Drug-induced modulation of gp130 signalling prevents articular cartilage degeneration and promotes repair.

OBJECTIVE: Human adult articular cartilage (AC) has little capacity for repair, and joint surface injuries often result in osteoarthritis (OA), characterised by loss of matrix, hypertrophy and chondrocyte apoptosis. Inflammation mediated by interleukin (IL)-6 family cytokines has been identified as a critical driver of proarthritic changes in mouse and human joints, resulting in a feed-forward process driving expression of matrix degrading enzymes and IL-6 itself. Here we show that signalling through glycoprotein 130 (gp130), the common receptor for IL-6 family cytokines, can have both context-specific and cytokine-specific effects on articular chondrocytes and that a small molecule gp130 modulator can bias signalling towards anti-inflammatory and antidegenerative outputs. METHODS: High throughput screening of 170 000 compounds identified a small molecule gp130 modulator termed regulator of cartilage growth and differentiation (RCGD 423) that promotes atypical homodimeric signalling in the absence of cytokine ligands, driving transient increases in MYC and pSTAT3 while suppressing oncostatin M- and IL-6-mediated activation of ERK and NF-κB via direct competition for gp130 occupancy. RESULTS: This small molecule increased proliferation while reducing apoptosis and hypertrophic responses in adult chondrocytes in vitro. In a rat partial meniscectomy model, RCGD 423 greatly reduced chondrocyte hypertrophy, loss and degeneration while increasing chondrocyte proliferation beyond that observed in response to injury. Moreover, RCGD 423 improved cartilage healing in a rat full-thickness osteochondral defect model, increasing proliferation of mesenchymal cells in the defect and also inhibiting breakdown of cartilage matrix in de novo generated cartilage. CONCLUSION: These results identify a novel strategy for AC remediation via small molecule-mediated modulation of gp130 signalling.

An injectable active hydrogel based on BMSC-derived extracellular matrix for cartilage regeneration enhancement.

Articular cartilage injury impairs joint function and necessitates orthopedic intervention to restore the structure and function of the cartilage. Extracellular matrix (ECM) scaffolds derived from bone marrow mesenchymal stem cells (BMSCs) can effectively promote cell adhesion, proliferation, and chondrogenesis. However, pre-shaped ECM scaffolds have limited applicability due to their poor fit with the irregular surface of most articular cartilage defects. In this study, we fabricated an injectable active ECM hydrogel from autologous BMSCs-derived ECM by freeze-drying, liquid nitrogen milling, and enzymatic digestion. Moreover, our in vitro and in vivo results demonstrated that the prepared hydrogel enhanced chondrocyte adhesion and proliferation, chondrogenesis, cartilage regeneration, and integration with host tissue, respectively. These findings indicate that active ECM components can provide trophic support for cell proliferation and differentiation, restoring the structure and function of damaged cartilage.

MiR-653-5p drives osteoarthritis pathogenesis by modulating chondrocyte senescence.

BACKGROUND: Due to the unclear pathogenesis of osteoarthritis (OA), effective treatment for this ailment is presently unavailable. Accumulating evidence points to chondrocyte senescence as a key driver in OA development. This study aims to identify OA-specific microRNAs (miRNAs) targeting chondrocyte senescence to alleviate OA progression. METHODS: We screened and identified miRNAs differentially expressed in OA and normal cartilage, then confirmed the impact of miR-653-5p on chondrocyte functions and senescence phenotypes through in vitro experiments with overexpression/silencing. We identified interleukin 6 (IL-6) as the target gene of miR-653-5p and confirmed the regulatory influence of miR-653-5p on the IL-6/JAK/STAT3 signaling pathway through gain/loss-of-function studies. Finally, we assessed the therapeutic efficacy of miR-653-5p on OA using a mouse model with destabilization of the medial meniscus. RESULTS: MiR-653-5p was significantly downregulated in cartilage tissues and chondrocytes from OA patients. Overexpression of miR-653-5p promoted chondrocyte matrix synthesis and proliferation while inhibiting chondrocyte senescence. Furthermore, bioinformatics target prediction and the luciferase reporter assays identified IL-6 as a target of miR-653-5p. Western blot assays demonstrated that miR-653-5p overexpression inhibited the protein expression of IL-6, the phosphorylation of JAK1 and STAT3, and the expression of chondrocyte senescence phenotypes by regulating the IL-6/JAK/STAT3 signaling pathway. More importantly, the cartilage destruction was significantly alleviated and chondrocyte senescence phenotypes were remarkably decreased in the OA mouse model treated by agomiR-653-5p compared to the control mice. CONCLUSIONS: MiR-653-5p showed a significant decrease in cartilage tissues of individuals with OA, leading to an upregulation of chondrocyte senescence phenotypes in the articular cartilage. AgomiR-653-5p emerges as a potential treatment approach for OA. These findings provide further insight into the role of miR-653-5p in chondrocyte senescence and the pathogenesis of OA.

Inactivation of a non-canonical gp130 signaling arm attenuates chronic systemic inflammation and multimorbidity induced by a high-fat diet.

Interleukin-6 (IL-6) is a major pro-inflammatory cytokine for which the levels in plasma demonstrate a robust correlation with age and body mass index (BMI) as part of the senescence-associated secretory phenotype. IL-6 cytokines also play a crucial role in metabolic homeostasis and regenerative processes, primarily via the canonical STAT3 pathway. Thus, selective modulation of IL-6 signaling may offer a unique opportunity for therapeutic interventions. Recently, we discovered that a non-canonical signaling pathway downstream of tyrosine (Y) 814 within the intracellular domain of gp130, the IL-6 co-receptor, is responsible for the recruitment and activation of SRC family of kinases (SFK). Mice with constitutive genetic inactivation of gp130 Y814 (F814 mice) show accelerated resolution of inflammatory response and superior regenerative outcomes in skin wound healing and posttraumatic models of osteoarthritis. The current study was designed to explore if selective genetic or pharmacological inhibition of the non-canonical gp130-Y814/SFK signaling reduces systemic chronic inflammation and multimorbidity in a high-fat diet (HFD)-induced model of accelerated aging. F814 mice showed significantly reduced inflammatory response to HFD in adipose and liver tissue, with significantly reduced levels of systemic inflammation compared to wild type mice. F814 mice were also protected from HFD-induced bone loss and cartilage degeneration. Pharmacological inhibition of gp130-Y814/SFK in mice on HFD mirrored the effects observed in F814 mice on HFD; furthermore, this pharmacological treatment also demonstrated a marked increase in physical activity levels and protective effects against inflammation-associated suppression of neurogenesis in the brain tissue compared to the control group. These findings suggest that selective inhibition of SFK signaling downstream of gp130 receptor represents a promising strategy to alleviate systemic chronic inflammation. Increased degenerative changes and tissue senescence are inevitable in obese and aged organisms, but we demonstrated that the systemic response and inflammation-associated multi-morbidity can be therapeutically mitigated.

Vitamin D and the regulation of placental inflammation.

The vitamin D-activating enzyme 1α-hydroxylase (CYP27B1) and vitamin D receptor (VDR) support anti-inflammatory responses to vitamin D in many tissues. Given the high basal expression of CYP27B1 and VDR in trophoblastic cells from the placenta, we hypothesized that anti-inflammatory effects of vitamin D may be particularly important in this organ. Pregnant wild type (WT) mice i.p. injected with LPS showed elevated expression of mouse Cyp27b1 (4-fold) and VDR (6-fold). Similar results were also obtained after ex vivo treatment of WT placentas with LPS. To assess the functional impact of this, we carried out ex vivo studies using placentas -/- for fetal (trophoblastic) Cyp27b1 or VDR. Vehicle-treated -/- placentas showed increased expression of IFN-γ and decreased expression of IL-10 relative to +/+ placentas. LPS-treated -/- placentas showed increased expression of TLR2, IFN-γ, and IL-6. Array analyses identified other inflammatory factors that are dysregulated in Cyp27b1(-/-) versus Cyp27b1(+/+) placentas after LPS challenge. Data highlighted enhanced expression of IL-4, IL-15, and IL-18, as well as several chemokines and their receptors, in Cyp27b1(-/-) placentas. Similar results for IL-6 expression were observed with placentas -/- for trophoblastic VDR. Finally, ex vivo treatment of WT placentas with the substrate for Cyp27b1, 25-hydroxyvitamin D(3), suppressed LPS-induced expression of IL-6 and the chemokine Ccl11. These data indicate that fetal (trophoblastic) vitamin D plays a pivotal role in controlling placental inflammation. In humans, this may be a key factor in placental responses to infection and associated adverse outcomes of pregnancy.

STAT3 promotes a youthful epigenetic state in articular chondrocytes.

Epigenetic mechanisms guiding articular cartilage regeneration and age-related disease such as osteoarthritis (OA) are poorly understood. STAT3 is a critical age-patterned transcription factor highly active in fetal and OA chondrocytes, but the context-specific role of STAT3 in regulating the epigenome of cartilage cells remain elusive. In this study, DNA methylation profiling was performed across human chondrocyte ontogeny to build an epigenetic clock and establish an association between CpG methylation and human chondrocyte age. Exposure of adult chondrocytes to a small molecule STAT3 agonist decreased DNA methylation, while genetic ablation of STAT3 in fetal chondrocytes induced global hypermethylation. CUT&RUN assay and subsequent transcriptional validation revealed DNA methyltransferase 3 beta (DNMT3B) as one of the putative STAT3 targets in chondrocyte development and OA. Functional assessment of human OA chondrocytes showed the acquisition of progenitor-like immature phenotype by a significant subset of cells. Finally, conditional deletion of Stat3 in cartilage cells increased DNMT3B expression in articular chondrocytes in the knee joint in vivo and resulted in a more prominent OA progression in a post-traumatic OA (PTOA) mouse model induced by destabilization of the medial meniscus (DMM). Taken together these data reveal a novel role for STAT3 in regulating DNA methylation in cartilage development and disease. Our findings also suggest that elevated levels of active STAT3 in OA chondrocytes may indicate an intrinsic attempt of the tissue to regenerate by promoting a progenitor-like phenotype. However, it is likely that chronic activation of this pathway, induced by IL-6 cytokines, is detrimental and leads to tissue degeneration.

Inhibition of a signaling modality within the gp130 receptor enhances tissue regeneration and mitigates osteoarthritis.

Adult mammals are incapable of multitissue regeneration, and augmentation of this potential may shift current therapeutic paradigms. We found that a common co-receptor of interleukin 6 (IL-6) cytokines, glycoprotein 130 (gp130), serves as a major nexus integrating various context-specific signaling inputs to either promote regenerative outcomes or aggravate disease progression. Via genetic and pharmacological experiments in vitro and in vivo, we demonstrated that a signaling tyrosine 814 (Y814) within gp130 serves as a major cellular stress sensor. Mice with constitutively inactivated Y814 (F814) were resistant to surgically induced osteoarthritis as reflected by reduced loss of proteoglycans, reduced synovitis, and synovial fibrosis. The F814 mice also exhibited enhanced regenerative, not reparative, responses after wounding in the skin. In addition, pharmacological modulation of gp130 Y814 upstream of the SRC and MAPK circuit by a small molecule, R805, elicited a protective effect on tissues after injury. Topical administration of R805 on mouse skin wounds resulted in enhanced hair follicle neogenesis and dermal regeneration. Intra-articular administration of R805 to rats after medial meniscal tear and to canines after arthroscopic meniscal release markedly mitigated the appearance of osteoarthritis. Single-cell sequencing data demonstrated that genetic and pharmacological modulation of Y814 resulted in attenuation of inflammatory gene signature as visualized by the anti-inflammatory macrophage and nonpathological fibroblast subpopulations in the skin and joint tissue after injury. Together, our study characterized a molecular mechanism that, if manipulated, enhances the intrinsic regenerative capacity of tissues through suppression of a proinflammatory milieu and prevents pathological outcomes in injury and disease.

A new mouse model of post-traumatic joint injury allows to identify the contribution of Gli1+ mesenchymal progenitors in arthrofibrosis and acquired heterotopic endochondral ossification.

Complex injury and open reconstructive surgeries of the knee often lead to joint dysfunction that may alter the normal biomechanics of the joint. Two major complications that often arise are excessive deposition of fibrotic tissue and acquired heterotopic endochondral ossification. Knee arthrofibrosis is a fibrotic joint disorder where aberrant buildup of scar tissue and adhesions develop around the joint. Heterotopic ossification is ectopic bone formation around the periarticular tissues. Even though arthrofibrosis and heterotopic ossification pose an immense clinical problem, limited studies focus on their cellular and molecular mechanisms. Effective cell-targeted therapeutics are needed, but the cellular origin of both knee disorders remains elusive. Moreover, all the current animal models of knee arthrofibrosis and stiffness are developed in rats and rabbits, limiting genetic experiments that would allow us to explore the contribution of specific cellular targets to these knee pathologies. Here, we present a novel mouse model where surgically induced injury and hyperextension of the knee lead to excessive deposition of disorganized collagen in the meniscus, synovium, and joint capsule in addition to formation of extra-skeletal bone in muscle and soft tissues within the joint capsule. As a functional outcome, arthrofibrosis and acquired heterotopic endochondral ossification coupled with a significant increase in total joint stiffness were observed. By employing this injury model and genetic lineage tracing, we also demonstrate that Gli1+ mesenchymal progenitors proliferate after joint injury and contribute to the pool of fibrotic cells in the synovium and ectopic osteoblasts within the joint capsule. These findings demonstrate that Gli1+ cells are a major cellular contributor to knee arthrofibrosis and acquired heterotopic ossification that manifest after knee injury. Our data demonstrate that genetic manipulation of Gli1+ cells in mice may offer a platform for identification of novel therapeutic targets to prevent knee joint dysfunction after chronic injury.

gp130/STAT3 signaling is required for homeostatic proliferation and anabolism in postnatal growth plate and articular chondrocytes.

Growth of long bones and vertebrae is maintained postnatally by a long-lasting pool of progenitor cells. Little is known about the molecular mechanisms that regulate the output and maintenance of the cells that give rise to mature cartilage. Here we demonstrate that postnatal chondrocyte-specific deletion of a transcription factor Stat3 results in severely reduced proliferation coupled with increased hypertrophy, growth plate fusion, stunting and signs of progressive dysfunction of the articular cartilage. This effect is dimorphic, with females more strongly affected than males. Chondrocyte-specific deletion of the IL-6 family cytokine receptor gp130, which activates Stat3, phenocopied Stat3-deletion; deletion of Lifr, one of many co-receptors that signals through gp130, resulted in a milder phenotype. These data define a molecular circuit that regulates chondrogenic cell maintenance and output and reveals a pivotal positive function of IL-6 family cytokines in the skeletal system with direct implications for skeletal development and regeneration.

Long-term repair of porcine articular cartilage using cryopreservable, clinically compatible human embryonic stem cell-derived chondrocytes.

Osteoarthritis (OA) impacts hundreds of millions of people worldwide, with those affected incurring significant physical and financial burdens. Injuries such as focal defects to the articular surface are a major contributing risk factor for the development of OA. Current cartilage repair strategies are moderately effective at reducing pain but often replace damaged tissue with biomechanically inferior fibrocartilage. Here we describe the development, transcriptomic ontogenetic characterization and quality assessment at the single cell level, as well as the scaled manufacturing of an allogeneic human pluripotent stem cell-derived articular chondrocyte formulation that exhibits long-term functional repair of porcine articular cartilage. These results define a new potential clinical paradigm for articular cartilage repair and mitigation of the associated risk of OA.

Modulation of Hedgehog Signaling by Kappa Opioids to Attenuate Osteoarthritis.

OBJECTIVE: Inhibition of hedgehog (HH) signaling prevents cartilage degeneration and promotes repair in animal models of osteoarthritis (OA). This study, undertaken in OA models and in human OA articular cartilage, was designed to explore whether kappa opioid receptor (KOR) modulation via the inhibition of HH signaling may have therapeutic potential for achieving disease-modifying activity in OA. METHODS: Primary human articular cartilage and synovial tissue samples from patients with knee OA undergoing total joint replacement and from healthy human subjects were obtained from the National Disease Research Interchange. For in vivo animal studies, a partial medial meniscectomy (PMM) model of knee OA in rats was used. A novel automated 3-dimensional indentation tester (Mach-1) was used to quantify the thickness and stiffness properties of the articular cartilage. RESULTS: Inhibition of HH signaling through KOR activation was achieved with a selective peptide agonist, JT09, which reduced HH signaling via the cAMP/CREB pathway in OA human articular chondrocytes (P = 0.002 for treated versus untreated OA chondrocytes). Moreover, JT09 markedly decreased matrix degeneration induced by an HH agonist, SAG, in pig articular chondrocytes and cartilage explants (P = 0.026 versus untreated controls). In vivo application of JT09 via intraarticular injection into the rat knee joint after PMM surgery significantly attenuated articular cartilage degeneration (60% improvement in the tibial plateau; P = 0.021 versus vehicle-treated controls). In JT09-treated rats, cartilage content, structure, and functional properties were largely maintained, and osteophyte formation was reduced by 70% (P = 0.005 versus vehicle-treated controls). CONCLUSION: The results of this study define a novel mechanism for the role of KOR in articular cartilage homeostasis and disease, providing a potential unifying mechanistic basis for the overlap in disease processes and features involving opioid and HH signaling. Moreover, this study identifies a potential novel therapeutic strategy in which KOR modulation can improve outcomes in patients with OA.

In vitro behavior of tendon stem/progenitor cells on bioactive electrospun nanofiber membranes for tendon-bone tissue engineering applications.

PURPOSE: In order to accelerate the tendon-bone healing processes and achieve the efficient osteointegration between the tendon graft and bone tunnel, we aim to design bioactive electrospun nanofiber membranes combined with tendon stem/progenitor cells (TSPCs) to promote osteogenic regeneration of the tendon and bone interface. METHODS: In this study, nanofiber membranes of polycaprolactone (PCL), PCL/collagen I (COL-1) hybrid nanofiber membranes, poly(dopamine) (PDA)-coated PCL nanofiber membranes and PDA-coated PCL/COL-1 hybrid nanofiber membranes were successfully fabricated by electrospinning. The biochemical characteristics and nanofibrous morphology of the membranes, as well as the characterization of rat TSPCs, were defined in vitro. After co-culture with different types of electrospun nanofiber membranes in vitro, cell proliferation, viability, adhesion and osteogenic differentiation of TSPCs were evaluated at different time points. RESULTS: Among all the membranes, the performance of the PCL/COL-1 (volume ratio: 2:1 v/v) group was superior in terms of its ability to support the adhesion, proliferation, and osteogenic differentiation of TSPCs. No benefit was found in this study to include PDA coating on cell adhesion, proliferation and osteogenic differentiation of TSPCs. CONCLUSION: The PCL/COL-1 hybrid electrospun nanofiber membranes are biocompatible, biomimetic, easily fabricated, and are capable of supporting cell adhesion, proliferation, and osteogenic differentiation of TSPCs. These bioactive electrospun nanofiber membranes may act as a suitable functional biomimetic scaffold in tendon-bone tissue engineering applications to enhance tendon-bone healing abilities.

Genetic ablation of adenosine receptor A3 results in articular cartilage degeneration.

Osteoarthritis (OA), the most common form of arthritis, is characterized by inflammation of joints and cartilage degradation leading to disability, discomfort, severe pain, inflammation, and stiffness of the joint. It has been shown that adenosine, a purine nucleoside composed of adenine attached to ribofuranose, is enzymatically produced by the human synovium. However, the functional significance of adenosine signaling in homeostasis and pathology of synovial joints remains unclear. Adenosine acts through four cell surface receptors, i.e., A1, A2A, A2B, and A3, and here, we have systematically analyzed mice with a deficiency for A3 receptor as well as pharmacological modulations of this receptor with specific analogs. The data show that adenosine receptor signaling plays an essential role in downregulating catabolic mechanisms resulting in prevention of cartilage degeneration. Ablation of A3 resulted in development of OA in aged mice. Mechanistically, A3 signaling inhibited cellular catabolic processes in chondrocytes including downregulation of Ca2+/calmodulin-dependent protein kinase (CaMKII), an enzyme that promotes matrix degradation and inflammation, as well as Runt-related transcription factor 2 (RUNX2). Additionally, selective A3 agonists protected chondrocytes from cell apoptosis caused by pro-inflammatory cytokines or hypo-osmotic stress. These novel data illuminate the protective role of A3, which is mediated via inhibition of intracellular CaMKII kinase and RUNX2 transcription factor, the two major pro-catabolic regulators in articular cartilage. KEY MESSAGES: Adenosine receptor A3 (A3) knockout results in progressive loss of articular cartilage in vivo. Ablation of A3 results in activation of matrix degradation and cartilage hypertrophy. A3 agonists downregulate RUNX2 and CaMKII expression in osteoarthritic human articular chondrocytes. A3 prevents articular cartilage matrix degradation induced by inflammation and osmotic fluctuations. A3 agonist inhibits proteolytic activity of cartilage-degrading enzymes.

Vitamin D-deficiency and sex-specific dysregulation of placental inflammation.

To investigate an immunomodulatory role for vitamin D in pregnancy we used mice raised on vitamin D-sufficient (SUFF), or -deficient (DEF) diets. At embryonic day 14, pregnant mice received intraperitoneal injection of lipopolysaccharide (LPS) or vehicle for 24h, with age-matched non-pregnant mice as controls. In non-pregnant mice, 6 serum analytes (IL-1ß, IL-18, MDC/CCL22, MIP-1α/CCL3, EGF, IgA) were lower in DEF mice. In pregnant DEF mice only GH was higher. In non-pregnant mice LPS induced 28 analytes, with 5 (IL-18, IP-10/CXCL10, MCP-1/CCL2, MIP-1ß/CCL4, MIP-3ß/CCL19) being highest in DEF mice. In pregnant SUFF mice 16 serum analytes increased with LPS, and 6 of these (IP-10/CXCL10, MCP-1/CCL2, SAP, TIMP-1, VCAM-1, vWF) were higher and 1 (GCP-2/CXCL6) lower in DEF mice. Parallel analysis of placental mRNAs showed elevated mRNA for Il-6, Ccl2 and Cxcl10 in placentae from male and female fetuses in LPS-DEF mice. However, LPS-induced expression of Ifnγ, Tnfα, and Cxcl6 was only observed in female placentae from DEF mice. LPS-DEF mice also showed smaller litter sizes relative to control SUFF mice. Numbers of female fetuses per dam were significantly lower for DEF mice with or without LPS challenge. LPS had no effect on numbers of male fetuses from DEF mothers, but significantly decreased male fetuses from SUFF mothers. These data indicate that vitamin D is an important component of anti-inflammatory immune responses during pregnancy, with the placenta and fetal sex playing pivotal roles in this process.

Mixed metastatic lung cancer lesions in bone are inhibited by noggin overexpression and Rank:Fc administration.

UNLABELLED: Lung cancer metastases to bone produce a primarily mixed osteolytic/osteoblastic lesion. The purpose of this study was to determine if blockade of both pathways would inhibit the formation these lesions in bone. Inhibition of the osteoblastic lesion with noggin and the osteolytic lesion with RANK:Fc was a successful treatment strategy to inhibit progression of mixed lung cancer lesions in bone. INTRODUCTION: Approximately 9-30% of patients with lung cancer develop bone metastases, leading to significant morbidity and mortality. A549 is a non-small-cell lung cancer (NSCLC) line that produces a mixed metastatic lesion in bone. We sought to determine if blockade of key components in both osteolytic and osteoblastic pathways would result in a reduction of a NSCLC tumor progression in a murine model of bony metastasis. MATERIALS AND METHODS: The study used a retroviral vector overexpressing noggin (RN), a specific inhibitor of BMP, and RANK:Fc, a chimeric protein that inhibits the RANK-RANKL interaction. A549 cells were transduced with RN before implantation in SCID mice. Cells were implanted in a subcutaneous model and tibial injection model. RANK:Fc was administered twice weekly at 15 mg/kg. There were five treatment groups: A549; A549 + RN; A549 + RANK:Fc; A549 + empty vector; and A549 + RN + RANK:Fc (n = 10/group). RESULTS: In SCID mice who underwent subcutaneous A549 tumor cell injection, animals treated with A549 + RN had significantly smaller subcutaneous tumor size at 8 weeks. In an intratibial model of bony metastasis, animals injected with A549 cells developed a mixed lytic/blastic lesion with cortical destruction at 8 weeks. Treatment with RANK:Fc inhibited the formation of osteoclasts, led to a smaller tumor volume in bone, and inhibited the lytic component of the mixed lesion. Animals treated with A549 + RN had a decreased number of osteoblasts in bone lesions, smaller tumor volume, and inhibition of the blastic component of the mixed lesions. Combination treatment inhibited both the lytic and blastic components of the lesion. CONCLUSIONS: The NSCLC cell line A549 forms a mixed osteolytic/osteoblastic lesion in vivo. Noggin overexpression inhibited the formation of the osteoblastic aspect of the lesion in bone and the tumor growth in vivo. Treatment with RANK:Fc limited the formation of the lytic aspect of the mixed lesion and also inhibited the rate of in vivo tumor growth. Inhibition of both pathways is necessary to effectively inhibit the progression of mixed metastatic lesions in bone.

Differential Responses to Vitamin D2 and Vitamin D3 Are Associated With Variations in Free 25-Hydroxyvitamin D.

25-Hydroxyvitamin D (25D) circulates bound primarily to serum vitamin D binding protein (DBP), with DBP showing higher binding affinity for 25D3 than 25D2. We therefore hypothesized that vitamin D2 (D2) promotes higher serum levels of unbound 25D (free 25D), with different functional responses, relative to vitamin D3 (D3). Week 3 C56BL/6 mice were placed on diets containing either D2 or D3 alone (both 1000 IU/kg). At week 8 and week 16, D2 mice had only 25D2 in circulation (26.6 ± 1.9 and 33.3 ± 4.4 ng/mL), and D3 mice had only 25D3 (28.3 ± 2.0 and 31.7 ± 2.1 ng/mL). At week 8 (44.5 ± 6.4 vs 62.4 ± 11.6 pg/mL, P < .05) and week 16 (78.4 ± 12.6 vs 95.5 ± 11.6), D2 mice had lower serum 1,25-dihydroxyvitamin D relative to D3 mice. By contrast, measured free 25D was significantly higher in D2 mice at week 8 (16.8 ± 0.65 vs 8.4 ± 0.63 pg/mL, P < .001) and week 16 (17.4 ± 0.43 vs 8.4 ± 0.44, P < .001). A two-way ANOVA of bone histomorphometry showed that week 8 D2 mice had significantly higher osteoclast surface/bone surface, eroded surface/bone surface, and mineral apposition rate compared with D3 mice. Osteoblast surface/bone surface was higher in week 8 D2 females but not week 8 D2 males. At week 16, D2 mice had significantly higher bone volume/total volume and trabecular number compared with D3 mice. Differences in bone phenotype were observed despite D2 mice reaching similar serum 25D levels and lower 1,25D levels compared with D3 mice. These data indicate that 25D2 binds less well to DBP than 25D3, with resulting higher levels of free 25D promoting differential effects on bone in mice exposed to D2 alone.

The effect of cyclooxygenase-2 (COX-2) inhibition on human prostate cancer induced osteoblastic and osteolytic lesions in bone.

BACKGROUND: The mechanism of bone formation by osteoblastic prostate cancer metastases is not well defined. Using knockout mice, it has been demonstrated that prostaglandins produced by COX-2 are critical for fracture repair. Therefore, our aim was to determine if COX-2 plays a role in the bone formation in osteoblastic prostate cancer metastases in bone. MATERIALS AND METHODS: We assessed the influence of pharmacologic COX-2 inhibition in a SCID mouse intratibial injection model of bone metastasis using two human prostate cancer cell lines that produce either osteoblastic lesions (LAPC-9) or osteolytic lesions (PC-3, negative control). SC-58236, a COX-2 specific inhibitor, was used at a dose of 3 mg/Kg intraperitoneally 3 times per week in the Treatment groups for 8 weeks until sacrifice. RESULTS: Western blot for COX-2 demonstrated that LAPC-9 cells expressed high levels of COX-2 while PC-3 cells did not. Treatment with SC-58236 significantly reduced the size of osteoblastic lesions after LAPC-9 injection based on both radiographic and histomorphometric criteria compared to the control group. In contrast, large osteolytic lesions were seen in both control and SC-58236 treated animals after PC-3 cell injections. The results of this study indicate that COX-2 inhibition can decrease the size of osteoblastic lesions produced by LAPC-9, a human prostate cancer cell line that expresses high levels of COX-2. This treatment had no effect on the osteolytic activity of PC-3 cells. CONCLUSION: These findings suggest that the progression of osteoblastic metastases induced by human prostate cancer cells may be limited by COX-2 inhibitors.

Vitamin D supplementation and antibacterial immune responses in adolescents and young adults with HIV/AIDS.

Human monocytes activated by toll-like receptor 2/1 ligand (TLR2/1L) show enhanced expression of the vitamin D receptor (VDR) and the vitamin D-activating enzyme 1α-hydroxylase (CYP27B1). The resulting intracrine conversion of precursor 25-hydroxyvitamin D3 (25OHD) to active 1,25-dihydroxyvitamin D (1,25(OH)2D) can stimulate expression of antibacterial cathelicidin (CAMP). To determine whether this response is functional in HIV-infected subjects (HIV+ ), serum from HIV+ subjects pre- and post-vitamin D supplementation was utilized in monocyte cultures with or without TLR2/1L. Expression of CYP27B1 and VDR was enhanced following treatment with TLR2/1L, although this effect was lower in HIV+ vs HIV- serum (p<0.05). CAMP was also lower in TLR2/1L-treated monocytes cultured in HIV+ serum (p<0.01). In a dose study, supplementation of HIV+ subjects with 4000IU or 7000IU vitamin D/day increased serum 25OHD from 17.3±8.0 and 20.6±6.2ng/ml (43nM and 51nM) at baseline to 41.1±12.0 and 51.9±23.1ng/ml (103nM and 130nM) after 12 weeks (both p<0.001). Greater percent change from baseline 25OHD was significantly associated with enhanced TLR2/1L-induced monocyte CAMP adjusted for baseline expression (p=0.009). In a randomized placebo-controlled trial, 7000IU vitamin D/day increased serum 25OHD from 18.0±8.6 to 32.7±13.8ng/ml (45nM and 82nM) after 12 weeks. Expression of CAMP increased significantly from baseline after 52 weeks of vitamin D-supplementation. At this time point, TLR2/1L-induced CAMP was positively associated with percent change from baseline in 25OHD (p=0.029 overall and 0.002 within vitamin D-supplemented only). These data indicate that vitamin D supplementation in HIV-infected subjects can promote improved antibacterial immunity, but also suggest that longer periods of supplementation are required to achieve this.

Dietary vitamin D restriction in pregnant female mice is associated with maternal hypertension and altered placental and fetal development.

Epidemiology has linked vitamin D deficiency with preeclampsia in humans. We hypothesized that low vitamin D status in pregnant mice may lead to symptoms of preeclampsia. Female BL6 mice were raised on vitamin D-sufficient or -deficient diets from weeks 4 of age and then mated with vitamin D-sufficient BL6 males at week 8. The resulting pregnant mice were either allowed to deliver pups and monitored for blood pressure (BP) and weight of offspring or euthanized at day 14 or 18 of gestation (E14 or E18) for analysis of serum, placental/kidney tissues, and fetuses. At E14 serum concentrations of 25-hydroxyvitamin D (30.1 ± 5.0 vs 1.8 ± 0.6 ng/mL, P < .001) and 1,25-dihydroxyvitamin D (119.5 ± 18.7 vs 37.4 ± 5.1 pg/mL, P < .01) were higher in sufficient vs deficient pregnant mice. At E14 BP was significantly elevated in vitamin D-deficient pregnant mice relative to vitamin D-sufficient mice for both systolic BP (124.89 ± 2.28 vs 105.34 ± 3.61 mm Hg, P < .001) and mean arterial pressure (115.33 ± 1.93 vs 89.33 ± 5.02 mm Hg, P < .001). This elevation continued through pregnancy until 7 days postpartum (PP7) but returned to baseline by PP14. Analysis of maternal kidneys showed increased expression of mRNA for renin and the angiotensin II receptor (3- and 4-fold, respectively) in vitamin D-deficient vs -sufficient mice at E14. Histological analysis of E14 placentas from vitamin D-deficient mice showed decreased vascular diameter within the labyrinth region. E14 and E18 fetuses from vitamin D-deficient mice were larger than those from vitamin D-sufficient mothers. However, by PP14 pups from vitamin D-deficient mothers weighed significantly less than those from vitamin D-sufficient mothers. Resupplementation of vitamin D periconceptually partially reversed the effects of vitamin D deficiency. These data provide further evidence that low vitamin D status may predispose pregnant women to dysregulated placental development and elevated blood pressure.