Basal and Oxidative Stress–Induced Expression of Metallothionein Is Decreased in Ascending Aortic Aneurysms of Bicuspid Aortic Valve Patients Julie A. Phillippi, PhD; Ekaterina A. Klyachko, PhD; John P. Kenny IV, BS; Michael A. Eskay, BS; Robert C. Gorman, MD; Thomas G. Gleason, MD 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. (Circulation. 2009;119:2498-2506.) Key Words: aorta  aneurysm  antioxidants  cells  genes  metalloproteinases  oxidative stress B icuspid aortic valve (BAV) is the most common congen- ital heart malformation, occurring in 1% to 2% of the population. 1,2 BAV patients are at increased risk for aortic dilatation, aneurysm, and dissection. 3–5 Its cause remains unknown, but the defect may arise during development of the aortic valvular cusps and aortic media from neural crest cells. 1,6,7 There appears to be a genetic basis for BAV with some proven linkages to chromosomal regions 18q, 5q, and 13q. 8 To date, no single gene has been identified; however, mutations in Notch 1 and 2 were shown to be associated with calcification of the aortic valve and aortic stenosis and suggest a potential association with BAV. 9 Editorial see p 2423 Clinical Perspective on p 2506 The molecular biological basis for aneurysm formation in BAV patients is unknown. We postulate that there is a marked reduction in the integrity of the extracellular matrix (ECM) in the aortic wall. Changes in gene expression of ECM proteins are known in other aortopathies. One specific example is mutation of the fibrillin-1 gene, the basis of the Marfan syndrome. 10 The integrity of the aortic ECM may be compromised by an increase in matrix-degrading enzymes such as the matrix metalloproteinase (MMP) family and/or a decrease in tissue inhibitors of MMPs. 11 MMPs control degradation of ECM proteins, including elastin and collagen, and play an important role in vascular biology. 11 MMPs are overexpressed in some types of aortic aneurysms. 1,4,12,13 Specifically, MMP-2 and MMP-9 have been implicated in thoracic aortic disease 14 –16 as a result of overproduction by smooth muscle cells (SMCs) of the aorta and macrophages. 17,18 MMP activity also is influenced by expression of their antagonists, the tissue inhibitors of MMPs, which are often downregulated. 13,15,16 However, the MMP/ Received February 1, 2008; accepted February 3, 2009. From the Thoracic Aortic Disease Research Laboratory, University of Pittsburgh, Pittsburgh, Pa (J.A.P., M.A.E., T.G.G.); Northwestern University Feinberg School of Medicine, Chicago, Ill (E.A.K., J.P.K.); and University of Pennsylvania School of Medicine, Philadelphia (R.C.G.). First author previously published as Julie A. Jadlowiec. Correspondence to Thomas G. Gleason, MD, UPMC Presbyterian Hospital, Suite C-718, 200 Lothrop St, Pittsburgh, PA 15213. E-mail gleasontg@upmc.edu © 2009 American Heart Association, Inc. Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.108.770776 2498 Vavular Heart Disease by guest on June 28, 2016 http://circ.ahajournals.org/ Downloaded from