Yi Chang

Expression of matrix metalloproteinase-9 in human platelets: regulation of platelet activation in in vitro and in vivo studies

The aim of this study was to identify the presence of matrix metalloproteinase‐9 (MMP‐9) in human platelets and systematically examine its inhibitory mechanisms of platelet activation. In this study, we report on an efficient method for the quantitative analysis of pro‐MMP‐9 in human platelets using capillary zone electrophoresis (CZE). To elucidate subcellular localization of MMP‐9 in human platelets, we investigated intraplatelet MMP‐9 by immunogold labeling and visualized it using electron microscopy. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated with fluorescein sodium. MMP‐9‐gold labeling was observed on the plasma membrane, α‐granules, open canalicular system, and within the cytoplasma both in resting and activated platelets. Furthermore, activated MMP‐9 concentration‐dependently (15–90 ng ml−1) inhibited platelet aggregation stimulated by agonists. Activated MMP‐9 (21 and 90 ng ml−1) inhibited phosphoinositide breakdown, intracellular Ca2+ mobilization, and thromboxane A2 formation in human platelets stimulated by collagen (1 μg ml−1). In addition, activated MMP‐9 (21 and 90 ng ml−1) significantly increased the formation of nitric oxide/cyclic GMP. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol‐12, 13‐dibutyrate (PDBu) (60 nM). This phosphorylation was markedly inhibited by activated MMP‐9 (21 and 90 ng ml−1). Activated MMP‐9 (1 μg g−1) significantly prolonged the latency period of inducing platelet plug formation in mesenteric venules. These results indicate that the antiplatelet activity of activated MMP‐9 may be involved in the following pathways. (1) Activated MMP‐9 may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown, protein kinase C activation, and thromboxane A2 formation, thereby leading to inhibition of intracellular Ca2+ mobilization. (2) Activated MMP‐9 also activated the formation of nitric oxide/cyclic GMP, resulting in inhibition of platelet aggregation. These results strongly indicate that MMP‐9 is a potent inhibitor of aggregation. It may play an important role as a negative feedback regulator during platelet activation.

Neuroprotective mechanisms of puerarin in middle cerebral artery occlusion-induced brain infarction in rats

Puerarin, a major isoflavonoid derived from the Chinese medical herb Radix puerariae (kudzu root), has been reported to be useful in the treatment of various cardiovascular diseases. In the present study, we examined the detailed mechanisms underlying the inhibitory effects of puerarin on inflammatory and apoptotic responses induced by middle cerebral artery occlusion (MCAO) in rats. Treatment of puerarin (25 and 50 mg/kg; intraperitoneally) 10 min before MCAO dose-dependently attenuated focal cerebral ischemia in rats. Administration of puerarin at 50 mg/kg, showed marked reduction in infarct size compared with that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in hypoxia-inducible factor-1α (HIF-1α), inducible nitric oxide synthase (iNOS), and active caspase-3 protein expressions as well as the mRNA expression of tumor necrosis factor-α (TNF-α) in ischemic regions. These expressions were markedly inhibited by the treatment of puerarin (50 mg/kg). In addition, puerarin (10~50 μM) concentration-dependently inhibited respiratory bursts in human neutrophils stimulated by formyl-Met-Leu-Phe. On the other hand, puerarin (20~500 μM) did not significantly inhibit the thiobarbituric acid-reactive substance reaction in rat brain homogenates. An electron spin resonance (ESR) method was conducted on the scavenging activity of puerarin on the free radicals formed. Puerarin (200 and 500 μM) did not reduce the ESR signal intensity of hydroxyl radical formation. In conclusion, we demonstrate that puerarin is a potent neuroprotective agent on MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by the inhibition of both HIF-1α and TNF-α activation, followed by the inhibition of inflammatory responses (i.e., iNOS expression), apoptosis formation (active caspase-3), and neutrophil activation, resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, puerarin treatment may represent a novel approach to lowering the risk of or improving function in ischemia-reperfusion brain injury-related disorders.

Protective Mechanisms of Inosine in Platelet Activation and Cerebral Ischemic Damage

Objective—Inosine is a naturally occurring nucleoside degraded from adenosine. Recent studies have demonstrated that inosine has potent immunomodulatory and neuroprotective effects. In the present study, we further investigated the inhibitory effects of inosine on platelet activation in vitro and in vivo, as well as in attenuating middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Methods and Results—Inosine concentration-dependently (0.5 to 6.0 mmol/L) inhibited platelet aggregation stimulated by agonists. Inosine (1.5 and 3.0 mmol/L) inhibited phosphoinositide breakdown, [Ca+2]i, and TxA2 formation in human platelets stimulated by collagen (1 &mgr;g/mL). In addition, inosine (1.5 and 3.0 mmol/L) markedly increased levels of cyclic guanylate monophosphate (GMP) and cyclic GMP-induced vasodilator-stimulated phosphoprotein Ser157 phosphorylation. Rapid phosphorylation of a platelet protein of molecular weight 47 000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 &mgr;g/mL). This phosphorylation was markedly inhibited by inosine (3.0 mmol/L). Inosine (1.5 and 3.0 mmol/L) markedly reduced hydroxyl radical in collagen (1 &mgr;g/mL)-activated platelets. In in vivo studies, inosine (400 mg/kg) significantly prolonged the latency period of inducing platelet plug formation in mesenteric venules of mice, and administration of 2 doses (100 mg/kg) or a single dose (150 mg/kg) of inosine significantly attenuated MCAO-induced focal cerebral ischemia in rats. Conclusions—Platelet aggregation contributes significantly to MCAO-induced focal cerebral ischemia. The most important findings of this study suggest that inosine markedly inhibited platelet activation in vitro and in vivo, as well as cerebral ischemia. Thus, inosine treatment may represent a novel approach to lowering the risk of or improving function in thromboembolic-related disorders and ischemia-reperfusion brain injury.

Inhibitory Effects of Ketamine on Lipopolysaccharide-Induced Microglial Activation

Microglia activated in response to brain injury release neurotoxic factors including nitric oxide (NO) and proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Ketamine, an anesthetic induction agent, is generally reserved for use in patients with severe hypotension or respiratory depression. In this study, we found that ketamine (100 and 250 μM) concentration-dependently inhibited lipopolysaccharide (LPS)-induced NO and IL-1β release in primary cultured microglia. However, ketamine (100 and 250 μM) did not significantly inhibit the LPS-induced TNF-α production in microglia, except at the higher concentration (500 μM). Further study of the molecular mechanisms revealed that ketamine markedly inhibited extracellular signal-regulated kinase (ERK1/2) phosphorylation but not c-Jun N-terminal kinase or p38 mitogen-activated protein kinase stimulated by LPS in microglia. These results suggest that microglial inactivation by ketamine is at least partially due to inhibition of ERK1/2 phosphorylation.

Therapeutic concentrations of propofol protects mouse macrophages from nitric oxide-induced cell death and apoptosis.

PurposeTo evaluate the potential effect of a clinically relevant concentration of propofol (PPF) on cell viability and nitric oxide-induced macrophage apoptosis.MethodsMouse macrophages (cell line Raw 264.7) were cultured and incubated with a nitric oxide donor sodium nitroprusside (SNP), PPF, and a combination of PPF and SNP for one, six and 24 hr. Cell viability was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Apoptotic cells were determined by analyzing the percentages of sub-G1 phase in macrophages. The amounts of nitric oxide were assayed.ResultsThe amounts of nitric oxide in macrophages were increased with time when incubated with SNP (P < 0.05). Simultaneously SNP caused cell death of macrophages in a concentration-and time-dependent manner (P < 0.05). PPFper se did not alter the amount of basal and SNP-provided nitric oxide in macrophages. A therapeutic concentration of PPF (30 μM) exhibited no cytotoxicity. After incubation with SNP for one and six hours, PPF could completely or partially block nitric oxide-induced cell death, respectively (P < 0.05).Administration of SNP to macrophages resulted in a time-dependent pattern of increase of apoptotic cells (P < 0.05). Similar to the results of the cell viability analyses, PPF was able to protect macrophages from nitric oxide-induced apoptosis in one and six hour-treated groups (P < 0.05) but not in the 24 hr treated group.ConclusionPPF at a therapeutic concentration, can protect mouse macrophagesin vitro from nitric oxide-induced cell apoptosis as well as cell death.RésuméObjectifÉvaluer l’effet potentiel d’une concentration thérapeutique de propofol (PPF) sur la viabilité cellulaire et l’apoptose des macrophages induite par l’oxyde nitrique.MéthodeDes macrophages de souris (souche cellulaire 264,7) ont été mis en culture et incubés avec un donneur d’oxyde nitrique, le nitroprussiate de sodium (NPS), du PPF et une combinaison de PPF et de NPS pendant une, six et 24 h. La viabilité cellulaire a été déterminée par une analyse colorimétrique du bromure 3-(4,5-diméthylthiazol-2-yl)-2, 5-diphényltétrazolium. Les cellules apoptotiques ont été déterminées en analysant les pourcentages de phase sous-G1 dans les macrophages. Les quantités d’oxyde nitrique ont été analysées.RésultatsLa quantité d’oxyde nitrique dans les macrophages a augmenté avec le temps dans le cas de l’incubation avec le NPS (P < 0,05). Simultanément, le NPS a causé la mort cellulaire des macrophages en fonction du temps et de la concentration (P < 0,05). Le PPF par luimême n’a pas modifié la quantité d’oxyde nitrique de base ou fournie par le NPS dans les macrophages. Une concentration thérapeutique de PPF (30 μM) n’a révélé aucune toxicité. Après l’incubation avec le NPS pendant une et six heures, le PPF a pu arrêter complètement ou partiellement la mort cellulaire induite par l’oxyde nitrique, respectivement (P < 0,05).L’administration de NPS aux macrophages a entraîné l’augmentation du nombre de cellules apoptotiques selon un modèle dépendant du temps (P < 0,05). Le PPF a pu protéger les macrophages de l’apoptose induite par l’oxyde nitrique dans les groupes traités pendant une et six heures (P < 0,05) mais non dans le groupe de 24 h.ConclusionLe PPF en concentration thérapeutique peut protéger les macrophages de souris in vitro de l’apoptose cellulaire aussi bien que de la mort cellulaire induites par l’oxyde nitrique.

Tetramethylpyrazine suppresses HIF-1α, TNF-α, and activated caspase-3 expression in middle cerebral artery occlusion-induced brain ischemia in rats

AbstractAim:To examine the detailed mechanisms underlying the inhibitory effect of tetramethylpyrazine (TMPZ) in inflammatory and apoptotic responses induced by middle cerebral artery occlusion (MCAO) in rats.Methods:MCAO-induced focal cerebral ischemia in rats was used in this study. The hypoxia-inducible factor-1α (HIF-1α), activation of caspase-3, and TNF-α mRNA transcription in ischemic regions were detected by immunoblotting and RT-PCR, respectively. Anti-oxidative activity was investigated using a thiobarbituric acid-reactive substance (TBARS) test in rat brain homogenate preparations.Results:We showed the statistical results of the infarct areas of solvent- and TMPZ (20 mg/kg)-treated groups at various distances from the frontal pole in MCAO-induced focal cerebral ischemia in rats. Treatment with TMPZ (20 mg/kg) markedly reduced the infarct area in all regions, especially in the third to fifth sections. MCAO-induced focal cerebral ischemia was associated with increases in HIF-1α and the activation of caspase-3, as well as TNF-α transcription in ischemic regions. These expressions were markedly inhibited by treatment with TMPZ (20 mg/kg). However, TMPZ (0.5-5 mmol/L) did not significantly inhibit TBARS reaction in rat brain homogenates.Conclusion:The neuroprotective effect of TMPZ may be mediated at least by a portion of the inhibition of HIF-1α and TNF-α activations, followed by the inhibition of apoptosis formation (active caspase-3), resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, TMPZ treatment may represent an ideal approach to lowering the risk of or improving function in ischemia-reperfusion brain injury-related disorders.

Mechanisms Involved in the Antiplatelet Activity of Ketamine in Human Platelets

The aim of this study was to systematically examine the inhibitory mechanisms of ketamine in platelet aggregation. In this study, ketamine concentration-dependently (100–350 µM) inhibited platelet aggregation both in washed human platelet suspensions and platelet-rich plasma stimulated by agonists. Ketamine inhibited phosphoinositide breakdown and intracellular Ca2+ mobilization in human platelets stimulated by collagen. Ketamine (200 and 350 µM) significantly inhibited thromboxane (Tx) A2 formation stimulated by collagen. Moreover, ketamine (200 and 350 µM) increased the fluorescence of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12,13-dibutyrate (100 nM). This phosphorylation was markedly inhibited by ketamine (350 µM). These results indicate that the antiplatelet activity of ketamine may be involved in the following pathways. Ketamine may change platelet membrane fluidity, with a resultant influence on activation of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and phosphorylation of P47, thereby leading to inhibition of intracellular Ca2+ mobilization and TxA2 formation, ultimately resulting in inhibition of platelet aggregation.

Brazilin isolated from Caesalpinia sappan L. acts as a novel collagen receptor agonist in human platelets

BackgroundBrazilin, isolated from the heartwood of Caesalpinia sappan L., has been shown to possess multiple pharmacological properties.MethodsIn this study, platelet aggregation, flow cytometry, immunoblotting analysis, and electron spin resonance (ESR) spectrometry were used to investigate the effects of brazilin on platelet activation ex vivo. Moreover, fluorescein sodium-induced platelet thrombi of mesenteric microvessels was also used in in vivo study.ResultsWe demonstrated that relatively low concentrations of brazilin (1 to 10 μM) potentiated platelet aggregation induced by collagen (0.1 μg/ml) in washed human platelets. Higher concentrations of brazilin (20 to 50 μM) directly triggered platelet aggregation. Brazilin-mediated platelet aggregation was slightly inhibited by ATP (an antagonist of ADP). It was not inhibited by yohimbine (an antagonist of epinephrine), by SCH79797 (an antagonist of thrombin protease-activated receptor [PAR] 1), or by tcY-NH2 (an antagonist of PAR 4). Brazilin did not significantly affect FITC-triflavin binding to the integrin αIIbβ3 in platelet suspensions. Pretreatment of the platelets with caffeic acid phenethyl ester (an antagonist of collagen receptors) or JAQ1 and Sam.G4 monoclonal antibodies raised against collagen receptor glycoprotein VI and integrin α2β1, respectively, abolished platelet aggregation stimulated by collagen or brazilin. The immunoblotting analysis showed that brazilin stimulated the phosphorylation of phospholipase C (PLC)γ2 and Lyn, which were significantly attenuated in the presence of JAQ1 and Sam.G4. In addition, brazilin did not significantly trigger hydroxyl radical formation in ESR analysis. An in vivo mouse study showed that brazilin treatment (2 and 4 mg/kg) significantly shortened the occlusion time for platelet plug formation in mesenteric venules.ConclusionTo the best of our knowledge, this study provides the first evidence that brazilin acts a novel collagen receptor agonist. Brazilin is a plant-based natural product, may offer therapeutic potential as intended anti-thrombotic agents for targeting of collagen receptors or to be used a useful tool for the study of detailed mechanisms in collagen receptors-mediated platelet activation.

Inhibitory mechanisms of cme-1, a novel polysaccharide from the mycelia of cordyceps sinensis, in platelet activation

OBJECTIVE CME-1 is a polysaccharide purified from the mycelia of medicinal mushroom Cordyceps sinensis, its molecular weight was determined to be 27.6 kDa by using nuclear magnetic resonance and gas chromatography-mass spectrometry. The initiation of arterial thromboses is relevant to various cardiovascular diseases (CVDs) and is believed to involve platelet activation. Our recent study exhibited that CME-1 has potent antiplatelet activity via the activation of adenylate cyclase/cyclic AMP ex vivo and in vivo. METHODS The aggregometry, and immunoblotting were used in this study. RESULTS In this study, the mechanisms of CME-1 in platelet activation is further investigated and found that CME-1 inhibited platelet aggregation as well as the ATP-release reaction, relative intracellular [Ca(+2)] mobilization, and the phosphorylation of phospholipase C (PLC)γ2 and protein kinase C (PKC) stimulated by collagen. CME-1 has no effects on inhibiting either convulxin, an agonist of glycoprotein VI, or aggretin, an agonist of integrin α2β1 stimulated platelet aggregation. Moreover, this compound markedly diminished thrombin and arachidonic acid (AA) induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 2, c-Jun N-terminal kinase 1, and Akt. Treatment with SQ22536, an inhibitor of adenylate cyclase, markedly diminished the CME-1-mediated increasing of cyclic AMP level and reversed prostaglandin E1- or CME-1-mediated inhibition of platelet aggregation and p38 MAPK and Akt phosphorylation stimulated by thrombin or AA. Furthermore, phosphodiesterase activity of human platelets was not altered by CME-1. CONCLUSION The crucial finding of this study is that the antiplatelet activity of CME-1 may initially inhibit the PLCγ2-PKC-p47 cascade, and inhibit PI3-kinase/Akt and MAPK phosphorylation through adenylate cyclase/ cyclic AMP activation, then inhibit intracellular [Ca(+2)] mobilization, and, ultimately, inhibit platelet activation. The novel role of CME-1 in antiplatelet activity indicates that this compound exhibits high therapeutic potential for treating or preventing CVDs.

Platonin inhibited PDGF-BB-induced proliferation of rat vascular smooth muscle cells via JNK1/2-dependent signaling

Aim:To examine the inhibitory actions of the immunoregulator platonin against proliferation of rat vascular smooth muscle cells (VSMCs).Methods:VSMCs were prepared from the thoracic aortas of male Wistar rats. Cell proliferation was examined using MTT assays. Cell cycles were analyzed using flow cytometry. c-Jun N-terminal kinase (JNK)1/2, extracellular signal-regulated kinase (ERK)1/2, AKT, and c-Jun phosphorylation or p27 expression were detected using immunoblotting.Results:Pretreatment with platonin (1–5 μmol/L) significantly suppressed VSMC proliferation stimulated by PDGF-BB (10 ng/mL) or 10% fetal bovine serum (FBS), and arrested cell cycle progression in the S and G2/M phases. The same concentrations of platonin significantly inhibited the phosphorylation of JNK1/2 but not ERK1/2 or AKT in VSMCs stimulated by PDGF-BB. Furthermore, platonin also attenuated c-Jun phosphorylation and markedly reversed the down-regulation of p27 expression after PDGF-BB stimulation.Conclusion:Platonin inhibited VSMC proliferation, possibly via inhibiting phosphorylation of JNK1/2 and c-Jun, and reversal of p27 down-regulation, thereby leading to cell cycle arrest at the S and G2/M phases. Thus, platonin may represent a novel approach for lowering the risk of abnormal VSMC proliferation and related vascular diseases.