Friday, April 25, 2025
4/25/2025
Regulation of Tendon Enthesis Development and Healing via HIF1 - ABSTRACT Achilles tendon ruptures are common musculoskeletal injuries, and the rate of rupture is rising with increased participation in sports. Vascular irregularities and degenerative changes to extracellular matrix (ECM) result in poor biomechanical properties that increase risk of tendon rupture. We have discovered that, following Achilles tendon rupture in mice, the tendon-bone enthesis exhibits profound cell loss, which impairs remodeling and further increases susceptibility to degeneration. This focal cell loss within the enthesis motivated our work to study enthesis cell survival during growth and following tendon injury. Our preliminary studies in mice have established the Achilles enthesis maintains a hypoxic niche during perinatal growth and depends on Hypoxia Inducible Factor-1a (HIF1a) for enthesis cell survival. Targeted loss of Hif1a in tendon and enthesis progenitor cells leads to focal cell death, disruption of enthesis ECM, and failed integration of tendon into bone. Conversely, overexpression of Hif1a rescues cell within the enthesis following Achilles tendon injury, suggesting its potential role in guiding tendon and enthesis regeneration. Therefore, we hypothesize hypoxia is critical for establishing the enthesis progenitor cell/ECM niche, and cell survival in this niche depends on and is enhanced by Hif1a. Our central goal is to define HIF1-dependent mechanisms of cell function and ECM production during growth and repair of the fibrocartilage enthesis. We hypothesize that HIF1a drives enthesis cell differentiation in part by regulating cell survival and ECM deposition. We will use innovative approaches (e.g., in vivo reporters; subcellular spatial RNA-sequencing; synthetic hydrogels; nascent protein labeling) to establish the role of HIF1a and hypoxia in maintaining, establishing, and protecting enthesis progenitors. Our long-term goal is to develop druggable therapeutics to prevent Achilles tendon rupture and improve tendon and enthesis healing. In Aim 1, we will establish the time course of progenitor cell survival, differentiation, and ECM deposition in the mouse Achilles enthesis using inducible and tissue-specific Hif1a-loss and gain of function models. Additionally, we will use synthetic 3D hydrogels to study the effects of HIF1a and hypoxia on tendon/enthesis progenitor cell survival, nascent ECM deposition, and mechanotransduction. In Aim 2, we will use parallel approaches to determine if and how HIF1a contributes to functional healing of Achilles tendon and enthesis using inducible HIF1a LOF and GOF mice with HIF (HIF1a- GFP and HIF2a-mCherry) reporters and structural/functional testing. We will also use novel HIF-targeting drugs to determine the therapeutic potential of HIF-agonists in tendon and enthesis healing. Together, we will identify the emergent role of HIF1a and hypoxia during tendon and enthesis development and repair following injury. Ultimately, this work will establish the potential therapeutic role of HIF1a in tendon and enthesis repair following injury, improving clinical care for the prevention and treatment of common musculoskeletal injuries.
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