Glycosaminoglycans affect endothelial to mesenchymal transformation, proliferation, and calcification in a 3D model of aortic valve disease

Abstract

Calcific nodules form in the fibrosa layer of the aortic valve in calcific aortic valve disease (CAVD). Glycosaminoglycans (GAGs), which are normally found in the valve spongiosa, are located local to calcific nodules. Previous work suggests that GAGs induce endothelial to mesenchymal transformation (EndMT), a phenomenon described by endothelial cells’ loss of the endothelial markers, gaining of migratory properties, and expression of mesenchymal markers such as alpha smooth muscle actin (α-SMA). EndMT is known to play roles in valvulogenesis and may provide a source of activated fibroblast with a potential role in CAVD progression. In this study, a 3D collagen hydrogel co-culture model of the aortic valve fibrosa was created to study the role of EndMT-derived activated valvular interstitial cell behavior in CAVD progression. Porcine aortic valve interstitial cells (PAVIC) and porcine aortic valve endothelial cells (PAVEC) were cultured within collagen I hydrogels containing the GAGs chondroitin sulfate (CS) or hyaluronic acid (HA). The model was used to study alkaline phosphatase (ALP) enzyme activity, cellular proliferation and matrix invasion, protein expression, and calcific nodule formation of the resident cell populations. CS and HA were found to alter ALP activity and increase cell proliferation. CS increased the formation of calcified nodules without the addition of osteogenic culture medium. This model has applications in the improvement of bioprosthetic valves by making replacements more micro-compositionally dynamic, as well as providing a platform for testing new pharmaceutical treatments of CAVD.

Free-aldehyde neutralized and oligohyaluronan loaded bovine pericardium with improved anti-calcification and endothelialization for bioprosthetic heart valves

“…Neutralizing the free aldehyde groups in Glut-fixed BHVs can enhance tissue durability, reduce calcification potential, and even stimulate partial endothelialization ( Gulbins et al, 2006 ). In addition, HA is a key component in valve tissue and is essential for maintaining the valve’s non-destructive movements and cell proliferation and differentiation ( Collins and Birkinshaw, 2013 ; Bramsen et al, 2022 ). However, the level of GAGs in bovine pericardium is relatively low ( Song et al, 2021 ).…”

Focusing on the Native Matrix Proteins in Calcific Aortic Valve Stenosis

“… 75 , 76 Glycosaminoglycans facilitate EndMT in ex vivo models of calcific aortic valve disease, via TGF-β1 and ERK1/2 pathway (a member of MAPK family). 6 , 74 Moreover, the mechanosensitive integrin-linked kinase, was downregulated in CAVS, and its silencing induced EndMT and RunX2-mediated osteoblastic differentiation in vitro, 75 while a similar role is proposed for the midkine pathway, which can be activated by several mechanosensitive and ECM-related receptors. 76 …”

“… 6 In particular, VECs exposed to a stiff matrix demonstrated increased EndMT under TGB-β1 stimulation, compared with a soft matrix. 74 Cyclic strain imposed ex vivo in a different direction to the cells alignment resulted in severe EndMT, via TGF-β or Wnt pathways, depending on the external force imposed. 75 , 76 Glycosaminoglycans facilitate EndMT in ex vivo models of calcific aortic valve disease, via TGF-β1 and ERK1/2 pathway (a member of MAPK family).…”