| Acute coronary syndromes (ACS), including acute myocardial infarction and unstable angina is a kind of acute cardiovascular events with high fatality and disability rate. Most acute coronary syndromes are triggered by vulnerable plaque rupture and the subsequent thrombus. In fact, the instabilities of atherosclerosis plaque are associated with many factors, such as with big lipid cores, thinner fibrous caps, more Macrophage infiltration, endothelial function damaged severely, increasing number of smooth muscle cells in plaque, platelet aggregation on plaque surface, bleeding in plaque, and so on. In these factors, fibrous caps becoming thinner may lead to plaque rupture under high blood flow conditions or with other stimulating factors. Extracellular matrix (ECM) is the main component of fibrous caps can be degraded by extracellular matrix metalloproteinase (MMPs) which can be induced by extracellular matrix metalloproteinase inducer (EMMPRIN). EMMPRIN, namely CD147, can induce many kinds of cells, such as smooth muscle cells, monocytes, to secrete MMPs, thereby degrading ECM to cause acute cardiovascular events.CD147was first identified as a surface protein on tumor cells and was found to be expressed constitutively on monocytes, granulocytes, endothelial cells, smooth muscle cells and lymphocytes. Recently CD147as a novel receptor, was recently reported to be localized in the open canalicular system (OCS) of platelets and a granules, and CD147expression is up-regulated on platelet surface after platelets are exposed to various stimuli (e.g., thrombin, ADP, and collagen). CD147on platelets surface can activate platelets in turn and stimulate MMP-9synthesis in monocytes and MMP-9can strongly degrade extracellular matrix. Meanwhile, membrane type1matrix metalloproteinase (MT1-MMP) from many kinds of cells induced by CD147can enhance the uptake of ox-LDL by monocytes and smooth muscle cells, which promote the progress of atherosclerosis.In addition, CD147is able to enhance platelet-monocyte interactions in vivo and to promote monocyte recruitment to the arterial wall. It has been shown that CD147expression on circulating platelets is positively associated with coronary heart disease (CHD). Compared with normal people, the patients with coronary heart disease have increasing platelet CD147expression. Moreover, platelet CD147expression increases with age.Ox-LDL can be involved in the formation of foam cells, to damage directly endothelial cells, promote synthesis of many inflammation factors, thereby promoting atherosclerosis progress. Although ox-LDL level is very low in normal blood, it increase markedly in patients with atherosclerosis or coronary heart disease, especially in the site of atherosclerosis plaque. That elevated levels of circulating oxidized LDL serve as a sensitive marker for coronary artery disease (CAD). Additionally, in apparently healthy people, circulating ox-LDL is associated with the progress of atherosclerosis. What is more, the level of circulating ox-LDL is associated with the severity of ACS.Circulating ox-LDL in patients with atherosclerosis can interact with platelet. It has been demonstrated that ox-LDL-platelet increase markedly in patients with ACS. Ox-LDL serving as independent stimuli can promote platelet aggregation, even degranulation. Although some studies reported that a low concentration of ox-LDL inhibited platelet activation, ox-LDL is generally thought to be bound to several receptors on platelets, including SRA, CD36, LOX-1and so on to activate platelet.Ox-LDL can induce coronary artery smooth muscle cells and monocytes to secrete CD147. However, it is unclear whether ox-LDL can induce platelet to express CD147. Platelet activation plays a vital role in ACS and ox-LDL is associated with the severity of ACS. CD147can lead to plaque rupture by induce MMPs which degrade the main component of fibrous caps. Therefore, to investigate the influence of ox-LDL on platelet CD147expression is very important.HDL can protect vessels by reverse cholesterol transport, protecting the endothelial function, anti-inflammation and so on. In addition, HDL is capable of inhibiting platelet activation via SR-BI on platelet. However, whether HDL has an inhibitory effect on platelet CD147expression is unclear.In the study, we used ox-LDL or HDL to treat washed platelets and examined the CD147, CD62P and CD63expression by flow cytometry and soluble EMMPRIN (sCD147) levels using enzyme-linked immunosorbent assay (ELISA). Laser scanning microscopy (LSM) and transmission electron microscope (TEM) was utilized to observe the morphological changes and granule releases of platelets. We found that ox-LDL induces the expressions of CD147, CD62P and CD63on platelets, whereas HDL inhibits the effect.[MATERIALS AND METHODS]1. experimental subjectTo choose healthy human volunteers from the305th hospital of Chinese People’s Liberation Army using fresh blood to prepare platelet. Inclusion criteria were:(1). free from hemostatic disorders, diabetes, and infectious, hepatic, renal, cardiovascular, and malignant diseases, hypertension;(2). non-smoking human volunteers;(3). did not take any medicine during the preceding two weeks;(4). did not ingest alcohol in the previous24hours;(5). Elbow vein exposure significantly and is thicker;(6). The blood samples were collected after overnight fasting (12hours at least).2. Experimental methods2.1.1Blood collectionBlood is collected via forearm venipunctures (Elbow vein) performed by a trained nurse using a21G needle without a tourniquet. The blood samples were collected after overnight fasting via forearm venipunctures performed by a trained nurse using a21G needle without a tourniquet. After discarding the initial4ml,24ml of blood was injected gently into two15-ml polypropylene tubes containing1volume of acid-citrate-dextrose (2.5%sodium citrate,1.4%citric acid, and2%glucose) for6volumes of blood, and the tubes were mixed by gentle inversion.2.1.2preparation of washed plateletsThe main steps include:(1). To obtain platelet-rich plasma(PRP): anti-coagulated blood was centrifuged at100x g for15minutes at24℃without braking. The supernatants were collected by foul straw into a clean centrifugal tube;(2). To wash the platelet:By centrifuging the PRP at500x g for8minutes in the presence of500nM PGE1, the platelets were isolated. The platelets were resuspended and washed twice with modified Tyrode’s solution (137mM NaCl,2.7mM KC1,12mM NaHCO3,0.4mM NaH2PO4,5mM HEPES,0.1%glucose, and0.35%bovine serum albumin, pH7.4). The washing procedure was also performed after the addition of500nM PGE1to the platelet suspension and performed for twice.2.1.3detection of platelet2.1.3.1To observe morphology of platelet by optical microscopeThe main steps include:(1). To observe whether platelet was activated via blood smears by Wright’s-Giemsa stain (morphology change);(2). After staining PRP (Wright’s stain), platelets were observed under the microscope to examine whether other cells were present or not;(3). To observe washed platelets2.1.3.2Flow cytometry (FCM) analysis of washed platelets The main steps includes:(1). The platelets were gated on the basis of light scatter and CD61-FITC expression and observe whether other cells were present;(2). To detect percentage of CD62P-positive platelets to examine whether platelet was activated by washing procedure;(3). To detect the function of platelet activation by the stimuli of thrombin.2.2Detections of CD147, CD62P and CD63by FCM2.2.1Washed platelets were stimulated with ox-LDLThe washed platelets were incubated with ox-LDL (50μg/ml or100μg/ml) for25minutes at37℃. After incubation, each sample was incubated with anti-CD61-FITC to gate platelets and then with anti-CD147, anti-CD62P and anti-CD63for20minutes in a dark box. After incubation with antibodies, each sample was added500μl Tyrode’s and then detected by FCM. In parallel, washed platelets were treated with PBS as a control.The isotype-matched control for nonspecific binding was utilized. The results were expressed as the percentage of antibody-positive platelets and CD147relative mean fluorescence intensity.2.2.2Platelets were pre-incubated with HDL prior to ox-LDLIn parallel with ox-LDL-treated platelets, platelets were treated with HDL (100μg/ml) prior to ox-LDL (50μg/ml、100μg/ml) at37℃for10minutes. Then the platelets were incubated with ox-LDL (50μg/ml or100μg/ml) for25minutes at37℃, incubated with anti-CD147,anti-CD62P and anti-CD63for20minutes in a dark box and then detected by FCM. The isotype-matched control for nonspecific binding was utilized. The results were expressed as the percentage of antibody-positive platelets and CD147relative mean fluorescence intensity.CD147relative mean fluorescence intensity was defined as monoclonal antibody/corresponding isotype control.2.3Soluble EMMPRIN (sCD147) levels were assessed by ELISA After incubation of the washed platelets with ox-LDL (50μg/ml or100μg/ml) for25minutes at37℃, the samples were centrifuged at750x g for20minutes. In parrel, PBS was added to washed platelets as control.The supematants were collected into clean EP tubes carefully, then using a commercially available human anti-CD147ELISA kit, according to the manufacturer’s instruction.2.4Influence of ox-LDL on platelets in morphology by LSMThe washed platelets either treated or not with ox-LDL (50μg/ml or100μg/ml) for25minutes at37℃were incubated with anti-CD61-FITC for25minutes (5μl per100μl sample). Samples were smeared on siliconized slides and viewed immediately via LSM.2.5Observe platelets by TEMWashed platelets were divided into4groups, namely,50μg/ml,100μg/ml ox-LDL-treated group, control group, HDL+50μg/ml ox-LDL-treated group.The main steps:(1). Firstly fixed with2.5%glutaraldehyde and2%paraformaldehyde,(2). Secondly fixed with osmium tetroxide,(3). washed twice with0.1M cacodylate buffer (pH7.4).(4). dehydrated with alcohol step by step,(5). embedded, sectioned,(6). double-stained with uranyl acetate and lead citrate.(7). The samples were observed with a transmission electron microscope.3. Statistical analysisAll experiments were performed at least five times with platelets from different donors. All data were presented as as means±SD. The expressions of CD147, CD62P and CD63of washed platelets treated with different concentrations of ox-LDL(control group, namely PBS group;50,100μg/ml) were assessed using one-way ANOVA and comparisons between two groups were performed by LSD-test (or Dunnell’s T3). Groups of platelets incubated with HDL or not were assessed using a paired Student’s t test. The statistics were analyzed using SPSS for Windows13.0. A value of P<0.05was considered statistically significant.[Results]1. Platelet CD147expression is up-regulated by ox-LDLThe percentages of platelets treated with50μg/ml or100μg/ml ox-LDL that were positive for CD147expression (8.06±3.90%, P=0.032;10.70±3.33%, P=0.006, respectively) were considerably higher than those of platelets untreated with ox-LDL (0.58±0.31%). However, no significant change in the expression of CD147on platelets was identified between the two groups of ox-LDL-treated platelets (50μg/ml vs.100μg/ml)(P>0.05). Similarly, the platelets treated with ox-LDL (50μg/ml or100μg/ml) had a significantly higher CD147relative mean fluorescence intensity (rMFI) than the untreated platelets (control group)(P<0.001). The soluble EMMPRIN (sCD147) levels increased in the100μg/ml ox-LDL-treated platelets compared with the untreated platelets (P=0.048); the difference between the50μg/ml ox-LDL-treated and untreated platelet sCD147levels was not significant (P=0.243). And the difference between the50μg/ml ox-LDL-treated and the100μg/ml ox-LDL-treated platelets was not significant (P=0.272). In particular, more intracellular a-granules were observed in the untreated platelets than in the ox-LDL-treated platelets by TEM, indicating that ox-LDL promoted platelet a-granule release.2. Ox-LDL induces platelet CD62P and CD63expressionThe percentage of CD62P-positive platelets was significantly higher in ox-LDL-stimulated platelets (50μg/ml or100μg/ml) than in non-stimulated platelets (P<0.001and P=0.004, respectively).The statistical difference between the two groups of different concentrations of ox-LDL in CD62P expression was not found. In parral, ox-LDL induces CD63expression (P<0.05). CD62P was located in a-granule and CD63in Lysosomal granules. We found that ox-LDL promoted platelet a-granule and Lysosomal granules release via TEM, indicating that ox-LDL up-regulates CD62P and CD63.3. HDL inhibits the ox-LDL-induced expression of platelet CD147, CD62P, and CD63platelets pre-incubated with HDL (100μg/ml) were stimulated with ox-LDL (50μg/ml or100μg/ml). Flow cytometry analysis suggested that HDL (100μg/ml) decreased CD147expression not only in the50μg/ml ox-LDL-stimulated platelets (P=0.027) but also in the100μg/ml ox-LDL-stimulated platelets (P=0.017). Pretreatment with HDL (100μg/ml) also attenuated the expression of CD62P and CD63on platelets stimulated with ox-LDL (50μg/ml or100μg/ml)(P<0.05).[Conclusions]The findings demonstrate that ox-LDL stimulates the expressions of CD147, CD62P and CD63on platelets and that the ox-LDL-stimulated effect is inhibited by HDL in vitro. These findings provide new insights into the relationship between ox-LDL/HDL and CD147in platelet in the context of plaque instability. |
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