Francisco Garcia Soriano

Diabetic endothelial dysfunction: the role of poly(ADP-ribose) polymerase activation

Diabetic patients frequently suffer from retinopathy, nephropathy, neuropathy and accelerated atherosclerosis. The loss of endothelial function precedes these vascular alterations. Here we report that activation of poly(ADP-ribose) polymerase (PARP) is an important factor in the pathogenesis of endothelial dysfunction in diabetes. Destruction of islet cells with streptozotocin in mice induced hyperglycemia, intravascular oxidant production, DNA strand breakage, PARP activation and a selective loss of endothelium-dependent vasodilation. Treatment with a novel potent PARP inhibitor, starting after the time of islet destruction, maintained normal vascular responsiveness, despite the persistence of severe hyperglycemia. Endothelial cells incubated in high glucose exhibited production of reactive nitrogen and oxygen species, consequent single-strand DNA breakage, PARP activation and associated metabolic and functional impairment. Basal and high-glucose-induced nuclear factor-κB activation were suppressed in the PARP-deficient cells. Our results indicate that PARP may be a novel drug target for the therapy of diabetic endothelial dysfunction.

Flagellin, a Novel Mediator of Salmonella-Induced Epithelial Activation and Systemic Inflammation: IκBα Degradation, Induction of Nitric Oxide Synthase, Induction of Proinflammatory Mediators, and Cardiovascular Dysfunction

Gram-negative sepsis is mediated by the actions of proinflammatory genes induced in response to microbes and their products. We report that flagellin, the monomeric subunit of flagella, is a potent proinflammatory species released by Salmonella. Flagellin (1 μg/ml) induces IκBα degradation, NF-κB nuclear translocation, and inducible NO synthase expression in cultured intestinal epithelial cells (IEC). Aflagellic Salmonella mutants do not induce NF-κB activation or NO production by cultured IEC. Antiserum to flagellin blocks NO production in IEC induced by medium conditioned by a variety of motile Gram-negative enteric pathogens (Escherichia coli, Salmonella muenchen, Serratia marcescens, Proteus mirabilis, and Proteus vulgaris). Flagellin, when injected systemically (∼10 μg/mouse), induces systemic inflammation characterized by the systemic expression of a range of proinflammatory cytokines and chemokines and of inducible NO synthase. At higher doses (∼300 μg/mouse), flagellin induces shock, characterized by hypotension, reduced vascular contractility in mice, and death. The effects of flagellin do not diminish in C3H/HeJ LPS-resistant mice, indicating that the Toll-like receptor-4 receptor is not involved in flagellin’s actions. In LPS-resistant mice, i.p. injection of S. dublin flagellin or medium conditioned by wild-type S. dublin induces serum IFN-γ and TNF-α, whereas medium conditioned by aflagellic mutants has no effect. Flagellin can be detected in the blood of rats with septic shock induced by live bacteria at approximately 1 μg/ml. We propose that flagellin released by Gram-negative pathogens may contribute to the inflammatory response by an LPS- and Toll-like receptor-4-independent pathway.

Novel phenanthridinone inhibitors of poly(adenosine 5'-diphosphate-ribose) synthetase: Potent cytoprotective and antishock agents

ObjectiveTo synthesize novel inhibitors of the nuclear enzyme poly(adenosine 5′-diphosphate [ADP]-ribose) synthetase (PARS), also known as poly(ADP-ribose) polymerase (PARP), and to test them in in vitro models of oxidant-induced cytotoxicity and in endotoxin and splanchnic occlusion-reperfusion-induced shock. DesignRandomized, prospective laboratory study. SettingResearch laboratory. SubjectsMurine macrophages, thymocytes, and endothelial cells; Balb/c mice and Wistar rats. InterventionsMacrophages and endothelial cells were treated with peroxynitrite and bleomycin to induce PARS activation, and thymocytes were treated with peroxynitrite to induce cell necrosis. Novel PARS inhibitors were synthesized and used to reduce PARS activation and to reverse cytotoxicity. Balb/c mice were subjected to splanchnic occlusion and reperfusion and were pretreated with various doses (1–10 mg/kg intraperitoneally) of PJ34, a selected, potent, water-soluble PARS inhibitor. The passage of fluorescein isothiocyanate-conjugated dextran (4 kDa) was analyzed in everted gut ileal sacs incubated ex vivo as an index of gut permeability. Wistar rats were subjected to Escherichia coli bacterial lipopolysaccharide (40 mg/kg intraperitoneally). PJ34 was also used at 10 mg/kg intraperitoneally, 1 hr before lipopolysaccharide or at 25 mg/kg intraperitoneally 1 hr after lipopolysaccharide treatment. Serum concentrations of indicators or multiple organ injury, concentrations of various proinflammatory mediators, and tissue concentrations of myeloperoxidase and malondialdehyde were measured. In addition, survival rates and vascular contractile and relaxant responses were recorded. Measurements and Main ResultsAppropriate modifications of the phenanthridinone core structure yielded significant increases in the potency of the compounds, both as PARS inhibitors and as cytoprotective agents. The compound N-(6-oxo-5,6-dihydro-phenanthridin-2-yl) -N,N-dimethylacetamide (designated as PJ34) was one of the potent PARS inhibitors of the series, and it dose-dependently protected against thymocyte necrosis, with a half-maximal restoration of cell viability of 35 nM and complete protection at 200 nM. PARS activation also was visualized by immunohistochemistry and was dose-dependently suppressed by PJ34. The effect of PJ34 was dose-dependently reversed by excess nicotinamide adenine dinucleotide (oxidized). The PARS inhibitors dose-dependently suppressed proinflammatory cytokine and chemokine production and restored viability in immunostimulated macrophages. PJ34 was selected for the subsequent in vivo studies. PJ34 significantly protected against splanchnic reperfusion-induced intestinal hyperpermeability in the mouse. PJ34 reduced peak plasma concentrations of tumor necrosis factor-&agr;, interleukin-1&bgr;, and nitrite/nitrate in the plasma of lipopolysaccharide-treated rats. PJ34 ameliorated the lipopolysaccharide-induced increases in indexes of liver and kidney failure and concentrations of myeloperoxidase and malondialdehyde in the lung and gut. Lipopolysaccharide elicited vascular dysfunction, which was normalized by PJ34. Lipopolysaccharide-induced mortality was reduced by PJ34 (both pre- and posttreatment). ConclusionsThe novel series of phenanthridinone PARS inhibitors have potent cytoprotective effects in vitro and significant protective effects in shock and reperfusion injury in rodent models in vivo.

Diabetic endothelial dysfunction: Role of reactive oxygen and nitrogen species production and poly(ADP-ribose) polymerase activation

Peroxynitrite and hydroxyl radicals are potent initiators of DNA single-strand breakage, which is an obligatory stimulus for the activation of the nuclear enzyme poly(ADP ribose) polymerase (PARP). In response to high glucose incubation medium in vitro, or diabetes and hyperglycemia in vivo, reactive nitrogen and oxygen species generation occurs. These reactive species trigger DNA single-strand breakage, which induces rapid activation of PARP. PARP in turn depletes the intracellular concentration of its substrate, NAD+, slowing the rate of glycolysis, electron transport, and ATP formation. This process results in acute endothelial dysfunction in diabetic blood vessels. Accordingly, inhibitors of PARP protect against endothelial injury under these conditions. In addition to the direct cytotoxic pathway regulated by DNA injury and PARP activation, PARP also appears to modulate the course of inflammation by regulating the activation of nuclear factor κB, and the expression of a number of genes, including the gene for intercellular adhesion molecule 1 and the inducible nitric oxide synthase. The research into the role of PARP in diabetic vascular injury is now supported by novel tools, such as new classes of potent inhibitors of PARP and genetically engineered animals lacking the gene for PARP. Pharmacological inhibition of PARP emerges as a potential approach for the experimental therapy of diabetic vascular dysfunction.

Resistance to acute septic peritonitis in poly(ADP-ribose) polymerase-1-deficient mice

Sepsis is associated with a widespread production of proinflammatory cytokines and various oxidant species. Activation of the enzyme poly(ADP-ribose) polymerase (PARP) has been shown to contribute to cell necrosis and organ failure in various diseases associated with inflammation and reperfusion injury. The aim of the current study was to elucidate the role of PARP activation in the multiple organ dysfunction complicating sepsis in a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Mice genetically deficient in PARP (PARP−/−) and their wild-type littermates (PARP+/+) were subjected to CLP. After 12 and 24 h, the proinflammatory cytokines TNF-&agr; and IL-6, as well as the anti-inflammatory cytokine IL-10, and nitrite/nitrate were measured in plasma samples. Organs were harvested for the measurement of myeloperoxidase (MPO) and malondialdehyde (MDA) levels, and immunohistochemical staining for nitrotyrosine and poly(ADP ribose) was performed in gut sections. PARP−/− mice, and their wild-type littermate showed a similar time-dependent increase in plasma nitrite/nitrate and in gut and lung MDA content, as well as the presence of nitrotyrosine in the gut. In contrast to wild-type mice showing a PARP activation in the gut, PARP−/− mice had no staining for poly(ADP ribose). PARP−/− mice had significantly lower plasma levels of TNF-&agr;, IL-6, and IL-10, and they exhibited a reduced degree of organ inflammation, indicated by decreased MPO activity in the gut and lung. These effects were associated with a significant improvement in the survival of CLP in PARP−/− mice. Thus, PARP activation has an important role in systemic inflammation and organ damage in the present model of polymicrobial septic shock.

Anti-inflammatory effects of a novel, potent inhibitor of poly (ADP-ribose) polymerase

Abstract.Objective and design: Oxygen- and nitrogen-derived free radicals and oxidants play an important role in the pathogenesis of various forms of inflammation. Recent work emphasizes the importance of oxidant-induced DNA strand breakage and activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) in the pathogenesis of various inflammatory diseases. We have recently demonstrated the efficacy of PJ34, a novel, potent phenanthridinone derivative PARP inhibitor, in rodent models of diabetic vascular dysfunction and stroke. Here we tested the efficacy of PARP inhibition in various models of local inflammation in rodents.¶Materials and methods: PJ34 (at doses of 0.03-30 mg/kg) was tested in rats and mice subjected to standard models of inflammation, with relevant parameters of inflammation measured using standard methods.¶Results: PJ34 treatment (s.c, i.p. and i.v.) dose-dependently suppressed neutrophil infiltration and nitric oxide (but not KC and IL-1β) production in peritonitis. In a model of systemic endotoxemia, PJ34 pretreatment significantly reduced plasma levels of TNF-α, IL-1β and nitrite/nitrate (breakdown products of nitric oxide) production. PJ34 treatment (oral gavage) induced a significant suppression of the inflammatory response in dextran sulfate colitis, multiple low dose streptozotocin diabetes and cyclophosphamide-accelerated autoimmune diabetes in the non-obese diabetic mice, and reduced the degree of mononuclear cell infiltration into the iris in an endotoxin-induced uveitis model. Delaying the start of PJ34 administration in the colitis model conferred significant protective effects, while in the arthritis model the post-treatment paradigm lacked protective effects.¶Conclusions: PJ34 provides significant, dose-dependent, anti-inflammatory effects in a variety of local inflammation models. Some of its actions are maintained in the post-treatment regimen and/or after discontinuation of treatment. We conclude that PARP inhibition offers a powerful means for reducing the severity of various forms of local inflammatory responses.

Flagellin from gram-negative bacteria is a potent mediator of acute pulmonary inflammation in sepsis.

Flagellin is a recently identified bacterial product that elicits immune response via toll-like receptor 5. Here, we demonstrate that flagellin is an extraordinarily potent proinflammatory stimulus in the lung during sepsis. In vitro, flagellin triggers the production of interleukin (IL)-8 by human lung epithelial (A549) cells, with 50% of the maximal response obtained at a concentration of 2 × 10−14 M. Flagellin also induces the expression of ICAM-1 in vitro. Intravenous administration of flagellin to mice elicited a severe acute lung inflammation that was significantly more pronounced than following lipopolysaccharide (LPS) administration. Flagellin induced a local release of proinflammatory cytokines, the accumulation of inflammatory cells, and the development of pulmonary hyperpermeability. These effects were associated with the nuclear translocation of the transcription NF-&kgr;B in the lung. Flagellin remained active in inducing pulmonary inflammation at doses as low as 10 ng/mouse. In the plasma of patients with sepsis, flagellin levels amounted to 7.1 ± 0.1 ng/mL. Plasma flagellin levels showed a significant positive correlation with the lung injury score, with the alveolar-arterial oxygen difference as well as with the duration of the sepsis. Flagellin emerges as a potent trigger of acute respiratory complications in gram-negative bacterial sepsis.

Poly (ADP-ribose) synthetase mediates intestinal mucosal barrier dysfunction after mesenteric ischemia

Peroxynitrite-mediated DNA strand breaks trigger poly (ADP-ribose) synthetase (PARS) activation, resulting in intracellular energetic failure and organ dysfunction. We investigated the role of PARS activation on the inflammatory and functional response of the intestine to mesenteric ischemia-reperfusion injury. Anesthetized rats exposed to 15 min occlusion of the superior mesenteric artery showed an increased mucosal PARS activity (ex vivo incorporation of radiolabelled NAD+ in gut mucosal scrapings) as soon as 10 min after reperfusion. During the first 30 min of reperfusion, significant mucosal damage developed, as well as mucosal hyperpermeability to a 4000 MW fluorescent dextran (FD4). These alterations were significantly reduced by treatment with the NO synthase inhibitor L-NMA, which blocks the production of peroxynitrite, as well as with the PARS inhibitors 3-aminobenzamide and nicotinamide, whereas they were markedly enhanced by the glutathione depletor L-buthionine-(S,R)-sulfoximine. Also, PARS inhibition significantly reduced ileal neutrophil infiltration (myeloperoxidase activity) at 3 h reperfusion. In a second set of experiments, the effects of 15 or 30 min ischemia followed by 3 h reperfusion were evaluated in PARS knockout and wild-type mice. Significant protection against histological damage, neutrophil infiltration, and mucosal barrier failure (evaluated by the mucosal-to-serosal FD4 clearance of everted ileal sacs incubated ex vivo) was noted in PARS knockout mice, who also showed reduced alterations in remote organs, as shown by lesser lipid peroxidation (malondialdehyde formation) and neutrophil infiltration in the lung and liver. In conclusion, PARS plays a crucial role in mediating intestinal injury and dysfunction in the early and late phases of mesenteric reperfusion. Pharmacological inhibition of PARS may be a novel approach to protect tissues from reperfusion-related damage.

Endothelial dysfunction in aging animals: the role of poly(ADP-ribose) polymerase activation

Recent work has demonstrated the production of reactive oxygen and nitrogen species in the vasculature of aging animals. Oxidant induced cell injury triggers the activation of nuclear enzyme poly(ADP ribose) polymerase (PARP) leading to endothelial dysfunction in various pathophysiological conditions (reperfusion, shock, diabetes). Here we studied whether the loss of endothelial function in aging rats is dependent upon the PARP pathway within the vasculature. Young (3 months‐old) and aging (22 months‐old) Wistar rats were treated for 2 months with vehicle or the PARP inhibitor PJ34. In the vehicle‐treated aging animals there was a significant loss of endothelial function, as measured by the relaxant responsiveness of vascular rings to acetylcholine. Treatment with PJ34, a potent PARP inhibitor, restored normal endothelial function. There was no impairment of the contractile function and endothelium‐independent vasodilatation in aging rats. Furthermore, we found no deterioration in the myocardial contractile function in aging animals. Thus, intraendothelial PARP activation may contribute to endothelial dysfunction associated with aging.

Potential role of poly(adenosine 5'-diphosphate-ribose) polymerase activation in the pathogenesis of myocardial contractile dysfunction associated with human septic shock.

Objective:Sepsis is associated with increased production of superoxide and nitric oxide, with consequent peroxynitrite generation. Cardiodepression is induced in the heart during oxidative stress associated with septic shock. Oxidative and nitrosative stress can lead to activation of the nuclear enzyme poly(adenosine 5′-diphosphate [ADP]-ribose) polymerase (PARP), with subsequent loss of myocardial contractile function. The aim of the study was to investigate whether cardiodepression found in septic patients is associated with plasma markers of myocardial necrosis and with myocardial PARP activation. Design:Prospective and observational study. Setting:University hospital intensive care unit for clinical and surgical patients. Patients:Twenty-five patients older than 18 yrs presenting with severe sepsis or septic shock. Patients with history of chronic heart failure, cancer, coronary artery disease, diabetes, or acquired immune deficiency syndrome were excluded. Interventions:Patients were followed for 28 days, and biochemical and hemodynamic data were collected on days 1, 3, and 6 of sepsis. The groups were survivors and nonsurvivors, defined only after the end of clinical patient evolution. Heart sections from patients who died were analyzed with hematoxylin-eosin and Picro Sirius-Red immunostaining and with electron microscopy. Measurements and Main Results:The study population included 25 individuals, of whom 12 (48%) died during the 6 days of follow-up. The initial data of the inflammation marker C-reactive protein and Acute Physiologic and Chronic Health. Evaluation severity were similar in both groups (nonsurvivors, 26 ± 2; survivors, 24 ± 5; NS). Overall, an increase in plasma troponin level was related to increased mortality risk. In patients who died, significant myocardial damage was detected, and histologic analysis of heart sections showed inflammatory infiltration, increased collagen deposition, and derangement of mitochondrial cristae. Immunohistochemical staining for poly(ADP-ribose) (PAR), the product of activated PARP, was demonstrated in septic hearts. There was a positive correlation between PAR staining densitometry and troponin I (r2 = 0.73; p < .05), and the correlation of PAR staining densitometry and left ventricular systolic stroke work index was r2 = 0.33 (p = .0509). Conclusion:There is significant PARP activation in the hearts of septic patients with impaired cardiac function. We hypothesize that PARP activation may be partly responsible for the cardiac depression seen in humans with severe sepsis.