Application of real-time RT-PCR to quantifying gene expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human abdominal aortic aneurysm.
BACKGROUND
The relative expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), key regulators in remodeling of extracellular matrix, are considered to play a pivotal role in the development of abdominal aortic aneurysm (AAA). However, few data exist regarding quantitative assessment of their expression in clinical settings.


METHODS
In 22 patients with AAA who underwent graft replacement, tissue samples of the AAA and non-dilated aorta were obtained. Using a real-time RT-PCR method that enabled quantitative measurement of mRNA levels in small tissue samples, we determined gene expression levels of MMPs and TIMPs relative to that of glutaraldehyde 3-phosphate dehydrogenase in each sample.


RESULTS
The expression levels of the MMP-1 and -3 genes were significantly augmented in AAA compared with non-dilated regions (4.48 +/- 2.01 versus 0.26 +/- 0.12, P < 0.01 and 1.89 +/- 1.00 versus 5.01 +/- 0.97, P < 0.05, respectively). Although genes for TIMP-1, -2 and -3 tended to be upregulated in AAA, relative expression levels of MMP-1 to TIMP-1, MMP-1 to TIMP-2, MMP-1 to TIMP-3, and MMP-3 to TIMP-2 were still higher in AAA than in non-dilated regions (1.12 +/- 0.63 versus 0.10 +/- 0.03, 4.13 +/- 1.12 versus 0.43 +/- 0.11, 1.61 +/- 0.59 versus 0.14 +/- 0.03, and 7.81 +/- 1.60 versus 2.56 +/- 0.76, respectively, P < 0.05).


CONCLUSIONS
These results demonstrate that the present real-time RT-PCR method is reliable for the determination of mRNA levels in small samples of vascular tissue and that disproportional expression of both MMP-1 and MMP-3 relative to TIMPs relates pathologically to the evolution of AAA.
Higashikata
Takeo
T
Division of Cardiovascular Medicine and Biotechnology, National Cardiovascular Center and Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan.
Yamagishi
Masakazu
M
Sasaki
Hiroaki
H
Minatoya
Kenji
K
Ogino
Hitoshi
H
Ishibashi-Ueda
Hatsue
H
Hao
Hiroyuki
H
Nagaya
Noritoshi
N
Tomoike
Hitonobu
H
Sakamoto
Aiji
A
D000368:Aged; D017544:Aortic Aneurysm, Abdominal / Q000201:enzymology*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D020782:Matrix Metalloproteinases / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis*; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000032:analysis*; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000032:analysis*
D019715:Tissue Inhibitor of Metalloproteinase-1; D019717:Tissue Inhibitor of Metalloproteinase-3; D019716:Tissue Inhibitor of Metalloproteinase-2; D020782:Matrix Metalloproteinases