Protein mineral particle-mediated cell and tissue calcification

• Protein-Mineralpartikel-vermittelte Zell- und Gewebekalzifizierung

Gorgels, Andrea; Jahnen-Dechent, Wilhelm (Thesis advisor); Blank, Lars M. (Thesis advisor)

Aachen : RWTH Aachen University (2021, 2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021

Abstract

Calciprotein particles (CPP) are blood-borne colloidal protein-mineral complexes comprising calcium, phosphate, fetuin-A, and major plasma proteins, which collectively stabilize mineral preventing crystal growth and precipitation. Higher numbers of CPP were found in patients suffering from chronic kidney disease (CKD) and the CPP number in these patients was directly associated with severity of vascular calcification. Protein-mineral particles including CPP are now considered a highly saturated mineral precursor for bone mineralization. On this note, CPP as a measurable entity may also be of interest regarding the mineralization paradox seen in CKD patients, namely decreased bone mineral mass and increased vascular mineral mass. One aim of this work was to analyze the involvement of CPP in pathological vascular calcification and physiological bone formation as well as the influence of specific blood alterations seen in CKD patients on CPP composition and action. CPP composition was altered by lowering the fetuin-A amount and by increasing lipids. Osteoblasts, smooth muscle cells and ex vivo aortic rings were cultured in the presence of CPP-based calcifying media and their calcification patterns were analyzed. A vessel-on-a-chip was developed to analyze the influence of endothelial barrier tightness on particle-induced calcification. It was shown that plasma fetuin-A deficiency was associated with increased calcification propensity as measured by accelerated ripening of CPP-1 into CPP-2. Lipids preferentially bound more crystalline particles corroborating the concept of lipidic calcified debris as a nidus of tissue calcification. Nevertheless, lipid binding did not directly influence calcification propensity. CPP calcified osteoblasts and smooth muscle cells comparable to calcium and phosphate. Detailed analysis of osteoblast and smooth muscle cell calcification revealed however, that active cell metabolism was necessary for calcification to occur in the presence of CPP. Both cell types transcytosed particles along the endolysosomal trajectory, ultimately enabling mineral deposition of the cellular surface or of surrounding extracellular matrix. CPP calcified smooth muscle cells but did not trigger calcification of aortic rings in tissue culture. The increased calcification propensity of CPP with reduced fetuin-A amount was reflected by increased calcification of smooth muscle cells, but not of aortic rings. Taken together, CPP are essential transport and clearance entities for excess mineral in the body, which mediate osteoblastic mineralization in physiology and vascular calcification in pathologic mineral metabolism. A tight endothelial barrier prevents vascular calcification induced by CPP. Endothelial integrity was thus identified a salient therapeutic target in CKD patients who are known to suffer from endothelial damage early on in the disease, and, in addition, cannot clear circulating CPP due to kidney failure and therefore have a strongly increased risk of vascular calcification.