Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals
Jian-Su Shao, … , Arleen P. Loewy, Dwight A. Towler
Jian-Su Shao, … , Arleen P. Loewy, Dwight A. Towler
Published May 2, 2005
Citation Information: J Clin Invest. 2005;115(5):1210-1220. https://doi.org/10.1172/JCI24140.
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Article Cardiology

Msx2 promotes cardiovascular calcification by activating paracrine Wnt signals

Abstract

In diabetic LDLR–/– mice, an ectopic BMP2-Msx2 gene regulatory program is upregulated in association with vascular calcification. We verified the procalcific actions of aortic Msx2 expression in vivo. CMV-Msx2 transgenic (CMV-Msx2Tg+) mice expressed 3-fold higher levels of aortic Msx2 than nontransgenic littermates. On high-fat diets, CMV-Msx2Tg+ mice exhibited marked cardiovascular calcification involving aortic and coronary tunica media. This corresponded to regions of Msx2 immunoreactivity in adjacent adventitial myofibroblasts, suggesting a potential paracrine osteogenic signal. To better understand Msx2-regulated calcification, we studied actions in 10T1/2 cells. We found that conditioned media from Msx2-transduced 10T1/2 cells (Msx2-CM) is both pro-osteogenic and adipostatic; these features are characteristic of Wnt signaling. Msx2-CM stimulated Wnt-dependent TCF/LEF transcription, and Msx2-transduced cells exhibited increased nuclear β-catenin localization with concomitant alkaline phosphatase induction. Msx2 upregulated Wnt3a and Wnt7a but downregulated expression of the canonical inhibitor Dkk1. Dkk1 treatment reversed osteogenic and adipostatic actions of Msx2. Teriparatide, a PTH1R agonist that inhibits murine vascular calcification, suppressed vascular BMP2-Msx2-Wnt signaling. Analyses of CMV-Msx2Tg+ mice confirmed that Msx2 suppresses aortic Dkk1 and upregulates vascular Wnts; moreover, TOPGAL+ (Wnt reporter); CMV-Msx2Tg+ mice exhibited augmented aortic LacZ expression. Thus, Msx2-expressing cells elaborated an osteogenic milieu that promotes vascular calcification in part via paracrine Wnt signals.

Authors

Jian-Su Shao, Su-Li Cheng, Joyce M. Pingsterhaus, Nichole Charlton-Kachigian, Arleen P. Loewy, Dwight A. Towler

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Expression of Msx2 in adventitial cells adjacent to segments of aortic a...
Expression of Msx2 in adventitial cells adjacent to segments of aortic and coronary medial calcification. Msx2 expression was visualized by immunohistochemistry in cardiovascular tissues from male CMV-Msx2Tg+ mice. Background signal arising from nonspecific binding of secondary antibody was assessed in the absence of primary antibody. Note expression of Msx2 transgene in adventitial cells (A) and cells at the aortic valve insertion (B). Msx2 expression was also observed in cardiac myofibroblasts (not shown). (C) Adjacent sections of cardiovascular tissues from male CMV-Msx2Tg+ mice were analyzed for Msx2 or ALP by immunohistochemistry. Alizarin red staining was used to determine patterns of tissue calcium deposition. While Msx2 is highly expressed in adventitial cells, ALP expression and calcification are most intense in the tunica media. (D) Western blot analysis of aortic proteins extracted from Msx2Tg+ compared with those from nontransgenic siblings. The expression of the osteocyte marker SOST was greater in aortas of CMV-Msx2Tg+ mice (lanes 2, 4, 6, and 8) as compared to SOST expression observed in aortas of nontransgenic siblings (lanes 1, 3, 5, and 7). All animals were fed high-fat diets as outlined in Methods. Arrows in A–C indicate nuclear staining. No 1° Ab, no primary antibody added.