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Background And Aims Of The Study: Previous research has demonstrated that tenascin-C, an extracellular matrix protein involved in bone development and mineralization, was specifically present in calcified aortic valves, always in association with matrix metalloproteinase (MMP)-2, and was not detectable in non-calcified human aortic valves. The aim of the present study was to identify downstream targets of tenascin-C in aortic valve interstitial cells.
Methods: Subtractive hybridization was performed using sheep aortic valve interstitial cells (SAVIC) grown on a substrate of collagen plus tenascin-C, versus cells grown on type I collagen alone.
Results: Subtractive hybridizations revealed that nearly 70% of the clones isolated contained the sequence for thymosin beta-4, an actin-binding protein, also associated with mineralization, regulation of MMPs and inflammation. In cell culture studies, it was shown that both thymosin beta4 and thymosin beta4 sulfoxide are produced by SAVIC. When aortic valve interstitial cells were grown on a substrate of tenascin-C, thymosin beta4 expression was up-regulated. The addition of thymosin beta4 to aortic valve interstitial cell cultures resulted in a re-orientation of cytoskeletal F-actin, and induction of MMP-2. However, thymosin beta4 antisense oligonucleotide transfection did not suppress MMP-2 expression.
Conclusion: Thymosin beta4 is up-regulated by tenascin-C, and may be involved in the primary initiation of valvular calcification.
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