Characterising novel mediators of vascular calcification.
Lead Research Organisation:
University of Edinburgh
Department Name: The Roslin Institute
Abstract
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.
Technical Summary
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.
People |
ORCID iD |
Vicky MacRae (Principal Investigator) |
Publications
Staines KA
(2012)
MEPE is a novel regulator of growth plate cartilage mineralization.
in Bone
Staines KA
(2012)
The importance of the SIBLING family of proteins on skeletal mineralisation and bone remodelling.
in The Journal of endocrinology
Patel JJ
(2018)
Inhibition of arterial medial calcification and bone mineralization by extracellular nucleotides: The same functional effect mediated by different cellular mechanisms.
in Journal of cellular physiology
Orriss IR
(2014)
Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats.
in International journal of molecular medicine
Macrae V
(2017)
Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification
in Journal of Visualized Experiments
Mackenzie NC
(2014)
miRNA-221 and miRNA-222 synergistically function to promote vascular calcification.
in Cell biochemistry and function
Mackenzie NC
(2011)
MOVAS-1 cell line: a new in vitro model of vascular calcification.
in International journal of molecular medicine
Mackenzie NC
(2012)
Altered bone development and an increase in FGF-23 expression in Enpp1(-/-) mice.
in PloS one
Mackenzie NC
(2012)
New insights into NPP1 function: lessons from clinical and animal studies.
in Bone
Mackenzie NC
(2011)
The role of cellular senescence during vascular calcification: a key paradigm in aging research.
in Current aging science
Description | During this fellowship I have characterised and employed the ectonucleotide pyrophosphatase /phosphodiesterase-1 (Enpp1) null mouse model of aortic calcification to examine novel pathways involved in vascular calcification. I have made key contributions to the vascular field, including the identification of specific microRNAs, FGF-23, BMP-9, Phospho1, IGF2, and sclerostin as novel mediators of vascular calcification. Additionally, I have developed innovative protocols to image vascular calcification ex vivo using micro-computed tomography. |
Exploitation Route | These findings have led to a better comprehension of the aetiology of vascular calcification, and may offer novel therapeutic strategies in the future. |
Sectors | Healthcare |
Description | Academic impact has been maximised primarily through peer-reviewed publications in high profile and open-access journals. To ensure that this dissemination was a dialogue with the scientific community, the academic impact has been augmented by presentations at national and international meetings. I have also prepared, displayed and manned exhibits on vascular calcification at the Roslin Institutes open doors days. |
First Year Of Impact | 2010 |
Title | Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification. |
Description | We have developed a new method of isolating primary rat valve interstitial cells |
Type Of Material | Model of mechanisms or symptoms - in vitro |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | online video available for researchers to view. |
Title | SkyScan bone micro CT data from Carmen Huesa and Neil Mackenzie |
Description | SkyScan bone micro CT data from Carmen Huesa and Neil Mackenzie |
Type Of Material | Database/Collection of data |
Year Produced | 2016 |
Provided To Others? | Yes |
Description | EuroSoftCalc European COST action |
Organisation | European Cooperation in Science and Technology (COST) |
Department | COST Action |
Country | Belgium |
Sector | Public |
PI Contribution | UK representative to the Management Committee and Working Group Leader (scientific exchanges and training) |
Collaborator Contribution | Clinical, research and training expertise in rare diseases of calcification. |
Impact | N/A |
Start Year | 2017 |
Description | Roslin open day |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | A range of activities, including soft play, jigsaws, models and colouring in to understand more about calcification. These activities all sparked questions and discussion with children and parents. |
Year(s) Of Engagement Activity | 2012,2013,2014,2015,2016,2017,2019 |
Description | School visit (Peebles, Scottish Borders) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | As part of Digital Learning Week at Kingsland Primary School, Peebles I spoke to 3 classes of 20-25 Primary 2 pupils about how I use digital technology in my scientific research . There were lots of questions from the children about both technology and working in science. |
Year(s) Of Engagement Activity | 2017 |
Description | online school visit "Meet a Roslin scientist" |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Schools |
Results and Impact | 15 pupils attended an online event where researchers from our lab discussed their job as a scientist |
Year(s) Of Engagement Activity | 2020,2021 |
URL | http://www.ed.ac.uk/easter-bush-campus/science-outreach-centre/news |
Description | public workshop for Anatomy Nights - matters of the heart |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | 20 adults and children attended for a talk, dissection demonstration, and viewing of experimental slides. This sparked questions and discussion throughout the event. |
Year(s) Of Engagement Activity | 2020 |