Chang-Hui Liao

Sphingosine‐1‐phosphate induces COX‐2 expression via PI3K/Akt and p42/p44 MAPK pathways in rat vascular smooth muscle cells

Sphingosine 1‐phosphate (S1P) has been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. S1P increases the expression of several proteins including COX‐2 in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating COX‐2 expression by S1P in VSMCs remain unclear. Western blotting and RT‐PCR analyses showed that S1P induced the expression of COX‐2 mRNA and protein in a time‐ and concentration‐dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and PI3K (wortmannin), and transfection with dominant negative mutants of p42/p44 mitogen‐activated protein kinases (ERK2) or Akt. These results suggested that both p42/p44 MAPK and PI3K/Akt pathways participated in COX‐2 expression induced by S1P in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt, which was attenuated by U0126, LY294002, or wortmannin, respectively. Furthermore, this up‐regulation of COX‐2 mRNA and protein was blocked by a selective NF‐κB inhibitor helenalin. Consistently, S1P‐stimulated translocation of NF‐κB into the nucleus was revealed by immnofluorescence staining. Moreover, S1P‐stimulated activation of NF‐κB promoter activity was blocked by phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002 and helenalin, but not by U0126, suggesting that involvement of PI3K/Akt in the activation of NF‐κB. COX‐2 promoter assay showed that S1P induced COX‐2 promoter activity mediated through p42/p44 MAPK, PI3K/Akt, and NF‐κB. These results suggested that in VSMCs, activation of p42/p44 MAPK, Akt and NF‐κB pathways was essential for S1P‐induced COX‐2 gene expression. Understanding the mechanisms involved in S1P‐induced COX‐2 expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis. J. Cell. Physiol.

Hydroxyethyl starch interferes with human blood ex vivo coagulation, platelet function and sedimentation.

BACKGROUND Hydroxyethyl starch (HES) solutions are widely used for intravascular volume expansion. In Taiwan, the medium molecular weight of HES 200/0.5 and HES 130/0.4 solutions are most commonly used. It has been demonstrated that HES may affect coagulation and platelet function significantly. However, the differential effects of each medium molecular weight HES on platelets remain poorly reported. Therefore, we studied the influence of the two HES solutions on platelet function in vitro by mixing whole blood with different proportions of HES 130 kD, HES 200 kD, and saline to determine the differences. METHODS Human blood samples for platelet function analyzer (PFA), aggregometry and blood/HES mixed test were drawn from the antecubital vein and put into test tubes containing 3.2% trisodium citrate (blood:citrate, 9:1). The specimens were divided into four groups, designated as whole blood, 10%, 20%, and 30% dilution with normal saline (N/S), HES130 or HES200 solution. The platelet function of each sample was measured by both PFA and platelet aggregometry. RESULTS The results showed that the PFA-100 closure times CEPI-CT and CADP-CT were significantly prolonged in the samples diluted with normal saline, HES130 and HES200 than in the controls. The ADP triggered whole blood aggregometry showed that attenuated impedance was observed in samples of 20% diluted with HES130 and HES200 groups. The blood/HES mixed sedimentation test showed significantly increased proportion of the upper liquid layer in the HES200 group than in other groups. CONCLUSION Our data demonstrated that HES200 and HES130 possess noticeably inhibitory effects on platelet function, especially when the HES replaced proportion was more than 20%. HES200 has a greater effect on blood cells and plasma separation than does HES130.

Hemeoxygenase-1 Upregulation Is Critical for Sirtinol-mediated Attenuation of Lung Injury After Trauma-hemorrhage in a Rodent Model

BACKGROUND: Hemeoxygenase-1 induction in response to adverse circulatory conditions is protective. Our recent study has shown that administration of sirtinol attenuates hepatic injury in male Sprague-Dawley rats after trauma-hemorrhage; however, the mechanism by which sirtinol produces the salutary effects remains unknown. We hypothesized that sirtinol administration in male Sprague-Dawley rats after trauma-hemorrhage decreases cytokine production and protects against lung injury through a hemeoxygenase-1 related pathway. METHODS: Male Sprague-Dawley rats (n = 8 per group) underwent trauma-hemorrhage (mean arterial blood pressure 40 mm Hg for 90 min, then resuscitation). A single dose of sirtinol (1 mg/kg of body weight) with or without a hemeoxygenase enzyme inhibitor (chromium-mesoporphyrin) or vehicle was administered IV during resuscitation. Twenty-four hours thereafter, myeloperoxidase activity (a marker of neutrophil sequestration) and tumor necrosis factor &agr;, interleukin-6, and interleukin-10 levels in the lung, protein concentrations in bronchoalveolar lavage fluid and tissue histology were measured. Lung hemeoxygenase-1 protein level was also determined. RESULTS: In the sirtinol-treated rats subjected to trauma-hemorrhage, there were significant improvements in lung myeloperoxidase activity (4.68 ± 0.31 vs 9.36 ± 1.03 U/mg protein, P < 0.05), tumor necrosis factor &agr; levels (710.7 ± 28 vs 1288 ± 40.69 pg/mg protein, P < 0.05), interleukin-6 levels (343.6 ± 18.41 vs 592.7 ± 22.3 pg/mg protein, P < 0.05), and protein concentrations (303.8 ± 24.54 vs 569.6 ± 34.82 &mgr;g/mL, P < 0.05) and lesser damage in histology. There was no statistically significant difference in interleukin-10 levels in the lung between sirtinol-treated trauma-hemorrhaged rats and vehicle-treated trauma-hemorrhaged rats (842.5 ± 54.18 vs 756.2 ± 41.34 pg/mg protein, respectively). Lung hemeoxygenase-1 protein levels were increased in rats receiving sirtinol treatment as compared with vehicle-treated trauma-hemorrhaged rats (5.18 ± 0.25 vs 2.70 ± 0.16, P < 0.05). Administration of the hemeoxygenase inhibitor chromium-mesoporphyrin prevented the sirtinol-induced attenuation of shock-induced lung damage. CONCLUSION: The salutary effects of sirtinol administration on attenuation of lung inflammation after trauma-hemorrhage are mediated via upregulation of hemeoxygenase-1 expression.

A new insight of anti-platelet effects of sirtinol in platelets aggregation via cyclic AMP phosphodiesterase

Sirtinol, a cell permeable six-membered lactone ring, is derived from naphthol and potent inhibitor of SIR2 and its naphtholic may have the inhibitory effects on platelets aggregation. In this study, platelet function was examined by collagen/epinephrine (CEPI) and collagen/ADP-induced closure times using the PFA-100 system reveal that CEPI-CT and CADP-CT were prolonged by sirtinol. The platelets aggregation regulated by physiological agonists such as: thrombin, collagen and AA and U46619 were significantly inhibited by sirtinol. Increases cAMP level was observed when sirtinol treated with Prostaglandin E1 in washed platelets. Moreover, sirtinol attenuated intracellular Ca(2+) release and thromboxane B2 formation stimulated by thrombin, collagen, AA and U46619 in human washed platelets. This study indicated that sirtinol could inhibit the platelet aggregation induced by physiological agonists, AA and U46619. The mechanism of action may include an increase of cAMP level with enhanced VASP-Ser157 phosphorylation via inhibition of cAMP phosphodiesterase activity and subsequent inhibition of intracellular Ca(2+) mobilization, thromboxane A2 formation, and ATP release during the platelet aggregation.

Secondary metabolites from the leaves of Neolitsea hiiranensis and the anti-inflammatory activity of some of them

Seven sesquiterpenoids, hiiranlactones A-D (1-4), (-)-ent-6α-methoxyeudesm-4(15)-en-1β-ol (5), (+)-villosine (6), hiiranepoxide (7), and one triterpenoid, hiiranterpenone (8), together with 22 known compounds, were isolated from the leaves of Neolitsea hiiranensis (Lauraceae). Their structures were elucidated by spectroscopic analysis and single crystal X-ray diffraction. Among the isolates, hiiranlactone B (2) and hiiranlactone D (4) exhibited inhibitory activity against fMLP-induced superoxide production by human neutrophils with IC(50) values of 21.86±3.97 and 25.78±4.77μM, respectively.

Platelet function analyzer (PFA-100) offers higher sensitivity and specificity than thromboelastography (TEG) in detection of platelet dysfunction.

BACKGROUND The development of advanced surgical procedures requires novel and early diagnostic techniques. One of the prime difficulties that need to be overcome is an abnormality in the coagulation system. The blood clot formation and fibrinolysis processes are very complicated and important perioperatively. However, most of the major surgeries, such as liver transplantation, use thromboelastography (TEG) for detection of coagulation abnormalities, even though TEG is not actually an ideal option. Therefore, we compared the sensitivity and specificity of the platelet function analyzer (PFA-100) and thromboelastogram (TEG) in predicting platelet dysfunction and bleeding risk. METHODS Human blood samples were drawn from healthy volunteers for this study. Levobupivacaine and CGS21680 have antiplatelet effects which were used as the detection target. The platelet counts before comparison, platelet aggregation, the closure time of PFA-100, and the parameters of TEG were examined for data analysis. RESULTS Platelet aggregations were suppressed by all levobupivacaine doses (10 microg/mL, 50 micropg/mL, 200 microg/mL) and CGS21680 (100 nM, 500 nM, 1 microM) in a dose-dependent manner. Levobupivacaine and CGS21680 at maximal test doses produced no significant alteration in any parameter in the TEG assay. In the samples measured with PFA-100, both levobupivacaine and CGS21680 at maximal test doses significantly prolonged the closure time in the PFA-100 assay. CONCLUSION We conclude that PFA-100 offers a higher sensitivity and specificity than TEG in detection of platelet dysfunction.

A New Dibenzofuran and Further Constituents from the Stems of Pourthiaea lucida with Inhibitory Activity on Superoxide Generation by Neutrophils

A new dibenzofuran, lucidafuran (1), was isolated from the stems of Pourthiaea lucida, together with eight known compounds. The structure of this new compound was determined through NMR and mass-spectrometric analyses. Among the isolated compounds, lucidafuran (1) and aucuparin (3) exhibited potent inhibitory activity against fMLP-induced superoxide (O(*-)(2)) production by human neutrophils with IC(50) values of 18.7+/-4.4 and 17.0+/-6.8 microM, resp.

Secondary metabolites from the root of Ehretia longiflora and their biological activities.

Bioassay-guided fractionation of the methanolic extract of the root of Ehretia longiflora (Boraginaceae) afforded eight compounds, ehretiquinone (1), ehretiolide (2), ehreticoumarin (3), ehretilactone A (4), ehretilactone B (5), ehretiamide (6), ehretine (7), and ehretiate (8), together with 12 known compounds (9-20). The relative configuration of 1 was determined by single crystal X-ray diffraction. Among the isolates, 1 and prenylhydroquinone (14) showed antitubercular activity against Mycobacterium tuberculosis strain H37Rv with MIC values of 25.0 and 26.2 μg/mL, respectively. Moreover, 1 exhibited inhibitory effects on N-formylmethionylleucylphenylalanine (fMLP)-induced superoxide production, with IC₅₀ value of 0.36±0.03μM.

Splitomicin suppresses human platelet aggregation via inhibition of cyclic AMP phosphodiesterase and intracellular Ca++ release

Splitomicin is derived from beta-naphthol and is an inhibitor of Silent Information Regulator 2 (SIR2). Its naphthoic moiety might be responsible for its inhibitory effects on platelets. The major goal of our study was to examine possible mechanisms of action of splitomicin on platelet aggregation in order to promote development of a novel anti-platelet aggregation therapy for cardiovascular and cerebrovascular diseases. To study the inhibitory effects of splitomicin on platelet aggregation, we used washed human platelets, and monitored platelet aggregation and ATP release induced by thrombin (0.1 U/ml), collagen (2 microg/ml), arachidonic acid (AA) (0.5 mM), U46619 (2 microM) or ADP (10 microM). Splitomicin inhibited platelet aggregation induced by thrombin, collagen, AA and U46619 with a concentration dependent manner. Splitomicin increased cAMP and this effect was enhanced when splitomicin (150 microM) was combined with PGE1 (0.5 microM). It did not further increase cAMP when combined with IBMX. This data indicated that splitomicin increases cAMP by inhibiting activity of phosphodiestease. In addition, splitomicin (300 microM) attenuated intracellular Ca(++) mobilization, and production of thromboxane B2 (TXB2) in platelets that was induced by thrombin, collagen, AA or U46619. The inhibitory mechanism of splitomicin on platelet aggregation may increase cyclic AMP levels via inhibition of cyclic AMP phosphodiesterase activity and subsequent inhibition of intracellular Ca(++) mobilization, TXB2 formation and ATP release.

A benzodiazepines derived compound, 4-(3-chlorophenyl)-1,3-dihydronaphtho [2,3-b][1,4]diazepin-2-one (ND700C), inhibits fMLP-induced superoxide anion release by activating protein phosphatase 2A in human neutrophils.

UNLABELLED We studied the mechanism underlying the inhibitory effect of a benzodiazepines derivative, 4-(3-chlorophenyl)-1,3-dihydronaphtho [2,3-b][1,4]diazepin-2-one (ND700C), on superoxide anion production induced by formly-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. ND700C inhibited the fMLP-induced superoxide anion production and cathepsin G release in a concentration-dependent manner with respective IC50 values of 5.0+/-0.5 and 8.7+/-0.8muM. In addition, ND700C was found to suppress fMLP-induced intracellular calcium mobilization and the phosphorylation of ERK and Akt. In another study, ND700C was observed to cause a rapid increase in intracellular cAMP level by up to threefold. Furthermore, when H89 was used to inhibit cAMP-dependent protein kinase A (PKA), we discovered that ND700Cs suppressive effects on calcium mobilization, phosphorylation, and superoxide anion production were abrogated. ND700C demonstrated additive effect on the PGE1-induced increase in cAMP. However, this additive effect was not demonstrated with the IBMX-induced rise in cAMP. Our results indicated that ND700C did not directly inhibit the activity of phosphodiesterase 4. In another set of experiments, calyculin A and okadaic acid (both protein phosphatase 2A inhibitors) were found to reverse ND700Cs positive effect on cAMP level. This observation suggested the involvement of protein phosphatase 2A in ND700Cs cAMP-elevating mechanism. We found that the activity of protein phosphatase 2A was activated by ND700C. Furthermore, protein phosphatase 2A was co-immunoprecipitated with phosphodiesterase 4 after ND700C treatment in human neutrophils. CONCLUSION ND700C inhibited fMLP-induced superoxide anion production through a PKA-dependent pathway. ND700C increased cAMP by activating protein phosphatase 2A, which subsequently inhibited phosphodiesterase 4.