stimulation of TRPV1 receptors on the sensory nerve endings in Guinea Pigs [50]. Furthermore, considering the emergence of data suggesting a degree of cross-talk between H₂S and epigenetic modifiers such as miRNAs [51, 52], and our own data suggesting broadly different epigenetic profiles between lung pathologies (including COPD) in ASM [4, 9, 10, 12, 13]. Hence, the difference between a COPD ASM cell and a ‘healthy’ ASM cell may incorporate one, or more likely, more of these H2S targets/activators. To address this further we intend to further these and other findings in our murine model of COPD [26].

In conclusion, we have shown for the first time that H₂S inhibits both human ASM proliferation and cytokine release induced by FCS, differentially between ASM cells isolated from non-smokers, smokers and patients with COPD. It is likely that exogenous H₂S targets the production of endogenous H₂S by inhibiting the transcription and subsequent translation of the CBS and MPST enzymes, and proliferation is controlled by H₂S through a negative-feedback pathway. H₂S inhibits the activity of the ERK-1/2 and p38 MAPKs, in the non-smokers and smokers, but with little effect in the COPD ASM cells. We propose that H₂S may provide a novel therapeutic avenue in the stabilization of ASM proliferation but that its effectiveness in COPD may be more limited.