scholarly journals Dynamics and mechanisms of intracellular calcium waves elicited by tandem bubble-induced jetting flow

2017 ◽  
Vol 115 (3) ◽  
pp. E353-E362 ◽  
Author(s):  
Fenfang Li ◽  
Chen Yang ◽  
Fang Yuan ◽  
Defei Liao ◽  
Thomas Li ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Intracellular Calcium ◽  
Cell Injury ◽  
Calcium Influx ◽  
Reaction Diffusion ◽  
Microfluidic System ◽  
Calcium Waves ◽  
Flow Shear ◽  
Cellular Mechanotransduction ◽  
Flow Shear Stress

One of the earliest events in cellular mechanotransduction is often an increase in intracellular calcium concentration associated with intracellular calcium waves (ICWs) in various physiologic or pathophysiologic processes. Although cavitation-induced calcium responses are believed to be important for modulating downstream bioeffects such as cell injury and mechanotransduction in ultrasound therapy, the fundamental mechanisms of these responses have not been elucidated. In this study, we investigated mechanistically the ICWs elicited in single HeLa cells by the tandem bubble-induced jetting flow in a microfluidic system. We identified two distinct (fast and slow) types of ICWs at varying degrees of flow shear stress-induced membrane deformation, as determined by different bubble standoff distances. We showed that ICWs were initiated by an extracellular calcium influx across the cell membrane nearest to the jetting flow, either primarily through poration sites for fast ICWs or opening of mechanosensitive ion channels for slow ICWs, which then propagated in the cytosol via a reaction−diffusion process from the endoplasmic reticulum. The speed of ICW (CICW) was found to correlate strongly with the severity of cell injury, with CICW in the range of 33 μm/s to 93 μm/s for fast ICWs and 1.4 μm/s to 12 μm/s for slow ICWs. Finally, we demonstrated that micrometer-sized beads attached to the cell membrane integrin could trigger ICWs under mild cavitation conditions without collateral injury. The relation between the characteristics of ICW and cell injury, and potential strategies to mitigate cavitation-induced injury while evoking an intracellular calcium response, may be particularly useful for exploiting ultrasound-stimulated mechanotransduction applications in the future.

Proteolytic Cleavage of Endothelial Cell-Bound Von Willebrand Factor Polymers by ADAMTS13 in the Absence of Flow Shear Stress

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3913-3913
Author(s):  
Shengyu Jin ◽  
Christopher G. Skipwith ◽  
Dezhi Shang ◽  
Chandrasekaran Nagaswami ◽  
John W. Weisel ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Cell Membrane ◽  
Cell Surface ◽  
Conditioned Medium ◽  
Function Analysis ◽  
Coagulation Factor ◽  
Proteolytic Cleavage ◽  
Flow Shear ◽  
Flow Shear Stress

Abstract Degradation of newly released unusually large (UL)-vWF on endothelial cells by plasma ADAMTS13 metalloprotease is considered to be critical for maintaining normal hemostasis. An inability to degrade the cell bound string-like UL-vWF multimers by ADAMTS13 may result in excessive platelet adhesion and aggregation on cell surface, leading to thrombotic thrombocytopenic purpura (TTP). However, the requirement for shear stress and potential cofactors in this process is not fully defined. By immunofluorescent microscopy, we showed that upon histamine stimulation the newly released UL-vWF rapidly formed strings and bundles on human umbilical vein endothelial cells (HUVECs). These strings and bundles were removed within 2~5 minutes by plasma-derived and recombinant ADAMTS13 at concentrations of between 2.5 and 10 nM in 20 mM HEPES buffer, pH 7.5 containing 150 mM NaCl and 1 mM CaCl. This process did not appear to depend upon flow shear stress and coagulation factor VIII that has been shown to markedly increase the proteolytic cleavage of soluble vWF under high shear stress. The removal of the cell-bound UL-vWF strings and bundles correlated with the increases of vWF antigen and proteolytic cleavage fragments (176K and 140K) in the conditioned medium as determined by an enzyme-linked immunoassay and Western blot, respectively. The proteolytic cleavage of the cell bound UL-vWF by ADAMTS13 was time- and concentration-dependent, with a half maximal concentration of ADAMTS13 of approximately 5 nM. No cleavage product was detected when the histamine-stimulated HUVECs were incubated with the buffer alone or plasma from patients with acquired TTP and inhibitors or with heat-inactivated recombinant ADAMTS13. These results suggest that the degradation of cell bound UL-vWF is specific for ADAMTS13. Unexpectedly, the vWF multimers in the conditioned medium of ADAMTS13-treated endothelial cells were quite similar in sizes to those in the endothelial cells treated with histamine in the absence of ADAMTS13, indicating that the proteolytic cleavage of UL-vWF by ADASMTS13 occurs at the site proximal to cell membrane. Scanning electron microscopy clearly demonstrated that the breaking point of UL-vWF strings and bundles by ADAMTS13 was located at approximately 2~5 μm from the cell membrane. Structure-function analysis further demonstrated that the Cys-rich and spacer domains of ADAMTS13 were required for proteolytic cleavage of the cell-bound UL-vWF. The more distal carboxyl-terminal domains including TSP1 2–8 and CUB domains of ADAMTS13 appeared to be dispensable. We conclude that the cell bound UL-vWF polymers adopt an open conformation that is sensitive to ADAMTS13 proteolysis, analogous to those observed under the denaturing conditions. We hypothesize that membrane binding and/or interaction of UL-vWF bundles with certain scaffold proteins may provide an access of ADAMTS13 metalloprotease to the cleavage (Tyr1605-Met1606) bond at the central A2 domain of the vWF proximal to cell surface. These findings provide novel insight into the biological function of ADAMTS13 in processing UL-vWF and help understand the pathogenesis of TTP.

scholarly journals Mechanotransduction in the Cardiovascular System: From Developmental Origins to Homeostasis and Pathology

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1607 ◽  
Author(s):  
Gloria Garoffolo ◽  
Maurizio Pesce
Keyword(s):  
Cardiovascular System ◽  
Mechanical Forces ◽  
Mechanical Stimuli ◽  
Flow Shear ◽  
Surrounding Matrix ◽  
Cellular Mechanotransduction ◽  
Flow Shear Stress ◽  
Sense And Respond ◽  
Metabolic Dysfunctions

With the term ‘mechanotransduction’, it is intended the ability of cells to sense and respond to mechanical forces by activating intracellular signal transduction pathways and the relative phenotypic adaptation. While a known role of mechanical stimuli has been acknowledged for developmental biology processes and morphogenesis in various organs, the response of cells to mechanical cues is now also emerging as a major pathophysiology determinant. Cells of the cardiovascular system are typically exposed to a variety of mechanical stimuli ranging from compression to strain and flow (shear) stress. In addition, these cells can also translate subtle changes in biophysical characteristics of the surrounding matrix, such as the stiffness, into intracellular activation cascades with consequent evolution toward pro-inflammatory/pro-fibrotic phenotypes. Since cellular mechanotransduction has a potential readout on long-lasting modifications of the chromatin, exposure of the cells to mechanically altered environments may have similar persisting consequences to those of metabolic dysfunctions or chronic inflammation. In the present review, we highlight the roles of mechanical forces on the control of cardiovascular formation during embryogenesis, and in the development and pathogenesis of the cardiovascular system.

Presence of P2X1 Purinoceptors in Human Platelets and Megakaryoblastic Cell Lines

1997 ◽  
Vol 78 (06) ◽  
pp. 1500-1504 ◽  
Author(s):  
Catherine Vial ◽  
Béatrice Hechier ◽  
Catherine Léon ◽  
Jean-Pierre Cazenave ◽  
Christian Gachet
Keyword(s):  
Intracellular Calcium ◽  
G Proteins ◽  
Cell Lines ◽  
Calcium Influx ◽  
Human Platelets ◽  
P2y1 Receptor ◽  
Calcium Response ◽  
Ionotropic Receptors ◽  
External Calcium

SummaryHuman platelets are thought to possess at least two subtypes of purinoceptor, one of which, coupled to G-proteins, could be the P2Y1 receptor (Léon et al. 1997). However, it has been suggested that the unique rapid calcium influx induced by ADP in platelets could involve P2X1 ionotropic receptors (MacKenzie et al. 1996) and the aim of this study was thus to investigate the presence of P2X purinoceptors in platelets and megakaryoblastic cells. Using PCR experiments, we found P2X1 mRNA to be present in human platelets and megakaryoblastic cell lines. In platelets, the selective P2X1 agonist αβMeATP induced a rise in intracellular calcium only in the presence of external calcium and this effect was antagonized by suramin and PPADS. Repeated addition of a�MeATP desensitized the P2X1 purinoceptor but only slightly affected the ADP response, while no calcium response to αβMeATP was observed in megakaryoblastic cells. These results support the existence of functional P2X1 purinoceptors on human platelets and the presence of P2X1 transcripts in megakaryoblastic cell lines.

scholarly journals The Effect of Fluid Flow Shear Stress and Substrate Stiffness on Yes-Associated Protein (YAP) Activity and Osteogenesis in Murine Osteosarcoma Cells

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3128
Author(s):  
Thomas R. Coughlin ◽  
Ali Sana ◽  
Kevin Voss ◽  
Abhilash Gadi ◽  
Upal Basu-Roy ◽  
...  
Keyword(s):  
Shear Stress ◽  
Fluid Flow ◽  
Bone Cells ◽  
Bone Cancer ◽  
Substrate Stiffness ◽  
Flow Shear ◽  
Fluid Movement ◽  
Flow Shear Stress ◽  
Fluid Flow Shear Stress ◽  
New Treatments

Osteosarcoma (OS) is an aggressive bone cancer originating in the mesenchymal lineage. Prognosis for metastatic disease is poor, with a mortality rate of approximately 40%; OS is an aggressive disease for which new treatments are needed. All bone cells are sensitive to their mechanical/physical surroundings and changes in these surroundings can affect their behavior. However, it is not well understood how OS cells specifically respond to fluid movement, or substrate stiffness—two stimuli of relevance in the tumor microenvironment. We used cells from spontaneous OS tumors in a mouse engineered to have a bone-specific knockout of pRb-1 and p53 in the osteoblast lineage. We silenced Sox2 (which regulates YAP) and tested the effect of fluid flow shear stress (FFSS) and substrate stiffness on YAP expression/activity—which was significantly reduced by loss of Sox2, but that effect was reversed by FFSS but not by substrate stiffness. Osteogenic gene expression was also reduced in the absence of Sox2 but again this was reversed by FFSS and remained largely unaffected by substrate stiffness. Thus we described the effect of two distinct stimuli on the mechanosensory and osteogenic profiles of OS cells. Taken together, these data suggest that modulation of fluid movement through, or stiffness levels within, OS tumors could represent a novel consideration in the development of new treatments to prevent their progression.

Temporal gradient in shear-induced signaling pathway: involvement of MAP kinase, c-fos, and connexin43

2000 ◽  
Vol 278 (5) ◽  
pp. H1598-H1605 ◽  
Author(s):  
Xuping Bao ◽  
Craig B. Clark ◽  
John A. Frangos
Keyword(s):  
Shear Stress ◽  
Mrna Expression ◽  
Signaling Pathway ◽  
Steady Shear ◽  
Flow Shear ◽  
Temporal Gradient ◽  
Step Flow ◽  
Shear Component ◽  
Flow Shear Stress ◽  
Impulse Flow

The effect of a temporal gradient in shear and steady shear on the activity of extracellular signal-regulated protein kinases 1 and 2 (ERK1/ERK2), c- fos, and connexin43 (Cx43) in human endothelial cells was investigated. Three laminar flow profiles (16 dyn/cm2), including impulse flow (shear stress abruptly applied for 3 s), ramp flow (shear stress smoothly transitioned at flow onset), and step flow (shear stress abruptly applied at flow onset) were utilized. Relative to static controls, impulse flow stimulated the phosphorylation of ERK1/ERK2 8.5- to 7.5-fold, respectively at 10 min, as well as the mRNA expression of c- fos 51-fold at 30 min, and Cx43 8-fold at 90 min. These high levels of mRNA expression were sustained for at least 4 h. In contrast, ramp flow was unable to significantly induce gene expression and even inhibited the activation of ERK1/ERK2. Step flow, which contains both a sharp temporal gradient in shear stress and a steady shear component, elicited only moderate and transient responses, indicating the distinct role of these fluid shear stimuli in endothelial signal transduction. The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 inhibited impulse flow-induced c -fos and Cx43 mRNA expression. Thus these findings implicate the involvement of ERK1/ERK2, c -fos, and Cx43 in the signaling pathway induced by the temporal gradient in shear.

scholarly journals Mechanosensitive calcium signaling promotes epithelial tight junction remodeling by activating RhoA

2021 ◽  
Author(s):  
Saranyaraajan Varadarajan ◽  
Rachel E. Stephenson ◽  
Eileen R. Misterovich ◽  
Jessica L. Wu ◽  
Ivan S. Erofeev ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Intracellular Calcium ◽  
Tight Junctions ◽  
Calcium Influx ◽  
Cell Junctions ◽  
Mechanical Stimuli ◽  
Local Repair ◽  
Transient Activation ◽  
Epithelial Tight Junction ◽  
Sense And Respond

Epithelia maintain an effective barrier by remodeling cell-cell junctions in response to mechanical stimuli. Cells often respond to mechanical stress through activating RhoA and remodeling actomyosin. Previously, we found that local leaks in the barrier are rapidly repaired by localized, transient activation of RhoA – ″Rho flares″ – but how Rho flares are initiated remains unknown. Here, we discovered that intracellular calcium flashes occur in Xenopus laevis epithelial cells undergoing Rho flare-mediated remodeling of tight junctions. Calcium flashes originate at the site of barrier leaks and propagate into the cell. Depletion of intracellular calcium or inhibition of mechanosensitive calcium channels (MSC) reduced the amplitude of calcium flashes and diminished the activation of Rho flares. Furthermore, MSC-dependent calcium influx was necessary to maintain global barrier function by regulating local repair of tight junctions through efficient junction contraction. We propose that MSC-dependent calcium flashes are an important mechanism allowing epithelial cells to sense and respond to local leaks induced by mechanical stimuli.

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