Inflammation & Allergy-Drug Targets (v.10, #2)

Small molecules gaseous neuro-transmitters are a family of biologically relevant mediators that orchestrate the activity of a myriad of biologically essential functions in mammals [1-4]. After the discovery of nitric oxide (NO), awarded by the Nobel prize in 1998, at least two other gaseous mediators have been added to this family, i.e. carbon monoxide (CO) and hydrogen sulfide (H2S). These agents are increasingly investigated for their biological activities and are exploited to generate novel chemical entities as drug candidates in a variety of pharmacological settings. An essential requirement for a gas to be considered a and#x201C;physiologicaland#x201D; mediator is that it should be generated endogenously by enzymatic steps making possible to tune its generation by acting on enzymatic reactions. In the case of NO, three isoforms of NO synthase (NOS), derived from three distinct genes, convert arginine into NO and citrulline, with neuronal NOS (nNOS) highly localized to the brain and peripheral nerves as well as a few non-neural tissues; endothelial NOS (eNOS) generating NO in blood stream, and inducible NOS (iNOS) occurring ubiquitously throughout the body, but with highest densities in inflammatory cells such as macrophages [1]. CO has long been known to be generated by heme oxygenase (HO) which exists in two different isoforms: HO-1 is a markedly inducible enzyme whose formation is stimulated by diverse stressors, including heme, and is abundant in liver, kidney and spleen; organs responsible for degradation and heme catabolism of aged red blood cells. By contrast, HO-2, localized to neurons in the brain and the endothelial layer of blood vessels, is constitutive and activated by calcium calmodulin, much like nNOS and eNOS [1-4]. For H2S the endogenous enzymatic pathways is mediated by cystathionine and#947;-lyase (CSE) and cystathionine-and#946;-synthase (CBS) [2]. The cover of this issue is an art drawing by Dr. Sabrina Cipriani. The artist work was intended as a conceptual resume of the main physiological roles H2S exerts in vasculature, liver and central nervous system (CNS), highlighting the complexities of interactions this mediator entertains with a number of physiologically relevant activities. Acting as the introduction to the Special Issue, the review by Dr. Stefano Fiorucci [5] surveys the functional role of H2S from its discovery to the latest exploitation in the field of medicinal chemistry. The author describes the different mechanisms through which H2S might exert its biochemical effects and how investigators have designed pharmacologically active H2S donors. However, despite the rapid growth of the field, the understanding of H2S physiology and pharmacology is still in its infancy. The signaling network of H2S, beyond the activation of KATP channels, remains to be identified. The relevance of the interaction with the NO-cGMP pathway for H2S physiology and pharmacology awaits further definition before H2S can be accepted as a fully independent drug target. There is a growing perception that alike NO, H2S might be protective in a number of such conditions, but the molecular basis of these effects still await to be fully discovered. The review article by Dr. Barbara Renga [6] examines the molecular biology of CBS and CSE, the key enzymes involved in H2S generation and highlight the potential therapeutic relevance of regulation of these enzymes. In mammals, CBS gene transcription is highly regulated. In fact, the CBS enzyme contains a heme cofactor that functions as a redox sensor and utilizes S-adenosylmethionine (SAM) as an allosteric activator. Impaired CB activity causes hyperhomocystinuria and hyperhomocysteinemia, both risk factors for cardiovascular diseases. Murine CSE gene regulation is well characterized but little is known about the human counterpart. Recently it has been demonstrated that CSE transcription is regulated by the nuclear receptor Farnesoid X Receptor (FXR). Mutations that decrease the activity of CSE cause cystathioninuria, hypercystathioninemia and increase the risk of developing atherosclerosis and bladder cancer. This review focuses on the recent aspects of the molecular regulation of both CBS and CSE and highlights the possibility that members of the nuclear receptors superfamily might be involved in the regulation of H2S metabolism. The review article by Sabrina Cipriani and Andrea Mencarelli [7] provides an in deep analysis of the physiological role of H2S in regulating essential aspects of gastrointestinal and liver physiology. H2S has been shown to be an important mediator that regulates liver microcirculation in normal states and in setting of altered liver microarchitecture including liver cirrhosis. Because the production of H2S by intestinal microbiota exposes the intestine to high concentrations of H2S generated through the reduction of unabsorbed intestinal inorganic sulphate, the review examines the contribution of this source of H2S to the gastrointestinal physiology and pathology. The review from Dr. Mariarosaria Bucci and Dr. Giuseppe Cirino [8] is focused on the regulatory effects H2S exerts in cardiovascular system. In mammalian cardiovascular system, H2S joins carbon monoxide (CO) and endothelial derived vasorelaxing factors (EDRFs)-NO, as the third gasotrasmitter. In the vasculature CSE is the main enzyme responsible for H2S biosynthesis starting from the substrate e.g. L-cysteine. There is a growing body of evidence that supports a role for H2S in regulating the vascular homeostasis. H2S is known to induce a concentration-dependent relaxation of large conduit arteries. Interestingly, H2S relaxes also peripheral resistance vessels such as mesenteric arteries suggesting a role for H2S also in the regulation of vascular resistance and systemic blood pressure. This vasodilatory effect is mediated by the activation of KATP channels. However, a cross-talk exists between the L-Argine/ NO and L-cysteine/H2S pathways. H2S acts also as an endogenous non selective inhibitor of phosphodiesterase activity. Compelling evidence are linking H2S to regulation of erectile function while remains unclear whether the L-cysteine/H2S pathway plays a pathogenetic role in erectile dysfunction. Despite the rapid growth of the field, it should be noted that several aspects of H2S physiology in the cardiovascular system remains unsolved and the availability of reliable inhibitors and donors remain a major limitations. In addition to its pathophysiological relevance in cardiovascular and neuronal disorders, there is considerable interest in the significance of H2S in inflammation. This controversial issue is discussed by Akhil Hegde and Madhav Bhatia [9]. A number of preclinical studies using H2S donors and inhibitors of its endogenous synthesis, have provided evidence for both pro- and antiinflammatory character of H2S. But so far, there is a significant lack of support from relevant clinical studies. One of the major contentious issues being variable dose and sampling time, controversies exist on the precise friend or foe nature of this gaseous transmitter. This review focuses on the intriguing effects of H2S in some of the inflammatory conditions such as acute pancreatitis, sepsis, burn injuries and local inflammation of the joints. The review describes ongoing investigations undertaken to elucidate the mechanisms of action of H2S in inflammation, including neurogenic inflammation and interaction with other biological mediators and pathways. The review form Dr. Weifang Rong, Dr. Hideo Kimura and Dr. David Grundy [10] examines the role of H2S as neurotransmitter in the central nervous system. Authors report that in the CNS, H2S, generated mainly by CBS in astrocytes and in neurons, appears to participate in cognition, memory, regulation of the cardiopulmonary functions and neuroprotection. In the peripheral nervous system, evidence suggests that H2S may be involved in autonomic control of the cardiopulmonary and gastrointestinal functions as well pain and inflammation. However, until now research in the field has relied mainly on the use of H2S donors and non-selective enzyme inhibitors. The physiological and pathophysiological roles of H2S as a gasotransmitter warrant further investigation. The review by Dr. Eleonora Distrutti [11] discusses the role of H2S in modulation of pain. Results from preclinical models of pain, including experimentally-induced somatic, neurophatic and visceral pain have provided contradictory evidence and H2S has been reported to exert either pronociceptive and antinociceptive effects. Several biochemical explanations might account for these differences: thus, H2S-induced antinociception appears linked to activation of T-type Ca2+ channels while analgesia is due to KATP channels opening. Moreover, local administration of H2S causes pain and, in contrast, systemic H2S administration results into antinociception. Dr. Distrutti concludes that, in the view of possibility to use H2S-realising drugs or compounds that block H2S synthesis for pain treatment, additional studies are needed to exploit the therapeutic potential of H2S in pain signalling. The latest review from Dr. Stefano Fiorucci and Dr. Luca Santucci [12] examines the role of H2S releasing agents in the treatment of pain and compares the efficacy of these agents to currently available non-steroidal anti-inflammatory drugs (NSAIDs) selective cyclooxygenase 2 inhibitor, the coxibs, and NO-donating drugs, CINOD. NSAIDs, are effective treatment for pain, fever and inflammation. However their use associates with a significant risk to develop gastrointestinal and cardiovascular complications. In the gastrointestinal tract the use of aspirin and NSAIDs is associated with a 4-6 fold increase in the risk of gastrointestinal bleeding. Selective inhibitors of COX-2, the coxibs, spares the gastrointestinal while exert anti-inflammatory and analgesic effects. However, their use associates with an increased risk of thrombo-embolic events. NO and H2S are vasodilatory agents that maintain mucosal integrity in the gastrointestinal tract. In the last decade hybrid molecules that release NO or H2S have been coupled with non-selective NSAIDs to generate new classes of anti-inflammatory and analgesic agents with the potential to spare the gastrointestinal and cardiovascular system. Naproxcinod has been the first, and so far the only, CINOD extensively investigated in clinical trials. Despite its promising profile, however, the approval of this drug was recently rejected by the Food and Drug Administration that asked for long-term controlled studies to assess the cardiovascular and gastrointestinal safety. NSAIDs that releases H2S as a mechanism to support an enhanced gastrointestinal and cardiovascular safety profile are being investigated in preclinical studies. Either naproxen and diclofenac coupled to an H2S releasing moiety have been reported to cause loess gastrointestinal and cardiovascular injury than parent NSAIDs. The review concludes that clinical studies are need to establish whether H2S-donating NSAIDs might have utility in clinical settings.

Gaseous neurotransmitters are a growing family of enzimatically generated gaseous mediators that exert regulatory functions in mammals. It is now widely recognized that hydrogen sulfide (H2S), along with nitric oxide (NO) and carbon monoxide (CO), is an important signaling molecule in cardiovascular, nervous, gastrointestinal, liver and lung physiology and pharmacology. The production of H2S from L-cysteine is catalysed primarily by two enzymes, cystathionine-and#947;-lyase and cystathionine and#946;-synthase. Evidence is accumulating to demonstrate that H2S delivered exogenously exerts beneficial effects in animal models of inflammation and pain highlighting the potential for the therapeutic exploitation of H2S. Several hybrids have been developed coupling an H2S-releasing moiety to conventional drugs. These molecular hybrids are currently evaluated for efficacy in animal models of gastrointestinal, cardiovascular and neurogical disorders and erectile dysfunction. The anti-inflammatory activity of H2S has also been exploited for generating anti-platelets and anti-inflammatory agents that inhibit cyclo-oxygenases while sparing the gastrointestinal and cardiovascular tract.

Cystathionine-and#946;-synthase (CBS) and cystathionine-and#947;-lyase (CSE) are two key enzymes involved in the synthesis of hydrogen sulfide (H2S). CBS catalyzes the pyridoxal 5'-phosphate (PLP)-dependent conversion of homocysteine in cystathionine whilst CSE the pyridoxal 5'-phosphate (PLP)-dependent synthesis of L-cysteine from cystathionine. In mammals, CBS gene transcription is poorly investigated and the activity of the enzyme is highly regulated. In fact, the CBS enzyme contains a heme cofactor that functions as a redox sensor and utilizes S-adenosylmethionine (SAM) as an allosteric activator. Impaired CBS activity causes hyperhomocystinuria and hyperhomocysteinemia, both risk factors for cardiovascular diseases. Murine CSE gene regulation is well characterized but little is known about the human counterpart and there is no information regarding the enzyme activity regulation. Recently it has been demonstrated that CSE transcription is regulated by the nuclear receptor Farnesoid X Receptor (FXR). Mutations that decrease the activity of CSE cause cystathioninuria, hypercystathioninemia and increase the risk of developing atherosclerosis and bladder cancer. This review focuses on the recent aspects of the molecular regulation of both CBS and CSE and highlights the possibility that members of the nuclear receptors superfamily might be involved in the regulation of hydrogen sulfide metabolism.

Hydrogen Sulfide in Gastrointestinal and Liver Physiopathology by Sabrina Cipriani, Andrea Mencarelli (92-102).
Hydrogen sulfide (H2S) is a gas that can be formed by the action of two enzymes, cystathionine gamma lyase (CSE) and cystathionine beta synthase (CBS). H2S has been known for hundreds of years for its poisoning effect, however the idea that H2S is not only a poison, but can exert a physiological role in mammalian organisms, originates from the evidence that this gaseous mediator is produced endogenously. In addition to H2S synthesis by gastrointestinal tissue, the intestinal mucosa, particularly in the large intestine, is regularly exposed to high concentrations of H2S that are generated by some species of bacteria and through the reduction of unabsorbed intestinal inorganic sulphate. This review reports on the effects of H2S in the gastrointestinal tract and liver and provides information on the therapeutic applications of H2Sdonating drugs.

Hydrogen Sulphide in Heart and Systemic Circulation by Mariarosaria Bucci, Giuseppe Cirino (103-108).
In the mammalian cardiovascular system, H2S joins carbon monoxide (CO) and endothelial derived relaxing factors, (EDRFs)-nitric oxide (NO), as the third gasotransmitter. In the vasculature, cystathionine-and#947;-lyase (CSE) is the main enzyme responsible for H2S biosynthesis starting from the substrate e.g. L-cysteine. There is a growing body of evidence that supports a role for H2S in regulating the vascular homeostasis. H2S (NaHS) is known to induce a concentration-dependent relaxation of large conduit arteries. Interestingly, H2S also relaxes peripheral resistance vessels such as mesenteric arteries suggesting a role for H2S also in the regulation of vascular resistance and systemic blood pressure. This vasodilatory effect is dependent on the activation of KATP channels. However, a cross-talk exists between the L-Argine/NO and L-cysteine/H2S pathways. Furthermore, it has been shown that H2S acts as an endogenous non-selective inhibitor of phosphodiesterase activity. Compelling evidence links H2S to regulation of erectile function while it remains unclear whether the L-cysteine/H2S pathway plays a pathogenetic role in erectile dysfunction. Despite the rapid growth of the field, it should be noted that several aspects of H2S physiology in the cardiovascular system remain unsolved and the lack of reliable inhibitors and donors remains a major limitation.

The Neurophysiology of Hydrogen Sulfide by Weifang Rong, Hideo Kimura, David Grundy (109-117).
Hydrogen sulfide (H2S) has emerged as the third endogenous gaseous mediator in the central and peripheral nervous system. H2S is generated by three enzymes, cystathionine-and#946;-synthase (CBS), cystathionine-and#947;-lyase (CSE) and 3- mercaptopyruvate sulfurtransferase (3MST). In the CNS, H2S, generated mainly by CBS in astrocytes and 3MST in neurons, appears to participate in cognition, memory, regulation of the cardiopulmonary functions and neuroprotection. In the peripheral nervous system, evidence suggests that H2S may be involved in autonomic control of the cardiopulmonary and gastrointestinal functions as well pain and inflammation.

Hydrogen Sulfide in Inflammation: Friend or Foe? by Akhil Hegde, Madhav Bhatia (118-122).
Hydrogen sulfide (H2S), the gaseous mediator produced by various cells in our body, was recently discovered to play a major role in human physiology despite its toxic nature known for centuries. In addition to its pathophysiological relevance in cardiovascular and neuronal disorders, there is considerable interest in the significance of H2S in inflammation. A number of preclinical studies in our laboratory as well as by others, using H2S donors and inhibitors of its endogenous synthesis, have provided evidence for both pro- and anti-inflammatory character of H2S. But so far, there is a significant lack of support from relevant clinical studies. One of the major contentious issues being variable dose and sampling time, controversies exist on the precise friend or foe nature of this gaseous transmitter. However, it is well accepted that once a clearer picture of the whole story of H2S in inflammation emerges, potential for therapeutic manipulations in this field are immense. This review focuses on the intriguing effects of H2S in some of the inflammatory conditions such as acute pancreatitis, sepsis, burn injuries and local inflammation of the joints. Active research projects have been undertaken to elucidate the mechanisms of action of H2S in inflammation, including neurogenic inflammation and interaction with other biological mediators and pathways. The early and fragmentary evidence obtained holds promise for a successful drug intervention for these inflammatory diseases.

Hydrogen Sulphide and Pain by Elenora Distrutti (123-132).
Physiopathological mechanisms and treatment of pain remain a significant challenge. In the last decade, the gasotransmitter hydrogen sulphide (H2S) has received wide attention for its ability to act as a multilevel regulatory molecule in a variety of biologic functions in mammals including modulation of pain processing. Results from preclinical models of pain, including experimentally-induced somatic, neurophatic and visceral pain have provided non univocal finding and, depending on the model, H2S has been reported to exert either pronociceptive and antinociceptive effects. Several biochemical explanations might account for these differences: thus, H2S-induced pronociception appears linked to activation of T-type Ca2+ channels while analgesia is due to KATP channels opening. Moreover, local administration of H2S causes pain and, in contrast, systemic H2S administration results into antinociception. In the view of possibility to use H2S-realising drugs or compounds that block H2S synthesis for pain treatment, additional studies are needed to exploit the therapeutic potential of H2S in pain signalling.

Hydrogen Sulfide-Based Therapies: Focus on H2S Releasing NSAIDs by Stefano Fiorucci, Luca Santucci (133-140).
Nonsteroidal anti-inflammatory pain medications, commonly referred to as NSAIDs, are effective treatment for pain, fever and inflammation. However their use associates with a 4-6 fold increase in the risk of gastrointestinal bleeding. The basic mode of action of NSAIDs lies in the inhibition of cyclooxygenases (COXs), a family of enzymes involved in the generation of prostaglandins (PGs). The COX exists at least in two isoforms, COX-1 and COX-2, with PGs mediating inflammation at site of injury generated by the COX-2, while COX-1 produces PGs that are essential in maintaining integrity in the gastrointestinal tract. Selective inhibitors of COX-2, the coxibs, spare the gastrointestinal tract while exerting anti-inflammatory and analgesic effects. However, their use has been linked to an increased risk of thromboembolic events. Nitric oxide (NO) and hydrogen sulfide (H2S), are potent vasodilatory agents that maintain mucosal integrity in the gastrointestinal tract. In the last decade hybrid molecules that release NO or H2S have been coupled with non-selective NSAIDs to generate new classes of anti-inflammatory and analgesic agents with the potential to spare the gastrointestinal and cardiovascular system. These agents, the NO-releasing NSAIDs, or CINOD, and the H2S-releasing NSAIDs are currently investigated as a potential alternative to NSAIDs and coxibs. Naproxcinod has been the first, and so far the only, CINOD extensively investigated in clinical trials. Despite its promising profile, the approval of this drug was recently rejected by the Food and Drug Administration because the lack of long-term controlled studies. NSAIDs that release H2S as a mechanism to support an enhanced gastrointestinal and cardiovascular safety are being investigated in preclinical studies. Either naproxen or diclofenac coupled to an H2S releasing moiety has been reported to cause less gastrointestinal and cardiovascular injury than parent NSAIDs in preclinical models.

The Implication of Pseudomonas aeruginosa Biofilms in Infections by Morten T. Rybtke, Peter O. Jensen, Niels Hoiby, Michael Givskov, Tim Tolker-Nielsen, Thomas Bjarnsholt (141-157).
Biofilm formation by bacteria is recognized as a major problem in chronic infections due to their recalcitrance against the immune defense and available antibiotic treatment schemes. The opportunistic pathogen Pseudomonas aeruginosa has drawn special attention in this regard due to its severity of infection in the lungs of cystic fibrosis patients and in chronic wounds. In this review we address the molecular basis of biofilm development by P. aeruginosa as well as the mechanisms employed by this bacterium in the increased tolerance displayed against antimicrobials. The complex build-up of the extracellular matrix encasing the biofilm-associated bacteria as well as the elaborate signaling mechanisms employed by the bacterium enables it to withstand the continuous stresses imposed by the immune defense and administered antibiotics resulting in a state of chronic inflammation that damages the host. The immune response leading to this chronic inflammation is described. Finally, novel treatment strategies against P. aeruginosa are described including quorum-sensing inhibition and induced biofilm-dispersion. The tolerance towards currently available antimicrobials calls for development of alternative treatment strategies where the underlying targets are less prone for resistance development as bacteria, in retrospect, have a unique ability to evade the actions of classic antibiotics.