Phosphorylation of Vasodilator-Stimulated Phosphoprotein (VASP) dampens hepatic ischemia-reperfusion injury

Recent work has demonstrated that the formation of platelet neutrophil complexes (PNCs) affects inflammatory tissue injury. Vasodilator-stimulated phosphoprotein (VASP) is crucially involved into the control of PNC formation and myocardial reperfusion injury. Given the clinical importance of hepatic IR injury we pursued the role of VASP during hepatic ischemia followed by reperfusion. We report here that VASP−/− animals demonstrate reduced hepatic IR injury compared to wildtype (WT) controls. This correlated with serum levels of lactate dehydrogenase (LDH), aspartate (AST) and alanine (ALT) aminotransferase and the presence of PNCs within ischemic hepatic tissue and could be confirmed using repression of VASP through siRNA. In studies employing bone marrow chimeric mice we identified hematopoietic VASP to be of crucial importance for the extent of hepatic injury. Phosphorylation of VASP on Ser153 through Prostaglandin E1 or on Ser235 through atrial natriuretic peptide resulted in a significant reduction of hepatic IR injury. This was associated with a reduced presence of PNCs in ischemic hepatic tissue. Taken together, these studies identified VASP and VASP phosphorylation as crucial target for future hepatoprotective strategies

Proapoptotic activity of Ukrain is based on Chelidonium majus L. alkaloids and mediated via a mitochondrial death pathway

BACKGROUND: The anticancer drug Ukrain (NSC-631570) which has been specified by the manufacturer as semisynthetic derivative of the Chelidonium majus L. alkaloid chelidonine and the alkylans thiotepa was reported to exert selective cytotoxic effects on human tumour cell lines in vitro. Few clinical trials suggest beneficial effects in the treatment of human cancer. Aim of the present study was to elucidate the importance of apoptosis induction for the antineoplastic activity of Ukrain, to define the molecular mechanism of its cytotoxic effects and to identify its active constituents by mass spectrometry. METHODS: Apoptosis induction was analysed in a Jurkat T-lymphoma cell model by fluorescence microscopy (chromatin condensation and nuclear fragmentation), flow cytometry (cellular shrinkage, depolarisation of the mitochondrial membrane potential, caspase-activation) and Western blot analysis (caspase-activation). Composition of Ukrain was analysed by mass spectrometry and LC-MS coupling. RESULTS: Ukrain turned out to be a potent inducer of apoptosis. Mechanistic analyses revealed that Ukrain induced depolarisation of the mitochondrial membrane potential and activation of caspases. Lack of caspase-8, expression of cFLIP-L and resistance to death receptor ligand-induced apoptosis failed to inhibit Ukrain-induced apoptosis while lack of FADD caused a delay but not abrogation of Ukrain-induced apoptosis pointing to a death receptor independent signalling pathway. In contrast, the broad spectrum caspase-inhibitor zVAD-fmk blocked Ukrain-induced cell death. Moreover, over-expression of Bcl-2 or Bcl-x(L )and expression of dominant negative caspase-9 partially reduced Ukrain-induced apoptosis pointing to Bcl-2 controlled mitochondrial signalling events. However, mass spectrometric analysis of Ukrain failed to detect the suggested trimeric chelidonine thiophosphortriamide or putative dimeric or monomeric chelidonine thiophosphortriamide intermediates from chemical synthesis. Instead, the Chelidonium majus L. alkaloids chelidonine, sanguinarine, chelerythrine, protopine and allocryptopine were identified as major components of Ukrain. Apart from sanguinarine and chelerythrine, chelidonine turned out to be a potent inducer of apoptosis triggering cell death at concentrations of 0.001 mM, while protopine and allocryptopine were less effective. Similar to Ukrain, apoptosis signalling of chelidonine involved Bcl-2 controlled mitochondrial alterations and caspase-activation. CONCLUSION: The potent proapoptotic effects of Ukrain are not due to the suggested "Ukrain-molecule" but to the cytotoxic efficacy of Chelidonium majus L. alkaloids including chelidonine

Gαi2- and Gαi3-deficient mice display opposite severity of myocardial ischemia reperfusion injury.

G-protein-coupled receptors (GPCRs) are the most abundant receptors in the heart and therefore are common targets for cardiovascular therapeutics. The activated GPCRs transduce their signals via heterotrimeric G-proteins. The four major families of G-proteins identified so far are specified through their α-subunit: Gαi, Gαs, Gαq and G12/13. Gαi-proteins have been reported to protect hearts from ischemia reperfusion injury. However, determining the individual impact of Gαi2 or Gαi3 on myocardial ischemia injury has not been clarified yet. Here, we first investigated expression of Gαi2 and Gαi3 on transcriptional level by quantitative PCR and on protein level by immunoblot analysis as well as by immunofluorescence in cardiac tissues of wild-type, Gαi2-, and Gαi3-deficient mice. Gαi2 was expressed at higher levels than Gαi3 in murine hearts, and irrespective of the isoform being knocked out we observed an up regulation of the remaining Gαi-protein. Myocardial ischemia promptly regulated cardiac mRNA and with a slight delay protein levels of both Gαi2 and Gαi3, indicating important roles for both Gαi isoforms. Furthermore, ischemia reperfusion injury in Gαi2- and Gαi3-deficient mice exhibited opposite outcomes. Whereas the absence of Gαi2 significantly increased the infarct size in the heart, the absence of Gαi3 or the concomitant upregulation of Gαi2 dramatically reduced cardiac infarction. In conclusion, we demonstrate for the first time that the genetic ablation of Gαi proteins has protective or deleterious effects on cardiac ischemia reperfusion injury depending on the isoform being absent

Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway-12

<p><b>Copyright information:</b></p><p>Taken from "Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway"</p><p>BMC Cancer 2006;6():14-14.</p><p>Published online 17 Jan 2006</p><p>PMCID:PMC1379651.</p><p>Copyright © 2006 Habermehl et al; licensee BioMed Central Ltd.</p> extract adjusted to the respective chelidonine concentration. Apoptosis induction was analysed by fluorescence microscopy upon combined staining with Hoechst33342 and propidium iodide. The percentage of cells with characteristic apoptotic nuclear morphology was determined by counting a minimum of 250 cells per data point in each of at least three independent experiments. Data represent the percentage of cells with apoptotic nuclear morphology (means ± SD, n = 3). . Jurkat Vector cells were treated for 24 h with medium supplemented with solvent or 50 μM extract adjusted to the respective chelidonine-concentration. Apoptosis induction was analysed by fluorescence microscopy upon combined staining with Hoechst33342 and propidium iodide. Characteristic micrographs of nuclear morphology are shown. . Jurkat vector cells were treated for 24 h with medium supplemented with solvent or 1, 5, 10 and 50 μM L. extract adjusted to the respective chelidonine-concentration. Induction of apoptosis was then measured using flow cytometry (cell shrinkage: white bars; breakdown of mitochondrial membrane potential: grey bars; caspase-activation: black bars). Data show means ± SD (n ≥ 3)

Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway-5

<p><b>Copyright information:</b></p><p>Taken from "Proapoptotic activity of Ukrain is based on L. alkaloids and mediated via a mitochondrial death pathway"</p><p>BMC Cancer 2006;6():14-14.</p><p>Published online 17 Jan 2006</p><p>PMCID:PMC1379651.</p><p>Copyright © 2006 Habermehl et al; licensee BioMed Central Ltd.</p>medium or 10 μg/ml Ukrain, for 12 h with medium or 25 μM Etoposide and for 48 h with medium or irradiated with 10 Gy 48 h prior to determination of apoptosis. Apoptosis was quantified by fluorescence microscopy upon Hoechst33342-staining by counting cells with apoptotic nuclear morphology. Data show specific apoptosis (apoptosis rates of treated cells minus apoptosis rates of untreated cells) as means ± SD (n = 3). One-way ANOVA was performed using GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California USA, . . Jurkat Vector, Jurkat Bcl-2 as well as Jurkat Caspase-9 DN cells were treated for 24 h with medium or 5, 10 and 50 μg/ml Ukrain. Apoptosis induction was then quantified by determination of the mitochondrial membrane potential (Δψm) (right panel). Data show induction of specific apoptosis (means ± SD; n ≥ 3). One-way ANOVA was performed using GraphPad InStat version 3.00 for Windows 95, GraphPad Software, San Diego California USA, . . Expression of Bcl-2 in Jurkat Vector and Bcl-2 overexpressing Jurkat cells (Jurkat Bcl-2) (upper panel) as well as expression of caspase-9 in Jurkat Vector and Jurkat cells expressing a dominant negative caspase-9 (Jurkat Caspase-9 DN) (lower panel) were verified by Western blot analysis of cytotsolic extracts with the respective antibodies. Jurkat Vector cells, Jurkat Bcl-2 cells as well as Jurkat Caspase-9 DN cells were treated for 0, 6, 12 and 24 h with medium or 10 μg/ml Ukrain. Caspase-activation was then determined by Western blot analysis of cytosolic extracts with antibodies against full length and active caspase-3, caspase-8 as well as PARP and cleaved PARP. Data from one representative experiment are shown