Elastic fibers reconstructed using adenovirus-mediated expression of tropoelastin and tested in the elastase model of abdominal aortic aneurysm in rats.
OBJECTIVE
An adenoviral vector carrying a recombinant tropoelastin (TE) gene with a Green Fluorescent Protein (GFP) tag adenoviral tropoelastin green fluorescent protein (AdTE-GFP) was transferred to aortic vascular smooth muscle cells (VSMCs) for studying the expression of recombinant elastin in vitro and the reconstruction of elastic fibers in vivo in experimental abdominal aortic aneurysm (AAA).


METHODS
The AAAs were induced in rats by perfusing the arteries with porcine pancreatic elastase, and after AAA formation, adenoviral vectors were perfused directly into the aneurysmal lumen. VSMCs transfected with AdTE-GFP in vitro and in vivo were detected by fluorescence microscopy. The TE mRNA levels and the level of 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 reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. The aortic diameters (AD) of the aneurysms were measured at three time points (immediately, 2 weeks, and 4 weeks post-perfusion) and comparisons were made among the AdTE-GFP transfected group, an empty adenovirus (AdNull) transfected group, and a phosphate buffered saline (PBS) perfused group. Formation of new elastic fibers in vivo was assessed by histologic analysis.


RESULTS
AdTE-GFP transfection reversed AAA formation (mean +/- standard error), with the controls showing increased ADs (a 48.50 +/- 16.55% increase for the PBS perfusion group and a 39.84 +/- 15.59% increase for the AdNull treated group) and the AdTE-GFP transfected group showing a decreased AD (23.04 +/- 14.49%, P < .01). Histological studies showed that the AdTE-GFP transfected group also had reconstructed elastic fibers in the aneurysmal wall. Real-time PCR for TE mRNA demonstrated a significant increase upon transfection of AdTE-GFP in vitro and in vivo. Western blot analysis for GFP demonstrated elastin-GFP expression only upon transfection of AdTE-GFP, although the amount of elastin-GFP protein tended to be lower in vivo than in vitro. Elastin von-Giesson stain combined with GFP antibody immunohistochemistry demonstrated new elastic fibers in the transfected aneurysmal VSMCs.


CONCLUSIONS
VSMCs were transfected efficiently with a special AdTE-GFP vector, enabling recombinant elastin to be produced in these VSMCs in vitro and in vivo. This expression of a recombinant elastin and the related reconstruction of elastic fibers within the aneurysmal tissue appeared to prevent or reverse the aneurysm dilatation.
Xiong
Jiang
J
Department of Vascular Surgery, Institute of Vascular Surgery, The First Affiliated Hospital, Sun Yet-Sen University, Guangzhou, China.
Wang
Shen Ming
SM
Chen
Liu Hua
LH
Lin
Ying
Y
Zhu
Yi Fan
YF
Ye
Cai Sheng
CS
D000256:Adenoviridae; D000818:Animals; D017544:Aortic Aneurysm, Abdominal / Q000628:therapy*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004547:Elastic Tissue; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D010196:Pancreatic Elastase; D051381:Rats; D017207:Rats, Sprague-Dawley; D014333:Tropoelastin / Q000096:biosynthesis*
D014333:Tropoelastin; D010196:Pancreatic Elastase