ox-LDL对人血单核细胞组织基质蛋白抑制物表达的影响

目的 :探讨氧化修饰低密度脂蛋白 （ox- L DL）对人单核细胞源巨噬细胞 （HMDM）组织基质金属蛋白酶抑制物（TIMPs）的基因和蛋白表达的影响。方法 :分别应用 RT- PCR和 Western blot检测 TIMPs（TIMP- 1和 TIMP- 2 ）基因和蛋白表达。结果 :ox- L DL 可明显抑制 HMDM TIMP- 1m RNA及其蛋白的表达 ,但对 TIMP- 2 m RNA及其蛋白的表达无明显影响。结论 :ox- L DL 可抑制 HMDM TIMP- 1m RNA及其蛋白的表达 ,可能间接增强基质金属蛋白酶 （MMPs）活性     

VEGF对人血单核细胞基质金属蛋白酶表达及活性的影响

目的 :探讨血管内皮生长因子 （VEGF）对人单核细胞源巨噬细胞 （HMDM）基质金属蛋白酶 （MMPs）的表达及其活性的影响。方法 :分别应用 RT- PCR和 Western blot检测 MMPs（MMP- 2和 MMP- 9）基因和蛋白表达 ,Zym ography活性分析检测 MMPs活性。结果 :VEGF可明显增加人单核细胞源巨噬细胞 （HMDM） MMP- 2、MMP- 9m RNA及其蛋白表达和活性。结论 :VEGF可刺激 HMDM MMP- 2、MMP- 9m RNA及其蛋白的表达 ,增强其活性 ,因而有可能促进动脉粥样硬化斑块内基质降解 ,进而引起粥样硬化斑块破裂。     

普罗布考对巨噬细胞分泌基质金属蛋白酶9的抑制作用

通过观察普罗布考对THP 1细胞基质金属蛋白酶 9表达的影响 ,初步探讨普罗布考稳定斑块的分子机制。运用逆转录聚合酶链反应、免疫印迹、明胶酶图等方法 ,检测氧化型低密度脂蛋白诱导后的人单核细胞 巨噬细胞系THP 1细胞基质金属蛋白酶 9mRNA表达、蛋白分泌和酶活性。实验发现 ,在氧化型低密度脂蛋白 (80mg L)刺激下 ,THP 1细胞基质金属蛋白酶 9的表达和酶活性明显增强 ;予普罗布考 2 0、4 0、6 0 μmol L处理 ,均能在对细胞活性无影响的前提下 ,显著抑制基质金属蛋白酶 9的蛋白分泌量及明胶降解活性 ,且这种作用呈浓度依赖性增强 ,其中 6 0 μmol L的普罗布考抑制基质金属蛋白酶 9的蛋白分泌和明胶降解活性分别达 74 .0 %± 2 .4 %和 4 8.0 %±5 .1% (P <0 .0 5 )。实验结果说明 ,普罗布考能有效抑制巨噬细胞的基质金属蛋白酶 9的蛋白分泌及其活性 ,这一作用可能是其抗动脉粥样硬化和稳定斑块的重要机制之一。     

罗格列酮对急性冠状动脉综合征患者外周血单核细胞源性巨噬细胞表达基质金属蛋白酶9和组织型基质金属蛋白酶抑制剂1的影响

目的 探讨过氧化体增殖物激活型受体γ在调节急性冠状动脉综合征患者外周血单核细胞源性巨噬细胞表达基质金属蛋白酶9、组织型基质金属蛋白酶抑制剂1中的作用.方法 用不同浓度罗格列酮干预急性冠状动脉综合征患者和对照者单核细胞源性巨噬细胞,然后测定各组上清液中基质金属蛋白酶9、组织型基质金属蛋白酶抑制剂l浓度及单核细胞源性巨噬细胞过氧化体增殖物激活型受体γ基质金属蛋白酶9和组织型基质金属蛋白酶抑制剂1 mRNA的表达.结果 干预后,急性冠状动脉综合征组及对照组过氧化体增殖物激活型受体γmRNA表达上调,表达强度与罗格列酮浓度呈正变关系;基质金属蛋白酶9表达下调,在急性冠状动脉综合征组其下调程度与罗格列酮浓度呈反变关系;组织型基质金属蛋白酶抑制剂1的表达无明显变化.结论 罗格列酮干预可上调单核细胞源性巨噬细胞过氧化体增殖物激活型受体γ表达,下调基质金属蛋白酶9表达,在急性冠状动脉综合征组尤其明显,对组织型基质金属蛋白酶抑制剂1表达无明显影响;可能存在稳定动脉粥样斑块的作用.     

粒细胞—巨噬细胞集落刺激因子对人内皮细胞基质金属蛋白酶分泌及活性的影响

为探讨粒细胞—巨噬细胞集落刺激因子对人内皮细胞基质金属蛋白酶分泌及活性的影响，体外培养人脐静脉内皮细胞，分别应用蛋白印迹法和酶谱法检测基质金属蛋白酶2的含量和活性。结果发现，与对照组相比，不同浓度(5、25和50μg/L)粒细胞—巨噬细胞集落刺激因子可使人内皮细胞基质金属蛋白酶2的蛋白表达和活性增强，且随粒细胞—巨噬细胞集落刺激因子剂量增加而增强。提示粒细胞—巨噬细胞集落刺激因子可通过刺激人内皮细胞基质金属蛋白酶2蛋白的表达，并增强其活性，促进内皮细胞对细胞外基质的降解，从而易于动脉粥样硬化斑块内炎性细胞的渗入，内皮细胞的迁移及新血管的形成，从而对斑块的稳定性产生影响。     

氧化型低密度脂蛋白对大鼠血管平滑肌细胞基质金属蛋白酶-2和9表达的影响

为探讨氧化型低密度脂蛋白致动脉粥样硬化的作用是否与基质金属蛋白酶表达活性改变有关,应用Northern blot、Dot blot、Westorn blott和明胶酶图分析方法观察氧化型低密度脂蛋白对体外培养的大鼠血管平滑肌细胞表达基质金属蛋白酶－2和9的影响。结果显示,10mg/L氧化型低密度脂蛋白作用于血管平滑骨细胞24h,可明显增强基质金属蛋白酶－2和9mRNA的表达、蛋白分泌和酶的活性,高浓度的氧化型低密度脂蛋白对基质金属蛋白酶－2和9表达的影响不同,氧化型低密度脂蛋白浓度为50mg／L时,基质金属蛋白酶－9表达活性仍然高于对照细胞,但于10mg／L氧化型低密度脂蛋白的作用强度无显著差别;相同条件下,基质金属蛋白酶－2的基因表达和蛋白分泌明显降低。以上结果提示,氧化型低密度脂蛋白可诱导血管平滑肌细胞基质金属蛋白酶－2和9的表达,并可促进细胞外基质降解。     

糖基化终产物对小鼠巨噬细胞基质金属蛋白酶诱导物表达、分泌及基质金属蛋白酶9活性的影响

目的 探讨糖基化终产物对小鼠巨噬细胞基质金属蛋白酶诱导物表达、分泌及基质金属蛋白酶9活性的影响.方法在培养的小鼠巨噬细胞株(J774A.1)中分别加入不同浓度(50、100、200及400 mg/L)的糖基化终产物干预24 h和同一浓度(200 mg/L)的糖基化终产物干预12、24及48 h,以无血清培养基和相应浓度的牛血清白蛋白为对照.用逆转录聚合酶链方法检测基质金属蛋白酶诱导物mRNA的表达,用酶联免疫吸附法检测上清中基质金属蛋白酶诱导物蛋白水平,用酶谱法检测上清中基质金属蛋白酶9的活性.结果 糖基化终产物干预组的基质金属蛋白酶诱导物mRNA表达水平和上清中蛋白水平与对照组比较差异有显著性,且随时间和浓度增加而增加(P<0.05).糖基化终产物干预组的上清中基质金属蛋白酶9的活性与对照组比较差异有显著性,且随时间和浓度增加而增加(P<0.05).结论 糖基化终产物促进小鼠巨噬细胞基质金属蛋白酶诱导物mRNA表达、蛋白分泌及基质金属蛋白酶9的活性;提示糖基化终产物可能通过调节巨噬细胞基质金属蛋白酶诱导物表达、分泌及基质金属蛋白酶9的活性而影响糖尿病动脉粥样硬化斑块的稳定性.     

急性冠状动脉综合征患者巨噬细胞表达过氧化体增殖物激活型受体γ及炎症相关因子的变化

目的 探讨急性冠状动脉综合征患者外周血单核细胞源性巨噬细胞表达过氧化体增殖物激活型受体γ核因子κB、基质金属蛋白酶9和组织型基质金属蛋白酶抑制剂1的变化及其间的关系.方法 选取急性冠状动脉综合征患者48例、稳定型心绞痛患者22例为研究对象,抽取外周动脉血20 mL,分离单个核细胞,加巨噬细胞集落刺激因子培养得单核细胞源性巨噬细胞;用CD40配体刺激后,酶联免疫吸附法测定上清液中基质金属蛋白酶9和组织型基质金属蛋白酶抑制剂1浓度,逆转录聚合酶链反应检测过氧化体增殖物激活型受体γ、基质金属蛋白酶9和组织型基质金属蛋白酶抑制剂1 mRNA表达,免疫组织化学法检测核因子κ亚单位P65表达.结果 急性冠状动脉综合征组过氧化体增殖物激活型受体γ mRNA表达低于稳定型心绞痛组(0.24±0.04比0.39±0.06,P<0.001),核因子κ P65表达高于稳定型心绞痛组(0.42±0.06比0.27±0.02,P<0.001),基质金属蛋白酶9在上清液中的浓度及其mRNA表达明显高于稳定型心绞痛组(231.11±51.47μg/L比126.02±13.26μg/L和0.674±0.11比0.24±0.05,P<0.001),组织型基质金属蛋白酶抑制剂1在上清液中的浓度及其mRNA表达强度高于稳定型心绞痛组(139.80±31.54μg/L比112.25±12.68μg/L和0.42±0.09比0.33±0.06,P<0.05).过氧化体增殖物激活型受体γ mRNA表达与核因子κ P65和基质金属蛋白酶9的表达强度呈负相关(P<0.001),与组织型基质金属蛋白酶抑制剂1的表达强度不相关(P>0.05);核因子κ P65与基质金属蛋白酶9和组织型基质金属蛋白酶抑制剂1的表达强度呈正相关(P<0.001).结论 急性冠状动脉综合征患者外周血单核细胞源性巨噬细胞过氧化体增殖物激活型受体γ表达下调,核因子κ活性增强,基质金属蛋白酶9和组织型基质金属蛋白酶抑制剂1表达上调;过氧化体增殖物激活型受体γ可能通过调节核因子κ活性而调节基质金属蛋白酶9基因转录;但组织型基质金属蛋白酶抑制剂1表达可能不受过氧化体增殖物激活型受体γ调节.     

白藜芦醇抑制可溶性CD40配体作用下巨噬细胞基质金属蛋白酶-9的表达

目的 探讨白藜芦醇抑制可溶性CD40配体(sCD40L)作用下对巨噬细胞基质金属蛋白酶-9(MMP-9)表达的影响.方法 佛波酯诱导人单核细胞株(THP-1)细胞分化为巨噬细胞.依次给予白藜芦醇和可溶性sCD40L孵育细胞,利用半定量反转录-聚合酶链反应(RT-PCR)、蛋白免疫印迹法、明胶酶谱法检测巨噬细胞内MMP-9和组织基质金属蛋白酶抑制因子-1(TIMP-1)基因的转录、蛋白表达和酶活性.结果 给予sCD40L刺激后巨噬细胞内MMP-9基因转录增多(1.53±0.04与0.75±0.01,P<0.05),蛋白分泌明显增加(244 930.8±31 268.6与192 976.8±20 223.1,P<0.05);白藜芦醇可抑制巨噬细胞MMP-9基因转录及蛋白分泌(P<0.01),降低MMP-9酶活性(P<0.05),升高巨噬细胞TIMP-1基因转录和蛋白分泌(P<0.05).结论 白藜芦醇可以抑制CD40途径活化的巨噬细胞内MMP-9的表达,调节MMP-9的活性,这可能是其抗动脉粥样硬化、稳定粥样斑块的作用机制之一.     

氨氯地平对ox- LDL诱导人单核巨噬细胞MMP-9表达的影响

目的 研究氨氯地平对氧化低密度脂蛋白(ox - LDL)诱导健康人单核巨噬细胞基质金属蛋白酶-9(MMP -9)mRNA及蛋白表达影响.方法 采用体外密度离心法分离健康人单核细胞培养并传至4代后,以佛波酯(40 ng/mL)诱导为巨噬细胞,培养48 h后用于实验.根据实验要求分为空白对照组、ox- LDL组、不同剂量(0.1 μmol/L,1 μmol/L,10 μmol/L)氨氯地平组.RT- PCR检测各组MMP-9 mRNA的表达情况.Elisa检测各组MMP -9蛋白的表达.结果 单核巨噬细胞经100 ug/mL ox - LDL诱导后,与空白对照组比较MMP-9 mRNA和蛋白表达显著增加；经ox- LDL诱导后禾同剂量氨氯地平组与ox - LDL组比较MMP -9mRNA和蛋白表达显著降低,并呈浓度效应依赖关系,差异具有统计学意义(P＜0.05).结论 ox- LDL可使人单核巨噬细胞MMP -9的表达增加,氨氯地平可呈浓度效应依赖性地减少ox- LDL诱导人单核巨噬细胞MMP -9的表达,从而在稳定斑块,减少急性冠脉事件发生中起作用.     

PPARγ基因转录与单核巨噬细胞来源的泡沫细胞形成

目的 :探讨PPARγ基因转录在动脉粥样病变局部的作用。方法 :观察动脉粥样硬化局部PPARγ基因表达与巨噬细胞来源的泡沫细胞、清道夫受体A(SRA)分布的关系 ;分析PPARγ配体对氧化低密度脂蛋白 (ox LDL)介导的巨噬细胞SRA、PPARγmRNA表达、肿瘤坏死因子α(TNF α)和基质金属蛋白酶 9(MMP 9)释放的影响。结果 :PPARγ基因表达与巨噬细胞来源的泡沫细胞、清道夫受体A(SRA)的分布相似 ;PPARγ配体在增加巨噬细胞PPARγmRNA表达的同时 ,显著抑制了ox LDL介导的巨噬细胞SRAmRNA表达和TNF α、MMP 9的释放。结论 :PPARγ基因激活可抑制巨噬细胞SRAmRNA表达及细胞因子释放     

Metalloproteinase expression in monocytes and macrophages and its relationship to atherosclerotic plaque instability

Matrix metalloproteinases (MMPs) can degrade strength-giving collagens and other structural proteins of the arterial extracellular matrix. Overproduction of MMPs by monocyte/macrophages could therefore promote atherosclerotic plaque rupture and myocardial infarction. Freshly-recruited monocyte macrophages appear to use a prostaglandin (PG)-dependent pathway to coordinately upregulate a broad and potentially highly-destructive spectrum of MMPs. Differentiated macrophages rely on a series of distinct pathways to selectively upregulate groups of MMPs. Moreover, recent evidence suggests that different macrophage phenotypes express characteristically different spectra of MMPs and their inhibitors. New therapies may result from targeting matrix MMP overproduction.     

普伐他汀对培养巨噬细胞表达基质金属蛋白酶活性的影响

目的　探讨普伐他汀对基质金属蛋白酶 (matrixmetalloproteinases ,MMPs)活性的影响及其与粥样斑块稳定性的关系。方法　从SD大鼠腹腔取巨噬细胞体外培养 ,接种于 2 4孔板中 ,逐孔加入普伐他汀 ,终浓度分别为 10 - 3、10 - 4及 10 - 5mol L ,每种浓度 3孔 ,分别在 2 4、4 8及 72h收集上清液 ,未加药孔为空白对照 ,采用酶谱分析法测量上清液中MMPs的活性。结果　巨噬细胞上清液中有MMP 2及MMP 9的活性表达 ,以 2 4h时活性最强。普伐他汀可以降低其活性 ,随着浓度的增加 ,抑制作用越明显。结论　普伐他汀可以使巨噬细胞产生的MMPs活性降低 ,可能使纤维帽中胶原的降解减少 ,从而增加粥样斑块的稳定性     

Monocyte matrix and ADAM metalloproteinase expression in type 2 diabetes after aspirin therapy

The matrix metalloproteinase system (MMP and the TIMP inhibitors), and the ADAM metalloproteinases, have roles in maintaining vascular plaque stability and the shedding of cell surface molecules, such as TNF-alpha and adhesion molecules; aspirin suppresses MMP expression and ADAM activity from some cell lines in vitro. In a randomised prospective controlled study, we examined peripheral venous monocyte MMP-9, TIMP-1 and ADAM mRNA levels, and protein expression, in subjects with type 2 diabetes (n=10) and controls (n=14) before and after oral aspirin therapy (150mg daily for 14 days) or no active intervention. Baseline monocyte TIMP-1 mRNA levels were significantly lower in the diabetes group (p=0.0014), although monocyte MMP-9 mRNA, and MMP-9 and TIMP-1 protein expression after culture did not differ significantly between groups. Plasma MMP-9 (p=0.027) and TIMP-1 (p=0.016) concentrations were significantly greater, and the ratio of plasma TIMP-1:MMP-9 concentrations significantly lower, in the diabetes group (p=0.023). ADAM mRNA levels did not differ significantly between groups and oral aspirin therapy had no significant effect on any variable. Type 2 diabetes is characterised by reduced monocyte TIMP-1 mRNA levels, and a lower plasma MMP-9 to TIMP-1 protein ratio compared to controls, a pattern that would promote coronary plaque instability if reproduced within vascular plaque. Monocyte ADAM mRNA levels do not differ between group and oral aspirin has no significant effect on these variables.     

Antioxidant properties of macrophages toward low-density lipoprotein.

Oxidative modification of low-density lipoprotein (LDL) has been implicated in atherosclerosis. Intensive scientific efforts over the last two decades have focused on the elucidation of the mechanisms by which LDL is oxidized in vivo. A wealth of in vitro studies has demonstrated that the cell types present in atherosclerotic lesions, including monocyte/macrophages, quantitatively one of the most important cell types in plaque development, promote LDL oxidation. The mechanisms of cellular prooxidant activities have been extensively investigated. Fewer studies have addressed possible protective properties of the cells in LDL oxidation. This review summarizes recent observations of antioxidant, and potentially antiatherogenic, activities of macrophages toward LDL, including macrophage-mediated detoxification of lipid and protein hydroperoxides, metal sequestration and the generation of compounds with antioxidant properties. These activities could contribute to the net effect of macrophages on deleterious LDL oxidation and to the complex role of these cells in lesion development.     

Nitric oxide regulates matrix metalloproteinase-9 activity by guanylyl-cyclase-dependent and -independent pathways

Matrix metalloproteinases (MMPs) are of central importance in the proteolytic remodeling of matrix and the generation of biologically active molecules. MMPs are distinguished by a conserved catalytic domain containing a zinc ion, as well as a prodomain that regulates enzyme activation by modulation of a cysteine residue within that domain. Because nitric oxide (NO) and derived reactive nitrogen species target zinc ions and cysteine thiols, we assessed the ability of NO to regulate MMPs. A dose-dependent, biphasic regulatory effect of NO on the activity of MMPs (MMP-9, -1, and -13) secreted from murine macrophages was observed. Low exogenous NO perturbed MMP/tissue inhibitor of metalloproteinase (TIMP)-1 levels by enhancing MMP activity and suppressing the endogenous inhibitor TIMP-1. This was cGMP-dependent, as confirmed by the cGMP analog 8-bromo-cGMP, as well as by the NO-soluble guanylyl cyclase-cGMP signaling inhibitor thrombospondin-1. Exposure of purified latent MMP-9 to exogenous NO demonstrated a concentration-dependent activation and inactivation of the enzyme, which occurred at higher NO flux. These chemical reactions occurred at concentrations similar to that of activated macrophages. Importantly, these results suggest that NO regulation of MMP-9 secreted from macrophages may occur chemically by reactive nitrogen species-mediated protein modification, biologically through soluble guanylyl-cyclase-dependent modulation of the MMP-9/TIMP-1 balance, or proteolytically through regulation of MMP-1 and -13, which can cleave the prodomain of MMP-9. Furthermore, when applied in a wound model, conditioned media exhibiting peak MMP activity increased vascular cell migration that was MMP-9-dependent, suggesting that MMP-9 is a key physiologic mediator of the effects of NO in this model.     

Increased secretion of tissue inhibitors of metalloproteinases 1 and 2 from the aortas of cholesterol fed rabbits partially counterbalances increased metalloproteinase activity.

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) plays an important role in extracellular matrix turnover and thereby modulates atherosclerotic plaque development. MMP-1, -2, -3, and -9 activity is increased by atherosclerosis, but the status of TIMPs is less clear. We therefore compared secretion of TIMPs-1 and -2 from cultured aortic explants derived from arch, middle, and distal portions of thoracic aortas of normal rabbits and rabbits fed a 1% cholesterol diet for 8 weeks, using reverse zymography of conditioned media. Cholesterol feeding significantly increased secretion of TIMP-1 from arch and middle portions (both 2.6-fold), accompanied by 2.0- and 2.7-fold increases in TIMP-2, respectively. Atherosclerotic aortas exhibited increased immunoreactive TIMP-1 and TIMP-2 in endothelial cells, smooth muscle cells, and macrophages. Staining of extracellular matrix was also prominent within the noncellular boundary region between fibrous cap and the lipid core, and within the lipid core. Increased TIMP-2 staining was also found in the media subjacent to the lipid core. In situ gelatin zymography demonstrated excess MMP activity within the plaque with partial inhibition in the lipid core base and subjacent media, consistent with the distribution of TIMPs. Casein zymography and in situ zymography demonstrated that increased caseinolytic activity was confined to the pericellular zones of macrophages within the lipid core, again consistent with its restriction by TIMPs. In summary, atherosclerosis increases TIMP expression, which counterbalances, in part, increased MMP activity.