A Potential Mechanism of High-Dose Ticagrelor in Modulating Platelet Activity and Atherosclerosis Mediated by Thymic Stromal Lymphopoietin Receptor.

Abnormal expression of thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR) was found in patients with acute coronary syndrome. Ticagrelor, an oral platelet ADP P2Y12 receptor antagonist, is widely used in these patients. The aim of this study was to verify whether different doses of ticagrelor regulated plaque progression and platelet activity by modulating TSLP/TSLPR. Seventy-five ApoE-/- mice were randomly divided into five groups: (1) high-cholesterol diet (HCD, n = 15); (2) HCD plus ticagrelor 25 mg/kg/d (T1, n = 15); (3) HCD plus ticagrelor 50 mg/kg/d (T2, n = 15); (4) HCD plus ticagrelor 100 mg/kg/d (T3, n = 15); and (5) a normal diet group (ND, n = 15). At day 0 and at week 16, blood lipids and serum TSLP levels, expression of TSLPR, CD62, and CD63, platelet aggregation, platelet ATP release, PI3K/Akt signaling pathway, and plaque morphology were assessed. HCD increased TSLPR expression and atherosclerosis progression but high-dose ticagrelor (100 mg/kg) moderated this trend. TSLPR was positively correlated with Akt1, platelet aggregation, corrected plaque area, and vulnerability index in the T3 group (P<0.01). In conclusion, low-dose ticagrelor only inhibited platelet activity. Besides this inhibition, high-dose ticagrelor modulated platelet activity and atherosclerosis mediated by TSLPR, potentially through the PI3K/Akt signal pathway.

Expansion of myeloid-derived suppressor cells in patients with acute coronary syndrome.

AIM: The aim of this study was to explore whether the circulating frequency and function of myeloid-derived suppressor cells (MDSCs) are altered in patients with acute coronary syndrome (ACS). METHODS: The frequency of MDSCs in peripheral blood was determined by flow cytometry, and mRNA expression in purified MDSCs was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). The suppressive function of MDSCs isolated from different groups was also determined. The plasma levels of certain cytokines were determined using Bio-Plex Pro™ Human Cytokine Assays. RESULTS: The frequency of circulating CD14(+)HLA-DR(-/low) MDSCs; arginase-1 (Arg-1) expression; and plasma levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, and IL-33 were markedly increased in ACS patients compared to stable angina (SA) or control patients. Furthermore, MDSCs from ACS patients were more potent suppressors of T-cell proliferation and IFN-γ production than those from the SA or control groups at ratios of 1:4 and 1:2; this effect was partially mediated by Arg-1. In addition, the frequency of MDSCs was positively correlated with plasma levels of IL-6, IL-33, and TNF-α. CONCLUSIONS: We observed an increased frequency and suppressive function of MDSCs in ACS patients, a result that may provide insights into the mechanisms involved in ACS.

Regulatory T cells protect fine particulate matter-induced inflammatory responses in human umbilical vein endothelial cells.

OBJECTIVE: To investigate the role of CD4(+)CD25(+) T cells (Tregs) in protecting fine particulate matter (PM-) induced inflammatory responses, and its potential mechanisms. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with graded concentrations (2, 5, 10, 20, and 40 µg/cm(2)) of suspension of fine particles for 24h. For coculture experiment, HUVECs were incubated alone, with CD4(+)CD25(-) T cells (Teff), or with Tregs in the presence of anti-CD3 monoclonal antibodies for 48 hours, and then were stimulated with or without suspension of fine particles for 24 hours. The expression of adhesion molecules and inflammatory cytokines was examined. RESULTS: Adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and inflammatory cytokines, such as interleukin (IL-) 6 and IL-8, were increased in a concentration-dependent manner. Moreover, the adhesion of human acute monocytic leukemia cells (THP-1) to endothelial cells was increased and NF- κ B activity was upregulated in HUVECs after treatment with fine particles. However, after Tregs treatment, fine particles-induced inflammatory responses and NF- κ B activation were significantly alleviated. Transwell experiments showed that Treg-mediated suppression of HUVECs inflammatory responses impaired by fine particles required cell contact and soluble factors. CONCLUSIONS: Tregs could attenuate fine particles-induced inflammatory responses and NF- κ B activation in HUVECs.

GABA and topiramate inhibit the formation of human macrophage-derived foam cells by modulating cholesterol-metabolism-associated molecules.

AIMS: γ-aminobutyric acid (GABA), the principal inhibitory neurotransmitter, acts on GABA receptors to play an important role in the modulation of macrophage functions. The present study examined the effects of GABA and a GABA receptor agonist on modulating cholesterol-metabolism-associated molecules in human monocyte-derived macrophages (HMDMs). METHODS: ORO stain, HPLC, qRT-PCR, Western blot and EMSA were carried out using HMDMs exposed to ox-LDL with or without GABAergic agents as the experimental model. RESULTS: GABA and topiramate reduced the percentage of cholesterol ester in lipid-laden HMDMs by down-regulating SR-A, CD36 and LOX-1 expression and up-regulating ABCA1, ABCG1 and SR-BI expression in lipid-laden HMDMs. The production of TNF-α was decreased in GABA-and topiramate-treated lipid-laden HMDMs, and levels of interleukin (IL)-6 did not change. The activation of two signaling pathways, p38MAPK and NF-κB, was repressed by GABA and topiramate in lipid-laden HMDMs. CONCLUSION: GABA and topiramate inhibit the formation of human macrophage-derived foam cells and may be a possibility for macrophage targeted therapy of atherosclerotic lesions.

Association between genetic variations in TFR2 gene and coronary heart disease in Chinese: a case-control study.

AIMS: Studies indicated that body iron stores were associated with coronary heart disease (CHD). Type 2 transferrin receptor (TFR2) participates in cellular iron overload and is related to cardiovascular disease. No studies investigated the associations between variants in TFR2 gene and CHD risk. METHODS: We sought to investigate this association in a Chinese Han population and performed a case-control study recruiting 1264 CHD patients and 1264 age and sex frequency matched controls. TaqMan single nucleotide polymorphisms (SNP) allelic discrimination was used to examine genotypes of the tagging single nucleotide polymorphisms (tagSNPs) of TFR2. The plasma ferritin levels were measured by ELISA. RESULTS: We did not find significant associations between variants of TFR2 gene (including tagSNPs rs2075674 and rs7385804) and the risk of CHD. After adjustment for the conventional risk factors of CHD, such as smoking and age, the results did not materially alter. Interaction analyses indicated that there were no significant interactions between conventional risk factors of CHD and these two tagSNPs on CHD risk. Among different genotypes of these two tagSNPs, no significant differences in plasma ferritin levels were found. CONCLUSION: In summary, the variants of rs2075674 and rs7385804 in TFR2 gene were not associated with CHD risk in a Chinese Han population.

Specific Kv1.3 blockade modulates key cholesterol-metabolism-associated molecules in human macrophages exposed to ox-LDL.

Cholesterol-metabolism-associated molecules, including scavenger receptor class A (SR-A), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), CD36, ACAT1, ABCA1, ABCG1, and scavenger receptor class B type I, can modulate cholesterol metabolism in the transformation from macrophages to foam cells. Voltage-gated potassium channel Kv1.3 has increasingly been demonstrated to play an important role in the modulation of macrophage function. Here, we investigate the role of Kv1.3 in modulating cholesterol-metabolism-associated molecules in human acute monocytic leukemia cell-derived macrophages (THP-1 macrophages) and human monocyte-derived macrophages exposed to oxidized LDL (ox-LDL). Human Kv1.3 and Kv1.5 channels (hKv1.3 and hKv1.5) are expressed in macrophages and form a heteromultimeric channel. The hKv1.3-E314 antibody that we had generated as a specific hKv1.3 blocker inhibited outward delayed rectifier potassium currents, whereas the hKv1.5-E313 antibody that we had generated as a specific hKv1.5 blocker failed. Accordingly, the hKv1.3-E314 antibody reduced percentage of cholesterol ester and enhanced apoA-I-mediated cholesterol efflux in THP-1 macrophages and human monocyte-derived macrophages exposed to ox-LDL. The hKv1.3-E314 antibody downregulated SR-A, LOX-1, and ACAT1 expression and upregulated ABCA1 expression in THP-1 macrophages and human monocyte-derived macrophages. Our results reveal that specific Kv1.3 blockade represents a novel strategy modulating cholesterol metabolism in macrophages, which benefits the treatment of atherosclerotic lesions.

Curcumin serves as a human kv1.3 blocker to inhibit effector memory T lymphocyte activities.

Curcumin, the principal active component of turmeric, has long been used to treat various diseases in India and China. Recent studies show that curcumin can serve as a therapeutic agent for autoimmune diseases via a variety of mechanisms. Effector memory T cells (T(EM), CCR7⁻ CD45RO⁺ T lymphocyte) have been demonstrated to play a crucial role in the pathogenesis of T cell-mediated autoimmune diseases, such as multiple sclerosis (MS) or rheumatoid arthritis (RA). Kv1.3 channels are predominantly expressed in T(EM) cells and control T(EM) activities. In the present study, we examined the effect of curcumin on human Kv1.3 (hKv1.3) channels stably expressed in HEK-293 cells and its ability to inhibit proliferation and cytokine secretion of T(EM) cells isolated from patients with MS or RA. Curcumin exhibited a direct blockage of hKv1.3 channels in a time-dependent and concentration-dependent manner. Moreover, the activation curve was shifted to a more positive potential, which was consistent with an open-channel blockade. Paralleling hKv1.3 inhibition, curcumin significantly inhibited proliferation and interferon-γ secretion of T(EM) cells. Our findings demonstrate that curcumin is able to inhibit proliferation and proinflammatory cytokine secretion of T(EM) cells probably through inhibition of hKv1.3 channels, which contributes to the potency of curcumin for the treatment of autoimmune diseases. This is probably one of pharmacological mechanisms of curcumin used to treat autoimmune diseases.

The antibody targeting the E314 peptide of human Kv1.3 pore region serves as a novel, potent and specific channel blocker.

Selective blockade of Kv1.3 channels in effector memory T (T(EM)) cells was validated to ameliorate autoimmune or autoimmune-associated diseases. We generated the antibody directed against one peptide of human Kv1.3 (hKv1.3) extracellular loop as a novel and possible Kv1.3 blocker. One peptide of hKv1.3 extracellular loop E3 containing 14 amino acids (E314) was chosen as an antigenic determinant to generate the E314 antibody. The E314 antibody specifically recognized 63.8KD protein stably expressed in hKv1.3-HEK 293 cell lines, whereas it did not recognize or cross-react to human Kv1.1(hKv1.1), Kv1.2(hKv1.2), Kv1.4(hKv1.4), Kv1.5(hKv1.5), KCa3.1(hKCa3.1), HERG, hKCNQ1/hKCNE1, Nav1.5 and Cav1.2 proteins stably expressed in HEK 293 cell lines or in human atrial or ventricular myocytes by Western blotting analysis and immunostaining detection. By the technique of whole-cell patch clamp, the E314 antibody was shown to have a directly inhibitory effect on hKv1.3 currents expressed in HEK 293 or Jurkat T cells and the inhibition showed a concentration-dependence. However, it exerted no significant difference on hKv1.1, hKv1.2, hKv1.4, hKv1.5, hKCa3.1, HERG, hKCNQ1/hKCNE1, L-type Ca(2+) or voltage-gated Na(+) currents. The present study demonstrates that the antibody targeting the E314 peptide of hKv1.3 pore region could be a novel, potent and specific hKv1.3 blocker without affecting a variety of closely related K(v)1 channels, KCa3.1 channels and functional cardiac ion channels underlying central nervous system (CNS) disorders or drug-acquired arrhythmias, which is required as a safe clinic-promising channel blocker.