Physiological concentrations of calciprotein particles trigger activation and pro-inflammatory response in endothelial cells and monocytes

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Abstract

Supraphysiological concentrations of calciprotein particles (CPPs), which are indispensable scavengers of excessive Ca2+ and PO43– ions in blood, induce pro-inflammatory activation of endothelial cells (ECs) and monocytes. Here, we determined physiological levels of CPPs (10 μg/mL calcium, corresponding to 10% increase in Ca2+ in the serum or medium) and investigated whether the pathological effects of calcium stress depend on the calcium delivery form, such as Ca2+ ions, albumin- or fetuin-centric calciprotein monomers (CPM-A/CPM-F), and albumin- or fetuin-centric CPPs (CPP-A/CPP-F). The treatment with CPP-A or CPP-F upregulated transcription of pro-inflammatory genes (VCAM1, ICAM1, SELE, IL6, CXCL8, CCL2, CXCL1, MIF) and promoted release of pro-inflammatory cytokines (IL-6, IL-8, MCP-1/CCL2, and MIP-3α/CCL20) and pro- and anti-thrombotic molecules (PAI-1 and uPAR) in human arterial ECs and monocytes, although these results depended on the type of cell and calcium-containing particles. Free Ca2+ ions and CPM-A/CPM-F induced less consistent detrimental effects. Intravenous administration of CaCl2, CPM-A, or CPP-A to Wistar rats increased production of chemokines (CX3CL1, MCP-1/CCL2, CXCL7, CCL11, CCL17), hepatokines (hepassocin, fetuin-A, FGF-21, GDF-15), proteases (MMP-2, MMP-3) and protease inhibitors (PAI-1) into the circulation. We concluded that molecular consequences of calcium overload are largely determined by the form of its delivery and CPPs are efficient inducers of mineral stress at physiological levels.

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D. K. Shishkova

Research Institute for Complex Issues of Cardiovascular Diseases

Email: kytiag@kemcardio.ru

Department of Experimental Medicine

Russian Federation, 650002 Kemerovo

V. E. Markova

Research Institute for Complex Issues of Cardiovascular Diseases

Email: kytiag@kemcardio.ru

Department of Experimental Medicine

Russian Federation, 650002 Kemerovo

Y. O. Markova

Research Institute for Complex Issues of Cardiovascular Diseases

Email: kytiag@kemcardio.ru

Department of Experimental Medicine

Russian Federation, 650002 Kemerovo

M. Yu. Sinitsky

Research Institute for Complex Issues of Cardiovascular Diseases

Email: kytiag@kemcardio.ru

Department of Experimental Medicine

Russian Federation, 650002 Kemerovo

A. V. Sinitskaya

Research Institute for Complex Issues of Cardiovascular Diseases

Email: kytiag@kemcardio.ru

Department of Experimental Medicine

Russian Federation, 650002 Kemerovo

V. G. Matveeva

Research Institute for Complex Issues of Cardiovascular Diseases

Email: kytiag@kemcardio.ru

Department of Experimental Medicine

Russian Federation, 650002 Kemerovo

E. A. Torgunakova

Research Institute for Complex Issues of Cardiovascular Diseases

Email: kytiag@kemcardio.ru

Department of Experimental Medicine

Russian Federation, 650002 Kemerovo

A. I. Lazebnaya

Research Institute for Complex Issues of Cardiovascular Diseases

Email: kytiag@kemcardio.ru

Department of Experimental Medicine

Russian Federation, 650002 Kemerovo

A. D. Stepanov

Research Institute for Complex Issues of Cardiovascular Diseases

Email: kytiag@kemcardio.ru

Department of Experimental Medicine

Russian Federation, 650002 Kemerovo

A. G. Kutikhin

Research Institute for Complex Issues of Cardiovascular Diseases

Author for correspondence.
Email: kytiag@kemcardio.ru

Department of Experimental Medicine

Russian Federation, 650002 Kemerovo

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3. Fig. 1. Micrographs of albumin CPCs (CPC-A), fetuin CPCs (CPC-F), CPCs from atherosclerotic plaques (CPC-B) and serum (CPC-S), obtained by scanning electron microscopy. Secondary electron mode; accelerating voltage – 10 kV (CPC-A) or 30 kV (other types of CPCs); magnification ×30,000; scale bar – 1 μm

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4. Fig. 2. Increase in the concentration of ionized calcium (Ca2+) in the culture medium (a) and rat blood serum (b) upon addition of increasing amounts of CaCl2. Abscissa axis – concentration of added calcium; ordinate axis – increase in molar concentration of Ca2+ relative to the control medium or serum without added calcium. A 10% increase in the Ca2+ concentration (blue dotted line) was achieved by adding 10 μg calcium per 1 ml of medium or serum (red circle).

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5. Fig. 3. Internalization of FITC-labeled CPM (FITC-CPM) and CPC (FITC-CPC) by HCAEC and HITAEC.

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6. Fig. 4. Bright-field microscopy (BF-A/BF-A, top) and phase-contrast microscopy (BF-F/BF-F, bottom) of HCAEC (left) and HITAEC (right) incubated with PBS (control), free Ca2+ ions, BF (BF-A, top; BF-F, bottom) or BF (BF-A, top; BF-F, bottom) (10 μg calcium per 1 ml serum-free culture medium) for 24 h; magnification ×200; scale bar – 100 μm

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7. Fig. 5. Cytotoxicity test after incubation of HCAEC (left panels) and HITAEC (right panels) with PBS (control), free Ca2+ ions (Ca2+-A or Ca2+-P), CPM (CPM-A or CPM-P) or CPC (CPC-A or CPC-P) (10 μg calcium per 1 ml of serum-free culture medium) for 24 h.

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8. Fig. 6. Assessment of IL-6 (upper panel), IL-8 (middle panel) and MCP-1/CCL2 (lower panel) levels in non-concentrated serum-free culture medium from HCAEC and HITAEC incubated with PBS (control; black), free Ca2+ (blue), CPM-A (green) and CPC-A (red) (10 μg calcium per 1 ml medium) for 24 h (ELISA).

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9. Fig. 7. Assessment of IL-6 (upper panel), IL-8 (middle panel) and MCP-1/CCL2 (lower panel) levels in non-concentrated serum-free culture medium from HCAEC and HITAEC incubated with PBS (control; black), free Ca2+ (blue), KPM-F (green) and KPC-F (red) (10 μg calcium per 1 ml medium) for 24 h (ELISA).

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10. Fig. 8. Cytokine profiling in concentrated (3-fold) serum-free culture medium from HCAEC (upper panel) and HITAEC (lower panel) incubated with PBS (control), free Ca2+ ions, CPM-A or CPC-A (10 μg calcium per 1 ml of culture medium) for 24 h (dot blot).

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11. Fig. 9. Cytokine profiling in concentrated (7-fold) serum-free culture medium from human monocytes incubated with PBS (control), free Ca2+ ions, CPM-A and CPC-A (10 μg calcium per 1 ml of culture medium) for 24 h (dot blotting).

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12. Fig. 10. Profiling of cytokines in rat serum after intravenous administration of FSBD (control), free Ca2+ ions, CPM-A and CPC-A (10 μg calcium per 1 ml of blood) for 1 hour.

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