Molecular Mechanisms of Calcification
The processes of aortic valve stenosis and calcification share many similarities with atherosclerosis, and the pathologies of both conditions have similar risk factors and histopathology. 2 Activation of VICs and pathways of calcific aortic stenosis is the result of mechanical and shear stress, endothelial damage and deposition of LDLs, triggering inflammatory events and attracting inflammatory cells (monocytes, macrophages and T cells).
These cells produce cytokines, including TGF- β , which regulates cell proliferation and differentiation; tumour necrosis factor- α , whose primary function is the regulation of the immune cells; and interleukin 2, which is produced by activated T lymphocytes with growth factor activity.1
VICs activated by the inflammatory process are designated myofibroblasts.5 These cells will develop angiogenic activity and produce matrix metalloproteinases, proteins that are involved in tissue remodelling and support VIC activation and transformation.96,97 During this process activated VICs differentiate into osteoblasts.
In Vitro Studies
Initial studies in our laboratory have involved the establishment and validation of porcine VIC isolation, culture and calcification procedures and the effect of denosumab on in vitro calcification. During the characterisation of porcine VICs, the first objective was to determine the expression level of a common marker of myofibroblast phenotype, α -actin, to demonstrate that active VICs were present in the samples. The expression of RUNX2, a major regulator of osteoblast differentiation, was analysed to corroborate that CardiologyCardiology the effect of the complete transdifferentiation of VICs had taken place and that the osteoblast phenotype was present. Furthermore, changes in the expression of TGF- β (a promoter of osteogenesis), were detected and recorded. Additionally, RhoA, a regulator of nodule formation in myofibroblasts, was analysed, followed by examining changes in the expression of RANKL, a key regulator of bone metabolism. Finally, calponin, a protein with potential capability to inhibit bone formation, was measured to complete the genetic studies. TGF- β can increase calcium and collagen deposition.98 It is known that TGF- β can also stimulate the expression of RANK on pre-osteoclastic cells, and in this way increase osteoclastic sensitivity to RANKL.99 RANKL is expressed in the membrane of osteoblasts and monocytes. As yet there is still no evidence that TGF- β promotes calcification in porcine VICs by increasing RANK expression levels.
Our recent unpublished studies demonstrated the upregulation of key molecules during the spontaneous calcification of porcine VICs with an increase of calcium, collagen and alkaline phosphatase (ALP) activity. In vitro calcification was determined using standard staining and enzyme activity assays. Calcification in pig VICs was induced with sodium phosphate. The cells expressed markers for both vascular smooth muscle cells and osteoblasts, suggesting a transdifferentiation of the phenotype. Upregulation of α -actin, RUNX2, TGF- β and RhoA and downregulation of calponin were noted, with no changes seen in RANKL expression. Sodium phosphate increased nodular formation by day 7 and ALP activity of porcine VICs by day 14. The findings suggest that porcine VICs may be a good model to study the process of CAVD.100
Denosumab as a Potential Inhibitor of VIC Calcification In Vitro
Denosumab is a human IgG 2 monoclonal antibody designed to target RANKL,101 which is expressed on the membrane of the osteoblasts and osteoclasts. Denosumab is used in the treatment of osteoporosis. Additionally, owing to its mechanism that blocks the receptor RANKL, it neutralises the activation of RANK receptors on the membrane of pre-osteoclast cells.More research is needed to address the interaction between RANK receptor and denosumab in porcine VICs.
Our recent unpublished studies showed that 50 μ g/mL denosumab inhibited induced calcium deposition to basal levels in porcine VIC culture.100 Although associated with bone loss and shown to reduce vascular calcification, the effect of denosumab on calcification of human VICs is unknown. Recently, denosumab has been shown to reduce calcium deposition in the aorta, although the mechanisms by which it affects ectopic calcification are poorly understood.102 Furthermore, osteoprotegerin (a signalling protein receptor and a member of the tumour necrosis factor receptor family) has been shown to stop ectopic calcification in vitro via a similar mechanism to denosumab, but there is still not enough evidence of any effect in reverting the process of calcification. Osteprotegerin’s mechanism of action is to block RANKL-RANK receptor interaction. 94,95 A fuller understanding of the mechanisms of action of denosumab may identify a novel therapeutic approach for clinical treatment, supplementing the current surgical approach. It should be noted that extrapolation of the results obtained in an in vitro porcine model to humans should be cautious, as species variations are likely to exist. Although it is not possible to include all mechanisms involved in CAVD in a single model, experimental models can contribute towards identifying the role several factors may play in the development of CAVD.