Calciprotein Particles Link Disturbed Mineral Homeostasis with Cardiovascular Disease by Causing Endothelial Dysfunction and Vascular Inflammation

Abstract

An association between high serum calcium/phosphate and cardiovascular events or death is well-established. However, a mechanistic explanation of this correlation is lacking. Here, we examined the role of calciprotein particles (CPPs), nanoscale bodies forming in the human blood upon its supersaturation with calcium and phosphate, in cardiovascular disease. The serum of patients with coronary artery disease or cerebrovascular disease displayed an increased propensity to form CPPs in combination with elevated ionised calcium as well as reduced albumin levels, altogether indicative of reduced Ca2+-binding capacity. Intravenous administration of CPPs to normolipidemic and normotensive Wistar rats provoked intimal hyperplasia and adventitial/perivascular inflammation in both balloon-injured and intact aortas in the absence of other cardiovascular risk factors. Upon the addition to primary human arterial endothelial cells, CPPs induced lysosome-dependent cell death, promoted the release of pro-inflammatory cytokines, stimulated leukocyte adhesion, and triggered endothelial-to-mesenchymal transition. We concluded that CPPs, which are formed in the blood as a result of altered mineral homeostasis, cause endothelial dysfunction and vascular inflammation, thereby contributing to the development of cardiovascular disease.

Calciprotein Particles Cause Physiologically Significant Pro-Inflammatory Response in Endothelial Cells and Systemic Circulation

“…Calciprotein particles (CPPs) represent self-assembling scavengers of excessive Ca 2+ and PO 4 3− ions, which are formed in the human serum under the guidance of acidic proteins termed mineral chaperones (e.g., fetuin-A and albumin) [ 1 , 2 , 3 , 4 , 5 ]. While protecting the human organism from extraskeletal calcification, circulating CPPs are internalised by Kupffer cells [ 6 , 7 , 8 ] and endothelial cells (ECs) [ 7 , 8 , 9 , 10 , 11 ] and induce calcium stress upon their dissolution in lysosomes, thereby causing a pro-inflammatory response through the inflammasome-dependent and inflammasome-independent mechanisms [ 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 ]. In patients with hyperphosphatemia (e.g., those with end-stage renal disease), a considerable proportion of primary amorphous CPPs transform into secondary crystallised CPPs, which instigate stronger cytokine release and indicate critical depletion of mineral buffering systems [ 15 , 16 ].…”

“…In patients with hyperphosphatemia (e.g., those with end-stage renal disease), a considerable proportion of primary amorphous CPPs transform into secondary crystallised CPPs, which instigate stronger cytokine release and indicate critical depletion of mineral buffering systems [ 15 , 16 ]. A number of reports documented that the serum of patients with chronic kidney disease [ 17 , 18 ], arterial hypertension [ 18 , 19 ], coronary artery disease [ 11 ], cerebrovascular disease [ 11 ], and autoimmune disorders [ 20 , 21 ] shows increased CPP generation that reflects uncurbed mineral stress in these clinical scenarios and emphasizes the need in the development of anti-CPP therapies. For the experiments, CPPs are routinely synthesised in vitro by the supersaturation of serum-supplemented cell culture medium with Ca 2+ and PO 4 3− ions [ 8 , 9 ], as artificially generated CPPs are similar to those isolated from calcified human tissues [ 9 ].…”

“…Although previous studies delineated cytokines overproduced by ECs under CPP treatment (i.e., interleukin-6, interleukin-8, and monocyte chemoattractant protein 1) [ 9 , 10 , 11 , 12 ] and itemised other pathological consequences of CPP internalisation (i.e., hampered NO biosynthesis, endothelial-to-mesenchymal transition, and impaired mechanotransduction) [ 10 , 11 ], a little is known about the proteomic signatures of ECs to calcium stress and whether CPP-mediated cytokine elevation is itself able to elicit pathological effects in the intact ECs. Further, the impact of blood cells, in particular leukocyte populations, into the systemic inflammation after encountering circulating CPPs remains obscure.…”

Excessive Adventitial and Perivascular Vascularisation Correlates with Vascular Inflammation and Intimal Hyperplasia

“…With the aim to investigate whether the endothelial dysfunction enhances VV and MPO + clusters expansion in the context of pre-existing vascular injury, in 10 out of 50 rats balloon angioplasty was followed by daily tail vein injections of either calciprotein particles (CPPs, 900 µL of particles per injection, equal to 600 µg calcium, n = 5) or equal volume of physiological saline for 5 days ( n = 5). CPPs were produced artificially as in our previous work [ 42 , 43 , 44 , 70 ]. Five weeks post-operation, all rats were euthanised by an intraperitoneal injection of a sodium pentobarbital (100 mg/kg body weight).…”

Endothelial Dysfunction in the Context of Blood–Brain Barrier Modeling

“…The endothelial-to-mesenchymal transition itself should also be defined as a continuum of events but not as an accomplished fact of the mesenchymal phenotype acquisition. Since the endothelial-to-mesenchymal transition is associated with both pro-inflammatory endothelial activation (leukocyte adhesion to endothelial cells and release of pro-inflammatory cytokines [ 83 , 142 , 143 ]) and is induced by chronic inflammation [ 144 , 145 ], turbulent flow [ 135 ] and impaired endothelial mechanotransduction [ 146 , 147 ] endothelial-to-mesenchymal transition can also be considered as a mandatory feature of endothelial dysfunction.…”