Biology subjects

Hilton, M. J.

Publications and source records attributed to Hilton, M. J..

7 recordsLinked to original sources

bioRxiv2026

Specific non-myogenic mesenchymal cells contribute to rotator cuff tear fibrosis and myosteatosis revealing novel therapeutic options

The rotator cuff is a group of four muscles in the shoulder, which aid in movement and rotation of the upper arm. Rotator cuff tears (RCTs) within tendons of these muscles are common musculoskeletal injuries, often resulting in intramuscular fat, fibrosis, and muscle atrophy. Fatty infiltration specifically correlates with high rates of retear following repair. The cellular sources and molecular cues that cause these pathologies are unknown and therefore non-surgical cell/drug therapies for RCTs do not exist. Thus, we first sought to determine the cellular source(s) and molecular underpinnings of fatty atrophy and fibrosis associated with massive RCTs. Using a murine model of massive RCTs combined with lineage tracing, we demonstrate that muscle resident Pdgfra+ non-myogenic mesenchymal cells (NMMCs) are responsible for the fatty and fibrotic RCT pathologies. Utilizing sorted Pdgfra+ cells from rotator cuff muscles and "deep" single cell RNA-sequencing, we identified a specific Dpp4+ cell population associated with RCT-induced fibrosis, while Gfra1+ nerve-associated NMMCs are drivers of the RCT-induced intramuscular fat pathology. Finally, we demonstrate that RCT-induced fatty infiltration occurs at least partially via the loss of GDNF-GFRA1-RET signaling, since local treatment of murine RCTs with a small molecule RET agonist reduces development of the RCT-induced intramuscular fat.

cell biology↗

bioRxiv2021

Interleukin-6 Signaling Mediates Cartilage Degradation and Pain in Post-Traumatic Osteoarthritis

Osteoarthritis (OA) and post-traumatic OA (PTOA) are prevalent joint disorders and leading causes of chronic pain. The disease pathology of OA/PTOA is caused by imbalanced catabolic and anabolic responses and pro-inflammatory changes; however, their connection to pain is not well studied. Since IL-6 is involved in cartilage degradation and conditions of inflammatory pain, we set out to identify whether IL-6 and IL-6 signaling mechanisms contribute to both PTOA-associated cartilage degradation and pain. We performed a modified destabilization of the medial meniscus (DMM) surgery, a model of PTOA, on conventional IL-6 KO and control mice and assessed both cartilage degradation and pain-associated phenotypes. Genetic removal of Il6 in males attenuates PTOA-associated cartilage catabolism, decreases innervation of soft tissues associated with the knee joint, and reduces nociceptive pain signaling, without improving subchondral bone sclerosis or chondrocyte apoptosis. We further demonstrate that specific downstream mediators of IL-6 signaling, the Janus kinases (JAKs), are critical in regulating both cartilage catabolism and pain signaling. We identified STAT3 as a key regulator of cartilage catabolism downstream of JAK; however, inhibition of STAT3 decreases cartilage anabolism while enhancing pain signals. ERK was found to be important for neurite outgrowth and pain signaling; however, inhibition of ERK was less effective in reducing cartilage catabolism. Therefore, our data demonstrate that IL-6 mediates both PTOA-associated cartilage degradation and pain, and provides critical details regarding the downstream mediators of IL-6 signaling as therapeutic targets for disease-modifying osteoarthritis drugs. Single Sentence Summary IL-6 mediates PTOA-associated cartilage degradation and pain via specific downstream signaling mechanisms in a gender specific manner.

pathology↗

bioRxiv2021

Hypertrophic Chondrocytes Serve as a Reservoir for Unique Marrow Associated Skeletal Stem and Progenitor Cells, Osteoblasts, and Adipocytes During Skeletal Development

Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; R26-tdTomatof/+ and Col10a1Cre; R26-tdTomatof/+) in combination with a PDGFRa-H2B-GFP transgenic line, single cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.

developmental biology↗

bioRxiv2020

Activation of Notch1 drives the development of radiation-induced thymic lymphoma in p53 wild-type mice

Mouse models of radiation-induced thymic lymphoma are widely used to study the development of radiation-induced blood cancers and to gain insights into the biology of human T-lymphoblastic leukemia/lymphoma. Here, we aimed to determine key oncogenic drivers for the development of radiation-induced thymic lymphoma by performing whole-exome sequencing using tumors and paired normal tissues from mice with and without irradiation. Thymic lymphomas from irradiated wild-type (WT), p53+/- and KrasLA1 mice were not observed to harbor significantly higher numbers of non-synonymous somatic mutations compared to thymic lymphomas from unirradiated p53-/- mice. However, we observed distinct patterns of recurrent mutations in genes that control the Notch1 signaling pathway based on the mutational status of p53. Preferential activation of Notch1 signaling in p53 WT lymphomas was also observed at the RNA and protein level. Using reporter mice for activation of the Notch1 signaling we observed that TBI enriched Notch1hi CD44+ thymocytes, which are capable of self-renewal in vivo. Mechanistically, genetic inhibition of Notch1 signaling in thymocytes prevented the formation of radiation-induced thymic lymphoma in p53 WT mice. Taken together, our results demonstrate a critical role of activated Notch1 signaling in driving multi-step carcinogenesis of thymic lymphoma following total-body irradiation in p53 WT mice.

cancer biology↗

bioRxiv2020

HES1 is a critical mediator of the SHH-GLI3 axis in regulating digit number

Sonic Hedgehog/GLI3 signaling is critical in regulating digit number, such that Gli3-deficiency results in polydactyly and Shh-deficiency leads to digit number reductions. Anterior-posterior SHH/GLI3 signaling gradients regulate cell cycle factors controlling mesenchymal cell proliferation, while simultaneously regulating Grem1 to coordinate BMP-induced chondrogenesis. SHH/GLI3 also coordinates the expression of additional genes, however their importance in digit formation remain unknown. Utilizing genetic and molecular approaches, we identified HES1 as a key transcriptional regulator downstream of SHH/GLI signaling capable of inducing preaxial polydactyly (PPD), required for Gli3-deficient PPD, and capable of overcoming digit number constraints of Shh-deficiency. Our data indicate that HES1, a direct SHH/GLI signaling target, induces mesenchymal cell proliferation via suppression of Cdkn1b, while inhibiting chondrogenic genes and the anterior autopod boundary regulator, Pax9. These findings fill gaps in knowledge regarding digit number and patterning, while creating a comprehensive framework for our molecular understanding of critical mediators of SHH/GLI3 signaling.

developmental biology↗

bioRxiv2020

Hypoxia Depletes Contaminating CD45+ Hematopoietic Cells from Bone Marrow Stromal Cell (BMSC) Cultures: Methods for BMSC Culture Purification

Culture expanded bone marrow stromal cells (BMSCs) are easily isolated, can be grown rapidly en masse, and contain both skeletal stem cells (SSCs) and multipotent mesenchymal progenitors (MMPs). Despite this functional heterogeneity, BMSC cultures continue to be utilized for many applications due to the lack of definitive and universally accepted markers to prospectively identify and purify SSCs. Isolation of BMSCs is widely based on their adherence to tissue culture plastic; however, this method poses unique challenges when working with murine models. In particular, hematopoietic cell contamination is a significant impediment during the isolation or murine BMSCs. Several strategies to reduce hematopoietic contamination have been developed with varying levels of success. In this study, we found that up to 60% of cells isolated in standard BMSC cultures expressed the hematopoietic marker CD45. Remarkably, when cultured at a physiological oxygen tension of 1% O2, there was a 10-fold reduction in hematopoietic cells associated with a concomitant increase in PDGFR+ expressing stromal cells. This finding was due, in part, to a differential response of the two populations to low oxygen tension, or hypoxia. Specifically, in standard tissue culture conditions of 21% O2, CD45+ hematopoietic cells showed increased proliferation coupled with no observable changes in cell death when compared to their counterparts grown at 1% O2. In contrast, PDGFR+ stromal cells responded to hypoxia by increasing proliferation and exhibiting a 10-fold decrease in cell death. In summary, we describe a simple and reliable method exploiting the divergent biological response of hematopoietic and stromal cells to hypoxia to significantly increase the PDGFR+ stromal cell population in murine BMSC cultures.

cell biology↗

bioRxiv2018

Chondrocyte-specific RUNX2 Overexpression Causes Chondrodysplasia During Development, but is Not Sufficient to Induce OA-like Articular Cartilage Degeneration in Adult Mice Without Injury

RUNX2 is a transcription factor critical for chondrocyte maturation and normal endochondral bone formation. It promotes the expression of factors catabolic to the cartilage extracellular matrix and is shown to be upregulated in human osteoarthritic cartilage and in murine articular cartilage following joint injury. To date, in vivo studies of RUNX2 overexpression in cartilage have been limited to forced expression in osteochondroprogenitor cells preventing investigation into the effects of chondrocyte-specific RUNX2 overexpression during development or in postnatal articular cartilage. Here, we used the Rosa26Runx2 allele in combination with the inducible Col2a1CreERT2 transgene or the inducible AcanCreERT2 knock-in allele to achieve chondrocyte-specific RUNX2 overexpression (OE) during embryonic development or in the postnatal articular cartilage of adult mice, respectively. RUNX2 OE was induced at E13.5 for all developmental studies and resulted in a phenotype resembling chondrodysplasia at E18.5. Histology and in situ hybridization analyses suggest an early onset of chondrocyte hypertrophy and accelerated terminal maturation in the limbs of the RUNX2 OE embryos compared to control embryos. Additionally, RUNX2 OE resulted in enhanced TUNEL staining indicative of increased chondrocyte apoptosis throughout all regions of the growth plate. For all postnatal studies, RUNX2 OE was induced at 2 months of age. Surprisingly, no histopathological signs of OA or cartilage catabolism were observed even six months following induction of RUNX2 OE in postnatal animals. Using the meniscal/ligamentous injury (MLI), a surgical model of knee joint destabilization and meniscal injury, however, we found that chondrocyte-specific RUNX2 OE accelerates the progression of OA pathogenesis following joint trauma. Histomorphometry and OARSI scoring confirmed decreased cartilage area two months following injury in the RUNX2 OE joints compared to control joints. Further, the numbers of MMP13-positive and TUNEL-positive chondrocytes were significantly greater in the articular cartilage of the RUNX2 OE joints compared to control joints one month following injury. Collectively, our data support that RUNX2 OE in growth plate chondrocytes is sufficient to promote their hypertrophy and terminal maturation during development. While RUNX2 overexpression alone is surprisingly insufficient to induce catabolic changes to the postnatal articular cartilage, it can accelerate the progression of post-traumatic OA. These results suggest that although increased RUNX2 expression may predetermine the rate of OA onset and/or progression following traumatic joint injury, alone this change is not sufficient to initiate the OA degenerative process.

pathology↗