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

ABSTRACT: Calciprotein particles (CPPs) represent an inherent mineral buffering system responsible for the scavenging of excessive Ca²⁺ and PO₄^3- ions in order to prevent extraskeletal calcification, although contributing to the development of endothelial dysfunction during the circulation in the bloodstream. Here, we performed label-free proteomic profiling to identify the functional consequences of CPP internalisation by endothelial cells (ECs) and found molecular signatures of significant disturbances in mitochondrial and lysosomal physiology, including oxidative stress, vacuolar acidification, accelerated proteolysis, Ca²⁺ cytosolic elevation, and mitochondrial outer membrane permeabilisation. Incubation of intact ECs with conditioned medium from CPP-treated ECs caused their pro-inflammatory activation manifested by vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) upregulation and elevated release of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1/ C-C motif ligand 2 (MCP-1/CCL2). Among the blood cells, monocytes were exclusively responsible for CPP internalisation. As compared to the co-incubation of donor blood with CPPs in the flow culture system, intravenous administration of CPPs to Wistar rats caused a considerably higher production of chemokines, indicating the major role of monocytes in CPP-triggered inflammation. Upregulation of sICAM-1 and IL-8 also suggested a notable contribution of endothelial dysfunction to systemic inflammatory response after CPP injections. Collectively, our results demonstrate the pathophysiological significance of CPPs and highlight the need for the development of anti-CPP therapies.

SUBMITTER: Shishkova D

PROVIDER: S-EPMC9738209 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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

Shishkova Daria D Lobov Arseniy A Zainullina Bozhana B Matveeva Vera V Markova Victoria V Sinitskaya Anna A Velikanova Elena E Sinitsky Maxim M Kanonykina Anastasia A Dyleva Yulia Y Kutikhin Anton A

International journal of molecular sciences 20221129 23

Calciprotein particles (CPPs) represent an inherent mineral buffering system responsible for the scavenging of excessive Ca2+ and PO43- ions in order to prevent extraskeletal calcification, although contributing to the development of endothelial dysfunction during the circulation in the bloodstream. Here, we performed label-free proteomic profiling to identify the functional consequences of CPP internalisation by endothelial cells (ECs) and found molecular signat ...[more]

PMID: 36499266

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Project description:Calciprotein particles (CPPs) represent self-assembling scavengers of excessive Ca2+ and PO43- ions which are formed in the human serum under the guidance of acidic proteins termed mineral chaperones (e.g., fetuin-A and albumin). While protecting the human organism from extraskeletal calcification, circulating CPPs are internalised by Kupffer cells and endothelial cells (ECs) and induce calcium stress upon their dissolution in lysosomes, thereby causing a pro-inflammatory response through the inflammasome-dependent and inflammasome-independent mechanisms. In patients with hyperphosphatemia (e.g., those with end-stage renal disease), a considerable proportion of primary amorphous CPPs transforms into secondary crystallised CPPs which instigate stronger cytokine release and indicate critical depletion of mineral buffering systems. A number of reports documented that serum of patients with chronic kidney disease, arterial hypertension, coronary artery disease, cerebrovascular disease, and autoimmune disorders 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 Ca2+ and PO43- ions, as artificially generated CPPs are similar to those isolated from calcified human tissues. Approximate concentration of CPPs in the human blood is 250 particles per µL (2.5 × 108 per L), as measured by fluorescent-labeled bisphosphonate OsteoSense 680EX (IVISense Osteo 680 fluorescent probe). The kinetics of CPP assembling and clearance is far from being fully understood, albeit it evidently depends on the amount of Ca2+ and PO43- ions and acidic serum proteins (especially fetuin-A and albumin), and several reports specified a role of Mg2+ ions, HCO3- ions, and pyrophosphate in these processes as intrinsic Ca2+ antagonists. Although previous studies delineated cytokines overproduced by ECs under CPP treatment (i.e., interleukin-6, interleukin-8, and monocyte chemoattractant protein 1) and itemised other pathological consequences of CPP internalisation (i.e., hampered NO biosynthesis, endothelial-to-mesenchymal transition, and impaired mechanotransduction), a little is known about the proteomic signatures of ECs to calcium stress. Here we for the first time performed an unbiased proteomic profiling of ECs treated with CPPs and revealed relative enrichment of the protein categories related to mitochondrial and lysosomal physiology after the CPP treatment.

Project description:Patients with chronic inflammatory diseases (CID) experience accelerated loss of bone mineral density, which is often accompanied by increased vascular calcification. These disturbances can be attenuated by therapies for inflammation, such as the tumor necrosis factor inhibitor infliximab. Calciprotein particles (CPP) are circulating colloidal aggregates of calcium and phosphate together with the mineral-binding protein fetuin-A, which have emerged as potential mediators of vascular calcification. The precise origins of serum CPP are unclear, but bone turnover may be an important source. In this longitudinal observational study, we studied patients with CID undergoing treatment with infliximab to assess the temporal relationship between bone turnover and circulating CPP. Ten patients with active CID receiving infliximab induction therapy and an additional 3 patients with quiescent CID on maintenance infliximab therapy were studied for 8 weeks with repeated measures of bone turnover markers as well as CPP (calciprotein monomers [CPM], primary CPP [CPP-I], and secondary CPP [CPP-II]). Therapeutic response was appraised using validated disease activity scores. At baseline, those with active CID had elevated markers of bone resorption and suppressed bone formation markers as well as higher CPM and CPP-I compared with those with quiescent CID. In responders, there was an early but transient reduction in resorption markers by week 1, but a more sustained increase in bone formation markers compared with non-responders at week 8. This was accompanied by reductions in CPM (β = -6.5 × 103 AU [95% CI -11.1, -1.8], p = 0.006) and CPP-I (β = -23.4 × 104 particles/mL [95% CI -34.8, -11.9], p < 0.001). In contrast, no significant changes in any markers were observed in non-responders or those receiving maintenance therapy. Thus, CPP have a dynamic association with changes in bone turnover in response to infliximab therapy, adding to accumulating evidence of the role of bone as a determinant of systemic levels. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Project description:Calciprotein particles (CPPs), assembled as a result of molecular interactions between fetuin-A and nascent calcium phosphate clusters, are indispensable scavengers of excessive Ca2+ and PO43– ions, thus representing an elegant mechanism which regulates mineral homeostasis. Generation of CPPs represents an evolutionary mechanism to prevent blood supersaturation with Ca2+ and PO43– ions (e.g., as a result of bone resorption) and to thwart extraskeletal calcification, a pathological condition which is frequent in patients with chronic kidney disease. It is believed that formation of CPPs accompanied evolution from the appearance of bony fish; the existence of CPPs in more ancient animals remains uncertain. Shortly after emergence, amorphous, spherical, and submicrometer-sized (30-100 nm) primary CPPs (CPP-P) evolve into crystalline, spindle- or needle-shaped, and micrometer-sized (100-300 nm) secondary CPPs (CPP-S). Upon executing their function of aggregating Ca2+ and PO43– ions, CPPs are removed from the circulation by endothelial cells (ECs), monocytes, and liver or spleen macrophages. Internalisation and digestion of CPPs by ECs induce a chain of detrimental events including an increase in cytosolic Ca2+, mitochondrial and endoplasmic reticulum stress, nuclear factor (NF)-κB-mediated transcriptional response and cytokine release, ultimately contributing to the development of pro-inflammatory endothelial activation, chronic low-grade inflammation, and inflammaging. Pathological permeability of dysfunctional endothelium promotes lipid retention and leukocyte extravasation, together contributing to the development of vascular inflammation, proteolytic environment, degradation of basement membrane and internal elastic lamina, and contractile-to-synthetic switch of vascular smooth muscle cells. Such an ensemble of molecular events ultimately induces intimal hyperplasia, vascular remodeling, and atherosclerotic progression. Hence, CPPs act as a double-edged sword which rescues the human body from an acute life-threatening disease (i.e., extraskeletal calcification) at the cost of promoting long-lasting conditions (i.e., endothelial dysfunction and atherosclerosis). Among the several forms of calcium transfer (free Ca2+ ions, colloidal calciprotein monomers, and particulate CPPs), CPPs are the most efficient in delivering calcium stress to ECs because of their deposition and dissolution in lysosomes, followed by a lysosomal membrane permeabilisation and an excessive Ca2+ migration into the cytosol. The molecular pattern of CPP-induced calcium stress within the ECs is well defined and includes elevated expression of cell adhesion molecules (VCAM1, ICAM1, and E-selectin) and endothelial-to-mesenchymal transition transcription factors (SNAI1, SNAI2, TWIST1, and ZEB1) in combination with an endothelial nitric oxide synthase (eNOS) uncoupling. However, our knowledge on extracellular signatures of such endothelial response remains limited to an augmented release of pro-inflammatory cytokines (IL-6, IL-8, MCP-1/CCL2, MIF, CXCL1, and MIP-3α/CCL20) and pro- or anti-thrombotic molecules (serpin E1/PAI-1 and uPAR). As EC-secreted bioactive factors (termed angiokines) control vascular tone, maintain hemostasis, and regulate instructive signaling governing local homeostasis within the most organs, decryption and interpretation of endothelial secretome in physiological and pathological conditions is of utmost importance. Discovery of a circulating biomarkers specific for endothelial dysfunction, which can be measured by a routine enzyme-linked immunosorbent assay, might inform on specific disease states and prompt to the respective pharmacological interventions. Here, we aimed at deciphering the secretome of ECs treated with either CPP-P or CPP-S, employing ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) for the analysis of serum-free cell culture supernatant. For the objective comparison of heterogeneous arterial ECs, we utilised human coronary artery endothelial cells (HCAEC) and human internal thoracic artery endothelial cells (HITAEC) which comprise an innermost lining in atheroprone and atheroresistant blood vessels (coronary artery and internal thoracic artery, respectively).

Project description:BackgroundExposure to traffic-derived particulate matter (PM), such as diesel exhaust particles (DEP), is a leading environmental cause of cardiovascular disease (CVD), and may contribute to endothelial dysfunction and development of atherosclerosis. It is still debated how DEP and other inhaled PM can contribute to CVD. However, organic chemicals (OC) adhered to the particle surface, are considered central to many of the biological effects. In the present study, we have explored the ability of OC from DEP to reach the endothelium and trigger pro-inflammatory reactions, a central step on the path to atherosclerosis.ResultsExposure-relevant concentrations of DEP (0.12 μg/cm2) applied on the epithelial side of an alveolar 3D tri-culture, rapidly induced pro-inflammatory and aryl hydrocarbon receptor (AhR)-regulated genes in the basolateral endothelial cells. These effects seem to be due to soluble lipophilic constituents rather than particle translocation. Extractable organic material of DEP (DEP-EOM) was next fractionated with increasing polarity, chemically characterized, and examined for direct effects on pro-inflammatory and AhR-regulated genes in human microvascular endothelial (HMEC-1) cells and primary human endothelial cells (PHEC) from four healthy donors. Exposure-relevant concentrations of lipophilic DEP-EOM (0.15 μg/cm2) induced low to moderate increases in IL-1α, IL-1β, COX2 and MMP-1 gene expression, and the MMP-1 secretion was increased. By contrast, the more polar EOM had negligible effects, even at higher concentrations. Use of pharmacological inhibitors indicated that AhR and protease-activated receptor-2 (PAR-2) were central in regulation of EOM-induced gene expression. Some effects also seemed to be attributed to redox-responses, at least at the highest exposure concentrations tested. Although the most lipophilic EOM, that contained the majority of PAHs and aliphatics, had the clearest low-concentration effects, there was no straight-forward link between chemical composition and biological effects.ConclusionLipophilic and semi-lipophilic chemicals seemed to detach from DEP, translocate through alveolar epithelial cells and trigger pro-inflammatory reactions in endothelial cells at exposure-relevant concentrations. These effects appeared to be triggered by AhR agonists, and involve PAR-2 signaling.

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

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

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