Endogenous CCN family member WISP1 inhibits trauma-induced heterotopic ossification
Ginny Ching-Yun Hsu, Simone Marini, Stefano Negri, Yiyun Wang, Jiajia Xu, Chase Pagani, Charles Hwang, David Stepien, Carolyn A. Meyers, Sarah Miller, Edward McCarthy, Karen M. Lyons, Benjamin Levi, Aaron W. James
Ginny Ching-Yun Hsu, Simone Marini, Stefano Negri, Yiyun Wang, Jiajia Xu, Chase Pagani, Charles Hwang, David Stepien, Carolyn A. Meyers, Sarah Miller, Edward McCarthy, Karen M. Lyons, Benjamin Levi, Aaron W. James
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Research Article Bone biology Stem cells

Endogenous CCN family member WISP1 inhibits trauma-induced heterotopic ossification

Abstract

Heterotopic ossification (HO) is defined as abnormal differentiation of local stromal cells of mesenchymal origin, resulting in pathologic cartilage and bone matrix deposition. Cyr61, CTGF, Nov (CCN) family members are matricellular proteins that have diverse regulatory functions on cell proliferation and differentiation, including the regulation of chondrogenesis. However, little is known regarding CCN family member expression or function in HO. Here, a combination of bulk and single-cell RNA sequencing defined the dynamic temporospatial pattern of CCN family member induction within a mouse model of trauma-induced HO. Among CCN family proteins, Wisp1 (also known as Ccn4) was most upregulated during the evolution of HO, and Wisp1 expression corresponded with chondrogenic gene profile. Immunohistochemistry confirmed WISP1 expression across traumatic and genetic HO mouse models as well as in human HO samples. Transgenic Wisp1LacZ/LacZ knockin animals showed an increase in endochondral ossification in HO after trauma. Finally, the transcriptome of Wisp1-null tenocytes revealed enrichment in signaling pathways, such as the STAT3 and PCP signaling pathways, that may explain increased HO in the context of Wisp1 deficiency. In sum, CCN family members, and in particular Wisp1, are spatiotemporally associated with and negatively regulate trauma-induced HO formation.

Authors

Ginny Ching-Yun Hsu, Simone Marini, Stefano Negri, Yiyun Wang, Jiajia Xu, Chase Pagani, Charles Hwang, David Stepien, Carolyn A. Meyers, Sarah Miller, Edward McCarthy, Karen M. Lyons, Benjamin Levi, Aaron W. James

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Figure 5

Transgenic Wisp1-knockout mice demonstrated increased posttraumatic heterotopic ossification.

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Transgenic Wisp1-knockout mice demonstrated increased posttraumatic hete...
Heterotopic ossification (HO) induction was performed in Wisp1–/– (Wisp1LacZ/LacZ) mice, in which in which exons 1–5 of the alleles are replaced by a LacZ-neo cassette. Wisp1+/+ littermates were used as control animals. (A and B) Representative Safranin O red/fast green staining within the injury site, 3 weeks after tenotomy. Cartilage appears orange, while tendon or bone appear green. n = 6 mice per genotype. (C and D) Histomorphometric quantification of relative cartilage among Wisp1+/+ and Wisp1–/– injury sites. Analysis performed on n = 6representative samples per genotype. (E and F) Masson’s trichrome staining within the site 9 weeks after tenotomy. Bone appears dark blue, while tendon appears light blue. (G and H) μCT reconstruction of bone formation within the injured tendon among Wisp1+/+ and Wisp1–/– animals. Reconstruction shown from a sagittal perspective. Heterotopic bone appears orange, while native bone appears gray. (I) μCT quantification of heterotopic bone volume (BV). Each dot indicates an individual mouse, with mean and SD indicated. n = 12 mice per genotype. (J–O) Immunohistochemical staining among Wisp1+/+ and Wisp1–/– animals at the sites 9 weeks after tenotomy, including (J and K) type II collagen (COL2), (L and M) aggrecan (ACAN), (N and O) Runt-related transcription factor 2 (RUNX2). *P < 0.05, **P < 0.01, ***P < 0.001 in comparison with Wisp1+/+ animals. Differences were calculated between groups by Student’s t test. Data represent mean ± SD. Scale bar: 100 μm; original magnification, ×20 (bottom images, J–O).