Collagen Organization of Mouse Tendons and Mechanotransduction Gene Expression of Mouse Tendon Fibroblasts are Dependent on HIF1-alpha and Oxygen tension

Abstract

Tendons are essential for transmitting skeletal muscle loads to bone yet tendons are unable to regenerate following load-induced injury.1–3 A major challenge in regenerating tendons lies in their low cell number, poor vascularization, and extracellular matrix (ECM) dense environment. The primary cells in tendons are fibroblasts (TFs) which form and organize the nascent collagen-rich ECM necessary for mechanical function and mechanotransduction. Eventually, TFs become embedded in this dense ECM with limited access to nutrients, where these cells also experience low levels of turnover. Development and elongation of tendon depends on the transcription factor, Scleraxis (Scx), which regulates the proliferation and differentiation of TF progenitors under mechanical loading while also promoting collagen deposition and organization.4-9 Collagen deposition is also controlled by hypoxia inducible factor-1a ( Hif1a), and we have recently shown that mouse Achilles tendons are under hypoxic stress during embryonic development, and the maturation of tendon enthesis relies on expression of Hif1a10,11. Hif1a regulates expression of Sox9 and genes associated with ECM deposition, such as Col1a1 and Col1a2.12 Yet, if and how hypoxia and Hif1a influences tendon organization and mechanotransductive gene expression remains unknown. In this study, we tested the hypothesis that both hypoxia and loss of Hif1a contribute to ECM organization in vivo and mechanotransductive gene expression in vitro. Hif1afl/wt wildtype (WT) and Hif1afl/fl; ScxCre+ ( Hif1acKO) mice were generated to study tendon ECM organization of developing (Postnatal day 14, P14) and mature (P56) Achilles tendons. Mouse hindlimbs were fixed, decalcified, paraffn sectioned, and stained with Picrosirius red to visualize collagen organization (n=3/genotype at both P14 and P56). Midsubstance Achilles tendons were imaged using brightfield and circular polarized light microscopy on an epifluorescent microscope (Leica). Images were acquired using a polarization camera and ThorCam software (ThorLabs Inc.) for quantitative polarized light microscopy (qPLI). Gene expression related to ECM ( Col1a1, Col1a2), progenitor markers ( Scx, Sox9), hypoxia ( Hif1a, Hif2a, Hilpda, Plin2), and mechanotransduction (Igfbp2, Igfbp3, and Igfbp9) was assessed using isolated and cultured mouse tail TFs from adult Hif1acKO and WT mice (n=3-4/group, age 4-6 months of age). Cells were cultured in normoxic (20% O2) or hypoxic (1% O2) conditions (for 1-wk) or were treated with a HIF stabilizer Roxadustat (FG-4592) to induce ‘pseudo-hypoxia’ or treated with a vehicle (DMSO) for 16h. RNA was isolated after 16h or 1-wk in culture and cDNA was used in SYBR-based quantitative PCR (qPCR). Data were normalized to 20% O2 conditions for fold change comparisons (2−ΔΔCq; Actb and Polr2a reference genes). Hif1acKO Achilles tendons had poor ECM organization compared to WT tendons at both ages. TFs from Hif1acKO mice had lower Hif1a expression compared to WT cells in both 20% and 1% O2. For WT TFs, Scx and Igfbp9 were downregulated in hypoxia compared to 20% O2 conditions, and the effect of hypoxia on Hif1acKO cells further downregulated Igfbp9 compared to 20% O2. Loss of Hif1a reduced Hilpda expression compared to WT TFs in hypoxia. FG treatment also resulted in increased Hilpda expression compared to hypoxia. Our findings support our hypothesis that Hif1a and hypoxia are regulators of ECM organization and mechanotransduction in mouse tendons. Identifying regulators of tendon ECM deposition and organization will inform therapeutic strategies for guided regeneration. REFERENCES: 1Fu C, et al. J Orthop Res. 2021.2Leong NL, et al. J Orthop Res. 2020. 3Shah SA, et al. J Orthop Res. 2017. 4Murchison ND, et al. Development. 2007. 5Killian ML, et al. FASEB j. 2016. 6Ideo K, et al. PLoS ONE. 2020.7Blitz E, et al. Development. 2013. 8Huang AH, et al. Development. 2019. 9Schweitzer R, et al. Development. 2001. 10Tata Z, et al. 2022, Proceedings of the ORS Annual Meeting. 11Phillips, et al. 2023, Proceedings of the ORS Annual Meeting.12Amarilio R, et al. Development. 2007. ACKNOWLEDGEMENTS: NIH NIAMS (R01AR079367, MLK; P30AR069620), NSF (CAREER 1944448, MLK). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.