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

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BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

Fig.  5. Cytotoxicity assay after incubation of HCAECs (left panel) and HITAEC (right panel) with DPBS (control), free Ca

ions, CPMs (CPM-A or CPM-F), or CPPs (CPP-A or CPP-F) (10  µg of calcium per 1  mL serum-free cell culture medium)

for 24  h: a) WST-8 colorimetric assay (evaluation of WST-8 reduction by intracellular dehydrogenases to a water-soluble

orange- yellow formazan compound with the maximum absorption at 450  nm). b) Annexin V and propidium iodide assay

(lower left quadrant Q2-LL, normal cells; lower right quadrant Q2-LR, early apoptotic cells; upper right quadrant Q2-UR,

late apoptotic cells; upper left quadrant Q2-UL, necrotic cells). Top panel, statistical analysis of the content of intact and

late apoptotic cells. Bottom panel: representative flow cytometry plots.

SHISHKOVA et al.142

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

synthesis produced a significantly higher fluorescence

signal comparison to those that incorporated FITC-

BSA during their synthesis (Fig. 3a). Similar fluores-

cence intensity for CPM-A and CPP-A suggested that

FITC-BSA had the same affinity for both these spe-

cies (Fig. 3a). Identification of FITC-labeled CPMs/CPPs

in lysosomes stained with the pH sensor LysoTrack-

er Red confirmed internalization of CPM-A, CPM-F,

CPP-A, and CPP-F by HCAECs and HITAECs after 4  h

of incubation, while FITC-BSA itself did not enter ECs

(Fig. 3b).

To compare the pathological effects of different

calcium delivery forms on ECs, we added Ca

, CPM-A/

CPM-F, or CPP-A/CPP-F (10  µg/mL) to HCAECs and

HITAECs. Using bright-field and phase contrast mi-

croscopies, we observed pathological alterations (i.e.,

loss of intercellular contacts, cell shrinkage and de-

tachment) in ECs after incubation with CPP-A or CPP-F,

but not with Ca

or CPM-A/CPM-F (Fig. 4).

To further investigate the cytotoxicity of CPP-A

and CPP-F, we assessed a decrease in the cell viability

and metabolism in different modes of calcium stress

using WST-8 colorimetric assay. After 24-hour incuba-

tion with CPP-A or CPP-F, the intensity of cell metab-

olism dropped in both HCAECs and HITAECs (Fig. 5a).

Flow cytometry analysis of cell death using annexin V

and propidium iodide staining identified that a signifi-

cant proportion of ECs underwent apoptosis after 24  h

of treatment with CPP-A or CPP-F (Fig. 5b).

RT-qPCR demonstrated a significant increase in

the expression of genes encoding cell adhesion mol-

ecules (VCAM1, ICAM1, and SELE) and pro-inflam-

matory cytokines (IL6, CXCL8, CCL2, and CXCL1) in

HCAECs treated with CPP-A (Table  1). Exposure to

CPP-F triggered a similar response, which included el-

evated expression of VCAM1, SELP, IL6, and MIF genes

along with a trend towards a significant increase in

the expression of ICAM1 and SELE genes (Table  2).

Table 1. Relative gene expression (ΔCt; fold change; p-value) in HCAECs and HITAECs treated with DPBS (control),

free Ca

ions, CPM-A, or CPP-A (10  µg of calcium per 1  mL of serum-free cell culture medium) for 24  h

Gene Metrics HCAEC HITAEC

DPBS Ca

CPM-A CPP-A DPBS Ca

CPM-A CPP-A

VCAM1

ΔCt

0.0003 ±

0.0006

0.0001 ±

0.0001

0.0002 ±

0.0001

0.0015 ±

0.0010

0.0003 ±

0.0002

0.0011 ±

0.0011

0.0006 ±

0.0004

0.0010 ±

0.0008

fold

change

1 0.52 0.76 6.00 1 3.67 2.00 3.33

p-value 1.00 0.904 0.985 0.001 1.00 0.009 0.463 0.029

ICAM1

ΔCt

0.0148 ±

0.0066

0.0372 ±

0.0210

0.0118 ±

0.0034

0.1169 ±

0.0837

0.0338 ±

0.0213

0.0503 ±

0.0339

0.0404 ±

0.0244

0.0432 ±

0.0201

fold

change

1 2.52 0.80 7.90 1 1.49 1.20 1.28

p-value 1.00 0.320 0.994 0.001 1.00 0.148 0.782 0.559

SELE

ΔCt

0.0056 ±

0.0026

0.0089 ±

0.0036

0.0020 ±

0.0009

0.0134 ±

0.0065

0.0595 ±

0.0351

0.1049 ±

0.1387

0.0909 ±

0.1203

0.0943 ±

0.0872

fold

change

1 1.59 0.36 2.39 1 1.76 1.53 1.58

p-value 1.00 0.041 0.026 0.001 1.00 0.415 0.687 0.619

SELP

ΔCt

0.0077 ±

0.0067

0.0027 ±

0.0008

0.0015 ±

0.0009

0.0026 ±

0.0023

0.0009 ±

0.0006

0.0054 ±

0.0058

0.0056 ±

0.0055

0.0025 ±

0.0030

fold

change

1 0.35 0.19 0.34 1 6.00 6.22 2.78

p-value 1.00 0.001 0.001 0.001 1.00 0.008 0.005 0.567

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Table 1 (cont.)

Gene Metrics HCAEC HITAEC

DPBS Ca

CPM-A CPP-A DPBS Ca

CPM-A CPP-A

IL6

ΔCt

0.0182 ±

0.0131

0.0058 ±

0.0023

0.0072 ±

0.0045

0.1197 ±

0.0837

0.0085 ±

0.0043

0.0132 ±

0.0155

0.0197 ±

0.0214

0.0247 ±

0.0272

fold

change

1 0.32 0.40 6.58 1 1.55 2.32 2.91

p-value 1.00 0.729 0.796 0.001 1.00 0.803 0.196 0.035

CXCL8

ΔCt

0.0371 ±

0.0260

0.0441 ±

0.0152

0.0250 ±

0.0105

2.1412 ±

1.5287

0.1396 ±

0.0561

0.1801 ±

0.2005

0.1825 ±

0.1871

0.3279 ±

0.3681

fold

change

1 1.19 0.67 57.71 1 1.29 1.31 2.35

p-value 1.00 0.999 0.999 0.001 1.00 0.914 0.901 0.045

CCL2

ΔCt

0.7514 ±

0.6502

0.4398 ±

0.4293

0.6965 ±

0.6669

1.3616 ±

1.0636

0.8908 ±

0.4072

1.2866 ±

1.5286

1.4987 ±

1.6929

1.6162 ±

1.9876

Fold

change

1 0.59 0.93 1.81 1 1.44 1.68 1.81

p-value 1.00 0.448 0.992 0.042 1.00 0.777 0.494 0.353

CXCL1

ΔCt

0.1267 ±

0.0562

0.0436 ±

0.0408

0.0444 ±

0.0343

0.3486 ±

0.1551

0.0647 ±

0.0279

0.1520 ±

0.1842

0.0944 ±

0.0885

0.0983 ±

0.1052

fold

change

1 0.34 0.35 2.75 1 2.35 1.46 1.52

p-value 1.00 0.017 0.018 0.001 1.00 0.069 0.782 0.715

MIF

ΔCt

0.3853 ±

0.1660

0.2309 ±

0.1040

0.2731 ±

0.0839

0.4170 ±

0.2857

0.2753 ±

0.1576

0.7919 ±

0.9280

0.5270 ±

0.4019

0.4618 ±

0.4146

fold

change

1 0.60 0.71 1.08 1 2.88 1.91 1.68

p-value 1.00 0.031 0.155 0.910 1.00 0.018 0.386 0.618

NOS3

ΔCt

0.0094 ±

0.0063

0.0069 ±

0.0036

0.0069 ±

0.0033

0.0091 ±

0.0087

0.0031 ±

0.0018

0.0093 ±

0.0090

0.0119 ±

0.0126

0.0060 ±

0.0037

fold

change

1 0.73 0.73 0.97 1 3.00 3.84 1.94

p-value 1.00 0.473 0.475 0.997 1.00 0.050 0.005 0.547

SNAI1

ΔCt

0.0168 ±

0.0101

0.0100 ±

0.0040

0.0124 ±

0.0065

0.0344 ±

0.0291

0.0049 ±

0.0020

0.0129 ±

0.0110

0.0140 ±

0.0124

0.0094 ±

0.0100

fold

change

1 0.60 0.74 2.05 1 2.63 2.86 1.92

p-value 1.00 0.487 0.771 0.009 1.00 0.042 0.018 0.371

SHISHKOVA et al.144

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Table 1 (cont.)

Gene Metrics HCAEC HITAEC

DPBS Ca

CPM-A CPP-A DPBS Ca

CPM-A CPP-A

SNAI2

ΔCt

0.0129 ±

0.0103

0.0038 ±

0.0009

0.0065 ±

0.0046

0.0047 ±

0.0032

0.0009 ±

0.0007

0.0033 ±

0.0022

0.0099 ±

0.0160

0.0030 ±

0.0055

fold

change

1 0.29 0.50 0.36 1 3.67 11.00 3.33

p-value 1.00 0.001 0.005 0.001 1.00 0.784 0.015 0.841

TWIST1

ΔCt

0.0015 ±

0.0012

0.0003 ±

0.0002

0.0002 ±

0.0001

0.0009 ±

0.0008

0.0004 ±

0.0004

0.0018 ±

0.0026

0.0037 ±

0.0077

0.0016 ±

0.0016

fold

change

1 0.20 0.13 0.60 1 4.50 9.25 4.00

p-value 1.00 0.001 0.001 0.150 1.00 0.742 0.170 0.874

ZEB1

ΔCt

0.2376 ±

0.1200

0.0779 ±

0.0561

0.1607 ±

0.0596

0.3277 ±

0.2237

0.1438 ±

0.0686

0.3697 ±

0.4382

0.4552 ±

0.4601

0.3559 ±

0.4082

fold

change

1 0.33 0.68 1.38 1 2.57 3.17 2.47

p-value 1.00 0.002 0.209 0.117 1.00 0.201 0.049 0.244

Note. Genes encoding pro-inflammatory cell adhesion molecules (VCAM1, ICAM1, SELE, SELP), pro-inflammatory cytokines

(IL6, CXCL8, CCL2, CXCL1, MIF), endothelial nitric oxide synthase (NOS3), and endothelial-to-mesenchymal transition tran-

scription factors (SNAI1, SNAI2, TWIST1, ZEB1) were analyzed. Significant fold change values and p-values are shown in

bold; ΔCt is shown as mean ± SD.

The same gene expression pattern was observed for

HITAECs, including CPP-A-induced activation of ex-

pression of VCAM1, IL6, and CXCL8 genes (Table 1),

while incubation with CPP-F upregulated expression

of SELE, SELP, CXCL1, and MIF genes (Table 2). Col-

lectively, these molecular signatures pointed towards

the development of pro-inflammatory endothelial

activation and suggested elevation in the content of

pro-inflammatory cytokines in cell culture medium.

In contrast to CPP-A/CPP-F, free Ca

ions and CPM-A/

CPM-F induced a stochastic rather than a consis-

tent response which did not reflect endothelial dys-

function.

Treatment with CPP-A significantly increased ex-

pression of inducible endothelial pro-inflammatory

cytokines (IL-6, IL-8, and MCP-1/CCL2) in the cell cul-

ture medium for both HCAECs and HITAECs (Fig.  6),

while addition of free Ca

ions and CPM-A caused no

stable pathological response at the protein level, thus

corroborating the results of gene expression profiling

(Fig. 6). Incubation with CPP-F also promoted expres-

sion of above-mentioned cytokines in HCAECs and

HITAECs, whereas free Ca

ions and CPM-F caused

a cytokine response in HITAECs (Fig.  7).

To further explore the release of cytokines upon

calcium overload, we performed a semi-quantitative

dot blotting analysis of serum-free cell culture me-

dium from HCAECs and HITAECs treated with Ca

CPM-A, and CPP-A. CPP-A increased production of

PAI-1 (also called serpin E1), chemokine (C-X-C mo-

tif) ligand 1 (CXCL1), MCP-1/CCL2, IL-8, macrophage

migration inhibitory factor (MIF), and soluble CD105

and CD147 proteins has been elevated in HCAECs, as

well as upregulated the synthesis of ST2 (suppression

of tumorigenicity  2) and RANTES/CCL5 [regulated

on activation, normal T  cell expressed and secreted/

chemokine (C-C motif) ligand  5] in HITAECs (Fig.  8

and Table S1 in the Online Resource 1). The levels of

soluble uPAR and MIP-3α/CCL20 were elevated in the

cell culture supernatant in both EC lines after incuba-

tion with CPP-A (Fig.  8 and Table  1), i.e., exposure to

CPP-A induced the release of 11 cytokines (Fig.  8 and

TableS1 in the Online Resource1). Six of these pro-in-

flammatory molecules (CXCL1, MCP-1/CCL2, MIF, uPAR,

sCD147, and ST2 protein) were also overrepresented

in the cell culture supernatant collected from CPM-A-

treated ECs, while five proteins [CXCL1, sCD147, ST2

protein, platelet-derived growth factor AA (PDGF-AA),

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Table  2. Relative gene expression (ΔCt, fold change, and p-value) in HCAECs and HITAECs treated with control

DPBS, free Ca

ions, CPM-F, and CPP-F (5  µg of calcium per 1  mL of serum-free cell culture medium) for 24  h

Gene Metrics HCAEC HITAEC

DPBS Ca

CPM-F CPP-F DPBS Ca

CPM-F CPP-F

VCAM1

ΔCt

0.0020 ±

0.0015

0.0019 ±

0.0007

0.0018 ±

0.0009

0.0042 ±

0.0032

0.0004 ±

0.0003

0.0004 ±

0.0002

0.0005 ±

0.0003

0.0006 ±

0.0005

fold

change

1 0.95 0.90 2.1 1 0.95 1.28 1.54

p-value 1.00 0.998 0.984 0.008 1.00 0.995 0.628 0.113

ICAM1

ΔCt

0.0540 ±

0.0510

0.0165 ±

0.0133

0.0208 ±

0.0266

0.1011 ±

0.1201

0.0646 ±

0.0282

0.0906 ±

0.0503

0.0820 ±

0.0306

0.0885 ±

0.0519

fold

change

1 0.31 0.39 1.87 1 1.40 1.27 1.37

p-value 1.00 0.229 0.319 0.098 1.00 0.159 0.454 0.213

SELE

ΔCt

0.0048 ±

0.0037

0.0082 ±

0.0107

0.0028 ±

0.0019

0.0091 ±

0.0053

0.0018 ±

0.0007

0.0084 ±

0.0055

0.0069 ±

0.0032

0.0058 ±

0.0061

fold

change

1 1.71 0.58 1.90 1 4.67 3.83 3.22

p-value 1.00 0.441 0.879 0.375 1.00 0.001 0.003 0.023

SELP

ΔCt

0.0119 ±

0.0131

0.0060 ±

0.0060

0.0089 ±

0.0079

0.0318 ±

0.0276

0.0050 ±

0.0023

0.0032 ±

0.0016

0.0035 ±

0.0017

0.0078 ±

0.0053

fold

change

1 0.50 0.75 2.67 1 0.64 0.70 1.56

-value 1.00 0.534 0.895 0.002 1.00 0.227 0.374 0.025

IL6

ΔCt

0.0072 ±

0.0029

0.0100 ±

0.0097

0.0063 ±

0.0046

0.0233 ±

0.0188

0.0044 ±

0.0016

0.0045 ±

0.0045

0.0031 ±

0.0012

0.0064 ±

0.0042

fold

change

1 1.39 0.88 3.24 1 1.02 0.70 1.45

p-value 1.00 0.783 0.988 0.001 1.00 0.999 0.463 0.191

CXCL8

ΔCt

0.1330 ±

0.0572

0.2582 ±

0.1501

0.1892 ±

0.0573

0.1632 ±

0.0898

0.0294 ±

0.0175

0.0636 ±

0.0714

0.0366 ±

0.0185

0.0522 ±

0.0411

fold

change

1 1.94 1.42 1.23 1 2.16 1.24 1.78

p-value 1.00 0.001 0.203 0.668 1.00 0.053 0.924 0.272

CCL2

ΔCt

1.1073 ±

0.3168

2.8331 ±

1.9144

1.1255 ±

0.3747

1.3772 ±

0.8673

0.3096 ±

0.0759

0.5079 ±

0.3852

0.4625 ±

0.1941

0.5145 ±

0.3698

Fold

change

1 2.56 1.02 1.24 1 1.64 1.49 1.66

p-value 1.00 0.001 0.999 0.795 1.00 0.105 0.265 0.090

SHISHKOVA et al.146

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Table 2 (cont.)

Gene Metrics HCAEC HITAEC

DPBS Ca

CPM-F CPP-F DPBS Ca

CPM-F CPP-F

CXCL1

ΔCt

0.3756 ±

0.1000

0.6205 ±

0.3781

0.4173 ±

0.1658

0.5612 ±

0.3499

0.0543 ±

0.0156

0.1053 ±

0.0885

0.0831 ±

0.0437

0.1477 ±

0.1538

fold

change

1 1.65 1.11 1.49 1 1.94 1.53 2.72

p-value 1.00 0.026 0.942 0.117 1.00 0.235 0.668 0.009

MIF

ΔCt

2.8076 ±

0.9811

2.6168 ±

1.6847

2.2889 ±

0.8340

5.5963 ±

3.2599

0.2999 ±

0.0856

0.4214 ±

0.3609

0.3627 ±

0.1458

0.6572 ±

0.5581

fold

change

1 0.93 0.82 1.99 1 1.41 1.21 2.19

p-value 1.00 0.983 0.763 0.001 1.00 0.583 0.904 0.007

NOS3

ΔCt

0.1599 ±

0.0597

0.1075 ±

0.0592

0.1686 ±

0.0643

0.2636 ±

0.0972

0.0176 ±

0.0056

0.0187 ±

0.0174

0.0170 ±

0.0060

0.0347 ±

0.0382

fold

change

1 0.67 1.05 1.65 1 1.06 0.97 1.97

p-value 1.00 0.082 0.969 0.001 1.00 0.997 0.999 0.050

SNAI1

ΔCt

0.0595 ±

0.0214

0.0513 ±

0.0284

0.0650 ±

0.0257

0.0943 ±

0.0377

0.0017 ±

0.0008

0.0020 ±

0.0017

0.0015 ±

0.0008

0.0031 ±

0.0024

fold

change

1 0.86 1.09 1.58 1 1.18 0.88 1.82

p-value 1.00 0.728 0.892 0.002 1.00 0.937 0.969 0.031

SNAI2

ΔCt

0.0162 ±

0.0088

0.0097 ±

0.0056

0.0196 ±

0.0100

0.0521 ±

0.0311

0.0003 ±

0.0003

0.0005 ±

0.0006

0.0002 ±

0.0001

0.0003 ±

0.0003

fold

change

1 0.60 1.21 3.22 1 1.57 0.63 1.01

p-value 1.00 0.596 0.901 0.001 1.00 0.451 0.856 0.999

TWIST1

ΔCt

0.0019 ±

0.0010

0.0016 ±

0.0009

0.0022 ±

0.0011

0.0029 ±

0.0014

0.00013 ±

0.0001

0.0002 ±

0.0001

0.0001 ±

0.0001

0.0003 ±

0.0003

fold

change

1 0.84 1.16 1.53 1 1.54 0.85 2.31

p-value 1.00 0.757 0.794 0.086 1.00 0.992 0.981 0.094

ZEB1

ΔCt

0.3158 ±

0.2627

0.2493 ±

0.0424

0.2738 ±

0.0783

0.2246 ±

0.2089

0.0468

± 0.0208

0.0734 ±

0.0970

0.0227 ±

0.0064

0.0589 ±

0.0651

fold

change

1 0.16 0.23 0.71 1 1.57 0.49 1.26

p-value 1.00 0.294 0.323 0.276 1.00 0.403 0.477 0.875

Note. For the analyzed genes, see note to Table 1.

CALCIPROTEIN PARTICLES 147

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Fig. 6. Assessment of IL-6 (top panel), IL-8 (middle panel),

and MCP-1/CCL2 (bottom panel) in non-concentrated se-

rum-free cell culture medium from HCAECs and HITAECs

treated with DPBS (control; black), free Ca

ions (blue),

CPM-A (green), and CPP-A (red) (10 µg of calcium per 1 mL

of serum-free cell culture medium) for 24 h (ELISA).

Fig.  7. Assessment of IL-6 (top panel), IL-8 (middle panel),

and MCP-1/CCL2 (bottom panel) in non-concentrated se-

rum-free cell culture medium from HCAECs and HITAECs

treated with DPBS (control, black), free Ca

ions (blue),

CPM-F (green), and CPP-F (red) (10 µg of calcium per 1 mL

of serum-free cell culture medium) for 24 h (ELISA).

and RANTES/CCL5] were upregulated after addition of

CaCl

(Fig. 8 and Table S1 in the Online Resource 1).

The most upregulated molecules were soluble CD147

(also called extracellular matrix metalloproteinase

inducer, EMMPRIN or basigin; fold change, 11.41 in

HCAECs after exposure to CPP-A) and MIP-3α/CCL20

(fold change, 12.52 in HITAECs, also after exposure

to CPP-A).

Likewise, incubation of monocytes with CPP-A in-

duced release of serpin E1/PAI-1, CXCL1/growth regu-

lated protein alpha (GROα), chemokine (C-X-C motif)

ligand  5/epithelial neutrophil-activating protein  78

(CXCL5/ENA-78), adiponectin, neutrophil gelatinase-as-

sociated lipocalin (NGAL)/lipocalin-2, IL-6, chitinase  3-

like  1, apolipoprotein A-I, uPAR, MIP-3α/CCL20, and

MMP-9, in contrast to Ca

and CPM-A, which triggered

SHISHKOVA et al.148

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

Fig.  8. Cytokine profiling of concentrated (3-fold) serum-free cell culture medium from HCAECs (top) and HITAECs (bottom)

treated with DPBS (control), free Ca

ions, CPM-A, or CPP-A (10  µg of calcium per 1  mL of cell culture medium) for 24  h

(dot blotting). Green, serpin E1/PAI-1; light brown, CXCL1/GROα; gray, CD105/endoglin; dark blue, MCP-1/CCL2; red, IL-8;

violet, MIF; dark brown, uPAR; gold, MIP-3α/CCL20; light blue, CD147/ EMMPRIN/basigin; azure, ST2; pink, PDGF-AA; dark

green, RANTES/CCL5. Short, medium, and long arrows indicate fold change from 1.20 to 1.49, from 1.50 to 1.99, and ≥2.00,

respectively, as compared with the DPBS group.

stochastic alterations of cytokine release (Fig.  9 and

Table S2 in the Online Resource 1). CPP-A caused an

increased release of eleven abovementioned cytokines

into the milieu, while the effects of Ca

and CPM-A

were limited to the induction of NGAL/lipocalin-2,

chitinase  3-like  1, and MMP-9 (Fig.  9 and Table S2

in the Online Resource  1). CXCL1, adiponectin, IL-6,

and apolipoprotein A-I were exclusively expressed

in the monocyte-derived culture medium upon the

incubation with CPP-P (amorphous primary CPPs).

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BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

Fig.  9. Cytokine profiling of concentrated (7-fold) serum-free cell culture medium of human monocytes treated with DPBS

(control), free Ca

ions, CPM-A, and CPP-A (10  µg of calcium per 1  mL of cell culture medium) for 24 hours (dot blotting).

Green, serpin E1/PAI-1; light brown, CXCL1/GROα); red, CXCL5/ENA-78; light blue, adiponectin; violet, NGAL/lipocalin-2;

dark blue, IL-6; sky blue, chitinase 3-like 1; pink, apolipoprotein A-I; brown, uPAR; gold, MIP-3α; dark green, MMP-9.

Short, medium, and long arrows indicate fold change from 1.20 to 1.49, 1.50 to 1.99, and ≥2.00, respectively, as compared

with the DPBS group.

Hence, we found that CPP-A caused pro-inflammatory

activation of ECs and monocytes, as evidenced by the

upregulation of expression of multiple cytokine genes

and increased release of corresponding proteins. Al-

though free Ca

ions and CPM-A also induced release

of several cytokines by ECs, their pro-inflammatory

effects were less pronounced as compared to CPP-A

regardless of the cell line (HCAECs, HITAECs, and

monocytes).

All tested calcium delivery forms induce sys-

temic inflammatory response in rats in the ab-

sence of other cardiovascular risk factors. Finally,

we examined in vivo effects of various calcium stress

modalities after intravenous administration of CaCl

CPM-A, and CPP-A to normolipidemic and normoten-

sive Wistar rats (10  µg of calcium per 1  mL of blood).

In contrast to the in  vitro findings, all calcium delivery

forms caused an elevation in the content of pro-in-

flammatory cytokines in rat serum as evidenced by dot

blotting (22, 30, and 24 cytokines in the case of Ca

CPM-A, and CPP-A injections, respectively; Fig.  10 and

Table S3 in the Online Resource  1). Among these mol-

ecules were granulocyte-macrophage colony-stimulat-

ing factor (GM-CSF), chemokines [chemokine (C-X3-C

motif) ligand  1 (CX3CL1, fractalkine), MCP-1/CCL2,

chemokine (C-X-C motif) ligand  7 (CXCL7), C-C motif

chemokine  11 (CCL11, eotaxin), C-C motif chemok-

ine  17 (CCL17)], serpin E1/PAI-1, matrix metallopro-

teinase  2 (MMP-2), matrix metalloproteinase  3 (MMP-3),

hepatokines [hepassocin, fetuin-A, fibroblast growth

factor (FGF-21), growth/differentiation factor  15

(GDF-15)], and proteins with pleiotropic effects [re-

ceptor for advanced glycation end-products (RAGE/

AGER), adiponectin, fibulin-3, galectin-1, and galec-

tin-3] (Fig.10 and Table S3 in the Online Resource 1).

Prolactin, GM-CSF, hepassocin, ciliary neurotroph-

ic factor (CNTF), MMP-3, CX3CL1/fractalkine, FGF-21,

fibuin-3, and GDF-15 were upregulated after all three

types of calcium intervention (Fig.  10 and Table  3

in the Online Resource  1). Yet, RAGE/AGER, fetuin-A,

MCP-1/CCL2, MMP-9, CCL17, and galectin-3 were up-

regulated exclusively after injections of CPM-A and

CPP-A, while release of hepatocyte growth factor

(HGF), CCL11/eotaxin, and galectin-1 was stimulated

only by CPM-A; serpin E1/PAI-1 was upregulated sole-

ly by CPP-A (Fig.  10 and Table  S3 in the Online Re-

source1). Among the cytokines overrepresented in the

cell culture medium from the CPM-A-treated ECs and

monocytes, MCP-1/CCL2 and MMP-9 remained elevat-

ed in the serum of rats injected with either CPM-A or

CPP-A (Table 3). The content of three proteins (uPAR,

CXCL1/GROα, and MIP-3α/CCL20) was increased

SHISHKOVA et al.150

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

Fig.  10. Cytokine profiling of nonconcentrated serum from rats treated with DPBS (control), free Ca

ions, CPM-A, and

CPP-A (10  µg of calcium per 1  mL of blood) for 1  h. Top panel (red box): green, prolactin; dark green, GM-CSF; black,

RAGE/AGER; light blue, hepassocin/fibrinogen-like protein  1 (FGL-1); violet, fetuin-A; dark blue, ciliary neurotrophic factor

(CNTF); gold, MMP-3; pink, hepatocyte growth factor (HGF); brown, chemokine (C-X3-C motif) ligand 1 (CX3CL1)/fractalkine;

red, MCP-1/CCL2; azure, MMP-9. Bottom panel (blue box): green, fibroblast growth factor 21 (FGF21); dark green, chemo-

kine (C-X-C motif) ligand 7 (CXCL7); black, fibulin-3; light blue, cysteine-rich angiogenic inducer 61/CCN family member  1

(Cyr61/CCN1); violet, C-C motif chemokine 11 (CCL11)/eotaxin; dark blue, serpin E1/PAI-1; gold, C-C motif chemokine  17

(CCL17)/thymus and activation-regulated chemokine (TARC); pink, galectin-1; brown, galectin-3; red, growth/differentiation

factor  15 (GDF-15); azure, Pref-1/delta like non-canonical Notch ligand  1 (DLK1)/fetal antigen-1 (FA1). Short, medium, and

long arrows indicate fold change from 1.20 to 1.49, 1.50 to 1.99, and ≥2.00, respectively, as compared with the DPBS group.

in the cell culture medium from ECs and monocytes

treated with CPP-A (Table 3). uPAR and MIP-3α/CCL20

were upregulated in the culture medium from all

three types of cells (HCAECs, HITAECs, and mono-

cytes), but not in the rat serum. Serpin E1/PAI-1 was

the only molecule that was upregulated in all samples

(EC- and monocyte-derived cell culture medium and

rat serum) after CPP-A treatment (Table 3).

DISCUSSION

Disturbed mineral homeostasis, e.g., reduction

in serum albumin and increase in serum calcium or

phosphate, is an independent cardiovascular risk fac-

tor [11]. It a manifestation of chronic kidney disease

which also promotes endothelial dysfunction via ele-

vating serum concentrations of urea and creatinine

[53,  54]. In addition to these biochemical triggers, en-

dothelial dysfunction is exacerbated by internalization

of CPPs by vascular ECs [8-15] and liver sinusoidal

ECs [17,  18] resulting in the development of chronic

low-grade inflammation [13,  14]. CPP internalization

is associated with the increased levels of pro-inflam-

matory cytokines, dysregulated balance between va-

soconstriction and vasodilation (including decreased

NO production), and endothelial-to-mesenchymal tran-

sition [55]. In particular, internalization of CPPs by ECs

and monocytes triggers the release of inducible endo-

thelial cytokines (IL-6, IL-8, MCP-1/CCL2, and soluble

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Table 3. Specific and common cytokines elevated in EC- or monocyte-derived cell culture medium or rat serum

upon the treatment with free Ca

ions, CPM-A, and CPP-A (10 µg of calcium per 1 mL of cell culture medium

or blood) for 24 h (cells) or 1 h (rats) compared with the control (DPBS)

Calcium stress inducer

In vitro (serum-free culture

medium from HCAECs

and HITAECs)

In vitro (serum-free

culture medium

from monocytes)

In vivo (rat serum)

Free Ca

ions

delivered by CaCl

CXCL1/GROα,

CD147/EMMPRIN/basigin,

ST2, PDGF-AA, MIP-3α/CCL20

NGAL/lipocalin-2,

chitinase 3-like 1,

MMP-9

prolactin, GM-CSF,

hepassocin/FGL1,

CNTF, MMP-3,

CX3CL1/fractalkine,

FGF-21, CXCL7, fibulin-3,

Cyr61/CCN, GDF-15

CPM-A

MCP-1/CCL2, CXCL1/GROα,

MIF, uPAR,

CD147/EMMPRIN/basigin,

ST2

MMP-9,

NGAL/lipocalin-2,

chitinase  3-like 1

MCP-1/CCL2, MMP-9,

prolactin, GM-CSF,

RAGE/AGER,

hepassocin/FGL1, fetuin-A,

CNTF, MMP-3, HGF,

CX3CL1/fractalkine,

FGF-21, CXCL7, fibulin-3,

Cyr61/CCN, CCL11/eotaxin,

CCL17/TARC, galectin-1,

galectin-3, GDF-15

CPP-A

Serpin E1/PAI-1, uPAR,

CXCL1/GROα, MIP-3α/CCL20,

MCP-1/CCL2,

CD105/endoglin, IL-8, MIF,

CD147/EMMPRIN/basigin,

ST2, RANTES/CCL5

Serpin E1/PAI-1,

uPAR, CXCL1/GROα,

MIP-3α/CCL20,

MMP-9, CXCL5/ENA-78,

adiponectin,

NGAL/lipocalin-2, IL-6,

chitinase  3-like  1,

apolipoprotein A-I

Serpin E1/PAI-1,

MCP-1/CCL2, MMP-9,

prolactin, GM-CSF,

RAGE/AGER,

hepassocin/FGL1, fetuin-A,

CNTF, MMP-3,

CX3CL1/fractalkine,

FGF-21, fibulin-3,

CCL17/TARC, galectin-3,

GDF-15, Pref-1/DLK1/FA1

CPM-A: upregulated in ECs

and rats

MCP-1/CCL2

CPM-A: upregulated

in monocytes and rats

MMP-9

CPP-A: upregulated in ECs

and monocytes

uPAR, CXCL1/GROα, MIP-3α/CCL20

CPP-A: upregulated in ECs

and rats

MCP-1/CCL2

CPP-A: upregulated

in monocytes and rats

MMP-9

CPP-A: upregulated in ECs,

monocytes, and rats

Serpin E1/PAI-1

intercellular adhesion molecule sICAM-1) and mono-

cyte-derived cytokines [MIP-1α, MIP-3α, cytokine-in-

duced neutrophil chemoattractant-1 (CINC-1), cyto-

kine-induced neutrophil chemoattractant-3 (CINC-3),

and C-X-C motif chemokine ligand  10 (CXCL10)] [13].

Since chronic low-grade inflammation and endothelial

dysfunction mutually promote each other [56-59], it is

difficult to distinguish the contribution of CPP-stimu-

lated ECs and monocytes, as well as specific cytokines

produced by these cells, to systemic inflammation.

According to the recent concepts, nanometer-

sized (~9-10  nm) CPMs act as building blocks for

SHISHKOVA et al.152

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

submicrometer-sized (~30-100  nm) primary CPPs (or

CPP-I), which, in turn, undergo aggregation and amor-

phous-to-crystalline transition to micrometer-sized

(100-300  nm) secondary CPPs (or CPP-II) [18,  27,  60,

61]. Initially amorphous and spherical, primary CPPs

mature into spindle- or needle-shaped crystalline sec-

ondary CPPs [18, 27, 60, 61]. While CPMs and primary

CPPs consist of amorphous calcium phosphate, second-

ary CPPs are composed of carbonate hydroxyapatite,

also called bioapatite. All these calcium and phosphate

scavengers can adsorb proteins from the surrounding

medium (e.g., circulating proteins from the blood) [18,

27,  60,  61]. Calcium and phosphate react with each

other in the presence of the mineral chaperone fe-

tuin-A and other acidic serum proteins with the for-

mation of amorphous calcium phosphate (CPMs or

primary CPPs) or carbonate hydroxyapatite (second-

ary CPPs) [22, 62-64]. CPPs act as mineral scavengers;

phosphate as an inherent component of hydroxyapa-

tite is an essential constituent of these compounds [22,

62-64]. It was reported that high phosphate concentra-

tions inhibit production of nitric oxide, thus impairing

vasodilation [65-67], induce apoptosis of endothelial

cells [65, 67], and promote systemic inflammation

in a dose-dependent manner by triggering oxidative

stress and stimulating expression of pro-inflammatory

cytokines [68]. Hence, high phosphate is considered as

an independent factor of endothelial dysfunction and

chronic low-grade inflammation [69-71].

Although it has been shown that exhaustion of

the serum Ca

-binding capacity leads to the precip-

itation of CPPs and that mineral stress affects the

development of endothelial dysfunction and systemic

inflammation, it remained unclear whether the con-

sequences of calcium overload are solely determined

by the calcium amount or also by the form of calcium

delivery (free Ca

ions, CPMs, and CPPs). Typically,

CPMs and CPPs are generated artificially by combin-

ing excessive concentrations of calcium and phosphate

with a protein source in a buffering solution. The

above question can be answered by applying a holis-

tic approach (by adding serum as a protein source)

or a reductionist approach (by adding a major serum

protein, e.g., albumin or fetuin-A, as a protein source).

The holistic approach recapitulates a scenario occur-

ring in human serum, while the reductionist approach

allows to analyze the mineral-buffering capacity of

each serum protein and to avoid potential negative

effects of other serum components. To better address

the task of modeling mineral stress, here we applied

the reductionist approach to synthesize simultaneous-

ly CPMs and CPPs. We chose BSA as a protein source

for CPM-A and CPP-A because (i)  the lower quartile

of serum albumin content is associated with cardio-

vascular disease and correlates with increased serum

calcification propensity  [11]; (ii)  the concentration of

serum albumin positively correlates with the concen-

tration of serum CPPs (measured with fluorescently

labeled bisphosphonate) and total calcium  [11]; (iii)  al-

bumin represents one of the two primary scavengers

of ionized calcium along with fetuin-A  [7]; (iv)  albu-

min is convenient to use as its yellow color facilitates

visual quality control. Beside BSA, we also used BSF

to synthesize CPM-F and CPP-F because fetuin-A is a

primary mineral chaperone that maintains generation

of CPMs and CPPs in human body [1-4].

When physiological concentrations of BSA were

mixed with supraphysiological concentrations of calci-

um and phosphate, the ratio (%) between the ionized,

protein-bound (CPMs), and phosphate-bound (CPPs)

calcium was 50  :  20  :  30, respectively. This validated

the physiological relevance of the implemented miner-

al stress model (as the distribution of ionized to bound

calcium was 1  :  1) and confirmed that circulating min-

eral depots – CPMs and CPPs – are able to maintain

their calcium-binding function at the physiological lev-

el even upon severe mineral stress. During mineral

stress, the amount of calcium in CPPs exceeded that

in CPMs, thus highlighting the primary role of CPPs

as mineral scavengers buffering the human blood

and controlling the level of ionized calcium. These

results are in accordance with the earlier reports on

the calcium ratio in CPPs and CPMs (1  :  1) [18,  72].

They suggest that CPPs represent an ultimate buffer

that aggregates excessive calcium and phosphate to

prevent blood supersaturation with ionized calcium

when other mineral buffers are exhausted. A relative-

ly low proportion of albumin bound to CPPs (~15%)

even at supraphysiological conditions of mineral stress

indicated that CPP generation, probably, does not af-

fect the functions of albumin in a living organism.

To compare the effects of different calcium vehi-

cles (CaCl

as a donor of Ca

ions, CPM-A/CPM-F, and

CPP-A/CPP-F), we selected ECs and monocytes, which

are the first cell populations encountering CPPs in

an organism. In this study, we determined and used

the physiological dose of calcium (10  µg/mL), as this

parameter is strictly regulated in a body in order to

prevent arrhythmia and extraskeletal calcification

[73-75]. This dose induced a 10% increase in the ion-

ized calcium concentration, which corresponded to

the interquartile range of plasma ionized calcium in

the population (from 0.10 to 0.14 mmol/L, i.e., from

4.0 to 5.6  µg/mL) [11]. Internalization of CPPs is a

mandatory pre-requisite of their detrimental effects

[8-19]. We showed that CPMs are internalized by the

ECs in a flow similarly to CPPs [10, 13]. In accordance

with our previous findings [9-15], incubation of arte-

rial ECs with CPP-A upregulated expression of genes

encoding pro-inflammatory cell adhesion molecules

(VCAM1, ICAM1, and SELE) and pro-inflammatory cy-

tokines (IL6, CXCL8, CCL2, and CXCL1). The treatment

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BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

of arterial ECs with CPP-F promoted expression of

VCAM1, ICAM1, SELE, SELP, IL6, MIF, and CXCL1

genes. Such molecular reconfiguration is typical for

dysfunctional ECs [55,  76,  77] and indicates develop-

ment of chronic low-grade inflammation [56-59] and

senescence-associated secretory phenotype [78-80].

Incubation of ECs and monocytes with CPP-A

or CPP-F (10  µg of calcium per 1 mL of cell culture

medium) promoted release of pro-inflammatory cyto-

kines. Among the molecules upregulated in arterial

ECs and monocytes after their exposure to CPP-A were

cytokines and chemokines (IL-6, IL-8, MCP-1/CCL2,

CXCL1/GROα, MIP-3α/CCL20), as well as pro- and an-

ti-thrombotic molecules (serpin E1/PAI-1 and uPAR).

Of these, uPAR, MIP-3α/CCL20, and serpin E1/PAI-1

were consistently upregulated in HCAECs, HITAECs,

and monocytes. To summarize the results of in  vitro

experiments, exposure to CPPs triggered the pro-in-

flammatory response characterized by the activation

of cytokine release and corresponding changes in the

gene expression. Although Ca

, CPM-A, and CPM-F

also stimulated production of several cytokines by

ECs, this response was less pronounced and suggest-

ed limited pathogenic effects of these calcium sources.

Cytokine profiling by ELISA revealed a statistical-

ly significant elevation in the IL-6, IL-8, and MCP-1/

CCL2 production by both HCAECs and HITAECs after

their incubation with CPP-A. Yet, dot blot experiments

found an increase in the IL-8 and MCP-1/CCL2 content

exclusively in HCAECs; no IL-6 was detected in the

cell culture supernatant even after concentrating it

3-fold. The most probable reason for this discrepancy

is a limited sensitivity of semi-quantitative chemilu-

minescent dot blotting, as IL-6 levels did not exceed

175pg/mL (in comparison with 250-500pg/mL for IL-8

and 2500-6000pg/mL for MCP-1/CCL2). This suggestion

was partially confirmed in our previous study [13],

in which the concentration of IL-6 after 24-hour cul-

turing of CPP-treated ECs reached 300-450 pg/mL and

could be detected by dot blotting.

Previous studies consistently reported cytotox-

ic and pro-inflammatory (primarily NLRP3 inflam-

masome-mediated) effects of CPPs mediated by the

calcium and osmotic stress that resulted from a sharp

rise in the cytosolic Ca

following the dissolution of

calcium in the lysosomes [11,  81,  82]. Bafilomycin A1,

a specific inhibitor of vacuolar H

ATPase (V-ATPase),

rescued ECs  [11] and vascular smooth muscle cells

[81] from the CPP-induced lysosome-dependent cell

death by preventing lysosomal acidification and

CPP dissolution. Similar cytoprotective effects were

reached by using inhibitors of calcium-sensing recep-

tor NLRP3 or caspase-1 during the calcium stress [82].

Likewise, pharmacological inhibition of cathepsin  B,

aprominent lysosomal protease, ameliorated the CPP-

related release of IL-1β by the macrophages  [25,  82].

Gene set enrichment analysis revealed an upregulation

of lysosome-related proteins, in particular, lysosomal

membrane proteins, in arterial ECs [13]. Moreover,

molecular terms related to the lysosome-mediated

calcium dissolution (e.g., vacuolar acidification, pH

regulation, regulation of proteolysis, Ca

elevation

in cytosol, and mitochondrial outer membrane per-

meabilization) were also upregulated in CPP-treated

HCAECs and HITAECs [13]. Further proteomic analysis

suggested that lysosomal response to CPP internaliza-

tion involves pre-existing protein machinery rather

than employs transcriptional, post-transcriptional, and

translational regulation [15]. Although some studies

reported lysosomal alkalization and reduced hydro-

lase activity during CPP dissolution because of Ca

overflow [26, 28], here we able to detected CPP-A

and CPP-F in EC lysosomes using standard pH sensor

(LysoTracker Red). Taken together lysosome-specific

distribution and cytotoxic and pro-apoptotic effects of

CPP-A and CPP-F, we suggested that their pathogenic

profile is similar to those of atherosclerotic plaque-

and serum-derived CPPs [8], as well as to CPP-P and

CPP-S (crystalline secondary CPPs) [9-13, 15].

Intravenous administration of Ca

, CPM-A, or

CPP-A to Wistar rats free of other cardiovascular risk

factors, also triggered systemic inflammatory response

primarily mediated by chemokines (MCP-1/CCL2,

CX3CL1, CXCL7, CCL11, and CCL17), hepatokines (he-

passocin, fetuin-A, FGF-21, and GDF-15), proteases

(MMP-2 and MMP-3), and protease inhibitors (serpin

E1/PAI-1). Hence, our in  vitro and in  vivo findings sup-

ported a pronounced pro-inflammatory effect of CPPs.

For this study, we selected a 1-hour time point based

on the results of our previous works [13] and taking

into account rapid utilization of excessive calcium

from the blood through its clearance by acidic serum

proteins acting as Ca

scavengers, removal of CPMs

by kidneys, and recycling of CPPs in the liver [5].

Notably, ionized calcium concentration represents one

of the most tightly regulated biochemical parameters in

the human blood (which can even be compared to pH),

as stable hypercalcemia is a relatively rare condition

and even transient hypercalcemia might lead to ar-

rhythmia [83-86]. Hence, the model of transient hyper-

calcemia, a condition that frequently occurs in patients

with hyperparathyroidism or excessive vitamin  D in-

take, is quite relevant [83-86]. The pathological effects

of calcium stress observed 1  h after the intravenous

administration of CaCl

, CPMs, or CPPs to Wistar rats,

were related to the low-grade systemic inflammation

defined as a minor to moderate increase in the cyto-

kine levels in the circulation. Such calcium stress-de-

rived transient increase leads to a pro-inflammatory

state which might negatively affect vascular health

by sustaining endothelial activation. If uncurbed (e.g.,

in elderly patients having more than one comorbid

SHISHKOVA et al.154

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

condition, such as diabetes mellitus or chronic kidney

disease), these transient elevations of pro-inflamma-

tory cytokines can contribute to the frailty syndrome,

in which biological age (i.e., age-related functional de-

cline) exceeds the chronological age [56-59].

Our results are in agreement with the data ob-

tained by Wilhelm Jahnen–Dechent’s group, who

showed the absence of CPM toxicity or pro-inflam-

matory effects after internalization by sinusoidal liver

ECs and proximal tubular epithelial cells, in contrast

to CPPs, which exhibited significant cytotoxic effects

and triggered rapid assembly of NLRP3 inflammasome

as early as within 2  h after CPP addition [18]. Com-

bined analysis of in  vitro and in  vivo results revealed

that serpin E1/PAI-1 was the only molecule upregulat-

ed in ECs, monocytes, and rat serum after CPP-A treat-

ment. The reasons behind this stable upregulation of

serpin E1/PAI-1 might include its relative abundance

(which permits to detect and increase in its release by

dot blotting) and calcium-dependent regulation. Other

cytokines overrepresented in the cell culture superna-

tant from the CPP-A/CPP-F-treated ECs or monocytes

or in the serum of CPP-A/CPP-F-treated rats (uPAR,

CXCL1/GROα, MIP-3α/CCL20, MCP-1/CCL2, and MMP-9)

exhibited less consistent expression across the exper-

imental models. For instance, uPAR and CXCL1/GROα

produced only a moderate signal in the cell culture su-

pernatant from either ECs or monocytes, while MIP-3α/

CCL20 and MMP-9 were highly expressed in mono-

cytes but not in the control ECs. Similar to serpin E1/

PAI-1, MCP-1/CCL2 showed a relatively high expression

in all models but was not upregulated in monocytes.

Further, serpin E1/PAI-1 is produced [87,  88] and even

its activity is maintained [89] in a calcium-dependent

manner, suggesting an existence of a mechanism ensur-

ing activation of its release after the treatment with

CPP-A or CPP-F. Unfortunately, previous studies have

not examined production of serpin E1/PAI-1 bythe cell

populations after exposure to CPPs.

Future studies might further investigate the ef-

fects of CPMs and CPPs, considering a higher affin-

ity of fetuin-A for calcium and a unique function of

this protein as a mineral chaperone governing the

CPP formation, although the average fetuin-A level in

the serum (1  g/L) is significantly lower than that of

albumin (34  g/L) [90-93]. It might be promising to in-

vestigate combined effects of Ca

and CPMs or CPPs,

as Ca

-dependent calcium-sensing receptor promotes

internalization of CPPs  [39], which might exacerbate

their pathogenic effects. Another task is to define the

hierarchy in the mineral-binding ability of acidic se-

rum proteins (albumin, fetuin-A, osteonectin, osteo-

protegerin, osteopontin, matrix Gla protein, Gla-rich

protein, alpha-1-acid glycoprotein, transferrin, hapto-

globin, fibrinogen, ceruloplasmin, alpha-2-macroglob-

ulin, immunoglobulin  A, fibronectin, and antithrom-

bin  III). From the diagnostic viewpoint, differential

detection of CPMs and CPPs might be performed us-

ing the flow cytometry approach with fluorescently

labeled bisphosphonate (e.g., IVISense Osteo 680) and

artificially synthesized CPMs and CPPs used to set the

CPM- and CPP-specific gates. Measuring serum concen-

trations of CPMs and CPPs in healthy individuals and

in various diseases states might help to better under-

stand the pathophysiological importance of these pa-

rameters.

Our findings suggest that the adverse effects of

calcium stress are determined by the calcium delivery

mode rather than simply by the amount of calcium.

This might indicate the necessity to reconsider the

approaches for CPP quantification, albeit alternative

techniques (fluorescent labeling in combination with

flow cytometry, turbidimetry, nephelometry, dynam-

ic light scattering, and scanning electron microscopy)

are less standardized and their application for CPP

quantification in vitro is currently debated. Dynamic

light scattering and scanning electron microscopy are

time-consuming, while turbidimetry depends on the

particle-size distribution. Apparently, future studies

will need to address the development of new methods

for CPP quantification.

CONCLUSIONS

We found that physiological increase in the Ca

concentration (by 10%, which is equal to the inter-

quartile range in a population) was achieved by add-

ing 10  µg of calcium to 1  mL of serum-free medium

or rat serum. Incubation of ECs and monocytes with

such amount of CPP-A or CPP-F initiated their pro-in-

flammatory activation that was manifested as tran-

scriptional reprogramming and increased release of

endothelium-derived (IL-6, IL-8, MCP-1/CCL2, uPAR,

MIP-3α/CCL20, serpin E1/PAI-1) and monocyte-derived

(IL-6, IL-8, MIP-1α/1β, MIP-3α/CCL20, uPAR, serpin E1/

PAI-1, CXCL1, CXCL5) cytokines. Addition of free Ca

and CPM-A induced limited detrimental effects in ECs

and monocytes, although CPMs were internalized by

ECs in a flow similar to CPPs. All forms of calcium

delivery (free Ca

ions, colloidal CPM-A, and cor-

puscular CPP-A) caused systemic inflammatory re-

sponse in normolipidemic and normotensive Wistar

rats (Ca

: 22 cytokines, CPM-A: 30 cytokines, CPP-A:

24 cytokines). Serpin E1/PAI-1 was the only mole-

cule upregulated in all tested cells (ECs and mono-

cytes) and rat serum after the treatment with CPP-A.

The increase in the release of chemokines (CX3CL1,

MCP-1/CCL2, CXCL7, CCL11, CCL17) and hepatokines

(hepassocin, fetuin-A, FGF-21, GDF-15) at the back-

ground of upregulated cytokine expression suggested

chemokine burst and release of liver injury markers.

CALCIPROTEIN PARTICLES 155

BIOCHEMISTRY (Moscow) Vol. 90 No. 1 2025

Supplementary information. The online version

containing supplementary material is available at

https://doi.org/10.1134/S0006297924604064.

Contributions. Conceptualization, D.Sh. and A.K.

developed the study concept and performed data vali-

dation; D.Sh., Victoria M., Yu.M., M.S., A.S., Vera M., E.T.,

A.L., and A.S. developed the methodology, performed

the experiments, and analyzed the data; A.K. analyzed

and curated the data, acquired the funding, supervised

the project, prepared the figures, wrote and edited the

text of the article. All authors have read and agreed

to the published version of the manuscript.

Funding. This study was funded by the Russian

Science Foundation (project no. 22-15-00107 “Circula-

tion of calciprotein particles in human blood: patho-

genic consequences and molecular mechanisms”

toA.K.; https://rscf.ru/en/project/22-15-00107/).

Ethics approval and consent to participate.

Allanimal study protocols were approved by the Local

Ethical Committee of the Research Institute for Com-

plex Issues of Cardiovascular Diseases (protocol code:

042/2023; date of approval, April 4, 2023). All animal

experiments were performed in accordance with the

European Convention for the Protection of Vertebrate

Animals (Strasbourg, 1986) and Directive 2010/63/EU

of the European Parliament on the protection of ani-

mals used for scientific purposes.

Conflict of interest. The authors of this work de-

clare that they have no conflict of interest.

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