Effects Of FXa On The Stability Of Atherosclerotic Lesion And The Underline Mechanisms

Atherosclerosis is a progressive inflammatory disease that can lead to sudden cardiac events by plaque rupture and subsequent thrombosis. FXa not only occupies a crucial position in the coagulation cascade responsible for thrombin generation, but also has pro-inflammatory effects. Hence, in the present study, we assess the effects of FXa on the development and stability of atherosclerotic lesions and explore the involved mechanisms.Objective:1. To assess the effects of fondaparinux, the specific indirect FXa inhibitor, on the development and stability of atherosclerotic lesions in apolipoprotein E-deficient mice;2. To explore the involved mechanisms that FXa affects the stability of atherosclerotic lesions;3. To investigate the pro-inflammatory effect of FXa in RAW 264.7 macrophage and explore the mechanisms.Method:1. Male ApoE-/- mice were placed on a lard-containing diet comprising 21% lard and 0.15% added cholesterol at 6 weeks of age. Fondaparinux was intraperitoneally administered at a dose of 5mg/kg/day from week 20 to 24. At the age of 24 weeks, mice were euthanized, blood samples were centrifuged at 1,500rpm for 10min at 4℃ to obtain plasma for the detection of plasma concentrations of total cholesterol、triglycerides、high-density lipoprotein and low-density lipoprotein cholesterol. The innominate arteries were dissected out, embedded in paraffin and serially sectioned (5μm). Every fifth section was stained with hematoxylin and eosin to evaluate the total lesion area、maximum lesion thickness、percentage of stenosis、thickness of fibrous cap and ratio of necrotic core. To identify vascular calcification, van Kossa stain was performed on adjacent slices. To detect intraplaque hemorrhage, additional sections were evaluated following Perls stain. To detect collagen, Masson stain was performed on adjacent slices. The expression of Factor X、a-SMA、Mac-2、VCAM-1、CD-31、 MMP-9、PAR-1 and PAR-2 in atherosclerotic lesions was detected by immunohistochemistry. Zymography was used to detect the enzyme activity of MMP-9. PAR-1 and PAR-2 signaling were examined by western blot. Total RNA was extracted from thoracic aortas and reverse transcribed with a RevertAid First Strand cDNA Synthesis Kit. The expression of inflammatory cytokine, Egr-1、 TNF-α、IFN-γ、IL-6、IL-10、MCP-1, was detected by Real-time PCR.2. The expression of PAR-1 and PAR-2 in RAW 264.7 macrophage was detected by western blot. Cells were stimulated with thrombin (specific PAR-1 agonist) and trypsin (specific PAR-2 agonist) respectively, then we examined the phosphorylation of ERK1/2 to assess whether the expression of PAR-1 and PAR-2 was involved in functional responses in RAW264.7 macrophage. Cells were exposed to different concentrations of FXa ranging from 0.25-1 U/ml, then we examined the phosphorylation of ERK1/2 to detect the maximal signal level. Cells were pre-incubated with TAP (specific FXa inhibitor) or hirudin (specific thrombin inhibitor), then stimulated with FXa, we examined the phosphorylation of ERK1/2 to verify that FXa-induced signal transduction is specific. Cells were pre-incubated with PBS (negative control) or ATAP2 (PAR-1 neutralizing antibody), then stimulated with FXa or thrombin, we examined the phosphorylation of ERK1/2 to verify that PAR-1 activation is not essential in FXa-induced signal transduction. Cells were exposed to thrombin (PAR-1 desensitization) or trypsin (PAR-2 desensitization) for 150 minutes prior to stimulation with FXa for indicated time points, then we examined the phosphorylation of ERK1/2 to verify that PAR-2 mediates FXa-induced signal transduction in the RAW 264.7 macrophage. Cells were pretreated with TAP、 SAM11 and U0126, then stimulated with FXa, then we extracted the total RNA of cells and detected the expression of pro-inflammatory cytokines, TNF-α、IFN-y、 IL-6 and MCP-1 by Real-time PCR to assess the pro-inflammatory effect of FXa in RAW 264.7 macrophage.Result:1. The administration of fondaparinux did not cause significant differences in body weight, total cholesterol、triglycerides、high-density lipoprotein and low-density lipoprotein cholesterol between experimental and control group. Administration of fondaparinux did not significantly reduce the total lesion area、maximum lesion thickness or percentage of stenosis. However, administration of fondaparinux increased the thickness of the fibrous cap remarkably (56.78±6.18μm vs 42.35±2.62μm of the control group, P=0.049) and decreased the ratio of necrotic core significantly (34.8±0.91% vs 41.32±1.44% of the control group, P=0.001). Moreover, The ratio of collagen to total lesion area had a significant increase (30.79±0.84% vs 23.52±1.04% of the control group, P=0.006) owing to the fondaparinux administration.2. Fondaparinux decreased the expression of Mac-2 (3.89±0.41 vs 5.24±0.35 of the control group, P=0.017)、a-SMA (5.67±0.54 vs 8.10±0.52 of the control group, P=0.002)、PAR-1 (5.19±0.43 vs 7.78±0.56 of the control group, P=0.001)、PAR-2 (3.13±0.30 vs 4.77±0.40 of the control group,P=0.003)、CD-31 (4.33±0.53 vs 6.33±0.60 of the control group, P=0.024)、MMP-9 (4.22±0.33 vs 7.45±0.44 of the control group, P=0.000) and VCAM-1 (6.22±0.60 vs 8.67±0.93 of the control group, P=0.041) in atherosclerotic lesion. There was no statistical significant difference in the staining against Factor X between experimental and control group. Fondaparinux reduced the enzyme activity of MMP9 (0.74±0.04 vs 1.00±0.07 of the control group, P=0.032) significantly. The PAR-1 and PAR-2 signaling was reduced significantly in the fondaparinux group.3. The mRNA expression of inflammatory mediators, such as Egr-1、TNF-α、IFN-γ、 IL-6、IL-10 and MCP-1, had a significant reduction in the group of fondaparinux-treated mice compared with controls.4. PAR-1 and PAR-2 were expressed and functional in RAW 264.7 macrophages.5. FXa elicited signal transduction in RAW 264.7 macrophage, which is specific, independent of thrombin formation. The maximal signal level was obtained when stimulated with 0.75 U/ml FXa.6. PAR-1 neutralization, ATAP2 didn’t impact the strong phosphorylation of ERK1/2 induced by FXa, but inhibited thrombin-induced ERK1/2 phosphorylation. FXa still induced ERK1/2 phosphorylation in thrombin-desensitized cells, in contrast, trypsin-desensitized cells failed to respond to subsequent FXa stimulation.7. FXa enhanced the expression of pro-inflammatory mediators, TNF-α、IFN-γ、L-6 and MCP-1 in RAW 264.7 macrophage. However, TAP、SAM11 and U0126 blocked FXa-induced expression of pro-inflammatory mediators.Conclusion:1. The inhibition of FXa does not affect body weight、serum lipid level and the progression of atherosclerosis development, but decreases inflammation、 neovascularization、expression and enzyme activity of MMP-9 in atherosclerotic lesion, it also increases the thickness of the fibrous cap and the ratio of collagen to total lesion area, reduces the ratio of necrotic core.2. The inhibition of FXa can promote the stability of atherosclerotic lesion, which is closely related to PAR-1/2 signal regulation.3. FXa induces PAR-2-dependent pro-inflammatory activity in RAW 264.7 macrophages through the ERK1/2 pathway.