Biology subjects

Abraham, A. C.

Publications and source records attributed to Abraham, A. C..

9 recordsLinked to original sources

bioRxiv2025

The developing tendon and enthesis are hypoxic and rely on hypoxia-inducible factor 1a (Hif1a) during postnatal development

The tendon-bone enthesis is a specialized fibrocartilaginous tissue crucial for muscle-to-bone force transmission, yet its postnatal development is not fully understood. Emerging evidence suggests hypoxia plays a pivotal role in enthesis maturation akin to its function in skeletal growth plates, with Hypoxia Inducible Factor 1 alpha (HIF-1) acting as a key regulator of cellular adaptation (e.g., cell survival, extracellular matrix (ECM) deposition). Here, we investigated the spatial and temporal dynamics of hypoxia in the murine Achilles tendon enthesis and elucidated the role of Hif1a in enthesis cell survival and ECM formation using Scleraxis-lineage conditional knockout (ScxCre; Hif1a cKO) mice. We found that while neonatal tendons rapidly resolve hypoxia after birth, the enthesis maintains a hypoxic niche through postnatal day 5, mirroring a gradient observed in growth plates. Disruption of HIF-1 in enthesis-resident cells resulted in pronounced deficits in grip strength, abnormal tendon-bone attachment morphology, disrupted calcaneal architecture, impaired mineralization, and significant ECM disorganization. Histological analyses revealed persistent cell death and loss of the characteristic fibrocartilaginous gradient in cKO entheses, including dysregulated collagen alignment. In vitro, HIF-1-deficient tendon fibroblasts exhibited blunted transcriptional responses to hypoxia, altered metabolic gene expression, and changes in ECM deposition. Collectively, our findings illuminate hypoxia as a sustained niche in the postnatal enthesis, with HIF-1 critically required for cell survival, ECM organization, and enthesis structural integrity. This work advances our understanding of enthesis biology and provides insights relevant to tendon-bone attachment disorders and regenerative strategies.

developmental biology↗

bioRxiv2025

Candida auris skin colonization is mediated by Als4112 and interactions with host extracellular matrix proteins

Candida auris is an often multidrug-resistant fungal pathogen notorious for persistent skin colonization and transmission in healthcare settings. However, the mechanisms driving its adherence to skin remain poorly understood. Here, we developed in vitro systems to allow for detailed analysis of early skin colonization events and identified critical host and pathogen mediators of attachment. Across multiple strains and clades of C. auris, we identified that Als4112, a conserved adhesin, is required for skin colonization via keratinocyte attachment and direct interactions with host extracellular matrix (ECM) proteins, especially basement membrane proteins such as laminin. In a murine epicutaneous infection and human skin explants, deletion of ALS4112 significantly reduced skin colonization, underscoring its essential role in establishing cutaneous persistence. Als4112 also contributes to systemic infection, highlighting the connection between adherence and pathogenicity in this organism. Finally, coating plastic and catheter surfaces with collagen I or III markedly inhibited C. auris attachment and biofilm formation, offering an approach to curb nosocomial transmission. Our study highlights the critical role of Als4112 in C. auris colonization and virulence in vivo, making it an attractive target for future vaccine development. This study also explores the potential of specific collagen coatings as a novel strategy to prevent C. auris adherence to abiotic surfaces, offering new therapeutic avenues to control the spread of C. auris in healthcare settings.

microbiology↗

bioRxiv2025

AMPK signaling is dysregulated in human tendinopathy and loss of AMPKα1 leads to cell, matrix and mechanical dysfunction in mouse Achilles tendon

Tendinopathy is a debilitating tendon disorder affecting millions of people, characterized by pain, swelling, and diminished biomechanical properties. While the precise mechanisms underlying tendon homeostasis remain unclear, metabolic regulation plays a critical role. In this study, we combine transcriptomic analysis of human tendinopathic samples with a conditional mouse model in which Prkaa1 (encoding AMPK1) is selectively deleted in tendon progenitors to elucidate the role of AMPK signaling in tendon homeostasis. RNA sequencing of diseased human tendons revealed downregulation of key metabolic genes, including several involved in the mitochondrial electron transport chain and AMPK signaling pathways, alongside an increase in markers associated with senescence and a secretory inflammatory profile. In parallel, mice with loss of Prkaa1 function exhibited normal postnatal development; however, by one month of age, tendons demonstrated widespread transcriptional alterations related to cell cycle regulation and ECM organization. By three months, AMPK1-deficient tendons showed significant reductions in mechanical strength and increased expression of senescence markers p21 and p16, progressing to prominent ectopic calcification with age. In vitro studies further confirmed that tendon fibroblasts lacking AMPK1 have altered ECM substrate adhesion profiles. Importantly, voluntary exercise partially rescued these deficits by enhancing ECM organization and reducing senescence marker expression. Collectively, our findings demonstrate that AMPK1 is critical for maintaining energy balance, regulating ECM remodeling, and preventing premature cellular senescence in tendon. These insights highlight AMPK signaling as a promising therapeutic target and underscore the beneficial role of exercise in mitigating tendinopathic changes.

pathology↗

bioRxiv2024

Functional Changes to Achilles Tendon and Enthesis in a Mouse Model of an Adolescent Masculine Gender-Affirming Hormone Treatment

Many transgender youth seek gender affirming care, such as puberty suppression, to prolong decision-making and to align their physical sex characteristics with their gender identity. During peripubertal growth, connective tissues such as tendon rapidly adapt to applied mechanical loads (e.g., exercise) yet if and how tendon adaptation is influenced by sex and gender affirming hormone therapy during growth remains unknown. The goal of this study was to understand the how pubertal suppression influences the structural and functional properties of the Achilles tendon using an established mouse model of transmasculine gender affirming hormone therapy. C57BL/6N female-born mice were assigned to experimental groups to mimic gender-affirming hormone therapy in human adolescents, and treatment was initiated prior to the onset of puberty (at postnatal day 26, P26). Experimental groups included controls and mice serially treated with gonadotropin release hormone analogue (GnRHa), delayed Testosterone (T), or GnRHa followed by T. We found that puberty suppression using GnRHa, with and without T, improved the overall tendon load capacity in female-born mice. Treatment with T resulted in an increase in the maximum load that tendon can withstand before failure. Additionally, we found that GnRHa, but not T, treatment resulted in a significant increase in cell density at the Achilles enthesis. NEW & NOTEWORTHYThese findings demonstrate that puberty suppression or testosterone does not negatively influence tendon structural or functional properties in a mouse model of transmasculine gender affirming care. In all treatment groups, the ability of the tendon to withstand load was significantly increased. Puberty suppression with GnRHa significantly increased enthesis cell density, suggesting an extended growth phase. These findings elucidate the effects of gender affirming care on the structural and functional properties of the tendon and enthesis.

physiology↗

bioRxiv2024

Overexpression of enhanced yellow fluorescent protein fused with Channelrhodopsin-2 causes contractile dysfunction in skeletal muscle

Skeletal muscle activation using optogenetics has emerged as a promising technique for inducing noninvasive muscle contraction and assessing muscle function both in vivo and in vitro. Transgenic mice overexpressing the optogenetic fusion protein, Channelphodopsin2-EYFP (ChR2-EYFP) in skeletal muscle are widely used; however, overexpression of fluorescent proteins can negatively impact the functionality of activable tissues. In this study, we characterized the contractile properties of ChR2-EYFP skeletal muscle and introduced the ChR2-only mouse model that expresses light-responsive ChR2 without the fluorescent EYFP in their skeletal muscles. We found a significant reduction in the contractile ability of ChR2-EYFP muscles compared to ChR2-only and WT mice, observed under both electrical and optogenetic stimulation paradigms. Bulk RNAseq identified downregulation of genes associated with transmembrane transport and metabolism in ChR2-EYFP muscle, while the ChR2-only muscle did not demonstrate any notable deviations from WT muscle. The RNAseq results were further corroborated by a reduced protein-level expression of ion-channel-related HCN2 in ChR2-EYFP muscles and gluconeogenesis-modulating FBP2 in both ChR2-EYFP and ChR2-only muscles. Overall, this study reveals an intrinsic skeletal dysfunction in the widely used ChR2-EYFP mice model and underscores the importance of considering alternative optogenetic models, such as the ChR2-only, for future research in skeletal muscle optogenetics.

physiology↗

bioRxiv2023

Nuclear Factor Kappa B Over-Activation in the Intervertebral Disc Leads to Macrophage Recruitment and Severe Disc Degeneration

ObjectiveLow back pain (LBP) is the leading cause of global disability and is thought to be driven primarily by intervertebral disc (IVD) degeneration (DD). Persistent upregulation of catabolic enzymes and inflammatory mediators have been associated with severe cases of DD. Nuclear factor kappa B (NF-{kappa}B) is a master transcription regulator of immune responses and is over expressed during inflammatory-driven musculoskeletal diseases, including DD. However, its role in triggering DD is unknown. Therefore, this study investigated the effect of NF-{kappa}B pathway over-activation on IVD integrity and DD pathology. MethodsUsing skeletally mature mouse model, we genetically targeted IVD cells for canonical NF-{kappa}B pathway activation via expression of a constitutively active form of inhibitor of {kappa}B kinase B (IKK{beta}), and assessed changes in IVD cellularity, structural integrity including histology, disc height, and extracellular matrix (ECM) biochemistry, biomechanics, expression of inflammatory, catabolic, and neurotropic mediators, and changes in macrophage subsets, longitudinally up to 6-months post activation. ResultsProlonged NF-{kappa}B activation led to severe structural degeneration, with a loss of glycosaminoglycan (GAG) content and complete loss of nucleus pulposus (NP) cellularity. Structural and compositional changes decreased IVD height and compressive mechanical properties with prolonged NF-{kappa}B activation. These alterations were accompanied by increases in gene expression of inflammatory molecules (Il1b, Il6, Nos2), chemokines (Mcp1, Mif), catabolic enzymes (Mmp3, Mmp9, Adamts4), and neurotrophic factors (Bdnf, Ngf) within IVD tissue. Increased recruitment of activated F4/80+ macrophages exhibited a greater abundance of pro-inflammatory (CD38+) over inflammatory-resolving (CD206+) macrophage subsets in the IVD, with temporal changes in the relative abundance of macrophage subsets over time, providing evidence for temporal regulation of macrophage polarization in DD in vivo, where macrophages participate in resolving the inflammatory cascade but promote fibrotic transformation of the IVD matrix. We further show that NF-{kappa}B driven secretory factors from IVD cells increase macrophage migration and inflammatory activation, and that the secretome of inflammatory-resolving macrophages mitigates effects of NF-{kappa}B overactivation. ConclusionOverall the observed results suggest prolonged NF-{kappa}B activation can induce severe DD, acting through increases in inflammatory cytokines, chemotactic proteins, catabolic enzymes, and the recruitment and inflammatory activation of a macrophage cell populations, that can be mitigated with inflammatory-resolving macrophage secretome.

bioengineering↗

bioRxiv2023

Optogenetic-Induced Muscle Loading Leads to Mechanical Adaptation of the Achilles Tendon Enthesis in Mice

The growth of the skeleton depends on the transmission of contractile muscle forces from tendon to bone across the extracellular matrix-rich enthesis. Loss of muscle loading leads to significant impairments in enthesis development. However, little is known about how the enthesis responds to increased loading during postnatal growth. To study the cellular and matrix adaptations of the enthesis in response to increased muscle loading, we used optogenetics to induce skeletal muscle contraction and unilaterally load the Achilles tendon and enthesis in young (i.e., during growth) and adult (i.e., mature) mice. In young mice, daily bouts of unilateral optogenetic loading led to expansion of the calcaneal apophysis and growth plate, as well as increased vascularization of the normally avascular enthesis. Daily loading bouts, delivered for 3 weeks, also led to a mechanically weaker enthesis with increased molecular-level accumulation of collagen damage in young mice. However, adult mice did not exhibit impaired mechanical properties or noticeable structural adaptations to the enthesis. We then focused on the transcriptional response of the young tendon and bone following optogenetic-induced loading. After 1 or 2 weeks of loading, we identified, in tendon, transcriptional activation of canonical pathways related to glucose metabolism (glycolysis) and inhibited pathways associated with cytoskeletal remodeling (e.g., RHOA and CREB signaling). In bone, we identified activation of inflammatory signaling (e.g., NFkB and STAT3 signaling) and inhibition of ERK/MAPK and PTEN signaling. Thus, we have demonstrated the utility of optogenetic-induced skeletal muscle contraction to elicit structural, functional, and molecular adaptation of the enthesis in vivo especially during growth.

bioengineering↗

bioRxiv2022

The role of TGFβ signaling in Gli1+ tendon and enthesis cells

The development of musculoskeletal tissues such as tendon, enthesis, and bone relies on proliferation and differentiation of mesenchymal progenitor cells. Gli1+ cells have been described as putative stem cells in several tissues and are presumed to play critical roles in tissue formation and maintenance. For example, the enthesis, a fibrocartilage tissue that connects tendon to bone, is mineralized postnatally by a pool of Gli1+ progenitor cells. These cells are regulated by hedgehog signaling, but it is unclear if TGF{beta} signaling, necessary for tenogenesis, also plays a role in their behavior. To examine the role of TGF{beta} signaling in Gli1+ cell function, the receptor for TGF{beta}, TbR2, was deleted in Gli1-lineage cells in mice at P5. Decreased TGF{beta} signaling in these cells led to defects in enthesis formation by P56, including deficient bone morphometry underlying the enthesis and decreased mechanical properties. In vitro experiments using Gli1+ cells isolated from mouse tail tendons demonstrated that TGF{beta} controls cell proliferation and differentiation through canonical and non-canonical pathways and that TGF{beta} directly controls the tendon transcription factor scleraxis by binding to its distant enhancer. These results have implications in the development of treatments for tendon and enthesis pathologies.

developmental biology↗

bioRxiv2021

Toughening mechanisms for the attachment of architectured materials: The mechanics of the tendon enthesis

Architectured materials offer tailored mechanical properties but are limited in engineering applications due to challenges in maintaining toughness across their attachments. The enthesis connects tendon and bone, two vastly different architectured materials, and exhibits toughness across a wide range of loadings. Understanding the mechanisms by which this is achieved could inform the development of engineered attachments. Integrating experiments, simulations, and novel imaging that enabled simultaneous observation of mineralized and unmineralized tissues, we identified putative mechanisms of enthesis toughening in a mouse model and then manipulated these mechanisms via in vivo control of mineralization and architecture. Imaging uncovered a fibrous architecture within the enthesis that controls trade-offs between strength and toughness. In vivo models of pathology revealed architectural adaptations that optimize these trade-offs through cross-scale mechanisms including nanoscale protein denaturation, milliscale load-sharing, and macroscale energy absorption. Results suggest strategies for optimizing architecture for tough bimaterial attachments in medicine and engineering. TeaserThe architecture of the tendon-to-bone attachment is designed for toughness.

bioengineering↗