Article

ISSN 0006-2979, Biochemistry (Moscow), 2025, Vol. 90, No. 1, pp. 132-160 © Pleiades Publishing, Ltd., 2025.

Published in Russian in Biokhimiya, 2025, Vol. 90, No. 1, pp. 144-172.

132

Physiological Concentrations of Calciprotein Particles

Trigger Activation and Pro-Inflammatory Response

in Endothelial Cells and Monocytes

Daria Shishkova

1,a

, Victoria Markova

1,b

, Yulia Markova

1,c

, Maxim Sinitsky

1,d

Anna Sinitskaya

1,e

, Vera Matveeva

1,f

, Evgenia Torgunakova

1,g

Anastasia Lazebnaya

1,h

, Alexander Stepanov

1,i

, and Anton Kutikhin

1,k

Department of Experimental Medicine,

Research Institute for Complex Issues of Cardiovascular Diseases, 650002 Kemerovo, Russia

e-mail: shidk@kemcardio.ru

e-mail: markve@kemcardio.ru

e-mail: markyo@kemcardio.ru

e-mail: sinimu@kemcardio.ru

e-mail: cepoav@kemcardio.ru

e-mail: matvvg@kemcardio.ru

e-mail: torgea@kemcardio.ru

e-mail: lazeai@kemcardio.ru

e-mail: stepav@kemcardio.ru

e-mail: kytiag@kemcardio.ru

Received November 13, 2024

Revised December 3, 2024

Accepted December 5, 2024

Abstract— Supraphysiological concentrations of calciprotein particles (CPPs), which are indispensable scav-

engers of excessive Ca

and PO

3−

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 Ca

in the serum or medium) and investigated whether the pathological effects of calcium

stress depend on the calcium delivery form, such as Ca

ions, albumin- or fetuin-centric calciprotein mono-

mers (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 Ca

ions and CPM-A/CPM-F induced less

consistent detrimental effects. Intravenous administration of CaCl

, 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.

DOI: 10.1134/S0006297924604064

Keywords: calciprotein particles, calciprotein monomers, calcium ions, calcium stress, mineral stress, endothe-

lial cells, monocytes, endothelial dysfunction, endothelial activation, systemic inflammatory response

Abbreviations: CPMs, calciprotein monomers; CPPs, concentrations of calciprotein particles; ECs, endothelial cells.

* To whom correspondence should be addressed.

INTRODUCTION

Calciprotein particles (CPPs) and calciprotein

monomers (CPMs) are formed through the molecular

interactions between fetuin-A and nascent calcium

phosphate clusters. They scavenge of excessive Ca

and PO

3−

ions, thus representing an elegant mech-

anism for the mineral homeostasis regulation [1-6].

While albumin (by far the most abundant serum pro-

tein) is mostly responsible for the clearance of circu-

lating Ca

ions [5, 7], fetuin-A operates as a mineral

chaperone that either stabilizes calcium phosphate

CALCIPROTEIN PARTICLES 133

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

as a colloid by forming CPMs or secures its physio-

logical aggregation into corpuscular CPPs [5, 7]. CPPs

are then removed from the circulation by endothelial

cells (ECs) [8-15], monocytes [13], and liver or spleen

macrophages [16-19]. Generation of CPMs and CPPs is

an evolutionary mechanism aimed to prevent blood

supersaturation with Ca

and PO

3−

ions (e.g., as a

result of bone resorption) and to avert extraskele-

tal calcification, a pathological condition that is fre-

quent in patients with chronic kidney disease [20-22].

Yet, internalization of CPPs by ECs and monocytes/

macrophages and their digestion in lysosomes induce

a chain of detrimental events including an increase

in cytosolic Ca

, mitochondrial and endoplasmic re-

ticulum stress, nuclear factor (NF)-κB-mediated tran-

scriptional response, and release of pro-inflammatory

cytokines. such as interleukin (IL)-6, IL-8, and mono-

cyte chemoattractant protein  1/chemokine (C-C motif)

ligand  2 (MCP-1/CCL2), ultimately contributing to the

development of chronic low-grade inflammation [8-19,

23-26]. Treatment with infliximab, a selective inhibitor

of tumor necrosis factor (TNF)-α, reduced CPM and

CPP count in the serum of patients with autoimmune

diseases (inflammatory bowel disease, inflammatory

arthritis) [27], suggesting an efficacy of anti-inflam-

matory therapies in reducing CPP-related endothelial

and monocyte/macrophage activation.

Currently, experimental studies employ a variety

of CPP concentrations, from 25  µg/mL [13, 15] to 100

or 200  µg/mL calcium [16-18, 25, 28], depending on the

cell type and duration of exposure. Above-median lev-

els of ionized serum calcium (Ca

) have been shown

as a significant risk factor of cardiovascular death, as

well as myocardial infarction and ischemic stroke [11,

29,  30]. The last two life-threatening conditions are

driven by atherosclerosis, the development of which

is triggered by endothelial activation and impaired en-

dothelial integrity [31-35]. Average interquartile range

between the risk (upper) and protective (lower) quar-

tiles of ionized calcium is 0.12  mmol/L (i.e., 10% of the

average reference value, or 4.8  µg/mL) [11], suggesting

that in order to obtain clinically relevant results, the

amount of calcium introduced to the cell culture or

experimental animals should not exceed these values.

Hence, an adequate quantification of CPP and CPM

physiological doses should include their recalculation

according to the respective mass of ionized calcium

(e.g., added as CaCl

) in order to reach a 10% increase

in the ionized calcium content in the medium.

Albeit the adverse consequences of calcium stress

have been well described [36-38], it remains unclear

whether its deleterious effects are determined by

a calcium source (free Ca

ions, colloidal CPMs, or

corpuscular CPPs) or solely depend on the amount

of calcium in the microenvironment. Earlier studies

have reported that stimulation of calcium-sensing

receptor by increasing the concentration of extracel-

lular Ca

promoted internalization of CPPs, leading

to the activation of NLRP3 (NLR family pyrin domain

containing3) inflammasome and IL-1β signaling path-

way [39]. Pathological effects of CPPs largely depend

on their crystallinity (amorphous primary CPPs and

crystalline secondary CPPs) and density (high-den-

sity CPPs that are precipitated at ≤16,000g and low-

density CPPs that are not precipitated at this centrif-

ugal force) [40]. The serum levels of high-density CPPs

are independently and positively associated with the

content of the pro-inflammatory cytokine eotaxin,

whereas the levels of low-density CPPs are negatively

associated with another potent inflammation inducer,

IL-8 [40]. Likewise, a higher hydrodynamic radius of

CPPs, which correlates with a reduced kidney function

and age-dependent vascular remodeling, is associated

with the cardiovascular mortality in patients with pe-

ripheral artery disease [41], as well as with vascular

calcification [42] and all-cause mortality in patients

with the end-stage renal disease [43]. The content of

primary and secondary CPPs is associated with the

vascular remodeling pathways, including those in-

volved in collagen assembly and extracellular matrix

formation [44]. CPP-induced vascular remodeling in-

cludes osteochondrogenic reprogramming of vascular

smooth muscle cells, which strongly depends on the

particle-size distribution, mineral composition, and

crystallinity of CPPs [45]. Recent studies demonstrat-

ed an association between increased CPP counts or

accelerated primary-to-secondary CPP transition with

chronic kidney disease  [44], ST-segment elevation myo-

cardial infarction  [46], and cardiovascular death in pa-

tients with the end-stage renal disease  [47] or type  2

diabetes mellitus  [48]. Removal of CPPs from blood

using specific columns ameliorated chronic inflamma-

tion, endothelial dysfunction, left ventricular hypertro-

phy, and vascular calcification [49]. Similarly, inhibi-

tion of primary-to-secondary CPP transition prevented

high phosphate-induced rat aortic calcification [50].

As indicated above, quantification of CPPs pri-

marily relies on determining the concentration of

calcium (µg) per unit volume (mL) [12,  14,  16]. Arti-

ficially synthesized calcium-free magnesiprotein par-

ticles (MPPs) did not exhibit any significant toxicity

after their introduction to cultured ECs cultures or

animals  [11], suggesting that calcium concentration

is a leading factor determining the consequences of

mineral stress. However, the spatiotemporal patterns

of intracellular calcium distribution might differ de-

pending on the calcium vehicle – from a steady and

controlled entry of Ca

ions through the cell mem-

brane [51,  52] to a sharp and uncurbed influx of Ca

ions into the cytosol after partial digestion of CPPs in

the lysosomes [11]. These features of calcium metabo-

lism may significantly affect transcriptional programs,

SHISHKOVA et al.134

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

and better understanding of cellular response to cir-

culating Ca

ions, CPMs, and CPPs is required to elu-

cidate the pathophysiology of mineral homeostasis

disorders.

Here, we investigated whether the calcium deliv-

ery form dictates the response of ECs and monocytes

to physiologically relevant mineral stress that which

was achieved by adding 10  µg/mL calcium (an amount

sufficient to gain a 10% increase in ionized calcium) to

either cell culture medium or rat serum. We found that

incubation of primary human arterial ECs with albu-

min-centric CPPs (CPP-A) initiated their pro-inflamma-

tory activation manifested as an elevated production

of pro-inflammatory cytokines [IL-6, IL-8, MCP-1/CCL2,

macrophage inflammatory protein-3 alpha (MIP-3α),

plasminogen activator inhibitor-1 (PAI-1), and uroki-

nase-type plasminogen activator receptor (uPAR)] and

verified by an increased expression of genes encoding

cell adhesion molecules (VCAM1, ICAM1, E-selectin)

and pro-inflammatory cytokines [IL-6, CXCL8 (chemo-

kine (C-X-C motif) ligand 8), CCL2, and CXCL1]. Incu-

bation with fetuin-centric CPPs (CPP-F) also promoted

release of IL-6, IL-8, and MCP-1/CCL2 and upregulated

expression of genes coding for cell adhesion molecules

(VCAM1, ICAM1, SELE, and SELP) and pro-inflammato-

ry cytokines (IL6, CXCL1, and MIF). Likewise, incuba-

tion of monocytes with CPP-A in the flow culture sys-

tem promoted release of IL-6, IL-8, MIP-1α/1β, MIP-3α,

CXCL1, CXCL5, PAI-1, uPAR, lipocalin-2, and matrix

metalloproteinase-9 (MMP-9). However, addition of

free Ca

ions and CPM-A caused only mild alterations

in the transcriptional program and cytokine release

by primary arterial ECs and monocytes. Intravenous

administration of excessive Ca

ions (CaCl

), CPM-A,

or CPP-A to Wistar rats precipitated systemic inflam-

matory response including an elevation in the con-

tent of multiple cytokines, hepatokines, and proteases.

Wesuggest that the pathological effects of CPPs in  vitro

are determined by a local calcium overload, as CPPs

represent calcium vehicles with a single destination

(lysosomes). In contrast, the inflammatory response

to the intravenous calcium bolus is less dependent

on the form of calcium delivery. Nevertheless, even

physiological doses of CPPs induced pro-inflammatory

activation of ECs and monocytes, as well as systemic

inflammatory response in vivo.

MATERIALS AND METHODS

Synthesis and quantification of CPMs and CPPs.

To prepare a mixture for the synthesis of CPMs and

CPPs, 340  mg bovine serum albumin (BSA; Sigma-

Aldrich, USA) or 8  mg bovine serum fetuin-A (BSF;

Sigma- Aldrich) were dissolved in 4  mL of physiolog-

ical saline with the subsequent addition of 2  mL of

HPO

(24mmol/L; Sigma-Aldrich) and 2  mL of CaCl

(40 mmol/L; Sigma-Aldrich). The mixture was resus-

pended after addition of each reagent. The final con-

centrations of reagents in the mixture were 42  mg/mL

for BSA or 1  mg/mL for BSF (equal to the median se-

rum level in a human population [11]), 10mmol/L for

CaCl

(3.2  mg of calcium), and 6mmol/L for Na

HPO

The suspension was then aliquoted into 8 microtubes

(1  mL per tube) that were placed into pre-heated

(37°C) heating block (Thermit, DNA-Technology, Rus-

sia) and incubated for 10  min. After this procedure,

the mixture contained three calcium sources: free Ca

ions, CPMs (either CPM-A or CPM-F), and CPPs (either

CPMs-F or CPPs-F).

The resulting suspension was then aliquoted into

four ultracentrifuge tubes (2  mL per tube; Beckman

Coulter, USA) and centrifuged at 200,000g (OPTIMA

MAX-XP, Beckman Coulter) for 1  h to sediment CPP-A/

CPP-F which were then resuspended in sterile deion-

ized water and visualized by scanning electron mi-

croscopy (S-3400N, Hitachi, Japan) at an accelerating

voltage of 10 or 30  kV after 1  :  200 dilution. To com-

pare CPPs-A and CPPs-F with primary CPPs generated

from tissue extracts or biological fluids, we employed

atherosclerotic plaque-derived and serum-derived

CPPs that had been generated in T-25 flasks (Wuxi

NEST Biotechnology, China) for 6 weeks after adding

either 3  mL of plaque extract or 3  mL of human se-

rum, 1  mmol/L CaCl

, and 1  mmol/L Na

HPO

to 7  mL

of Dulbecco’s Modified Eagle’s Medium (DMEM; Pan-

Eco, Russia) containing 10% fetal bovine serum (FBS,

Capricorn Scientific, Germany), 1% L-glutamine–pen-

icillin–streptomycin solution (Thermo Fisher Scien-

tific, USA), and 0.4% amphotericin B (Thermo Fisher

Scientific). Plaque extracts were obtained as described

in  [8]. After incubation for 6  weeks, CPPs were sedi-

mented, prepared for scanning electron microscopy,

and visualized as described in [8]. The supernatant

with CPM-A/CPM-F and free Ca

ions was transferred

into centrifugal filters with a 30-kDa molecular weight

cutoff (Guangzhou Jet Bio-Filtration, China) and cen-

trifuged at 1800g for 25  min to separate CPM-A/CPM-F

(retentate) and free Ca

ions (filtrate).

The concentration of calcium in CPP-A/CPP-F,

CPM-A/CPM-F and of free Ca

ions was measured by

using o-cresolphthalein complexone and diethanol-

amine-based colorimetric assay (CalciScore, AppScience

Products, Russia) after 1  :  30, 1  :  10, and 1  :  10 dilution,

respectively. Albumin concentration was measured us-

ing BCA Protein Assay Kit (Thermo Fisher Scientific)

after 1  :  200 dilution of the CPM-containing retentate;

the filtrate containing free Ca

ions was not dilut-

ed before the measurement as it was expected to be

devoid of albumin. The results of colorimetric assays

were detected by spectrophotometry (Multiskan Sky,

Thermo Fisher Scientific) at 575  nm (calcium) and

CALCIPROTEIN PARTICLES 135

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

562  nm (albumin). All procedures were performed

under sterile conditions.

Dosage estimation. The amount calcium required

for a 10% increase in the ionized calcium content in

the milieu was estimated by adding 5, 10, 15, or 20  µg

of calcium (in a form of CaCl

) dissolved in aque-

ous BSA solution (300 mg/mL, average albumin con-

centration in the retentate) or aqueous BSF solution

(28  mg/mL, average fetuin-A concentration in the re-

tentate) per 1  mL of serum-free EndoLife cell culture

medium (EL1, AppScience Products) or by adding 10,

15, 20, or 40  µg of calcium dissolved in aqueous BSA

solution (300  µg/mL) per 1  mL rat serum. The mix-

ture was briefly resuspended and incubated for 1  h,

after which the concentration of ionized calcium Ca

was measured (Konelab 70i, Thermo Fisher Scientific).

EndoLife medium and rat serum without CaCl

addi-

tion were used as respective controls. According to our

previous study, a 10% increase in the ionized calci-

um content [0.10-0.14mmol/L (from 4.0 to 5.6  µg/mL);

average, 0.12mmol/L (4.8  µg/mL) for human serum] is

equal to the interquartile range between the highest

(risk) and the lowest (protective) quartiles.

Cell culture. Primary human coronary artery

endothelial cells (HCAECs, Cell Applications, USA)

and human internal thoracic artery endothelial cells

(HITAECs, Cell Applications) were grown in T-75 flasks

according to the manufacturer’s protocol in EndoBoost

Medium (EB1, AppScience Products) using 0.25% tryp-

sin-EDTA solution (PanEco), and 10% FBS for trypsin

inhibition during subculturing. Immediately before

the experiments, EndoBoost Medium we replaced

with serum-free EndoLife Medium, during which the

cells were washed s twice with warm (37°C) Ca

-and

-free Dulbecco’s Phosphate Buffered Saline (DPBS)

(pH7.4, BioLot) to remove the residual serum compo-

nents. HCAECs and HITAECs were grown in parallel,

were seeded into flow culture chambers (Ibidi, Germa-

ny) or 6-well plates (Wuxi NEST Biotechnology), and

grown until reaching confluence.

Monocytes have been isolated from 5 healthy

volunteers (the authors of this study) by consecutive

extraction of peripheral blood mononuclear cells using

a Ficoll density gradient centrifugation (Ficoll solution,

1077  g/cm

; PanEco) and positive magnetic separation

of CD14

cells with an EasySep Magnet kit (STEMCELL

Technologies, USA) and monocyte isolation kit (STEM-

CELL Technologies) according to the manufacturer’s

instructions under sterile conditions. Monocyte count

was performed with an automated Countess  II cell

counter (Thermo Fisher Scientific) and cell counting

chamber slides (Thermo Fisher Scientific).

Internalization assay. To analyze internalization

of CPMs and CPPs by ECs, CPM-A and CPP-A were

labeled with fluorescein 5-isothiocyanate-conjugated

BSA (FITC-BSA, Thermo Fisher Scientific) either during

CPM/CPP synthesis (by adding 750  µg of FITC-BSA at

a 5  µg/µL concentration) or after the synthesis by in-

cubation of sedimented CPP-A with 125  µg (25  µL) of

FITC-BSA for 1  h at 4°C and subsequent incubation

of 500  µL of retentate (CPM-A) with 250  µg (50  µL) of

FITC-BSA for 1  h at 4°C after vortexing. The synthesis

of CPM-A and CPP-A was performed in the dark less

than 24  h before the experiment. After the labeling,

sedimented CPP-A were resuspended in DPBS, centri-

fuged at 13,000g (Microfuge 20R, Beckman Coulter) for

10  min to wash CPP-A from unbound FITC-BSA, and

resuspended in 400  µL of DPBS.

Laminar flow was established using Ibidi Pump

System Quad system (Ibidi) equipped with four sep-

arate flow culture units and Perfusion Set Yellow/

Green (Ibidi). Before starting the experiment, HCAECs

and HITAECs were cultured until confluence in flow

culture chambers (350,000 cells per chamber) and

exposed to a laminar flow (15  dyn/cm

) using a se-

rum-free EndoLife cell culture medium during 24  h.

Next, FITC-labeled CPM-A and CPP-A were added into

the system (10  µg of calcium per 1  mL medium; 150  µg

of calcium per unit). In total, three consecutive runs

were performed: (i)  with CPM-A and CPP-A labeled

during their synthesis; (ii)  with CPM-A and CPP-A

labeled after the synthesis; and (iii)  with unlabeled

CPM-A and CPP-A. ECs were incubated with CPM-A

and CPP-A for 1  h; nuclei were counterstained with

Hoechst 33342 (2  µg/mL, Thermo Fisher Scientific) for

5  min. FITC-labeled CPM-A and CPP-A were visualized

after thorough washing by confocal microscopy (LSM

700, Carl Zeiss, Germany).

To investigate colocalization of lysosomes and

FITC-labeled CPMs and CPPs, CPM-A, CPM-F, CPP-A,

and CPP-F were labeled with FITC after their synthesis

as described above. FITC-labeled CPM-A, CPM-F, CPP-A,

and CPP-F (10  µg calcium per 1  mL medium, 4  µg cal-

cium per well) were added to confluent of HCAECs

and HITAECs seeded into 8-well chambers (80826,

Ibidi) for 3  h, and then replaced the medium with a

fresh one containing the pH sensor LysoTracker Red

(1  µmol/L; Thermo Fisher Scientific) for 1  h. Unbound

FITC-BSA (60  µg) was used as a control; nuclei were

counterstained with Hoechst 33342 for 10  min. FITC-

labeled CPM-A, CPM-F, CPP-A, and CPP-F were visual-

ized after thorough washing by confocal microscopy.

Treatment of ECs and monocytes with free Ca

ions, CPMs, and CPPs. To investigate the response of

ECs to equal calcium concentrations delivered by dif-

ferent distinct vehicles, we added DPBS (control), free

ions (CaCl

as a vehicle), CPMs (either CPM-A or

CPM-F), or CPPs (either CPP-A or CPP-F) (10  µg of cal-

cium per 1  mL cell culture medium; 20  µg calcium per

well of a 6-well plate; n  =  18 wells per group) to con-

fluent HCAEC and HITAEC cultures for 24  h. We also

added BSA (12  mg; i.e., average mass of albumin

SHISHKOVA et al.136

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

in added CPM-A) or BSF (0.33  mg; i.e., average mass

of fetuin-A in CPM-F) to all wells in the respective

experiments for negating potential protective effects

of these proteins. Serum-supplemented EndoBoost

medium was replaced with serum-free EndoLife me-

dium immediately before starting the experiment.

After incubation for 24  h, the cells were examined

by phase contrast microscopy; cell culture medium

was removed, and the cells were washed with ice-

cold (4°C) DPBS and lysed in TRIzol reagent (Thermo

Fisher Scientific) to extract RNA according to the man-

ufacturer’s protocols. Cell culture medium was centri-

fuged at 2000g (MiniSpin Plus, Eppendorf, Germany)

to remove cell debris, transferred into new tubes, and

frozen at −80°C.

To evaluate the cytotoxicity of different modali-

ties of calcium stress, we conducted colorimetric as-

say using water-soluble tetrazolium salt (WST)-8 and

annexin V/propidium iodide staining followed by flow

cytometry. For the WST-8 assay, HCAECs and HITAECs

were grown in 96-well plates (Wuxi NEST Biotechnol-

ogy) to confluency serum-supplemented EndoBoost

medium; next, the culture medium was replaced with

serum-free EndoLife medium, and added DPBS (con-

trol), free Ca

ions (as CaCl

), CPMs (either CPM-A or

CPM-F), or CPPs (either CPP-A or CPP-F) were added

to the wells (10  µg calcium per 1  mL cell culture me-

dium; 2  µg calcium per well of 96-well plate; n = 12

wells per group) for 24  h. Next, the medium was re-

placed with 100  µL of fresh serum-free EndoLife me-

dium and 10  µL of WST-8 reagent (Wuhan Servicebio

Technology, China) was added for 2  h. The products

of reaction were detected spectrophotometrically

at 450 nm.

For annexin V/propidium iodide staining, HCAECs

and HITAECs were seeded into 6-well plates (Wuxi

NEST Biotechnology) and grown to confluency in se-

rum-supplemented EndoBoost medium. Next, the me-

dium was replaced with serum-free EndoLife medium,

and DPBS (control), free Ca

ions (using CaCl

as a

vehicle), CPMs (either CPM-A or CPM-F), or CPPs (ei-

ther CPP-A or CPP-F) were added to the wells (10  µg

calcium per 1  mL cell culture medium, 20  µg calcium

per well of 6-well plate) for 24  h. The cells were then

detached using Accutase (Capricorn Scientific) and an-

alyzed by the annexin V/propidium iodide assay using

a respective kit (ab14085, Abcam, United Kingdom) ac-

cording to the manufacturer’s protocol. Flow cytome-

try was conducted with a CytoFlex instrument using

the CytExpert software (Beckman Coulter).

To study the monocyte response, we incubat-

ed monocytes (350,000 cells per unit) in serum-free

EndoLife medium with equal concentrations of free

ions (CaCl

), CPM-A, or CPP-A (10  µg calcium per

1  mL culture medium; 150  µg calcium per unit; n = 5

donors/runs per group) in a flow culture system using

the above-mentioned perfusion set for 24  h. Similar

to the previous experiment, DPBS was used as a con-

trol and BSA (87  mg, an average mass of albumin in

added CPM-A) was added to all units for negating its

potential protective effects. Four experimental groups

(DPBS, Ca

, CPM-A, and CPP-A) were distributed across

four units of the flow culture system. The experiment

was performed under sterile conditions. After 24  h of

incubation, cell culture medium was collected, centri-

fuged at 220g (5804R, Eppendorf) to sediment mono-

cytes and then at 2000g to remove cell debris, and

then frozen at −80°C.

Gene expression analysis. Gene expression in

, CPM-A/CPM-F, or CPP-A/CPP-F-treated HCAECs

and HITAECs was analyzed by reverse transcrip-

tion-polymerase chain reaction (RT-qPCR). Briefly,

cDNA was synthesized with M-MuLV–RH First Strand

cDNA Synthesis Kit (R01-250, Evrogen, Russia) and

reverse transcriptase M-MuLV–RH (R03-50, Evrogen),

and RT-qPCR was carried out with customized prim-

ers (500nmol/L each, Evrogen, TableS1 in the Online

Resource  1), (20  ng), and BioMaster HS-qPCR Lo-ROX

SYBR Master Mix (MHR031-2040, Biolabmix, Russia)

according to the manufacturer’s protocol. The levels

of mRNAs (VCAM1, ICAM1, SELE, SELP, IL6, CXCL8,

CCL2, CXCL1, MIF, NOS3, SNAI1, SNAI2, TWIST1, and

ZEB1 genes) were quantified by calculating ΔCt using

the 2

−ΔΔCt

method and normalized to the average ex-

pression level of three housekeeping genes (GAPDH,

ACTB, and B2M) and to the DPBS-treated group (2

−ΔΔCt

Administration of free Ca

ions, CPMs, and

CPPs to Wistar rats. The animal study protocol was

approved by the Local Ethical Committee of the Re-

search Institute for Complex Issues of Cardiovascular

Diseases (protocol code, 042/2023; date of approval,

April 4, 2023). Animal experiments were performed in

accordance with the European Convention for the Pro-

tection of Vertebrate Animals (Strasbourg, 1986) and

Directive 2010/63/EU of the European Parliament on

the protection of animals used for scientific purpos-

es. Male Wistar rats (body weight, ~300  g; estimated

blood volume, ~20  mL, i.e., 6.5% of body weight) were

used in the experiments. To investigate the response

to the intravenous administration of various calcium

sources, DPBS (control), free Ca

ions (CaCl

), CPM-A,

or CPP-A (10  µg calcium per 1  mL rat blood; 200  µg

calcium per rat; n  =  5 rats per group, n  =  20 rats in

total) were injected into the rat tail vein. BSA was

added to all injections (average mass of albumin add-

ed to CPM-A, 120  mg,) for adjustment of the possible

immune response to BSA. After 1  h, all rats were eu-

thanized by intraperitoneal injection of sodium pento-

barbital (100  mg/kg body weight). Serum was obtained

by centrifuging rat blood at 1700g for 15  min.

Dot blotting and enzyme-linked immunosor-

bent assay (ELISA). Protein levels in the cell culture

CALCIPROTEIN PARTICLES 137

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

medium were measured by dot blotting and ELISA.

Dot blotting was conducted using Proteome Profiler

Human XL Cytokine Array Kit (ARY022B, R&D Sys-

tems, USA) and Proteome Profiler Rat XL Cytokine

Array (ARY030, R&D Systems) according to the manu-

facturer’s instructions; proteins were visualized using

chemiluminescence detection with an Odyssey XF im-

aging system (LI-COR Biosciences, USA). Densitometric

quantification was performed using the ImageJ soft-

ware (National Institutes of Health, USA). To increase

dot blotting sensitivity, cell culture medium was con-

centrated using HyperVAC-LITE vacuum centrifugal

concentrators (Gyrozen, Republic of Korea) before

the measurements. Rat serum was assessed without

preliminary concentrating. All culture medium sam-

ples were concentrated to the same extent: 7-fold from

medium from monocytes (from 14  mL to 2  mL) and

3-fold for medium from endothelial cells (from 3  mL

to 1  mL). Next, 1  mL of the concentrated medium or

non-concentrated rat serum were loaded for dot blot-

ting. The content of IL-8, IL-6, and MCP-1/CCL2 was

determined by ELISA using the corresponding kits

(A-8768, A-8762, and A-8782, Vector-Best, Russia) ac-

cording to the manufacturer’s protocols. Colorimetric

detection of ELISA results was conducted spectro-

photometrically at 450 nm. For ELISA measurement,

100  µL of non-concentrated cell culture medium was

used for all samples.

Statistical analysis was performed with Graph-

Pad Prism 8 (GraphPad Software, USA). For RT-qPCR,

the data are presented as mean ± standard deviation

(SD). Four independent groups were compared by the

ordinary one-way analysis of variance (ANOVA) and

subsequent Dunnett’s multiple comparison test with

a single pooled variance. The results of ELISA mea-

surements are presented as median, 25th and 75th

percentiles, and range. Four independent groups were

compared by the Kruskal–Wallis test with subsequent

Dunn’s multiple comparison test. The differences were

considered as statistically significant at p ≤ 0.05.

RESULTS

Physiological relevance of CPM and CPP syn-

thesis under conditions of mineral stress. To inves-

tigate the effects of different calcium delivery forms

on ECs and monocytes, we created a rection mixture

containing physiological concentration of BSA, phys-

iological saline (NaCl), and supraphysiological levels

of Na

HPO

, and CaCl

for simultaneous generation

of albumin-centric CPMs (CPM-A) and CPPs (CPP-A).

Previously, similar mineral stress conditions have

been used to produce fetuin-centric CPMs (CPM-F)

and CPPs (CPP-F) [18]. Next, we used ultracentrifuga-

tion to isolate CPPs followed by ultrafiltration to sep-

arate CPMs (yellow retentate) from free ions and salts

(transparent filtrate). Therefore, calcium was repre-

sented by (i)free Ca

ions, (ii) CPMs (colloidal form),

and (iii) CPPs (corpuscular form). We used albumin

to assemble CPMs (CPM-A) and CPPs (CPP-A) because

a below-median content of serum albumin has been

demonstrated as an independent risk factor for the

coronary artery disease and ischemic stroke (in con-

junction with above-median serum levels of Ca

) [11].

Low serum albumin levels were found to correlate

with a higher serum calcification propensity (i.e., CPP

precipitation), while the content of albumin showed

a positive correlation with total calcium (fetuin and

phosphate did not display such associations) [11]. How-

ever, because fetuin-A plays a pivotal role as a mineral

chaperone and governs formation of CPMs and CPPs in

human blood, we also used CPM-F and CPP-F in most

of the experiments. CPM-F and CPP-F were synthesized

using the protocol described except that bovine serum

fetuin (BSF) was used instead of BSA.

Scanning electron microscopy of CPP-A showed

their sponge-like structure and irregular shape, which

differed from spherical and needle-shaped appearance

of primary and secondary blood-derived CPPs, respec-

tively (Fig.1). CPP-F had a spherical shape and sponge-

like structure, thus closely resembling atherosclerotic

plaque- and serum-derived primary CPPs [11]. These

observations were in agreement with our previous

data on the comparison of albumin-centric, fetuin-cen-

tric, plaque-derived, and serum-derived CPPs [8] and

can be explained by the different cooperation of acidic

serum proteins during CPP generation in the blood.

CPPs and CPMs absorbed ~30 and ~20% of calci-

um, respectively, whereas ~50% of calcium remained

in the solution as free Ca

ions. This distribution was

in agreement with the physiological ratio between ion-

ized calcium (Ca

) and protein- and phosphate-bound

calcium in human serum (1  :  1). CPPs contained from

11 to 17% of total albumin, whereas 83 to 89% of al-

bumin remained in the retentate, thus retaining the

-binding ability. The filtrate contained no BSA or

BSF, which confirmed the efficiency of the ultrafiltra-

tion procedure. Taken together, these data confirmed

the physiological relevance of the procedure devel-

oped for artificial synthesis of CPMs and CPPs under

conditions of mineral stress.

Physiological concentrations of CPPs cause

pro-inflammatory activation of ECs and monocytes.

To determine the amount of calcium that has to be

added to ensure physiological elevation in the ion-

ized calcium content, we calculated the dose-response

curve. Thus, an addition of 10  µg of calcium per 1  mL

of serum-free cell culture medium (Fig.  2a) or rat se-

rum (Fig.  2b) was sufficient to achieve a 10% increase

in the concentration of ionized calcium (i.e., the in-

terquartile range between the risk and protective

SHISHKOVA et al.138

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

Fig.  1. Scanning electron microscopy images of albumin-centric (CPP-A), fetuin-centric (CPP-F), calcified atherosclerotic

plaque-derived (CPP-PD), and serum-derived (CPP-SD) CPPs. Secondary electron mode; acceleration voltage, 10  kV (CPP-A)

or 30 kV (CPP-F, CPP-PD, and CPP-SD); magnification, ×30,000; scale bar: 1 µm.

Fig. 2. Increase in the ionized calcium (Ca

) concentration in (a) cell culture medium and (b) rat serum upon addition

of increasing amounts of CaCl

; x-axis, concentration of added calcium; y-axis, increase in Ca

concentration relative to

the control medium or serum without calcium addition. An increase in the Ca

concentration by 10% (blue dashed line)

was achieved by the addition of 10 µg of calcium per 1 mL of cell culture medium or rat serum (red circle).

CALCIPROTEIN PARTICLES 139

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

Fig. 3. Internalization of FITC-BSA-labeled CPMs (FITC-CPMs) and CPPs (FITC-CPPs) by HCAECs and HITAECs. a) Comparison

of signal intensities of internalized FITC-CPMs and FITC-CPPs obtained by two different labeling techniques. ECs were treat-

ed for 1  h with unlabeled CPMs and CPPs (left panel), CPMs and CPPs that incorporated FITC-BSA during their synthesis

(central panel), and CPMs and CPPs that were incubated with FITC-BSA after their synthesis (right paned). Nuclei were

counterstained with Hoechst 33342. Confocal microscopy; magnification, ×630; scale bar, 5  µm. b) Lysosomes stained with

LysoTracker Red (LTR) in ECs treated for 4 h with CPMs (FITC-CPM-A and FITC-CPM-F) or CPPs (FITC-CPP-A and FITC-CPP-F):

left panel, free FITC-BSA; central panel: CPM-A and CPM-F co-incubated with FITC-BSA during their synthesis; right panel,

CPP-A and CPP-F co-incubated with FITC-BSA after their synthesis. Yellow arrows indicate CPP-A and CPP-F inside the cells.

Nuclei were counterstained with Hoechst 33342. Confocal microscopy; magnification, ×200; scale bar, 50  µm.

SHISHKOVA et al.140

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

Fig.  4. Bright-field microscopy (CPM-A/CPP-A, top) and phase-contrast microscopy (CPM-F/CPP-F, bottom) of HCAECs (left

panels) and HITAECs (right panels) treated with DPBS (control), free Ca

ions, CPMs (CPM-A, top; CPM-F, bottom), or CPPs

(CPP-A, top; CPP-F, bottom) (10 µg of calcium per 1 mL serum-free cell culture medium) for 24 h; magnification, ×200; scale

bar, 100 µm.

quartiles in the population). Hence, we selected 10  µg/mL

as the optimal calcium concentration to model clini-

cally relevant mineral stress. Further experiments in-

cluded four groups: 1)  control (DPBS); 2)  free Ca

ions

delivered as CaCl

; 3)  either CPM-A or CPM-F; 4)  either

CPP-A or CPP-F.

We then asked whether CPMs are internalized in

a flow system in a similar manner as CPPs. To address

this question, we labeled CPM-A and CPP-A with FITC-

BSA either during CPM-A/CPP-A generation (by adding

FITC-BSA to the solution) or after their formation by

incubation of sedimented CPP-A and separated CPM-A

with FITC-BSA. An intense green fluorescence was ev-

ident in ECs already 1  h after addition of FITC-labeled

CPM-A and CPP-A to the flow culture system (Fig. 3a).

CPM-A and CPP-A incubated with FITC-BSA after their