| Background and Research StrategyAdvances in recent research have provided greater understanding of the underlying pathophysiology of atherosclerosis. Evidence now supports that atherosclerosis is a progressive, dynamic, inflammatory process. Low-density lipoprotein cholesterol is recognized as a major cause of atherosclerosis. LDL particles in hyper-cholesterolemic patients provide the initial inflammatory response leading to thrombus formation. The higher the LDL-C level, the greater the risk of atherosclerotic heart disease, the most direct evidence is that the statins could down-regulate the coronary events by lowing the LDL-C. However, a latest research indicated that the extent of down-regulated LDL-C don't accord with the degree of improvement on ASIt had been regarded that the platelets don't actively participate in immune responses, for it is anuclear cell. But in recent years, it has been found that the activated platelets actively participate in inflammatory reactions, immune responses via expressing membrane protein such asαIIbβ3(PAC-1),CD40L,P-seletcin and secreting TNF-a, IL-Iβ, IL-3, IL-6 and adhesion molecules(e.g. CD36) when tissue injured. Blood platelets not only play critical roles in homeostasis but also are engaged in cell's interaction. Platelet's micro-particles facilitate the interaction of mononuclear cells with endothelial cells and stimulate the mononuclear cells secreting adhesion molecules. Importantly, the discovery that platelets express CD40 and CD40 Ligand on its surface makes sure that the platelets may play important role in inflammatory reaction and immune response, for it's widely conceded that CD40-CD40L is part of whole organic immune network. Now it is conceded that activated platelets involve in atherosclerotic vascular disease, inflammatory reaction and immune response via expressing CD40 and CD40L on its surface. Through CD40-CD40L pathway, platelets participate in initiation and regulation of atherosclerosis, DM, inflammatory disease and other systemic diseases. It becomes a new hotspot of research field now.Vascular endothelium plays a crucial role in atherosclerosis, endothelial cells injury and dysfunction may be the most important reason for initiation and progression of atherosclerosis; recently, some studies demonstrated that several genes of vascular endothelial cells are correlated with the formation and progression of atherosclerosis. For example, Kotowicz's demonstrated endothelia, smooth muscle and mononuclear/macrophage cells expressed CD40L and CD40 as these cells were cultured in serum-free medium in vitro, and the expression increased apparently when these cultured cells stimulated by IL-1β, TNF-α, INF-αor ox-LDL, the interaction of CD40 with CD40L up-regulated the expression of adhesion molecules, cytokines, matrix metalloproteinase (MMP), tissue factor (TF). Take another example for atherosclerosis-related genes; PPARαis a nuclear receptor, named Peroxisome proliferators-activated receptorα. PPARαis expressed in atherosclerotic plaque, endothelial cells, and smooth muscle cells and mononuclear/macrophage cells in atherosclerotic plaques also have up-regulated expression of PPARα. Activated PPARαhas been reported of inhibiting production of iNOS, MMP and SR-A in macrophage and foam cells, and of inhibiting the recruitment of mononuclear cells to injured or atherosclerotic endothelial cells. The third gene we talk about is COX-2, COX-2 also participate in formation and progression of atherosclerosis, there have notably up-regulated expression of COX-2 in atherosclerotic plaque, but it's argued for its forward or backward effects of COX-2 on atherosclerosis, but it's sure that COX-2 is up-regulated in the atherosclerosis plaque and regulated by NF-κB. Metzner J have demonstrated that the cox-2 promote the formation of atherosclerosis in the early stage in ApoE deficient mouse model. With evolvement of the study, more and more results support that COX-2 promote the formation and progression of atherosclerosis.Basing on the argument effects of LDL on atherosclerosis, basing on the new discoveries of immune and inflammatory functions of platelets and the new effects of PPARαand COX-2 genes on the atherosclerosis, we suppose what changes of platelet's immune and inflammatory function would be in hyperlipidemia patients and what changes of the function would be if platelets are placed in the hyperlipidemia plasma or high LDL culture mediums and what the change would be if interfered by statins? What effects would be produced on the expression of COX-2 and PPARαin endothelial cells if immune activated platelets are co-cultured with the endothelial cells? What effect would be produced by adding the LDL into the co-culture medium and what is the potential mechanism or pathway? There are few researches about these, so we try to do some works on these aspects.Detect CD40L and PAC-1 expression of platelets from clinic simple hyperlipoproteinaemia patients before treatment and after treatment of Fluvastatin for 4 weeks. Culture platelets with simple hyperlipoproteinaemia patient's plasma in vitro, observe the effects of hyperlipoproteinaemia plasma and fluvastatin on the expression of PAC-1 and CD40L on platelets, subsequently, stimulate platelets with LDL or LDL and ADP, detect the expression of CD40L and PAC-1 and observe the effects of LDL and fluvastatin on the expression of CD40L and PAC-1.Co-culture activated platelets with endothelial cells of human umbilical vein (HUVECs) and interfer in it with LDL in needed group, observe the expression and activity of PPARαand COX-2 genes in HUVECs by real time RT-PCR, Western Blot, ELISA and activity detection of nuclear factor. Try to obseve the effects of activated platelets and LDL on the expression and activity of PPARαand COX-2 in HUVECs. In third part, we use the CD40L monoclonal antibody to block the pathway of CD40-CD40L, use reconstituted human CD40 agonist to activate the CD40-CD40L pathway, observe the expression and activity of COX-2 and PPARαgenes by the same detection methods described above. Try to study the potential mechanisms.Part one: The effect of blood lipid on immune and inflammatory functions of platelets. ObjectiveTry to study on the CD40L and PAC-1 expression of platelets from clinic simple hyperlipoproteinaemia patients by detecting the platelets before and 4 weeks after treatment of Fluvastatin, culture the platelets with simple hyperlipoproteinaemia patients plasma in vitro, observe the direct effect of hyperlipoproteinaemia plasma on the immune activation of platelets, stimulate the platelets with LDL or LDL and ADP, observe the expression of CD40L and the concentration of sCD40L by RT-PCR, Western Blot, Flow Cytometry and ELISA as the immune activation marker.Methods1. Firstly take further leukocytes filtration of aphaeresis platelets before preservation and culture of platelets.2. Select 20 cases of clinic simple hyperlipoproteinaemia patients, treat these patients with fluvastatin 40 mg per day for 4 weeks, and detect PAC-1 and CD40L expression on platelets by Flow Cytometry before and after treatment.3. Collect the patient's plasma according to design requirements, culture healthy platelets with the hyperlipoproteinaemia plasma and interfere with Fluvastatins in needed group. Detect expression of PAC-1 and CD40L on platelets by Flow cytometry. Try to observe the effects of hyperlipoproteinaemia patient's plasma and fluvastatin on the expression of CD40L and PAC-1 on platelets.4. Culture the platelets and interfere with LDL or LDL with ADP, observe the effects of LDL on the expression of CD40L mRNA and protein of platelets,try to further explore the effects of LDL and fluvastatin on the immune activation of platelets.5. groupingSection one: expression of PAC-1 and CD40L on platelets in hyperlipoproteinaemia patients and effects of fluvastatin on it(1) Control group: 20 cases of healthy person with normal blood lipid(2) Pre-treatment of Hyperlipoproteinaemia group: 20 cases of simple hyperlipoproteinaemia patients with LDL-C>4.0mmol/L, TG<1.7mmol/L. Liver disease, renal disease, Hypertension, DM, hyperthyroidism, infectious disease and chronic inflammatory disease were excluded. (3)Post-treatment of Hyperlipoproteinaemia group: 20 cases of patients above were treated with Fluvastatins 40 mg per day for 4 weeks.Section two: effects of Hyperlipoproteinaemia (high LDL-C) plasma on the immune activation of platelets and the effects of fluvastatin on it(1) Control group: Uncultured healthy platelets brought from blood center are detected in 12 hours(2) Normal LDL-C group: Normal platelets are cultured in Normal LDL-C plasma at 22±2℃and gently oscillating state for 4 days. Chose 3 cases of normal LDL-C plasma from healthy person and do 6 times culture per plasma.(3)High LDL-C group: Normal platelets are cultured in High LDL-C plasma at 22±2℃and gently oscillating state for 4 days. Chose 3 cases of high LDL-C plasma from hyperlipoproteinaemia person and do 6 times culture per plasma.(4) Fluvastatin-added high LDL-C plasma group: Normal platelets are cultured in Fluvastatin-added high LDL-C plasma at 22±2℃and gently oscillating state for 4 days. The high LDL-C plasma comes from group 3.Section three: effect of low-density lipoprotein (LDL) on CD40L expression of platelets.According to requirements, platelets are divided into 6 groups and cultured in incubator at 37℃, 5%CO2.(1) Control group: Platelets are cultured in RPM 1640 medium for 12 hours.(2)LDL (5μg/mL) group: Platelets are cultured in RPM 1640 medium and stimulated with LDL at final concentration of 5μg/mL for 12 hours.(3) LDL (50μg/mL) group: Platelets are cultured in RPM 1640 medium and stimulated with LDL at final concentration of 50μg/mL for 12 hours.(4) ADP control group: Platelets are cultured in RPM 1640 medium and stimulated ADP at final concentration of 1μmol/L for 12 hours.(5)ADP-added LDL (50μg/mL) group: Platelets are cultured in RPM 1640 medium and stimulated with LDL and ADP at final concentration of 50μg/mL and 1μmol/L respectively for 12 hours. (6) Fluvastatin group: Platelets are cultured in Fluvastatin-added RPM 1640 medium and stimulated with same LDL and ADP as group 5 for 12 hours, the final concentration of fluvastatin is 10-2mol/L.6. Parameters for detection(1) Detect and estimate the purity and concentration of platelets by direct cell counting under microscope and Flow Cytometry.(2) Detect positive platelets expressed PAC-1 or CD40L by Flow Cytometry.(3) The expression of CD40L mRNA is measured by RT-PCR(4) The expression of CD40L protein is measured by Western Blot(5) The concentration of soluble CD40L (sCD40L) in culture medium is measured by ELISA,ResultsSection 1: expression of PAC-1 and CD40L on platelets in hyperlipoproteinaemia patients and effects of fluvastatin on the expression.(1) Compared pre-treatment of Hyperlipoproteinaemia group with post-treatment of Hyperlipoproteinaemia group, LDL-C concentration decreased significantly in the post-treatment group(3.98±0.57 VS 4.97±0.67,P<0.01).(2) Compared pre-treatment of Hyperlipoproteinaemia group with control group, percentage of PAC-1 expressed platelets increased apparently in the pre-treatment group(9.47±1.96 VS 5.73±1.20,P<0.01);Compared the post-treatment group with pre-treatment of Hyperlipoproteinaemia group, the percentage of PAC-1 expressed platelets decreased significantly in post-treatment group(7.29±1.35 VS 9.47±1.96,P<0.01).(3) Compared the pre-treatment group with control group, percentage of CD40L expressed platelets increased apparently in the pre-treatment group(3.04±0.62 VS 1.87±0.49,P<0.01);Compared the pre-treatment group with the post-treatment group, the percentage of PAC-1 expressed platelets decreased significantly in the post-treatment group(2.17±0.53 VS 3.04±0.62,P<0.01).(4) The concentration of LDL-C has linear correlation with the percentage of PAC-1 expressed platelets(r=0.497, n=60, P<0.01) and with the percentage of CD40L expressed platelets (r=0.473, n=60, P<0.01).Section 2: effects of Hyperlipoproteinaemia plasma on immune activation of platelets in vitro(1) In normal LDL-C plasma culture group, percentage of PAC-1 expressed platelets increased significantly compared with control group(6.39±1.23 VS 3.75±1.06, P<0.01), Compared high LDL-C plasma culture group with the normal LDL-C group, the percentage increased apparently in the high LDL-C group(10.47±1.39 VS 6.39±1.23,P<0.01), in fluvastatin-added high LDL-C plasma culture group, the percentage didn't change obviously compared with the high LDL-C group(10.39±1.41 VS 10.47±1.39,P>0.05).(2) In normal LDL-C group, percentage of CD40L expressed platelets increased significantly compared with control group(1.87±0.47 VS 0.75±0.41,P<0.01), Compared high LDL-C group with normal LDL-C group, the percentage increased apparently in high LDL-C group(3.19±0.73 VS 1.87±0.47, P<0.01), in fluvastatin-added high LDL-C group, the percentage didn't change obviously either compared with high LDL-C group(3.21±0.69 VS 3.19±0.73,P>0.05)(3) Linear regression analysis of concentration of LDL-C with percentage of PAC-1 or CD40L expressed platelets indicated that the concentration of LDL-C has linear correlation with the percentage of PAC-1 expressed platelets and CD40L expressed platelets (Pall<0.05).Section 3: effects of LDL and fluvastatin on CD40L expression of platelets.(1) Effects of LDL or LDL and ADP on PAC-1 expression on platelets and possible effects of fluvastatin on the expression.Compared LDL (5μg/mL) group with control group, percentage of PAC-1 positive platelets detected by Flow Cytometry did not change obviously(3.84±1.71 VS 3.87±1.63,P>0.05); Compared LDL (50μg/mL) group with control group, the percentage of PAC-1 positive platelets did not change obviously either(3.99±1.74 VS 3.87±1.63,P>0.05); Compared ADP control group with control group, the percentage of PAC-1 positive platelets increased significantly in ADP control group(11.31±2.12 VS 3.87±1.63,P<0.01); compared ADP-added LDL (50μg/mL) group with ADP control group, the percentage increased too in ADP-added LDL group(17.73±2.09 VS 11.31±2.12, P<0.05);In fluvastatin group, the percentage of PAC-1 positive platelets didn't decrease compared with ADP-added LDL (50μg/mL) group(17.81±2.15 VS 17.73±2.09,P>0.05).(2) Effects of LDL or LDL and ADP on CD40L expression on platelets and possible effects of fluvastatin on the expression.Just like the expression of PAC-1, Compared LDL (5μg/mL) group with control group, percentage of CD40L expressed platelets detected by Flow Cytometry did not change obviously(0.78±0.47 VS 0.77±0.44,P>0.05); Compared LDL (50μg/mL) group with control group, the percentage did not change obviously either(0.83±0.46 VS 0.77±0.44,P>0.05); Compared ADP control group with control group, the percentage increased significantly in ADP control group (2.91±0.69 VS 0.77±0.44,P<0.01); compared ADP-added LDL (50μg/mL) group with ADP control group, the percentage increased too in ADP-added LDL group(4.17±0.71 VS 2.91±0.69,P<0.05);In fluvastatin group, the percentage didn't decrease compared with ADP-added LDL (50μg/mL) group(4.09±0.76 VS 4.17±0.71,P>0.05).(3) Effects of LDL or LDL and ADP on concentration of soluble CD40L (sCD40L) in culture medium and possible effects of fluvastatin on itCompared LDL (5μg/mL) group with control group, concentration of sCD40L in culture medium did not change obviously(4.14±1.47μg/L VS 3.81±1.27μg/L,P>0.05); Compared LDL (50μg/mL) group with control group, the concentration increased seemingly in LDL (50μg/mL) group, but it's not significant in statistics(4.37±1.54μg/L VS 3.81±1.27μg/L,P>0.05); Compared ADP control group with control group, the concentration increased significantly in ADP control group (9.31±2.12μg/L VS 3.81±1.27μg/L ,P<0.01); compared ADP-added LDL (50μg/mL) group with ADP control group, the concentration also increased in ADP-added LDL(50μg/mL) group (11.38±2.19μg/L VS 9.31±2.12μg/L,P<0.05);In fluvastatin group, the concentration didn't decrease compared with ADP-added LDL (50μg/mL) group(11.27±2.11μg/L VS 11.38±2.19μg/L,P>0.05).(4) Effects of LDL or LDL and ADP on CD40L mRNA expression in platelets and possible effects of fluvastatin on it.Compared LDL (5μg/mL) group with control group, CD40L mRNA expression in platelets did not change obviously(0.97±0.08 VS 0.99±0.07, P>0.05); Compared LDL (50μg/mL) group with control group, the CD40L mRNA expression in LDL (50μg/mL) group did not increase in statistics(1.10±0.12 VS 0.99±0.07,P>0.05); Compared ADP control group with control group, the CD40L mRNA expression didn't upregulated in ADP control group (1.02±0.09 VS 0.99±0.07,P>0.05); the expression of CD40L mRNA in ADP-added LDL (50μg/mL) group didn't upregulated either compared with control group, (0.98±0.08 VS 0.99±0.07,P>0.05);In fluvastatin group, the expression of CD40L mRNA didn't change obviously compared with ADP-added LDL (50μg/mL) group(1.04±0.10 VS 0.98±0.08,P>0.05).(5): effects of LDL or LDL and ADP on CD40L protein expression in platelets and the possible effects of fluvastatin on it.Compared LDL (5μg/mL) group with control group, CD40L protein expression in platelets did not change obviously(0.90±0.12 VS 0.96±0.10,P>0.05); Compared LDL (50μg/mL) group with control group, the CD40L protein expression did not increase in statistics(1.05±0.17 VS 0.96±0.10,P>0.05); Compared ADP control group with control group, the CD40L protein expression increased significantly in ADP control group (1.40±0.21 VS 0.96±0.10,P<0.01); compared ADP-added LDL (50μg/mL) group with ADP control group, the expression increased in ADP-added LDL (50μg/mL) group (1.63±0.19 VS 1.40±0.21,P<0.05);In fluvastatin group, the expression of CD40L protein didn't decrease obviously compared with ADP-added LDL (50μg/mL) group(1.59±0.21 VS 1.63±0.19,P>0.05).Conclusionsimmune activated Platelets express several membrane proteins and receptors engaged in immune response and inflammatory reaction, such as GPⅡb /Ⅲa (PAC-1), CD40 Ligand, CD40 receptor,P-selectin, on the contrary, inactivated platelets do not express these proteins and receptors or express a little, so as platelets were stimulated by some cytokines or activators and expressed these membrane proteins and receptors, it was called immune activation of platelets. Our study demonstrated that Compared pre-treatment of Hyperlipoproteinaemia group with control group, the percentage of PAC-1 and CD40L expressed platelets increased apparently in the pre-treatment group, which indicated platelets are partly activated on immune and inflammatory function in Hyperlipoproteinaemia patients. Linear correlation analysis of LDL-C concentration with percentage of PAC-1 or CD40L expressed platelets demonstrated the concentration of LDL-C has linear correlation with the percentage of PAC-1 expressed platelets and CD40L expressed platelets, which indicated the LDL-C may be correlated with the immune activation of platelets. After 4 weeks treatment of fluvastatin, the percentage of PAC-1 and CD40L expressed platelets decreased followed the decreased concentration of LDL-C, which indicated the fluvastatin could decrease the concentration of LDL-C in patients and partly inhibit the immune activation of platelets at the same time.By culturing healthy platelets in plasma contained high concentration of LDL-C in vitro, discovered that the percentage of PAC-1 and CD40L expressed platelets in high LDL-C plasma culture group increased apparently compared with normal LDL-C plasma culture group, which indicated the LDL-C may be correlated with the immune activation of platelets in vitro too. However, the fluvastatin didn't down-regulate the percentage of CD40L and PAC-1 expressed platelets in vitro, which indicated fluvastatin may not be able to inhibit the immune activation of platelets directly.Platelets express LDL receptor on its surface, for further study of whether the LDL-C could directly activate the platelets; we cultured heathy platelets in RPM 1640 medium and stimulated the platelets with LDL. Fond LDL at concentration of 5μg/mL or 50μg/mL didn't increase the expression of PAC-1 and CD40L on and in platelets, however LDL of 50μg/mL further up-regulated the ADP activated expression of PAC-1 and CD40L of platelets, which indicated LDL may not be able to directly increase the immune activation of platelets, it may increase the sensitivity of platelets to other activator and increase the immune activation indirectly. These make us deduce that the increased immune activation of platelets in hyperlipoproteinaemia patients may not be directly induced by LDL-C in plasma; it may be induced by ox-LDL, inflammatory cytokines or other activators; the LDL-C may increase the sensitivity of platelets to other activators and indirectly up-regulate the immune activation of platelets.Part 2: effects of immune activated platelets on expression and activity of COX-2 and PPARαof HUVECsObjectiveIn this part, study effects of immune activated platelets on COX-2 and PPARαexpression of HUVECs by co-culturing HUVECs with platelets in vitro and study the possible effect of LDL in the co-culture process by adding LDL in necessary group.Methods1 Culture of HUVECsRevivified HUVECs were cultured and proliferated in cells incubator at 37℃and 5%CO2 conditions, after the revivified HUVECs have been cultured for 24 hours, the shape of the cells changed to thin and flat, the cell volume expanded, the nuclear become clear and quantity of the nuclear increased, part of the nuclear changed shape to polygon, aggregated cells dispersed and intercellular space have been enlarged. The densely covered monolayer of cells formed after the cells have been cultured for 48 to 72 hours. The survival rate of cells was over 95% by trypan blue. Adjusted the concentration of cells to 2×106/ml by cell counting chamber and transferred these cells to six well plates, grouped and interfered with the HUVECs as HUVECs adhered to bottom of six well plates.2 Culture of platelets and co-culture of platelets with HUVECsFirstly take further leukocytes filtration of aphaeresis platelets brought from province blood center before preservation and culture of it, subsequently, we detect the content of red blood cells and white blood cells in platelet's plasma and make sure that the concentration of red and white blood cells are lower enough. The filtrated platelets firstly cultured and persevered at condition of 22℃and gently oscillating state. Before the experiment, the platelets were washed with warm PBS buffer solution at 22℃and centrifuged at 4000G and 22℃for 10 minutes and recovered the platelets, Repeated the process above twice again. In the end, adjusted the concentration of platelets to 2×107/ml and co-cultured with HUVECs at the concentration of 2×106/ml in the incubator at the conditions of 37℃and 5%CO2 for short time, the co-culture medium was RMP1640.3 Method for activating plateletsWhen platelets were stimulated by ADP, the expression of PAC-1 and CD40L of platelets increased prominently in short time. As we know, the CD40L of platelets engaged in immune response and inflammatory reaction broadly, so we called the increased expression of CD40L of platelets immune activation of platelets. For getting enough activation of platelets, we activated the platelets by adding ADP at concentration of 5μmol/L4 GroupingAccording to design, six groups are divided (and n=6).(1) Control group: HUVECs are cultured in incubator at condition of 37℃, 5%CO2 for 8 hours(2) Platelet control group: Platelets are co-cultured with HUVECs at the ratio of 2×107:2×106 at the condition above for 8 hours(3) ADP control group: Add ADP into the culture medium to final concentration of 5μmol/L and culture HUVECs at the condition above for 8 hours.(4) LDL control group: Add LDL into the culture medium to final concentration of 50μg/mL and culture HUVECs at the condition above for 8 hours.(5) Platelets activated group: Add ADP into the co-culture medium to final concentration of 5μmol/L and co-culture HUVECs with platelets at the condition above for 8 hours.(6) Platelets activated plus LDL group: Firstly add LDL into the co-culture medium to final concentration of 50μg/ml for 30 minutes, secondarily, add the ADP into the co-culture medium to final concentration of 5μmol/L and co-culture at the condition above for 8 hours.5 Parameters for detection(1) CD40 mRNA expression of HUVECs is detected by RT-PCR.(2) Expression of COX-2 and PPARαmRNA is detected by RT-PCR(3) Expression of COX-2 and PPARαprotein is detected by Western Blot(4) The concentration of PGE2 in co-culture medium is measured by ELISA (5) PPARαactivity of HUVECs is detected by EIAResults(1) Effect of immune activated platelets on expression of COX-2 mRNA and possible role LDL played.HUVECS cultured in vitro had a little expression of COX-2 mRNA, compared platelet control group or ADP control group with control group, expression of COX-2 mRNA in HUVECs didn't change obviously (0.68±0.21 VS 0.53±0.17,0.77±0.25 VS 0.53±0.17,Pall>0.05); compared LDL control group with control group, the expression of COX-2 mRNA increased seemingly in LDL control group, but it's not significant in statistics(0.83±0.26 VS 0.53±0.17,P>0.05); compared platelets activated group with ADP control group or platelet control group, the expression of COX-2 mRNA increased significantly in platelets activated group (1.49±0.27 VS 0.53±0.17,1.49±0.27 VS 0.68±0.21,Pall<0.01); in platelets activated plus LDL group, the expression of COX-2 mRNA increased significantly compared with platelet activated group(1.79±0.20 VS 1.49±0.27,P<0.05).(2) Effect of immune activated platelets on expression of PPARαmRNA and possible role LDL played.HUVECS cultured in vitro had a little PPAR mRNA expression in HUVECs, the expression of PPARαmRNA in platelet control group, ADP control group and LDL control group didn't change obviously compared with control group(1.17±0.16 VS 1.13±0.17,1.19±0.19 VS 1.13±0.17 and 1.12±0.14 VS 1.13±0.17,Pall>0.05); the expression of PPARαmRNA in platelets activated group up-regulated significantly compared with ADP control group or platelet control group(1.45±0.21 VS 1.19±0.19,1.45±0.21 VS 1.17±0.16,Pall<0.01); in platelets activated plus LDL group, the expression of PPAR mRNA increased significantly compared with platelet activated group(1.74±0.23 VS 1.45±0.21,P<0.01).(3) Effect of immune activated platelets on expression of COX-2 protein and possible role LDL played.HUVECS cultured in vitro had a little COX-2 protein expression in HUVECs, all the expression of COX-2 protein in platelet control group, ADP control group and LDL control group didn't change obviously compared with control group(0.70±0.07 VS 0.57±0.07,0.71±0.07 VS 0.57±0.07 and 0.58±0.070 VS 0.57±0.07,Pall >0.05); the expression of COX-2 protein in platelets activated group up-regulated significantly compared with ADP control group or platelet control group(1.60±0.15 VS 0.70±0.07,1.60±0.15 VS 0.71±0.07,Pall <0.01); in platelets activated plus LDL group, the expression of COX-2 protein increased significantly compared with platelet activated group(1.97±0.15 VS 1.60±0.15,P<0.05).(4) Effect of immune activated platelets on expression of PPARαprotein and possible role LDL played.HUVECS cultured in vitro had a little expression of PPARαprotein in HUVECs, both the expression of PPARαprotein in platelet control group and ADP control group didn't change obviously compared with control group(0.96±0.07 VS 0.98±0.08,0.95±0.09 VS 0.98±0.08, Pall >0.05); compared LDL control group with control group, the expression of PPARαprotein increased seemingly in LDL control group, but it's not significant in statistics(1.12±0.012 VS 0.98±0.08,P>0.05); expression of PPARαprotein in platelets activated group up-regulated significantly compared with ADP control group or platelet control group(1.63±0.14 VS 0.96±0.07,1.63±0.14 VS 0.95±0.09,Pall <0.01); in platelets activated plus LDL group, the expression of PPAR protein increased significantly compared with platelet activated group(1.95±0.16 VS 1.63±0.14,P<0.05). (5) Effect of immune activated platelets on COX-2 activity and possible role LDL played.We got the COX-2 activity in HUVECs by measuring the concentration of PGE2 in culture or co-culture medium. By calculating and analyzing these measured data, we fond that the mono-culture medium of HUVECs had low concentration of PGE2(21.31±2.34pg/ml), the concentration of PGE2 in platelet control group, ADP control group and LDL control group didn't change obviously compared with control group(23.73±2.53 pg/ml VS 21.31±2.34pg/ml,22.26±2.27pg/ml VS 21.31±2.34pg/ml, 25.74±3.12 pg/ml VS 21.31±2.34pg/ml, Pall>0.05); the concentration of PGE2 in platelets activated group increased significantly compared with platelet control group or ADP control group(42.46±5.57 pg/ml VS 23.73±2.53 pg/ml, 42.46±5.57 pg/ml VS 22.26±2.27pg/ml, Pall>0.05); in platelets activated plus LDL group, the concentration of PGE2 increased significantly compared with platelet activated group(64.35±6.19 pg/ml VS 42.46±5.57 pg/ml, P<0.01).(6) Effect of immune activated platelets on PPARαactivity of HUVECs and possible role LDL played.PPARαin HUVECs cultured in vitro had a little ability binding to the PPREs of DNA, the basic binding activity was 0.56±0.14; the binding activity of PPARαin platelet control group, ADP control group and LDL control group didn't change obviously compared with control group(0.61±0.14 VS 0.56±0.14, 0.58±0.13 VS 0.56±0.14 and 0.64±0.16 VS 0.56±0.14, Pall>0.05); the binding activity of PPARαin platelets activated group increased seemingly compared with control group, but it's not significant in statistics(0.73±0.18 VS 0.56±0.14, P>0.05). The binding activity of PPARαin platelets activated plus LDL group didn't increase either compared with control group(0.76±0.17 VS 0.56±0.14,P>0.05).Conclusions Vascular endothelia cell injury or dysfunction is one important reason forinitiation and progression of atherosclerosis; COX-2 and PPARαin vascular endothelia cells are engaged in formation and progression of atherosclerosis, several latest studies support that COX-2 has strong correlation with inflammatory reaction and prompt the initiation and progression of atherosclerosis. On the contrary, activated PPARαhas been reported of inhibiting the production of iNOS, MMP and SR-A in macrophage and foam cells, and of inhibiting the recruitment of mononuclear cells to injured endothelial cells. The platelets could adhere to endothelial cells or recruit other cells adhering to endothelial cells by the receptor or ligand expressed on its surface, these function enlarge quickly when platelets are immune activated. In our study, we observed the effects of immune activated platelets on HUVECs by co-culturing immune activated platelets with HUVECs, especially observed the effects of immune activated platelets on expression and activity of COX-2 and PPARαof HUVECs and observed the role of LDL played in the co-culture process. Our study demonstrated that the activated platelets remarkably up-regulated COX-2 and PPARαexpression of HUVECs; the up-regulated expression of COX-2 accompanied by increased activity of COX-2 but the up-regulated expression of PPARαdidn't follow increased binding activity of PPARαin HUVECs. These results indicated the immune activated platelets may be engaged in the initiation or progression of atherosclerosis by up-regulating the expression and activity of COX-2 in vascular endothelia cells. The up-regulated expression of PPARαhadn't increased binding activity, which indicated the up-regulated PPARαmay not be able to exert adequate effects of inhibiting atherosclerosis by limiting inflammatory reaction. In our study, we fond the LDL itself at concentration of 50μg/mL didn't up-regulate the COX-2 and PPAR?...
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