Search

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.

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.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 β2-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.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 β2-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 β2-integrin, and F-actin polymerization.Cytoplasmic translocation of PAD4 by CXCL1 induces neutrophil adhesion to vascular endothelial cells and citrullination of PDIA1.

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.

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.

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.

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)-κB 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 P 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-κB 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.

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.

Wire injury of an artery has been recognized as a standard model of vascular inflammation and atherosclerosis; however, the mechanism of leukocyte recruitment has not been studied in this model. In this study, we documented the recruitment of leukocytes to the murine femoral artery after a wire injury. A transluminal mechanical injury was generated by insertion of a wire into the femoral artery of male C57BL/6J mice. The mice were anesthetized and ventilated after tracheotomy and protected from hypothermia by a warming lamp. Body temperature and blood pH did not significantly change during the experiment. The interaction between rhodamine 6G-labeled leukocytes and the injured femoral artery was monitored using an epifluorescent microscope, and the images were evaluated using a computer-assisted image analysis program. In the absence of injury, virtually no leukocyte adhesion was observed. In contrast, the number of adherent leukocytes increased 4 and 24 h after injury and declined 72 h after injury. The rolling flux of leukocytes increased 4 h after injury and remained high up to 7 days, but it was faster 72 h after injury. We identified another peak of leukocyte adhesion 7 days after injury. Injection of anti-P-selectin antibody significantly reduced leukocyte adhesion at the early and later phases. In conclusion, we have established a novel experimental system for direct observation of leukocyte recruitment to the injured femoral artery. Our system revealed a previously undetected, unique profile of leukocyte recruitment during vascular injury.

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.

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.

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.

Background Although inflammation within adipose tissues is known to play a role in metabolic syndrome, the causative connection between inflamed adipose tissue and atherosclerosis is not fully understood. In the present study, we examined the direct effects of adipose tissue on macro-vascular inflammation using intravital microscopic analysis of the femoral artery after adipose tissue transplantation. Methods and Results We obtained subcutaneous (SQ) and visceral (VIS) adipose tissues from C57BL/6 mice fed normal chow (NC) or a high fat diet (HF), then transplanted the tissues into the perivascular area of the femoral artery of recipient C57/BL6 mice. Quantitative intravital microscopic analysis revealed an increase in adherent leukocytes after adipose tissue transplantation, with VIS found to induce significantly more leukocyte accumulation as compared to SQ. Moreover, adipose tissues from HF fed mice showed significantly more adhesion to the femoral artery. Simultaneous flow cytometry demonstrated upregulation of CD11b on peripheral granulocyte and monocytes after adipose tissue transplantation. We also observed dominant expressions of the inflammatory cytokine IL-6, and chemokines MCP-1 and MIP-1β in the stromal vascular fraction (SVF) of these adipose tissues as well as sera of recipient mice after transplantation. Finally, massive accumulations of pro-inflammatory and dendritic cells were detected in mice with VIS transplantation as compared to SQ, as well as in HF mice as compared to those fed NC. Conclusion Our in vivo findings indicate that adipose tissue stimulates leukocyte accumulation in the femoral artery. The underlying mechanisms involve upregulation of CD11b in leukocytes, induction of cytokines and chemokines, and accumulation of pro-inflammatory cells in the SVF.

Recent studies have demonstrated a potential synergistic effect of the combination of amlodipine with atorvastatin to reduce acute inflammation. The intraluminal wire injury of the mouse femoral artery induced significant leukocyte recruitment to the injured area and oxidative stress within 24 h. Administration of low-dose amlodipine (0.5 mg kg(-1) per day) or atorvastatin (1 mg kg(-1) per day) alone for 7 days failed to modulate leukocyte adhesion, whereas their co-administration for 7 days significantly inhibited leukocyte adhesion. Moreover, flow cytometric analysis showed that injury-induced oxidative stress and CD11b expression in three leukocyte fractions were elevated after injury and then reduced after the co-administration. Next, adoptive transfer of mononuclear cells (MNCs) was performed and MNCs were harvested from mice after wire injury exhibited adhesion to the recipient injured artery. Furthermore, the co-administration of low-dose atorvastatin and amlodipine to MNCs or the vasculature reduced the recruitment of MNCs to the injured artery. Our findings indicate that amlodipine and atorvastatin synergistically inhibit vascular inflammation. The underlying mechanisms of their effect involve, at least in part, stabilizing oxidative stress at the point of injury, suggesting the clinical efficacy of this drug combination for the treatment of vascular diseases.

Leukocyte recruitment plays a pivotal role during inflammation after vascular injury. The importance of oxidative stress in vascular injury and its modulation by angiotensin II receptor blockers (olmesartan) have been demonstrated. We examined the contribution of leukocyte-associated oxidative stress in acute-phase leukocyte recruitment and its modulation by olmesartan. Male mice were treated with olmesartan (5 mg/kg per day) or vehicle for 7 days before the transluminal wire injury of the femoral artery. Intravital microscopy of the artery revealed that the mechanical injury increased adherent leukocytes at both 24 hours and 7 days after the injury, which was significantly reduced by olmesartan treatment. Dihydroethidium-associated fluorescence intensity observed in vehicle-treated mice was significantly diminished under olmesartan treatment. Apocynin, a nicotinamide-adenine dinucleotide phosphate oxidase inhibitor, showed a similar inhibitory effect on the leukocyte adhesion. Adoptive transfer of mononuclear cells, harvested from mice after wire injury, but not from those without wire injury, exhibited adhesion to the recipient injured artery. Furthermore, olmesartan treatment of mononuclear cells, but not of injured vasculature, reduced their recruitment to the injured artery. These data indicate that leukocyte recruitment to the mechanically injured artery is mediated by oxidative stress in leukocytes but not in vasculatures. Treatment with olmesartan blocked leukocyte recruitment by antagonizing mononuclear cells-associated oxidative stress.

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.