Cardiovascular diseases are the leading cause of morbidity and mortality worldwide and the estimated costs of current treatments are 260 billion dollars. Pathologic calcification occurs in a variety of cardiovascular diseases as an end-stage process. The mechanisms of this type of calcification are incompletely understood and no therapy is available to prevent calcification. Elastin, one of the major structural proteins present in the ECM or arterial walls, is prone to calcification in a number of diseases such as advanced atherosclerosis, age related arterial hardening, and bioprosthetic heart valve (BPHV) clacification. The overall objectives of this research are the following: (A) Understanding the basic mechanism of elastin calcification as it has important implications in arteriosclerosis and BPHV calcification. (B) To discover therapies for preventing calcification based on the basic understanding of the mechanism. (C) Understanding ECM function in cardiovascular calcification using elastin as a model structural protein. The PI has developed a rat subdermal implant model, where a purified elastin implant undergoes severe pathologic calcification within 21 days. He has shown that such elastin calcification also occurs in a circulatory rat aortic allograft model. The following hypotheses will be studied using these two animal models. I: Post-implant extracellular matrix (ECM) remodeling events have a mechanistic role is elastin oriented calcification. II: Blocking of specific ECM signaling events in elastin calcification process will inhibit calcification. This will include site-specific delivery of lactose (to supress elastin receptor activation), MMP inhibitor (to suppress general MMP activity), adenovirus AdCMV.hTIMP-2 (to overexpress tissue inhibitor of matrix metalloproteinase-2 or TIMP-2), and anti-sense oligonuclotide for translation initiation site of tenascin mRNA (to suppress tenascin expression). III: Aluminum ions bind to elastin and alter its structure such that post-implant ECM remodeling events are modulated leading to inhibition of calcification. The experiments will include rat subdermal implantation of purified elastin (extracted from porcine aorta) and rat aortic allograft implants. The time-specific ECM remodeling events will be studied from 1 day to 90 days in terms of MMP activity (MMP-2 and MMP-9 by immunohistochemistry and zymography) TN-C expression (by immunohistochemistry, western blots and RNAase protection assays), and alkaline phosphatase activity and elastin degradation. The in vitro studies will include elastin conformational studies with FT-IR, NMR and circular dichroism spectroscopies, study of elastin-MMP interactions by surface plasmon resonance analysis and elastolysis experiments with MMPS.
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