Validations in vitro will be performed as a way to further prioritize and narrow down selected compounds, advancing only a limited number of the most promising compounds to in vivo inference studies. This reduces animal experiments to the absolute minimum in line with the “Three R” strategy to reduce, refine and replace the use of animals in experiments.
Depending on the targets identified, appropriate simple cellular models related to cardiovascular disease progression, either vascular endothelial, smooth-muscle or inflammatory cells will be used. The in vivo animal models with the highest similarity to human disease will be further used for the selected target validation.
The final aim is to obtain data on the effects of around 5 promising compounds in long-term studies. These compounds will be investigated for plaque formation, inflammation, and cardiac function.
The potential toxicity of the candidate compounds will be investigated by well-established assays, analysing membrane integrity and total protein content, among others. Interference compounds leading to toxic effects will not be suitable for in vivo use. All analyses will include dose-response studies to estimate the most suitable dose(s) for the subsequent in vivo studies.
Efficiency of the interference assays in altering the expression or activity of the target as well as of the most relevant and easily measurable down-stream molecules will be studied.
A short term in vivo optimization phase will be implemented to allow for the detection of severe side-effects and the elimination of a specific compound or to a compound dosage change. Time points for administration of the compound will vary for each model, to allow for the investigation of treatment efficiency at various stages of the disease progression.