Effect Of Free Fatty Acids On The Prothrombotic State In Metabolic Syndrome

BackgroudMetabolic syndrome displays a significant prothrombotic state with increased coagulation, inhibited fibronolysis and increased platelet activation. The prothrombotic state induces the increased risk of pathological thrombosis, and renders patients highly susceptible to myocardial infarction, ischemic stroke and peripheral vascular diseases. Although many clinical studies showed that the prothrombotic state is a key feature of metabolic syndrome, the pathogenesis and mechanisms of the state is not completely understood.Protein C, a key endogenous anticoagulant molecule, is efficiently activated on the surface of endothelial cells by the binding of thrombin to the endothelial transmembrane glycoprotein, Thrombomodulin. Once activated, protein C, together with its cofactor protein S, inhibits coagulation by proteolytic degradation of factor Villa and factor Va. In addition, activated protein C (APC) promotes fibrin degradation by inhibiting the activity of plasminogen activator inhibitor—1 (PAI-1). Moreover, APC has significant anti-inflammatory and anti-apoptotic functions. Thus, APC not only regulates hemostasis and inflammation, but also provides additional levels of cellular protection. Thrombomodulin and endothelial protein C receptor (EPCR) control the protein C anticoagulation pathway. Maximal rates of protein C activation require thrombin binding to thrombomodulin and protein C binding to EPCR. Loss of thrombomodulin disrupts the protein C anticoagulant pathway and causes juvenile-onset thrombosis. Localized loss of thrombomodulin from endothelium has been associated with arteriosclerotic lesions, therefore, thrombomodulin may act in a vasoprotective manner to prevent cardiovascular diseases. Deregulation of this system may be responsible for the prothrombotic state in metabolic syndrome.The prothrombotic state in metabolic syndrome has a complex pathogenesis. Metabolic syndrome is often characterized by high circulating concentrations of FFAs. Thus, the vasculature of patients with metabolic syndrome is constantly exposed to high levels of FFAs. Clinical studies have shown that high FFA levels directly impair vascular functions are associated with myocardial infarction, stroke, and sudden death. Long-term increases in FFA levels may have an adverse effect on the regulation of thrombosis. But it is not understood whether FFAs can regulate TM expression and the mechanisms involved.In this study, we hypothesize that the metabolic stress of high concentrations of FFAs may deregulate the thrombomodulin-APC system, which in turn may be responsible for the development of the prothrombotic state in metabolic syndrome. Our study may be helpful to understand the mechanisms of the prothrombotic state in metabolic syndrome, and supply a new therapeutic target in treating and preventing cardiovascular diseases in metabolic syndrome.Objectives1. Elucidate the effects of high serum FFAs level on the prothrombotic state in metabolic syndrome.2. Examine the effects of FFAs on the expression of TM-protion C system and the possible mechanisms involved.Methods1. Animal model8 to 10 weeks of male wide-type mice were divided into two groups. One group of mice were fed a chow diet containing 10% fat, while the other group of mice were fed a high-fat diet containing 50% fat for 20 weeks. The aortas were rapidly excised and rinsed in ice-cold saline, and then stored for histology and further use.2. Tail-bleeding timeMice were anesthetized and placed on a 37℃heating pad. The tail was transected with a sterile scalpel at a point where the tail diameter was approximately 1 mm wide. After transection, the tail was immediately placed in a 50 mL tube containing 0.9% NaCl warmed to 37℃. The time between bleeding to stop was recorded.3. FeCl3 induced carotid artery thrombosis modelFeCl3 induced thrombosis was used in this study. In brief, mice were anesthetized. An incision was made, and a segment of the right common carotid artery was exposed with blunt dissection. A small piece of filter paper(2 mm X 3 mm) was saturated with 10% FeCl3 was deposited on the isolated artery for 3 min, followed by washing with saline. Blood flow was monitored using a Doppler flow probe for 30 min after FeCl3 application. The occlusion time was recorded as the first image that showed 0 flow.4. Metabolic factors measurementsBlood samples were collected from retio-orbital sinus. Circulating free fatty acids, glocuse, insulin, TG, VLDL, LDL, HDL were measured.5. Histological and morphology analysesSections were stained with hematoxylin and eosin(H&E). TM, PAI-1, TF were detected with immunostaining.6. Cell culturePrimary human aortic endothelial cells (HAECs) were cultured at 37℃in 5% CO2 in endothelial cell growth medium-2 (EGM-2). Cells between passages 5 to 9 were used for all of the experiments. The medium was changed every two or three days.7. Preparation of fatty acid-albumin complexes.Saturated PA, polyunsaturated LA, and monounsaturated OA were used in this study. Lipid-containing media were prepared by conjugation of FFAs with BSA using a modification of the method described previously. Briefly, FFAs were first dissolved in ethanol at 200mM, and then combined with 10% FFA-free low endotoxin BSA to final concentrations of 1-5mM. The pH of all solutions was adjusted to approximately 7.5, and the stock solutions were filter-sterilized and stored at-20℃until use. Control solution containing ethanol and BSA was prepared similarly. Working solutions were prepared fresh by diluting stock solution (1:10) in 2% FCS-EBM.8. Protein C activation.HAECs in 96-well plates were treated with different kind and different dose of FFAs for 24 hours.Then the activation of protein C was detected according to the manufacturer's instructions.9. siRNA-induced gene sliencingSliencing gene expression wsas achieved by using specific siRNAs. HAECs were transfected with siRNAs using Lipofectamine2000 according to the manufacture's instructions.10. Western blot analysisCell extracts and mice aortas were prepared with lysis buffer. Equal amounts of protein were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Following blocking with 5% non-fat milk, the blots were incubated with a primary antibody at 4℃overnight. Then the blots were washed with TBST and incubated with HRP-conjugated secondary antibody. The blots were then visualized by use of enhanced chemiluminescence.11. RT-PCRTotal RNA was extracted from HAECs with Trizol according to the manufacturer's instructions. Signal-strand cDNA was synthesized with iScript cDNA synthesis kit. Semi-quantitative real-time PCR was performed with iCycler iQ real-time PCR detection system.12. Plasmid DNA transfectionHAECs were transfected with different types of plasmids including wide type, constitutively active and dominant-negative by using LipofectAMINETM 2000 according to the manufacturer's instructions. Transfected cells were then treated with PA. Results1. Mice modelMice fed a high-fat diet showed notable characteristics of metabolic syndrome with higher body weight and elevated blood levels of FFAs, TG, VLDLs, glucose and insulin(P<0.05).2. Prothrombotic state in mice fed a high-fat dietThe tail bleeding time was significantly lower in mice fed a high-fat diet than that in chow diet. Furthermore, the occlusion time following FeCl3 injury of the carotid artery was significantly shorter in mice fed a high-fat diet compared to that in control mice, indicating that the development of prothrombotic state in mice fed a high-fat diet(P<0.01).3. Reduced expression of thrombomodulin and increased expression of PAI-1 and TF in mice fed a high-fat dietWe examined the expression of TM, PAI-1 and TF in the aorta of obese mice. Immunostaining of the vascular wall showed significantly decreased expression of thrombomodulin(P<0.001), and increased expression of PAI-1 and TF(P<0.01) on the surface of the endothelium in the aorta of mice fed a high-fat diet. This finding is consistent with the results of Western blot. These results show that thrombomodulin is down-regulated in obese mice, which may contribute to the hypercoagulable state in obesity and metabolic syndrome.4. JNK and p38 MAPK stress signaling pathways were activated in mice fed a high-fat dietWestern blot showed that JNK and p38 MAPK stress pathways were activated in mice fed a high-fat diet(P<0.01), indicating that the activation of stree pathways may contribute to the prothrombotic state in these mice.5. Suppression of thrombomodulin and EPCR expression and increased expression of TF by FFAs.To determine the potential role of FFAs in thrombosis dysregulation, we examined the effect of FFAs on the expression of prothrombotic factors and antithrombotic factors in endothelial cells. Saturated fatty acid PA, polyunsaturated fatty acid LA, and monounsaturated fatty acid oleic acid (OA) were used for the study. PA and LA significantly decreased the expression of thrombomodulin in a dose-dependent fashion(P<0.01), whereas OA had a minimal effect on thrombomodulin(P>0.05). Similarly, treatment with PA and LA substantially inhibited EPCR expression and increased to varying degrees the expression of the prothrombotic factor, PAI-1(P<0.01). PA significantly decreased mRNA for thrombomodulin in a dose-dependent manner.6. FFAs inhibited endothelial-mediated protein C activationWe compared the activation of protein C in FFA-treated and untreated HAECs. PA and LA caused a significant dose-dependent decrease in protein C activation(P<0.01), whereas OA had no effect(P>0.05).7. JNK and p38 stress pathways were involved in PA's inhibitory effect on the expression of TM.Silencing JNK and p38 with specific siRNAs decreased PA-induced thrombomodulin suppression(P<0.01), indicating that JNK and p38 pathways mediated PA-induced inhibition of thrombomodulin expression. Furthermore, wide type of JNK plasmid can enhance PA's inhibitory effect on TM expression, while dominant-negative of JNK plasmid can abrogate the effect.8. Foxol was involved in PA-inhibited TM expressionWhen HAECs cells were transiently transfected with Foxol-specific siRNAs before treated with PA, PA-induced inhibition of TM expression was significantly decreased(P<0.001). Wide type and constitutively active of Foxol plasmids enhanced PA's inhibitory effect of TM expression(P<0.05), while dominant-negative of Foxol plasmid can abrogate the effect(P>0.05).Conclusions1. A high-fat diet induced high elevated circulating FFAs level, the shortened bleeding time, indicating that the prothrombotic state in metabotic syndrome mice.2. The aortas from mice fed a high-fat diet showed decreased TM expression, and increased expression of PAI-1 and TF, indicating that the imbalance of coagulation and anticoagulation.3. PA and LA inhibited the expression of TM and EPCR, and increased PAI-1 expression in endothelial cells, indicating that FFAs can induce the imbalance of coagulation and anticoagulation in endothelial cells.4. JNK and p38 stress pathways mediated PA-inhibited TM expression in HAECs.5. Foxol was involved in PA-inhibited TM expression in HAECs.6. JNK, p38/Foxol pathway mediated free fat acid's inhibitory effect of thrombomodulin expression, which may be a new therapeutic target in treating cardiovascular and cerebrovascular complications. BackgroudMetabolic syndrome is associated with a prothrombotic state, which contributes to the atherothrombotic complications, such as myocardial infarction, stroke, and peripheral vascular complications. Antithrombotic therapy improves survival in acute cardiac and cerebrovascular complications patients. Although the dysregulation in coagulation and fibrinolysis has been reported, the development of prothrombotic state is not well understood.Endothelium plays an active role in regulating pro-coagulation and anti-coagulation balance by generating several active regulatory molecules, such as von Willebrand factor (vWF), thrombomodulin (TM), tissue plasminogen activator (t-PA), and plasminogen activator inhibitor (PAI-1). Among these factors, thrombomodulin-protein C pathway is a major physiological anticoagulation system of the endothelium. Endothelial dysfunction can cause coagulation dysregulation and promote vascular thrombosis.Endothelium plays an active role in regulating pro-coagulation and anti-coagulation balance by generating several active regulatory molecules, such as von Willebrand factor (vWF), thrombomodulin (TM), tissue plasminogen activator (t-PA), and plasminogen activator inhibitor (PAI-1). Among these factors, thrombomodulin-protein C pathway is a major physiological anticoagulation system of the endothelium. Endothelial dysfunction can cause coagulation dysregulation and promote vascular thrombosis.Thrombomodulin, a glycoprotein on the surface of endothelial cells, is a key factor in protein C activation3. When bound to thrombin, TM triggers the activation of protein C by facilitating the conversion of circulating protein C to activated protein C (APC). Activated protein C can inhibit coagulation by degradingⅧa and factor Va and enhance fibrinolysis by inactivating PAI-1. TM plays a key role in anticoagulation, and mutation or down-regulation of TM promotes while overexpression of TM prevents arterial thrombosis. In addition, TM functions as an anti-inflammatory and anti-apoptotic molecule. It has been shown that TM inhibited inflammatory response and blocked cell apoptosis. TM is down-regulated in vascular diseases including atherosclerotic lesions, and TM is negatively regulated by inflammatory factors, wall tension and oxidized lipids. However, many reports showed that the down-regulated expression of TM was associated with the pathologic thrombosis, the mechanisms involved in the down-regulation of TM expression are not completely understood.Mitogen-activated protein kinases (MAPKs) are serine/threonine-specific protein kinases that contribute to extracellular stimuli and regulate various cellular activities, such as gene expression, mitosis, transformation, apoptosis. Stress signaling JNK and p38 pathways are activated in many cardiovascular diseases including atherosclerosis and are involved in pathphysiological changes in these conditions. JNK and p38 signaling pathways are activated by metabolic stress and inflammatory factors. Previous study showed that JNK and p38 can be activated by free fatty acids (FFA) and were involved in vascular insulin resistance. We have found that JNK and p38 pathways are involved in the dowm-regulation of TM, but the detailed mechanisms involved are not well understood.How stress signaling down-regulates TM expression? ATF-2 is a downstream target transcription factor of JNK and p38 pathways. Previous study has reported that ATF-2 mediated LPS-induced TF expression and may be involved in thrombosis dysregulation. But whether transcriptional factor ATF-2 is involved in the regulation of TM expression is still unclear.So in this study, we examined the effects of transcriptional factor ATF-2 on the regulation of TM expression, which may be helpful for the development of therapeutic intervention for pathologic thrombosis and cardiovascular complications.Objectives1. Identify the effects of transcriptional factor ATF-2 in the regulation of TM expression in HAECs.2. Examine the possible mechanisms how ATF-2 mediated TM expression.Metarials and Methods1. Cell culturePrimary human aortic endothelial cells (HAECs) were cultured at 37℃in 5% CO2 in endothelial cell growth medium-2 (EGM-2) containing 2% FBS, FGF-2, VEGF, IGF-1, EGF. Cells between passages 5 to 9 were used for all of the experiments. The medium was changed every two or three days. The endothelial cells were plated on 6-well plates, and then treated with different doses of palmitic acid or transfected with siRNAs.2. Preparation of palmitic acids:Preparation of PA was carried out as previously. Briefly, PA was dissolved in ethanol at 200mM, and then combined with 10% FFA-free low endotoxin BSA to final concentrations of 1-5mM. The pH of all solutions was adjusted to approximately 7.5, and the stock solutions were stored at-20℃.3. siRNA transfectionSilencing gene expression was achieved using specific siRNA. HAECs were transfected with siRNAs using Lipofectamine2000 according to the manufacturer's instructions. Transfected cells were then treated with palmitic acid for 24h.4. Western blot analysisCell extracts were prepared with lysis buffer, and boiled with 5 min. Protein samples(15μg per lane) and protein marker were seperated by SDS-PAGE gel electropohresis and transferred to PVDF membranes. The membranes were blocked with blocking buffer, and then incubated with the primary antibody overnight. The membranse were washed with PBST, and then incubated with the HRP-conjugated secondary antibody. The blots were then visualized with enhanced chemiluminescene. The expression of cytokine was demonstrated by the ration of integral optical density between cytokine and p-actin5. RNA extration and real-time quantitative PCRTotal RNA was extracted from HAECs with Trizol according to the manufacturer's instructions. Signal-strand cDNA was synthesized with iScript cDNA synthesis kit. Semi-quantitative real-time PCR was performed with iCycler iQ real-time PCR detection system. The primers for human thrombomodulin mRNA were as follows:forward:5'-CCGATGTCATTTCCTTGCTA-3'; reverse:5'-GTTGTCTCCCGTAACCCACT-3'. The mRNA levels were estimated from the value of the threshold cycle (Ct) of the real-time PCR adjusted by that ofβ-actin.6. Chromatin immunoprecipitation assayThe ChIP assay kit (Upstate) was used according to the manufacturer's instructions. Treated HAECs were first incubated with 1% formaldehyde at 37℃for 15 min to cross-link DNA-protein complexes. Cells were then rinsed and lysed. Cell lysates were sonicated and centrifuged to produce chromatin fragments. The supernatants were pre-cleared with a mixture of salmon sperm DNA/protein A/protein G, followed by immunoprecipitation with antibody-protein A-agarose slurry. (IgG served as the negative control.) The DNA was recovered by extraction with the phenol/chloroform/isoamyl alcohol mixture. The immunoprecipitated DNA was used as a template for PCR. The PCR products were separated by 1.5% agarose gel.7. ImmunoprecipitationImmunoprecipitation was conducted as described previously.Treated cells were lysed for 60 min in ice-cold extraction buffer. For immunoprecipitation, cleared cell lysates were incubated with the appropriate antibody precoupled to protein A/G-agarose beads (Santa Cruz Biotechnology) at 4℃overnight. The beads were washed twice with extraction buffer and twice with extraction buffer containing 0.5 M LiCl. Proteins were eluted directly in SDS sample buffer for Western blot analysis.8. Statistical analysisData are presented as mean±SEM. One-way ANOVA was used to analyze the differences among groups. P values< 0.05 were considered statistically significant.Results:1. Free fatty acids suppressed thrombomodulin expression in HAECs.HAECs were incubated with different concentrations of PA for 24h. Western blotting showed that PA significantly suppressed the expression of thrombomodulin in a dose-dependent manner. Furthermore, PA significantly inhibited the expression of thrombomodulin mRNA, indicating that PA suppressed thrombomodulin expression at the transcriptional level.2. Free fatty acids activated ATF-2 pathway.HAECs were treated with different doses of PA for 24h. The phosphorylated protein and total protein of ATF-2 were examined by Western blot. The activation of pathways was demonstrated by the ratio of phosphorylated and total protein. Consistent with previous observation, ATF-2 was activated by PA in a dose-dependent manner.3. Transcriptional factor ATF-2 binded directly to the TM promoter.The promoter region in the TM gene contained many ATF-2 binding sites(agTGACGgatt at-1288/-1277, gcTGACTcgct at-1026/-1016, and ccTGACAgtgt at-939/-929). ChiP assay was used to examine whether ATF-2 can bind to the TM promoter. The results of ChiP assay showed that ATF-2 can bind to the TM promoter at the gcTGACTcgct (-1026/-1016) and ccTGACAgtgt (-939/-929) sites. Importantly, the binding was significantly increased by PA treatment, indicating that binding of ATF-2 to TM promoter may be involved in PA-induced suppression of thrombomodulin transcription. 4. ATF-2 was involved in PA-inhibited TM expression.We then examined whether ATF-2 can regulate TM expression. When HAECs cells were transiently transfected with ATF-2-specific siRNAs before treated with PA, PA-induced inhibition of TM expression was significantly prevented, which showed that a critical role for ATF-2 transcription factor in PA-induced down-regulation of thrombomodulin transcription (P<0.05).5. Recruitment of HDAC4 and formation of ATF-2/HDAC4 transcription repressor complex in the TM promoter.We finally investigated how the transcription factor ATF-2 can bind to the TM promoter and inhibit gene expression. ChiP assay showed that PA treatment significantly increased transcription repressor HDAC4 binding to the TM promoter. The double-chip assay showed that ATF-2 and HDAC4 were in the same transcription repression complex in the TM promoter.Conclusions1. PA significantly inhibited thrombomodulin expression in human aortic endothelial cells(HAECs).2. ATF-2 was involved in the down-regulation of thrombomodulin expression.3. ATF-2 was mediated TM suppression by recruitment of HDAC4 and formation of ATF-2/HDAC4 transcription repressor complex in the TM promoter.