Characterising novel mediators of vascular calcification.

According to the World Health Organisation (WHO), an estimated 17 million people die each year of cardiovascular diseases, particularly heart attacks and strokes. A significant risk factor in the development of cardiovascular disease isVascular calcification. The process of vascular calcification shares many similarities with that of skeletal mineralisation, and involves the deposition of calcium phosphate mineral in arteries, heart valves, and cardiac muscle. Vascular calcification has severe clinical consequences, however, the mediators and mechanisms of vascular calcification have yet to be fully elucidated. A family of self-regulating proteins which alter the circulating levels of the hormone fibroblastic growth factor-23 (FGF23) have been recently shown to exert direct effects on skeletal mineralisation, along with FGF23 itself. These proteins include DMP1 (dentin matrix protein 1), PHEX (phosphate regulating neutral endopeptidase on chromosome X) and MEPE (matrix extracellular phosphoglycoprotein). This fellowship proposal will examine whether these newly discovered mediators of skeletal mineralisation also form a regulatory network in vascular calcification. Initially, the gene and protein expression patterns of FGF23, PHEX, DMP1 and MEPE will be determined during the calcification of vascular smooth muscle cells (VSMCs) (vascular calcification model) and compared to osteoblasts (skeletal mineralisation model). These observations will be confirmed in mice models of vascular calcification. Gene knockdown and over expression studies will investigate the function of FGF23, PHEX, DMP1 and MEPE in VSMCs and osteobasts. To discover the underpinning mechanisms, the regulation of key signaling pathways by FGF23, PHEX, DMP1 and MEPE during vascular calcification will be studied. To assess potential therapeuric strategies against vascular calcification, the study of novel inhibitors or inducers of FGF23, PHEX and DMP1 and MEPE will also be undertaken. Finally, a large scale analysis of all the genes and microRNAs (which can regulate the amount of protein expressed by the genes) altered during vascular calcification will be undertaken. This will identify new mediators of vascular calcification and potential therapeutic targets.

The process of vascular calcification (VC) shares many similarities with that of skeletal mineralisation, and involves the deposition of hydroxyapaptite crystals in arteries and cardiac muscle. VC has several clinical consequences, however, the mediators and mechanisms have yet to be fully elucidated. FGF23, DMP1, PHEX and MEPE have been recently shown to exert direct effects on skeletal mineralisation. This project will examine whether they also mediate vascular calcification. Expression patterns of FGF23, PHEX, DMP1 and MEPE will be Initially determined during the calcification of vascular smooth muscle cells (VSMCs) (VC model) and osteoblasts (skeletal mineralisation model) using quantitative PCR (qPCR) and immunoblotting. These observations will be confirmed by immunohistochemistry and in-situ hybridisation studies in enpp1-/- and ank/ank mice, in which VC is observed. To investigate FGF23, PHEX, DMP1 and MEPE function, microRNA and overexpression constructs will be cloned into the pSLIK lentiviral vector, permitting manipulation of gene expression at pre-determined time points during the culture period. This will allow the elucidation of the roles of these genes in both the initiation and propagation of calcification. Analysis of cell samples will include qPCR and immunoblotting. The calcification ability of matrix vesicles derived from the cell cultures will also be determined. To elucidate the underpinning mechanisms, the regulation of the Gas6/Axl, PI3kinase/Akt, ERK1/2, p38 MAPK and c-Jun N-terminal kinase signal transduction pathways by FGF23, PHEX, DMP1 and MEPE during VC will be studied in transfected cells. To assess potential therapeuric strategies against VC, inhibitors of DMP1 (DRB) and PHEX (MEPE-ASARM peptides) and an inducer of FGF23 (Dec-RVKR-CMK) will be studied in cells derived from enpp1-/- and ank/ank mice. Microarray and microRNA array systems analysis will identify novel mediators of VC and new potential therapeutic targets.