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.

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.

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.

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.

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.

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.

Ezetimibe improves liver steatosis and insulin resistance in obese rat model of metabolic syndrome.

Non-alcoholic fatty liver disease (NAFLD) is associated with the metabolic syndrome characterized by dislipidemia and insulin resistance. We hypothesized that ezetimibe, an inhibitor of NPC1L1, improves these metabolic disorders in Zucker obese fatty rats (ZOF). Ezetimibe significantly lowered total cholesterol and triglycerides in ZOF with prominent reduction in the remnant lipoprotein fraction and small dense low density lipoprotein fraction. Moreover, lipid deposition and fibrosis of liver were decreased by ezetimibe. Interestingly, ezetimibe improved insulin and plasma glucose response after intraperitoneal glucose injection. Further, ezetimibe enhanced insulin signaling in cultured hepatocytes. Our results indicate the potential of ezetimibe in treating the metabolic syndrome and NAFLD.

Functional impact of IgA nephropathy-associated selectin gene haplotype on leukocyte-endothelial interaction.

Previously, we discovered single-nucleotide polymorphisms (SNPs) associated with Immunoglobulin A (IgA) nephropathy in selectin genes, which were 712C>T(P238S) in L selectin, -642A>G in the promoter region of L selectin, and 1402C>T(H468Y) in E selectin. Interestingly, these SNPs were in nearly complete linkage disequilibrium, thus two haplotypes, disease-associated TGT and wild-type (Wt) CAC, were constructed. To investigate the functional significance of TGT haplotype, a stable CHO transfectant expressing a P238S-L-selectin variant (CHO-varL) and a recombinant adenovirus vector containing an H468Y-E-selectin variant (Ad-varE) were established and compared to their Wt counterparts. Under flow, CHO-varL exhibited significantly less adhesion over IL-1beta-activated HUVEC monolayers compared to CHO-wtL. Furthermore, a luciferase reporter construct, containing a promoter region of the L-selectin variant (luc-varL), exhibited significantly less transcription activity compared to Wt (luc-wtL). These results suggest that the adhesive interactions and expression level of L selectin in disease-associated haplotypes are significantly compromised, indicating a potential role of these SNPs in the pathogenesis of inflammatory diseases, including IgA nephropathy.