Search

ObjectiveNRF2 is a master transcription factor that regulates the stress response. NRF2 is frequently mutated and activated in human esophageal squamous cell carcinoma (ESCC), which drives resistance to chemotherapy and radiation therapy. Therefore, a great need exists for NRF2 inhibitors for targeted therapy of NRF2highESCC.DesignWe performed high-throughput screening of two compound libraries from which hit compounds were further validated in human ESCC cells and a genetically modified mouse model. The mechanism of action of one compound was explored by biochemical assays.ResultsUsing high-throughput screening of two small molecule compound libraries, we identified 11 hit compounds as potential NRF2 inhibitors with minimal cytotoxicity at specified concentrations. We then validated two of these compounds, pyrimethamine and mitoxantrone, by demonstrating their dose- and time-dependent inhibitory effects on the expression of NRF2 and its target genes in twoNRF2Muthuman ESCC cells (KYSE70 and KYSE180). RNAseq and qPCR confirmed the suppression of global NRF2 signaling by these two compounds. Mechanistically, pyrimethamine reduced NRF2 half-life by promoting NRF2 ubiquitination and degradation in KYSE70 and KYSE180 cells. Expression of anNrf2E79Qallele in mouse esophageal epithelium (Sox2CreER;LSL-Nrf2E79Q/+) resulted in an NRF2highphenotype, which included squamous hyperplasia, hyperkeratinization, and hyperactive glycolysis. Treatment with pyrimethamine (30mg/kg/day,p.o.) suppressed the NRF2highesophageal phenotype with no observed toxicity.ConclusionWe have identified and validated pyrimethamine as an NRF2 inhibitor that may be rapidly tested in the clinic as a radiation and chemotherapy sensitizer for NRF2highESCC.SummaryWhat is already known on this topic – summarise the state of scientific knowledge on this subject before you did your study and why this study needed to be doneMutational activation of the NRF2 transcription factor drives ESCC progression and therapeutic resistance. Targeted therapies to block NRF2 have not yet been realized, despite great needs.What this study adds – summarise what we now know as a result of this study that we did not know beforeA screen of >35,000 small molecules identified eleven potential NRF2 inhibitors. Pyrimethamine and mitoxantrone were validated to inhibit the expression of NRF2 and NQO1 in human ESCC cells in both dose- and time-dependent manners.Pyrimethamine enhanced NRF2 protein ubiquitination and degradation, resulting a decreased half-life.A genetically modified mouse model was established to express theNrf2E79Qmutant allele in the mouse esophageal epithelium upon tamoxifen induction. Pyrimethamine suppressed the NRF2highesophageal phenotype induced by the mutant allele.How this study might affect research, practice or policy – summarise the implications of this studyAs an FDA-approved drug, Pyrimethamine has the potential for immediate translation to a clinical trial on NRF2highESCC in humans.Further exploration of its mechanisms of action may lead to more potent NRF2 inhibitors for future use.

Pegloticase is an enzyme used to reduce serum uric acid levels in patients with chronic, treatment-refractory gout. Clinically, about 40% of patients develop high titers of anti-PEG antibodies (APA) after initial treatment, which in turn quickly eliminate subsequent doses of pegloticase from the systemic circulation and render the treatment ineffective. We previously found that pre-infusion with high MW free PEG (40 kDa) can serve as a decoy to saturate circulating APA, preventing binding to a subsequently administered dose of PEG-liposomes and restoring their prolonged circulation in mice, without any detectible toxicity. Here, we investigated the use of 40 kDa free PEG to restore the circulation of radio-labeled pegloticase in mice using longitudinal Positron Emission Tomography (PET) imaging over 4 days. Mice injected with pegloticase developed appreciable APA titers by Day 9, which further increased through Day 14. Compared to naive mice, mice with pegloticase-induced APA rapidly cleared 89Zr-labeled pegloticase, with ~75% lower pegloticase concentrations in the circulation at four hours after treatment. The 96-h AUC in APA+ mice was less than 30% of the AUC in naive mice. In contrast, pre-infusion of free PEG into PEG-sensitized mice restored the AUC of pegloticase to ~80% of that seen in naive mice, resulting in a similar biodistribution to pegloticase in naive mice over time. These results suggest that pre-infusion of free PEG may be a promising strategy to enable the safe and efficacious use of pegloticase and other PEGylated drugs in patients that have previously failed therapy due to induced APA.

Reduced knee weight-bearing from prescription or sedentary lifestyles are associated with cartilage degradation; effects on the meniscus are unclear. Rodents exposed to spaceflight or hind limb unloading (HLU) represent unique opportunities to evaluate this question. This study evaluated arthritic changes in the medial knee compartment that bears the highest loads across the knee after actual and simulated spaceflight, and recovery with subsequent full weight-bearing. Cartilage and meniscal degradation in mice were measured via microCT, histology, and proteomics and/or biochemically after: (1) ~ 35 days on the International Space Station (ISS); (2) 13-days aboard the Space Shuttle Atlantis; or (3) 30 days of HLU, followed by a 49-day weight-bearing readaptation with/without exercise. Cartilage degradation post-ISS and HLU occurred at similar spatial locations, the tibial-femoral cartilage-cartilage contact point, with meniscal volume decline. Cartilage and meniscal glycosaminoglycan content were decreased in unloaded mice, with elevated catabolic enzymes (e.g., matrix metalloproteinases), and elevated oxidative stress and catabolic molecular pathway responses in menisci. After the 13-day Shuttle flight, meniscal degradation was observed. During readaptation, recovery of cartilage volume and thickness occurred with exercise. Reduced weight-bearing from either spaceflight or HLU induced an arthritic phenotype in cartilage and menisci, and exercise promoted recovery.

© 2019 American Society for Investigative Pathology Mitogen-activated protein kinases, including c-Jun NH2-terminal kinase (JNK), play an important role in the development and function of a large variety of tissues. The skeletal phenotype of JNK1 and JNK2 double-knockout (dKO) mice (JNK1 fl/fl Col2-Cre/JNK2 −/− ) and control genotypes were analyzed at different embryonic and postnatal stages. JNK1/2 dKO mice displayed a severe scoliotic phenotype beginning during development that was grossly apparent around weaning age. Alcian blue staining at embryonic day 17.5 showed abnormal fusion of the posterior spinal elements. In adult mice, fusion of vertebral bodies and of spinous and transverse processes was noted by micro–computed tomography, Alcian blue/Alizarin red staining, and histology. The long bones developed normally, and histologic sections of growth plate and articular cartilage revealed no significant abnormalities. Histologic sections of the vertebral column at embryonic days 15.5 and 17.5 revealed an abnormal organization of the annulus fibrosus in the dKOs, with chondrocyte-like cells and fusion of dorsal processes. Spinal sections in 10-week–old dKO mice showed replacement of intervertebral disk structures (annulus fibrosus and nucleus pulposus) by cartilage and bone tissues, with cells staining for markers of hypertrophic chondrocytes, including collagen X and runt-related transcription factor 2. These findings demonstrate a requirement for both JNK1 and JNK2 in the normal development of the axial skeleton. Loss of JNK signaling results in abnormal endochondral bone formation and subsequent severe scoliosis.

The age at which astronauts experience microgravity is a critical consideration for skeletal health and similarly has clinical relevance for musculoskeletal disuse on Earth. While astronauts are extensively studied for bone and other physiological changes, rodent studies enable direct evaluation of skeletal changes with microgravity. Yet, mouse spaceflight studies have predominately evaluated tissues from young, growing mice. We evaluated bone microarchitecture in tibiae and femurs from Young (9-week-old) and Mature (32-weeks-old) female, C57BL/6N mice flown in microgravity for ~2 and ~3 weeks, respectively. Microgravity-induced changes were both compartment- and site-specific. Changes were greater in trabecular versus cortical bone in Mature mice exposed to microgravity (−40.0% Tb. BV/TV vs −4.4% Ct. BV/TV), and bone loss was greater in the proximal tibia as compared to the distal femur. Trabecular thickness in Young mice increased by +25.0% on Earth and no significant difference following microgravity. In Mature mice exposed to microgravity, trabecular thickness rapidly decreased (−24.5%) while no change was detected in age-matched mice that were maintained on Earth. Mature mice exposed to microgravity experienced greater bone loss than Young mice with net skeletal growth. Moreover, machine learning classification models confirmed that microgravity exposure-driven decrements in trabecular microarchitecture and cortical structure occurred disproportionately in Mature than in Young mice. Our results suggest that age of disuse onset may have clinical implications in osteoporotic or other at-risk populations on Earth and may contribute to understanding bone loss patterns in astronauts.

While joint damage is the primary co-morbidity of hemophilia, osteoporosis and osteopenia are also observed. Coagulation factor VIII deficient (FVIII−/−) mice develop an osteoporotic phenotype in the absence of induced hemarthrosis that is exacerbated two weeks after an induced joint injury. Here we have compared comprehensively the bone health of clotting factor VIII, factor IX, and Von Willebrand Factor knockout (FVIII−/−, FIX−/−, and VWF−/− respectively) mice both in the absence of joint hemorrhage and following induced joint injury. We found FVIII−/− and FIX−/− mice, but not VWF−/− mice, developmentally have an osteoporotic phenotype. Unilateral induced hemarthrosis causes further bone damage in both FVIII−/− and FIX−/− mice, but has little effect on VWF−/− bone health, indicating that the FVIII.VWF complex is not required for normal bone remodeling in vivo. To further investigate the bone healing following hemarthrosis in hemophilia we examined a two week time course using microCT, serum chemistry, and histological analysis. Elevated ratio of osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL), increased osterix+ osteoblastic cells, and decreased smoothness of the cortical bone surface were evident within several days of injury, indicative of acute heterotopic mineralization along the cortical surface. This was closely followed by increased interleukin-6 (IL-6) levels, increased osteoclast numbers, and significant trabecular bone loss. Uncoupled and disorganized bone formation and resorption continued for the duration of the study resulting in significant deterioration of the joint. Further elucidation of the shared mechanisms underlying abnormal bone homeostasis in the absence of FVIII or FIX is needed to guide evidence-based approaches to the screening and treatment of the prevalent bone defects in hemophilia A and B.

Radiation therapy and estrogen deficiency can damage healthy bone and lead to an increased fracture risk. The goal of this study is to develop a mouse model for radiation therapy using a fractionated biologically equivalent dose for cervical cancer treatment in both pre- and postmenopausal women. Thirty-two female C57BL/6 mice 13 weeks of age were divided into four groups: Sham + non-irradiated (SHAM + NR), Sham + irradiated (SHAM + IRR), ovariectomy + non-irradiated (OVX + NR) and ovariectomy + irradiated (OVX + IRR). The irradiated mice received a 6 Gy dose of X-rays to the hindlimbs at Day 2, Day 4 and Day 7 (18 Gy total). Tissues were collected at Day 35. DEXA, microCT analysis and FEA were used to quantify structural and functional changes at the proximal tibia, midshaft femur, proximal femur and L1 vertebra. There was a significant (p

The long-term adaptations to microgravity and other spaceflight challenges within the confines of a spacecraft, and readaptations to weight-bearing upon reaching a destination, are unclear. While post-flight gait change in astronauts have been well documented and reflect multi-system deficits, no data from rodents have been collected. Thus, the purpose of this study was to evaluate gait changes in response to spaceflight. A prospective collection of gait data was collected on 3 groups of mice: those who spent~35 days in orbit (FLIGHT) aboard the International Space Station (ISS); a ground-based control with the same habitat conditions as ISS (Ground Control; GC); and a vivarium control with typical rodent housing conditions (VIV). Pre-flight and post-flight gait measurements were conducted utilizing an optimized and portable gait analysis system (DigiGait, Mouse Specifics, Inc). The total data acquisition time for gait patterns of FLIGHT and control mice was 1.5–5 min/mouse, allowing all 20 mice per group to be assessed in less than an hour. Patterns of longitudinal gait changes were observed in the hind limbs and the forelimbs of the FLIGHT mice after ~35 days in orbit; few differences were observed in gait characteristics within the GC and VIV controls from the initial to the final gait assessment, and between groups. For FLIGHT mice, 12 out of 18 of the evaluated gait characteristics in the hind limbs were significantly changed, including: stride width variability; stride length and variance; stride, swing, and stance duration; paw angle and area at peak stance; and step angle, among others. Gait characteristics that decreased included stride frequency, and others. Moreover, numerous forelimb gait characteristics in the FLIGHT mice were changed at post-flight measures relative to pre-flight. This rapid DigiGait gait measurement tool and customized spaceflight protocol is useful for providing preliminary insight into how spaceflight could affect multiple systems in rodents in which deficits are reflected by altered gait characteristics.

Computed tomography (CT) images can potentially provide insights into bone structure for diagnosis of disorders and diseases. However, evaluation of trabecular bone structure and whole bone shape is often qualitative or semi-quantitative. This limits inter-study comparisons and the ability to detect subtle bone quality variations during early disease onset or in response to new treatments. In this work, we enable quantitative characterization of bone diseases through bone morphometry, texture analysis, and shape analysis methods. The potential of our analysis methods to identify the impact of hemophilia is validated in a mouse femur wound model. In our results, shape localizes and characterizes the formation of spurious bone, and our texture and bone morphometry analysis results provide extra information about the composition of that bone. Some of our one-dimensional (1D) textural features were able to significantly differentiate our injured femurs from our healthy femurs, even with this small sample size demonstrating the potential of the proposed analysis framework. While trabecular bone morphometrics have been a pillar in 3D microCT bone research for decades, the proposed analysis framework augments how we define and understand phenotypical presentation of bone disease. The contributed open source software is exposed to the medical image analysis community through 3D Slicer extensions to ensure both robustness and reproducibility.

As the duration of manned missions outside of the Earth's protective shielding increase, astronauts are at risk for exposure to space radiation. Various organ systems may be damaged due to exposure. This study investigates the bone strength changes using finite element modeling of Long Evans rats (n=85) subjected to graded, head-only proton (0, 10, 25, and 100 cGy, 150 MeV/n) and 28silicon (0, 10, 25, and 50 cGy, 300 MeV/n) radiation. The strength of the femoral neck will be examined due its clinical relevance to hip fractures. It has been shown in previous studies that bone mineral density was not reduced at the site of fracture. These findings question whether measurements of bone mineral density may be used to assess risk of hip fracture. The mechanisms leading to the irregular relationship between bone density and strength are still uncertain within literature and investigated to greater extent in clinical applications. Finite element analysis within this study simulated physiological loading of the femoral neck. No significant changes in femoral neck strength were found across doses of proton or 28silicon head-only radiation. Future work includes performing mechanical testing of the bone samples. Moving from mouse to larger animal models may also provide the increased lifespan for assessing the long-term outcomes of radiation exposure.

Chronic kidney disease (CKD) increases bone fracture risk. While the causes of bone fragility in CKD are not clear, the disrupted mineral homeostasis inherent to CKD may cause material quality changes to bone tissue. In this study, 11-week old male C57Bl/6J mice underwent either 5/6th nephrectomy (5/6 Nx) or sham procedures. Mice were fed a normal chow diet and euthanized 11 weeks post-surgery. Moderate CKD with high bone turnover was established in the 5/6 Nx group as determined through serum chemistry and bone gene expression assays. We compared nanoindentation modulus and mineral volume fraction (assessed through quantitative backscattered scanning electron microscopy) at matched sites in arrays placed on the cortical bone of the tibia mid-diaphysis. Trabecular and cortical bone microarchitecture (μCT) and whole bone strength were also evaluated. We found that moderate CKD minimally affected bone microarchitecture and did not influence whole bone strength. Meanwhile, bone material quality decreased with CKD; a pattern of altered tissue maturation was observed with 5/6 Nx whereby the newest 60 micrometers of bone tissue adjacent to the periosteal surface had lower indentation modulus and mineral volume fraction than more interior, older bone. The variance of modulus and mineral volume fraction were also altered following 5/6 Nx, implying that tissue-scale heterogeneity may be negatively affected by CKD. The observed lower bone material quality may play a role in the decreased fracture resistance that is clinically associated with human CKD.

Bone loss associated with microgravity exposure poses a significant barrier to long-duration spaceflight. Osteoprotegerin-Fc (OPG-Fc) is a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor that causes sustained inhibition of bone resorption after a single subcutaneous injection. We tested the ability of OPG-Fc to preserve bone mass during 12 days of spaceflight (SF). 64-day-old female C57BL/6J mice (n=12/group) were injected subcutaneously with OPG-Fc (20 mg/kg) or an inert vehicle (VEH), 24 hours prior to launch. Ground control (GC) mice (VEH or OPG-Fc) were maintained under environmental conditions that mimicked those in the space shuttle middeck. Age-matched baseline (BL) controls were sacrificed at launch. GC/VEH, but not SF/VEH mice, gained tibia BMD and trabecular volume fraction (BV/TV) during the mission (p

The microgravity conditions of prolonged spaceflight are known to result in skeletal muscle atrophy that leads to diminished functional performance. To assess if inhibition of the growth factor myostatin has potential to reverse these effects, mice were treated with a myostatin antibody while housed on the International Space Station. Grip strength of ground control mice increased 3.1% compared to baseline values over the 6 weeks of the study, whereas grip strength measured for the first time in space showed flight animals to be -7.8% decreased in strength compared to baseline values. Control mice in space exhibited, compared to ground-based controls, a smaller increase in DEXA-measured muscle mass (+3.9% vs +5.6% respectively) although the difference was not significant. All individual flight limb muscles analyzed (except for the EDL) weighed significantly less than their ground counterparts at the study end (range -4.4% to -28.4%). Treatment with myostatin antibody YN41 was able to prevent many of these space-induced muscle changes. YN41 was able to block the reduction in muscle grip strength caused by spaceflight and was able to significantly increase the weight of all muscles of flight mice (apart from the EDL). Muscles of YN41-treated flight mice weighed as much as muscles from Ground IgG mice, with the exception of the soleus, demonstrating the ability to prevent spaceflight-induced atrophy. Muscle gene expression analysis demonstrated significant effects of microgravity and myostatin inhibition on many genes. Gamt and Actc1 gene expression was modulated by microgravity and YN41 in opposing directions. Myostatin inhibition did not overcome the significant reduction of microgravity on femoral BMD nor did it increase femoral or vertebral BMD in ground control mice. In summary, myostatin inhibition may be an effective countermeasure to detrimental consequences of skeletal muscle under microgravity conditions.

Background Following induced joint hemorrhage, hemophilia B results in the abnormal persistence of iron deposition, inflammation, and neovascularity of the synovial tissue, as well as deterioration of the bone articular surface and strength. Previously, we demonstrated that a factor IX (FIX) replacement protein with extended circulating FIX activity, glycoPEGylated FIX nonacog beta pegol (N9-GP), could improve synovial and osteochondral parameters in F9 knockout mice when administered after joint injury. Objective We explored the use of N9-GP prior to unilateral joint hemorrhage and compared to unmodified recombinant FIX (rFIX). Methods Pharmacodynamics, histology, and microcomputed tomography were used to assess the effects of prophylactic administration of glycoPEGylated FIX. Results In comparison to rFIX, N9-GP significantly improved soft tissue histological parameters, as well as bone outcome at 2 weeks post injury, while performing equally in reduction of blood present in the joint space assessed 1 day after injury. Conclusions These results indicate that, in comparison to rFIX, the prophylactic use of extended half-life FIX provides superior protection from bleeding-induced joint damage, manifested by improved correction of histologic parameters.

While chronic degenerative arthropathy is the main morbidity of haemophilia, a very high prevalence of low bone density is also seen in men and boys with haemophilia. This study investigates bone degradation in the knee joint of haemophilic mice resulting from haemarthrosis and the efficacy of aggressive treatment with factor VIII in the period surrounding injury to prevent bone pathology. Skeletally mature factor VIII knock-out mice were subjected to knee joint haemorrhage induced by puncture of the left knee joint capsule. Mice received either intravenous factor VIII treatment or placebo immediately prior to injury and at hours 4, 24, 48, 72 and 96 after haemorrhage. Mice were killed 2-weeks after injury and the joint morphology and loss of bone in the proximal tibia was assessed using microCT imaging. Quantitative microCT imaging of the knee joint found acute bone loss at the proximal tibia following injury including loss of trabecular bone volumetric density and bone mineral density, as well as trabecular connectivity density, number and thickness. Unexpectedly, joint injury also resulted in calcification of the joint soft tissues including the tendons, ligaments, menisci and cartilage. Treatment with factor VIII prevented this bone and soft tissue degeneration. Knee joint haemorrhage resulted in acute changes in adjacent bone including loss of bone density and mineralization of joint soft tissues. The rapid calcification and loss of bone has implications for the initiation and progression of osteoarthritic degradation following joint bleeding.

During spaceflight, mice are housed in specially designed cages called the Animal Enclosure Module (AEM). Utilization of this flight hardware may affect the skeletal properties of housed animals, independent of microgravity considerations. To address this issue, we studied the effect of 13 days of AEM housing versus standard vivarium enclosure on female C57BL/6J mice (n=12/group). The effects of AEM housing were most pronounced in the trabecular compartment. AEM mice had 44% and 144% greater trabecular bone volume fraction and connectivity density, respectively, versus vivarium. A similar response was seen at the proximal humerus. We noted a decrease in proximal tibia osteoclast surface (-65%) and eroded surface (-73%) for AEM versus vivarium, while tibia trabecular mineralizing surface (MS/BS) was nearly three-fold greater. Surprisingly, there was also decreased osteoblast surface, as well as lower osteoid volume, surface, and thickness at this site. The effects of AEM housing on femur cortical bone were modest: there was greater periosteal MS/BS, with no effect at the endocortical surface, and lower femur stiffness. Taken together, we have demonstrated significant effects of AEM housing on ground control mice, particularly in the trabecular bone compartment. These findings suggest that an early increase in bone formation, perhaps due to altered behavior and loading in this unique housing environment, was followed by decreased bone formation and resorption as the animals adapted to their new environment. Characterization of spaceflight animal housing is critical to elucidating the true effects of microgravity on skeletal parameters and for the proper selection of ground-based controls.

Wound healing requires interactions between coagulation, inflammation, angiogenesis, cellular migration, and proliferation. Healing in dermal wounds of hemophilia B mice is delayed when compared with hemostatically normal wild-type (WT) mice, with abnormal persistence of iron deposition, inflammation, and neovascularity. We observed healing following induced joint hemorrhage in WT and factor IX (FIX) knockout (FIX-/-) mice, examining also parameters previously studied in an excisional skin wound model. Hemostatically normal mice tolerated this joint bleeding challenge, cleared blood from the joint, and healed with minimal pathology, even if additional autologous blood was injected intra-articularly at the time of wounding. Following hemarthrosis, joint wound healing in hemophilia B mice was impaired and demonstrated similar abnormal histologic features as previously described in hemophilic dermal wounds. Therefore, studies of pathophysiology and therapy of hemophilic joint bleeding performed in hemostatically normal animals are not likely to accurately reflect the healing defect of hemophilia. We additionally explored the hypothesis that the use of a FIX replacement protein with extended circulating FIX activity could improve synovial and osteochondral wound healing in hemophilic mice, when compared with treatment with unmodified recombinant FIX (rFIX) in the established joint bleeding model. Significantly improved synovial wound healing and preservation of normal osteochondral architecture are achieved by extending FIX activity after hemarthrosis using glycoPEGylated FIX when compared with an equivalent dose of rFIX. These results suggest that treating joint bleeding only until hemostasis is achieved may not result in optimal joint healing, which is improved by extending factor activity.

Rapid bone loss during spaceflight is a well-established and continuing medical issue for astronauts. It has been reported that astronauts have displayed bone loss at rates of up to 2.7%/month in weight-bearing bones, or about 6 times that of post-menopausal women [1]. Rodent models have provided a means to further our understanding of the effects of microgravity on bone quality, both from studies in which rodents have flown aboard space missions and those in which weightlessness is simulated on earth through musculoskeletal unloading [2]. Such studies have the potential to not only further our understanding of the cause of decreased bone integrity in space, but also provide an accelerated model for the study of osteo-degenerative diseases affecting the general public, leading to improved treatment methods for both spaceflight and age or illness related osteoporosis.

Background Recurrent bleeding into joints results in a crippling arthritis, the consequence of intra-articular (IA) pathologic lysed red blood cell iron and inflammatory signals from macrophages/monocytes. Hemophilic mice that experience joint bleeding develop synovial and osteochondral changes that mimic the changes seen in human hemophilia. We previously examined in hemophilia B mice the efficacy of conventional recombinant factor IX (rFIX) compared to a half-life extended polyethyleneglycol-conjugated factor IX (N9-GP) to support wound healing when administered "on demand" after the initiation of induced joint hemorrhage. In the "on-demand" scenario, N9-GP improved wound healing when compared to rFIX, although neither treatment group demonstrated completely normal joint healing (as observed in hemostatically normal mice that experienced the same injury). We test the hypothesis that the prophylactic use of N9-GP may support wound healing in synovium, cartilage, and bone and may do so more effectively than rFIX. Method: FIX deficient mice were treated prophylactically with either rFIX or N9-GP at the dose of 250 IUnits human factor IX/kg 30 minutes, 24hr or 72hr prior to the induction of unilateral hemarthosis. Injured hemostatically normal (iWT) mice were treated with I.V. normal saline as the controls. Joint diameter was measured daily to quantify joint swelling. Plasma and tissues were harvested from cohorts of mice (n = 8-10/group) at two weeks after hemarthrosis. Pathologic synovial hyperplasia, neovascularization, iron deposition and macrophage infiltration of the synovium were quantified by histologic examination. Integrity of trabecular bone neighboring the hemarthrosis was quantified by microComputed Tomography (mCT). Result: WT mice developed no detectable joint swelling at days 1-14 after the joint bleeding challenge, whereas mice treated prophylactically with rFIX were protected from joint swelling only in the cohort treated 24 hours prior to hemarthrosis. Joint swelling did not develop in groups that received prophylactic N9-GP using any of the 3 schedules. No mouse treated with rFIX (n = 28 total) or N9-GP (n=27 total) developed FIX inhibitors or FIX-binding IgG. Synovitis was graded 0 to 10 by Valentino mouse hemophilic synovitis score, with a mean score in the iWT mice of 1.0±0.5 and in the injured untreated FIX-/- mice of 4.9±1.4. Synovitis was greatly diminished although not eliminated by prophylactic N9-GP given 30 minutes prior to injury (1.7±1.2), and this result contrasted with the relatively less good healing provided by rFIX given 30 minutes prior to injury (3.9±1.1). Synovitis, iron staining, and persistence of macrophages within the joint were each diminished by N9-GP and rFIX prophylaxis. The degree of protection diminished as the interval between dosing and injury increased. Better protection was seen using N9-GP when compared to rFIX at each timepoint. Strikingly, rFIX at this dose provided minimal or no prophylactic protection from synovial neoangiogenesis (vessels/hpf) at two weeks after hemarthrosis even when given at the shortest interval preceding the injury. Injured, untreated FIX-/- mice developed 17.6±5.1 vessels/hpf compared to rFIX given 30 minutes prior to injury (16.1±4.8), 24 hours prior (17.8±5.4) or 72 hours prior (17.1±6.3). In contrast, iWT mice displayed 7.1±3.1 at two weeks after injury, which was comparable to N9-GP given 30 minutes prior to injury (9.4±2.4) and better than N9-GP given 24 hours prior (12.6±3.3) or 72 hours prior (13.1±5.6). Consistent with previous reports, hemophilic mice when compared to iWT mice demonstrated acute losses of bone mineral density in the bone adjacent to the joint experiencing bleeding, as well as structural defects in the number, thickness, and spacing of the bone trabeculae. Prophylactic rFIX or N9-GP given at 30 minutes or 24 hours prior to injury resulted in protection that did not differ statistically from findings in iWT mice. When prophylaxis was given at 72 hours prior to injury, significant volumetric bone mineral density loss of at least 20% was evident in the rFIX group (vBMD 98 mgHA) compared to iWT (131 mgHA) and N9-GP given 72 hours prior (125 mgHA). Conclusion: The support of normal wound healing in joint and bone following hemarthrosis was significantly improved by N9-GP prophylaxis when compared to rFIX in hemophilia B mice, with neoangiogenesis particularly improved. Disclosures Knudsen: Novo Nordisk: Employment. Monroe:Novo Nordisk: Honoraria, Research Funding. Ezban:Novo Nordisk: Employment. Bateman:Novo Nordisk: Honoraria, Research Funding; Baxter: Honoraria, Research Funding. Monahan:CSL Behring: Consultancy, Honoraria; Pfizer: Honoraria; Chatham LLC: Consultancy; Baxter/Baxalta: Consultancy, Honoraria, Research Funding; Prolor: Research Funding; Bayer: Consultancy; Novo Nordisk: Consultancy, Honoraria, Research Funding; Asklepios BioPharmaceutical: Consultancy, Patents & Royalties: Author I.P. licensed by UNC to AskBio, Research Funding.