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The presence of blood during ophthalmic surgery is problematic, as it can obstruct a surgeon’s view of the operative field. This is particularly true when performing trabeculectomy surgery to enhance ocular fluid outflow and reduce intraocular pressure as a treatment for glaucoma, one of the most common vision loss conditions worldwide. In this study, we investigated the performance of a transparent, self-assembling peptide gel (SPG-178) and its ability to maintain visibility during trabeculectomy surgery. Unlike the hyaluronic acid gel commonly used in ophthalmic surgery, SPG-178 did not permit the ingress of blood into the gel itself. Rather, it forced blood to flow peripherally to the gel. Moreover, if bleeding occurred under the SPG-178 gel, perfusion with saline was able to effectively flush the blood away along the interface between the SPG-178 and the ocular tissue (in this case scleral) to clear the surgical field of view. In experimental trabeculectomy surgeries with mitomycin C used as an adjuvant, there were no differences in the postoperative recovery of intraocular pressure or bleb morphology with or without the use of SPG-178. SPG-178, therefore, when used in a gel formulation, represents a new material for use in intraocular surgery to ensure a clear operative field with likely beneficial treatment outcomes.

Caveolae are defined as 50–100 nm wide pits in the plasma membrane containing oligomeric caveolin proteins. They have been implicated in endocytosis (including phagocytosis), transcytosis, calcium signalling, and numerous other signal transduction events. Caveolin-1, a major structural component of caveolae, enhances Rab5 activity. In this study, we examined the effect of a synthetic cell-permeable peptide of the caveolin-1 scaffolding domain (CSD) on phagocytosis. Treatment with the CSD peptide increased Rab5 activity, Rab5-early endosome antigen 1 (EEA1) interaction, and phagocytosis of Escherichia coli. The results suggest that the synthetic cell-permeable CSD peptide is an activator of phagocytosis.

To determine senescence-associated changes in the gingival tissues of aged mice and gingival fibroblast cultures.The production of senescence-associated β-galactosidase (SA-β-gal) and mRNA expression of p16, p21, interleukin (IL)-1β, and tumor necrosis factor α (TNF-α) were evaluated in gingival tissues, gingival fibroblasts of 10- and 20-month-old C57BL/6NCrl mice, and multiple-passaged and hydrogen peroxide-stimulated human gingival fibroblasts (HGFs). Changes in molecular expression in HGF cultures due to senescent cell elimination by the senolytic drug ABT-263 (Navitoclax) were analyzed.Compared to 10-week-old mice, the 20-month-old mice had higher numbers of M1 macrophages. The proportion of cells expressing SA-β-gal were also higher in 20- month-old mice than in 10-week-old-mice. Gingival fibroblasts in 20-month-old mice expressed less collagen 1a1, collagen 4a1, and collagen 4a2 mRNA than those in 10-week-old mice. Compared to control cells, H2O2 treated HGF cells expressed higher levels of SA-β-gal and p16, p21, IL-1β, and TNF-α. Furthermore, ABT-263 suppressed HGF cell expression of cytokines after senescence induction.Senescence-associated changes were observed in the gingival tissues of aged mice and HGF cultures. In addition, the potential of senolytic drugs to modify aging-related changes in the gingiva was shown.

Common age-related changes in the human eye contribute to the development of dry eye, including decreases in aqueous tear production. Although the infiltration of lymphocytes into the lacrimal glands occurs with age, age-related increases in tear production have also been observed in mice; however, the mechanisms underlying this increase remain unclear. We herein demonstrated that increases in tear production were not dependent on body weight gain or systemic conditions, such as insulin resistance, using aged mice and high-fat diet-fed mice. The results obtained also showed that senescence-associated T (SA-T) cells accumulated in the lacrimal glands of aged mice, particularly females. Expression levels of the nuclear transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) in whole lacrimal glands and epithelial cells isolated from lacrimal glands were significantly higher in aged mice than in young mice. The expression levels of adiponectin and one of its receptors, AdipoR2, also increased in the lacrimal glands of aged mice, but not in those of high-fat diet-fed mice. Collectively, the present results indicate that PPARγ and adiponectin-mediated signaling contribute to age-related increases in tear production in mice and have potential as therapeutic targets for the treatment of dry eye in humans.

Objectives Tooth bleaching has become one of the most frequently requested esthetic procedures in dental practice. A side effect of bleaching is gingival irritation. This study examined the efficacy of propolis to treat gingival irritation caused by bleaching in vivo and in vitro. Methods Gingival irritation was mimicked by a mild burn injury to oral mucosa in young (10 week old) and aged (18 month old) mice. Propolis ointment was immediately applied to the burn area. After 24 h, gingiva was collected to determine the efficacy of propolis by hematoxylin and eosin staining and real-time polymerase chain reaction (PCR). Results Topical application of propolis ointment reduced the infiltration of inflammatory cells at irritated sites and promoted the repair of the mucosal epithelium in young and aged mice. It also suppressed the expression of IL-1 β and TNF-α and increased keratin 1 and 5 expression in the irritated gingiva. Propolis suppressed an increase in IL-1β and TNF- α upon stimulation with H2O2 in young and SA- β -gal-expressing senescent human gingival fibroblasts (HGFs) cultures. Conclusions Propolis may be effective for mucosal repair in gingival irritation as it suppresses the expression of proinflammatory cytokines and promotes keratin expression.

To determine the contribution of the ON and OFF cone bipolar cell pathways to the electroretinograms (ERGs) elicited by ultraviolet (UV) and middle wavelength light in mice.The experiments were performed on 8- to 10-week-old C57BL/6J mice. The ERGs elicited by single-flash and flickering UV light stimuli were compared to those elicited by green light stimuli under photopic conditions. Pharmacological agents were used to selectively block the ON and OFF pathways contributing to the ERGs. Saline was used as a control. The flicker ERGs elicited by UV light were compared to the ERGs elicited by green light after the injection of the pharmacological agents to determine the contribution of the cone ON and OFF pathways to the ERGs.The photopic single-flash and flicker ERGs were more sensitive to the UV light stimuli than to those elicited by green light stimuli. The flicker ERG responses elicited by both UV and green light stimuli at stimulus frequencies lower than 15-Hz decreased after L-2-amino-4-phosphobutyric acid was injected. The ERGs elicited by UV light at 30-Hz and by green light at frequencies lower than 15-Hz decreased significantly after the intravitreal injection of cis-2, 3-piperidine-dicarboxylic acid. An analysis of the ON and OFF components of the flicker ERGs showed that there might be pharmacological differences between the UV light-sensitive responses and the green light-sensitive responses.These results suggest that the UV light-sensitive cones connect to both the ON and OFF bipolar cells differently than that of the green light-sensitive cones.

Summary: In the vertebrate retina, cone photoreceptors play crucial roles in photopic vision by transmitting light-evoked signals to ON- and/or OFF-bipolar cells. However, the mechanisms underlying selective synapse formation in the cone photoreceptor pathway remain poorly understood. Here, we found that Lrit1, a leucine-rich transmembrane protein, localizes to the photoreceptor synaptic terminal and regulates the synaptic connection between cone photoreceptors and cone ON-bipolar cells. Lrit1-deficient retinas exhibit an aberrant morphology of cone photoreceptor pedicles, as well as an impairment of signal transmission from cone photoreceptors to cone ON-bipolar cells. Furthermore, we demonstrated that Lrit1 interacts with Frmpd2, a photoreceptor scaffold protein, and with mGluR6, an ON-bipolar cell-specific glutamate receptor. Additionally, Lrit1-null mice showed visual acuity impairments in their optokinetic responses. These results suggest that the Frmpd2-Lrit1-mGluR6 axis regulates selective synapse formation in cone photoreceptors and is essential for normal visual function. : Ueno et al. finds that Lrit1 plays an important role in regulating the synaptic connection between cone photoreceptors and cone ON-bipolar cells. The Frmpd2-Lrit1-mGluR6 axis is crucial for selective synapse formation in cone photoreceptors and for development of normal visual function. Keywords: retina, circuit, synapse formation, cone photoreceptor cell, ON-bipolar cell, visual acuity

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Corticosteroids, which are widely used for AD treatment, have adverse effects, and alternative treatments are urgently needed. This study examined the effect of topical application of high-dose glucose on inflamed skin in a murine model of AD. High-dose glucose treatment on the ear reduced dermatitis scores and ear thicknesses in mite antigen-treated NC/Nga mice. The levels of thymus and activation-regulated chemokine (TARC), Th cytokines (interleukin (IL)-4, IL-5, IL-12, IL-13, and (interferon) IFN-γ), and IgE were decreased in the serum of high-dose glucose-treated mice. Expression of claudin-1 and filaggrin was reduced in the ear epithelium in the NC/Nga mice. However, the reduced expression was restored by topical treatment with high-dose glucose. High-dose glucose also induced the expression of claudin-1 and filaggrin in cultured human skin keratinocytes. Co-stimulation with IL-4, IL-13, and thymic stromal lymphoprotein downregulated the expression of filaggrin in culture. However, high-dose glucose treatment restored the reduced expression of filaggrin. These results suggest that high-dose glucose treatment suppresses inflammation in the skin lesions by improving the skin barrier function.

The morbidity of temporomandibular joint osteoarthritis (TMJOA) increases with age. Condylar articular cartilage degradation, which causes TMJOA, is known to be involved in articular chondrocyte metabolic imbalances in the temporomandibular joint (TMJ) and in other joints of the body. Epigenetic regulation, such as the chemical modification of DNA and histones, is implicated in cartilage homeostasis. However, few studies have been conducted on the epigenetic regulation of condylar articular cartilage degradation. The present study investigated the regulation of histone H3 lysine 9 (H3K9) methylation and its effects on the pathogenesis of degenerative TMJ cartilage disorders. The histone H3K9 methylation level was decreased in degenerated condylar articular cartilage in aged mice. Treatment with chaetocin (a selective H3K9 methylation inhibitor) reduced cell viability and promoted caspase‑3/7 activity in ATDC5 mouse chondroprogenitor cells. The inhibition of H3K9 methylation increased matrix metalloproteinase (Mmp)1 and Mmp13 mRNA expression in these cells. Furthermore, the expression levels of Sox9 and collagen α1(II) (Col2a1) mRNA, which are anabolic factors for chondrogenic differentiation, were also decreased by treatment with chaetocin, which is an inhibitor of histone methyltransferases. These results indicated that histone H3K9 methylation regulates chondrocyte homeostasis in terms of cell growth, apoptosis and gene expression, and highlighted a possible future therapy option for TMJOA.

Immunosenescence is characterized by age-associated changes in immunological functions. Although age- and autoimmune-related sialadenitis cause dry mouth (xerostomia), the roles of immunosenescence and cellular senescence in the pathogenesis of sialadenitis remain unknown. We demonstrated that acquired immune cells rather than innate immune cells infiltrated the salivary glands (SG) of aged mice. An analysis of isolated epithelial cells from SG revealed that the expression levels of the chemokine CXCL13 were elevated in aged mice. Senescence-associated T cells (SA-Ts), which secrete large amounts of atypical pro-inflammatory cytokines, are involved in the pathogenesis of metabolic disorders and autoimmune diseases. The present results showed that SA-Ts and B cells, which express the CXCL13 receptor CXCR5, accumulated in the SG of aged mice, particularly females. CD4+ T cells derived from aged mice exhibited stronger in vitro migratory activity toward CXCL13 than those from young mice. In a mouse model of Sjögren’s syndrome (SS), SA-Ts also accumulated in SG, presumably via CXCL12-CXCR4 signaling. Collectively, the present results indicate that SA-Ts accumulate in SG, contribute to the pathogenesis of age- and SS-related sialadenitis by up-regulating chemokines in epithelial cells, and have potential as therapeutic targets for the treatment of xerostomia caused by these types of sialadenitis.

Recent identification of platelet/megakaryocyte-biased hematopoietic stem/repopulating cells requires revision of the intermediate pathway for megakaryopoiesis. Here, we show a unipotent megakaryopoietic pathway bypassing the bipotent megakaryocyte/erythroid progenitors (biEMPs). Cells purified from mouse bone marrow by CD42b (GPIbα) marking were demonstrated to be unipotent megakaryocytic progenitors (MKPs) by culture and transplantation. A subpopulation of freshly isolated CD41+ cells in the lineage Sca1+cKit+ (LSK) fraction (subCD41+LSK) differentiated only into MKP and mature megakaryocytes in culture. Although CD41+LSK cells as a whole were capable of differentiating into all myeloid and lymphoid cells in vivo, they produced unipotent MKP, mature megakaryocytes, and platelets in vitro and in vivo much more efficiently than Flt3+CD41−LSK cells, especially at the early phase after transplantation. In single cell polymerase chain reaction and thrombopoietin (TPO) signaling analyses, the MKP and a fraction of CD41+LSK, but not the biEMP, showed the similarities in mRNA expression profile and visible TPO-mediated phosphorylation. On increased demand of platelet production after 5-FU treatment, a part of CD41+LSK population expressed CD42b on the surface, and 90% of them showed unipotent megakaryopoietic capacity in single cell culture and predominantly produced platelets in vivo at the early phase after transplantation. These results suggest that the CD41+CD42b+LSK are straightforward progenies of megakaryocytes/platelet-biased stem/repopulating cells, but not progenies of biEMP. Consequently, we show a unipotent/highly biased megakaryopoietic pathway interconnecting stem/repopulating cells and mature megakaryocytes, the one that may play physiologic roles especially in emergency megakaryopoiesis. Stem Cells 2015;33:2196–2207

Interleukin-33 (IL-33) is an IL-1 cytokine family member that is involved in the development of chronic inflammatory diseases and the initiation of allergic inflammation in response to pathogens. Porphyromonas gingivalis is a primary pathogen that is involved in chronic periodontitis and its bacterial components induce inflammatory responses. Dendritic cells (DCs) recognize pathogen- associated molecular patterns by expression of pattern-recognition receptors, such as Toll-like receptors (TLRs). DCs play an essential role in resistance to infection and maintenance of mucosal immune system. In this study, we investigated whether P. gingivalis increases the expression of IL-33 in mouse bone marrow-derived DCs (BMDCs). BMDCs exhibited an increased expression of IL-33 mRNA upon stimulation with P. gingivalis whole cells. Furthermore, fimbriae and lipopeptide derived from P. gingivalis exhibited higher IL-33 mRNA expression than P. gingivalis whole cells. In contrast, lipopolysaccharide derived from P. gingivalis did not induce IL-33 mRNA expression in BMDCs. The IL-33 mRNA expression after stimulation with fimbriae or lipopeptide was up-regulated in BMDCs from wild-type mice but not from TLR2-deficient (TLR2

The protection of telomeres 1 (POT1) protein is a 75-kDa protein that plays an important role in telomere protection, which is related to telomere elongation. Although POT1 is present in and acts in the nuclei, little is known about the functions of POT1 in the cytosol. We here examined the role of POT1b in phagocytosis in a macrophage-like RAW 264 cell line. We found that POT1 was present in the cytosol, where it was bound to Rab5, which is a protein important for endocytosis. POT1b knockdown in RAW 264 cells increased Rab5 activity and facilitated the phagocytosis of whole cells of Escherichia coli and Staphylococcus aureus. Furthermore, POT1b knockdown enhanced the expression of inducible nitric oxide synthase (iNOS), followed by the promotion of nitric oxide (NO) generation in response to stimulation by bacterial whole cells. These results suggest that POT1b negatively regulates phagocytosis by controlling Rab5 activity and thereby modulates bacteria-induced NO generation. These findings suggest that POT1b participates in innate immune responses.

Interleukin (IL)-31 is important for innate immunity in mucosal tissues and skin, and increased IL-31 expression participates in the pathogenesis of chronic inflammatory diseases affecting the skin, airways, lungs, and intestines. We investigated the contribution of mast cells to the induction of IL-31 production following infection with the periodontal pathogen, Porphyromonas gingivalis. We found that oral infection with P. gingivalis increased IL-31 expression in the gingival tissues of wild-type mice but not in those of mast cell-deficient mice. The P. gingivalis-induced IL-31 production by human mast cells occurred through the activation of the JNK and NF-κB signalling pathways and was dependent on the P. gingivalis lysine-specific protease gingipain-K. P. gingivalis infection induced IL-31 receptor α and oncostatin M receptor β expression in human gingival epithelial cells. Notably, the P. gingivalis-induced IL-31 production by mast cells led to the downregulation of claudin-1, a tight junction molecule, in gingival epithelial cells, resulting in an IL-31-dependent increase in the paracellular permeability of the gingival epithelial barrier. These findings suggest that IL-31 produced by mast cells in response to P. gingivalis infection causes gingival epithelial barrier dysfunction, which may contribute to the chronic inflammation observed in periodontitis.

1,5-anhydro- d -fructose (1,5-AF), a monosaccharide formed from starch and glycogen, exhibits antioxidant and antibacterial activity, and inhibits cytokine release by attenuating NF-κB activation in LPS-stimulated mice. The present study examined whether 1,5-AF inhibits lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) in vitro and in vivo. We found that 1,5-AF significantly blocked the production of NO, and protein and mRNA expression of iNOS, and up-regulated IL-10 production in vitro. We also investigated the effects of 1,5-AF on acute lung inflammation in C57BL/6J mice. We found that protein and mRNA expression of iNOS in lung tissues were inhibited by 1,5-AF pretreatment. In addition, the serum level of IL-10 was upregulated by 1,5-AF. Collectively, the iNOS transcriptional and translational inhibitory effects of 1,5-AF seem to be prolonged and enhanced by the production of IL-10. These results suggest that 1,5-AF could be a useful adjunct in the treatment of acute lung inflammation.

Aim: To achieve complete regeneration of dental pulp in vivo by stem/progenitor cells obtained from a fraction of side population (SP) cells from canine pulp. Materials & methods: A subfraction of SP cells, CD31-/CD146- SP cells, were isolated by flow cytometry from canine dental pulp. The efficiency of this subfraction of SP cells was evaluated in an experimental model of pulp injury in the dog. Results: The fractionated SP cells formed extensive networks of tube-like structures in vitro. Transplantation of the SP cells into an in vivo model of amputated pulp resulted in complete regeneration of pulp tissue with capillaries and neuronal cells within 14 days. Gene-expression studies demonstrated the expression of pro-angiogenic factors, implying trophic action on endothelial cells. Conclusions: This investigation demonstrates the potential utility of fractionated SP cells as a source of cells for total pulp regeneration complete with angiogenesis and vasculogenesis.

Aquaporin 3 (AQP3) is the predominant water channel protein in human keratinocytes and acts as an inflammatory mediator in some lesions. A chronic, inflammatory process of periodontitis is related with a dramatic change of surrounding fluid homeostasis to plasma extravasation. The exact pattern of aquaporin (AQP) water channel expression and its mechanism in periodontal disease is still unknown. We describe herein an up-regulated AQP3 expression in the epithelial lesion with chronic periodontitis and its functional role. The levels of AQP3 expression in inflamed gingival epithelial tissues were significantly higher than those of healthy subjects. Consistent with these results, AQP3 expression (i.e., levels of mRNA and protein) in cultured rat primary gingival epithelial cells and the human gingival epithelial cell line Ca9-22 were strongly increased in response to TNF-alpha treatment through the 55 kDa TNF-alpha receptor (TNFR I). In this context, small interfering RNA- (siRNA)-mediated "aqp-3 gene silencing," which could reduce AQP3 expression by more than 65%, significantly attenuated selected proinflammatory events of ICAM-1 expression induced by TNF-alpha in Ca9-22. A sixfold increase in leukocyte adherence to TNF-alpha-stimulated epithelial cells was demonstrated by an adherence assay (P < 0.001) and pretreatment with AQP3 siRNA and anti-ICAM-1 antibody reduced leukocyte retention by 85% (P < 0.001). Our study indicates for the first time a novel important mode in the regulation of the inflammatory response through TNF-alpha/TNFR I ligation at the site of epithelial lesions by specialized membrane channel AQP3 and ICAM-1 protein, which is closely implicated in the development of periodontitis mechanisms.

To investigate the effect of electrical stimulation (ES) on the induction of insulin-like growth factor 1 (IGF-1) in cultured retinal Müller cells.Müller cells were isolated from rat retinas. ES was applied to Müller cells of passage 1 with biphasic pulses (duration, 1 ms; frequency, 20 Hz; current, 0-10 mA) for 30 min. The mRNA level of IGF-1 was determined by reverse transcription-polymerase chain reaction (RT-PCR) immediately to 2 h after ES. The change of intracellular calcium concentration ([Ca(2+)]) induced by ES was monitored by Ca(2+) imaging with Fura 2-AM. Ca(2+) imaging and RT-PCR were performed with and without the application of l muM nifedipine, an L-type calcium channel blocker.The mRNA level of IGF-1 was increased significantly (P0.05) by about 1.3-fold immediately after 10 mA ES. [Ca(2+)] began to increase immediately after the start of ES, reached a maximum of approximately 1.8-fold, and continued to increase until about 20 min after the ES. The inductions of IGF-1 transcription and Ca(2+) influx were suppressed by nifedipine.These results indicate that the enhancement of IGF-1 transcription by ES in cultured Müller cells depends largely on Ca(2+) influx through L-type Ca(2+) channels.

Osterix, a zinc-finger-containing transcription factor, is required for osteoblast differentiation and bone formation. Osterix is also expressed in dental mesenchymal cells of the tooth germ. However, transcriptional regulation by Osterix in tooth development is not clear. Genetic studies in osteogenesis place Osterix downstream of Runx2 (Runt-related 2). The expression of Osterix in odontoblasts overlaps with Runx3 during terminal differentiation in vivo. Runx3 down-regulates Osterix expression in mouse DPCs (dental pulp cells). Therefore the regulatory role of Runx3 on Osterix expression in tooth development was investigated. Enforced expression of Runx3 down-regulated the activity of the Osterix promoter in the human embryonic kidney 293 cell line. When the Runx3 responsive element on the Osterix promoter, located at −713 to −707 bp (site 3, AGTGGTT) relative to the cap site, was mutated, this down-regulation was abrogated. Furthermore, electrophoretic mobility-shift assay and chromatin immunoprecipitation assays in mouse DPCs demonstrated direct functional binding of Runx3 to the Osterix promoter. These results demonstrate the transcriptional regulation of Osterix expression by Runx3 during differentiation of dental pulp cells into odontoblasts during tooth development.

Purpose: Recently, 14-member macrolide antibiotics such as clarithromycin and roxithromycin have been shown to have anticancer and antiangiogenic effects. We investigated the suppressive effect of roxithromycin on accelerated hepatocellular carcinoma growth in a rat hepatocarcinogenetic model and compared results with effects from TNP-470.Experimental Design: Tumor was induced by oral diethylnitrosamine administration for 17 weeks. Normal saline, TNP-470 (50 mg/kg), or roxithromycin (40 or 100 mg/kg) was administered i.p. thrice per week from week 10 to 17.Results: Carcinomatous tissue growing outside dysplastic nodules and a marked expression of placental glutathione S-transferase were detected in rats with induced carcinogenesis. Tumor growth was accompanied by augmented expression of inducible nitric oxide synthase, activation of nuclear factor κB, and increased lipid peroxidation level. All these effects were absent in animals that received roxithromycin or TNP-470. The inhibitory effect of roxithromycin was dose dependent and no clear differences were noted between groups given roxithromycin 100 mg/kg and TNP-470 50 mg/kg.Conclusions: Our results indicate that roxithromycin inhibits oxidative stress, nitric oxide production, and nuclear factor κB activation induced by experimental hepatocarcinogenesis. The data provide additional evidence for the potential use of roxithromycin in treatment of hepatocellular carcinoma prevention.

Although an excess of reactive oxygen species (ROS) can damage the vasculature, low concentrations of ROS mediate intracellular signal transduction pathways. We hypothesized that hydrogen peroxide plays a beneficial role in the vasculature by inhibiting endothelial exocytosis that would otherwise induce vascular inflammation and thrombosis. We now show that endogenous H(2)O(2) inhibits thrombin-induced exocytosis of granules from endothelial cells. H(2)O(2) regulates exocytosis by inhibiting N-ethylmaleimide sensitive factor (NSF), a protein that regulates membrane fusion events necessary for exocytosis. H(2)O(2) decreases the ability of NSF to hydrolyze adenosine triphosphate and to disassemble the soluble NSF attachment protein receptor complex. Mutation of NSF cysteine residue C264T eliminates the sensitivity of NSF to H(2)O(2), suggesting that this cysteine residue is a redox sensor for NSF. Increasing endogenous H(2)O(2) levels in mice decreases exocytosis and platelet rolling on venules in vivo. By inhibiting endothelial cell exocytosis, endogenous H(2)O(2) may protect the vasculature from inflammation and thrombosis.

Endothelial exocytosis is an early stage in the process of leukocyte trafficking. N-ethylmaleimide-sensitive factor (NSF) plays a critical role in regulating exocytosis. We hypothesized that inhibitors of NSF decrease endothelial exocytosis and vascular inflammation. We designed a novel fusion polypeptide consisting of a human immunodeficiency virus transactivator of transcription (TAT) protein transduction domain joined to a NSF homohexamerization domain. We show that this TAT-NSF polypeptide inhibits the ATPase activity and the disassembly activity of NSF. Furthermore, the TAT-NSF polypeptide decreases endothelial cell exocytosis and reduces leukocyte adherence to endothelial cells in culture. Finally, the TAT-NSF polypeptide inhibits leukocyte rolling on murine venules in vivo and inhibits leukocyte trafficking into the peritoneal cavity in a murine model of experimental peritonitis. These data suggest that NSF is a critical regulator of leukocyte trafficking in vivo. Novel compounds that inhibit the exocytic machinery in endothelial cells may be useful anti-inflammatory drugs.

N-Ethyl-maleimide-sensitive factor (NSF) plays a critical role in the regulation of exocytosis. NSF regulates exocytosis by interacting with a complex containing soluble NSF attachment protein receptor (SNARE) molecules, hydrolyzing ATP, and disassembling the SNARE complex. We hypothesized that peptide inhibitors of NSF would decrease exocytosis. We now report the development of a novel set of peptides that block exocytosis by inhibiting NSF activity. These NSF inhibitors are fusion polypeptides composed of an 11 amino acid human immunodeficiency virus transactivating regulatory protein (TAT) domain fused to a 22 amino acid NSF domain. These TAT-NSF fusion polypeptides cross endothelial cell membranes, inhibit NSF hydrolysis of ATP, decrease NSF disassembly of SNARE molecules, and block exocytosis of von Willebrand factor. Control peptides have no effect on exocytosis. TAT-NSF inhibitors administered to mice prolong the bleeding time. Blood concentrations of these TAT-NSF peptides rapidly decrease within 5 min after injection and then remain constant from 10 to 60 min after injection. These TAT-NSF compounds may be useful in the treatment of a variety of diseases in which exocytosis plays a prominent role, including myocardial infarction, stroke, thrombosis, and autoimmune disorders.

Although peroxynitrite is harmful to the host, the beneficial effects of peroxynitrite are less well understood. We explored the role of peroxynitrite in the host immune response to Coxsackievirus infection. Peroxynitrite inhibits viral replicationin vitro, in part by inhibiting viral RNA entry into the host cell. Nitrotyrosine, a marker for peroxynitrite production, is colocalized with viral antigens in the hearts of infected mice but not control mice. Nitrotyrosine coprecipitates with the viral polypeptide VP1 as well. Guanidinoethyl disulfide, a scavenger of peroxynitrite, blocks peroxynitrite inhibition of viral replicationin vitroand permits an increase in viral replicationin vivo. These data suggest that peroxynitrite is an endogenous effector of the immune response to viruses.

The mechanism underlying anti-inflammatory effects of macrolide antibiotics remains uncertain. In this study, we first show the evidences concerning the possible link between leukocytic cyclic adenosine monophosphate (cAMP) signaling and the mechanism of anti-inflammatory, cytoprotective actions of macrolides. The clinical range of macrolides (i.e., erythromycin, roxithromycin, and clarithromycin) preferentially inhibited nuclear factor-κB activation mediated by reactive oxygen intermediates, inducing cAMP-dependent signaling [i.e., cAMP and cAMP-responsive element-binding protein (CREB)] by “primed” but not “resting” leukocytes. In this context, cAMP/CREB inhibition with adenosine 3′:5′-cyclic monophosphothioate, rp-isomer (rp-cAMPs) and CREB decoy oligonucleotides reduced the anti-inflammatory actions of macrolides. These results thus indicate that macrolide-induced cAMP/CREB signaling, selectively by primed leukocytes, plays a major role in the mechanism of anti-inflammatory actions of macrolides.

ATP-sensitive K(+) (K(ATP)) channels comprise the pore-forming subunit (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptors (SUR1 or SUR2). K(ATP) channels with different combinations of these subunits are present in various tissues and regulate cellular functions. From the analysis of mouse models with targeted deletion of the gene encoding the pore-forming subunit Kir6.1 or Kir6.2, functional roles of K(ATP) channels in various organs have been clarified. Kir6.1(-/-) mice showed sudden death associated with ST elevation and atrioventricular block in ECG, a phenotype resembling Prinzmetal angina in humans. Kir6.2(-/-) mice were more susceptible to generalized seizure during hypoxia than wild-type (WT) mice, suggesting that neuronal K(ATP) channels, probably composed of Kir6.2 and SUR1, play a crucial role for the protection of the brain against lethal damage due to seizure. In Kir6.2(-/-) mice lacking the sarcolemmal K(ATP) channel activity in cardiac cells, ischemic preconditioning failed to reduce the infarct size, suggesting that sarcolemmal K(ATP) channels play an important role in cardioprotection against ischemia/reperfusion injuries in the heart. Mitochondrial K(ATP) channels have been also proposed to play a crucial role in cardioprotection, although the molecular identity of the channel has not been established. Nicorandil and minoxidil, K(+) channel openers activating mitochondrial K(ATP) channels, decreased the mitochondrial membrane potential, thereby preventing the Ca(2+) overload in the mitochondria of guinea-pig ventricular cells. SURs are the receptors for K(+) channel openers and the activating effects on sarcolemmal K(ATP) channels in cardiovascular tissues could be modulated by the interaction of nucleotides. Due to the molecular diversity of the accessory and pore subunits of K(ATP) channels, there would be considerable differences in the tissue selectivity of K(ATP) channel-acting drugs. Studies of Kir6.1 and Kir6.2 knockout mice indicate that K(ATP) channels are involved in the mechanisms of the protection against metabolic stress. Further clarification of physiological as well as pathophysiological roles of K(ATP) channels may lead to a new therapeutic strategy to improve the quality of life.

1. The inwardly rectifying K+ channel current (IK(IR)) recorded from isolated retinal pigmented epithelial (RPE) cells showed poor dependence on external K+ ([K+]o) and low sensitivity to block by Ba2+. We examined the molecular identity and specific subcellular localization of the KIR channel in RPE cells. 2. The Kir7.1 channel current heterologously expressed in HEK293T cells (human embryonic kidney cell line) showed identical properties to those of the RPE IK(IR), i.e. poor dependence on [K+]o and low sensitivity to Ba2+ block. 3. Expression of Kir7.1 mRNA and protein was detected in RPE cells by RT-PCR and immunoblot techniques, respectively. 4. Immunohistochemical studies including electron microscopy revealed that the Kir7.1 channel was localized specifically at the proximal roots of the apical processes of RPE cells, where Na+,K+-ATPase immunoreactivity was also detected. 5. The middle-distal portions of apical processes of RPE cells in the intact tissue exhibited immunoreactivity of Kir4.1, a common KIR channel. In the isolated RPE cells, however, Kir4.1 immunoreactivity was largely lost, while Kir7.1 immunoreactivity remained. 6. These data indicate that the only IK(IR) recorded in isolated RPE cells is derived from the functional Kir7.1 channel localized at the root of apical processes. Co-localization with Na+,K+-ATPase suggests that the Kir7.1 channel may provide the pathway for recycling of K+ to maintain pump activity and thus is essential for K+ handling in RPE cells.

Abstract —The ATP-sensitive K + (K ATP ) channels are composed of the pore-forming K + channel Kir6.0 and different sulfonylurea receptors (SURs). SUR1, SUR2A, and SUR2B are sulfonylurea receptors that are characteristic for pancreatic, cardiac, and vascular smooth muscle–type K ATP channels, respectively. The structural elements of SURs that are responsible for their different characteristics have not been entirely determined. Here we report that the 42 amino acid segment at the C-terminal tail of SURs plays a critical role in the differential activation of different SUR-K ATP channels by ADP and diazoxide. In inside-out patches of human embryonic kidney 293T cells coexpressing distinct SURs and Kir6.2, much higher concentrations of ADP were needed to activate channels that contained SUR2A than SUR1 or SUR2B. In all types of K ATP channels, diazoxide increased potency but not efficacy of ADP to evoke channel activation. Replacement of the C-terminal segment of SUR1 with that of SUR2A inhibited ADP-mediated channel activation and reduced diazoxide modulation. Point mutations of the second nucleotide-binding domains (NBD2) of SUR1 and SUR2B, which would prevent ADP binding or ATP hydrolysis, showed similar effects. It is therefore suggested that the C-terminal segment of SUR2A possesses an inhibitory effect on NBD2-mediated ADP-induced channel activation, which underlies the differential effects of ADP and diazoxide on K ATP channels containing different SURs.

The ribosomes from BCG strongly induced delayed type hypersensitivity (DTH) skin reactions in guinea pigs immunized with live BCG or heat killed Mycobacterium tuberculosis H37Rv, and also induced lymphocyte proliferative response in mice immunized with ribosomes. In contrast, neither ribosomal proteins nor RNA alone induced both DTH skin reactions and lymphocyte proliferative responses. Particle form consisted of ribosomal proteins and RNAs might be absolutely required for the activation of immune responses.

Treatment of deep caries with pulpitis is a major challenge in dentistry. Stem cell therapy represents a potential strategy to regenerate the dentin-pulp complex, enabling conservation and restoration of teeth. The objective of this study was to assess the efficacy and safety of pulp stem cell transplantation as a prelude for the impending clinical trials. Clinical-grade pulp stem cells were isolated and expanded according to good manufacturing practice conditions. The absence of contamination, abnormalities/aberrations in karyotype, and tumor formation after transplantation in an immunodeficient mouse ensured excellent quality control. After autologous transplantation of pulp stem cells with granulocyte-colony stimulating factor (G-CSF) in a dog pulpectomized tooth, regenerated pulp tissue including vasculature and innervation completely filled in the root canal, and regenerated dentin was formed in the coronal part and prevented microleakage up to day 180. Transplantation of pulp stem cells with G-CSF yielded a significantly larger amount of regenerated dentin-pulp complex compared with transplantation of G-CSF or stem cells alone. Also noteworthy was the reduction in the number of inflammatory cells and apoptotic cells and the significant increase in neurite outgrowth compared with results without G-CSF. The transplanted stem cells expressed angiogenic/neurotrophic factors. It is significant that G-CSF together with conditioned medium of pulp stem cells stimulated cell migration and neurite outgrowth, prevented cell death, and promoted immunosuppression in vitro. Furthermore, there was no evidence of toxicity or adverse events. In conclusion, the combinatorial trophic effects of pulp stem cells and G-CSF are of immediate utility for pulp/dentin regeneration, demonstrating the prerequisites of safety and efficacy critical for clinical applications.

We investigated the effect of tryptophan (Trp) on exocrine secretory function, using isolated rat pancreatic acini. Trp inhibited cholecystokinin-octapeptide (CCK-8)-stimulated amylase secretion, causing a downward shift in the dose-response curve. The inhibitory effect of Trp was dose-dependent and was observed only on the sustained secretion, there being no effect on the initial phase of amylase secretion. Trp (10 mM) also inhibited amylase secretion in response to carbachol and bombesin, as well as fluoride, a potent activator of guanine-nucleotide binding proteins. Since Ca2+ influx is necessary for sustained secretion, we examined the effect of Trp on Ca2+ influx and efflux. Trp increased the CCK-8-stimulated Ca2+ influx rate without affecting Ca2+ efflux, suggesting that Trp elevates intracellular Ca2+ levels. Increasing intracellular Ca2+ levels with A23187 resulted in the inhibition of CCK-8-stimulated amylase secretion. These results indicate that Trp inhibits CCK-stimulated sustained amylase secretion, in part by increasing Ca2+ influx into acinar cells.

Background/Aims: Insulin is shown to exert various regulatory effects on the exocrine pancreatic function. We investigated the direct effect of insulin on exocrine pancreatic secretion. Methods: The effects of insulin on amylase release, 125I-secretin binding and Na+- and K+-activated adenosine triphosphate phosphohydrolase (Na+,K+-ATPase) activity were measured using the isolated rat pancreatic acini. Results: Insulin potentiated the amylase release elicited by secretin plus cholecystokinin (CCK), but not by either secretin or CCK alone. The potentiating effect of insulin was dependent on the concentration and preincubation time. Insulin had no effect on 125I-secretin binding. Ouabain, a specific Na+,K+-ATPase inhibitor, caused a concentration-dependent inhibition of the potentiated secretion by insulin without affecting the secretory response to secretin plus CCK. In membranes prepared from acini treated with insulin, Na+,K+-ATPase activity was significantly increased. Similar results were obtained when acini were treated with insulin in combination with secretin plus CCK. Conclusions: Insulin exerts a direct effect on pancreatic acinar cells and potentiates exocrine secretion elicited by secretin in combination with CCK, in part, by increasing Na+,K+-ATPase activity.

High-mobility group box 1 (HMGB1) is a nuclear factor and a secreted protein. During inflammation, HMGB1 is secreted into the extracellular space where it can interact with the receptor for advanced glycation end products and trigger proinflammatory signals. Extracellular HMGB1 plays a critical role in several inflammatory diseases such as sepsis and rheumatoid arthritis. Valproic acid (VPA) is one of the most frequently prescribed antiepileptic drugs. The present study was undertaken to investigate the effect of VPA on secretion of HMGB1 in systemic inflammatory responses induced by lipopolysaccharide. Pretreatment with VPA increased the susceptibility of mice to lipopolysaccharide in endotoxemia. Valproic acid induced HMGB1 release and nuclear factor κB activation in RAW-blue cells. Valproic acid promoted the phosphorylation of ERK1/2 but not that of p38 or JNK. The MEK1/2 inhibitor PD98059 also suppressed HMGB1 release and activation of nuclear factor κB induced by VPA. Valproic acid induced expression of γ-aminobutyric acid receptors in macrophages, and picrotoxin, a γ-aminobutyric acid A receptor antagonist, inhibited the VPA-activated phosphorylation of ERK and VPA-induced HMGB1 release. These results suggest that VPA may exacerbate innate immune responses to endotoxin through enhanced release of HMGB1.

Loss of pulp due to caries and pulpitis leads to loss of teeth and reduced quality of life. Thus, there is an unmet need for regeneration of pulp. A promising approach is stem cell therapy. Autologous pulp stem/progenitor (CD105(+)) cells were transplanted into a root canal with stromal cell-derived factor-1 (SDF-1) after pulpectomy in mature teeth with complete apical closure in dogs. The root canal was successfully filled with regenerated pulp including nerves and vasculature by day 14, followed by new dentin formation along the dentinal wall. The newly regenerated tissue was significantly larger in the transplantation of pulp CD105(+) cells with SDF-1 compared with those of adipose CD105(+) cells with SDF-1 or unfractionated total pulp cells with SDF-1. The pulp CD105(+) cells highly expressed angiogenic/neurotrophic factors compared with other cells and localized in the vicinity of newly formed capillaries after transplantation, demonstrating its potent trophic effects on neovascularization. Two-dimensional electrophoretic analyses and real-time reverse transcription-polymerase chain reaction analyses demonstrated that the qualitative and quantitative protein and mRNA expression patterns of the regenerated pulp were similar to those of normal pulp. Thus, this novel stem cell therapy is the first demonstration of complete pulp regeneration, implying novel treatment to preserve and save teeth.

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg(-/-) mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg(-/-) OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg(-/-) mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg(-/-) OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg(-/-) mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.

Regenerative therapy using stem cells is a promising approach for the treatment of stroke. Recently, we reported that CD31⁻/CD146⁻ side population (SP) cells from porcine dental pulp exhibit highly vasculogenic potential in hindlimb ischemia. In this study, we investigated the influence of CD31⁻/CD146⁻ SP cells after transient middle cerebral artery occlusion (TMCAO). Adult male Sprague-Dawley rats were subjected to 2 h of TMCAO. Twenty-four hours after TMCAO, CD31⁻/CD146⁻ SP cells were transplanted into the brain. Motor function and infarct volume were evaluated. Neurogenesis and vasculogenesis were determined with immunochemical markers, and the levels of neurotrophic factors were assayed with real-time reverse transcription-polymerase chain reaction. In the cell transplantation group, the number of doublecortin-positive cells increased twofold, and the number of NeuN-positive cells increased eightfold, as compared with the control phosphate-buffered saline group. The vascular endothelial growth factor level in the ischemic brain with transplanted cells was 28 times higher than that in the normal brain. In conclusion, CD31⁻/CD146⁻ SP cells promoted migration and differentiation of the endogenous neuronal progenitor cells and induced vasculogenesis, and ameliorated ischemic brain injury after TMCAO.

In most mammals and all primates, visual function is dependent on an intraretinal vascular system that is highly adapted to the unique challenge of supplying oxygen and nutrients to a tissue whose translucency is essential for function. Consistent with the requirement that blood vessels minimally interfere with light, the retina has a relatively low density of capillaries.1 However, this paucity of microvessels leaves little functional reserve for the vital task of matching local perfusion to metabolic demand. Thus, efficient coupling of blood flow to meet local needs is particularly important in the retina. As a likely adaptation to increase the efficient control of local perfusion, the circulatory system of the retina appears to be especially well developed for the decentralized regulation of blood flow. Indicative of this, the capillaries of the retina have the highest density of abluminal pericytes,2 whose contractions and relaxations regulate lumen diameter and, thereby, capillary perfusion.3,4 Although decentralization is an important operational feature of the retina's circulatory system, much remains to be learned about how its microvasculature is functionally organized. To address this gap in knowledge, we characterized the distribution and regulation of functional L-type voltage-dependent calcium channels (VDCCs) within the feeder vessel/capillary unit of the normal and diabetic retina. This distal portion of the retinal microvasculature was of particular interest because evidence is accumulating that a capillary network plus the feeder vessel linking it with a myocyte-encircled arteriole constitute a functional unit in which cell-to-cell transmission by way of gap junctions is highly efficient.5,6,7 We focused on VDCCs because though they are known to be critically important in the generation of voltage-driven vasomotor responses in arteries and arterioles, the location and regulation of these ion channels within the retinal microvasculature are not well characterized. In this study, we also assessed the effect of diabetes on VDCC function because dysfunction of these channels may contribute to blood flow dysregulation and sight-threatening complications in the diabetic retina. To facilitate the assessment of VDCC-dependent changes in ionic currents, intracellular calcium, abluminal cell contractility, and lumen diameter in the feeder vessel/capillary unit, we studied microvascular complexes that were freshly isolated from the adult rat retina and included an arteriole whose outer diameter measured 12 to 18 μm, a precapillary feeder vessel with an outer diameter of 7 to 10 μm, and a network of capillaries with outer diameters of

Cell therapy with stem cells and endothelial progenitor cells (EPCs) to stimulate vasculogenesis as a potential treatment for ischemic disease is an exciting area of research in regenerative medicine. EPCs are present in bone marrow, peripheral blood, and adipose tissue. Autologous EPCs, however, are obtained by invasive biopsy, a potentially painful procedure. An alternative approach is proposed in this investigation. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. We isolated a highly vasculogenic subfraction of side population (SP) cells based on CD31 and CD146, from dental pulp. The CD31−;CD146− SP cells, demonstrating CD34+ and vascular endothelial growth factor-2 (VEGFR2)/Flk1+, were similar to EPCs. These cells were distinct from the hematopoietic lineage as CD11b, CD14, and CD45 mRNA were not expressed. They showed high proliferation and migration activities and multilineage differentiation potential including vasculogenic potential. In models of mouse hind limb ischemia, local transplantation of this subfraction of SP cells resulted in successful engraftment and an increase in the blood flow including high density of capillary formation. The transplanted cells were in proximity of the newly formed vasculature and expressed several proangiogenic factors, such as VEGF-A, G-CSF, GM-CSF, and MMP3. Conditioned medium from this subfraction showed the mitogenic and antiapoptotic activity on human umbilical vein endothelial cells. In conclusion, subfraction of SP cells from dental pulp is a new stem cell source for cell-based therapy to stimulate angiogenesis/vasculogenesis during tissue regeneration. Disclosure of potential conflicts of interest is found at the end of this article.

Endothelial exocytosis of granules is a rapid response to vascular injury. However, the molecular machinery that regulates exocytosis in endothelial cells is not well understood. Recently developed techniques have defined the endothelial proteins that control vesicle and granule trafficking in endothelial cells. These techniques have revealed that syntaxin 4, synaptobrevin 3, and N-ethylmaleimide-sensitive factor (NSF) play a critical role in endothelial granule exocytosis. Additional studies have shown that nitric oxide regulates exocytosis by chemically modifying NSF. Further characterization of the factors that regulate exocytosis will lead to novel treatments for vascular diseases such as myocardial infarction and stroke.

Nitric oxide (NO) has been thought to regulate the immune system through S nitrosylation of the transcriptional factor NF-kappaB. However, regulatory effects of NO on innate immune responses are unclear. Here, we report that NO has a capability to control Toll-like receptor-mediated signaling through S nitrosylation. We found that the adaptor protein MyD88 was primarily S nitrosylated, depending on the presence of endothelial NO synthase (eNOS). S nitrosylation at a particular cysteine residue within the TIR domain of MyD88 resulted in slight reduction of the NF-kappaB-activating property. This modification could be restored by the antioxidant glutathione. Through S nitrosylation, NO could negatively regulate the multiple steps of MyD88 functioning, including translocation to the cell membrane after LPS stimulation, interaction with TIRAP, binding to TRAF6, and induction of IkappaBalpha phosphorylation. Interestingly, glutathione could reversely neutralize such NO-derived effects. We also found that an acute febrile response to LPS was precipitated in eNOS-deficient mice, indicating that eNOS-derived NO exerts an initial suppressive effect on inflammatory processes. Thus, NO has a potential to retard induction of MyD88-dependent signaling events through the reversible and oxidative modification by NO, by which precipitous signaling reactions are relieved. Such an effect may reflect appropriate regulation of the acute-phase inflammatory responses in living organisms.

Dental pulp has the potential to form dentin as a regenerative response to caries. This regeneration is mediated by stem/progenitor cells. Thus, stem cell therapy might be of potential utility in induction of reparative dentin. We isolated side population (SP) cells from dental pulp based on the exclusion of the DNA binding dye Hoechst 33342 by flow cytometry and compared its self-renewal capacities and multipotency with non-SP cells and primary pulp cells. The cumulative cell number of the SP cells was greater than the non-SP cells and primary pulp cells. Bmi1 was continuously expressed in SP cells, suggesting longer proliferative lifespan and self-renewal capacity of SP cells. Next, the maintenance of the multilineage differentiation potential of pulp SP cells was investigated. Expression of type II collagen and aggrecan confirmed chondrogenic conversion (30%) of SP cells. SP cells expressed peroxisome proliferator-activated receptor gamma and adaptor protein 2, showing adipogenic conversion. Expression of mRNA and proteins of neurofilament and neuromodulin confirmed neurogenic conversion (90%). These results demonstrate that pulp SP cells maintain multilineage differentiation potential. We further examined whether bone morphogenetic protein 2 (BMP2) could induce differentiation of pulp SP cells into odontoblasts. BMP2 stimulated the expression of dentin sialophosphoprotein (Dspp) and enamelysin in three-dimensional pellet cultures. Autogenous transplantation of the Bmp2-supplemented SP cells on the amputated pulp stimulated the reparative dentin formation. Thus, adult pulp contains SP cells, which are enriched for stem cell properties and useful for cell therapy with BMP2 for dentin regeneration.

Three-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors protect the vasculature from inflammation and atherosclerosis by cholesterol dependent and cholesterol independent mechanisms. We hypothesized that HMG-CoA reductase inhibitors decrease exocytosis of Weibel-Palade bodies, endothelial cell granules whose contents promote thrombosis and vascular inflammation. We pretreated human aortic endothelial cells with simvastatin for 24 hours, then stimulated the cells with thrombin, and measured the amount of vWF released into the media. We then measured the effect of simvastatin on myocardial infarction in mice. Simvastatin decreased thrombin-stimulated Weibel-Palade body exocytosis by 89%. Simvastatin inhibited exocytosis in part by increasing synthesis of nitric oxide (NO), which S-nitrosylated N-ethylmaleimide sensitive factor (NSF), a critical regulator of exocytosis. Simvastatin treatment attenuated myocardial infarct size by 58% in wild-type but not eNOS knockout mice. Furthermore, simvastatin decreased endothelial exocytosis and neutrophil infiltration into ischemic-reperfused myocardium, which was mediated in part by P-selectin contained in Weibel-Palade bodies. However, simvastatin did not affect exocytosis and inflammation in myocardial infarcts of eNOS knockout mice. Inhibition of endothelial exocytosis is a novel mechanism by which HMG-CoA reductase inhibitors may reduce vascular inflammation, inhibit thrombosis, and protect the ischemic myocardium. These findings may explain part of the pleiotropic effects of statin therapy for patients with cardiovascular disease.

Recently, 14-member macrolide antibiotics such as Clarithromycin and Roxithromycin (RXM) have been shown to have anti-cancer and anti-angiogeneic effects. However, it is not fully understood whether and how RXM suppresses angiogenesis in human hepatoma, which is a well-known hypervascular tumor.In the present study, we examined the effects of RXM on tumor angiogenesis in the human hepatoma cell line, HepG2. In vivo, angiogenesis was examined using a mouse dorsal air sac model.The inhibitory effect of RXM was dose-dependent and the angiogenesis index of 100mg/kg/day of RXM administered intraperitoneally twice a day was significantly lower than the control. Next, we examined the effect of RXM on vascular endothelial growth factor (VEGF) mRNA expression and its protein level in HepG2 cells. When 100 microM of RXM were added, VEGF mRNA expression in HepG cells was inhibited and its protein level reduced.These results suggest that RXM inhibits tumor angiogenesis in human hepatoma, and that VEGF alteration may be involved in the mechanism of this inhibitory effect. Because RXM is widely used in clinical practice, it may represent an effective new strategy for human hepatoma therapy.

Nitric oxide (NO) inhibits vascular inflammation, but the molecular basis for its anti-inflammatory properties is unknown. We show that NO inhibits exocytosis of Weibel-Palade bodies, endothelial granules that mediate vascular inflammation and thrombosis, by regulating the activity of N-ethylmaleimide-sensitive factor (NSF). NO inhibits NSF disassembly of soluble NSF attachment protein receptor (SNARE) complexes by nitrosylating critical cysteine residues of NSF. NO may regulate exocytosis in a variety of physiological processes, including vascular inflammation, neurotransmission, thrombosis, and cytotoxic T lymphocyte cell killing.

Vascular endothelial growth factor (VEGF) has recently attracted attention as a potent inducer of vascular permeability and angiogenesis. Aberrant angiogenesis is often associated with lesion formation in chronic periodontitis. The aim of the present study was to investigate the properties of VEGF expression in human gingival fibroblasts (HGF) culture. HGF were stimulated with lipopolysaccharide (LPS), vesicle (Ve) and outer membrane protein (OMP) from Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. HGF constitutively produced VEGF and levels were significantly enhanced (P < 0.01) by stimulation with Ve and OMP from A. actinomycetemcomitans and P. gingivalis at concentrations of 10 microg/ml or higher. On the other hand, VEGF levels were not increased by LPS stimulation. VEGF mRNA expression was also observed in Ve- and OMP-stimulated HGF. A vascular permeability enhancement (VPE) assay was performed using guinea pigs to ascertain whether supernatant from cultures of Ve- and OMP-stimulated HGF enhance vascular permeability in vivo. Supernatant from cultures of Ve- and OMP-stimulated HGF strongly induced VPE. This was markedly suppressed upon simultaneous injection of anti-VEGF polyclonal antibodies with the supernatant. Heating and protease treatment of the stimulants reduced the VEGF enhancing levels in Ve and OMP in vitro. These results suggest that Ve and OMP may be crucial heat-labile and protease-sensitive components of periodontal pathogens that enhance VEGF expression. In addition, VEGF might be associated with the etiology of periodontitis in its early stages according to neovascularization stimulated by periodontal pathogens causing swelling and edema.

ATP-sensitive K + (K ATP ) channels are composed of sulfonylurea receptors (SURs) and inwardly rectifying Kir6.2-channels. The C-terminal 42 amino acid residues (C42) of SURs are responsible for ADP-induced differential activation of K ATP channels in SUR-subtypes. By examining ADP-effect on K ATP channels containing various chimeras of SUR2A and SUR2B, we identified a segment of 7 residues at central portion of C42 critical for this phenomenon. A 3-D structure model of the region containing the second nucleotide-binding domain (NBD2) of SUR and C42 was developed based on the structure of HisP, a nucleotide-binding protein forming the bacterial Histidine transporter complex. In the model, the polar and charged residues in the critical segment located within a distance that allows their electrostatic interaction with Arg1344 at the Walker-A loop of NBD2. Therefore, the interaction might be involved in the control of ADP-induced differential activation of SUR2-subtype K ATP channels.

1. In native cardiac myocytes, there is a time dependence to the G protein-gated inwardly rectifying K(+) (K(G)) channel current during voltage steps that accelerates as the concentration of acetylcholine is increased. This phenomenon has been called 'relaxation' and is not reproduced in the reconstituted Kir3.1/Kir3.4 channel in Xenopus oocytes. We have shown that RGS4, a regulator of G protein signalling, restores relaxation to the reconstituted Kir3.1/Kir3.4 channel. In this study, we examined the mechanism of this phenomenon by expressing various combinations of membrane receptors, G proteins, Kir3.0 subunits and mutants of RGS4 in Xenopus oocytes. 2. RGS4 restored relaxation to K(G) channels activated by the pertussis toxin (PTX)-sensitive G protein-coupled m(2)-muscarinic receptor but not to those activated by the G(s) protein-coupled beta(2)-adrenergic receptor. 3. RGS4 induced relaxation not only in heteromeric K(G) channels composed of Kir3.1 and Kir3.4 but also in homomeric assemblies of either an active mutant of Kir3.1 (Kir3.1/F137S) or an isoform of Kir3.2 (Kir3.2d). 4. Truncation mutants of RGS4 showed that the RGS domain itself was essential to reproduce the effect of wild-type RGS4 on the K(G) channel. 5. The mutation of residues in the RGS domain which interact with the alpha subunit of the G protein (G(alpha)) impaired the effect of RGS4. 6. This study therefore shows that interaction between the RGS domain and PTX-sensitive G(alpha) subunits mediates the effect of RGS4 on the agonist concentration-dependent relaxation of K(G) channels.

1. In the eye, different substances and ions including potassium (K+) are transported between neural retina and choroid via the subretinal space. Inwardly rectifying K+ channels (Kir) on the apical membrane of retinal pigment epithelial (RPE) cells are thought to play an essential role in K+ transport in the subretinal space. 2. Single-channel recordings from the apical membrane of RPE cells exhibited functional expression of a Kir channel with properties identical to those of Kir4.1, while recordings from the basolateral membrane showed no detectable Kir channel currents. 3. The expression of Kir4.1 mRNA in RPE cells was confirmed by RT-PCR analysis and in situ hybridization. Furthermore, using immunohistochemistry, we found that Kir4.1 was prominently expressed in RPE cells and localized specifically on the processes on their apical membrane. 4. Developmental studies revealed that expression of Kir4.1 started to appear 10 days or later after birth in RPE cells, in parallel with the maturation of retinal neuronal activity as represented by the a- and b-waves of the electroretinogram. 5. These data suggest that Kir4.1 is one of the Kir channels involved in RPE-mediated control of K+ ions in the subretinal space.

We investigated whether vascular endothelial growth factor (VEGF) production by human pulp cells (HPC) is regulated by lipopolysaccharide (LPS) in relation to the pathogenesis of pulpitis. Although HPC incubated with medium alone only marginally expressed VEGF mRNA and produced a low level of VEGF as detected by enzyme-linked immunosorbent assay, the VEGF mRNA expression and VEGF production were markedly enhanced upon stimulation with LPS from Escherichia coli. Prevotella intermedia LPS, phorbol 12-myristate 13-acetate, and interleukin-6 also induced VEGF mRNA expression in HPC. A simian virus 40-infected HPC line also exhibited increased VEGF mRNA expression in response to E. coli LPS, but lung and skin fibroblasts did not. Fetal bovine serum (FBS) increased the sensitivity of HPC to LPS in a dose-dependent manner. HPC did not express membrane CD14 on their surfaces. However, the anti-CD14 monoclonal antibody MY4 inhibited VEGF induction upon stimulation with LPS in HPC cultures in the presence of 10% FBS but not in the absence of FBS. LPS augmented the VEGF production in HPC cultures in the presence of recombinant human soluble CD14 (sCD14). To clarify the mechanisms of VEGF induction by LPS, we examined the possible activation of the transcription factor AP-1 in HPC stimulated with LPS, by a gel mobility shift assay. AP-1 activation in HPC was clearly observed, whereas that in skin fibroblasts was not. The AP-1 inhibitor curcumin strongly inhibited LPS-induced VEGF production in HPC cultures. In addition, a protein synthesis inhibitor, cycloheximide, inhibited VEGF mRNA accumulation in response to LPS. These results suggest that the enhanced production of VEGF in HPC induced by LPS takes place via an sCD14-dependent pathway which requires new protein synthesis and is mediated in part through AP-1 activation.

Vinculin, a 116-kDa membrane cytoskeletal protein, is an important molecule for cell adhesion; however, little is known about its other cellular functions. Here, we demonstrated that vinculin binds to Rab5 and is required for Staphylococcus aureus (S. aureus) uptake in cells. Viunculin directly bound to Rab5 and enhanced the activation of S. aureus uptake. Over-expression of active vinculin mutants enhanced S. aureus uptake, whereas over-expression of an inactive vinculin mutant decreased S. aureus uptake. Vinculin bound to Rab5 at the N-terminal region (1-258) of vinculin. Vinculin and Rab5 were involved in the S. aureus-induced phosphorylation of MAP kinases (p38, Erk, and JNK) and IL-6 expression. Finally, vinculin and Rab5 knockdown reduced infection of S. aureus, phosphorylation of MAPKs and IL-6 expression in murine lungs. Our results suggest that vinculin binds to Rab5 and that these two molecules cooperatively enhance bacterial infection and the inflammatory response.