Involvement of the visfatin/toll-like receptor 4 signaling axis in human dental pulp cell senescence: Protection via toll-like receptor 4 blockade.

Background: /purpose: Dental pulp plays an important role in the maintenance of tooth homeostasis and repair. The aging of dental pulp affects the functional life of the tooth owing to the senescence of dental pulp cells. Toll-like receptor 4 (TLR4) is involved in regulating cellular senescence in dental pulp. We have recently demonstrated that visfatin induces the senescence of human dental pulp cells (hDPCs). Here, we explored the association of TLR4 with visfatin signaling in cellular senescence in hDPCs. Materials and methods: mRNA levels were determined using reverse transcription polymerase chain reaction (PCR) and quantitative real time-PCR. Protein levels were determined using immunofluorescence staining and Western blot analysis. Gene silencing was performed using small interfering RNA. The degree of cellular senescence was measured by senescence-associated-ß-galactosidase (SA-ß-gal) staining. Oxidative stress was determined by measurement of NADP/NADPH levels and intracellular reactive oxygen species (ROS) levels. Results: Neutralizing anti-TLR4 antibodies or TLR4 inhibitor markedly blocked visfatin-induced hDPCs senescence, as revealed by an increase in the number of SA-ß-gal-positive hDPCs and upregulation of p21 and p53 proteins. Moreover, visfatin-induced senescence was associated with excessive ROS production; NADPH consumption; telomere DNA damage induction; interleukin (IL)-1ß, IL-6, IL-8, cyclooxygenase-2, and tumor necrosis factor-α upregulation; and nuclear factor-κB and mitogen-activated protein kinase activation. All of these alterations were attenuated by TLR4 blockade. Conclusion: Our findings indicate that TLR4 plays an important role in visfatin-induced senescence of hDPCs and suggest that the visfatin/TLR4 signaling axis can be a novel therapeutic target for the treatment of inflammaging-related diseases, including pulpitis.

Proteomics Analysis of Antitumor Activity of Agrimonia pilosa Ledeb. in Human Oral Squamous Cell Carcinoma Cells.

Oral cancer is a malignant neoplasm of oral cavity. It accounts for approximately 5% of all malignant tumors. Approximately 97% of all oral cancers are squamous cell carcinomas, followed by adenocarcinomas, and rarely malignant melanomas. It occurs particularly in males (twice as common in males than in females) of middle age (above 40 years). Agrimonia pilosa Ledeb. has traditionally been known for its effective antitumor activity and is currently used in China for cancer therapy. A. pilosa Ledeb. has been traditionally used for the treatment of abdominal pain, sore throat, headache, blood discharge, parasitic infections, and eczema in Korea and other Asian countries. Most studies on A. pilosa Ledeb. are related to the leaves and a few investigated the roots of the plant. However, detailed mechanisms of antitumor activity of A. pilosa Ledeb. have not been fully elucidated. Furthermore, to date, there have been no reports on the antitumor effect of A. pilosa Ledeb. in oral squamous cells. In this study, we used proteomic technology to observe changes in proteins related to anticancer activity of A. pilosa Ledeb. and identified target proteins among altered proteins to reveal the underlying mechanism of action.

Odontogenic Differentiation-Induced Tooth Regeneration by Psoralea corylifolia L.

Psoralea corylifolia L. (P. corylifolia) has been used as an oriental phytomedicine to treat coldness of hands and feet in bone marrow injury. Hydroxyapatite is usually used for tooth regeneration. In this study, the role of P. corylifolia and bakuchiol, a compound originated from P. corylifolia as differentiation-inducing substances for tooth regeneration, was determined by monitoring odontogenic differentiation in human dental pulp stem cells (hDPSCs). We confirmed that P. corylifolia extracts and bakuchiol increased the odontogenic differentiation of hDPSCs. In addition, the expression of the odontogenic differentiation marker genes alkaline phosphatase (APL), Runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), and dentin matrix acidic phosphoprotein-1 (DMP-1) was proved by real-time polymerase chain reaction, and protein expression of dentin matrix acidic phosphoprotein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) was proved by western blotting. Further, by confirming the increase in small mothers against decapentaplegia (SMAD) 1/5/8 phosphorylation, the SMAD signaling pathway was found to increase the differentiation of odontoblasts. This study confirmed that P. corylifolia L. extracts and bakuchiol alone promote odontogenic differentiation in hDPSCs. These results suggest that bakuchiol from P. corylifolia is responsible for odontogenic differentiation, and they encourage future in vivo studies on dentin regeneration.

Natural Herb Mixture Extract Accelerates Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells by Activating the SMAD Pathway.

We aimed to analyze the effects and explore the molecular mechanisms of a natural herb mixture extract (NME) on osteoblasts during differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs). We tried to confirm the regulation of osteogenic differentiation during NME treatment. Alkaline phosphatase assay and Alizarin red S staining were performed to evaluate the regulation of osteogenic differentiation. Real-time polymerase chain reaction was performed to analyze the expression of osteoblast maker genes, and Western blot was used to verify the signaling pathway. Signaling pathway conformation, selective bone morphogenetic protein receptor inhibitor, and dorsomorphin homolog 1 were used as pretreatments before inducing osteogenic differentiation. We determined that MME (natural herb mixture extract) was a safe material and significantly increased osteoblast differentiation and that SMAD phosphorylation is a key signaling pathway that regulates osteogenic differentiation in hBMSCs.

FK866 Protects Human Dental Pulp Cells against Oxidative Stress-Induced Cellular Senescence.

FK866 possesses various functional properties, such as anti-angiogenic, anti-cancer, and anti-inflammatory activities. We previously demonstrated that premature senescence of human dental pulp cells (hDPCs) was induced by hydrogen peroxide (H2O2). The present study aimed to investigate whether H2O2-induced premature senescence of hDPCs is affected by treatment with FK866. We found that FK866 markedly inhibited the senescent characteristics of hDPCs after exposure to H2O2, as revealed by an increase in the number of senescence-associated ß-galactosidase (SA-ß-gal)-positive hDPCs and the upregulation of the p21 and p53 proteins, which acts as molecular indicators of cellular senescence. Moreover, the stimulatory effects of H2O2 on cellular senescence are associated with oxidative stress induction, such as excessive ROS production and NADPH consumption, telomere DNA damage induction, and upregulation of senescence-associated secretory phenotype factors (IL-1ß, IL-6, IL-8, COX-2, and TNF-α) as well as NF-κB activation, which were all blocked by FK866. Thus, FK866 might antagonize H2O2-induced premature senescence of hDPCs, acting as a potential therapeutic antioxidant by attenuating oxidative stress-induced pathologies in dental pulp, including inflammation and cellular senescence.

Agrimonia pilosa Ledeb Root Extract: Anti-Inflammatory Activities of the Medicinal Herb in LPS-Induced Inflammation.

Inflammation regulation is essential for maintaining healthy functions and normal homeostasis of the body. Porphyromonas gingivalis (P. gingivalis) is a gram-negative anaerobic bacterium and a major pathogen that causes oral inflammation and other systemic inflammations. This study aims to examine the anti-inflammatory effects of Agrimonia pilosa Ledeb root extracts (APL-ME) in Porphyromonas gingivalis LPS-induced RAW 264.7 cells and find anti-inflammatory effect compounds of APL-ME. The anti-inflammatory effects of APL-ME were evaluated anti-oxidant activity, cell viability, nitrite concentration, pro-inflammatory cytokines (interleukin-1[Formula: see text], interleukin-6, tumor necrosis factor (TNF)-[Formula: see text], and anti-inflammatory cytokine (interleukin-10 (IL-10)). Also, Inflammation related genes and proteins, cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), expression were decreased by APL-ME and mitogen-activated protein kinase (MAPK) signaling proteins expression was regulated by APL-ME. Liquid chromatography-mass spectrometer (LC/MS)-MS analysis results indicated that several components were detected in APL-ME. Our study indicated that APL-ME suppressed nitrite concentrations, pro-inflammatory cytokines such as IL-1[Formula: see text], IL-6 and TNF-[Formula: see text] in P. gingivalis LPS induced RAW 264.7 cells. However, IL-10 expression was increased by ALP-ME. In addition, protein expressions of COX-2 and iNOS were inhibited APL-ME extracts dose-dependently. According to these results, APL-ME has anti-inflammatory effects in P. gingivalis LPS induced RAW 264.7 cells.

Visfatin Induces Senescence of Human Dental Pulp Cells.

Dental pulp plays an important role in the health of teeth. The aging of teeth is strongly related to the senescence of dental pulp cells. A novel adipokine, visfatin, is closely associated with cellular senescence. However, little is known about the effect of visfatin on the senescence of human dental pulp cells (hDPCs). Here, it was found that in vivo visfatin levels in human dental pulp tissues increase with age and are upregulated in vitro in hDPCs during premature senescence activated by H2O2, suggesting a correlation between visfatin and senescence. In addition, visfatin knockdown by small interfering RNA led to the reduction in hDPC senescence; however, treatment with exogenous visfatin protein induced the senescence of hDPCs along with increased NADPH consumption, which was reversed by FK866, a chemical inhibitor of visfatin. Furthermore, visfatin-induced senescence was associated with both the induction of telomere damage and the upregulation of senescence-associated secretory phenotype (SASP) factors as well as NF-κB activation, which were all inhibited by FK866. Taken together, these results demonstrate, for the first time, that visfatin plays a pivotal role in hDPC senescence in association with telomere dysfunction and the induction of SASP factors.

Decreasing Effect of Lidocaine·HCl on the Thickness of the Neuronal and Model Membrane.

This study examined the mechanism of action of a local anesthetic, lidocaine·HCl. Energy transfer between the surface fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid, and the hydrophobic fluorescent probe, 1,3-di(1-pyrenyl) propane, was used to determine the effect of lidocaine·HCl on the thickness (D) of the synaptosomal plasma membrane vesicles (SPMV) isolated from the bovine cerebral cortex, and liposomes of the total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. The thickness (D) of the intact SPMV, SPMVTL and SPMVPL were 1.044±0.008, 0.914±0.005 and 0.890±0.003 (arbitrary units, n=5) at 37℃ (pH 7.4), respectively. Lidocaine·HCl decreased the thickness of the neuronal and model membrane lipid bilayers in a dose-dependent manner with a significant decrease in the thickness, even at 0.1 mM. The decreasing effect of lidocaine·HCl on the membrane thickness might be responsible for some, but not all of its anesthetic action.

Mutant p53-Notch1 Signaling Axis Is Involved in Curcumin-Induced Apoptosis of Breast Cancer Cells.

Notch1 has been reported to be highly expressed in triple-negative and other subtypes of breast cancer. Mutant p53 (R280K) is overexpressed in MDA-MB-231 triple-negative human breast cancer cells. The present study aimed to determine whether the mutant p53 can be a potent transcriptional activator of the Notch1 in MDA-MB-231 cells, and explore the role of this mutant p53-Notch1 axis in curcumin-induced apoptosis. We found that curcumin treatment resulted in an induction of apoptosis in MDA-MB-231 cells, together with downregulation of Notch1 and its downstream target, Hes1. This reduction in Notch1 expression was determined to be due to the decreased activity of endogenous mutant p53. We confirmed the suppressive effect of curcumin on Notch1 transcription by performing a Notch1 promoter-driven reporter assay and identified a putative p53-binding site in the Notch1 promoter by EMSA and chromatin immunoprecipitation analysis. Overexpression of mutant p53 increased Notch1 promoter activity, whereas knockdown of mutant p53 by small interfering RNA suppressed Notch1 expression, leading to the induction of cellular apoptosis. Moreover, curcumin-induced apoptosis was further enhanced by the knockdown of Notch1 or mutant p53, but it was decreased by the overexpression of active Notch1. Taken together, our results demonstrate, for the first time, that Notch1 is a transcriptional target of mutant p53 in breast cancer cells and suggest that the targeting of mutant p53 and/or Notch1 may be combined with a chemotherapeutic strategy to improve the response of breast cancer cells to curcumin.

Melatonin inhibits visfatin-induced inducible nitric oxide synthase expression and nitric oxide production in macrophages.

Aberrant expression of inducible nitric oxide synthase (iNOS) in macrophages, which has been reported to be suppressed by melatonin, has an important contribution in the development of pathological inflammation. Visfatin, an adipokine, regulates the expression of various inflammatory factors, leading to inflammation; however, the influence of visfatin on iNOS-driven processes in macrophages is unclear. Here, we report the assessment of the role of visfatin in the regulation of iNOS gene expression in macrophages. Our data show that the levels of iNOS protein in peritoneal macrophages as well as nitric oxide (NO) in blood plasma were significantly lower after lipopolysaccharide treatment in visfatin(+/-) mice than those in the WT mice. In addition, visfatin increases iNOS mRNA and protein levels in RAW 264.7 cells, along with increasing production of NO. The enhancement of iNOS expression was prevented by treating the cells with inhibitors of the Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3), nuclear factor (NF)-κB, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase pathways. Our results also show that visfatin-induced iNOS expression and NO production were significantly inhibited by melatonin, an effect that was closely associated with a reduction in phosphorylated JAK2/STAT3 levels and with the inhibition of p65 translocation into nucleus. In conclusion, our data show, for the first time, that melatonin suppresses visfatin-induced iNOS upregulation in macrophages by inhibiting the STAT3 and NF-κB pathways. Moreover, our data suggest that melatonin could be therapeutically useful for attenuating the development of visfatin-iNOS axis-associated diseases.

Glycyrrhetinic acid inhibits Porphyromonas gingivalis lipopolysaccharide-induced vascular permeability via the suppression of interleukin-8.

OBJECTIVE: Porphyromonas gingivalis is a major periodontopathogen that plays a role in the pathogenesis of periodontal disease. In this study, we investigated the effect of 18alpha-glycyrrhetinic acid (18α-GA), a natural triterpenoid compound derived from licorice root extract, on P. gingivalis lipopolysaccharide (LPS)-induced vascular permeability, which is a hallmark of inflammatory diseases such as periodontitis. METHODS: The inhibitory effects of 18α-GA on endothelial permeability were determined by measuring in vivo and in vitro endothelial permeability. Endothelial cells were pretreated with 18α-GA before exposure to P. gingivalis LPS, and total RNA or proteins were extracted and analyzed by reverse transcription polymerase chain reaction or western blotting. RESULTS: Porphyromonas gingivalis LPS-induced endothelial permeability was significantly inhibited by 18α-GA both in vivo and in vitro. 18α-GA reduces P. gingivalis LPS-induced gap formation of endothelial cells. Importantly, 18α-GA modulated the expression and secretion of interleukin-8 (IL-8), a key inducer of vascular permeability, by downregulating nuclear factor-κB (NF-κB). 18α-GA suppressed P. gingivalis LPS-stimulated inhibitor of kappa B (IκB) kinase activation, IκBα phosphorylation, and nuclear translocation of NF-κB. CONCLUSIONS: Overall, these findings suggest that 18α-GA significantly reduces P. gingivalis LPS-induced vascular permeability by repressing NF-κB-dependent endothelial IL-8 production, suggesting its therapeutic potential in P. gingivalis-related vascular diseases.

The Effect of Lidocaine · HCl on the Fluidity of Native and Model Membrane Lipid Bilayers.

The purpose of this study is to investigated the mechanism of pharmacological action of local anesthetic and provide the basic information about the development of new effective local anesthetics. Fluorescent probe techniques were used to evaluate the effect of lidocaine·HCl on the physical properties (transbilayer asymmetric lateral and rotational mobility, annular lipid fluidity and protein distribution) of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex, and liposomes of total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from the SPMV. An experimental procedure was used based on selective quenching of 1,3-di(1-pyrenyl)propane (Py-3-Py) and 1,6-diphenyl-1,3,5-hexatriene (DPH) by trinitrophenyl groups, and radiationless energy transfer from the tryptophans of membrane proteins to Py-3-Py. Lidocaine·HCl increased the bulk lateral and rotational mobility of neuronal and model membrane lipid bilayes, and had a greater fluidizing effect on the inner monolayer than the outer monolayer. Lidocaine·HCl increased annular lipid fluidity in SPMV lipid bilayers. It also caused membrane proteins to cluster. The most important finding of this study is that there is far greater increase in annular lipid fluidity than that in lateral and rotational mobilities by lidocaine·HCl. Lidocaine·HCl alters the stereo or dynamics of the proteins in the lipid bilayers by combining with lipids, especially with the annular lipids. In conclusion, the present data suggest that lidocaine, in addition to its direct interaction with proteins, concurrently interacts with membrane lipids, fluidizing the membrane, and thus inducing conformational changes of proteins known to be intimately associated with membrane lipid.

The effect of methanol on the structural parameters of neuronal membrane lipid bilayers.

The structures of the intact synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortexs, and the outer and the inner monolayer separately, were evaluated with 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1,3-di(1-pyrenyl)propane (Py-3-Py) as fluorescent reporters and trinitrophenyl groups as quenching agents. The methanol increased bulk rotational and lateral mobilities of SPMVs lipid bilayers. The methanol increased the rotational and lateral mobilities of the outer monolayers more than of the inner monolayers. n-(9-Anthroyloxy)stearic acid (n-AS) were used to evaluate the effect of the methanol on the rotational mobility at the 16, 12, 9, 6, and 2 position of aliphatic chains present in phospholipids of the SPMVs outer monolayers. The methanol decreased the anisotropy of the 16-(9-anthroyloxy)palmitic acid (16-AP), 12-(9-anthroyloxy)stearic acid (12-AS), 9-(9-anthroyloxy)stearic acid (9-AS), and 6-(9-anthroyloxy)stearic acid (6-AS) in the SPMVs outer monolayer but it increased the anisotropy of 2-(9-anthroyloxy)stearic acid (2-AS) in the monolayers. The magnitude of the increased rotational mobility by the methanol was in the order at the position of 16, 12, 9, and 6 of aliphatic chains in phospholipids of the outer monolayers. Furthermore, the methanol increased annular lipid fluidity and also caused membrane proteins to cluster. The important finding is that was far greater increase by methanol in annular lipid fluidity than increase in lateral and rotational mobilities by the methanol. Methanol alters the stereo or dynamics of the proteins in the lipid bilayers by combining with lipids, especially with the annular lipids. In conclusion, the present data suggest that methanol, in additions to its direct interaction with proteins, concurrently interacts with membrane lipids, fluidizing the membrane, and thus inducing conformational changes of proteins known to be intimately associated with membranes lipids.

Upregulation of thromboxane synthase mediates visfatin-induced interleukin-8 expression and angiogenic activity in endothelial cells.

Thromboxane synthase (TXAS) is an enzyme that catalyzes the synthesis of thromboxane A(2) (TXA(2)). Overexpression of TXAS is associated with a variety of vascular diseases. Recently, we reported that visfatin, a novel adipokine, exhibits angiogenic actions. In this study, we showed that visfatin increased mRNA and protein levels of TXAS and stimulated TXA(2) biosynthesis in vascular endothelial cells. In addition, visfatin induced the expression and secretion of interleukin-8 (IL-8), which is blocked by a TXAS inhibitor and by the transfection of siRNA specific for TXAS. Furthermore, the inhibition of TXAS activity and blockade of the IL-8 receptor attenuated visfatin-induced endothelial angiogenesis. Together, these results showed that visfatin promoted IL-8 production by upregulation of TXAS, leading to angiogenic activation in endothelial cells.

Curcumin down-regulates visfatin expression and inhibits breast cancer cell invasion.

Obesity is frequently associated with breast cancer. Such associations are possibly mediated by adipokines. Visfatin, an adipokine, has recently been shown to be related to the development and progression of breast cancer. Therefore, the down-regulation of visfatin may be a novel strategy for breast cancer therapy. Curcumin has anticancer activities by modulating multiple signaling pathways and genes. The purpose of this study was to investigate whether visfatin gene expression is affected by curcumin in human breast cancer cells and to characterize the functional role of visfatin in breast cancer. We found that the mRNA and protein levels of visfatin were down-regulated by curcumin in MDA-MB-231, MDA-MB-468, and MCF-7 breast cancer cells, along with decreased activity of constitutive nuclear factor (NF)-κB. We confirmed the repressive effect of curcumin on visfatin transcription by performing a visfatin promoter-driven reporter assay and identified two putative NF-κB-binding sites on visfatin promoter that are important for this effect. EMSA and chromatin immunoprecipitation analysis indicated the binding of p65 to the visfatin promoter, which was effectively blocked by curcumin. Enforced expression of p65 protein increased visfatin promoter activity, whereas blocking NF-κB signaling suppressed visfatin gene expression. Visfatin could enhance the invasion of MDA-MB-231 cells and also attenuate curcumin-induced inhibition of cell invasion; on the other hand, visfatin knockdown by small interfering RNA led to the reduction of cell invasion. Our data demonstrate, for the first time, that curcumin down-regulates visfatin gene expression in human breast cancer cells by a mechanism that is, at least in part, NF-κB dependent and suggest that visfatin may contribute to breast cancer cell invasion and link obesity to breast cancer development and progression.

Neuromedin B receptor antagonist suppresses tumor angiogenesis and tumor growth in vitro and in vivo.

Neuromedin B (NMB), a member of the mammalian bombesin-like peptide family, and its receptor were aberrantly expressed in vascularized solid tumors. Here, the NMB receptor (NMB-R) antagonist PD168368 specifically inhibited both NMB-induced in vivo and in vitro angiogenesis. In addition, PD168368 showed growth inhibitory effects on MDA-MB-231 breast cancer cells by inducing cell cycle arrest and apoptosis. Furthermore, PD168368 effectively suppressed tumor growth in a xenograft model of breast tumor in vivo. Overall, NMB-R antagonist exhibited a significant antitumor activity by simultaneously inhibiting neovascularization and cancer cell growth, thereby suggesting that NMB-R could be a potential therapeutic target for cancer treatment.

Visfatin induces neurite outgrowth in PC12 cells via ERK1/2 signaling pathway.

The angiogenic and inflammatory functions of visfatin and its effect on vascular cells, are fairly well known. However, its role within the nervous system remains largely unclear. To gain insight into this area, we studied the neuritogenic effect of visfatin on PC12 rat pheochromocytoma cells. We investigated whether visfatin gene expression, which is upregulated by hypoxia in cancer cells, is associated with neuritogenesis in PC12 cells. Using RT-PCR, Western blot analysis, ELISA, morphological observations, and immunostaining, we initially showed that CoCl(2), a hypoxic mimetic agent, upregulated visfatin gene expression along with neurite outgrowth in PC12 cells. We also showed that visfatin stimulated neurite outgrowth in PC12 cells. Moreover, in PC12 cells, visfatin evoked the activation of the extracellular signal-regulated kinase 1/2 (ERK1/2), which is closely linked to neuritogenesis. Visfatin-induced outgrowth of neurites was prevented by inhibition of the ERK1/2 pathway. Taken together, our results demonstrate for the first time that visfatin induces neurite outgrowth in PC12 cells via the activation of an ERK-dependent pathway, and suggest that visfatin may exert various biological, physiological, and pathological functions in not only the vascular system but also the nervous system.

Angiogenic role of orexin-A via the activation of extracellular signal-regulated kinase in endothelial cells.

Orexin-A, a neuropeptide originally discovered in the hypothalamus, is found in peripheral organs, as well as in the central nervous system, and is involved in the regulation of food intake, energy homeostasis, and cardiovascular functions. In this study, we report that orexin-A induces invivo neovascularization in a mouse Matrigel plug and ex vivo sprouting of endothelial cells in rat aortic rings. We also show that orexin-A increases migration and tube formation in human umbilical vein endothelial cells (HUVECs), and this effect is mediated by orexin receptors on endothelial cells. Moreover, orexin-A activates the extracellular signal-regulated kinase 1/2 (ERK1/2) in HUVECs, which is closely linked to angiogenic responses. The inhibition of ERK activation significantly suppresses orexin-A-stimulated endothelial angiogenesis. Taken together, our results indicate that orexin-A functions as a new proangiogenic peptide and requires MEK/ERK-dependent pathway for its angiogenic actions. These results suggest orexin-A and its receptor may act as important modulators of angiogenesis under pathophysiological conditions.

Mutually opposite effects of dopamine.HCl and chlorpromazine. HCl on the thickness of liposomal lipid bilayers.

The aim of this study was to provide a basis for examining the molecular mechanism for the pharmacological action of dopamine.HCl (DA) and chlorpromazin.HCl (CPZ). Radiationless energy transfer from the surface fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid, to the hydrophobic fluorescent probe, bispyrenylpropane (Py-Py), was used to examine the effects of DA and CPZ on the thickness (D) of the liposomal lipid bilayers prepared with total lipids (SPMVTL) and phospholipids (SPMVPL) extracted from neuronal membranes. The thickness (D) of intact SPMVTL and SPMVPL (37 degrees C, pH 7.4) were 0.914 +/- 0.010 and 0.886 +/- 0.009 (arbitrary units, n = 5), respectively. DA decreased the thickness of both SPMVTL and SPMVPL in a dose-dependent manner with a significant decrease in thickness observed even at 40 x 10(-9) M and 40 x 10(-9) M, respectively. On the other hand, CPZ increased the thickness of both SPMVTL and SPMVPL in a dose-dependent manner with a significant increase in thickness observed even at 35 x 10(-5) M and 35 x 10(-5) M, respectively. The sensitivities to the decreasing and increasing effect of the membrane lipid bilayers thickness by DA and CPZ, respectively, differed according to the liposomes in descending order of SPMVPL and SPMVTL. The decreasing and increasing action of DA and CPZ, respectively, on the membrane thickness had many effects that may be responsible for the dopaminergic receptor-DA and -CPZ interaction as well as the protein-lipid interaction.

Thromboxane A(2) increases endothelial permeability through upregulation of interleukin-8.

Thromboxane A(2) (TXA(2)), a major prostanoid formed from prostaglandin H(2) by thromboxane synthase, is involved in the pathogenesis of a variety of vascular diseases. In this study, we report that TXA(2) mimetic U46619 significantly increases the endothelial permeability both in vitro and in vivo. U46619 enhanced the expression and secretion of interleukin-8 (IL-8), a major inducer of vascular permeability, in endothelial cells. Promoter analysis showed that the U46619-induced expression of IL-8 was mainly regulated by nuclear factor-kappaB (NF-kappaB). U46619 induced the activation of NF-kappaB through IkappaB kinase (IKK) activation, IkappaB phosphorylation and NF-kappaB nuclear translocation. Furthermore, the inhibition of IL-8 or blockade of the IL-8 receptor attenuated the U46619-induced endothelial cell permeability by modulating the cell-cell junctions. Overall, these results suggest that U46619 promotes vascular permeability through the production of IL-8 via NF-kappaB activation in endothelial cells.