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.2021 Feb;122(2):249-258.
doi: 10.1002/jcb.29847. Epub 2020 Sep 9.

Serotonin receptor type 2B activation augments TNF-α-induced matrix mineralization in murine valvular interstitial cells

Affiliations

Serotonin receptor type 2B activation augments TNF-α-induced matrix mineralization in murine valvular interstitial cells

Felicia Fong et al. J Cell Biochem.2021 Feb.

Abstract

Calcification, fibrosis, and chronic inflammation are the predominant features of calcific aortic valve disease, a life-threatening condition. Drugs that induce serotonin (5-hydroxytryptamine [5-HT]) are known to damage valves, and activated platelets, which carry peripheral serotonin, are known to promote calcific aortic valve stenosis. However, the role of 5-HT in valve leaflet pathology is not known. We tested whether serotonin mediates inflammation-induced matrix mineralization in valve cells. Real-time reverse transcription-polymerase chain reaction analysis showed that murine aortic valve interstitial cells (VICs) expressed both serotonin receptor types 2A and 2B (Htr2a and Htr2b). Although Htr2a expression was greater at baseline, Htr2b expression was induced several-fold more than Htr2a in response to the pro-calcific tumor necrosis factor-α (TNF-α) treatment. 5-HT also augmented TNF-α-induced osteoblastic differentiation and matrix mineralization of VIC, but 5-HT alone had no effects. Inhibition of serotonin receptor type 2B, using specific inhibitors or lentiviral knockdown in VIC, attenuated 5-HT effects on TNF-α-induced osteoblastic differentiation and mineralization. 5-HT treatment also augmented TNF-α-induced matrix metalloproteinase-3 expression, which was also attenuated by Htr2b knockdown. Htr2b expression in aortic roots and serum levels of peripheral 5-HT were also greater in the hyperlipidemic Apoe-/- mice than in control normolipemic mice. These findings suggest a new role for serotonin signaling in inflammation-induced calcific valvulopathy.

Keywords: HTR2B; calcification; inflammation; serotonin; valve.

© 2020 Wiley Periodicals LLC.

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Conflict of interest statement

CONFLICT OF INTEREST

None

Figures

Figure 1.
Figure 1.. Expression of 5-HT receptor subtypes in VIC at baseline and in response to TNF-α.
(A) Realtime RT-qPCR analyses of VIC and vascular smooth muscle cells (SMC) for baseline expression of type-2 serotonin receptors (presented in log10 scale).(B) Realtime RT-qPCR analyses for Htr2a and Htr2b expression in VIC treated with TNF-α (25 ng/mL) for 11 days. *p < 0.0001.
Figure 2.
Figure 2.. Effects of 5-HT on TNF-α induction of matrix mineralization in VIC.
(A) 5-HT effects on phosphorylation of p44/42 MAPK, assessed by Western blot, in VIC treated with vehicle (control) or 5-HT for 5, 8 or 10 min. , and p44/42 MAPK was used as a loading control.(B, left) Qualitative analysis of matrix mineralization, assessed by alizarin red staining, in VIC treated with vehicle (control), TNF-α and/or 5-HT, as indicated, for 11 days. Magnification 2X.(B, right) Quantitative analysis of matrix mineralization, assessed byo-cresolphthalein complexone, in VIC treated with control, TNF-α (25 ng/mL) and/or 5-HT (10 μM), as indicated, for 14 days. *p < 0.001.(C) Quantitative analysis of matrix mineralization, in VIC treated with vehicle, TNF-α+5-HT, SB (25 μM; SB204741), or ketanserin (1 μM), as indicated. *p < 0.0001, **p = 0.003.(D) Quantitative analysis of matrix mineralization, in control VIC and in Htr2a or Htr2b knocked down VIC treated with vehicle or TNF-α+5-HT, as indicated. *p < 0.0001.
Figure 2.
Figure 2.. Effects of 5-HT on TNF-α induction of matrix mineralization in VIC.
(A) 5-HT effects on phosphorylation of p44/42 MAPK, assessed by Western blot, in VIC treated with vehicle (control) or 5-HT for 5, 8 or 10 min. , and p44/42 MAPK was used as a loading control.(B, left) Qualitative analysis of matrix mineralization, assessed by alizarin red staining, in VIC treated with vehicle (control), TNF-α and/or 5-HT, as indicated, for 11 days. Magnification 2X.(B, right) Quantitative analysis of matrix mineralization, assessed byo-cresolphthalein complexone, in VIC treated with control, TNF-α (25 ng/mL) and/or 5-HT (10 μM), as indicated, for 14 days. *p < 0.001.(C) Quantitative analysis of matrix mineralization, in VIC treated with vehicle, TNF-α+5-HT, SB (25 μM; SB204741), or ketanserin (1 μM), as indicated. *p < 0.0001, **p = 0.003.(D) Quantitative analysis of matrix mineralization, in control VIC and in Htr2a or Htr2b knocked down VIC treated with vehicle or TNF-α+5-HT, as indicated. *p < 0.0001.
Figure 3.
Figure 3.. Effects of 5-HT on TNF-α-induced Bmp2 expression in VIC.
(A) Realtime RT-qPCR analyses for Bmp2 expression in VIC treated with vehicle, TNF-α (25 ng/mL) and/or 5-HT (10 μM) for 8 days. *p < 0.0001.(B) Realtime RT-qPCR analyses for Bmp2 expression in control VIC and in Htr2a-knockdown VIC, or in Htr2b-knockdown VIC treated with vehicle or TNF-α+5-HT, as indicated for 9 days. *p = 0.0003.
Figure 4.
Figure 4.. Effects of 5-HT on TNF-α-induced Mmp3 expression in VIC.
(A) Realtime RT-qPCR analyses for Mmp3 expression in VIC treated with vehicle, TNF-α (25 ng/mL) and/or 5-HT (10 μM) for 4 days. *p < 0.0001.(B) Realtime RT-qPCR analyses for Mmp3 expression in control VIC and in Htr2a-knockdown VIC or in Htr2b-knockdown VIC, treated with vehicle, TNF-α+5-HT, as indicated. *p = 0.0001.
Figure 5.
Figure 5.. Serum 5-HT levels and expression of HTR2A and HTR2B in mouse atherosclerotic lesions.
(A) Peripheral 5-HT levels, assessed by ELISA, in WT (C57BL/6) and hyperlipidemic (Apoe−/−) mice.(B) Immunohistochemical analysis of HTR2A (a and c) and HTR2B (b, d, e and f) in mouse aortic roots (counterstained with hematoxylin). Calcium mineral is stained purple (white arrow), serotonin receptor immunopositivity is indicated in brown (black arrows), and cholesterol clefts are identified by arrowheads.
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