RESUMEN

End stage liver disease is marked by portal hypertension, systemic elevations in ammonia, and development of hepatocellular carcinoma (HCC). While these clinical consequences of cirrhosis are well described, it remains poorly understood whether hepatic insufficiency and the accompanying elevations in ammonia contribute to HCC carcinogenesis. Using preclinical models, we discovered that ammonia entered the cell through the transporter SLC4A11 and served as a nitrogen source for amino acid and nucleotide biosynthesis. Elevated ammonia promoted cancer stem cell properties in vitro and tumor initiation in vivo. Enhancing ammonia clearance reduced HCC stemness and tumor growth. In patients, elevations in serum ammonia were associated with an increased incidence of HCC. Taken together, this study forms the foundation for clinical investigations using ammonia lowering agents as potential therapies to mitigate HCC incidence and aggressiveness.

RESUMEN

Radiotherapy induces a type I interferon-mediated (T1IFN-mediated) antitumoral immune response that we hypothesized could be potentiated by a first-in-class ataxia telangiectasia mutated (ATM) inhibitor, leading to enhanced innate immune signaling, T1IFN expression, and sensitization to immunotherapy in pancreatic cancer. We evaluated the effects of AZD1390 or a structurally related compound, AZD0156, on innate immune signaling and found that both inhibitors enhanced radiation-induced T1IFN expression via the POLIII/RIG-I/MAVS pathway. In immunocompetent syngeneic mouse models of pancreatic cancer, ATM inhibitor enhanced radiation-induced antitumoral immune responses and sensitized tumors to anti-PD-L1, producing immunogenic memory and durable tumor control. Therapeutic responses were associated with increased intratumoral CD8+ T cell frequency and effector function. Tumor control was dependent on CD8+ T cells, as therapeutic efficacy was blunted in CD8+ T cell-depleted mice. Adaptive immune responses to combination therapy provided systemic control of contralateral tumors outside of the radiation field. Taken together, we show that a clinical candidate ATM inhibitor enhances radiation-induced T1IFN, leading to both innate and subsequent adaptive antitumoral immune responses and sensitization of otherwise resistant pancreatic cancer to immunotherapy.

Asunto(s)

Ataxia Telangiectasia , Interferón Tipo I , Neoplasias Pancreáticas , Piridinas , Quinolonas , Animales , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/radioterapia , Neoplasias Pancreáticas/patología , Inmunidad

RESUMEN

Introduction: HIV-related comorbidities appear to be related to chronic inflammation, a condition characterizing people living with HIV (PLWH). Prior work indicates that cannabidiol (CBD) might reduce inflammation; however, the genetics underpinning of this effect are not well investigated. Our main objective is to detect gene expression alterations in human peripheral blood mononuclear cells (PBMCs) from PLWH after at least 1 month of CBD treatment. Materials and Methods: We analyzed ∼41,000 PBMCs from three PLWH at baseline and after CBD treatment (27-60 days) through single-cell RNA sequencing. Results: We obtained a coherent signature, characterized by an anti-inflammatory activity, of differentially expressed genes in myeloid cells. Conclusions: Our study shows how CBD is associated with alterations of gene expression in myeloid cells after CBD treatment. Clinical Trial Registration: NCT05209867.

Asunto(s)

Cannabidiol , Infecciones por VIH , Células Mieloides , Humanos , Cannabidiol/farmacología , Cannabidiol/administración & dosificación , Cannabidiol/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Masculino , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Adulto , Persona de Mediana Edad , Femenino , Antiinflamatorios/farmacología , Antiinflamatorios/administración & dosificación , Administración Oral , Transcriptoma/efectos de los fármacos , Inflamación/tratamiento farmacológico , Inflamación/genética

RESUMEN

OBJECTIVE: To characterize the role of neutrophil extracellular traps (NETs) in heterotopic ossification (HO) formation and progression and to use mechanical and pharmacological methods to decrease NETosis and mitigate HO formation. BACKGROUND: Traumatic HO is the aberrant osteochondral differentiation of mesenchymal progenitor cells after traumatic injury, burns, or surgery. While the innate immune response has been shown to be necessary for HO formation, the specific immune cell phenotype and function remain unknown. Neutrophils, one of the earliest immune cells to respond after HO-inducing injuries, can extrude DNA, forming highly inflammatory NETs. We hypothesized that neutrophils and NETs would be diagnostic biomarkers and therapeutic targets for the detection and mitigation of HO. METHODS: C57BL6J mice underwent burn/tenotomy (a well-established mouse model of HO) or a non-HO-forming sham injury. These mice were either (1) ambulated ad libitum, (2) ambulated ad libitum with daily intraperitoneal hydroxychloroquine, ODN-2088 (both known to affect NETosis pathways), or control injections, or (3) had the injured hind limb immobilized. Single-cell analysis was performed to analyze neutrophils, NETosis, and downstream signaling after the HO-forming injury. Immunofluorescence microscopy was used to visualize NETosis at the HO site and neutrophils were identified using flow cytometry. Serum and cell lysates from HO sites were analyzed using enzyme-linked immunosorbent assay for myeloperoxidase-DNA and ELA2-DNA complexes to identify NETosis. Micro-computerized tomography was performed on all groups to analyze the HO volume. RESULTS: Molecular and transcriptional analyses revealed the presence of NETs within the HO injury site, which peaked in the early phases after injury. These NETs were highly restricted to the HO site, with gene signatures derived from both in vitro NET induction and clinical neutrophil characterizations showing a high degree of NET "priming" at the site of injury, but not in neutrophils in the blood or bone marrow. Cell-cell communication analyses revealed that this localized NET formation coincided with high levels of toll-like receptor signaling specific to neutrophils at the injury site. Reducing the overall neutrophil abundance within the injury site, either pharmacologically through treatment with hydroxychloroquine, the toll-like receptor 9 inhibitor OPN-2088, or mechanical treatment with limb offloading, results in the mitigation of HO formation. CONCLUSIONS: These data provide a further understanding of the ability of neutrophils to form NETs at the injury site, clarify the role of neutrophils in HO, and identify potential diagnostic and therapeutic targets for HO mitigation.

Asunto(s)

Trampas Extracelulares , Neutrófilos , Animales , Ratones , Neutrófilos/metabolismo , Hidroxicloroquina/metabolismo , Trampas Extracelulares/metabolismo , Inmunidad Innata , ADN/metabolismo

RESUMEN

Effective therapies are lacking for patients with advanced colorectal cancer (CRC). The CRC tumor microenvironment has elevated metabolic waste products due to altered metabolism and proximity to the microbiota. The role of metabolite waste in tumor development, progression, and treatment resistance is unclear. We generated an autochthonous metastatic mouse model of CRC and used unbiased multi-omic analyses to reveal a robust accumulation of tumoral ammonia. The high ammonia levels induce T cell metabolic reprogramming, increase exhaustion, and decrease proliferation. CRC patients have increased serum ammonia, and the ammonia-related gene signature correlates with altered T cell response, adverse patient outcomes, and lack of response to immune checkpoint blockade. We demonstrate that enhancing ammonia clearance reactivates T cells, decreases tumor growth, and extends survival. Moreover, decreasing tumor-associated ammonia enhances anti-PD-L1 efficacy. These findings indicate that enhancing ammonia detoxification can reactivate T cells, highlighting a new approach to enhance the efficacy of immunotherapies.

Asunto(s)

Amoníaco , Neoplasias Colorrectales , Animales , Ratones , Agotamiento de Células T , Linfocitos T , Neoplasias Colorrectales/patología , Inmunoterapia , Microambiente Tumoral

RESUMEN

CRISPR screening is a powerful tool that links specific genetic alterations to corresponding phenotypes, thus allowing for high-throughput identification of novel gene functions. Pooled CRISPR screens have enabled discovery of innate and adaptive immune response regulators in the setting of viral infection and cancer. Emerging methods couple pooled CRISPR screens with parallel high-content readouts at the transcriptomic, epigenetic, proteomic, and optical levels. These approaches are illuminating cancer immune evasion mechanisms as well as nominating novel targets that augment T cell activation, increase T cell infiltration into tumors, and promote enhanced T cell cytotoxicity. This review details recent methodological advances in high-content CRISPR screens and highlights the impact this technology is having on tumor immunology.

Asunto(s)

Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteómica , Epigenómica

RESUMEN

Transforming growth factor-ß1 (TGF-ß1) plays a central role in normal and aberrant wound healing, but the precise mechanism in the local environment remains elusive. Here, using a mouse model of aberrant wound healing resulting in heterotopic ossification (HO) after traumatic injury, we find autocrine TGF-ß1 signaling in macrophages, and not mesenchymal stem/progenitor cells, is critical in HO formation. In-depth single-cell transcriptomic and epigenomic analyses in combination with immunostaining of cells from the injury site demonstrated increased TGF-ß1 signaling in early infiltrating macrophages, with open chromatin regions in TGF-ß1-stimulated genes at binding sites specific for transcription factors of activated TGF-ß1 (SMAD2/3). Genetic deletion of TGF-ß1 receptor type 1 (Tgfbr1; Alk5), in macrophages, resulted in increased HO, with a trend toward decreased tendinous HO. To bypass the effect seen by altering the receptor, we administered a systemic treatment with TGF-ß1/3 ligand trap TGF-ßRII-Fc, which resulted in decreased HO formation and a delay in macrophage infiltration to the injury site. Overall, our data support the role of the TGF-ß1/ALK5 signaling pathway in HO.

Asunto(s)

Osificación Heterotópica , Factor de Crecimiento Transformador beta1 , Humanos , Cromatina/metabolismo , Ligandos , Macrófagos/metabolismo , Osificación Heterotópica/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta1/metabolismo , Cicatrización de Heridas , Factor de Crecimiento Transformador beta/metabolismo

RESUMEN

Tumor-associated macrophages (TAMs) are a major cellular component in the tumor microenvironment (TME). However, the relationship between the phenotype and metabolic pattern of TAMs remains poorly understood. We performed single-cell transcriptome profiling on hepatic TAMs from mice bearing liver metastatic tumors. We find that TAMs manifest high heterogeneity at the levels of transcription, development, metabolism, and function. Integrative analyses and validation experiments indicate that increased purine metabolism is a feature of TAMs with pro-tumor and terminal differentiation phenotypes. Like mouse TAMs, human TAMs are highly heterogeneous. Human TAMs with increased purine metabolism exhibit a pro-tumor phenotype and correlate with poor therapeutic efficacy to immune checkpoint blockade. Altogether, our work demonstrates that TAMs are developmentally, metabolically, and functionally heterogeneous and purine metabolism may be a key metabolic feature of a pro-tumor macrophage population.

Asunto(s)

Neoplasias Hepáticas , Microambiente Tumoral , Animales , Perfilación de la Expresión Génica , Neoplasias Hepáticas/patología , Macrófagos/metabolismo , Ratones , Macrófagos Asociados a Tumores

RESUMEN

Pain is a central feature of soft tissue trauma, which under certain contexts, results in aberrant osteochondral differentiation of tissue-specific stem cells. Here, the role of sensory nerve fibers in this abnormal cell fate decision is investigated using a severe extremity injury model in mice. Soft tissue trauma results in NGF (Nerve growth factor) expression, particularly within perivascular cell types. Consequently, NGF-responsive axonal invasion occurs which precedes osteocartilaginous differentiation. Surgical denervation impedes axonal ingrowth, with significant delays in cartilage and bone formation. Likewise, either deletion of Ngf or two complementary methods to inhibit its receptor TrkA (Tropomyosin receptor kinase A) lead to similar delays in axonal invasion and osteochondral differentiation. Mechanistically, single-cell sequencing suggests a shift from TGFß to FGF signaling activation among pre-chondrogenic cells after denervation. Finally, analysis of human pathologic specimens and databases confirms the relevance of NGF-TrkA signaling in human disease. In sum, NGF-mediated TrkA-expressing axonal ingrowth drives abnormal osteochondral differentiation after soft tissue trauma. NGF-TrkA signaling inhibition may have dual therapeutic use in soft tissue trauma, both as an analgesic and negative regulator of aberrant stem cell differentiation.

Asunto(s)

Diferenciación Celular , Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Transducción de Señal , Heridas y Lesiones/metabolismo , Animales , Axones/metabolismo , Cartílago/metabolismo , Ratones , Ratones Endogámicos C57BL , Factor de Crecimiento Nervioso/genética , Osteogénesis , Células Madre/metabolismo , Heridas y Lesiones/patología

RESUMEN

Heterotopic ossification (HO) is a devastating condition in which ectopic bone forms inappropriately in soft tissues following traumatic injuries and orthopedic surgeries as a result of aberrant mesenchymal progenitor cell (MPC) differentiation. HO leads to chronic pain, decreased range of motion, and an overall decrease in quality of life. While several treatments have shown promise in animal models, all must be given during early stages of formation. Methods for early determination of whether and where endochondral ossification/soft tissue mineralization (HO anlagen) develop are lacking. At-risk patients are not identified sufficiently early in the process of MPC differentiation and soft tissue endochondral ossification for potential treatments to be effective. Hence, a critical need exists to develop technologies capable of detecting HO anlagen soon after trauma, when treatments are most effective. In this study, we investigate high frequency spectral ultrasound imaging (SUSI) as a noninvasive strategy to identify HO anlagen at early time points after injury. We show that by determining quantitative parameters based on tissue organization and structure, SUSI identifies HO anlagen as early as 1-week postinjury in a mouse model of burn/tenotomy and 3 days postinjury in a rat model of blast/amputation. We analyze single cell RNA sequencing profiles of the MPCs responsible for HO formation and show that the early tissue changes detected by SUSI match chondrogenic and osteogenic gene expression in this population. SUSI identifies sites of soft tissue endochondral ossification at early stages of HO formation so that effective intervention can be targeted when and where it is needed following trauma-induced injury. Furthermore, we characterize the chondrogenic to osteogenic transition that occurs in the MPCs during HO formation and correlate gene expression to SUSI detection of the HO anlagen.

Asunto(s)

Modelos Animales de Enfermedad , Osificación Heterotópica/diagnóstico por imagen , Osificación Heterotópica/genética , Ultrasonografía/métodos , Animales , Quemaduras/diagnóstico por imagen , Quemaduras/genética , Diferenciación Celular/genética , Condrogénesis/genética , Perfilación de la Expresión Génica/métodos , Ontología de Genes , Humanos , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Osteogénesis/genética , RNA-Seq/métodos , Ratas Sprague-Dawley , Roedores , Análisis de la Célula Individual/métodos , Tenotomía , Microtomografía por Rayos X/métodos

RESUMEN

Heterotopic ossification (HO) is a form of pathological cell-fate change of mesenchymal stem/precursor cells (MSCs) that occurs following traumatic injury, limiting range of motion in extremities and causing pain. MSCs have been shown to differentiate to form bone; however, their lineage and aberrant processes after trauma are not well understood. Utilizing a well-established mouse HO model and inducible lineage-tracing mouse (Hoxa11-CreERT2;ROSA26-LSL-TdTomato), we found that Hoxa11-lineage cells represent HO progenitors specifically in the zeugopod. Bioinformatic single-cell transcriptomic and epigenomic analyses showed Hoxa11-lineage cells are regionally restricted mesenchymal cells that, after injury, gain the potential to undergo differentiation toward chondrocytes, osteoblasts, and adipocytes. This study identifies Hoxa11-lineage cells as zeugopod-specific ectopic bone progenitors and elucidates the fate specification and multipotency that mesenchymal cells acquire after injury. Furthermore, this highlights homeobox patterning genes as useful tools to trace region-specific progenitors and enable location-specific gene deletion.

Asunto(s)

Huesos/metabolismo , Diferenciación Celular , Linaje de la Célula , Células Madre Mesenquimatosas/metabolismo , Osificación Heterotópica/genética , Osificación Heterotópica/metabolismo , Osteogénesis , Adipocitos/metabolismo , Animales , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Expresión Génica Ectópica , Epigenómica , Femenino , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , Osificación Heterotópica/patología , Osteoblastos/metabolismo , Análisis de la Célula Individual , Tendones/metabolismo

RESUMEN

Ischemia reperfusion (IR) injury results in devastating skeletal muscle fibrosis. Here, we recapitulate this injury with a mouse model of hindlimb IR injury which leads to skeletal muscle fibrosis. Injury resulted in extensive immune infiltration with robust neutrophil extracellular trap (NET) formation in the skeletal muscle, however, direct targeting of NETs via the peptidylarginine deiminase 4 (PAD4) mechanism was insufficient to reduce muscle fibrosis. Circulating levels of IL-10 and TNFα were significantly elevated post injury, indicating toll-like receptor (TLR) signaling may be involved in muscle injury. Administration of hydroxychloroquine (HCQ), a small molecule inhibitor of TLR7/8/9, following injury reduced NET formation, IL-10, and TNFα levels and ultimately mitigated muscle fibrosis and improved myofiber regeneration following IR injury. HCQ treatment decreased fibroadipogenic progenitor cell proliferation and partially inhibited ERK1/2 phosphorylation in the injured tissue, suggesting it may act through a combination of TLR7/8/9 and ERK signaling mechanisms. We demonstrate that treatment with FDA-approved HCQ leads to decreased muscle fibrosis and increased myofiber regeneration following IR injury, suggesting short-term HCQ treatment may be a viable treatment to prevent muscle fibrosis in ischemia reperfusion and traumatic extremity injury.

Asunto(s)

Trampas Extracelulares/metabolismo , Músculo Esquelético/metabolismo , Enfermedades Musculares/metabolismo , Neutrófilos/metabolismo , Daño por Reperfusión/metabolismo , Transducción de Señal/fisiología , Receptores Toll-Like/metabolismo , Animales , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , Fibrosis/metabolismo , Interleucina-10/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Arginina Deiminasa Proteína-Tipo 4/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Cells sense the extracellular environment and mechanical stimuli and translate these signals into intracellular responses through mechanotransduction, which alters cell maintenance, proliferation, and differentiation. Here we use a mouse model of trauma-induced heterotopic ossification (HO) to examine how cell-extrinsic forces impact mesenchymal progenitor cell (MPC) fate. After injury, single-cell (sc) RNA sequencing of the injury site reveals an early increase in MPC genes associated with pathways of cell adhesion and ECM-receptor interactions, and MPC trajectories to cartilage and bone. Immunostaining uncovers active mechanotransduction after injury with increased focal adhesion kinase signaling and nuclear translocation of transcriptional coactivator TAZ, inhibition of which mitigates HO. Similarly, joint immobilization decreases mechanotransductive signaling, and completely inhibits HO. Joint immobilization decreases collagen alignment and increases adipogenesis. Further, scRNA sequencing of the HO site after injury with or without immobilization identifies gene signatures in mobile MPCs correlating with osteogenesis, and signatures from immobile MPCs with adipogenesis. scATAC-seq in these same MPCs confirm that in mobile MPCs, chromatin regions around osteogenic genes are open, whereas in immobile MPCs, regions around adipogenic genes are open. Together these data suggest that joint immobilization after injury results in decreased ECM alignment, altered MPC mechanotransduction, and changes in genomic architecture favoring adipogenesis over osteogenesis, resulting in decreased formation of HO.

Asunto(s)

Extremidades/lesiones , Células Madre Mesenquimatosas/patología , Células Madre Mesenquimatosas/fisiología , Osificación Heterotópica/etiología , Restricción Física , Aciltransferasas , Adipogénesis/genética , Animales , Diferenciación Celular , Linaje de la Célula , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Quinasa 1 de Adhesión Focal/deficiencia , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Masculino , Mecanotransducción Celular/genética , Mecanotransducción Celular/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osificación Heterotópica/patología , Osificación Heterotópica/fisiopatología , Osteogénesis/genética , Restricción Física/efectos adversos , Restricción Física/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Heterotopic ossification (HO) is defined as ectopic bone formation around joints and in soft tissues following trauma, particularly blast-related extremity injuries, thermal injuries, central nerve injuries, or orthopaedic surgeries, leading to increased pain and diminished quality of life. Current treatment options include pharmacotherapy with non-steroidal anti-inflammatory drugs, radiotherapy, and surgical excision, but these treatments have limited efficacy and have associated complication profiles. In contrast, small molecule inhibitors have been shown to have higher specificity and less systemic cytotoxicity. Previous studies have shown that bone morphogenetic protein (BMP) signaling and downstream non-canonical (SMAD-independent) BMP signaling mediated induction of TGF-ß activated kinase-1 (TAK1) contributes to HO. In the current study, small molecule inhibition of TAK1, NG-25, was evaluated for its efficacy in limiting ectopic bone formation following a rat blast-associated lower limb trauma and a murine burn tenotomy injury model. A significant decrease in total HO volume in the rat blast injury model was observed by microCT imaging with no systemic complications following NG-25 therapy. Furthermore, tissue-resident mesenchymal progenitor cells (MPCs) harvested from rats treated with NG-25 demonstrated decreased proliferation, limited osteogenic differentiation capacity, and reduced gene expression of Tac1, Col10a1, Ibsp, Smad3, and Sox2 (P < 0.05). Single cell RNA-sequencing of murine cells harvested from the injury site in a burn tenotomy injury model showed increased expression of these genes in MPCs during stages of chondrogenic differentiation. Additional in vitro cell cultures of murine tissue-resident MPCs and osteochondrogenic progenitors (OCPs) treated with NG-25 demonstrated reduced chondrogenic differentiation by 10.2-fold (P < 0.001) and 133.3-fold (P < 0.001), respectively, as well as associated reduction in chondrogenic gene expression. Induction of HO in Tak1 knockout mice demonstrated a 7.1-fold (P < 0.001) and 2.7-fold reduction (P < 0.001) in chondrogenic differentiation of murine MPCs and OCPs, respectively, with reduced chondrogenic gene expression. Together, our in vivo models and in vitro cell culture studies demonstrate the importance of TAK1 signaling in chondrogenic differentiation and HO formation and suggest that small molecule inhibition of TAK1 is a promising therapy to limit the formation and progression of HO.

Asunto(s)

Osificación Heterotópica , Osteogénesis , Animales , Condrogénesis , Extremidad Inferior , Ratones , Osificación Heterotópica/tratamiento farmacológico , Calidad de Vida , Ratas

RESUMEN

Heterotopic ossification (HO), the formation of ectopic bone in soft tissues, has been extensively studied in its two primary forms: post-traumatic HO (tHO) typically found in patients who have experienced musculoskeletal or neurogenic injury and in fibrodysplasia ossificans progressiva (FOP), where it is genetically driven. Given that in both diseases HO arises via endochondral ossification, the molecular mechanisms behind both diseases have been postulated to be manifestations of similar pathways including those activated by BMP/TGFß superfamily ligands. A significant step towards understanding the molecular mechanism by which HO arises in FOP was the discovery that FOP causing ACVR1 variants trigger HO in response to activin A, a ligand that does not activate signaling from wild type ACVR1, and that is not inherently osteogenic in wild type settings. The physiological significance of this finding was demonstrated by showing that activin A neutralizing antibodies stop HO in two different genetically accurate mouse models of FOP. In order to explore the role of activin A in tHO, we performed single cell RNA sequencing and compared the expression of activin A as well as other BMP pathway genes in tHO and FOP HO. We show that activin A is expressed in response to injury in both settings, but by different types of cells. Given that wild type ACVR1 does not transduce signal when engaged by activin A, we hypothesized that inhibition of activin A will not block tHO. Nonetheless, as activin A was expressed in tHO lesions, we tested its inhibition and compared it with inhibition of BMPs. We show here that anti-activin A does not block tHO, whereas agents such as antibodies that neutralize ACVR1 or ALK3-Fc (which blocks osteogenic BMPs) are beneficial, though not completely curative. These results demonstrate that inhibition of activin A should not be considered as a therapeutic strategy for ameliorating tHO.

Asunto(s)

Miositis Osificante , Osificación Heterotópica , Receptores de Activinas Tipo I/genética , Activinas , Animales , Humanos , Ratones , Miositis Osificante/genética

RESUMEN

Perivascular mural cells surround capillaries and microvessels and have diverse regenerative or fibrotic functions after tissue injury. Subsynovial fibrosis is a well-known pathologic feature of osteoarthritis, yet transgenic animals for use in visualizing perivascular cell contribution to fibrosis during arthritic changes have not been developed. Here, inducible Pdgfra-CreERT2 reporter mice were subjected to joint-destabilization surgery to induce arthritic changes, and cell lineage was traced over an 8-week period with a focus on the joint-associated fat pad. Results showed that, at baseline, inducible Pdgfra reporter activity highlighted adventitial and, to a lesser extent, pericytic cells within the infrapatellar fat pad. Joint-destabilization surgery was associated with marked fibrosis of the infrapatellar fat pad, accompanied by an expansion of perivascular Pdgfra-expressing cellular descendants, many of which adopted α-smooth muscle actin expression. Gene expression analysis of microdissected infrapatellar fat pad confirmed enrichment in membrane-bound green fluorescent protein/Pdgfra-expressing cells, along with a gene signature that corresponded with injury-associated fibro-adipogenic progenitors. Our results highlight dynamic changes in joint-associated perivascular fibro-adipogenic progenitors during osteoarthritis.

Asunto(s)

Adipocitos/patología , Fibroblastos/patología , Osteoartritis/patología , Tejido Adiposo/patología , Animales , Linaje de la Célula , Articulación de la Rodilla/patología , Ratones , Ratones Transgénicos , Células Madre

RESUMEN

Myeloid cells are critical to the development of fibrosis following muscle injury; however, the mechanism of their role in fibrosis formation remains unclear. In this study, we demonstrate that myeloid cell-derived TGF-ß1 signaling is increased in a profibrotic ischemia reperfusion and cardiotoxin muscle injury model. We found that myeloid-specific deletion of Tgfb1 abrogates the fibrotic response in this injury model and reduces fibro/adipogenic progenitor cell proliferation while simultaneously enhancing muscle regeneration, which is abrogated by adaptive transfer of normal macrophages. Similarly, a murine TGFBRII-Fc ligand trap administered after injury significantly reduced muscle fibrosis and improved muscle regeneration. This study ultimately demonstrates that infiltrating myeloid cell TGF-ß1 is responsible for the development of traumatic muscle fibrosis, and its blockade offers a promising therapeutic target for preventing muscle fibrosis after ischemic injury.

Asunto(s)

Fibrosis/inmunología , Fibrosis/patología , Macrófagos/inmunología , Músculo Esquelético/inmunología , Músculo Esquelético/patología , Células Mieloides/inmunología , Factor de Crecimiento Transformador beta1/inmunología , Animales , Cardiotoxinas , Fibrosis/complicaciones , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Mieloides/patología , Fenotipo , Daño por Reperfusión/inducido químicamente , Daño por Reperfusión/complicaciones , Daño por Reperfusión/inmunología

RESUMEN

Heterotopic ossification (HO) is an aberrant regenerative process with ectopic bone induction in response to musculoskeletal trauma, in which mesenchymal stem cells (MSC) differentiate into osteochondrogenic cells instead of myocytes or tenocytes. Despite frequent cases of hospitalized musculoskeletal trauma, the inflammatory responses and cell population dynamics that regulate subsequent wound healing and tissue regeneration are still unclear. Here we examine, using a mouse model of trauma-induced HO, the local microenvironment of the initial post-injury inflammatory response. Single cell transcriptome analyses identify distinct monocyte/macrophage populations at the injury site, with their dynamic changes over time elucidated using trajectory analyses. Mechanistically, transforming growth factor beta-1 (TGFß1)-producing monocytes/macrophages are associated with HO and aberrant chondrogenic progenitor cell differentiation, while CD47-activating peptides that reduce systemic macrophage TGFß levels and help ameliorate HO. Our data thus implicate CD47 activation as a therapeutic approach for modulating monocyte/macrophage phenotypes, MSC differentiation and HO formation during wound healing.

Asunto(s)

Quemaduras/patología , Monocitos/patología , Osificación Heterotópica/patología , Cicatrización de Heridas/fisiología , Animales , Antígeno CD47/metabolismo , Diferenciación Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Macrófagos/patología , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Péptidos/farmacología , Fagocitosis , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo

RESUMEN

Heterotopic ossification (HO) is a debilitating condition characterized by the pathologic formation of ectopic bone. HO occurs commonly following orthopedic surgeries, burns, and neurologic injuries. While surgical excision may provide palliation, the procedure is often burdened with significant intra-operative blood loss due to a more robust contribution of blood supply to the pathologic bone than to native bone. Based on these clinical observations, we set out to examine the role of vascular signaling in HO. Vascular endothelial growth factor A (VEGFA) has previously been shown to be a crucial pro-angiogenic and pro-osteogenic cue during normal bone development and homeostasis. Our findings, using a validated mouse model of HO, demonstrate that HO lesions are highly vascular, and that VEGFA is critical to ectopic bone formation, despite lacking a contribution of endothelial cells within the developing anlagen.

RESUMEN

Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of transforming growth factor-ß activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by reactivation to elicit differentiation and extracellular matrix production. Although the current regenerative medicine paradigm is centered on the effects of drug treatment ("drug on"), the impact of drug withdrawal ("drug off") implicit in these regimens is unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dual-inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment ("drug on") and subsequent withdrawal ("drug off") through genetic modification, was used here to inactivate and reactivate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the "drug on" (Cre-mediated inactivation) and "drug off" (Flp-mediated reactivation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. Stem Cells 2019;37:766-778.

Asunto(s)

Regeneración Ósea/efectos de los fármacos , Fracturas Óseas/genética , Quinasas Quinasa Quinasa PAM/genética , Células Madre Mesenquimatosas/enzimología , Osteoblastos/enzimología , Cicatrización de Heridas/genética , Animales , Regeneración Ósea/genética , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN Nucleotidiltransferasas/genética , ADN Nucleotidiltransferasas/metabolismo , Femenino , Efecto Fundador , Fracturas Óseas/tratamiento farmacológico , Fracturas Óseas/enzimología , Fracturas Óseas/patología , Regulación de la Expresión Génica , Integrasas/genética , Integrasas/metabolismo , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/deficiencia , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Cráneo/efectos de los fármacos , Cráneo/lesiones , Cráneo/metabolismo , Cicatrización de Heridas/efectos de los fármacos