PAR1 Agonists Stimulate APC-Like Endothelial Cytoprotection and Confer Resistance to Thromboinflammatory Injury

Stimulation of protease-activated receptor 1 (PAR1) on endothelium by activated protein C (APC) is protective in several animal models of disease, and APC has been used clinically in severe sepsis and wound healing. Clinical use of APC, however, is limited by its immunogenicity and its anticoagulant activity. We show that a class of small molecules termed “parmodulins” that act at the cytosolic face of PAR1 stimulates APC-like cytoprotective signaling in endothelium. Parmodulins block thrombin generation in response to inflammatory mediators and inhibit platelet accumulation on endothelium cultured under flow. Evaluation of the antithrombotic mechanism showed that parmodulins induce cytoprotective signaling through Gβγ, activating a PI3K/Akt pathway and eliciting a genetic program that includes suppression of NF-κB–mediated transcriptional activation and up-regulation of select cytoprotective transcripts. STC1 is among the up-regulated transcripts, and knockdown of stanniocalin-1 blocks the protective effects of both parmodulins and APC. Induction of this signaling pathway in vivo protects against thromboinflammatory injury in blood vessels. Small-molecule activation of endothelial cytoprotection through PAR1 represents an approach for treatment of thromboinflammatory disease and provides proof-of-principle for the strategy of targeting the cytoplasmic surface of GPCRs to achieve pathway selective signaling

Glutathione treatment protects the rat liver against injury after warm ischemia and Kupffer cell activation

Background/Aim: The generation of reactive oxygen species by activated Kupffer cells (KC) may contribute to reperfusion injury of the liver during liver transplantation or resection. The aim of our present studies was to investigate (1) prevention of hepatic reperfusion injury after warm ischemia by administration of the antioxidant glutathione (GSH) and (2) whether GSH confers protection through influences on KC toxicity. Methods: Isolated perfused rat livers were subjected to 1 h of warm ischemia followed by 90 min of reperfusion without (n = 5) or with GSH or catalase (n = 4-5 each). Selective KC activation by zymosan (150 mug/ml) in continuously perfused rat livers was used to investigate KC-related liver injury. Results: Postischemic infusion of 0.1, 0.5, 1.0 and 2.0 mM GSH, but not 0.05 mM GSH prevented reperfusion injury after warm ischemia as indicated by a marked reduction of sinusoidal LDH efflux by up to 83 +/- 13% (mean +/- SD; p < 0.05) and a concomitant significant improvement of postischemic bile flow by 58 +/- 27% (p < 0.05). A similar protection was conveyed by KC blockade with gadolinium chloride indicating prevention of KC-related reperfusion injury by postischemic GSH treatment. Postischemic treatment with catalase (150 U/ml) resulted in a reduction of LDH efflux by 40 +/- 9% (p < 0.05). Accordingly, catalase as well as GSH (0.1-2.0 mM) nearly completely prevented the increase in LDH efflux following selective :KC activation by zymosan in continously perfused rat livers. Conclusion: Postischemic administration of GSH protects the liver against reperfusion injury after warm ischemia. Detoxification of KC-derived hydrogen peroxide seem to be an important feature of the protective mechanisms. Copyright (C) 2002 S. Karger AG, Basel

Astrocyte Apoptosis and HIV Replication Are Modulated in Host Cells Coinfected with Trypanosoma cruzi

The protozoan Trypanosoma cruzi is the etiological agent of Chagas disease. In immunosuppressed individuals, as it occurs in the coinfection with human immunodeficiency virus (HIV), the central nervous system may be affected. In this regard, reactivation of Chagas disease is severe and often lethal, and it accounts for meningoencephalitis. Astrocytes play a crucial role in the environment maintenance of healthy neurons; however, they can host HIV and T. cruzi. In this report, human astrocytes were infected in vitro with both genetically modified-pathogens to express alternative fluorophore. As evidenced by fluorescence microscopy and flow cytometry, HIV and T. cruzi coexist in the same astrocyte, likely favoring reciprocal interactions. In this context, lower rates of cell death were observed in both T. cruzi monoinfected-astrocytes and HIV-T. cruzi coinfection in comparison with those infected only with HIV. The level of HIV replication is significantly diminished under T. cruzi coinfection, but without affecting the infectivity of the HIV progeny. This interference with viral replication appears to be related to the T. cruzi multiplication rate or its increased intracellular presence but does not require their intracellular cohabitation or infected cell-to-cell contact. Among several Th1/Th2/Th17 profile-related cytokines, only IL-6 was overexpressed in HIV-T. cruzi coinfection exhibiting its cytoprotective role. This study demonstrates that T. cruzi and HIV are able to coinfect astrocytes thus altering viral replication and apoptosis.Fil: Urquiza, Javier Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Burgos, Juan Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ojeda, Diego Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Pascuale, Carla Antonela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Leguizamon, Maria Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Quarleri, Jorge Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentin

Use of Meldonium in the Treatment of Patients with Coronary Artery Disease and Concomitant Arterial Hypertension

Coronary artery disease (CAD) remains one of the leading causes of mortality and disability in Ukraine. Arterial hypertension (AH) is one of the most common diseases and a leading risk factor for coronary artery disease.The aim of the work is to evaluate the antianginal activity of meldonium in the complex therapy in patients with CAD with stable angina and concomitant AH.Materials and methods. The study included 82 patients with CAD, stable angina pectoris II–III functional class, including 52 patients with concomitant AH stage II. The patients were divided into 2 groups. Patients in group 1 were prescribed meldonium at a dose of 750 mg/d for 2 months in addition to basic therapy for the underlying disease. Patients in group 2 continued basic antianginal, disaggregant, hypolipidemic therapy.Results. The use of meldonium led to a decrease in the frequency of angina attacks and the need for nitroglycerin. From the 1st month of therapy and up to 2 months treatment decreased it consumption by 63 and 82.3 % respectively. Adding meldonium to basic therapy led to a likely reduction in shortness of breath, episodes of palpitations, tinnitus, and headache. In all patients, after the treatment, an increase in exercise tolerance was observed, which was more pronounced in the group where patients were receiving meldonium. In the group of patients receiving meldonium, normalisation of blood pressure was faster and more pronounced.Conclusions. Meldonium has antianginal activity, which is manifested by an increase in the physical tolerance of patients, a decrease in the frequency of angina attacks, the need for sublingual nitroglycerin intake and improvement in the well-being of patients. Additional use of meldonium promotes faster and better normalization of blood pressure. The use of meldonium in the complex therapy of patients with stable angina and concomitant AH allows to increase the effectiveness of traditional antianginal therapy and to improve the quality of life of such patients

Thioredoxin-1 maintains mechanistic target of rapamycin (mTOR) function during oxidative stress in cardiomyocytes

Thioredoxin 1 (Trx1) is a 12-kDa oxidoreductase that catalyzes thiol-disulfide exchange reactions to reduce proteins with disulfide bonds. As such, Trx1 helps protect the heart against stresses, such as ischemia and pressure overload. Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, metabolism, and survival. We have shown previously that mTOR activity is increased in response to myocardial ischemia-reperfusion injury. However, whether Trx1 interacts with mTOR to preserve heart function remains unknown. Using a substrate-trapping mutant of Trx1 (Trx1C35S), we show here that mTOR is a direct interacting partner of Trx1 in the heart. In response to H2O2 treatment in cardiomyocytes, mTOR exhibited a high molecular weight shift in non-reducing SDS-PAGE in a 2-mercaptoethanol-sensitive manner, suggesting that mTOR is oxidized and forms disulfide bonds with itself or other proteins. The mTOR oxidation was accompanied by reduced phosphorylation of endogenous substrates, such as S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1) in cardiomyocytes. Immune complex kinase assays disclosed that H2O2 treatment diminished mTOR kinase activity, indicating that mTOR is inhibited by oxidation. Of note, Trx1 overexpression attenuated both H2O2-mediated mTOR oxidation and inhibition, whereas Trx1 knockdown increased mTOR oxidation and inhibition. Moreover, Trx1 normalized H2O2-induced down-regulation of metabolic genes and stimulation of cell death, and an mTOR inhibitor abolished Trx1-mediated rescue of gene expression. H2O2-induced oxidation and inhibition of mTOR were attenuated when Cys-1483 of mTOR was mutated to phenylalanine. These results suggest that Trx1 protects cardiomyocytes against stress by reducing mTOR at Cys-1483, thereby preserving the activity of mTOR and inhibiting cell death

PIGMENTATION OF THE HEART IN THE BICHIR, POLYPTERUS SENEGALUS

El resumen aparece en el Program & Abstracts of the 10th International Congress of Vertebrate Morphology, Barcelona 2013. Anatomical Record, Volume 296, Special Feature — 1: P-078.The presence of melanin-containing cells in the heart has been documented in tetrapods, but not in fish. It has been even suggested that dark pigmented cells are exclusively associated with hearts having two atria and two ventricles. The aim here is to report the occurrence of pigment cells in the heart of the bichir, an extant representative of the polypteriformes, an ancient ray-finned fish lineage that split from the stem of the actinopterygians soon after their divergence from the sarcopterygians. The bichir heart is composed of sinus venosus, atrium, ventricle, conus arteriosus and bulbus arteriosus arranged sequentially within the pericardial cavity. Dendritic-shaped cells containing melanosomes were found in the five cardiac components of the 12 bichirs included in this study. Numerous melanophores were distributed regularly over the surface of all segments having myocardium in their walls, thus resulting in a marked pigmentation of the whole heart. The bulbus arteriosus, which in the bichir is reduced in size, showed an even more intense pigmentation. In all instances, the melanophores were localized in the subepicardial space. Pigment cells also occurred in the pericardium and ventral aorta. The functional role of melanocytes in the tetrapod heart remains obscure. Antiinflamatory activity, cytoprotection and effects on the viscoelastic properties of the cardiac tissue have been adduced as possible actions of such cells. The role of pigment cells in the bichir heart constitutes a new open question. Interestingly, however, the only cells that have been shown to form melanin-containing cells in the heart derive from the neural crest. If the melanophores of the bichir heart are indeed of neural crest origin, it would suggest a much more extensive contribution and persistence of elements from the neural crest in the primitive heart of jawed vertebrates as assumed so far in most papers devoted to vertebrate heart embryology.Proyecto CGL2010-16417/BOS; Fondos FEDER; Beca FPI ref. BES-2011-046901

Phosphorylation of survivin at threonine 34 inhibits its mitotic function and enhances its cytoprotective activity

Survivin is an essential chromosomal passenger protein required for mitotic progression. It is also an inhibitor of apoptosis and can prevent caspase-mediated cell death. In addition, survivin levels are elevated in cancer cells where its presence correlates with increased resistance to chemo- and radio-therapy, which makes it an attractive target for novel anti-cancer strategies. Interestingly, survivin is phosphorylated by the mitotic kinase, cdk1, and a non-phosphorylatable form, survivin(T34A), cannot inhibit apoptosis. Here we rigorously test the ability of survivin(T34A) and its corresponding phosphomimetic, survivin(T34E), to promote cell viability through survivin's dual roles. The effects of these mutations are diametrically opposed: survivin(T34A) accelerates cell proliferation and promotes apoptosis, whereas survivin(T34E) retards growth and promotes survival. Thus the phosphorylation status of survivin at T34 is pivotal to a cell's decision to live or die

Protective activity of aromatic amines and imines against oxidative nerve cell death

Oxidative stress is a widespread phenomenon in the pathology of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Neuronal cell death due to oxidative stress may causally contribute to the pathogeneses of these diseases. Therefore, neuroprotective antioxidants are considered to be a promising approach to slow down disease progression. We have investigated different aromatic amine and imine compounds for neuroprotective antioxidant functions in cell culture, and found that these compounds possess excellent cytoprotective potential in diverse paradigms of oxidative neuronal cell death, including clonal cell lines, primary cerebellar neurons, and organotypic hippocampal slice cultures. Aromatic amines and imines are effective against oxidative glutamate toxicity, glutathione depletion, and hydrogen peroxide toxicity. Their mode of action as direct antioxidants; was experimentally confirmed by electron spin resonance spectroscopy, cell-free brain lipid peroxidation assays, and intracellular peroxide measurements. With half-maximal effective concentrations of 20-75 nm in different neuroprotection experiments, the aromatic imines phenothiazine, phenoxazine, and iminostilbene proved to be about two orders of magnitude more effective than common phenolic antioxidants. This remarkable efficacy could be directly correlated to calculated properties of the compounds by means of a novel, quantitative structure-activity relationship model. We conclude that bridged bisarylimines with a single free NH-bond, such as iminostilbene, are superior neuroprotective antioxidants, and may be promising lead structures for rational drug development