Basal and oxidative stress-induced expression of metallothionein is decreased in ascending aortic aneurysms of bicuspid aortic valve patients

Abstract

Background: Bicuspid aortic valve (BAV) is a heritable condition that has been linked by an unknown mechanism to a predisposition for ascending aortic aneurysm. Matrix metalloproteinases have been implicated in this predisposition. Metallothionein is a poorly characterized, metal-binding protein that regulates matrix metalloproteinases and is an antioxidant known to be upregulated under oxidative stress.

Methods and results: To determine putative factors involved in the pathogenesis of aortic aneurysm in BAV patients, our first goal was to identify genes that are dysregulated in ascending aortic aneurysms of BAV patients compared with tricuspid aortic valve patients and nondiseased (control) donors. By microarray analysis (22,000 probe sets), 110 dysregulated genes were identified in BAV compared with tricuspid aortic valve patients and control donors; 8 were genes of the metallothionein family. Metallothionein gene expression and protein expression were significantly lower in aortic tissue and cultured aortic smooth muscle cells from BAV patients compared with control subjects. Matrix metalloproteinase-9 expression was increased in BAV aortic samples relative to controls. BAV aorta was more susceptible to oxidative stress, and induction of metallothionein under oxidative stress was reduced in BAV patients compared with control subjects.

Conclusions: These results demonstrate dysregulated metallothionein expression in ascending aortic smooth muscle cells of BAV patients that may contribute to an inadequate response to oxidative stress and provoke aneurysm formation. We hypothesize that metallothionein plays a pivotal role in the response of ascending aortic smooth muscle cells to oxidative stress cues normally involved in the maintenance of the extracellular matrix, including the regulation of matrix metalloproteinase expression.

Figure 1

Metallothionein gene expression was reduced in TAV and BAV patients vs control. Total RNA was harvested from aortic tissue and analyzed by qPCR for metallothionein isoforms. A, MT-1A; B, MT-1X; C, MT-1E; and D, MT-2A. Bars represent relative metallothionein expression in TAAs of BAV and TAV patients and normal donor patients normalized to the housekeeping gene, 18S rRNA (mean±SEM; n=12, 19, and 24 for control, TAV, and BAV, respectively). *Significantly different from control, P<0.001.

Figure 2

Metallothionein protein expression was reduced in aortic tissue from BAV patients vs control. A, Western blot analyses for 6-kDa MT-1/2 protein and the loading control, β-actin. B, Band intensities from A were quantified by densitometry and displayed as a ratio of metallothionein to β-actin. Bars represent mean±SEM metallothionein protein expression (n=12, 19, and 24 for control, TAV, and BAV, respectively). C, Metallothionein protein expression vs age. D, Metallothionein protein expression vs diameter (dia.) of the ascending aorta. Bars represent normalized metallothionein protein expression. *Significantly different from control and TAV, P<0.01.

Figure 3

Induction of metallothionein gene expression by Cd²⁺ is reduced in BAV patients vs control. SMCs were isolated from aortic tissue and cultured in the presence or absence of 5 μmol/L CdCl₂ for 24 hours. Total RNA was isolated and subjected to qPCR analysis for gene expression of metallothionein isoforms. A, MT-1A. B, MT-1X. Bars represent mean±SEM of 3 assay replicates. C, Western blot analyses of MT-1/2 protein expression and β-actin loading controls in SMCs as pooled triplicates cultured in the presence and absence of 5 μmol/L Cd²⁺ for 24 hours. D, Quantification of band intensities in C. MT-1/2 protein expression is normalized to β-actin loading controls.

Figure 4

Cell viability under oxidative stress conditions in vitro. SMCs isolated from BAV patients (dotted line) exhibit the least cell viability under oxidative stress relative to TAV (dashed line) and control (solid line) cells. SMCs from control, TAV, and BAV patients were cultured in the presence or absence of t-butyl hydroperoxide (tBHP; 0 to 9.6 mmol/L) for 3 hours. Cell viability was assessed by MTS assay. Lines represent mean±SEM; n=3, 3, and 2, respectively.

Figure 5

MMP gene expression was elevated in TAV and BAV patients vs control. RNA was harvested from aortic tissue and analyzed by qPCR for MMP-2 (A) and MMP-9 (B) gene expression. Bars represent mean±SEM MMP-2 or -9 gene expression for TAV and BAV relative to control; n=11, 19, and 20 for control, TAV, and BAV, respectively. *Significantly from control, P<0.05.