Objectives:
Endothelial cells and platelets are highly important during initiation of atherosclerosis. Our data indicates that adventitial progenitors are key drivers of vascular calcification. Crystalline calcium salts have been shown to induce the secretion of the pro-inflammatory cytokine interleukin 1β (IL1β) through the activation of the inflammasome nucleotide-binding domain leucine-rich repeat containing protein 3 (NLRP3) in macrophages. Since endothelial cells, platelets and adventitial progenitors also express NLRP3, secretion of IL1β can also be induced in these cell types. In platelets, IL1β is released associated with microvesicles after stimulation with LPS. We aim to identify microcalcifications as initiators of vascular inflammation through the release of IL1β and related leaderless inflammatory proteins such as IL-18, IL-33, HMGB1, or MIF and establish their detrimental role in heart, vessel and kidney disease.
Methodologies:
1. Defined calcium nanocrystals will be generated (cooperation with ESR12). Endothelial cells platelets and Gli1+ adventitial progenitors will be incubated with artificial calcium-protein nanocrystals. NLRP3 and caspase-1 activation, IL1β processing and release, as well as release of related non-conventionally secreted inflammatory proteins (LLSIPs) will be determined using mass spectrometry (ESR5) and mass spectrometry imaging (ESR8). The influence of factors that inhibit calcification e.g. fetuin A (ESR12) or calcification blocking factor (ESR5) on inflammasome activation with be investigated.
2. The pathophysiologic roles of microcalcification in animal models of vascular inflammation (ESR7), atherosclerosis (ESR1) and chronic kidney disease (ESR4) will be investigated in relation to calcification-induced inflammasome activation and inflammatory protein secretion using cell specific Cre drivers for endothelial cells, platelets and adventitial progenitors.
3. Establishing IL1β- and LLSIP-containing microvesicles as biomarkers in samples of patients with vascular inflammation (ESR4) and kidney disease (ESR15) using state-of-the-art mass spectrometry (ESR12) and imaging (ESR1) technologies.
