miRNAs regulation of Runx2-mediated osteoblast differentiation of valve interstitial cells in valve heart disease

Lead Research Organisation: University of Bristol
Department Name: Bristol Medical School

Abstract

Valve heart disease, mainly represented by aortic valve stenosis (AVS) (43%), is the 3rd commonest cardiovascular disease. It is prevalent in the elderly (4.5% of population >60 years old) and associated with 50% increased 5-year cardiovascular risk. In AVS, valve interstitial cells (VICs) undergo a heterotopic bone-like formation process. Runx2 is a master transcriptional factor instrumental to osteogenesis when located into the nuclei. Runx2 nuclear location depends on the binding of importins and co-activators/repressors to the nuclear location signal (NLS) and nuclear matrix targeting signal (NMTS), respectively. Therapeutic targeting of nuclear transport is emerging as a new strategy to treat multiple diseases. Bimax 1/2, M9M , cSN50.1 are NLS-transport regulators mainly screened for research purposes.

MicroRNAs (miRNAs) are endogenous small single-stranded non-coding RNAs regulating gene expression by complementary hybridation to multiple target mRNAs, often within the same pathway. Specific miRNA profiles have been identified in AVS phenotypes. Loss of miRNAs targeting Runx2 is associated with aberrant Runx2 expression and ectopic calcification (e.g. miR-133, miR-204, miR-26a). Control VICs can express Runx2,
while nuclear Runx2 is evidenced in calcifying counterparts. Studying miRNA-dependent regulation of importins and Runx2 co-activators/co-repressors might translate to mechanistic understanding of AVS that can be developed for tailored therapy.

The aim of this project is to target miRNAs regulating Runx2 activation to block VIC-to-osteoblast differentiation.

Objectives

O1. To identify miRNAs regulating the nuclear location/activation of Runx2 during the calcific AVS progression in human valves from AVS subsets (different calcification scores) and non-calcific controls.
O2. To decipher potential therapeutic targets in the signalling pathway and mechanistic understanding using hVICs as in vitro model of calcific AVS.
O3. To determine the possibility of blocking/reverting the progression of aberrant Runx2-dependent hVIC-to osteoblast differentiation and calcification in an in vitro model of human VIC (hVIC) calcification and ex vivo
model of aortic leaflet calcification by using specific miRNAs and NLS-transport regulators.

Methods

1. Customised microarrays of validated miRNAs targeting Runx2, importins (e.g. IPO8/9, KPNA4 or KPNB1/2) and Runx2 co-activators/repressors (e.g. p300, TAZ, WWP1 or HDACs, YAP1, Smad3/5) will be studied in aortic leaflets of AVS subsets (low/moderate/high calcification) and non-calcified controls (healthy/aortic insufficiency) (e.g. miR-133, miR-204, miR-193b, miR-455-3p/5p, miR-708, miR-2861, miR-26a, miR-181b, miR-223). Bioinformatic tools will identify miRNAs targeting NLS/NTMS-associated
proteins and predicted binding sites. GEO datasets (GSE87885, and GPL13764) will be screened to define VHD restricted miRNAs. KEGG pathways will be interrogated to identify miRNA/target pathways. Target validation will be done by qPCR, WB and immunohistochemistry.
2. Calcification in vitro models (inorganic phosphate) will be applied to primary hVICs to confirm miRNAs/target associations. AgomiR/antagomiR assays will confirm miRNA/mRNA regulation and pro/anti-osteogenic effects. Secreted NanoLuc RUNX2, COL1A1 and SP7/Osx promoter reporter vectors will address the real time effect of miRNAs on calcifying hVICs. Luciferase constructs containing miRNA binding sites will validate miRNA/mRNA associations. Runx2/miRNA-target interaction will be studied by proximity ligation assay.
3. Explanted swine valves will be used for ex vivo calcification studies. Ago/antagomiR assays will be conducted to inhibit the progression of calcification and that will be validated in human aortic leaflets.

NLS-regulators will be tested as potential AVS therapeutics

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/T517872/1 01/10/2020 30/09/2025
2444592 Studentship EP/T517872/1 01/10/2020 30/09/2024 Androulla Christodoulou