Danger signal extracellular calcium initiates differentiation of monocytes into SPP1/osteopontin-producing macrophages

The danger signal extracellular calcium is pathophysiologically increased in the synovial fluid of patients with rheumatoid arthritis (RA). Calcium activates the NLRP3-inflammasome via the calcium-sensing receptor in monocytes/macrophages primed by lipopolysaccharide, and this effect is mediated by the uptake of calciprotein particles (CPPs) formed out of calcium, phosphate, and fetuin-A. Aim of the study was to unravel the influence of calcium on monocytes when the priming signal is not present. Monocytes were isolated from the blood of healthy controls and RA patients. Macrophages were characterized using scRNA-seq, DNA microarray, and proteomics. Imaging flow cytometry was utilized to study intracellular events. Here we show that extracellular calcium and CPPs lead to the differentiation of monocytes into calcium-macrophages when the priming signal is absent. Additional growth factors are not needed, and differentiation is triggered by calcium-dependent CPP-uptake, lysosomal alkalization due to CPP overload, and TFEB- and STAT3-dependent increased transcription of the lysosomal gene network. Calcium-macrophages have a needle-like shape, are characterized by excessive, constitutive SPP1/osteopontin production and a strong pro-inflammatory cytokine response. Calcium-macrophages differentiated out of RA monocytes show a stronger manifestation of this phenotype, suggesting the differentiation process might lead to the pro-inflammatory macrophage response seen in the RA synovial membrane.

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

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.

Klotho and calciprotein particles as therapeutic targets against accelerated ageing

The klotho gene, named after a Greek goddess who spins the thread of life, was identified as a putative ‘ageing-suppressor’ gene. Klotho-deficient mice exhibit complex ageing-like phenotypes including hypogonadism, arteriosclerosis (vascular calcification), cardiac hypertrophy, osteopenia, sarcopenia, frailty, and premature death. Klotho protein functions as the obligate co-receptor for fibroblast growth factor-23 (FGF23), a bone-derived hormone that promotes urinary phosphate excretion in response to phosphate intake. Thus, Klotho-deficient mice suffer not only from accelerated ageing but also from phosphate retention due to impaired phosphate excretion. Importantly, restoration of the phosphate balance by placing Klotho-deficient mice on low phosphate diet rescued them from premature ageing, leading us to the notion that phosphate accelerates ageing. Because the extracellular fluid is super-saturated in terms of phosphate and calcium ions, an increase in the phosphate concentration can trigger precipitation of calcium-phosphate. In the blood, calcium-phosphate precipitated upon increase in the blood phosphate concentration is adsorbed by serum protein fetuin-A to form colloidal nanoparticles called calciprotein particles (CPPs). In the urine, CPPs appear in the renal tubular fluid when FGF23 increases phosphate load excreted per nephron. CPPs can induce cell damage, ectopic calcification, and inflammatory responses. CPPs in the blood can induce arteriosclerosis and non-infectious chronic inflammation, whereas CPPs in the urine can induce renal tubular damage and interstitial inflammation/fibrosis. Thus, we propose that CPPs behave like a pathogen that accelerates ageing and should be regarded as a novel therapeutic target against age-related disorders including chronic kidney disease.

Pathophysiological and clinical significance of mineral homeostasis disorders in the development of cardiovascular disease

A growing incidence, prevalence, morbidity and mortality from cardiovascular disease dictate an urgent need in identification of its risk factors and their pathogenetic links with coronary artery disease and stroke. Aging of the population is inevitably associated with an increasing prevalence of comorbid conditions. Among them are disorders of mineral homeostasis which, often being neglected, are clearly associated with major adverse cardiovascular events and cardiovascular death. Maintenance of mineral homeostasis in the human body is largely dependent on the formation of calciprotein particles (CPPs) which arise in the blood upon the binding of a mineral chaperone fetuin-A to nascent calcium phosphate crystals, thereby aggregating excessive calcium (Ca2+) and phosphate (PO4 3-), removing them from the bloodstream and preventing extraskeletal calcification. During the circulation, CPPs are internalised by arterial endothelial cells and provoke endothelial dysfunction through endothelial activation, endothelialto-mesenchymal transition and impairment of endothelial mechanotransduction. Animal studies demonstrated that regular intravenous injections of CPPs lead to intimal hyperplasia and adventitial/perivascular inflammation in the absence of any other cardiovascular risk factors, indicating pathophysiological importance of CPPs for cardiovascular disease. Further, a number of clinical studies suggested an association of an augmented serum calcification propensity or elevated CPP count with arterial hypertension, myocardial infarction, chronic brain ischemia, ischemic stroke and cardiovascular death in patients with chronic kidney disease (including those with end-stage renal disease as well as kidney transplant recipients) and individuals with a preserved renal function. Here, we critically discuss the pathophysiological consequences of CPP formation, mechanisms of their pathogenic effects, and potential therapeutic interventions.

Effects of fetuin-A-containing calciprotein particles on posttranslational modifications of fetuin-A in HepG2 cells

Fetuin-A is an inhibitor of ectopic calcification that is expressed mainly in hepatocytes and is secreted into the circulation after posttranslational processing, including glycosylation and phosphorylation. The molecular weight (MW) of fully modified fetuin-A (FM-fetuin-A) is approximately 60 kDa in an immunoblot, which is much higher than the estimated MW by amino acid sequence. Under conditions of calcification stress such as advanced stage chronic kidney disease, fetuin-A prevents calcification by forming colloidal complexes, which are referred to as calciprotein particles (CPP). Since the significance of CPP in this process is unclear, we investigated the effect of synthetic secondary CPP on the level of FM-fetuin-A in HepG2 cells. Secondary CPP increased the level of FM-fetuin-A in dose- and time-dependent manners, but did not affect expression of mRNA for fetuin-A. Treatment with O- and/or N-glycosidase caused a shift of the 60 kDa band of FM-fetuin-A to a lower MW. Preincubation with brefeldin A, an inhibitor of transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus, completely blocked the secondary CPP-induced increase in FM-fetuin-A. Treatment with BAPTA-AM, an intracellular calcium chelating agent, also inhibited the CPP-induced increase in the FM-fetuin-A level. Secondary CPP accelerate posttranslational processing of fetuin-A in HepG2 cells.

Calciprotein Particles

Hypercalcemia and hyperphosphatemia associate with an elevated risk of cardiovascular events, yet the pathophysiological basis of this association is unclear. Disturbed mineral homeostasis and the associated hypercalcemia and hyperphosphatemia may result in the formation of circulating calciprotein particles (CPPs) that aggregate the excessive calcium and phosphate ions. If not counteracted, the initially formed harmless amorphous spherical complexes (primary CPPs) may mature into damaging crystalline complexes (secondary CPPs). Secondary CPPs are internalized by vascular cells, causing a massive influx of calcium ions into the cytosol, leading to a proinflammatory response, cellular dysfunction, and cell death. Although the pathophysiological effects induced by CPPs in vascular cells receive increasing attention, a complete picture of how these particles contribute to the development of atherosclerosis and vascular calcification remains elusive. We here discuss existing knowledge on CPP formation and function in atherosclerosis and vascular calcification, techniques for investigating CPPs, and models currently applied to assess CPP-induced cardiovascular pathogenesis. Lastly, we evaluate the potential diagnostic value of serum CPP measurements and the therapeutic potential of anti-CPP therapies currently under development.