Objectives:
Extracellular vesicles (EVs) are considered major players in promoting macrophage (MO) and VSMC-mediated calcification in vitro and in vivo. EVs can be produced by living cells (micro- and matrix vesicles, exosomes) and dying cells (apoptotic bodies) and are present in calcified arteries of CKD patients. VSMC can generate EVs in vitro that differ in composition and calcifying properties depending on environmental factors such as Ca2+ and phosphate, and phenotype. VSMC normally produce EVs that contain inhibitors of calcification such as carboxylated-MGP and Fetuin-A. Elevated Ca2+ and phosphate, inflammatory cytokines and reactive oxygen species can cause phenotypic switching and a shift of production towards EVs lacking inhibitors and exposing pro-calcifying phosphatidylserine (PS) and annexin A5. Little is known about other lipid moieties of EVs and their role in VC, e.g. phosphorylcholine (PC) and malone dialdehyde (MDA) although both are implicated in atherosclerosis. ESR13 will design novel EV-targeting compounds for imaging and treatment of VC.
Methodologies:
1. Molecular Imaging – Design and production of near infrared imaging probes targeting PS exposing EVs (annexin A5 based). This part will be carried out in collaboration with ESR11 and ESR12. Probes will be tested in in vitro EV-production and calcification assay by cultured VSMC and in in vivo vascular calcification (apoE-/- on western type diet and i) warfarin, ii) high phosphate, and iii) unilateral nephrectomy). This part will be carried out in collaboration with ESR3, ESR9 and ESR11.
2. Targeted therapy – Growth of calcium precipitates will be inhibited by targeting the calcification inhibitor inhibiting Fetuin-A to EVs (in collaboration with ESR12). i) Chemical coupling of Fetuin-A to Gla-peptides (ESR9) and ii) fusing them to annexin A5. Effects of Annexin A5 itself and of antibodies against PC and MDA will be tested.
3. Targeted therapy – Removal of calcium precipitates by EV targeted reprogramming of VSMC and macrophages. i) Chemical coupling of RANKL to Gla-peptides and ii) fusion of RANKL to annexin A5.
4. Targeted compounds will be tested in in vitro cell culture assays (VSMC and macrophages) and in in vivo vascular calcification (apoE-/- on western type diet and i) warfarin, ii) high phosphate, and iii) unilateral nephrectomy).
