The Effect And Mechanism Of Tumor Suppressor Gene PDCD4in Atherosclerosis

ObjectiveAtherosclerosis (AS) is the chronic inflammation of arterial vessel wall caused by abnormal metabolism of lipid. It is the primary pathological inducement of acute coronary syndrome (ACS) including angina pectoris, acute myocardial infarction or sudden death. AS has been a common disease that severely affects the health of elderly in our country and there is an increasing trend annually. Although the pathogenesis of AS is still not clear, in recent years the inflammation theory that atherosclerosis is the chronic inflammatory disease of the arterial wall has been broadly approved. Accumulated evidence demonstrates that many cytokines and immune cells participate in the development of AS. That is to say, atherosclerosis is a vascular damage process in which vascular wall cells and blood cells interact with each other under the effects of various kinds of inflammatory and pathogenesis factors. Both monocytes/macrophages-mediated innate immunity and T cells-mediated adaptive immunity play important roles during this process.Tumor suppressor genes can control the excessive proliferation of cells and prevent the formation of tumor. In recent years, many tumor suppressor genes such as P53and PTEN not only have an intimate association with the development of tumors, but also are found to be involved in the physiological and pathological process of immune responses and inflammatory diseases. Programmed cell death4(PDCD4) is defined as a novel tumor suppressor gene which plays an important role in the suppression of multiple tumors development. However, its effects on inflammatory diseases are still unknown. Limited reports showed that PDCD4knockout (Pdcd4-/-) mice could resist autoimmune disease including Experimental Autoimmune Encephalomyelitis (EAE), Type Ⅰ diabetes and LPS-induced endotoxic shock. These prompt us that PDCD4not only suppresses the development of tumor, but also has a crucial effect on inflammatory diseases. Then, does PDCD4really affect on the development of atherosclerosis? What effects does PDCD4have on atherosclerosis? What is the mechanism of this? All questions above are still not clear.We explored the role of PDCD4in AS and how it works in this study, which may provide theoretical evidence and experimental foundation for clinical therapy of AS. Here we will investigate these questions from the following two aspects:1. The expression and function of PDCD4during the formation of ASWe detected that as atherosclerosis plaque developed in Apoe-/-mouse, the expression of PDCD4in local atherosclerotic plaque of Apoe-/-mice increased gradually, which indicates that PDCD4participates in the development of atherosclerosis. Then we adopted the double gene-knock out mouse (Pdcd4-/-Apoe-/-) to prove that the deficiency of PDCD4significantly reduce plaque formation, which demonstrates that endogenous PDCD4plays a catalytic role in atherosclerosis. We also found that deficiency of PDCD4could influence the T cell and macrophage infiltration in local plaque.2. The influence of PDCD4on immunocytes and its function in the process of atherogenesisFirst, using bone marrow transplantation method, we proved that immunocytes play important roles during the process of PDCD4regulating atherosclerosis. Then using experiments of in vivo and in vitro, we compared the percentage of T lymphocytes and macrophages subsets as well as various cytokines those cells secreted between the two genotype mice both in the local of plaques and in peripheral. We found that deficiency of PDCD4significantly increased the level of IL-10, which further proved that the increase of IL-10level resulting from deficiency of PDCD4is one of the most important reasons for plaque reduction.Methods1. The expression and function of PDCD4during the formation of AS (1) The expression of PDCD4during the formation of AS1) We detected the expression of PDCD4in local plaques in vessels of mice given high fat diet for0,8and16weeks respectively by using Western blot method.2) We determined the kinds of cells in which PDCD4was expressed by using immunohistochemistry method.(2) The function of PDCD4during the formation of AS1) Hybridize Pdcd4-/-mice with Apoe-/-mice to obtain double genes knockout mice (Pdcd4-/-Apoe-/-) as the experimental group, while Apoe-/-mice as the control group. Both groups were given high fat diet to induce the formation of plaques in vessels.2) After high fat diet for8or16weeks, we separated aortic roots to make frozen sections, which were stained with HE, oil red O and immunohistochemistry to compare the difference of plaque sizes, lipid deposition, components (including smooth muscle and collagen) and inflammatory infiltration between Pdcd4-/-Apoe-/-and Apoe-/-mice. Then we stained the aortic root and thoracoabdominal aorta en face using oil red O to compare the plaque quantities and sizes between these two genotype mice.2. The influence of PDCD4on immune cells and its function in the process of atherogenesis(1) To prove that immune cells play main roles during the process in which deficiency of PDCD4reduced the development of atherosclerosis1) We collected bone marrow cells from both femur and tibia of Pdcd4-/-and C57BL/6mice and transplanted them respectively by tail vein injection into Ldlr-/-mice which had accepted mortal dose of X-ray radiation. After giving high fat diet for12or16weeks, we separated aortic roots to make frozen sections which will then be stained with HE and oil red O to compare the sizes of plaques. Furthermore, the frozen sections will also be stained to compare the infiltrations of T cells and macrophages in the local of plaques by immunohistochemistry.2) We tested and compared different infiltrating conditions of both macrophages and T cells in local plaques by using immunohistochemistry method. 3) Using Western Blot method, we tested the PDCD4expression in vessel plaques of Ldlr-/-mice that had received high lipid diets respectively for2weeks,12weeks and16weeks after accepting bone marrow transplantation.(2) The influence of PDCD4deficiency on macrophages and T cell subsets in vivo1) Spleen was grinded to make cell suspension after high fat diet for8or16weeks. We tested and compared the related proportional changes of both macrophages and T cells subsets by flow cytometry. Then we analyzed relevance between the changed percentage of subsets and the size of plaques using linear regression.2) We stained the frozen sections by immnunohistochemical or immunofluorescence method to detect the proportion changes of CD4and CD8in local plaques.3. The influence of PDCD4deficiency on macrophage and T cell related cytokines in vivo(1) Inside the serum:We compared the changes of cytokines, including IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17, IL-10and TGF-(3, in serum of experimental and control mice after given high fat diet by flow cytometry staining with BD Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17Cytokine Kit. Then we analyzed the correlation between the concentration of cytokines and the areas of plaques by linear regression analysis.(2) In local plaques:1) We stained frozen sections of local plaques to detect the changes of IL-17and IL-10levels.2) The aortic arch and thoracoabdominal aorta which contain a large of plaques were extracted total RNA and proteins respectively after given high fat diet for16weeks of experimental and control groups mice, and detected the expression of cytokines by real time PCR or Western blot respectively.4. To study the effect of PDCD4on T cell functions and the mechanism in vitro(1) The effect on the proliferation of T cell1) Spleen cells were cultivated and stimulated in vitro. Then we compared the proliferation conditions between experimental and control groups by using CCK8kit. 2) We detected the proliferation of CD4+, CD8+T cells by flow cytometry.3) We detected the proportion changes of co-stimulatory molecules CD28and CD137and co-inhibitory molecule CTLA-4on CD4+and CD8+T cells by flow cytometry.(2) The effect on cytokine secreted by T cells:We also detected various changes of cytokines in supernatant of activated T cells between two genotype mice by flow cytometry staining with BD CBA Mouse Th1/Th2/Th17Cytokine Kit.5. To study the effect of PDCD4on macrophage functions and the mechanism in vitro:Peritoneal macrophages of Pdcd4-/-and C57BL/6mice were collected, cultured in vitro and stimulated to activation by oxLDL for different periods of time. Then we compared the concentrations of cytokines, including pro-inflammatory factors IL-6and TNF-α and inhibitory inflammationary factor IL-10by ELISA.6. Experiments in vivo to ensure the effect of IL-17and IL-10on formation of plaques:After given high fat diet for4weeks, PDCD4-/-Apoe-/-mice were divided into three groups and given respectively physiological saline, recombination IL-17cytokine and IL-10neutralizing antibodies by intraperitoneal injection once a week for five weeks, and Apoe-/-mice as control were also given physiological saline. Then the aortic root of every mouse was separated to make frozen sections and stained by HE and oil red O to compare the sizes of plaques.7. To explore that PDCD4regulates the expression of IL-10in transcription or translation level in molecule mechanism(1) RNA binding with PDCD4was acquired by RNA-Binding Protein Immunopreci-pitation Kit, from which IL-10was amplified with real time PCR. This experiment showed whether PDCD4binds with IL-10mRNA, which could prove whether PDCD4inhibits IL-10transcription by binding with it.(2) The total RNA of activated macrophages was extracted and detected the expression of IL-10in mRNA level by RT-PCR.(3) Proteins of activated macrophages were extracted and detected the activating conditions of MAPK (JNK, ERK1/2and P38) signal pathway in Pdcd4-/-and C57BL/6mice. Then we detected the expression of IL-10after inhibiting the pathway using ERK and P38inhibitor to demonstrate the role this signal pathway plays in the process of IL-10production.Results1. The expression and function of PDCD4during the formation of AS(1) Establishment of atherosclerosis mice modelWe gave Apoe-/-mice of8weeks old high fat diet to make a high fat environment which induced the plaque formation. Take the8weeks old mice as the no-plaque stage,16weeks old mice (receiving high-fat diet for8weeks) as the early plaque stage and24weeks old mice (receiving high-fat diet for16weeks) as the late plaque stage.(2) The expression of PDCD4during the formation of ASTo determine the expression of PDCD4during the development of AS, we extracted genome protein from the aortic arch and thoracoabdominal aorta of16and24weeks old Apoe-/-mice respectively. The no-plaque vessels from8weeks old mice were taken as controls. The expression of PDCD4was detected in them by western blot. The results showed that as time of high fat diet extended, the expression of PDCD4increased significantly. Using immunohistochemical method, we found that PDCD4expressed in immune cells mainly and it also has expression in smooth muscle cells, which indicates that PDCD4may promote the formation and development of AS.(3) The role of PDCD4during the formation of AS plaquesTo assess the role of PDCD4in the development of AS, we took the double gene knock-out mice Pdcd4-/-Apoe-/-mice and Apoe-/-mice as the experimental and control groups respectively to study the effect of PDCD4deficiency on AS formation. The results showed that Pdcd4-/-Apoe-/-mice that had accepted high fat diet for both8weeks and16weeks had significantly smaller size of plaques (p<0.01), lower levels of lipid deposition (p<0.01) and weaker infiltration of macrophages and T cells (p<0.01). The collagen components and smooth muscle cells also reduced significantly. The data above prompt that deficiency of PDCD4results in smaller size of plaques, which means that PDCD4promotes the development of atherosclerosis.2. The influence of PDCD4on immunocytes and its effect on AS(1) To prove that immune cells play main roles during the process of PDCD4deficiency reduced AS developmentWe have known that immunocytes play important roles during the AS development and PDCD4deficiency could induce the reduction of macrophage and T cell infiltration in plaque. To demonstrate that PDCD4affected the formation of plaques through immune cells, we collected bone marrow cells from femur and tibia of C57BL/6and Pdcd4-/-mice and then transplant them into Ldlr-/-mice respectively. Then these mice were given high-fat diet to induce plaque formation. We separated aortic root to make frozen sections which would be then stained by both HE and oil red O. We found that the plaque area of Ldlr-/-mice that had accepted bone marrow cells from Pdcd4-/-mice was significantly decreased compared with that accepted C5BL/6bone marrow cells (p<0.05). Then we dissected the aortic arch and the thoracoabdominal aorta under dissecting microscope and stained them by red oil O and found that Ldlr-/-mice that had accepted bone marrow cells from Pdcd4-/-mice had significantly decreased numbers and areas of plaques (p<0.01). Infiltration of macrophages and T cells in the plaques of Ldlr-/-mice that had accepted bone marrow cells from Pdcd4-/-mice was much weaker than that of the control group. Those data above demonstrate that immune cells play an important role during the process of PDCD4affecting the development of atherosclerosis.(2) PDCD4deficiency reduced CD8+T cells and increased Tregs in vivoTo further study the effect of PDCD4on immune cells, we tested the quantitive changes of macrophages (F4/80) and the subsets of T cells (CD4+, CD8+T cells and Tregs) by flow cytometry. We found that there was no significant macrophage proportion changes after the PDCD4deficiency, while the CD8+T cell proportions reduced significantly after high fat diet for8week and16week (p<0.01). CD4+T cell proportion had a reduced trend. CD4+Foxp3+regulatory T cell (Treg) ratio increased obviously (p<0.01) and the effective Th17, Th2, Th1cell proportion had no changes. Linear regression analysis showed that the quantitive of CD8+T cells and Tregs had respectively a positive and negative correlation with the size of plaques. Further chemical staining of immune cells in local plaques proved that the proportion of CD8+T cell numbers in area of Pdcd4-/-AopE-/-mice plaque was much smaller than that of the control group (p<0.01), while Treg (Foxp3+) cell proportion is much larger (p<0.05). These changes were consistent with those of the peripheral cells. We have known that CD8+T cells play a promote role during the formation of AS while Treg cells play a inhibit one. Our results together showed that CD8+T cell increase and Treg cell reduction caused by the PDCD4deficiency may be a main reason for plaque reduction.(3) PDCD4deficiency increased IL-10and decreased IL-17in vivoTo study the influence of PDCD4on cytokines secreted by macrophages and T cells, we obtained serum from Pdcd4-/-Apoe-/-and Apoe-/-mice that had accepted high fat diet for16weeks and detected the concentration of various cytokines, including IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17, IL-10and TGF-β by flow cytometry after BD CBA Mouse Th1/Th2/Th17Cytokine Kit staining and ELISA. The results showed that the levels of pro-inflammatory cytokines IL-10, IL-6and IFN-y were higher (p<0.05) and the level of IL-17and TGF-β much lower (p<0.05) in Pdcd4-/-Apoe-/-mice compared with those of the control group. We did not found obvious changes in other cytokines. To determine the relation between cytokine changes and plaque reduction, we made a correlation analysis between cytokines that changed significantly and the size of plaques. The results showed that only the pro-inflammatory cytokine IL-17and anti-inflammatory cytokine IL-10had respectively positive and negative correlation with the plaque area. The other cytokines had no obvious correlations with the area of plaque.To further demonstrate the influence of PDCD4deficiency on cytokines and its relation with plaque formation, we use different experimental methods including real time PCR, Western blot, immunohistochemical and immunofluroscene method to detect changes of various cytokines and found that the change trend of IL-17and IL-10expression was consistent with that of the peripheral region. The expression of IL-17in local plaque of Pdcd4-/-Apoe-/-mice was significantly lower (p<0.05) than that of the control group, while the IL-10expression was much higher (p<0.01). As different from the peripheral changes, the mRNA level of IL-6in local plaque of PDCD4-/-Apoe-/-mice was significantly lower (p<0.05) than that of the control group, while the TGF-β expression was much higher (p<0.001). We did not found obvious changes in other cytokines. We have known that IL-6and IL-17are pro-inflammatory cytokines that can promote the AS plaque formation, while IL-10and TGF-β are anti-inflammatory cytokines that can inhibit the AS plaque formation. Therefore we conclude that the PDCD4inducing cytokine profile changes may be another mechanism for the plaque reduction resulting from PDCD4deficiency.(4) To study the influence of PDCD4on T cells and the mechanism in vitro1) PDCD4deficiency reduced the proliferation of CD8+T cellsWe have demonstrated the effect of PDCD4on the percentage of T cell subsets. In order to further explore its effect on T cell function, we separated spleens of Pdcd4-/-Apoe-/-and Apoe-/-mice of8weeks old and grinded them to make single cell suspension. Then we cultivated the splenocytes in vitro and used anti-CD3and anti-CD28antibody to provide respectively first and second stimulatory signal of T cells activation. We detected the proliferation of T cells after3days using CCK8kit and found that the proliferative ability of spleen cells from Pdcd4-/-Apoe-/-mice was significantly weaker than that of the control group (p<0.001). Then we detected the proportion of proliferative CD4+and CD8+T cell subsets using flow cytometry and found that the proliferation of CD4+T cells did not show any difference while the proportion of CD8+T cells after proliferation in Pdcd4-/-Apoe-/-mice was much lower than that of the control group (p<0.05), which demonstrated that deficiency of PDCD4decreased the proliferative ability of CD8+T cells.Besides the first signal was provided by antigens, the activation of T cells also needs a second signal transferred by the co-stimulatory molecules. Therefore in order to make clear whether the reduced proliferous ability of CD8+T cells was resulted from the changes of co-stimulatory molecules, we detected the expressions of co-stimulatory molecules CD28and CD137and co-suppressive molecule CTLA-4 using flow cytometry. The results showed that the expressions of CD28and CD137providing activated signals did not change significantly on CD4+T cells but were much lower on CD8+T cells of Pdcd4-/-Apoe-/-mice compared with Apoe-/-mice. The expression of CTLA-4providing suppressive signals on both CD4+and CD8+T cells was significantly higher than that on the control group (p<0.05). These data above showed that the reduction number and proliferous ability of CD8+T cell induced by PDCD4deficiency may be resulted from the down-regulation of co-stimulatory molecules CD28and CD137and up-regulation of co-suppressive molecule CTLA-4after PDCD4deficiency.2) PDCD4deficiency reduced IL-17secreted by T cellWe detected the concentration of cytokines secreted by T cell in the cultured supernatant by flow cytometry after BD CBA Mouse Th1/Th2/Th17Cytokine Kit staining. We found that T cells from Pdcd4-/-Apoe-/-mice produced a much lower level of IL-17than that of the control group (p<0.05) while other cytokines did not show any differences, which was consistent with the changes of IL-17in serum that had been shown previously.(5) PDCD4deficiency leads to the high level of IL-10secreted by macrophages in vitroWe further studied in vitro the effect of PDCD4on the functions of macrophages. We collected peritoneal macrophages of C57BL/6and Pdcd4-/-mice, cultured them in vitro and stimulated with oxLDL to imitate high fat environment in vivo and activate macrophages. First, we collected cultured supernatant after stimulation respectively for6,12,24and48hours and detected the concentrations of cytokines including IL-6, TNF-a, IL-10and TGF-β by ELISA. The results showed that except for TGF-β which showed nearly no difference between two genotype mice, the other three cytokines from Pdcd4-/-mice all showed higher levels than those of the control group. However, the concentrations of IL-6and TNF-a reached a summit at12h after which the concentrations gradually reduced with the extension of time, while the concentration of IL-10continued increasing with the extension of time which prompted that anti-inflammatory function of IL-10may play a major role. (6) IL-10was the main reason for PDCD4deficiency attenuated AS by treatment with antibodyIn our previous study, we treated Apoe-/-mice with recombinant IL-17which could aggravated significantly atherosclerosis. To confirm that the decrease of IL-17and the increase of IL-10caused by PDCD4deficiency is the main reason of reduced atherosclerotic plaques, we applied the same treatment. We divided Pdcd4-/-Apoe-/-mice that had accepted high fat diet for5weeks into three groups and gave them normal saline, recombinant IL-17and IL-10neutralizing antibody respectively by intraperitoneal injection once a week for5weeks. Apoe-/-mice injected normal saline were taken as the normal controls. Then we separated aortic root to make frozen sections of these mice and stained them by HE and oil red O. The results showed that the plaque size of mice injected IL-17had no significant difference with ones injected normal saline, while the plaques of mice injected IL-10neutralizing antibody were much bigger than that ones injected normal saline (p<0.05). These data above prompted that the increase of IL-10was the main mechanism of the reduced atherosclerotic plaques caused by PDCD4deficiency.(7) To explore that PDCD4regulates the expression of IL-10in transcription level in molecule mechanismIn order to investigate whether PDCD4regulated the expression of IL-10in translation level, we acquired all RNA binding with PDCD4in activated macrophages by RNA-Binding Protein Immunoprecipitation Kit (RIP). Then, we amplified IL-10by real time PCR. The result showed that there was no IL-10production in RNA binding with neither PDCD4nor IgG control, which indicated PDCD4did not affect the expression of IL-10in translation level. So we considered PDCD4regulated the expression of IL-10in transcription.Then, we explored the effect of PDCD4on the expression of IL-10in transcription level. We collected macrophages at3h,6h and12h after stimulation and extracted their total RNA to detect the expression of IL-10at mRNA level by RT-PCR. We found that the mRNA level of IL-10was significantly increased in Pdcd4-/macrophages compared with the control group, which prompted that PDCD4 regulated the expression of IL-10at transcriptional level.In order to demonstrate the way by which PDCD4deficiency increased IL-10transcriptional level, we detected the expression of molecules by western blot during MAPK signal pathway in which macrophages produced IL-10after stimulated for6h,12h and24h respectively. The results showed that p-JNK had no significant difference between these two genotype mice while the phosphorylated activation of ERK1/2and P38in Pdcd4-/-mice are much higher than that of the control group. To determine the two effects of these two pathways, we stimulated macrophages into activation and at the same time added ERK1/2and P38inhibitor respectively. We found significant reduction of IL-10. These data above showed that deficiency of PDCD4may up-regulate the increase production of IL-10by increasing phosphorylated activation of ERK1/2and P38.Conclusions1. PDCD4is up-regulated during the formation of atheromatous plaques.2. Plaque area of Pdcd4-/-Apoe-/-mice reduces and infiltration of macrophages and T cells in local plaques also reduces.3. Using bone marrow transplantation, we proved that immune cells play important roles during the process that PDCD4affect the development of atherosclerosis.4. The proportion of CD8+T cells in peripheral region and local plaque both reduce while the Treg proportion increases; concentration of pro-inflammatory cytokine IL-17decreases while anti-inflammatory cytokine IL-10concentration increases.5. Deficiency of PDCD4may cause the decreased proliferative ability of CD8+T cells by down-regulating co-stimulatory molecules CD28and CD137and up-regulating co-suppressive molecule CTLA-4.6. Deficiency of PDCD4can also cause increased secretion of IL-10.7. Experiments in vivo confirm that increase of IL-10is the main reason of reduced plaque area caused by PDCD4deficiency.8. PDCD4regulated the expression of IL-10not in translation level, probably in transcription level through ERK and P38pathway.Originality and Significance1. We demonstrate for the first time that PDCD4not only as the tumor suppressor gene, but also play an important role in the development of atherosclerosis. We found that PDCD4was up-regulated during the development of atherosclerosis. We introduced Pdcd4-/-mice from abroad and hybridized them with Apoe-/-mice to get Pdcd4-/-Apoe-/-mice, and prepared the model of atherosclerosis using them. During the study we found that plaque areas of Pdcd4-/-Apoe-/-mice reduced obviously. This study provides theoretical basis and experimental evidence for clinical therapy of atherosclerosis by targeting PDCD4.2. We confirm the effects of immunocytes during the process that PDCD4affects atherosclerosis. And we put forward the first time that PDCD4can induce the quantitive and functional changes of both macrophages and T cell subsets.3. We found that the expression of IL-10changed after PDCD4knockout and this expression changes were achieved by ERK and P38pathway. We verified that IL-10plays a major role during the process of PDCD4affecting atherosclerosis. Our study not only demonstrates the mechanism of PDCD4affecting atherosclerosis, but also provides therapeutic target for atherosclerosis which is aggravated by PDCD4.Limitations of this study1. Due to technical limitations, we took only gene knockout mice as research objects, but did not care for the over expression system of PDCD4.2. Due to time limitations, we focus on the effect of PDCD4on immune cells, but did not care for whether PDCD4could affect the function of smooth muscle cells.