Cancer-Derived Succinate Promotes Macrophage Polarization and Cancer Metastasis via Succinate Receptor.

Macrophages form a major cell population in the tumor microenvironment. They can be activated and polarized into tumor-associated macrophages (TAM) by the tumor-derived soluble molecules to promote tumor progression and metastasis. Here, we used comparative metabolomics coupled with biochemical and animal studies to show that cancer cells release succinate into their microenvironment and activate succinate receptor (SUCNR1) signaling to polarize macrophages into TAM. Furthermore, the results from in vitro and in vivo studies revealed that succinate promotes not only cancer cell migration and invasion but also cancer metastasis. These effects are mediated by SUCNR1-triggered PI3K-hypoxia-inducible factor 1α (HIF-1α) axis. Compared with healthy subjects and tumor-free lung tissues, serum succinate levels and lung cancer SUCNR1 expression were elevated in lung cancer patients, suggesting an important clinical relevance. Collectively, our findings indicate that the secreted tumor-derived succinate belongs to a novel class of cancer progression factors, controlling TAM polarization and promoting tumorigenic signaling.

Extracellular Succinate: A Physiological Messenger and a Pathological Trigger.

When tissues are under physiological stresses, such as vigorous exercise and cold exposure, skeletal muscle cells secrete succinate into the extracellular space for adaptation and survival. By contrast, environmental toxins and injurious agents induce cellular secretion of succinate to damage tissues, trigger inflammation, and induce tissue fibrosis. Extracellular succinate induces cellular changes and tissue adaptation or damage by ligating cell surface succinate receptor-1 (SUCNR-1) and activating downstream signaling pathways and transcriptional programs. Since SUCNR-1 mediates not only pathological processes but also physiological functions, targeting it for drug development is hampered by incomplete knowledge about the characteristics of its physiological vs. pathological actions. This review summarizes the current status of extracellular succinate in health and disease and discusses the underlying mechanisms and therapeutic implications.

Cancer-derived extracellular succinate: a driver of cancer metastasis.

Succinate is a tricarboxylic acid (TCA) cycle intermediate normally confined to the mitochondrial matrix. It is a substrate of succinate dehydrogenase (SDH). Mutation of SDH subunits (SDHD and SDHB) in hereditary tumors such as paraganglioma or reduction of SDHB expression in cancer results in matrix succinate accumulation which is transported to cytoplasma and secreted into the extracellular milieu. Excessive cytosolic succinate is known to stabilize hypoxia inducible factor-1α (HIF-1α) by inhibiting prolyl hydroxylase. Recent reports indicate that cancer-secreted succinate enhances cancer cell migration and promotes cancer metastasis by activating succinate receptor-1 (SUCNR-1)-mediated signaling and transcription pathways. Cancer-derived extracellular succinate enhances cancer cell and macrophage migration through SUCNR-1 â†’ PI-3 K â†’ HIF-1α pathway. Extracellular succinate induces tumor angiogenesis through SUCNR-1-mediated ERK1/2 and STAT3 activation resulting in upregulation of vascular endothelial growth factor (VEGF) expression. Succinate increases SUCNR-1 expression in cancer cells which is considered as a target for developing new anti-metastasis drugs. Furthermore, serum succinate which is elevated in cancer patients may be a theranostic biomarker for selecting patients for SUCNR-1 antagonist therapy.

5-Methoxytryptophan attenuates postinfarct cardiac injury by controlling oxidative stress and immune activation.

AIMS: Myocardial infarction (MI) remains a major cause of heart failure. 5-Methoxytryptophan (5-MTP), a 5-methoxyindole metabolite of L-tryptophan, exerts anti-inflammatory and antifibrotic effects, but MI impairs the biosynthesis of cardiac 5-MTP. Therefore, we evaluated the effect of exogenous 5-MTP administration on rescuing post-MI cardiac injury. METHODS AND RESULTS: After a detailed pharmacokinetic analysis of 5-MTP, Sprague Dawley rats that had undergone left anterior descending coronary artery ligation received intraperitoneal administration of either 17 mg/kg 5-MTP or saline at 0.5 and 24 h after MI. Cardiac systolic function, infarction size, and fibrosis were evaluated using echocardiography, triphenyltetrazolium chloride staining, and Masson trichrome staining, respectively. Myocardial apoptosis was analyzed by staining for caspase-3 and cardiac troponin I. 5-MTP treatment decreased the infarct area and myocardial apoptosis; attenuated systolic dysfunction and left ventricular dilatation; and reduced cardiomyocyte hypertrophy, myocardial fibrosis, and infarct expansion. Crucially, 5-MTP alleviated oxidative stress by preserving mitochondrial antioxidant enzymes and downregulating reactive oxygen species-generating NADPH oxidase isoforms and endothelin-1. Consequently, 5-MTP-treated MI rat hearts exhibited lower levels of chemokines and cytokines, namely interleukin (IL)-1ß, IL-18, IL-6, C-C motif chemokine ligand (CCL)-2, and CCL5, accompanied by reduced infiltration of CD11b+ cells and CD4+ T cells. Notably, 5-MTP protected against H2O2-induced damage in HL-1 cardiomyocytes and human umbilical vein endothelial cells in vitro. CONCLUSION: 5-MTP prevented post-MI cardiac injury by promoting mitochondrial stabilization and controlling redox imbalance. This cytoprotective effect ameliorated macrophage and T-cell infiltration, thus reducing the infarct size, attenuating fibrosis, and restoring myocardial function.

Restoration of 5-methoxytryptophan protects against atherosclerotic chondrogenesis and calcification in ApoE-/- mice fed high fat diet.

BACKGROUND: Toll-like receptor-2 (TLR2) promotes vascular smooth muscle cell (VSMC) transdifferentiation to chondrocytes and calcification in a p38 MAPK-dependent manner. Vascular 5-methoxytryptophan (5-MTP) is a newly identified factor with anti-inflammatory actions. As 5-MTP targets p38 MAPK for its actions, we postulated that 5-MTP protects against vascular chondrogenesis and calcification. METHODS: High-fat diet-induced advanced atherosclerosis in mice were performed to investigate the effect of 5-MTP on atherosclerotic lesions and calcification. VSMCs were used to determine the role of 5-MTP in VSMC chondrogenic differentiation and calcification. Alizarin red S and Alcian blue staining were used to measure VSMC calcification and chondrogenic differentiation, respectively. RESULTS: 5-MTP was detected in aortic tissues of ApoE-/- mice fed control chow. It was reduced in ApoE-/- mice fed high-fat diet (HFD), but was restored in ApoE-/-Tlr2-/- mice, suggesting that HFD reduces vascular 5-MTP production via TLR2. Intraperitoneal injection of 5-MTP or its analog into ApoE-/- mice fed HFD reduced aortic atherosclerotic lesions and calcification which was accompanied by reduction of chondrogenesis and calcium deposition. Pam3CSK4 (Pam3), ligand of TLR2, induced SMC phenotypic switch to chondrocytes. Pretreatment with 5-MTP preserved SMC contractile proteins and blocked Pam3-induced chondrocyte differentiation and calcification. 5-MTP inhibited HFD-induced p38 MAPK activation in vivo and Pam3-induced p38 MAPK activation in SMCs. 5-MTP suppressed HFD-induced CREB activation in aortic tissues and Pam3-induced CREB and NF-κB activation in SMCs. CONCLUSIONS: These findings suggest that 5-MTP is a vascular arsenal against atherosclerosis and calcification by inhibiting TLR2-mediated SMC phenotypic switch to chondrocytes and the consequent calcification. 5-MTP exerts these effects by blocking p38 MAPK activation and inhibiting CREB and NF-κB transactivation activity.

Control of Mesenchymal Stromal Cell Senescence by Tryptophan Metabolites.

Cellular senescence contributes to aging and age-related disorders. High glucose (HG) induces mesenchymal stromal/stem cell (MSC) senescence, which hampers cell expansion and impairs MSC function. Intracellular HG triggers metabolic shift from aerobic glycolysis to oxidative phosphorylation, resulting in reactive oxygen species (ROS) overproduction. It causes mitochondrial dysfunction and morphological changes. Tryptophan metabolites such as 5-methoxytryptophan (5-MTP) and melatonin attenuate HG-induced MSC senescence by protecting mitochondrial integrity and function and reducing ROS generation. They upregulate the expression of antioxidant enzymes. Both metabolites inhibit stress-induced MSC senescence by blocking p38 MAPK signaling pathway, NF-κB, and p300 histone acetyltransferase activity. Furthermore, melatonin upregulates SIRT-1, which reduces NF-κB activity by de-acetylation of NF-κB subunits. Melatonin and 5-MTP are a new class of metabolites protecting MSCs against replicative and stress-induced cellular senescence. They provide new strategies to improve the efficiency of MSC-based therapy for diverse human diseases.

Cytoguardin: A Tryptophan Metabolite against Cancer Growth and Metastasis.

Cytoguardin was identified in the conditioned medium of fibroblasts as a tryptophan metabolite, 5-methoxytryptophan (5-MTP). It is synthesized via two enzymatic steps: tryptophan hydroxylase (TPH) and hydroxyindole O-methyltransferase (HIOMT). A truncated HIOMT isoform, HIOMT298, catalyzes 5-MTP synthesis. Cancer cells produce scarce 5-MTP due to defective HIOMT298 expression. 5-MTP inhibits cancer cell COX-2 expression and thereby reduces COX-2-mediated cell proliferation and migration. 5-MTP also inhibits MMP-9 expression and thereby reduces cancer cell invasion. 5-MTP exerts its anti-cancer effect by blocking p38 MAPK and p38-mediated NF-κB and p300 HAT activation. The stable transfection of A549 cells with HIOMT298 restores 5-MTP production which renders cancer cells less aggressive. The implantation of HIOMT-transfected A549 into subcutaneous tissues of a murine xenograft tumor model shows that HIOMT-transduced A549 cells form smaller tumors and generate fewer metastatic lung nodules than control A549 cells. HIOMT298 transfection suppresses aromatic amino acid decarboxylase (AADC) expression and serotonin production. Serotonin is a cancer-promoting factor. By restoring 5-MTP and suppressing serotonin production, HIOMT298 overexpression converts cancer cells into less malignant phenotypes. The analysis of HIOMT expression in a human cancer tissue array showed reduced HIOMT levels in a majority of colorectal, pancreatic, and breast cancer. HIOMT298 may be a biomarker of human cancer progression. Furthermore, 5-MTP has the potential to be a lead compound in the development of new therapy for the chemoprevention of certain cancers such as hepatocellular cancer.

5-methoxytryptophan: an arsenal against vascular injury and inflammation.

5-methoxytryptophan (5-MTP) is an endothelial factor with anti-inflammatory properties. It is synthesized from L-tryptophan via two enzymatic steps: tryptophan hydroxylase-1 (TPH-1) and hydroxyindole O-methyltransferase. Lipopolysaccharide (LPS) and pro-inflammatory cytokines suppress endothelial 5-MTP production by inhibiting TPH-1 expression. 5-MTP protects endothelial barrier function and promotes endothelial repair, while it blocks vascular smooth muscle cell migration and proliferation by inhibiting p38 MAPK activation. 5-MTP controls macrophage transmigration and activation by inhibiting p38 MAPK and NF-κB activation. 5-MTP administration attenuates arterial intimal hyperplasia, defends against systemic inflammation and prevents renal fibrosis in relevant murine models. Serum 5-MTP level is depressed in human sepsis as well as in mice with sepsis-like disorder. It is reduced in chronic kidney disease and acute myocardial infarction in humans. The reported data suggest that serum 5-MTP may be a theranostic biomarker. In summary, 5-MTP represents a new class of tryptophan metabolite which defends against inflammation and inflammation-mediated tissue damage and fibrosis. It may be a valuable lead compound for developing new drugs to treat complex human inflammatory disorders.

Restoration of hydroxyindole O-methyltransferase levels in human cancer cells induces a tryptophan-metabolic switch and attenuates cancer progression.

5-Methoxytryptophan (5-MTP) is a tryptophan metabolite with recently discovered anti-inflammatory and tumor-suppressing activities. Its synthesis is catalyzed by a hydroxyindole O-methyltransferase (HIOMT)-like enzyme. However, the exact identity of this HIOMT in human cells remains unclear. Human HIOMT exists in several alternatively spliced isoforms, and we hypothesized that 5-MTP-producing HIOMT is a distinct isoform. Here, we show that human fibroblasts and cancer cells express the HIOMT298 isoform as contrasted with the expression of the HIOMT345 isoform in pineal cells. Sequencing analysis of the cloned isoforms revealed that HIOMT298 is identical to the sequence of a previously reported truncated HIOMT isoform. Of note, HIOMT298 expression was reduced in cancer cells and tissues. Stable transfection of A549 cancer cells with HIOMT298 restored HIOMT expression to normal levels, accompanied by 5-MTP production. Furthermore, HIOMT298 transfection caused a tryptophan-metabolic switch from serotonin to 5-MTP production. To determine the in vivo relevance of this alteration, we compared growth and lung metastasis of HIOMT298-transfected A549 cells with those of vector- or untransfected A549 cells as controls in a murine xenograft model. Of note, the HIOMT298-transfected A549 cells exhibited slower growth and lower metastasis than the controls. Our findings provide insight into the crucial role of HIOMT298 in 5-MTP production in cells and in inhibiting cancer progression and highlight the potential therapeutic value of 5-MTP for managing cancer.

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.

Human mesenchymal stem cells (MSCs) for treatment towards immune- and inflammation-mediated diseases: review of current clinical trials.

Human mesenchymal stem cells (MSCs) are multilineage somatic progenitor/stem cells that have been shown to possess immunomodulatory properties in recent years. Initially met with much skepticism, MSC immunomodulation has now been well reproduced across tissue sources and species to be clinically relevant. This has opened up the use of these versatile cells for application as 3rd party/allogeneic use in cell replacement/tissue regeneration, as well as for immune- and inflammation-mediated disease entities. Most surprisingly, use of MSCs for in immune-/inflammation-mediated diseases appears to yield more efficacy than for regenerative medicine, since engraftment of the exogenous cell does not appear necessary. In this review, we focus on this non-traditional clinical use of a tissue-specific stem cell, and highlight important findings and trends in this exciting area of stem cell therapy.

Prostacyclin protects vascular integrity via PPAR/14-3-3 pathway.

Vascular integrity is protected by the lining endothelial cells (ECs) through structural and molecular protective mechanisms. In response to external stresses, ECs are dynamic in producing protective molecules such as prostacyclin (PGI2). PGI2 is known to inhibit platelet aggregation and controls smooth muscle cell contraction via IP receptors. Recent studies indicate that PGI2 defends endothelial survival and protects vascular smooth muscle cell from apoptosis via peroxisome-proliferator activated receptors (PPAR). PPAR activation results in 14-3-3 upregulation. Increase in cytosolic 14-3-3ɛ or 14-3-3ß enhances binding and sequestration of Akt-mediated phosphorylated Bad and reduces Bad-mediated apoptosis via the mitochondrial pathway. Experimental data indicate that administration of PGI2 analogs or augmentation of PGI2 production by gene transfer attenuates endothelial damage and organ infarction caused by ischemia-reperfusion injury. The protective effect of PGI2 is attributed in part to preserving endothelial integrity.

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.

TM hidden treasure: lectin-like domain.

In this issue of Blood, Kuo et al have used recombinant lectin-like domain of thrombomodulin domain 1 (TMD1) to demonstrate the action of lectin-like domain in blocking Lewis Y antigen (LeY)­mediated angiogenesis and control of tumor growth.

Influence of single nucleotide polymorphisms in factor VIII and von Willebrand factor genes on plasma factor VIII activity: the ARIC Study.

Factor VIII (FVIII) functions as a cofactor for factor IXa in the contact coagulation pathway and circulates in a protective complex with von Willebrand factor (VWF). Plasma FVIII activity is strongly influenced by environmental and genetic factors through VWF-dependent and -independent mechanisms. Single nucleotide polymorphisms (SNPs) of the coding and promoter sequence in the FVIII gene have been extensively studied for effects on FVIII synthesis, secretion, and activity, but impacts of non-disease-causing intronic SNPs remain largely unknown. We analyzed FVIII SNPs and FVIII activity in 10,434 healthy Americans of European (EA) or African (AA) descent in the Atherosclerosis Risk in Communities (ARIC) study. Among covariates, age, race, diabetes, and ABO contributed 2.2%, 3.5%, 4%, and 10.7% to FVIII intersubject variation, respectively. Four intronic FVIII SNPs associated with FVIII activity and 8 with FVIII-VWF ratio in a sex- and race-dependent manner. The FVIII haplotypes AT and GCTTTT also associated with FVIII activity. Seven VWF SNPs were associated with FVIII activity in EA subjects, but no FVIII SNPs were associated with VWF Ag. These data demonstrate that intronic SNPs could directly or indirectly influence intersubject variation of FVIII activity. Further investigation may reveal novel mechanisms of regulating FVIII expression and activity.

5-methoxyindole metabolites of L-tryptophan: control of COX-2 expression, inflammation and tumorigenesis.

Cyclooxygenase-2(COX-2) overexpression promotes inflammation and tumorigenesis. COX-2 expression in response to diverse stimuli is tightly controlled to avoid persistent overexpression. 5-methoxyindole metabolites of L-tryptophan represent a new class of compounds that control COX-2 expression at the transcriptional level. Two of the metabolites, the newly discovered 5-methoxytryptophan (5-MTP, also known as cytoguardin) and N-acetyl 5-methoxytryptamine (melatonin) are the focus of this review. 5-MTP is produced by mesenchymal cells such as fibroblasts via 5-hydroxytryptophan (5-HTP). It inhibits COX-2 transcriptional activation induced by diverse proinflammatory and mitogenic factors. Cancer cells are deficient in cytoguardin production which contributes to COX-2 overexpression. Fibroblast-generated 5-MTP is capable of restoring the control of COX-2 overexpression in cancer cells. 5-MTP blocks cancer cell migration and invasion in vitro and inhibits tumor growth and cancer metastasis in a xenograft model. Melatonin possesses similar COX-2 suppressing and anti-cancer properties albeit at supra-pharmacological concentrations. By contrast, 5-hydroxyindole metabolites of L-tryptophan such as 5-hydroxytryptamine (serotonin), 5-hydroxytryptophol and other serotonin catabolites do not control COX-2 expression. 5-hydroxytryptophan inhibits COX-2 expression through conversion to 5-MTP. The physiological relevance of 5-MTP as an endogenous regulator of inflammation and cancer metastasis remains to be investigated. On the other hand, 5-methoxyindole metabolites of tryptophan are valuable lead compounds for development of new anti-inflammatory drugs and cancer chemoprevention.

11ß-HSD1 inhibition reduces atherosclerosis in mice by altering proinflammatory gene expression in the vasculature.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is implicated in the etiology of metabolic syndrome. We previously showed that pharmacological inhibition of 11ß-HSD1 ameliorated multiple facets of metabolic syndrome and attenuated atherosclerosis in ApoE-/- mice. However, the molecular mechanism underlying the atheroprotective effect was not clear. In this study, we tested whether and how 11ß-HSD1 inhibition affects vascular inflammation, a major culprit for atherosclerosis and its associated complications. ApoE-/- mice were treated with an 11ß-HSD1 inhibitor for various periods of time. Plasma lipids and aortic cholesterol accumulation were quantified. Several microarray studies were carried out to examine the effect of 11ß-HSD1 inhibition on gene expression in atherosclerotic tissues. Our data suggest 11ß-HSD1 inhibition can directly modulate atherosclerotic plaques and attenuate atherosclerosis independently of lipid lowering effects. We identified immune response genes as the category of mRNA most significantly suppressed by 11ß-HSD1 inhibition. This anti-inflammatory effect was further confirmed in plaque macrophages and smooth muscle cells procured by laser capture microdissection. These findings in the vascular wall were corroborated by reduction in circulating MCP1 levels after 11ß-HSD1 inhibition. Taken together, our data suggest 11ß-HSD1 inhibition regulates proinflammatory gene expression in atherosclerotic tissues of ApoE-/- mice, and this effect may contribute to the attenuation of atherosclerosis in these animals.

Thromboxane A2 is a key regulator of pathogenesis during Trypanosoma cruzi infection.

Chagas' disease is caused by infection with the parasite Trypanosoma cruzi. We report that infected, but not uninfected, human endothelial cells (ECs) released thromboxane A(2) (TXA(2)). Physical chromatography and liquid chromatography-tandem mass spectrometry revealed that TXA(2) is the predominant eicosanoid present in all life stages of T. cruzi. Parasite-derived TXA(2) accounts for up to 90% of the circulating levels of TXA(2) in infected wild-type mice, and perturbs host physiology. Mice in which the gene for the TXA(2) receptor (TP) has been deleted, exhibited higher mortality and more severe cardiac pathology and parasitism (fourfold) than WT mice after infection. Conversely, deletion of the TXA(2) synthase gene had no effect on survival or disease severity. TP expression on somatic cells, but not cells involved in either acquired or innate immunity, was the primary determinant of disease progression. The higher intracellular parasitism observed in TP-null ECs was ablated upon restoration of TP expression. We conclude that the host response to parasite-derived TXA(2) in T. cruzi infection is possibly an important determinant of mortality and parasitism. A deeper understanding of the role of TXA(2) may result in novel therapeutic targets for a disease with limited treatment options.

Genetic determinants of plasma von Willebrand factor antigen levels: a target gene SNP and haplotype analysis of ARIC cohort.

von Willebrand factor (VWF) is an essential component of hemostasis and has been implicated in thrombosis. Multimer size and the amount of circulating VWF are known to impact hemostatic function. We associated 78 VWF single nucleotide polymorphisms (SNPs) and haplotypes constructed from those SNPs with VWF antigen level in 7856 subjects of European descent. Among the nongenomic factors, age and body mass index contributed 4.8% and 1.6% of VWF variation, respectively. The SNP rs514659 (tags O blood type) contributed 15.4% of the variance. Among the VWF SNPs, we identified 18 SNPs that are associated with levels of VWF. The correlative SNPs are either intronic (89%) or silent exonic (11%). Although SNPs examined are distributed throughout the entire VWF gene without apparent cluster, all the positive SNPs are located in a 50-kb region. Exons in this region encode for VWF D2, D', and D3 domains that are known to regulate VWF multimerization and storage. Mutations in the D3 domain are also associated with von Willebrand disease. Fifteen of these 18 correlative SNPs are in 2 distinct haplotype blocks. In summary, we identified a cluster of intronic VWF SNPs that associate with plasma levels of VWF, individually or additively, in a large cohort of healthy subjects.

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. CONCLUSIONS: 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.