key: cord-0014423-0fg7270u
authors: Merz, Tamara; Denoix, Nicole; Wepler, Martin; Gäßler, Holger; Messerer, David A. C.; Hartmann, Clair; Datzmann, Thomas; Radermacher, Peter; McCook, Oscar
title: H(2)S in acute lung injury: a therapeutic dead end(?)
date: 2020-12-18
journal: Intensive Care Med Exp
DOI: 10.1186/s40635-020-00324-0
sha: c76c513cf87c6133ab105297c4c51a425af57e89
doc_id: 14423
cord_uid: 0fg7270u

This review addresses the plausibility of hydrogen sulfide (H(2)S) therapy for acute lung injury (ALI) and circulatory shock, by contrasting the promising preclinical results to the present clinical reality. The review discusses how the narrow therapeutic window and width, and potentially toxic effects, the route, dosing, and timing of administration all have to be balanced out very carefully. The development of standardized methods to determine in vitro and in vivo H(2)S concentrations, and the pharmacokinetics and pharmacodynamics of H(2)S-releasing compounds is a necessity to facilitate the safety of H(2)S-based therapies. We suggest the potential of exploiting already clinically approved compounds, which are known or unknown H(2)S donors, as a surrogate strategy.

This review explores the plausibility of hydrogen sulfide (H 2 S) therapy for acute lung injury (ALI) and circulatory shock. H 2 S is a toxic gas with a characteristic smell of rotten eggs, and is also produced endogenously by three different enzymes: cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvatesulfurtransferase (MST) [1] . In 1996 and 1997, physiological roles of H 2 S in the brain and vascular smooth muscle, respectively [2, 3] , were discovered, which led to its classification as the third "endogenous gaso-transmitter" [4] , besides nitric oxide and carbon monoxide.

In 2005, in a hallmark study, Blackstone et al. demonstrated that inhaled H 2 S (80 ppm, ambient temperature 13°C) can induce a "suspended-animation" like state by reduction of the metabolic rate in spontaneously breathing mice. This was accompanied by a fall in body temperature down to 15°C [5] . The metabolic rate dropped by 90% after 6 h of H 2 S exposure. The effect was fully reversible upon transferring the mice into room air and room temperature [5] . These findings led to high hopes and a frenzy of speculation regarding the ability of H 2 S to induce a hypometabolic state which could be exploited in patient care [6] . However, the fact that this effect was first shown in experimental conditions (low ambient temperature, no maintenance of body temperature, and no anesthesia) that are contrary to the current clinical practice, some drawbacks have to be anticipated in translating this effect to critical care medicine. Interestingly, H 2 S-induced hypometabolism and hypothermia could be reproduced in mice at room temperature, but could not be confirmed in anesthetized sheep [7] . In anesthetized pigs, Simon et al. did report a sulfide-induced drop in metabolism in a model of aortic occlusion with intravenous sulfide administration [8] . However, in large animals, the effect seems to take longer to manifest and is not as pronounced as in rodents. Thus, the concept of H 2 S-induced "suspended animation" or hypometabolism should remain in the realm of science fiction (as suggested by Drabek et al. [9] ), but it is also true that potentially therapeutic effects of H 2 S independent of hypometabolism [10] [11] [12] : anti-inflammatory, antioxidant, organ-specific benefits, regulation of blood pressure, and glucose metabolism [13] [14] [15] [16] [17] , are encouraging for the clinical development of H 2 S donors and have not yet been fully explored [18] . After a brief introduction into the role of H 2 S in the lung, its role in chronic lung diseases and modes of exogenous H 2 S administration, we will review the current literature of exogenous H 2 S administration in preclinical models of acute lung injury (ALI, mostly rodents), translationally more relevant models of lung injury and circulatory shock (resuscitated large animal models), and finally conclude with the current status of clinical trials of H 2 S therapies and an outlook on future clinical development.

High levels of H 2 S gas have been shown to be an environmental hazard, entering the body through the lung and being further distributed via the bloodstream [17] . H 2 S as a byproduct of various industries and pollutant arising from sewers can cause a "knockdown" effect upon inhalation of > 500 ppm: pulmonary injury, loss of consciousness, cardiopulmonary arrest, and death [19] . Generally, 10-20 ppm of H 2 S are considered to be safe to inhale acutely [17] . The effects of a chronic low-level exposure to H 2 S on lung toxicity have not been well characterized, and epidemiological studies are controversial, either reporting no relevant effect [20] , or reduced lung function [21] . Bates et al. investigated the effects of naturally occurring H 2 S in geothermal areas on pulmonary health and found no detrimental effect and surprisingly even suggest a potential benefit on lung function [22] . H 2 S reportedly plays a role in lung development [23] , and a deficiency in the endogenous H 2 S enzymes impairs alveolarization [24] . In the adult lung, the expression of the endogenous enzymes has been identified in a variety of pulmonary compartments in different species: rodents [25] [26] [27] , bovine [28] , and humans [29] [30] [31] [32] . An upregulation of the endogenous H 2 S enzymes has been reported to play a role in the adaptive response to injury [27, 33] . However, the role of endogenous H 2 S in the adult lung is not well established.

Chronic pulmonary diseases have been found to be associated with reduced H 2 S serum levels in patients [34] and suppressed pulmonary CSE expression [31] . Even though a few preclinical studies report pro-inflammatory effects of H 2 S in general (e.g., [35, 36] ), it seems well established that the predominant H 2 S effect in the pathophysiology of chronic pulmonary diseases is anti-inflammatory [25, 31, 32, 37, 38] . Interestingly, low expression of the H 2 S-producing enzymes was shown to compromise the antiinflammatory effects of glucocorticoid therapy in asthma [31, 39] . Low levels of CSE expression and H 2 S production in early development have been correlated to a higher susceptibility to allergic asthma in young mice [40] . The protective role of H 2 S in chronic inflammatory lung diseases has been thoroughly reviewed by Chen and Wang ([41] : animal models [25, 37, 39, 42] and human studies [34, 43] ) and reported more recently (animal models: [38, 44] human: [31] , human in vitro: [32] ). There are numerous studies reporting a potential benefit of exogenous H 2 S administration in chronic lung diseases [25, 32, 38, 44, 45] .

The possible strategies for exogenous administration of H 2 S have been reviewed recently by Szabo and Papapetropoulos [17] and comprise the following: inhalation of gaseous H 2 S and intraperitoneal (i.p.) or intravenous (i.v.) administration of various H 2 S-releasing compounds: H 2 S-releasing salts (e.g., Na 2 S, NaHS) and slow H 2 S-releasing donors (GYY4137, AP39, diallyl-trisulfide (DATS)). Regarding the effects of exogenous H 2 S on inflammation reveals that short-term free sulfide levels as a consequence of the administration of H 2 S-releasing salts can have detrimental effects, whereas a slow continuous H 2 S release from slow-releasing donors attenuated inflammation (demonstrated in vitro by [46] and thoroughly reviewed by [13] ). An overview of currently available H 2 S-releasing compounds is given in Table 1 .

In the following subsections, 70 articles investigating the effects of exogenous H 2 S administration in various models of acute lung injury are reviewed. These articles were identified in a literature search on PubMed in August 2019 with the search term "hydrogen sulfide" in combination with either "acute lung injury" or "ventilator-induced lung injury" or "shock" and "lung." Articles that were not available in English or did not deal with exogenous H 2 S administration were excluded.

The effects of exogenous H 2 S in murine models of VILI are mostly reported to be antiinflammatory. Only one study reports an acceleration of VILI with 60 ppm of H 2 S gas administration as an inhaled gas [47] . However, in the same study, pre-treatment with an intra-arterial bolus of Na 2 S (0.55 mg/kg) before starting harmful ventilation could attenuate lung inflammation and oxidative stress [47] . The latter is well in accordance with the protective effects of H 2 S in VILI reported by Aslami et al. and Wang et al., who observed reduced inflammation and improved lung function in animals with VILI, treated with a continuous infusion of 2 mg/kg/h NaHS or DATS, respectively [48, 49] .

In contrast to the harmful effects of gaseous H 2 S administration (60 ppm) [47] , four separate reports from a different group all indicate a beneficial effect of 80 ppm of H 2 S: anti-inflammatory and anti-apoptotic effects [11] , attenuated lung damage [50] , antioxidant effects [51] , and prevention of edema formation, even with a reduced H 2 S administration time [52] . These contrasting results might be due to the fact that the latter group used a milder VILI protocol with a tidal volume of 12 ml/kg over a longer time (6 h) [11, [50] [51] [52] rather than 40 ml/kg for 4 h as [47] . In conclusion, these results suggest an overall beneficial effect of H 2 S in VILI.

Up to 1/3 of all pancreatitis patients develop ALI or acute respiratory distress syndrome (ARDS), which accounts for 60% of pancreatitis-related deaths [53] . Inhibition of cystathionine-γ-lyase (CSE) had anti-inflammatory effects in a murine model of pancreatitis-induced lung injury [54] . In a follow-up experiment, Bhatia et al. 2006 reported an induction of lung inflammation and histological damage in response to i.p. injection of 10 mg/kg NaHS in mice [55] . The effects were only present 1 h postinjection and by 3 and 6 h, the inflammatory state had returned to baseline [55] , suggesting that the toxic effects were a transitory consequence of NaHS-induced high peak sulfide concentrations, which were quickly cleared. Besides Bhatia et al. 2005 [54] , three more studies report a benefit of the inhibition of endogenous H 2 S production by CSE (either chemically or genetic deletion) on pancreatitis-induced ALI in murine models [56] [57] [58] . However, as mentioned previously, the effects of H 2 S on inflammation are controversial: in other studies, both the administration of ACS15 (H 2 S-releasing diclofenac) and NaHS pre-treatment (10-15 mg/kg) led to an attenuation of inflammation in pancreatitis-induced ALI [59, 60] . The context of H 2 S administration seems to be crucial: in a healthy animal, 10 mg/kg NaHS induces transient lung inflammation, whereas this kind of pre-treatment is anti-inflammatory in subsequent pancreatitisinduced ALI. Furthermore, the role of CBS in the CSE inhibition experiments is not clear-it could potentially be upregulated in response to CSE inhibition. Neither of the CSE inhibition experiments report pulmonary H 2 S levels; thus, no causal conclusions about the role of H 2 S itself in inflammation can be drawn from these studies.

Acute lung injury is common in burn injury patients and can also be aggravated by the inhalation of smoke. In a murine model of hot water-induced skin burn, Zhang et al. observed aggravated lung inflammation and histological damage in animals treated with NaHS (10 mg/kg) [61] , which could be mediated by transient toxic peak sulfide release, which has to be anticipated with this dose of NaHS. In contrast, in a similar model, Ahmad et al. report attenuated pulmonary cell infiltration and oxidative stress with the administration of AP39 [62] . However, confoundedly, another arm in this study was treated with AOAA, an inhibitor of endogenous H 2 S enzymes [63] , which had the same effects as AP39, prompting their conclusion of a "complex pathogenic role of H 2 S in burns" [62] . However, the authors neither report H 2 S levels nor the expression levels of the endogenous enzymes, which makes it difficult to interpret their data. In the lung, the upregulation of the endogenous H 2 S enzymes can represent an adaptive response to stress [27] . Thus, it is tempting to speculate that their apparently ambivalent results may be attributed to AOAA and AP39 having a similar regulatory effect on the endogenous H 2 S enzymes, which has not been investigated or reported yet. In fact, Han et al. report attenuated lung injury and antioxidant effects of spontaneous breathing of 80 ppm H 2 S in a rat model of cotton smoke-induced ALI [64] . In a combined model of smoke-and flame burn-induced lung injury, Esechie et al. were able to demonstrate attenuated inflammation and improved 5 days survival due to subcutaneous Na 2 S treatment [65] . They were also able to confirm this protective effect of Na 2 S in a large animal (ovine) model of smoke and burn injury, where a 24-h primed continuous i.v. infusion of Na 2 S after injury ameliorated pulmonary pathophysiological changes [66] .

Overall, H 2 S seems to mediate protective effects in burn-and/or smoke-induced ALI.

All studies investigating the effects of exogenous H 2 S in LPS-induced lung inflammation were performed in rodents and reported beneficial effects, regardless of the mode of LPS (locally or systemically) and H 2 S (salt, slow-releasing donor, inhalation) administration. Inhalation of 80 ppm H 2 S after intranasal LPS attenuated lung histological damage and had anti-inflammatory and antioxidative effects [67, 68] . Pretreatment with GYY4137 also attenuated lung injury and cell infiltration after LPS inhalation [69] . Both GYY4137 and NaHS pre-treatment also attenuated lung injury and inflammation after intratracheal LPS exposure [70, 71] . A therapeutic administration of H 2 S, either sodium thiosulfate (STS) or GYY4137, after intratracheal LPS ameliorated pulmonary inflammation as well [72, 73] . GYY4137 also attenuated cell infiltration in the lung after i.v. injection with LPS. Pre-treatment with GYY4137 had antioxidant and anti-inflammatory effects in i.p. injection of LPS. NaHS administration 3 h after i.v. LPS attenuated inflammation and oxidative stress and protected the mitochondria in the lung [74] .

In contrast to studies investigating endotoxin administration, the role of exogenous H 2 S in murine models of cecal ligation and puncture (CLP, abdominal sepsis) is controversial: both beneficial and detrimental effects have been reported. In a resuscitated murine model, 100 ppm of inhaled H 2 S had minor anti-inflammatory effects, though not mediating protective effects in CLP [75] . A variety of studies report aggravation of sepsis-induced lung injury by NaHS [76] [77] [78] [79] [80] [81] [82] . However, in all these models, NaHS was administered as an i.p. bolus and did not comprise any additional resuscitative measures. The route of administration might also be a confounding factor combined with the CLP. Furthermore, the dose of H 2 S that was used in these studies was much higher than the dose of the previously mentioned LPS experiments (i.e., 10 mg/kg during CLP versus 0.78-3.12 mg/kg i.p. NaHS during LPS). In fact, 1 h i.v. administration of NaHS at a rate of 1 and 3 mg/(kg × h) after CLP attenuated oxidative stress and cell infiltration in the lung [83] . High peak sulfide levels achieved by the bolus administration of a high dose of H 2 S can exert toxic detrimental effects, whereas achieving a less pronounced elevation of sulfide levels over a longer period of time could exert a benefit [13] . In a model of enterocolitis, the slow-releasing H 2 S donor GYY4137 attenuated lung inflammation and edema, whereas Na 2 S (20 mg/kg 3 times daily) had no effect [84] .

ALI is most commonly modeled in mice by an intravenous injection of oleic acid (OA) [85] . Studies investigating exogenous H 2 S administration in this model consistently report beneficial effects: attenuated edema formation, reduced cell infiltration, and antiinflammatory and antioxidant effects of NaHS pre-treatment [86] [87] [88] [89] .

In models of hyperoxia-or ozone-induced ALI, NaHS administration exerted antiinflammatory and antioxidative effects [90] [91] [92] . However, hyperoxia cannot only induce lung damage, depending on the experimental protocol: hyperoxia, as an experimental therapy in combined fracture healing and blunt chest trauma, exerted lung-protective effects. Interestingly, these protective effects were associated with an amelioration of the stress-induced upregulation of endogenous H 2 S enzymes and thus restoring the naive state of protein expression [27] .

Blunt chest trauma induces mechanical and inflammatory injury to the lung [93] . In a resuscitated, murine model of thoracic trauma, a continuous i.v. infusion of Na 2 S (0.2 mg/(kg × h)) had no effect on lung mechanics and gas exchange, but reduced apoptosis and cytokine production [33] . These effects were even more pronounced in combination with hypothermia [33] . Inhaled H 2 S (100 ppm) attenuated inflammation and cell infiltration in the lung in a non-resuscitated rat model of thoracic trauma [94] . However, in both these studies, the effects of H 2 S were rather weak and a clear benefit could not have been determined [33, 94] , in contrast to models of other types of injury. Interestingly, an upregulation of pulmonary CSE expression in response to combined acute on chronic lung disease, i.e., thoracic trauma after cigarette smoke exposure, was suggested to be an adaptive response to injury [27, 95] , in that a genetic deletion of CSE in the same kind of acute on chronic trauma was associated with aggravated ALI [96] .

In a rat model of lung transplantation, NaHS (0.7 mg/kg i.p.) improved lung function and reduced cell infiltration and oxidative stress [97] . NaHS pre-treatment was beneficial in limb I/R-induced lung injury, due to anti-inflammatory effects and attenuated edema formation [98] . GYY4137 pre-treatment has been tested in infrarenal aortic cross clamping, as well as lung I/R, and beneficial effects have been reported in both types of lung injury: anti-inflammatory and antioxidant activity, respectively [99, 100] . Results in models of hemorrhagic shock are controversial. One study found a beneficial effect of an i.p. bolus of NaHS in a rat model: attenuated edema formation, cell infiltration, and necrosis [101] . Another study of HS in mice determined pulmonary anti-inflammatory effects of AP39; however, the mortality rate in the treated arm of this study was very high due to profound vasodilation [102] . Using a lower dose of AP39 yielded no effects at all [102] . These opposite effects of exogenous H 2 S administration in these two experiments might be due to the different H 2 S-releasing compounds used or resuscitative measures. Chai et al. [101] performed the re-transfusion/resuscitation only with fluid administration, whereas Wepler et al. [102] used re-transfusion of shed blood and a full-scale small animal intensive care unit (ICU) setup (see below), which certainly changes the pathophysiology. In general, the role of H 2 S in hemorrhagic shock is controversial, with either a beneficial [103] [104] [105] [106] [107] [108] , harmful [109, 110] , or no impact [111, 112] .

Animal models with the purpose to identify relevant novel therapeutic strategies for patient care should reflect the clinical situation as closely as possible. In the context of ALI and shock research, the clinical practice for patient care in the ICU has to be reflected in experimental models to facilitate the translation from preclinical research to the clinical reality, i.e., temperature management, frequent blood gas analysis, lungprotective mechanical ventilation, hemodynamic monitoring, fluid administration, and catecholamine support titrated to the mean arterial pressure (MAP) [113] . Metabolic and organ-specific differences between small and large animals need to be taken into account [114, 115] , as well as the challenge of reproducing the patient's pathophysiology (e.g., comorbidities and premedication).

In particular for H 2 S, in a translational scenario, the implementation of intensive care measures (e.g., maintenance of body temperature, anesthesia, fluid resuscitation) might interfere with its effects, thus contributing to the lack of a hypometabolic effect in resuscitated rodent intensive care models [10, 33, 75, 102] . In large animals, the effects of H 2 S administration, in general, have been less robust, not only due to the intensive care measures, but also due to their large body size and different metabolic and thermoregulatory phenotype [114] . Large resuscitated animal studies reflect (i) no or very limited effects [8, 103, 112, [116] [117] [118] , (ii) organ-specific effects [66] , or (iii) beneficial effects restricted to a narrow timing and dosing window [119, 120] .

As aforementioned, the induction of suspended animation by H 2 S inhalation was successful in small animals [5] ; however, the translation to larger animals and eventually humans has proven to be challenging. Small animals have a much higher metabolic rate in relation to their body weight than large animals [121] ; thus, the induction of a hypometabolic state is much easier to perform in small animals [114] . To induce that same state in a larger animal, a much higher dose of H 2 S would be needed, harboring the risk of toxicity [114] . However, the challenges of measuring H 2 S/sulfide in biological samples make it difficult to perform dose-finding studies.

Nonetheless, several studies in large animal models explored the therapeutic potential in various types of ALI. Na 2 S in an ovine model of burn reduced mortality and improved gas exchange [66] . In porcine models, Na 2 S was further studied in hemorrhagic shock, where it attenuated lung damage when administered at the time of reperfusion, however largely unrelated to hypothermia [120] . Administration of STS in the acute phase of resuscitation (24 h) after hemorrhagic shock in a porcine comorbid atherosclerotic model showed only a limited effect by improved gas exchange and lung mechanics in comparison to vehicle-treated animals ( Table 2 , [122] ). Nußbaum et al. investigated the effects of GYY4137 during long-term resuscitated septic shock in pigs with atherosclerosis: GYY4137 treatment led to a preferential utilization of carbohydrates; however, they did not observe any major benefit of the treatment, gas exchange was not affected, and they did not further investigate lung tissue [117] . Unfortunately, none of the other large animal studies report lung function or lung histopathology. Still, it seems that exogenous H 2 S can mediate lung-protective effects in translationally relevant large animal models, when carefully timed and titrated.

To be able to answer the question posted in the title of this review, the clinical development of H 2 S-releasing compounds has to be taken into consideration as well. As we shift from large animal preclinical studies to clinical trials, a search on clinicaltrials.gov (August 2019) for the term "sulfide" revealed a total of 64 clinical trials (see Fig. 1 ). Only two trials were found, which focused on a lung pathology (i.e., asthma), falling into the category "observational" in Fig. 1 , investigating the potential use of H 2 S as a biomarker. There are no interventional clinical trials addressing the therapeutic potential of exogenous H 2 S in lung injury or lung disease. Of the 50 interventional trials identified, only 20 were evaluating H 2 S donors, 8 evaluated their intervention based on H 2 S as a biomarker, and 5 suggested H 2 S as a part of the mechanism of their intervention (see Fig. 1 ). The category "other" in Fig. 1 includes contrast agents, chemotherapeutics, and dietary supplements with a sulfide moiety. Only 6 of the 20 interventional Atherosclerotic pigs were surgically instrumented and, after a short recovery period, underwent 3 h of hemorrhagic shock (target mean arterial pressure 40 ± 5 mmHg). Seventy-two hours of resuscitation comprised re-transfusion of the shed blood and fluid and catecholamine administration targeted to the pre-shock mean arterial pressure. Further details about the experimental protocol can be found in [122] . STS was administered during the first 24 h of resuscitation after hemorrhagic shock. Effects on lung function were most pronounced at 48 h after hemorrhagic shock. Data shown are median (lower quartile, upper quartile) *Significant to control group #Significant to baseline (p < 0.05 in two-way ANOVA) trials with H 2 S donors are relevant to intensive care (see Fig. 2 ), excluding skin diseases, colonoscopy, and arthritis. IK-1001, a solution generated by bubbling H 2 S gas into an aqueous solution, was the first compound, designated to administer H 2 S, under investigation in clinical trials in 2009. The first trial of IK-1001 targeted "renal impairment" (NCT00879645) and was terminated prematurely (actual recruitment of 28 participants) because investigators were unable to determine sulfide levels. The issue of not being able to reliably measure sulfide is of course critical for clinical approval of a compound: how would one ever be able to determine the safety of a compound that cannot be measured? One complexity is the fact that exogenous sulfide is highly volatile and rapidly bound and/or metabolized in vivo [27] . Various sulfide pools are available in biological systems and sulfide engages in many different chemical reactions [123] , suggesting that these endogenous pools are highly dynamic. Exogenous administration of H 2 S might change the balance Fig. 1 Overview of clinical trials found with the search term "sulfide" listed on clinicaltrials.gov Fig. 2 Clinical trials found with the search term "sulfide" on clinicaltrials.gov and relevant to intensive care. Excluded: skin diseases, halitosis, dental conditions, colonoscopy, and similar of this whole system in ways that we do not fully understand yet. The second trial with IK-1001 in coronary artery bypass (NCT00858936) was terminated after recruiting 6 participants with reasons not reported. The third trial in ST-elevation myocardial infarct (STEMI, NCT01007461) was withdrawn by company decision-non-safety related.

As mentioned above, IK-1001 is an aqueous solution of physically dissolved H 2 S, thus resembling the characteristics of the administration of H 2 S-releasing salts or inhaled H 2 S (see also Table 1 ). Neither administration of H 2 S via inhalation nor injection of H 2 S-releasing salts will likely be ever used in clinical practice, due to airway mucosal damage and the potential of toxic peak sulfide concentrations, respectively [27] . In fact, inhalation of 300 ppm H 2 S, though sub-lethal, is used as a model to study lung injury [124, 125] . Efforts to avoid the airway irritation of gaseous H 2 S using extracorporeal membrane lung ventilation in a preclinical study were successful, but there was no improvement on the outcome from cardiopulmonary bypass [126] .

SG-1002, a mixture of organic sulfide-releasing compounds and salts, has been under investigation in heart failure. A phase I trial revealed the compound to be safe and well tolerated (NCT01989208); a follow-up phase II trial is still in progress with no results posted yet (NCT02278276).

An interesting perspective for H 2 S-based therapeutics is the reconsideration of compounds that are already clinically approved and have only recently been identified to be able to release H 2 S: (i) sodium thiosulfate (STS) [17, 127] , approved for cyanide detoxification and cisplatin overdosage; (ii) ammonium tetrathiomolybdate (ATTM) [128, 129] , approved for Wilson's disease, a copper metabolism disorder; and (iii) zofenopril [130] , an inhibitor of angiotensin converting enzyme approved for hypertension. These compounds all have been tested extensively and are known to have good safety profiles (see also Table 1 ).

For example, Dyson et al. showed ATTM led to a 50% reduction of infarct size in rat models of myocardial and cerebral I/R as well as improved survival after hemorrhagic shock [129] . The good safety profile of STS [131] in particular might be related to the fact that thiosulfate itself is an endogenous intermediate of oxidative H 2 S metabolism [127] and is suggested to be "a circulating 'carrier' molecule of beneficial effects of H 2 S" [132] , in particular under hypoxic conditions [127] . The clinical trial of IK-1001 in renal impairment even used thiosulfate as an indirect measure of H 2 S release from their compound (NCT00879645), although ultimately not successful. STS is currently under investigation in a phase 2 clinical trial to preserve cardiac function in STEMI (NCT02899364). With regard to the lung, as mentioned previously, STS was beneficial in murine models of intratracheal LPS and CLP [72] . Our own group's findings support these results from Sakaguchi et al.: we determined a beneficial effect of STS to the lung, i.e., improved gas exchange and lung mechanics in a translationally relevant large animal model of hemorrhagic shock (Table 2) . Thus, STS is a very promising compound for the development of therapeutic H 2 S administration in ALI in a clinical setting.

Exogenous H 2 S administration has been demonstrated to be beneficial in various preclinical models of lung injury. However, due to the narrow therapeutic window and width, and potentially toxic effects, the route, dosing, and timing of administration all have to be balanced out very carefully. The development of methods to determine H 2 S levels and/or the pharmacokinetics and pharmacodynamics of H 2 S-releasing compounds is absolutely necessary to facilitate the safety of H 2 S-based therapies. Awaiting the results of currently ongoing clinical trials and the re-evaluation of already approved H 2 S-releasing compounds for novel indications could likely help to prove that H 2 S is in fact not a therapeutic dead end [6] . 

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

The authors declare that they have no competing interests. 

Physiological implications of hydrogen sulfide: a whiff exploration that blossomed

The possible role of hydrogen sulfide as an endogenous neuromodulator

The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide

Two's company, three's a crowd: can H2S be the third endogenous gaseous transmitter?

H 2 S induces a suspended animation-like state in mice

Nothing rotten about hydrogen sulfide's medical promise

H 2 S induced hypometabolism in mice is missing in sedated sheep

Hemodynamic and metabolic effects of hydrogen sulfide during porcine ischemia/reperfusion injury

Hydrogen sulfide-curiouser and curiouser

Cardiac and metabolic effects of hypothermia and inhaled hydrogen sulfide in anesthetized and ventilated mice

Inhaled hydrogen sulfide protects against ventilatorinduced lung injury

Inhaled hydrogen sulfide prevents endotoxin-induced systemic inflammation and improves survival by altering sulfide metabolism in mice

Hydrogen sulfide and inflammation: the good, the bad, the ugly and the promising

Hydrogen sulfide increases glutathione production and suppresses oxidative stress in mitochondria

Hydrogen sulfide in hypertension

Decreased gluconeogenesis in the absence of cystathionine gamma-lyase and the underlying mechanisms

CII: pharmacological modulation of H 2 S levels: H2S donors and H2S biosynthesis inhibitors

Whatever happened to

Toxicology of hydrogen sulfide

Exposure to hydrogen sulphide and respiratory function

Respiratory effects of chronic hydrogen sulfide exposure

Investigation of hydrogen sulfide exposure and lung function, asthma and chronic obstructive pulmonary disease in a geothermal area of New Zealand

Systemic hydrogen sulfide administration partially restores normal alveolarization in an experimental animal model of bronchopulmonary dysplasia

The H 2 S-generating enzymes cystathionine β-synthase and cystathionine γ-lyase play a role in vascular development during normal lung alveolarization

Endogenous hydrogen sulfide reduces airway inflammation and remodeling in a rat model of asthma

Hydrogen sulfide and asthma

H 2 S during circulatory shock: some unresolved questions

Hypoxic pulmonary vasodilation: a paradigm shift with a hydrogen sulfide mechanism

Hydrogen sulfide inhibits proliferation and release of IL-8 from human airway smooth muscle cells

Hydrogen sulfide-induces DNA damage and changes in apoptotic gene expression in human lung fibroblast cells

Metabolic changes of H 2 S in smokers and patients of COPD which might involve in inflammation, oxidative stress and steroid sensitivity

The antiproliferative and anti-inflammatory response of COPD airway smooth muscle cells to hydrogen sulfide

) Cardiopulmonary, histologic, and inflammatory effects of intravenous Na 2 S after blunt chest trauma-induced lung contusion in mice

Endogenous hydrogen sulfide in patients with COPD

Hydrogen sulfide induces the synthesis of proinflammatory cytokines in human monocyte cell line U937 via the ERK-NF-kappaB pathway

Pro-inflammatory effects of hydrogen sulphide on substance P in caerulein-induced acute pancreatitis

Involvement of endogenous hydrogen sulfide in cigarette smoke-induced changes in airway responsiveness and inflammation of rat lung

The inhibitory role of hydrogen sulfide in airway hyperresponsiveness and inflammation in a mouse model of asthma

Regulative mechanism of budesonide on endogenous hydrogen sulfide, cystathionine-gamma-lyase and cystathionine-beta-synthase system in asthmatic rats

Age-dependent allergic asthma development and cystathionine gamma-lyase deficiency

The message in the air: hydrogen sulfide metabolism in chronic respiratory diseases

Hydrogen sulfide ameliorates tobacco smoke-induced oxidative stress and emphysema in mice

Effect of theophylline on endogenous hydrogen sulfide production in patients with COPD

Protective effect of exogenous hydrogen sulfide on pulmonary artery endothelial cells by suppressing endoplasmic reticulum stress in a rat model of chronic obstructive pulmonary disease

Hydrogen sulfide attenuates particulate matter-induced human lung endothelial barrier disruption via combined reactive oxygen species scavenging and Akt activation

The effect of hydrogen sulfide donors on lipopolysaccharide-induced formation of inflammatory mediators in macrophages

Protective and detrimental effects of sodium sulfide and hydrogen sulfide in murine ventilator-induced lung injury

Suspended animation inducer hydrogen sulfide is protective in an in vivo model of ventilator-induced lung injury

Intravenous controlled-release hydrogen sulfide protects against ventilator-induced lung injury

Genetic targets of hydrogen sulfide in ventilator-induced lung injury--a microarray study

Hydrogen sulfide prevents formation of reactive oxygen species through PI3K/Akt signaling and limits ventilator-induced lung injury

Pre-and posttreatment with hydrogen sulfide prevents ventilator-induced lung injury by limiting inflammation and oxidation

Lung injury in acute pancreatitis: mechanisms, prevention, and therapy

Role of hydrogen sulfide in acute pancreatitis and associated lung injury

Role of substance P in hydrogen sulfide-induced pulmonary inflammation in mice

Inhibition of hydrogen sulfide synthesis attenuates chemokine production and protects mice against acute pancreatitis and associated lung injury

The effect of CSE gene deletion in caerulein-induced acute pancreatitis in the mouse

Cystathionine-gamma-lyase inhibitor attenuates acute lung injury induced by acute pancreatitis in rats

Treatment with H 2 S-releasing diclofenac protects mice against acute pancreatitis-associated lung injury

Effects of hydrogen sulfide on inflammation in caerulein-induced acute pancreatitis

Role of hydrogen sulfide in severe burn injury-induced inflammation in mice

Both the H 2 S biosynthesis inhibitor aminooxyacetic acid and the mitochondrially targeted H 2 S donor AP39 exert protective effects in a mouse model of burn injury

Selectivity of commonly used pharmacological inhibitors for cystathionine β synthase (CBS) and cystathionine γ lyase (CSE)

Protective effects of hydrogen sulfide inhalation on oxidative stress in rats with cotton smoke inhalation-induced lung injury

Protective effect of hydrogen sulfide in a murine model of acute lung injury induced by combined burn and smoke inhalation

Beneficial effect of a hydrogen sulphide donor (sodium sulphide) in an ovine model of burn-and smoke-induced acute lung injury

Inhaled hydrogen sulfide protects against lipopolysaccharide-induced acute lung injury in mice

Hydrogen sulfide exerts anti-oxidative and anti-inflammatory effects in acute lung injury

Hydrogen sulfide limits neutrophil transmigration, inflammation, and oxidative burst in lipopolysaccharide-induced acute lung injury

Hydrogen sulfide ameliorated lipopolysaccharide-induced acute lung injury by inhibiting autophagy through PI3K/Akt/mTOR pathway in mice

Effects of CCK-8 and cystathionine γ-lyase/hydrogen sulfide system on acute lung injury in rats

Sodium thiosulfate attenuates acute lung injury in mice

GYY4137 attenuates LPS-induced acute lung injury via heme oxygenase-1 modulation

In vivo study of the effects of exogenous hydrogen sulfide on lung mitochondria in acute lung injury in rats

Inflammatory effects of hypothermia and inhaled H2S during resuscitated, hyperdynamic murine septic shock

Role of hydrogen sulfide in cecal ligation and puncture-induced sepsis in the mouse

Endogenous hydrogen sulfide regulates leukocyte trafficking in cecal ligation and puncture-induced sepsis

Hydrogen sulfide acts as an inflammatory mediator in cecal ligation and puncture-induced sepsis in mice by upregulating the production of cytokines and chemokines via NF-kappaB

Endogenous hydrogen sulfide regulates inflammatory response by activating the ERK pathway in polymicrobial sepsis

Hydrogen sulfide up-regulates substance P in polymicrobial sepsis-associated lung injury

Hydrogen sulfide promotes transient receptor potential vanilloid 1-mediated neurogenic inflammation in polymicrobial sepsis

Hydrogen sulfide upregulates cyclooxygenase-2 and prostaglandin E metabolite in sepsis-evoked acute lung injury via transient receptor potential vanilloid type 1 channel activation

Delayed treatment with sodium hydrosulfide improves regional blood flow and alleviates cecal ligation and puncture (CLP)-induced septic shock

Hydrogen sulfide provides intestinal protection during a murine model of experimental necrotizing enterocolitis

ARDS: clinical lessons from the oleic acid model of acute lung injury

Regulatory effects of hydrogen sulfide on IL-6, IL-8 and IL-10 levels in the plasma and pulmonary tissue of rats with acute lung injury

Effect of endogenous hydrogen sulfide on oxidative stress in oleic acidinduced acute lung injury in rats

Hydrogen sulfide donor regulates alveolar epithelial cell apoptosis in rats with acute lung injury

Regulatory effects of hydrogen sulfide on alveolar epithelial cell endoplasmic reticulum stress in rats with acute lung injury

Hydrogen sulfide prevents hyperoxia-induced lung injury by downregulating reactive oxygen species formation and angiopoietin-2 release

Treatment with exogenous hydrogen sulfide attenuates hyperoxia-induced acute lung injury in mice

Inhibitory effect of hydrogen sulfide on ozone-induced airway inflammation, oxidative stress, and bronchial hyperresponsiveness

The role of C5a in the innate immune response after experimental blunt chest trauma

Inhaled hydrogen sulfide induces suspended animation, but does not alter the inflammatory response after blunt chest trauma

In-depth characterization of the effects of cigarette smoke exposure on the acute trauma response and hemorrhage in mice

The role of cystathionine-γ-lyase in blunt chest trauma in cigarette smoke exposed mice

Inhibition of hydrogen sulfide generation contributes to lung injury after experimental orthotopic lung transplantation

H 2 S protecting against lung injury following limb ischemia-reperfusion by alleviating inflammation and water transport abnormality in rats

Hydrogen sulfide ameliorates acute lung injury induced by infrarenal aortic cross-clamping by inhibiting inflammation and angiopoietin 2 release

Hydrogen sulfide attenuates lung ischemia-reperfusion injury through SIRT3-dependent regulation of mitochondrial function in type 2 diabetic rats

Exogenous hydrogen sulfide protects against traumatic hemorrhagic shock via attenuation of oxidative stress

The mitochondria-targeted H 2 S-donor AP39 in a murine model of combined hemorrhagic shock and blunt chest trauma

Hydrogen sulfide improves resuscitation via non-hibernatory mechanisms in a porcine shock model

Chai W (2012) An exogenous hydrogen sulphide donor, NaHS, inhibits the nuclear factor κB inhibitor kinase/nuclear factor κb inhibitor/nuclear factor-κB signaling pathway and exerts cardioprotective effects in a rat hemorrhagic shock model

Effects of hydrogen sulfide on hemodynamics, inflammatory response and oxidative stress during resuscitated hemorrhagic shock in rats

Surviving blood loss using hydrogen sulfide

Effects of hyperoxia and mild therapeutic hypothermia during resuscitation from porcine hemorrhagic shock

Hyperoxia or therapeutic hypothermia during resuscitation from non-lethal hemorrhagic shock in swine

Role of hydrogen sulphide in haemorrhagic shock in the rat: protective effect of inhibitors of hydrogen sulphide biosynthesis

Hydrogen sulphide is pro-inflammatory in haemorrhagic shock

The effects of genetic 3-mercaptopyruvate sulfurtransferase deficiency in murine traumatic-hemorrhagic shock

Intravenous hydrogen sulfide does not induce hypothermia or improve survival from hemorrhagic shock in pigs

Preclinical septic shock research: why we need an animal ICU

Potential applications of hydrogen sulfide-induced suspended animation

Is pharmacological, H 2 S-induced 'suspended animation' feasible in the ICU?

Hydrogen sulfide does not increase resuscitability in a porcine model of prolonged cardiac arrest

Metabolic, cardiac, and renal effects of the slow hydrogen sulfide-releasing molecule GYY4137 during resuscitated septic shock in swine with pre-existing coronary artery disease

Intravenous hydrogen sulfide does not induce neuroprotection after aortic balloon occlusion-induced spinal cord ischemia/reperfusion injury in a human-like porcine model of ubiquitous arteriosclerosis

Effects of intravenous sulfide during porcine aortic occlusion-induced kidney ischemia/reperfusion injury

Effects of intravenous sulfide during resuscitated porcine hemorrhagic shock

Absence of hydrogen sulfide-induced hypometabolism in pigs: a mechanistic explanation in relation to small nonhibernating mammals

Effects of sodium thiosulfate (Na 2 S 2 O 3 ) during resuscitation from hemorrhagic shock in swine with preexisting atherosclerosis

Chemical aspects of hydrogen sulfide measurements in physiological samples

Myocardial and lung injuries induced by hydrogen sulfide and the effectiveness of oxygen therapy in rats

Dexamethasone ameliorates H 2 S-induced acute lung injury by alleviating matrix metalloproteinase-2 and -9 expression

Administration of hydrogen sulfide via extracorporeal membrane lung ventilation in sheep with partial cardiopulmonary bypass perfusion: a proof of concept study on metabolic and vasomotor effects

Thiosulfate: a readily accessible source of hydrogen sulfide in oxygen sensing

Ammonium tetrathiomolybdate as a water-soluble and slow-release hydrogen sulfide donor

Ammonium tetrathiomolybdate following ischemia/reperfusion injury: chemistry, pharmacology, and impact of a new class of sulfide donor in preclinical injury models

Zofenopril protects against myocardial ischemia-reperfusion injury by increasing nitric oxide and hydrogen sulfide bioavailability

Toxicity profile of delayed high dose sodium thiosulfate in children treated with carboplatin in conjunction with blood-brainbarrier disruption

Thiosulfate mediates cytoprotective effects of hydrogen sulfide against neuronal ischemia

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