Development of a quantitative analysis method for assessing patient body surface deformation using an optical surface tracking system.

This study aimed to develop a new method to quantitatively analyze body shape changes in patients during radiotherapy without additional radiation exposure using an optical surface tracking system. This method's accuracy was evaluated using a cubic phantom with a known shift. Surface images of three-dimensionally printed phantoms, which simulated the head and neck shapes of real patients before and after treatment, were used to create a deformation surface area histogram. The near-maximum deformation value covering an area of 2 cm2 in the surface image (Def-2cm2) was calculated. A volumetric modulated arc therapy (VMAT) plan was also created on the pre-treatment phantom, and the dose distribution was recalculated on the post-treatment phantom to compare the dose indices. Surface images of four patients were analyzed to evaluate Def-2cm2 and examine whether this method can be used in clinical cases. Experiments with the cubic phantom resulted in a mean deformation error of 0.08 mm. With head and neck phantoms, the Def-2cm2 value was 17.5 mm, and the dose that covered 95% of the planning target volume in the VMAT plan decreased by 11.7%, indicating that deformation of the body surface may affect the dose distribution. Although analysis of the clinical data showed no clinically relevant deformation in any of the cases, slight skin sagging and respiratory changes in the body surface were observed. The proposed method can quantitatively and accurately evaluate the deformation of a body surface. This method is expected to be used to make decisions regarding modifications to treatment plans.

Emerging roles of hypoxia-inducible factors and reactive oxygen species in cancer and pluripotent stem cells.

Eukaryotic organisms require oxygen homeostasis to maintain proper cellular function for survival. During conditions of low oxygen tension (hypoxia), cells activate the transcription of genes that induce an adaptive response, which supplies oxygen to tissues. Hypoxia and hypoxia-inducible factors (HIFs) may contribute to the maintenance of putative cancer stem cells, which can continue self-renewal indefinitely and express stemness genes in hypoxic stress environments (stem cell niches). Reactive oxygen species (ROS) have long been recognized as toxic by-products of aerobic metabolism that are harmful to living cells, leading to DNA damage, senescence, or cell death. HIFs may promote a cancer stem cell state, whereas the loss of HIFs induces the production of cellular ROS and activation of proteins p53 and p16(Ink4a), which lead to tumor cell death and senescence. ROS seem to inhibit HIF regulation in cancer cells. By contrast, controversial data have suggested that hypoxia increases the generation of ROS, which prevents hydroxylation of HIF proteins by inducing their transcription as negative feedback. Moreover, hypoxic conditions enhance the generation of induced pluripotent stem cells (iPSCs). During reprogramming of somatic cells into a PSC state, cells attain a metabolic state typically observed in embryonic stem cells (ESCs). ESCs and iPSCs share similar bioenergetic metabolisms, including decreased mitochondrial number and activity, and induced anaerobic glycolysis. This review discusses the current knowledge regarding the emerging roles of ROS homeostasis in cellular reprogramming and the implications of hypoxic regulation in cancer development.

A scoring algorithm for predicting the presence of adult asthma: a prospective derivation study.

BACKGROUND: To predict the presence of asthma in adult patients with respiratory symptoms, we developed a scoring algorithm using clinical parameters. METHODS: We prospectively analysed 566 adult outpatients who visited Kinki University Hospital for the first time with complaints of nonspecific respiratory symptoms. Asthma was comprehensively diagnosed by specialists using symptoms, signs, and objective tools including bronchodilator reversibility and/or the assessment of bronchial hyperresponsiveness (BHR). Multiple logistic regression analysis was performed to categorise patients and determine the accuracy of diagnosing asthma. RESULTS: A scoring algorithm using the symptom-sign score was developed, based on diurnal variation of symptoms (1 point), recurrent episodes (2 points), medical history of allergic diseases (1 point), and wheeze sound (2 points). A score of >3 had 35% sensitivity and 97% specificity for discriminating between patients with and without asthma and assigned a high probability of having asthma (accuracy 90%). A score of 1 or 2 points assigned intermediate probability (accuracy 68%). After providing additional data of forced expiratory volume in 1 second/forced vital capacity (FEV(1)/FVC) ratio <0.7, the post-test probability of having asthma was increased to 93%. A score of 0 points assigned low probability (accuracy 31%). After providing additional data of positive reversibility, the post-test probability of having asthma was increased to 88%. CONCLUSIONS: This pragmatic diagnostic algorithm is useful for predicting the presence of adult asthma and for determining the appropriate time for consultation with a pulmonologist.

Effects of a cysteinyl leukotriene dual 1/2 receptor antagonist on antigen-induced airway hypersensitivity and airway inflammation in a guinea pig asthma model.

BACKGROUND: Little is known about the role of the cysteinyl leukotriene (cysLT) 2 receptor in the pathophysiology of asthma. The aim of this study is to investigate the effects of a cysLT1 receptor antagonist (montelukast) and a dual cysLT1/2 receptor antagonist (BAY-u9773) on airway hypersensitivity and airway inflammation induced by antigen challenge in ovalbumin (OVA)-sensitized guinea pigs. METHODS: Male Hartley guinea pigs sensitized with OVA were intraperitoneally administered 0.1, 1, or 10 mg/kg of montelukast or 0.1 mg/kg of BAY-u9773 and then challenged with inhaled OVA. Airway reactivity to acetylcholine, inflammatory cells in bronchoalveolar lavage (BAL) fluid, and eosinophil infiltration in airway walls after OVA challenge were evaluated. RESULTS: Pretreatment with 1 or 10 mg/kg, but not 0.1 mg/kg, of montelukast significantly suppressed airway hypersensitivity and eosinophil infiltration into the BAL fluid. Moreover, 0.1 mg/kg of BAY-u9773 significantly suppressed the development of these markers. The suppressive effects of BAY-u9773, although not significantly different, trended toward being greater than those of montelukast. Although all of the doses of montelukast tested and 0.1 mg/kg of BAY-u9773 significantly suppressed eosinophil infiltration in airway walls, the suppressive effect of BAY-u9773 was significantly greater than that of 0.1 mg/kg of montelukast. CONCLUSION: Signaling may contribute to the pathophysiology of asthma via the cysLT1/2 receptor.

Annual change of respiratory functions in adult patients with asthma: the potential of antiasthma treatments for many years to repair irreversible changes of the airway.

BACKGROUND: Little is known regarding annual changes of respiratory functions among patients with asthma after asthma symptoms enter remission. OBJECTIVE: Annual changes of respiratory function and influence of patient characteristics and treatment variables on these changes were assessed in patients with adult asthma. METHODS: Respiratory function (pre- and postbronchodilator forced expiratory volume in one second [FEV1] and reversibility by short-acting ß2-agonist) and their changes were retrospectively investigated and relationships between these changes, after symptomatic remission, and patient characteristics and treatments were analyzed in adult outpatients with asthma who had undergone spirometry (including a reversibility test) ≥5 times in >5 years. RESULTS: In patients ≥40 years old, or with disease duration ≥10 years or receiving treatment for severe asthma (steps 4-5, high-dose inhaled glucocorticosteroids, or addition of other medications), both pre- and postbronchodilator FEV1 values were significantly lower (p < .05). Mean annual change of prebronchodilator FEV1 (Δpre-FEV1), annual change of postbronchodilator FEV1 (Δpost-FEV1), and annual change of reversibility (Δ reversibility) were -13.8 ± 59.7 ml/year, -25.9 ± 51.0 ml/year, and -0.56% ± 1.89%/year, respectively. Multivariate analysis after stepwise selection for variables in patient characteristics or treatments showed that disease duration ≥10 years contributed to annual improvement of respiratory functions (Δpre-FEV1: odds ratio [OR] 1.57, 95% confidence interval [CI] 1.01-2.46; Δpost-FEV1: OR 2.13, 95% CI 1.25-3.66), treatment with long-acting ß2-agonists (LABAs) contributed to annual improvement of respiratory function (Δpre-FEV1: OR 2.05, 95% CI 1.23-3.16; Δpost-FEV1: OR 1.78, 95% CI 1.11-2.87), and poor compliance contributed to annual worsening of respiratory functions (Δpre-FEV1: OR 0.43, 95% CI 0.24-0.76; Δpost-FEV1: OR 0.39, 95% CI 0.22-0.70). In addition, duration of disease ≥10 years and severe treatment (steps 4-5) from the beginning contributed to decreasing Δreversibility (OR 0.55, 95% CI 0.34-0.87 and OR 0.50, 95% CI 0.29-0.83, respectively). CONCLUSIONS: Long-term treatments for asthma are expected to normalize respiratory dysfunction, which cannot be repaired in the short term. Treatment with LABAs and patient compliance may be the most important factors associated with annual improvement of respiratory functions.

Evaluation of Chinese hamster ovary cell stability during repeated batch culture for large-scale antibody production.

Pharmaceutical manufacturing plants can be operated continuously for several months. It is therefore important to use cells with long-term stability for the production of active ingredients. We investigated the reliability and long-term stability of an antibody-producing cell line. A recombinant Chinese hamster ovary (CHO) cell line was cultivated in spinner flasks and reactors, including a practical production-scale reactor (1600 L), for 109 days to produce monoclonal antibodies against the HM1.24 antigen. During cultivation, the cells remained stable and there was an increase in the rate of cell proliferation, yielding viable cells at high density. A decrease in cell-specific productivity was associated with this increase in the rate of cell proliferation. The cells were genetically stable and other measures of cellular function remained consistent throughout the cultivation period.

Changes in the quality of antibodies produced by Chinese hamster ovary cells during the death phase of cell culture.

Although overproduction of recombinant proteins by mammalian cells is well established, little attention has been paid to analysis of the quality of the products. We focused on the quality of antibodies produced during the death phase of a recombinant Chinese hamster ovary (CHO) cell line. The quality of the monoclonal antibody against HM1.24 antigen and the post-translational characteristics of the subunits during CHO cell culture in a 160-L bioreactor were investigated. The culture supernatant of a stable cell line was collected and purified by affinity chromatography and then analyzed. There were no significant changes in gel-permeation chromatography variables, carbohydrate structure, or antibody-dependent cellular cytotoxicity activity during the death phase of cell culture. However, ion-exchange chromatography analysis revealed that antibody heterogeneity changed, as indicated by a decrease in cell viability. The results presented here provide useful information that will help in determining the time to end each batch culture.

Impact of implanted bone marrow progenitor cell composition on limb salvage after cell implantation in patients with critical limb ischemia.

OBJECTIVE: The aim of this study is to identify which factors influence limb salvage after bone marrow mononuclear cell implantation (BMI) in patients with chronic critical limb ischemia (CLI). METHODS: Thirteen no-option CLI patients treated with BMI were enrolled in the present study. Limb ischemia was assessed using the ankle-brachial index (ABI), transcutaneous oxygen tension (TcO(2)), and rest pain score. The cell populations among the implanted cells were determined by May-Giemsa staining and flow cytometry. RESULTS: Major lower extremity amputations after BMI were performed in seven patients. Before implantation, there were no significant differences between the amputation group (n=7) and the salvage group (n=6) in clinical characteristics, the ABI, the TcO(2) level, or the rest pain score. After implantation, there were no differences between the groups in the serum levels of angiogenic or inflammatory cytokines. The number of implanted BM cells was the same in the two groups, but the cells implanted in the limb salvage group were composed of significantly higher numbers of hematopoietic progenitors (erythroblasts and myeloblasts) and lymphocytes (p<0.05, respectively). The number of CD34-positive cells was somewhat greater in the salvage group than in the amputation group (p=0.09) and was positively associated with the number of erythroblasts (r(2)=0.29, p=0.06) and the number of myeloblasts (r(2)=0.59, p<0.01). CONCLUSIONS: The cellular composition of the BM cells injected may affect limb salvage after the implantation in patients with severe CLI. The favorable effects of BMI appear to reflect the impact of the progenitor cell doses.

The strategy for predicting future exacerbation of asthma using a combination of the Asthma Control Test and lung function test.

BACKGROUND: Various factors have been reported to be useful for predicting future exacerbations. OBJECTIVE: This study was intended to determine a usefulness of a combination of a patient-based questionnaire, such as the Asthma Control Test (ACT) score with objective assessments, such as forced expiratory volume in 1 second (FEV(1)) and/or exhaled nitric oxide (FE(NO)), for predicting future exacerbations in adult asthmatics. METHODS: We therefore enrolled 78 subjects with mild to moderate asthma, who were clinically stable for 3 months who all had been regularly receiving inhaled steroid treatment. All subjects underwent a routine assessment of asthma control including the ACT score, spirometry, and FE(NO), and then were followed up until a severe exacerbation occurred. The predictors of an increased risk of severe exacerbation were identified and validated using decision trees based on a classification and regression tree (CART) analysis. The properties of the developed models were the evaluated with the area under the ROC curve (AUC) (95% confidence interval [CI]). RESULTS: The CART analysis automatically selected the variables and cut-off points, the ACT score

Inhaled montelukast inhibits cysteinyl-leukotriene-induced bronchoconstriction in ovalbumin-sensitized guinea-pigs: the potential as a new asthma medication.

Oral cysteinyl-leukotriene (LT) receptor antagonists such as montelukast are used for reducing airway inflammation and exacerbations. However, inhaled therapy using LT receptor antagonists has not been studied. In the present study, the effect of inhaled montelukast was investigated on airway hyperresponsiveness measured by cysteinyl-LT induced bronchoconstriction in an animal model of asthma. Bronchoconstriction responses were induced by inhaled LTC4 and LTD4 (0.2 microg/ml each) or three doses of intravenous LTC4 and LTD4 (0.3, 1, 3 microg/kg) in ovalbumin (OVA)-sensitized Hartley male guinea-pigs. The response was measured by the change in peak pressure of airway opening (Pao). The effect of montelukast was evaluated by the comparison of bronchoconstriction responses between the groups of animals pre-treated with 15-min inhalation of 10mg/ml montelukast and saline. To evaluate the tissue injury which might be caused by montelukast inhalation, lung tissues were examined for the histology. The broncoconstriction responses induced by inhaled LTC4 and LTD4 were enhanced by OVA sensitization in the guinea-pigs. In sensitized animals, the significant increases in peak Pao were 18.5+/-2.1 cmH(2)O by LTC4 inhalation and 25.0+/-1.6 cmH(2)O by LTD4 inhalation on average. Prior treatment of inhaled montelukast potently suppressed the peak Pao increases induced by both inhaled and intravenous LTC4 and LTD4 (all P<0.01 vs. saline control). Moreover, the suppression of inhaled montelukast against LTD4-induced bronchoconstriction was observed for at least up to 24h. According to the histological examination, montelukast inhalation produced no injury to the lung tissue. Inhaled montelukast, a cysteinyl-LT receptor antagonist, was effective in inhibiting cysteinyl-LT-induced acute bronchoconstriction, and may have the potential for clinical use as a new asthma drug.

Increased plasma urotensin-II and carotid atherosclerosis are associated with vascular dementia.

AIM: Human urotensin-II (UII) is a cyclic neuropeptide with potent vasoconstrictive activity in the vasculature. The expression of UII and its receptor (UT) mRNA is detected at high levels in the brain. We evaluated the relationship between plasma UII levels and vascular dementia (VaD) caused by stroke or atherosclerotic small vessel disease. METHODS: Carotid artery intima-media thickness (IMT), plaques, plasma levels of immunoreactive UII (IR-UII), and atherosclerotic biomarkers were determined in 42 patients with VaD, 197 with Alzheimer's disease (AD), and 47 non-demented elderly controls. RESULTS: Age, gender, body mass index, systolic blood pressure (SBP), fasting plasma glucose, insulin, triglycerides, high-density lipoprotein cholesterol, leptin, and plasminogen activator inhibitor-1 levels were not significantly different among these groups. IR-UII, low-density lipoprotein (LDL) cholesterol, lipoprotein(a), lipid peroxides, interleukin-6, and high-sensitive C-reactive protein (hs-CRP) levels, and maximum IMT were significantly higher in VaD than in AD patients or controls. IR-UII level showed a significantly positive correlation with SBP or maximum IMT. Multivariate logistic regression analysis revealed a significantly independent association between IR-UII levels or increased maximum IMT (> or =1.1 mm) and VaD as compared with SBP, LDL cholesterol, and interleukin-6 levels. CONCLUSION: Increased plasma IR-UII levels and carotid atherosclerosis may be involved in the pathogenesis and progression of VaD.

Aldosterone increases osteopontin gene expression in rat endothelial cells.

Aldosterone is currently recognized as one of the important risk hormones for cardiovascular disease. However, the cellular mechanism by which aldosterone affects the process of cardiovascular injury has not been well understood. In the present study, we investigated whether aldosterone induces pro-inflammatory genes expression in rat aortic endothelial cells. Aldosterone significantly increased steady-state osteopontin mRNA and protein levels, but not those of adhesion molecules or chemokine. The stimulatory effect of aldosterone on osteopontin expression was time-dependent (3-24h) and dose-dependent (10(-10)-10(-6)M), and abolished by a mineralocorticoid receptor (MR) antagonist spironolactone, but not by a glucocorticoid receptor antagonist RU486. The aldosterone-induced osteopontin mRNA expression was completely blocked by a transcription inhibitor, actinomycin D, and a protein synthesis inhibitor, cycloheximide. Thus, the present study demonstrated for the first time that aldosterone directly acts on endothelial cells to induce osteopontin gene expression via MR-mediated genomic action, which may be responsible for the initiation of inflammation and fibrosis in cardiovascular tissue induced by aldosterone.

Time-course of changes in the levels of interleukin 6 in acutely decompensated heart failure.

BACKGROUND: Although cytokine elevation has been demonstrated in chronic heart failure, little attention has been focused on cytokine levels during the acute stage. We examined the changes of cytokine levels in patients with acutely decompensated heart failure to investigate their relationship with severity of heart failure. METHODS: Patients with acutely decompensated heart failure (73 patients; 72+/-2 years) were included. Blood samples were taken from the peripheral vein on admission before the start of drug therapy, at 12, 24, 48 and 72 h as well as 1, 2 and 4 weeks after admission. Control data were obtained from age-matched normal patients who had no cardiovascular disease. Serum IL-6, IL-1beta and TNF-alpha levels were measured using the ELISA method. RESULTS: Mean IL-6, IL-1beta and TNF-alpha levels on admission were significantly higher than those in the control patients (p<0.001). IL-6 peaked at 12 h and declined thereafter, whereas IL-1beta and TNF-alpha remained unchanged throughout the duration of the study. Peak IL-6 significantly correlated with pulmonary wedge pressure on admission (r=0.332, p=0.0041). % change of IL-6 levels between peak (12 h after admission) and 24 h was significantly correlated with that of pulmonary wedge pressure between peak (on admission) and 24 h (r=0.308, p=0.0081). Peak IL-6 in patients treated with mechanical ventilation on admission was significantly higher than that in patients who underwent no mechanical ventilation (p<0.05). CONCLUSIONS: IL-6 levels possibly reflect the severity of heart failure and thus may be useful for the evaluation of disease status in acutely decompensated heart failure.

Differential localization of Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 and CD47 and its molecular mechanisms in cultured hippocampal neurons.

Polarized localization of membrane proteins to axons or dendrites is important for a variety of neuronal functions, including neurite outgrowth and synaptogenesis during neural development. Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) and its ligand cluster of differentiation 47 (CD47), both of which are members of the Ig superfamily of proteins, are thought to constitute an intercellular communication system in the CNS, although the physiological functions of this CD47-SHPS-1 system remain unknown. To provide insight into these functions, we have now examined the localization of SHPS-1 and CD47 in cultured hippocampal neurons. Endogenous SHPS-1 was detected at the surface of both axons and dendrites, whereas endogenous CD47 was localized predominantly to the surface of dendrites. Forced expression of these two proteins confirmed their distinct localizations. The extracellular regions of SHPS-1 and CD47 were responsible, at least in part, for their axonal and dendritic localizations, respectively; however, the axonal localization of SHPS-1 was not mediated by any one of the three Ig domains in its extracellular region. Overexpression of SHPS-1 and CD47 in distinct neurons resulted in marked accumulation of these proteins at sites of contact between SHPS-1-expressing axons and CD47-expressing dendrites. Such contact sites exhibited an enlarged structure but did not contain the synaptic marker protein vesicle-associated membrane protein-2. These results suggest that differential localization of SHPS-1 and CD47 at axons and dendrites generates a directional intercellular communication system that potentially contributes to regulation of synaptogenesis and the formation of neural networks.

Concomitant expression of adrenomedullin and its receptor components in rat adipose tissues.

Adrenomedullin (AM) expressed by and secreted from a variety of cells plays pluripotent roles in an autocrine/paracrine fashion. The present study was undertaken to explore the expression of AM and its receptor genes in adipose tissues, their changes during the development of obesity, and the process of preadipocyte differentiation. Both mature adipocytes and stromal vascular cells constituting adipose tissue expressed AM transcript. AM and its receptor component [calcitonin receptor-like receptor and receptor activity-modifying protein-2 (CRLR/RAMP2)] mRNAs were expressed in a variety of rat adipose tissues, including epididymal, mesenteric, retroperitoneal, and subcutaneous adipose tissue. AM mRNA levels in rat and human epididymal adipose tissue were about one-tenth of those in the kidney. Steady-state mRNA levels of AM and CRLR/RAMP2 in epididymal, mesenteric, and retroperitoneal adipose tissues in rats fed a high-fat diet for 4 wk were far greater than those in rats with normal diet accompanied by increased plasma AM levels, whereas steady-state AM mRNA levels conversely decreased in other organs, such as kidney and liver. AM mRNA expressed in a mouse preadipocyte cell line (3T3-L1) transiently decreased by day 3, returned to basal level by day 6, and then increased by day 9 during preadipocyte differentiation, which paralleled AM secretion from the cells. However, the addition of either exogenous AM or AM receptor antagonist calcitonin gene-related peptide-(8-37), to block endogenous AM did not affect lipid droplet accumulation during preadipocyte differentiation. The present study demonstrates for the first time that AM and its receptor component (CRLR/RAMP2) mRNAs were concomitantly expressed in various adipose tissues, whose tissue-specific upregulation was induced during the development of obesity. These data suggest that AM may act as a new member of adipokines, although its functional role, as well as its pathophysiological significance in obesity, remains to be determined.

High molecular weight form insulin-like growth factor II-producing mesenteric sarcoma causing hypoglycemia.

An 81-year-old woman presented with frequent episodes of hypoglycemia. Her serum level of insulin was normal, but her serum insulin-like growth factor (IGF)-II level was high. She was found to have a spindle cell sarcoma originated from the mesentery of the sigmoid colon, which was completely resected. Postoperatively, hypoglycemia ameliorated with concomitant reduction in serum IGF-II levels. Immunohistochemical study revealed positive immunostaining for IGF-II in tumor cells, and the abundant expression of IGF-II mRNA was demonstrated by RT-PCR. The presence of high molecular weight (HMW) form IGF-II in patient's serum was confirmed by immunoblotting. This is the first report of a patient with HMW form IGF-II-producing mesenteric sarcoma causing hypoglycemia.

Ectodomain shedding of SHPS-1 and its role in regulation of cell migration.

SHPS-1 is a transmembrane protein whose cytoplasmic region undergoes tyrosine phosphorylation and then binds the protein-tyrosine phosphatase SHP-2. Formation of the SHPS-1-SHP-2 complex is implicated in regulation of cell migration. In addition, SHPS-1 and its ligand CD47 constitute an intercellular recognition system that contributes to inhibition of cell migration by cell-cell contact. The ectodomain of SHPS-1 has now been shown to be shed from cells in a reaction likely mediated by a metalloproteinase. This process was promoted by activation of protein kinase C or of Ras, and the released ectodomain exhibited minimal CD47-binding activity. Metalloproteinases catalyzed the cleavage of a recombinant SHPS-1-Fc fusion protein in vitro, and the primary cleavage site was localized to the juxtamembrane region of SHPS-1. Forced expression of an SHPS-1 mutant resistant to ectodomain shedding impaired cell migration, cell spreading, and reorganization of the actin cytoskeleton. It also increased the tyrosine phosphorylation of paxillin and FAK triggered by cell adhesion. These results suggest that shedding of the ectodomain of SHPS-1 plays an important role in regulation of cell migration and spreading by this protein.

Antioxidant effect of adrenomedullin on angiotensin II-induced reactive oxygen species generation in vascular smooth muscle cells.

Recent adrenomedullin (AM) gene-targeting studies have proposed a novel concept that AM plays a protective role against oxidative stress in vivo. The present study was undertaken to explore the underlying molecular mechanism of the putative antioxidant action of AM against angiotensin II (Ang II)induced reactive oxygen species (ROS) generation in rat vascular smooth muscle cells (VSMCs). Intracellular ROS levels were measured by dichlorofluoroscein fluorescence. Redox-sensitive c-Jun amino-terminal kinase (JNK) and ERK1/2 activation and gene expression induced by Ang II in VSMCs were also studied. AM dose-relatedly (10(-8)-10(-7) m) inhibited intracellular ROS generation stimulated by Ang II (10(-7) m), as mimicked by dibutyl-cAMP, the effect of which was inhibited by the pretreatment with N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride, a protein kinase A inhibitor, and calcitonin gene-related peptide(8-37), an AM/calcitonin gene-related peptide receptor antagonist. Ang II induced JNK and ERK1/2 activation via a redox-sensitive manner, whereas AM inhibited JNK, but not ERK1/2, activation by Ang II. Furthermore, AM inhibited Ang II-induced redox-sensitive gene expression (plasminogen activator inhibitor-1 and monocyte chemoattractant protein-1) in the same manner as N-acetyl-l-cysteine, a potent antioxidant. AM also inhibited Ang II-induced up-regulation of Nox1, a critical membrane-bound component of reduced nicotinamide adenine dinucleotide phosphate oxidase in VSMCs, in the same degree as N-acetyl-l-cysteine. Our study demonstrates for the first time that AM directly inhibits intracellular ROS generation via an AM receptor-mediated and c-AMP-protein kinase A-dependent mechanism in VSMCs and that AM with its potent antioxidant action inhibits redox-sensitive JNK activation and gene expression induced by Ang II. These data suggest that AM plays a protective role as an endogenous antioxidant in Ang II-induced vascular injury.

Endothelin-1 induces cyclooxygenase-2 expression and generation of reactive oxygen species in endothelial cells.

Since both endothelin-1 (ET-1) and aldosterone have been shown to induce expression of several pro-inflammatory genes, including cyclooxygenase-2 (COX-2), in the vasculature as a cardiovascular risk hormone, the present study was undertaken to examine the effects of ET-1 and aldosterone on COX-2 gene expression as measured by a real-time reverse transcriptase-polymerase chain reaction in aortic endothelial cells. Treatment with ET-1(10 M) markedly upregulated COX-2 mRNA levels in rat endothelial cells, whereas aldosterone (10 M) did not show any effect. The ET-1-induced COX-2 upregulation was inhibited by pretreatment with a non-selective endothelin receptor antagonist (TAK044), a protein kinase C inhibitor (GF109203X), and a MEK inhibitor (PD98059). Furthermore, ET-1 increased intracellular reactive oxygen species generation as estimated by the measurement of dichlorofluorescein fluorescence, whose effect was blocked by a COX-2 inhibitor (NS398). Our data show that ET-1 induces COX-2 upregulation in rat endothelial cells via a protein kinase C-dependent and extracellular signal-regulated kinase-dependent pathway, which may largely contribute to the generation of intracellular reactive oxygen species.