Amro A. Hussien

Fibrosis arises from aberrant tissue repair in systemic sclerosis and is characterized by progressive stiffening across multiple tissues, including tendons. How altered tissue mechanics sustain fibrotic remodeling remains poorly understood, in part because of limited experimental models that capture key biophysical features of fibrosis. Here we develop a modular cantilever-based mechano-culture platform that enables tendon-like constructs to be maintained under controlled static tension. We show that elevated matrix tension induces fibroblast-to-myofibroblast activation and scar-like phenotypes in vitro. Analysis of preclinical and clinical models of systemic sclerosis reveals that increased three-dimensional matrix stiffness inversely correlates with transcription of major profibrotic collagens, while positively regulating genes associated with stromal–immune interactions. Co-culture with bone marrow–derived macrophages overrides tension-dependent suppression of matrix gene expression, suggesting that immune cues can supersede mechanical checkpoints. These findings demonstrate how tissue mechanics orchestrates reciprocal interactions between stromal and immune compartments to drive progressive fibrosis, and establish a reductionist platform for dissecting mechano-regulatory pathways in fibrotic diseases.

Tendons are sparsely vascularized connective tissues that link muscles to bones, withstanding some of the highest mechanical stresses in the body. Mechanical overloading and tissue hypervascularity are implicated in tendinopathy, a common musculoskeletal disorder, yet their mechanistic roles remain unclear. Here, we identify hypoxia-inducible factor 1α (HIF1α) as not only a marker but also a driver of tendinopathy. Histological and multiomics evaluation of human tendinopathic samples revealed extensive extracellular matrix remodeling, including pathological collagen cross-linking coinciding with active hypoxic signaling. Hypothesizing a causal contribution of hypoxia signaling, we generated mice with tenocyte-targeted deletions of the von Hippel–Lindau ( Vhl ) gene, which controls hypoxia signaling by regulating HIFα degradation. Vhl inactivation was sufficient to induce pathological hallmarks of tendinopathy, such as collagen matrix disorganization, cross-linking, altered mechanics, and neurovascular ingrowth. This phenotype was HIF1α dependent given that codeleting HIF1α rescued tendon morphology and mechanics. Moreover, deleting vascular endothelial growth factor A ( Vegfa ) alongside VHL effectively suppressed neovascularization but failed to rescue extracellular matrix abnormalities or restore mechanical function, emphasizing a direct role of HIF1α in driving tendon disease independently of angiogenesis. Mechanistically, we found that HIF1α activation was strain dependent in primary cultured human tendon cells and induced by mechanical overload in murine tendon explants. Furthermore, genetically removing Hif1 α from tenocytes prevented aberrant tendon remodeling in response to chronic overload. These findings position HIF1α signaling as a central driver of tendinopathy that acts through a maladaptive tissue response to chronic overload, providing mechanistic insights that could be leveraged for therapeutic approaches.

Tendinopathy is a complex, painful condition that affects up to 5% of the general population in their lifetime. Antibiotic treatment with fluoroquinolones has been associated with the onset of tendinopathy and tendon rupture. The mechanisms behind fluoroquinolone induced tendinopathy remain unclear. To probe activation of potentially causative pathways, we treated juvenile mice with ciprofloxacin in drinking water for 4 weeks and performed RNA sequencing on tail tendons. We discovered that ciprofloxacin‐treated mice had upregulated genes relating to nerve development. Additionally, treated mice showed downregulation of genes associated with extracellular matrix (ECM) processes. We further explored ECM changes using histological and mechanical testing methods on patellar tendons. We found that ciprofloxacin treatment led to altered cell morphology and proteoglycan density. These changes translated to a decrease in mechanical properties of the patellar tendons. Furthermore, ciprofloxacin‐treated mice had a higher percentage of apoptotic cells, and we confirmed increased presence of nerve cells (Plexin B1 ⁺ ) in the patellar tendons compared to controls. Taken together, we showed that ciprofloxacin treatment in juvenile mice induces structural and biological phenotypes commonly associated with fluoroquinolone‐induced tendinopathy and identify the axis of pathological neural activation as a promising area for further exploration. Clinical significance: Oral administration of ciprofloxacin in mice presents a clinically relevant model for studying mechanisms of tendinopathy in humans.

Tendinopathies are debilitating diseases currently increasing in prevalence and associated costs. There is a need to deepen our understanding of the underlying cell signaling pathways to unlock effective treatments. In this work, we screen cell signaling pathways in human tendinopathies and find positively enriched IL-6/JAK/STAT signaling alongside signatures of cell populations typically activated by IL-6 in other tissues. In human tendinopathic tendons, we also confirm the strong presence and co-localization of IL-6, IL-6R, and CD90, an established marker of reparative fibroblasts. To dissect the underlying causalities, we combine IL-6 knock-out mice with an explant-based assembloid model of tendon damage to successfully connect IL-6 signaling to reparative fibroblast activation and recruitment. Vice versa, we show that these reparative fibroblasts promote the development of tendinopathy hallmarks in the damaged explant upon IL-6 activation. We conclude that IL-6 activates tendon fibroblast populations which then initiate and deteriorate tendinopathy hallmarks.

Tendons are mostly avascular dense connective tissues that link muscles to bones, withstanding some of the highest mechanical stresses in the body. Mechanical overload and tissue hypervascularity are implicated in tendinopathy, a common musculoskeletal disorder, but mechanistic understanding of their roles is largely lacking. Here, we identify HIF1α not only as a marker but as a driver of tendinopathy. Initial histological and multi-omics evaluation of human tendinopathic samples revealed extensive extracellular matrix remodeling, including pathological collagen crosslinking coinciding with active hypoxic signaling. Hypothesizing a causal contribution of hypoxia signaling, we generated mice with tenocyte-targeted deletions of the Von Hippel-Lindau (VHL) gene, which controls hypoxia signaling by regulating HIFα degradation. We demonstrated that VHL inactivation suffices to induce pathological hallmarks of tendinopathy, such as collagen matrix disorganization, crosslinking, altered mechanics and neuro-vascular ingrowth. This phenotype was HIF1α-dependent, since co-deleting HIF1α rescued tendon morphology and mechanics. Moreover, deleting vascular endothelial growth factor A (VEGFA) alongside VHL effectively decoupled the effects of vascular ingrowth from persistently aberrant extracellular matrix remodeling and mechanical dysfunction, emphasizing a direct role of HIF1α in driving tendon disease that is independent of angiogenesis. Mechanistically, we linked tendon mechanical overload to the onset of HIF1α signaling in primary cultured human tendon cells. Furthermore, genetically removing HIF1α from tenocytes prevented aberrant tendon remodeling in response to chronic overload. These findings position HIF1α signaling as a central driver of tendinopathy that acts through a maladaptive tissue response to chronic overload, providing mechanistic insights that could be leveraged for improved therapeutic approaches. One Sentence Summary HIF1α activation promotes tendinopathy and its inhibition prevents overload-induced maladaptation, suggesting therapeutic potential.

Tendinopathies are debilitating diseases currently increasing in prevalence and associated costs. There is a need to deepen our understanding of the underlying cell signaling pathways to unlock effective treatments. In this work, we screen cell signaling pathways in human tendinopathies and find positively enriched IL-6/JAK/STAT signaling alongside signatures of cell populations typically activated by IL-6 in other tissues. In human tendinopathic tendons, we also confirm the strong presence and co-localization of IL-6, IL6R, and CD90, an established marker of reparative fibroblasts. To dissect the underlying causalities, we combine IL-6 knock-out mice with an explant-based assembloid model of tendon damage to successfully connect IL-6 signaling to reparative fibroblast activation and recruitment. Vice versa, we show that these reparative fibroblasts promote the development of tendinopathy hallmarks in the damaged explant upon IL-6 activation. We conclude that IL-6 activates tendon fibroblast populations which then initiate and deteriorate tendinopathy hallmarks.

Tendinopathies are debilitating diseases currently increasing in prevalence and associated costs. There is a need to deepen our understanding of the underlying cell signaling pathways to unlock effective treatments. In this work, we screen cell signaling pathways in human tendinopathies and find positively enriched IL-6/JAK/STAT signaling alongside signatures of cell populations typically activated by IL-6 in other tissues. In human tendinopathic tendons, we also confirm the strong presence and co-localization of IL-6, IL6R, and CD90, an established marker of reparative fibroblasts. To dissect the underlying causalities, we combine IL-6 knock-out mice with an explant-based assembloid model of tendon damage to successfully connect IL-6 signaling to reparative fibroblast activation and recruitment. Vice versa, we show that these reparative fibroblasts promote the development of tendinopathy hallmarks in the damaged explant upon IL-6 activation. We conclude that IL-6 activates tendon fibroblast populations which then initiate and deteriorate tendinopathy hallmarks.

Extracellular matrix stiffness is a major regulator of cellular states. Stiffness‐sensing investigations are typically performed using cells that have acquired “mechanical memory” through prolonged conditioning in rigid environments, e.g., tissue culture plastic (TCP). This potentially masks the full extent of the matrix stiffness‐driven mechanosensing programs. Here, a biomaterial composed of 2D mechanovariant silicone substrates with simplified and scalable surface biofunctionalization chemistry is developed to facilitate large‐scale cell culture expansion processes. Using RNA sequencing, stiffness‐mediated mechano‐responses of human tendon‐derived stromal cells are broadly mapped. Matrix elasticity (E.) approximating tendon microscale stiffness range (E. ≈ 35 kPa) distinctly favors transcriptional programs related to chromatin remodeling and Hippo signaling; whereas compliant stiffnesses (E. ≈ 2 kPa) are enriched in processes related to cell stemness, synapse assembly, and angiogenesis. While tendon stromal cells undergo dramatic phenotypic drift on conventional TCP, mechanovariant substrates abrogate this activation with tenogenic stiffnesses inducing a transcriptional program that strongly correlates with established tendon tissue‐specific expression signature. Computational inference predicts that AKT1 and ERK1/2 are major stiffness‐sensing signaling hubs. Together, these findings highlight how matrix biophysical cues may dictate the transcriptional identity of tendon cells, and how matrix mechano‐reciprocity regulates diverse sets of previously underappreciated mechanosensitive processes in tendon fibroblasts.

Tendons are among the most mechanically stressed tissues of the body, with a functional core of type‐I collagen fibers maintained by embedded stromal fibroblasts known as tenocytes. The intrinsic load‐bearing core compartment of tendon is surrounded, nourished, and repaired by the extrinsic peritendon, a synovial‐like tissue compartment with access to tendon stem/progenitor cells as well as blood monocytes. In vitro tendon model systems generally lack this important feature of tissue compartmentalization, while in vivo models are cumbersome when isolating multicellular mechanisms. To bridge this gap, an improved in vitro model of explanted tendon core stromal tissue (mouse tail tendon fascicles) surrounded by cell‐laden collagen hydrogels that mimic extrinsic tissue compartments is suggested. Using this model, CD146⁺ tendon stem/progenitor cell and CD45⁺F4/80⁺ bone‐marrow derived macrophage activity within a tendon injury‐like niche are recapitulated. It is found that extrinsic stromal progenitors recruit to the damaged core, contribute to an overall increase in catabolic ECM gene expression, and accelerate the decrease in mechanical properties. Conversely, it is found that extrinsic bone‐marrow derived macrophages in these conditions adopt a proresolution phenotype that mitigates rapid tissue breakdown by outwardly migrated tenocytes and F4/80⁺ “tenophages” from the intrinsic tissue core.

Adolescent idiopathic scoliosis (AIS) is the most common spine disorder affecting children worldwide, yet little is known about the pathogenesis of this disorder. Here, we demonstrate that genetic regulation of structural components of the axial skeleton, the intervertebral discs, and dense connective tissues (i.e., ligaments and tendons) is essential for the maintenance of spinal alignment. We show that the adhesion G protein-coupled receptor ADGRG6, previously implicated in human AIS association studies, is required in these tissues to maintain typical spine alignment in mice. Furthermore, we show that ADGRG6 regulates biomechanical properties of tendon and stimulates CREB signaling governing gene expression in cartilaginous tissues of the spine. Treatment with a cAMP agonist could mirror aspects of receptor function in culture, thus defining core pathways for regulating these axial cartilaginous and connective tissues. As ADGRG6 is a key gene involved in human AIS, these findings open up novel therapeutic opportunities for human scoliosis.