Synergy by Ristocetin and CXCL12 in Human Platelet Activation: Divergent Regulation by Rho/Rho-Kinase and Rac.

CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase.

Successful treatment with spinal cord stimulation for pain due to eosinophilic granulomatosis with polyangiitis: a case report.

BACKGROUND: Although most patients of eosinophilic granulomatosis with polyangiitis (EGPA) experience a reduction in pain within several weeks to months of the initiation of immunotherapies, some suffer from residual neuropathic symptoms for a long time. CASE PRESENTATION: A 28-year-old woman diagnosed with EGPA visited. She had been treated with steroid pulse therapy, intravenous immunoglobulin, and mepolizumab (antiinterleukin-5 agent). Her symptoms other than peripheral neuropathy improved, but posterior lower thigh pain and weakness of the lower legs worsened. At the initial visit, she used crutches and complained of numb pain in both posterior lower thighs, especially the left one. She also presented with left foot drop and reported a decreased tactile sensation on the lateral sides of both lower thighs. We performed spinal cord stimulation (SCS) at the L1 level on both sides. Her pain remarkably decreased, her tactile sensation improved, her muscle strength increased, and she was able to walk without crutches. CONCLUSIONS: We herein report the first case of lower extremity pain being successfully treated with SCS in an EGPA patient who did not respond well to drug therapy. Because the cause of pain in EGPA is neuropathy induced by vasculitis, there is ample ability for SCS to improve this pain. When pain is neuropathic, whatever the cause, SCS may be worth trying, even for pain from disorders other than EGPA.

HSP70 inhibitors upregulate prostaglandin E1-induced synthesis of interleukin-6 in osteoblasts.

Interleukin-6 (IL-6) is a pro-inflammatory and bone-resorptive cytokine that also regulates bone formation. We previously showed that prostaglandin E1 (PGE1) induces the synthesis of IL-6 by activating p44/p42 mitogen-activated protein kinase (MAPK), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38 MAPK in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether heat shock protein 70 (HSP70), a molecular chaperone that coordinates protein folding and homeostasis, affects PGE1-stimulated IL-6 synthesis in MC3T3-E1 cells through the MAPK activation. The osteoblast-like MC3T3-E1 cells were treated with HSP70 inhibitors-VER-155008 and YM-08-, PD98059, SB203580 or SP600125 and then stimulated with PGE1. IL-6 synthesis was evaluated using an IL-6 enzyme-linked immunosorbent assay kit. IL-6 mRNA expression was measured by real-time RT-PCR. The phosphorylation of p38 MAPK was evaluated by Western blotting. We found that VER-155008, an HSP70 inhibitor, enhanced the PGE1-stimulated IL-6 release and IL-6 mRNA expression. YM-08, another HSP70 inhibitor, also enhanced PGE1-stimulated IL-6 release. PD98059, a p44/p42 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, upregulated PGE1-stimulated IL-6 release. On the other hand, SB203580, a p38 MAPK inhibitor, suppressed PGE1-stimulated IL-6 release. YM-08 stimulated the PGE1-induced phosphorylation of p38 MAPK. SB203580 suppressed the amplification by YM-08 of the PGE1-stimulated IL-6 release. Our results suggest that HSP70 inhibitors upregulate the PGE1-stimulated IL-6 synthesis through p38 MAPK in osteoblasts and therefore affect bone remodeling.

Comparison of regional cerebral oxygen saturation during one-lung ventilation under desflurane or propofol anesthesia: A randomized trial.

BACKGROUND: During one-lung ventilation (OLV), deterioration of pulmonary oxygenation reduces arterial oxygen saturation and cerebral oxygen saturation (rSO2). However, oxidative stress during OLV causes lung injury, so the fraction of inspiratory oxygen (FiO2) should be kept as low as possible. We investigated the changes in rSO2 under propofol or desflurane anesthesia while percutaneous oxygen saturation (SpO2) was kept as low as possible during OLV. METHODS: Thirty-six patients scheduled for thoracic surgery under OLV in the lateral decubitus position were randomly assigned to propofol (n = 19) or desflurane (n = 17) anesthesia. FiO2 was set to 0.4 at the start of surgery under two-lung ventilation (measurement point: T3) and then adjusted to maintain an SpO2 of 92% to 94% after the initiation of OLV. The primary outcome was the difference in the absolute value of the decrease in rSO2 from T3 to 30 minutes after the initiation of OLV (T5), which was analyzed by an analysis of covariance adjusted for the rSO2 value at T3. RESULTS: The mean rSO2 values were 61.5% ± 5.1% at T3 and 57.1% ± 5.3% at T5 in the propofol group and 62.2% ± 6.0% at T3 and 58.6% ± 5.3% at T5 in the desflurane group. The difference in the absolute value of decrease between groups (propofol group - desflurane group) was 0.95 (95% confidence interval, [-0.32, 2.2]; P = .152). CONCLUSIONS: Both propofol and desflurane anesthesia maintain comparable cerebral oxygenation and can be used safely, even when the SpO2 is kept as low as possible during OLV.

A practical guide for perioperative smoking cessation.

The perioperative management of patients who are smokers presents anesthesiologists with various challenges related to respiratory, circulatory, and other clinical problems. Regarding 30-day postoperative outcomes, smokers have higher risks of mortality and complications than non-smokers, including death, pneumonia, unplanned tracheal intubation, mechanical ventilation, cardiac arrest, myocardial infarction, and stroke. Given the benefits of smoking cessation and the adverse effects of smoking on perioperative patient management, patients should quit smoking long before surgery. However, anesthesiologists cannot address these issues alone. The Japanese Society of Anesthesiologists established guidelines in 2015 (published in a medical journal in 2017) to enlighten surgical staff members and patients regarding perioperative tobacco cessation. The primary objective of perioperative smoking cessation is to reduce the risks of adverse cardiovascular and respiratory events, wound infection, and other perioperative complications. Perioperative preparations constitute a powerful teachable moment, a "golden opportunity" for smoking cessation to achieve improved primary disease outcomes and prevent the occurrence of tobacco-related conditions. This review updates the aforementioned guidelines as a practical guide to cover the nuts and bolts of perioperative smoking cessation. Its goal is to assist surgeons, anesthesiologists, and other medical professionals and to increase patients' awareness of smoking risks before elective surgery.

Thrombopoietin and collagen in low doses cooperatively induce human platelet activation.

Aim: In acute medicine, we occasionally treat life-threatening conditions such as sepsis and trauma, which cause severe thrombocytopenia. Serum thrombopoietin levels have been reported to increase under the condition of thrombocytopenia related to severity. Collagen is a crucial activator of platelets, and Rho family members, such as Rho/Rho-kinase and Rac, play roles as active molecules involved in the intracellular signaling pathways in platelet activation. The present study aimed to elucidate the effects of thrombopoietin (TPO) on subthreshold low-dose collagen-stimulated human platelets in terms of Rho/Rho-kinase and Rac. Methods: Platelet-rich plasma donated from healthy volunteers was stimulated by the subthreshold low-dose of collagen after pretreatment with TPO and/or NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction, or Y27632, an inhibitor of Rho-kinase. Platelet aggregation was measured using an aggregometer based on laser-scattering methods. Proteins involved in intracellular signaling were analyzed using western blotting, and the secretion of platelet-derived growth factor-AB from activated platelets was determined using an enzyme-linked immunosorbent assay. Results: Under the existence of TPO, the low dose of collagen remarkably elicited the aggregation and platelet-derived growth factor-AB secretion of platelets, which were suppressed by NSC23766 and Y27632. The combination of TPO and collagen considerably induced a transient increase of guanosine triphosphate (GTP)-binding Rac and GTP-binding Rho followed by an increase of phosphorylated cofilin, a Rho-kinase substrate. Conclusion: These results strongly suggest that TPO and collagen in low doses cooperatively potentiate human platelet activation through both Rac and Rho/Rho-kinase mediated pathways.

Amplification by tramadol of PGD2-induced osteoprotegerin synthesis in osteoblasts: Involvement of µ-opioid receptor and 5-HT transporter.

Tramadol, a weak µ-opioid receptor (MOR) agonist with inhibitory effects on the reuptake of serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine, is an effective analgesic to chronic pains. Osteoprotegerin produced by osteoblasts is essential for bone remodeling to suppress osteoclastic bone resorption. We previously reported that prostaglandin D2 (PGD2) induces osteoprotegerin synthesis whereby p44/p42 mitogen-activated protein (MAP) kinase, p38 MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) are involved in osteoblast-like MC3T3-E1 cells. Herein, we investigated the mechanism underlying the effect of tramadol on the PGD2-induced osteoprotegerin synthesis in these cells. Tramadol enhanced the PGD2-induced release and mRNA expression of osteoprotegerin. Naloxone, a MOR antagonist, reduced the amplification by tramadol of the PGD2-stimulated osteoprotegerin release. Not the selective norepinephrine reuptake inhibitor reboxetine but the selective serotonin reuptake inhibitors fluvoxamine and sertraline upregulated the PGD2-induced osteoprotegerin release, which was further amplified by morphine. Tramadol enhanced PGD2-stimulated phosphorylation of p38 MAP kinase and SAPK/JNK, but not p44/p42 MAP kinase. Both SB203580 and SP600125 suppressed the tramadol effect to enhance the PGD2-stimulated osteoprotegerin release. Tramadol enhanced the PGE2-induced osteoprotegerin release as well as PGD2. These results suggest that tramadol amplifies the PGD2-induced osteoprotegerin synthesis at the upstream of p38 MAP kinase and SAPK/JNK in the involvement of both MOR and 5-HT transporter in osteoblasts.

Edaravone attenuates sustained pial arteriolar vasoconstriction independently of endothelial function after unclamping of the abdominal aorta in rabbits.

BACKGROUND: Cerebral blood flow (CBF) has direct effects on neuronal function and neurocognitive disorders. Oxidative stress from abdominal aortic surgery is important in the pathophysiology of CBF impairment. We investigated the effect of edaravone on the pial arteriolar diameter changes induced by abdominal aortic surgery and the involvement of the endothelium in the changes. METHODS: The closed cranial window technique was used in rabbits to measure changes in pial arteriolar diameter after the unclamping of abdominal aortic cross-clamping with an intravenous free radical scavenger, edaravone (control group [n = 6], edaravone 10 µg/kg/min [n = 6], 100 µg/kg/min [n = 6]). Pial vasodilatory responses to topical application of acetylcholine (ACh) into the cranial window were investigated before abdominal aortic cross-clamping and after unclamping with intravenous administration of edaravone (control group [n = 6], edaravone 100 µg/kg/min [n = 6]). RESULTS: Aortic unclamping-induced vasoconstriction was significantly attenuated by continuous infusion of edaravone at 100 µg/kg/min. Topical ACh after unclamping did not produce any changes in pial arteriolar responses in comparison to before aortic cross-clamping in the control or edaravone groups. The changes in the response to topical ACh after unclamping in the saline and edaravone groups did not differ significantly. CONCLUSIONS: Free radicals during abdominal aortic surgery might have contracted cerebral blood vessels independently of endothelial function in rabbits. Suppression of free radicals attenuated the sustained pial arteriolar vasoconstriction after aortic unclamping. Thus, the free radical scavenger might have some brain protective effect that maintains CBF independently of endothelial function.

Acetaminophen reduces osteoprotegerin synthesis stimulated by PGE2 and PGF2α in osteoblasts: attenuation of SAPK/JNK but not p38 MAPK or p44/p42 MAPK.

Acetaminophen is one of the most widely used analgesic and antipyretic medicines, whose long-period use has reportedly been associated with an increased risk of bone fracture. However, the mechanism underlying this undesired effect remains to be investigated. The homeostatic control of bone tissue depends on the interaction between osteoblasts and osteoclasts. Osteoprotegerin produced by osteoblasts is known to play an essential role in suppressing osteoclast induction. We have previously reported that prostaglandin (PG) E2 and PGF2α induce osteoprotegerin synthesis through p38 mitogen-activated protein kinase (MAPK), p44/p42 MAPK and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of acetaminophen on the osteoprotegerin synthesis induced by PGE2 and PGF2α in MC3T3-E1 cells. Acetaminophen significantly suppressed the osteoprotegerin release stimulated by PGE2 and PGF2α. The PGE2-induced expression of osteoprotegerin mRNA was also reduced by acetaminophen. Acetaminophen markedly downregulated the phosphorylation of SAPK/JNK stimulated by PGE2 and PGF2α, but not those of p38 MAPK or p44/p42 MAPK. SP600125, an inhibitor of SAPK/JNK, suppressed the levels of PGE2- and PGF2α-upregulated osteoprotegerin mRNA expression. Taken together, these results strongly suggest that acetaminophen reduces the PGE2- and PGF2α-stimulated synthesis of osteoprotegerin in osteoblasts, and that the suppressive effect is exerted via attenuation of SAPK/JNK. These findings provide a molecular basis for the possible effect of acetaminophen on bone tissue metabolism.

Duloxetine suppresses BMP-4-induced release of osteoprotegerin via inhibition of the SMAD signaling pathway in osteoblasts.

Duloxetine, a selective serotonin-norepinephrine reuptake inhibitor, is currently recommended for the treatment of chronic painful disorders such as fibromyalgia, chronic musculoskeletal pain, and diabetic peripheral neuropathy. We previously demonstrated that bone morphogenetic protein-4 (BMP-4) stimulates osteoprotegerin (OPG) production in osteoblast-like MC3T3-E1 cells, and that p70 S6 kinase positively regulates OPG synthesis. The present study aimed to investigate the effect of duloxetine on BMP-4-stimulated OPG synthesis in these cells. Duloxetine dose-dependently suppressed OPG release stimulated by BMP-4. Fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), reduced BMP-4-stimulated OPG release, whereas a selective and specific norepinephrine reuptake inhibitor, reboxetine, failed to affect OPG release. In addition, another SSRI sertraline also inhibited BMP-4-stimulated OPG release. On the other hand, siRNA of SMAD1 reduced the OPG release stimulated by BMP-4, indicating the involvement of the SMAD1/5/8 pathway in OPG release. Rapamycin inhibited BMP-4-stimulated p70 S6 kinase phosphorylation, and compound C suppressed the SMAD1/5/8 phosphorylation stimulated by BMP-4. Duloxetine did not affect BMP-4-induced phosphorylation of p70 S6 kinase but suppressed SMAD1/5/8 phosphorylation. Both fluvoxamine and sertraline also inhibited BMP-4-elicited phosphorylation of SMAD1/5/8. These results strongly suggest that duloxetine suppresses BMP-4-stimulated OPG release via inhibition of the Smad1/5/8 signaling pathway in osteoblasts.

HSP90 inhibitors strengthen extracellular ATP-stimulated synthesis of interleukin-6 in osteoblasts: Amplification of p38 MAP kinase.

Heat shock protein 90 (HSP90) is expressed ubiquitously in a variety of cell types including osteoblasts. HSP90 acts as a key driver of proteostasis under pathophysiological conditions. Here, we investigated the involvement of HSP90 in extracellular ATP-stimulated interleukin (IL)-6 synthesis and HSP90 downstream signalling in osteoblast-like MC3T3-E1 cells. In osteoblasts, extracellular ATP stimulates the synthesis of IL-6, a bone-remodelling agent. Geldanamycin, 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and onalespib, three different HSP90 inhibitors, amplified the ATP-stimulated IL-6 release. Geldanamycin increased IL-6 mRNA expression elicited by ATP. ATP enhanced the triiodothyronine-induced osteocalcin release, but HSP90 inhibitors suppressed the release. Extracellular ATP induced the phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK), p38 MAPK, c-Jun N-terminal kinase (JNK), p70 S6 kinase, Akt, and myosin phosphatase-targeting subunit (MYPT), a Rho-kinase substrate. SB203580, an inhibitor of p38 MAPK, suppressed ATP-stimulated IL-6 release. Inhibitors of MEK1/2 (PD98059), JNK (SP600125), upstream kinase of p70 S6 kinase (rapamycin) and Akt (deguelin), all increased IL-6 release. Y27632, a Rho-kinase inhibitor, failed to affect the IL-6 release stimulated by ATP. Geldanamycin and 17-AAG both amplified ATP-induced p38 MAPK phosphorylation, although geldanamycin inhibited the phosphorylation of Akt induced by ATP. In addition, SB203580 significantly reduced the amplification by geldanamycin of the IL-6 release. Taken together, our results strongly suggest that HSP90 inhibitors up-regulate extracellular ATP-stimulated IL-6 synthesis via amplification of p38 MAPK activation in osteoblasts. SIGNIFICANCE OF THE STUDY: Heat shock protein 90 (HSP90) acts as a key driver of proteostasis under pathophysiological conditions in a variety of cell types. We have previously shown that HSP90 is expressed at high levels in osteoblast-like MC3T3-E1 cells, even in their quiescent state, consistent with HSP90 performing an important physiological function in osteoblasts. In the present study, we investigated whether HSP90 is implicated in extracellular ATP-induced interleukin (IL)-6 synthesis in osteoblast-like MC3T3-E1 cells. Our results strongly suggest that HSP90 inhibitors up-regulate extracellular ATP-stimulated IL-6 synthesis via amplification of p38 mitogen-activated protein kinase activation in osteoblasts.

A Comparison of the Required Bronchial Cuff Volume Obtained by 2 Cuff Inflation Methods, Capnogram Waveform-Guided Versus Pressure-Guided: A Prospective Randomized Controlled Study.

BACKGROUND: Double-lumen endobronchial tubes (DLTs) are used for one-lung ventilation (OLV) during thoracic surgery. Overinflation into the bronchial cuff causes damage to the tracheobronchial mucosa, whereas underinflation leads to an incomplete collapse of the nonventilated lung or incomplete ventilation of the ventilated lung. However, how to determine the appropriate bronchial cuff volume and pressure during OLV is unclear. The objective of this study is to compare the required bronchial cuff volume for lung separation obtained by 2 different cuff inflation methods under closed- and open-chest conditions. METHODS: A total of 64 patients scheduled to undergo elective thoracic surgery requiring OLV were recruited. Left DLTs were used for both right- and left-sided surgery. The patients were randomly assigned to 1 of 2 inflation-type groups to estimate the bronchial cuff volume. In the capnogram waveform-guided bronchial cuff inflation group (capno group, n = 27), the bronchial cuff was inflated until a capnometer sampling gas containing CO2 from the nonventilated lung displayed a flat line. The corresponding bronchial cuff volume and pressure were then recorded. In the pressure-guided bronchial cuff inflation group (pressure group, n = 29), the bronchial cuff was inflated by a cuff inflator to a pressure of 20 cm H2O. Lung separation was confirmed when a flat line of a capnometer was observed after gas sampling from the nonventilated lung. RESULTS: Under closed-chest conditions, the bronchial cuff sealing volume for the capno group was significantly lower than that for the pressure group (mean [standard deviation {SD}], 1.00 [0.65] mL vs 1.44 [0.59] mL, mean difference, -0.44; 97.5% confidence interval [CI], -0.78 to -0.11; P = .010). Under open-chest conditions, the bronchial cuff sealing volume for the capno group was also significantly lower than that for the pressure group (mean [SD], 0.65 [0.66] mL vs 1.22 [0.45] mL, mean difference, -0.58; 97.5% CI, -0.88 to -0.27; P < .001). CONCLUSIONS: The lowest cuff volume providing an air-tight bronchial seal was obtained by the capnogram waveform-guided bronchial cuff inflation method. Since the cuff volume required to achieve an air-tight seal decreases after opening the chest, readjustment of the bronchial cuff volume to prevent bronchial cuff damage to the tracheobronchial mucosa after opening the chest may be advisable.

Synergistic effect of collagen and CXCL12 in the low doses on human platelet activation.

CXCL12, also known as stromal cell-derived factor-1, is a chemokine classified into CXC families, which exerts its function by binding to specific receptors called CXCR4 and CXCR7. Human platelets express CXCR4 and CXCR7 on the plasma membrane. It has been reported that CXCL12 potentiates to induce platelet aggregation in cooperation with agonists including collagen. However, the precise roles and mechanisms of CXCL12 in human platelet activation are not fully elucidated. In the present study, we investigated the effect of simultaneous stimulation with low doses of collagen and CXCL12 on the activation of human platelets. The simultaneous stimulation with collagen and CXCL12 induced the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble CD40 ligand (sCD40L) from human platelets in addition to their aggregation, despite the fact that the simultaneous stimulation with thrombin receptor-activating peptide (TRAP) or adenosine diphosphate (ADP), and CXCL12 had little effects on the platelet aggregation. The agonist of Glycoprotein (GP) Ⅵ convulxin and CXCL12 also induced platelet aggregation synergistically. The monoclonal antibody against CXCR4 but not CXCR7 suppressed the platelet aggregation induced by simultaneous stimulation with collagen and CXCL12. The phosphorylation of p38 mitogen-activated protein kinase (MAPK), but not p44/p42 MAPK, was induced by the simultaneous stimulation. In addition, the simultaneous stimulation with collagen and CXCL12 induced the phosphorylation of HSP27 and the subsequent release of phosphorylated-HSP27 from human platelets. SB203580, a specific inhibitor of p38 MAPK, attenuated the platelet aggregation, the phosphorylation of p38 MAPK and HSP27, the PDGF-AB secretion, the sCD40L release and the phosphorylated-HSP27 release induced by the simultaneous stimulation with collagen and CXCL12. These results strongly suggest that collagen and CXCL12 in low doses synergistically act to induce PDGF-AB secretion, sCD40L release and phosphorylated-HSP27 release from activated human platelets via p38 MAPK activation.

Cigarette Smoking Cessation Temporarily Enhances the Release of Phosphorylated-HSP27 from Human Platelets.

Objective Cigarette smoking is a risk factor for arteriopathy, including acute coronary syndrome, stroke and peripheral vascular disease. Thus, cessation is strongly recommended in order to reduce these risks. We recently demonstrated that smoking cessation causes temporary hyper-aggregability of human platelets. We previously showed that heat shock protein 27 (HSP27) is released from human platelets stimulated by collagen, accompanied by its phosphorylation. Accumulating evidence indicates potent roles of extracellular HSP27 as a modulator of inflammation. In the present study, using the stored samples obtained in the previous study, we investigated the effect of cigarette smoking cessation on the release of phosphorylated-HSP27 from collagen-activated human platelets (n=15 patients). Methods We enrolled patients who visited smoking cessation outpatient services between January 2012 and November 2014. Platelet-rich plasma, chronologically obtained before and after the cessation, was stimulated by collagen using a PA-200 aggregometer in the previous study. The levels of phosphorylated-HSP27 released from platelets were determined by an enzyme-linked immunosorbent assay. The phosphorylation of HSP27 in platelets was evaluated by a Western blot analysis. Results Cessation of cigarette smoking significantly upregulated the levels of collagen-stimulated release of phosphorylated-HSP27 at four and eight weeks after quitting smoking compared to before cessation. However, there was no significant difference between the levels before cessation and those at 12 weeks after cessation. The levels of phosphorylated-HSP27 stimulated by collagen in the platelets at four weeks after smoking cessation were remarkably enhanced compared to before cessation. Conclusion Cigarette smoking cessation temporarily enhances the collagen-stimulated release of phosphorylated-HSP27 from human platelets in the short term.

Olive polyphenol reduces the collagen-elicited release of phosphorylated HSP27 from human platelets.

Hydroxytyrosol (HT) and oleuropein (OLE) are natural polyphenols found in extra virgin olive oil. Accumulating evidence indicates that ingestion of olive oil contributes to reduce the risk of cardiovascular diseases and stroke. It has been reported that HT and OLE inhibit human platelet aggregation. We have shown that collagen induces the phosphorylation of heat shock protein 27 (HSP27) in human platelets, resulting in the release of HSP27, an extracellular pro-inflammatory agent. In this study, we investigated the effects of HT and OLE on the collagen-stimulated human platelet activation. The PDGF-AB secretion and the soluble CD40 ligand (sCD40L) release by collagen were reduced by HT or OLE. HT and OLE significantly suppressed the phosphorylation of HSP27 and the release of phosphorylated-HSP27. These findings suggest that olive polyphenol reduces the collagen-stimulated phosphorylation of HSP27 in human platelets and the release. Our results may provide a novel anti- inflammatory effect of olive polyphenol.

Smoking cessation affects human platelet activation induced by collagen.

It is firmly established that smoking is a risk factor of cardiovascular disease, stroke and peripheral vascular disease. Although smoking alters the hemostatic process, the influence of smoking on human platelet activation remains controversial. For patients undergoing surgery, cessation of smoking prior to the procedure is recommended as it increases the risk of postoperative morbidity or mortality. The presented study investigated the effects of smoking cessation on human platelet activation induced via collagen (n=19 patients). Blood samples were taken on four occasions: Before smoking cessation, and at 4, 8 and 12 weeks after smoking cessation. Platelet aggregation using citrated platelet-rich plasma (PRP) was monitored using a PA-200 aggregometer, which determined the size of platelet aggregates using laser scattering methods. A low dose of collagen (1 µg/ml) accelerated platelet aggregation at 4 or 8 weeks after smoking cessation when compared with results before cessation. After 12 weeks, levels of platelet aggregation induced by collagen were almost equal to those recorded prior to smoking cessation. The secretion levels of collagen-induced platelet-derived growth factor (PDGF)-AB at 4 or 8 weeks after smoking cessation were significantly higher than those before smoking was stopped. Furthermore, smoking cessation markedly strengthened the collagen-induced phosphorylation of p38 mitogen-activated protein (MAP) kinase after 4 weeks. The results of the current study indicated that smoking cessation causes temporary short-term human platelet hyper-activation. The further suggest that the incidence of complications due to human platelet hyper-reactivity may be lowered by considering the period of smoking abstinence.

Limitation by Rho-kinase and Rac of transforming growth factor-ß-induced interleukin-6 release from astrocytes.

Transforming growth factor (TGF)-ß stimulates release of interleukin (IL)-6, which is recognized to function as both a pro- and anti- inflammatory cytokine in the central nervous system, from astrocytes. It is generally recognized that effects of TGF-ß are mediated through Smad-independent as well as Smad-dependent pathways. Small GTPases regulate a variety of cell functions. In the present study, we investigated whether or not Rho-kinase, a downstream effector of Rho, and Rac are implicated in TGF-ß-stimulated IL-6 release from astrocytes (C8D1A cells). Y-27632 or fasudil (Rho-kinase inhibitors) or NSC23766 (an inhibitor of Rac-guanine nucleotide exchange factor interaction) significantly enhanced TGF-ß-stimulated IL-6 release from these cells. TGF-ß-stimulated IL-6 release was markedly upregulated in RhoA- or Rac-knockdown C8D1A cells. We found that SIS3 (a specific inhibitor of TGF-ß-dependent Smad3 phosphorylation) or LY364947 (a TGF-ß type I receptor kinase inhibitor) significantly reduced the IL-6 release. However, TGF-ß-induced-Smad2 and Smad3 phosphorylation was not affected by Y-27632, fasudil or NSC23766. In conclusion, our results strongly suggest that Rho-kinase and Rac limit TGF-ß-induced IL-6 release from astrocytes, and the suppressive effects are exerted independently of the Smad pathway or at a point downstream of Smad2/3 complex.

Endothelium-dependent vasodilation in the cerebral arterioles of rats deteriorates during acute hyperglycemia and then is restored by reducing the glucose level.

PURPOSE: Acute hyperglycemia in patients with traumatic brain injury correlates with a poor neurological outcome. We investigated the endothelium function of rat cerebral arterioles during acute hyperglycemia and after reducing the glucose levels using insulin. We also examined whether or not oxidative stress was involved in the cerebral arteriole response to acute hyperglycemia. METHODS: In isoflurane-anesthetized, mechanically ventilated rats, we used closed cranial window preparation to measure the changes in the pial arteriolar diameter following the topical application of acetylcholine (ACh) or adenosine. We examined the pial arteriolar vasodilator response before hyperglycemia, during hyperglycemia, and after reducing the glucose level using insulin. After intravenous pretreatment with an NADPH oxidase inhibitor (apocynin or diphenylene iodonium), we reexamined the pial arteriolar vasodilator response following the topical application of ACh. RESULTS: Under control conditions, the topical application of ACh dose-dependently dilated the cerebral arterioles. The vasodilatory responses to topical ACh were impaired during hyperglycemia and improved after the administration of insulin. The vasodilatory responses to topical adenosine were not affected by the glucose levels. In the apocynin or diphenylene iodonium pretreatment group, the topical application of ACh dilated the cerebral arterioles during hyperglycemia. CONCLUSION: Acute hyperglycemia induces a dysfunction of the endothelium-dependent vasodilation of rat cerebral arterioles. The dysfunction can be reversed by improving the acute hyperglycemia and it can be prevented entirely by the administration of NADPH oxidase inhibitors. These results could suggest that controlling the glucose levels works protectivity to endothelium function of cerebral arterioles.

Effect of blood pressure elevation on cerebral oxygen desaturation in the beach chair position.

OBJECTIVE: The beach chair position (BCP) during shoulder arthroscopy is a known risk factor for cerebral ischemia. We retrospectively investigated whether maintaining the arterial blood pressure (ABP) prevented the decrease in the regional cerebral tissue oxygen saturation (rSO2). METHODS: We analyzed 20 consecutive patients who underwent elective shoulder surgery in the BCP under general anesthesia. The bilateral rSO2 was monitored continuously throughout the procedure with the help of near-infrared spectroscopy (INVOS 5100 Cerebral Oximeter, Somanetics Corporation, Troy, MI, USA). Anesthesiologists maintained patient blood pressure while they were in the BCP, which was measured using an ABP transducer placed at the level of the external auditory meatus. We compared rSO2 measured in the supine position and in the BCP. RESULTS: Measurement of cortex level mean ABP (mABP) values in the BCP were >50 mmHg and over 60% higher than those noted for the supine position in most patients. Although all bilateral rSO2 values in the BCP were significantly lower than those in the supine position, the reductions was <20%. Further, 35% (7 of 20) patients that were part of the study experienced cerebral desaturation events at any given point during the procedure. None of the patients experienced clinical postoperative neurological complications. CONCLUSIONS: Although cortex level mABP in the BCP was >50 mmHg, a decrease was recorded in the rSO2 values. This rSO2 decrease was proportional to the reduction in the cortex level mABP induced by a postural change to the BCP. Therefore, despite appropriate blood pressure management, shoulder surgery in the BCP might involve certain risks for patients with cerebrovascular diseases.

AICAR reduces the collagen-stimulated secretion of PDGF-AB and release of soluble CD40 ligand from human platelets: Suppression of HSP27 phosphorylation via p44/p42 MAP kinase.

We have previously reported that collagen-induced phosphorylation of heat shock protein (HSP) 27 via p44/p42 mitogen-activated protein (MAP) kinase in human platelets is sufficient to induce the secretion of platelet-derived growth factor (PDGF)-AB and the release of soluble cluster of differentiation 40 ligand (sCD40L). Adenosine monophosphate-activated protein kinase (AMPK), which is known to regulate energy homeostasis, has a crucial role as an energy sensor in various eukaryotic cells. The present study investigated the effects of 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranosyl 5'-monophosphate (AICAR), which is an activator of AMPK, on the collagen-induced activation of human platelets. It was demonstrated that AICAR dose-dependently reduced collagen-stimulated platelet aggregation up to 1.0 µM. Analysis of the size of platelet aggregates demonstrated that AICAR decreased the ratio of large aggregates (50-70 µm), whereas the ratio of small aggregates (9-25 µm) was increased by AICAR administration. AICAR markedly attenuated the phosphorylation levels of p44/p42 MAP kinase and HSP27, which are induced by collagen. Furthermore, AICAR significantly decreased the secretion of PDGF-AB and the collagen-induced release of sCD40L. These results indicated that AICAR-activated AMPK may reduce the secretion of PDGF-AB and the collagen-induced release of sCD40L by inhibiting HSP27 phosphorylation via p44/p42 MAP kinase in human platelets.