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- Elastic fibers reconstructed using adenovirus-mediated expression of tropoelastin and tested in the elastase model of abdominal aortic aneurysm in rats
- 作者:熊江|发布时间:2008-11-16|浏览量:2069次
本文发表于美国血管外科官方杂志(影响因子:3.77),J Vasc Surg. 2008 Oct;48(4):965-73. Epub 2008 Jun 30.
Jiang Xiong, MD, Liu.Hua Chen, MD, Ying Lin, MD, Yi.Fan Zhu, MD, Cai.Sheng Ye, MD, Shen.Ming Wang, MD,FACS
From theDepartment of Vascular Surgery, Institute of Vascular Surgery, The First Affiliated Hospital, Sun Yet-sen University, Guangzhou, China.北京301医院血管外科熊江
Competition of interests: none.
Correspondence and reprint requests to:
Dr. Shenming Wang, Department of Vascular Surgery, Insitute of Vascular Surgery, The First Affiliated Hospital, Sun Yet-sen University, Zhongshan Road II, Guangzhou, China PR, 510080
E-mail: shenmingwang@sohu.com
This study was supported by National Natural Science Foundation of China (No. 30672054), China Postdoctoral Science Foundation (No. 20060390751) and Guangdong Province Science and Technology Project (No. 2007B030702011)
ABSTRACT:
Objective: Identification of factors involved in arterial wall remodeling after injury to elastic fibers elicited by inflammation and proteolysis is important towards the development of new therapies for abdominal aortic aneurysm (AAA). Adenoviral vector carrying tropoelastin (TE) gene with GFP tag (AdTE-GFP) transferred to aortic vascular smooth muscle cells (VSMCs) was used for expression of recombinant elastin in vitro and reconstruction of elastic fibers in vivo in experimental aneurysms.
Methods: The AAAs were induced in rats with the method of porcine pancreatic elastase perfusion and adenoviral vectors were perfused directly into the aneurysmal lumen after AAA formation. VSMCs transfected with AdTE-GFP in vitro and in vivo were detected by fluorescence microscope. mRNA leveles and recombinant elastin expression of AdTE-GFP transfected VSMCs in vitro (at 1, 3 and 5 days) and in vivo (at 2 and 4 weeks) were compared by real-time RT-PCR and Western blot analysis, respectively. Aortic diameters (AD) of aneurysms were measured at three time points (immediately, 2 weeks and 4 weeks post-perfusion) and compared between AdTE-GFP transfected group, empty adenovirus (AdNull) transfected group and PBS perfused group. Formation of neonatal elastic fibers in vivo was assessed by histologic analysis.
Results: The TE mRNA expression of the VSMCs transfected with AdTE-GFP at day 1, and day 3, was significantly higher (P<0.01) than that of AdNull transfected group and PBS group. The relative expression level of recombinant elastin-GFP of VSMCs in vitro at day 3 was 10-fold (P<0.01) of that at day 1, and that of recombinant elastin-GFP at day 5 was 3.2-fold (P<0.01) of that at day 3. In the aneurysm transfected with AdTE-GFP at 2 weeks and at 4 weeks, the relative expression level of TE mRNA was significantly higher (P<0.01 and P<0.05) than in the AdNull transfected group, the PBS perfused group, or the normal abdominal aorta (NAA) group. The relative expression level of recombinant elastin-GFP at 2 weeks was 7.7-fold (P<0.01) of that at 4 weeks. No recombinant elastin-GFP protein was found in aneurysm of AdNull transfected and the PBS perfused group. During of the 4-week duration of the experiment, the aneurysm of AdTE-GFP transfected group reduced in size but the aneurysm of AdNull transfected group and PBS perfused group dilated. The neonatal elastic fibers of aneurysm transfected with AdTE-GFP weredetected by elastin von-Giesson stain combined with GFP antibody immunohistochemistry.
Conclusions: VSMCs were transfected efficiently with AdTE-GFP and recombinant elastin was produced in vitro and in vivo. Expressing recombinant elastin and reconstructing elastic fibers within the aneurysmal tissue prevented aneurysm dilatation.
Keywords: Abdominal aortic aneurysm; Tropoelastin; Elastin; Adenovirus; Gene; Animal model; Vascular smooth muscle cell
Clinical relevance
Abdominal aortic aneurysms are associated with progressive destruction of aortic wall elastin, but methods to treat aneurysms remain undefined. The development of a gene therapy approach in an animal model in which to study recombinant elastin expression and reconstruction of elastic fibers within aneurysmal wall by VSMCs might lead to prevention of aneurysm dilatation and improvement of aneurysmal wall elasticity.
Abdominal aortic aneurysms (AAAs) are characterized by the progressive destruction of aortic wall connective tissue, most notably the elastic lamellae of the media layer.1-3 In contrast to stenotic atherosclerotic lesions where artery wall changes are localized predominantly in the intima and adventitia, changes in AAAs extend to the media layer where increased inflammation and elastin destruction are observed. On one hand, remaining vascular smooth muscle cells (VSMCs) in the media have been shown to produce matrix metalloproteinase-9 (MMP9), 4,5 suggesting that VSMCs may participate in extracellular matrix (ECM) destruction. On the other hand, VSMCs may produce elastin, collagen type I, collagen type III6and, thus, participate in wall reconstruction and AAA stabilization.7
During the development of the aorta, synthesis of elastin occurs very early, and its expression is regulated, in part, at the transcriptional level.8 Elastin is one of the major structural proteins of large arteries, contributing the physical properties of extensibility and elastic recoil9-13. Elastin is synthesized as a soluble monomeric precursor called tropoelastin (TE), which has an apparent molecular mass of 62?75 kDa, depending on animal species and isoforms.14,15 TE is subsequently secreted from the VSMC and assembled into a highly stable, insoluble, polymeric structure in the ECM through covalent cross-links derived from lysine residues.16-18 Elastic fibers are arranged into concentric rings of elastic lamellae and VSMCs. Elastic lamellae provide the resilience that enables arteries to absorb hemodynamic stress of cardiac systole and to release this energy in the form of sustained blood pressure during diastole. The number of lamellar units appears to be species-specific, fixed, and genetically predetermined.19 Expression of elastin in the aneurysmal wall may be able to change the composition of the ECM of the media and reconstruct the elasticity of the aneurysmal wall. We hypothesize that these alterations could regenerate the elastic structure of aneurysmal wall and stabilize the AAA. In the present study, we used a replication-deficient adenoviral vector carrying rat TE gene with a GFP tag (AdTE-GFP) as the delivery system in rat aortic VSMCs cultures as well as in the elastase-perfusion model of AAA in rats. TE mRNA and recombinant elastin were detected in AdTE-GFP transfected VSMCs in vitro (at day 1, 3 and 5) and in the rat AAA model in vivo (at 2 and 4 weeks) by real-time RT-PCR and Western blot analysis, respectively. Aortic diameters (AD) were found to be smaller with animals receiving the TE construct.
MATERIALS AND METHODS
Preparation of rat TE cDNA construct. Total ribonucleic acid (RNA) was isolated from aortas of male Sprague Dawley (SD) rats and used in a reverse transcription-polymerase chain reaction (RT-PCR) as a template to generate the complementary DNA (cDNA) of a segment of rat TE, corresponding to the sequence from 1,201 to 1,800 base-pair (bp) (Accession Number: M60647).22 The TE cDNA fragment was ligated into a plasmid vector with an N-terminal Green Fluorescent Protein (GFP) (pShuttle-CMV, Stratagene, Baltimore, Maryland, USA). The GFP was used for identification of synthesized recombinant elastin molecules in vitro and in vivo. The modified plasmids were transformed into Escherichia coli and the successfully transformed bacteria were selected with Luria Broth agar plates containing 100 μL/mL kanamycin. The selected recombinant constructs were sequenced to ensure that the correct fragment of the TE gene had been cloned into the constructs.
Transfection of VMSCs with rat TE cDNA. The VSMCs were isolated from aortas of 6- to 8-week-old male SD rats and were grown to confluency in M199, with L-glutamine, 10% fetal bovine serum, penicillin-streptomycin, and fungizone (GibcoBRL, Gaithersburg, Maryland, USA). The purity of the VSMCs was 90% by immunohistological evaluation with α-actin antibody (Dakopatt, Copenhagen, Denmark). Passages five to eight of cultured VSMCs were seeded into 12-well plates with 1×106 cells/ml and cultured until confluent. VSMCs were divided into three groups (n=9): those transfected with 1) AdTE-GFP (100 μg); 2) AdNull (100 μg) and 3) PBS. The transfected cells were then cultured in M199 with 10% fetal bovine serum.
Assessment of in vitro mRNA expression. Each group of cells was collected from 24 h, 3 d, and 5 d culture. After centrifugation, total RNA was isolated from VSMC samples using Trizol (Invitrogen, Carlsbad, California, USA) reagent. cDNA was synthesized from 4 μg of total RNA using random primers according to the manufacturer’s protocol (MBI Fermantas, Vilnius, Lithuania). The single-stranded cDNA was then used in a quantitative real-time PCR to evaluate the relative expression level of TE compared to β-actin. Two sets of primers were used for PCR. The TE primers were: 5’- ATCGGAGGTCCAGGCATTG and 5’- ACCAGCACCAACCCCGTAT, and the β-actin primers were 5’-ATGGTGGGTATGGGTCAGAAG and 5’- ATGGTGGGTATGGGTCAGAAG. Real-time PCR was performed and primers were designed according to the ABI protocols (Applied Biosystems, Perkin-Elmer, Foster City, California, USA). All samples were examined in duplicate in the Applied Biosystems (ABI7300) real-time PCR machine. The reactions were run for 40 cycles (96℃ 15s, 60℃ 60 s).
Assessment of in vitro gene expression. 1×106 cells and 1 ml of conditioned media from transfected cell cultures were collected from cultures at 24 h, 3 d, and 5 d. The cells were lysed in 0.5 mL of lysis buffer. After centrifugation, the supernatants of the cell lysate and the media were used for Western blot analysis. Ten ug of protein was separated by gel electrophoresis, and the gels were blotted onto nitrocellulose membranes. After blocking with 5% non-fat milk protein, the membranes were incubated with a primary rabbit anti-rat GFP antibody (Beyotime Institute of Biotechnology, Shanghai, China) and anti-β-actin (Cell Signaling Technology, Beverly, Massachusetts, USA) at 4℃ overnight. After washing, the membranes were incubated with goat anti-rabbit immunoglobulin G antibody (BioRad, Hercules, California, USA) at room temperature for 50 minutes. The membranes were then exposed to enhanced chemiluminescence reagents (Amersham, Boston, USA) and chemiluminescence assesses by x-ray film exposure. The bands from Western blots were analyzed by image acquisition and analysis software (LabWorks, Upland, California, USA). The integrated optical density (OD) of band (elastin-GFP/β-actin) represented the relative expression level of protein.
Animal model. Animals were housed and cared for according to a protocol approved by the SunYet- Sen University of Medicine Animal Studies Committee. Adult male SD rats weighing 350-400 g underwent aortic perfusion with porcine pancreatic elastase (PPE) as described by Anidjar.23 Briefly, rats were anesthetized with 5% pentobarbital (0.1 ml/100 g body weight) and a laparotomy was performed under sterile conditions. After clamping the aorta just below the left renal vein, ligation of the encompassing the distal aorta, and catheterization just above the aortic bifurcation, the infrarenal abdominal aorta was perfused continuously with 2 ml saline containing 25U/ml of PPE (type I, Sigma, St. Louis, USA) for 2 hr at 100 mmHg using a syringe pump (Sage Instruments, Boston, Massashusetts, USA). 24,25
A second surgical procedure was performed on day 14, each animal underwent relaparotomy at which time the AD was measured. All the rats in which the AD had increased more than 50% of the normal AD just above the left renal vein were considered having AAAs. Thirty six aneurysmal rats were divided into 3 groups (n=12). After clamping the proximal and distal aorta, the aorta of the three groups were perfused either with 1) 2 ml saline containing AdTE-GFP (200 μg); 2) AdNull (200 μg) or 3) PBS (0.2 ml) through a PE10 catheter introduced at the non-ligation aortic bifurcation.21 The AD was measured using a microscopic grid immediately post-perfusion at the second surgical procedure and before euthanasia at 2 weeks and 4 weeks after the second surgical procedure. Frozen sections of the aortic wall collected after 3 days of post-perfusion with AdTE-GFP were used to identify the expression of recombinant elastin-GFP by fluorescence microscope. Aortic tissue was excised and snapfrozen for subsequent mRNA and protein analyses. For histologic studies (two rats from each group), the tissue was perfusion-fixed in situ with 10% neutral buffered formalin.
Histology of aorta tissue. Aortic tissue samples were collected at 2 weeks and 4 weeks after the second surgical procedure. Perfusion-fixed specimens were immersed in 10% neutral buffered formalin at 4℃, dehydrated through graded ethanols, and embedded in paraffin. Specimens were cut in cross-section at 5 μm and attached to glass slides. Sections were stained with hematoxylin and eosin (HE) and elastic von-Giesson stain. Immunohistochemistry to identify recombinant elastin-GFP was performed using the Avidin-Biotin Complex technique and rabbit anti-rat GFP monoclonal antibody.
Assessment of in vivo mRNA and protein expression. Frozen aortic tissues were collected at 2 weeks and 4 weeks after the second surgical procedure. Samples for RNA analyses (approximately 50 μg) were pulverized using a mortar and pestle which were chilled with liquid nitrogen, and total RNA was isolated and used for mRNA analysis as described above.
Samples for protein analyses were minced and homogenized with lysis buffer. After centrifugation, the supernant was used for Western blot analysis as described above. The bands from Western blot were analyzed by image acquisition and analysis software (LabWorks, Upland, California, USA).
Data analysis. All results were presented as mean±standard error of the mean, which included the AD at three time points for each animal group, the data obtained from TE mRNA and recombinant elastin expression in vitro and in vivo. One-way ANOVA as implemented in the statistical software package SPSS Ver.11.0 (SPSS, Chicago, Illinois, USA) was used to compare all data between groups and inside groups in vitro and in vivo. A p-value of<0.05 was considered statistically significant.
RESULTS
Adenoviral vectors carrying a construct of TE were tested in the elastase-perfusion model of AAA in rats.Two and four weeks after perfusion, AdTE-GFP treated animals had significantly smaller aortic AD than AdNull treated and PBS perfused group (P<0.01). During of the 4-weeks duration after perfusion, the aneurysm of the PBS perfused and AdNull transfected group continued to dilate and comparison of AD measurements from immediately after perfusion to 2 weeks post-perfusion (P<0.05) as well as from 2 weeks to 4 weeks post-perfusion (P<0.05) were significantly different. However, the aneurysm of AdTE-GFP group became smaller from immediately after perfusion to 4 weeks post-perfusion (P<0.05)(see Fig 1 A). The recombinant elastin-GFP was visualized by fluorescence microscope in frozen sections of aneurysmal wall at 3 days after perfusion of AdTE-GFP (see Fig 1B and C). Histological sections of the aorta demonstrated that the elastic fibers were reconstructed in media of aneurysm transfected by AdTE-GFP, and no neonatal elastic fibers were identified at any time point in the AdNull transfected group.At 2 weeks post-perfusion, the recombinant elastin was assembled into intermittent fragments in the media(see Fig 1 D and E). At 4 weeks post-perfusion, the elastic fibers were arranged into partial concentric rings of elastic lamellae in the media (see Fig 1 F and G). The number of brown cells at 4 weeks was more than that at 2 weeks. Animals receiving AdNull demonstrated reconstruction of elastic fibers failed at 4 weeks post-perfusion (see Fig 1 H and I).
In vitro and in vivo TE mRNA expression. The relative expression level of TE mRNA in the VSMCs transfected with AdTE-GFP was significantly higher (P<0.01) than that of the AdNull transfected group at day 1 and 3 (see Fig 2A). However, at day 5, the relative expression level of TE mRNA in the AdTE-GFP transfected group declined to a low level, which was not significantly different (P>0.05) from the AdNull transfected group at day 3, day 5, or the PBS group. At 2 weeks the rats transfected with AdTE-GFP had significantly higher (P<0.01) relative expression level of TE mRNA than the AdNull transfected group, the PBS group or the normal abdominal aorta (NAA) group. The situation was similar, although not as pronounced at 4 weeks (P<0.05). There was no significant difference between AdNull transfected group, the PBS group or the normal abdominal aorta (NAA) group at any time point during the 4-weeks duration of the in vivo study (see Fig 2B).
In vitro and in vivo protein expression. The mRNA of TE-GFP construct used in this study was about 1.44 kb long, and it encodes for the recombinant elastin-GFP of ~480 amino acids and the corresponding protein is expected to migrate at ~52 kDa. The recombinant elastin-GFP protein was detected in both the cell lysates and the culture media of the cells transfected with elastin gene, but not in the cells transfected with an empty vector. The recombinant elastin-GFP (molecular weight was between 47 kDa and 55kDa) was detected at day 1, 3 and 5 after transfecting VSMCs with AdTE-GFP, but no recombinant elastin-GFP was found in VSMCs transfected with AdNull or in the PBS group at any time point during the 5-day duration of the in vitro study (see Fig 3A). The green fluorescent cells which expressed GFP were observed in AdTE-GFP transfected VSMCs (see Fig 3E, F, G). The amount of recombinant elastin-GFP at day 3 was estimated to be 10-fold higher (P<0.01) than at day 1, and the amount of recombinant elastin-GFP at day 5 was 3.2-fold higher (P<0.01) than at day 3 (see Fig 3C). The recombinant elastin-GFP protein was detected 2 and 4 weeks after adenoviral transfection (see Fig 3B). The amount of elastin-GFP protein at 2 weeks was 7.7-fold higher (P<0.01) than at 4 weeks. However, no recombinant elastin-GFP protein was found in rats transfected with AdNull or in the PBS group at any time during the 4-week duration of the in vivo study (see Fig 3D).
DISCUSSION
Identification of factors promoting arterial wall repair after ECM injury elicited with proteolysis would be of major importance in developing new therapies for aneurysms. In vivo manipulations of arteries constantly resulting in wall structure reconstruction, enable development of predictive biology, because tissue analysis can be performed before major wall remodeling occurs21 Recent evidence that overexpression or blocking of expression of some signaling molecules may exert part of this effect on remodeling through alterations of the ECM, which may occur both locally and systemically by paracrine mechanism involving inhibition of MMPs by their tissue inhibitors26,27. We, therefore, hypothesized, that the alterations of ECM after transfection with adenoviral vector carrying TE gene might improve elastic structure in the AAA wall by controlling remodeling.
Because elastic fibers consist of TE core surrounded by fibrillin and microfibrils, and are the basis of elastic properties of arteries and other structures, we evaluated the effect of elastin transgene on AAA. It would be difficult to clone and express the full length TE (2,595 bp). 22 According to Mizuno’s report22, the extensile and resilient properties of elastin are localized to a small fragmentof approximately 600 bp of the rat TE cDNA22. This fragment was cloned into an adenoviral vector, which was transfected into rat aortic VSMCs in vitro, with high transfection efficiency.
We chose VSMCs for the study, because VSMCs are the cellular component of the media in normal arteries and in AAAs, which are the crucial paracrine cells taking part in artery wall repair after pathological or mechanical injury28. By modulating ECM structure, VSMCs participate in wall reconstruction and AAA stabilization.29 During the development of the aorta, synthesis of elastin occurs very early, and its expression is regulated, in part, at the transcriptional level8. Such studies have highlighted the role of ECM signaling in the vascular wall and have shown that elastin and collagen not only display elasticity or rigidity but also are involved in the control of VSMC function30. Moreover, VSMCs do not express recombinant elastin under the normal condition either in vitro or in vivo, so we could compare the amount of the recombinant elastin in Adneo-virus infected VSMCs to the endogenous elastin in this study.
We were able to induce detectable overexpression of the elastin transgene in vitro and in vivo for up to 4 weeks after transfection with the AdTE-GFP, though the expression at 4 weeks was significantly decreased. The limited duration of elastin production may have a prolonged influence on arterial wall remodeling because elastin molecules are known to stay for long time. Histological examination of aneurysmal wall in the AdTE-GFP transfected group showed accumulation of elastin in the media of aneurysmal wall and the elastin fibers were assembled into a lamella pattern. In contrast, the aneurysmal wall of the AdNull transfected and PBS group had substantial loss of elastic fibers. The accumulated elastic fibers in the AdTE-GFP transfected group could be contributed by the production of recombinant elastin by VSMCs transfected with AdTE-GFP. It is possible that elastic fibers have the capacity of altering the physical properties of the aneurysmal wall, which may have added elastic recoil to the aneurysmal wall. The fact that in the AdTE-GFP transfected group, the diameter of AAA gradually decreased from immediately after the elastase-perfusion to 2 weeks post-perfusion (P<0.05) and from 2 weeks to 4 weeks post-perfusion (P<0.05), which may be explained by the elastic fibers assembled within aneurysmal wall. Future biochemical measurement and biomechanical analysis may provide a more reliable quantitative assessment of elasticity function of aneurysmal wall after administration of recombinant elastin. In addition, long-term studies will be required to determine the long-term effects of limited recombinant elastin production.
This study was intended to evaluate the proof-of-concept of elastin expression in aneurysmal wall. This preliminary study suggests that increasing the elastic fibers in aneurysmal wall will prevent aneurysm dilatation.
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