PURPOSE: Dexmedetomidine, a full agonist of alpha2B-adrenoceptors, is used for analgesia and sedation in the intensive care units. Dexmedetomidine produces an initial transient hypertension due to the activation of post-junctional alpha2B-adrenoceptors on vascular smooth muscle cells (SMCs). The aims of this in vitro study were to identify mitogen-activated protein kinase (MAPK) isoforms that are primarily involved in full, alpha2B-adrenoceptor agonist, dexmedetomidine-induced contraction of isolated rat aortic SMCs. MATERIALS AND METHODS: Rat thoracic aortic rings without endothelium were isolated and suspended for isometric tension recording. Cumulative dexmedetomidine (10(-9) to 10(-6) M) dose-response curves were generated in the presence or absence of extracellular signal-regulated kinase (ERK) inhibitor PD 98059, p38 MAPK inhibitor SB 203580, c-Jun NH2-terminal kinase (JNK) inhibitor SP 600125, L-type calcium channel blocker (verapamil and nifedipine), and alpha2-adrenoceptor inhibitor atipamezole. Dexmedetomidine-induced phosphorylation of ERK, JNK, and p38 MAPK in rat aortic SMCs was detected using Western blotting. RESULTS: SP 600125 (10(-6) to 10(-5) M) attenuated dexmedetomidine-evoked contraction in a concentration-dependent manner, whereas PD 98059 had no effect on dexmedetomidine-induced contraction. SB 203580 (10(-5) M) attenuated dexmedetomidine-induced contraction. Dexmedetomidine-evoked contractions were both abolished by atipamezole and attenuated by verapamil and nifedipine. Dexmedetomidine induced phosphorylation of JNK and p38 MAPK in rat aortic SMCs, but did not induce phosphorylation of ERK. CONCLUSION: Dexmedetomidine-induced contraction involves a JNK- and p38 MAPK-mediated pathway downstream of alpha2-adrenoceptor stimulation in rat aortic SMCs. In addition, dexmedetomidine-induced contractions are primarily dependent on calcium influx via L-type calcium channels.
Adrenergic alpha-2 Receptor Agonists/*pharmacology ; Animals ; Anthracenes/pharmacology ; Aorta/cytology ; Dexmedetomidine/*pharmacology ; Enzyme Inhibitors/pharmacology ; Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors/physiology ; Flavonoids/pharmacology ; Imidazoles/pharmacology ; JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors/*physiology ; Male ; *Muscle Contraction ; Muscle, Smooth, Vascular/drug effects/enzymology/*physiology ; Protein Isoforms/antagonists & inhibitors/physiology ; Pyridines/pharmacology ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/physiology

BACKGROUND: Recent clinical observation reported that there was a significant correlation between change in circulating vascular endothelial growth factor (VEGF) levels and the occurrence of severe acute graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the action mechanisms of VEGF in GVHD have not been demonstrated. METHODS: This study investigated whether or not blockade of VEGF has an effect on acute GVHD in a lethally irradiated murine allo-HSCT model of B6 (H-2b)-->B6D2F1 (H-2b/d). Syngeneic or allogeneic recipient mice were injected subcutaneously with anti-VEGF peptides, dRK6 (50 microg/dose) or control diluent every other day for 2 weeks (total 7 doses). RESULTS: Administration of the dRK6 peptide after allo-HSCT significantly reduced survival with greaterclinical GVHD scores and body weight loss. Allogeneic recipients injected with the dRK6 peptide exhibited significantly increased circulating levels of VEGF and expansion of donor CD3+ T cells on day +7 compared to control treated animals. The donor CD4+ and CD8+ T-cell subsets have differential expansion caused by the dRK6 injection. The circulating VEGF levels were reduced on day +14 regardless of blockade of VEGF. CONCLUSION: Together these findings demonstrate that the allo-reactive responses after allo-HSCT are exaggerated by the blockade of VEGF. VEGF seems to be consumed during the progression of acute GVHD in this murine allo-HSCT model.
Animals ; Body Weight ; Graft vs Host Disease ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Humans ; Mice ; Oligopeptides ; Peptides ; T-Lymphocyte Subsets ; T-Lymphocytes ; Tissue Donors ; Vascular Endothelial Growth Factor A

AMP-activated protein kinase (AMPK), an enzyme involved in energy homeostasis, regulates inflammatory responses, but its precise mechanisms are not fully understood. Recent evidence has shown that resveratrol (RES), an AMPK activator, reduces prostaglandin E2 production in lipopolysaccharide (LPS)-treated microglia. Here, we examined the effect of RES on nuclear factor kappa B (NF-kappaB) dependent cyclooxygenase (COX)-2 activation in LPS-treated RWA 264.7 macrophages. We found that treatment with RES increased AMPK activation. AMPK and acetyl CoA carboxylase phosphorylation were attenuated in cells treated with LPS+RES, compared to cells treated with LPS alone. RES inhibited tumor necrosis factor (TNF)-alpha and TNF receptor 1 in LPS-treated cells. Finally, RES inhibited LPS-induced NF-kappaB translocation into the nucleus and COX-2 expression. Moreover, the effects of 5-aminoimidazole-4-carboxamide ribose and compound C were consistent with the effects of RES in LPS-treated cells. Taken together, these results suggest that the anti-inflammatory action of RES in RAW 264.7 macrophages is dependent on AMPK activation and is associated with inhibition of the LPS-stimulated NF-kappaB-dependent COX-2 signaling pathway.
Acetyl-CoA Carboxylase ; AMP-Activated Protein Kinases ; Dinoprostone ; Homeostasis ; Macrophages ; Microglia ; NF-kappa B ; Phosphorylation ; Prostaglandin-Endoperoxide Synthases ; Receptors, Tumor Necrosis Factor ; Ribose ; Stilbenes ; Tumor Necrosis Factor-alpha

BACKGROUND: The intravenous administration of indigo carmine has been reported to produce transiently increased blood pressure in patients. The goal of this in vitro study was to examine the effect of indigo carmine on phenylephrine-induced contractions in an isolated rat aorta and to determine the associated cellular mechanism with particular focus on the endothelium-derived vasodilators. METHODS: The concentration-response curves for phenylephrine were generated in the presence or absence of indigo carmine. Phenylephrine concentration-response curves were generated for the endothelium-intact rings pretreated independently with a nitric oxide synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME), a cyclooxygenase inhibitor, indomethacin, and a low-molecular-weight superoxide anion scavenger, tiron, in the presence or absence of indigo carmine. The fluorescence of oxidized dichlorofluorescein was measured in rat aortic vascular smooth muscle cells cultured in the control, indigo carmine alone and tiron plus indigo carmine. RESULTS: Indigo carmine (10(-5) M) increased the phenylephrine-induced maximum contraction in the endothelium-intact rings with or without indomethacin, whereas indigo carmine produced a slight leftward shift in the phenylephrine concentration-response curves in the endothelium-denuded rings and L-NAME-pretreated endothelium-intact rings. In the endothelium-intact rings pretreated with tiron (10(-2) M), indigo carmine did not alter phenylephrine concentration-response curves significantly. Indigo carmine (10(-5) M) increased the fluorescence of oxidized dichlorofluorescein in the vascular smooth muscle cells, whereas tiron abolished the indigo carmine-induced increase in oxidized dichlorofluorescein fluorescence. CONCLUSIONS: Indigo carmine increases the phenylephrine-induced contraction mainly through an endothelium-dependent mechanism involving the inactivation of nitric oxide caused by the increased production of reactive oxygen species.
1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt ; Administration, Intravenous ; Animals ; Aorta ; Blood Pressure ; Contracts ; Fluorescence ; Humans ; Indigo Carmine ; Indoles ; Indomethacin ; Muscle, Smooth, Vascular ; Nitric Oxide ; Nitric Oxide Synthase ; Phenylephrine ; Prostaglandin-Endoperoxide Synthases ; Rats ; Reactive Oxygen Species ; Superoxides

Vascular inflammation process has been suggested to be an important risk factor in the development of atherosclerosis. Recently we reported that induction of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) selectively inhibits vascular cell adhesion molecule-1 (VCAM-1) but not intercellular cell adhesion molecule-1 (ICAM-1) in tumor necrosis factor (TNF)-alpha-activated human umbilical vein endothelial cells (HUVEC). In this study, we investigated whether genipin inhibits expression of cellular adhesion molecules, which is relevant to inflammation. Pretreatment with genipin reduced reactive oxygen species (ROS) production and expression of VCAM-1, but not ICAM-1 in TNF-alpha-activated HUVEC. Genipin dose- and time-dependently increased PPAR-gamma expression and inhibited TNF-alpha-induced phosphorylation of Akt and PKC with different degrees. Finally, genipin prevented TNF-alpha-induced adhesion of U937 monocytic cells to HUVEC. Taken together, these results indicate that upregualtion of PPAR-gamma by genipin selectively inhibits TNF-alpha-induced expression of VCAM-1, in which regulation of Akt and/or PKC play a key role. We concluded that genipin can be used for the treatment of cardiovascular disorders such as atherosclerosis.
Atherosclerosis ; Cell Adhesion ; Endothelial Cells ; Human Umbilical Vein Endothelial Cells ; Humans ; Inflammation ; Intercellular Adhesion Molecule-1 ; Iridoids ; Peroxisomes ; Phosphorylation ; Reactive Oxygen Species ; Risk Factors ; Tumor Necrosis Factor-alpha ; Up-Regulation ; Vascular Cell Adhesion Molecule-1

OBJECTIVE: Aberrant expression of the cell surface proteoglycan, syndecan-1, is found in many malignancies. The current study describes the immunohistochemical study of syndecan-1 expression in normal, hyperplastic, and malignant endometrial tissues for evaluation of application as a parameter of cancer progression in patients with endometrial hyperplasia. METHODS: Immunohistochemical staining of syndecan-1 was performed in 101 formalin fixed, paraffin embedded sections of normal, hyperplastic, and malignant endometrial tissues. We analyzed specimens from patients with normal endometrium (NE, N=10) as controls, and those of simple hyperplasia (SH, N=20), complex hyperplasia without atypia (CH, N=20), atypical hyperplasia (AH, N=20), and endometrial cancer (EC, N=31). RESULTS: The mean rank of expression scores based on the frequency of syndecan-1 staining were 31.6, 20.5, 52.9, 72.1, and 62.1 for NE, SH, CH, AH and EC, respectively (p<0.001). Syndecan-1 expression was significantly greater in CH (p<0.001) or AH (p<0.001) than in SH, and significantly greater in AH compared to CH (p=0.028). Syndecan-1 is more frequently expressed in CH (p=0.042), AH (p<0.001), or EC (p=0.002) than in NE. Syndecan-1 expression did not differ significantly between NE and SH (p=0.248). CONCLUSION: Syndecan-1 expression appears to be useful as a predictive indicator in endometrial hyperplasia.
Endometrial Hyperplasia ; Endometrial Neoplasms ; Endometrium ; Female ; Formaldehyde ; Humans ; Hyperplasia ; Paraffin ; Proteoglycans ; Syndecan-1

Inflammatory processes of vascular endothelial cells play a key role in the development ofatherosclerosis. We determined the anti-inflammatory effects and mechanisms of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on LPS-treated human umbilical vein endothelial cells (HUVECs) to evaluate their cardioprotective potential. Cells were pretreated with DHA, EPA, or troglitazone prior to activation with LPS. Expression of COX-2, prostaglandin E2 (PGE2) and IL-6 production, and NF-kappaB activity were measured by Western blot, ELISA, and luciferase activity, respectively. Results showed that EPA, DHA, or troglitazone significantly reduced COX-2 expression, NF-kappaB luciferase activity, and PGE2 and IL-6 production in a dose-dependent fashion. Interestingly, low doses (10 micrometer) of DHA and EPA, but not troglitozone, significantly increased the activity of NF-kappaB in resting HUVECs. Our study suggests that while DHA, EPA, and troglitazone may be protective on HUVECs under inflammatory conditions in a dose-dependent manner. However there may be some negative effects when the concentrations are abnormally low, even in normal endothelium.
Blotting, Western ; Chromans ; Cyclooxygenase 2 ; Dinoprostone ; Eicosapentaenoic Acid ; Endothelial Cells ; Endothelium ; Enzyme-Linked Immunosorbent Assay ; Human Umbilical Vein Endothelial Cells ; Humans ; Interleukin-6 ; Luciferases ; NF-kappa B ; Thiazolidinediones

A brief ischemic insult induces significant protection against subsequent massive ischemic events. The molecular mechanisms known as preconditioning (PC)-induced ischemic tolerance are not completely understood. We investigated whether kinetic changes of cyclooxygenase (COX)-2 during reperfusion time-periods after PC were related to ischemic tolerance. Rats were given PC by occlusion of middle cerebral artery (MCAO) for 10 min and sacrificed after the indicated time-periods of reperfusion (1, 2, 4, 8, 12, 18 or 24 h). In PC-treated rats, focal ischemia was induced by occlusion of MCA for 24 h and brain infarct volume was then studied to determine whether different reperfusion time influenced the damage. We report that the most significant protection against focal ischemia was obtained in rats with 8 h reperfusion after PC. Administration of indomethacin (10 mg/kg, oral) or rofecoxib (5 mg/kg, oral) 48 h prior to PC counteracted the effect of PC. Immunohistochemical analysis showed that COX-2 and HO-1 protein were induced in PC-treated rat brain, which was significantly inhibited by rofecoxib. Taken together, we concluded that the kinetic changes of COX-2 expression during the reperfusion period after PC might be partly responsible for ischemic tolerance.
Animals ; Brain ; Heme Oxygenase (Decyclizing) ; Indomethacin ; Ischemia ; Ischemic Preconditioning ; Lactones ; Middle Cerebral Artery ; Prostaglandin-Endoperoxide Synthases ; Rats ; Reperfusion ; Stroke ; Sulfones

PURPOSE: To study the effect of recombinant human epidermal growth factor (rhEGF) on oral mucositis induced by cisplatin and radiotherapy in a mouse model. MATERIALS AND METHODS: Twenty-four ICR mice were divided into three groups? the normal control group, the no rhEGF group (treatment with cisplatin and radiation) and the rhEGF group (treatment with cisplatin, radiation and rhEGF). A model of mucositis induced by cisplatin and radiotherapy was established by injecting mice with cisplatin (10 mg/kg) on day 1 and with radiation exposure (5 Gy/day) to the head and neck on days 1~5. rhEGF was administered subcutaneously on days -1 to 0 (1 mg/kg/day) and on days 3 to 5 (1 mg/kg/day). Evaluation included body weight, oral intake, and histology. RESULTS: For the comparison of the change of body weight between the rhEGF group and the no rhEGF group, a statistically significant difference was observed in the rhEGF group for the 5 days after day 3 of the experiment. The rhEGF group and no rhEGF group had reduced food intake until day 5 of the experiment, and then the mice demonstrated increased food intake after day 13 of the of experiment. When the histological examination was conducted on day 7 after treatment with cisplatin and radiation, the rhEGF group showed a focal cellular reaction in the epidermal layer of the mucosa, while the no rhEGF group did not show inflammation of the oral mucosa. CONCLUSION: These findings suggest that rhEGF has a potential to reduce the oral mucositis burden in mice after treatment with cisplatin and radiation. The optimal dose, number and timing of the administration of rhEGF require further investigation.
Animals ; Body Weight ; Cisplatin* ; Eating ; Epidermal Growth Factor* ; Head ; Humans* ; Inflammation ; Mice* ; Mice, Inbred ICR ; Mouth Mucosa ; Mucositis ; Mucous Membrane ; Neck ; Radiotherapy* ; Stomatitis*

Chinese herb medicines have traditionally been used to treat or alleviate the symptom of various diseases. The rationale for use of certain herbs to certain disorder is now getting unveiled by modern technology. In the present study, we investigated whether herb mix extract (HMX), which is alleged to be useful for gastric ulcer, protects stomach from oxidative stress. Rats were allowed to normal diet with and without HMX (1, 5, 10 mg/kg) for 30 days. To induce gastric ulcer, ethanol (75%, 1.5 ml) or acidified aspirin (100 mg/kg in 0.2 N HCl) was administered by oral route in 24 h-fasted rats and examined the gastric ulceration (bleeding) by measuring the size 1 h after the treatment. Results indicated the area of gastric bleeding was significantly less in HMX fed rats than in normal diet fed ones, and it was dependent on the duration and amount of HMX. To investigate the underlying mechanism by which HMX protects stomach from oxidative stress, expression of enzymes like heme oxygenase (HO), cyclooxygenase (COX), and inducible nitric oxide (iNOS) were investigated in MKN-74 cells, where aspirin or H. pylori was introduced. The results were compared with RAW 264.7 cells to check if there's cell specificities exist. The expression of HO-1 but not COX-2, iNOS was significantly increased by HMX. Furthermore, HO-1 inhibitor, SnPP IX reduced the HO-1 activity and reversed the survival rate in HMX-treated MKN-74 cells. There's no difference between RAW 264.7 cells and MKN-74 cells. We, thus, concluded that HMX is beneficial for protection from oxidative injury, and induction of HO-1 by HMX in gastric cells is, at least, responsible for protection from oxidative stress such as ethanol, aspirin and possibly H. pylori infection.
Animals ; Asian Continental Ancestry Group ; Aspirin* ; Cell Survival* ; Diet ; Ethanol* ; Heme Oxygenase (Decyclizing) ; Heme Oxygenase-1* ; Heme* ; Hemorrhage* ; Humans ; Nitric Oxide ; Oxidative Stress ; Prostaglandin-Endoperoxide Synthases ; Rats* ; Reactive Oxygen Species ; Stomach Ulcer ; Stomach* ; Survival Rate