Chien Sung Tsai

Force-specific activation of Smad1/5 regulates vascular endothelial cell cycle progression in response to disturbed flow.

Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress, but the mechanism of force-specific activation of their signaling to modulate cellular function remains unclear. We have demonstrated that bone morphogenetic protein receptor (BMPR)-specific Smad1/5 can be force-specifically activated by oscillatory shear stress (OSS) in ECs to cause cell cycle progression. Smad1/5 is highly activated in ECs of atherosclerotic lesions in diseased human coronary arteries from patients with end-stage heart failure undergoing heart transplantation and from apolipoprotein E-deficient mice. Application of OSS (0.5 ± 4 dyn/cm2) causes the sustained activation of Smad1/5 in ECs through activations of mammalian target of rapamycin and p70S6 kinase, leading to up-regulation of cyclin A and down-regulations of p21CIP1 and p27KIP1 and, hence, EC cycle progression. En face examination of rat aortas reveals high levels of phospho-Smad1/5 in ECs of the straight segment of thoracic aorta and the inner, but not the outer, curvature of aortic arch. Immunohistochemical and en face examinations of the experimentally stenosed abdominal aorta in rats show high levels of phospho-Smad1/5 in ECs at poststenotic sites, where OSS occurs. These OSS activations of EC Smad1/5 in vitro and in vivo are not inhibited by the BMP-specific antagonist Noggin and, hence, are independent of BMP ligand. Transfecting ECs with Smad1/5-specific small interfering RNAs inhibits the OSS-induced EC cycle progression. Our findings demonstrate the force-specificity of the activation of Smad1/5 and its contribution to cell cycle progression in ECs induced by disturbed flow.

Salvianolic acid B inhibits low-density lipoprotein oxidation and neointimal hyperplasia in endothelium-denuded hypercholesterolaemic rabbits

BACKGROUNDnAtherosclerosis and restenosis are inflammatory responses involving free radicals and lipid peroxidation and may be prevented/cured by antioxidant-mediated lipid peroxidation inhibition. Salvianolic acid (Sal B), a water-soluble antioxidant obtained from a Chinese medicinal herb, is believed to have multiple preventive and therapeutic effects against human vascular diseases. In this study the in vitro and in vivo inhibitory effects of Sal B on oxidative stress were determined.nnnRESULTSnIn human aortic endothelial cells (HAECs), Sal B reduced oxidative stress, inhibited low-density lipoprotein (LDL) oxidation and reduced oxidised LDL-induced cytotoxicity. Sal B inhibited Cu(2+) -induced LDL oxidation in vitro (with a potency 16.3 times that of probucol) and attenuated HAEC-mediated LDL oxidation as well as reactive oxygen species (ROS) production. In cholesterol-fed New Zealand White rabbits (with probucol as positive control), Sal B intake reduced Cu(2+) -induced LDL oxidation, lipid deposition in the thoracic aorta, intimal thickness of the aortic arch and thoracic aorta and neointimal formation in the abdominal aorta.nnnCONCLUSIONnThe data obtained in this study suggest that Sal B protects HAECs from oxidative injury-mediated cell death via inhibition of ROS production. The antioxidant activity of Sal B may help explain its efficacy in the treatment of vascular diseases.

Cilostazol Attenuates MCP-1 and MMP-9 Expression In Vivo in LPS-Administrated Balloon-Injured Rabbit Aorta and In Vitro in LPS-Treated Monocytic THP-1 Cells

Monocyte chemoattractant protein‐1 (MCP‐1) and matrix metalloproteinase‐9 (MMP‐9) are involved in vascular inflammation. We tested the hypothesis, and explored the underlining mechanisms that cilostazol, a phosphodiesterase 3 inhibitor with antiplatelet and antithrombotic properties, inhibits lipopolysaccharide (LPS)‐induced MCP‐1 and MMP‐9 expression. In a rabbit aorta balloon‐injury model, administration of LPS increased macrophage infiltration and MCP‐1 and MMP‐9 expression; cilostazol supplementation prevented this phenomenon and reduced intimal hyperplasia. In contrast, the reverse zymography showed that cilostazol did not affect TIMP‐1 expression in serum. In monocytic THP‐1 cells, cilostazol and N6,O2′‐dibutyryl‐cAMP (dioctanoyl‐cAMP, a cAMP analog) dose‐dependently inhibited LPS‐induced MCP‐1 protein expression and MMP‐9 activation, but did not affect the tissue inhibitor of metalloproteinase‐1. Quantitative real‐time polymerase chain reaction (PCR) showed that cilostazol inhibited MCP‐1 and MMP‐9 mRNA expression. Cilostazol significantly inhibited LPS‐induced activation of p38, JNK, and nuclear factor‐κB, and the respective inhibitors of p38 and JNK greatly reduced the level of LPS‐induced MCP‐1 and MMP‐9, suggesting the involvement of the p38 and JNK pathways. In conclusion, cilostazol administered with LPS in vivo reduced neointimal hyperplasia and macrophage infiltration in the balloon‐injured rabbit aorta; in vitro, cilostazol inhibits LPS‐induced MCP‐1 and MMP‐9 expression. These data suggest that cilostazol may play an important role in preventing endotoxin‐ and injured‐mediated vascular inflammation. J. Cell. Biochem. 103: 54–66, 2008.

Expression of thrombomodulin on monocytes is associated with early outcomes in patients with coronary artery bypass graft surgery.

Thrombomodulin (TM) mediates blood coagulation and inflammation and is expressed constitutively on resting monocytes. This expression might be a key regulator of monocyte-related inflammation. Conventional cardiopulmonary bypass (CPB), beating-heart CPB, and off-pump techniques have been used widely in cardiac surgery. Although beating-heart CPB and off-pump techniques have reduced postoperative inflammation significantly, the underlying mechanisms remain unclear. Whether CPB affects the expression of TM and changes the actual immune capacity of monocytes is also unknown. In this study, we analyzed TM expression on monocytes and in plasma among patients undergoing elective coronary artery bypass graft surgery. The days spent in an intensive care unit (ICU) and incidence of fever in the ICU were significantly lower in the beating-heart CPB and off-pump groups than in the conventional CPB group. Enzyme-linked immunosorbent assay showed a significant increase in TM at 30 min after the commencement of CPB and at the end of surgery in the conventional CPB group, whereas the level increased less markedly in the beating-heart CPB group. Flow cytometry showed that conventional CPB markedly reduced the expression of TM on monocytes. Based on monocyte chemotaxis analysis and an actin polymerization assay, we propose that TM expression on monocytes is associated with systemic inflammation. We conclude that the beating-heart CPB and off-pump techniques have a lower impact on patients than conventional CPB. The reduced incidence of fever and shorter ICU stay seem to be associated predominantly with the lower concentration of TM in plasma and with a higher expression of TM on monocytes.

Porphyromonas gingivalis GroEL induces osteoclastogenesis of periodontal ligament cells and enhances alveolar bone resorption in rats.

Porphyromonas gingivalis is a major periodontal pathogen that contains a variety of virulence factors. The antibody titer to P. gingivalis GroEL, a homologue of HSP60, is significantly higher in periodontitis patients than in healthy control subjects, suggesting that P. gingivalis GroEL is a potential stimulator of periodontal disease. However, the specific role of GroEL in periodontal disease remains unclear. Here, we investigated the effect of P. gingivalis GroEL on human periodontal ligament (PDL) cells in vitro, as well as its effect on alveolar bone resorption in rats in vivo. First, we found that stimulation of PDL cells with recombinant GroEL increased the secretion of the bone resorption-associated cytokines interleukin (IL)-6 and IL-8, potentially via NF-κB activation. Furthermore, GroEL could effectively stimulate PDL cell migration, possibly through activation of integrin α1 and α2 mRNA expression as well as cytoskeletal reorganization. Additionally, GroEL may be involved in osteoclastogenesis via receptor activator of nuclear factor κ-B ligand (RANKL) activation and alkaline phosphatase (ALP) mRNA inhibition in PDL cells. Finally, we inoculated GroEL into rat gingiva, and the results of microcomputed tomography (micro-CT) and histomorphometric assays indicated that the administration of GroEL significantly increased inflammation and bone loss. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to osteoclastogenesis of PDL cells and resulting in periodontal disease with alveolar bone resorption.

Ginkgo biloba extract suppresses endotoxin-mediated monocyte activation by inhibiting nitric oxide- and tristetraprolin-mediated toll-like receptor 4 expression

Monocytes expressing toll-like receptor 4 (TLR4) play a major role in regulating the innate immune response and are involved in systemic inflammation. Previous studies have shown that Ginkgo biloba extract (GBE) may act as a therapeutic agent for some cardiovascular and neurological disorders. The objective of this study was to determine whether GBE could modulate immunity in human cells. The monocytic cell line THP-1 was used. Enzyme-linked immunosorbent assay results showed that lipopolysaccharide (LPS) induces the expression of monocyte chemotactic protein-1 (MIP-1), tumor necrosis factor-α, stromal cell-derived factor-1, and MIP-1α, and this induction may be repressed by GBE treatment due to TLR4 blockade. The Griess reagent assay and western blot analysis showed that GBE-mediated inhibition of TLR4 expression was associated with the activation of mitogen-activated protein kinase and production of nitric oxide (NO). Actinomycin D chase experiments demonstrated that GBE decreased the TLR4 mRNA stability in cells. Confocal microscopy and real-time polymerase chain reaction showed that GBE induced the expression of intracellular tristetraprolin (TTP). Transfection with TTP siRNA reversed the effects of GBE in naïve or TLR4-overexpressing cells. Treatment with SNAP (an NO donor) may increase intracellular TTP expression in cells. Immunoprecipitation analysis showed that GBE mediates TTP activation and increases the interaction of TTP with the 3 untranslated region (UTR) of TLR4 mRNA by regulating NO production. Our findings indicate that GBE could decrease the sensitivity of monocytes to LPS. Utilizing TTP to control TLR4 expression may be a promising approach for controlling systemic inflammation, and GBE may have potential applications in the clinical treatment of immune diseases.

The role of calpain-myosin 9-Rab7b pathway in mediating the expression of toll-like receptor 4 in platelets: A novel mechanism involved in α-granules trafficking

Toll-like receptors (TLRs) plays a critical role in innate immunity. In 2004, Aslam R. and Shiraki R. first determined that murine and human platelets express functional TLRs. Additionally, Andonegui G. demonstrated that platelets express TLR4, which contributes to thrombocytopenia. However, the underlying mechanisms of TLR4 expression by platelets have been rarely explored until now. The aim of this study was to identify the mechanism of TLR4 expression underlying thrombin treatment. The human washed platelets were used in this study. According to flowcytometry and western blot analysis, the surface levels of TLR4 were significantly enhanced in thrombin-activated human platelets and decreased by TMB-8, calpeptin, and U73122, but not Y27632 (a Rho-associated protein kinase ROCK inhibitor) indicating that thrombin-mediated TLR4 expression was modulated by PAR/PLC pathway, calcium and calpain. Co-immunoprecipitation (co-IP) assay demonstrated that the interaction between TLR4 and myosin-9 (a substrate of calpain) was regulated by calpain; cleavage of myosin-9 enhanced TLR4 expression in thrombin treated platelets. Transmission electron microscope data indicated that human platelets used α-granules to control TLR4 expression; the co-IP experiment suggested that myosin-9 did not coordinate with Rab7b to negatively regulate TLR4 trafficking in thrombin treated platelets. In summary, phospholipase Cγ-calpain-myosin 9-Rab7b axis was responsible for the mechanism underlying the regulation of TLR4 containing α-granules trafficking in thrombin-stimulated platelets, which was involved in coagulation.

On-pump beating-heart coronary artery bypass provides efficacious short- and long-term outcomes in hemodialysis patients

BACKGROUNDnOn-pump beating-heart coronary artery bypass grafting surgery (CABG) is beneficial due to the elimination of cardioplegic arrest. However, there are few reports regarding its efficacy in chronic hemodialysis patients. This study investigated the potential benefits of on-pump beating-heart CABG in chronic hemodialysis patients.nnnMETHODSnFrom January 2002 to January 2010, 186 patients with chronic hemodialysis underwent CABG in our institution. In total, 82 patients underwent conventional CABG with cardioplegic arrest, 56 underwent off-pump CABG and 48 underwent on-pump beating-heart CABG. The early results and long-term outcomes were compared among these three groups.nnnRESULTSnOn-pump beating-heart CABG significantly reduced the duration of cardiopulmonary bypass (CPB) compared with conventional CABG. The post-operative pericardial drainage amount (P < 0.01), length of hospital stay (P < 0.001) and length of post-operative intensive care unit stay (P < 0.001) were significantly lower in the on-pump beating-heart and off-pump CABG groups than in the conventional CABG group. No significant difference was found regarding 30-day mortality and morbidity rates including stroke, pneumonia, arrhythmia, intestinal complication and low cardiac output syndrome. There were no statistical differences in the freedom from cardiac events (P = 0.323), but on-pump beating-heart CABG provided better long-term survival than conventional CABG (P = 0.009).nnnCONCLUSIONSnOn-pump beating-heart CABG is a safe procedure that provides optimal operative exposure in chronic hemodialysis patients. The use of CPB and the elimination of cardioplegic arrest may be beneficial for the short- and long-term survival of chronic hemodialysis patients.

TNF-α inhibits toll-like receptor 4 expression on monocytic cells via tristetraprolin during cardiopulmonary bypass

Toll-like receptor 4 (TLR4) plays a major role in regulating the innate immune response, which is related to postoperative complications. Although inflammatory capacity and TNF-&agr; synthesis were altered on monocytes after cardiopulmonary bypass (CPB), whether the CPB and the CPB-induced TNF-&agr; affect TLR4 expression on monocytes have not yet clarified. We speculate that the changing of TNF-&agr; level during CPB may be involved in monocytic TLR4 expression. As previous report, our enzyme-linked immunosorbent assay showed that CPB elevated the plasma level of TNF-&agr;, whereas off-pump cardiac surgery does not. Flow cytometry reported decreased levels of monocytic TLR4 in patients undergoing CPB but not undergoing off-pump cardiac surgery. To elucidate whether the CPB-induced TNF-&agr; is related to TLR4 down-regulation, we used human monocytic THP-1 cells. Actinomycin D chase experiments demonstrated that TNF-&; decreased TLR4 expression and TLR4 mRNA stability on THP-1. Confocal microscopy and real-time polymerase chain reaction showed that TNF-&agr; induced intracellular tristetraprolin (TTP) expression. Transfection with TTP siRNA reversed the down-regulation of TLR4 in TNF-&agr;-stimulated THP-1. Treatment with ERK1/2 inhibitor and SAPK/JNK inhibitor decreased TNF-&agr;-induced TTP expression. Immunoprecipitation and Western blot analysis showed that the TNF-&agr;-mediated activation of TTP might be inhibited by p38 mitogen-activated protein kinase inhibitor and by PD98059. We also demonstrated in clinical samples with confocal microscopy and flow cytometry that CPB led to an elevation of TTP in monocytes. In conclusion, CPB and TNF-&agr; decrease TLR4 expression on monocytes; TTP expression and mitogen-activated protein kinase-signaling pathways play critical roles in CPB- and TNF-&agr;-mediated decreases of TLR4 on monocytes. Our results suggest that using TTP to control cytokine message decay rate may be a promising approach for controlling system inflammation and preventing post-CPB complications.

TNF-α-decreased thrombomodulin expression in monocytes is inhibited by propofol through regulation of tristetraprolin and human antigen R activities.

Thrombomodulin (TM) is expressed on the surface of monocytes and is a key regulator of actual immune capacity. Propofol is an anesthetic agent that exerts anti-inflammatory effects. The objective of this study was to determine whether propofol could modulate TM in TNF-&agr;-stimulated monocytes. THP-1 cells and male New Zealand rabbits were used in this study. The results showed that TNF-&agr; decreases the TM expression by mediating posttranscriptional modification, and this inhibition may be repressed by treatment with propofol. Immunofluorescence, immunoprecipitation, and pull-down assays were used to demonstrate that Rac1-dependent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Cdc42, and p38 mitogen-activated protein kinase activation, as well as tristetraprolin (TTP) expression, all contributed to the downregulation of TM in TNF-&agr;-treated cells. Propofol reversed the effects of TNF-&agr; on TM downregulation. Propofol mediated the expression of intracellular TTP and the distribution of cytosolic human antigen R (HuR) and changed their interactions with the 3&vprime;-untranslated region of TM mRNA regulating by Cdc42 and Rac1. In addition, the animal study showed that propofol regulates TM, TTP, and HuR expression on monocytes in TNF-&agr;-treated rabbits. In conclusion, the inhibition of TM expression in TNF-&agr;-treated monocytes was mediated by the activation of NADPH oxidase and the expression of TTP. Propofol may inhibit the downregulation of TM by mediating NADPH oxidase and TTP inactivation and through the activation of HuR in vitro and in vivo. Utilizing TTP and HuR to control TM expression may be a promising approach for controlling systemic inflammation, and propofol may possess potential implications for the clinical immunity of monocytes after anesthesia or surgery.