A novel therapy for fracture healing by increasing lymphatic drainage.

Background: The musculoskeletal system contains an extensive network of lymphatic vessels. Decreased lymph flow of the draining collecting lymphatics usually occurs in clinic after traumatic fractures. However, whether defects in lymphatic drainage can affect fracture healing is unclear. Methods: To investigate the effect of lymphatic dysfunction on fracture healing, we used a selective VEGFR3 tyrosine kinase inhibitor to treat tibial fractured mice for 5 weeks versus a vehicle-treated control. To ensure successfully establishing deceased lymphatic drainage model for fractured mice, we measured lymphatic clearance by near infrared indocyanine green lymphatic imaging (NIR-ICG) and the volume of the draining popliteal lymph nodes (PLNs) by ultrasound at the whole phases of fracture healing. In addition, hindlimb edema from day 0 to day 7 post-fracture, pain sensation by Hargreaves test at day 1 post-fracture, bone histomorphometry by micro-CT and callus composition by Alcian Blue-Hematoxylin/Orange G staining at day 14 post-fracture, and bone quality by biomechanical testing at day 35 post-fracture were applied to evaluate fracture healing. To promote fracture healing via increasing lymphatic drainage, we then treated fractured mice with anti-mouse podoplanin (PDPN) neutralizing antibody or isotype IgG antibody for 1 week to observe lymphatic drainage function and assess bone repair as methods described above. Results: Compared to vehicle-treated group, SAR-treatment group significantly decreased lymphatic clearance and the volume of draining PLNs. SAR-treatment group significantly increased soft tissue swelling, and reduced bone volume (BV)/tissue volume (TV), trabecular number (Tb.N), woven bone and biomechanical properties of fracture callus. In addition, anti-PDPN treated group significantly reduced the number of CD41+ platelets in PLNs and increased the number of pulsatile lymphatic vessels, lymphatic clearance and the volume of PLNs. Moreover, anti-PDPN treated group significantly reduced hindlimb edema and pain sensation and increased BV/TV, trabecular number (Tb.Th), woven bone and biomechanical properties of fracture callus. Conclusions: Inhibition of proper lymphatic drainage function delayed fracture healing. Use of a anti-PDPN neutralizing antibody reduced lymphatic platelet thrombosis (LPT), increased lymphatic drainage and improved fracture healing. The translational potential of this article: (1) We demonstrated lymphatic drainage function is crucial for fracture healing. (2) To unblock the lymphatic drainage and prevent the risk of bleeding and mortality by blood thinner, we demonstrated PDPN neutralizing antibody is a novel and safe way forward in the treatment of bone fracture healing by eliminating LPT and increasing lymphatic drainage.

Reduced expression of semaphorin 3A in osteoclasts causes lymphatic expansion in a Gorham-Stout disease (GSD) mouse model. / ç ´éª¨ç»†èƒžä¸­ä¿¡å·ç´ 3Aè¡¨è¾¾çš„å‡å°‘å¯¼è‡´å°é¼ GSDæ¨¡åž‹ä¸­æ·‹å·´ç®¡æ‰©å¼ .

Gorham-Stout disease (GSD) is a sporadic chronic disease characterized by progressive bone dissolution, absorption, and disappearance along with lymphatic vessel infiltration in bone-marrow cavities. Although the osteolytic mechanism of GSD has been widely studied, the cause of lymphatic hyperplasia in GSD is rarely investigated. In this study, by comparing the RNA expression profile of osteoclasts (OCs) with that of OC precursors (OCPs) by RNA sequencing, we identified a new factor, semaphorin 3A (Sema3A), which is an osteoprotective factor involved in the lymphatic expansion of GSD. Compared to OCPs, OCs enhanced the growth, migration, and tube formation of lymphatic endothelial cells (LECs), in which the expression of Sema3A is low compared to that in OCPs. In the presence of recombinant Sema3A, the growth, migration, and tube formation of LECs were inhibited, further confirming the inhibitory effect of Sema3A on LECs in vitro. Using an LEC-induced GSD mouse model, the effect of Sema3A was examined by injecting lentivirus-expressing Sema3A into the tibiae in vivo. We found that the overexpression of Sema3A in tibiae suppressed the expansion of LECs and alleviated bone loss, whereas the injection of lentivirus expressing Sema3A short hairpin RNA (shRNA) into the tibiae caused GSD-like phenotypes. Histological staining further demonstrated that OCs decreased and osteocalcin increased after Sema3A lentiviral treatment, compared with the control. Based on the above results, we propose that reduced Sema3A in OCs is one of the mechanisms contributing to the pathogeneses of GSD and that expressing Sema3A represents a new approach for the treatment of GSD.

Distinct mast cell subpopulations within and around lymphatic vessels regulate lymph flow and progression of inflammatory-erosive arthritis in TNF-transgenic mice.

Objective: Inflammatory-erosive arthritis is exacerbated by dysfunction of joint-draining popliteal lymphatic vessels (PLVs). Synovial mast cells are known to be pro-inflammatory in rheumatoid arthritis (RA). In other settings they have anti-inflammatory and tissue reparative effects. Herein, we elucidate the role of mast cells on PLV function and inflammatory-erosive arthritis in tumor necrosis factor transgenic (TNF-tg) mice that exhibit defects in PLVs commensurate with disease progression. Methods: Whole mount immunofluorescent microscopy, toluidine blue stained histology, scanning electron microscopy, and in silico bioinformatics were performed to phenotype and quantify PLV mast cells. Ankle bone volumes were assessed by µCT, while corresponding histology quantified synovitis and osteoclasts. Near-infrared indocyanine green imaging measured lymphatic clearance as an outcome of PLV draining function. Effects of genetic MC depletion were assessed via comparison of 4.5-month-old WT, TNF-tg, MC deficient KitW-sh/W-sh (cKit-/-), and TNF-tg x cKit-/- mice. Pharmacological inhibition of mast cells was assessed by treating TNF-tg mice with placebo or cromolyn sodium (3.15mg/kg/day) for 3-weeks. Results: PLVs are surrounded by MCT+/MCPT1+/MCPT4+ mast cells whose numbers are increased 2.8-fold in TNF-tg mice. The percentage of peri-vascular degranulating mast cells was inversely correlated with ICG clearance. A population of MCT+/MCPT1-/MCPT4- mast cells were embedded within the PLV structure. In silico single-cell RNA-seq (scRNAseq) analyses identified a population of PLV-associated mast cells (marker genes: Mcpt4, Cma1, Cpa3, Tpsb2, Kit, Fcer1a & Gata2) with enhanced TGFß-related signaling that are phenotypically distinct from known MC subsets in the Mouse Cell Atlas. cKit-/- mice have greater lymphatic defects than TNF-tg mice with exacerbation of lymphatic dysfunction and inflammatory-erosive arthritis in TNF-tg x cKit-/- vs. TNF-Tg mice. Cromolyn sodium therapy stabilized PLV mast cells, increased TNF-induced bone loss, synovitis, and osteoclasts, and decreased ICG clearance. Conclusions: Mast cells are required for normal lymphatic function. Genetic ablation and pharmacological inhibition of mast cells exacerbates TNF-induced inflammatory-erosive arthritis with decreased lymphatic clearance. Together, these findings support an inflammatory role of activated/degranulated peri-PLV mast cells during arthritic progression, and a homeostatic role of intra-PLV mast cells, in which loss of the latter dominantly exacerbates arthritis secondary to defects in joint-draining lymphatics, warranting investigation into specific cellular mechanisms.

Lymphatic platelet thrombosis limits bone repair by precluding lymphatic transporting DAMPs.

Lymphatic vessels (LVs) interdigitated with blood vessels, travel and form an extensive transport network in the musculoskeletal system. Blood vessels in bone regulate osteogenesis and hematopoiesis, however, whether LVs in bone affect fracture healing is unclear. Here, by near infrared indocyanine green lymphatic imaging (NIR-ICG), we examined lymphatic draining function at the tibial fracture sites and found lymphatic drainage insufficiency (LDI) occurred as early as two weeks after fracture. Sufficient lymphatic drainage facilitates fracture healing. In addition, we identified that lymphatic platelet thrombosis (LPT) blocks the draining lymphoid sinus and LVs, caused LDI and then inhibited fracture healing, which can be rescued by a pharmacological approach. Moreover, unblocked lymphatic drainage decreased neutrophils and increased M2-like macrophages of hematoma niche to support osteoblast (OB) survival and bone marrow-derived mesenchymal stem cell (BMSC) proliferation via transporting damage-associated molecular patterns (DAMPs). These findings demonstrate that LPT limits bone regeneration by blocking lymphatic drainage from transporting DAMPs. Together, these findings represent a novel way forward in the treatment of bone repair.

Hepatorenal pathologies in TNF-transgenic mouse model of rheumatoid arthritis are alleviated by anti-TNF treatment.

OBJECTIVE: To examine and quantify liver and kidney lesions and their response to anti-tumor necrosis factor (TNF) therapy in a TNF-Tg mouse model of rheumatoid arthritis (RA). METHODS: Female TNF-Tg (Tg3647) mice were used as the animal model for chronic RA. Ultrasound, immunofluorescence, histological staining, serology tests, and real-time RT-PCR were used to examine the pathological changes in the liver and kidney. RESULTS: TNF-Tg mice showed a significant decrease in the body weight and a dramatic increase in the volumes of the gallbladder, knee cavity, and popliteal lymph nodes. The liver and kidneys of TNF-Tg mice showed increased chronic inflammation and accumulation of immune cells and fibrosis, compared to wild-type (WT) mice. Moreover, upregulation of inflammatory factors and impaired normal function were observed in the liver and kidneys of TNF-Tg mice. Inflammatory infiltration and fibrosis of the liver and kidneys of female TNF-Tg mice were improved after anti-TNF treatment, and better treatment effects were achieved at 4.5-month-old mice when they were received 8 weeks of intervention. CONCLUSIONS: We found that TNF drives the development of liver and kidney pathology in female TNF-Tg mice and that there are limitations to the loss of utility of anti-TNF for the prolonged treatment of RA-associated hepatic and renal injury. This study provides a reliable and clinically relevant animal model for further studies exploring the molecular mechanisms and drug discovery for hepatorenal pathologies in RA.

Multi-omics analysis identifies IgG2b class-switching with ALCAM-CD6 co-stimulation in joint-draining lymph nodes during advanced inflammatory-erosive arthritis.

Introduction: Defective lymphatic drainage and translocation of B-cells in inflamed (Bin) joint-draining lymph node sinuses are pathogenic phenomena in patients with severe rheumatoid arthritis (RA). However, the molecular mechanisms underlying this lymphatic dysfunction remain poorly understood. Herein, we utilized multi-omic spatial and single-cell transcriptomics to evaluate altered cellular composition (including lymphatic endothelial cells, macrophages, B-cells, and T-cells) in the joint-draining lymph node sinuses and their associated phenotypic changes and cell-cell interactions during RA development using the tumor necrosis factor transgenic (TNF-Tg) mouse model. Methods: Popliteal lymph nodes (PLNs) from wild-type (n=10) and TNF-Tg male mice with "Early" (5 to 6-months of age; n=6) and "Advanced" (>8-months of age; n=12) arthritis were harvested and processed for spatial transcriptomics. Single-cell RNA sequencing (scRNAseq) was performed in PLNs from the TNF-Tg cohorts (n=6 PLNs pooled/cohort). PLN histopathology and ELISPOT along with ankle histology and micro-CT were evaluated. Histopathology of human lymph nodes and synovia was performed for clinical correlation. Results: Advanced PLN sinuses exhibited an increased Ighg2b/Ighm expression ratio (Early 0.5 ± 0.1 vs Advanced 1.4 ± 0.5 counts/counts; p<0.001) that significantly correlated with reduced talus bone volumes in the afferent ankle (R2 = 0.54, p<0.001). Integration of single-cell and spatial transcriptomics revealed the increased IgG2b+ plasma cells localized in MARCO+ peri-follicular medullary sinuses. A concomitant decreased Fth1 expression (Early 2.5 ± 0.74 vs Advanced 1.0 ± 0.50 counts, p<0.001) within Advanced PLN sinuses was associated with accumulation of iron-laden Prussian blue positive macrophages in lymph nodes and synovium of Advanced TNF-Tg mice, and further validated in RA clinical samples. T-cells were increased 8-fold in Advanced PLNs, and bioinformatic pathway assessment identified the interaction between ALCAM+ macrophages and CD6+ T-cells as a plausible co-stimulatory mechanism to promote IgG2b class-switching. Discussion: Collectively, these data support a model of flare in chronic TNF-induced arthritis in which loss of lymphatic flow through affected joint-draining lymph nodes facilitates the interaction between effluxing macrophages and T-cells via ALCAM-CD6 co-stimulation, initiating IgG2b class-switching and plasma cell differentiation of the expanded Bin population. Future work is warranted to investigate immunoglobulin clonality and potential autoimmune consequences, as well as the efficacy of anti-CD6 therapy to prevent these pathogenic events.

Nuclear Factor-Kappa B Regulation of Osteoclastogenesis and Osteoblastogenesis.

Maintenance of skeletal integrity requires the coordinated activity of multinucleated bone-resorbing osteoclasts and bone-forming osteoblasts. Osteoclasts form resorption lacunae on bone surfaces in response to cytokines by fusion of precursor cells. Osteoblasts are derived from mesenchymal precursors and lay down new bone in resorption lacunae during bone remodeling. Nuclear factorkappa B (NF-κB) signaling regulates osteoclast and osteoblast formation and is activated in osteoclast precursors in response to the essential osteoclastogenic cytokine, receptor activator of NF-κB ligand (RANKL), which can also control osteoblast formation through RANK-RANKL reverse signaling in osteoblast precursors. RANKL and some pro-inflammatory cytokines, including tumor necrosis factor (TNF), activate NF-κB signaling to positively regulate osteoclast formation and functions. However, these cytokines also limit osteoclast and osteoblast formation through NF-κB signaling molecules, including TNF receptor-associated factors (TRAFs). TRAF6 mediates RANKL-induced osteoclast formation through canonical NF-κB signaling. In contrast, TRAF3 limits RANKL- and TNF-induced osteoclast formation, and it restricts transforming growth factor ß (TGFß)-induced inhibition of osteoblast formation in young and adult mice. During aging, neutrophils expressing TGFß and C-C chemokine receptor type 5 (CCR5) increase in bone marrow of mice in response to increased NF-κB-induced CC motif chemokine ligand 5 (CCL5) expression by mesenchymal progenitor cells and injection of these neutrophils into young mice decreased bone mass. TGFß causes degradation of TRAF3, resulting in decreased glycogen synthase kinase-3ß/ß-catenin-mediated osteoblast formation and age-related osteoporosis in mice. The CCR5 inhibitor, maraviroc, prevented accumulation of TGFß+/CCR5+ neutrophils in bone marrow and increased bone mass by inhibiting bone resorption and increasing bone formation in aged mice. This paper updates current understanding of how NF-κB signaling is involved in the positive and negative regulation of cytokine-mediated osteoclast and osteoblast formation and activation with a focus on the role of TRAF3 signaling, which can be targeted therapeutically to enhance bone mass.

The roles of bone remodeling in normal hematopoiesis and age-related hematological malignancies.

Prior research establishing that bone interacts in coordination with the bone marrow microenvironment (BMME) to regulate hematopoietic homeostasis was largely based on analyses of individual bone-associated cell populations. Recent advances in intravital imaging has suggested that the expansion of hematopoietic stem cells (HSCs) and acute myeloid leukemia cells is restricted to bone marrow microdomains during a distinct stage of bone remodeling. These findings indicate that dynamic bone remodeling likely imposes additional heterogeneity within the BMME to yield differential clonal responses. A holistic understanding of the role of bone remodeling in regulating the stem cell niche and how these interactions are altered in age-related hematological malignancies will be critical to the development of novel interventions. To advance this understanding, herein, we provide a synopsis of the cellular and molecular constituents that participate in bone turnover and their known connections to the hematopoietic compartment. Specifically, we elaborate on the coupling between bone remodeling and the BMME in homeostasis and age-related hematological malignancies and after treatment with bone-targeting approaches. We then discuss unresolved questions and ambiguities that remain in the field.

Molecular signatures distinguish senescent cells from inflammatory cells in aged mouse callus stromal cells.

Cellular senescence plays important roles in age-related diseases, including musculoskeletal disorders. Senescent cells (SCs) exert a senescence-associated secretory phenotype (SASP) by producing SASP factors, some of which overlap with factors produced by inflammatory cells (Inf-Cs). However, the differences between SCs and Inf-Cs and how they interact with each other during fracture repair have not been well studied. Here, we analyzed single cell RNA sequencing data of aged mouse fracture callus stromal cells. We defined Inf-Cs as cells that express NF-κB Rela/Relb, SCs as cells that express the senescence genes, Cdkn1a, Cdkn2a or Cdkn2c, and inflammatory SCs (Inf-SCs) as cells that express both NF-κB and senescence genes. Differentially expressed genes and pathway analyses revealed that Inf-SCs and SCs had a similar gene expression profile and upregulated pathways that are related to DNA damage/oxidation-reduction and cellular senescence, while Inf-Cs expressed different gene signatures and pathways from SCs and Inf-SCs, mainly related to inflammation. Cellchat software analysis indicated that SCs and Inf-SCs are potential ligand-producing cells that affect Inf-Cs as target cells. Cell culture experiments demonstrated that SC conditioned medium promoted inflammatory gene expression by callus-derived mesenchymal progenitor cells, and Inf-Cs had reduced osteoblast differentiation capacity. In summary, we have identified three cell subclusters associated with inflammation and senescence in callus stromal cells, predicted potential effects of Inf-SCs and SCs on Inf-Cs by production of active ligands, and demonstrated that when mesenchymal progenitors acquire inflammatory phenotypes their osteogenic potential is reduced.

Implementation of automated behavior metrics to evaluate voluntary wheel running effects on inflammatory-erosive arthritis and interstitial lung disease in TNF-Tg mice.

BACKGROUND: Although treatment options and algorithms for rheumatoid arthritis (RA) have improved remarkably in recent decades, there continues to be no definitive cure or pharmacologic intervention with reliable long-term efficacy. For this reason, the combination of medications and healthy lifestyle modifications are essential for controlling joint disease, and extra-articular manifestations of RA, such as interstitial lung disease (ILD) and other lung pathologies, which greatly impact morbidity and mortality. Generally, exercise has been deemed beneficial in RA patients, and both patients and clinicians are motivated to incorporate effective non-pharmacologic interventions. However, there are limited evidence-based and specific exercise regimens available to support engagement in such activities for RA patients. Here, we provided the continuous opportunity for exercise to mice and implemented automated recording and quantification of wheel running behavior. This allowed us to describe the associated effects on the progression of inflammatory-erosive arthritis and ILD in the tumor necrosis factor transgenic (TNF-Tg) mouse model of RA. METHODS: Wild-type (WT; males, n=9; females, n=9) and TNF-Tg (males, n=12; females, n=14) mice were singly housed with free access to a running wheel starting at 2 months until 5 to 5.5 months of age. Measures of running included distance, rate, length, and number of run bouts, which were derived from continuously recorded data streams collected automatically and in real-time. In vivo lung, ankle, and knee micro-computed tomography (micro-CT), along with terminal micro-CT and histology were performed to examine the association of running behaviors and disease progression relative to sedentary controls. RESULTS: TNF-Tg males and females exhibited significantly reduced running distance, rate, length, and number of run bouts compared to WT counterparts by 5 months of age (p<0.0001). Compared to sedentary controls, running males and females showed increased aerated lung volumes (p<0.05) that were positively correlated with running distance and rate in female mice (WT: Distance, ρ=0.705/rate, ρ=0.693 (p<0.01); TNF-Tg: ρ=0.380 (p=0.06)/ρ=0.403 (p<0.05)). Talus bone volumes were significantly reduced in running versus sedentary males and negatively correlated with running distance and rate in TNF-Tg mice (male: ρ=-903/ρ=-0.865; female: ρ=-0.614/ρ=-0.594 (p<0.001)). Histopathology validated the lung and ankle micro-CT findings. CONCLUSIONS: Implementation of automated wheel running behavior metrics allows for evaluation of longitudinal activity modifications hands-off and in real-time to relate with biomarkers of disease severity. Through such analysis, we determined that wheel running activity increases aerated lung volumes, but exacerbates inflammatory-erosive arthritis in TNF-Tg mice. To the end of a clinically relevant model, additional functional assessment of these outcomes and studies of pain behavior are warranted.

Targeting Synovial Lymphatic Function as a Novel Therapeutic Intervention for Age-Related Osteoarthritis in Mice.

OBJECTIVE: The synovial lymphatic system (SLS) removes catabolic factors from the joint. Vascular endothelial growth factor C (VEGF-C) and its receptor, VEGFR-3, are crucial for lymphangiogenesis. However, their involvement in age-related osteoarthritis (OA) is unknown. This study was undertaken to determine whether the SLS and the VEGF-C/VEGFR-3 pathway contribute to the development and progression of age-related OA, using a murine model of naturally occurring joint disease. METHODS: SLS function was assessed in the knees of young (3-month-old) and aged (19-24-month-old) male and female C57BL/6J mice via a newly established in vivo IVIS-dextran imaging approach, which, in addition to histology, was used to assess the effects of VEGF-C treatment on SLS function and OA pathology in aged mice. RNA-sequencing of synovial tissue was performed to explore molecular mechanisms of the disease in the mouse knee joints. RESULTS: Results showed that aged mice had impaired SLS function, including decreases in joint clearance (mean T1/2 of signal intensity clearance, 2.8 hours in aged mice versus 0.5 hours in young mice; P < 0.0001), synovial influx (mean ± SD 1.7 ± 0.8% in aged mice versus 4.1 ± 1.9% in young mice; P = 0.0004), and lymph node draining capacity (mean ± SD epifluorescence total radiant intensity ([photons/second]/[µW/cm2 ]) 1.4 ± 0.8 in aged mice versus 3.7 ± 1.2 in young mice; P < 0.0001). RNA-sequencing of the synovial tissue showed that Vegf-c and Vegfr3 signaling genes were decreased in the synovium of aged mice. VEGF-C treatment resulted in improvements in SLS function in aged mice, including increased percentage of signal intensity joint clearance (mean ± SD 63 ± 9% in VEGF-C-treated aged mice versus 52 ± 15% in vehicle-treated aged mice; P = 0.012), increased total articular cartilage cross-sectional area (mean ± SD 0.38 ± 0.07 mm2 in VEGF-C-treated aged mice versus 0.26 ± 0.07 mm2 in vehicle-treated aged mice; P < 0.0001), and decreased percentage of matrix metallopeptidase 13-positive staining area within total synovial area in 22-month-old VEGF-C-treated mice versus 22-month-old vehicle-treated mice (mean ± SD decrease 7 ± 2% versus 4 ± 1%; P = 0.0004). CONCLUSION: SLS function is reduced in the knee joints of aged mice due to decreased VEGF-C/VEGFR-3 signaling. VEGF-C treatment attenuates OA joint damage and improves synovial lymphatic drainage in aged mice. The SLS and VEGF-C/VEGFR-3 signaling represent novel physiopathologic mechanisms that could potentially be used as therapeutic targets for age-related OA.

TGFß1+CCR5+ neutrophil subset increases in bone marrow and causes age-related osteoporosis in male mice.

TGFß1 induces age-related bone loss by promoting degradation of TNF receptor-associated factor 3 (TRAF3), levels of which decrease in murine and human bone during aging. We report that a subset of neutrophils (TGFß1+CCR5+) is the major source of TGFß1 in murine bone. Their numbers are increased in bone marrow (BM) of aged wild-type mice and adult mice with TRAF3 conditionally deleted in mesenchymal progenitor cells (MPCs), associated with increased expression in BM of the chemokine, CCL5, suggesting that TRAF3 in MPCs limits TGFß1+CCR5+ neutrophil numbers in BM of young mice. During aging, TGFß1-induced TRAF3 degradation in MPCs promotes NF-κB-mediated expression of CCL5 by MPCs, associated with higher TGFß1+CCR5+ neutrophil numbers in BM where they induce bone loss. TGFß1+CCR5+ neutrophils decreased bone mass in male mice. The FDA-approved CCR5 antagonist, maraviroc, reduced TGFß1+CCR5+ neutrophil numbers in BM and increased bone mass in aged mice. 15-mon-old mice with TGFßRII specifically deleted in MPCs had lower numbers of TGFß1+CCR5+ neutrophils in BM and higher bone volume than wild-type littermates. We propose that pharmacologic reduction of TGFß1+CCR5+ neutrophil numbers in BM could treat or prevent age-related osteoporosis.

The Enigmas of Lymphatic Muscle Cells: Where Do They Come From, How Are They Maintained, and Can They Regenerate?

Lymphatic muscle cell (LMC) contractility and coverage of collecting lymphatic vessels (CLVs) are integral to effective lymphatic drainage and tissue homeostasis. In fact, defects in lymphatic contractility have been identified in various conditions, including rheumatoid arthritis, inflammatory bowel disease, and obesity. However, the fundamental role of LMCs in these pathologic processes is limited, primarily due to the difficulty in directly investigating the enigmatic nature of this poorly characterized cell type. LMCs are a unique cell type that exhibit dual tonic and phasic contractility with hybrid structural features of both vascular smooth muscle cells (VSMCs) and cardiac myocytes. While advances have been made in recent years to better understand the biochemistry and function of LMCs, central questions regarding their origins, investiture into CLVs, and homeostasis remain unanswered. To summarize these discoveries, unexplained experimental results, and critical future directions, here we provide a focused review of current knowledge and open questions related to LMC progenitor cells, recruitment, maintenance, and regeneration. We also highlight the high-priority research goal of identifying LMC-specific genes towards genetic conditional- inducible in vivo gain and loss of function studies. While our interest in LMCs has been focused on understanding lymphatic dysfunction in an arthritic flare, these concepts are integral to the broader field of lymphatic biology, and have important potential for clinical translation through targeted therapeutics to control lymphatic contractility and drainage.

Current understanding of osteoarthritis pathogenesis and relevant new approaches.

Osteoarthritis (OA) is the most common degenerative joint disease that causes painful swelling and permanent damage to the joints in the body. The molecular mechanisms of OA are currently unknown. OA is a heterogeneous disease that affects the entire joint, and multiple tissues are altered during OA development. To better understand the pathological mechanisms of OA, new approaches, methods, and techniques need to be used to understand OA pathogenesis. In this review, we first focus on the epigenetic regulation of OA, with a particular focus on DNA methylation, histone modification, and microRNA regulation, followed by a summary of several key mediators in OA-associated pain. We then introduce several innovative techniques that have been and will continue to be used in the fields of OA and OA-associated pain, such as CRISPR, scRNA sequencing, and lineage tracing. Next, we discuss the timely updates concerning cell death regulation in OA pathology, including pyroptosis, ferroptosis, and autophagy, as well as their individual roles in OA and potential molecular targets in treating OA. Finally, our review highlights new directions on the role of the synovial lymphatic system in OA. An improved understanding of OA pathogenesis will aid in the development of more specific and effective therapeutic interventions for OA.

Persistent popliteal lymphatic muscle cell coverage defects despite amelioration of arthritis and recovery of popliteal lymphatic vessel function in TNF-Tg mice following anti-TNF therapy.

While rheumatoid arthritis patients and tumor necrosis factor transgenic (TNF-Tg) mice with inflammatory-erosive arthritis display lymphatic drainage deficits, the mechanisms responsible remain unknown. As ultrastructural studies of joint-draining popliteal lymphatic vessels (PLVs) in TNF-Tg mice revealed evidence of lymphatic muscle cell (LMC) damage, we aimed to evaluate PLV-LMC coverage in TNF-Tg mice. We tested the hypothesis that alpha smooth muscle actin (αSMA)+ PLV-LMC coverage decreases with severe inflammatory-erosive arthritis, and is recovered by anti-TNF therapy facilitated by increased PLV-LMC turnover during amelioration of joint disease. TNF-Tg mice with established disease received anti-TNF monoclonal antibody (mAb) or placebo IgG isotype control mAb therapy (n = 5) for 6-weeks, while wild-type (WT) littermates (n = 8) received vehicle (PBS). Bromodeoxyuridine (BrdU) was also administered daily during the treatment period to monitor PLV-LMC turnover. Effective anti-TNF therapy was confirmed by longitudinal assessment of popliteal lymph node (PLN) volume via ultrasound, PLV contraction frequency via near-infrared imaging of indocyanine green, and ankle bone volumes via micro-computed tomography (micro-CT). Terminal knee micro-CT, and ankle and knee histology were also performed. PLVs were immunostained for αSMA and BrdU to evaluate PLV-LMC coverage and turnover, respectively, via whole-mount fluorescent microscopy. Anti-TNF therapy reduced PLN volume, increased talus and patella bone volumes, and reduced tarsal and knee synovial areas compared to placebo treated TNF-Tg mice (p < 0.05), as expected. Anti-TNF therapy also increased PLV contraction frequency at 3-weeks (from 0.81 ± 1.0 to 3.2 ± 2.0 contractions per minute, p < 0.05). However, both anti-TNF and placebo treated TNF-Tg mice exhibited significantly reduced αSMA+ PLV-LMC coverage compared to WT (p < 0.05). There was no correlation of αSMA+ PLV-LMC coverage restoration with amelioration of inflammatory-erosive arthritis. Similarly, there was no difference in PLV-LMC turnover measured by BrdU labeling between WT, TNF-Tg placebo, and TNF-Tg anti-TNF groups with an average of < 1% BrdU+ PLV-LMCs incorporated per week. Taken together these results demonstrate that PLV-LMC turnover in adult mice is limited, and that recovery of PLV function during amelioration of inflammatory-erosive arthritis occurs without restoration of αSMA+ LMC coverage. Future studies are warranted to investigate the direct and indirect effects of chronic TNF exposure, and the role of proximal inflammatory cells on PLV contractility.

Age-associated callus senescent cells produce TGF-ß1 that inhibits fracture healing in aged mice.

Cellular senescence plays an important role in human diseases, including osteoporosis and osteoarthritis. Senescent cells (SCs) produce the senescence-associated secretory phenotype to affect the function of neighboring cells and SCs themselves. Delayed fracture healing is common in the elderly and is accompanied by reduced mesenchymal progenitor cells (MPCs). However, the contribution of cellular senescence to fracture healing in the aged has not to our knowledge been studied. Here, we used C57BL/6J 4-month-old young and 20-month-old aged mice and demonstrated a rapid increase in SCs in the fracture callus of aged mice. The senolytic drugs dasatinib plus quercetin enhanced fracture healing in aged mice. Aged callus SCs inhibited the growth and proliferation of callus-derived MPCs (CaMPCs) and expressed high levels of TGF-ß1. TGF-ß-neutralizing Ab prevented the inhibitory effects of aged callus SCs on CaMPCs and promoted fracture healing in aged mice, which was associated with increased CaMPCs and proliferating cells. Thus, fracture triggered a significant cellular senescence in the callus cells of aged mice, which inhibited MPCs by expressing TGF-ß1. Short-term administration of dasatinib plus quercetin depleted callus SCs and accelerated fracture healing in aged mice. Senolytic drugs represent a promising therapy, while TGF-ß1 signaling is a molecular mechanism for fractures in the elderly via SCs.

Single-cell transcriptomics of popliteal lymphatic vessels and peripheral veins reveals altered lymphatic muscle and immune cell populations in the TNF-Tg arthritis model.

BACKGROUND: Lymphatic dysfunction exists in tumor necrosis factor transgenic (TNF-Tg) mice and rheumatoid arthritis (RA) patients. While joint-draining TNF-Tg popliteal lymphatic vessels (PLVs) have deficits in contractility during end-stage arthritis, the nature of lymphatic muscle cells (LMCs) and their TNF-altered transcriptome remain unknown. Thus, we performed single-cell RNA-sequencing (scRNAseq) on TNF-Tg LMCs in PLVs efferent to inflamed joints versus wild-type (WT) controls. METHODS: Single-cell suspensions of PLVs were sorted for smooth muscle cells (SMCs), which was validated by Cspg4-Cre;tdTomato reporter gene expression. Single-cell RNA-seq was performed on a 10x Genomics platform and analyzed using the Seurat R package. Uniform Manifold Approximation and Projections (UMAPs) and Ingenuity Pathway Analysis software were used to assess cell clusters and functional genomics in WT vs. TNF-Tg populations. RESULTS: Fluorescent imaging of Cspg4-Cre;tdTomato vessels demonstrated dim PLVs and strong reporter gene expression in the adjacent superficial saphenous vein, which was corroborated by flow cytometry of LMCs and vascular smooth muscle cells (VSMCs) from these vessels. Due to their unique morphology, these populations could also be readily detected by scatter analysis of cells from non-fluorescent mice. Bioinformatics analysis of flow sorted WT and TNF-Tg cells identified 20 unique cell clusters that together were 22.4% LMCs, 15.0% VSMCs, and 62.6% non-muscle cells of 8879 total cells. LMCs and M2-macrophages were decreased, while inflammatory monocytes were increased in TNF-Tg lower limb vasculature. SMC populations were defined by Cald1, Tpm1, and Pdgfrb expression and were enriched in myofibroblast-like gene expression. TNF-Tg LMCs exhibited enhanced functional genomics associated with cell death, phagocyte recruitment, and joint inflammation. Among the most prominent TNF-induced genes in SMCs were Mmp3, Cxcl12, and Ccl19, and the most downregulated genes were Zbtb16, Galnt15, and Apod. CONCLUSIONS: Single-cell RNA-seq can be used to investigate functional genomics of lower limb vasculature in mice. Our findings confirm the inflammatory transcriptome of TNF-Tg vessels and altered gene expression in SMC populations. This study further supports a potential role of mesenchymal stromal cells in inflammatory-erosive arthritis pathogenesis, and warrants future studies to define the effects of this TNF-altered transcriptome on PLV function and joint homeostasis.

Single-cell RNA landscape of the osteoimmunology microenvironment in periodontitis.

Single-cell RNA sequencing (scRNA-seq) enables specific profiling of cell populations at single-cell resolution. The osteoimmunology microenvironment in the occurrence and development of periodontitis remains poorly understood at the single-cell level. In this study, we used single-cell transcriptomics to comprehensively reveal the complexities of the molecular components and differences with counterparts residing in periodontal tissues. Methods: We performed scRNA-seq to identify 51248 single cells from healthy controls (n=4), patients with severe chronic periodontitis (n=5), and patients with severe chronic periodontitis after initial periodontal therapy within 1 month (n=3). Uniform manifold approximation and projection (UMAP) were further conducted to explore the cellular composition of periodontal tissues. Pseudotime cell trajectory and RNA velocity analysis, combined with gene enrichment analysis were used to reveal the molecular pathways underlying cell fate decisions. CellPhoneDB were performed to identify ligand-receptor pairs among the major cell types in the osteoimmunology microenvironment of periodontal tissues. Results: A cell atlas of the osteoimmunology microenvironment in periodontal tissues was characterized and included ten major cell types, such as fibroblasts, monocytic cells, endothelial cells, and T and B cells. The enrichment of TNFRSF21+ fibroblasts with high expression of CXCL1, CXCL2, CXCL5, CXCL6, CXCL13, and IL24 was detected in patients with periodontitis compared to healthy individuals. The fractions of CD55+ mesenchymal stem cells (MSCs), APOE+ pre-osteoblasts (pre-OBs), and IBSP+ osteoblasts decreased significantly in response to initial periodontal therapy. In addition, CXCL12+ MSC-like pericytes could convert their identity into a pre-OB state during inflammatory responses even after initial periodontal therapy confirmed by single-cell trajectory. Moreover, we portrayed the distinct subtypes of monocytic cells and abundant endothelial cells significantly involved in the immune response. The heterogeneity of T and B cells in periodontal tissues was characterized. Finally, we mapped osteoblast/osteoclast differentiation mediators to their source cell populations by identifying ligand-receptor pairs and highlighted the effects of Ephrin-Eph signaling on bone regeneration after initial periodontal therapy. Conclusions: Our analyses uncovered striking spatiotemporal dynamics in gene expression, population composition, and cell-cell interactions during periodontitis progression. These findings provide insights into the cellular and molecular underpinning of periodontal bone regeneration.

Proteasome inhibition-enhanced fracture repair is associated with increased mesenchymal progenitor cells in mice.

The ubiquitin/proteasome system controls the stability of Runx2 and JunB, proteins essential for differentiation of mesenchymal progenitor/stem cells (MPCs) to osteoblasts. Local administration of proteasome inhibitor enhances bone fracture healing by accelerating endochondral ossification. However, if a short-term administration of proteasome inhibitor enhances fracture repair and potential mechanisms involved have yet to be exploited. We hypothesize that injury activates the ubiquitin/proteasome system in callus, leading to elevated protein ubiquitination and degradation, decreased MPCs, and impaired fracture healing, which can be prevented by a short-term of proteasome inhibition. We used a tibial fracture model in Nestin-GFP reporter mice, in which a subgroup of MPCs are labeled by Nestin-GFP, to test our hypothesis. We found increased expression of ubiquitin E3 ligases and ubiquitinated proteins in callus tissues at the early phase of fracture repair. Proteasome inhibitor Bortezomib, given soon after fracture, enhanced fracture repair, which is accompanied by increased callus Nestin-GFP+ cells and their proliferation, and the expression of osteoblast-associated genes and Runx2 and JunB proteins. Thus, early treatment of fractures with Bortezomib could enhance the fracture repair by increasing the number and proliferation of MPCs.

Bone-Targeted Bortezomib Inhibits Bortezomib-Resistant Multiple Myeloma in Mice by Providing Higher Levels of Bortezomib in Bone.

Limited treatment options exist for cancer within the bone, as demonstrated by the inevitable, pernicious course of metastatic and blood cancers. The difficulty of eliminating bone-residing cancer, especially drug-resistant cancer, necessitates novel, alternative treatments to manipulate tumor cells and their microenvironment, with minimal off-target effects. To this end, bone-targeted conjugate (BP-Btz) was generated by linking bortezomib (Btz, an anticancer, bone-stimulatory drug) to a bisphosphonate (BP, a targeting ligand) through a cleavable linker that enables spatiotemporally controlled delivery of Btz to bone under acidic conditions for treating multiple myeloma (MM). Three conjugates with different linkers were developed and screened for best efficacy in mouse model of MM. Results demonstrated that the lead candidate BP-Btz with optimal linker could overcome Btz resistance, reduced tumor burden, bone destruction, or tumor metastasis more effectively than BP or free Btz without thrombocytopenia and neurotoxicity in mice bearing myeloma. Furthermore, pharmacokinetic and pharmacodynamic studies showed that BP-Btz bound to bone matrix, released Btz in acidic conditions, and had a higher local concentration and longer half-life than Btz in bone. Our findings suggest the potential of bone-targeted Btz conjugate as an efficacious Btz-resistant MM treatment mechanism. © 2021 American Society for Bone and Mineral Research (ASBMR).