Experimental Studies On The Effect Of Phospholipid Transfer Protein On Development And Stability Of Atherosclerotic Plaques

BackgroundThe multifunctional role of phospholipid transfer protein (PLTP) in lipoprotein metabolism has come into notice. PLTP could not only facilitate the transfer of phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and sphingomyeline, but also bind to these phospholipids directly. In plasma, PLTP mainly binds to HDL. Besides, PLTP is also involved in the transmission of vitamin E between lipoproteins and cellular membranes. Low PLTP activity results in the accumulation of vitamin E in apoB-containing lipoproteins. PLTP is also involved in the transportation of lipopolysaccharides, enhancing the immune responses to inflammation. PLTP could facilitate the transfer of phospholipids and free cholesterol from the surface of triglyceride-rich lipoprotein towards HDL particles. PLTP can also promote the formation of preβ-HDL, which can promote the cholesterol diversion.In addition to these potential anti-atherosclerosis effects, several researches mentioned the potential pro-atherosclerosis functions of PLTP as well. Inflammatory factors in the arterial walls were obviously decreased when the gene of PLTP was knocked out. Efflux of cholesterol and reverse cholesterol transport in macrophages are damaged in PLTP-/-ApoE-/-mice.Atherosclerosis is a complex pathological process caused by multiple factors. It is characterized by infiltration of macrophages, proliferation and migration of smooth muscle cells, dysfunction of endothelial cells and accumulation of intracellular and extracellular lipids. The pathological process begins with the dysfunction of intima, following with the involved pathological changes occurring in turns. Previous studies about PLTP mainly focused on metabolisms of lipids in plasma and macrophages. It is still unknown whether PLTP has other effects during the pathological process of atherosclerosis, such as those on atherogenic lipids, collagens, and the amount of macrophages and smooth muscles cells. In order to clarify these possible effects, we constructed the recombinant PLTP adenovirus, transfected ApoE-/-mice with various doses of the virus via different approaches, and observed whether PLTP affects the composition of atherosclerotic plaques.Objectives:1. To study the effects of PLTP over-expressing on the development of atherosclerosis in ApoE-/-mice;2. To study whether the effects of PLTP on atherosclerosis are dose-dependent.Methods:1. Construction of a recombinant adenovirus expressing PLTP (Ad-PLTP)A cDN A clone of murine PLTP was subcloned into pDC316-mCMV-EGFP vector for adenovirus packaging in293A cells. The adenovirus expressing EGFP alone (Ad-GFP) was used as control.2. Animal experimental protocolSixty ApoE-/-mice (male,8-week old) were involved in this study and all were fed on a western diet. Atherosclerotic plaques were induced by placement of a perivascular collar around the common carotid artery at the end of week2. All mice were randomly divided into the following five groups at the end of week10, i.e.8weeks after collar placement (n=12for each group):Group A, which were injected with normal saline; Group B, which were transfected with Ad-GFP; Group C, which were transfected with Ad-PLTP by local incubation around collar-induced plaques; Group D, which were transfected with Ad-PLTP via tail injection in a low dose; Group E transfected with a high-dose Ad-PLTP via tail injection. All mice were euthanized at the end of week12.3. Serum lipid profileThe serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using commercial kits.4. Histological and morphological analysisThe whole aorta was stained with oil red O. Serial transverse cryosections of5-μm thick carotid plaques were obtained, and were selectively stain with hematoxylin and eosin (HE) for plaque morphology, oil red O for lipids, picrosirius red for collagen. Immunohistochemical staining for SMCs, MOMA-2, MCP-1and P4Hα1was performed with specific antibody.5. Molecular biological analysisThe mRNA expression of MCP-1, ICAM-1and MMP-9was measured by quantified real-time PCR.6. Statistical analysisQuantitative values are expressed as mean±standard error of the mean (SEM) and analyzed by unpaired t-est or one-way ANOVA as appropriate. A level of P<0.05was considered significant.Results1. Serum lipid levelCompared to the two control groups, HDL-C concentration was markedly increased in mice transfected with Ad-PTLP no matter by local incubation or via tail injection (P<0.05). There were no significantly differences in the serum level of TC, TG, LDL-C, and body weight among groups.2. The efficiency of Adenovirus transfectionGFP-positive cells was enriched in carotid plaques7days after transfection, and the transfection efficiency reached to70%.3. Plaque burdenPhotographs of the appearance of carotid arteries in each group of ApoE-/-mice showed that the growth of carotid artery plaques was significantly increased in mice transfected with PLTP adenovirus locally or systemically (P<0.05).Cross sectional staining revealed that plaque area was significantly increased in mice transfected with Ad-PLTP, compared with those in the control groups (P<0.05). Furthermore, mice transfected with a high dose of Ad-PLTP displayed a much larger plaque burden than those with a low dose of Ad-PLTP, which indicated that PLTP might have a dose-dependent effects on plaque burden (P<0.05).The gross oil red O staining revealed that the atherosclerotic area in the whole aorta was also increased in mice transfected with Ad-PLTP compared with those in control groups, and mice transfected with a high dose of Ad-PLTP showed a much larger oil red-positive area than those with a low dose of Ad-PLTP, consistent with the carotid plaques (P<0.05).4. Histological and morphological analysisSpecific staining and immuno-staining revealed that carotid plaques of mice transfected with Ad-PLTP exhibited increased contents of collagen fibers and smooth muscle cells and decreased macrophage, compared with those in control groups (P<0.05). Furthermore, mice transefected with a high dose of Ad-PLTP showed a more decrease of contents of collagen fibers and smooth muscle cells than those with a low dose of Ad-PLTP (P<0.05), indicating that PLTP showed a dose-dependent effects on plaque property.5. P4Hα1expressionImmuno-staining revealed that the expression of P4Ha1in carotid plaques was markedly reduced in mice transfected with Ad-PLTP, compared with those in control groups (P<0.05). Mice transefected with a high dose of Ad-PLTP showed more decreased P4Ha1expression than those with a low dose of Ad-PLTP (P<0.05).6. Expression of inflammatory factorsQuantified RT-PCR revealed that carotid plaques of mice transfected with Ad-PLTP showed a much higher expression of inflammatory factors including MCP-1, ICAM-1and MMP-9than those in control groups (P<0.05). Mice transefected with a high dose of Ad-PLTP showed more increased expression of inflammatory factors than those with a low dose of Ad-PLTP (P<0.05). Conclusions1. PLTP promoted atheroslerosis development by altering plaque composition and the expression of inflammatory factors in the ApoE-/-mice;2. The effects of PLTP on atherosclerotic plaques was in a dose-dependent manner in ApoE-/-mice. BackgroundThe mechanism of atherosclerosis is complex and remains unclear. Currently, the most acknowledged theories believe that atherosclerotic plaques are lipid-derived and the results of chronic inflammation. Whatever the theories are, they ultimately come down to the pathological changes of components in the atherosclerotic plaque, such as endothelial cells, smooth muscle cells and macrophages. Dysfunction of endothelial cells, secretion of large amounts of inflammatory factors and adhesion molecules, migration of smooth muscle cells to the intima, transformation of macrophages into foam cells and increased apoptosis of macrophages, all of these contribute to atherosclerosis.Reactive oxygen species (ROS) are the key mediators of vascular inflammation in atherosclerosis, involving in the whole pathological process from lipid streaks to rupture of mature plaques. It is confirmed in different animal models that ROS do function in the pathological process of atherosclerosis and other vascular diseases. ROS accelerate the progression of atherosclerosis through activation of matrix metalloproteinases, oxidative modification of LDL and so on. Phospholipid transfer protein (PLTP) is a multifunctional extracellular lipid transfer protein, which transfers phospholipids and vitamin E between lipoproteins and cell membranes, or between different lipoproteins. PLTP is closely related to a variety of diseases, involving in the pathological processes of diseases such as inflammation, cancers, nervous system diseases, impaired glucose tolerance, abnormal lipid metabolism and so on. The relationship between PLTP and atherosclerosis is more and more concerned. Accumulate evidences prove that PLTP transfers cholesterol out of macrophages. Our preliminary study found that over-expression of PLTP promotes atherosclerosis, but with no obvious change of lipid in plaques. Therefore, we propose that PLTP can increase oxidative stress in macrophages to produce ROS, thus promoting the secretion of inflammatory factors and oxidation of lipoproteins, which accelerate the development of atherosclerosis.Establishment of appropriate animal models, especially proper gene knockout mice models, can help us directly observe the effect of PLTP on atherosclerosis in vivo and study the mechanism involved. In order to clarify the relationship between PLTP and atherosclerosis, we established PLTP-/-ApoE-/-mouse model, over-expressed PLTP via adenovirus and observed the impact of PLTP on atherosclerotic plaques. We hope to find new targets for clinical intervention of atherosclerosis and provide theoretical basis for clinical drug screening.Objectives1. To confirm the feasibility of establishing PLTP-/-ApoE-/-mouse model based on ApoE-/-mouse;2. To observe the impact of PLTP on atherosclerotic plaques;3. To explore the function of ROS in the process when PLTP affects the development of atherosclerosis plaques.Methods1. Establishment of PLTP-/-ApoE-/-mouse modelThe identified ApoE-/-mice and PLTP+/-mice were then put in one cage following the principle of1female mouse and2male mice, which were from different parents. The offspring mice were considered as the Fl generation, of which the male ones were separated from the female ones. Toes of the mice were clipped to mark and tails were cut to extract DNA at the age of4weeks old to identify their genotypes. Double heterozygous mice of the Fl generation were put in one cage following the above principle. When the F2generation was born, they were marked and identified as mentioned above. Finally, PLTP-/-ApoE-/-mice were picked out and put in one cage to breed, the male offspring were used for the follow-up experiments.2. In vivo experiments2.1Animal experimental process:The8-week-old male mice were divided into four groups on the basis of genotypes and interventions:ApoE-/-mice+Ad-GFP group (n=18); ApoE-/-mice+Ad-PLTP group (n=18); PLTP-/-ApoE-/-mice+Ad-GFP group (n=16); PLTP-/-ApoE-/-mice+Ad-PLTP group (n=16). After8weeks high-fat diet, each group was transfected with Ad-GFP or Ad-PLTP via tail vein injection. Two weeks after transfection, all mice were euthanized.2.2Serum lipid and inflammatory factors profile At the end of experiment, peritoneal blood of the mice was collected immediately. The serum was frozen and kept at-80℃. Concentrations of Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were detected by enzymatic methods. Concentrations of MCP-1, TNF-aand IL-6were detected by ELISA assays.2.3Histological and Morphology AnalysesThe whole aortas were stained with oil red O to observe the aortic plaque growth. Cross cryosections of aortic roots were stained with HE for morphology and oil red O for lipid. Immunohistochemical staining for SMCs, MOMA-2, MCP-1ICAM-land MMP9was performed with specific antibody.2.4Assessment of ROS in plaquesThe aortic roots cryopreserved in liquid nitrogen were homogenized after thawing, and then added10μmol/L DCFH-DA, incubated at37℃for30minutes in dark. Washed3times with PBS, the DCFH were measured by the fluorescence microplate reader.3. In vitro experiments3.1Primary culture of peritoneal macrophages3%sodium thioglycollate was injected into peritoneal cavity of the mice, which induces the generation of macrophages. Mice peritoneal were cut open under sterile conditions and the enterocoelia macrophages were harvested three days later. The MOMA-2antibody was used to identify the extracted primary macrophages via immunofluorescence test.3.2Experiment groups:3.2.1Grouping of the peritoneal macrophages according to different genotypes and interventionsIn order to observe the impact of different levels of PLTP on ROS, LDL oxidation and inflammatory factors, cells were randomly divided into4groups:A:ApoE-/-+Ad-GFP group; B:ApoE-/-+Ad-PLTP group; C:PLTP-/-ApoE-/-+Ad-GFP group; D:PLTP-/-ApoE-/-+Ad-PLTP group.3.2.2Grouping of RAW264.7cells according to whether the antioxidant N-acetyl cysteine (NAC) or PLTP adenovirus intervenesIn order to observe whether ROS are involved in the different expression of the inflammatory cytokines induced by PLTP, the cells were divided into4groups:blank control group, Ad-PLTP group, NAC group, and Ad-PLTP+NAC group.3.3Detection of intracellular ROS levelsDCFH-DA diluted with serum-free medium (1:1000) was added to the cells prewashed with PBS, incubated at37℃for30minutes in dark. Then the cells were washed3times with serum-free medium and observed by the fluorescence microplate reader, with the excitation wave of488nm and emission wavelength of525nm.3.4Detection of LDL oxidationLDL was added to intervened cells, and the oxidation level of LDL was detected via assessing the malondialdehyde concentration (MDA).3.5Western blot analysisAt the end of intervention, the proteins in the cells were extracted, and the expressions of MCP-1, ICAM-land MMP-9were detected with specific antibodies.3.6Gelatin zymographyThe proteins in the macrophages were extracted and measured after intervention, and the activity of MMP9was assessed by gelatin zymography.Results1. Identification of PLTP-/-ApoE-/-micePLTP-/-ApoE-/-mouse model was obtained after multi-generation co-cage hybridization. The genotypes of the mice were identified according to electrophoretic bands.PLTP gene interpretation:The bright bars of PLTP-/-mice are at240bp, the bright bars of PLTP+/+mice are at300bp, the bright bars of PLTP+/-mice are at both240bp and300bp; ApoE gene interpretation:the bright bars of ApoE-/-mice are at243bp, the bright bars of ApoE+/+mice are at155bp, the bright bars of ApoE+/-mice are at both155bp and243bp.2. In vivo experiment2.1Serum lipid levelIn comparison with ApoE-/-+Ad-GFP group, HDL-C was decreased significantly in both ApoE-/-+Ad-PLTP group and PLTP-/-ApoE-/-+Ad-GFP group (P<0.05), while increased significantly in PLTP-/-ApoE-/-+Ad-PLTP group compared with PLTP-/-ApoE-/-+Ad-GFP group(P<0.05). There were no significant differences in TC, TG or LDL-C levels among groups. PLTP-/-ApoE-/-mice showed a decreased body weight compared to ApoE-/-mice.2.2serum levels of inflammatory factorsThe MCP-1concentration was markedly increased in both ApoE-/-+Ad-PLTP mice and PLTP-/-ApoE-/-+Ad-PLTP mice (P<0.05). Compared with ApoE-/-mice, the MCP-1concentration was evidently increased in PLTP-/-ApoE-/-mice (P<0.05). IL-6showed similar alterations with MCP-1among groups of mice, except for the ApoE-/-+Ad-PLTP group which showed no significant difference compared with ApoE-/-+Ad-GFP mice. The TNF-a level did not differ among groups of mice(P>0.05).2.3Plaque burden Compared to ApoE-/-+Ad-GFP mice, growth of carotid artery plaques was significantly reduced in PLTP-/-ApoE-/-+Ad-GFP mice. Compared with the Ad-GFP groups, growth of the carotid artery plaque was more obvious in both PLTP-/-ApoE-/-+Ad-PLTP group and ApoE-/-+Ad-PLTP group. The aortic oil red O staining showed that the arterial atherosclerotic plaque burden was obviously reduced in PLTP-/-ApoE-/-mice compared with ApoE-/-mice (P<0.05), while evidently increased in both ApoE-/-+Ad-PLTP group and PLTP-/-ApoE-/-+Ad-PLTP group compared with the Ad-GFP groups(P<0.05).Cross cryosectional HE staining showed that the aortic root plaque area was significantly decreased in PLTP-/-ApoE-/-mice compared with ApoE-/-mice (P<0.05), while markedly increased in ApoE-/-+Ad-PLTP mice (P<0.05) and PLTP-/-ApoE-/-+Ad-PLTP mice (P<0.05) compared with the Ad-GFP groups.2.4Pathologic analysis of aortic root plaque compositionComponents of lipids showed no significant difference among groups of mice (P>0.05). Macrophages were remarkably decreased in PLTP-/-ApoE-/-+Ad-GFP mice compared with ApoE-/-+Ad-GFP mice (P<0.05), and significantly increased in both ApoE-/-+Ad-PLTP mice and PLTP-/-ApoE-/-+Ad-PLTP mice compared with the Ad-GFP groups (P<0.05). The amount of SMCs did not differ significantly among groups of mice (P>0.05).2.5Expression of MCP-1, ICAM-1and MMP-9in aortic plaquesConcentrations of MCP-1, ICAM-1and MMP-9in plaques were obviously decreased in the PLTP-/-ApoE-/-mice compared with those in the ApoE-/-mice (P <0.05), and increased in aortic root plaques of the ApoE-/-+Ad-PLTP mice and the PLTP-/-ApoE-/-+Ad-PLTP mice compared with the Ad-GFP groups (P<0.05).2.6ROS level in aortic plaquesThe ROS level in plaques was obviously decreased in the PLTP-/-ApoE-/-mice compared with that in the ApoE-/-mice (P<0.05), and increased in plaques of the ApoE-/-+Ad-PLTP mice and the PLTP-/-ApoE-/-+Ad-PLTP mice compared with the Ad-GFP groups (P<0.05).3. In vitro experiment 3.1Identification of enterocoelia macrophagesThe immunofluorescence test proved that the purity of enterocoelia macrophages was more than95%.3.2The impact of PLTP on ROS in macrophagesThe concentration of ROS was remarkably decreased in PLTP-/-ApoE-/-+Ad-GFP mice originated macrophages compared with that in ApoE-/-+Ad-GFP mice originated ones (P<0.05), and significantly increased in both ApoE-/-+Ad-PLTP mice and PLTP-/-ApoE-/-+Ad-PLTP mice originated macrophages compared with the Ad-GFP mice originated ones (P<0.05).3.3Measurement of the ability of macrophages of different PLTP levels to oxide LDLThe concentration of MDA, representing the ability of macrophages to oxide LDL, was remarkably decreased in PLTP-/-ApoE-/-+Ad-GFP mice originated macrophages compared with that in ApoE-/-+Ad-GFP mice originated ones (P<0.05), and significantly increased in both ApoE-/-+Ad-PLTP mice and PLTP-/-ApoE-+Ad-PLTP mice originated macrophages compared with the Ad-GFP mice originated ones (P<0.05).3.4The impact of PLTP on the expression of MCP-1, ICAM and MMP-9and theactivity of MMP-9in macrophagesThe expression of MCP-1, ICAM and MMP-9in macrophages were remarkably increased in both ApoE-/-+Ad-PLTP mice and PLTP-/-ApoE-/-+Ad-PLTP mice compared with the Ad-GFP groups (P<0.05). Compared with ApoE-/-+Ad-GFP mice, the expression of MCP-1, ICAM and MMP-9was significantly decreased (P<0.05). The activity of MMP-9coincided with the expression of it.3.5The impact of NAC on variations of inflammatory factors induced by PLTPCompared with control group, the expression of MCP-1, ICAM-1and MMP-9was remarkably increased in Ad-PLTP group (P<0.05), but showed no significant difference in NAC group (P>0.05). Compared with Ad-PLTP group, the expression of MCP-1, ICAM-1and MMP-9was evidently decreased in Ad-PLTP+NAC group (P<0.05). Conclusions1. The establishment of PLTP-/-ApoE-/-mouse model is feasible, and the model could be applied to study the relationship between PLTP and atherosclerosis;2. Compared with that in the ApoE-/-mice, atherosclerosis observed in the PLTP-/-ApoE-/-mice is reduced, which indicated that PLTP promoted atherosclerosis development;3. PLTP could promote atherosclerosis through increasing the level of ROS in macrophages, which enhances the oxidation of LDL and the production of inflammatory factors. BackgroundCoronary Heart Diseases (CHD) are considered the most important danger to the health of human beings. One of the main causes of acute cardiovascular events is thrombosis resulted from the rupture of atherosclerotic plaque. A variety of factors could destabilize and finally rupture the plaques, but the involved mechanisms are still unclear. Vulnerability of atherosclerotic plaques is a hot spot for research all over the world. Further studying the mechanisms of plaque vulnerability and interfering the related factors could provide theoretical basis and new targets for clinical prevention and intervention of acute cardiovascular events.Apoptosis of macrophages in the mature plaques is an important pathological process affecting stability of atherosclerotic plaques. Cell fragments resulted from macrophages apoptosis aggravate inflammation, recruitment of mononuclear cells in the plaques, which promotes the formation of foam cells. As the plaques grow, foamed macrophages aggregate in necrotic cores and shoulders of plaques, produce and release matrix metalloproteinases, which degrade collagens in plaques, thus reducing the thickness of the fibrous caps, damaging the structure of plaques and promoting the vulnerability of the plaques.JAK kinase could couple with the downstream signal transducer and activator of transcription (STAT) and regulate cellular functions. JAK/STAT signaling pathway plays an important role in cellular apoptosis. It is proved that JAK2functions in the apoptosis of smooth muscle cells induced by hydrogen peroxide, which could be inhibited by the JAK2inhibitor AG490.Our previous studies confirmed that over expressing PLTP could promote the formation of atherosclerotic plaques in ApoE-/-mice, which was dose-dependent, while changes of PLTP level in the gene knockout mice could affect the formation of atherosclerotic plaques as well. However, whether or not could PLTP affect the stability of atherosclerotic plaques and what exactly is the mechanism involved are still unrevealed, and little is known about the role of PLTP in signal pathways. It is reported that PLTP could bind with ABCA1(ATP-binding cassette transporter Al) to activate JAK2as an imitator of apolipoproteins, thus increasing the outflow of lipids. It is also proved that PLTP could enhance the cellular apoptosis induced by TRL. Based on all the previous work, in order to further clarify the mechanism involved in the effect of PLTP on atherosclerotic plaques, we propose that PLTP could promote the apoptosis of macrophages to protect the stability of artery atherosclerotic plaques, and the JAK2-STAT signaling pathway might be one of cellular mechanisms.Objectives:1. To clarify the impact of PLTP on atherosclerotic plaque stability;2.To explore the effect of PLTP on macrophage apoptosis and study the mechanism.Methods:1. In vivo experiment1.1Animal experimental process:The8-week-old male mice were divided into four groups according to different genotypes and interventions:ApoE-/-+Ad-GFP group (n=16); ApoE-/-+Ad-PLTP group (n=16); PLTP-/-ApoE-/-+Ad-GFP group (n=16); PLTP-/-ApoE-/-+Ad-PLTP group (n=16). After2weeks high-fat diet, all mice were performed on the right common carotid artery collar casing surgery to induce te formation of atherosclerotic plaques. Eight weeks after the surgery, groups of mice were injected different viruses respectively through tail veins. Two weeks later, the mice were sacrificed by anesthesia, and the samples were collected.1.2Measurement of lipids and body weight:At the end of the experiment, the peritoneal blood was collected immediately. The serum was frozen and kept at-80℃. Concentrations of Total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were detected by Enzymatic methods.1.3Histological and Morphology Analyses:Carotid artery frozen sections were used for HE staining (plaque morphology) and oil red O staining (lipid content). The expressions of SMC, MOMA-2, MCP-1, ICAM-1, MMP9and RIP3in carotid plaques were detected via immunohistochemistry staining. Vulnerable index was calculated, vulnerable index=(the percentage of macrophage positive area+the percentage of lipid positive area)/(the percentage of smooth muscle cell positive area+the percentage of collagen positive area). Fibrous cap thickness, ratio of cap to nuclear, intima-media thickness and plaque area were measured by the image analysis software Image-Pro Plus6.0.1.4Measurement of apoptosis-related indexes:The expression of cleaved caspase3in plaques was detected by immunofluorescence test. Apoptosis in plaques was detected by TUNEL test. Types of apoptotic cells were identified by co-localization via both immunofluorescence and TUNEL test.2. In vitro experiment2.1Primary culture of peritoneal macrophages:The same as that in paper II.2.2Experiment groups:2.2.1In order to observe the effect of PLTP on the expression of JAK2-STAT1/3, RIP3and cleaved caspase3, the macrophages were divided into4groups according to different origins of the cells and interventions:ApoE-/-+Ad-GFP group; PLTP-/-ApoE-/-+Ad-GFP group; ApoE-/-+Ad-PLTP group; PLTP-/-ApoE-/-+Ad-PLTP group.2.2.2In order to observe whether JAK2/STAT3signal pathway is involved in the effect of PLTP on cleaved caspase3, the RWA264.7cells were divided into four groups:control group (Ad-GFP); Ad-PLTP group; AG490group; Ad-PLTP+AG490group.2.2.3In order to observe whether RIP3participates in the effect of PLTP on cleaved caspase3, the RWA264.7cells were randomly divided into four groups according to different interventions:control group (Ad-GFP); Ad-PLTP group; RIP3siRNA group; Ad-PLTP+RIP3siRNA group.In order to observe whether ROS are involved in the different expression of the cleaved caspase3induced by PLTP, the cells were divided into4groups:blank control group, Ad-PLTP group, NAC group, and Ad-PLTP+NAC group.2.3Western blot analysis:Cells were collected at the end of interventions, the proteins of the cells were extracted and the expressions of cleaved caspase3, p-JAK2, p-STAT3and RIP3were measured.2.4Detection of intracellular ROS levels:The same as that in paper Ⅱ.Results1. In vivo experiments:1.1Differences of blood lipids and body mass among groups of mice:Detection of blood lipids showed no significant difference in TC, TG or LDL-C levels among groups of mice (P>0.05), but an obvious difference in HDL-C: compared with ApoE-/-+Ad-GFP group, HDL-C was decreased significantly in both ApoE-/-+Ad-PLTP group and PLTP-/-ApoE-/-+Ad-GFP group (P<0.05), while increased significantly in PLTP-/-ApoE-/-+Ad-PLTP group compared with PLTP-/-ApoE-/-+Ad-GFP group(P<0.05). In contrast to ApoE-/-mice, the body weight of PLTP-/-ApoE-/-mice was decreased significantly (P<0.05). No obvious effect of PLTP adeno virus intervention on body weight of the mice was observed.1.2Carotid plaque growth in each group of mice:HE sections staining showed that the carotid artery plaque area was significantly decreased in PLTP-/-ApoE-/-mice compared with ApoE-/-mice (P<0.05), while obviously increased in ApoE-/-+Ad-PLTP mice (P<0.05) and PLTP-/-ApoE-/-+Ad-PLTP mice (P<0.05) compared with the Ad-GFP groups.1.3Comparison of carotid plaque morphological characteristics among groups of Measurement of selected indicators using image analysis software (Image-Pro Plus6.0software) showed that the cap thickness and cap-nuclear ratio were significantly decreased, and the intima-media thickness was obviously increased in ApoE-/-+Ad-PLTP mice and PLTP-/-ApoE-/-+Ad-PLTP mice compared with the Ad-GFP groups (P<0.05). Compared to ApoE-/-mice, the cap thickness was increased, intima-media thickness deceased and cap-nuclear ratio enlarged in PLTP-/-ApoE-/-mice (P<0.05).1.4Pathology analysis of carotid plaque composition in different groups of mice:Compared with ApoE-/-mice, the collagen content and the number of smooth muscle cells were significantly increased in the carotid plaques of PLTP-/-ApoE-/-mice, while macrophages were obviously decreased (P<0.05). Compared with the empty virus groups, the collagen content and smooth muscle cells were remarkably decreased in both ApoE-/-+Ad-PLTP and PLTP-/-ApoE-/-+Ad-PLTP mice, while macrophages were obviously increased (P<0.05). No visible difference of lipids was observed among different groups of mice (P>0.05). Compared with ApoE-/-mice, the plaque vulnerable index was significantly reduced in PLTP-/-ApoE-/-mice (P<0.05); while the plaque vulnerable index was markedly increased in ApoE-/-+Ad-PLTP mice and PLTP-/-ApoE-/-+Ad-PLTP mice compared with the Ad-GFP groups.1.5Differences of MCP-1, ICAM-1and MMP-9in plaques among groups of mice:Compared with ApoE-/-mice, the MCP-1, ICAM-1and MMP-9content were obviously decreased in PLTP-/-ApoE-/-mice in carotid plaques (P<0.05). The MCP-1, ICAM-1and MMP-9content were significantly increased in carotid plaques in both ApoE-/-+Ad-PLTP mice and PLTP-/-ApoE-/-+Ad-PLTP mice compared with the Ad-GFP groups (P<0.05).1.6Detection of apoptosis-related indexes and apoptosis location in plaques of each group of mice:TUNEL was applied to measure apoptosis:apoptosis was evidently reduced in PLTP-/-ApoE-/-mice compared with ApoE-/-mice, but significantly increased in both ApoE-/-+Ad-PLTP mice and PLTP-/-ApoE-/-+Ad-PLTP mice compared with Ad-GFP groups.Co-localization of the apoptotic cells and macrophages showed that apoptosis mainly occurs where macrophages aggregate.The expression of RIP3and cleaved caspase3in plaques was obviously lower in PLTP-/-ApoE-/-mice than that in ApoE-/-mice (P<0.05). Compared with Ad-GFP groups, expression of RIP3and cleaved caspase3in carotid plaque was notably increased both in ApoE-/-+Ad-PLTP mice and PLTP-/-ApoE-/-+Ad-PLTP mice (P <0.05).2. In vitro experiment2.1Identification of enterocoelia macrophages:The immunofluorescence test proved that the purity of enterocoelia macrophages was more than95%.2.2The effect of PLTP on the cleaved caspase3expression:Compared with ApoE-/-+Ad-GFP group, cleaved caspase3expression was significantly decreased in PLTP-/-ApoE-/-+Ad-GFP group (P<0.05). Compared with ApoE-/-+Ad-GFP group, cleaved caspase3expression was remarkably increased in ApoE-/-+Ad-PLTP group (P<0.05). Compared with PLTP-/-ApoE-/-+Ad-GFP group, cleaved caspase3expression was significantly increased as well in PLTP-/-ApoE-/-+Ad-PLTP group (P<0.05)2.3PLTP could regulate cleaved caspase-3expression through JAK2-STAT3signal pathway:Compared with ApoE-/-+Ad-GFP group, phosphorylation of JAK2/STAT3was significantly enhanced in ApoE-/-+Ad-PLTP group (P<0.05). Compared with PLTP-/-ApoE-/-+Ad-GFP group, phosphorylation of Jak2/STAT3was remarkably enhanced in PLTP-/-ApoE-/-+Ad-PLTP group (P<0.05). Compared with PLTP-/-ApoE-/-+Ad-GFP group, activity of STAT1was remarkably reduced in ApoE-/-+Ad-GFP group, but phosphorylation of JAK2-STAT3showed no significant difference (P<0.05)JAK2inhibitor (AG490) can significantly impact the effect of PLTP on cleaved caspase-3. The cleaved caspase-3was remarkably enhanced in Ad-PLTP group, while AG490had no significant effect on the cleaved caspase-3expression. In Ad-PLTP+AG490group, the increase of cleaved caspase-3induced by PLTP was significantly inhibited (P<0.05).2.4PLTP could regulate the cleaved caspase-3expression by RIP3/ROS:Compared with ApoE-/-+Ad-GFP group, the expression of RIP3was significantly decreased in PLTP-/-ApoE-/-+Ad-GFP group (P<0.05). Compared with ApoE-/-+Ad-GFP group, the RIP3expression was remarkably increased in ApoE-/-+Ad-PLTP (P<0.05). Compared with PLTP-/-ApoE-/-+Ad-GFP group, the RIP3expression was significantly increased in PLTP-/-ApoE-/-+Ad-PLTP group (P<0.05)Intervention by RIP3siRNA resulted in a significant inhibition of the increase of cleaved caspase-3caused by PLTP. C...