Using a novel human 3D spheroid model to study mechanisms and therapeutics for vascular calcification

Vascular calcification (VC) is a serious and widespread clinical problem manifesting in atherosclerosis, chronic kidney disease (CKD), diabetes and ageing. It is an independent risk factor for cardiovascular mortality in all disease contexts. Currently, there are no treatments to prevent or regress VC. Therefore, there is a serious unmet clinical need to understand the molecular mechanisms driving the calcification process, and to identify novel treatment strategies. VC is a cell-mediated process, driven by vascular smooth muscle cells (VSMCs). Signalling pathways activated in response to stressors cause VSMCs to undergo phenotypic change and conversion to osteogenic-like cells capable of orchestrating the calcification process. The development of VC is also highly dependent on the extracellular matrix (ECM) environment surrounding the VSMCs. The ECM undergoes changes in ageing and disease that significantly modify the niche occupied by the cells and these modifications promote osteogenic change and mineralisation. Current mechanistic and preclinical research into VC relies predominantly on 2D in vitro models of rodent VSMCs grown in the absence of ECM. Calcification can only be induced by addition of supraphysiological levels of minerals such as calcium and/or phosphate leading to rapid mineralisation that does not mimic the in vivo situation where mineralisation is a slow process occurring in the context of a modified ECM. Similarly, rodent models used in mechanistic studies and preclinical drug testing, rely on toxic mineral overload or diets often coupled with complex surgeries such as nephrectomy to induce calcification. This is because rodents are resistant to the induction of calcification and even when induced, the process does not follow the same course as in man, as it is rapid and cartilaginous rather than osteogenic and the ECM environment is healthy. Our aim is to replace animal use with human models of VC. To this end we have developed two novel human 3D in vitro models of VC; a spheroid model and a modified ECM model. Here, VSMC osteogenic differentiation and mineralisation occurs without the need for additional stimuli and in the context of the native ECM. We propose to characterise and refine these models to determine mechanisms of calcification and to optimise them for use in high throughput screening (HTS) to identify drugs that inhibit calcification processes. Importantly, these two models address different aspects of calcification and only require commercially available human VSMC cultures. Thus they will be widely accessible to the research community and easily established in any laboratory.