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[Preprint]. 2024 Apr 6:2024.04.05.588239.
doi: 10.1101/2024.04.05.588239.

Cyr61 delivery promotes angiogenesis during bone fracture repair

Annemarie Lang  1 , Emily A Eastburn  1   2 , Mousa Younesi  2 , Madhura Nijsure  1   2 , Carly Siciliano  1   2 , Annapurna Pranatharthi Haran  1   2 , Christopher J Panebianco  1 , Elizabeth Seidl  1   2 , Rui Tang  3 , Eben Alsberg  3 , Nick J Willett  4   5 , Riccardo Gottardi  2   6 , Dongeun Huh  2 , Joel D Boerckel  1   2
Affiliations

Cyr61 delivery promotes angiogenesis during bone fracture repair

Annemarie Lang et al. bioRxiv. .

Update in

  • CYR61 delivery promotes angiogenesis during bone fracture repair.
    Lang A, Eastburn EA, Younesi M, Nijsure MP, Siciliano C, Pranatharthi Haran A, Panebianco CJ, Seidl E, Tang R, Alsberg E, Willett NJ, Gottardi R, Huh D, Boerckel JD. Lang A, et al. NPJ Regen Med. 2025 Apr 22;10(1):20. doi: 10.1038/s41536-025-00398-y. NPJ Regen Med. 2025. PMID: 40263309 Free PMC article.

Abstract

Compromised vascular supply and insufficient neovascularization impede bone repair, increasing risk of non-union. Cyr61, Cysteine-rich angiogenic inducer of 61kD (also known as CCN1), is a matricellular growth factor that is regulated by mechanical cues during fracture repair. Here, we map the distribution of endogenous Cyr61 during bone repair and evaluate the effects of recombinant Cyr61 delivery on vascularized bone regeneration. In vitro, Cyr61 treatment did not alter chondrogenesis or osteogenic gene expression, but significantly enhanced angiogenesis. In a mouse femoral fracture model, Cyr61 delivery did not alter cartilage or bone formation, but accelerated neovascularization during fracture repair. Early initiation of ambulatory mechanical loading disrupted Cyr61-induced neovascularization. Together, these data indicate that Cyr61 delivery can enhance angiogenesis during bone repair, particularly for fractures with stable fixation, and may have therapeutic potential for fractures with limited blood vessel supply.

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Conflict of interest statement

Competing interests: Authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.. Spatial expression of YAP, CCN1/CYR61, EMCN and OSX in the fractured bone at 14 dpf.
(A) Exemplary Movat’s Pentachrome staining 14 dpf; black box indicates ROI as magnified in B, C and D. (B) Magnification of fracture gap and adjacent bone marrow areas (ROI from A; black box indicates ROI for G). (C, D) Overview stainings. (E, F) Quantifications. One-way ANOVA with matched pairs was used to determine the statistical significance; p-values are indicated with *p < 0.05; ***p < 0.001. (G) Magnifications from endochondral part of fracture gap. Scale bars indicate 1 mm (A) and 500 μm (B-D) and 100 μm (G).
Figure 2.
Figure 2.. Cyr61 accelerates in vitro angiogenesis, but not chondrogenesis or osteogenesis of hMSCs.
(A) Experimental setup. (B) Representative images of Safranin-O-staining and (C) quantitative measurement of chondrogenic pellet diameter at 2 weeks. (D) Relative mRNA expression of RUNX2, SP7, COL1A1 and CYR61 after 3 weeks of osteogenic differentiation normalized to housekeeping gene and control. (E) 3D in vitro angiogenesis assay combing HUVECs and hMSCs. (F) Exemplary images of tube formation at 3 days. (G) Quantification of relative GFP+ cell area, relative tube number and tube length. Mean ± SEM and individual data points. One-way ANOVA was used to determine the statistical significance; p-values are indicated with *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars indicate 500 μm (B) and 200 μm (F).
Figure 3.
Figure 3.. Cyr61 treatment had no effect on fracture callus bone or cartilage formation at 14 dpf.
(A) Experimental setup. (B) 3D Representative reconstruction images of microCT analysis. (C) MicroCT - quantification of bone volume (BV) and bone volume fraction (bone volume/BV; total callus volume/TV). (D) Exemplary images of Movat’s Pentachrome staining – yellow = mineralized bone; green = cartilage; magenta = bone marrow; red = muscle tissue. (E) Quantification of mineralized bone and cartilage area in gap. Mean ± SEM and individual data points. One-way ANOVA was used to determine the statistical significance. Scale bars indicate 500 μm (D).
Figure 4.
Figure 4.. Treatment with endogenous Cyr61 promotes revascularization in the fracture gap 14 dpf.
(A) Representative images of EMCN and OSX staining and (B) quantifications. (C) Quantifications and (D) representative images of F4/80 and SOX9 staining. Mean ± SEM and individual data points. One-way ANOVA was used to determine the statistical significance; p-values are indicated with *p < 0.05; ***p < 0.001. Scale bars indicate 200 μm (A, D).
Figure 5.
Figure 5.. Cyr61 promotes vascular maturation with or without loading in vitro
(A) Experimental setup. (B) Representative images. (C-F) Quantifications of (C) vessel length, (D) vascular area, (E) number of vessel junctions and (F) number of vessel endpoints as measure for vascular connectivity. Absolute values are given for the whole chip area. Mean ± SEM and individual data points. One-way ANOVA was used to determine the statistical significance; p-values are indicated with *p < 0.05; **p < 0.01; ***p < 0.001. Scale bars indicate 500 μm.
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References

    1. Phelps E. A., García A. J., Engineering more than a cell: vascularization strategies in tissue engineering. Curr Opin Biotechnol 21, 704–709 (2010). - PMC - PubMed
    1. van Gastel N., Stegen S., Eelen G., Schoors S., Carlier A., Daniëls V. W., Baryawno N., Przybylski D., Depypere M., Stiers P. J., Lambrechts D., Van Looveren R., Torrekens S., Sharda A., Agostinis P., Lambrechts D., Maes F., Swinnen J. V., Geris L., Van Oosterwyck H., Thienpont B., Carmeliet P., Scadden D. T., Carmeliet G., Lipid availability determines fate of skeletal progenitor cells via SOX9. Nature 579, 111–117 (2020). - PMC - PubMed
    1. Maes C., Kobayashi T., Selig M. K., Torrekens S., Roth S. I., Mackem S., Carmeliet G., Kronenberg H. M., Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev Cell 19, 329–344 (2010). - PMC - PubMed
    1. Street J., Bao M., deGuzman L., Bunting S., Peale F. V. Jr., Ferrara N., Steinmetz H., Hoeffel J., Cleland J. L., Daugherty A., van Bruggen N., Redmond H. P., Carano R. A., Filvaroff E. H., Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover. Proc Natl Acad Sci U S A 99, 9656–9661 (2002). - PMC - PubMed
    1. Fayaz H. C., Giannoudis P. V., Vrahas M. S., Smith R. M., Moran C., Pape H. C., Krettek C., Jupiter J. B., The role of stem cells in fracture healing and nonunion. International Orthopaedics 35, 1587–1597 (2011). - PMC - PubMed

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