| BackgroundsTraditionally, adipose has been regarded as a simple energy storage organ, but mounting evidence suggests it produces and secretes many bioactive substances, collectively referred to adipocytokines such as adiponectin (APN), resistin, TNFα, etc. Adiponectin has been found to play an important role in preventing atherosclerosis in which it reduces the size of atherosclerotic lesions, inhibits neointimal thickening and proliferation of vascular smooth muscle cells in injured arteries, and suppresses expression of vascular adhesion molecules.Secreted by adipocytes, adiponectin is a collagen-like protein whose encoding gene is located in chromosome 3q27 and named as apM 1 gene in human beings. There are 17 kbp in apM 1 gene including 3 exons and 2 introns and the complete apM 1mRNA has 4517 bp. The molecular structure of adiponectin is like complement C1q. Adiponectin is induced in the process of adipocytes differentiation and its secretion is regulated by insulin.Adiponectin has significant roles in regulating metabolism of glucose and fatty acids. Low plasma adiponectin levels are associated with insulin resistance and risk of type 2 diabetes and high plasma adiponectin levels decrease the risk of type 2 diabetes. The adiponectin-knockout mice exhibit significant insulin resistance and decreased carbohydrate tolerance.New Zealand rabbit is an ideal model in the research on atherosclerosis, but there are some limitations due to the rabbit adiponectin gene sequence has not been reported at present. With this consideration in our mind, the present study was designed. Research Objectives1. Cloning and sequencing of New Zealand rabbit adiponectin gene.2. Expressing in vitro and sequencing of New Zealand rabbit adiponectin protein.3. Investigating the relationship between New Zealand rabbit adiponectin gene expression and age.4. Investigating the relationship betwene New Zealand rabbit adiponectin gene expression and high-cholesterol diet.Methods1. Cloning of the rabbit adiponectin gene fragments(1) To clone the gene fragments, cervical back fat (brown fat) was obtained from 3-month-old male rabbits freely consuming a standard diet.(2) Total RNA was extracted from adipocytes by use of Trizol reagent following the manufacturer's instruction.(3) Degenerative primers were designed from highly conserved regions of mouse and human adiponectin sequences and used to amplify adiponectin cDNA (forward primer, 5'-ACACCTCCAGGGCTCAGGATGCT-3'; reverse primer, 5'-TCAGTTGGTATCATGG TAGAGAAG-3'). The PCR product was separated and purified with use of the EZNA Gel Extraction Kit according to the manufacturer's protocol. The PCR product was cloned into a pGEM-T Vector System I and named pGEM-T-APN.(4) The pGEM-T-APN was transformed into E. coli JM109 and the positive transformants were selected.(5) The positive transformants were amplified at 37℃ with shaking and then sequenced (Invitrogen, Shanghai, China).2. Protein expression in Pichia pastorisExpression and purification of the protein were carried out with a pGAPZα Pichia Expression Kit (Invitrogen) following the manufacturer's instruction.(1) The cDNA containing the coding region of mature rabbit adiponectin was amplified by PCR (containing EcoR I and Not I sites), then inserted into the pGEM-T vector by T/A cloning strategy, leading to a subcloning vector pGEM/APN (containing adiponectin gene). By sequencing, the pGEM/APN was confirmed to contain adiponectin gene.(2) The pGEM/APN was then inserted between the EcoR I and Not I sites of pGAPZα (containing an amino-terminal His-tag) leading to pGAPZα-APN.(3) The pGAPZα-APN was transformed into E. Coli DH5α.(4) After the above transformation, plate transformation was mixed onto low salt LB plates with 25 μg/ml ZeocinTM (Invitrogen) and ZeocinYM resistant colonies were selected. The pGAPZα-APN was transformed into the GS115 yeast by electroporation.(5) The recombinant yeasts were cultured in YPD medium and grown in flask at 30 °C with shaking overnight. The supernatant was transferred to a separate tube and stored at -80 °C until ready to assay. Sample proteins were separated by SDS-PAGE and analyzed with Western blot.(6) Measurement of adiponectin level in the supernatant of the recombinant yeasts medium was performed by using a rat anti-human adiponectin ELISA kit.3. The effect of age on the expression of New Zealand rabbit adiponectin gene About 100mg subcutaneous adipose tissue was obtained for extraction of totaladipocytic RNA from each rabbit of 2-week old, 1-month old, 3-month old, 6-month old and 12-month old. Adipose tissue mRNA transcription levels were measured by a real-time PCR procedure and the plasma adiponectin concentrations were determined by using a rat anti-human ELISA kit.4. The effect of age on the expression of New Zealand rabbit adiponectin gene Twelve New Zealand rabbits of 3-month old were fed on diet containing 1%cholesterol. The subcutaneous adipose tissues and bood samples were harvested on different timepoint (before high-cholesterol diet, 1-month, 2-month, 3-month, and 4-month after high-cholesterol diet). As above, real-time PCR and ELISA assay were carried out.5. Statistical analysesData are present as mean±SEM and were analysed by using ANOVA procedure. A value of P<0.05 was considered as significant. Results1. Cloning of rabbit adiponectinTo investigate whether rabbit adiponectin gene might have the same function as that in humans and mice, we used RT-PCR and adipocytes RNA to clone rabbit adiponectin with translation start code (atg) and stop code (tga) (GenBank DQ334867). The open reading frame for rabbit adiponectin is 735 bp long, and when translated, yields a protein of 244 amino acids. Rabbit adiponectin is highly homologous to that of humans (86.4%; GenBank NM004797) and mice (81.5%; GenBank NM009605), with 85.7% and 85.3% identity at the amino acid level, respectively.2. Expression of rabbit adiponectin in vitroWestern blotting with the anti-His tag monoclonal antibody or adiponectin polyclonal antibody indicated a single predominant band at approximately 42 kDa (containing 9.3 kDa α-factor signal peptide and 2.5 kDa C-terminal His-tag). Aiponectin level in the supernatant of cultured Pichia pastoris was 0.29±0.02 μg/ml.3. Effect of age on the expression of rabbit adiponectinThe results of real-time PCR showed that adiponectin mRNA expression of aidpose tissue in different age rabbits was significantly different (P<0.01). It was highest in 1-month old group and a decending trend relevant to age could be seen. The adiponectin plasma levels exhibited an alike result as the mRNA expression.4. Effect of high-cholesterol diet on rabbit adiponectin expressionThe high-cholesterol diet played a significant role on the expression of rabbit adiponectin gene. Adiponectin mRNA transcription was positively correlated with the serum high cholesterol concentrations (Pearson Correlation=0.469,P=0.001) and no such relationship was seen in the control rabbits fed on normal diet. The mRNA transcription levels were significantly different among different timepoint no matter in the high-cholesterol diet rabbits or in the control (Repeated Measurements Analysis of Variance, P<0.01). No significant difference was observed in adiponectin gene expression before diet intervention between the two groups (P>0.05). The mRNA transcription of adipose tissue adiponectin on fixed timepoint were significantly different between high-cholesterol diet rabbits and the control (P<0.01). The adiponectin plasma levels exhibited an alike result as the mRNA expression. Conclutions1 The rabbit adiponectin cDNA sequence shared a high homology with those of human beings and mouse.2 Rabbit adiponectin gene expression among different age rabbits was significantly different.3 The high-cholesterol diet played a significant role on the expression of rabbit adiponectin gene. Adiponectin mRNA transcription was positively correlated with the serum high cholesterol concentrations. BackgroundsAdiponectin has been found to play an important role in preventing atherosclerosis in which it reduces the size of atherosclerotic lesions, inhibits neointimal thickening and proliferation of vascular smooth muscle cells in injured arteries, and suppresses expression of vascular adhesion molecules.Through inhibiting nuclear factor KB, adiponectin inhibits the expression of adhesion molecules induced by TNF a and inhibits mitogen activated protein pathway. And then adiponectin plays a role of repressing smooth muscle cells induced by growth factors. Adiponectin suppresses the formation of foam cells by inhibiting the expression of scavenger receptor in macrophagus. Therefore, adiponectin assembles in injured arteries and plays a protective role of anti-atherosclerosis.Although there are a lot of researches on the relationship of adiponectin to insulin resistance and atherosclerosis, many problems need to elucidate such as:1. Adiponectin inhibits neointimal thickening and proliferation of vascular smooth muscle cells in injured arteries, but how does it produce this effect?2. Is there a pathway that adiponectin prevents atherosclerosis by adventitia?3. What in vivo morphological changes of blood vessels could be found after local transfer of adiponectin-producing adenovirus?All above questions are main design route and objective of our present study. Research Objectives1. To investigate the role and mechanism of adiponectin in preventing atherosclerosis. 2. To investigate the effect and mechanism of adiponectin in preventing atherosclerosis via adventitia. Methods1. Construction of atherosclerotic rabbit modelsWe created the atherosclerotic model as described. Briefly, a balloon catheter was inserted into the abdominal aorta of rabbits (n=70) through the right femoral artery, and the aorta was injured by engorged balloons. All rabbits were fed on 1% high-cholesterol diet.2. Local transfer of adenovirus and intravascular ultrasonography analysesAfter received 3 months of high-cholesterol diet, all rabbits were performed intravascular ultrasonography (IVUS) analyses and then were randomly divided into the following 4 groups: 1) Ad-APN transfer via intima, 2) Ad-βgal (adenovirus expressing β galactosidase gene) transfer via intima, 3) Ad-APN transfer via adventitia and 4) Ad-βgal transfer via adventitia. Ad-APN or Ad-βgal was transferred to abdominal aortic intima or adventitia.3. ELISA assayAiponectin levels in rabbit serum and in the supernatant of cultured Pichia pastoris were quantitatively determined via immunoassays. A kit consisting of rat anti-human adiponectin enzyme was applied and the measurements were performed following the manufacturer's instructions.4. Histochemical analysesFor the histochemical analyses, the 3 sections (100μm apart) from each rabbit on the 14th day after adenovirus transfer were stained with hematoxylin and eosin, Oil Red O (ORO), and picrosirius red. The lesion size was quantified with use of Image-Pro Plus 5.0 software. For immunohistochemical analyses, paraffin-imbedded cross-sections (5 μm thick) were incubated with either rabbit adiponectin polyclonal antibody, mouse vascular cell adhesion molecule-1 (VCAM-1) monoclonal antibody, mouse intercellular adhesion molecule-1 (ICAM-1) monoclonal antibody,a-actin monoclonal antibody, RAM 11 monoclonal antibody, mouse collagen I monoclonal antibody, or mouse collagen III monoclonal antibody. 5. Real-time PCR analysesVCAM-1, ICAM-1 and type I and III collagen cDNA was cloned with total RNA of abdominal aortas by RT-PCR. The relative quantitation of target gene expression was determined by use of LightCycler following the manufacturer's protocol. To quantify the expression of VCAM-1 and ICAM-1, real-time PCR involved use of the SYBR Green kit according to the manufacturer's instructions. For quantification of type I and III collagen level, TaqMan hydrolysis probes were utilized.6. Statistical analysesData were analyzed using an ANOVA procedure. A value of P<0.05 was considered statistically significant. Data are presented as mean±SEM. SPSS for Windows Version 13.0 was used for statistical analysis. Results1. Atherosclerosis was reduced by regional transfer of Ad-APNSerum samples were collected from rabbits before aorta injury, before and after adenovirus transfer, and subjected to ELESA assay. Serum adiponectin concentrations in rabbits after Ad-APN intima and adventitia transfer increased about 9 times as much as those before transfer (intima: 9.17±0.61 versus 1.02±0.06 μg/ml, P<0.01; adventitia: 9.27±0.23 versus 1.02±0.06 μg/ml, P<0.01). After transfer, the serum adiponectin concentrations elevated to a level about 10 times higher than the level of endogenous adiponectin in Ad-βgal treated rabbits (intima: 9.17±0.61 versus 0.92±0.05 μg/ml, P<0.01; adventitia: 9.27±0.23 versus 0.90±0.05 μg/ml, P<0.01), and about 4 times higher than those in aorta non-injured rabbits on normal cholesterol diet (2.45±0.17μg/ml, P<0.01).Ultrasonography revealed the atherosclerotic plaque area in abdominal aortas of rabbits infected via intima with Ad-APN was significantly reduced, by 35.2% compared with the area before treatment (3.98±0.32 versus 2.58±0.12 mm2, P<0.01), and by 35.8% compared with that in Ad-βgal-treated rabbits (4.02±0.31 versus 2.58±0.12 mm2, P<0.01). The lumen area stenosis (LAS) was also reduced, by 32.7% (35.07±2.43 versus 23.60±1.97%, P<0.01) and 24.1% (31.09±2.03 versus 23.60±1.97%, P<0.01), respectively. In rabbits infected via adventitia, Ad-APN treatment reduced plaque area by 28.9% as compared with the area before treatment (4.08±0.41 versus 2.90±0.23 mm2, P<0.01) and 25.6% compared with that in Ad-βgal-treated rabbits (3.90±0.16 versus 2.90±0.23 mm2, P<0.01). Likewise, the LAS was reduced, by 23.6% (34.19±2.12 versus 26.13±1.88%, P<0.01) and 19.4% (32.43±1.54 versus 26.13±1.88%, P<0.05), respectively.In rabbits with Ad-APN infection via intima, the atherosclerotic plaque area as seen on ORO staining was reduced significantly, by 35.8% (2.57±0.30 versus 1.65±0.15 mm2, P<0.01) and the maximal thickness of plaque was reduced, by 26.7% (0.75±0.04 versus 0.55±0.03 mm, P<0.01) as compared with before treatment. In rabbits infected via adventitia, the plaque area and maximal thickness of plaque was reduced by a similar, significant level.2. Adiponectin inhibits mRNA expression of VCAM-1 and ICAM-1 Immunohistochemical analyses revealed that adenovirus-derived rabbit adiponectinabundantly adhered to cells in the atherosclerotic plaque of rabbits via intima transfer. In rabbits treated via adventitia, adiponectin adhered to cells among adventitia.Compared with Ad-βgal treatment, infection with Ad-APN via intima showed significantly suppressed mRNA expression of VCAM-1, by 18.5% (20.70±1.29 versus 16.87±0.88%, P<0.05) and expression of ICAM-1 by 40.7% (2.70±0.68 versus 1.60±0.19%, P<0.01). Rabbits infected via adventitia showed significantly suppressed mRNA expression of VCAM-1 by 26.9% (21.70±0.79 versus 15.87±0.58%, P<0.01) and expression of ICAM-1 by 30.7% (2.70±0.21 versus 1.87±0.19%, P<0.01) compared with Ad-βgal treated rabbits. However, rabbits infected via intima or adventitia showed no significant difference in atherosclerotic plaque area, LAS, plaque maximal thickness or mRNA level of VCAM-1 and ICAM-1 in the abdominal aorta.3. Local transfer of Ad-APN had no effect on expression of collagen Ad-APN-treated and Ad-βgal-treated rabbits did not differ in expression of type Iand III collagen, regardless of infection via intima or adventitia.Compared with Ad-βgal treatment, infection with Ad-APN via intima showed no significant difference in mRNA expression of type I collagen (1.82±0.26 versus 1.55±0.17%, P>0.05) and type III collagen (26.96±5.12 versus 25.15±3.67%, P>0.05). As well, compared with Ad-βgal treatment, infection with Ad-APN via adventitia showed no significant difference in mRNA expression of type I collagen (2.51± 1.07 versus 1.86±0.28%, P>0.05) and type III collagen (19.98±3.20 versus 23.07±4.07%, P>0.05).4. Effect of adiponectin on the vulnerobility index of atherosclerotic plaqueCompared with Ad-βgal treatment, the area of α-actin in abdominal aortas of rabbits infected via intima with Ad-APN was significantly incresed by 28.88 (P<0.01), that of Oil Red staining reduced by 24.48% (P<0.01), and RAM 11 by 28.29% (P<0.01). So the vulnerability index of plaque was reduced by 33.78% (P<0.01).Compared with Ad-βgal treatment, the area of α-actin in abdominal aortas of rabbits infected via intima with Ad-APN was significantly incresed by 23.88 (P<0.01), that of Oil Red staining reduced by 25.54% (P<0.01), and RAM 11 by 32.119% (P<0.01). So the vulnerability index of plaque was reduced by 27.03% (P<0.01). Conclusions1 After adenovirus transfer, the serum adiponectin concentrations of Ad-APN-treated rabbits notably increased and the atherosclerotic area was significantly reduced by inhibiting the expression of adhesion molecules (VCAM-1 and ICAM-1) in the vascular walls.2 Local transfer of Ad-APN had no effect on the expression of type I and III collagen in the vascular walls.3 Local transfer of Ad-APN reduced the vulnerability of plaque by increasing the amount of smooth muscle cells and decreasing the amount of lipids and macrophage cells in vascular walls.
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