| Background:Atherosclerosis is the primary cause of many cardiovascular, cerebrovascular and peripheral vascular diseases. Atherosclerosis can lead to ischemia of the heart, brain and extremities by narrowing of arteries, rupture of atherosclerotic plaques and thrombotic occlusion of arteries. Although atherosclerosis is manly considered as a chronic inflammatory disease, it is also a complex, multi-factorial disease initiated by focal lipid deposition in the arterial wall. Endothelial dysfunction, infiltrating of leukocytes, oxidative stress, cell apoptosis, lipid accumulation, angiogenesis, collagen content and many other factors contribute to atherogenesis. However, at the early stage of atherosclerosis, endothelial dysfunction plays a crucial role and is probably a starting point. Therefore, it is important to maintain normal endothelial function for preventing atherosclerosis.Poly(ADP-ribose) polymerase (PARP) family is the most abundant nuclear enzyme in eukaryotic cells, among which PARP-1has more than90%catalytic activity. PARP catalyzes ADP-ribose units from their substrate β-nicotinamide adenine dinucleotide covalently to themselves and other nuclear chromatin-associated proteins. DNA strand breaks induced by ROS or RNS lead to the activation of PARP. PARP overactivation leads to rapid depletion of intracellular ATP, energy crisis and even cell death. PARP activation has been reported to broadly participate in the pathophysiological process of numerous cardiovascular diseases, including atherosclerosis, heart failure, ischemia-repeprgusion and dilated cardiomyopathy.PARP inhibition effectively attenuates atherogenesis through several mechanisms. PARP inhibition reduces NFκB activation and prevents the expression of proinflammatory molecules. PARP inhibition also decreases monocyte recruitment into atherosclerotic lesions. PARP inhibition increases TIMP-3expression and prevents collagen degradation. PARP inhibition also reduced cell apoptosis and promotes stability of atherosclerotic plaques. Moreover, PARP inhibition maintains normal endothelial function.NO plays multiple biologic activities in the endothelium, including regulation of vascular tone, blood flow, suppression of vascular smooth muscle cell proliferation, leukocyte-endothelial interactions and anti-thrombosis effect. NO is a critical molecule for endothelial function. Previous studies have reported that PARP regulates activation of eNOS and expression of iNOS. However, how PARP regulates NO production in different cells remains unknown.Lipid peroxidation plays a crucial role in atherosclerosis. Aldehydes are the major end products of lipid peroxidation. They can react with proteins and nucleic acids to generate various adducts that result in the inactivation of proteins and DNA damage which induce cellular or tissue dysfunction and promote atherogenesis. Aldehydes can be metabolized into less toxic chemical species by aldehyde dehydrogenases among which ALDH2displays the highest affinity for acetaldehyde. Whether PARP activation regulates the expression and activity of ALDH2remains obscure.Objectives:1. We determined the effect of PARP inhibition on atherogenesis using PARP inhibitors or PARP-1gene deficiency.2. We determined the effect of PARP inhibition on NO production3. We tried to find out the underlying molecular mechanism how PARP inhibition regulates NO production in mouse aortic endothelial cells.4. We explored the effect of PARP activation and inhibition on the expression and activation of ALDH2and the underlying mechanism.Methods:1. Generation of apoeE-/-PARP-1-/-mice PARP-1-/-mice was purchased from Jackson Lab. Before breeding with apoE-/mice, PARP-1-/-mice was backcrossed to C57BL/6J at least7generations. Then PARP-1-/-mice (C57BL/6J background) was crossed with apoE-/-mice to genenrate apoE+/-PARP-1+/-mice. All experiment mice were got by breeding above heterozygotes.2. Animal model for atherosclerosisApoE-/-mice and apoE-/-PARP-1-/-mice were divided into four groups. Group1: apoE-/-mice were fed normal diet (ND group); Group2:apoE-/-mice were fed high cholesterol diet (HD group); Group3:apoE-/-mice were fed high cholesterol diet and injected with3mg/kg DPQ daily (HD+DPQ group); Group4:apoE-/-ARP-1-/-mice were fed high cholesterol diet (DKO group).12weeks later, aortas were collected and analyzed.3. Histological stainingTo quantify the area of atherosclerotic plaques, the whole aorta and sections of aortic sinuses were stained with Oil Red O to visualize atherosclerotic lesions.4. ImmunochemistryAortic sinuses was embedded in paraffin and sliced into5μm sections. After deparafinization and rehydration, sections were incubated with primary antibodies over night at4℃. At second day, secondary antibodies were used according to primary antibodies. Finally, a3,3’-diaminobenzidine (DAB) Kit was used to visualize the primary antibodies and analyzed by use of Image Pro-Plus software.5. NO assayNO production in aortas and cells was determined with use of the Total Nitric Oxide Assay Kit. Nitrate was converted to nitrite by nitrate reductase and nitrite was tested by reaction with Griess reagent. The concentration of NO was expressed as μmol/mg protein.6. Western BlottingProtein was extracted from mouse aortas and cultured cells with RIPA buffer containing1mM protease inhibitor PMSF. Then protein was separated by12%SDS-PAGE gel and transferred to nitrocellulose membranes. Primary antibodies were used to bind corresponding protein. After incubating with secondary antibodies, protein bands were visualized with enhanced chemiluminescence plus reagents and analyzed with Image J software.7. Arginase activity assayAortas and cells were lyzed with lysis buffer and centrifuged for30min at14000g,4℃. Lysates were heated for10min at56℃and incubated with0.5M arginine for60minutes at37℃, the reaction was stopped with400μl stop buffer (96%H2SO4:85%H3PO4:H2O,1:3:7, v/v). Finally,25μl9%α-isonitrosopropiophenone was added and heated at100℃for45min. Arginase activity were detected at540nm.8. Isolation and culture of mouse peritoneal macrophagesPeritoneal macrophages were isolated from C57BL/6J and PARP-1-/-mice. Mice were peritoneally injected with2ml4%thioglycollate.3days later, the peritoneal cavity were opened and rinsed with RPMI-1640medium. After centrifugation at1000rpm for10minutes, peritoneal macrophages were isolated and responded with RPMI-1640medium containing10%fetal bovine serum. Then macrophages were cultured in6-well plates.9. Isolation and culture of mouse aortic endothelial cellsThoracic aortas were isolated from C57BL/6J and PARP-1-/-mice and completely cleaned of adventitia. Then aortas were coronally cut into6segments and washed in PBS. The segments were placed endothelial side down onto neutralized collagen gel (mixture of2.2ml type Ⅰ rat-tail collagen,4ml DMEM containing10%fetal bovine serum and0.36ml0.1M NaOH). After5days, cellular outgrowth from the aortic segments was digested with0.3%collagenase H solution and the released endothelial cells were passaged in a tissue culture flask with EGM-210. Culture of human aortic endothelial cells and experimentsHAECs were cultured in37℃,5%CO2hood with ECM medium.Results:1. PARP inhibition attenuates formation of atherosclerotic plaques:Using en face oil red O staining, we analyzed the size of atherosclerosis on whole aortas. Also, we analyzed the area of atherosclerotic lesions on aortic sinuses. We found PARP inhibition could effectively inhibit the progress of atherogenesis.2. PARP inhibition does not change blood cell counts, lipid profiles.3. PARP inhibition reduces reactive oxygen stress and protein nitration: Immnohistochemistry staining of8-oxo-dG and3-nitrotyrosine indicated that reactive oxygen stress and reactive nitric stress can be reduced by PARP inhibition.4. PARP inhibition increases NO production in aortas:Compared to mice on a high cholesterol diet, NO production was increased in mice with PARP inhibition with inhibitor or gene knockout.5. PARP inhibition decreases expression of Arg Ⅱ but not Arg Ⅰ:Ox-LDL treatment increased both Arg Ⅰ and Arg Ⅱprotein expression in mice peritoneal macrophages, but only Arg Ⅱ protein expression was reduced by PARP-1gene deficiency.6. PARP inhibition increases NO production in MAECs:Ox-LDL treatment decreased NO production in wild type peritoneal macrophages, but in PARP-1deficient macrophages, ox-LDL almost failed to reduced NO production.7. PARP inhibition increased the activity of ALDH2in HAFCs:Ox-LDL inhibited the activity of ALDH2in a time and concentration dependent manner without alteration of protein expression of ALDH2. PARP inhibition partly prevented the inhibitory effect of ox-LDL on ALDH activity.Conclusion:PARP inhibitor or PARP-1-/-gene deficiency effectively attenuates atherogenesis. PARP inhibition increases NO production and maintains normal endothelial function. Moreover. PARP inhibition prevents the inhibitory effect of ox-LDL on ALDH2activity. Background:Leukocyte rolling and adhesion on vascular endothelium plays a crucial role in atherosclerosis. Atherosclerosis initiates from endothelial dysfunction or endothelial injury. Under stimulation of TNFa, LPS, dyslipidmia or physical injury, P-selectin glycoprotein ligand-1(PSGL-1), vascular cell adhesion molecule-1(VCAM-1) and other proinflammatory molecules are induced to express in endothelial cells or collagen under endothelium is exposed, then leukocytes or platelets rolls or adheres on the endothelial surface by the help of the interaction among proinflammatory molecules. After adhering on endothelium, leukocytes will infiltrate into subendothelium where leukocytes will change into macrophages which will phagocytoses lipid or dead cells, finally local lesion is formed and promotes the progression of atherosclerosis.Platelets derive from megakarocytes which own intact cellular membrane but have not nuclei. Previous views emphasize the function of platelets as mediators during blood coagulation, hemostasis and injured vessel reparation. Recently, besides the above function, platelets also play a very important role in atherosclerosis, inflammation, immunity, cancer and other diseases. Stimulators such as ADP, thrombin, thromboxane A2, collagen, can induce platelet activation, release, aggregation or adhesion. For atherosclerosis, platelets participate in the progression of leukocyte rolling and adhesion through two pathways:1. Platelets adheres on the endothelial surface through the interaction between GPⅠbα, GPⅥ, α2β1, αⅡbβ3, JAM-A, JAM-C, CX3CR1and von Willebrand factor(vWF) or collagen, then under the interaction between P-selectin on platelets and PSGL-1on leukocytes, Mac-1is activated, platelets and leukocytes can bind to form aggregates by the binding of platelet GPIba and leukocyte Mac-1which will enhance the adhesion of leukocytes on vessel wall;2. Activated platelets can interact with leukocytes and can transfer P-selectin, CCL5to leukocytes which will enhance the adhering ability of leukocytes.Nuclear factor kappa B(NF-κB) is the key regulator of inflammation, immunity, apoptosis and cell proliferation. It has been shown to be involved in the transcriptional regulation of more than150genes. Usually, NF-κB binds together with its inhibitor IκBα and cannot function as a transcription factor. But, when IκBα is phosphorylated by IκB kinase(IKK) and is degraded by ptoteases, NF-κB is dissociated from IκBα and freely migrates into the nucleus and activates the expression of target genes. IKK have four subtyes:α, β, γ and ε. Among them, IKKβ plays a major role in phosphorylation of IκBα. Previous studies reported that NF-κB played a different role in different cells during atherosclerosis using conditional IKKβ knockout mice. Endothelial IKKβ-deficient mice expressed reduced atherosclerosis, but macrophage IKKβ-deficient mice expressed exacerbated atherosclerosis.Recent studies found that besides nucleated cells, platelets also contain functional NF-κB. It has been reported that activated platelets promote atherosclerosis and the formation of neointima. However, how platlet NF-κB effects on atherosclerosis remains unkown. This studies aims to study the function of NF-κB during platelet activation, release and aggregation and the effect on atherosclerosis and neointima formation using platelet IKKβ-deficient mice.Objectives:1. Using loxp-Cre system, to generate platelet IKKβ-deficient mice;2. After12weeks western diet, to examine the size of spontaneous lesion;3. To observe the effect of IKKβ-deficient platelets on neointima formation of injured carotid artery with a guide wire;4. To explore the effect of IKKβ deficiency on platelet function and the underlying mechanisms. Methods:1. Generation of platelet IKKβ-deficient miceBreeding floxed IKKβ mice with PF4-Cre mice, IKKβfl/flPF4cr emice were generated and then bred with Ldlr-/-mice to generate Ldlr-/-IKKβfl/flPF4cre mice and their littermate control Ldlr-/-IKKβfl/fl mice.2. Animal model of spontaneous atherosclerosisAfter12weeks western diet, the size of spontaneous lesion on aortas were calculated.3. Animal model of neointima formationWith a guide wire, left carotid arteries of mice which has been on western diet for2weeks were injured.4weeks later, left carotid arteries were collected and analyzed.4. Lipid profile and hemogramPlasma triglycerides (TG), total cholesterol (TC), low-desity lipoprotein (LDL) and high-density lipoprotein (HDL) were measured via an automated enzyme technique. Blood cell counts were quantified using an automated blood cell counter.5. Bone marrow transplantationAfter receiving lethal irradiation, mice immediately received a injection of bone marrow cells from donor mice via tail vein.4weeks later, mice were ready for other experiment.6. Neutrophil depietionTo decide the role of neutrophil on neointima formation in platelet IKKβ-deficient mice, anti-PMN was intraperitoneally injected to deplete blood neutrophils.7. Movat stainingTo evaluate the size of neointima, Movat staining was performed.8. ImmunochemistryUsing anti-F4/80antibody, macrophages in plaque lesions were analyzed9. Leukocyte adhesion on injured carotid arteriesAfter injured by a guide wire, the leukocyte adhesion on carotid artery was analyzed under a intravital epifluorescence microscope system.10. Leukocyte adhesion on activated plateletsBlood was carefully collected from mouse heart with sodium citrate. Platelets were isolated and diluted to2×109/ml. Then, platelets were loaded into a rectangular glass capillary tube. After adhering to the surface of the capillary tube,0.1U/ml thrombin was used to activate platelets. Blood from the carotid artery was passed through the micro-flow chamber and the leukocyte rolling and adhesion were analyzed.11. Platelet activation, aggregation and releaseWashed platelets were stimulated by thrombin, collagen or ADP to determine the effect of IKKP deficiency on platelet activation, aggregation and release.12. Flow cytometryThe purity of isolated platelets and the expression of P-selectin, GPIba, GPV, GPVI, GPIX, αⅡbβ3were determined by flow cytometry.13. Platelet electron microscopyPlatelets were observed under a Philips301electron microscope to determine the aggregation and release of a granule.14. Western blottingPlatelets were lysed in a RIPA lysis buffer and protein was isolate in a SDS-PAGE gel. GPIba, AD AM17, P38and p-P38were conjugated with their primary antibody.Results:1. Platelet IKKβ deficiency did not change the size of spontaneous atherosclerosis in Ldlr-/-mice.2. Platelet IKKβ deficiency accelerated neointima formation in Ldlr-/-mice and increased the content of macrophages within plaque lesions. Bone marrow transplantation further supported this evidence.3. Platelet IKKβ deficiency did not change platelet adhesion but increased leukocyte adhesion on injured carotid artery. With a intravital epifluorescence microscope, we found platelet IKKβ deficiency did not affect leukocyte rolling, but obviously increased leukocyte adhesion on injured carotid artery or activated platelets.4. Platelet IKKβ deficiency attenuated platelet activation, aggregation and release. Under stimulation of thrombin, we found the expression of P-selectin was inhibited by IKKβ deficiency. Through analyzing the release of ATP and electron microscope observation, we found platelet aggregation and release were reduced in IKKβdeficient platelets.5. Platelet IKKβ deficiency decreased the shedding of GPIba and GPV, but not GPVI. GPIX and αⅡbβ3.6. By western blotting, we found platelet IKKβ deficiency prevented the phosphorylation of P-38and inhibited the maturation of ADAM17which led to reduced GPIbα shedding.Conclusions:1. Platelet IKKβ deficiency promotes neointma formation of injured carotid artery.2. Platelet IKKβ deficiency enhances the interaction between platelets and leukocytes and increases leukocyte adhesion on injured carotid artery.3. Platelet IKKβ deficiency inhibits platelet activation, aggregation and release by thrombin.4. Platelet IKKβ deficiency reduces the shedding of GPIbα.5. Platelet IKKβ deficiency prevents the phosphorylation of P-38and inhibits the maturation of ADAM17... |
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