PRO-INFLAMMATORY ACTIVATION OF ENDOTHELIAL CELLS AND MONOCYTES AT CALCIUM STRESS

Highlights

• Pathophysiological calcium stress (5 μg calcium per 1 mL) causes the release of pro-inflammatory cytokines, soluble membrane receptor forms, and pro-angiogenic factors into the cell culture medium by endothelial cells regardless of the stress source (free calcium ions, calciprotein monomers, and calciprotein particles).
• Exposure of monocytes to fetuin-centric calciprotein monomers and calciprotein particles (5 μg calcium per 1 mL) increased the expression of endothelial cell-ligating receptors, elevated the release of pro-inflammatory and pro-thrombotic molecules, and induced adhesion of monocytes to endothelial cells under the pulsatile flow.
• Intravenous administration of fetuin-centric calciprotein monomers and calciprotein particles (5 μg calcium per 1 mL) to Wistar rats induced higher pro-inflammatory response then free calcium ions, and increases the release of soluble endothelial receptor forms, pro-inflammatory molecules, pro-thrombotic molecules, bioactive matrikines, and hepatokines.

Aim. To define the pathological effects of free calcium ions (Ca²⁺), fetuin-centric calciprotein monomers (CPM-F), and fetuin-centric calciprotein particles (CPP-F) on endothelial cells (ECs) and monocytes.

Methods. Primary human coronary artery endothelial cells (HCAEC) and human internal thoracic artery endothelial cells (HITAEC) and human monocytes were incubated with Ca²⁺ (using CaCl₂ as a donor), calciprotein monomers, and calciprotein particles to induce pathophysiological calcium stress (5 μg per 1 mL, equal to ≈ 10% increase in Ca²⁺) with the following analysis of the gene expression and measurement of the pro-inflammatory molecules in the cell culture medium by dot blot profiling. Adhesion of ECs to monocytes was performed after their co-culture under the 24 hours of pulsatile flow. Systemic inflammatory response was conducted by the intravenous injections of CaCl₂, CPM-F, and CPP-F to Wistar rats with the subsequent measurement of pro-inflammatory molecules in the serum by dot blotting.

Results. Regardless of the calcium stress mode (Ca²⁺, CPM-F, or CPP-F), it has led to the increased release of: 1) pro-inflammatory cytokines (IL-6, IL-8, MCP-1/CCL2, CCL5/RANTES, GM-CSF, CXCL1/GROα, MIF); 2) soluble endothelial receptor forms (CD31/PECAM-1, CD105/ENG, CD147/BSG, CD106/VCAM-1) indicating cell death or pathological shedding of these receptors; 3) pro-angiogenic factors (angiogenin, angiopoietin-2, HGF, PDGF, SDF-1α) into the cell culture medium by ECs. Exposure of monocytes to CPP-F elevated the expression of the genes encoding EC-ligating receptors (ITGB1, ITGA4, SELPG), whilst treatment with CPM-F and CPP-F increased the release of pro-inflammatory (lipocalin-2/NGAL, MMP-9, and myeloperoxidase), pro-angiogenic (angiogenic, BDNF), and pro-thrombotic (uPAR, PAI-1, thrombospondin-1) molecules by monocytes. After 24 hours of co-incubation in the pulsatile flow system, monocytes showed an increased adhesion to ECs upon the treatment with Ca²⁺, CPM-F, or CPP-F. Intravenous administration of CPM-F or CPP-F to Wistar rats elevated an increased release of soluble endothelial receptor forms (VCAM-1/CD106, ICAM-1/CD54), pro-inflammatory molecules (CXCL7, CCL11/eotaxin, FLT3LG), pro-thrombotic molecules (PAI-1), matrikines (fibulin-3, osteopontin, endostatin, CCN3, MMP-3), hepatokines (hepassocin, fetuin-А, RBP4, IGF-1, IGFBP-2/3/5/6), and proteins of metabolic pathways (resistin, RAGE, lipocalin-2/NGAL).

Conclusion. Our results indicate the pathophysiological relevance of endothelial and monocyte activation under calcium stress in context of endothelial dysfunction.

1. Smith ER, Holt SG. The formation and function of calciprotein particles. Pflugers Arch. 2025;477(6):753-772. doi: 10.1007/s00424-025-03083-7

2. Kutikhin AG, Feenstra L, Kostyunin AE, Yuzhalin AE, Hillebrands JL, Krenning G. Calciprotein Particles: Balancing Mineral Homeostasis and Vascular Pathology. Arterioscler Thromb Vasc Biol. 2021;41(5):1607-1624. doi: 10.1161/ATVBAHA.120.315697.

3. Jahnen-Dechent W, Heiss A, Schäfer C, Ketteler M. Fetuin-A regulation of calcified matrix metabolism. Circ Res. 2011;108(12):1494-509. doi: 10.1161/CIRCRESAHA.110.234260.

4. Koeppert S, Ghallab A, Peglow S, Winkler CF, Graeber S, Büscher A, Hengstler JG, Jahnen-Dechent W. Live Imaging of Calciprotein Particle Clearance and Receptor Mediated Uptake: Role of Calciprotein Monomers. Front Cell Dev Biol. 2021;9:633925. doi: 10.3389/fcell.2021.633925.

5. Shishkova D, Markova V, Markova Y, Sinitsky M, Sinitskaya A, Matveeva V, Torgunakova E, Lazebnaya A, Stepanov A, Kutikhin A. Physiological Concentrations of Calciprotein Particles Trigger Activation and Pro-Inflammatory Response in Endothelial Cells and Monocytes. Biochemistry (Mosc). 2025;90(1):132-160. doi: 10.1134/S0006297924604064.

6. Zeper LW, Bos C, Leermakers PA, Franssen GM, Raavé R, Hoenderop JGJ, de Baaij JHF. Liver and spleen predominantly mediate calciprotein particle clearance in a rat model of chronic kidney disease. Am J Physiol Renal Physiol. 2024;326(4):F622-F634. doi: 10.1152/ajprenal.00239.2023.

7. Köppert S, Büscher A, Babler A, Ghallab A, Buhl EM, Latz E, Hengstler JG, Smith ER, Jahnen-Dechent W. Cellular Clearance and Biological Activity of Calciprotein Particles Depend on Their Maturation State and Crystallinity. Front Immunol. 2018;9:1991. doi: 10.3389/fimmu.2018.01991.

8. Herrmann M, Schäfer C, Heiss A, Gräber S, Kinkeldey A, Büscher A, Schmitt MM, Bornemann J, Nimmerjahn F, Herrmann M, Helming L, Gordon S, Jahnen-Dechent W. Clearance of fetuin-A--containing calciprotein particles is mediated by scavenger receptor-A. Circ Res. 2012;111(5):575-84. doi: 10.1161/CIRCRESAHA.111.261479.

9. Shishkova D, Lobov A, Zainullina B, Matveeva V, Markova V, Sinitskaya A, Velikanova E, Sinitsky M, Kanonykina A, Dyleva Y, Kutikhin A. Calciprotein Particles Cause Physiologically Significant Pro-Inflammatory Response in Endothelial Cells and Systemic Circulation. Int J Mol Sci. 2022;23(23):14941. doi: 10.3390/ijms232314941.

10. Kutikhin AG, Velikanova EA, Mukhamadiyarov RA, Glushkova TV, Borisov VV, Matveeva VG, Antonova LV, Filip'ev DE, Golovkin AS, Shishkova DK, Burago AY, Frolov AV, Dolgov VY, Efimova OS, Popova AN, Malysheva VY, Vladimirov AA, Sozinov SA, Ismagilov ZR, Russakov DM, Lomzov AA, Pyshnyi DV, Gutakovsky AK, Zhivodkov YA, Demidov EA, Peltek SE, Dolganyuk VF, Babich OO, Grigoriev EV, Brusina EB, Barbarash OL, Yuzhalin AE. Apoptosis-mediated endothelial toxicity but not direct calcification or functional changes in anti-calcification proteins defines pathogenic effects of calcium phosphate bions. Sci Rep. 2016;6:27255. doi: 10.1038/srep27255.

11. Shishkova D, Velikanova E, Sinitsky M, Tsepokina A, Gruzdeva O, Bogdanov L, Kutikhin A. Calcium Phosphate Bions Cause Intimal Hyperplasia in Intact Aortas of Normolipidemic Rats through Endothelial Injury. Int J Mol Sci. 2019;20(22):5728. doi: 10.3390/ijms20225728.

12. Shishkova D, Markova V, Sinitsky M, Tsepokina A, Velikanova E, Bogdanov L, Glushkova T, Kutikhin A. Calciprotein Particles Cause Endothelial Dysfunction under Flow. Int J Mol Sci. 2020;21(22):8802. doi: 10.3390/ijms21228802.

13. Shishkova DK, Velikanova EA, Bogdanov LA, Sinitsky MY, Kostyunin AE, Tsepokina AV, Gruzdeva OV, Mironov AV, Mukhamadiyarov RA, Glushkova TV, Krivkina EO, Matveeva VG, Hryachkova ON, Markova VE, Dyleva YA, Belik EV, Frolov AV, Shabaev AR, Efimova OS, Popova AN, Malysheva VY, Kolmykov RP, Sevostyanov OG, Russakov DM, Dolganyuk VF, Gutakovsky AK, Zhivodkov YA, Kozhukhov AS, Brusina EB, Ismagilov ZR, Barbarash OL, Yuzhalin AE, Kutikhin AG. Calciprotein Particles Link Disturbed Mineral Homeostasis with Cardiovascular Disease by Causing Endothelial Dysfunction and Vascular Inflammation. Int J Mol Sci. 2021;22(22):12458. doi: 10.3390/ijms222212458.

14. Heiss A, DuChesne A, Denecke B, Grötzinger J, Yamamoto K, Renné T, Jahnen-Dechent W. Structural basis of calcification inhibition by alpha 2-HS glycoprotein/fetuin-A. Formation of colloidal calciprotein particles. J Biol Chem. 2003;278(15):13333-41. doi: 10.1074/jbc.M210868200.

15. Heiss A, Eckert T, Aretz A, Richtering W, van Dorp W, Schäfer C, Jahnen-Dechent W. Hierarchical role of fetuin-A and acidic serum proteins in the formation and stabilization of calcium phosphate particles. J Biol Chem. 2008;283(21):14815-25. doi: 10.1074/jbc.M709938200.

16. Shishkova D.K., Markova V.E., Markova Yu.O., Torgunakova E.A., Kondratiev E.A., Dyleva Yu.A., Kutikhin A.G. Patterns of calcium distribution by biochemical serum compartments in vitro modeling of mineral stress in endothelial dysfunction. Complex Issues of Cardiovascular Diseases = Kompleksnye problemy serdečno-sosudistyh zabolevanij. 2024. Vol. 13. № 2. P. 60-71. doi: 10.17802/2306-1278-2024-13-2-60-71.

17. Jerka D, Bonowicz K, Piekarska K, Gokyer S, Derici US, Hindy OA, Altunay BB, Yazgan I, Steinbrink K, Kleszczyński K, Yilgor P, Gagat M. Unraveling Endothelial Cell Migration: Insights into Fundamental Forces, Inflammation, Biomaterial Applications, and Tissue Regeneration Strategies. ACS Appl Bio Mater. 2024;7(4):2054-2069. doi: 10.1021/acsabm.3c01227.

18. Lee HW, Shin JH, Simons M. Flow goes forward and cells step backward: endothelial migration. Exp Mol Med. 2022;54(6):711-719. doi: 10.1038/s12276-022-00785-1.

19. Lin A, Miano JM, Fisher EA, Misra A. Chronic inflammation and vascular cell plasticity in atherosclerosis. Nat Cardiovasc Res. 2024;3(12):1408-1423. doi: 10.1038/s44161-024-00569-y.

20. Peña OA, Martin P. Cellular and molecular mechanisms of skin wound healing. Nat Rev Mol Cell Biol. 2024;25(8):599-616. doi: 10.1038/s41580-024-00715-1.

21. Hilgendorf I, Frantz S, Frangogiannis NG. Repair of the Infarcted Heart: Cellular Effectors, Molecular Mechanisms and Therapeutic Opportunities. Circ Res. 2024;134(12):1718-1751. doi: 10.1161/CIRCRESAHA.124.323658.

22. Amersfoort J, Eelen G, Carmeliet P. Immunomodulation by endothelial cells - partnering up with the immune system? Nat Rev Immunol. 2022;22(9):576-588. doi: 10.1038/s41577-022-00694-4.

23. Wu X, Reboll MR, Korf-Klingebiel M, Wollert KC. Angiogenesis after acute myocardial infarction. Cardiovasc Res. 2021;117(5):1257-1273. doi: 10.1093/cvr/cvaa287.

24. Yan F, Liu X, Ding H, Zhang W. Paracrine mechanisms of endothelial progenitor cells in vascular repair. Acta Histochem. 2022;124(1):151833. doi: 10.1016/j.acthis.2021.151833.

25. Chong MS, Ng WK, Chan JK. Concise Review: Endothelial Progenitor Cells in Regenerative Medicine: Applications and Challenges. Stem Cells Transl Med. 2016;5(4):530-8. doi: 10.5966/sctm.2015-0227.