过氧化体增殖物激活型受体γ对巨噬细胞脂质蓄积及CD36表达的影响

目的观察过氧化体增殖物激活型受体γ激动剂和拮抗剂对THP-1巨噬细胞胆固醇蓄积及CD36表达的影响。方法实验分对照组、氧化型低密度脂蛋白组、Ciglitazone处理组和GW9662处理组,后两组用50 mg/L氧化型低密度脂蛋白分别与过氧化体增殖物激活型受体γ激动剂Ciglitazone(10μmol/L)及拮抗剂GW9662(10μmol/L)共同孵育24 h,高效液相色谱分析法检测细胞总胆固醇蓄积情况,RT-PCR和Western blot分别检测THP-1巨噬细胞CD36 mRNA和蛋白的表达。结果与对照组(76.28±10.36 mg/g)相比,氧化型低密度脂蛋白(121.63±13.32 mg/g)能使细胞总胆固醇含量显著增加,而Ciglitazone能使氧化型低密度脂蛋白处理的细胞总胆固醇含量进一步增加(136.23±14.78 mg/g),GW9662能使氧化型低密度脂蛋白处理的细胞总胆固醇含量减少(98.52±11.45 mg/g)。过氧化体增殖物激活型受体γ拮抗剂GW9662使巨噬细胞CD36 mRNA和蛋白的表达下调及胆固醇蓄积减少,过氧化体增殖物激活型受体γ激动剂Ciglitazone使巨噬细胞CD36 mRNA和蛋白的表达上调及胆固醇蓄积增多。结论过氧化体增殖物激活型受体γ拮抗剂使THP-1巨噬细胞胆固醇蓄积减少及氧化型低密度脂蛋白诱导的CD36表达下调。     

肺炎衣原体通过上调清道夫受体A1诱导THP-1源性泡沫细胞形成

目的观察清道夫受体A1和CD36在肺炎衣原体诱导THP-1源性泡沫细胞形成中的作用。方法给予不同浓度的肺炎衣原体(1×105～1×106IFU)感染THP-1源性巨噬细胞0～72h。运用油红O染色观察细胞质内脂滴的变化,酶荧光学法检测细胞内胆固醇酯含量的变化。分别运用逆转录聚合酶链反应和Western-Blot检测清道夫受体A1和CD36的mRNA和蛋白表达。结果高浓度的肺炎衣原体(5×105和1×106IFU)感染负荷低密度脂蛋白的THP-1源性巨噬细胞48h后,细胞质内的脂滴明显增多,胆固醇酯占总胆固醇百分比明显增加(>50%)。在负荷低密度脂蛋白的THP-1源性巨噬细胞上,肺炎衣原体感染呈浓度和时间依赖性地上调道夫受体A1 mRNA和蛋白表达,但不影响CD36 mRNA和蛋白表达。结论清道夫受体A1表达上调是肺炎衣原体诱导THP-1源性泡沫细胞形成的机制之一,这可能为进一步阐明肺炎衣原体感染促进动脉粥样硬化发生发展提供一个新的理论依据。     

24-乙酰泽泻醇A对氧化型低密度脂蛋白诱导巨噬细胞脂代谢因子ABCA1、CD36及炎症因子CD147、MMP-9的影响

目的通过体外实验探讨24-乙酰泽泻醇A对氧化型低密度脂蛋白(ox-LDL)诱导大鼠腹腔巨噬细胞脂代谢因子ATP结合盒转运体A1(ABCA1)、B族清道夫受体(CD36)和炎症因子细胞外基质金属蛋白酶诱导因子(CD147)、基质金属蛋白酶9(MMP-9)蛋白表达的影响。方法分别采用50 mg/L ox-LDL和10 mg/L Dil-ox-LDL诱导巨噬细胞,10 mg/L 24-乙酰泽泻醇A进行干预。荧光显微镜观察细胞内Dil-ox-LDL蓄积情况;蛋白免疫印迹检测细胞ABCA1、CD36、CD147、MMP-9蛋白的表达。结果 10 mg/L Dil-ox-LDL诱导后大鼠腹腔巨噬细胞内有大量的Dil-ox-LDL蓄积,10 mg/L 24-乙酰泽泻醇A干预后,细胞内Dil-ox-LDL蓄积明显减轻。与对照组比较,50 mg/L ox-LDL诱导后大鼠腹腔巨噬细胞ABCA1、CD36和CD147、MMP-9蛋白表达明显增加,10 mg/L 24-乙酰泽泻醇A干预后,ABCA1蛋白表达进一步上升(P0.01),CD36、CD147和MMP-9蛋白表达被明显抑制(P0.05或P0.01)。结论 24-乙酰泽泻醇A上调巨噬细胞的脂代谢因子ABCA1和抑制CD36的表达,减少胆固醇蓄积,同时抑制炎症因子CD147和MMP-9的分泌。     

CD36介导氧化型低密度脂蛋白诱导U937细胞泡沫化和凋亡

为探讨清道夫受体ＣＤ３６在氧化型低密度脂蛋白诱导Ｕ９３７细胞泡沫化和凋亡中的作用,用氧化型低密度脂蛋白温育Ｕ９３７细胞,观察Ｕ９３７细胞泡沫化过程中ＣＤ３６的表达时序和泡沫细胞的凋亡;用ＣＤ３６单克隆抗体阻断Ｕ９３７细胞的吞噬作用,观察Ｕ９３７细胞吞噬蓄积胆固醇和细胞凋亡的改变。细胞胆固醇以修饰的酶荧光法测定;用流式细胞术检测异硫氰酸荧光素－抗ＣＤ３６单克隆抗体特异标记的ＣＤ３６和细胞的凋亡情况;用逆转录聚合酶链反应检测ＣＤ３６ｍＲＮＡ的表达。结果发现,８０ｍｇ／Ｌ氧化型低密度脂蛋白与Ｕ９３７细胞温育２４ｈ可增加细胞内总胆固醇,４８ｈ时可形成典型的泡沫细胞;ＣＤ３６表达呈现时序性改变,６ｈ即可检出ＣＤ３６表达增高,２４ｈ达到最高值,４８ｈ表达略有降低,ＣＤ３６ｍＲＮＡ的转录与ＣＤ３６的表达一致。用２００ｍｇ／Ｌ     

Expression of macrophage (Mphi) scavenger receptor, CD36, in cultured human aortic smooth muscle cells in association with expression of peroxisome proliferator activated receptor-gamma, which regulates gain of Mphi-like phenotype in vitro, and its implication in atherogenesis

CD36 is one of the major receptors for oxidized low density lipoproteins belonging to macrophage (Mphi) scavenger receptor (SR) class B and is thought to play an important role in the foam cell formation from monocyte-Mphi in the atherosclerotic lesions. Although it has been hypothesized that smooth muscle cells (SMCs) may be the other origin of foam cells in vivo, supporting data are still very limited. In the present study, we have tested the expression of a variety of SRs, including CD36, in 8 lots of primary human aortic SMCs (HASMCs) explanted from 8 different donors. Functional CD36 was expressed in cultured HASMCs, and the levels of expression were widely ranged between the lots. SR class A (SR-A) was expressed abundantly in CD36-negative lots. Other Mphi markers, such as CD32 and CD68, were expressed in all lots tested. These data suggest that the cultured HASMCs gained an Mphi-like phenotype. To determine the mechanism for the above-described phenotypic change, we have tested the expression of a nuclear receptor, peroxisome proliferator activated receptor-gamma, in those cells. This nuclear receptor was abundantly expressed in CD36-positive lots, whereas c-fms was expressed abundantly in CD36-negative/SR-A-positive lots. The synthetic ligand of peroxisome proliferator activated receptor-gamma, troglitazone, upregulated the expression of CD36 only in CD36-positive lots. These observations demonstrate that cultured HASMCs can gain an Mphi-like phenotype, possibly classified by the expression of CD36 or SR-A. The present study may support the possibilities of transformation of HASMCs into foam cells in vivo.     

外源性Rb基因对U937细胞泡沫化过程中CD36表达的影响

为研究抑癌基因Rb基因在U937细胞泡沫化过程中的表达，应用重组腺病毒载体导入外源性Rb基因。采用逆转录聚合酶链反应和流式细胞术检测氧化型低密度脂蛋白致U937细胞泡沫化过程中清道夫受体CD36和Rb基因的表达和外源性Rb基因导入U937细胞后CD36和Rb基因的表达。结果发现，氧化型低密度脂蛋白致U937细胞泡沫化过程中，Rb mRNA和蛋白的表达随作用时间延长呈下调趋势，CD36 mRNA和蛋白的表达则呈上调趋势；随着外源性Rb导入U937细胞并表达，CD36表达呈下调趋势。结果提示，外源性Rb基因的导入可以下调清道夫受体CD36的表达。     

EPA and DHA attenuate ox-LDL-induced expression of adhesion molecules in human coronary artery endothelial cells via protein kinase B pathway

Uptake of oxidized low-density lipoprotein (ox-LDL) by endothelial cells is a critical step for the initiation and development of atherosclerosis. Adhesion molecules are inflammatory makers, which are upregulated by ox-LDL and play a pivotal role in atherogenesis. A number of studies suggest that fish and its constituents can reduce inflammation and decrease atherosclerosis. We hypothesized that fish oil constituents namely docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) may reduce expression of adhesion molecules induced by ox-LDL. Cultured human coronary artery endothelial cells (HCAECs) were incubated with ox-LDL for 24 h. Parallel groups of cells were pretreated with DHA or EPA (10 or 50 microM) overnight before incubation with ox-LDL. Ox-LDL markedly increased the expression of P-selectin and intracellular adhesion molecule-1 (ICAM-1) (both protein and mRNA) in HCAECs, and enhanced the adhesion of monocytes to the cultured HCAECs. Both EPA and DHA decreased ox-LDL-induced upregulation of expression of P-selectin and ICAM-1, and the enhanced adhesion of monocytes to HCAECs. To determine the role of protein kinase B (PKB) as an intracellular-signaling pathway, HCAECs were treated with the PKB upstream inhibitor wortmannin (100 nM) or transfected with plasmids encoding dominant-negative mutants of PKB (PKB-DN) before treatment with DHA. Ox-LDL alone downregulated the activity of PKB; DHA attenuated this effect of ox-LDL, and both wortmannin and PKB-DN blocked the effect of DHA. The present study in human coronary endothelial cells suggests that both EPA and DHA attenuate ox-LDL-induced expression of adhesion molecules, and the adhesion of monocytes to HCAECs by modulation of PKB activation. These effects may be important mechanisms of anti-atherosclerotic effects of fish and fish oils.     

Ox-LDL induces endothelial cell apoptosis via the LOX-1-dependent endoplasmic reticulum stress pathway

Objective

To investigate the effect of lectin-like ox-LDL receptor-1 (LOX-1) on oxidized low-density lipoprotein (ox-LDL)-induced apoptosis and the involvement of the endoplasmic reticulum (ER) stress response pathway.

Methods and results

Human umbilical vein endothelial cells were treated with 50, 100, or 200 μg/ml ox-LDL and cultured for 12, 24, or 48 h for concentration- and time-dependent studies. Cells were transfected with LOX-1 or Nox-4 shRNAs, and target proteins were inhibited with the corresponding antibodies for mechanistic studies. Active proteins and mRNAs were analyzed by Western blotting and RT-PCR, respectively. Cell apoptosis was analyzed by Annexin and Hoechst staining assays. Ox-LDL induced both apoptosis and protein expression of LOX-1 and Nox-4 through activation of ER stress sensors IRE1 and PERK, and nuclear translocation of ATF6 and their subsequent pathways were indicated by JNK, eukaryotic initiation factor 2 phosphorylation, XBP-1, and chaperone GRP78 expression; up-regulation of proapoptotic proteins CHOP and Bcl-2; and caspase-12 activity. LOX-1 gene silencing and treatment with an anti-LOX-1 antibody attenuated the effects of ox-LDL. Pretreatment with irestatin 9389, salubrinal, or AEBSF also blocked ox-LDL-induced expression of CHOP and Bcl-2 and activation of caspase-12 activity, leading to an attenuation of endothelial cell apoptosis. Furthermore, Nox-4 siRNA attenuated the up-regulated expression of GRP78, PERK, IRE1, and XBP-1 to reduce ox-LDL-induced endothelial cell apoptosis.

Conclusions

LOX-1 plays a critical role in ox-LDL-induced endothelial cell apoptosis via the ER stress pathway.     

腹部脂肪组织分泌抵抗素样分子α促进清道夫受体表达

目的探讨腹部脂肪组织抵抗素样分子α(RELMα)mRNA及蛋白表达情况,研究其对与动脉粥样硬化密切相关的巨噬细胞清道夫受体B(CD36)及平滑肌细胞清道夫受体A(SR-A)表达的影响。方法取C57BL/6J小鼠腹部脂肪组织,体外培养巨噬细胞及平滑肌细胞,用氧化型低密度脂蛋白(ox-LDL,ox-LDC组)以及终浓度为3×10~(-6)mmol/L(A组)、9×10~(-6)mmol/L(B组)、2.7×10~(-5)mmol/L(C组)的RELMα刺激培养细胞,对照组加等量生理盐水刺激细胞。采用RT-PCR及免疫组织化学检测脂肪细胞内RELMα表达,采用流式细胞仪检测CD36及SR-A表达。结果小鼠腹部脂肪组织可见RELMαmRNA蛋白阳性表达。与对照组比较,ox-LDL组CD36荧光强度明显增强,SR-A表达阳性率明显升高(P<0.01);与ox-LDL组比较,A、B、C组SR-A表达阳性率明显升高(P<0.05)。结论小鼠腹部脂肪组织能分泌RELMα,其不影响巨噬细胞CD36的表达,但能促进ox-LDL诱导的平滑肌细胞SR-A表达,提示腹部脂肪组织可能通过RELMα,影响SR-A表达从而促进动脉粥样硬化进展。     

Oxidized-LDL through LOX-1 increases the expression of angiotensin converting enzyme in human coronary artery endothelial cells

BACKGROUND AND OBJECTIVES: Our previous studies have shown that oxidized low-density lipoprotein (ox-LDL) and angiotensin II (Ang II) influence each other's action in endothelial cells. This study was designed to examine the regulation by ox-LDL of the expression of angiotensin converting enzyme (ACE) gene in human coronary artery endothelial cells (HCAECs). In addition, we studied the effect of the HMG CoA reductase inhibitor simvastatin on this interaction. METHODS AND RESULTS: Cultured HCAECs were incubated with ox-LDL (10-80 microg/ml) for 1-24 h. Ox-LDL increased the expression of ACE in a concentration- and time-dependent fashion. The upregulation of ACE expression in response to ox-LDL was mediated by its endothelial receptor LOX-1, since pretreatment of HCAECs with a blocking antibody to LOX-1 prevented the expression of ACE (P<0.01). Native-LDL had no significant effect on ACE expression. In this process, ox-LDL-induced activation of mitogen-activated protein kinase (MAPK p42/44) played an important role, since pretreatment of HCAECs with the MAPK p42/44 inhibitor (PD98059, 10 microM) inhibited MAPK activation and subsequently attenuated the expression of ACE (P<0.01 vs. ox-LDL alone). In other experiments, we pretreated HCAECs with simvastatin (10 microM) and then exposed the cells to ox-LDL. Simvastatin markedly attenuated ox-LDL-induced MAPK activation, and concurrently reduced ACE expression (P<0.01 vs. ox-LDL alone). CONCLUSIONS: Our observations provide direct evidence that ox-LDL via LOX-1 activation induces ACE gene expression in HCAECs, and MAPK activation plays a signal transduction role in this process. Simvastatin, which inhibits MAPK activation, also blocks ox-LDL-mediated upregulation of ACE.     

小檗碱对2型糖尿病大鼠巨噬细胞ox-LDL、CD36和PPARγ的影响

目的探讨小檗碱对T2DM大鼠脂代谢、腹腔巨噬细胞（PM）及肺泡巨噬细胞（AM）氧化低密度脂蛋白（ox-LDL）、CD36和过氧化物酶体增殖物激活受体γ（PPARγ）表达的影响。方法SD大鼠分为正常对照组、高脂组、糖尿病组、小檗碱治疗组。实验结束后测定各组BG、Ins、血脂,PM及AM内ox-LDL含量、CD36与PPARγ的蛋白与mRNA表达水平。结果与正常对照组比较，糖尿病组BG、Ins、TC、TG、LDL-C显著升高；PM及AM内ox-LDL含量、CD36与PPARγ的蛋白和mRNA表达显著升高。与糖尿病组比较，小檗碱治疗组BG、Ins及LDL-C水平降低，TC、TG显著下降，HDL-C升高，PM及AM内ox-LDL含量下降，CD36的蛋白和mRNA表达降低，PPARγ mRNA表达降低。结论小檗碱降低T2DM大鼠血糖，改善胰岛素抵抗和脂代谢紊乱，其机制可能与降低巨噬细胞内ox-LDL，抑制CD36和PPARγ的表达有关。     

Role of caspases in Ox-LDL-induced apoptotic cascade in human coronary artery endothelial cells

Oxidized low-density lipoprotein (ox-LDL) induces apoptosis in endothelial cells. However, steps leading to ox-LDL-induced apoptosis remain unclear. We examined the role of ox-LDL and its newly described receptor LOX-1 in the expression of intracellular pro- and antiapoptotic proteins and caspase pathways in human coronary artery endothelial cells (HCAECs). Cells were cultured and treated with different concentrations (10 to 80 microg/mL) of ox-LDL for different times (2 to 24 hours). Ox-LDL induced apoptosis in HCAECs in a concentration- and time-dependent manner. Ox-LDL also activated caspase-9 and caspase-3, but not caspase-8. After ox-LDL treatment, there was a significant release of activators of caspase-9, including cytochrome c and Smac from mitochondria to cytoplasmic compartment, and their release was not affected by treatment of cells with inhibitors of either caspase-8 or caspase-9. Ox-LDL also decreased expression of antiapoptotic proteins Bcl-2 and c-IAP (inhibitory apoptotic protein)-1, which are involved in the release of cytochrome c and Smac and activation of caspase-9, in a concentration- and time-dependent manner. On the other hand, ox-LDL did not change the expression of Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP) and proapoptotic protein Fas, which are required for the activation of caspase-8. Further, ox-LDL did not cause the truncation of Bid, which implies the activation of caspase-8. In other experiments, pretreatment of HCAECs with the caspase-9 inhibitor z-LEHD-fmk, but not the caspase-8 inhibitor z-IETD-fmk, blocked ox-LDL-induced activation of caspase-3 and apoptosis. As expected, pretreatment with the caspase-3 inhibitor DEVD-CHO inhibited ox-LDL-induced activation of caspase-3 and resultant apoptosis. The proapoptotic effects of ox-LDL were mediated by its receptor LOX-1, because pretreatment of HCAECs with antisense-LOX-1, but not sense-LOX-1, blocked these effects of ox-LDL. These findings suggest that ox-LDL through its receptor LOX-1 decreases the expression of antiapoptotic proteins Bcl-2 and c-IAP-1. This is followed by activation of apoptotic signaling pathway, involving release of cytochrome c and Smac and activation of caspase-9 and then caspase-3.     

孤儿核受体Nur77通过转录激活功能调控巨噬细胞脂质沉积

目的:探讨孤儿核受体Nur77调控巨噬细胞脂质沉积与其转录激活功能的关系。方法:建立稳定表达GFP、GFP-Nur77、GFP-Nur77/ΔDBD和GFP-Nur77/ΔTAD c DNA的小鼠巨噬细胞RAW264.7单克隆细胞系,40μg/ml氧化型低密度脂蛋白(ox-LDL)刺激24 h后,油红"O"染色定性观察细胞内脂质沉积,液相色谱-质谱联用法(LC-MS)定量检测细胞内胆固醇酯,荧光实时定量PCR(RT-PCR)检测CD36和ABCA1 mRNA水平的变化,流式细胞仪检测CD36蛋白表达,Western blot方法检测ABCA1蛋白表达。结果:与对照GFP-RAW264.7相比,高表达Nur77能够显著减少ox-LDL诱导引起的巨噬细胞内脂质沉积,同时降低CD36的mRNA及蛋白表达,上调ABCA1的mRNA及蛋白表达。但GFP-Nur77/ΔDBD RAW264.7和GFP-Nur77/ΔTAD RAW264.7细胞内胆固醇都明显升高,并伴随CD36的mRNA及蛋白表达的降低和ABCA1的mRNA及蛋白表达的升高。结论:孤儿核受体Nur77通过减少脂质摄取和增加脂质排出而减轻巨噬细胞内脂质沉积,这一作用与Nur77 DNA结合能力以及转录活性有关。     

Increased frequency of thymic T follicular helper cells in myasthenia gravis patients with thymoma

Background

To investigate the presence of T follicular helper (TFH) cells and their associated molecules in myasthenia gravis (MG) patients with thymoma.

Methods

TFH cells are detected in thymus around the thymoma region of 50 patients and atrophic thymus in 10 patients as control. The percentage of TFH cells among CD4⁺ T cells and the expression level of surface markers CXC chemokine receptor 5 (CXCR5), inducible co-stimulator (ICOS), programmed cell death 1 and the cytoplasmic marker B cell lymphoma 6 (Bcl-6) were analyzed by immunohistochemistry (IHC) staining, immunofluorescence (IF) and western blotting (WB).

Results

Higher percentage of thymic TFH cells was found in MG patients with thymoma compared with both thymoma patients without MG and control group. The expression levels of the four markers in thymoma of MG patients were significantly higher than thymoma patients without MG and control group. No significant difference was found in the levels of programmed cell death 1 (PD-1) and Bcl-6 between thymoma patients without MG and the control, while the levels of CXCR5 and ICOS in thymoma patients without MG were higher than control group.

Conclusions

These results suggested thymic TFH cells might involve in the pathogenesis of MG with thymoma. However, it needs further study to test if the inhibition of the function of TFH cells could effectively alleviate the severity of MG.     

氧化型低密度脂蛋白对可诱导共刺激分子在人外周血T细胞表达的影响

目的:探讨可诱导共刺激分子(ICOS)在人外周血T细胞的表达及氧化低密度脂蛋白(ox-LDL)的干预作用。方法:以人外周血T细胞为实验模型,通过间接免疫荧光法、RT-PCR和Western blot等方法,观察ox-LDL对人外周血T细胞表达ICOS的影响。结果:①ICOS表达于人外周血T细胞膜,荧光信号呈点状散在分布于细胞表面。RT-PCR检测显示,ICOS mRNA的扩增片段位于相当于Marker 368 bp的位置。Western blot检测显示,ICOS的分子量约为55 kD。②ox-LDL刺激组ICOS的吸光度(A)值高于空白对照组,提示ox-LDL能够明显增加人外周血T细胞中的ICOS mRNA和其蛋白的表达(P0.05)。结论:ICOS表达于人外周血T细胞表面,ox-LDL能够上调ICOSmRNA和其蛋白的表达,这可能是动脉粥样硬化的免疫学发病机制之一。     

不同阶段泡沫细胞模型的建立与鉴定

目的建立不同阶段的泡沫细胞,并对其生物特性进行鉴定。方法体外培养人THP-1来源的巨噬细胞（THP-M）,由ox-LDL诱导其转化为不同阶段泡沫细胞,ELISA方法检测不同阶段泡沫细胞内总胆固醇和胆固醇酯含量;透射电镜和油红O染色光镜观察细胞内脂质,噻唑蓝染色法测定不同阶段泡沫细胞的活性变化规律。Western blot方法测定II型微管相关蛋白1轻链3（LC3II）蛋白表达水平。结果 24h开始,THP-M细胞质内出现大量的红染颗粒或脂质空泡,总胆固醇和胆固醇酯均较对照组增加,细胞胆固醇酯含量大于总胆固醇的50%,泡沫细胞已经形成。噻唑蓝染色结果显示,自48h开始少量泡沫细胞出现死亡现象（存活率93.21±3.66%）,60h和72h细胞存活率率分别为72.15±2.23%和63.06±1.83%。电镜扫描结果发现,24h细胞内脂滴聚集,24h内细胞浆自噬小体逐渐增多。36h、48h细胞内脂滴大小和形态各异,可见吞噬脂滴后尚未消化完全的自噬溶酶体,部分细胞膜破裂;60h和72h,细胞内自噬小体数量明显减少,细胞大量崩解。Western blot检测结果表明自噬标志性蛋白LC3II表达水平变化规律与电镜观察自噬小体结果一致。结论利用oxLDL诱导THP-M24h、36h-48h、60h-72h后,细胞形态和细胞内脂含量分别符合早期、中期和晚期泡沫细胞的生物特征,成功建立了早、中、晚期泡沫细胞模型,其中细胞活力变化可能与II型凋亡（自噬性死亡）相关。为不同病程AS研究提供支持。     

Different effect of statins on platelet oxidized-LDL receptor (CD36 and LOX-1) expression in hypercholesterolemic subjects.

Hydroxymethyl-glutaryl-CoA-reductase inhibitors (statins) reduce cardiovascular mortality by decreasing cholesterol as well as by non-lipid-related actions. Oxidized low-density lipoproteins (ox-LDL) are pro-atherogenic molecules and potent platelet agonists. CD36 and lectin-like ox-LDL receptor-1 (LOX-1) are specific ox-LDL receptors also expressed in platelets. This study was planned to address whether treatment with atorvastatin 10 mg/day, pravastatin 40 mg/day or simvastatin 20 mg/day could affect platelet CD36 and LOX-1 expression. Twenty-four patients for each treatment were evaluated after 3, 6, and 9 days and at 6 weeks for complete lipid profile (chromogenic), ox-LDL (ELISA), platelet P-selectin (P-sel), CD36, LOX-1 (FACS), and intracellular citrullin recovery (iCit) (HPLC). Data show hyperactivated platelets (P-sel absolute values, percent variation in activated cells, all p < 0.001), and CD36 and LOX-1 overexpression (all p < 0.001) in patients at baseline. P-sel, CD36, and LOX-1 were significantly decreased by atorvastatin and simvastatin (all p < 0.01) and related with iCit increase (r = 0.58, p < 0.001) and platelet-associated ox-LDL (r = 0.51, p < 0.01) at 9 days. Pravastatin reduced LOX-1 and P-sel (p < 0.05) at 6 weeks in relation with decreased LDL and ox-LDL (r = 0.39, p < 0.01 and r = 0.37, p < 0.01, respectively). These data suggest that atorvastatin and simvastatin reduce platelet activity by exposure of CD36 and LOX-1 before significant LDL reduction, whereas pravastatin action is detected later and in relation with LDL and ox-LDL lowering. Rapid and consistent reduction of CD36 and LOX-1 could be considered a direct anti-atherothrombotic mechanism related to the role of ox-LDL in platelet activation, platelet-endothelium interactions, and NO synthase activity.     

Contribution of monocytes Siglec-1 in stimulating T cells proliferation and activation in atherosclerosis

ObjectiveAtherosclerosis (AS) is widely accepted as an inflammatory disease and monocytes are particularly important in inflammatory immune responses. As an important biomarker of monocytes activation, Siglec-1 is highly expressed on circulating monocytes and atherosclerotic plaques of coronary artery disease (CAD) patients, but the exact role of Siglec-1 has not been elucidated.MethodsM-CSF, INF-α, IFN-γ, TNF-α and ox-LDL alone or in combination were used to stimulate Siglec-1 expression on monocytes, whereas small interfering RNA (si-RNA) or blocking antibody was used to down-regulate Siglec-1. Meanwhile, the role of Siglec-1 in chemokines secretion was determined. Then monocytes from CAD patients or healthy controls were cocultured with CD4+ or CD8+ T cells from a third healthy individual, and lymphocyte proliferation and activation were determined.ResultsAll the stimuluses could enhance Siglec-1 expression on monocytes in a dose-dependent manner, and M-CSF could synergistically stimulate Siglec-1 expression with ox-LDL. Moreover, the secretion of MCP-1, MIP-1α and MIP-2 were enhanced when Siglec-1 was up-regulated and down to normal level when Siglec-1 was blocked. More importantly, increased Siglec-1 expression on monocytes was related to the increased T cell proliferation and pro-inflammatory cytokines secretion in CAD patients. However, down-regulation of Siglec-1 could attenuate proliferation and activation of cocultured CD4+ and CD8+ T cells.ConclusionSiglec-1 can promote chemokines and pro-inflammatory cytokines secretion and influence the inflammatory process of AS.     

Effect of High Glucose on Human Alveolar Macrophage Phenotype and Phagocytosis of Mycobacteria

Purpose

Diabetes mellitus (DBM) reduces immunological activity and increases susceptibility to various infections, including tuberculosis (TB). Human alveolar macrophage (hAM) functions are altered in DBM.

Methods

To mimic hyperglycemic conditions in the lung alveolus, we co-cultured a hAM cell line (Daisy cell line) with human umbilical vein endothelial cells for 48 h in the presence of culture media alone, normal glucose (5 mM), and high glucose (22 mM). Using flow cytometry, immunophenotype characterization included cell surface markers CD 11c, CD14, CD16, CD86, CD163, CD169, CD206, CX3CR-1, CSF-1R, and matrix metalloproteinase-9 (MMP9). Phagocytic function was measured by immunofluorescence microscopy at 24 h after inoculation of cells with GFP-expressing Mycobacterium smegmatis.

Results

Direct exposure of AMs to high glucose and exposure in the co-culture system yield different results for the same phenotypic markers. MMP9 expression was increased under both conditions. CD169 and CX3CR1 expressions were decreased when AMs were exposed directly to high glucose but increased under co-culture. Immunofluorescence assay revealed that phagocytosis decreased in AMs when directly exposed to increased glucose levels from 2.5 mM to normal glucose (5 mM), yet AMs under co-culture did not show decreased phagocytosis until concentrations were raised to 25 mM.

Conclusion

Alteration in the expression of certain receptors may contribute to defective sentinel function of AMs, promoting susceptibility to TB in a diabetic host. Variability in cell surface marker expression under direct glucose exposure compared to exposure via co-culture reveals that cell signaling between endothelial cells and AMs may play a crucial role in the phenotypic expression of AMs.