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

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.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.Our findings indicated that SRPO inhibits vascular inflammation in obesity via inactivation of platelets and leukocytes, and improvement of obese

THP-1 cell adhesion to HUVECs (Flow chamber assay) and expression of E-selectin in HUVECs.

<p>(A) SRPO significantly reduced the number of PRP-induced THP-1 cell that adhered to HUVECs. HUVEC monolayers were stimulated with 3 ng/ml TNF-α for 3.5 h and exposed to PPP or PRP for 20 min. PRP was pretreated or not with SRPO (10 μM) just before addition to the HUVECs. THP-1 cells were perfused over activated HUVEC monolayers at a flow rate of 1.0 dyne/cm². Adhesion assays were performed as described in Materials and Methods. Data are the mean ± SD of three independent experiments in each group. (B) SRPO significantly reduced the expression of PRP-induced E-selectin in HUVECs. E-selectin expression was determined by FIA as described in Materials and Methods. Data are the mean ± SD of 6 independent experiments in each group.</p

Effects of SRPO on HFFD-induced obesity.

<p>(A) Body weight (n = 17, 28, 29). (B) Epididymal fat weight (n = 13, 18, 18). (C) Liver weight (n = 17, 23, 25). Values are the mean ± SE in each group. (Abbreviations: NC = normal chow; HFFD = high-fat high-fructose diet; VEH = vehicle; SRPO = Sarpogrelate hydrochloride.)</p

SRPO significantly decreased leukocyte-endothelial interactions and serum MCP-1 level.

<p>(A) Image of leukocyte-endothelial interactions in the femoral arteries of mice (with the margins of vessels indicated by dotted lines). Arrows indicate adhered or rolling leukocytes. (B) The numbers of rolling (left) and adherent cells (right) in all groups were calculated as described in Methods. Values are the mean ± SE (n = 10, 16, 18). (C) Effect of SRPO on serum MCP-1 levels in HFFD-induced obesity. Values are the mean ± SE (n = 13, 13, 11). Serum MCP-1 level of the HFFD + VEH group was higher than in the NC group, and SRPO prevented the increase in serum MCP-1 level on the HFFD + VEH group.</p

THP-1 cell adhesion to HUVECs (Non-static rotational assay) and expression of PKCα in THP-1 cells.

<p>(A) SRPO significantly reduced PMA-triggered THP-1 cell adhesion to the HUVECs. THP-1 cells were pretreated with SRPO (10 μM) for 1 h and stimulated with PMA (10 nM) for 10 min before the assay. Preliminary experiments with trypan blue staining demonstrated that THP-1 cells were not damaged by SRPO (10 μM, 1h) treatment (data not shown). Adhesion assays were performed as described in Materials and Methods. Data are the mean ± SD of three independent experiments in each group. (B) SRPO attenuated PMA-induced PKCα activation in THP-1 cells. THP-1 cells were incubated in the presence or absence of SRPO (10 μM) for 1 h and stimulated with PMA (10 nM) for 10 min before the assay, and membrane proteins and total PKCα protein were detected by immunoblotting. Data are representative of three independent experiments.</p