Triglycidylamine crosslinking of porcine aortic valve cusps or bovine pericardium results in improved biocompatibility, biomechanics, and calcification resistance: chemical and biological mechanisms.

We investigated a novel polyepoxide crosslinker that was hypothesized to confer both material stabilization and calcification resistance when used to prepare bioprosthetic heart valves. Triglycidylamine (TGA) was synthesized via reacting epichlorhydrin and NH(3). TGA was used to crosslink porcine aortic cusps, bovine pericardium, and type I collagen. Control materials were crosslinked with glutaraldehyde (Glut). TGA-pretreated materials had shrink temperatures comparable to Glut fixation. However, TGA crosslinking conferred significantly greater collagenase resistance than Glut pretreatment, and significantly improved biomechanical compliance. Sheep aortic valve interstitial cells grown on TGA-pretreated collagen did not calcify, whereas sheep aortic valve interstitial cells grown on control substrates calcified extensively. Rat subdermal implants (porcine aortic cusps/bovine pericardium) pretreated with TGA demonstrated significantly less calcification than Glut pretreated implants. Investigations of extracellular matrix proteins associated with calcification, matrix metalloproteinases (MMPs) 2 and 9, tenascin-C, and osteopontin, revealed that MMP-9 and tenascin-C demonstrated reduced expression both in vitro and in vivo with TGA crosslinking compared to controls, whereas osteopontin and MMP-2 expression were not affected. TGA pretreatment of heterograft biomaterials results in improved stability compared to Glut, confers biomechanical properties superior to Glut crosslinking, and demonstrates significant calcification resistance.

Transcription factor Egr-1 in calcific aortic valve disease.

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.