9 research outputs found
The Potential Role of Megakaryocytes in Mechanically Mediated Bone Adaptation.
Maintenance of bone mass and geometry is heavily dependent upon mechanical stimuli. Current paradigms suggest that osteocytes embedded within the mineralized matrix and osteoblasts on the bone surfaces are the primary responders to physical forces. However, other cells within the marrow cavity are also subject to a mechanically active environment. Megakaryocytes (MKs), cells which produce platelets, may physiologically be exposed to fluid shear forces. Recent studies have highlighted the potent effects MKs have on osteoblast proliferation as well as bone formation in vivo. We hypothesize that MKs are capable of responding to physical forces and that the interactions between these cells and osteoblasts can be influenced by mechanical stimulation.
We have demonstrated that two MK cell lines respond to fluid shear stress in culture. Furthermore, we isolated MKs from histologic sections of murine tibiae that were exposed to compressive loads in vivo using laser capture microdissection. C-fos, a transcription factor shown to be upregulated in response to load in various tissue types, was increased in MKs from loaded relative to non-loaded limbs at a level comparable to that of osteocytes from the same limbs.
To assess the functional outcomes of this mechanoresponsiveness, we first set out to determine whether animals with elevated numbers of MKs demonstrated altered adaptation to mechanical loading. GATA-1low mice, a transgenic mouse model with arrested MK maturation leading to an elevated number of immature MKs within the marrow, were shown to have a minimally altered response to load compared to wild-type littermates. Mice injected with thrombopoietin, a potent inducer of MK proliferation and differentiation, showed no difference in response to load compared with vehicle-injected mice.
Finally, we developed a co-culture system to address whether mechanical stimulation of MKs in culture would impact osteoblast proliferation and differentiation. The presence of MKs in culture, but not conditioned media, has dramatic effects on osteoblast proliferation. Our data suggests a minimal, but non-significant, decrease in proliferation as well as an increase in mineralization capacity of osteoblasts co-cultured with MKs exposed to shear compared to co-cultures with unstimulated MKs. Further studies are necessary to investigate the mechanism driving this phenomenon.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/75996/1/cpagedas_1.pd
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Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line
The role of mechanosensitive (MS) Ca2+-permeable ion channels in platelets is unclear, despite the importance of shear stress in platelet function. We sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca2+ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to microscope slides within flow chambers that allowed application of physiological and pathological shear stress. Arterial shear (1002.6s-1) induced a sustained increase in intracellular calcium ([Ca2+]i) in Meg-01 cells and enhanced the frequency of repetitive Ca2+ transients by 80% in platelets. These Ca2+ increases were abrogated by the MS channel inhibitor GsMTx-4 or by chelation of extracellular Ca2+. Thrombus formation was studied on collagen-coated surfaces using 3,3'-dihexyloxacarbocyanine iodide (DiOC6)-stained platelets. In addition, [Ca2+]i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4 independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation and on Ca2+ influx via TRPC6 or Orai1 channels, and caused only a minor inhibition of P2X1-dependent Ca2+ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca2+ transients by 170%. Piezo1 mRNA transcripts and protein were detected in both platelets and Meg-01 cells using qRT-PCR and Western blotting. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca2+ entry and thrombus formation under arterial shear stress
miR-33-5p, a novel mechano-sensitive microRNA promotes osteoblast differentiation by targeting Hmga2
Bone mechanobiology in mice: toward single-cell in vivo mechanomics
ISSN:1617-7959ISSN:1617-794