Prolonged Cadmium Exposure and Osteoclastogenesis: A Mechanistic Mouse and in Vitro Study.

BACKGROUND: Cadmium (Cd) is a highly toxic and widespread environmental oxidative stressor that causes a myriad of health problems, including osteoporosis and bone damage. Although nuclear factor erythroid 2-related factor 2 (NRF2) and its Cap 'n' Collar and basic region Leucine Zipper (CNC-bZIP) family member nuclear factor erythroid 2-related factor 1 (NRF1) coordinate various stress responses by regulating the transcription of a variety of antioxidant and cytoprotective genes, they play distinct roles in bone metabolism and remodeling. However, the precise roles of both transcription factors in bone loss induced by prolonged Cd exposure remain unclear. OBJECTIVES: We aimed to understand the molecular mechanisms underlying Cd-induced bone loss, focusing mainly on the roles of NRF2 and NRF1 in osteoclastogenesis provoked by Cd. METHODS: Male wild-type (WT), global Nrf2-knockout (Nrf2-/-) and myeloid-specific Nrf2 knockout [Nrf2(M)-KO] mice were administered Cd (50 or 100 ppm) via drinking water for 8 or 16 wk, followed by micro-computed tomography, histological analyses, and plasma biochemical testing. Osteoclastogenesis was evaluated using bone marrow-derived osteoclast progenitor cells (BM-OPCs) and RAW 264.7 cells in the presence of Cd (10 or 20 nM) with a combination of genetic and chemical modulations targeting NRF2 and NRF1. RESULTS: Compared with relevant control mice, global Nrf2-/- or Nrf2(M)-KO mice showed exacerbated bone loss and augmented osteoclast activity following exposure to 100 ppm Cd in drinking water for up to 16 wk. In vitro osteoclastogenic analyses suggested that Nrf2-deficient BM-OPCs and RAW 264.7 cells responded more robustly to low levels of Cd (up to 20 nM) with regard to osteoclast differentiation compared with WT cells. Further mechanistic studies supported a compensatory up-regulation of long isoform of NRF1 (L-NRF1) and subsequent induction of nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1 (NFATc1) as the key molecular events in the Nrf2 deficiency-worsened and Cd-provoked osteoclastogenesis. L-Nrf1 silenced (via lentiviral means) Nrf2-knockdown (KD) RAW cells exposed to Cd showed dramatically different NFATc1 and subsequent osteoclastogenesis outcomes compared with the cells of Nrf2-KD alone exposed to Cd, suggesting a mitigating effect of the Nrf1 silencing. In addition, suppression of reactive oxygen species by exogenous antioxidants N-acetyl-l-cysteine (2 mM) and mitoquinone mesylate (MitoQ; 0.2µM) mitigated the L-NRF1-associated effects on NFATc1-driven osteoclastogenesis outcomes in Cd-exposed Nrf2-KD cells. CONCLUSIONS: This in vivo and in vitro study supported the authors' hypothesis that Cd exposure caused bone loss, in which NRF2 and L-NRF1 responded to Cd and osteoclastogenic stimuli in a cooperative, but contradictive, manner to coordinate Nfatc1 expression, osteoclastogenesis and thus bone homeostasis. Our study suggests a novel strategy targeting NRF2 and L-NRF1 to prevent and treat the bone toxicity of Cd. https://doi.org/10.1289/EHP13849.

Causal relationship between the gut microbiome and basal cell carcinoma, melanoma skin cancer, ease of skin tanning: evidence from three two-sample mendelian randomisation studies.

Objectives: The present study used publicly available genome-wide association study (GWAS) summary data to perform three two-sample Mendelian randomization (MR) studies, aiming to examine the causal links between gut microbiome and BCC, melanoma skin cancer, ease of skin tanning. Methods: SNPs associated with exposures to basal cell carcinoma, melanoma skin cancer and ease of skin tanning from the genome-wide association study data of UK Biobank and MRC-IEU (MRC Integrative Epidemiology Unit), and the meta-analysis data from Biobank and MRC-IEU were used as instrumental variables (IVs). The casual estimates were assessed with a two-sample Mendelian randomisation test using the inverse-variance-weighted (IVW) method, Wald ratio, MR-Egger method, maximum likelihood, weighted median, simple mode, and weighted mode. Results: After the application of MR analysis, diffirent effects of multiple groups of gut microbiota was observed for BCC, melanoma skin cancer and ease of skin tanning. The relationships between the gut microbiome and BCC, melanoma skin cancer, ease of skin tanning were supported by a suite of sensitivity analyses, with no statistical evidence of instrument heterogeneity or horizontal pleiotropy. Further investigation is required to explore the relationship between between the gut microbiome and BCC, melanoma skin cancer, ease of skin tanning. Conclusion: Our study initially identified potential causal roles between the gut microbiome and BCC, melanoma skin cancer, ease of skin tanning, and highlighted the role of gut microbiome in the progression of basal cell carcinoma, melanoma skin cancer, ease of skin tanning.

Nrf2 Mitigates RANKL and M-CSF Induced Osteoclast Differentiation via ROS-Dependent Mechanisms.

Nuclear factor-erythroid 2-related factor 2 (Nrf2) has been shown to be a negative regulator of osteoclast differentiation, but the precise mechanisms have not yet been established. We examined the precise roles of Nrf2 in regulating antioxidants and reactive oxygen species (ROS) levels, especially the cytoplasmic and mitochondrial ROS during osteoclastogenesis in vitro. In the current study, we found that the absence of Nrf2 promotes osteoclast differentiation in bone-marrow-derived macrophages (BMMs) and RAW 264.7 cells. The receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) significantly lowered the levels of Nrf2 and its downstream antioxidant enzymes at mRNA and/or protein levels during osteoclast differentiation in the BMMs of mice and RAW 264.7 mouse leukemic monocytes. Compared to the wild-type cells, Nrf2-deficient cells exhibited heightened sensitivity to both transient RANKL-induced cytoplasmic ROS and prolonged RANKL and M-CSF-induced cytoplasmic and mitochondrial ROS accumulation. Furthermore, exogenous antioxidant agents, including N-acetyl-cysteine (NAC), diphenyleneiodonium chloride (DPI), and mitoquinone mesylate (MitoQ), exhibited substantial capability to suppress the elevation of ROS levels during osteoclast differentiation induced by Nrf2 deficiency, and they consequently inhibited osteoclast differentiation augmented by the lack of Nrf2. The activation of phosphorylated c-FOS resulting from elevated ROS promoted osteoclast differentiation. The inhibition of c-FOS blocked osteoclast differentiation, which was elevated by Nrf2-deficiency. Taken together, these data reveal that Nrf2 effectively decreased the accumulation of intracellular ROS and the phosphorylation of c-FOS during osteoclastic differentiation by regulating antioxidant enzymes and subsequently inhibited RANKL-induced osteoclast differentiation.

Rationally Designed Enzyme-Resistant Peptidic Assemblies for Plasma Membrane Targeting in Cancer Treatment.

Peptides are being increasingly important for subcellular targeted cancer treatment to improve specificity and reverse multidrug resistance. However, there has been yet any report on targeting plasma membrane (PM) through self-assembling peptides. A simple synthetic peptidic molecule (tF4) is developed. It is revealed that tF4 is carboxyl esterase-resistant and self-assembles into vesical nanostructures. Importantly, tF4 assemblies interact with PM through orthogonal hydrogen bonding and hydrophobic interaction to regulate cancer cellular functions. Mechanistically, tF4 assemblies induce stress fiber formation, cytoskeleton reconstruction, and death receptor 4/5 (DR4/5) expression in cancer cells. DR4/5 triggers extrinsic caspase-8 signaling cascade, resulting in cell death. The results provide a new strategy for developing enzyme-resistant and PM-targeting peptidic molecules against cancer.

GE11 peptide-decorated acidity-responsive micelles for improved drug delivery and enhanced combination therapy of metastatic breast cancer.

Nanotechnology-mediated drug delivery systems suffer from insufficient retention in tumor tissues and unreliable drug release at specific target sites. Herein, we developed an epidermal growth factor receptor-targeted multifunctional micellar nanoplatform (GE11-DOX+CEL-M) by encapsulating celecoxib into polymeric micelles based on the conjugate of GE11-poly(ethylene glycol)-b-poly(trimethylene carbonate) with doxorubicin to suppress tumor growth and metastasis. The polymeric micelles maintained stable nanostructures under physiological conditions but quickly disintegrated in a weakly acidic environment, which is conducive to controlled drug release. Importantly, GE11-DOX+CEL-M micelles effectively delivered the drug combination to tumor sites and enhanced tumor cell uptake through GE11-mediated active tumor targeting. Subsequently, GE11-DOX+CEL-M micelles dissociated in response to intracellular slightly acidic microenvironmental stimuli, resulting in rapid release of celecoxib and doxorubicin to synergistically inhibit the proliferation and migration of tumor cells. Systemic administration of GE11-DOX+CEL-M micelles into mice bearing subcutaneous 4T1 tumor models resulted in higher tumor growth suppression and decreased lung metastasis of tumor cells compared with micelles without GE11 decoration or delivering only doxorubicin. Furthermore, the micelles effectively reduced the systemic toxicity of the chemotherapy drugs. This nanotherapeutic system provides a promising strategy for safe and effective cancer therapy.

Characterization and assessment of lung and bone marrow derived endothelial cells and their bone regenerative potential.

Angiogenesis is important for successful fracture repair. Aging negatively affects the number and activity of endothelial cells (ECs) and subsequently leads to impaired bone healing. We previously showed that implantation of lung-derived endothelial cells (LECs) improved fracture healing in rats. In this study, we characterized and compared neonatal lung and bone marrow-derived endothelial cells (neonatal LECs and neonatal BMECs) and further asses3sed if implantation of neonatal BMECs could enhance bone healing in both young and aged mice. We assessed neonatal EC tube formation, proliferation, and wound migration ability in vitro in ECs isolated from the bone marrow and lungs of neonatal mice. The in vitro studies demonstrated that both neonatal LECs and neonatal BMECs exhibited EC traits. To test the function of neonatal ECs in vivo, we created a femoral fracture in young and aged mice and implanted a collagen sponge to deliver neonatal BMECs at the fracture site. In the mouse fracture model, endochondral ossification was delayed in aged control mice compared to young controls. Neonatal BMECs significantly improved endochondral bone formation only in aged mice. These data suggest BMECs have potential to enhance aged bone healing. Compared to LECs, BMECs are more feasible for translational cell therapy and clinical applications in bone repair. Future studies are needed to examine the fate and function of BMECs implanted into the fracture sites.

Use of Composite Acellular Dermal Matrix-Ultrathin Split-Thickness Skin in Hand Hot-Crush Injuries: A One-Step Grafting Procedure.

Background: Hot-crush injuries to the hands can be devastating, and early debridement and coverage with skin autograft remains the golden standard of wound treatment. However, this type of treatment is not feasible or unlikely to succeed due to limited donor sites and wound characteristics of hot-crush injuries on hands. Thus, the composite grafting of acellular dermal matrix (ADM) and split-thickness skin graft (STSG) as a novel alternative method has been attempted. In this series, the results are presented to demonstrate the feasibility and effectiveness of the use of one-stage procedure for early reconstruction in hand hot-crush injuries. Methods: All consecutive patients with hand hot-crush injuries, who underwent one-stage procedure of ADM and ultrathin STSG for soft tissue coverage at our institution from December 2018 to November 2019, were retrospectively analyzed. Wound dressings were opened on 7 days after operation to examine graft survival and complications. Patients were followed up for at least 9 months to evaluate their hand profiles. Results: Samples of 14 patients with a total of 23 wounds were involved in the study. Thirteen of the 23 third-fourth-degree wounds had varying degrees of tendon exposure. On 7 days postoperation, the composite grafts survived in 12 patients with minimal focal graft losses and liquefaction and necrosis in 2 patients, which achieved successful healing following new coverage of ultrathin STSG. All the wounds healed with hospital stays ranging from 9 days to 32 days (median: 24.5 days). At the final follow-up (from 9 months to 20 months), all patients achieved excellent or good total active motion grade and good scar quality (Vancouver scar scale scored 1-3) with no revision surgery. Conclusions: One-stage composite grafting of ADM and ultrathin STSG is a reliable alternative for early reconstruction in hand hot-crush injuries, which delivers good functional outcomes and a good cosmetic appearance.

Role of lncSLCO1C1 in gastric cancer progression and resistance to oxaliplatin therapy.

BACKGROUND: Gastric carcinoma (GC) is one of the most deadly diseases due to tumour metastasis and resistance to therapy. Understanding the molecular mechanism of tumour progression and drug resistance will improve therapeutic efficacy and develop novel intervention strategies. METHODS: Differentially expressed long non-coding RNAs (lncRNAs) in clinical specimens were identified by LncRNA microarrays and validated in different clinical cohorts by quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridisation and bioinformatics analysis. Biological functions of lncRNA were investigated by using cell proliferation assays, migration assays, xenograft tumour models and bioinformatics analysis. Effects of lncSLCO1C1 on GC cell survival were assessed by comet assays and immunofluorescence assays. Underlying molecular mechanisms were further explored by using a number of technologies including RNA pull-down, mass spectrometry analysis, RNA immunoprecipitation, co-immunoprecipitation, miRNA sequencing, luciferase reporter assays and molecular modelling. RESULTS: LncSLCO1C1 was highly upregulated in GC tissue samples and associated with GC patients' poor overall survival. Overexpression of lncSLCO1C1 promoted proliferation and migration, whereas decreased lncSLCO1C1 expression produced the opposite effects. lncSLCO1C1 also mediated tumour resistance to chemotherapy with oxaliplatin by reducing DNA damage and increasing cell proliferation. Despite sequence overlapping between lncSLCO1C1 and PDE3A, alternations of PDE3A expression had no effect on the GC cell progression, indicating that lncSLCO1C1, not PDE3A, related with the progression of GC cells. Mechanistically, lncSLCO1C1 serves as a scaffold for the structure-specific recognition protein 1 (SSRP1)/H2A/H2B complex and regulates the function of SSRP1 in reducing DNA damage. Meanwhile, lncSLCO1C1 functions as a sponge to adsorb miR-204-5p and miR-211-5p that target SSRP1 mRNA, and thus increases SSRP1 expression. Patients with high expressions of both lncSLCO1C1 and SSRP1 have poor overall survival, highlighting the role of lncSLCO1C1 in GC progression. CONCLUSIONS: LncSLCO1C1 promotes GC progression by enhancing cell growth and preventing DNA damage via interacting and scaffolding the SSRP1/H2A/H2b complex and absorbing both miR-211-5p and miR-204-5p to increase SSRP1 expression.

Downregulation of Manic fringe impedes angiogenesis and cell migration of renal carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a highly angiogenic cancer. Manic fringe (MFng) is elevated in ccRCC compared to the normal kidney. However, its role in ccRCC tumour angiogenesis remains elusive. This study seeks to determine the expression pattern of MFng in ccRCC blood vessels and its role in angiogenesis. The association between MFng and the blood vessels was established through online compendia, immunohistochemistry and qPCR analyses. The anti-angiogenic potential of lentiviral-mediated MFng knockdown in endothelial cells (EC shMFng) was assessed for viability, proliferation, apoptosis, migration, adhesion, cell cycle, vessel sprouting, and molecular expression of adhesion and apoptosis markers. Finally, EC shMFng were co-cultured with 786-0 renal cancer cells to determine their impact on cancer cell migration. The online dataset analyses and immunostaining on ccRCC tissues revealed high expression of MFng in ECs. MFng and CD31/PECAM-1 genes were up-regulated in ccRCC tissue samples compared to normal kidney tissues. EC shMFng demonstrated decreased cell viability due to G1 cell cycle arrest and reduced Ki-67 protein expression. In addition, shMFng down-regulated endothelial adhesion molecules and hindered EC migration, network formation and sprouting, compared to their respective empty vector (EV) controls. Co-culture assay of EC shMFng with 786-0 renal cancer cells inhibited cancer cell migration. These findings underscore the potential role of MFng in ECs in influencing renal cancer cell migration, thus opening an avenue for anti-angiogenic strategy targeting MFng to treat ccRCC.

Effects of diet, BMP-2 treatment, and femoral skeletal injury on endothelial cells derived from the ipsilateral and contralateral limbs.

Type 2 diabetes (T2D) results in physiological and structural changes in bone, contributing to poor fracture healing. T2D compromises microvascular performance, which can negatively impact bone regeneration as angiogenesis is required for new bone formation. We examined the effects of bone morphogenetic protein-2 (BMP-2) administered locally at the time of femoral segmental bone defect (SBD) surgery, and its angiogenic impacts on endothelial cells (ECs) isolated from the ipsilateral or contralateral tibia in T2D mice. Male C57BL/6 mice were fed either a low-fat diet (LFD) or high-fat diet (HFD) starting at 8 weeks. After 12 weeks, the T2D phenotype in HFD mice was confirmed via glucose and insulin tolerance testing and echoMRI, and all mice underwent SBD surgery. Mice were treated with BMP-2 (5 µg) or saline at the time of surgery. Three weeks postsurgery, bone marrow ECs were isolated from ipsilateral and contralateral tibias, and proliferation, angiogenic potential, and gene expression of the cells was analyzed. BMP-2 treatment increased EC proliferation by two fold compared with saline in LFD contralateral tibia ECs, but no changes were seen in surgical tibia EC proliferation. BMP-2 treatment enhanced vessel-like structure formation in HFD mice whereas, the opposite was observed in LFD mice. Still, in BMP-2 treated LFD mice, ipsilateral tibia ECs increased expression of CD31, FLT-1, ANGPT1, and ANGPT2. These data suggest that the modulating effects of T2D and BMP-2 on the microenvironment of bone marrow ECs may differentially influence angiogenic properties at the fractured limb versus the contralateral limb.

The Effects of SRT1720 Treatment on Endothelial Cells Derived from the Lung and Bone Marrow of Young and Aged, Male and Female Mice.

Angiogenesis is critical for successful fracture healing. Age-related alterations in endothelial cells (ECs) may cause impaired bone healing. Therefore, examining therapeutic treatments to improve angiogenesis in aging may enhance bone healing. Sirtuin 1 (SIRT1) is highly expressed in ECs and its activation is known to counteract aging. Here, we examined the effects of SRT1720 treatment (SIRT1 activator) on the growth and function of bone marrow and lung ECs (BMECs and LECs, respectively), derived from young (3-4 month) and old (20-24 month) mice. While aging did not alter EC proliferation, treatment with SRT1720 significantly increased proliferation of all LECs. However, SRT1720 only increased proliferation of old female BMECs. Vessel-like tube assays showed similar vessel-like structures between young and old LECs and BMECs from both male and female mice. SRT1720 significantly improved vessel-like structures in all LECs. No age, sex, or treatment differences were found in migration related parameters of LECs. In males, old BMECs had greater migration rates than young BMECs, whereas in females, old BMECs had lower migration rates than young BMECs. Collectively, our data suggest that treatment with SRT1720 appears to enhance the angiogenic potential of LECs irrespective of age or sex. However, its role in BMECs is sex- and age-dependent.

The effects of bone morphogenetic protein 2 and thrombopoietin treatment on angiogenic properties of endothelial cells derived from the lung and bone marrow of young and aged, male and female mice.

With an aging world population, there is an increased risk of fracture and impaired healing. One contributing factor may be aging-associated decreases in vascular function; thus, enhancing angiogenesis could improve fracture healing. Both bone morphogenetic protein 2 (BMP-2) and thrombopoietin (TPO) have pro-angiogenic effects. The aim of this study was to investigate the effects of treatment with BMP-2 or TPO on the in vitro angiogenic and proliferative potential of endothelial cells (ECs) isolated from lungs (LECs) or bone marrow (BMECs) of young (3-4 months) and old (22-24 months), male and female, C57BL/6J mice. Cell proliferation, vessel-like structure formation, migration, and gene expression were used to evaluate angiogenic properties. In vitro characterization of ECs generally showed impaired vessel-like structure formation and proliferation in old ECs compared to young ECs, but improved migration characteristics in old BMECs. Differential sex-based angiogenic responses were observed, especially with respect to drug treatments and gene expression. Importantly, these studies suggest that NTN1, ROBO2, and SLIT3, along with angiogenic markers (CD31, FLT-1, ANGPT1, and ANGP2) differentially regulate EC proliferation and functional outcomes based on treatment, sex, and age. Furthermore, treatment of old ECs with TPO typically improved vessel-like structure parameters, but impaired migration. Thus, TPO may serve as an alternative treatment to BMP-2 for fracture healing in aging owing to improved angiogenesis and fracture healing, and the lack of side effects associated with BMP-2.

Nuclear and stromal expression of Manic fringe in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common type of kidney cancer and has the highest mortality rate among genitourinary cancers. Despite the advances in molecular targeted therapies to treat RCC, the inevitable emergence of resistance has delineated the need to uncover biomarkers to prospectively identify patient response to treatment and more accurately predict patient prognosis. Fringe is a fucose specific ß1, 3N-acetylglucosaminyltransferase that modifies the Notch receptors. Given the link between its function and aberrant Notch activation in RCC, Fringe may be implicated in this disease. The Fringe homologs comprise of Lunatic fringe (LFng), Manic fringe (MFng) and Radical fringe (RFng). MFng has been reported to play a role in cancer. MFng is also essential in the development of B cells. However, the expression profile and clinical significance of MFng, and its association with B cells in RCC are unknown. CD20 is a clinically employed biomarker for B cells. This pilot study aimed to determine if MFng protein expression can be utilized as a prospective biomarker for therapeutics and prognosis in RCC, as well as to determine its association with CD20+ B cells. Analysis of publicly available MFng gene expression datasets on The Cancer Genome Atlas Netlwork (TCGA) identified MFng gene expression to be up-regulated in Kidney Clear Cell Renal Carcinoma (KIRC) patients. However there was no significant association between the patient survival probability and the level of MFng expression in this cohort. Immunohistochemistry performed on a tissue microarray containing cores from 64 patients revealed an elevated MFng protein expression in the epithelial and stromal tissues of RCC compared to the normal kidney, suggesting a possible role in tumorigenesis. Our study describes for the first time to our knowledge, the protein expression of MFng in the nuclear compartment of normal kidney and RCC, implicating a prospective involvement in gene transcription. At the cellular level, cytoplasmic MFng was also abundant in the normal kidney and RCC. However, MFng protein expression in the malignant epithelial and stromal tissue of RCC had no positive correlation with the patients' overall survival, progression-free survival and time to metastasis, as well as the gender, age, tumor stage and RCC subtype, indicating that MFng may not be an appropriate prognostic marker. The association between CD20+ B cells and epithelial MFng was found to approach borderline insignificance. Nonetheless, these preliminary findings may provide valuable information on the suitability of MFng as a potential therapeutic molecular marker for RCC, thus warrants further investigation using a larger cohort.

Tspan5 promotes epithelial-mesenchymal transition and tumour metastasis of hepatocellular carcinoma by activating Notch signalling.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide due to a high rate of tumour metastasis and disease recurrence. In physiological conditions, tetraspanins interact with specific partner proteins in tetraspanin-enriched microdomains and regulate their subcellular localization and function. However, the function of Tspan5 in pathological processes, particularly in cancer biology and its clinical significance, are still unclear. Here, we describe that a high expression of Tspan5 is significantly associated with some clinicopathological features including invasive length, vascular invasion, clinical stage and poor overall survival of HCC patients. Alterations of Tspan5 expression by lentivirus transductions in HCC cells demonstrated that Tspan5 promotes wound healing and cell migration in vitro and tumour metastasis of HCC cells in vivo. Mechanistic studies revealed that Tspan5 promoted cell migration and tumour metastasis by increasing the enzymatic maturation of ADAM10 and activating Notch signalling via the increase of the cleavage of the Notch1 receptor catalysed by the γ-secretase complex. Activation of Notch signalling by Tspan5 was shown further to enhance the epithelial-mesenchymal transition (EMT) and actin skeleton rearrangement of tumour cells. In clinical HCC samples, Tspan5 expression is strongly correlated with many key molecules acting in Notch signalling and EMT, highlighting the role of Tspan5 in the regulation of Notch signalling, EMT and tumour metastasis of HCC. Our findings provide new insights into the mechanism of tumour metastasis and disease progression of HCC and may facilitate the development of novel clinical intervention strategies against HCC.

The effects of high fat diet, bone healing, and BMP-2 treatment on endothelial cell growth and function.

Angiogenesis is a vital process during the regeneration of bone tissue. The aim of this study was to investigate angiogenesis at the fracture site as well as at distal locations from obesity-induced type 2 diabetic mice that were treated with bone morphogenetic protein-2 (BMP-2, local administration at the time of surgery) to heal a femoral critical sized defect (CSD) or saline as a control. Mice were fed a high fat diet (HFD) to induce a type 2 diabetic-like phenotype while low fat diet (LFD) animals served as controls. Endothelial cells (ECs) were isolated from the lungs (LECs) and bone marrow (BMECs) 3 weeks post-surgery, and the fractured femurs were also examined. Our studies demonstrate that local administration of BMP-2 at the fracture site in a CSD model results in complete bone healing within 3 weeks for all HFD mice and 66.7% of LFD mice, whereas those treated with saline remain unhealed. At the fracture site, vessel parameters and adipocyte numbers were significantly increased in BMP-2 treated femurs, irrespective of diet. At distal sites, LEC and BMEC proliferation was not altered by diet or BMP-2 treatment. HFD increased the tube formation ability of both LECs and BMECs. Interestingly, BMP-2 treatment at the time of surgery reduced tube formation in LECs and humeri BMECs. However, migration of BMECs from HFD mice treated with BMP-2 was increased compared to BMECs from HFD mice treated with saline. BMP-2 treatment significantly increased the expression of CD31, FLT-1, and ANGPT2 in LECs and BMECs in LFD mice, but reduced the expression of these same genes in HFD mice. To date, this is the first study that depicts the systemic influence of fracture surgery and local BMP-2 treatment on the proliferation and angiogenic potential of ECs derived from the bone marrow and lungs.

Electrical stimulation of hindlimb skeletal muscle has beneficial effects on sublesional bone in a rat model of spinal cord injury.

Spinal cord injury (SCI) results in marked atrophy of sublesional skeletal muscle and substantial loss of bone. In this study, the effects of prolonged electrical stimulation (ES) and/or testosterone enanthate (TE) on muscle mass and bone formation in a rat model of SCI were tested. Compared to sham-transected animals, a significant reduction of the mass of soleus, plantaris and extensor digitorum longus (EDL) muscles was observed in animals 6 weeks post-SCI. Notably, ES or ES + TE resulted in the increased mass of the EDL muscles. ES or ES + TE significantly decreased mRNA levels of muscle atrophy markers (e.g., MAFbx and MurF1) in the EDL. Significant decreases in bone mineral density (BMD) (-27%) and trabecular bone volume (-49.3%) at the distal femur were observed in animals 6 weeks post injury. TE, ES and ES + TE treatment significantly increased BMD by +6.4%, +5.4%, +8.5% and bone volume by +22.2%, and +56.2% and+ 60.2%, respectively. Notably, ES alone or ES + TE resulted in almost complete restoration of cortical stiffness estimated by finite element analysis in SCI animals. Osteoblastogenesis was evaluated by colony-forming unit-fibroblastic (CFU-F) staining using bone marrow mesenchymal stem cells obtained from the femur. SCI decreased the CFU-F+ cells by -56.8% compared to sham animals. TE or ES + TE treatment after SCI increased osteoblastogenesis by +74.6% and +67.2%, respectively. An osteoclastogenesis assay revealed significantly increased TRAP+ multinucleated cells (+34.8%) in SCI animals compared to sham animals. TE, ES and TE + ES treatment following SCI markedly decreased TRAP+ cells by -51.3%, -40.3% and -46.9%, respectively. Each intervention greatly reduced the ratio of RANKL to OPG mRNA of sublesional long bone. Collectively, our findings demonstrate that after neurologically complete paralysis, dynamic muscle resistance exercise by ES reduced muscle atrophy, downregulated genes involved in muscle wasting, and restored mechanical loading to sublesional bone to a degree that allowed for the preservation of bone by inhibition of bone resorption and/or by facilitating bone formation.

The loss of STAT3 in mature osteoclasts has detrimental effects on bone structure.

Signal Transducer and Activator of Transcription 3 (STAT3) has recently been shown to be involved in bone development and has been implicated in bone diseases, such as Job's Syndrome. Bone growth and changes have been known for many years to differ between sexes with male bones tending to have higher bone mass than female bones and older females tending to lose bone mass at faster rates than older males. Previous studies using conditional knock mice with Stat3 specifically deleted from the osteoblasts showed both sexes exhibited decreased bone mineral density (BMD) and strength. Using the Cre-Lox system with Cathepsin K promotor driving Cre to target the deletion of the Stat3 gene in mature osteoclasts (STAT3-cKO mice), we observed that 8-week old STAT3-cKO female femurs exhibited significantly lower BMD and bone mineral content (BMC) compared to littermate control (CN) females. There were no differences in BMD and BMC observed between male knock-out and male CN femurs. However, micro-computed tomography (µCT) analysis showed that both male and female STAT3-cKO mice had significant decreases in bone volume/tissue volume (BV/TV). Bone histomorphometry analysis of the distal femur, further revealed a decrease in bone formation rate and mineralizing surface/bone surface (MS/BS) with a significant decrease in osteoclast surface in female, but not male, STAT3-cKO mice. Profiling gene expression in an osteoclastic cell line with a knockdown of STAT3 showed an upregulation of a number of genes that are directly regulated by estrogen receptors. These data collectively suggest that regulation of STAT3 differs in male and female osteoclasts and that inactivation of STAT3 in osteoclasts affects bone turnover more in females than males, demonstrating the complicated nature of STAT3 signaling pathways in osteoclastogenesis. Drugs targeting the STAT3 pathway may be used for treatment of diseases such as Job's Syndrome and osteoporosis.

Author Correction: Epstein-Barr virus-encoded microRNA BART1 induces tumour metastasis by regulating PTEN-dependent pathways in nasopharyngeal carcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Development of Therapeutic Anti-JAGGED1 Antibodies for Cancer Therapy.

The role of Notch signaling and its ligand JAGGED1 (JAG1) in tumor biology has been firmly established, making them appealing therapeutic targets for cancer treatment. Here, we report the development and characterization of human/rat-specific JAG1-neutralizing mAbs. Epitope mapping identified their binding to the Notch receptor interaction site within the JAG1 Delta/Serrate/Lag2 domain, where E228D substitution prevented effective binding to the murine Jag1 ortholog. These antibodies were able to specifically inhibit JAG1-Notch binding in vitro, downregulate Notch signaling in cancer cells, and block the heterotypic JAG1-mediated Notch signaling between endothelial and vascular smooth muscle cells. Functionally, in vitro treatment impaired three-dimensional growth of breast cancer cell spheroids, in association with a reduction in cancer stem cell number. In vivo testing showed variable effects on human xenograft growth when only tumor-expressed JAG1 was targeted (mouse models) but a more robust effect when stromal-expressed Jag1 was also targeted (rat MDA-MB-231 xenograft model). Importantly, treatment of established triple receptor-negative breast cancer brain metastasis in rats showed a significant reduction in neoplastic growth. MRI imaging demonstrated that this was associated with a substantial improvement in blood-brain barrier function and tumor perfusion. Lastly, JAG1-targeting antibody treatment did not cause any detectable toxicity, further supporting its clinical potential for cancer therapy.