Refine
Has Fulltext
- yes (17)
Is part of the Bibliography
- yes (17)
Year of publication
Document Type
- Journal article (17)
Language
- English (17)
Keywords
- Mesenchymal stem cells (2)
- Multiple myeloma (2)
- in-vitro (2)
- mechanotransduction (2)
- 2',7'-dichlorofluorescin (1)
- Angiopoietin-like 4 (1)
- Bioreaktor (1)
- Bisphosphonates (1)
- Bone disease (1)
- Breast cancer cells (1)
Institute
- Lehrstuhl für Orthopädie (17)
- Medizinische Klinik und Poliklinik II (4)
- Theodor-Boveri-Institut für Biowissenschaften (3)
- Lehrstuhl für Tissue Engineering und Regenerative Medizin (2)
- Fakultät für Mathematik und Informatik (1)
- Institut für Molekulare Infektionsbiologie (1)
- Institut für Organische Chemie (1)
- Institut für Pharmakologie und Toxikologie (1)
- Institut für Pharmazie und Lebensmittelchemie (1)
- Institut für Virologie und Immunbiologie (1)
EU-Project number / Contract (GA) number
- 241719 (1)
- EU-1650-0006 (1)
The KISS1 Receptor as an In Vivo Microenvironment Imaging Biomarker of Multiple Myeloma Bone Disease
(2016)
Multiple myeloma is one of the most common hematological diseases and is characterized by an aberrant proliferation of plasma cells within the bone marrow. As a result of crosstalk between cancer cells and the bone microenvironment, bone homeostasis is disrupted leading to osteolytic lesions and poor prognosis. Current diagnostic strategies for myeloma typically rely on detection of excess monoclonal immunoglobulins or light chains in the urine or serum. However, these strategies fail to localize the sites of malignancies. In this study we sought to identify novel biomarkers of myeloma bone disease which could target the malignant cells and/or the surrounding cells of the tumor microenvironment. From these studies, the KISS1 receptor (KISS1R), a G-protein-coupled receptor known to play a role in the regulation of endocrine functions, was identified as a target gene that was upregulated on mesenchymal stem cells (MSCs) and osteoprogenitor cells (OPCs) when co-cultured with myeloma cells. To determine the potential of this receptor as a biomarker, in vitro and in vivo studies were performed with the KISS1R ligand, kisspeptin, conjugated with a fluorescent dye. In vitro microscopy showed binding of fluorescently-labeled kisspeptin to both myeloma cells as well as MSCs under direct co-culture conditions. Next, conjugated kisspeptin was injected into immune-competent mice containing myeloma bone lesions. Tumor-burdened limbs showed increased peak fluorescence compared to contralateral controls. These data suggest the utility of the KISS1R as a novel biomarker for multiple myeloma, capable of targeting both tumor cells and host cells of the tumor microenvironment.
Objectives
The long head of the biceps (LHB) is often resected in shoulder surgery and could therefore serve as a cell source for tissue engineering approaches in the shoulder. However, whether it represents a suitable cell source for regenerative approaches, both in the inflamed and non-inflamed states, remains unclear. In the present study, inflamed and native human LHBs were comparatively characterized for features of regeneration.
Methods
In total, 22 resected LHB tendons were classified into inflamed samples (n = 11) and non-inflamed samples (n = 11). Proliferation potential and specific marker gene expression of primary LHB-derived cell cultures were analyzed. Multipotentiality, including osteogenic, adipogenic, chondrogenic, and tenogenic differentiation potential of both groups were compared under respective lineage-specific culture conditions.
Results
Inflammation does not seem to affect the proliferation rate of the isolated tendon-derived stem cells (TDSCs) and the tenogenic marker gene expression. Cells from both groups showed an equivalent osteogenic, adipogenic, chondrogenic and tenogenic differentiation potential in histology and real-time polymerase chain reaction (RT-PCR) analysis.
Conclusion
These results suggest that the LHB tendon might be a suitable cell source for regenerative approaches, both in inflamed and non-inflamed states. The LHB with and without tendinitis has been characterized as a novel source of TDSCs, which might facilitate treatment of degeneration and induction of regeneration in shoulder surgery.
The signal modelling framework JimenaE simulates dynamically Boolean networks. In contrast to SQUAD, there is systematic and not just heuristic calculation of all system states. These specific features are not present in CellNetAnalyzer and BoolNet. JimenaE is an expert extension of Jimena, with new optimized code, network conversion into different formats, rapid convergence both for system state calculation as well as for all three network centralities. It allows higher accuracy in determining network states and allows to dissect networks and identification of network control type and amount for each protein with high accuracy. Biological examples demonstrate this: (i) High plasticity of mesenchymal stromal cells for differentiation into chondrocytes, osteoblasts and adipocytes and differentiation-specific network control focusses on wnt-, TGF-beta and PPAR-gamma signaling. JimenaE allows to study individual proteins, removal or adding interactions (or autocrine loops) and accurately quantifies effects as well as number of system states. (ii) Dynamical modelling of cell–cell interactions of plant Arapidopsis thaliana against Pseudomonas syringae DC3000: We analyze for the first time the pathogen perspective and its interaction with the host. We next provide a detailed analysis on how plant hormonal regulation stimulates specific proteins and who and which protein has which type and amount of network control including a detailed heatmap of the A.thaliana response distinguishing between two states of the immune response. (iii) In an immune response network of dendritic cells confronted with Aspergillus fumigatus, JimenaE calculates now accurately the specific values for centralities and protein-specific network control including chemokine and pattern recognition receptors.
Background: Anti-resorptive bisphosphonates (BP) are used for the treatment of osteoporosis and bone metastases. Clinical studies indicated a benefit in survival and tumor relapse in subpopulations of breast cancer patients receiving zoledronic acid, thus stimulating the debate about its anti-tumor activity. Amino-bisphosphonates in nM concentrations inhibit farnesyl pyrophosphate synthase leading to accumulation of isopentenyl pyrophosphate (IPP) and the ATP/ pyrophosphate adduct ApppI, which induces apoptosis in osteoclasts. For anti-tumor effects μM concentrations are needed and a sensitizer for bisphosphonate effects would be beneficial in clinical anti-tumor applications. We hypothesized that enhancing intracellular pyrophosphate accumulation via inhibition of probenecid-sensitive channels and transporters would sensitize tumor cells for bisphosphonates anti-tumor efficacy.
Methods: MDA-MB-231, T47D and MCF-7 breast cancer cells were treated with BP (zoledronic acid, risedronate, ibandronate, alendronate) and the pyrophosphate channel inhibitors probenecid and novobiocin. We determined cell viability and caspase 3/7 activity (apoptosis), accumulation of IPP and ApppI, expression of ANKH, PANX1, ABCC1, SLC22A11, and the zoledronic acid target gene and tumor-suppressor KLF2.
Results: Treatment of MDA-MB-231 with BP induced caspase 3/7 activity, with zoledronic acid being the most effective. In MCF-7 and T47D either BP markedly suppressed cell viability with only minor effects on apoptosis. Co-treatment with probenecid enhanced BP effects on cell viability, IPP/ApppI accumulation as measurable in MCF-7 and T47D cells, caspase 3/7 activity and target gene expression. Novobiocin co-treatment of MDA-MB-231 yielded identical results on viability and apoptosis compared to probenecid, rendering SLC22A family members as candidate modulators of BP effects, whereas no such evidence was found for ANKH, ABCC1 and PANX1.
Conclusions: In summary, we demonstrate effects of various bisphosphonates on caspase 3/7 activity, cell viability and expression of tumor suppressor genes in breast cancer cells. Blocking probenecid- and novobiocin-sensitive channels and transporters enhances BP anti-tumor effects and renders SLC22A family members good candidates as BP modulators. Further studies will have to unravel if treatment with such BP-sensitizers translates into preclinical and clinical efficacy.
The canonical Wnt/beta-catenin pathway plays a key role in the regulation of bone remodeling in mice and humans. Two transmembrane proteins that are involved in decreasing the activity of this pathway by binding to extracellular antagonists, such as Dickkopf 1 (Dkk1), are the low-density lipoprotein receptor related protein 5 (Lrp5) and Kremen 2 (Krm2). Lrp 5 deficiency (Lrp5(-/-)) as well as osteoblast-specific overexpression of Krm2 in mice (Col1a1-Krm2) result in severe osteoporosis occurring at young age. In this study, we analyzed the influence of Lrp5 deficiency and osteoblast-specific overexpression of Krm2 on fracture healing in mice using flexible and semi-rigid fracture fixation. We demonstrated that fracture healing was highly impaired in both mouse genotypes, but that impairment was more severe in Col1a1-Krm2 than in Lrp5(-/-) mice and particularly evident in mice in which the more flexible fixation was used. Bone formation was more reduced in Col1a1-Krm2 than in Lrp5(-/-) mice, whereas osteoclast number was similarly increased in both genotypes in comparison with wild-type mice. Using microarray analysis we identified reduced expression of genes mainly involved in osteogenesis that seemed to be responsible for the observed stronger impairment of healing in Col1a1-Krm2 mice. In line with these findings, we detected decreased expression of sphingomyelin phosphodiesterase 3 (Smpd3) and less active beta-catenin in the calli of Col1a1-Krm2 mice. Since Krm2 seems to play a significant role in regulating bone formation during fracture healing, antagonizing KRM2 might be a therapeutic option to improve fracture healing under compromised conditions, such as osteoporosis.
Metabolic glycoengineering enables a directed modification of cell surfaces by introducing target molecules to surface proteins displaying new features. Biochemical pathways involving glycans differ in dependence on the cell type; therefore, this technique should be tailored for the best results. We characterized metabolic glycoengineering in telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) as a model for primary hMSC, to investigate its applicability in TERT-modified cell lines. The metabolic incorporation of N-azidoacetylmannosamine (Ac\(_4\)ManNAz) and N-alkyneacetylmannosamine (Ac\(_4\)ManNAl) into the glycocalyx as a first step in the glycoengineering process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency (osteogenic and adipogenic differentiation potential) was maintained under these adapted culture conditions. In the second step, glycoengineered cells were modified with fluorescent dyes using Cu-mediated click chemistry. In these analyses, the two mannose derivatives showed superior incorporation efficiencies compared to glucose and galactose isomers. In time-dependent experiments, the incorporation of Ac\(_4\)ManNAz was detectable for up to six days while Ac\(_4\)ManNAl-derived metabolites were absent after two days. Taken together, these findings demonstrate the successful metabolic glycoengineering of immortalized hMSC resulting in transient cell surface modifications, and thus present a useful model to address different scientific questions regarding glycosylation processes in skeletal precursors.
CCN family member 1 (CCN1), also known as cysteine-rich angiogenic inducer 61 (CYR61), belongs to the extracellular matrix-associated CCN protein family. The diverse functions of these proteins include regulation of cell migration, adhesion, proliferation, differentiation and survival/apoptosis, induction of angiogenesis and cellular senescence. Their functions are partly overlapping, largely non-redundant, cell-type specific, and depend on the local microenvironment. To elucidate the role of CCN1 in the crosstalk between stromal cells and myeloma cells, we performed co-culture experiments with primary mesenchymal stem cells (MSC) and the interleukin-6 (IL-6)-dependent myeloma cell line INA-6. Here we show that INA-6 cells display increased transcription and induction of splicing of intron-retaining CCN1 pre-mRNA when cultured in contact with MSC. Protein analyses confirmed that INA-6 cells co-cultured with MSC show increased levels of CCN1 protein consistent with the existence of a pre-mature stop codon in intron 1 that abolishes translation of unspliced mRNA. Addition of recombinant CCN1-Fc protein to INA-6 cells was also found to induce splicing of CCN1 pre-mRNA in a concentration-dependent manner. Only full length CCN1-Fc was able to induce mRNA splicing of all introns, whereas truncated recombinant isoforms lacking domain 4 failed to induce intron splicing. Blocking RGD-dependent integrins on INA-6 cells resulted in an inhibition of these splicing events. These findings expand knowledge on splicing of the proangiogenic, matricellular factor CCN1 in the tumor microenvironment. We propose that contact with MSC-derived CCN1 leads to splicing and enhanced transcription of CCN1 which further contributes to the translation of angiogenic factor CCN1 in myeloma cells, supporting tumor viability and myeloma bone disease.
Muscle and bone interact via physical forces and secreted osteokines and myokines. Physical forces are generated through gravity, locomotion, exercise, and external devices. Cells sense mechanical strain via adhesion molecules and translate it into biochemical responses, modulating the basic mechanisms of cellular biology such as lineage commitment, tissue formation, and maturation. This may result in the initiation of bone formation, muscle hypertrophy, and the enhanced production of extracellular matrix constituents, adhesion molecules, and cytoskeletal elements. Bone and muscle mass, resistance to strain, and the stiffness of matrix, cells, and tissues are enhanced, influencing fracture resistance and muscle power. This propagates a dynamic and continuous reciprocity of physicochemical interaction. Secreted growth and differentiation factors are important effectors of mutual interaction. The acute effects of exercise induce the secretion of exosomes with cargo molecules that are capable of mediating the endocrine effects between muscle, bone, and the organism. Long-term changes induce adaptations of the respective tissue secretome that maintain adequate homeostatic conditions. Lessons from unloading, microgravity, and disuse teach us that gratuitous tissue is removed or reorganized while immobility and inflammation trigger muscle and bone marrow fatty infiltration and propagate degenerative diseases such as sarcopenia and osteoporosis. Ongoing research will certainly find new therapeutic targets for prevention and treatment.
In situ guided tissue regeneration, also addressed as in situ tissue engineering or endogenous regeneration, has a great potential for population-wide “minimal invasive” applications. During the last two decades, tissue engineering has been developed with remarkable in vitro and preclinical success but still the number of applications in clinical routine is extremely small. Moreover, the vision of population-wide applications of ex vivo tissue engineered constructs based on cells, growth and differentiation factors and scaffolds, must probably be deemed unrealistic for economic and regulation-related issues. Hence, the progress made in this respect will be mostly applicable to a fraction of post-traumatic or post-surgery situations such as big tissue defects due to tumor manifestation. Minimally invasive procedures would probably qualify for a broader application and ideally would only require off the shelf standardized products without cells. Such products should mimic the microenvironment of regenerating tissues and make use of the endogenous tissue regeneration capacities. Functionally, the chemotaxis of regenerative cells, their amplification as a transient amplifying pool and their concerted differentiation and remodeling should be addressed. This is especially important because the main target populations for such applications are the elderly and diseased. The quality of regenerative cells is impaired in such organisms and high levels of inhibitors also interfere with regeneration and healing. In metabolic bone diseases like osteoporosis, it is already known that antagonists for inhibitors such as activin and sclerostin enhance bone formation. Implementing such strategies into applications for in situ guided tissue regeneration should greatly enhance the efficacy of tailored procedures in the future.
Aseptic loosening of total hip and knee joint replacements is the most common indication for revision surgery after primary hip and knee arthroplasty. Research suggests that exposure and uptake of wear by mesenchymal stromal cells (MSC) and macrophages results in the secretion of proinflammatory cytokines and local osteolysis, but also impaired cell viability and regenerative capacity of MSC. Therefore, this in vitro study compared the regenerative and differentiation capacity of MSC derived from patients undergoing primary total hip arthroplasty (MSCprim) to MSC derived from patients undergoing revision surgery after aseptic loosening of total hip and knee joint implants (MSCrev). Regenerative capacity was examined by measuring the cumulative population doubling (CPD) in addition to the number of passages until cells stopped proliferating. Osteogenesis and adipogenesis in monolayer cultures were assessed using histological stainings. Furthermore, RT‐PCR was performed to evaluate the relative expression of osteogenic and adipogenic marker genes as well as the expression of markers for a senescence‐associated secretory phenotype (SASP). MSCrev possessed a limited regenerative capacity in comparison to MSCprim. Interestingly, MSCrev also showed an impaired osteogenic and adipogenic differentiation capacity compared to MSCprim and displayed a SASP early after isolation. Whether this is the cause or the consequence of the aseptic loosening of total joint implants remains unclear. Future research should focus on the identification of specific cell markers on MSCprim, which may influence complication rates such as aseptic loosening of total joint arthroplasty to further individualize and optimize total joint arthroplasty.