Cheng-Chin Kuo

TLR 2 Induces Vascular Smooth Muscle Cell Migration Through cAMP Response Element−Binding Protein−Mediated Interleukin-6 Production

Objective—Migration of vascular smooth muscle cells (VSMCs) from the media into intima contributes to the development of atherosclerosis. Gene deletion experiments implicate a role for toll-like receptor 2 (TLR2) in atherogenesis. However, the underlying mechanisms remain unclear. We postulate that TLR2 promotes VSMC migration by enhancing interleukin (IL)-6 production. Methods and Results—Migration assays revealed that TLR2 agonists promoted VSMC migration but not cell proliferation or viability. TLR2 deficiency or inhibition of TLR2 signaling with anti-TLR2 antibody suppressed TLR2 agonist–induced VSMC migration and IL-6 production, which was mediated via p38 mitogen-associated protein kinase and extracellular signal–regulated kinase 1/2 signaling pathways. Neutralizing anti−IL-6 antibodies impaired TLR2-mediated VSMC migration and formation of filamentous actin fiber and lamellipodia. Blockade of p38 mitogen-associated protein kinase or extracellular signal–regulated kinase 1/2 activation inhibited TLR2 agonist pam3CSK4-induced phosphorylation of cAMP response element−binding protein, which regulates IL-6 promoter activity through the cAMP response element site. Moreover, cAMP response element−binding protein small interfering RNA inhibited pam3CSK4-induced IL-6 production and VSMC migration. Additionally, Rac1 small interfering RNA inhibited pam3CSK4-induced VSMC migration but not IL-6 production. Conclusion—Our results suggest that on ligand binding, TLR2 activates p38 mitogen-associated protein kinase and extracellular signal–regulated kinase 1/2 signaling in VSMCs. These signaling pathways act in concert to activate cAMP response element−binding protein and subsequent IL-6 production, which in turn promotes VSMC migration via Rac1-mediated actin cytoskeletal reorganization.

Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan

Cyclooxygenase-2 (COX-2) expression is induced by mitogenic and proinflammatory factors. Its overexpression plays a causal role in inflammation and tumorigenesis. COX-2 expression is tightly regulated, but the mechanisms are largely unclear. Here we show the control of COX-2 expression by an endogenous tryptophan metabolite, 5-methoxytryptophan (5-MTP). By using comparative metabolomic analysis and enzyme-immunoassay, our results reveal that normal fibroblasts produce and release 5-MTP into the extracellular milieu whereas A549 and other cancer cells were defective in 5-MTP production. 5-MTP was synthesized from l-tryptophan via tryptophan hydroxylase-1 and hydroxyindole O-methyltransferase. 5-MTP blocked cancer cell COX-2 overexpression and suppressed A549 migration and invasion. Furthermore, i.p. infusion of 5-MTP reduced tumor growth and cancer metastasis in a murine xenograft tumor model. We conclude that 5-MTP synthesis represents a mechanism for endogenous control of COX-2 overexpression and is a valuable lead for new anti-cancer and anti-inflammatory drug development.

Bortezomib suppresses focal adhesion kinase expression via interrupting nuclear factor-kappa B.

AIMS Bortezomib is a potent proteasome inhibitor currently used to treat various malignancies with promising results. To explore the role of bortezomib in reducing cancer cell migration and inducing apoptosis, we evaluated the effects of bortezomib on the expression of focal adhesion kinase (FAK). MAIN METHODS Various types of cancer cells including lung cancer A549, H1299; a breast cancer MCF7; a hepatocellular carcinoma Huh7, and a tongue squamous cell carcinoma SCC-25 were treated with different concentrations of bortezomib or MG-132 as indicated for 24h. Protein and mRNA levels were determined by Western blotting and real-time PCR. Apoptosis was analyzed by caspase 3 cleavage and activity. FAK promoter and NFkappaB binding activities were measured by luciferase-reporter method. NFkappaB subunit p65 binding capacity was determined by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis. KEY FINDINGS Both bortezomib and another proteasome inhibitor, MG-132, significantly reduced FAK expression, suppressed cancer cell migration and increased cell apoptosis. Results of real-time PCR and promoter activity assay revealed that bortezomib decreased FAK expression through transcriptional inactivation. Results of FAK promoter activity and ChIP assays in A549 and H1299 cells indicated that bortezomib suppressed FAK activity through a p53-independent pathway. Furthermore, reduction of NFkappaB binding capacity demonstrated by EMSA and ChIP assay suggested that NFkappaB plays an important role in bortezomib suppressing FAK expression. SIGNIFICANCE These results suggested that FAK is downregulated by bortezomib through a proteasome-dependent NFkappaB inhibitory mechanism. Thus, FAK could be a potential molecular target of bortezomib for therapeutic strategy.

Endothelium-Derived 5-Methoxytryptophan Is a Circulating Anti-inflammatory Molecule that Blocks Systemic Inflammation

RATIONALE Systemic inflammation has emerged as a key pathophysiological process that induces multiorgan injury and causes serious human diseases. Endothelium is critical in maintaining cellular and inflammatory homeostasis, controlling systemic inflammation, and progression of inflammatory diseases. We postulated that endothelium produces and releases endogenous soluble factors to modulate inflammatory responses and protect against systemic inflammation. OBJECTIVE To identify endothelial cell-released soluble factors that protect against endothelial barrier dysfunction and systemic inflammation. METHODS AND RESULTS We found that conditioned medium of endothelial cells inhibited cyclooxgenase-2 and interleukin-6 expression in macrophages stimulated with lipopolysaccharide. Analysis of conditioned medium extracts by liquid chromatography-mass spectrometry showed the presence of 5-methoxytryptophan (5-MTP), but not other related tryptophan metabolites. Furthermore, endothelial cell-derived 5-MTP suppressed lipopolysaccharide-induced inflammatory responses and signaling in macrophages and endotoxemic lung tissues. Lipopolysaccharide suppressed 5-MTP level in endothelial cell-conditioned medium and reduced serum 5-MTP level in the murine sepsis model. Intraperitoneal injection of 5-MTP restored serum 5-MTP accompanied by the inhibition of lipopolysaccharide-induced endothelial leakage and suppression of lipopolysaccharide- or cecal ligation and puncture-mediated proinflammatory mediators overexpression. 5-MTP administration rescued lungs from lipopolysaccharide-induced damages and prevented sepsis-related mortality. Importantly, compared with healthy subjects, serum 5-MTP level in septic patients was decreased by 65%, indicating an important clinical relevance. CONCLUSIONS We conclude that 5-MTP belongs to a novel class of endothelium-derived protective molecules that defend against endothelial barrier dysfunction and excessive systemic inflammatory responses.

Heme oxygenase-1 deficiency exacerbates angiotensin II-induced aortic aneurysm in mice.

Abdominal aortic aneurysm (AAA) is a chronic but often fatal disease in elderly population. Heme oxygenase-1 (HO-1) is a stress response protein with antioxidative and anti-inflammatory properties. HO-1 has been shown to protect against atherogenesis and arterial intimal thickening. Emerging evidences suggest that AAA and arterial occlusive disease have distinct pathogenic mechanisms. Thus, in this study we investigated the role of HO-1 in angiotensin II-induced AAA formation in HO-1+/+apoE−/− and HO-1−/−apoE−/− mice. We found that complete loss of HO-1 increased AAA incidence and rupture rate, and drastically increased aneurysmal area and severity, accompanied with severe elastin degradation and medial degeneration. Interestingly, we often observed not only AAA but also thoracic aortic aneurysm in HO-1−/−apoE−/− mice. Furthermore, reactive oxygen species levels, vascular smooth muscle cell (VSMC) loss, macrophage infiltration, matrix metalloproteinase (MMP) activity were markedly enhanced in the aneurysmal aortic wall in HO-1−/−apoE−/− mice. In addition, HO-1−/−apoE−/− VSMCs were more susceptible to oxidant-induced cell death and macrophages from HO-1−/−apoE−/− mice had aggravated responses to angiotensin II with substantial increases in inflammatory cytokine productions and MMP9 activity. Taken together, our results demonstrate the essential roles of HO-1 in suppressing the pathogenesis of AAA. Targeting HO-1 might be a promising therapeutic strategy for AAA.

Quiescent and Proliferative Fibroblasts Exhibit Differential p300 HAT Activation through Control of 5-Methoxytryptophan Production

Quiescent fibroblasts possess unique genetic program and exhibit high metabolic activity distinct from proliferative fibroblasts. In response to inflammatory stimulation, quiescent fibroblasts are more active in expressing cyclooxygenase-2 and other proinflammatory genes than proliferative fibroblasts. The underlying transcriptional mechanism is unclear. Here we show that phorbol 12-myristate 13-acetate (PMA) and cytokines increased p300 histone acetyltransferase activity to a higher magnitude (> 2 fold) in quiescent fibroblasts than in proliferative fibroblasts. Binding of p300 to cyclooxygenase-2 promoter was reduced in proliferative fibroblasts. By ultrahigh-performance liquid chromatography coupled with a quadrupole time of flight mass spectrometer and enzyme-immunoassay, we found that production of 5-methoxytryptophan was 2–3 folds higher in proliferative fibroblasts than that in quiescent fibroblasts. Addition of 5-methoxytryptophan and its metabolic precursor, 5-hydroxytryptophan, to quiescent fibroblasts suppressed PMA-induced p300 histone acetyltransferase activity and cyclooxygenase-2 expression to the level of proliferative fibroblasts. Silencing of tryptophan hydroxylase-1 or hydroxyindole O-methyltransferase in proliferative fibroblasts with siRNA resulted in elevation of PMA-induced p300 histone acetyltransferase activity to the level of that in quiescent fibroblasts, which was rescued by addition of 5-hydroxytryptophan or 5-methoxytryptophan. Our findings indicate that robust inflammatory gene expression in quiescent fibroblasts vs. proliferative fibroblasts is attributed to uncontrolled p300 histone acetyltransferase activation due to deficiency of 5-methoxytryptophan production. 5-methoxytryptophan thus is a potential valuable lead compound for new anti-inflammatory drug development.

A Novel Protective Function of 5-Methoxytryptophan in Vascular Injury

5-Methoxytryptophan (5-MTP), a 5-methoxyindole metabolite of tryptophan metabolism, was recently shown to suppress inflammatory mediator-induced cancer cell proliferation and migration. However, the role of 5-MTP in vascular disease is unknown. In this study, we investigated whether 5-MTP protects against vascular remodeling following arterial injury. Measurements of serum 5-MTP levels in healthy subjects and patients with coronary artery disease (CAD) showed that serum 5-MTP concentrations were inversely correlated with CAD. To test the role of 5-MTP in occlusive vascular disease, we subjected mice to a carotid artery ligation model of neointima formation and treated mice with vehicle or 5-MTP. Compared with vehicle-treated mice, 5-MTP significantly reduced intimal thickening by 40% 4 weeks after ligation. BrdU incorporation assays revealed that 5-MTP significantly reduced VSMC proliferation both in vivo and in vitro. Furthermore, 5-MTP reduced endothelial loss and detachment, ICAM-1 and VCAM-1 expressions, and inflammatory cell infiltration in the ligated arterial wall, suggesting attenuation of endothelial dysfunction. Signaling pathway analysis indicated that 5-MTP mediated its effects predominantly via suppressing p38 MAPK signaling in endothelial and VSMCs. Our data demonstrate a novel vascular protective function of 5-MTP against arterial injury-induced intimal hyperplasia. 5-MTP might be a therapeutic target for preventing and/or treating vascular remodeling.

TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function

Migration of vascular smooth muscle cells (VSMCs) into the intima is considered to be a vital event in the pathophysiology of atherosclerosis. Despite substantial evidence supporting the pathogenic role of Toll-like receptor 4 (TLR4) in the progression of atherogenesis, its function in the regulation of VSMC migration remains unclear. The goal of the present study was to elucidate the mechanism by which TLR4 regulates VSMC migration. Inhibitor experiments revealed that TLR4-induced IL-6 secretion and VSMC migration were mediated via the concerted actions of MyD88 and TRIF on the activation of p38 MAPK and ERK1/2 signaling. Neutralizing anti-IL-6 antibodies abrogated TLR4-driven VSMC migration and F-actin polymerization. Blockade of p38 MAPK or ERK1/2 signaling cascade inhibited TLR4 agonist-mediated activation of cAMP response element binding protein (CREB). Moreover, siRNA-mediated suppression of CREB production repressed TLR4-induced IL-6 production and VSMC migration. Rac-1 inhibitor suppressed TLR4-driven VSMC migration but not IL-6 production. Importantly, the serum level of IL-6 and TLR4 endogenous ligand HMGB1 was significantly higher in patients with coronary artery diseases (CAD) than in healthy subjects. Serum HMGB1 level was positively correlated with serum IL-6 level in CAD patients. The expression of both HMGB1 and IL-6 was clearly detected in the atherosclerotic tissue of the CAD patients. Additionally, there was a positive association between p-CREB and HMGB1 in mouse atherosclerotic tissue. Based on our findings, we concluded that, upon ligand binding, TLR4 activates p38 MAPK and ERK1/2 signaling through MyD88 and TRIF in VSMCs. These signaling pathways subsequently coordinate an additive augmentation of CREB-driven IL-6 production, which in turn triggers Rac-1-mediated actin cytoskeleton to promote VSMC migration.

5-methoxytryptophan is a potential marker for post-myocardial infarction heart failure - a preliminary approach to clinical utility.

BACKGROUND Ventricular remodeling following myocardial infarction (MI) is closely associated with cyclooxygenase-2 (COX-2) expression. 5-methoxytryptophan (5-MTP) was reported to control COX-2 expression. OBJECTIVES To investigeate the association between 5-MTP and post-MI left ventricular remodeling. METHODS This prospective study enrolled 26 non-diabetic patients with first-time ST segment elevation myocardial infarction (STEMI), and 58 controls. Levels of 5-MTP, N-terminal of pro-brain natriuretic peptide (NT-proBNP), aminoterminal propeptide of type III procollagen, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1 were measured at day 1, day 3, 3months, 6months, and 1year post-MI. Echocardiography was performed during the acute stage (within 72h) and 3months, 6months, and 1year post-MI. RESULTS The STEMI patients had a significantly lower plasma 5-MTP level at day 1 which reached a nadir at 3months post-MI. The level of 5-MTP at day 3 post-MI was significantly correlated with the level of NT-proBNP 1year post-MI, suggesting that the level of plasma 5-MTP in the early phase after MI may predict subsequent cardiac stress and failure. Receiver operating characteristic curve analysis revealed that plasma 5-MTP had the best area under the curve value to predict plasma NT-proBNP 1year post-MI. Further analysis using net reclassification improvement and integrated discrimination improvement models confirmed that plasma 5-MTP at day 3 post-MI significantly improved the predictive power of each of the parameters. CONCLUSION In non-diabetic STEMI patients, plasma 5-MTP levels were associated with biomarkers of post-MI left ventricular remodeling and damage.

Rho-associated kinase inhibitors promote the cardiac differentiation of embryonic and induced pluripotent stem cells

BACKGROUND Rho-associated kinase (ROCK) plays an important role in maintaining embryonic stem (ES) cell pluripotency. To determine whether ROCK is involved in ES cell differentiation into cardiac and hematopoietic lineages, we evaluated the effect of ROCK inhibitors, Y-27632 and fasudil on murine ES and induced pluripotent stem (iPS) cell differentiation. METHODS Gene expression levels were determined by real-time PCR, Western blot analysis and immunofluorescent confocal microscopy. Cell transplantation of induced differentiated cells were assessed in vivo in a mouse model (three groups, n=8/group) of acute myocardial infarction (MI). The cell engraftment was examined by immunohistochemical staining and the outcome was analyzed by echocardiography. RESULTS Cells were cultured in hematopoietic differentiation medium in the presence or absence of ROCK inhibitor and colony formation as well as markers of ES, hematopoietic stem cells (HSC) and cells of cardiac lineages were analyzed. ROCK inhibition resulted in a drastic change in colony morphology accompanied by loss of hematopoietic markers (GATA-1, CD41 and β-Major) and expressed markers of cardiac lineages (GATA-4, Isl-1, Tbx-5, Tbx-20, MLC-2a, MLC-2v, α-MHC, cTnI and cTnT) in murine ES and iPS cells. Fasudil-induced cardiac progenitor (Mesp-1 expressing) cells were infused into a murine MI model. They engrafted into the peri-infarct and infarct regions and preserved left ventricular function. CONCLUSIONS These findings provide new insights into the signaling required for ES cell differentiation into hematopoietic as well as cardiac lineages and suggest that ROCK inhibitors are useful in directing iPS cell differentiation into cardiac progenitor cells for cell therapy of cardiovascular diseases.