Predictors for cerebral edema in acute ischemic stroke treated with intravenous thrombolysis

Cerebral edema (CED) is a severe complication of acute ischemic stroke. There is uncertainty regarding the predictors for the development of CED after cerebral infarction. We aimed to determine which baseline clinical and radiological parameters predict development of CED in patients treated with intravenous thrombolysis. We used an image-based classification of CED with 3 degrees of severity (less severe CED 1 and most severe CED 3) on postintravenous thrombolysis imaging scans. We extracted data from 42 187 patients recorded in the SITS International Register (Safe Implementation of Treatments in Stroke) during 2002 to 2011. We did univariate comparisons of baseline data between patients with or without CED. We used backward logistic regression to select a set of predictors for each CED severity. CED was detected in 9579/42 187 patients (22.7%: 12.5% CED 1, 4.9% CED 2, 5.3% CED 3). In patients with CED versus no CED, the baseline National Institutes of Health Stroke Scale score was higher (17 versus 10; P<0.001), signs of acute infarct was more common (27.9% versus 19.2%; P<0.001), hyperdense artery sign was more common (37.6% versus 14.6%; P<0.001), and blood glucose was higher (6.8 versus 6.4 mmol/L; P<0.001). Baseline National Institutes of Health Stroke Scale, hyperdense artery sign, blood glucose, impaired consciousness, and signs of acute infarct on imaging were independent predictors for all edema types. The most important baseline predictors for early CED are National Institutes of Health Stroke Scale, hyperdense artery sign, higher blood glucose, decreased level of consciousness, and signs of infarct at baseline. The findings can be used to improve selection and monitoring of patients for drug or surgical treatment

Characterization of human glutathione-dependent microsomal prostaglandin E synthase-1

Prostaglandins (PGs) are lipid mediators, which act as local hormones. PGs are formed in most calls and are synthesized de novo from membrane-released arachidonic acid (AA) upon cell activation. Prostaglandin H synthase (PGHS) -1 or 2, also referred to as COX-1 and COX-2, metabolize AA to PGH2, which is subsequently converted in a cell-specific manner by downstream enzymes to biologically active prostanoids, i.e. PGE2, PGD2, PGF2alpha, PGI2 or TXA2. PGHS-1 is constitutively expressed in many calls and is mainly involved in housekeeping functions, such as vascular homeostasis, whereas PGHS-2 can be induced by proinflammatory cytokines at sites of inflammation. Prostaglandin E synthase (PGES) specifically catalyzes the conversion of PGH2 to PGE2, which is a biologically potent prostaglandin involved in several pathological conditions; including pain, favor, inflammation and possibly some forms of cancers and neurodegenerative diseases. mPGES-1 was initially identified as a homologue to microsomal glutathione transferase-1 (MGST1) with 37% identity on the amino acid sequence level and referred to as MGST1-like 1 (MGST1-L1). Based on the properties of MGST1-L1, regarding size, amino acid sequence, hydropathy and membrane localization, the protein was identified as a member of the MAPEG-superfamily (membrane-associated proteins in eicosanoid and glutathione metabolism). The superfamily consists of 16- 18 kDa, integral membrane proteins with typical hydropathy profiles and diverse functions. The MAPEG family comprises six human members, which in addition to mPGES-1 are; 5-lipoxygenase activating protein (FLAP), leukotriene C4 synthase (LTC4S), MGST1, MGST2 and MGST3. MGST1 -2 and -3 are glutathione transferases as well as glutathione-dependent peroxidases, while FLAP and LTC4S are crucial for leukotriene biosynthesis. Human mPGES-1 was cloned and characterized as a 16 kDa, inducible GSH-dependent microsomal PGE synthase. Northern dot blot analysis of mPGES-1 mRNA demonstrated a low expression in most tissues, medium expression in reproductive organs and a high expression in two cancer cell lines (A549 and HeLa). A549 cells had been used earlier as a model system to study PGHS-2 induction by the proinflammatory cytokine IL-1beta and mPGES-1 was also induced by IL-1beta in these calls. A protein of similar size was detected in microsomes from sheep vesicular glands, which are known to contain a highly efficient microsomal PGES, indicating that mPGES-1 was the long-sought membrane bound PGES. Furthermore, a time study of PGHS-2 and mPGES-1 expression revealed a coordinate induction of these enzymes, which was correlated with increased PGES activity in the microsomal fraction. Tumor necrosis factor-alpha (TNF-alpha) also induced mPGES-1 in these cells and dexamethasone was found to counteract the effect of these cytokines on mPGES-1 induction. A method based on RP-HPLC and UV-detection was developed to efficiently quantify PGES activity. A small set of potential mPGES-1 inhibitors were tested and NS-398, Sulindac sulfide and LTC4 were found to inhibit PGES activity with IC50-values of 20 µm, 80 µm and 5 µm, respectively. The human mPGES-1 gene structure was investigated. The mPGES-1 gene span a region of approximately 15 kb, is divided into three exons, and is localized on chromosome 9q34.3. A 682 bp fragment directly upstream of the translation start site exhibited promoter activity when transfected in A549 calls. The putative promoter is GC-rich, lacks a TATA box at a functional site and contains numerous potential transcription factor binding-sites. Two GC-boxes, two tandem Barble-boxes and an aryl hydrocarbon response element were identified. The putative promoter region of mPGES1 was transcriptionally active and reporter constructs were regulated by IL-1beta and phenobarbital. The expression of mPGES-1 was investigated in synovial tissues from patients suffering from rheumatoid arthritis (RA). Primary synovial cells obtained from patients with RA were treated with IL-1beta or TNF-alpha. Both cytokines were found to induce mPGES-1 mRNA from low basal levels to maximum levels after 24 hours and the induction by IL-1beta was inhibited by dexamothasone in a dose-dependent manner. The protein expression of mPGES-1 was also induced by IL-1beta with a linear increase up to 72 h. In contrast, the PGHS-2 induction demonstrated an earlier peak expression (4-8 h). Furthermore, the protein expression of mPGES-1 was correlated with increased microsomal PGES activity. In these biochemical experiments any significant contribution of cytosolic PGES or other cytosolic or nonn-inducible membrane bound PGE syntheses was ruled out. A purification protocol for mPGES-1 was developed. Human mPGES-1 was expressed with a histidine tag in Eschericha coli, solubilized by Triton X-100 and purified by a combination of hydroxyapatite and immobilized metal affinity chromatography. mPGES-1 catalyzed a rapid GSH-dependent conversion of PGH2 to PGE2 (170 µmol/min mg). The enzyme, also displayed a high GSH-dependent activity against PGG2, forming 15hydroperoxy PGE2 (250 µmol/min mg). In addition, mPGES-1 possessed several other activities; glutathionedependent peroxidase activity towards cumene hydroperoxide, 5-HpETE and 15-hydroperoxy-PGE2, as well as conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) to GSH. These activities likely reflect the relationship with other MAPEG enzymes. Two-dimensional crystals of purified mPGES-1 were obtained and a 10 A projection map was determined by electron crystallography. Hydrodynamic studies were also performed on the mPGES-1-Triton X-100 complex to investigate the oligomeric state of the protein. Electron crystallography and hydrodynamic studies independently demonstrated a trimeric organization of mPGES-1. Together with other studies published to date, mPGES-1 has been verified biologically as a drug target and the next stop in this validation process requires specific inhibitors to be tested in animal disease models

Expression of microsomal prostaglandin E synthase-1 in intestinal type gastric adenocarcinoma and in gastric cancer cell lines

Gastrointestinal carcinomas synthesize elevated levels of prostaglandin E(2) (PGE(2)), which has been mechanistically linked to carcinogenesis. Recently, microsomal prostaglandin E synthase-1 (mPGES-1) was cloned, which seems to be inducible and linked to cyclooxygenase-2 (Cox-2) in the biosynthesis of PGE(2). We examined expression of mPGES-1 in intestinal type gastric adenocarcinomas and in gastric cancer cell lines. The transcript for mPGES-1 was elevated in 57% (4/7) of gastric carcinomas as detected by Northern blot analysis. Moderate to strong mPGES-1 immunoreactivity was observed in 56% (5/9) of the carcinomas as detected by immunohistochemistry. Furthermore, mPGES-1 mRNA, protein and microsomal PGES activity were detected in gastric adenocarcinoma cell lines that originated from intestinal type tumors (MKN-7 and MKN-28). In contrast to Cox-2, however, expression of mPGES-1 mRNA or protein were not induced by phorbol 12-myristate 13-acetate (PMA) or interleukin-1beta (IL-1beta) in any of the gastric cancer cell lines tested (MKN-1, -7, -28, -45 and -74). Two gastric cancer cell lines (MKN-45 and MKN-74) did not express mPGES-1 and lacked microsomal PGES activity, but were still able to synthesize PGE(2). Because all gastric cell lines expressed cPGES as detected by immunoblotting, it is possible that Cox-2 can interact with cPGES or with some other yet unidentified PGES in gastric cancer cells. Furthermore, our data show that regulatory mechanisms that drive expression of mPGES-1 and Cox-2 dissociate in gastric cancer cell line