Exploration And Intervention Study Of Inflammatory Targets For Atherosclerotic Plaque Rupture In ApoE Deficient Mice

Atherosclerosis affects the cerebral,coronary and peripheral vasculature and represents the most common cause of morbidity and mortality worldwide.Pathology and clinical studies showed that plaque rupture contributes to about 75% of the thrombi responsible for acute coronary syndromes,which leading to occurring of the major clinical events（MACE）.Last century,most people considered atherosclerosis to be a cholesterol storage disease.On such basis,lipid lowering drugs have been developed.Among these drugs,statins are by far the most clinically and financially successful ones.However,even adherence to contemporary therapies does not completely eliminate the MACE.Indeed,a meta-analysis of secondary prevention trials with statin therapy estimated the residual risk of major adverse cardiovascular events to be as high as 21% at 5 years.Atherosclerosis is now recognized as a chronic and progressive disease,which driven by lipid and inflammatory-immune response.The residual risk above mentioned is related to the failure of current therapies to adequately resolve inflammation.It is promising for further declining MACE in patients with atherosclerosis to define specific inflammatory targets associated with plaque rupture and develop corresponding therapeutic strategy.An animal model with high spontaneous plaque rupture is of great importance for evaluating the value of candidate targets and therapeutic strategies.Our team developed a vulnerable plaque model in left common carotid artery of Apo E deficient mice by means of combined partial ligation of the left renal artery and left common carotid artery.At present,the “R+C” model with the highest（80%）incidence of plaque rupture among current atherosclerotic models has been widely accepted.The study aimed to find specific inflammatory targets associated with plaque rupture and evaluate corresponding therapeutic strategy in the R+C model.Firstly,investigated whether the unstable plaques in our R+C mouse model also respond to pharmacological intervention,we evaluated the effects of statin therapy on plaque vulnerability in R+C model.We also studied the anti-inflammatory mechanism of statins.Secondly,we studied the association between sub-cellular location of nuclear receptor Nur77 and inflammation activity in macrophage and illustrated the role of Nur77 in development and progression of atherosclerosis plaque in Apo E mice.Finally,we studied the effect of RXRα antagonist K-80003 on inflammation and plaque stability and then evaluated its safety.In sum,we studied the underlying mechanism and inflammatory targets of atherosclerotic plaque rupture and implemented therapeutic tragedies.Our study provided new direction and basis for developing new anti-inflammatory drugs to treat atherosclerosis.Part One Atorvastatin improves plaque stability in Apo E-knockout mice by regulating chemokines and chemokine receptors Objective An animal model of plaque instability/rupture should include responsiveness to pharmacological agents known to reduce the risk of plaque rupture in humans.It is well documented that statins protect atherosclerotic patients from inflammatory changes and plaque instability in coronary arteries.We investigated the effect of atorvastatin on plaque morphology in our R+C model independent of its anti-lipidemic effects.We further studied the anti-inflammatory mechanism of statins to exploit other potential targets associated with plaque stability.Methods Thirty female Apo E-knockout(ApoE^-/-)mice underwent combined partial ligation of the left renal artery and left common carotid artery at the age of 8-10 weeks.Then mice were randomly divided into two groups: control（n = 15）and atorvastatin（10 mg/kg/day）（n = 15）.Isosmotic saline or atorvastatin was infused via a stomach tube after surgery for 8 weeks.Mice were anesthetized then blood samples and carotid arteries were collected.The carotid arteries Cross-cryosections（5-μm）were prepared and stained with H&E and immunohistochemical staining for examining lesion area,plaque morphology,cellular components,matrix metalloproteinase（MMP）activity and chemokines expression.Peripheral blood monocytes were analyzed by FACS to measure the expression levels of the chemokine receptors levels.ELISAs were used to detect plasma levels of inflammatory markers according to the manufacturers’ instructions.Results（1）Vulnerable plaque numbers were significantly reduced in R+C model under the treatment of atorvastatin,while there was no difference in total cholesterol level and lesion area.（2）Atorvastatin significantly decreased macrophage infiltration and subendothelial lipid deposition,reduced intimal collagen content,also elevated collagenase activity and expression of matrix metalloproteinases（MMPs）.（3）Atorvastatin downregulated the expression of chemokines MCP-1,CX3CL1 and their receptors CCR2 and CX3CR1.（4）Levels of the plasma inflammatory markers CRP and TNF-α also significantly decreased in atorvastatin-treated mice.Conclusion Atorvastatin exerts anti-inflammatory effects and improves plaque stability in R+C mouse model independent of plasma cholesterol level.Through modulating levels of chemokines and their receptors,statins decrease macrophage infiltration to exert protective effects.R+C model is a novel mouse model of atherosclerotic plaque instability for drug testing and mechanistic/therapeutic discoveries.Part Two Effect and Mechanism of Nuclear Receptor Nur77 on Progress of Atherosclerotic PlaqueObjective Nuclear Receptor Nur77 is an important transcription factor which has a crucial function in regulation of atherosclerosis.Recent experimental data demonstrated that Nur77 migrates to cytoplasm to exert “transcription independent effect” in certain pathophysiology settings.This part investigated the sub-cellular location of Nur77 in macrophages in different inflammatory state of atherosclerotic plaques to illustrate the role of Nur77 translocation in inflammation activation.We also investigated the effect of Nur77 deficiency in progress and rupture of atherosclerotic plaque in apo E knockout mice.Methods To study the role of Nur77 translocation in inflammation activation,double immunofluorescent staining against Nur77 and CD68 was performed in different inflammatory state of atherosclerotic plaques.Nur77+/+ApoE^-/-（Apo E group for short）and Nur77-/-ApoE^-/-（DKO group for short）mice with the same litter underwent combined partial ligation of the left renal artery and left common carotid artery at the age of 8-10 weeks and were randomly divided into group 1w,2w,6w and 8w respectively.At corresponding endpoint,carotid arteries harvested and cross-cryosections were prepared.H&E.and immunohistochemical staining were performed for examining lesion area,plaque morphology and cellular components both in Apo E and DKO groups.Results（1）.Nur77 resided in nucleus of macrophages in the initial stage of atherosclerotic plaque with slight inflammation,while in vulnerable plaque with severe inflammation,Nur77 migrated into cytoplasm.This reflected the close relation between Nur77 translocation and inflammation activation.（2）.There was no difference between Apo E and DKO group in lesion area and plaque feature in the 1w and 2w groups which representing initial lesions.Nur77-/-apo E-/-carotid artery displayed dramatically decreased lesion area,plaque stability and incidence of plaque rupture in 6w and 8w groups which revealing advanced atherosclerotic lesion with a high incidence of plaque rupture in apo E deficient mice.（3）.Immunofluorescence analysis of cryosections showed elevated vascular smooth muscle cells and collagen content,reduced expression of matrix metalloproteinases（MMPs）in DKO group.There was also a diminished trend in macrophages and lipid content in plaque of DKO group.Conclusion（1）.Cytoplasmic translocation of orphan nuclear receptor Nur77 in macrophages augments inflammation and promotes the development of vulnerable atherosclerotic plaques and incidence of plaque rupture.（2）.Translocation of Nur77 from the nucleus to cytoplasm represents a promising target in vulnerable plaque treatment.Part Three Effect of RXRα inhibitor K-80003 on Inflammation and Stability of Atherosclerotic Plaque in Apo E Deficient MiceObjective Retinoid X receptor（RXR）plays an important role in regulating atherosclerosis progress.The sulindac analog K-80003 has high affinity for RXR and effectively inhibits RXR activity with tiny cyclooxygenase inhibitory effect.This study aimed to investigate the effect of K-80003 on atherosclerosis progression and plaque stability.We also evaluated its anti-inflammatory effect and drug safety to define its value on clinical application.Methods Forty Female Apo E-knockout(ApoE^-/-)mice underwent combined partial ligation of the left renal artery and left common carotid artery at the age of 8-10 weeks.Then mice were randomly divided into two groups: control and K-80003 group.The solvent or K-80003（15 mg/kg/day）was respectively infused via a stomach tube after surgery every day.At the end point,mice were euthanized and then blood samples and carotid arteries were collected.The carotid arteries’ Cross-cryosections（5-μm）were prepared and stained with H&E and immuno-histochemical staining for revealing the effect of K-80003 on lesion area,plaque morphology,cellular components,and matrix metalloproteinase（MMP）expression.Then we assessed the effect of K-80003 on inflammation markers in atherosclerotic plaque and serum by means of immunofluorescence staining and ELISA.Biochemical indexes of serum were compared to evaluate the safety of K-80003.Result（1）Cytoplasmic translocation of orphan nuclear receptor Nur77 in macrophages augments inflammation.Lesion area and vulnerable plaque incidence were significantly reduced after 8 weeks’ medication of K-80003.（2）K-80003 significantly decreased macrophage infiltration,sub-endothelial lipid deposition and expression of matrix metalloproteinases（MMPs）,while intimal collagen content and the number of vascular smooth muscle cell were elevated.（3）K80003 inhibited the NF-κB activation and expression of TNF-α,IL-6,MCP-1 in carotid atherosclerotic plaque.Serum proinflammatory markers hs-CRP and TNF-α were also down-regulated.（4）.K80003 had no effect on serum lipid,aminotransferase,urea nitrogen and creatinine level and also with unaffected arachidonic acid metabolite levels.Conclusion（1）RXRα represents a promising target in vulnerable plaque treatment.（2）Sulindac analog K-80003 inhibits atherosclerotic plaque progression and improves plaque stability through its anti-inflammatory effects.（3）K-80003 had no effect on serum lipid,aminotransferase,urea nitrogen and creatinine level and also with unaffected arachidonic acid metabolite levels in Apo E deficient mice.K-80003 has a promising application prospect in treating atherosclerosis.Summary The present study aimed to define specific inflammatory targets associated with plaque rupture and asses corresponding therapeutic strategy by the application of R+C model in apo E deficient mice and human femoral atherosclerotic lesions.The first part we investigated the responsiveness of our R+C mouse model to statin to assess its application value for drug testing and mechanistic/therapeutic discoveries.The second part investigated the association between sub-cellular translocation of nuclear receptor Nur77 and progress of atherosclerosis plaque in Apo E mice.Finally we evaluated the anti-inflammatory and atheroprotective effect of RXRα inhibitor K-80003 and studied its drug safety.The study revealed that（1）Atorvastatin exerts anti-inflammatory effects and improves plaque stability in R+C mouse model independent of plasma cholesterol level.So our R+C model is a novel mouse model of atherosclerotic plaque instability for drug testing and mechanistic/therapeutic discoveries Through modulating levels of chemokines and their receptors,statins decrease macrophage infiltration to exert protective effects.（2）Cytoplasmic translocation of orphan nuclear receptor Nur77,RXRα in macrophages augments inflammation and Nur77-/-apo E-/-carotid artery displayed dramatically decreased lesion area,plaque stability and incidence of plaque rupture after 6 or 8 weeks of R+C model surgery.（3）Sulindac analog K-80003 efficiently and safely exerts the anti-inflammatory and atheroprotective function by its anti-inflammatory effects.These findings elucidate new mechanism involving in vulnerable plaque and provided new direction and basis for developing new anti-inflammatory drugs to treat atherosclerosis.