Int J Med Sci 2020; 17(17):2703-2717. doi:10.7150/ijms.43184

Research Paper

Indoxyl Sulfate-induced Vascular Calcification is mediated through Altered Notch Signaling Pathway in Vascular Smooth Muscle Cells

Kazutoshi Yamaguchi1, Maimaiti Yisireyili1, Sumie Goto2, Katsuhiro Kato1, Xian Wu Cheng3,4, Takayuki Nakayama5, Tadashi Matsushita6,7, Toshimitsu Niwa8, Toyoaki Murohara1, Kyosuke Takeshita1,6,9✉

1. Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
2. Biomedical Research Laboratories, Kureha Co., Tokyo, Japan.
3. Department of Cardiology/Hypertension and Heart Center, Yanbian University Hospital, Yanji, Jilin, China.
4. Department of Community Health and Geriatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.
5. Department of Blood Transfusion, Aichi Medical University Hospital, Nagakute, Japan.
6. Department of Clinical Laboratory, Nagoya University Hospital, Nagoya, Japan.
7. Department of Blood Transfusion, Nagoya University Hospital, Nagoya, Japan.
8. Shubun University, Ichinomiya, Aichi, Japan.
9. Department of Clinical Laboratory, Saitama Medical Centre, Saitama Medical University, Kawagoe, Japan.

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Citation:
Yamaguchi K, Yisireyili M, Goto S, Kato K, Cheng XW, Nakayama T, Matsushita T, Niwa T, Murohara T, Takeshita K. Indoxyl Sulfate-induced Vascular Calcification is mediated through Altered Notch Signaling Pathway in Vascular Smooth Muscle Cells. Int J Med Sci 2020; 17(17):2703-2717. doi:10.7150/ijms.43184. Available from http://www.medsci.org/v17p2703.htm

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Abstract

Introduction: The aim of this study was to determine the role of Notch in indoxyl sulfate (IS)-induced vascular calcification (VC).

Materials and methods: VC and expression of Notch-related and osteogenic molecules were examined in Dahl salt-sensitive (DS), DS hypertensive (DH), and DH IS-treated rats (DH+IS). The effects of IS on expression of Notch receptors, apoptotic activity, and calcification were examined in cultured aortic smooth muscle cells (SMCs).

Results: Medial calcification was noted only in aortas and coronary arteries of DH+IS rats. Notch1, Notch3, and Hes-1 were expressed in aortic SMCs of all rats, but only weakly in the central areas of the media and around the calcified lesions in DH+IS rats. RT-PCR and western blotting of DH+IS rat aortas showed downregulation of Notch ligands, Notch1 and Notch3, downstream transcriptional factors, and SM22, and conversely, overexpression of osteogenic markers. Expression of Notch1 and Notch3 in aortic SMCs was highest in incubation under 500 μM IS for 24hrs, and then decreased time- and dose-dependently. Coupled with this decrease, IS increased caspase 3/7 activity and TUNEL-positive aortic SMCs. In addition, pharmacological Notch signal inhibition with DAPT induced apoptosis in aortic SMCs. ZVAD, a caspase inhibitor abrogated IS-induced and DAPT-induced in vitro vascular calcification. Knockdown of Notch1 and Notch3 cooperatively increased expression of osteogenic transcriptional factors and decreased expression of SM22.

Conclusion: Our results suggested that IS-induced VC is mediated through suppression of Notch activity in aortic SMCs, induction of osteogenic differentiation and apoptosis.

Keywords: keyword 1 indoxyl sulfate, keyword 2 vascular calcification, keyword 3 Notch signal.