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...

The cytoskeletal and/or nuclear matrix molecules responsible for morphological changes associated with apoptosis were identified using monoclonal antibodies (mAbs). We developed mAbs against Triton X-100-insoluble components of HL-60 cells pretreated with all-trans retinoic acid. In particular, one mAb recognized a 22-kDa protein that exhibited intriguing behavior by forming an aggregate and appearing as a speck during apoptosis induced by retinoic acid and other anti-tumor drugs. Cloning and sequencing of its cDNA revealed that this protein comprises 195 amino acids and that its Cterminal half has a caspase recruitment domain (CARD) motif, characteristic of numerous proteins involved in apoptotic signaling. We referred to this protein as ASC (apoptosis-associated speck-like protein containing a CARD). The ASC gene was mapped on chromosome 16p11.2-12. The antisense oligonucleotides of ASC were found to reduce the expression of ASC, and consequently, etoposide-mediated apoptosis of HL-60 cells was suppressed. Our results indicate that ASC is a novel member of the CARD-containing adaptor protein family.

Mutations in cryopyrin and pyrin proteins are responsible for several autoinflammatory disorders in humans, suggesting that these proteins play important roles in regulating inflammation. Using a HEK293 cell-based reconstitution system that stably expresses ASC and procaspase-1 we demonstrated that neither cryopyrin nor pyrin or their corresponding disease-associated mutants could significantly activate NF-jB in this system. However, both cryopyrin and two disease-associated cryopyrin mutants induced ASC oligomerization and ASC-dependent caspase-1 activation, with the disease-associated mutants being more potent than the wild-type (WT) cryopyrin, because of increased selfoligomerization. Contrary to the proposed anti-inflammatory activity of WT pyrin, our results demonstrated that pyrin, like cryopyrin, can also assemble an inflammasome complex with ASC and procaspase-1 leading to ASC oligomerization, caspase-1 activation and interleukin-1b processing. Thus, we propose that pyrin could function as a proinflammatory molecule.

The j31-6 structure of N-linked oligosaccharides, formed by j8-1,6-N-acetylglucosaminyltransferase (GnT-V), is associated with metastatic potential. We established a highly metastatic subclone, B16-hm, from low metastatic B16-F1 murine melanoma cells. The gene encoding 18-1,4-N-acetylglucosaminyltransferase (GnT-III) was introduced into the B16-hm cells, and three clones that stably expressed high GnT-III activity were obtained. In these transfectants, the affinity to leukoagglutinating phytohemagglutinin was reduced, whereas the binding to erythroagglutinating phytohemagglutinin was increased, indicating that the level of 31-6 structure was decreased due to competition for substrate between intrinsic GnT-V and ectopically expressed GnT-III.Lung metastasis after intravenous injection of the transfectants into syngeneic and nude mice was significantly suppressed, suggesting that the decrease in 81-6 structure suppressed metastasis via a mechanism independent of the murine system. These transfectants also displayed decreased invasiveness into Matrigel and inhibited cell attachment to collagen and laminin. Cell growth was not affected. Our results demonstrate a causative role for p1-6 branches in invasion and cell attachment in the extravasation stage of metastasis.Malignant transformation is highly associated with alterations of N-linked oligosaccharides. The extensive and persistent changes in the population of protein-bound oligosaccharides in malignant cells have been proposed to disturb cell division, decrease intercellular adhesiveness, and mask immunogenicity (1). The glycopeptides in polyoma-transformed baby hamster kidney cells have unusually high molecular weights due to the presence of external sialic acid as well as galactose and GlcNAc (2). The predominant surface glycopeptide of baby hamster kidney cells transformed by Rous sarcoma virus was determined to be a triantennary, completely sialylated, complex glycopeptide containing a core region of Man, GlcNAc, and Fuc (3). Synthesis of these elongated branches is initiated by 13-1,6-N-acetylglucosaminyltransferase (GnT-V; EC 2.4.1.155), which catalyzes the formation of the (31-6 branch (4). GnT-V activity correlated well with the metastatic potential of rastransformed Rat2 fibroblasts, SP1 mammary carcinoma cells, the MDAY-D2 lymphoma cell line (5, 6), and human colon cancer cells (7). Rat2 fibroblasts transfected with H-ras or v-frs exhibited metastatic potential and had elevated GnT-V activity and increased (1-6 branches (8), whereas a mutant with decreased GnT-V activity from a highly metastatic tumor cell line had a decreased potential for metastasis in mice (6). These observations have suggested a positive correlation between ,B1-6 branching and metastatic capacity.One approach to analyzing the role of GnT-V and its product, (31-6 branches, in metastasis is to reduce GnT-V activity in malignant cells with a high tendency to metastasize. As shown in Fig. 1, both 1-1,4-N-acetylglucosaminyltransferase (GnT-III; EC 2.4.1.144) and GnT-V use the t...

Carcinogenesis is a multistep process accompanied by genetic alterations of precancerous cells and by simultaneous construction of the tumor microenvironment.1 Clinical and experimental evidence have evaluated the importance of interactions between cancer cells and the surrounding stroma to facilitate tumor progression. 2,3One of the cellular components in the intratumoral stroma is a subpopulation of fibroblasts. 4 These activated fibroblasts, sometimes termed tumor-associated fibroblasts, are capable of modulating the tumor microenvironment during tumor development and progression. A specific contribution of the tumor-associated fibroblasts is to sup-

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Toll-like receptors (TLRs

Despite the importance of stromal cells in tumor progression, our overall understanding of the molecular signals that regulate the complex cellular interactions within tumor stroma is limited. Here, we provide multiple lines of evidence that tumor-associated macrophages (TAM) preferentially traffic to stromal areas formed within tumors in a manner dependent on a hyaluronan (HA)-rich tumor microenvironment. To address the role of stroma-derived HA in macrophage recruitment, we disrupted the HA synthase 2 (Has2) gene in stromal fibroblasts using conditional gene targeting. The Has2 null fibroblasts showed severe impairment in recruiting macrophages when inoculated with tumor cells into nude mice, which shows the contribution of stroma-derived HA in intratumoral macrophage mobilization. Furthermore, a deficiency in stromal HA attenuated tumor angiogenesis and lymphangiogenesis concomitantly with impaired macrophage recruitment. Taken together, our results suggest that stromal HA serves as a microenvironmental signal for the recruitment of TAMs, which are key regulatory cells involved in tumor neovascularization. Cancer Res; 70(18); 7073-83. ©2010 AACR.