Endoplasmic Reticulum Stress Contributes To The Instability Of Atherosclerotic Plaques Induced By Cold Stress

BackgroundThe sudden rupture of vulnerable plaque of atherosclerosis is an importantpathophysiological basis of acute coronary syndromes (ACS). Characterizationsof vulnerable plaques are thin fibrous caps, large lipid cores, neovascularization,and a large number of inflammatory cells, etc. The vulnerability of the plaque isaffected by various types of stress as mental stress and cold stress. Cold stresscan lead to hypertension, in consequence to the increased morbidity andmortality of ACS in winter. All these indicate cold stress may trigger ACS.It’s necessary to establish an ideal atherosclerosis animal model for furtherstudy of the relation between cold stress and rupture of vulnerable plaque.Although atherosclerotic plaque model induced by the high-fat diet plus ballooninjury in rabbits, and the model established by high fat diet in ApoE knock-outmice were usually applied, there were no appropriate standard models.In recent years, reports showed that apoptosis of macrophages maycontribute to the vulnerably of atherosclerotic plaques through ERS. Animalexperiments abroad have confirmed that CHOP pathway of ERS in macrophages contributes to the instability of atherosclerotic plaques.This study was designed to establish the atherosclerotic plaque model, thenobserve the impact and possible mechanisms of cold stress on the plaqueinstability。MethodsPart Ⅰ: The establishment of atherosclerotic plaque model in rabbits.42male New Zealand white rabbits were randomly divided into two groups:Control group and balloon injury group. Balloon injury group fed on high-fatdiet for2weeks following by balloon injury of abdominal aorta.20th week isthe end of the experiment. At week20, the initiation and progression ofatherosclerotic were observed by ultrasonography. Meanwhile, serum levels ofblood lipid, as TC, TG and LDL were determined by Enzyme-LinkedImmunosorbent Assay (ELISA) method. Pathological changes of atheroscleroticplaques were evaluated by HE staining.Part Ⅱ: Cold stress increased the instability of artery atherosclerotic plaquein rabbits with induction of ER stress.40male New Zealand white rabbits fed on high-fat diet for2weeksfollowing by balloon injury of abdominal aorta were randomly divided into twogroups, control group and cold-stress group. We applied cold stress to adapt theprevious model established by high-fat diet plus balloon injury in rabbits asfollows and exposition to cold (4℃) for1hour per day. Pathological changes ofatherosclerotic plaques among these groups were evaluated at week20. Theprogression of atherosclerotic was observed by ultrasonography, theangiogenesis was observed by contrast-enhanced ultrasonography andimmunohistochemistry. Plaque size, shape and tissue composition changes wereobserved by HE, Masson, oil red O and Sirius red staining. Meanwhile, serumlevels of blood lipid, oxidized low density protein（ox-LDL）, hypersensitiveC-reaction protein（hs-CRP）and interleukin (IL)-8were determined by ELISA.Macrophage infiltration was assessed by immunohistochemistry. GRP78, CHOP, JNK and p-JNK expression were determined by Western-blot analysis.Part Ⅲ: ERS plays an important role in macrophage transformation intofoam cells and apoptosis.Different treatments were given to macrophage by ox-LDL (25,50,100mg/L),100mg/L plus sp600125(JNK inhibitor) and tunicamycin (endoplasmicreticulum agonist) in vitro. After24h, the intracellular lipid accumulation inmacrophage was determined by Oil Red O staining, the apoptosis wasdetermined by TUNEL. GRP78, CHOP and JNK, p-JNK and Caspase-3of theindicator of ERS expression were determined by the Western-blot analysis.Results1. The model of atherosclerosis plaque in rabbit was established successfully.Compared with control group, TG, TC, LDL (all P<0.05vs. control) in balloongroup were significantly increased, and the ultrasonography results displayedintima-media thickness (IMT)(P<0.05vs. control)were significantly increased.2. Compared with control group, ox-LDL (P<0.05vs.control group),hs-CRP (P<0.05vs.control group) and IL-8(P<0.05vs.control group) weresignificantly increased after cold-stress treatment, ultrasonography resultsshowed IMT (P<0.05vs. control group) were significantly increased.Contrast-enhanced ultrasonography and immunohistochemical staining showedthat neovascularization in plaques after cold stress increased significantly(P<0.05vs.control group). HE staining revealed that after cold stress, plaquelipid cores were greater and plaque fibrous caps were thinner, less collagenfibers assessed by Masson staining and Sirius red staining were found in coldstress group, while more macrophages were observed in CS group by Oil red Ostaining and immunohistochemical staining of macrophages (P<0.05vs.controlgroup). Western-blot showed that the ERS markers of GRP78, CHOP, JNK andp-JNK expression in CS group were increased significantly (both P<0.05vs.control group).3. Oil red O staining showed macrophages would transform into foam cells in vitro if they were associated with the amount of ox-LDL (P<0.05vs. control),Western-blot and TUNEL staining showed the expression levels GRP78(P<0.05vs. control), CHOP (P<0.05vs. control), phosphorylation levels ofJNK (P<0.05vs. control) and apoptosis (P<0.05vs. control) were significantlyincreased, while JNK inhibitor lowered these effects (all P <0.05vs.100mg/Lgroup).Conclusions1. High fat fed and balloon injury in the abdominal aorta could induce theatherosclerotic plaque formation in rabbits.2. Under cold stress, atherosclerotic lesion were aggravated，and theinstability of atherosclerotic plaque were increased.3. Under cold stress, endoplasmic reticulum stress were induced, and itsdownstream JNK pathway were activated4. Macrophages transformed into foam cells and apoptosis of macrophageswere significantly increased, with the JNK pathway activation downstream ofendoplasmic reticulum stress, while JNK inhibitor decreased these effects.