TrkA + sensory neurons regulate osteosarcoma proliferation and vascularization to promote disease progression.

Bone pain is a presenting feature of bone cancers such as osteosarcoma (OS), relayed by skeletal-innervating peripheral afferent neurons. Potential functions of tumor-associated sensory neurons in bone cancers beyond pain sensation are unknown. To uncover neural regulatory functions, a chemical-genetic approach in mice with a knock-in allele for TrkA was used to functionally perturb sensory nerve innervation during OS growth and disease progression. TrkA inhibition in transgenic mice led to significant reductions in sarcoma-associated sensory innervation and vascularization, tumor growth and metastasis, and prolonged overall survival. Single-cell transcriptomics revealed that sarcoma denervation was associated with phenotypic alterations in both OS tumor cells and cells within the tumor microenvironment, and with reduced calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor (VEGF) signaling. Multimodal and multi-omics analyses of human OS bone samples and human dorsal root ganglia neurons further implicated peripheral innervation and neurotrophin signaling in OS tumor biology. In order to curb tumor-associated axonal ingrowth, we next leveraged FDA-approved bupivacaine liposomes leading to significant reductions in sarcoma growth, vascularity, as well as alleviation of pain. In sum, TrkA-expressing peripheral neurons positively regulate key aspects of OS progression and sensory neural inhibition appears to disrupt calcitonin receptor signaling (CALCR) and VEGF signaling within the sarcoma microenvironment leading to significantly reduced tumor growth and improved survival. These data suggest that interventions to prevent pathological innervation of osteosarcoma represent a novel adjunctive therapy to improve clinical outcomes and survival.

Requirement of Pdgfrα+ cells for calvarial bone repair.

Platelet-derived growth factor receptor α (PDGFRα) is often considered as a general marker of mesenchymal cells and fibroblasts, but also shows expression in a portion of osteoprogenitor cells. Within the skeleton, Pdgfrα+ mesenchymal cells have been identified in bone marrow and periosteum of long bones, where they play a crucial role in participating in fracture repair. A similar examination of Pdgfrα+ cells in calvarial bone healing has not been examined. Here, we utilize Pdgfrα-CreERTM;mT/mG reporter animals to examine the contribution of Pdgfrα+ mesenchymal cells to calvarial bone repair through histology and single-cell RNA sequencing (scRNA-Seq). Results showed that Pdgfrα+ mesenchymal cells are present in several cell clusters by scRNA-Seq, and by histology a dramatic increase in Pdgfrα+ cells populated the defect site at early timepoints to give rise to healed bone tissue overtime. Notably, diphtheria toxin-mediated ablation of Pdgfrα reporter+ cells resulted in significantly impaired calvarial bone healing. Our findings suggest that Pdgfrα-expressing cells within the calvarial niche play a critical role in the process of calvarial bone repair.

TrkA-mediated sensory innervation of injured mouse tendon supports tendon sheath progenitor cell expansion and tendon repair.

Peripheral neurons terminate at the surface of tendons partly to relay nociceptive pain signals; however, the role of peripheral nerves in tendon injury and repair remains unclear. Here, we show that after Achilles tendon injury in mice, there is new nerve growth near tendon cells that express nerve growth factor (NGF). Conditional deletion of the Ngf gene in either myeloid or mesenchymal mouse cells limited both innervation and tendon repair. Similarly, inhibition of the NGF receptor tropomyosin receptor kinase A (TrkA) abrogated tendon healing in mouse tendon injury. Sural nerve transection blocked the postinjury increase in tendon sensory innervation and the expansion of tendon sheath progenitor cells (TSPCs) expressing tubulin polymerization promoting protein family member 3. Single cell and spatial transcriptomics revealed that disruption of sensory innervation resulted in dysregulated inflammatory signaling and transforming growth factor-ß (TGFß) signaling in injured mouse tendon. Culture of mouse TSPCs with conditioned medium from dorsal root ganglia neuron further supported a role for neuronal mediators and TGFß signaling in TSPC proliferation. Transcriptomic and histologic analyses of injured human tendon biopsy samples supported a role for innervation and TGFß signaling in human tendon regeneration. Last, treating mice after tendon injury systemically with a small-molecule partial agonist of TrkA increased neurovascular response, TGFß signaling, TSPC expansion, and tendon tissue repair. Although further studies should investigate the potential effects of denervation on mechanical loading of tendon, our results suggest that peripheral innervation is critical for the regenerative response after acute tendon injury.

ZIC1 Dictates Osteogenesis Versus Adipogenesis in Human Mesenchymal Progenitor Cells Via a Hedgehog Dependent Mechanism.

Numerous intrinsic factors regulate mesenchymal progenitor commitment to a specific cell fate, such as osteogenic or adipogenic lineages. Identification and modulation of novel intrinsic regulatory factors represent an opportunity to harness the regenerative potential of mesenchymal progenitors. In the present study, the transcription factor (TF) ZIC1 was identified to be differentially expressed among adipose compared with skeletal-derived mesenchymal progenitor cells. We observed that ZIC1 overexpression in human mesenchymal progenitors promotes osteogenesis and prevents adipogenesis. ZIC1 knockdown demonstrated the converse effects on cell differentiation. ZIC1 misexpression was associated with altered Hedgehog signaling, and the Hedgehog antagonist cyclopamine reversed the osteo/adipogenic differentiation alterations associated with ZIC1 overexpression. Finally, human mesenchymal progenitor cells with or without ZIC1 overexpression were implanted in an ossicle assay in NOD-SCID gamma mice. ZIC1 overexpression led to significantly increased ossicle formation in comparison to the control, as assessed by radiographic and histologic measures. Together, these data suggest that ZIC1 represents a TF at the center of osteo/adipogenic cell fate determinations-findings that have relevance in the fields of stem cell biology and therapeutic regenerative medicine.

CNTNAP4 signaling regulates osteosarcoma disease progression.

Improved treatment strategies for sarcoma rely on clarification of the molecular mediators of disease progression. Recently, we reported that the secreted glycoprotein NELL-1 modulates osteosarcoma (OS) disease progression in part via altering the sarcomatous extracellular matrix (ECM) and cell-ECM interactions. Of known NELL-1 interactor proteins, Contactin-associated protein-like 4 (Cntnap4) encodes a member of the neurexin superfamily of transmembrane molecules best known for its presynaptic functions in the central nervous system. Here, CRISPR/Cas9 gene deletion of CNTNAP4 reduced OS tumor growth, sarcoma-associated angiogenesis, and pulmonary metastases. CNTNAP4 knockout (KO) in OS tumor cells largely phenocopied the effects of NELL-1 KO, including reductions in sarcoma cell attachment, migration, and invasion. Further, CNTNAP4 KO cells were found to be unresponsive to the effects of NELL-1 treatment. Transcriptomic analysis combined with protein phospho-array demonstrated notable reductions in the MAPK/ERK signaling cascade with CNTNAP4 deletion, and the ERK1/2 agonist isoproterenol restored cell functions among CNTNAP4 KO tumor cells. Finally, human primary cells and tissues in combination with sequencing datasets confirmed the significance of CNTNAP4 signaling in human sarcomas. In summary, our findings demonstrate the biological importance of NELL-1/CNTNAP4 signaling axis in disease progression of human sarcomas and suggest that targeting the NELL-1/CNTNAP4 signaling pathway represents a strategy with potential therapeutic benefit in sarcoma patients.

Neurovascular coupling in bone regeneration.

The mammalian skeletal system is densely innervated by both neural and vascular networks. Peripheral nerves in the skeleton include sensory and sympathetic nerves. The crosstalk between skeletal and neural tissues is critical for skeletal development and regeneration. The cellular processes of osteogenesis and angiogenesis are coupled in both physiological and pathophysiological contexts. The cellular and molecular regulation of osteogenesis and angiogenesis have yet to be fully defined. This review will provide a detailed characterization of the regulatory role of nerves and blood vessels during bone regeneration. Furthermore, given the importance of the spatial relationship between nerves and blood vessels in bone, we discuss neurovascular coupling during physiological and pathological bone formation. A better understanding of the interactions between nerves and blood vessels will inform future novel therapeutic neural and vascular targeting for clinical bone repair and regeneration.

NELL1 Regulates the Matrisome to Promote Osteosarcoma Progression.

Sarcomas produce an abnormal extracellular matrix (ECM), which in turn provides instructive cues for cell growth and invasion. Neural EGF like-like molecule 1 (NELL1) is a secreted glycoprotein characterized by its nonneoplastic osteoinductive effects, yet it is highly expressed in skeletal sarcomas. Here, we show that genetic deletion of NELL1 markedly reduces invasive behavior across human osteosarcoma (OS) cell lines. NELL1 deletion resulted in reduced OS disease progression, inhibiting metastasis and improving survival in a xenograft mouse model. These observations were recapitulated with Nell1 conditional knockout in mouse models of p53/Rb-driven sarcomagenesis, which reduced tumor frequency and extended tumor-free survival. Transcriptomic and phosphoproteomic analyses demonstrated that NELL1 loss skews the expression of matricellular proteins associated with reduced FAK signaling. Culturing NELL1 knockout sarcoma cells on wild-type OS-enriched matricellular proteins reversed the phenotypic and signaling changes induced by NELL1 deficiency. In sarcoma patients, high expression of NELL1 correlated with decreased overall survival. These findings in mouse and human models suggest that NELL1 expression alters the sarcoma ECM, thereby modulating cellular invasive potential and prognosis. Disruption of NELL1 signaling may represent a novel therapeutic approach to short-circuit sarcoma disease progression. SIGNIFICANCE: NELL1 modulates the sarcoma matrisome to promote tumor growth, invasion, and metastasis, identifying the matrix-associated protein as an orchestrator of cell-ECM interactions in sarcomagenesis and disease progression.

Acetabular Reaming Is a Reliable Model to Produce and Characterize Periarticular Heterotopic Ossification of the Hip.

Heterotopic ossification (HO) is a pathologic process characterized by the formation of bone tissue in extraskeletal locations. The hip is a common location of HO, especially as a complication of arthroplasty. Here, we devise a first-of-its-kind mouse model of post-surgical hip HO and validate expected cell sources of HO using several HO progenitor cell reporter lines. To induce HO, an anterolateral surgical approach to the hip was used, followed by disclocation and acetabular reaming. Animals were analyzed with high-resolution roentgenograms and micro-computed tomography, conventional histology, immunohistochemistry, and assessments of fluorescent reporter activity. All the treated animals' developed periarticular HO with an anatomical distribution similar to human patients after arthroplasty. Heterotopic bone was found in periosteal, inter/intramuscular, and intracapsular locations. Further, the use of either PDGFRα or scleraxis (Scx) reporter mice demonstrated that both cell types gave rise to periarticular HO in this model. In summary, acetabular reaming reproducibly induces periarticular HO in the mouse reproducing human disease, and with defined mesenchymal cellular contributors similar to other experimental HO models. This protocol may be used in the future for further detailing of the cellular and molecular mediators of post-surgical HO, as well as the screening of new therapies.

NGF-p75 signaling coordinates skeletal cell migration during bone repair.

Bone regeneration following injury is initiated by inflammatory signals and occurs in association with infiltration by sensory nerve fibers. Together, these events are believed to coordinate angiogenesis and tissue reprogramming, but the mechanism of coupling immune signals to reinnervation and osteogenesis is unknown. Here, we found that nerve growth factor (NGF) is expressed following cranial bone injury and signals via p75 in resident mesenchymal osteogenic precursors to affect their migration into the damaged tissue. Mice lacking Ngf in myeloid cells demonstrated reduced migration of osteogenic precursors to the injury site with consequently delayed bone healing. These features were phenocopied by mice lacking p75 in Pdgfra+ osteoblast precursors. Single-cell transcriptomics identified mesenchymal subpopulations with potential roles in cell migration and immune response, altered in the context of p75 deletion. Together, these results identify the role of p75 signaling pathway in coordinating skeletal cell migration during early bone repair.

NGF-TrkA signaling dictates neural ingrowth and aberrant osteochondral differentiation after soft tissue trauma.

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.

Human perivascular stem cells prevent bone graft resorption in osteoporotic contexts by inhibiting osteoclast formation.

The vascular wall stores mesenchymal progenitor cells which are able to induce bone regeneration, via direct and paracrine mechanisms. Although much is known regarding perivascular cell regulation of osteoblasts, their regulation of osteoclasts, and by extension utility in states of high bone resorption, is not known. Here, human perivascular stem cells (PSCs) were used as a means to prevent autograft resorption in a gonadectomy-induced osteoporotic spine fusion model. Furthermore, the paracrine regulation by PSCs of osteoclast formation was evaluated, using coculture, conditioned medium, and purified extracellular vesicles. Results showed that PSCs when mixed with autograft bone induce an increase in osteoblast:osteoclast ratio, promote bone matrix formation, and prevent bone graft resorption. The confluence of these factors resulted in high rates of fusion in an ovariectomized rat lumbar spine fusion model. Application of PSCs was superior across metrics to either the use of unpurified, culture-defined adipose-derived stromal cells or autograft bone alone. Under coculture conditions, PSCs negatively regulated osteoclast formation and did so via secreted, nonvesicular paracrine factors. Total RNA sequencing identified secreted factors overexpressed by PSCs which may explain their negative regulation of graft resorption. In summary, PSCs reduce osteoclast formation and prevent bone graft resorption in high turnover states such as gonadectomy-induced osteoporosis.

NLRX1 suppresses tumorigenesis and attenuates histiocytic sarcoma through the negative regulation of NF-κB signaling.

Histiocytic sarcoma is an uncommon malignancy in both humans and veterinary species. Research exploring the pathogenesis of this disease is scarce; thus, diagnostic and therapeutic options for patients are limited. Recent publications have suggested a role for the NLR, NLRX1, in acting as a tumor suppressor. Based on these prior findings, we hypothesized that NLRX1 would function to inhibit tumorigenesis and thus the development of histiocytic sarcoma. To test this, we utilized Nlrx1-/- mice and a model of urethane-induced tumorigenesis. Nlrx1-/- mice exposed to urethane developed splenic histiocytic sarcoma that was associated with significant up-regulation of the NF-κB signaling pathway. Additionally, development of these tumors was also significantly associated with the increased regulation of genes associated with AKT signaling, cell death and autophagy. Together, these data show that NLRX1 suppresses tumorigenesis and reveals new genetic pathways involved in the pathobiology of histiocytic sarcoma.

Mono(2-ethylhexyl) phthalate induces apoptosis in p53-silenced L02 cells via activation of both mitochondrial and death receptor pathways.

Mono(2-ethylhexyl) phthalate (MEHP) is one of the main metabolites of di(2-ethylhexyl) phthalate. The evidence shows that DEHP may exert its toxic effects primarily via MEHP, which is 10-fold more potent than its parent compound in toxicity in vitro. MEHP-induced apoptosis is mediated by either p53-dependent or -independent pathway. However, the detailed mechanism of its toxicity remains unclear. In this study, immortalized normal human liver cell line L02 was chosen, as an in vitro model of nonmalignant liver, to elucidate the role of p53 in MEHP-induced apoptosis. The cells were treated with MEHP (6.25, 12.50, 25.00, 50.00, and 100.00 µM) for 24 and 36 h, then small interfering RNA (siRNA) was used to specifically silence p53 gene of L02 cells. The results indicated that MEHP caused oxidative DNA damage and apoptosis in L02 cells were associated with the p53 signaling pathway. Further study found that MEHP (50.00 and 100.00 µM) induced apoptosis in p53-silenced L02 cells, along with the up-regulations of Fas and FasL proteins as well as increased the Bax/Bcl-2 ratio and Caspase 3, 8, and 9 activities. Additionally, both FasL inhibitor (AF-016) and Caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp- fluoromethylketone (Z-VAD-FMK) could prevent the cell apoptosis induced by MEHP. The findings suggested that MEHP-induced apoptosis in L02 cells involving a Caspases-mediated mitochondrial signaling pathway and/or death receptor pathway. p53 was not absolutely necessary for MEHP-induced L02 cell apoptosis.

Activation of the PI3K-AKT-mTOR signaling pathway promotes DEHP-induced Hep3B cell proliferation.

Hep3B cells were treated with DEHP at various concentrations (62.5, 125.0, 250.0, 500.0 and 1000.0 µM). After 24 h exposure to DEHP only, increased Hep3B cell viability was observed (p<0.05 or p<0.01). However, after 24 h co-exposure to DEHP at indicated concentrations plus 50.0 µM LY294002 (PI3K inhibitor), cell viability was significantly decreased compared to the corresponding DEHP treated groups. DEHP increased mitochondrial membrane potential level and induced oxidative DNA damage in Hep3B cells, DEHP also increased DNA replication rate and accelerated the cell cycle. The PI3K inhibitor LY294002 could recover the mitochondrial membrane potential and attenuate the oxidative stress in Hep3B cells; however, it could not protect the cells from oxidation of DNA damage. The findings showed that LY294002 attenuated DEHP-induced up-regulation of the selected genes (pi3k, akt, mtor and p70s6k) involved in PI3K-AKT-mTOR signaling pathway at both mRNA and protein levels thus inhibited the cell abnormal proliferation.

Mono-2-ethylhexyl phthalate induced loss of mitochondrial membrane potential and activation of Caspase3 in HepG2 cells.

L02 and HepG2 cells were exposed to mono-(2-ethylhexyl) phthalate (MEHP) at concentrations of 6.25-100µM. After 48h treatment, MEHP decreased HepG2 cell viability in a concentration-dependent manner and L02 cell viability in the 50 and 100µM groups (p<0.01). Furthermore, at 24 and 48h after treatment, MEHP decreased the glutathione levels of HepG2 cells in all treatment groups and in the ΔΨ(m) in L02 and HepG2 cells with MEHP≥25µM (p<0.05 or p<0.01). At 24h after treatment, MEHP induced activation of caspase3 in all treated HepG2 and L02 cells (p<0.05 or p<0.01) except the 100µM MEHP treatment group. The increase in the Bax to Bcl-2 ratio suggests that Bcl-2 family involved in the control of MEHP-induced apoptosis in these two cell types. The data suggest that MEHP could induce apoptosis of HepG2 cells through mitochondria- and caspase3-dependent pathways.