Anti-atherosclerosis Mechanism Of TIPE2by Negatively Regulating VSMCs Functions The Detection Of TIPE1Protein In Murine Tissues And TIPE1,TIPE2mRAN In Cell Lines

Atherosclerosis, characterized by lipid deposition in aorta, hyperplasia of smooth muscle and connective tissue, intimal fibrous thickening and plague formation, has considered as a common disease which badly threats to human health. However, its pathogenesis is still unknown today. Generally, most people believe that endothelial injury and the following lipid uptake by macrophages are the initial events of atherogenesis, while dysfunction of vascular smooth muscle cells (VSMCs) including phenotype switching, migration, abnormal proliferation and cytokine excessive secretion was considered as the key step of atherogenesis. In addition, VSMCs-derived foam cells are important component of plague and myointimal cells (proliferative VSMCs in intima) could promote plague formation by synthesis of collagen, elastin and other glycoproteins. Therefore, studies on functional regulation of VSMCs have theoretical and practical significance on AS therapy and pathogenesis research.TIPE2, tumor necrosis factor-alpha (TNF-α) induced protein-8-like2, was identified as a novel immunological negative regulator by Sun et al in2008, which was selectively expressed in lymphoid organs, inflammatory tissues and other non-lymphoid tissues, such as endocrine tissues and reproductive organs. TIPE2plays a key role in the maintenance of immune homeostasis through negatively regulating innate and adaptive immunity. Further studies indicated that TIPE2was a potential atheroprotective factor through negatively regulating macrophage functions. It is known that VSMCs play vital roles through the whole process of AS. The question is that whether TIPE2could play roles during atherosclerosis by regulating VSMCs function. And if so. what roles will it play? To address this issue, this project will focus on whether TIPE2could regulate VSMCs function and how to do. Our results provided a further evidence on TIPE2as a atheroprotetive factor and suggested that TIPE2may be a novel potential new drug target for AS.Materials and Methods1. Atherosclerotic lesions induced by carotid collar placement1.1Carotid collar placement surgery. TIPE2-/-mice, sex-and age-matched ApoE-/-mice were anesthetized by peritoneal injection of Nembutal (40mg/kg) and then conducted carotid collar placement. After surgery, mice were given high-fat diet until they were sacrificed.1.2Tissue Harvesting and Preparation. Mice were anesthetized, and blood samples were collected from the left ventricle. In situ perfusion fixation through the left cardiac ventricle was performed by normal saline instillation for15minutes, followed by infusion of4%paraformldehyde (Sigma) for30min. Subsequently, both carotid bifurcations and common carotid arteries were removed and left in10%formalin overnight before freezing. The specimens were stored at-20℃until further use. Transverse7μm cryosections were prepared in a proximal direction from the carotid bifurcation and mounted in order on a parallel series of slides.1.3Histology and Optometry. Cryosections were routinely stained with hematoxylin (Sigma Diagnostics) and eosin (Merck Diagnostica) and with oil red O (Sigma Diagnostics) for lipid visualization. Corresponding sections on separate slides were stained immunohistochemically with antibodies against smooth muscle α-actin (SMα, dilution1:200, Millipore, Billerica, MA, USA). The images were taken with an Olympus microscope and SMa-positive-areas were analyzed by Imagepro Plus6software.1.4Cholesterol and triglyceride Assay. Blood was collected after overnight fasting to determine plasma cholesterol and triglyceride concentrations. Total plasma cholesterol and triglyceride levels were determined using commercial kits (Roche Diagnostics, Indianapolis. IN), according to the manufacturer’s guidelines.2. Isolation, culture and identification of VSMCsMurine VSMCs used for this study were obtained from the aortaventralis of8-week-old male C57BL/6J or TIPE2-/-mice by enzymatic digestion(type I Collagenase and type III Elastase, Sigma), cultured in high-glucose version of DMEM medium (GIBCO-BRL, Carlsbad, CA. USA) supplemented with15%FBS at37℃/5%CO2. When cultured in medium, all cells were SMa-positive (smooth muscle α-actin). For experiments, VSMCs of third to tenth passages were used. Cells were made quiescent by incubation with serum-free DMEM for24h before use.3. Effect of ox-LDL on TIPE2expression in VSMCsWT VSMCs were treated with ox-LDL, and then both mRNA and protein of TIPE2were detected by RT-PCR, real-time PCR and Western blot, respectively.4. The detection of TIPE2in phenotype switch of VSMCsThe aortaventralis were isolated from WT and TIPE2-/-mice and differentiated VSMC-special marker genes, including smooth muscle α-actin (SMa), smooth muscle myosin heavy chain(SM-MHC), calponin (CAL), were determined by real-time PCR.In vitro, WT and TIPE2-/-VSMCs were exposed to20μg/ml ox-LDL, and differentiated VSMC-special marker genes described above were detected by real-time PCR. In addition, SMa protein was measured by western blot. After TIPE2over-expression or silence, marker genes expression was also measured.5. The study of TIPE2in VSMCs migrationWT and TIPE2-/-VSMCs or VSMCs transfected with TIPE2vectors and negative control cells were treated with20μg/ml ox-LDL, and cell migration was measured by TRANSWELL and wound healing assay. Additionally, mRNA of MMP-9was detected by real-time PCR.6. The study of TIPE2in VSMCs proliferationCCK-8assay and cell cycle analysis were performed to measure cell proliferation. Furthermore, expression of PCNA and cell cycle related proteins including CyclinDl, CyclinE, P21and P27were detected by western blot.7. Effect of TIPE2on synthetic function of VSMCsWT and TIPE2-/-VSMCs were exposed to20μg/ml ox-LDL for variety time, and mRNA of PDGF-A, PDGFR-α, IL-1β, IL-6, IL-8, TNF-α, MCP-1and TGF-β were detected by real-time PCR.8. Mechanism for the functional alteration of VSMCs due to TIPE2WT and TIPE2-/-VSMCs were stimulated with ox-LDL for variety time and phosphorylation of ERK1/2and P38were detected by western blot. Furthermore, specific MAPK inhibitors (20μM PD98059plus30μM SB203580) were used to verify whether ERK1/2and P38signal pathways were involved in VSMCs functional modulation. And RAF-1and RAC-1inhibitors (10μM GW5074,50μM NSC23766, respectively) were used to find out the pathway interfered by TIPE2and further affected VSMC function.Results1. Effect of TIPE2on plaque formation in vivo1.1TIPE2deficiency accelerated atherosclerosis developmentThe extent of atherosclerotic plaques was determined using hematoxylin and eosin-stained and Oil Red O-stained tissue sections. Significant increase of lesion area was observed in TIPE2-/-mice compared with ApoE-/-mice (1.01×105±5.174×103vs.5.65×104±5.844×103μm2, p=0.0005). Furthermore, the SMa-positive area in lesions from TIPE2-/-mice were significant larger than that in ApoE-/-mice (3.78×104×5.56×103vs.2.33×104±0.88×103μm2,p=0.0422), indicating that TIPE2deficiency in VSMCs promoted cell proliferation. Additionally, VSMCs irregularly distributed in the whole lesions of TIPE2-/-mice, and displayed less distribution in media but more in lesion, unlike that of ApoE-/-mice which mainly distributed in media and the edge of lesions. These data suggested that TIPE2deficiency may also increase VSMCs migration.1.2TIPE2deficiency had no effect on lipid metabolismThe sera of TIPE2competent (including C57BL/6and ApoE-/-) or deficient mice were analyzed for differences in lipid levels. When fed with normal diet, TIPE2-/-mice had no significantly different serum total cholesterol and triglyceride with C57BL/6mice, but had significantly lower serum total cholesterol than ApoE-/-mice. However, when fed with high-fat diet, both serum total cholesterol and triglyceride of TIPE2-/-mice had no significantly change but hypercholesterolemia was observed in ApoE-/-mice. These data suggested that TIPE2deficiency had no effect on lipid metabolism in mice.2. TIPE2expressed in cultured VSMCs and was upregulated by ox-LDLWT VSMCs were treated with ox-LDL. As expected, both TIPE2mRNA and protein levels were up-regulated by ox-LDL in a dose-and time-dependent manner.3. TIPE2negatively regulated VSMCs function in vitro.3.1TIPE2inhibited VSMCs switching from contractile to synthetic phenotype VSMCs are uniquely plastic, as they can oscillate between contractile (a quiescent, more differentiated state) or synthetic phenotype (a proliferative, low differentiated state). TIPE2deficiency down-regulated differentiated VSMC-special marker genes expression, including SMa, SM-MHC and Calponin, While TIPE2over-expression up-regulated these genes expression, indicating that TIPE2could prevent VSMCs phenotype switching.3.2TIPE2decreased MMP-9expression and inhibited VSMCs migration in vitroTIPE2deficiency enhanced VSMCs migration and up-regulated MMP-9expression either in absence or presence of ox-LDL. However, TIPE2over-expression significantly declined VSMCs migration and suppressed MMP-9expression.3.3TIPE2negatively regulated VSMCs proliferationTIPE2deficiency increased VSMCs proliferation and PCNA expression. Cell cycle analysis showed that TIPE2prevented the down-regulation of the cyclin-dependent kinase inhibitors p21cipl and p27kipl, as well as up-regulation of cyclinDl and cyclinE, which are essential for the progression through the G0/G1phase, resulting cell cycle arrest in G0/G1phase.3.4TIPE2affected synthetic function of VSMCsTIPE2deficiency up-regulated mRNA levels of PDGF-A. PDGFR-α, IL-1β, IL-6, IL-8, TNF-α, MCP-1, which were reported as atherogenic factors. However, TGF-β, an atheroprotective factor which suppressed effector leukocytes and promoted VSMCs differentiation, was down-regulated.4. Mechanism for the functional alteration of VSMCs due to TIPE2deficiencyBoth under normal and ox-LDL-stimulated condition. TIPE2-/- VSMCs showed stronger phosphorylation of ERK1/2and P38compared with WT VSMCs. Therefore, PD98059plus SB203580were used to blocked ERK1/2and P38signal in VSMCs and determined whether the enhanced signal due to TIPE2deficiency was involved in VSMCs dysfunction. As expected. VSMCs dysfunction including phenotype switch, hyper-proliferation and abnormal migration, were revived after blockade of ERK1/2and P38signal. Furthermore, we found that both RAC-1and RAF-1contributed to ERK1/2and P38activation in VSMCs, but the enhanced activation due to TIPE2deficiency were normalized by NSC23766, but not affected by GW5074, indicating that TIPE2inhibited ERK1/2and P38activation through interfering Rac-1but not Raf-1cascades.Conclusions1. TIPE2deficiency accelerated atherogenesis, promoted VSMCs proliferation and migration in vivo, suggesting that TIPE2is an atheroprotective factor.2. TIPE2expressed in VSMCs and up-regulated by ox-LDL stimulation, indicating that TIPE2may involved in atherosclerosis development via functional modulation in VSMCs.3. TIPE2involved in atherosclerosis development may mainly through regulating VSMC phenotype switching, proliferation, migration and synthetic functions.4. TIPE2modulated VSMCs function via negatively regulating ERK1/2and P38activations.5. TIPE2inhibited ERK1/2and P38activation through interfering with RAC-1but not RAF-1signaling cascades.6. The atheroprotective role of TIPE2was further confirmed in this study. It may also lead to the development of TIPE2-based strategies for treating the disease. Innovations and significances1. It is the first time to figure out that TIPE2play a regulatory role in VSMC function, which is associated with atherosclerosis initial and development.2. Our study proved that TIPE2is a novel athero-protective factor, which retards atherogenesis via negatively regulating VSMCs function. Otherwise, it is the first time to show that novel anti-inflammatory protein TIPE2could also play regulatory roles in non-immune cells function.3. Systematically study on VSMCs functional alteration caused by TIPE2deletion. It may provide a new strategy for the prevention and treatment of atherosclerosis and other VSMC dysfunction-related diseases such as angioplasty restenosis. Members of the tumor necrosis factor-alpha-induced protein-8(TNFAIP8or TIPE) family play important roles in immune homeostasis and cancer. Both TIPE1(TNFAIP8-like1) and TIPE2(TNFAIP8-like2) are new members of TIPE family. TIPE1plays important role in cell death regulation while TIPE2is a newly discovered negative regulator of innate immunity and cellular immunity. TIPE2deficiency in mice causes fetal inflammatory diseases and TIPE2downregulation in humans is associated with systemic autoimmunity. However, the other functions of TIPE1and TIPE2are not yet clear. Due to the lack of a suitable antibody, the nature of cells and tissues that express TIPE1protein has not been determined. In this study, we generated highly specific antibody to TIPE1, then examined expressions of TIPE1protein in various murine tissues by immunohistochemistry and TIPE1, TIPE2mRNA in cell lines by RT-PCR and real-time PCR.Materials and Methods1. Antibody preparation and specificity verificationAnti-TIPE1Abs were generated by JinSiTe Corp (China) and used to detect expressions of TIPE1in various murine tissues after verifying specificity by western blot.2. Tissues preparationThe animals were anesthetized and exanguinated by femoral artery transection. In situ perfusion fixation through the left cardiac ventricle was performed by normal saline instillation for15min, followed by infusion of4%paraformldehyde for30min. Subsequently, the specimens were removed and performed for paraffin embedding. Then3-5um sections were prepared.3. ImmunohistochemistryParaffin-embedded sections were deparaffinized and gradually rehydrated, and then treated with0.3%hydrogen peroxide and10%methanol to block endogenous peroxidase activity. Nonspecific staining was blocked by incubation in goat serum. Sections were incubated for4h at4℃with Abs (anti-TIPE1,1:200, or isotype-matched control antibodies,1:200). After they were washed in PBS, tissues were incubated with the biotinylated goat anti-rabbit IgG mAb. After washing, sections were incubated with HRP-streptavidin, washed again, and stained with diaminobenzidene in0.07%hydrogen peroxide. Finally, the sections were counterstained with hematoxylin, and dehydrated in graded concentrations of alcohol and dimethylbenzene before mounting in neutral balsam. The sections were examined under an optical microscopy.4. Expressions of TIPE1and TIPE2mRNA in human and murine cell linesCells grew in common medium were collected and used to extract total RNA, then detected the expressions of TIPE1and TIPE2by RT-PCR and real-time PCR.Results1. The antibodies we generated bind specifically to TIP1proteinsThe specificity of antibodies were measured by western blot and results indicated that the antibodies we generated bound specifically to TIPE1, and cannot bind to TIPE which shared considerable sequence homology with TIPE1. 2. TIPE1expression in mice tissues2.1TIPE1expression in the renal systemBoth in the ciliated epithelial cells constituting the proximal convoluted tubules within the renal cortex and the tubular epithelial cells at the corticomedullary junction, TIPE1expression was the strongest. Moderate level staining was detected in the distal convoluted tubules and collecting ducts. However, TIPE1protein was not detectable in the glomeruli and fat cells within the renal medulla.In the urinary bladder, an intense level of TIPE1protein was detected in transitional epithelia.2.2TIPE1expression in lymphoid tissuesThe expression pattern of TIPE1protein in the lymph node was similar to that in the spleen. TIPE1protein was mainly observed in monocytes in the T cell zone, but not in primary follicles or cells within germinal centers. Among the hematopoietic cells of the bone marrow, moderate level of TIPE1protein was observed in megakaryocytes. However, both erythroid precursors and mature erythroid cells were negative.2.3TIPE1expression in reproductive organsIn the testes of adult male mice, TIPE1protein can be detected within the spermatogonia, primary spermatocytes, secondary spermatocytes and spermatids. The staining was the strongest in spermatogonia, but the weakest in spermatids. No staining of TIPE1protein was detectable in the spermatozoa. Intense level of TIPE1protein was detected in the ciliated columnar epithelial cells of the epididymal ducts, which expressed TIPE1more strongly than epithelial cells of the ductus of deferens. Moderate staining was observed in epitheliumof prostate.In adult females, moderate levels of TIPE1protein were observed within the granulose cells of primordial, primary, secondary and mature follicles, but not in the connective tissue between the follicles. A low level staining of TIPE1protein was detected in cells of corpus luteum. In uterus, TIPE1protein was detectable in the uterine glands, but not in smooth muscle cells and the epithelial cells of the uterus. Moderate levels of TIPE1protein were observed in the simple columnar ciliated epithelium of the oviduct and there was an intense staining detectable in mammary glands of the secretory phase of the female.2.4TIPE1expression in the endocrine systemIntense levels of TIPE1protein were detected in the chromaffin cells located in adrenal medulla. Very low level staining was observed in the cells of the adrenal cortex, including aldosterone-producing cells of zona glomerulosa, glucocorticoid-secreting cells of zona fasciculate, and androgen-secreting cells of zona reticularis.In the pancreas, an intense level of TIPE1protein was observed in the endocrine cells of the islets, but not found in the pancreatic acini cells that are responsible for the secretion in the zymogen granular and the columnar epithelial cells lining the pancreatic ducts.Very weak staining of TIPE1protein was observed in cells of parathyroid gland, but not seen in the cuboidal epithelium of the thyroid follicles.2.5TIPE1expression in the gastrointestinal and respiratory systemsTIPE1protein was observed mainly in the glands of the gastrointestinal system. Intense levels of TIPE1protein were detected in the acini and ducts of salivary glands and the glandular portion of the stomach. In the stomach, the staining of TIPE1protein was obvious in epithelium and lamina propria. Parietal cells of fundic glands showed a stronger staining, whereas, TIPE1protein was not detected in the mucosa, submucosa, and serosa of the nonglandular portion of the stomach. In the small intestine, moderate staining of TIPE1protein was detected in the simple columnar epithelial cells and crypt cells of the intestinal villi. Intense levels of TIPEl protein expression were observed in the cells of the lamina propria. which showed morphological characteristics of granulocytes.Very weak staining of TIPE1protein was detected in hepatic cells in the liver.In the respiratory system, an intense level of TIPE1staining was detected in the pseudostratified ciliated columnar epithelium and chondrocytes in hyaline cartilage in the trachea, but not detectable in the alveolar epithelium of the lung.2.6TIPE1expression in the central nervous system and other organsIn the nervous system, TIPE1protein was mainly observed in neurons in the gray matter and a very weak staining or no detectable of TIPEl protein were seen in glial, astrocytes, and oligodendrocytes. Moderate level of TIPE1protein was detected both in the granular layer and molecular layer in the cerebellum. Neuronal cells of the hippocampus were stained with TIPE1antibodies, whereas, TIPE1protein was not observed in the endothelial cells of blood vessels in the brain.TIPE1protein was intensively expressed in muscular tissues, e.g. skeletal muscle and cardiomyocytes were marked by strong staining. In skin, TIPE1protein was not observed in the epidermis and dermis, but was intensively expressed in the hair follicles and sebaceous glands.3. TIPE1expression in cultured cell linesThe highest level of TIPE1protein was detected in HMy2.CIR cells which originated from human B lymphoblast transformed with Eptein-Barr Viral DNA and HepG2.2.15cells, a human hepatoma cell line integrated of the HBV genome, but not detectable in HepG2cells. Moderate or low levels of TIPE1staining were detected in most human carcinoma cell lines. No detectable or very week expression of TIPE1protein were seen in cell lines which originated from the stomach, such as MKN-45, MKN-28, SGC-7901, AGS, and hepatoma cell lines BEL-7402, SMMC-7721and HepG2, but is highly expressed in transformed cells such as HEK293and MOVAS cells. Moderate levels of TIPE1were detected in murine macrophage lines RAW264.7and J774A.1, and human cervix carcinoma Hela cells. Relatively low levels of TIPE1were observed in the transitional cell carcinoma T24, which originates from the urinary bladder. K-562originates from chronic myelogenous leukemia and the human monocyte THP1cell line, but it is not detectable in the lung carcinoma cell line A549, U-87MG, which are cell lines originated from astrocytoma, and T47D cell line originated from ductal carcinoma.4. TIPE2expression in cultured cell linesThe highest level of TIPE2was detected in THP1cells originated from human monocyte and the mouse macrophage lines RAW264.7and J774A.1. TIPE2is not detected or weakly expressed in most human carcinoma cell lines, such as U-87MG and U251cell lines originated from astrocytoma and hepatoma cell line BEL-7402, but is highly expressed in transformed cells such as HEK293and MOVAS cells. Moderate level of TIPE2was detected in transitional cell carcinoma T24originated from urinary bladder. Relatively low levels of TIPE2were detected in hepatocellular carcinoma cell line HepG2, lung carcinoma cell line A549, and K-562originated from chronic myelogenous leukemia, and cervix cell line HeLa. A high level of TIPE2was detected in SK-OV-3originated from ovarian adenocarcinoma, but not detected in3AO and A2780cell lines derived from ovarian epithelial carcinoma. These studies demonstrate that TIPE2is mainly expressed in monocytic cell lines and some epithelia-derived cell lines with secretion function.Conclusion1. TIPE1protein was detected in a wide variety of tissues in C57BL/6mice, such as neurons in brain, hepatocytes, germ cells of female and male reproductive organs, muscular tissues, and a variety of cells of the epithelial origin, particularly those with secretory functions.2. TIPE1protein was not expressed in mature T or B lymphocytes, but detectable in human B lymphoblast cell line HMy2.CIR and murine T cell line EL4.3. High levels of TIPE1mRNA were detected in most human carcinoma cell lines, especially in cells transformed with viral genomes.4. High levels of TIPE2were detected in monocyte/macrophage derived cell lines and ovarian adenocarcinoma cells, but not detectable or weakly expressed in most human carcinoma cell lines.5. Our study reported the expression of TIPE1and TIPE2in murine tissues and cell lines and furthermore predict their functions, which may provide theoretical guidance and experimental basis for TIPE1and TIPE2functional research.Innovations and significances1. It is the first time to detect the expression of TIPE1protein in murine tissues and TIPE1, TIPE2mRNA in cell lines, and furthermore predict their function.2. Our study may provide theoretical guidance and experimental basis for TIPE1and TIPE2functional research, which is benefit for further study on TIPE1and TIPE2function.