David Hernández-Espinosa

Inhibition of poly(ADP-ribose) polymerase attenuates the severity of acute pancreatitis and associated lung injury

The severity of acute pancreatitis results from the transmigration and activation of leukocytes within the pancreas and the local synthesis and release of proinflammatory-soluble mediators that transform a local injury into a systemic inflammatory response. Poly(ADP-ribose)polymerase-1 (PARP-1) is a nuclear DNA-binding protein that has been shown to play a relevant role in cell necrosis and organ failure in various diseases associated with inflammation. Therefore, we set out to investigate whether the genetic deletion of PARP-1 or PARP-2 (a new member of the PARP family) genes, or pharmacological inhibition of PARP activity might affect the development and severity of acute pancreatitis and pancreatitis-associated lung injury. Secretagogue-induced acute pancreatitis was achieved by 12 hourly intraperitoneal injections of cerulein in mice deficient in PARP-1 or PARP-2 genes, and wild-type (WT) littermate mice untreated or treated with PARP activity inhibitors. The severity of pancreatitis was assessed by measurements of serum amylase, lipase, interleukin-1β and IL-6, pancreatic water content, histologic grading and pancreas myeloperoxidase (MPO) activity. Lung injury was evaluated by quantifying MPO activity and morphological changes. We found that the severity of acute pancreatitis and pancreatitis-associated lung injury was significantly attenuated in mice lacking PARP-1, but not PARP-2, compared with WT mice. Interestingly, administration of PARP inhibitors, 3-aminobenzamide or PJ34 (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethyacetamide HCl), in WT mice markedly decreased acute pancreatitis severity and pulmonary-associated injury in a larger extension than genetic deletion of PARP-1. Our results support the potential therapeutic application of PARP inhibitors in the development and severity of acute pancreatitis and associated lung injury.

Protein Z/Z‐dependent protease inhibitor (PZ/ZPI) anticoagulant system and thrombosis

A new anticoagulant system involving a serpin has been recently characterised. The protein Z/Z‐dependent protease inhibitor (PZ/ZPI) system inhibits activated factors X, XI and IX by different mechanisms. By homology with other anticoagulant systems (antithrombin or the protein C/protein S), deficiency of the serpin (ZPI) or its cofactor (PZ) might imbalance the haemostatic system with thrombotic consequences. Evidence supports the in vivo anticoagulant role of this complex and the thrombotic consequences of its deficiency. Non‐sense variations of the ZPI (W303X and R67X) have been associated with increased risk of venous thrombosis. Moreover, PZ deficient mice carrying the FV Leiden have a thrombotic phenotype. Finally, some reports suggest that PZ deficiency might increase the risk of thrombosis. However, other studies question the thrombotic relevance of both ZPI and PZ deficiencies. This system could play a redundant role in haemostasis that explains the conflicting results on its thrombotic potential, which might be exacerbated in combination with other prothrombotic factors.

Therapeutic treatment with poly(ADP-ribose) polymerase inhibitors attenuates the severity of acute pancreatitis and associated liver and lung injury

The mortality associated with acute pancreatitis (AP) is largely attributable to abnormalities that occur in distant organs and supportive care remains the only treatment for patients with these complications. Recently, prophylactic pharmacological blockade of poly(ADP‐ribose) polymerase (PARP) enzymes has been shown to attenuate the severity of the disease. However, the clinical relevance of PARP inhibitors administered after the onset of AP remains uncertain. The aim of the present study was to investigate the therapeutic effects of PARP inhibitors in established AP.

Role of lipopolysaccharide and cecal ligation and puncture on blood coagulation and inflammation in sensitive and resistant mice models.

The hemostatic system is severely disturbed during endotoxemia, leading to a hypercoagulable state. However, it remains uncertain to what extent hypercoagulability is the critical factor in determining the clinical course rather than just the consequence of a severe systemic inflammatory response. To answer this question, we evaluated the evolution of hemostatic and inflammatory markers, as well as histological features, in mice sensitive and resistant to two models of endotoxemia: lipopolysaccharide-injection and cecal ligation puncture. Genetic (knockout mice) and pharmacological (PJ34) blockade of the nuclear enzyme PARP-1 was used to achieve resistance to the endotoxemia. In both models, endotoxemia resulted in antithrombin deficiency, decreased platelets, and fibrin deposition in organs, which were similar in all groups of mice. By contrast, proinflammatory mediators, inflammatory cell infiltration (especially that mediated by mononuclear cells), and organ degeneration were more intense in sensitive animals. Further studies supported a negative role for the triggering of the coagulation cascade in the mortality associated with the endotoxic shock. Hirudin had a minor effect on cell infiltration and organ damage, despite causing a potent inhibition of fibrin deposition. On the other hand, a sublethal dose of lipopolysaccharide yielded significant fibrin deposition but weak activation of the inflammatory response. Our results suggest that activation of coagulation by endotoxemia is severe and independent of the inflammatory response. However, such activation may act with fibrin deposition to have a minor influence on survival in sepsis.

Poly(ADP-ribose) polymerase-1 inhibition increases expression of heat shock proteins and attenuates heat stroke-induced liver injury.

Objective: Heat stroke is a life-threatening illness characterized by an increased core body temperature as a result of exposure to high ambient temperature. Despite advances in supportive care, heat stroke is often fatal, and no specific and effective therapies exist. The pathophysiological responses to heat stroke involve a systemic inflammatory response and a disseminated intravascular coagulation in the host, which lead to a multiorgan dysfunction syndrome. Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear DNA-binding protein that has been shown to play a relevant role in cell necrosis and organ failure in various diseases associated with inflammation. Therefore, we set out to investigate whether inhibition of PARP activity might affect the heat stroke-induced injury. Design: Controlled animal study. Setting: Research laboratory of an academic institution. Subjects: PARP-1–deficient mice (Parp-1−/−) and wild-type mice (C57BL/6J). Interventions: Wild-type mice untreated or treated with either PJ34 or 3-AB, two generic PARP inhibitors, and Parp-1−/− mice were subjected to heat exposure as a model to study heat stroke. Measurements and Main Results: We measured rectal temperature, serum interleukin-1&bgr; and interleukin-6, liver histology, and heat shock proteins expression. We found that the heat stroke-induced injury was attenuated in mice lacking PARP-1 and was markedly reduced in wild-type mice treated with PARP inhibitors. Interestingly, heat-induced expression of heat shock proteins 27 and 70 was boosted after PARP inhibition. Indeed, PARP inhibition increased expression of heat shock proteins 27 and 70 even in the absence of heat exposure. Accordingly, PARP inhibition increased thermal tolerance that may contribute to attenuate the clinical effects of heat stroke, resulting in increased survival. Conclusions: Our results find a new protective function of PARP inhibitors and support their potential therapeutic application in the treatment of heat stroke.

Factors with conformational effects on haemostatic serpins: Implications in thrombosis

Serpins are key actors of systems involving proteolytic reactions, such as the haemostatic system, as they are irreversible suicide inhibitors of serine proteases. The structural flexibility and physical properties of serpins that are required for their efficient inhibitory mechanism also make them especially vulnerable to even minor factors that induce conformational changes in the native form of these molecules, leading to a number of inactive conformations, such as latent, cleaved or polymers. Increasing numbers of conformational mutations affecting haemostatic serpins, mainly antithrombin, the main endogenous anticoagulant, have been described. These mutations cause circulating deficiencies of the molecules, in most cases due to intracellular retention, which may be associated with a hyper-coagulable state. Indeed, conformational mutations in antithrombin have been identified in patients with severe venous thrombosis, which has led to the hypothesis that these disorders might be included in the group of conformational diseases. Moreover, we have recently demonstrated that other factors, including both drugs, such as the treatment with L-asparaginase, or environmental factors, such as high temperatures or hyperlipidemia, may also have conformational consequences on hepatic antithrombin, thus resulting in intracellular aggregation and plasma deficiency, which may increase the risk of thrombosis. In this study, we review the causes of deficiency of haemostatic serpins that may be explained by conformational mechanisms, and their association with an increased risk of venous thrombosis.

Dexamethasone induces a heat‐stress response that ameliorates the conformational consequences on antithrombin of l‐asparaginase treatment

Summary.  Background: l‐asparaginase (l‐ASP) treatment of patients with acute lymphoblastic leukemia causes a severe antithrombin deficiency by intracellular retention of this serpin within the endoplasmic reticulum (ER) of hepatic cells, and a subsequent risk of thrombosis. Interestingly, co‐administration of dexamethasone with l‐ASP seems to reduce the risk of thrombosis. Objectives: We have investigated the effect of two corticoids, dexamethasone and prednisone, on the conformational consequences of l‐ASP treatment on antithrombin. Patients/methods: Levels, activity, conformation and immunohistological features of antithrombin were studied in patients, cell and mice models. Because of the importance of the steroid receptor‐heat stress response (HSR) axis, and the role of unfolded protein response (UPR) in conformational diseases, we also evaluated Hsp27, Hsp70, Hsp90, HSF‐1 and ER chaperons (Grp78 and Grp94). Results: In all models, l‐ASP alone or in combination with prednisone caused the intracellular retention of antithrombin associated with a severe deficiency. In contrast, the combination of l‐ASP with dexamethasone ameliorated both the deficiency and intracellular retention of the serpin, which is associated with increased expression of heat shock proteins and ER‐chaperons. Conclusions: These results suggest a protective effect of dexamethasone on the conformational consequences of l‐ASP on antithrombin as a result of exacerbated HSR and UPR that help to explain the reduced risk of thrombosis reported in patients that follow this scheme of treatment.

Latent and polymeric antithrombin : Clearance and potential thrombotic risk

Antithrombin, the most potent anticoagulant in vivo, displays a significant conformational flexibility. The native five-stranded anticoagulant form transforms under different conditions or mutations to inactive six-stranded conformations: latent or polymer. However, the function, potential deleterious effects, and clearance of these forms are not completely known. The dimerization of latent antithrombin with a native molecule has been suggested to have thrombotic potential. We have assessed the potential thrombogenicity of high amounts of latent and polymeric antithrombin by experiments performed in mice and human plasma. Moreover, we have analyzed the clearance of 125I-labeled native, latent, polymer, and thrombin-complexed antithrombins in rat, as well as the clearance of latent antithrombin from plasma of patients treated with commercial concentrates. Our results show that high plasma levels of latent or polymeric antithrombin do not interfere with the anticoagulant function of native antithrombin. Moreover, we confirm that all monomeric forms of antithrombin have similar turnover. Finally, we show that polymers have the longest half-life of all conformers, being in circulation for prolonged periods of time. In conclusion, our data support that latent and polymeric antithrombin would not likely have a thrombotic effect, thus dispelling doubts about the potential harmful effect of latent antithrombin present in commercial concentrates for therapeutic use. Moreover, the suggested antiangiogenic role of latent antithrombin, together with its stability in plasma and its negligible thrombogenicity raises the possibility of its use as a new antiangiogenic drug.

In vivo effects of hyperthermia on the functional and conformational characteristics of antithrombin

Summary.  Background: High temperatures produce in vitro transitions of antithrombin to its inactive latent and polymeric forms. Accordingly, high body temperatures might contribute in vivo to conformational changes in antithrombin associated with increased thrombotic risk. Methods: We assessed the in vivo effects of different hyperthermic stimuli on antithrombin. We studied two mouse models of hyperthermia. (i) Febrile syndrome induced by turpentine. (ii) Heat stroke generated by exposure to 42 °C. Body temperatures were measured. Antigen, anti‐factor Xa activity and conformational features of plasma antithrombin were studied. Furthermore, structural and ultrastructural features from livers were analyzed. Intracellular retention of serpins (antithrombin and α1‐antitrypsin) was studied by western‐blotting, immunohistochemistry, and immunogold‐labeling‐electron microscopy. Results: Hyperthermic stimuli caused a moderate deficiency of circulating antithrombin and a slight increase in its latent form. Moreover, hyperthermia caused intracellular retention of antithrombin into aggregates within the lumen of the endoplasmic reticulum of hepatocytes. This effect was similar for α1‐antitrypsin. Conclusion: Hyperthermia causes minor conformational changes on circulating antithrombin in vivo, although it has severe consequences for intracellular antithrombin and other hepatic serpins, inducing the intracellular retention of the nascent protein. These effects may contribute to the moderate plasma deficiency of antithrombin and the increased thrombotic risk detected in hyperthermic conditions.

Intracellular retention of hepatic serpins caused by severe hyperlipidemia

Abstract: Background: High levels of circulating lipids contribute to both the development of non‐alcoholic liver steatosis (NALS) and peripheral arterial disease, leading to increased thrombotic risk. However, the effects of hyperlipidemia on hepatic proteins have barely been studied. Antithrombin is a hepatic serpin with anticoagulant and anti‐inflammatory roles. The conformational flexibility of antithrombin renders it susceptible to both, genetic and posttranslational modifications. Thus, mutations and environmental factors have been shown to alter this molecule.