Background-Inflammation plays a key role in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury; however, the mechanism by which myocardial I/R induces inflammation remains unclear. Recent evidence indicates that a sterile inflammatory response triggered by tissue damage is mediated through a multiple-protein complex called the inflammasome. Therefore, we hypothesized that the inflammasome is an initial sensor for danger signal(s) in myocardial I/R injury. Methods and Results-We demonstrate that inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, is crucially involved in the initial inflammatory response after myocardial I/R injury. We found that inflammasomes are formed by I/R and that its subsequent activation of inflammasomes leads to interleukin-1␤ production, resulting in inflammatory responses such as inflammatory cell infiltration and cytokine expression in the heart. In mice deficient for apoptosis-associated speck-like adaptor protein and caspase-1, these inflammatory responses and subsequent injuries, including infarct development and myocardial fibrosis and dysfunction, were markedly diminished. Bone marrow transplantation experiments with apoptosis-associated speck-like adaptor protein-deficient mice revealed that inflammasome activation in bone marrow cells and myocardial resident cells such as cardiomyocytes or cardiac fibroblasts plays an important role in myocardial I/R injury. In vitro experiments revealed that hypoxia/reoxygenation stimulated inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, and that hypoxia/reoxygenation-induced activation was mediated through reactive oxygen species production and potassium efflux. Conclusions-Our results demonstrate the molecular basis for the initial inflammatory response after I/R and suggest that the inflammasome is a potential novel therapeutic target for preventing myocardial I/R injury. (Circulation. 2011;123:594-604.)Key Words: cytokine Ⅲ heart Ⅲ hypoxia Ⅲ inflammation Ⅲ leukocyte I ncreasing evidence indicates that inflammation is involved in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury. 1 One prominent and early mediator for inflammation in I/R injury is interleukin-1␤ (IL-1␤). 2,3 I/R induces IL-1␤ expression in the heart, and the inhibition of IL-1␤ prevents myocardial injury after I/R, 3 suggesting that the deleterious effects of myocardial I/R are mediated, at least in part, by IL-1␤. In the generation of IL-1␤, pro-IL-1␤, an inactive precursor, undergoes proteolysis by the converting enzyme caspase-1. Caspase-1 is activated within a cytosolic multiprotein complex, the inflammasome. The inflammasome contains cytoplasmic receptors of the NACHT leucine-rich-repeat protein family that are associated with the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which in turn recruits and activates caspase-1. 4,5 Increasing evidence indicates that several sterile inflammatory responses triggered by tissue damage are mediated by th...

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to addressins expressed in the high endothelial venules (HEV) of secondary lymphoid organs. Peripheral node addressin recognized by the MECA-79 antibody is apparently part of the L-selectin ligand, but its chemical nature has been undefined. We now identify a sulfated extended core1 mucin-type O-glycan, Gal beta 1-->4(sulfo-->6)GlcNAc beta 1-->3Gal beta 1-->3GalNAc, as the MECA-79 epitope. Molecular cloning of a HEV-expressed core1-beta 1,3-N-acetylglucosaminyltransferase (Core1-beta 3GlcNAcT) enabled the construction of the 6-sulfo sialyl Lewis x on extended core1 O-glycans, recapitulating the potent L-selectin-mediated, shear-dependent adhesion observed with novel L-selectin ligands derived from core2 beta1,6-N-acetylglucosaminyltransferase-I null mice. These results identify Core1-beta 3GlcNAcT and its cognate extended core1 O-glycans as essential participants in the expression of the MECA-79-positive, HEV-specific L-selectin ligands required for lymphocyte homing.

Helicobacter pylori infects the stomachs of nearly a half the human population, yet most infected individuals remain asymptomatic, which suggests that there is a host defense against this bacterium. Because H. pylori is rarely found in deeper portions of the gastric mucosa, where O-glycans are expressed that have terminal alpha1,4-linked N-acetylglucosamine, we tested whether these O-glycans might affect H. pylori growth. Here, we report that these O-glycans have antimicrobial activity against H. pylori, inhibiting its biosynthesis of cholesteryl-alpha-D-glucopyranoside, a major cell wall component. Thus, the unique O-glycans in gastric mucin appeared to function as a natural antibiotic, protecting the host from H. pylori infection.

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to unique carbohydrate ligands, sulfated sialyl Lewis(x), which are expressed on high endothelial venules (HEV) in secondary lymphoid organs. The nature of the sulfotransferase(s) that contribute to sulfation of such L-selectin counterreceptors has been uncertain. We herein describe a novel L-selectin ligand sulfotransferase, termed LSST, that directs the synthesis of the 6-sulfo sialyl Lewis(x) on L-selectin counterreceptors CD34, GlyCAM-1, and MAdCAM-1. LSST is predominantly expressed in HEV and exhibits striking catalytic preference for core 2-branched mucin-type O-glycans as found in natural L-selectin counterreceptors. LSST enhances L-selectin-mediated adhesion under shear compared to nonsulfated controls. LSST therefore corresponds to an HEV-specific sulfotransferase that contributes to the biosynthesis of L-selectin ligands required for lymphocyte homing.

Two human epithelial cell lines, trophoblastic teratocarcinoma HT-H and endometrial adenocarcinoma SNG-M cells, adhere to each other at their respective apical cell surfaces in a divalent cation-independent manner. Two novel molecules responsible for the adhesion between these two cell types were identified by expression eDNA cloning. One, named trophinin, is an intrinsic membrane protein and mediates homophilic self-binding. Another, named tastin, is a cytoplasmic protein and is necessary for trophinin to function as a cell adhesion molecule. Trophinin and tastin appear to be associated with the cytoskeleton in HT-H and SNG-M cells. These molecules are normally not expressed in various types of human cells in tissues, with the exception of macrophages. Strong expression of these molecules was detected in the trophectoderm surface of monkey blastocyst. These molecules are also expressed in human endometrial surface epithelium on day 16/17 at the early secretory phase of human endometrium, the time consistent with that expected for the "implantation window."

Lymphocyte homing is mediated by specific interactions between L-selectin on lymphocytes and sulfated carbohydrates restricted to high endothelial venules in lymph nodes. Here we generated mice deficient in both N-acetylglucosamine-6-O-sulfotransferase 1 (GlcNAc6ST-1) and GlcNAc6ST-2 and found that mutant mice had approximately 75% less homing of lymphocytes to the peripheral lymph nodes than did wild-type mice. Consequently, these mice had lower contact hypersensitivity responses than those of wild-type mice. Carbohydrate structural analysis showed that 6-sulfo sialyl Lewis X, a dominant ligand for L-selectin, was almost completely absent from the high endothelial venules of these mutant mice, whereas the amount of unsulfated sialyl Lewis X was much greater. These results demonstrate the essential function of GlcNAc6ST-1 and GlcNAc6ST-2 in L-selectin ligand biosynthesis in high endothelial venules and their importance in immune surveillance.

Macular corneal dystrophy (MCD; MIM 217800) is an autosomal recessive hereditary disease in which progressive punctate opacities in the cornea result in bilateral loss of vision, eventually necessitating corneal transplantation. MCD is classified into two subtypes, type I and type II, defined by the respective absence and presence of sulphated keratan sulphate in the patient serum, although both types have clinically indistinguishable phenotypes. The gene responsible for MCD type I has been mapped to chromosome 16q22, and that responsible for MCD type II may involve the same locus. Here we identify a new carbohydrate sulphotransferase gene (CHST6), encoding an enzyme designated corneal N-acetylglucosamine-6-sulphotransferase (C-GlcNAc6ST), within the critical region of MCD type I. In MCD type I, we identified several mutations that may lead to inactivation of C-GlcNAc6ST within the coding region of CHST6. In MCD type II, we found large deletions and/or replacements caused by homologous recombination in the upstream region of CHST6. In situ hybridization analysis did not detect CHST6 transcripts in corneal epithelium in an MCD type II patient, suggesting that the mutations found in type II lead to loss of cornea-specific expression of CHST6.

Pancreatic adenocarcinoma is an aggressive human malignancy and is characterized by resistance to apoptosis. Recently, NADPH oxidase (Nox) 4-mediated generation of intracellular reactive oxygen species (ROS) was proposed to confer antiapoptotic activity and thus a growth advantage to pancreatic cancer cells. The signaling mechanism by which Nox4 transmits cell survival signals remains unclear. Here, we show that both a flavoprotein inhibitor, diphenylene iodonium (DPI), and small interfering RNAs designed to target Nox4 mRNA (siNox4R-NAs) inhibited superoxide production in PANC-1 pancreatic cancer cells, and depletion of ROS by DPI or siNox4RNAs induced apoptosis. Parallely, DPI treatment and siNox4RNA transfection blocked activation of the cell survival kinase AKT by attenuating phosphorylation of AKT. Furthermore, AKT phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) on Ser-83 was reduced by DPI and siNox4RNAs. When ASK1Ser83Ala (an AKT phosphorylation-defective ASK1 mutant) was introduced into PANC-1 cells, this mutant alone induced apoptosis. But, addition of DPI or co-transfection of siNox4RNA had no additive effect, indicating that the mutant can substitute for these reagents in apoptosis induction. Taken together, these findings suggest that ROS generated by Nox4, at least in part, transmit cell survival signals through the AKT-ASK1 pathway in pancreatic cancer cells and their depletion leads to apoptosis.