Participation of lipopolysaccharide in hyperplasic adipose expansion: Involvement of NADPH oxidase/ROS/p42/p44 MAPK-dependent Cyclooxygenase-2.

Obesity is a world-wide problem, especially the child obesity, with the complication of various metabolic diseases. Child obesity can be developed as early as the age between 2 and 6. The expansion of fat mass in child age includes both hyperplasia and hypertrophy of adipose tissue, suggesting the importance of proliferation and adipogenesis of preadipocytes. The changed composition of gut microbiota is associated with obesity, revealing the roles of lipopolysaccharide (LPS) on manipulating adipose tissue development. Studies suggest that LPS enters the circulation and acts as a pro-inflammatory regulator to facilitate pathologies. Nevertheless, the underlying mechanisms behind LPS-modulated obesity are yet clearly elucidated. This study showed that LPS enhanced the expression of cyclooxygenase-2 (COX-2), an inflammatory regulator of obesity, in preadipocytes. Pretreating preadipocytes with the scavenger of reactive oxygen species (ROS) or the inhibitors of NADPH oxidase or p42/p44 MAPK markedly decreased LPS-stimulated gene expression of COX-2 together with the phosphorylation of p47phox and p42/p44 MAPK, separately. LPS activated p42/p44 MAPK via NADPH oxidase-dependent ROS accumulation in preadipocytes. Reduction of intracellular ROS or attenuation of p42/p44 MAPK activation both reduced LPS-mediated COX-2 expression and preadipocyte proliferation. Moreover, LPS-induced preadipocyte proliferation and adipogenesis were abolished by the inhibition of COX-2 or PEG2 receptors. Taken together, our results suggested that LPS enhanced the proliferation and adipogenesis of preadipocytes via NADPH oxidase/ROS/p42/p44 MAPK-dependent COX-2 expression.

Targeting MAPK/NF-κB Pathways in Anti-Inflammatory Potential of Rutaecarpine: Impact on Src/FAK-Mediated Macrophage Migration.

Studies have discovered that different extracts of Evodia rutaecarpa and its phytochemicals show a variety of biological activities associated with inflammation. Although rutaecarpine, an alkaloid isolated from the unripe fruit of E. rutaecarpa, has been exposed to have anti-inflammatory properties, the mechanism of action has not been well studied. Thus, this study investigated the molecular mechanisms of rutaecarpine (RUT) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. RUT reserved the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-α), and interleukin (IL)-1ß in the LPS-induced macrophages. RUT showed an inhibitory effect on the mitogen-activated protein kinases (MAPKs), and it also inhibited nuclear transcription factor kappa-B (NF-κB) by hindering IκBα and NF-κB p65 phosphorylation and p65 nuclear translocation. The phospho-PI3K and Akt was concentration-dependently suppressed by RUT. However, RUT not only suggestively reduced the migratory ability of macrophages and their numbers induced by LPS but also inhibited the phospho-Src, and FAK. Taken together, these results indicate that RUT participates a vital role in the inhibition of LPS-induced inflammatory processes in RAW 264.7 macrophages and that the mechanisms involve PI3K/Akt and MAPK-mediated downregulation of NF-κB signaling pathways. Notably, reducing the migration and number of cells induced by LPS via inhibiting of Src/FAK pathway was also included to the anti-inflammatory mechanism of RUT. Therefore, RUT may have potential benefits as a therapeutic agent against chronic inflammatory diseases.

Rutaecarpine, an Alkaloid from Evodia rutaecarpa, Can Prevent Platelet Activation in Humans and Reduce Microvascular Thrombosis in Mice: Crucial Role of the PI3K/Akt/GSK3ß Signal Axis through a Cyclic Nucleotides/VASP-Independent Mechanism.

The role of activated platelets in acute and chronic cardiovascular diseases (CVDs) is well established. Therefore, antiplatelet drugs significantly reduce the risk of severe CVDs. Evodia rutaecarpa (Wu-Chu-Yu) is a well-known Chinese medicine, and rutaecarpine (Rut) is a main bioactive component with substantial beneficial properties including vasodilation. To address a research gap, we investigated the inhibitory mechanisms of Rut in washed human platelets and experimental mice. At low concentrations (1-5 µM), Rut strongly inhibited collagen-induced platelet aggregation, whereas it exerted only a slight or no effect on platelets stimulated with other agonists (e.g., thrombin). Rut markedly inhibited P-selectin expression; adenosine triphosphate release; [Ca2+]i mobilization; hydroxyl radical formation; and phospholipase C (PLC)γ2/protein kinase C (PKC), mitogen-activated protein kinase, and phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3ß (GSK3ß) phosphorylation stimulated by collagen. SQ22536 (an adenylate cyclase inhibitor) or ODQ (a guanylate cyclase inhibitor) did not reverse Rut-mediated antiplatelet aggregation. Rut was not directly responding to vasodilator-stimulated phosphoprotein phosphorylation. Rut significantly increased the occlusion time of fluorescence irradiated thrombotic platelet plug formation. The findings demonstrated that Rut exerts a strong effect against platelet activation through the PLCγ2/PKC and PI3K/Akt/GSK3ß pathways. Thus, Rut can be a potential therapeutic agent for thromboembolic disorders.

Wide-Angle Coronary Bifurcation Stenotic Lesions Treated With One Drug-Eluting Stent and Sequential Balloon Technique: A Better Strategy?

BACKGROUND: Clinically significant bifurcation lesions account for up to 20% of percutaneous coronary intervention (PCI) procedures, and present technical challenges due to the potential for occlusion of the side branch vessel. Percutaneous coronary intervention using final kissing ballooning (FKB) plays a major role in treating bifurcation lesions, but sequential dilatation (SD) is a less complicated PCI technique with a shallower learning curve. Previous studies have shown no benefit of FKB over SD, but wide-angle (>70°) bifurcation lesions may respond differently to narrow-angle bifurcation lesions. METHODS: Retrospective analysis was carried out to compare outcomes of FKB and SD stenting specifically for wide-angle bifurcation lesions: 7,582 PCIs performed at a single medical centre between 1 January 2009 and 31 May 2016 were screened. This yielded 112 SD and 102 FKB cases for comparative analysis, which was conducted with respect to major adverse cardiac event (MACE)-free survival and target lesion revascularisation (TLR)-free survival rates. RESULTS: The comparative analysis was achieved using the log-rank test and presented as Kaplan-Meier curves. All baseline characteristics were balanced among the groups. The mean procedure and fluoroscopy times were significantly longer for patients with FKB than SD. Patients with SD had slightly better MACE and TLR rates than those with FKB in both the drug-eluting stent (DES) and bare metal stent (BMS) groups. In addition, patients with DES had slightly lower MACE and TLR rates than those with BMS in both the FKD and SD groups. Major adverse cardiac event-free survival and TLR-free survival rates were also slightly higher in patients with DES than those with BMS in both the FKD and SD groups. However, these differences were not statistically significant. CONCLUSIONS: These results suggest that the most applicable procedure for PCI of wide-angulated bifurcation stenosis would be a combination of DES and SD.

Platelet MicroRNA 365-3p Expression Correlates with High On-treatment Platelet Reactivity in Coronary Artery Disease Patients.

PURPOSE: The expression level of platelet microRNAs (miRNAs) correlates with heart disease and may be altered by antiplatelet therapy. This study aims to assess whether certain miRNAs are associated with treatment response by platelets in patients who received percutaneous coronary intervention and antiplatelet therapy. The dynamic expression of certain miRNAs in patients receiving different antiplatelet regimens was also investigated. METHODS: Healthy subjects (N = 20) received no-stent or antiplatelet therapy (as control), and patients (N = 155) who underwent stent implant and received treatment regimens that included aspirin plus clopidogrel, ticagrelor, or cilostazol were included. The association of miR-96-5p, miR-495-3p, miR-107, miR-223-3p, miR-15a-5, miR-365-3p, and miR-339-3p levels with treatment response, SYNTAX score, and HTPR was determined. RESULTS: Of the different treatment regimens, ticagrelor was the most efficacious. At 24 h following drug administration, ROC analysis revealed that miR-339-3p and miR-365-3p had the highest sensitivity (74.3% and 90.0%, respectively) and specificity (71.4% and 93.3%) for detecting HTPR compared with the five other miRNAs. The SYNTAX score positively correlated with miR-223-3p and miR-365-3p levels at 24 h (P ≤ 0.006) and with miR-365-3p levels 7 days following drug administration (P = 0.014). The expression of all three miRNAs reached the highest levels in hyperresponsive (P2Y12 reaction unit < 85) followed by hyporesponsive (P2Y12 reaction unit ≥ 208) and then normoreactive. The normoreactive value was very close to that of controls. CONCLUSIONS: Our data suggest that miR-365-3p expression level correlates with the antiplatelet treatment response. CLINICAL TRIAL REGISTRATION: NCT02101437.

Lipopolysaccharide promoted proliferation and adipogenesis of preadipocytes through JAK/STAT and AMPK-regulated cPLA2 expression.

The proliferation and adipogenesis of preadipocytes played important roles in the development of adipose tissue and contributed much to the processes of obesity. On the other hand, lipopolysaccharide (LPS), also known as endotoxin, is a key outer membrane component of gram-negative bacteria in the gut microbiota, and has a dominant role in linking inflammation to high-fat diet-induced metabolic syndrome. Studies suggested the potential roles of LPS in hepatic steatosis and in obese mice models. However, the molecular mechanisms underlying LPS-regulated obesity remained largely unknown. Here we reported that LPS stimulated expression of cyosolic phospholipase A2 (cPLA2), one of inflammation regulators of obesity, in the preadipocytes. Pretreatment the inhibitors of JAK2, STAT3, STAT5 or AMPK significantly reduced LPS-increased mRNA and protein expression of cPLA2 together with phosphorylation of JAK2, STAT3, STAT5 and AMPK, separately. Similarly, transfection of siRNA against JAK2 or AMPK abolished expression of cPLA2 and phosphorylation of JAK2 or AMPK together with downregulated expression of JAK2 and AMPK protein. LPS enhanced activation of STAT3 and STAT5 via JAK2-dependent manner in the preadipocytes. Transfection of JAK2 or AMPK siRNA further proofed the independence of JAK2 and AMPK in LPS-treated preadipocytes. In addition, LPS-increased DNA synthesis, cell numbers and cell viability of preadipocytes were attenuated by AACOCF3, AG490, BML-275, cPLA2 siRNA, JAK2 siRNA or AMPK siRNA. Attenuation JAK2/STAT or AMPK-dependent cPLA2 expression reduced LPS-mediated adipogenesis of preadipocytes. Stimulation of arachidonic acid or AMPK activator, A-769662, increased cell numbers and cell viability and promoted differentiation of preadipocytes. Collectively, these results indicated that LPS increased preadipocytes proliferation and adipogenesis via JAK/STAT and AMPK-dependent cPLA2 expression. The mechanisms of LPS-stimulated cPLA2 expression may be a link between bacteria and obesity and provides the molecular basis for preventing metabolic syndrome or hyperplasic obesity.

Esculetin, a Coumarin Derivative, Prevents Thrombosis: Inhibitory Signaling on PLCγ2-PKC-AKT Activation in Human Platelets.

Esculetin, a bioactive 6,7-dihydroxy derivative of coumarin, possesses pharmacological activities against obesity, diabetes, renal failure, and cardiovascular disorders (CVDs). Platelet activation plays a major role in CVDs. Thus, disrupting platelet activation represents an attractive therapeutic target. We examined the effect of esculetin in human platelet activation and experimental mouse models. At 10-80 µM, esculetin inhibited collagen- and arachidonic acid-induced platelet aggregation in washed human platelets. However, it had no effects on other agonists such as thrombin and U46619. Esculetin inhibited adenosine triphosphate release, P-selectin expression, hydroxyl radical (OH·) formation, Akt activation, and phospholipase C (PLC)γ2/protein kinase C (PKC) phosphorylation, but did not diminish mitogen-activated protein kinase phosphorylation in collagen-activated human platelets. Platelet function analysis indicated that esculetin substantially prolonged the closure time of whole blood. In experimental mice, esculetin significantly increased the occlusion time in thrombotic platelet plug formation and reduced mortality associated with acute pulmonary thromboembolism. However, it did not prolong the bleeding time. This study demonstrates that esculetin inhibits human platelet activation via hindering the PLCγ2-PKC cascade, hydroxyl radical formation, Akt activation, and ultimately suppressing platelet activation. Therefore, esculetin may act as an essential therapeutic agent for preventing thromboembolic diseases.

High Levels of Serum Adipocyte Fatty Acid-binding Protein Predict Cardiovascular Events in Coronary Artery Disease Patients.

Background: Adipocyte fatty acid-binding protein (A-FABP) is a cardiometabolic predictor of cardiovascular (CV) disease in humans. We evaluated the association between serum A-FABP levels and future CV events in patients with coronary artery disease (CAD). Methods: A total of 106 CAD patients were enrolled in this study between January and December 2012 and were followed-up until June 30, 2017. The primary endpoint was the incidence of major adverse CV events. Results: During a median follow-up period of 53 months, 44 CV events occurred. Patients with CV events presented higher systolic blood pressure (p = 0.020), total serum cholesterol (p = 0.047), and serum A-FABP levels (p < 0.001) compared with patients without CV events. Kaplan-Meier analysis showed that the cumulative incidence of CV events in the high A-FABP group (median A-FABP concentration of >17.63 ng/mL) was higher than that in the low A-FABP group (log-rank p < 0.001). Multivariate Cox analysis showed that triglycerides (hazard ratio (HR): 1.008, 95% confidence interval (CI): 1.001-1.016, p = 0.026) and serum A-FABP levels (HR: 1.027, 95% CI: 1.009-1.047, p = 0.004) were independently associated with CV events. Conclusion: Serum A-FABP level is a biomarker for future CV events in patients with CAD. Further prospective studies are needed to confirm the mechanisms underlying this association.

Neuroprotective Effects of Platonin, a Therapeutic Immunomodulating Medicine, on Traumatic Brain Injury in Mice after Controlled Cortical Impact.

Traumatic brain injury (TBI) is one of the leading causes of mortality worldwide and leads to persistent cognitive, sensory, motor dysfunction, and emotional disorders. TBI-caused primary injury results in structural damage to brain tissues. Following the primary injury, secondary injuries which are accompanied by neuroinflammation, microglial activation, and additional cell death subsequently occur. Platonin, a cyanine photosensitizing dye, has been used to treat trauma, ulcers, and some types of acute inflammation. In the present study, the neuroprotective effects of platonin against TBI were explored in a controlled cortical impact (CCI) injury model in mice. Treatment with platonin (200 µg/kg) significantly reduced the neurological severity score, general locomotor activity, and anxiety-related behavior, and improved the rotarod performance of CCI-injured mice. In addition, platonin reduced lesion volumes, the expression of cleaved caspase-3, and microglial activation in TBI-insulted brains. Platonin also suppressed messenger (m)RNA levels of caspase-3, caspase-1, cyclooxygenase-2, tumor necrosis factor-α, interleukin-6, and interleukin-1ß. On the other hand, free radical production after TBI was obviously attenuated in platonin-treated mice. Treatment with platonin exhibited prominent neuroprotective properties against TBI in a CCI mouse model through its anti-inflammatory, anti-apoptotic, and anti-free radical capabilities. This evidence collectively indicates that platonin may be a potential therapeutic medicine for use with TBIs.

Structure-Antiplatelet Activity Relationships of Novel Ruthenium (II) Complexes: Investigation of Its Molecular Targets.

The regulation of platelet function by pharmacological agents that modulate platelet signaling has proven to be a positive approach to the prevention of thrombosis. Ruthenium complexes are fascinating for the development of new drugs, as they possess numerous chemical and biological properties. The present study aims to evaluate the structure-activity relationship (SAR) of newly synthesized ruthenium (II) complexes, TQ-1, TQ-2 and TQ-3 in agonists-induced washed human platelets. Silica gel column chromatography, aggregometry, immunoblotting, NMR, and X-ray analyses were performed in this study. Of the three tested compounds, TQ-3 showed a concentration (1-5 µM) dependent inhibitory effect on platelet aggregation induced by collagen (1 µg/mL) and thrombin (0.01 U/mL) in washed human platelets; however, TQ-1 and TQ-2 had no response even at 250 µM of collagen and thrombin-induced aggregation. TQ-3 was effective with inhibiting collagen-induced ATP release, calcium mobilization ([Ca2+]i) and P-selectin expression without cytotoxicity. Moreover, TQ-3 significantly abolished collagen-induced Lyn-Fyn-Syk, Akt-JNK and p38 mitogen-activated protein kinases (p38 MAPKs) phosphorylation. The compound TQ-3 containing an electron donating amino group with two phenyl groups of the quinoline core could be accounted for by its hydrophobicity and this nature might be the reason for the noted antiplatelet effects of TQ-3. The present results provide a molecular basis for the inhibition by TQ-3 in collagen-induced platelet aggregation, through the suppression of multiple machineries of the signaling pathway. These results may suggest that TQ-3 can be considered a potential agent for the treatment of vascular diseases.

In-Hospital Implementation of Evidence-Based Medications is Associated with Improved Survival in Diabetic Patients with Acute Coronary Syndrome - Data from TSOC ACS-DM Registry.

BACKGROUND: Patients with acute coronary syndrome (ACS) and diabetes mellitus (DM) receive less aggressive treatment and have worse outcomes in Taiwan. We sought to explore whether the current practices of prescribing guideline-directed medical therapy (GDMT) for ACS and clinical outcomes have improved over time. METHODS: A total of 1534 consecutive diabetic patients with ACS were enrolled between 2013 and 2015 from 27 hospitals in the nationwide registry initiated by the Taiwan Society of Cardiology (the TSOC ACS-DM Registry). Baseline and clinical demographics, treatment, and clinical outcomes were compared to those of 1000 ACS patients with DM recruited in the Taiwan ACS-full spectrum (ACS-FS) Registry, which was performed between 2008 and 2010. RESULTS: Compared to the DM patients in the Taiwan ACS-FS Registry, even though reperfusion therapy was carried out in significantly fewer patients, the primary percutaneous coronary intervention (PCI) rate for ST-segment elevation myocardial infarction (STEMI) and the prescription rates of GDMT for ACS including P2Y12 inhibitors, renin-angiotensin blockers, beta-blockers, and statins were significantly higher in those in the TSOC ACS-DM Registry. Moreover, significant reductions in 1-year mortality, recurrent nonfatal MI and stroke were observed compared to those of the DM patients in the Taiwan ACS-FS Registry. Multivariate analysis identified reperfusion therapy in combination with GDMT as a strong predictor of better 1-year outcomes [hazard ratio (95% confidence interval) = 0.54 (0.33-0.89)]. CONCLUSIONS: Marked improvements in performing primary PCI for STEMI and prescribing GDMT for ACS were observed over time in Taiwan. This was associated with improved 1-year event-free survival in the diabetic patients with ACS.

Change of scaling-induced proinflammatory cytokine on the clinical efficacy of periodontitis treatment.

Proinflammatory cytokines are key inflammatory mediators in periodontitis. This study aimed to investigate the relationship between proinflammatory cytokines in saliva and periodontal status. To investigate the usefulness of cytokines in the therapeutic approach for periodontal disease, the relationship between stimulated cytokine changes and the periodontitis treatment outcome was investigated in this study. Saliva was obtained from 22 patients diagnosed by dentists as having chronic periodontitis. The proinflammatory cytokine (interleukin-1α (IL-1α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and tumor necrosis factor ß (TNF-ß)) levels were determined using a commercially available kit. The IL-1ß and IL-6 levels increased, whereas the TNF-ß levels decreased with the severity of periodontitis (4 mm pocket percentage). Poststimulation IL-1α, IL-6, and IL-8 levels were higher in patients who had an improved treatment outcome. The differences of IL-6 levels (cut point: 0.05 µg/g) yielded a sensitivity and specificity of 90.0% and 81.82%, respectively, for predicting the periodontitis treatment outcome. Among the proinflammatory cytokines, stimulated IL-6 was an excellent marker for predicting the periodontitis treatment outcome.

Hinokitiol Exerts Anticancer Activity through Downregulation of MMPs 9/2 and Enhancement of Catalase and SOD Enzymes: In Vivo Augmentation of Lung Histoarchitecture.

Melanoma is extremely resistant to chemotherapy and the death rate is increasing hastily worldwide. Extracellular matrix promotes the migration and invasion of tumor cells through the production of matrix metalloproteinase (MMP)-2 and -9. Evidence has shown that natural dietary antioxidants are capable of inhibiting cancer cell growth. Our recent studies showed that hinokitiol, a natural bioactive compound, inhibited vascular smooth muscle cell proliferation and platelets aggregation. The present study is to investigate the anticancer efficacy of hinokitiol against B16-F10 melanoma cells via modulating tumor invasion factors MMPs, antioxidant enzymes in vitro. An in vivo mice model of histological investigation was performed to study the patterns of elastic and collagen fibers. Hinokitiol inhibited the expression and activity of MMPs-2 and -9 in B16-F10 melanoma cells, as measured by western blotting and gelatin zymography, respectively. An observed increase in protein expression of MMPs 2/9 in melanoma cells was significantly inhibited by hinokitiol. Notably, hinokitiol (1-5 µM) increased the activities of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) from the reduction in melanoma cells. Also, hinokitiol (2-10 µM) concentration dependently reduced in vitro Fenton reaction induced hydroxyl radical (OH·) formation. An in vivo study showed that hinokitiol treatment increased elastic fibers (EF), collagens dispersion, and improved alveolar alterations in the lungs of B16/F10 injected mice. Overall, our findings propose that hinokitiol may be a potent anticancer candidate through down regulation of MMPs 9/2, reduction of OH· production and enhancement of antioxidant enzymes SOD and CAT.

Andrographolide, a Novel NF-κB Inhibitor, Inhibits Vascular Smooth Muscle Cell Proliferation and Cerebral Endothelial Cell Inflammation.

BACKGROUND: Aberrant vascular smooth muscle cell (VSMC) proliferation and cerebral endothelial cell (CEC) dysfunction contribute significantly in the pathogenesis of cardiovascular diseases. Therefore, inhibition of these cellular events would be by candidate agents for treating these diseases. In the present study, the mechanism of anti-proliferative and anti-inflammatory effects of andrographolides, a novel nuclear factor-κB inhibitor, was investigated in VSMC and CEC cells. METHODS: VSMCs and CECs were isolated from rat artery and mouse brain, respectively, and cultured before experimentation. The effect of andro on platelet-derived growth factor-BB (PDGF-BB) induced VSMC cell proliferation was evaluated by cell number, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of extracellular signal regulated kinase 1/2 (ERK1/2), proliferating cell nuclear antigen (PCNA), and the effects on lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and, cyclooxygenase-2 (COX2) were detected by Western blotting. RESULTS: Andro significantly inhibited PDGF-BB (10 ng/ml) induced cell proliferation in a concentration (20-100 µM) dependent manner, which may be due to reducing the expression of ERK1/2, and by inhibiting the expression of PCNA. Andro also remarkably diminished LPS-induced iNOS and COX2 expression. CONCLUSIONS: The results of this study suggested that the effects of andro against VSMCs proliferation and CECs dysfunction may represent a promising approach for treatment of vascular diseases. KEY WORDS: Andrographolide; CECs; COX2/iNOS; ERK/PCNA; LPS; PDGF-BB; VSMCs.

Effects of treatment with clopidogrel with or without proton pump inhibitor omeprazole on the risk of ischemic stroke: a nationwide cohort study.

Most proton pump inhibitors (PPIs) inhibit the bioactivation of clopidogrel to its active metabolite. There is controversy concerning whether PPIs alter the effectiveness of clopidogrel in reducing the risk of ischemic stroke (IS). We therefore aimed to examine the risk of IS associated with concomitant use of clopidogrel and omeprazole, a PPI commonly used in clinical settings. We conducted a retrospective cohort study using the National Health Insurance Research Database of Taiwan dated from 2000 to 2013. The study cohorts comprised 407 patients diagnosed with acute coronary syndrome (ACS) and with concomitant use of clopidogrel and omeprazole (the exposed cohort), 814 ACS patients with single use of clopidogrel (the comparison cohort), and 230 ACS patients with concurrent use of clopidogrel and pantoprazole (the reference cohort). The primary outcome was incident IS. The hazard ratios (HRs) and 95% confidence intervals (CIs) derived from the time-dependent Cox regression model were used to assess the association between concomitant use of clopidogrel and omeprazole and the risk of IS. The incidence rate of IS was significantly higher in the exposed cohort (81.67 per 1000 person-years) than in the comparison cohort (57.45 per 1000 person-years), resulting in an adjusted HR of 1.39 (95% CI 1.03-1.74). By contrast, there was no significant difference in the risk of IS between the exposed and reference cohorts (adjusted HR 1.11; 95% CI 0.81-1.52). The present study revealed that patients taking both clopidogrel and omeprazole was associated with an increased risk of IS.

Platelet protease-activated receptor (PAR)4, but not PAR1, associated with neutral sphingomyelinase responsible for thrombin-stimulated ceramide-NF-κB signaling in human platelets.

Thrombin activates platelets mainly through protease-activated receptor (PAR)1 and PAR4. However, downstream platelet signaling between PAR1 and PAR4 is not yet well understood. This study investigated the relationship between nSMase/ceramide and the NF-κB signaling pathway in PARs-mediated human platelet activation. The LC-MS/MS, aggregometry, flow cytometry, immunoprecipitation, and mesenteric microvessels of mice were used in this study. Human platelets stimulated by thrombin, 3-OMS (a neutral sphingomyelinase [nSMase] inhibitor) and Bay11-7082 (an NF-κB inhibitor) significantly inhibited platelet activation such as P-selectin expression. Thrombin also activated IκB kinase (IKK)ß and IκBα phosphorylation; such phosphorylation was inhibited by 3-OMS and SB203580 (a p38 MAPK inhibitor). Moreover, 3-OMS abolished platelet aggregation, IKKß, and p38 MAPK phosphorylation stimulated by PAR4-AP (a PAR4 agonist) but not by PAR1-AP (a PAR1 agonist). Immunoprecipitation revealed that nSMase was directly associated with PAR4 but not PAR1 in resting platelets. In human platelets, C24:0-ceramide is the predominant form of ceramides in the LC/MS-MS assay; C24:0-ceramide increases after stimulation by thrombin or PAR4-AP, but not after stimulation by PAR1-AP. We also found that C2-ceramide (a cell-permeable ceramide analog) activated p38 MAPK and IKKß phosphorylation in platelets and markedly shortened the occlusion time of platelet plug formation in vivo. This study demonstrated that thrombin activated nSMase by binding to PAR4, but not to PAR1, to increase the C24:0-ceramide level, followed by the activation of p38 MAPK-NF-κB signaling. Our results showed a novel physiological significance of PAR4-nSMase/ceramide-p38 MAPK-NF-κB cascade in platelet activation.

A novel bioactivity of andrographolide from Andrographis paniculata on cerebral ischemia/reperfusion-induced brain injury through induction of cerebral endothelial cell apoptosis.

CONTEXT: Andrographolide, extracted from the leaves of Andrographis paniculata (Burm. f.) Nees (Acanthaceae), is a labdane diterpene lactone. It is widely reported to possess anti-inflammatory and antitumorigenic activities. Cerebral endothelial cells (CECs) play a crucial role in supporting the integrity and the function of the blood-brain barrier (BBB). However, no data are available concerning the effects of andrographolide in CECs. The aim of this study was to examine the detailed mechanisms of andrographolide on CECs. OBJECTIVE: This study investigated a novel bioactivity of andrographolide on cerebral ischemia/reperfusion-induced brain injury. MATERIALS AND METHODS: CECs were treated with andrographolide (20-100 µΜ) for the indicated times (0-24 h). After the reactions, cell survival rate and cytotoxicity were tested by the MTT assay and the lactate dehydrogenase (LDH) test, respectively. Western blotting was used to detect caspase-3 expression. In addition, analysis of cell cycle and apoptosis using PI staining and annexin V-FITC/PI labeling, respectively, was performed by flow cytometry. We also investigated the effect of andrographolide on middle cerebral artery occlusion (MCAO)/reperfusion-induced brain injury in a rat model. RESULTS: In the present study, we found that andrographolide (50-100 µΜ) markedly inhibited CEC growth according to an MTT assay and caused CEC damage according to a LDH test. Our data also revealed that andrographolide (50 µM) induced CEC apoptosis and caspase-3 activation as respectively detected by PI/annexin-V double staining and western blotting. Moreover, andrographolide arrested the CEC cell cycle at the G0/G1 phase by PI staining. In addition, andrographolide (5 mg/kg) caused deterioration of MCAO/reperfusion-induced brain injury in a rat model. CONCLUSIONS: These data suggest that andrographolide may disrupt BBB integrity, thereby deteriorating MCAO/reperfusion-induced brain injury, which are, in part, associated with its capacity to arrest cell-cycle and induce CEC apoptosis.

Garcinol acts as a novel integrin αIIbß3 inhibitor in human platelets.

AIMS: Platelet activation plays a central role in arterial thrombosis. Platelets are activated by adhesive proteins (i.e., collagen) or soluble agonists (i.e., thrombin), the respective receptor-specific signaling cause inside-out signaling, leading to the binding of fibrinogen to integrin αIIbß3. This binding triggers outside-in signaling, resulting in platelet aggregation. Garcinol, a polyisoprenylated benzophenone, is extracted from the fruit rind of Garcinia indica. Although garcinol exhibits considerable bioactivities, few studies have investigated the effect of garcinol on platelet activation. MAIN METHODS: Aggregometry, immunoblotting, flow cytometer, confocal microscopic analysis, fibrin clot retraction, animal studies such as fluorescein-induced platelet plug formation in mesenteric microvessels, acute pulmonary thromboembolism, and tail bleeding time were performed in this study. KEY FINDINGS: This study indicates that garcinol inhibited platelet aggregation stimulated by collagen, thrombin, arachidonic acid, and U46619. Garcinol reduced integrin αIIbß3 inside-out signaling, including ATP release; cytosolic Ca2+ mobilization; P-selectin expression; and Syk, PLCγ2/PKC, PI3K/Akt/GSK3ß, MAPKs, and NF-κB activation stimulated by collagen. Garcinol directly inhibited integrin αIIbß3 activation by interfering with FITC-PAC-1 and FITC-triflavin by collagen. Additionally, garcinol affected integrin αIIbß3-mediated outside-in signaling, such as decreasing platelet adhesion and the single-platelet spreading area; suppressing integrin ß3, Src, FAK, and Syk phosphorylation on immobilized fibrinogen; and inhibiting thrombin-stimulated fibrin clot retraction. Garcinol substantially reduced mortality caused by pulmonary thromboembolism and prolonged the occlusion time of thrombotic platelet plug formation without extending bleeding time in mice. SIGNIFICANCE: This study identified that garcinol, a novel antithrombotic agent, acts as a naturally occurring integrin αIIbß3 inhibitor.

Mechanisms of glabridin inhibition of integrin αIIbß3 inside-out signals and NF-κB activation in human platelets.

BACKGROUND: Platelets play a crucial role in cardiovascular diseases (CVDs) and are activated by endogenous agonists like collagen. These agonists initiate signal transduction through specific platelet receptors, resulting in platelet aggregation. Glabridin, a prenylated isoflavonoid found in licorice root, is known for its significance in metabolic abnormalities. Glabridin has been observed to inhibit collagen-induced platelet aggregation, but the precise mechanisms, specifically concerning NF-κB activation and integrin αIIbß3 signaling, are not yet fully understood. METHODS: In this study, platelet suspensions were prepared from healthy human blood donors, and the aggregation ability was observed using a lumi-aggregometer. The inhibitory mechanisms of glabridin in human platelets were evaluated through immunoblotting and confocal microscopy. The anti-thrombotic effects of glabridin were assessed by histological analysis of lung sections in acute pulmonary thromboembolism and by examining fluorescein-induced platelet plug formation in mesenteric microvessels in mice. RESULTS: Glabridin inhibited integrin αIIbß3 inside-out signals such as Lyn, Fyn, Syk, and integrin ß3 activation and NF-κB-mediated signal events, with similar potency to classical inhibitors BAY11-7082 and Ro106-9920. Glabridin and BAY11-7082 inhibited IKK, IκBα, and p65 phosphorylation and reversed IκBα degradation, while Ro106-9920 only reduced p65 phosphorylation and reversed IκBα degradation. BAY11-7082 reduced Lyn, Fyn, Syk, integrin ß3, phospholipase Cγ2 and protein kinase C activation. Glabridin reduced platelet plug formation in mesenteric microvessels and occluded vessels in thromboembolic lungs of mice. CONCLUSION: Our study revealed a new pathway for activating integrin αIIbß3 inside-out signals and NF-κB, which contributes to the antiplatelet aggregation effect of glabridin. Glabridin could be a valuable prophylactic or clinical treatment option for CVDs.

A novel role of sesamol in inhibiting NF-κB-mediated signaling in platelet activation.

BACKGROUND: Platelet activation is relevant to a variety of coronary heart diseases. Our previous studies revealed that sesamol possesses potent antiplatelet activity through increasing cyclic AMP formation. 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 sesamol in NF-κB-mediated platelet function. METHODS: Platelet aggregation, Fura 2-AM fluorescence, and immunoblotting analysis were used in this study. RESULTS: NF-κB signaling events, including IKKß phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 µg/ml) in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 µM). Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 µM)-mediated inhibitory effects of IKKß phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA) inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLC)γ2 phosphorylation, protein kinase C (PKC) activation, [Ca(2+)]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca(2+)]i mobilization. CONCLUSIONS: Sesamol activates cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca(2+)]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data demonstrating that inhibition of NF-κB interferes with platelet function may have a great impact when these types of drugs are considered for the treatment of cancer and various inflammatory diseases.