Ceramides: a potential therapeutic target in pulmonary emphysema

Chronic Obstructive Pulmonary Disease (COPD) is a common and increasing source of morbidity and mortality in the developed world [1] and is associated with a large cost burden [2]. Pulmonary emphysema, an important component of COPD, is caused by permanent destruction of alveoli, airflow obstruction and lung hyperinflation, leading to a decreased lung function and breathlessness. The pathogenesis is related to smoke exposure, but why only a minority of all smokers develops emphysema remains unclear. Reports suggest that inflammation is partly responsible. The inflammatory infiltrate seen in emphysema is similar to that found in infection and correlates to the extent of emphysema [3], while the degree of cell death correlates to the amount of inflammation [4]. In humans, airway inflammation persists for many years after smoking cessation [5]. Sphingolipids are important structural components of biological membranes and have recently been shown to serve as messenger molecules in cell proliferation, apoptosis, cell contact and adhesion, endothelial barrier function and during inflammation [6‐14]. Ceramide is the central molecule in the sphingolipid pathway [15] and is formed either de novo from the condensation of palmitate with serine via the activity of a serine palmitoyltransferase (SPT), by the salvage pathway via sphingosine or by degradation of sphingomyelin by sphingomyelinase (SMase) [16]. Ceramide is degraded by ceramidase to sphingosine which can be phosphorylated to sphingosine-1-phosphate (S1P) [17]. Ceramide and S1P form a rheostat [17, 18] whereby ceramide stimulates apoptosis and cell cycle arrest while S1P stimulates cell survival and proliferation. Sphingolipid metabolism has been shown to be altered in a variety of diseases, including cystic fibrosis [19, 20] and asthma [21]. Ceramide has been shown to trigger apoptosis in an experimental mouse model of emphysema [22], and increased levels of apoptosis have been found in the lungs of patients with severe cigarette-induced emphysema [23]. Increased ceramide levels have also been shown to influence surfactant production [24] and activity [25]. Since ceramide levels are increased in the lungs of patients with smoke-induced emphysema [22] ceramide upregulation might be an important pathogenetic element in emphysema development. We investigated ceramide profiles in the lungs and examined the effect of pharmacological interventions targeting SMases and SPT in an animal model of elastase-induced emphysema.

In this study we showed that intratracheal elastase caused a transient inflammatory response with influx of erythrocytes and inflammatory cells in the alveolar spaces, combined with an increase in multiple ceramide and dihydroceramide species in BAL fluid which peaked at day 1 and 2 and returned to control levels 3 days after elastase instillation. Ceramide expression was primarily localized to the epithelium and to inflammatory cells. Sphingomyelinase inhibitors did not prevent or reduce the effects of intratracheal elastase. In contrast, SPT inhibition reduced sphingolipid and protein levels and ameliorated lung function changes after elastase instillation.

Apoptosis of alveolar cells has been shown to play a crucial role in the development of emphysema [30‐32]. Petrache et al. showed that direct intratracheal instillation of a synthetic short chain ceramide caused emphysema-like defects [33] and observed increased ceramide levels in the lungs of individuals with cigarette smoke-induced emphysema [33]. Our study shows for the first time that ceramide levels are increased in the first few days following the intratracheal instillation of elastase in mice. Earlier studies have described an increase in apoptosis in the elastase-induced emphysema model [34‐37]. Long chain ceramides (Cer16:0, Cer18:0) have been shown in cancer cells to exhibit anti-proliferative and pro-apoptotic effects [38]. Our study shows that long chain ceramides are increased a few days after elastase instillation and we speculate that they were responsible for triggering apoptosis. Long chain ceramides have also been shown to increase the permeability of the lung endothelium after cigarette-smoke [39] and thereby enhance endothelial leakage leading to edema formation [40]. In our model we found evidence of significant edema formation at day 2 and 3 after elastase, as measured by increased IgM and protein levels in the BAL fluid. Our observation of long chain ceramides being increased at those time-points is suggestive of a causal role of ceramides in our model. The elastase-induced emphysema model does not mimic the low-grade, long-term inflammatory pathophysiology of smoke-induced pulmonary emphysema in patients [41]. However, it is an excellent model to accurately reproduce the histological damage seen in emphysematous lungs and to evaluate the effect of interventions. The short developmental time of the model when compared to genetic or cigarette smoke models makes it easier to do perform multiple experiments in a relatively short time period.

Staining of lung sections from patients with cystic fibrosis or lung emphysema for ceramides showed that ceramide formation is increased in the airway epithelium [20]. By immunofluorescent staining we indeed found increased ceramides in the airway epithelium, in agreement with the increased ceramide levels in BAL which mainly contains products from epithelial cells. We also found positive immunofluorescence signals for ceramide in alveolar macrophages, which could be due to uptake of apoptotic alveolar epithelial cells, or internal ceramide production [42], triggered by the inflammatory response after elastase.

Sphingomyelinase inhibition has been examined in a variety of animal models. In a model of cystic fibrosis, acid sphingomyelinase inhibitors (desipramine and amitriptyline) and glycosphingolipid inhibitors (Miglustat) decreased pulmonary inflammation [14, 43]. In spinal cord injury an inhibitor of acid sphingomyelinase (NB6) and an inhibitor of de novo synthesis of ceramide (Fumonisin B1) reduced spinal cell apoptosis and inflammation after injury, and improved motor function [44]. Zoledronic acid has been shown to specifically inhibit acid sphingomyelinase [45]. Sphingolactone has previously shown to ameliorate LPS-induced acute lung injury in mice by inhibiting neutral sphingomyelinase, thereby decreasing ceramide levels In the present study, treatment with myriocin (SPT inhibitor) produced a marked reduction in overall sphingolipid levels, which suggests that de-novo ceramide synthesis was responsible for their increase. The de novo pathway was also implicated in VEGF-inhibition-induced emphysema, demonstrating the importance of de novo ceramide generation in the process of alveolar destruction [33]. This is further corroborated by the observation that neither acid- nor neutral sphingomyelinase inhibitors were able to block the increase in ceramides. Inadequate dosing of these inhibitors seems unlikely since similar doses have been used successfully in other animal models [46‐48]. Since inhibition of synthesis of very long acyl chain (C22-C24) ceramides in Cer synthase 2 knockout mice has been shown to elevate C16-ceramide and sphinganine levels [49], it is possible that inhibition of one specific step in the complex ceramide pathway leads to unintended alterations in individual ceramide metabolites. In the present study, desipramine exhibited an unexpected effect, namely upregulation of very long chain ceramides in both vehicle and elastase-treated mice. However, zoledronic acid, another inhibitor of acid sphingomyelinase, did not have this effect. Desipramine has previously been described to not only inhibit ASMase, but also acid ceramidase and other lysosomal enzymes [45, 50]. This non-specific inhibitory effect could account for the different effect on sphingolipid levels compared to zoledronic acid. Acid sphingomyelinase protein levels were transiently upregulated after elastase instillation, suggesting that it may have contributed to the transient increase in ceramides. However, the contribution must have been minimal as we found no effect of acid sphingomyelinase inhibitors. The increased ceramide levels after elastase instillation could also be due to reduced degradation by ceramidases. A major role in the transient rise in ceramides, however, is unlikely since ceramide returned to normal 5 days after elastase instillation while acid ceramidase levels were still decreasing. The initial decrease of neutral ceramidase after elastase instillation may have contributed to the early increase in ceramide levels but neutral ceramidase levels were already increased and normalized at the time (day 2) ceramide levels peaked. Thus, contribution of neutral ceramidase to the elevation in ceramides after elastase treatment is most likely minimal.

Blocking the de-novo pathway of ceramide synthesis by myriocin had a positive effect on lung function but no effect on histology. A possible explanation for this structure-function discrepancy is that ceramides reduce surfactant synthesis, due to downregulation of Thyroid Transcription Factor-1 (TTF-1), and this may affect lung function without a measurable morphological alteration [24, 51]. However, our observation of no changes in saturated phosphatidylcholine content in BAL of elastase-treated and control mice (data not shown) makes this explanation less likely. Clinical studies in humans also show that structural abnormalities of the lungs and airways are at best weakly correlated with changes in lung function, and this is especially true for localized or inhomogeneous structural abnormalities, as seen in our model [52, 53]. Alternatively, the morphometric methodology may be insufficiently sensitive to demonstrate relatively small changes that might underlie significant functional improvement, or more sensitive approaches to the analysis of lung structure might be needed. Such approaches may include, but are not limited to, micro CT and advanced functional MRI imaging techniques.

It could be argued that desipramine, being a tricyclic antidepressant, and zoledronic acid, a biphosphonate, have limited specificity to influence ceramide metabolism. Desipramine is known to inhibit the re-uptake of norepinephrine and to a lesser extent serotonin [54]; however, both catecholamines are unlikely to affect ceramide levels in BAL. Zoledronic acid binds and blocks farnesyl diphosphate synthase in the HMG-CoA reductase pathway, preventing the formation of metabolites essential for sub-cellular protein trafficking [55]. As a bisphosphonate it may bind to calcium in bone, thereby preventing the functioning of osteoclasts, the only bone-resorbing cells in the body [56]. Again, it is very unlikely that these additional actions of zoledronic acid interfered with our ceramide outcomes. Importantly, we showed that neither desipramine nor zoledronic acid influenced BAL ceramide levels, which argues against a significant contribution of SMases in the production of ceramides in our elastase model [57].

In conclusion, we demonstrated the involvement of ceramides and dihydroceramides in elastase-induced emphysema. Our findings suggest an important role for ceramides in the acute phase of elastase-induced emphysema. Further experiments should be undertaken to better evaluate the preventive and therapeutic potencies of ceramide inhibition in this emphysema model, and ultimately in humans with COPD and emphysema.

This work was supported by an operating grant (MOP-86472) from the Canadian Institute of Health Research and infrastructure grants (CCURE, CSCCD) from the Canadian Foundation for Innovation.