The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro.

The development of diabetic vascular complications is initiated, at least in part, by mitochondrial reactive oxygen species (ROS) production in endothelial cells. Hyperglycemia induces superoxide production in the mitochondria and initiates changes in the mitochondrial membrane potential that leads to mitochondrial dysfunction. Hydrogen sulfide (H2S) supplementation has been shown to reduce the mitochondrial oxidant production and shows efficacy against diabetic vascular damage in vivo. However, the half-life of H2S is very short and it is not specific for the mitochondria. We have therefore evaluated two novel mitochondria-targeted anethole dithiolethione and hydroxythiobenzamide H2S donors (AP39 and AP123 respectively) at preventing hyperglycemia-induced oxidative stress and metabolic changes in microvascular endothelial cells in vitro. Hyperglycemia (HG) induced significant increase in the activity of the citric acid cycle and led to elevated mitochondrial membrane potential. Mitochondrial oxidant production was increased and the mitochondrial electron transport decreased in hyperglycemic cells. AP39 and AP123 (30-300nM) decreased HG-induced hyperpolarisation of the mitochondrial membrane and inhibited the mitochondrial oxidant production. Both H2S donors (30-300nM) increased the electron transport at respiratory complex III and improved the cellular metabolism. Targeting H2S to mitochondria retained the cytoprotective effect of H2S against glucose-induced damage in endothelial cells suggesting that the molecular target of H2S action is within the mitochondria. Mitochondrial targeting of H2S also induced >1000-fold increase in the potency of H2S against hyperglycemia-induced injury. The high potency and long-lasting effect elicited by these H2S donors strongly suggests that these compounds could be useful against diabetic vascular complications.

Screening of a composite library of clinically used drugs and well-characterized pharmacological compounds for cystathionine ß-synthase inhibition identifies benserazide as a drug potentially suitable for repurposing for the experimental therapy of colon cancer.

Cystathionine-ß-synthase (CBS) has been recently identified as a drug target for several forms of cancer. Currently no potent and selective CBS inhibitors are available. Using a composite collection of 8871 clinically used drugs and well-annotated pharmacological compounds (including the LOPAC library, the FDA Approved Drug Library, the NIH Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the 'Killer Plates' collection and a small custom collection of PLP-dependent enzyme inhibitors), we conducted an in vitro screen in order to identify inhibitors for CBS using a primary 7-azido-4-methylcoumarin (AzMc) screen to detect CBS-derived hydrogen sulfide (H2S) production. Initial hits were subjected to counterscreens using the methylene blue assay (a secondary assay to measure H2S production) and were assessed for their ability to quench the H2S signal produced by the H2S donor compound GYY4137. Four compounds, hexachlorophene, tannic acid, aurintricarboxylic acid and benserazide showed concentration-dependent CBS inhibitory actions without scavenging H2S released from GYY4137, identifying them as direct CBS inhibitors. Hexachlorophene (IC50: ∼60µM), tannic acid (IC50: ∼40µM) and benserazide (IC50: ∼30µM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: ∼3µM) and NSC67078 (IC50: ∼1µM), while aurintricarboxylic acid (IC50: ∼3µM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: ∼1µM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50 of ∼6µM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: ∼6µM), tannic acid (IC50: ∼20µM), benserazide (IC50: ∼20µM), and NSC67078 (IC50: ∼0.3µM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: ∼300µM). In contrast, although a CBS inhibitor in the cell-free assay, aurintricarboxylic acid failed to inhibit HCT116 proliferation at lower concentrations, and stimulated cell proliferation at 300µM. Copper-containing compounds present in the libraries, were also found to be potent inhibitors of recombinant CBS; however this activity was due to the CBS inhibitory effect of copper ions themselves. However, copper ions, up to 300µM, did not inhibit HCT116 cell proliferation. Benserazide was only a weak inhibitor of the activity of the other H2S-generating enzymes CSE and 3-MST activity (16% and 35% inhibition at 100µM, respectively) in vitro. Benserazide suppressed HCT116 mitochondrial function and inhibited proliferation of the high CBS-expressing colon cancer cell line HT29, but not the low CBS-expressing line, LoVo. The major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity and suppressed HCT116 cell proliferation in vitro. In an in vivo study of nude mice bearing human colon cancer cell xenografts, benserazide (50mg/kg/days.q.) prevented tumor growth. In silico docking simulations showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.

The HIV Protease Inhibitor Saquinavir Inhibits HMGB1-Driven Inflammation by Targeting the Interaction of Cathepsin V with TLR4/MyD88.

Extracellular high-mobility group box 1 (HMGB1) (disulfide form), via activation of toll-like receptor 4 (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1-induced TLR4 activation, as assayed by the production of TNF-α by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334 or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver ischemia/reperfusion (I/R) models, both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4-driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside. Disulfide HMGB1 drives pathologic inflammation in many models by activating signaling through TLR4. Cell-based screening identified the mammalian protease cathepsin V as a novel therapeutic target to inhibit TLR4-mediated inflammation induced by extracellular HMGB1 (disulfide form). We identified two protease inhibitors (PIs) that block cathepsin V and thereby inhibit disulfide HMGB1-induced TLR4 activation: saquinavir (SQV), a first-generation PI targeting viral HIV protease and STO33438 (334), targeting mammalian proteases. We discovered that cathepsin V binds TLR4 under basal and HMGB1-stimulated conditions, but dissociates in the presence of SQV over time. Thus cathepsin V is a novel target for first-generation HIV PIs and represents a potential therapeutic target of pathologic inflammation.

Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation.

Hydrogen sulfide (H(2)S) is a unique gasotransmitter, with regulatory roles in the cardiovascular, nervous, and immune systems. Some of the vascular actions of H(2)S (stimulation of angiogenesis, relaxation of vascular smooth muscle) resemble those of nitric oxide (NO). Although it was generally assumed that H(2)S and NO exert their effects via separate pathways, the results of the current study show that H(2)S and NO are mutually required to elicit angiogenesis and vasodilatation. Exposure of endothelial cells to H(2)S increases intracellular cyclic guanosine 5'-monophosphate (cGMP) in a NO-dependent manner, and activated protein kinase G (PKG) and its downstream effector, the vasodilator-stimulated phosphoprotein (VASP). Inhibition of endothelial isoform of NO synthase (eNOS) or PKG-I abolishes the H(2)S-stimulated angiogenic response, and attenuated H(2)S-stimulated vasorelaxation, demonstrating the requirement of NO in vascular H(2)S signaling. Conversely, silencing of the H(2)S-producing enzyme cystathionine-γ-lyase abolishes NO-stimulated cGMP accumulation and angiogenesis and attenuates the acetylcholine-induced vasorelaxation, indicating a partial requirement of H(2)S in the vascular activity of NO. The actions of H(2)S and NO converge at cGMP; though H(2)S does not directly activate soluble guanylyl cyclase, it maintains a tonic inhibitory effect on PDE5, thereby delaying the degradation of cGMP. H(2)S also activates PI3K/Akt, and increases eNOS phosphorylation at its activating site S1177. The cooperative action of the two gasotransmitters on increasing and maintaining intracellular cGMP is essential for PKG activation and angiogenesis and vasorelaxation. H(2)S-induced wound healing and microvessel growth in matrigel plugs is suppressed by pharmacological inhibition or genetic ablation of eNOS. Thus, NO and H(2)S are mutually required for the physiological control of vascular function.

Modulation of poly(ADP-ribose) polymerase-1 (PARP-1)-mediated oxidative cell injury by ring finger protein 146 (RNF146) in cardiac myocytes.

Poly(ADP-ribose) polymerase-1 (PARP-1) activation is a hallmark of oxidative stress-induced cellular injury that can lead to energetic failure and necrotic cell death via depleting the cellular nicotinamide adenine dinucleotide (NAD(+)) and ATP pools. Pharmacological PARP-1 inhibition or genetic PARP-1 deficiency exert protective effects in multiple models of cardiomyocyte injury. However, the connection between nuclear PARP-1 activation and depletion of the cytoplasmic and mitochondrial energy pools is poorly understood. By using cultured rat cardiomyocytes, here we report that ring finger protein 146 (RNF146), a cytoplasmic E3-ubiquitin ligase, acts as a direct interactor of PARP-1. Overexpression of RNF146 exerts protection against oxidant-induced cell death, whereas PARP-1-mediated cellular injury is augmented after RNF146 silencing. RNF146 translocates to the nucleus upon PARP-1 activation, triggering the exit of PARP-1 from the nucleus, followed by rapid degradation of both proteins. PARP-1 and RNF146 degradation occurs in the early phase of myocardial ischemia-reperfusion injury; it precedes the induction of heat shock protein expression. Taken together, PARP-1 release from the nucleus and its rapid degradation represent newly identified steps of the necrotic cell death program induced by oxidative stress. These steps are controlled by the ubiquitin-proteasome pathway protein RNF146. The current results shed new light on the mechanism of necrotic cell death. RNF146 may represent a distinct target for experimental therapeutic intervention of oxidant-mediated cardiac injury.

Hydrogen sulfide replacement therapy protects the vascular endothelium in hyperglycemia by preserving mitochondrial function.

The goal of the present studies was to investigate the role of changes in hydrogen sulfide (H(2)S) homeostasis in the pathogenesis of hyperglycemic endothelial dysfunction. Exposure of bEnd3 microvascular endothelial cells to elevated extracellular glucose (in vitro "hyperglycemia") induced the mitochondrial formation of reactive oxygen species (ROS), which resulted in an increased consumption of endogenous and exogenous H(2)S. Replacement of H(2)S or overexpression of the H(2)S-producing enzyme cystathionine-γ-lyase (CSE) attenuated the hyperglycemia-induced enhancement of ROS formation, attenuated nuclear DNA injury, reduced the activation of the nuclear enzyme poly(ADP-ribose) polymerase, and improved cellular viability. In vitro hyperglycemia resulted in a switch from oxidative phosphorylation to glycolysis, an effect that was partially corrected by H(2)S supplementation. Exposure of isolated vascular rings to high glucose in vitro induced an impairment of endothelium-dependent relaxations, which was prevented by CSE overexpression or H(2)S supplementation. siRNA silencing of CSE exacerbated ROS production in hyperglycemic endothelial cells. Vascular rings from CSE(-/-) mice exhibited an accelerated impairment of endothelium-dependent relaxations in response to in vitro hyperglycemia, compared with wild-type controls. Streptozotocin-induced diabetes in rats resulted in a decrease in the circulating level of H(2)S; replacement of H(2)S protected from the development of endothelial dysfunction ex vivo. In conclusion, endogenously produced H(2)S protects against the development of hyperglycemia-induced endothelial dysfunction. We hypothesize that, in hyperglycemic endothelial cells, mitochondrial ROS production and increased H(2)S catabolism form a positive feed-forward cycle. H(2)S replacement protects against these alterations, resulting in reduced ROS formation, improved endothelial metabolic state, and maintenance of normal endothelial function.

Identification of agents that reduce renal hypoxia-reoxygenation injury using cell-based screening: purine nucleosides are alternative energy sources in LLC-PK1 cells during hypoxia.

Acute tubular necrosis is a clinical problem that lacks specific therapy and is characterized by high mortality rate. The ischemic renal injury affects the proximal tubule cells causing dysfunction and cell death after severe hypoperfusion. We utilized a cell-based screening approach in a hypoxia-reoxygenation model of tubular injury to search for cytoprotective action using a library of pharmacologically active compounds. Oxygen-glucose deprivation (OGD) induced ATP depletion, suppressed aerobic and anaerobic metabolism, increased the permeability of the monolayer, caused poly(ADP-ribose) polymerase cleavage and caspase-dependent cell death. The only compound that proved cytoprotective either applied prior to the hypoxia induction or during the reoxygenation was adenosine. The protective effect of adenosine required the coordinated actions of adenosine deaminase and adenosine kinase, but did not requisite the purine receptors. Adenosine and inosine better preserved the cellular ATP content during ischemia than equimolar amount of glucose, and accelerated the restoration of the cellular ATP pool following the OGD. Our results suggest that radical changes occur in the cellular metabolism to respond to the energy demand during and following hypoxia, which include the use of nucleosides as an essential energy source. Thus purine nucleoside supplementation holds promise in the treatment of acute renal failure.

Cardioprotective effects of hydrogen sulfide.

The gaseous mediator hydrogen sulfide (H(2)S) is synthesized mainly by cystathionine γ-lyase in the heart and plays a role in the regulation of cardiovascular homeostasis. Here we first overview the state of the art in the literature on the cardioprotective effects of H(2)S in various models of cardiac injury. Subsequently, we present original data showing the beneficial effects of parenteral administration of a donor of H(2)S on myocardial and endothelial function during reperfusion in a canine experimental model of cardiopulmonary bypass. Overview of the literature demonstrates that various formulations of H(2)S exert cardioprotective effects in cultured cells, isolated hearts and various rodent and large animal models of regional or global myocardial ischemia and heart failure. In addition, the production of H(2)S plays a role in myocardial pre- and post-conditioning responses. The pathways implicated in the cardioprotective action of H(2)S are multiple and involve K(ATP) channels, regulation of mitochondrial respiration, and regulation of cytoprotective genes such as Nrf-2. In the experimental part of the current article, we demonstrate the cardioprotective effects of H(2)S in a canine model of cardiopulmonary bypass surgery. Anesthetized dogs were subjected hypothermic cardiopulmonary bypass with 60 min of hypothermic cardiac arrest in the presence of either saline (control, n=8), or H(2)S infusion (1 mg/kg/h for 2 h). Left ventricular hemodynamic variables (via combined pressure-volume-conductance catheter) as well as coronary blood flow, endothelium-dependent vasodilatation to acetylcholine and endothelium-independent vasodilatation to sodium nitroprusside were measured at baseline and after 60 min of reperfusion. Ex vivo vascular function and high-energy phosphate contents were also measured. H(2)S led to a significantly better recovery of preload recruitable stroke work (p<0.05) after 60 min of reperfusion. Coronary blood flow was also significantly higher in the H(2)S group (p<0.05). While the vasodilatory response to sodium nitroprusside was similar in both groups, acetylcholine resulted in a significantly higher increase in coronary blood flow in the H(2)S-treated group (p<0.05) both in vivo and ex vivo. Furthermore, high-energy phosphate contents were better preserved in the H(2)S group. Additionally, the cytoprotective effects of H(2)S were confirmed also using in vitro cell culture experiments in H9c2 cardiac myocytes exposed to hypoxia and reoxygenation or to the cytotoxic oxidant hydrogen peroxide. Thus, therapeutic administration of H(2)S exerts cardioprotective effects in a variety of experimental models, including a significant improvement of the recovery of myocardial and endothelial function in a canine model of cardiopulmonary bypass with hypothermic cardiac arrest.

Reduction of liver ischemia-reperfusion injury via glutamine pretreatment.

BACKGROUND: Surgical methods that reduce bleeding during major hepatic resections lead to warm ischemia-reperfusion (I-R) injury of the liver. This is well known to have a considerable impact on the postoperative outcome. Much research work has been done to develop possible protective techniques. We aimed to investigate the effectivity of L-alanyl-L-glutamine dipeptide pretreatment in an animal model of hepatic I-R injury. MATERIALS AND METHODS: Male Wistar rats underwent normothermic, 60 min segmental liver ischemia followed by 24 h of reperfusion. The animals (n=30) were divided into three experimental groups: sham operated, I-R, and glutamine (Gln) pretreated. Twenty-four h prior to I-R injury, rats in the Gln group received 500 mg/kg Dipeptiven infusion as glutamine pretreatment. Hepatic microcirculation during the first hour of reperfusion was monitored by noninvasive laser Doppler flowmeter. After a 24-h reperfusion period, liver tissue was analyzed by histologic and immunohistochemical assessments. Serum necroenzyme and antioxidant levels were measured. RESULTS: In the Gln group, the integral of the reperfusion curve (RA) and the plateau maximum (PM(10)) of the flow graph showed improving tendency (RA: P=0.096; PM(10): P=0.084). Severity of histologic damage was reduced. Serum necroenzymes (ALT: P=0.042, AST: P=0.044) were significantly lower. Chemiluminescent intensity of liver and plasma was significantly decreased (P=0.0003 and P=0.0496). Further spectrophotometric analysis of liver homogenate samples also showed significant improvement of the redox homeostasis. CONCLUSIONS: Our results suggest that L-alanyl-L-glutamine dipeptide pretreatment given 24 h prior to I-R injury could be an effective method to reduce liver damage caused by hepatic inflow occlusion.

Regulation of kinase cascade activation and heat shock protein expression by poly(ADP-ribose) polymerase inhibition in doxorubicin-induced heart failure.

Cardiomyopathy is one of the most severe side effects of the chemotherapeutic agent doxorubicin (DOX). The formation of reactive oxygen species plays a critical role in the development of cardiomyopathies, and the pathophysiological cascade activates nuclear enzyme poly(ADP-ribose) polymerase (PARP), and kinase pathways. We characterized the effects of the PARP-inhibitor and kinase-modulator compound L-2286 in DOX-induced cardiac injury models. We studied the effect of the established superoxide dismutase-mimic Tempol and compared the effects of this agent with those of the PARP inhibitor. In the rat H9C2 cardiomyocytes, in which DOX-induced poly(ADP-ribosyl)ation, L-2286 protected them from the DOX-induced injury in a concentration-dependent manner. In the in vivo studies, mice were pretreated (for 1 week) with L-2286 or Tempol before the DOX treatment. Both the agents improved the activation of cytoprotective kinases, Akt, phospho-specific protein kinase C ϵ, ζ/λ and suppressed the activity of cell death promoting kinases glycogen synthase kinase-3ß, JNK, and p38 mitogen-activated protein kinase, but the effect of PARP inhibitor was more pronounced and improved the survival as well. L-2286 activated the phosphorylation of proapoptotic transcription factor FKHR1 and promoted the expression of Hsp72 and Hsp90. These data suggest that the mode of the cytoprotective action of the PARP inhibitor may include the modulation of kinase pathways and heat shock protein expression.

[Experimental food-induced fatty liver and its adjuvant therapy with natural bioactive substances]. / Alimentáris eredetu kísérletes zsírmáj és adjuváns kezelése természetes eredetu bioaktív hatóanyagokkal.

UNLABELLED: Changes of redox-homeostasis generate cytokines, and free radicals influence many intracellular signaling pathways in different liver diseases. Liophylised table beet and carrot powder (GPS Powder Kft. 1361/004/2003BFÁÉÉÁ) containing bioactive components such as betaine, betanins, betaxanthins, flavonoids, polyphenols, glutamine, beta carotene, vitamins and folic acid may induce changes in various cellular pathways. AIM: The aim of this study was to determine the protecting effects of bioactive agents of the liophylised table beet and carrot powder on fatty liver in a "short term" experiment. METHOD: Male Wistar rats were fed with chow with or without high fat (2% cholesterol, 0.5% cholic acid, 20% sunflower oil) and treated with 0.1 or 1 g/bwkg/day natural product for ten days parallel with the feedings. Cyclooxygenase-2, inducible nitric oxide synthase and tumor necrosis factor-α mRNA levels were determined using molecular biologic methods. Free radicals, H-donating activity, reducing power and free SH-group concentrations were determined by luminometry and spectrophotometry. Mobilized methyl groups were assayed by high pressure liquid chromatography method in liver homogenates. RESULTS: It was found that the higher dose of the natural product better decreased the induced free radical reactions, cyclooxygenase-2, inducible nitric oxide synthase and tumor necrosis factor-α mRNA-levels both in normal and fatty liver tissues. Although treatments failed to exert significant changes in all global antioxidant parameters, mobilized methyl group concentrations were higher after treatments in fatty liver. Favorable tendencies were also noted in the redox-homeostasis of the fatty liver after treatment. CONCLUSIONS: As expected, lyophylised table beet and carrot proved to be a "functional food" in rats with alimentary fat induced fatty liver. It cannot be ruled out that this beneficial effect may have clinical relevance.

Improvement of aging-associated cardiovascular dysfunction by the orally administered copper(II)-aspirinate complex.

BACKGROUND: Aging-associated nitro-oxidative stress causes tissue injury and activates proinflammatory pathways that play an important role in the pathogenesis of aging-associated cardiovascular dysfunction. It has been recently reported, that the copper(II)-aspirinate complex (CuAsp) exerts not only the well-known anti-inflammatory and platelet antiaggregating effects of aspirin, but, due to its superoxide dismutase mimetic activity, it acts as a potent antioxidant as well. In this study we investigated the effects of CuAsp on aging-associated myocardial and endothelial dysfunction. METHODS AND RESULTS: Aging and young rats were treated for 3 weeks with vehicle, or with CuAsp (200 mg/kg per day per os). Left ventricular pressure-volume relations were measured by using a microtip pressure-volume conductance catheter, and indexes of contractility (e.g., slope of end-systolic pressure-volume relationships [ESPVR] [E(es)], and dP/dt(max) - end-diastolic volume [EDV]) were calculated. In organ bath experiments for isometric tension with isolated aortic rings, endothelium-dependent and -independent vasorelaxation were investigated by using acetylcholine and sodium nitroprusside. When compared to the young controls, aging rats showed impaired left ventricular contractility (E(es), 0.51 +/- 0.04 vs. 2.16 +/- 0.28 mmHg/microL; dP/dt(max) - EDV, 10.71 +/- 2.02 vs. 37.23 +/- 4.18 mmHg/sec per microL; p < 0.05) and a marked endothelial dysfunction (maximal relaxation to acetylcholine: 66.66 +/- 1.30 vs. 87.09 +/- 1.35%; p < 0.05). Treatment with CuAsp resulted in reduced nitro-oxidative stress, improved cardiac function (E(es), 1.21 +/- 0.17 vs. 0.51 +/- 0.04 mmHg/microL; dP/dt(max) - EDV, 23.40 +/- 3.34 vs. 10.71 +/- 2.02 mmHg/sec per microL; p < 0.05) and higher vasorelaxation to acetylcholine in aging animals (94.83 +/- 0.73 vs. 66.66 +/- 1.30%; p < 0.05). The treatment did not influence the cardiovascular functions of young rats. CONCLUSIONS: Our results demonstrate that oxidative stress and inflammatory pathways contribute to the pathogenesis of cardiovascular dysfunction in the aging organism, which can be reversed by CuAsp.

Development of a stretch-induced neurotrauma model for medium-throughput screening in vitro: identification of rifampicin as a neuroprotectant.

BACKGROUND AND PURPOSE: We hypothesized that an in vitro, stretch-based model of neural injury may be useful to identify compounds that decrease the cellular damage in neurotrauma. EXPERIMENTAL APPROACH: We screened three neural cell lines (B35, RN33B and SH-SY5Y) subjected to two differentiation methods and selected all-trans-retinoic acid-differentiated B35 rat neuroblastoma cells subjected to rapid stretch injury, coupled with a subthreshold concentration of H2 O2 , for the screen. The model induced marked alterations in gene expression and proteomic signature of the cells and culminated in delayed cell death (LDH release) and mitochondrial dysfunction [reduced 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) conversion]. Follow-up studies utilized human stem cell-derived neurons subjected to rapid stretch injury. KEY RESULTS: From screening of a composite library of 3500 drugs, five drugs (when applied in a post-treatment regimen relative to stretch injury) improved both LDH and MTT responses. The effects of rifampicin were investigated in further detail. Rifampicin reduced cell necrosis and apoptosis and improved cellular bioenergetics. In a second model (stretch injury in human stem cell-derived neurons), rifampicin pretreatment attenuated LDH release, protected against the loss of neurite length and maintained neuron-specific class III ß-tubulin immunoreactivity. CONCLUSIONS AND IMPLICATIONS: We conclude that the current model is suitable for medium-throughput screening to identify compounds with neuroprotective potential. Rifampicin, when applied either in pre- or post-treatment, improves the viability of neurons subjected to stretch injury and protects against neurite loss. Rifampicin may be a candidate for repurposing for the therapy of traumatic brain injury. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

The peroxynitrite decomposition catalyst FP15 improves ageing-associated cardiac and vascular dysfunction.

Overproduction of oxidants and free radicals in ageing tissues induces nitro-oxidative stress, which has recently been implicated in the pathogenesis of cardiovascular dysfunction associated with ageing. Peroxynitrite, a strong cytotoxic oxidant damages proteins and DNA and activates several pathways causing tissue injury, including the peroxynitrite-poly(ADP-ribose) polymerase (PARP) pathway. In this study, we investigated the effectiveness of the peroxynitrite decomposition catalyst FP15 on ageing-associated cardiac and vascular dysfunction. Young and ageing rats were treated with vehicle or FP15 intraperitoneally. Using a microtip Millar pressure catheter we performed left ventricular blood pressure analysis to assess systolic and diastolic function. Endothelium-dependent and -independent vasorelaxation of isolated aortic rings were investigated by using acetylcholine and sodium nitroprusside. Ageing animals showed a marked reduction of systolic and diastolic cardiac function and loss of endothelium-dependent relaxant responsiveness of aortic rings. FP15-treatment significantly improved cardiac performance and endothelial function. Immunohistochemical staining confirmed that FP15 effectively reduced nitrosative stress and prevented the activation of PARP in the aortic wall of ageing rats. Our results demonstrate the importance of endogenous peroxynitrite-overproduction in the pathogenesis of ageing-associated cardiovascular dysfunction. Pharmacological decomposition of peroxynitrite by FP15 may represent a novel therapeutic utility to improve cardiac and vascular dysfunction associated with ageing.

Single dose treatment with PARP-inhibitor INO-1001 improves aging-associated cardiac and vascular dysfunction.

Overproduction of reactive oxygen species in aging tissues has been implicated in the pathogenesis of aging-associated cardiovascular dysfunction. Oxidant-induced DNA-damage activates the poly(ADP-ribose) polymerase (PARP) pathway, leading to tissue injury. In this study we investigated the acute effects of the PARP inhibitor INO-1001 on aging-associated cardiac and endothelial dysfunction. Using a pressure-volume conductance catheter, left ventricular pressure-volume analysis of young and aging rats was performed before and after a single injection of INO-1001. Endothelium-dependent and -independent vasorelaxation of isolated aortic rings were investigated by using acetylcholine and sodium nitroprusside. Aging animals showed a marked reduction of myocardial contractility and endothelium-dependent relaxant responsiveness of aortic rings. Single dose INO-1001-treatment resulted in acute improvement in their cardiac and endothelial function. Immunohistochemistry for nitrotyrosine and poly(ADP-ribose) confirmed enhanced nitro-oxidative stress and PARP-activation in aging animals. Acute treatment with INO-1001 decreased PARP-activation, but did not affect nitrotyrosine-immunoreactivity. Our results demonstrate that the aging-associated chronic cardiovascular dysfunction can be improved, at least, short term, by a single treatment course with a PARP-inhibitor, supporting the role of the nitro-oxidative stress -- PARP -- pathway in the age-related functional decline of the cardiovascular system. Pharmacological inhibition of PARP may represent a novel therapeutic utility to improve aging-associated cardiovascular dysfunction.

Poly(ADP-ribose) polymerase inhibition improves endothelial dysfunction induced by reactive oxidant hydrogen peroxide in vitro.

Reactive oxygen species, such as hydrogen peroxide (H(2)O(2)) induce oxidative stress and DNA-injury. The subsequent activation of poly(ADP-ribose) polymerase (PARP) has been implicated in the pathogenesis of various cardiovascular diseases including ischaemia-reperfusion injury, circulatory shock, diabetic complications and atherosclerosis. We investigated the effect of PARP-inhibition on endothelial dysfunction induced by H(2)O(2). In vascular reactivity measurements on isolated rat aortic rings we investigated the phenylephrine-induced contraction, and endothelium-dependent and -independent vasorelaxation by using cumulative concentrations of acetylcholine and sodium nitroprusside. Endothelial dysfunction was induced by exposing the rings to H(2)O(2) (200 and 400 muM) for 30 min. In the treatment group, rings were preincubated with the potent PARP-inhibitor INO-1001. DNA strand breaks were assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. Immunohistochemical analysis was performed for poly(ADP-ribose) (the enzymatic product of PARP) and for apoptosis inducing factor (a pro-apoptotic factor regulated by PARP). Exposure to H(2)O(2) resulted in reduced contraction forces and a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings (maximal relaxation to acetylcholine: 86.21+/-1.574% control vs. 72.55+/-1.984% H(2)O(2) 200 muM vs. 66.86+/-1.961% H(2)O(2) 400 muM; P<0.05). PARP-inhibition significantly improved the acetylcholine-induced vasorelaxation (77.75+/-3.019% vs. 66.86+/-1.961%; P<0.05), while the contractility remained unaffected. The dose-response curves of endothelium-independent vasorelaxation to sodium nitroprusside did not differ in any groups studied. In the H(2)O(2) groups immunohistochemical analysis showed enhanced PARP-activation and nuclear translocation of apoptosis inducing factor, which were prevented by INO-1001. Our results demonstrate that PARP activation contributes to the pathogenesis of H(2)O(2)-induced endothelial dysfunction, which can be prevented by PARP inhibitors.

Poly(ADP-Ribose) polymerase inhibition improves endothelial dysfunction induced by hypochlorite.

Reactive oxygen species, such as myeloperoxidase-derived hypochlorite, induce oxidative stress and DNA injury. The subsequent activation of the DNA-damage-poly(ADP-ribose) polymerase (PARP) pathway has been implicated in the pathogenesis of various diseases, including ischemia-reperfusion injury, circulatory shock, diabetic complications, and atherosclerosis. We investigated the effect of PARP inhibition on the impaired endothelium-dependent vasorelaxation induced by hypochlorite. In organ bath experiments for isometric tension, we investigated the endothelium-dependent and endothelium-independent vasorelaxation of isolated rat aortic rings using cumulative concentrations of acetylcholine and sodium nitro-prusside. Endothelial dysfunction was induced by exposing rings to hypochlorite (100-400 microM). In the treatment group, rings were preincubated with the PARP inhibitor INO-1001. DNA strand breaks were assessed by the TUNEL method. Immunohistochemistry was performed for 4-hydroxynonenal (a marker of lipid peroxidation), nitrotyrosine (a marker of nitrosative stress), and poly(ADP-ribose) (an enzymatic product of PARP). Exposure to hypochlorite resulted in a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings, which was significantly improved by PARP inhibition, whereas the endothelium-independent vasorelaxation remained unaffected. In the hypochlorite groups we found increased DNA breakage, lipidperoxidation, and enhanced nitrotyrosine formation. The hypochloride-induced activation of PARP was prevented by INO-1001. Our results demonstrate that PARP activation contributes to the pathogenesis of hypochlorite-induced endothelial dysfunction, which can be prevented by PARP inhibitors.

Short-term alanyl-glutamine dipeptide pretreatment in liver ischemia-reperfusion model: effects on microcirculation and antioxidant status in rats.

BACKGROUND & AIMS: Ischemia-reperfusion (I-R) injury is responsible for the morbidity associated with liver surgery. Production of toxic free radicals influences the microcirculation. The aim of our study was to examine the effect of glutamine (Gln) supplementation--adminstered in alanyl-glutamine dipeptide form--on liver function, immuno/histopathology and the oxidative state of the liver after injury. METHODS: Two-hundred and fifty grams male Wistar rats underwent normothermic, 60 min, segmental liver ischemia followed by 6 h of reperfusion. The animals (n = 45) were divided into three groups: sham operated, I-R and parenteral Gln pretreatment. Hepatic microcirculation was monitored by laser Doppler flowmetry. At the 6 h of reperfusion, histological alterations, TUNEL reaction, active caspase-3 reaction, serum and liver tissue antioxidant levels, serum ALAT, ASAT and TNF-alpha levels were measured. RESULTS: Upon reperfusion, the Gln group had significantly (p<0.05) higher flow rates than the I-R group and, at the end of the 6h of reperfusion, significantly (p<0.05) lower serum ALAT and ASAT levels. The liver chemiluminescent intensity was lower, free SH-groups were elevated, while the reducing power was decreased in the Gln-pretreated group. Positive staining for caspase-3 after Gln pretreatment was significantly increased in contrast to the control tissues. CONCLUSION: Glutamine pretreatment is beneficial in supporting hepatic microcirculation and can prevent hepatocellular necrosis in liver reperfusion injury.

Oxidant-induced cardiomyocyte injury: identification of the cytoprotective effect of a dopamine 1 receptor agonist using a cell-based high-throughput assay.

Myocyte injury due to myocardial reperfusion injury plays a crucial role in the pathogenesis of acute myocardial infarction even after successful coronary revascularization. Identification of compounds that reduce reperfusion-associated myocyte death is important. Therefore, we developed an in vitro model of myocardial reperfusion injury in H9c2 rat cardiomyocytes and applied a cell-based high-throughput approach to screen a standard library of pharmacologically active compounds (LOPAC) in order to identify drugs with cardioprotective effects. Oxidative stress was induced with hydrogen peroxide (H2O2) treatment, which resulted in approximately 50% reduction in cell viability. Test compounds were added at a 3-microM final concentration as a pretreatment or in a delayed fashion (30 min after the peroxide challenge in order to imitate pharmacological treatment following angioplasty). Cells were cultured for 3 or 24 h. Viability was quantitated with the methylthiazolyldiphenyl-tetrazolium bromide method. Cytotoxicity and cytoprotection were also evaluated by measuring the lactate dehydrogenase activity in the cell culture supernatant. The screening identified a number of compounds with cytoprotective action, including molecules that are known to interfere with components of DNA repair and cell cycle progression, e.g. poly(ADP-ribose) polymerase (PARP) inhibitors, topoisomerase inhibitors, and cyclin dependent kinase inhibitors, or reduce energy consumption by interfering with cardiac myofilament function. A number of dopamine D1 receptor agonists also provided significant cytoprotection at 3 h, but only three of them showed a similar effect at 24 h: chloro- and bromo-APB and chloro-PB hydrobromide. Chloro-APB hydrobromide significantly reduced peroxide-induced PARP activation in the myocytes independently of its action on dopamine D1 receptors, but lacked PARP inhibitor capacity in a cell-free PARP assay system. In conclusion, the pattern of cytoprotective drugs identified in the current assay supports the overall validity of our model system. The findings demonstrate that cytoprotective agents, including novel indirect inhibitors of cellular PARP activation can be identified with the method, chloro-APB hydrobromide being one such compound. The current experimental setting can be employed for cell-based high-throughput screening of various compound libraries.

Role of the peroxynitrite-poly (ADP-ribose) polymerase pathway in the pathogenesis of liver injury.

Oxidative and nitrosative stress triggers DNA strand breakage, which then activates the nuclear enzyme poly(ADP-ribose) polymerase (PARP). One of the key triggers of DNA single strand breakage in pathophysiological conditions is peroxynitrite, a reactive species produced from the reaction of nitric oxide and superoxide. Activation of PARP can dramatically lower the intracellular concentration of its substrate, nicotinamide adenine dinucleotide, thus slowing the rate of glycolysis, electron transport and subsequently ATP formation. This process can result in cell dysfunction and cell death. Here we review the role of PARP in various forms of liver injury.