Joen R. Sheu

Inhibitory mechanisms of resveratrol in platelet activation: pivotal roles of p38 MAPK and NO/cyclic GMP

Resveratrol has been reported to have antiplatelet activity; however, the detailed mechanisms have not yet been resolved. This study aimed to systematically examine the detailed mechanisms of resveratrol in the prevention of platelet activation in vitro and in vivo. Resveratrol (0·05–0·25u2003μmol/l) showed stronger inhibition of platelet aggregation stimulated by collagen (1u2003μg/ml) than other agonists. Resveratrol (0·15 and 0·25u2003μmol/l) inhibited collagen‐induced platelet activation accompanied by [Ca+2]i mobilization, thromboxane A2 (TxA2) formation, phosphoinositide breakdown, and protein kinase C (PKC) activation. Resveratrol markedly increased levels of NO/cyclic guanosine monophosphate (GMP), and cyclic GMP‐induced vasodilator‐stimulated phosphoprotein phosphorylation. Resveratrol markedly inhibited p38 mitogen‐activated protein kinase (MAPK) but not Jun N‐terminal kinase or extracellular signal‐regulated kinase‐2 phosphorylation in washed platelets. Resveratrol‐reduced hydroxyl radical (OH−) formation in the electron spin resonance study. In an in vivo study, resveratrol (5u2003mg/kg) significantly prolonged platelet plug formation of mice. In conclusion, the main findings of this study suggest that the inhibitory effects of resveratrol possibly involve (i) inhibition of the p38 MAPK‐cytosolic phospholipase A2‐arachidonic acid‐TxA2‐[Ca+2]i cascade and (ii) activation of NO/cyclic GMP, resulting in inhibition of phospholipase C and/or PKC activation. Resveratrol is likely to exert significant protective effects in thromboembolic‐related disorders by inhibiting platelet aggregation.

Mechanisms of resveratrol-induced platelet apoptosis

AIMSnApoptotic events have recently been found to occur in platelets, which are anuclear. Resveratrol is present in red wine and has various biological activities, including inhibition of platelet aggregation. Although considerable evidence is available as to the induction of tumour cell apoptosis by resveratrol, resveratrols effects on platelet apoptosis have not yet been investigated. In the present study, we demonstrate that resveratrol also markedly stimulates apoptosis in washed human platelets.nnnMETHODS AND RESULTSnResveratrol (5-25 microM) completely inhibited platelet aggregation stimulated by collagen. Furthermore, resveratrol time- and concentration-dependently stimulated dissipation of the mitochondrial membrane potential (DeltaPsim), activation of caspases-9, -3, and -8, gelsolin and actin cleavage, Bid cleavage into truncated Bid, Bax translocation, cytochrome c release, and phosphatidylserine exposure but not P-selectin expression in washed human platelets. The presence of z-IETD-fmk, a caspase-8 inhibitor, markedly reversed tBid formation and caspase activation and partially reversed the dissipation of platelet DeltaPsim stimulated by resveratrol. In addition, resveratrol also directly evoked dissipation of DeltaPsim and release of cytochrome c from isolated mitochondria. Furthermore, resveratrol shortened platelet survival or enhanced platelet clearance in an in vivo study.nnnCONCLUSIONnThis study demonstrates for the first time that resveratrol simultaneously inhibits platelet aggregation and stimulates platelet apoptosis. Stimulation of platelet apoptosis by resveratrol may represent the increased therapeutic potential for patients suffering from thrombotic conditions or thrombocytosis to promote platelet destruction and thus prevent pathological clotting. Furthermore, this study also provides a novel conception that rigorous surveillance of platelet numbers may be important during resveratrol treatment in the clinic.

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.

Combined cord blood stem cells and gene therapy enhances angiogenesis and improves cardiac performance in mouse after acute myocardial infarction

Backgroundu2002 Gene and stem cell therapies hold promise for the treatment of ischaemic cardiovascular disease. However, combined stem cell and angiogenic growth factor gene therapy for acute ischaemic myocardium has not been previously reported. This study hypothesized that combined stem cell and gene therapy would not only augment new vessels formation but also improve myocardial function in acute ischaemic myocardium.

Andrographolide Enhances Nuclear Factor-κB Subunit p65 Ser536 Dephosphorylation through Activation of Protein Phosphatase 2A in Vascular Smooth Muscle Cells

Recent studies have demonstrated that transcription factor nuclear factor (NF)-κB inhibition may contribute to the protective anti-inflammatory actions of andrographolide, an abundant component of plants of the genus Andrographis. However, the precise mechanism by which andrographolide inhibits NF-κB signaling remains unclear. We thus investigated the mechanism involved in andrographolide suppression of NF-κB signaling in rat vascular smooth muscle cells (VSMCs) exposed to proinflammatory stimuli, LPS, and IFN-γ. Andrographolide was shown to suppress LPS/IFN-γ-induced inducible nitric-oxide synthase and matrix metalloprotease 9 expression in rat VSMCs. Andrographolide also inhibited LPS/IFN-γ-induced p65 nuclear translocation, DNA binding activity, p65 Ser536 phosphorylation, and NF-κB reporter activity. However, IKK phosphorylation and downstream inhibitory κBα phosphorylation and degradation were not altered by the presence of andrographolide in LPS/IFN-γ-stimulated VSMCs. These andrographolide inhibitory actions could be prevented by selective inhibition of neutral sphingomyelinase and protein phosphatase 2A (PP2A). Furthermore, andrographolide was demonstrated to increase ceramide formation and PP2A activity in VSMCs and to inhibit neointimal formation in rat carotid injury models. These results suggest that andrographolide caused neutral sphingomyelinase-mediated ceramide formation and PP2A activation to dephosphorylate p65 Ser536, leading to NF-κB inactivation and subsequent inducible nitric-oxide synthase down-regulation in rat VSMCs stimulated by LPS and IFN-γ.

Suppression of NF-κB signaling by andrographolide with a novel mechanism in human platelets: regulatory roles of the p38 MAPK-hydroxyl radical-ERK2 cascade.

Andrographolide, a novel nuclear factor-κB (NF-κB) inhibitor, is isolated from leaves of Andrographis paniculata. Platelet activation is relevant to a variety of coronary heart diseases. Our recent studies revealed that andrographolide possesses potent antiplatelet activity by activating the endothelial nitric oxide synthase (eNOS)-NO-cyclic GMP pathway. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of andrographolide in NF-κB-mediated events in platelets. In this study, NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were time-dependently activated by collagen in human platelets, and these signaling events were attenuated by andrographolide (35 and 75 μM). ODQ and KT5823, respective inhibitors of guanylate cyclase and cyclic GMP-dependent kinase (PKG), strongly reversed andrographolide-mediated inhibition of platelet aggregation, relative [Ca(2+)]i mobilization, and IKKβ, and p65 phosphorylation. In addition, SB203580 (an inhibitor of p38 MAPK), but not PD98059 (an inhibitor of ERKs), markedly abolished IKKβ and p65 phosphorylation. SB203580, NAC (a free-radical scavenger), and BAY11-7082 (an inhibitor of NF-κB) all diminished ERK2 phosphorylation, whereas PD98059, BAY11-7082, and NAC had no effects on p38 MAPK phosphorylation. Furthermore, SB203580, but not BAY11-7082 or PD98059, reduced collagen-induced hydroxyl radical ((·)HO) formation. KT5823 also markedly reversed andrographolide-mediated inhibition of p38 MAPK and ERK2 phosphorylation, and hydroxyl radical formation in platelets. In conclusion, this study demonstrated that andrographolide may involve an increase in cyclic GMP/PKG, followed by inhibition of the p38 MAPK/(·)HO-NF-κB-ERK2 cascade in activated platelets. Therefore, andrographolide may have a high therapeutic potential to treat thromboembolic disorders and may also be considered for treating various inflammatory diseases.

Homogenous distribution of fast response action potentials in canine pulmonary vein sleeves: a contradictory report

Pulmonary veins may serve as source of ectopic focus (or foci) in initiating atrial tachyarrhythmias in human beings. However, the animal model for such focal atrial fibrillation is still lacking and cellular mechanism for arrhythmias remains to be studied. Recently, a series of reports of cellular electrophysiological characterization of pulmonary vein sleeves demonstrated an extremely high incidence of automaticity (varied from 40 to 76%) and triggered activity (from 0 to 44%) in normal healthy control dogs and rabbits. The present study was therefore designed to re-investigate the cellular electrophysiological properties of canine pulmonary veins. Intracellular action potentials were characterized in pulmonary vein sleeves in 50 normal healthy dogs. Conventional glass microelectrode recording technique was used. Experiments were focused on the incidence of automaticity and triggered activity in pulmonary vein sleeve tissues. Surprisingly, our results showed that all pulmonary vein sleeves tissues in these dogs displayed fast-response action potentials under the well-controlled experimental condition (100%, n=50). No spontaneous pacemaking activities, early or delayed afterdepolarisations were observed (0%, n=50). No high-frequency spikes or irregular rhythm could be recorded in all experiments (0%, n=50). Both the frequency response and membrane responsiveness of the pulmonary vein action potentials were characterized. No electrophysiological inhomogeneity between the distal and the proximal part of the sleeves was observed. In conclusion, canine pulmonary vein sleeves do not display arrhythmogenic activities under normal physiological conditions. The possible explanations for the controversy in pulmonary veins electrophysiology were discussed.

Mechanisms Involved in the Antiplatelet Activity of Midazolam in Human Platelets

Background Midazolam is widely used as a sedative and anesthetic induction agent. The aim of this study was to systematically examine the inhibitory mechanisms of midazolam in platelet aggregation. Methods The inhibitory mechanisms of midazolam in platelet aggregation were explored by means of analysis of the platelet glycoprotein IIb–IIIa complex, phosphoinositide breakdown, intracellular Ca+2 mobilization, measurement of membrane fluidity, thromboxane B2 formation, and protein kinase C activity. Results In this study, midazolam dose-dependently (6–26 &mgr;m) inhibited platelet aggregation in human platelets stimulated by agonists. Midazolam also dose-dependently inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. Midazolam (6–26 &mgr;m) significantly inhibited thromboxane A2 formation stimulated by collagen in human platelets. Moreover, midazolam (15 and 26 &mgr;m) dose-dependently decreased 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 collagen (2 &mgr;g/ml). This phosphorylation was markedly inhibited by midazolam (26 &mgr;m). Conclusions These results indicate that the antiplatelet activity of midazolam may be involved in the following pathways: the effects of midazolam may initially be caused by induction of conformational changes in platelet membrane, leading to a change in the activity of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca+2 mobilization and phosphorylation of P47 protein.

Amyloid beta peptide-activated signal pathways in human platelets

Amyloid beta peptide (amyloid-beta), which accumulates in the cerebral microvessels in an age-dependent manner, plays a key role in the pathogenesis of cerebral amyloid angiopathy. Platelets are an important cellular element in vasculopathy of various causes. Amyloid-beta may activate or potentiate platelet aggregation. The present study explored the signaling events that underlie amyloid-beta activation of platelet aggregation. Platelet aggregometry, immunoblotting and assays to detect activated cellular events were applied to examine the signaling processes of amyloid-beta activation of platelets. Exogenous amyloid-beta (1-2 microM) potentiated platelet aggregation caused by collagen and other agonists. At higher concentrations (5-10 microM), amyloid-beta induced platelet aggregation which was accompanied by an increase in thromboxane A2 (TxA2) formation. These amyloid-beta actions on platelets were causally related to amyloid-beta activation of p38 mitogen-activated protein kinase (MAPK). Inhibitors of p38 MAPK and its upstream signaling pathways including proteinase-activated receptor 1 (PAR1), Ras, phosphoinositide 3-kinase (PI3-kinase), or Akt, but not extracellular signal-regulated kinase 2 (ERK2)/c-Jun N-terminal kinase 1 (JNK1), blocked amyloid-beta-induced platelet activation. These findings suggest that the p38 MAPK, but not ERK2 or JNK1 pathway, is specifically activated in amyloid-beta-induced platelet aggregation with the following signaling pathway: PAR1 --> Ras/Raf --> PI3-kinase --> Akt --> p38 MAPK --> cytosolic phospholipase A2 (cPLA2)--> TxA2. In conclusion, this study demonstrates amyloid-beta activation of a p38 MAPK signaling pathway in platelets leading to aggregation. Further studies are needed to define the specific role of amyloid-beta activation of platelets in the pathogenesis of vasculopathy including cerebral amyloid angiopathy.

The antiplatelet activity of PMC, a potent α-tocopherol analogue, is mediated through inhibition of cyclo-oxygenase

PMC, a potent α‐tocopherol derivative, dose‐dependently (5–25u2003μM) inhibited the ATP‐release reaction and platelet aggregation in washed human platelets stimulated by agonists (collagen and ADP). PMC also dose‐dependently inhibited the intracellular Ca2+ mobilization, whereas it did not inhibit phosphoinositide breakdown in human platelets stimulated by collagen. PMC (10 and 25u2003μM) significantly inhibited collagen‐stimulated thromboxane A2 (TxA2) formation in human platelets. On the other hand, PMC (25 and 100u2003μM) did not increase the formation of cyclic AMP or cyclic GMP in platelets. Moreover, PMC (25, 100, and 200u2003μM) did not affect the thromboxane synthetase activity of aspirin‐treated platelet microsomes. PMC (10 and 25u2003μM) markedly inhibited the exogenous arachidonic acid (100u2003μM)‐induced prostaglandin E2 (PGE2) formation in the presence of imidazole (600u2003μM) in washed human platelets, indicating that PMC inhibits cyclo‐oxygenase activity. We conclude that PMC may exert its anti‐platelet aggregation activity by inhibiting cyclo‐oxygenase activity, which leads to reduced prostaglandin formation; this, in turn, is followed by a reduction of TxA2 formation, and finally inhibition of [Ca2+]i mobilization and ATP‐release.