Metabolic systems analysis of LPS induced endothelial dysfunction applied to sepsis patient stratification.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked FilesEndothelial dysfunction contributes to sepsis outcome. Metabolic phenotypes associated with endothelial dysfunction are not well characterised in part due to difficulties in assessing endothelial metabolism in situ. Here, we describe the construction of iEC2812, a genome scale metabolic reconstruction of endothelial cells and its application to describe metabolic changes that occur following endothelial dysfunction. Metabolic gene expression analysis of three endothelial subtypes using iEC2812 suggested their similar metabolism in culture. To mimic endothelial dysfunction, an in vitro sepsis endothelial cell culture model was established and the metabotypes associated with increased endothelial permeability and glycocalyx loss after inflammatory stimuli were quantitatively defined through metabolomics. These data and transcriptomic data were then used to parametrize iEC2812 and investigate the metabotypes of endothelial dysfunction. Glycan production and increased fatty acid metabolism accompany increased glycocalyx shedding and endothelial permeability after inflammatory stimulation. iEC2812 was then used to analyse sepsis patient plasma metabolome profiles and predict changes to endothelial derived biomarkers. These analyses revealed increased changes in glycan metabolism in sepsis non-survivors corresponding to metabolism of endothelial dysfunction in culture. The results show concordance between endothelial health and sepsis survival in particular between endothelial cell metabolism and the plasma metabolome in patients with sepsis.RANNIS Landspitali Reykjavik Rigshospitalet Copenhage

eNOS activation mediated by AMPK after stimulation of endothelial cells with histamine or thrombin is dependent on LKB1.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldReports on the role of AMP-activated protein kinase (AMPK) in thrombin-mediated activation of endothelial nitric-oxide synthase (eNOS) in endothelial cells have been conflicting. Previously, we have shown that under culture conditions that allow reduction of ATP-levels after stimulation, activation of AMPK contributes to eNOS phosphorylation and activation in endothelial cells after treatment with thrombin. In this paper we examined the signaling pathways mediating phosphorylation and activation of eNOS after stimulation of cultured human umbilical vein endothelial cells (HUVEC) with histamine and the role of LKB1-AMPK in the signaling. In Morgan's medium 199 intracellular ATP was lowered by treatment with histamine or the ionophore A23187 while in medium RMPI 1640 ATP was unchanged after identical treatment. In medium 199 inhibition of Ca(+2)/CaM kinase kinase (CaMKK) by STO-609 only partially inhibited AMPK phosphorylation but after gene silencing of LKB1 with siRNA there was a total inhibition of AMPK phosphorylation by STO-609 after treatment with either histamine or thrombin, demonstrating phosphorylation of AMPK by both upstream kinases, LKB1 and CaMKK. Downregulation of AMPK with siRNA partially inhibited eNOS phosphorylation caused by histamine in cells maintained in medium 199. Downregulation of LKB1 by siRNA inhibited both phosphorylation and activity of eNOS and addition of the AMPK inhibitor Compound C had no further effect on eNOS phosphorylation. When experiments were carried out in medium 1640, STO-609 totally prevented the phosphorylation of AMPK without affecting eNOS phosphorylation. AMPKα2 downregulation resulted in a loss of the integrity of the endothelial monolayer and increased expression of GRP78, indicative of endoplasmic reticular (ER) stress. Downregulation of AMPKα1 had no such effect. The results show that culture conditions affect endothelial signal transduction pathways after histamine stimulation. Under conditions where intracellular ATP is lowered by histamine, AMPK is activated by both LKB1 and CaMKK and, in turn, mediates eNOS phosphorylation in an LKB1 dependent manner. Both AMPKα1 and -α2 are involved in the signaling. Under conditions where intracellular ATP is unchanged after histamine treatment, CaMKK alone activates AMPK and eNOS is phosphorylated and activated independent of AMPK

Thrombin or Ca(++)-ionophore-mediated fall in endothelial ATP levels independent of poly(ADP-Ribose) polymerase activity and NAD levels--comparison with the effects of hydrogen peroxide.

To access publisher's full text version of this article click on the hyperlink at the bottom of the pageTo test the hypothesis that a fall in cellular ATP following stimulation of endothelial cells with thrombin is secondary to a decrease in NAD levels caused by poly(ADP-Ribose)polymerase (PARP), we measured the levels of NAD and ATP in endothelial cells after treatment with thrombin, the Ca(++)-ionophore A23187, or hydrogen peroxide (H2O2), and compared the effects of inhibitors of PARP, NAD synthesis, and ADP-ribose breakdown on these responses. Neither thrombin nor A23187 caused a reduction in endothelial NAD levels and A23187 affected ATP levels independently of NAD levels or PARP activity. H2O2 induced lowering of NAD caused modest lowering of ATP but marked additional ATP-lowering, independent of PARP and NAD, was also demonstrated. We conclude that in endothelial cells ATP levels are largely independent of NAD and PARP, which do not play a role in thrombin or Ca(++)-ionophore-mediated lowering of ATP. H2O2 caused ATP lowering through a similar mechanism as thrombin and A23187 but, additionally, caused a further ATP lowering through its intense stimulation of PARP and marked lowering of NAD.University of Iceland Research Fund Research Fund of Landspitali, University Hospital Helga Jonsdottir and Sigurlidi Kristjansson Memorial fun

Role of ADP-ribosylation in endothelial signal transduction and prostacyclin production

AbstractADP-ribosylation of proteins by the enzymatic transfer of ADP-ribose from NAD has been implicated in a number of biological processes. We report that inhibitors of ADP-ribosylation, most notably the novel inhibitor of arginine specific cellular mono(ADP-ribosyl) transferase, meta-iodobenzylguanidine (MIBG) as well as nicotinamide, l-arginine methyl ester (LAME) and guanyltyramine, inhibit histamine-induced endothelial production of inositol phosphates, release of arachidonic acid and production of prostacyclin (PGI2). Those same responses were unaffected by MIBG when triggered by thrombin or leukotriene C4. These findings suggest that ADP-ribosylation serves a role in histamine-induced production of prostacyclin and imply differences in transduction pathways employed by the different agonists

Sleep-wake dynamics under extended light and extended dark conditions in adult zebrafish.

To access publisher's full text version of this article click on the hyperlink at the bottom of the pageWe characterize the effects of sleep deprivation on sleep-wake behavior, neurogenesis and stress in adult zebrafish, and describe light-induced changes in gene expression. Sleep deprivation was performed using two stimuli: mild electroshock and light. Comparisons were made between five groups of fish: naïve; electroshock sleep-deprived and yoked-control; fish exposed to constant light (increasing wakefulness); and fish exposed to constant darkness (increasing sleep). Behavioral parameters assessed were sleep percentage, number of sleep-wake transitions, and sleep and wake bout length. Using microarray technology, light-dark modulation of gene expression was examined. In parallel with gene expression, neurogenesis was measured and stress following sleep deprivation was assessed behaviorally and physiologically. Our results indicate that sleep duration is most effectively altered by varying exposure to ambient light. Further, while the sleep-wake dynamics are comparable to those observed in mammals, zebrafish may exhibit weaker sleep homeostasis and sleep pressure than do mammals; and sleep deprivation does not significantly alter their stress responses. Finally, modulation of gene expression by light and dark was observed. Genes upregulated during the dark period are broadly related to growth, morphogenesis, energy balance, and lipid synthesis. Genes upregulated during light are broadly related to synaptic plasticity and cell proliferation

Bráð miðeyrnabólga

Neðst á síðunni er hægt að nálgast greinina í heild sinni með því að smella á hlekkinn View/OpenSýkingar í efri öndunarvegum eru algengar hér á landi (1,2), sem erlendis (3-5). Vitað er að bráðar miðeyrnabólgur koma oft í kjölfar slíkra sýkinga (6,7) og faraldsfræðilegar rannsóknir sýna að yfir 60% barna hafa fengið bráða miðeymabólgu einu sinni eða oftar við tveggja ára aldur (2,8,9). Læknar hér á landi meðhöndla bráða miðeyrnabólgu á mismunandi hátt og svo virðist sem lyfjaval ákvarðist að einhverju leyti af því hvar þeir fá menntun sína (10). Í Bandaríkjunum er hefð fyrir því að meðhöndla eyrnabólgu með breiðvirku sýklalyfi (3,11), en í Svíþjóð, Danmörku og Finnlandi er að jafnaði gefið fenoxýmetýlpenisillín (5,12,13). Í Hollandi eru hins vegar aðeins mm 30% tilfella meðhöndluð með sýklalyfjum (14). Í þessum löndum hafa ráðleggingar um meðferð verið studdar rannsóknum og reynslu undanfarinna ára. Hér á landi skortir enn haldgóðar upplýsingar um tíðni og næmi helstu orsakavalda eyrnabólgu, en nýlegar athuganir gefa þó vísbendingu um þá helstu (15). Enda þótt ekki sé hægt að gefa algildar læknisfræðilegar ráðleggingar um greiningu og meðferð við eyrnabólgu þykir æskilegt að samræma sem mest læknisfræðilegar ákvarðanir þar að lútandi hér á landi. Slík samræming getur stuðlað að samhæfðari kennslu læknanema, auðveldari samskiptum lækna úr mismunandi sérgreinum, einfaldari fræðslu til sjúklinga og betri áætlanagerð varðandi lyfjanotkun og kostnað. Að frumkvæði landlæknisembættisins var í mars 1991 haldið þing um efri öndunarfærasýkingar með þátttöku sérfróðra aðila úr hinum ýmsu sérgreinum læknisfræðinnar. Höfundar þessarar greinar tóku að sér að leggja mat á þær rannsóknir sem fjalla um bráða miðeyrnabólgu og reynslu lækna hér á landi og freista þess að túlka þær miðað við íslenskar aðstæður. Sérstök áhersla var lögð á að safna saman og leggja mat á íslenskar rannsóknir bæði birtar og óbirtar

Bráð miðeyrnabólga

Neðst á síðunni er hægt að nálgast greinina í heild sinni með því að smella á hlekkinn View/OpenSýkingar í efri öndunarvegum eru algengar hér á landi (1,2), sem erlendis (3-5). Vitað er að bráðar miðeyrnabólgur koma oft í kjölfar slíkra sýkinga (6,7) og faraldsfræðilegar rannsóknir sýna að yfir 60% barna hafa fengið bráða miðeymabólgu einu sinni eða oftar við tveggja ára aldur (2,8,9). Læknar hér á landi meðhöndla bráða miðeyrnabólgu á mismunandi hátt og svo virðist sem lyfjaval ákvarðist að einhverju leyti af því hvar þeir fá menntun sína (10). Í Bandaríkjunum er hefð fyrir því að meðhöndla eyrnabólgu með breiðvirku sýklalyfi (3,11), en í Svíþjóð, Danmörku og Finnlandi er að jafnaði gefið fenoxýmetýlpenisillín (5,12,13). Í Hollandi eru hins vegar aðeins mm 30% tilfella meðhöndluð með sýklalyfjum (14). Í þessum löndum hafa ráðleggingar um meðferð verið studdar rannsóknum og reynslu undanfarinna ára. Hér á landi skortir enn haldgóðar upplýsingar um tíðni og næmi helstu orsakavalda eyrnabólgu, en nýlegar athuganir gefa þó vísbendingu um þá helstu (15). Enda þótt ekki sé hægt að gefa algildar læknisfræðilegar ráðleggingar um greiningu og meðferð við eyrnabólgu þykir æskilegt að samræma sem mest læknisfræðilegar ákvarðanir þar að lútandi hér á landi. Slík samræming getur stuðlað að samhæfðari kennslu læknanema, auðveldari samskiptum lækna úr mismunandi sérgreinum, einfaldari fræðslu til sjúklinga og betri áætlanagerð varðandi lyfjanotkun og kostnað. Að frumkvæði landlæknisembættisins var í mars 1991 haldið þing um efri öndunarfærasýkingar með þátttöku sérfróðra aðila úr hinum ýmsu sérgreinum læknisfræðinnar. Höfundar þessarar greinar tóku að sér að leggja mat á þær rannsóknir sem fjalla um bráða miðeyrnabólgu og reynslu lækna hér á landi og freista þess að túlka þær miðað við íslenskar aðstæður. Sérstök áhersla var lögð á að safna saman og leggja mat á íslenskar rannsóknir bæði birtar og óbirtar