Central role of endogenous Toll-like receptor-2 activation in regulating inflammation, reactive oxygen species production, and subsequent neointimal formation after vascular injury

Background

It is now evident that inflammation after vascular injury has significant impact on the restenosis after revascularization procedures such as angioplasty, stenting, and bypass grafting. However, the mechanisms that regulate inflammation and repair after vascular injury are incompletely understood. Here, we report that vascular injury-mediated cytokine expression, reactive oxygen species (ROS) production, as well as subsequent neointimal formation requires Toll-like receptor-2 (TLR-2) mediated signaling pathway in vivo.

Methods and results

Vascular injury was induced by cuff-placement around the femoral artery in non-transgenic littermates (NLC) and TLR-2 knockout (TLR-2KO) mice. After cuff-placement in NLC mice, expression of TLR-2 was significantly increased in both smooth muscle medial layer and adventitia. Interestingly, we found that inflammatory genes expression such as tumor necrosis factor-α, interleukin-1β (IL-1β), IL-6, and monocyte chemoattractant protein-1 were markedly decreased in TLR-2KO mice compared with NLC mice. In addition, ROS production after vascular injury was attenuated in TLR-2KO mice compared with NLC mice. Since we observed the significant role of endogenous TLR-2 activation in regulating inflammatory responses and ROS production after vascular injury, we determined whether inhibition of endogenous TLR-2 activation can inhibit neointimal proliferation after vascular injury. Neointimal hyperplasia was markedly suppressed in TLR-2KO mice compared with WT mice at both 2 and 4 weeks after vascular injury.

Conclusions

These findings suggested that endogenous TLR-2 activation might play a central role in the regulation of vascular inflammation as well as subsequent neointimal formation in injured vessels.     

Regulation of mitochondrial morphology and cell survival by Mitogenin I and mitochondrial single-stranded DNA binding protein

We found that a mouse homolog of human DNA polymerase delta interacting protein 38, referred to as Mitogenin I in this paper, and mitochondrial single-stranded DNA-binding protein (mtSSB), identified as upregulated genes in the heart of mice with juvenile visceral steatosis, play a role in the regulation of mitochondrial morphology. We demonstrated that overexpression of Mitogenin I or mtSSB increased elongated or fragmented mitochondria in mouse C2C12 myoblast cells, respectively. On the other hand, the silencing of Mitogenin I or mtSSB by RNA interference led to an increase in fragmented or elongated mitochondria in the cells, respectively, suggesting that Mitogenin I and mtSSB are involved in the processes of mitochondrial fusion and fission, respectively. In addition, we showed that the silencing of Mitogenin I resulted in an increase in the number of trypan blue-positive cells and the silencing of mtSSB resulted in an enhancement of the sensitivity of the cells to apoptotic stimulation by etoposide. The present results demonstrated that these proteins play a role in cell survival.     

Evidence for covalent lipoyl adduction with dopaquinone following tyrosinase-catalyzed oxidation

Previous studies have examined the conjugation of sulfhydryl compounds such as L-cysteine and glutathione with DOPA-quinone following the oxidation of tyrosine and DOPA by tyrosinase. These covalent reactions play a key role in the regulation and metabolism of pigment cells. We report on the first direct evidence for the formation of lipoyl adducts in reactions of thiol groups with DOPA-quinone in dihydrolipoic acid (6,8-dimercaptooctanoic acid [DHLA]). Incubating DHLA with DOPA-quinone followed by tyrosinase-catalyzed oxidation resulted in the three products predicted by HPLC-UV and LC-ESI(-)-MS analyses for DHLA DOPA conjugates. In the current study, we identified 5-S-lipoyl-DOPA among the principal products isolated by HPLC and characterized by FAB(-)-MS, ESI(-)-MS/MS, and 1H NMR, 2D-COSY studies. Collectively, these results suggest that DHLA undergoes sulfhydryl conjugation with DOPA-quinone, pointing to the involvement of thiol-reactive metabolites.     

Mu-calpain is involved in the regulation of TNF-alpha-induced matrix metalloproteinase-3 release in a rheumatoid synovial cell line

Calpain is secreted by intra-articular synovial cells and degrades the main components of cartilage matrix proteins, proteoglycan, and collagen, causing cartilage destruction. Matrix metalloproteinase-3 (MMP-3) has also been detected in synovial fluid and serum, and is involved in the development and progression of rheumatoid arthritis by degradation of the extracellular matrix and cartilage destruction. To investigate the relationship between calpain and MMP-3 in rheumatic inflammation, we utilized the rheumatic synovial cell line, MH7A. Tumor necrosis factor (TNF-alpha) stimulation-induced increased expression of mu-calpain, m-calpain, and MMP-3 in these cells, as well as the release of calpain and MMP-3 into the culture medium. The calpain inhibitors, ALLN (calpain inhibitor I) and calpeptin, did not affect the intracellular expression of MMP-3, but reduced the secretion of MMP-3 in a concentration-dependent manner. Down-regulation of mu- but not m-calpain by small interfering RNAs abolished TNF-alpha-induced MMP-3 release from the synovial cells. These findings suggest that calpain, particularly mu-calpain, regulates MMP-3 release by rheumatic synovial cells, in addition to exerting its own degradative action on cartilage.     

An N-glycan structure correlates with pulmonary metastatic ability of cancer cells

N-Glycan structures on the surface of cancer cells have diverse structures and play significant roles in metastatic process. However, little is known about their roles in organ-selective metastasis. Our study revealed that an alpha1,6-fucosylated biantennary N-glycan structure designated A2G2F is characteristic of lungs, with far more abundant expression in normal human and murine lungs than in other organs. In this study, we further examined the role of A2G2F in pulmonary metastasis. We stained metastatic cancers by alpha1,6-fucose-specific Lens culinaris agglutinin lectin and revealed that pulmonary metastatic nodules more abundantly expressed alpha1,6-fucosylated N-glycans than hepatic metastatic nodules from common primary cancers. The most specific alpha1,6-fucosylated N-glycan structure in pulmonary metastatic cancer was identified to be A2G2F. Using a B16 melanoma cell metastasis model, we showed that A2G2F-rich B16 cells formed more pulmonary metastatic nodules than A2G2F-poor cells. Our results suggest that A2G2F plays a critical role in pulmonary metastasis.     

Femtoliter compartment in liposomes for in vitro selection of proteins

The aqueous compartment in liposomes provides a reaction resembling the cell and therefore is used as a microcompartment in which to study enzymatic reactions. However, regardless of their method of preparation, the heterogeneity in size of cell-size liposomes limits their potential uses. We established a strategy to estimate the internal aqueous volume of cell-size liposomes using a fluorescence-activated cell sorter (FACS). Reactions inside individual liposomes can be measured in a high-throughput format provided that the encapsulated proteins give rise to a fluorescent signal such as by exhibiting fluorescence themselves or by catalyzing production of a fluorescent compound. The strategy of volume estimation was applied to in vitro selection experiments. The green fluorescent protein (GFP) gene was encapsulated into liposomes together with an in vitro translation system. Here liposomes carrying a single copy of the gene were identified using the internal aqueous volume information of individual liposomes, and those exhibiting higher green fluorescence intensity were sorted by the FACS machine. This system was able to enrich those encoding GFP with higher fluorescence intensity over those with lower intensity. These results suggest the possibility of performing evolutionary experiments in an environment that mimics the cell.     

The Caenorhabditis elegans homologue of deleted in azoospermia is involved in the sperm/oocyte switch

The Deleted in Azoospermia (DAZ) gene family encodes putative translational activators that are required for meiosis and other aspects of gametogenesis in animals. The single Caenorhabditis elegans homologue of DAZ, daz-1, is an essential factor for female meiosis. Here, we show that daz-1 is important for the switch from spermatogenesis to oogenesis (the sperm/oocyte switch), which is an essential step for the hermaphrodite germline to produce oocytes. RNA interference of the daz-1 orthologue in a related nematode, Caenorhabditis briggsae, resulted in a complete loss of the sperm/oocyte switch. The C. elegans hermaphrodite deficient in daz-1 also revealed a failure in the sperm/oocyte switch if the genetic background was conditional masculinization of germline. DAZ-1 could bind specifically to mRNAs encoding the FBF proteins, which are translational regulators for the sperm/oocyte switch and germ stem cell proliferation. Expression of the FBF proteins seemed to be lowered in the daz-1 mutant at the stage for the sperm/oocyte switch. Conversely, a mutation in gld-3, a gene that functionally counteracts FBF, could partially restore oogenesis in the daz-1 mutant. Together, we propose that daz-1 plays a role upstream of the pathway for germ cell sex determination.     

Helicobacter pylori eradication therapy on histologic change in the distal esophagus

BACKGROUND: Although cases of reflux esophagitis (RE) developing after treatment to eradicate Helicobacter pylori have been discussed in some detail, no reports are available concerning the histologic examination of RE both before and after eradication therapy. MATERIALS AND METHODS: Sixty-one patients and 111 specimens were investigated using endoscopic and histologic techniques. The histologic findings including basal zone height, papillar height, Ki-67 labeling index, and COX-2 expression before and after treatment for H. pylori infection were compared with those in normal controls and patients with endoscopic RE. RESULTS: Twelve months after eradication therapy, the incidence of newly developed endoscopic RE was 20% (5/25). Basal zone height and papillar height had increased at 1 month, but had returned to pretreatment levels after 12 months of eradication therapy. The Ki-67 labeling index was significantly increased 1 and 12 months after eradication therapy compared to values before treatment. COX-2 expression gradually increased after the treatment. The phenomena linked to esophagitis appeared after eradication therapy. However, the severity and extent of these signs were not so high after the treatment of H. pylori than those in patients with overt reflux esophagitis. Focusing on the patients with hiatal hernia, papillar height and Ki-67 labeling index increased significantly after eradication therapy, values being almost the same as those in the patients with endoscopic RE. CONCLUSIONS: Hiatal hernia plays an important role in the possible occurrence of hidden RE after treatment for a H. pylori infection.     

Changes in sperm motility in response to osmolality/Ca2+ in three Indonesian fresh water teleosts: goby (Oxyeleotris marmorata), Java carp (Puntius javanicus), and catfish (Clarias batrachus)

Sperm of most fresh water teleosts become motile when released into the hypotonic fresh water environment, but the role of osmolality and Ca2+ on sperm motility is not clear. Osmotic pressure and Ca2+ concentrations increase from fresh water to brackish water. Java carp Puntius javanicus and catfish Clarias batrachus live and reproduce only in fresh water. On the other hand, goby Oxyeleotris marmorata can acclimate and reproduce from fresh water to brackish water. In the present study, sperm motility and trajectory were compared among these three Indonesian endemic species. Sperm of Java carp, goby, and catfish begun to move in the hypotonic condition (< 200 mOsm/kg). However, the response to Ca2+ was different among these teleosts. In the presence of Ca2+, Java carp sperm swam in circular paths and immediately become quiescent, suggesting that Java carp sperm motility is activated in hypotonic aquatic environment without Ca2+. Goby sperm swam straightforward in the presence or absence of Ca2+. Percentages of motile sperm increased in 100-200 mOsm/kg but suppressed by removal of Ca2+. Regarding sperm motility and trajectory, no response was found in catfish sperm. These results suggest that a response to Ca2+ is different among sperm of the three species and suited to their habitat.     

Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible genes in human amniotic epithelial cells

Background  

Exposure to dioxins results in a broad range of pathophysiological disorders in human fetuses. In order to evaluate the effects of dioxins on the feto-placental tissues, we analyzed the gene expression in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated primary cultures of human amniotic epithelial cells.