RESUMO
BACKGROUND AND AIM OF THE STUDY: Previous immunohistochemistry studies have shown that the transcription factor, Egr-1, is increased in human atherosclerotic lesions but is absent from the normal adjacent aortic wall. The hypothesis was investigated that Egr-1 is also increased in calcified heart valve cusps because of the unique presence in these tissues of proteins known to be regulated by Egr-1, such as tenascin C (TN-C). METHODS: Non-calcified and calcified human aortic valves were obtained at autopsy or from cardiac surgery. Egr-1 immunohistochemical studies were performed. The effects of Egr-1 on cellular proliferation and on mechanisms of calcification were also investigated using sheep aortic valve interstitial cell (SAVIC) cultures. Signal transduction pathways involving Egr-1 were studied with specific inhibitors. RESULTS: Immunohistochemical studies revealed that calcific aortic stenosis cusps contained a significantly higher level of Egr-1 in the spindle-shaped interstitial cells of calcified human aortic valves, but not white blood cells. By comparison, Egr-1 was detected at very low levels in the interstitial cells of non-calcified human aortic valve cusps. SAVIC cultivated on denatured versus native collagen substrates demonstrated a marked increase in Egr-1 levels (by Western blotting), and an absence of calcification in these cultures, compared to SAVIC grown on native collagen which calcified severely with little Egr-1 expression. Parallel increases in TN-C and osteopontin (OPN), both of which are proteins associated with heart valve calcification, were observed (by Western blotting) in SAVIC grown on denatured collagen. Furthermore, a protein kinase-C (PKC) inhibitor blocked the up-regulation of Egr-1 and TN-C, implicating PKC-dependent signaling control of Egr-1 and TN-C up-regulation. CONCLUSION: Egr-1 is up-regulated in human calcific aortic stenosis cusps compared to non-calcified normal cusps. Egr-1 up-regulation involves a PKC-dependent signaling pathway. TN-C and OPN appear to be co-regulated with Egr-1. Furthermore, in SAVIC cultures on denatured collagen, Egr-1 up-regulation was associated with inhibition of calcification. Taken together, these results suggest that complex Egr-1 mechanisms may be operative in calcific aortic stenosis.
