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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.

Visual Abstract, Highlights • Neutrophil adhesion to the vascular endothelium in the femoral artery of low-density lipoprotein receptor–null mice fed with HFD was significantly enhanced compared with wild-type mice fed with HFD or low-density lipoprotein receptor–null mice fed with NC. • HFD induced citrullinated histone H3 of neutrophils in LDL receptor–null mice through the up-regulation of CXCL1 in blood. • Hypercitrullination of histone H3 in neutrophils by CXCL1 enhanced neutrophil adhesion in vitro and in vivo. • Pemafibrate decreased neutrophil adhesion and citrullination of histone H3 through the reduction of CXCL1., Summary 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.

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.

Recent studies have reported that dipeptidyl-peptitase IV (DPP-IV) is correlated with diabetic conditions and also with dyslipidemia caused by overnutrition, especially a high fat diet. However, the role of DPP-IV in diabetes during dyslipidemia has been unclear. We utilized a lymph fistula rat model to determine whether intestinal lymph, which absorbs dietary fats, is affected by a chronic high-fat and high-cholesterol diet (HFHC). HFHC diet rats showed significantly higher DPP-IV activity in intestinal lymph and plasma compared to rats receiving a normal chow diet. In addition, HFHC diet rats showed significantly increased DPP-IV mRNA expression in the intestine. However, DPP-IV mRNA in the lymphocytes isolated from intestinal lymph and mesenteric lymph nodes did not show significant differences from that in the normal diet rats. In conclusion, HFHC diets increased DPP-IV expression in intestinal lymph; these results indicate the applicability of a previously unrecognized role for DPP-IV in metabolic disorders, including diabetes.

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.

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 ?B 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 ?B 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. Conclusion— 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.

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.

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-β (PKCβ). Because PKCβ 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 PKCβ in human umbilical vein endothelial cells, resulting in IRS-1 dysfunction via serine phosphorylation. ApoCIII also activated mitogen-activated protein kinase through PKCβ. The impaired insulin signaling was restored by PKCβ 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 PKCβ 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 PKCβ, which inhibits the IRS-1/PI3K/Akt/eNOS pathway. Conclusion— 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.

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.

Objective. To monitor the anti-inflammatory effect of rosuvastatin in leukocyte endothelial interactions in the atheroprone femoral artery in vivo.Methods and Results. Male Apolipoprotein E null mice (ApoE−/− mice, 6 weeks old) were fed a high-fat diet (20% fat, 1.25% cholesterol) with or without the HMG CoA reductase inhibitor rosuvastatin (10 mg/kg/day) for 6 weeks. Significant leukocyte adhesion was observed in the femoral artery of ApoE−/− mice, but not of wild type mice, in the absence of rosuvastatin. Interestingly, no obvious plaque formation was observed in the artery at this time point. The number of adherent leukocytes was dramatically diminished in ApoE−/− mice treated with rosuvastatin. DHE-associated oxidative stress and the expression of gp91-phox, a component of NADPH oxidase, were induced in ApoE−/− mice and were abolished by rosuvastatin treatment.Conclusion. Our data documented leukocyte recruitment prior to lipid accumulation and subsequent inhibition by rosuvastatin. The underlying mechanism seemed to involve oxidative stress and an anti-inflammatory effect on the endothelium of atheroprone vessels.

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.

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±9.3/10−2mm2), whereas that was significantly reduced in the mice with aliskiren (18.4±4.4,PPConclusions—Our in vivo and in vitro findings indicate a pivotal role for renin inhibition in vascular inflammation independent of blood pressure.

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.

Objective Apolipoprotein (apo) CIII, a prominent component of atherogenic dyslipidemia, is elevated in patients with type2 diabetes and insulin resistance, and independently predicts cardiovascular disease (CVD). Endothelial dysfunction is an important component of the pathophysiology of atherosclerosis, and is associated with insulin resistance. We previously reported that apoCIII has a direct effect on vascular endothelium, activating endothelial cells to recruit monocytes through PKCβ and NF-κB-dependent mechanisms. In the present study, we investigated the effect of apoCIII on insulin-dependent endothelial nitric oxide synthase (eNOS) function in endothelial cells and in the aortas of C57BL/6J mice. Methods and Results ApoCIII treatment (100μg/ml, 30 minutes) inhibited insulin-induced ISR-1/phosphatidylinositol 3-kinase (PI3K)/Akt activation in human umbilical vein endothelial cells (HUVECs). Furthermore, apoCIII reduced insulin-stimulated eNOS activation and NO release into the media. ApoCIII induced PKCβII activation in HUVECs, resulting in IRS-1 phosphorylation at pSer 616 and ERK activation. Impaired insulin-stimulated eNOS activation and NO production were restored by PKCβ inhibitor and MEK1 inhibitor to a lesser extent. In addition, treatment of C57BL/6J mice with apoCIII resulted in impaired insulin-dependent activation of PI3K/Akt/eNOS pathway in the aorta. Pretreatment with PKCβ inhibitor attenuated inhibitory effects of apoCIII. ApoCIII resides on VLDL fraction in the plasma, especially in the postprandial state. Injection of apoCIII-rich VLDL, but not apoCIII-deficient VLDL, also impaired these processes. Conclusion Our data suggest that apoCIII impairs insulin stimulation of NO production by vascular endothelium. This adverse effect of apoCIII is mediated by its activation of PKCβII which inhibits the IRS-1/PI3K/Akt/eNOS pathway. These results indicate that apoCIII not only modulates lipoprotein metabolism, but also may directly contribute to the development of diabetic complications through endothelial dysfunction. Finally, our results may point to a novel mechanism that links dyslipidemia, insulin resistance and endothelial dysfunction.