Clemens Löwik

Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease.

The development of novel anti-cancer strategies requires more sensitive and less invasive methods to detect and monitor in vivo minimal residual disease in cancer models. Bone marrow metastases are indirectly detected by radiography as osteolytic and/or osteosclerotic lesions. Marrow micrometastases elude radiographic detection and, therefore, more sensitive methods are needed for their direct identification. Injection of cancer cells into the left cardiac ventricle of mice closely mimics micrometastatic spread. When luciferase-transfected cells are used, whole-body bioluminescent reporter imaging can detect microscopic bone marrow metastases of approximately 0.5 mm(3) volume, a size below the limit in which tumors need to induce angiogenesis for further growth. This sensitivity translates into early detection of intramedullary tumor growth, preceding the appearance of a radiologically evident osteolysis by approximately 2 weeks. Bioluminescent reporter imaging also enables continuous monitoring in the same animal of growth kinetics for each metastatic site and guides end-point analyses specifically to the bones affected by metastatic growth. This model will accelerate the understanding of the molecular events in metastasis and the evaluation of novel therapies aiming at repressing initial stages of metastatic growth.

Inducible production of nitric oxide in osteoblast-like cells and in fetal mouse bone explants is associated with suppression of osteoclastic bone resorption.

Nitric oxide (NO) has been suggested to be involved in the regulation of osteoclast activity. Since osteoblasts, through the release of various factors, are the main regulators of osteoclastic resorption, first we have investigated whether osteoblast-like cells and fetal mouse long bone explants are able to produce NO. Second, we have assessed the effect of NO on osteoclastic resorption in whole bone cultures. In this study we show that primary rat osteoblast-like cells as well as the clonal rat osteoblast-like cell line UMR-106, stimulated with IFN-gamma together with TNF-alpha and LPS, produce NO, measured as nitrite production. IL-1 alpha enhanced while TGF-beta 2 inhibited TNF-alpha + IFN-gamma + LPS-stimulated NO production in UMR-106 cells dose dependently. Both the cytokines, however, had no effect when given alone. The competitive inhibitor of NO production, NG-monomethyl-arginine (L-NMMA), and cycloheximide abolished the increase in nitrite production induced by TNF-alpha + IFN-gamma + LPS, while hydrocortisone had no effect, as previously reported for chondrocytes. Calciotropic hormones had either no effect [1,25(OH)2D3] or had a small inhibitory effect (parathyroid hormone) on stimulated NO production. Furthermore, we found that in cultured fetal mouse long bone explants the combination of TNF-alpha + IFN-gamma + LPS as well as the NO donor sodium nitroprusside could inhibit osteoclastic resorption, measured as 45Ca release. The inhibition of resorption was prevented by concurrent administration of L-NMMA. Histological evaluation revealed that the TNF-alpha + IFN-gamma + LPS-induced inhibition of 45Ca release was associated with a decrease in the number of tartrate-resistant acid phosphatase-positive osteoclasts. We propose that the NO production by osteogenic cells (osteoblasts and chondrocytes) may represent an important regulatory mechanism of osteoclastic activity especially under pathological conditions characterized by release of bone-resorbing inflammatory cytokines.

Binding and antiresorptive properties of heterocycle-containing bisphosphonate analogs : Structure-activity relationships

To define structure-activity relationships for bisphosphonate activity, we examined the bone binding and antiresorptive properties of heterocycle-containing analogs of risedronate, a pyridylbisphosphonate, in cultures of mouse fetal bone explants. Our studies indicated that hydroxybisphosphonates with the nitrogen molecule in the pyridyl ring were very potent inhibitors of osteoclastic resorption. Changing the place of the nitrogen in the ring structure of risedronate or its methylation did not significantly alter antiresorptive potency in relation to risedronate. Extension of the R2 chain, however, reduced efficacy. In binding experiments, we found that all heterocyclic bisphosphonates with a hydroxyl group in R1 had comparable affinity for bone mineral and inhibited calcium incorporation into bone explants to a similar extent. The affinity of a risedronate analog without R1 was markedly reduced. We also examined the properties of a risedronate analog (NE-10790) belonging to the group of phosphonocarboxylates in which one of the phosphonate groups is substituted by a carboxyl group. NE-10790 had strongly reduced binding affinity, but still retained some antiresorptive activity. Interestingly, the continuous presence of NE-10790 in cultures of fetal mouse metacarpal bones increased its antiresorptive efficacy by about 40-fold compared with 24 h preincubation, whereas, under the same conditions, the potency of high-affinity hydroxybisphosphonates did not change or only slightly increased. This may be explained by the differences in pharmacokinetic behavior between compounds of high and of low affinity for bone mineral. These data show that, as with alkylbisphosphonates, heterocycle-containing bisphosphonates with a nitrogen functionality in the R2 chain are potent antiresorptive agents and a hydroxyl substitution in the R1 chain confers high affinity for bone mineral, probably due to tridentate configuration. The group of phosphonocarboxylates, with strongly reduced bone affinity, provides an interesting therapeutic option.

Molecular imaging of tumor angiogenesis using αvβ3-integrin targeted multimodal quantum dots

Molecular imaging of angiogenesis is urgently needed for diagnostic purposes such as early detection, monitoring of (angiostatic) therapy and individualized therapy. Multimodality molecular imaging is a promising and refined technique to study tumor angiogenesis, which has so far been largely unexplored due to the lack of suitable multimodal contrast agents. Here, we report on the application of a novel αvβ3-specific quantum dot-based nanoparticle, which has been optimized for both optical and magnetic resonance detection of tumor angiogenesis. Upon intravenous injection of RGD-pQDs in tumor-bearing mice, intravital microscopy allowed the detection of angiogenically activated endothelium at cellular resolution with a small scanning window and limited penetration depth, while magnetic resonance imaging was used to visualize angiogenesis at anatomical resolution throughout the entire tumor. Fluorescence imaging allowed whole-body investigation of angiogenic activity. Using these quantum dots and the aforementioned imaging modalities, the angiogenic tumor vasculature was readily detected with the highest angiogenic activity occurring in the periphery of the tumor. This nanoparticle may be employed for multimodality imaging of a variety of diseases that are accompanied by activation of endothelial cells. Furthermore, the current technology might be developed for molecular imaging of other pathophysiological processes.

Dose-dependent effects of phytoestrogens on bone.

Phytoestrogens have the potential to maintain bone health and delay or prevent osteoporosis. This review focuses on their dose-dependent effects on bone and their possible mechanisms of action. Phytoestrogens exert biphasic dose-dependent effects on osteoblasts and osteoprogenitor cells, stimulating osteogenesis at low concentrations and inhibiting osteogenesis at high concentrations. They inhibit osteoclast formation and activity. Recent data show that the balance between estrogen receptors and peroxisome proliferator-activated receptors, which are dose-dependently activated by phytoestrogens, determines their biological effects on bone. This review provides a new understanding of the mechanism of action of phytoestrogens and could be important for future studies to find precise beneficial doses in vivo and in clinical trials.

Sclerostin in Mineralized Matrices and van Buchem Disease

Sclerostin is an inhibitor of bone formation expressed by osteocytes. We hypothesized that sclerostin is expressed by cells of the same origin and also embedded within mineralized matrices. In this study, we analyzed (a) sclerostin expression using immunohistochemistry, (b) whether the genomic defect in individuals with van Buchem disease (VBD) was associated with the absence of sclerostin expression, and (c) whether this was associated with hypercementosis. Sclerostin was expressed by cementocytes in mouse and human teeth and by mineralized hypertrophic chondrocytes in the human growth plate. In individuals with VBD, sclerostin expression was absent or strongly decreased in osteocytes and cementocytes. This was associated with increased bone formation, but no overt changes in cementum thickness. In conclusion, sclerostin is expressed by all 3 terminally differentiated cell types embedded within mineralized matrices: osteocytes, cementocytes, and hypertrophic chondrocytes.

Effects of exogenous prostanoids on the proliferation of osteoblast-like cells in vitro

The effects of several prostaglandins on the proliferation of secondary cultures of osteoblast-like cells, as measured by the incorporation of [3H]-thymidine into DNA and total DNA content of the cultures, were studied. PGE2 in the concentration range of 10(-8) to 10(-5) M caused a direct, dose-related stimulation of proliferation, while PGF2 alpha and PGD2 were less effective. PGA2 and 6-keto-PGF1 alpha were inactive in the osteoblasts in concentrations of 10(-7) to 10(-6) M. A similar stimulation profile was observed for the induction of ornithine decarboxylase (ODC, L-ornithine decarboxy-lyase, EC 4.1.1.17): the order of potency of the different prostaglandins in the induction of the ODC activity was PGE2 greater than PGF2 alpha = PGD2; again, PGA2 and 6-keto-PGF1 alpha were without effect in concentrations up to 10(-6) M. These results show that the primary prostaglandins, in order of potency PGE2 greater than PGF2 alpha = PGD2, can have a direct, stimulatory effect on the proliferation of osteoblasts, which is closely related to the induction of ODC activity.

Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8

Abstract Until now, many extracellular matrix proteins, e.g. osteopontin and osteonectin, have been used to determine a cell’s osteogenic maturation. The disadvantage in evaluation of these proteins is their relative wide-ranging appearance throughout the osteogenic differentiation process. Thus, the aim of this study was to establish an immunohistochemical setup using E11, a marker that binds selectively to cells of the late osteogenic cell lineage. In addition, the histochemical expression of the bone matrix proteins osteonectin, osteopontin and fibronectin was compared to that of E11 using monoclonal antibodies. For light microscopical detection of osteogenic markers in cultured cells we developed a simple paraffin technique using a fibrin glue as embedding medium. This allows the handling of cultured cells such as a tissue sample and includes the use of stored biological specimens for further immunohistochemical experiments. We used newborn rat calvariae for whole tissue preparations and for isolation and cultivation of bone cells. In addition, we included the rat osteosarcoma cell line ROS 17/2.8 in this study. For the first time, we have localised E11 in osteocytes of rat calvaria preparations at the electron microscopical level. E11 was detected at plasma membranes of osteocytes and their processes, but not at those of osteoblasts. Accompanying experiments with cultured newborn rat calvaria cells and ROS 17/2.8 cells revealed E11 reactivity on a subset of cells. The results obtained confirm the suitability of the differentiation marker E11 as a sensitive instrument for the characterisation of bone cell culture systems.

First missense mutation in the SOST gene causing sclerosteosis by loss of sclerostin function.

Sclerosteosis is a rare bone dysplasia characterized by greatly increased bone mass, especially of the long bones and the skull. Patients are tall, show facial asymmetry and often have syndactyly. Clinical complications are due to entrapment of cranial nerves. The disease is thought to be due to loss‐of‐function mutations in the SOST gene. The SOST gene product, sclerostin, is secreted by osteocytes and transported to the bone surface where it inhibits osteoblastic bone formation by antagonizing Wnt signaling. In a small Turkish family with sclerosteosis, we identified a missense mutation (c.499T>C; p.Cys167Arg) in exon 2 of the SOST gene. This type of mutation has not been previously reported and using different functional approaches, we show that it has a devastating effect on the biological function of sclerostin. The affected cysteine is the last cysteine residue of the cystine‐knot motif and loss of this residue leads to retention of the mutant protein in the ER, possibly as a consequence of impaired folding. Together with a significant reduced ability to bind to LRP5 and inhibit Wnt signaling, the p.Cys167Arg mutation leads to a complete loss of function of sclerostin and thus to the characteristic sclerosteosis phenotype.

Inhibition of Gsk3β in cartilage induces osteoarthritic features through activation of the canonical Wnt signaling pathway

OBJECTIVE In the past years, the canonical Wnt/β-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. In this pathway, glycogen synthase kinase-3β (GSK3β) down-regulates transduction of the canonical Wnt signal by promoting degradation of β-catenin. In this study we wanted to further investigate the role of Gsk3β in cartilage maintenance. DESIGN Therefore, we have treated chondrocytes ex vivo and in vivo with GIN, a selective GSK3β inhibitor. RESULTS In E17.5 fetal mouse metatarsals, GIN treatment resulted in loss of expression of cartilage markers and decreased chondrocyte proliferation from day 1 onward. Late (3 days) effects of GIN included cartilage matrix degradation and increased apoptosis. Prolonged (7 days) GIN treatment resulted in resorption of the metatarsal. These changes were confirmed by microarray analysis showing a decrease in expression of typical chondrocyte markers and induction of expression of proteinases involved in cartilage matrix degradation. An intra-articular injection of GIN in rat knee joints induced nuclear accumulation of β-catenin in chondrocytes 72 h later. Three intra-articular GIN injections with a 2 days interval were associated with surface fibrillation, a decrease in glycosaminoglycan expression and chondrocyte hypocellularity 6 weeks later. CONCLUSIONS These results suggest that, by down-regulating β-catenin, Gsk3β preserves the chondrocytic phenotype, and is involved in maintenance of the cartilage extracellular matrix. Short term β-catenin up-regulation in cartilage secondary to Gsk3β inhibition may be sufficient to induce osteoarthritis-like features in vivo.