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Patients with back pain comprise a large proportion of the outpatient practice among physiatrists. Diagnostic tools are limited to clinical history, physical examinations, and imaging. Nonsurgical treatments are largely empirical, encompassing medications, physical therapy, manual treatments, and interventional spinal procedures. A body of literature is emerging confirming elevated levels of biomarkers including inflammatory cytokines in patients with back pain and/or radiculopathy, largely because the protein assay sensitivity has increased. These biomarkers may serve as tools to assist diagnosis and assess outcomes.The presence of inflammatory mediators in the intervertebral disc tissues and blood helped to confirm the inflammatory underpinnings of back pain related to intervertebral disc degeneration. Literature reviewed here suggests that biomarkers could assist clinical diagnosis and monitor physiological outcomes during and after treatments for spine-related pain. Biomarkers must be measured in a large and diverse asymptomatic population, in the context of age and comorbidities to prevent false-positive tests. These levels can then be rationally compared with those in patients with back disorders including discogenic back pain, radiculopathy, and spinal stenosis. While studies reviewed here used "candidate marker" approaches, future nonbiased approaches in clearly defined patient populations could uncover novel biomarkers in clinical management of patients.

Posttraumatic osteoarthritis (PTOA) results in joint pain, loss of joint function, and impaired quality of daily life in patients with limited treatment options. We previously demonstrated that epidermal growth factor receptor (EGFR) signaling is essential for maintaining chondroprogenitors during articular cartilage development and homeostasis. Here, we used a nonsurgical, loading-induced PTOA mouse model to investigate the protective action of EGFR signaling. A single bout of cyclic tibial loading at a peak force of 6 N injured cartilage at the posterior aspect of lateral femoral condyle. Similar loading at a peak force of 9 N ruptured the anterior cruciate ligament, causing additional cartilage damage at the medial compartment and ectopic cartilage formation in meniscus and synovium. Constitutively overexpression of an EGFR ligand, heparin binding EGF-like growth factor (HBEGF), in chondrocytes significantly reduced cartilage injury length, synovitis, and pain after 6 N loading and mitigated medial side cartilage damage and ectopic cartilage formation after 9 N loading. Mechanistically, overactivation of EGFR signaling protected chondrocytes from loading-induced apoptosis and loss of proliferative ability and lubricant synthesis. Overexpressing HBEGF in adult cartilage starting right before 6 N loading had similar beneficial effects. In contrast, inactivating EGFR in adult cartilage led to accelerated PTOA progression with elevated cartilage Mankin score and synovitis score and increased ectopic cartilage formation. As a therapeutic approach, we constructed a nanoparticle conjugated with the EGFR ligand TGFα. Intra-articular injections of this nanoconstruct once every 3 weeks for 12 weeks partially mitigated PTOA symptoms in cartilage and synovium after 6 N loading. Our findings demonstrate the anabolic actions of EGFR signaling in maintaining articular cartilage during PTOA development and shed light on developing a novel nanomedicine for PTOA. © 2022 American Society for Bone and Mineral Research (ASBMR).

Angiogenesis associates with chondrocytes differentiation in inflammatory arthritis. Interleukin (IL)- 1β stimulated SW1353 cells have a phenotype similar to this kind of chondrocytes. IL-17A, a target in T helper 17 (Th17)/IL-17 signaling pathways, was expressed by SW1353 cells. The study aimed to explore the role of IL-35 on angiogenesis in IL-1β stimulated SW1353 cells and its related signaling pathways.Microarray dataset was downloaded from the Gene Expression Omnibus database of arthritis cartilage. The protein-protein interaction (PPI) was analyzed for IL-35, pro-angiogenic factors and the differentially expressed genes (DEGs). We studied the effects of IL-35 on proliferation and apoptosis in IL-1β stimulated SW1353 cells using cell counting kit-8 (CCK-8) assay and flow cytometry. The expression of pro-angiogenic factors and IL-17A were assessed by western blot and real-time PCR. Added plumbagin (inhibitor of IL-17A) to repeat the above experiment. The secretion of IL-17A was assessed by ELISA.IL-35, pro-angiogenic factors interacted with DEGs to affect the function of arthritis chondrocytes. IL-35 promoted IL-1β-stimulated SW1353 cells proliferation, inhibited apoptosis, and decreased pro-angiogenic molecules and IL-17A expression in a concentration dependent manner. IL-35 inhibited IL-17A secretion in the supernatants of these cells. Blocking the Th17/IL-17 related pathways with plumbagin abolished the effects of IL-35 on IL-1β-stimulated SW1353 cells.These results suggested that IL-35 regulated differentiation and pro-angiogenic molecules expression in IL-1β stimulated SW1353 cells via Th17/IL-17 related signaling pathways. Our findings may reveal the mechanisms of novel angiogenesis molecules in inflammatory chondrocyte lesion.

Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native nucleus pulposus cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived nucleus pulposus cells in the postnatal mouse disc. Specifically, we established the existence of early and late stage nucleus pulposus cells, corresponding to notochordal progenitor and mature cells, respectively. Late stage cells exhibited significantly higher expression levels of extracellular matrix genes including aggrecan, and collagens II and VI, along with elevated TGF-β and PI3K-Akt signaling. Additionally, we identified Cd9 as a novel surface marker of late stage nucleus pulposus cells, and demonstrated that these cells were localized to the nucleus pulposus periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich matrix. Finally, we used a goat model to show the Cd9+ nucleus pulposus cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy nucleus pulposus extracellular matrix. Improved understanding of the developmental mechanisms underlying regulation of ECM deposition in the postnatal NP may inform improved regenerative strategies for disc degeneration and associated low back pain.

Figure S6 Lower levels of TGF-β1 are induced in F-PRT-treated mouse skin compared to S-PRT. (A) and (B) depict the immunofluorescent evaluation of TGF-β1 in mouse skin derived from 2 additional representative sets of mice irradiated with F-PRT or S-PRT (30 Gy) (Scale bar, 100 Î1/4m; Magnification, 10x).Â

Supplemental Methods

Table S2. Genes that are upregulated in the S-PRT-treated bone revealed by RNASeq analysis

In studies of electron and proton radiotherapy, ultrahigh dose rates of FLASH radiotherapy appear to produce fewer toxicities than standard dose rates while maintaining local tumor control. FLASH-proton radiotherapy (F-PRT) brings the spatial advantages of PRT to FLASH dose rates (>40 Gy/second), making it important to understand if and how F-PRT spares normal tissues while providing antitumor efficacy that is equivalent to standard-proton radiotherapy (S-PRT). Here we studied PRT damage to skin and mesenchymal tissues of muscle and bone and found that F-PRT of the C57BL/6 murine hind leg produced fewer severe toxicities leading to death or requiring euthanasia than S-PRT of the same dose. RNA-seq analyses of murine skin and bone revealed pathways upregulated by S-PRT yet unaltered by F-PRT, such as apoptosis signaling and keratinocyte differentiation in skin, as well as osteoclast differentiation and chondrocyte development in bone. Corroborating these findings, F-PRT reduced skin injury, stem cell depletion, and inflammation, mitigated late effects including lymphedema, and decreased histopathologically detected myofiber atrophy, bone resorption, hair follicle atrophy, and epidermal hyperplasia. F-PRT was equipotent to S-PRT in control of two murine sarcoma models, including at an orthotopic intramuscular site, thereby establishing its relevance to mesenchymal cancers. Finally, S-PRT produced greater increases in TGFβ1 in murine skin and the skin of canines enrolled in a phase I study of F-PRT versus S-PRT. Collectively, these data provide novel insights into F-PRT-mediated tissue sparing and support its ongoing investigation in applications that would benefit from this sparing of skin and mesenchymal tissues.Significance:These findings will spur investigation of FLASH radiotherapy in sarcoma and additional cancers where mesenchymal tissues are at risk, including head and neck cancer, breast cancer, and pelvic malignancies.

Supplemental Materials & Methods

Skeletal fragility is associated with type 2 diabetes mellitus (T2D), but the underlying mechanism is not well understood. Here, in a mouse model for youth-onset T2D, we show that both trabecular and cortical bone mass are reduced due to diminished osteoblast activity. Stable isotope tracing in vivo with13C-glucose demonstrates that both glycolysis and glucose fueling of the TCA cycle are impaired in diabetic bones. Similarly, Seahorse assays show suppression of both glycolysis and oxidative phosphorylation by diabetes in bone marrow mesenchymal cells as a whole, whereas single-cell RNA sequencing reveals distinct modes of metabolic dysregulation among the subpopulations. Metformin not only promotes glycolysis and osteoblast differentiation in vitro, but also improves bone mass in diabetic mice. Finally, targeted overexpression of Hif1a or Pfkfb3 in osteoblasts of T2D mice averts bone loss. The study identifies osteoblast-intrinsic defects in glucose metabolism as an underlying cause of diabetic osteopenia, which may be targeted therapeutically.

Genome-wide association studies (GWASs) have revolutionized our understanding of the genetics of complex diseases, such as osteoporosis; however, the challenge has been converting associations to causal genes. Studies have demonstrated the utility of transcriptomics data in linking disease-associated variants to genes; though for osteoporosis, few population transcriptomics datasets have been generated on bone or bone cells, and an even smaller number have profiled individual cell-types. To begin to evaluate approaches to address this challenge, we profiled the transcriptomes of bone marrow-derived stromal cells (BMSCs) cultured under osteogenic conditions, a popular model of osteoblast differentiation and activity, from five Diversity Outbred (DO) mice using single-cell RNA-seq (scRNA-seq). The goal of the study was to determine if BMSCs could serve as a model for the generation of cell-type specific transcriptomic profiles of mesenchymal lineage cells derived from large populations of mice to inform genetic studies. We demonstrate that dissociation of BMSCs from a heavily mineralized matrix had little effect on viability or their transcriptomic signatures. Furthermore, we show that BMSCs cultured under osteogenic conditions are diverse and consist of cells with characteristics of mesenchymal progenitors, marrow adipogenic lineage precursors (MALPs), osteoblasts, osteocyte-like cells, and immune cells. Importantly, all cells were nearly identical from a transcriptomic perspective to cells isolated directly from bone. We also demonstrated the ability to multiplex single cells and subsequently assign cells to their “mouse-of-origin” using demultiplexing approaches based on genotypes inferred from coding SNPs. We employed scRNA-seq analytical tools to confirm the biological identity of profiled cell-types. SCENIC was used to reconstruct gene regulatory networks (GRNs) and we showed that identified cell-types show GRNs expected of osteogenic and pre-adipogenic lineage cells. Further, CELLECT analysis showed that osteoblasts, osteocyte-like cells, and MALPs captured a significant component of BMD heritability. Together, these data suggest that BMSCs cultured under osteogenic conditions coupled with scRNA-seq can be used as a scalable and biologically informative model to generate cell-type specific transcriptomic profiles of mesenchymal lineage cells in large mouse, and potentially human, populations.

Insufficient bone fracture repair represents a significant clinical burden, and identification of novel therapeutics enhancing repair would have substantial clinical and societal impact. Activin A is a TGF-β protein superfamily member known to stimulate ectopic bone formation, but its roles in fracture repair and its therapeutic potentials remain unclear. Using two mouse tibia fracture repair models, here we mapped activin A expression at the tissue and single cell levels, tested its requirement for normal repair and evaluated its ability to enhance repair when provided exogenously. Activin A was minimally expressed in periosteum of intact bones but was markedly upregulated in developing callus soon after fracture. Single cell RNA-sequencing revealed that the activin A-encoding gene Inhba marked a unique, highly proliferative progenitor cell (PPC) population with a myofibroblast character which emerged over repair time and lay at the center of a developmental trajectory bifurcation producing cartilage and bone cells within callus. Systemic administration of a neutralizing activin A antibody impaired fracture repair and its endochondral and intramembranous phases, whereas local delivery of recombinant activin A enhanced repair. Activin A delivery also induced SMAD2 phosphorylation in vivo and increased the fraction of αSMA+ myofibroblasts within fracture callus. Gain- and loss-of-function experiments in vitro showed that activin A directly stimulated myofibroblast differentiation, chondrogenesis and osteogenesis in periosteal progenitor cells. Together, these data identify a unique population of Inhba-expressing proliferative progenitor cells that give rise to chondrocytes and osteoblasts during fracture healing and establish activin A as a potential new therapeutic tool to enhance it.One Sentence SummaryDeficits in bone fracture repair remain a clinical challenge and the present study provides evidence for the therapeutic potentials of activin A

Colony stimulating factor 1 (Csf1) is an essential growth factor for osteoclast progenitors and thus an important regulator for bone resorption. It remains elusive which mesenchymal cells synthesize Csf1 stimulating osteoclastogenesis. We recently identified a novel mesenchymal cell population, marrow adipogenic lineage precursors (MALPs), in bone. Single cell RNA- sequencing indicated specific expression of Csf1 in MALPs, which is further increased during aging. To investigate its role, we constructed Csf1 CKO mice using Adipoq-Cre. These mice showed increased femoral trabecular bone over time, but their cortical bone appeared normal. In comparison, depletion of Csf1 in the entire mesenchymal lineage using Prx1-Cre led to a more striking high bone mass phenotype, suggesting that additional mesenchymal subpopulations secrete Csf1. TRAP staining revealed diminished osteoclasts in the femoral secondary spongiosa region of Csf1 CKOAdipoq mice, but not at the chondral-osseous junction nor at the endosteal surface of cortical bone. Moreover, Csf1 CKOAdipoq mice were resistant to LPS-induced calvarial osteolysis. Bone marrow cellularity, hematopoietic progenitors, and macrophages were also reduced in these mice. Taken together, our studies demonstrate that MALPs are a critical player in controlling bone remodeling and hematopoiesis.

Skeletal muscle has remarkable regenerative ability after injury. Mesenchymal fibro-adipogenic progenitors (FAPs) are necessary, active participants during this repair process, but the molecular signatures of these cells and their functional relevance remain largely unexplored. Here, using a lineage tracing mouse model (Gli1-CreER Tomato), we demonstrate that Gli1 marks a small subset of muscle-resident FAPs with elevated Hedgehog (Hh) signaling. Upon notexin muscle injury, these cells preferentially and rapidly expanded within FAPs. Ablation of Gli1+ cells using a DTA mouse model drastically reduced fibroblastic colony-forming unit (CFU-F) colonies generated by muscle cells and impaired muscle repair at 28 days. Pharmacologic manipulation revealed that Gli1+ FAPs rely on Hh signaling to increase the size of regenerating myofiber. Sorted Gli1+ FAPs displayed superior clonogenicity and reduced adipogenic differentiation ability in culture compared to sorted Gli1- FAPs. In a glycerol injury model, Gli1+ FAPs were less likely to give rise to muscle adipocytes compared to other FAPs. Further cell ablation and Hh activator/inhibitor treatments demonstrated their dual actions in enhancing myogenesis and reducing adipogenesis after injury. Examining single-cell RNA-sequencing dataset of FAPs from normal mice indicated that Gli1+ FAPs with increased Hh signaling provide trophic signals to myogenic cells while restrict their own adipogenic differentiation. Collectively, our findings identified a subpopulation of FAPs that play an essential role in skeletal muscle repair. © 2021 American Society for Bone and Mineral Research (ASBMR).

OBJECTIVE The aim was to identify the source of cells within the center of the abnormal fibrocartilage tissue of the degenerative intervertebral disc after injury. DESIGN Cross-breeding of mice with an inducible type II promoter collagen construct (Col2CreER) to Rosa26-TdTomato mice has been shown to result in Cre-recombinase activity and Tomato expression in inner annulus fibrosus cells after tamoxifen injection. To investigate the role of the inner annulus fibrosus in the intervertebral disc injury response, tail intervertebral discs of Col2CreER/tdTomato mice were punctured with a needle and examined 1-4 wks after injury. N-cadherin was examined by immunostaining. RESULTS After the injury, the fibrocartilage in the degenerative intervertebral disc consisted of residual diseased nucleus pulposus cells and encroaching inner annulus fibrosus cells. The residual nucleus pulposus cells had lost their epithelial cell-like morphology and instead became oval shaped, with reduced adhesion to neighboring nucleus pulposus cells. This change in cellular morphology coincided with a loss of N-cadherin, which contributes to maintenance of healthy nucleus pulposus cell morphology. As expected, injured tail intervertebral discs showed reduced compressive properties as determined by biomechanical assessments. CONCLUSIONS The cellular composition of the degenerative intervertebral disc has been defined here, which is an important step in developing future treatments.

We showed previously that inflammatory mediators, including IL8, in intervertebral disc tissues from patients with discogenic back pain may play a key role in back pain. To investigate the molecular mechanism of IL8 signaling in back pain, we generated a mouse model that conditionally expresses human (h) IL8. We hypothesized that hIL8 levels affect mouse activity and function. Briefly, hIL8 cDNA was inserted into the pCALL2 plasmid, linearized, and injected into mouse embryos. Resulting pCALL2–hIL8 mice were then bred with GDF5–Cre mice to express the transgene in cartilage and intervertebral disc (IVD) tissues. Functional capacities including nest-making and other natural behaviors were measured. Both male and female mice expressing hIL8 showed lower nesting scores than did littermates that did not express hIL8 (n = 14 to 16 per group). At 28 wk of age, mice expressing hIL8 (n = 35) spent more time immobile and eating during each night than littermate controls (n = 33). Furthermore, hIL8-expressing mice traveled shorter distances and at a lower average speed than littermate controls. Thus, in an initial effort to investigate the relationship between this chemokine and mouse behavior, we have documented changes in normal activities in mice conditionally expressing hIL8.

To facilitate rational experimental design and fulfill the NIH requirement of including sex as a biologic variable, we examined the influences of genetic background and sex on responses to intervertebral disc (IVD) injury in the mouse tail. The goal of this study was to compare gene expression and histologic changes in response to a tail IVD injury (needle puncture) in male and female mice on the DBA and C57BL/6 (B6) backgrounds. We hypothesized that extracellular matrix gene expression in response to IVD injury differs between mice of different genetic backgrounds and sex. Consistent changes were detected in gene expression and histologic features after IVD injury in mice on both genetic backgrounds and sexes. In particular, expression of col1a1 and adam8 was higher in the injured IVD of DBA mice than B6 mice. Conversely, col2a1 expression was higher in B6 mice than DBA mice. Sex-associated differences were significant only in B6 mice, in which col2a1 expression was greater in male mice than in female. Histologic differences in response to injury were not apparent between DBA and B6 mice or between males and females. In conclusion, mouse tail IVD showed sex- and strain-related changes in gene expression and histology after needle puncture. The magnitude of change in gene expression differed with regard to genetic background and, to a lesser degree, sex.

Oxidative stress and the reactive oxygen species (ROS) have important roles in osteoarthritis (OA) development and progression. Scavenging ROS by exogenous antioxidant enzymes could be a promising approach for OA treatment. However, the direct use of antioxidant enzymes, such as superoxide dismutase (SOD), is challenging due to a lack of effective drug delivery system to knee joints. This study utilized a highly efficient antioxidative nanoparticle based on SOD-loaded porous polymersome nanoparticles (SOD-NPs) for delivery of SOD to mouse knee joints. The resultant SOD-NPs had prolonged mouse joint retention time with predominant accumulation in synovium but not in articular cartilage. Examining human synovial explants revealed that SOD-NPs minimize oxidative damages induced by OA-like insults. Intra-articular injections of SOD-NPs in mice receiving OA surgery were effective in attenuating OA initiation and preventing its further progression. Mechanistically, SOD-NPs reduced ROS production and the synthesis of catabolic proteases in both articular cartilage and synovium. Hence, our work demonstrates the therapeutic potential of SOD-NPs and indicate that targeting synovium holds a great promise for OA therapy.

Cartilage is essential throughout vertebrate life. It starts developing in embryos when osteochondroprogenitor cells commit to chondrogenesis, activate a pancartilaginous program to form cartilaginous skeletal primordia, and also embrace a growth-plate program to drive skeletal growth or an articular program to build permanent joint cartilage. Various forms of cartilage malformation and degeneration diseases afflict humans, but underlying mechanisms are still incompletely understood and treatment options suboptimal. The transcription factor SOX9 is required for embryonic chondrogenesis, but its postnatal roles remain unclear, despite evidence that it is down-regulated in osteoarthritis and heterozygously inactivated in campomelic dysplasia, a severe skeletal dysplasia characterized postnatally by small stature and kyphoscoliosis. Using conditional knockout mice and high-throughput sequencing assays, we show here that SOX9 is required postnatally to prevent growth-plate closure and preosteoarthritic deterioration of articular cartilage. Its deficiency prompts growth-plate chondrocytes at all stages to swiftly reach a terminal/dedifferentiated stage marked by expression of chondrocyte-specific (Mgp) and progenitor-specific (Nt5e and Sox4) genes. Up-regulation of osteogenic genes (Runx2, Sp7, and Postn) and overt osteoblastogenesis quickly ensue. SOX9 deficiency does not perturb the articular program, except in load-bearing regions, where it also provokes chondrocyte-to-osteoblast conversion via a progenitor stage. Pathway analyses support roles for SOX9 in controlling TGFβ and BMP signaling activities during this cell lineage transition. Altogether, these findings deepen our current understanding of the cellular and molecular mechanisms that specifically ensure lifelong growth-plate and articular cartilage vigor by identifying osteogenic plasticity of growth-plate and articular chondrocytes and a SOX9-countered chondrocyte dedifferentiation/osteoblast redifferentiation process.

Defective or insufficient bone repair and regeneration are common in patients as a result of major trauma or severe disease. Cell therapy with periosteal mesenchymal progenitors, which can be limited in severe injury, serves as a promising approach; however, its efficacy is limited due to a repair-hostile ischemic tissue microenvironment after traumatic fracture. Here we report that plasminogen (Plg), a factor that is upregulated in these environments, is critical for fracture healing. Plg knockout mice had impaired trabecular and cortical bone structure and exhibited delayed and incomplete fracture healing. Interestingly, Plg deficiency greatly reduced the thickness of expanded periosteum, suggesting a role of Plg in periosteal mesenchymal progenitor-mediated bone repair. In culture, Plg increased cell proliferation and migration in periosteal mesenchymal progenitors and inhibited cell death under ischemic conditions. Mechanistically, we revealed that Plg cleaved and activated Cyr61 to regulate periosteal progenitor function. Thus, our study uncovers a cellular mechanism underlying fracture healing, by which Plg activates Cyr61 to promote periosteal progenitor proliferation, survival, and migration and improves bone repair after fracture. Targeting Plg may offer a rational and effective therapeutic opportunity for improving fracture healing. © 2021 American Society for Bone and Mineral Research (ASBMR).

Meniscal tears are associated with a high risk of osteoarthritis but currently have no disease-modifying therapies. Using a Gli1 reporter line, we found that Gli1+ cells contribute to the development of meniscus horns from 2 weeks of age. In adult mice, Gli1+ cells resided at the superficial layer of meniscus and expressed known mesenchymal progenitor markers. In culture, meniscal Gli1+ cells possessed high progenitor activities under the control of Hh signal. Meniscus injury at the anterior horn induced a quick expansion of Gli1-lineage cells. Normally, meniscal tissue healed slowly, leading to cartilage degeneration. Ablation of Gli1+ cells further hindered this repair process. Strikingly, intra-articular injection of Gli1+ meniscal cells or an Hh agonist right after injury accelerated the bridging of the interrupted ends and attenuated signs of osteoarthritis. Taken together, our work identified a novel progenitor population in meniscus and proposes a new treatment for repairing injured meniscus and preventing osteoarthritis.

To investigate serum interleukin (IL)- 35 levels in patients with rheumatoid arthritis (RA) and to describe the association between serum IL-35 levels and clinical parameters: erythrocyte sedimentation rate (ESR), C reactive protein (CRP), global health on Visual Analog Scale, Disease Activity Score in 28 joints based on ESR (DAS28-ESR), rheumatoid factor (RF) and anticyclic citrullinated peptide antibodies (ACPAs). The study included 129 patients with RA and 83 healthy controls. Serum IL-35 levels were detected by ELISA. ESR and CRP were measured by the Westergren method and the immune transmission turbidity method, respectively. RF and ACPA were measured using immunoturbidimetric assays and chemiluminescence analysis, respectively. The results showed that serum IL-35 levels were elevated in patients with RA. Univariate and multivariate analyses showed that the high serum IL-35 levels were correlated with low ESR and DAS28-ESR. These suggested that IL-35, an important anti-inflammatory cytokine, may participate in the regulation of the pathogenesis of RA, especially with disease activity.

We aimed to investigate the effects of interleukin (IL)-35 on proangiogenic factors in IL-1β-pretreated chondrocyte-like SW1353 cells and screen-related genes that participated in osteoarthritis (OA) cartilage with IL-35, proangiogenic factors, and P38 mitogen-activated protein kinase (MAPK) signaling pathway. Different concentrations of IL-35 incubated with IL-1β stimulated SW1353 cells with or without SB203580 (inhibitor of P38 MAPK). Proangiogenic molecule expression was assessed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Microarray datasets were downloaded from the Gene Expression Omnibus database of OA cartilage. Protein-protein interaction of genes was visualized by Search Tool for the Retrieval Interacting Genes and Cytoscape. Database for Annotation, Visualization, and Integrated Discovery was used to screen biological processes and pathways. IL-35 inhibited mRNA expression of proangiogenic factors in IL-1β-stimulated SW1353 cells through the P38 MAPK signaling pathway. IL-35 inhibited angiopoietin-2 secretion. We found that 8 related genes, 18 biological processes, and 6 pathways may associate with IL-35, P38 MAPK signaling pathway, and cartilage angiogenesis. IL-35 regulated the expression of proangiogenic factors through P38 MAPK signaling pathway in IL-1β-stimulated SW1353 cells. IL-35 and P38 MAPK pathway may participate in neovascularization of cartilage. Our findings may provide molecular mechanisms and possible genes target treatment for OA.

In studies of electron and proton radiotherapy, ultrahigh dose rates of FLASH radiotherapy appear to produce fewer toxicities than standard dose rates while maintaining local tumor control. FLASH-proton radiotherapy (F-PRT) brings the spatial advantages of PRT to FLASH dose rates (>40 Gy/second), making it important to understand if and how F-PRT spares normal tissues while providing antitumor efficacy that is equivalent to standard-proton radiotherapy (S-PRT). Here we studied PRT damage to skin and mesenchymal tissues of muscle and bone and found that F-PRT of the C57BL/6 murine hind leg produced fewer severe toxicities leading to death or requiring euthanasia than S-PRT of the same dose. RNA-seq analyses of murine skin and bone revealed pathways upregulated by S-PRT yet unaltered by F-PRT, such as apoptosis signaling and keratinocyte differentiation in skin, as well as osteoclast differentiation and chondrocyte development in bone. Corroborating these findings, F-PRT reduced skin injury, stem cell depletion, and inflammation, mitigated late effects including lymphedema, and decreased histopathologically detected myofiber atrophy, bone resorption, hair follicle atrophy, and epidermal hyperplasia. F-PRT was equipotent to S-PRT in control of two murine sarcoma models, including at an orthotopic intramuscular site, thereby establishing its relevance to mesenchymal cancers. Finally, S-PRT produced greater increases in TGFβ1 in murine skin and the skin of canines enrolled in a phase I study of F-PRT versus S-PRT. Collectively, these data provide novel insights into F-PRT-mediated tissue sparing and support its ongoing investigation in applications that would benefit from this sparing of skin and mesenchymal tissues. Significance: These findings will spur investigation of FLASH radiotherapy in sarcoma and additional cancers where mesenchymal tissues are at risk, including head and neck cancer, breast cancer, and pelvic malignancies.

Radiation causes a collapse of bone marrow cells and elimination of microvasculature. To understand how bone marrow recovers after radiation, we focused on mesenchymal lineage cells that provide a supportive microenvironment for hematopoiesis and angiogenesis in bone. We recently discovered a nonproliferative subpopulation of marrow adipogenic lineage precursors (MALPs) that express adipogenic markers with no lipid accumulation. Single-cell transcriptomic analysis revealed that MALPs acquire proliferation and myofibroblast features shortly after radiation. Using an adipocyte-specific Adipoq-Cre, we validated that MALPs rapidly and transiently expanded at day 3 after radiation, coinciding with marrow vessel dilation and diminished marrow cellularity. Concurrently, MALPs lost most of their cell processes, became more elongated, and highly expressed myofibroblast-related genes. Radiation activated mTOR signaling in MALPs that is essential for their myofibroblast conversion and subsequent bone marrow recovery at day 14. Ablation of MALPs blocked the recovery of bone marrow vasculature and cellularity, including hematopoietic stem and progenitors. Moreover, VEGFa deficiency in MALPs delayed bone marrow recovery after radiation. Taken together, our research demonstrates a critical role of MALPs in mediating bone marrow repair after radiation injury and sheds light on a cellular target for treating marrow suppression after radiotherapy.

ObjectiveOsteoporosis is a common metabolic bone disease mainly involving bone remodeling and blood vessels. The current study aimed to explore the suppressive role of interleukin (IL)-35 in nuclear factor kappa-B ligand receptor activator (RANKL) and macrophage colony stimulating factor (M-CSF)-induced osteoclastogenesis and angiogenesis in osteoclasts.MethodsOsteoclasts differentiation were induced by incubation of mouse leukemic monocyte/macrophage cell line RAW264.7 cells in the presence of RANKL and M-CSF and was assessed with tartrate-resistant acid phosphatase (TRAP) staining assay. The viability and apoptosis of RAW264.7 was measured using CCK-8 assay and flow cytometry, respectively. The expression of angiogenic genes and proteins were measured using RT-PCR, Western blots and ELISA. The inhibition of Th17/IL-17 axis was examined using plumbagin, which was demonstrated as an IL-17A related signaling pathway inhibitor.ResultsIL-35 inhibited the viability of RAW264.7 cells and promoted the apoptosis of RAW264.7 cells in a dose-dependent manner. Furthermore, IL-35 dose-dependently suppressed the expression of angiogenic markers including VEGF and its receptor. The suppressive effect of IL-35 was confirmed through the activation of Th17/IL-17 axis.ConclusionsWe demonstrated for the first time the immuno-suppressive function of IL-35 on RANKL and M-CSF-induced osteoclastogenesis and angiogenesis through Th17/IL-17 axis. Therapeutic approach involving augmentation of IL-35 regulatory response may serve as a novel treatment option for osteoporosis, especially by suppressing bone resorption and angiogenesis.

Heterotopic ossification (HO) is a common, potentially debilitating pathology that is instigated by inflammation caused by tissue damage or other insults, which is followed by chondrogenesis, osteogenesis, and extraskeletal bone accumulation. Current remedies are not very effective and have side effects, including the risk of triggering additional HO. The TGF-β family member activin A is produced by activated macrophages and other inflammatory cells and stimulates the intracellular effectors SMAD2 and SMAD3 (SMAD2/3). Because HO starts with inflammation and because SMAD2/3 activation is chondrogenic, we tested whether activin A stimulated HO development. Using mouse models of acquired intramuscular and subdermal HO, we found that blockage of endogenous activin A by a systemically administered neutralizing antibody reduced HO development and bone accumulation. Single-cell RNA-seq analysis and developmental trajectories showed that the antibody treatment reduced the recruitment of Sox9+ skeletal progenitors, many of which also expressed the gene encoding activin A (Inhba), to HO sites. Gain-of-function assays showed that activin A enhanced the chondrogenic differentiation of progenitor cells through SMAD2/3 signaling, and inclusion of activin A in HO-inducing implants enhanced HO development in vivo. Together, our data reveal that activin A is a critical upstream signaling stimulator of acquired HO in mice and could represent an effective therapeutic target against forms of this pathology in patients.