Mineralizing Fibroblast-Colony-Forming Assays

Scutt, Andrew; Reading, L.; Scutt, Nanette; Still, Karen

doi:10.1385/1-59259-366-6:29

Cited by 13 publications

(12 citation statements)

References 18 publications

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“…To determine the effect of cinaciguat on osteoblast precursors, we used murine bone marrow stromal cells; when switched to osteoblastic differentiation medium, these cells develop into ALP-positive colonies as a measure of early osteoblast differentiation (31). We found that cinaciguat increased the area occupied by ALP-positive colonies derived from these cells, similar to the effect of 8-pCPT-cGMP, suggesting enhanced osteoblastic differentiation from mesenchymal stem cells ( Figure 1K).…”

Section: Cinaciguat Increases the Intracellular Cgmp Concentration Anmentioning

confidence: 64%

Soluble Guanylate Cyclase as a Novel Treatment Target for Osteoporosis

et al. 2014

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Osteoporosis is a major health problem leading to fractures that cause substantial morbidity and mortality. Current osteoporosis therapies have significant drawbacks, creating a need for novel bone-anabolic agents. We previously showed that the nitric oxide/cyclic GMP (cGMP)/protein kinase G pathway mediates some of the anabolic effects of estrogens and mechanical stimulation in osteoblasts and osteocytes, leading us to hypothesize that cGMP-elevating agents may have bone-protective effects. We tested cinaciguat, a prototype of a novel class of soluble guanylate cyclase activators, in a mouse model of estrogen deficiency-induced osteoporosis. Compared with sham-operated mice, ovariectomized mice had lower serum cGMP concentrations, which were largely restored to normal by treatment with cinaciguat or low-dose 17β-estradiol. Microcomputed tomography of tibiae showed that cinaciguat significantly improved trabecular bone microarchitecture in ovariectomized animals, with effect sizes similar to those obtained with estrogen replacement therapy. Cinaciguat reversed ovariectomy-induced osteocyte apoptosis as efficiently as estradiol and enhanced bone formation parameters in vivo, consistent with in vitro effects on osteoblast proliferation, differentiation, and survival. Compared with 17β-estradiol, which completely reversed the ovariectomy-induced increase in osteoclast number, cinaciguat had little effect on osteoclasts. Direct guanylate cyclase stimulators have been extremely well tolerated in clinical trials of cardiovascular diseases, and our findings provide proof-of-concept for this new class of drugs as a novel, anabolic treatment strategy for postmenopausal osteoporosis, confirming an important role of nitric oxide/cGMP/protein kinase G signaling in bone.

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Section: Cinaciguat Increases the Intracellular Cgmp Concentration Anmentioning

confidence: 64%

Soluble Guanylate Cyclase as a Novel Treatment Target for Osteoporosis

et al. 2014

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“…Each colony represents the clonal expansion of one mesenchymal cell; thus, the assay reflects the number of mesenchymal cells present in the original bone marrow cell suspension and their clonogenic potential. The number of alkaline phosphatase-positive (ALP-positive) colonies is proportional to the number of progenitors with osteoblastic potential within the mesenchymal cell population (36).…”

Section: Mice With Reduced Vegf Expression In Osteoblastic Lineage Cementioning

confidence: 99%

Intracellular VEGF regulates the balance between osteoblast and adipocyte differentiation

Li¹,

Berendsen²,

Jia³

et al. 2012

J. Clin. Invest.

316

283

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Osteoporotic bones have reduced spongy bone mass, altered bone architecture, and increased marrow fat. Bone marrow stem cells from osteoporotic patients are more likely to differentiate into adipocytes than control cells, suggesting that adipocyte differentiation may play a role in osteoporosis. VEGF is highly expressed in osteoblastic precursor cells and is known to stimulate bone formation. Here we tested the hypothesis that VEGF is also an important regulator of cell fate, determining whether differentiation gives rise to osteoblasts or adipocytes. Mice with conditional VEGF deficiency in osteoblastic precursor cells exhibited an osteoporosis-like phenotype characterized by reduced bone mass and increased bone marrow fat. In addition, reduced VEGF expression in mesenchymal stem cells resulted in reduced osteoblast and increased adipocyte differentiation. Osteoblast differentiation was reduced when VEGF receptor 1 or 2 was knocked down but was unaffected by treatment with recombinant VEGF or neutralizing antibodies against VEGF. Our results suggested that VEGF controls differentiation in mesenchymal stem cells by regulating the transcription factors RUNX2 and PPARγ2 as well as through a reciprocal interaction with nuclear envelope proteins lamin A/C. Importantly, our data support a model whereby VEGF regulates differentiation through an intracrine mechanism that is distinct from the role of secreted VEGF and its receptors.

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“…MCFU-f cultures assays were performed essentially as previously described by Scutt et al [31]. Nucleated bone marrow cells from three female rats were seeded in DMEM/10% fetal calf serum (FCS) (EuroClone, Devon, UK) with 10 nM dexamethasone (Sigma, Oslo, Norway), 50 μ g/mL ascorbic acid (Sigma), and 2.0 mM β -glycerophosphate, in 6 well-plates (1.0 × 10 6 cells/well).…”

Section: Methodsmentioning

confidence: 99%

Skeletal Effects of the Saturated 3-Thia Fatty Acid Tetradecylthioacetic Acid in Rats

et al. 2011

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This study explores the skeletal effects of the peroxisome proliferator activated receptor (PPAR)pan agonist tetradecylthioacetic acid (TTA). Rats, without (Study I) and with ovariectomy (OVX) or sham operation (Study II), were given TTA or vehicle daily for 4 months. Bone markers in plasma, whole body and femoral bone mineral density and content (BMD and BMC), and body composition were examined. Histomorphometric and biomechanical analyses (Study I) and biomechanical and μCT analyses (Study II) of the femur were performed. Normal rats fed TTA had higher femoral BMD and increased total and cortical area in femur compared to controls. The ovariectomized groups had decreased BMD and impaired microarchitecture parameters compared to SHAM. However, the TTA OVX group maintained femoral BMC, trabecular thickness in the femoral head, and cortical volume in the femoral metaphysis as SHAM. TTA might increase BMD and exert a light preventive effect on estrogen-related bone loss in rats.

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Mineralizing Fibroblast-Colony-Forming Assays

Cited by 13 publications

References 18 publications

Soluble Guanylate Cyclase as a Novel Treatment Target for Osteoporosis

Soluble Guanylate Cyclase as a Novel Treatment Target for Osteoporosis

Intracellular VEGF regulates the balance between osteoblast and adipocyte differentiation

Skeletal Effects of the Saturated 3-Thia Fatty Acid Tetradecylthioacetic Acid in Rats

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