Coordinated demethylation of H3K9 and H3K27 is required for rapid inflammatory responses of endothelial cells.

Histone H3 lysine-9 di-methylation (H3K9me2) and lysine-27 tri-methylation (H3K27me3) are linked to repression of gene expression, but the functions of repressive histone methylation dynamics during inflammatory responses remain enigmatic. Here, we report that lysine demethylases 7A (KDM7A) and 6A (UTX) play crucial roles in tumor necrosis factor (TNF)-α signaling in endothelial cells (ECs), where they are regulated by a novel TNF-α-responsive microRNA, miR-3679-5p. TNF-α rapidly induces co-occupancy of KDM7A and UTX at nuclear factor kappa-B (NF-κB)-associated elements in human ECs. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and are both required for activation of NF-κB-dependent inflammatory genes. Chromosome conformation capture-based methods furthermore uncover increased interactions between TNF-α-induced super enhancers at NF-κB-relevant loci, coinciding with KDM7A and UTX recruitments. Simultaneous pharmacological inhibition of KDM7A and UTX significantly reduces leukocyte adhesion in mice, establishing the biological and potential translational relevance of this mechanism. Collectively, these findings suggest that rapid erasure of repressive histone marks by KDM7A and UTX is essential for NF-κB-dependent regulation of genes that control inflammatory responses of ECs.

Neutrophil Protein Kinase R Mediates Endothelial Adhesion and Migration by the Promotion of Neutrophil Actin Polymerization.

Neutrophils protect against bacterial and fungal infections, but tight regulation of cell activation is essential for avoiding tissue damage in autoimmune disorders. Protein kinase R (PKR) is a serine/threonine kinase originally characterized by its role in the defense mechanisms against viral infection. Although PKR is involved in the signaling pathways of neurodegenerative diseases and metabolic disorders, its function in neutrophils is not well delineated. In this study, we demonstrate that human neutrophil PKR mediates adhesion to endothelial cells under physiological flow conditions but does not mediate rolling on those cells. Also, neutrophil PKR activation contributes to migration toward chemoattractants. Mechanistically, neutrophil PKR mediates the cell spreading and binding to ICAM-1 in static condition. Moreover, Ab microarray reveals that calcium/calmodulin-dependent protein kinase II is phosphorylated downstream of PKR and affects actin polymerization that is a cytoskeleton rearrangement indispensable for neutrophil migration induced by fMLF. In vivo, neutrophil recruitment into the dorsal air pouch of mice is reduced by PKR inhibitor treatment. Also, in mice with nephrotoxic serum nephritis, the compound treatment suppresses neutrophil accumulation in kidney glomerulus and subsequent development of albuminuria. Thus, in vascular inflammation, neutrophil PKR plays a critical role in the recruitment process, including endothelial adhesion and migration via leukocyte actin polymerization.

Oral administration of the milk casein-derived tripeptide Val-Pro-Pro attenuates high-fat diet-induced adipose tissue inflammation in mice.

Inflammation of adipose tissue triggers the metabolic syndrome, atherosclerosis and CHD. In the present study, we investigated whether the milk casein-derived tripeptide valine-proline-proline (VPP) has an anti-inflammatory effect on the adipose tissue of high-fat diet (HFD)-fed mice. Male C57BL/6J mice (7 weeks of age) were fed ad libitum with either a HFD and plain tap water (HFD group) or a HFD and water containing 0·3 mg VPP/ml (HFD+VPP group) for 10 weeks. The results showed that the expression level of CD18 in the peripheral blood monocytes of the HFD+VPP group was significantly decreased compared with the level observed in those of the HFD group. Activated monocytes and pro-inflammatory macrophages were accumulated in the stromal vascular fractions of the adipose tissue from HFD-fed mice, which were significantly decreased in those supplemented with VPP. The formation of crown-like structures rich in pro-inflammatory macrophages was also significantly reduced in the adipose tissue of mice administered with VPP. Real-time PCR analysis revealed that the expression of monocyte chemoattractant protein-1 and that of the pro-inflammatory cytokine IL-6 in adipose tissue tend to be lower in the HFD+VPP group than in the HFD group. These observations indicate that oral administration of VPP exerts an anti-inflammatory effect on the adipose tissue of HFD-fed mice, which may eventually lead to the primary prevention of chronic inflammation-related diseases.

CXCL1-Triggered PAD4 Cytoplasmic Translocation Enhances Neutrophil Adhesion through Citrullination of PDIA1.

AIMS: Vascular inflammation is critical for the development and progression of atherosclerosis. Previously, we reported that neutrophils adhere to the vascular endothelium in low-density lipoprotein receptor null mice fed a high-fat diet through hypercitrullination of histone H3 by peptidylarginine deiminase 4 (PAD4) in neutrophils. However, the involvement of PAD4 and citrullination of proteins other than histone H3 in neutrophil adhesion is not well known. In this study, we investigated the function of PAD4 and identified citrullinated proteins during vascular inflammation. METHODS: We pefformed flow assay under physiological flow conditions using differentiated HL-60 (dHL-60) cells stimulated with CXCL1 and human umbilical vein endothelial cells (HUVECs). Furthermore, phalloidin stain for dHL-60 stimulated with CXCL1 to observe F-actin polymerization and immunohistochemistry for the activated ß2-integrin was conducted. To identify a target of citrullination in the cytoplasm of dHL-60 cells, liquid chromatography-mass spectrometry (LC-MS/MS) for dHL-60 stimulated with CXCL1 was performed. RESULTS: Inhibition or knockdown of PAD4 significantly decreased adhesion of under physiological flow conditions. Thr-Asp-F-amidine trifluoroacetate salt (TDFA), a PAD4 inhibitor, inhibited cytoplasmic translocation of PAD4 by CXCL1. TDFA or knockdown of PAD4 significantly decreased expression of ß2-integrin and F-actin polymerization activated by CXCL1. Moreover, LC-MS/MS identified protein disulfide isomerase A1 (PDIA1) as a target of citrullination in the cytoplasm of dHL-60 cells. Knockdown of PDIA1 significantly decreased adhesion of dHL-60 cells to HUVECs, expression of ß2-integrin, and F-actin polymerization. CONCLUSIONS: Cytoplasmic translocation of PAD4 by CXCL1 induces neutrophil adhesion to vascular endothelial cells and citrullination of PDIA1.

The complement C3-complement factor D-C3a receptor signalling axis regulates cardiac remodelling in right ventricular failure.

Failure of the right ventricle plays a critical role in any type of heart failure. However, the mechanism remains unclear, and there is no specific therapy. Here, we show that the right ventricle predominantly expresses alternative complement pathway-related genes, including Cfd and C3aR1. Complement 3 (C3)-knockout attenuates right ventricular dysfunction and fibrosis in a mouse model of right ventricular failure. C3a is produced from C3 by the C3 convertase complex, which includes the essential component complement factor D (Cfd). Cfd-knockout mice also show attenuation of right ventricular failure. Moreover, the plasma concentration of CFD correlates with the severity of right ventricular failure in patients with chronic right ventricular failure. A C3a receptor (C3aR) antagonist dramatically improves right ventricular dysfunction in mice. In summary, we demonstrate the crucial role of the C3-Cfd-C3aR axis in right ventricular failure and highlight potential therapeutic targets for right ventricular failure.

Indoxyl sulfate induces leukocyte-endothelial interactions through up-regulation of E-selectin.

Despite a positive correlation between chronic kidney disease and atherosclerosis, the causative role of uremic toxins in leukocyte-endothelial interactions has not been reported. We thus examined the effects of indoxyl sulfate, a uremic toxin, on leukocyte adhesion to activated endothelial cells and the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with indoxyl sulfate significantly enhanced the adhesion of human monocytic cells (THP-1 cell line) to TNF-α-activated HUVEC under physiological flow conditions. Treatment with indoxyl sulfate enhanced the expression level of E-selectin, but not that of ICAM-1 or VCAM-1, in HUVEC. Indoxyl sulfate treatment enhanced the activation of JNK, p38 MAPK, and NF-κB in TNF-α-activated HUVEC. Inhibitors of JNK and NF-κB attenuated indoxyl sulfate-induced E-selectin expression in HUVEC and subsequent THP-1 adhesion. Furthermore, treatment with the NAD(P)H oxidase inhibitor apocynin and the glutathione donor N-acetylcysteine inhibited indoxyl sulfate-induced enhancement of THP-1 adhesion to HUVEC. Next, we examined the in vivo effect of indoxyl sulfate in nephrectomized chronic kidney disease model mice. Indoxyl sulfate-induced leukocyte adhesion to the femoral artery was significantly reduced by anti-E-selectin antibody treatment. These findings suggest that indoxyl sulfate enhances leukocyte-endothelial interactions through up-regulation of E-selectin, presumably via the JNK- and NF-κB-dependent pathway.

Apolipoprotein CIII induces monocyte chemoattractant protein-1 and interleukin 6 expression via Toll-like receptor 2 pathway in mouse adipocytes.

OBJECTIVE: To examine the direct effect of apolipoprotein CIII (apoCIII) on adipokine expressions that are involved in obesity, insulin resistance, or metabolic syndrome. METHODS AND RESULTS: ApoCIII in triglyceride-rich lipoproteins is elevated in patients with obesity, insulin resistance, or metabolic syndrome. Its level is also associated with proinflammatory adipokines. Fully differentiated mouse 3T3L1 adipocytes were incubated with apoCIII. ApoCIII activated nuclear factor κB of 3T3L1 adipocytes and induced the expression of monocyte chemoattractant protein (MCP) 1 and interleukin (IL) 6. ApoCIII also activated extracellular signal-regulated kinase and p38. Mitogen-activated protein kinase kinase (MEK)-1 inhibitor PD98059, but not p38 inhibitor SB203580, inhibited apoCIII-induced upregulation of MCP-1 and IL-6. Previously, it was shown that apoCIII activates proinflammatory signals through toll-like receptor (TLR) 2. TLR2-blocking antibody abolished activation of nuclear factor κB and extracellular signal-regulated kinase induced by apoCIII and inhibited apoCIII-induced upregulation of MCP-1 and IL-6. ApoCIII also reduced adiponectin expression of 3T3L1 adipocytes, which was recovered by TLR2-blocking antibody. ApoCIII induced the expression of MCP-1 and IL-6 in TLR2-overexpressed human embryonic kidney 293 cells but not wild-type human embryonic kidney 293 cells without TLR2. ApoCIII induced the expression of MCP-1 and IL-6 and decreased adiponectin expression in white adipose tissue of wild-type mice but not of TLR2-deficient mice in vivo. CONCLUSIONS: ApoCIII may activate extracellular signal-regulated kinase and nuclear factor kB through TLR2 and induce proinflammatory adipokine expression in vitro and in vivo. Thus, apoCIII links dyslipidemia to inflammation in adipocytes, which, in turn, may contribute to atherosclerosis.

Complement C5a-triggered differentiated HL-60 stimulates migration of THP-1 monocytic leukocytes via secretion of CCL2.

Leukocytes play an important role in vascular inflammation prior to atherosclerosis. In particular, monocyte adhesion and migration to the endothelium contribute to the development of vascular inflammation. Previously, we showed the importance of neutrophils and complement C5a in the early phase of vascular inflammation in mice fed a high-fat diet. However, the relationship between monocytes and neutrophils is not well understood. In this study, we elucidated the involvement of neutrophils in the migration of monocytes. We observed that C5a induces CCL2 expression in neutrophil-like dHL-60 cells. To investigate the physiological significance of CCL2 secretion, we performed a chemotaxis assay. Interestingly, dHL-60 culture supernatant in the presence of C5a enhanced the migration of THP-1 in comparison with the absence of C5a. Furthermore, CCL2 expression and secretion significantly increased in C5a-stimulated dHL-60 through the phosphorylation of NF-κB p65. Actin polymerization on THP-1 was enhanced by the presence of C5a compared with the absence of C5a when stimulated by a dHL-60-cultured medium. These results suggest that crosstalk between neutrophils and monocytes via CCL2 may play an important role in vascular inflammation.

High-Fat Diet Enhances Neutrophil Adhesion in LDLR-Null Mice Via Hypercitrullination of Histone H3.

Neutrophil adhesion on the atheroprone femoral artery of high-fat diet-fed low-density lipoprotein receptor-null mice was enhanced more than in wild-type mice. The inhibition of histone H3 citrullination of neutrophils reversed the enhancement of neutrophil adhesion, suggesting that hypercitrullination contributes to enhanced neutrophil adhesion. Furthermore, pemafibrate reduced the citrullination of histone H3 in these mice. Therefore, the hypercitrullination of histone H3 in neutrophils contributes to atherosclerotic vascular inflammation.

Toll-like receptor 2 mediates apolipoprotein CIII-induced monocyte activation.

Apolipoprotein (apo)CIII predicts risk for coronary heart disease. We recently reported that apoCIII directly activates human monocytes. Recent evidence indicates that toll-like receptor (TLR)2 can contribute to atherogenesis through transduction of inflammatory signals. Here, we tested the hypothesis that apoCIII activates human monocytoid THP-1 cells through TLR2. ApoCIII induced the association of TLR2 with myeloid differentiation factor 88, activated nuclear factor (NF)-kappaB in THP-1 cells, and increased their adhesion to human umbilical vein endothelial cells (HUVECs). Anti-TLR2 blocking antibody, but not anti-TLR4 blocking antibody or isotype-matched IgG, inhibited these processes (P<0.05). ApoCIII bound with high affinity to human recombinant TLR2 protein and showed a significantly higher (P<0.05) and saturable binding to 293 cells overexpressing human TLR2 than to parental 293 cells with no endogenous TLR2. Overexpression of TLR2 in 293 cells augmented apoCIII-induced NF-kappaB activation and beta(1) integrin expression, processes inhibited by anti-apoCIII antibody as well as anti-TLR2 antibody. Exposure of peripheral blood monocytes isolated from C57BL/6 (wild-type) mice to apoCIII activated their NF-kappaB and increased their adhesiveness to HUVECs. In contrast, apoCIII did not activate monocytes from TLR2-deficient mice. Finally, intravenous administration to C57BL/6 mice of apoCIII-rich very-low-density lipoprotein (VLDL), but not of apoCIII-deficient VLDL, activated monocytes and increased their adhesiveness to HUVECs, processes attenuated by anti-TLR2 or anti-apoCIII antibody. ApoCIII-rich VLDL did not activate monocytes from TLR2-deficient mice. In conclusion, apoCIII activated monocytes at least partly through a TLR2-dependent pathway. The present study identifies a novel mechanism for proinflammatory and proatherogenic effects of apoCIII and a role for TLR2 in atherosclerosis induced by atherogenic lipoproteins.

Dynamic observation of mechanically-injured mouse femoral artery reveals an antiinflammatory effect of renin inhibitor.

OBJECTIVE: The renin-angiotensin-aldosterone system (RAS) plays a central role in atherosclerosis. To investigate the effects of a direct renin inhibitor aliskiren on vascular inflammation, we conducted leukocyte adhesion assays in vivo and in vitro using a novel real-time imaging system. METHODS AND RESULTS: Aliskiren (10 mg/kg/d) or PBS was administered to C57BL/6 mice (6-7 weeks of age; Oriental Yeast, Tokyo, Japan) for 2 weeks via an osmotic pump. Blood pressure was not significantly changed in the 2 groups throughout the experimental period. A perivascular cuff injury was then introduced to the femoral artery and real-time intravital microscopic observation was conducted 24 hours after injury. The number of adherent leukocytes was elevated in the injured mice without aliskiren (43.8+/-9.3/10(-2) mm(2)), whereas that was significantly reduced in the mice with aliskiren (18.4+/-4.4, P<0.05). Treatment of human umbilical vein endothelial cells (HUVECs) with aliskiren significantly reduced the adhesion of THP-1 cells to TNF-alpha-activated HUVECs (P<0.05). Interestingly, TNF-alpha-induced renin activity and angiotensin II production in HUVECs were also blunted by aliskiren. Furthermore, exogenous renin and angiotensin II abrogated the aliskiren-mediated reduction of THP-1 cell adhesion to HUVECs. CONCLUSIONS: Our in vivo and in vitro findings indicate a pivotal role for renin inhibition in vascular inflammation independent of blood pressure.

HFD-induced hepatic lipid accumulation and inflammation are decreased in Factor D deficient mouse.

Excessive intake of fat causes accumulation of fat in liver, leading to non-alcoholic fatty liver disease (NAFLD). High-fat diet (HFD) upregulates the expression of Factor D, a complement pathway component, in the liver of mice. However, the functions of Factor D in liver are not well known. Therefore, the current study investigated the relationship between Factor D and hepatic lipid accumulation using CRISPR/Cas9-mediated Factor D knockout (FD-KO) mice. Factor D deficiency downregulated expression of genes related to fatty acid uptake and de novo lipogenesis in the liver. Furthermore, Factor D deficiency reduced the expression of inflammatory factors (Tnf and Ccl2) and fibrosis markers and decreased accumulation of F4/80-positive macrophages. These data suggest that the Factor D deficiency improved hepatic lipid accumulation and hepatic inflammation in HFD-fed mice.

Apolipoprotein CIII links hyperlipidemia with vascular endothelial cell dysfunction.

BACKGROUND: Apolipoprotein CIII (apoCIII) is a component of some triglyceride-rich very-low-density and low-density lipoprotein and is elevated in dyslipidemia with insulin resistance and the metabolic syndrome. We previously reported that apoCIII directly activates proinflammatory and atherogenic signaling in vascular endothelial cells through protein kinase C-beta (PKCbeta). Because PKCbeta impairs the response of vascular endothelial cells to insulin, we tested the hypothesis that apoCIII affects insulin signaling in vascular endothelial cells and its function in vitro and in vivo. METHODS AND RESULTS: ApoCIII inhibited insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), decreasing phosphatidylinositol 3-kinase (PI3K)/Akt activation in human umbilical vein endothelial cells. These effects of apoCIII led to reduced endothelial nitric oxide synthase (eNOS) activation and NO release into the media. ApoCIII activated PKCbeta in human umbilical vein endothelial cells, resulting in IRS-1 dysfunction via serine phosphorylation. ApoCIII also activated mitogen-activated protein kinase through PKCbeta. The impaired insulin signaling was restored by PKCbeta inhibitor or MEK1 inhibitor. ApoCIII-rich very-low-density lipoprotein and apoCIII impaired insulin signaling in the aorta of C57BL/6J mice and in human umbilical vein endothelial cells, which was recovered by PKCbeta inhibitor. They also inhibited endothelium-dependent relaxation of the aortas of C57BL/6J mice. In summary, apoCIII in very-low-density lipoprotein impaired insulin stimulation of NO production by vascular endothelium and induced endothelial dysfunction in vivo. This adverse effect of apoCIII was mediated by its activation of PKCbeta, which inhibits the IRS-1/PI3K/Akt/eNOS pathway. CONCLUSIONS: Our results suggest that apoCIII is a crucial link between dyslipidemia and insulin resistance in vascular endothelial cells with consequential deleterious effects on their atheroprotective functions.

7-Ketocholesterol enhances leukocyte adhesion to endothelial cells via p38MAPK pathway.

7-Ketocholesterol is a major dietary cholesterol oxidation product found in high concentrations in atherosclerotic plaques, which contribute to the development of atherosclerosis. This study aimed to investigate the effects of 7-ketocholesterol on endothelial inflammation, as well as the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with 7-ketocholesterol significantly enhanced the total interactions between human monocytic cells (THP-1 cell line) and TNFα-activated HUVECs under physiological flow conditions, compared to pretreatment with cholesterol (TNFα+50 µM cholesterol: 13.1 ± 0.54 cells/CPF, TNFα+50 µM 7-ketocholesterol: 18.9 ± 0.35 cells/CPF, p < 0.01). 7-Ketocholesterol enhanced the expression of E-selectin, ICAM-1, and VCAM-1 proteins. It also activated p38 mitogen-activated protein kinase (MAPK), and treatment with a p38 MAPK inhibitor inhibited both E-selectin expression via ATF-2 activation and 7-ketocholesterol-induced THP-1 adhesion to HUVECs. These findings suggest that 7-ketocholesterol enhances leukocyte-endothelial interactions by upregulating the expression of adhesion molecules, presumably via the p38 MAPK-dependent pathway.

Roles for Cell-Cell Adhesion and Contact in Obesity-Induced Hepatic Myeloid Cell Accumulation and Glucose Intolerance.

Obesity promotes infiltration of inflammatory cells into various tissues, leading to parenchymal and stromal cell interaction and development of cellular and organ dysfunction. Liver sinusoidal endothelial cells (LSECs) are the first cells that contact portal blood cells and substances in the liver, but their functions in the development of obesity-associated glucose metabolism remain unclear. Here, we find that LSECs are involved in obesity-associated accumulation of myeloid cells via VLA-4-dependent cell-cell adhesion. VLA-4 blockade in mice fed a high-fat diet attenuated myeloid cell accumulation in the liver to improve hepatic inflammation and systemic glucose intolerance. Ex vivo studies further show that cell-cell contact between intrahepatic leukocytes and parenchymal hepatocytes induces gluconeogenesis via a Notch-dependent pathway. These findings suggest that cell-cell interaction between parenchymal and stromal cells regulates hepatic glucose metabolism and offers potential strategies for treatment or prevention of obesity-associated glucose intolerance.

Crucial Role of the Aryl Hydrocarbon Receptor (AhR) in Indoxyl Sulfate-Induced Vascular Inflammation.

AIM: The aryl hydrocarbon receptor (AhR), a ligand-inducible transcription factor mediating toxic effects of dioxins and uremic toxins, has recently emerged as a pathophysiological regulator of immune-inflammatory conditions. Indoxyl sulfate, a uremic toxin, is associated with cardiovascular disease in patients with chronic kidney disease and has been shown to be a ligand for AhR. The aim of this study was to investigate the potential role of AhR in indoxyl sulfate-induced leukocyte-endothelial interactions. METHODS: Endothelial cell-specific AhR knockout (eAhR KO) mice were produced by crossing AhR floxed mice with Tie2 Cre mice. Indoxyl sulfate was administered for 2 weeks, followed by injection of TNF-α. Leukocyte recruitment to the femoral artery was assessed by intravital microscopy. Vascular endothelial cells were transfected with siRNA specific to AhR (siAhR) and treated with indoxyl sulfate, followed by stimulation with TNF-α. RESULTS: Indoxyl sulfate dramatically enhanced TNF-α-induced leukocyte recruitment to the vascular wall in control animals but not in eAhR KO mice. In endothelial cells, siAhR significantly reduced indoxyl sulfate-enhanced leukocyte adhesion as well as E-selectin expression, whereas the activation of JNK and nuclear factor-κB was not affected. A luciferase assay revealed that the region between －153 and －146 bps in the E-selectin promoter was responsible for indoxyl sulfate activity via AhR. Mutational analysis of this region revealed that activator protein-1 (AP-1) is responsible for indoxyl sulfate-triggered E-selectin expression via AhR. CONCLUSION: AhR mediates indoxyl sulfate-enhanced leukocyte-endothelial interactions through AP-1 transcriptional activity, which may constitute a new mechanism of vascular inflammation in patients with renal disease.

Inhibitory Effect of Serotonin Antagonist on Leukocyte-Endothelial Interactions In Vivo and In Vitro.

BACKGROUND: Although 5-HT2A serotonergic antagonists have been used to treat vascular disease in patients with diabetes mellitus or obesity, their effects on leukocyte-endothelial interactions have not been fully investigated. In this study, we assessed the effects of sarpogrelate hydrochloride (SRPO), a 5-HT2A receptor inverse agonist, on leukocyte-endothelial cell interactions in obesity both in vivo and in vitro. METHODS AND FINDINGS: In the in vivo experiment, C57BL/6 mice were fed a high-fat high-fructose diet (HFFD), comprising 20% fat and 30% fructose, with or without intraperitoneal injection of 5 mg/kg/day SRPO for 4 weeks. The body weight, visceral fat weight, and serum monocyte chemoattractant protein-1 levels in the mice increased significantly with the HFFD, but these effects were prevented by chronic injections of SRPO. Intravital microscopy of the femoral artery detected significant leukocyte-endothelial interactions after treatment with HFFD, but these leukocyte-endothelial interactions were reduced in the mice injected with SRPO. In the in vitro experiment, pre-incubation of activated human umbilical vein endothelial cells (HUVECs) with platelet-rich plasma (PRP) induced THP-1 cell adhesion under physiological flow conditions, but the adhesion was reduced by pretreatment of PRP with SRPO. A fluorescent immunobinding assay showed that PRP induced significant upregulation of E-selectin in HUVECs, but this upregulation was reduced by pretreatment of PRP with SRPO. In other in vitro conditions, pre-incubation of THP-1 cells with phorbol 12-myristate 13-acetate increased the adhesion of THP-1 cells to activated HUVECs under rotational conditions, but this adhesion was reduced by pretreatment with SRPO. Western blotting analysis showed that protein kinase C α activation in THP-1 cells was inhibited by SRPO. CONCLUSION: Our findings indicated that SRPO inhibits vascular inflammation in obesity via inactivation of platelets and leukocytes, and improvement of obese.

Critical role of the C5a-activated neutrophils in high-fat diet-induced vascular inflammation.

Exceed and chronic high-fat diet (HFD) contributes to the diagnosis and development of atherosclerosis, obesity, and metabolic syndrome. However, the key molecular component(s) triggered by HFD responsible for initiating vascular inflammation remain unknown. We observed that feeding HFD for 4 weeks is sufficient to induce leukocyte recruitment in the femoral artery of wild-type mice. Neutrophil- and monocyte-depletion analyses confirmed the preferential recruitment of neutrophils in these mice. Protein analysis of sera from HFD-fed mice revealed a marked elevation of complement component C5a levels. Exogenous C5a alone induced leukocyte recruitment, which was abolished by a C5a-receptor antagonist. We also examined the role of neutrophil-derived MCP-1 in accumulation of leukocytes in the artery. These results demonstrated a previously unrecognized role for C5a and neutrophils in the early onset of HFD-induced vascular inflammation. Further study may help in elucidating a novel regulatory pathway to control diet-induced inflammation such as that in case of atherosclerosis.

Ezetimibe reduced hepatic steatosis induced by dietary oxysterols in nonhuman primates.

Oxidized cholesterol (oxysterols) plays an important and multifaceted role in lipid metabolism. Here we examined whether dietary oxysterols accelerate hepatic lipid accumulation and inflammation in nonhuman primates. We also examined the effect of the Niemann-Pick C1-like1 inhibitor, ezetimibe (Ez). Macaca fascicularis (5-year-old males) were fed either regular cholesterol + high-fat diet (control-HFD) or oxysterols + high-fat diet (ox-HFD; with 0.015% of oxysterols cholesterol) for 24 weeks. Compared with control-HFD, ox-HFD did not affect plasma lipid levels, but it did affect hepatic lipid levels [total cholesterol, 40.9 mg·g-1 (ox-HFD) versus 3.2 (control-HFD) mg·g-1; triglycerides, 28.0 (ox-HFD) versus 5.7 (control-HFD) mg·g-1]. Ox-HFD increased lipid accumulation as well as recruitment of inflammatory cells when compared to control-HFD. We then examined the effects of Ez, 0.2 mg·kg-1·day-1 for 12 weeks. In addition to a significant reduction in dyslipidemia, Ez alleviated biochemical and pathological aspects of steatosis. Dietary oxysterols aggravate steatosis in nonhuman primates. Treatment with Ez may be a novel therapeutic approach to NAFLD by alleviating dyslipidemia.

Cell density impacts epigenetic regulation of cytokine-induced E-selectin gene expression in vascular endothelium.

Growing evidence suggests that the phenotype of endothelial cells during angiogenesis differs from that of quiescent endothelial cells, although little is known regarding the difference in the susceptibility to inflammation between both the conditions. Here, we assessed the inflammatory response in sparse and confluent endothelial cell monolayers. To obtain sparse and confluent monolayers, human umbilical vein endothelial cells were seeded at a density of 7.3 × 10(3) cells/cm(2) and 29.2 × 10(3) cells/cm(2), respectively, followed by culturing for 36 h and stimulation with tumor necrosis factor α. The levels of tumor necrosis factor α-induced E-selectin protein and mRNA expression were higher in the confluent monolayer than in the sparse monolayer. The phosphorylation of c-jun N-terminal kinase and p38 mitogen-activated protein kinase or nuclear factor-κB activation was not involved in this phenomenon. A chromatin immunoprecipitation assay of the E-selectin promoter using an anti-acetyl-histone H3 antibody showed that the E-selectin promoter was highly and specifically acetylated in the confluent monolayer after tumor necrosis factor α activation. Furthermore, chromatin accessibility real-time PCR showed that the chromatin accessibility at the E-selectin promoter was higher in the confluent monolayer than in the sparse monolayer. Our data suggest that the inflammatory response may change during blood vessel maturation via epigenetic mechanisms that affect the accessibility of chromatin.