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Mutations in the parkin gene are related with early-onset Parkinson's disease. Parkin is identified as an E3-ligase that combines target proteins with ubiquitin. alpha-Synuclein and synphilin-1 are substrates for E3-ligase activity of parkin and considered to be involved in the pathogenesis of Parkinson's disease. We previously demonstrated both alpha-synuclein and synphilin-1 are expressed in vascular endothelial cells (VEC). In the present study, we addressed possible expression of parkin in VEC. Parkin immunoreactivity was detected in vascular endothelial cells in postmortem human brain. Expressions of parkin mRNA and protein in human umbilical vein endothelial cells (HUVEC) were demonstrated by reverse-transcription polymerase-chain reaction (RT-PCR) and western blotting. Expression of parkin in HUVEC was not altered with tunicamycin treatment, which exerts unfolded protein stress on cells. We conclude that parkin is expressed in VEC, and that unfolded protein stress may not regulate its expression.

Heparin is primarily used as an anticoagulant but has many biological functions as well. It binds with high affinity to a range of cytokines including interferon-gamma (IFN-gamma) and members of chemokine superfamily. IFN-gamma is a proinflammatory cytokine that plays a pivotal role in immune and inflammatory responses; and in endothelial cells, it regulates the expression of fractalkine/CX3CL1 that is a potent agonist for the chemotaxis and adhesion of monocytes and lymphocytes. We have investigated the effect of heparin on the fractalkine expression in human umbilical vein endothelial cells (HUVEC) in culture. HUVEC were treated with approximately 100 mg/mL heparin and the expression of the IFN-gamma-induced fractalkine mRNA and protein were measured by reverse transcription-PCR and western blotting. The IFN-gamma-induced expressions of fractalkine mRNA and protein were inhibited by heparin in a concentration-dependent manner. Heparin also inhibited adhesion of mononuclear cells (MNC) to HUVEC monolayers stimulated with IFN-gamma, but it did not inhibit the MNC adhesion to the monolayers stimulated with interleukin-1beta. Electrophoretic analysis demonstrated direct binding of heparin to IFN-gamma and heparin was found to partially block the binding of IFN-gamma to IFN-gamma receptor (IFN-gamma R). Heparin may play a regulatory role in inflammatory and immune responses by modulating the interaction between leukocytes and the vascular endothelium.

Growth related oncogene protein-alpha (GRO-alpha) is a member of C-X-C chemokine and plays an important role in inflammatory responses. Expression of GRO gene family is regulated by a number of factors at both transcriptional and posttranscriptional levels. In the present study, we have addressed the possible regulation of GRO-alpha expression by ubiquitin-proteasome system. Cultures of human umbilical vein endothelial cells were treated with a proteasome inhibitor, MG132, and the levels of GRO-alpha mRNA were analyzed by reverse transcription-polymerase chain reaction or northern blotting. Levels of GRO-alpha protein in the cell-conditioned medium were determined by enzyme-linked immunosorbent assay. MG132 alone increased the levels of GRO-alpha mRNA and protein; however, it did not affect the GRO-alpha mRNA induced by lipopolysaccharide (LPS) and inhibited the LPS-induced decrease in IkappaB levels. Other proteasome inhibitors, MG115 and lactacystin, also induced the expression of GRO-alpha mRNA. MG132 induced the phosphorylation of p38 MAPK, MEK and JNK. Pretreatment of the cells with SB203580, an inhibitor of p38 MAPK, suppressed the MG132-induced GRO-alpha expression, but pretreatment of the cells with U0126, PD98059 or SP600125, inhibitors of MEK1/2 or JNK, did not influence the effect of MG132. We conclude that MG132 upregulates GRO-alpha expression in vascular endothelial cells, at least in part, through the activation of p38 MAPK.

Epithelial neutrophil-activating peptide-78 (ENA-78) is a member of CXC chemokines. It is produced by endothelial cells stimulated with interleukin-1 (IL-1), along with other CXC chemokines such as IL-8 and growth-related oncogene protein-alpha (GRO-alpha). IL-1-induced ENA-78 production by endothelial cells may be important for the regulation of neutrophil activation. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a natural ligand for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and affects the expression of various genes. We examined the effect of 15d-PGJ(2) on the expression of ENA-78 in cultured endothelial cells stimulated with IL-1beta. 15d-PGJ(2) inhibited the IL-1beta-induced expression of ENA-78, but not the expression of IL-8 or GRO-alpha in response to IL-1. Ciglitazone, another agonist for PPAR-gamma, had no effect on the expression of ENA-78, suggesting that 15d-PGJ(2) may inhibit the expression of ENA-78 in a PPAR-gamma-independent manner. 15d-PGJ(2) may modulate inflammatory reactions by regulating the balance of CXC chemokines in endothelial cells.

alpha-Synuclein is known to be a major component of Lewy bodies and glial cytoplasmic inclusions in the brains of patients with alpha-synucleinopathies. Synphilin-1, an alpha-synuclein-associated protein, is also present in these inclusions. However, little is known about the post-translational modifications of synphilin-1. In the present study, it is reported that synphilin-1 is phosphorylated by glycogen synthase kinase-3beta in vitro. It is well known that protein phosphorylation is involved in various physiological phenomena, including signal transduction and protein degradation. Therefore, phosphorylation of synphilin-1 may play an important role in the function of this protein in the brain.

CX3CL1/fractalkine is a chemokine with a unique CX3C motif. Hypoxia mediates the expression of various genes, such as vascular endothelial growth factor (VEGF), cyclooxygenase-2, and plasminogen-activator inhibitor-1, in vascular endothelial cells. We studied the effect of hypoxia on the expression of fractalkine induced by interferon-gamma (IFN-gamma) in endothelial cells. Human umbilical vein endothelial cells were cultured, and the stimulation of the cells with IFN-gamma was found to induce the expression of fractalkine. Hypoxia inhibited the expression of fractalkine mRNA and protein by IFN-gamma, and this effect was observed with concomitant increase in VEGF expression. Desferrioxamine, an iron chelator that mimics hypoxia in vitro, also inhibited the fractalkine production induced by IFN-gamma. Hypoxia did not affect the degradation of fractalkine mRNA. The inhibition of fractalkine expression by hypoxia was reversed on returning the cultures to reoxygenation condition. Inhibition of IFN-induced fractalkine expression by hypoxia was not affected by the presence of a radical scavenger, N-acetyl-L-cysteine, and the involvement of reactive oxygen species may be excluded. Inhibition of fractalkine expression by hypoxia may be involved in the pathophysiology of ischemic diseases.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), one of major hematopoietic growth factors, activates mature leukocytes. GM-CSF is produced by endothelial cells stimulated with lipopolysaccharide (LPS), and the LPS-induced GM-CSF production may play an important role in the activation of neutrophils on the endothelial surface. 15-Deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) is a ligand for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and modulates inflammatory reactions by regulating the expression of various genes. We studied the effect of 15d-PGJ2 on the LPS-induced GM-CSF expression in endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured and the expressions of GM-CSF mRNA and protein were analyzed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. 15d-PGJ2 inhibited the LPS-induced GM-CSF expression in a concentration-dependent manner; but ciglitazone, another agonist for PPAR-gamma, had no effect. This suggests that 15d-PGJ2 inhibits GM-CSF expression through a mechanism unrelated to PPAR-gamma. 15d-PGJ2 induced, by itself, the expression of interleukin-8, a potent proinflammatory chemokine, in HUVEC. 15d-PGJ2 may regulate inflammatory reactions by controlling the balance of various cytokines.

Background: Galectin-9 is involved in chemotaxis and adhesion of eosinophils, and is induced in vascular endothelial cells by interferon-gamma (IFN-gamma). 15-deoxy-delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is a ligand for peroxisome proliferator-activated receptor-gamma (PPAR-gamma), and known to modulate the expression of various genes., Methods: We have studied the effect of 15d-PGJ(2) on the IFN-gamma-induced galectin-9 expression in human umbilical vein endothelial cells (HUVEC) in culture., Results: 15d-PGJ(2) inhibited the IFN-gamma-induced galectin-9 expression in a PPAR-gamma-independent manner, and also inhibited the adhesion of EoL-1 cells to an HUVEC monolayer treated with IFN-gamma. 15d-PGJ(2) partially inhibited IFN-gamma-induced phosphorylation of STAT-1 in HUVEC., Conclusions: 15d-PGJ(2) may regulate inflammatory reactions through the inhibition of galectin-9 expression., (Copyright 2003 S. Karger AG, Basel)

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma)is a member of nuclear hormone receptor superfamily, and is knownto play a role in various biological processes including inflammatoryresponses and adipocyte differentiation. CX3CL1/fractalkineis a potent agonist for chemotaxis and adhesion of monocytes and lymphocytes. Endothelial cells produce fractalkine when stimulated with cytokinessuch as interleukin-1 (IL-1), tumour necrosis factor-alpha andinterferon-gamma (IFN-gamma). We herein report that 15-deoxy-n12,14 -prostaglandinJ2 (15d-PGJ2), a PPAR-gamma agonist,inhibits the expression of fractalkine induced by IFN-gamma orIL-1beta in human endothelial cells. Agonist for PPAR-alpha (WY14643)or PPAR-gamma (ciglitazone) did not inhibit the cytokine-inducedfractalkine expression, and the effect of 15d-PGJ2 maybe independent of PPAR. 15-Deoxy-D12,14 prostaglandinJ2 also inhibited the adhesion of blood mononuclear cellsto endothelial monolayers treated with IFN-gamma or IL-1beta. The data suggest that 15d-PGJ2 regulates inflammatoryreactions, at least in part, through the inhibition of fractalkineexpression and leucocyte traffic through the endothelium.

Background: Cyclin E and p27 play pivotal roles in cancer development and progression. We investigated whether the prognosis in cases of non-small cell lung cancer without lymph node metastases that underwent complete resection could be associated with tissue expression of cyclin E, p27, and Ki-67., Methods: Tumors from 62 patients at least 5 years after surgery were assessed by immunohistochemistry for expression of cyclin E, p27, and Ki-67. Disease-free survival (DFS) after surgery was used to evaluate disease prognosis. We also investigated the relationship between expression of these factors and postoperative recurrence., Results: In non-small cell lung cancer, p27-negative expression and pT factor were significantly unfavorable prognostic factors in multivariate analysis. The DFS rate in cyclin E-positive expression was significantly lower than in cyclin E-negative expression. Similarly, p27-negative expression and high Ki-67 expression correlated with a shortened DFS rate. In combinations of expression of cyclin E and p27, the cyclin E-negative/p27-positive group had a significantly higher DFS rate than did the other groups. According to histological type, there were correlations between the risk of postoperative recurrence and expression of these three biological factors, especially in adenocarcinoma., Conclusion: By analyzing the expression of cyclin E, p27, and Ki-67 of tumor cells, it was possible to extract the patient group for whom closer follow-up and postoperative treatment is necessary to improve survival rate.

Alpha-synuclein was originally identified as the presynaptic nerve terminal protein. Recently, we reported that alpha-synuclein is also expressed in cultured human astrocytes and that its levels are increased by stimulation with interleukin-1beta, suggesting that it may be involved in inflammatory processes. We therefore investigated the effect of inflammatory stimuli on alpha-synuclein expression in human macrophages. Alpha-synuclein mRNA and protein were detected in cultured human macrophages and levels of alpha-synuclein protein were increased by stimulation with lipopolysaccharide and interleukin-1beta in a time- and concentration-dependent manner. Immunofluorescent staining showed that alpha-synuclein protein was expressed within the cytoplasm and nucleus. Furthermore, alpha-synuclein immunoreactivity was present in alveolar macrophages from human lung tissues. These findings suggest that the function of alpha-synuclein is not exclusive to the nervous system and that alpha-synuclein may play a role in inflammatory processes and immune responses.

Galectin-9 is a member of the galectin family and has been identified as an eosinophil chemoattractant produced by activated T lymphocytes. Vascular endothelial cells play an important role in the initial step of eosinophil recruitment and activation in immune and inflammatory responses. We have addressed the stimulation of galectin-9 expression in endothelial cells. Galectin-9 was detected in membrane and cytosolic fractions of human umbilical vein endothelial cells stimulated with interferon-gamma (IFN-gamma). IFN-gamma also enhanced the adhesion of human eosinophilic leukemia-1 cells to endothelial monolayers, and it was inhibited by the presence of lactose. Interleukin-4, which induces eotaxin expression, did not affect the expression of galectin-9. The in situ endothelium from patients with inflammatory diseases was found to express galectin-9. IFN-gamma-induced production of galectin-9 by endothelial cells may play an important role in immune responses by regulating interactions between the vascular wall and eosinophils.

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells. To examine whether platelet-activating factor (PAF) induces the expression of VEGF in human astrocytes, we stimulated cultured normal astrocytes with PAF and performed semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) or real-time quantitative PCR for VEGF mRNA and enzyme-linked immunosorbent assay for VEGF protein. PAF increased the expression of VEGF in astrocytes in time- and dose-dependent manners. After 24-h stimulation, 10 nM PAF increased the levels of VEGF protein in astrocyte-conditioned medium by 1.3-fold. When the cells were subjected to hypoxia, the PAF-induced production of VEGF was enhanced by 6.7-fold as compared to the unstimulated cells incubated under normoxia. Dexamethasone was found to inhibit the enhanced VEGF production in response to the stimulation with PAF under hypoxia. We conclude that PAF induces VEGF gene expression in human astrocytes, and the PAF-induced increase in the expression of VEGF may modulate nervous tissue injury due to hypoxia.

The non-amyloid beta component of Alzheimer's disease amyloid (NAC) is detected in cerebral amyloid angiopathy; and the precursor of NAC is now known to be identical to alpha-synuclein (alpha-S), a major component of Lewy bodies in Parkinson's disease. We studied if cerebral vascular cells express alpha-S. Immunohistochemical studies of human cerebral tissues from control and cerebral amyloid angiopathy patients revealed the expression of alpha-S in vascular endothelial and smooth muscle cells. Then we studied the expression of alpha-S in vitro using cultures of vascular cells. Cultures of human umbilical vein endothelial cells and umbilical artery smooth muscle cells were found to constitutively express alpha-S messenger RNA and protein. alpha-S is normally expressed in vascular cells and may play some physiological role in the vascular wall.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in inflammatory reactions. We have addressed the possible regulation of IL-6 expression by the ubiquitin-protease system in human umbilical vein endothelial cells. Cultured endothelial cells were treated with MG-132, a protease inhibitor, and the levels of IL-6 mRNA and protein were measured by reverse transcription-PCR and ELISA. MG-132 increased the expression of IL-6 mRNA and protein;and this effect was abolished by the pretreatment of the cells with U0126, an inhibitor of MAP or ERK kinases (MEK 1/2). MG-132 treatment was also found to enhance the level of phosphorylated MEK 1/2. Treatment of the cells with actinomycin D inhibited IL-6 expression in response to MG-132, suggesting the transcriptional upregulation of IL-6 under proteasomal inhibition. We conclude that a protease inhibitor MG-132 upregulates IL-6 expression in vascular endothelial cells, at least in part, through the activation of MEK 1/2.

Plasminogen activator inhibitor-1 (PAI-1) is one of the target genes of hypoxia inducible factor-1alpha (HIF-1alpha). Besides being an important physiological regulator of the fibrinolytic system PAI-1 is also involved in cancer invasiveness. HIF-1alpha is expressed in various types of pulmonary cells, but the relation of PAI-1 to HIF-1alpha under hypoxic condition in these cells are not fully elucidated. We, therefore, studied the effect of hypoxia on the expression of PAI-1 in a lung cancer cell line EBC-1. The expression of HIF-1alpha protein in EBC-1 cells was enhanced by hypoxia, and this was associated with increased secretion of PAI-1. Hypoxia did not affect the levels of HIF-1alpha mRNA but enhanced the PAI-1 mRNA. Pretreatment of the cells with MG132, which inhibits the proteasomal degradation of HIF-1alpha, increased the production of PAI-1 under both normoxia and hypoxia. We conclude that hypoxia induces PAI-1 expression, in EBC-1 cells, through the stabilization of HIF-1 complex and this may be related to cancer metastasis.