The Effect And Mechanism Of Poly(ADP-ribose)Polymerase1on Low Shear Stress Induced Atherosclerosis

BackgroudAtherosclerosis is a chronic inflammatory disease, which primarily occurred at the inner wall of curvatures and outer wall of bifurcations with disturbed flow. Local hemodynamic forces have been proved to be closely associated with the site-specific predilection, and one of them is called wall shear stress (WSS). Wall shear stress is a frictional force exerted by the flowing blood to the vessel intima. It is directly proportional to the velocity of blood stream, and inversely proportional to the radius of the artery. The value of physiological WSS ranges from0.5Pa to1.2Pa, and values below and above this range are called low and high WSS, respectively. WSS can activate a variety of cell signaling molecules, such as protein kinase C (PKC), mitogen-activated protein kinases (MAPK) and so on. Moreover, low shear stress (LSS) can cause atherosclerosis by promoting the transport of atherogenic substances between the lumen and the vessel wall. Poly(ADP-ribose) polymerase1(PARP-1) is a highly conserved DNA binding protein. As a nuclear protein, PARP-1takes part in the repair of damaged DNA. Once binding to the damaged DNA, PARP-1can be activated and catalyze nicotinamide adenine dinucleotide (NAD+) into nicotinamide and ADP, which plays a critical role in DNA repair, gene transcription, cell cycle, cell death, chromosome function, and gene stability. However, excessive activation of PARP-1can cause depletion of intracellular NAD+and ATP, thereby resulting in cellular energy crisis, irreversible cytotoxicity, and even cell death. It has been demonstrated that oxidative stress induced overactivation of PARP-1is closely associated with the pathogenesis of a number of diseases, such as endothelium dysfunction, shock, diabetes, hypertension, atherosclerosis, and so on. Moreover, as a co-activator of nuclear factor kappa B (NF-κB), PARP-1can regulate the expression of a variety of key inflammatory cytokines, including monocyte chemotactic protein-1(MCP-1), intercellular adhesion molecule-1(ICAM-1), and inducible nitric oxide synthase (iNOS).Based on the previous studies, we make the following assumptions:1) Low shear stress can induce oxidative stress and DNA damage to cause PARP-1activation;2) PARP-1plays an indispensable role in LSS induced inflammation.Based on the above ideas, we stimulate human umbilical vein cells by low shear stress in vitro to validate the hypothesis in the present study. We aim to investigate the role and mechanism of PARP-1in LSS induced inflammation in HUVECs. We try to look for new protective and therapeutic targets in LSS associated inflammatory disease. Material and Methods1. Cell Culture and Shear Stress Model:Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell medium (ECM). HUVECs were stimulated by low shear stress (0.4Pa) to induce inflammation, and cells under static were considered to be the control.2. Genes Expression Interference in HUVECs:PARP-1expression was inhibited by PARP-1inhibitor ABT888or transient transfection with PARP-1siRNA in our experiments. The activity of JNK, p38, and ERK was inhibited by SP600125, SB203580, and U0126, respectively.3. Real-time RT-PCR:Total RNA was extracted from HUVECs. In the in vitro experiment, the mRNA expression of iNOS and ICAM-1in HUVECs was analyzed.4. Western Blotting:Total proteins were extracted from HUVECs. The protein expression of ICAM-1, iNOS, PARP-1, PAR, nitrotyrosine, H2A-X, p-H2A-X, c-Jun, p-c-Jun, MAPKAPK-2, p-MAPKAPK-2, ERK, p-ERK, Sirtl, IκBα, and p-p65was analyzed in our experiment.5. Immunocytofluorescence:To indicate the production of superoxide union, the expression and translocation of subunit of NF-κB p65, immunocytofluorescence was performed.6. Spectrophotometry:Spectrophotometry was performed to detect the intracellular NAD+level and Sirtl activity.7. Comet Assay:HUVECs were collected and comet assay was performed to assess the DNA damage.8. Electrophoretic Mobility Shift Assay (EMSA):Nuclear protein was extracted from HUVECs and electrophoretic mobility shift assay was performed to assess the NF-κB activity.Results1. The mRNA and protein expression of iNOS and ICAM-1were regulated by LSS in a time-dependent manner.HUVECs were stimulated by LSS for0,4h,8h,12h16h,24h, and36h. The RT-PCR and WB showed that LSS could induce the expression of iNOS and ICAM-1in HUVECs compared with the control. The mRNA and protein expression of iNOS began to elevate after HUVECs were stimulated by LSS for4h, and it reached peak at12h, while the mRNA and protein expression of ICAM-1was also in a high level at the same time.2. LSS stimulation increased PARP-1expression and activity in HUVECs.After HUVECs were stimulated by LSS for12h, PARP-1expression and activity were assessed by western blotting. Compared with the control, LSS increased the protein expression and activity of PARP-1, while PARP-1siRNA decreased it and PARP-1inhibitor ABT888only reduced PARP-1activity (the PAR formation).3. LSS stimulation increased the production of superoxide anion (O2-) and3-nitrotyrosine (3-NT).The production of O2-and3-NT was assessed to investigate the intracellular oxidative stress. Compared with the control, LSS significantly increased the O2-production and3-NT expression as assessed by DHE staining and western blotting in HUVECs.4. LSS stimulation induced DNA damage in HUVECs.The comet assay was performed to investigate LSS induced DNA damage. The results showed that LSS could significantly increase the content of DNA in tail, while there was almost no DNA in tail in static cells. Meanwhile, compared with the control, the phosphorylation of H2A-X was significantly increased by LSS. These results suggested that LSS could induce DNA damage.5. PARP-1could be activated by LSS via MEK/ERK pathway.Compared with the control, LSS could activate the JNK, p38, and ERK pathways. The use of SP600125, SB203580, and U0126reduced the phosphorylation of JNK, p38, and ERK. However, PARP-1activation could be decreased only by the MEK/ERK inhibitor U0126.6. PARP-1inhibition reduced the protein expression of iNOS and ICAM-1.The western blotting showed that compared with the control, LSS could significantly increase the protein expression of iNOS and ICAM-1, while the expression of inflammatory cytokines was decreased after PARP-1was inhibited by ABT888or PARP-1siRNA.7. PARP-1inhibition increased Sirtl activity by upregulation of intracellular NAD+Compared with the control, LSS significantly reduced the intracellular NAD+level and Sirtl activity. However, after PARP-1was inhibited by ABT888or siRNA, the intracellular level of NAD+was increased as well as the Sirtl activity, with no effect on the Sirtl expression.8. PARP-1inhibition decreased inflammatory cytokines expression by inhibiting the translocation and activity of NF-kB.The immunofluorescence showed that the subunit of NF-κB p65was almost cytoplasmic under static condition, while LSS could induce its nuclear translocation. However, LSS induced NF-κB translocation was significantly inhibited after PARP-1inhibition. Moreover, the EMS A result demonstrated that LSS increased the transcription activity of NF-κB, while PARP-1inhibition decreased it. The western blotting showed that LSS could reduce the expression of IkBa and phosphorylation of p65, while PARP-1inhibition decreased LSS induced phosphorylation of p65, with no effect on IkBa expression.Conclusion1. Low shear stress could induce the inflammatory response in HUVECs.2. Low shear stress could induce oxidative stress and DNA damage in HUVECs.3. Low shear stress could activate PARP-1via MEK/ERK pathway.4. PARP-1inhibition decreased LSS induced inflammatory cytokines expression, which was accomplished by two mechanisms:(1) PARP-1inhibition increased Sirtl activity by upregulation of intracellular level of NAD+.(2) PARP-1inhibition suppressed NF-κB nuclear translocation and activity, as well as its phosphorylation.5. In the experiment, we investigated the role and mechanism of PARP-1in LSS induced inflammation in HUVECs. The results suggested that PARP-1might be a target gene for treatment of inflammation response during atherosclerosis in the future. BackgroundIn the western developed countries, atherosclerotic diseases remain the leading causes of morbidity and mortality. Atherosclerosis has become the main cause of death in our country with the improvement of life quality and change of eating habits. The causes of atherosclerosis are multifactorial and it would be of great help to prevent and detect the disease early if we can figure out the pathogenesis.Atherosclerosis is usually regarded as a systemic disease and its pathogenesis is closely associated with several classic risk factors, such as hypertension, hyperlipidemia, diabetes mellitus, and cigarette smoking. Atherosclerotic plaque usually occurs in regions of curvature, bifurcation, and branching of the vessels, which suggests that hemodynamics and vessel geometry may play critical roles in the plaque formation. A lot of experiments in vivo and vitro have studied the flow patterns in blood vessels and looked for an association between preferential areas of plaque formation and hemodynamics. Wall shear stress attracts more attention in the pathogenesis of atherosclerosis as researches go on.In a straight vessel, blood does not flow at the same velocity at every point. Instead, the velocity of blood flow is the fastest at the center and the slowest close to the wall. The flowing blood can create friction which leads to a tangential force on the intima surface of the artery and is defined as the wall shear stress. Wall shear stress is a particularly important hemodynamic force that can stimulate the release of vaso-active substances and change gene expression, cell metabolism, and cell morphology.Poly(ADP-ribose) polymerase1(PARP-1), a highly conserved nuclear enzyme, is the most abundant member of PARP family. PARP-1can be activated by DNA damage due to oxidative stress. In the nucleus, activated PARP-1catalyzes the covalent transfer of ADP-ribose from NAD+to target proteins. Under normal condition, PARP-1takes part in DNA damage repair. However, under pathological condition, overactivation of PARP-1can induce intracellular depletion of NAD+and ATP, resulting in energy crisis, cell dysfunction, and even cell death. Numerous researches have demonstrated that overactivation of PARP-1is closely associated with various diseases, such as shock, heart failure, ischemic reperfusion injury, and diabetes.Based on previous studies, we make the following hypothesis:1) Inhibition of PARP-1can reduce low shear stress induced the formation of atherosclerotic plaque in the early stage of atherosclerosis;2) PARP-1inhibition reduces low shear stress induced the formation of atherosclerotic plaque through suppressing inflammation of endothelial cells.Based on the above ideas, we design the experiment in vivo to validate the hypothesis in the present study. Our studies try to demonstrate the role of PARP-1in low shear stress induced formation of atherosclerotic plaque and look for new opportunities and therapeutic method for plaque formation and acute cardiovascular events prevention.Meterials and Methods1. Experiment Animal:ApoE-/-PARP-1-/-mice were generated by crossing the offspring of ApoE+/-PARP-1+/-mice that resulted from mating their respective homozygous knockouts and were genotyped by PCR.2. Animal Model:Male ApoE-/-and ApoE-/-PARP-1-/-gene knockout mice (8weeks old) were fed with high fat diet and placed a silastic perivascular collar around the right common carotid artery in all mice to induce atherosclerotic lesions. All animals were randomly divided into three groups:the control group,15ApoE-/-mice that received intragastic administration of normal saline; the inhibitor group,15ApoE-/-mice that received intragastic administration of PARP-1inhibitor ABT888; the double knockout group,15ApoE/PARP-1gene knockout mice.3. Poly(ADP-ribose) polymerase1Gene Intervention:Poly(ADP-ribose) polymerase1expression was reduced by gene knockout or PARP-1inhibitor ABT888in our experiments.4. Heart Rate and Blood Pressure:The heart rate and blood pressure were assessed by a noninvasive method.5. Blood Lipid Parameters:All mice were starved overnight and blood samples were collected before euthanasia in the experiment. Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were obtained by the use of an enzymatic assay.6. H&E Staining and Oil Red O Staining:In the animals, the common carotid arteries were collected and consecutive sections (5μm) were obtained from each mouse. H&E staining and oil red O staining were used to indicate the morphology and lipid content of the plaque.7. Immunofluorescence Analysis:In the animals, the common carotid arteries were collected and consecutive sections (5μm) were obtained from each mouse for immunofluorescence analysis to show the expression of ICAM-1.8. Western Blotting:The mice common carotid arteries were collected and protein was extracted. The protein expression of PARP-1, PAR (the PARP-1activity), and ICAM-1was analyzed.9. DHE Staining:The common carotid arteries were collected and consecutive sections (5um) were obtained for DHE staining. The method was used to indicate the superoxide anion production of the plaque.Results1. The general characteristics of mice:The heart rate, the blood pressure and the lipid profiles were assessed after the experiment. There was no significant difference in heart rate, blood pressure, serum levels of TC, TG, LDL-C and HDL-C among the three groups of mice. These results suggested that PARP-1gene inhibition had no effect on blood lipid profiles.2. Inhibition of PARP-1can reduce low shear stress induced plaque formation:The morphology and lipid content were assessed by H&E staining and oil red O staining after experiment. Compared with the undisturbed shear stress (USS) in the left artery of control group, low shear stress (LSS, proximal part of right carotid) could significantly increase the plaque formation and lipid content (p＜0.05, VS. USS). Meanwhile, compared with the low shear stress in the control group, PARP-1inhibition or knockout could significantly reduce the plaque formation and lipid content in the experiment group (p<0.05, VS. LSS of control).3. Inhibition of PARP-1can reduce LSS induced endothelial inflammation:The protein expression of ICAM-1was assessed by immunofluorescence and western blotting after experiment. The immunofluorescence showed that in the control group, low shear stress could increase ICAM-1protein expression in the endothelium compared with the undisturbed shear stress (p＜0.05, VS. USS). However, compared with low shear stress in control group, PARP-1inhibition or knockout significantly reduced the protein expression of ICAM-1in low shear stress of experiment group (p＜0.05, VS. LSS of control). The western blotting showed the similar results, and PARP-1gene knockout had a greater effect than PARP-1inhibition.4. Low shear stress increased PARP-1expression and activity:The PARP-1expression and PAR formation (PARP-1activity) were assessed by western blotting after experiment. The results showed that compared with undisturbed shear stress in control group, low shear stress could significantly increase PARP-1expression and PAR formation (p＜0.05, VS. USS). Meanwhile, PARP-1inhibition by ABT888decreased PAR formation without an effect on PARP-1expression, while PARP-1knockout significantly reduced PARP-1expression and PAR formation (p＜0.05, VS. LSS of control).5. Low shear stress increased oxidative stress:Superoxide anion was assessed by DHE staining. The DHE staining showed that compared with undisturbed shear stress in control group, low shear stress increased superoxide anion formation (p＜0.05, VS. USS). Meanwhile, PARP-1inhibition or knockout had no significant effect on superoxide anion formation.Conclusion1. PARP-1inhibition reduced low shear stress induced atherosclerotic plaque formation;2. PARP-1inhibition reduced low shear stress induced endothelial inflammation;3. Low shear stress increased PARP-1expression and PAR formation;4. Low shear stress increased oxidative stress;5. The experiment further elucidated the effect and mechanism of PARP-1on atherosclerotic plaque formation and provided new opportunities and therapeutic method for acute cardiovascular events prevention. BackgroudDiabetes mellitus is a kind of metabolic syndrome, which is characterized by hyperglycemia owing to a relative or absolute lack of insulin or insulin resistance. Diabetes mellitus currently affects more than180million people around the world, and the number of patients is anticipated to rise to300million by2025. As the living conditions improve and life style changes, the morbidity of diabetes in China has increased to a high level, which is approximate9.7%.Cardiovascular disease, which is mainly caused by coronary artery disease (CAD) and hypertension, is the main cause of death in diabetic patients. However, scientific researches have demonstrated that diabetes mellitus can result in heart failure in the absence of CAD and hypertension, which is termed as diabetic cardiomyopathy in clinical. Diabetic cardiomyopathy (DCM), as an early complication of diabetes, is manifested by diastolic and systolic dysfunction. Although the precise mechanism of DCM is still obscure, oxidative stress plays an important role in its pathogenesis. Hyperglycemia-induced oxidative stress induces the development of micro-vascular pathogenesis, which results in myocardial cell death, hypertrophy, fibrosis, and dysfunction. Moreover, oxidative stress induces DNA damage and activation of poly(ADP-ribose) polymerase1(PARP-1).PARP-1is a wildly studied nuclear enzyme in PARP family. PARP-1has3functional domains:a DNA-binding domain, an automodification domain, and a catalytic domain. Normally, PARP-1takes part in DNA damage repair and the maintenance of genomic stability. However, PARP-1rapidly uses NAD+to transfer poly(ADP-ribose)(PAR) to target proteins when it is overactivated. To resynthesize NAD+, the cell unduly consumes its ATP pools, resulting in an energy crisis and cell death.PARP-1inhibition can limit cellular energy depletion to reduce cell death. Moreover, recent evidence suggests that inhibition of PARP-1activates a prosurvival signaling cascade through Akt phosphorylation. In the heart, insulin growth factor1(IGF-1) confers protection against cardiac dysfunction via phosphorylation of IGF-1receptor (IGF-1R), which can result in the activation of the PI3K/Akt axis.Based on the previous studies, we make the following assumptions:1) PARP-1inhibition can reduce high glucose induced cardiomyocyte apoptosis;2) PARP-1inhibition reduces apoptosis through activating IGF-1R/Akt signaling pathway.Based on the above ideas, we design the experiment in vitro to validate the hypothesis in the present study. We aim to investigate the role and underlying mechanism of PARP-1in cardiomyocyte apoptosis during diabetic cardiomyopathy and look for new prevention and therapeutic targets on DCM.Material and Methods1. Cell Culture and Cell Model:Rat H9c2cardiomyocytes were cultured in DMEM and stimulated by normal glucose (5.5mM, NG) and high glucose (33mM, HG) in our experiments.2. Interference of Gene and Protein Expression:In vitro, transient transfection with PARP-1siRNA was performed to inhibit PARP-1expression; IGF-1and PPP were used to promote or inhibit the phosphorylation of IGF-1R in our experiments.3. Laser Confocal Microscopy:To determine the intracellular content of superoxide anion and reactive oxygen species, laser confocal microscopy was used in our experiments.4. Flowcytometry (FCM):FCM was used to determine the apoptosis of cardiomyocyte in all groups.5. Real-time RT-PCR:Real-time RT-PCR was performed to determine the gene expression of PARP-1.6. Western Blotting (WB):WB was performed to determine the protein expression of PARP-1, the phosphorylation of IGF-1R and Akt in cells.Results1. The oxidative stress in H9c2cardiomyocyte was induced by high glucose in a time dependent manner.The rat H9c2cardiomyocytes were stimulated by high glucose (33mM, HG) for24h,36h and48h, then cells were stained by DHE and DCF, the intracellular content of superoxide anion and reactive oxygen species were determined by laser confocal microscopy. The results showed that after cardiomyocytes were stimulated by HG for24h, the oxidative stress was the same as that in the control group (5.5mM); after cardiomyocytes were stimulated by HG for36h. compared with the control group, the intracellular content of superoxide anion and reactive oxygen species were increased by nearly1fold and3fold (p＜0.05, VS. control). After cardiomyocytes were stimulated by HG for48h, the intracellular content of superoxide anion and reactive oxygen species were increased by nearly3fold and4fold (p＜0.01, VS. control).2. HG increased the PARP-1expression and activity in H9c2cardiomyocyte.The PARP-1expression and activity were determined after cardiomyocytes were stimulated by HG for24h,36h and48h. RT-PCR showed that compared with the normal control group, there was no significant change in PARP-1gene expression after HG stimulation for24h, while HG stimulation for36h and48h could significantly increase the PARP-1gene expression. WB showed that the protein expression of PARP-1and PAR had no significant change after HG stimulation for24h; while their expression was significantly increased after HG stimulation for36h and48h, and the increase was in a time dependent manner. These results showed that HG stimulation could increase the expression and activity of PARP-1.3. The gene and protein expression of PARP-1were reduced by PARP-1siRNA.In our experiments, PARP-1siRNA was used to inhibit PARP-1expression and the interference efficiency was determined by RT-PCR and WB. RT-PCR showed that PARP-1gene expression was decreased by siRNA compared with the control; WB showed that PARP-1protein expression was reduced to50%by PARP-1siRNA compared with the control. The negative control of siRNA had no significant effect on PARP-1expression. These results showed that PARP-1siRNA could effectively inhibit PARP-1expression.4. PARP-1inhibition reduced the assumption of intracellular NAD+induced by HG.PARP-1used intracellular NAD+as the substrate for poly(ADP-ribose) reaction once activated. The intracellular level of NAD+was determined by NAD+/NADH assay kit. Spectrophotometry showed that HG reduced the intracellular level of NAD+compared with the control; while PARP-1inhibition could significantly reduce the assumption of NAD+induced by HG. These results showed that PARP-1played a critical role in HG induced NAD+assumption.5. PARP-1inhibition reduced HG induced cardiomyocytes apoptosis.The apoptosis of cardiomyocytes was assessed by flowcytometry. The result of FCM showed that HG increased the cardiomyocyte apoptosis compared with the normal control; IGF-1could reduce HG induced apoptosis, while PPP aggravated it; HG induced cardiomyocyte apoptosis was significantly reduced by PARP-1inhibition. These results showed that just like IGF-1, PARP-1inhibition could also reduce HG induced apoptosis.6. PARP-1inhibition increased the phosphorylation of Akt.In the following experiment, we investigated the underlying mechanism. Considering the critical role of Akt in anti-apoptosis, we determined the expression of Akt. WB showed that there was no significantly difference in total Akt among all groups. Compared with the HG group, IGF-1and PARP-1 inhibition could significantly increase the phosphorylation of Akt, while PPP had the adverse effect. These results suggested that Akt was closely associated with the anti-apoptosis mechanism.7. PARP-1inhibition increased the phosphorylation of IGF-1R.Then we examined the expression of IGF-1R. WB showed that there was no significantly difference in total IGF-1R expression among all groups. However, compared with the HG group, PPP administration reduced the phosphorylation of IGF-1R, while IGF-1and PARP-1inhibition increased it. These results suggested that PARP-1inhibition reduced apoptosis through the IGF-1R/Akt signaling pathway.Conclusion1. PARP-1inhibition can reduce high glucose induced cardiomyocytes apoptosis.2. PARP-1inhibition reduces apoptosis through activating IGF-1R/Akt signaling pathway.3. After we do some research on the high glucose induced cardiomyocyte apoptosis, we find that PARP-1may be a new therapeutic target for the treatment of diabetic cardiomyopathy.