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Objectives and questions: Physiological levels of calcium and vitamin D as well as their pharmacological supplementation as in osteoporotic patients have been extensively studied, yet their effects on cardiovascular outcomes remain debated. To prevent adverse effects of either insufficient or excessive supplementation, we focus on elucidating the processes involved in human smooth muscle cell (hSMC) mineralization that is causal for vascular calcification.

Material and methods: Using an in vitro hSMC calcification model, we examined proliferation, cytotoxicity, and mineralization under homeostatic and pro-osteogenic conditions, supplemented with calcitriol (DECOSTRIOL inject) and/or calcium gluconate.

Results: High doses of calcium (5mM) and calcitriol (50ng/ml) were cytotoxic, whereas lower concentrations did not affect hSMC proliferation. Alizarin red staining revealed that 2 mM calcium, with or without 10 ng/ml calcitriol, enhanced mineralized matrix deposition in both homeostatic and pro-osteogenic cultures. Time-lapse imaging indicated interconnected active and passive mineralization processes in these cultures, consistent with an early decrease in supernatant phosphate levels. Notably, 10 ng/ml calcitriol alone attenuated hSMC mineralization in pro-osteogenic cultures. Purinergic gene expression was differentially modulated by supplementation, depending on environmental conditions. In pro-osteogenic cultures, 2 mM calcium promoted NT5E (CD73) expression, while 10ng/ml calcitriol inhibited the upregulation of NT5E (CD73), ENPP1, and ADORA2B—genes linked to calcification. Conversely, in homeostatic cultures, the combination of 2 mM calcium and 10 ng/ml calcitriol upregulated NT5E (CD73) and ADORA2B expression, potentially promoting mineralization. Functional experiments to explore purinergic signaling in calcitriol- and calcium-mediated hSMC mineralization are ongoing.

Discussion and conclusions: Our findings highlight the need for caution regarding high medical doses of both supplements, suggest a modulating role of purinergic signaling in calcitriol- and calcium-associated hSMC mineralization, and indicate a potential adverse interaction between both supplements in vascular calcification.