Inhibition of Matrix Metalloproteinase Activity Attenuates Tenascin-C Production and Calcification of Implanted Purified Elastin in Rats

Abstract

Elastin, a major extracellular matrix protein present in arterial walls provides elastic recoil and resilience to arteries. Elastin is prone to calcification in a number of cardiovascular diseases including atherosclerosis and bioprosthetic heart valve mineralization. We have recently shown that purified elastin when implanted subdermally in rats undergoes severe calcification. In the present study, we used this elastin implant model to investigate the molecular mechanisms underlying elastin calcification. Intense matrix metalloproteinase (MMP-2) and tenascin-C (TN-C) expression were seen in the proximity of the initial calcific deposits at 7 days. Gelatin zymography studies showed both MMP-2 (latent and active form) and MMP-9 expression within the implants. To investigate the role of MMPs in calcification, rats were adminis- Elastin is an extracellular matrix protein present in a variety of tissues including the arterial wall and heart valves. 1 Pathological calcification of elastin occurs in a number of disease processes including atherosclerosis, cardiac valve disease, and bioprosthetic heart valve calcification. [2][3][4] Despite the importance of elastin calcification in cardiovascular disease, the mechanisms underlying this process are not fully understood. We recently characterized a rat subdermal implant model to study calcification of purified elastin. 5 Explants from these animals showed deposition of poorly crystalline hydroxyapatite on implanted elastin fibers, comparable to pathological cardiovascular calcification. 5 This system is therefore useful for determining the cellular and molecular mechanisms leading to elastin-oriented calcification.Although the elastic fibers can be considered physiologically inert during adult life, a wide range of insults to elastic tissue can result in either chronic loss or excess accumulation. 6 Matrix metalloproteinases (MMPs) are involved in elastolysis. In particular, both MMP-2 and MMP-9 are known to bind to insoluble elastin, 7 and each has been shown to be actively involved in elastin degradation. 8,9 Exuberant production of MMPs is a hallmark of many destructive diseases, such as arthritis, chronic ulceration, and tumor formation. 10 -12 With respect to calcification, MMPs have also been detected in association with calcification of bioprostheses. 13,14 For example, subdermally implanted glutaraldehyde-treated bovine parietal pericardium contains an array of extracellular matrix protein-degrading proteinases including serine proteinases and MMPs. 13,14 High concentrations of MMPs are also present in atherosclerotic plaques 15 and in restenotic lesions. 16

In vivo cellular repopulation of tubular elastin scaffolds mediated by basic fibroblast growth factor

“…EL-Gel-FGF and EL-Gel samples were implanted subdermally (n = 6 per group per time point) in adult male Sprague Dawley CD rats weighing 250-300g, placed under general anesthesia, as described earlier [11]. Briefly, a 1 cm wide skin incision was made on the back of the rats, and two subcutaneous pouches were created in each rat by blunt dissection.…”

Elastin Calcification in the Rat Subdermal Model Is Accompanied by Up-Regulation of Degradative and Osteogenic Cellular Responses

“…Each rat received three 30-to 40-mg elastin implants (one per pouch), which were rehydrated in sterile saline 1 to 2 hours before implantation. Four rats were sacrificed by CO 2 asphyxiation at each time point (3,7,14, and 21 days after implantation), and the implants were retrieved along with the surrounding fibrous capsule. One explant from each rat was frozen in OCT (Sakura Finetek, Zoeterwoude, The Netherlands) on dry ice and stored at Ϫ80°C for immunohistochemistry; another explant was frozen on dry ice for protein analysis.…”

Elastin Degradation and Calcification in an Abdominal Aorta Injury Model

“…4 We have shown earlier that pure elastin undergoes extensive degeneration and calcification when implanted subdermally in juvenile rats 5 and that local delivery of a synthetic MMP inhibitor significantly reduced elastin calcification in the rat subdermal model. 6 Moreover, we have shown that treatment of elastin with aluminum ions before implantation completely abolished elastin calcification by mechanisms that include inhibition of elastolysis by MMPs. 7 These results strongly point to a direct correlation between elastin degradation and calcification.…”