Role of type 2 cannabinoid receptor in bone metabolism

Cannabinoid receptors play an important role in regulating bone mass and bone turnover. Studies in our laboratories have shown that young mice lacking type 1 cannabinoid receptor (CNR1-/-) had increased bone mass and were resistant to ovariectomy-induced bone loss. Other workers have reported that type 2 cannabinoid receptor knockout mice (CNR2-/-) develop age-related osteoporosis. The aim of this PhD thesis was to further investigate the role of CNR2 in bone metabolism in vitro and in vivo, using genetic and pharmacological approaches. This study showed that CNR2-/- mice had normal bone mass and bone turnover at 3 months of age, but following ovariectomy, CNR2-/- mice were partially protected from bone loss, because of a mild defect in osteoclast formation and bone resorption. In keeping with this, studies in vitro showed that RANKL-stimulated bone marrow cultures from CNR2-/- mice had fewer osteoclasts than cultures from wild type littermates. The CNR2-selective antagonist/inverse agonist AM630, inhibited osteoclast formation in wild type bone marrow cultures in vitro and prevented ovariectomy-induced bone loss in wild type mice in vivo. In contrast, osteoclast cultures from CNR2-/- mice were resistant to the inhibitory effects of AM630 at low concentrations and CNR2-/- ovariectomised mice did not respond to its protective effects at low doses, consistent with a CNR2- mediated effect. These results indicate that CNR2 regulates bone loss under conditions of increased bone turnover, such as ovariectomy, by affecting osteoclast differentiation and function. CNR2-deficient mice developed accelerated age-related osteoporosis and by 12 months of age they had a significant reduction in osteoblast numbers and bone formation, whereas osteoclast numbers remained comparable to wild type littermates. In agreement with this, osteoblasts derived from bone marrow of CNR2-/- mice had reduced PTHstimulated alkaline phosphatase activity and ability to form bone nodules, when compared with wild type cultures. The CNR2-selective agonist, HU308, stimulated bone nodule formation in wild type calvarial osteoblast cultures in vitro and reversed ovariectomy-induced bone loss in wild type mice in vivo. HU308 had blunted effects on bone nodule formation in cultures from CNR2-/- mice and no significant effects on ovariectomy-induced bone loss in CNR2-/- mice, indicating a CNR2-mediated effect. These studies demonstrate that CNR2 protects against age-related bone loss by mainly enhancing osteoblast differentiation and bone formation. In conclusion, type 2 cannabinoid receptors protect from bone loss by maintaining bone remodelling at balance. In addition, type 2 cannabinoid receptor agonists show evidence of anabolic activity, whereas antagonists/inverse agonists show evidence of antiosteoclastic activity in vitro and in vivo

Probiotics inhibit cartilage damage and progression of osteoarthritis in mice

Increasing interest has focussed on the possible role of alterations in the microbiome in the pathogenesis of metabolic disease, inflammatory disease, and osteoporosis. Here we examined the role of the microbiome in a preclinical model of osteoarthritis in mice subjected to destabilisation of medical meniscus (DMM). The intestinal microbiome was depleted by broad-spectrum antibiotics from 1 week before birth until the age of 6 weeks when mice were subjected reconstitution of the microbiome with faecal microbial transplant (FMT) followed by the administration of a mixture of probiotic strains Lacticaseibacillus paracasei 8700:2, Lactiplantibacillus plantarum HEAL9 and L. plantarum HEAL19 or vehicle. All mice were subjected to DMM at the age of 8 weeks. The severity of osteoarthritis was evaluated by histological analysis and effects on subchondral bone were investigated by microCT analyses. The combination of FMT and probiotics significantly inhibited cartilage damage at the medial femoral condyle such that the OARSI score was 4.64 ± 0.32 (mean ± sem) in the FMT and probiotic group compared with 6.48 ± 0.53 in the FMT and vehicle group (p = 0.007). MicroCT analysis of epiphyseal bone from the femoral condyle showed that the probiotic group had higher BV/TV, increased Tb.Th, and moderately thicker subchondral bone plates than the control group. There was no difference between groups in joint inflammation or in serum concentrations of inflammatory cytokines and chemokines. We conclude that treatment with probiotics following FMT in mice where the microbiome has been depleted inhibits DMM-induced cartilage damage and impacts on the structure of subchondral bone particularly at the femoral condyle. While further studies are required to elucidate the mechanism of action, our research suggests that these probiotics may represent a novel intervention for the treatment of osteoarthritis

Heavy cannabis use is associated with low bone mineral density and an increased risk of fractures

Purpose: To investigate possible associations between recreational cannabis use and bone health in humans.  Methods: Cross-sectional study of individuals recruited from primary care in the UK between 2011 and 2014. Cases were regular smokers of cannabis divided into moderate (n=56) and heavy user (n=144) subgroups depending on whether they reported fewer or more than 5000 cannabis smoking episodes during their lifetime. Controls comprised 114 cigarette smokers.  Results: Heavy cannabis users had lower total hip bone mineral density (mean ± SD Z-score: -0.20±0.9 vs. +0.2±0.9, p<0.0005), lower spine bone mineral density (-0.5±1.2 vs. 0.0±1.2, p<0.0005) and lower BMI (26.5±6.0 vs 29.0±7.0, p=0.01) than controls. Fracture rate was also increased in heavy users (rate ratio=2.17, 95% confidence interval 1.59 to 2.95; p<0.001). When compared with controls, CTX serum concentrations were raised in heavy cannabis users (0.3±0.1 vs. 0.2±0.1 pg/ml, p=0.045) as were P1NP concentrations (47.1±19.2 vs. 41.2±17.8 pg/ml, p=0.01). Serum 25(OH)D concentrations were reduced in heavy users compared with controls (25.3±16.8 vs. 36.9±26.7 nmol/l, p=0.002). Multiple regression analysis revealed that heavy cannabis use was an independent predictor of spine bone mineral density accounting for 5.4% of the variance (p=0.035) and total hip bone mineral density accounting for 5.8% of the variance (p=0.001) but mediation analysis suggested that the effect on spine bone mineral density was indirect and mediated through low BMI.  Conclusion: Heavy cannabis use is associated with low bone mineral density, low BMI, high bone turnover and an increased risk of fracture. Heavy cannabis use negatively impacts on bone health both directly and indirectly through an effect on BMI

Raman Spectroscopy as a Predictive Tool for Monitoring Osteoporosis Therapy in a Rat Model of Postmenopausal Osteoporosis

Pharmacological therapy of osteoporosis reduces bone loss and risk of fracture in patients. Modulation of bone mineral density cannot explain all effects. Other aspects of bone quality affecting fragility and ways to monitor them need to be better understood. Keratinous tissue acts as surrogate marker for bone protein deterioration caused by oestrogen deficiency in rats. Ovariectomised rats were treated with alendronate (ALN), parathyroid hormone (PTH) or estrogen (E2). MicroCT assessed macro structural changes. Raman spectroscopy assessed biochemical changes. Micro CT confirmed that all treatments prevented ovariectomy-induced macro structural bone loss in rats. PTH induced macro structural changes unrelated to ovariectomy. Raman analysis revealed ALN and PTH partially protect against molecular level changes to bone collagen (80% protection) and mineral (50% protection) phases. E2 failed to prevent biochemical change. The treatments induced alterations unassociated with the ovariectomy; increased beta sheet with E2, globular alpha helices with PTH and fibrous alpha helices with both ALN and PTH. ALN is closest to maintaining physiological status of the animals, while PTH (comparable protective effect) induces side effects. E2 is unable to prevent molecular level changes associated with ovariectomy. Raman spectroscopy can act as predictive tool for monitoring pharmacological therapy of osteoporosis in rodents. Keratinous tissue is a useful surrogate marker for the protein related impact of these therapies. The results demonstrate utility of surrogates where a clear systemic causation connects the surrogate to the target tissue. It demonstrates the need to assess broader biomolecular impact of interventions to examine side effects. [Figure not available: see full text.]

Raman Spectroscopy Predicts the Link between Claw Keratin and Bone Collagen Structure in a Rodent Model of Oestrogen Deficiency

Osteoporosis is a common disease characterized by reduced bone mass and an increased risk of fragility fractures. Low bone mineral density is known to significantly increase the risk of osteoporotic fractures; however, the majority of non-traumatic fractures occur in individuals with a bone mineral density too high to be classified as osteoporotic. Therefore, there is an urgent need to investigate aspects of bone health, other than bone mass, that can predict the risk of fracture. Here, we successfully predicted association between bone collagen and nail keratin in relation to bone loss due to oestrogen deficiency using Raman spectroscopy. Raman signal signature successfully discriminated between ovariectomised rats and their sham controls with a high degree of accuracy for the bone (sensitivity 89%, specificity 91%) and claw tissue (sensitivity 89%, specificity 82%). When tested in an independent set of claw samples the classifier gave 92% sensitivity and 85% specificity. Comparison of the spectral changes occurring in the bone tissue with the changes occurring in the keratin showed a number of common features that could be attributed to common changes in the structure of bone collagen and claw keratin. This study established that systemic oestrogen deficiency mediates parallel structural changes in both the claw (primarily keratin) and bone proteins (primarily collagen). This strengthens the hypothesis that nail keratin can act as a surrogate marker of bone protein status where systemic processes induce changes

Targeted sequencing of the Paget's disease associated 14q32 locus identifies several missense coding variants in RIN3 that predispose to Paget's disease of bone

Paget's disease of bone (PDB) is a common disorder with a strong genetic component characterized by increased but disorganized bone remodelling. Previous genome-wide association studies identified a locus on chromosome 14q32 tagged by rs10498635 which was significantly associated with susceptibility to PDB in several European populations. Here we conducted fine-mapping and targeted sequencing of the candidate locus to identify possible functional variants. Imputation in 741 PDB patients and 2699 controls confirmed that the association was confined to a 60 kb region in the RIN3 gene and conditional analysis adjusting for rs10498635 identified no new independent signals. Sequencing of the RIN3 gene identified a common missense variant (p.R279C) that was strongly associated with the disease (OR = 0.64; P = 1.4 × 10(−9)), and was in strong linkage disequilibrium with rs10498635. A further 13 rare missense variants were identified, seven of which were novel and detected only in PDB cases. When combined, these rare variants were over-represented in cases compared with controls (OR = 3.72; P = 8.9 × 10(−10)). Most rare variants were located in a region that encodes a proline-rich, intrinsically disordered domain of the protein and many were predicted to be pathogenic. RIN3 was expressed in bone tissue and its expression level was ∼10-fold higher in osteoclasts compared with osteoblasts. We conclude that susceptibility to PDB at the 14q32 locus is mediated by a combination of common and rare coding variants in RIN3 and suggest that RIN3 may contribute to PDB susceptibility by affecting osteoclast function

Role of type 2 cannabinoid receptor in bone metabolism

Cannabinoid receptors play an important role in regulating bone mass and bone turnover. Studies in our laboratories have shown that young mice lacking type 1 cannabinoid receptor (CNR1-/-) had increased bone mass and were resistant to ovariectomy-induced bone loss. Other workers have reported that type 2 cannabinoid receptor knockout mice (CNR2-/-) develop age-related osteoporosis. The aim of this PhD thesis was to further investigate the role of CNR2 in bone metabolism in vitro and in vivo, using genetic and pharmacological approaches. This study showed that CNR2-/- mice had normal bone mass and bone turnover at 3 months of age, but following ovariectomy, CNR2-/- mice were partially protected from bone loss, because of a mild defect in osteoclast formation and bone resorption. In keeping with this, studies in vitro showed that RANKL-stimulated bone marrow cultures from CNR2-/- mice had fewer osteoclasts than cultures from wild type littermates. The CNR2-selective antagonist/inverse agonist AM630, inhibited osteoclast formation in wild type bone marrow cultures in vitro and prevented ovariectomy-induced bone loss in wild type mice in vivo. In contrast, osteoclast cultures from CNR2-/- mice were resistant to the inhibitory effects of AM630 at low concentrations and CNR2-/- ovariectomised mice did not respond to its protective effects at low doses, consistent with a CNR2- mediated effect. These results indicate that CNR2 regulates bone loss under conditions of increased bone turnover, such as ovariectomy, by affecting osteoclast differentiation and function. CNR2-deficient mice developed accelerated age-related osteoporosis and by 12 months of age they had a significant reduction in osteoblast numbers and bone formation, whereas osteoclast numbers remained comparable to wild type littermates. In agreement with this, osteoblasts derived from bone marrow of CNR2-/- mice had reduced PTHstimulated alkaline phosphatase activity and ability to form bone nodules, when compared with wild type cultures. The CNR2-selective agonist, HU308, stimulated bone nodule formation in wild type calvarial osteoblast cultures in vitro and reversed ovariectomy-induced bone loss in wild type mice in vivo. HU308 had blunted effects on bone nodule formation in cultures from CNR2-/- mice and no significant effects on ovariectomy-induced bone loss in CNR2-/- mice, indicating a CNR2-mediated effect. These studies demonstrate that CNR2 protects against age-related bone loss by mainly enhancing osteoblast differentiation and bone formation. In conclusion, type 2 cannabinoid receptors protect from bone loss by maintaining bone remodelling at balance. In addition, type 2 cannabinoid receptor agonists show evidence of anabolic activity, whereas antagonists/inverse agonists show evidence of antiosteoclastic activity in vitro and in vivo.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Management Implications at a Protected, Peri-Urban, Salt Lake Ecosystem: The Case of Larnaca’s Salt Lakes (Cyprus)

Larnaca’s salt lakes constitute one of the most important protected areas in Cyprus and in the southeast Mediterranean. It is in continuity with the urban area of Larnaca city, being the major green infrastructure in the area, supporting (among others) biodiversity, recreation, culture, and food production. The present study aims to provide an overview of the area’s ecosystem types and their potential to provide ecosystem services, focusing on assessing the water ecosystem condition and drafting the relevant baseline accounts. The results of the study include an ecosystem type map of the area, followed by potential supply maps of ecosystem services, for the three major categories (i.e., provisioning, regulating and maintenance, and cultural) and the estimation and assessment of ecosystem condition variables for wetlands, as proposed by the EU-wide methodology to map and assess the ecosystem condition, in line with the System of Environmental Economic Accounting—Ecosystem Accounting (SEEA EA). A critical exceedance (over 10%) of the imperviousness and the increase in the soil moisture deficit within the wider salt lakes area have been identified and can constitute useful variables associated with the anthropogenic, climatic, and other effects on the condition of the salt lakes. Discussion for integrating this information to existing knowledge is provided toward supporting scientifically informed decision and policymaking for the sustainable development of protected areas

Conditional deletion of E11/Podoplanin in bone protects against ovariectomy-induced increases in osteoclast formation and activity

E11/Podoplanin (Pdpn) is implicated in early osteocytogenesis and the formation of osteocyte dendrites. This dendritic network is critical for bone modeling/remodeling, through the production of the receptor for receptor activator of nuclear factor ?B (RANK)-ligand (RANKL). Despite this, the role of Pdpn in the control of bone remodeling is yet to be established in vivo. Here we utilised bone-specific Pdpn conditional knockout mice (cKO) to examine the role of Pdpn in the bone loss associated with ovariectomy (OVX). MicroCT revealed that Pdpn deletion had no significant effect on OVX-induced changes in trabecular microarchitecture. Significant differences between genotypes were observed in the trabecular pattern factor (