Fluid shear stress remodels expression and function  of junctional proteins in cultured bone cells

Thi, Mia M.; Kojima, Takashi; Cowin, Stephen C.; Weinbaum, Sheldon; Spray, David C.

doi:10.1152/ajpcell.00052.2002

Cited by 89 publications

(76 citation statements)

References 64 publications

(83 reference statements)

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“…These results suggest that HSSGs play only a secondary role in the reorganization of actin filaments and their associated linker protein vinculin. Previous studies (17,21) have shown that ZO-1 and Cx43 expression is sensitive to fluid shear stress in USS and HSSG regions, because stable junctions are not able to form. Recent studies suggest that, in addition to organizing TJ proteins, such as occludin and ZO-2, and enabling Cx43 to dock at the cell surface, ZO-1 serves as a linker between TJ proteins and F-actin (39,40).…”

Section: Discussionmentioning

confidence: 93%

“…To detect the changes in stress fibers (SFs) and dense peripheral actin bands (DPABs) [F-actin rhodamine-labeled phalloidin (Sigma)], focal adhesions (FAs) [Vinculin and Paxillin (Chemicon)], and tight junction [TJ; ZO-1 (Zymed)] and gap junction complexes (Cx43, courtesy of E. Hertzberg, Albert Einstein College of Medicine), immunofluorescence analyses were performed on both controls and shear stress-exposed cells (21). Fifteen milliunits per milliliter of heparinase III (Sigma) was used to digest the glycocalyx (11), and HSPG antibody (US Biological, Swampscott, MA) and CellTracker orange (Molecular Probes) were used to assess heparan sulfate removal.…”

Section: Methodsmentioning

confidence: 99%

“…The flow loop consisted of a parallel plate flow chamber and a recirculating flow circuit, as described in ref. 21. Spatial variations of shear stress gradients in the entire chamber cross section were calculated by using an infinite series solution of the 2D steady Navier-Stokes equation.…”

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The role of the glycocalyx in reorganization of the actin cytoskeleton under fluid shear stress: A “bumper-car” model

Thi

Tarbell

Weinbaum

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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285

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We propose a conceptual model for the cytoskeletal organization of endothelial cells (ECs) based on a major dichotomy in structure and function at basal and apical aspects of the cells. Intracellular distributions of filamentous actin (F-actin), vinculin, paxillin, ZO-1, and Cx43 were analyzed from confocal micrographs of rat fat-pad ECs after 5 h of shear stress. With intact glycocalyx, there was severe disruption of the dense peripheral actin bands (DPABs) and migration of vinculin to cell borders under a uniform shear stress (10.5 dyne͞cm 2 ; 1 dyne ‫؍‬ 10 N). This behavior was augmented in corner flow regions of the flow chamber where high shear stress gradients were present. In striking contrast, no such reorganization was observed if the glycocalyx was compromised. These results are explained in terms of a ''bumper-car'' model, in which the actin cortical web and DPAB are only loosely connected to basal attachment sites, allowing for two distinct cellular signaling pathways in response to fluid shear stress, one transmitted by glycocalyx core proteins as a torque that acts on the actin cortical web (ACW) and DPAB, and the other emanating from focal adhesions and stress fibers at the basal and apical membranes of the cell. mechanotransduction ͉ actin cortical web ͉ dense peripheral actin band H emodynamic shearing stresses on endothelial cells (ECs) are widely recognized as playing a vital role in the regulation of vessel wall remodeling, cellular signaling, mass transport, red and white cell interaction, and atherogenesis (1-3). The possible roles of the endothelial glycocalyx (EG) in this regulation as a molecular sieve, as a barrier and modulator of interactions between blood cells and ECs, and as a mechanotransducer of fluid shear stress have been studied more recently (4-7). Relatively little is known about the specific proteins in the EG, although hyaluronan, chondroitin, and heparan sulfate play a significant role in its assembly (8, 9). In the early 1990s, investigators first observed that the shear-induced dilation of small arteries was abolished when sialic acids were removed from the EG by neuraminidase (10). Florian et al. (11) recently verified the presence of heparan sulfate proteoglycan (HSPG) in the glycocalyx of cultured bovine aortic ECs and demonstrated that partial removal of HSPG with heparinase completely blocked shear-induced NO release. A puzzling and still not understood consequence of EG degradation was the observation that shear-induced NO production was greatly inhibited without apparent effect on shear-dependent vasodilation due to prostaglandin I 2 release (12). Squire et al. (13) showed that the ultrastructural organization of the EG was quasiperiodic, anchored to a geodesic-like scaffold of hexagonally arranged filamentous actin (F-actin) filaments forming an actin cortical web (ACW) (14) just beneath the plasmalemma. A fundamental question addressed in ref. 7 is how fluid shear stresses acting at the surface of the EG are transmitted to this ACW if there is essentially ...

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Section: Discussionmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

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The role of the glycocalyx in reorganization of the actin cytoskeleton under fluid shear stress: A “bumper-car” model

Thi

Tarbell

Weinbaum

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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285

308

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“…Opening of membrane channels for the passage of signaling molecules initiated by pN-level forces is detectable by whole-cell patch-clamp electrophysiology with high temporal resolution. Previous culture studies have shown that networks of osteocytes respond to forces imposed by fluid shear stress and substrate stretching, as well as to cell indentation of a much higher magnitude, with the release of nucleotides, prostaglandin E2 (PGE2), and nitric oxide (NO) into the extracellular space and elevated intercellular Ca 2+ levels owing to receptor activation (20)(21)(22)(23)(24)(25). The osteocyte mechanotransducer is likely a protein complex that includes integrins, pannexin, or connexin hemichannels (21,(26)(27)(28)(29) in addition to ligand-gated receptors, particularly purinergic receptors (30,31) and hormone receptors (32).…”

Section: Discussionmentioning

confidence: 99%

Matrix-dependent adhesion mediates network responses to physiological stimulation of the osteocyte cell process

Schaffler

Weinbaum

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Osteocytes are bone cells that form cellular networks that sense mechanical loads distributed throughout the bone tissue. Interstitial fluid flow in the lacunar canalicular system produces focal strains at localized attachment sites around the osteocyte cell process. These regions of periodic attachment between the osteocyte cell membrane and its canalicular wall are sites where pN-level fluid-flow induced forces are generated in vivo. In this study, we show that focally applied forces of this magnitude using a newly developed Stokesian fluid stimulus probe initiate rapid and transient intercellular electrical signals in vitro. Our experiments demonstrate both direct gap junction coupling and extracellular purinergic P2 receptor signaling between MLO-Y4 cells in a connected bone cell network. Intercellular signaling was initiated by pN-level forces applied at integrin attachment sites along both appositional and distal unapposed cell processes, but not initiated at their cell bodies with equivalent forces. Electrical coupling was evident in 58% of all cell pairs tested with appositional connections; coupling strength increased with the increasing number of junctional connections. Apyrase, a nucleotidedegrading enzyme, suppressed and abolished force-induced effector responses, indicating a contribution from ATP released by the stimulated cell. This work extends the understanding of how osteocytes modulate their microenvironment in response to mechanical signals and highlights mechanisms of intercellular relay of mechanoresponsive signals in the bone network.cell signaling | electrophysiology | mechanotransduction | junctional conductance I ncreasing evidence indicates that bone homeostasis and osteogenic remodeling require mechanical loading of whole bone tissue (1-4). Interstitial fluid in bone passes through hierarchical levels of porosity when bone tissue is deformed by external loads. The interstitial fluid is the medium that translates endogenous whole tissue mechanical loads to cellular level forces in bone. Measuring the magnitude of endogenous forces acting on osteocytes in vivo has been a major challenge experimentally, but is of significance for investigating force-induced cell signals in vitro. Mathematical models of in vivo forces acting within the bone porosity of the lacunar-canalicular system (LCS) containing osteocytes have theoretically predicted forces between 1 and 10 pN acting on attachment sites along the osteocyte cell process membrane (5-7). These predictions guided the development of a Stokesian fluid stimulus probe (SFSP) used to focally stimulate osteocytes in vitro at in vivo-level forces (8). In this study, we examined the intercellular electrical signals induced by pN-level forces in interconnected MLO-Y4 bone cell networks, and measured the intercellular signaling through gap junction channels and through changes in nonjunctional conductance. Signaling through nonjunctional membranes likely involves ATP release and binding to purinergic receptors (P2Rs), as demonstrated by the partia...

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“…65 Many researcher have suggested that fluid flow induced shear stress is the primary mechanism of informing bone cells of loading conditions (osteocytes and osteoclasts more than osteoblasts). 66,67 Fluid flow also induces an electric field from movement of charged particles. Osteoclasts have been shown to migrate and act based on electrical charges caused by these fluid flows as well.…”

Section: Bone Mechanics and Mechanobiologymentioning

confidence: 99%

Bone tissue engineering: A review in bone biomimetics and drug delivery strategies

Porter

Ruckh

Popat

2009

Biotechnology Progress

672

499

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Critical‐sized defects in bone, whether induced by primary tumor resection, trauma, or selective surgery have in many cases presented insurmountable challenges to the current gold standard treatment for bone repair. The primary purpose of a tissue‐engineered scaffold is to use engineering principles to incite and promote the natural healing process of bone which does not occur in critical‐sized defects. A synthetic bone scaffold must be biocompatible, biodegradable to allow native tissue integration, and mimic the multidimensional hierarchical structure of native bone. In addition to being physically and chemically biomimetic, an ideal scaffold is capable of eluting bioactive molecules (e.g., BMPs, TGF‐βs, etc., to accelerate extracellular matrix production and tissue integration) or drugs (e.g., antibiotics, cisplatin, etc., to prevent undesired biological response such as sepsis or cancer recurrence) in a temporally and spatially controlled manner. Various biomaterials including ceramics, metals, polymers, and composites have been investigated for their potential as bone scaffold materials. However, due to their tunable physiochemical properties, biocompatibility, and controllable biodegradability, polymers have emerged as the principal material in bone tissue engineering. This article briefly reviews the physiological and anatomical characteristics of native bone, describes key technologies in mimicking the physical and chemical environment of bone using synthetic materials, and provides an overview of local drug delivery as it pertains to bone tissue engineering is included. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009

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Fluid shear stress remodels expression and function of junctional proteins in cultured bone cells

Cited by 89 publications

References 64 publications

The role of the glycocalyx in reorganization of the actin cytoskeleton under fluid shear stress: A “bumper-car” model

The role of the glycocalyx in reorganization of the actin cytoskeleton under fluid shear stress: A “bumper-car” model

Matrix-dependent adhesion mediates network responses to physiological stimulation of the osteocyte cell process

Bone tissue engineering: A review in bone biomimetics and drug delivery strategies

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