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. 2019 Jun 3;9:8193. doi: 10.1038/s41598-019-44668-4

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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BMP-2 expression is increased in calcifying carotid arteries from vitamin D₃-injected rats. Male SD rats were injected with vehicle or 5 mg/kg vitamin D₃ (VitD₃) for 3 consecutive days, and carotid arteries were harvested 14 days later. (A) Immunohistochemical staining showing increased BMP-2 expression in calcifying areas after vitamin D₃ injection. (B) qPCR results showing increased BMP-2 mRNA levels in vitamin D₃ injected rats. (C) Western blotting results showed that vitamin D₃ injection increased BMP-2 protein levels. (D) Vitamin D₃ injection increased cell proliferation in calcifying areas but did not cause neointimal hyperplasia. Neointimal hyperplasia was evaluated by vessel wall thickness. Elastin integrity was examined by VVG staining. Calcification was assessed by Von Kossa staining. Cell proliferation was determined by PCNA immunohistochemical staining. (E) Quantitative data showing increased PCNA-positive cells in vitamin D₃-injected rats.