Fluid Shear Stress Stimulation Research Articles

Zinc finger protein 36 like 2-histone deacetylase 1 axis is involved in the bone responses to mechanical stress

The molecular mechanism underlying the promotion of fracture healing by mechanical stimuli remains unclear. The present study aimed to investigate the role of zinc finger protein 36 like 2 (ZFP36L2)-histone deacetylase 1 (HDAC1) axis on the osteogenic responses to moderate mechanical stimulation. Appropriate stimulation of fluid shear stress (FSS) was performed on MC3T3-E1 cells transduced with ZFP36L2 and HDAC1 recombinant adenoviruses, aiming to validate the influence of mechanical stress on the expression of ZFP36L2-HDAC1 and the osteogenic differentiation and mineralization. The results showed that moderate FSS stimulation significantly upregulated the expression of ZFP36L2 in MC3T3-E1 cells (p < 0.01). The overexpression of ZFP36L1 markedly enhanced the levels of osteogenic differentiation markers, including bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), Osterix, and collagen type I alpha 1 (COL1A1) (p < 0.01). ZFP36L2 accelerated the degradation of HDAC1 by specifically binding to its 3’ UTR region, thereby fulfilling its function at the post-transcriptional regulatory gene level and promoting the osteogenic differentiation and mineralization fate of cells. Mechanical unloading notably diminished/elevated the expression of ZFP36L2/HDAC1, decreased bone mineral density and bone volume fraction, hindered the release of osteogenic-related factors and vascular endothelial growth factor in callus tissue (p < 0.01), and was detrimental to fracture healing. Collectively, proper stress stimulation plays a crucial role in facilitating osteogenesis through the promotion of ZFP36L2 and subsequent degradation of HDAC1. Targeting ZFP36L2-HDAC1 axis may provide promising insights to enhance bone defect healing.

Fluid shear force and hydrostatic pressure jointly promote osteogenic differentiation of BMSCs by activating YAP1 and NFAT2.

Natural bone tissue features a complex mechanical environment, with cells responding to diverse mechanical stimuli, including fluid shear stress (FSS) and hydrostatic pressure (HP). However, current in vitro experiments commonly employ a singular mechanical stimulus to simulate the mechanical environment in vivo. The understanding of the combined effects and mechanisms of multiple mechanical stimuli remains limited. Hence, this study constructed a mechanical stimulation device capable of simultaneously applying FSS and HP to cells. This study investigated the impact of FSS and HP on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and examined the distinctions and interactions between the two mechanisms. The results demonstrated that both FSS and HP individually enhanced the osteogenic differentiation of BMSCs, with a more pronounced effect observed through their combined application. BMSCs responded to external FSS and HP stimulation through the integrin-cytoskeleton and Piezo1 ion channel respectively. This led to the activation of downstream biochemical signals, resulting in the dephosphorylation and nuclear translocation of the intracellular transcription factors Yes Associated Protein 1 (YAP1) and nuclear factor of activated T cells 2 (NFAT2). Activated YAP1 could bind to NFAT2 to enhance transcriptional activity, thereby promoting osteogenic differentiation of BMSCs more effectively. This study highlights the significance of composite mechanical stimulation in BMSCs' osteogenic differentiation, offering guidance for establishing a complex mechanical environment for in vitro functional bone tissue construction.

Effect of Hydrogel Matrix and Fluid Shear Stress on the Behavioral Regulation of Mesenchymal Stem Cells

Development of a micromodel that recapitulates multiple mechanical properties to mimic the complex mechanical microenvironment is crucial for cell‐based research. Herein, a microsystem combining structure of hydrogel matrix and acoustic streaming (AS) to mimic the cellular microenvironment is proposed, which can realize multiplex cellular mechanical cues, including matrix stiffness, fluid shear stress (FSS) generated by AS, and matrix roughness. The results demonstrate that the cell spreading area enlarges with the increase of matrix stiffness, and cell spreading is encouraged by integrin β1 cluster to polymerize actin when combines the hydrogel matrix with FSS. In addition, FSS has the influence on the roughness of the hydrogel, which further affects the cell morphology and mechanical properties, inducing mesenchymal stem cells (MSCs) differentiation into astrocytes rapidly. Meanwhile, cell migration is also enhanced by FSS stimulation, particularly, undifferentiated cells at 22 kPa hydrogel have the fastest migration speed, and change the movement model from contact inhibition to contact stimulation migration. Especially, matrix roughness and stiffness dominantly control of cell spreading and differentiation, whereas FSS affects cell migration. In conclusion, the microsystem in this work shows superior performance in regulating the spreading, differentiation, and migration of MSCs, which provides a new tool for cell‐microenvironment study.

Activation of Dendritic Cells Isolated from the Blood of Patients with Prostate Cancer by Ex Vivo Fluid Shear Stress Stimulation.

Prostate cancer is one of the most common cancers among men in the United States and a leading cause of cancer-related death in men. Treatment options for patients with advanced prostate cancer include hormone therapies, chemotherapies, radioligand therapies, and immunotherapies. Provenge (sipuleucel-T) is an autologous cancer-vaccine-based immunotherapy approved for men with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). Administration of sipuleucel-T involves leukapheresis of patient blood to isolate antigen-presenting cells (APCs), including dendritic cells (DCs), and subsequent incubation of isolated APCs with both an antigen, prostatic acid phosphatase (PAP), and granulocyte macrophage-colony stimulating factor (GM-CSF) before their infusion back into the patient. Although sipuleucel-T has been shown to improve overall survival, other meaningful outcomes, such as prostate-specific antigen (PSA) levels and radiographic response, are inconsistent. This lack of robust response may be due to limited ex vivo activation of DCs using current protocols. Earlier studies have shown that many cell types can be activated ex vivo by external forces such as fluid shear stress (FSS). We hypothesize that novel fluid shear stress technologies and methods can be used to improve ex vivo efficacy of prostate cancer DC activation in prostate cancer. Herein, we report a new protocol for activating DCs from patients with prostate cancer using ex vivo fluid shear stress. Ultimately, the goal of these studies is to improve DC activation to expand the efficacy of therapies such as sipuleucel-T. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample collection and DC isolation Basic Protocol 2: Determination and application of fluid shear stress Basic Protocol 3: Flow cytometry analysis of DCs after FSS stimulation.

Piezo Mediates the Mechanosensation and Injury-Repair of Pulpo-Dentinal Complex

ObjectivesThe aim of this research was to investigate the functions of Piezo channels in dentin defect, including mechanical signalling and odontoblast responses. MethodsRat dentin-defect models were constructed, and spatiotemporal expression of Piezo proteins was detected in the pulpo-dentinal complex. Real-time polymerase chain reaction (rtPCR) was used to investigate the functional expression pattern of Piezo channels in odontoblasts. Moreover, RNA interference technology was employed to uncover the underlying mechanisms of the Piezo-driven inflammatory response and repair under fluid shear stress (FSS) conditions in vitro. ResultsPiezo1 and Piezo2 were found to be widely expressed in the odontoblast layer and dental pulp in the rat dentin-defect model during the end stage of reparative dentin formation. The expression levels of the Piezo1 and Piezo2 genes in MDPC-23 cells were high in the initial stage under FSS loading and then decreased over time. Moreover, the expression trends of inflammatory, odontogenic, and mineralisation genes were generally contrary to those of Piezo1 and Piezo2 over time. After silencing of Piezo1/Piezo2, FSS stimulation resulted in significantly higher expression of inflammatory, odontogenesis, and mineralisation genes in MDPC-23 cells. Finally, the expression of genes involved in the integrin β1/ERK1 and Wnt5b/β-catenin signalling pathways was changed in response to RNA silencing of Piezo1 and Piezo2. ConclusionsPiezo1 and Piezo2 may be involved in regulating the expression of inflammatory and odontogenic genes in odontoblasts stimulated by FSS.

Knockdown of TRPV2 inhibits the migration of RAW264.7 cells toward low fluid shear stress region.

Our previous studies have demonstrated that macrophages (RAW264.7) have a special ability for sensing the gradient of fluid shear stress (FSS) and migrate toward the low-FSS region. However, the molecular mechanism regulating this phenomenon is still unclear. In this study, we examined the transcriptome genes in RAW264.7 cells, MC3T3-E1 osteoblasts, mesenchymal stem cells, canine renal epithelial cells, and periodontal ligament cells. The expression levels of genes related to cell migration, force transfer, and force sensitivity in the Ca2+ signaling pathway were analyzed. We observed that the transient receptor potential cation channel type 2 (TRPV2) was highly expressed in RAW264.7 cells. Furthermore, we used lentiviral transfection to knockdown TRPV2 expression in RAW264.7 cells and studied the effect of TRPV2 on the migration of RAW264.7 cells under a gradient FSS field. The results showed that compared with normal cells, TRPV2-knockdown cells had impaired ability for sensing FSS gradient to migrate toward the low-FSS region and lower intracellular calcium response to FSS stimulation. This study may reveal the molecular mechanism of regulating the directional migration of macrophages under a gradient FSS field.

Assessing effects of mechanical stimulation of fluid shear stress on inducing matrix Metalloproteinase-9 in cultured corneal epithelial cells

Blinking is regarded as mechanical stimulation of fluid shear stress on the corneal epithelial cells. Therefore, we evaluated whether fluid shear stress affects matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in cultured human corneal epithelial cells (HCECs). No other study has shown the influence of fluid shear stress on HCECs regarding mRNA expression and the protein levels of MMPs. Cultured HCECs were exposed to shear stress (0, 1.2, 12 dyne/cm2) for 12 and 24 h with the parallel-plate type of flow chamber. Gene expression of MMPs and TIMPs was measured by real-time polymerase reaction. Concentrations of MMP-1 and MMP-9 in cell lysates were determined using bead-based amplified luminescent proximity homogenous assay-linked immunosorbent assay. The expression of MMP-9 and MMP-1 in HCECs exposed to low and high flow for 12 and 24 h, respectively, increased significantly compared with those under static conditions. The expression of MMP-9 in the cells exposed to high flow for 24 h increased significantly compared with those under static and low flow conditions. Levels of MMP-9 in cell lysates exposed to fluid flow for 24 h were elevated significantly with increasing shear stress. Fluid shear stress exerted on HCECs affected MMPs, which was associated with inflammation and pathogenesis. Mechanical stress induced by blinking might influence expression of MMPs on the ocular surface. Further studies are warranted to establish the molecular mechanism of shear stress-induced alternations of MMPs.

Fluid shear stress and endothelial cells synergistically promote osteogenesis of mesenchymal stem cells via integrin β1-FAK-ERK1/2 pathway.

Prevascularization and mechanical stimulation have been reported as effective methods for the construction of functional bone tissue. However, their combined effects on osteogenic differentiation and its mechanism remain to be explored. Here, the effects of fluid shear stress (FSS) on osteogenic differentiation of rat bone-marrow-derived mesenchymal stem cells (BMSCs) when cocultured with human umbilical vein endothelial cells (HUVECs) were investigated, and underlying signaling mechanisms were further explored. FSS stimulation for 1–4 h/day increased alkaline phosphatase (ALP) activity and calcium deposition in coculture systems and promoted the proliferation of cocultured cells. FSS stimulation for 2 h/day was selected as the optimized protocol according to osteogenesis in the coculture. In this situation, the mRNA levels of ALP, runt-related transcriptional factor 2 (Runx2) and osteocalcin (OCN), and protein levels of OCN and osteopontin (OPN) in BMSCs were upregulated. Furthermore, FSS and coculture with HUVECs synergistically increased integrin β1 expression in BMSCs and further activated focal adhesion kinases (FAKs) and downstream extracellular signal-related kinase (ERK), leading to the enhancement of Runx2 expression. Blocking the phosphorylation of FAK abrogated FSS-induced ERK phosphorylation and inhibited osteogenesis of cocultured BMSCs. These results revealed that FSS and coculture with HUVECs synergistically promotes the osteogenesis of BMSCs, which was mediated by the integrin β1-FAK-ERK signaling pathway.

Effects of compound stimulation of fluid shear stress plus ultrasound on stem cell proliferation and osteogenesis.

Bone tissue with strong adaptability is often in a complex dynamical microenvironment in vivo, which is associated with the pathogenesis and treatment of orthopedic diseases. Therefore, it is of great significance to investigate the effects of corresponding compound stimulation on cell behaviors. Herein, a fluid shear stress (FSS) plus ultrasound stimulation platform suitable for cell studies based on a microfluidic chip was constructed and bone marrow mesenchymal stem cell (BMSC) was chosen as a model cell. The proliferation and osteogenesis of BMSCs under the compound stimulation of FSS plus ultrasound in growth medium without any soluble induction factors were firstly investigated. Single FSS stimulation and static culture conditions were also examined. Results illustrated that suitable single FSS stimulation (about 0.06 dyn/cm2) could significantly enhance cell proliferation and osteogenesis simultaneously when compared to the static control, while greater FSS mitigated or even restricted these enhancing effects. Interestingly, ultrasound stimulation combined with this suitable FSS stimulation further accelerated cell proliferation as the intensity of ultrasound increasing. As for the osteogenesis under compound stimulation, it was relatively restricted under lower ultrasound intensity (about 0.075 W/cm2), while promoted when the intensity became higher (about 1.75 W/cm2). This study suggests that both the cell proliferation and osteogenesis are very responsive to the magnitudes of FSS and ultrasound stimulations and can be both significantly enhanced by proper combination strategies. Moreover, these findings will provide valuable references for the construction of effective cell bioreactors and also the treatment of orthopedic diseases.

Fluid shear stress generates a unique signaling response by activating multiple TGFβ family type I receptors in osteocytes.

Bone is a dynamic tissue that constantly adapts to changing mechanical demands. The transforming growth factor beta (TGFβ) signaling pathway plays several important roles in maintaining skeletal homeostasis by both coupling the bone‐forming and bone‐resorbing activities of osteoblasts and osteoclasts and by playing a causal role in the anabolic response of bone to applied loads. However, the extent to which the TGFβ signaling pathway in osteocytes is directly regulated by fluid shear stress (FSS) is unknown, despite work suggesting that fluid flow along canaliculi is a dominant physical cue sensed by osteocytes following bone compression. To investigate the effects of FSS on TGFβ signaling in osteocytes, we stimulated osteocytic OCY454 cells cultured within a microfluidic platform with FSS. We find that FSS rapidly upregulates Smad2/3 phosphorylation and TGFβ target gene expression, even in the absence of added TGFβ. Indeed, relative to treatment with TGFβ, FSS induced a larger increase in levels of pSmad2/3 and Serpine1 that persisted even in the presence of a TGFβ receptor type I inhibitor. Our results show that FSS stimulation rapidly induces phosphorylation of multiple TGFβ family R‐Smads by stimulating multimerization and concurrently activating several TGFβ and BMP type I receptors, in a manner that requires the activity of the corresponding ligand. While the individual roles of the TGFβ and BMP signaling pathways in bone mechanotransduction remain unclear, these results implicate that FSS activates both pathways to generate a downstream response that differs from that achieved by either ligand alone.

Online Analysis of Drug Toxicity to Cells with Shear Stress on an Integrated Microfluidic Chip.

Mechanical stimulation, especially fluid shear stress (FSS), is essential for a cell to regulate regular behaviors. A high-throughput platform to provide varying FSS for cell research is desperately required for better mimicking of the complex fluidic microenvironment. This work reports an integrated microfluidic chip that could afford five different FSS gradients consistently to investigate drug toxicity on cells with the stimulation of FSS. Compared with traditional methods to provide FSS, this device would be easier to operate, have higher throughput, and could eliminate interference factors from the culture environment for cells (apart from the unique variable FSS). On such a multi-FSS platform, effects of drugs toxicity on cells were exhibited, which would be more intense than that under static conditions. The results indicated that FSS enhanced the drug toxicity. The designed biochip provides an easy and high-throughput platform to evaluate the toxicity of drugs in the more authentic microenvironment and could be promisingly applied in future drug screening tests.

Fluid shear stress improves morphology, cytoskeleton architecture, viability, and regulates cytokine expression in a time-dependent manner in MLO-Y4 cells.

The effects of load-induced interstitial fluid shear stress (FSS) on instantaneous signaling response of osteocytes (e.g., calcium signaling) have been well documented. FSS can also initiate the release of many important messenger molecules of osteocytes (e.g., ATP and PGE2 ). However, the effects of FSS on cellular function and bone metabolism-modulating cytokine expression of osteocytes have not been fully identified (some inconsistent/conflicting results have been documented). Herein, osteocyte-like MLO-Y4 cells were stimulated with 1 Pa, 2-h FSS, and the effects of FSS on cellular morphology, cytoskeletal microstructure, biological activity, and gene and protein expression of important cytokines were investigated. SEM and cytoskeleton staining revealed that FSS induced well-organized cytoskeleton and increased filopodia processes. The osteocytic viability was sustained and apoptosis was inhibited via flow cytometry. FSS promoted Wnt3a and β-catenin gene and protein expression in 0-, 3-, and 6-h (sample collection time post FSS) groups. The FSS-stimulated cells in the 3-h group exhibited more significant effects on the promotion of OCN and Cx43 and inhibition of DKK1 and SOST expression than the 0- and 6-h groups. The 3-h group with FSS stimulation also showed the most prominent effects on suppressing RANKL and RANKL/OPG gene and protein expression. This study revealed a direct regulatory effect of FSS on osteocytic morphology and apoptotic characteristics, and showed that osteocyte-secreted bone metabolism-modulating molecule expression was regulated by FSS in a time-dependent manner. This study not only enriches our basic knowledge for understanding osteocytic mechanotransduction, but also provides important evidence for more scientific experimental design.

Fluid shear stress promotes osteoblast proliferation through the NFATc1-ERK5 pathway

ABSTRACTPurpose: Extracellular-regulated kinase 5 (ERK5) is thought to regulate osteoblast proliferation. To further understand how ERK5 signaling regulates osteoblast proliferation induced by fluid shear stress (FSS), we examined some potential signaling targets associated with ERK5 in MC3T3-E1 cells.Methods: MC3T3-E1 cells were treated with XMD8-92 (an ERK5 inhibitor) or Cyclosporin A (CsA, a nuclear factor of activated T cells (NFAT) c1 inhibitor) and/or exposed to 12 dyn/cm2 FSS. Phosphorylated-ERK5 (p-ERK5) and expression levels of NFATc1, ERK5, E2F2, and cyclin E1 were analyzed by western blot. The mRNA levels of genes associated with cell proliferation were analyzed by Polymerase Chain Reaction (PCR) array. Subcellular localization of p-ERK5 and NFATc1 were determined by immunofluorescence. Cell proliferation was evaluated by MTT assay.Results: NFATc1 expression was up-regulated by FSS. XMD8-92 only blocked ERK5 activation; however, CsA decreased NFATc1 and p-ERK5 levels, including after FSS stimulation. Exposure to NFATc1 inhibitor or ERK5 inhibitor resulted in decreased E2F2 and cyclin E1 expression and proliferation by proliferative MC3T3-E1 cells. Furthermore, immunofluorescence results illustrated that NFATc1 induced ERK5 phosphorylation, resulting in p-ERK5 translocation to the nucleus.Conclusions: Our results reveal that NFATc1 acts as an intermediate to promote the phosphorylation of ERK5 induced by FSS. Moreover, activated NFATc1-ERK5 signaling up-regulates the expression of E2F2 and cyclin E1, which promote osteoblast proliferation.

Transforming Growth Factor-β Signaling Cascade Induced by Mechanical Stimulation of Fluid Shear Stress in Cultured Corneal Epithelial Cells.

Because blinking is regarded as mechanical stimulation of fluid shear stress on the corneal epithelial cells, we investigated the effects of fluid shear stress on cultured human corneal epithelial cells (HCECs). The HCECs were exposed to shear stress (0, 1.2, 12 dyne/cm2) with the parallel-plate type of flow chamber. Wound healing, cellular proliferation, growth factor expression, TGF-β1 concentration in the culture supernatant, and phosphorylation of SMAD2 were investigated. Monolayers of HCECs exposed to shear stress had delayed wound healing and decreased proliferation compared with those of the static control (0 dyne/cm2). With increasing shear stress, TGF-β1 expression and phosphorylation of SMAD2 increased significantly, but the levels of total TGF-β1 in the culture supernatant decreased significantly. Delayed wound healing, decreased proliferation, and phosphorylation of the SMAD2 by shear stress were canceled out with a TGF-β receptor inhibitor. Fluid shear stress on the HCECs affected TGF-β signaling, which was associated with delayed wound healing. Mechanical stress by blinking might involve TGF-β signaling, and activation of TGF-β might be a key factor in wound healing of the corneal epithelium. Further studies should investigate the molecular mechanism of shear stress-induced activation of TGF-β.

Fluid shear stress enhances the cell volume decrease of osteoblast cells by increasing the expression of the ClC-3 chloride channel.

ClC-3 is a volume-sensitive chloride channel that is responsible for cell volume adjustment and regulatory cell volume decrease (RVD). In order to evaluate the effects of fluid shear stress (FSS) stimulation on the osteoblast ClC-3 chloride channel, MC3T3-E1 cells were stimulated by FSS in the experimental group. Fluorescence quantitative polymerase chain reaction was used to detect changes in ClC-3 mRNA expression, the chloride ion fluorescent probe N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) was used to detect the chloride channel activity, and whole-cell patch clamping was used to monitor the changes in the volume-sensitive chloride current activated by a hypotonic environment following mechanical stimulation. The results show that the expression of the osteoblast chloride channel ClC-3 was significantly higher in the FSS group compared with the control group. MQAE fluorescence intensity was significantly reduced in the FSS group compared to the control group, suggesting that mechanical stimulation increased chloride channel activity and increased the efflux of intracellular chloride ions. Image analysis of osteoblast volume changes showed that osteoblast RVD was enhanced by mechanical stimulation. Whole-cell patch clamping showed that the osteoblast volume-sensitive chloride current was larger in the stimulated group compared to the control group, suggesting that elevated ClC-3 chloride channel expression results in an increased volume-sensitive chloride current. In conclusion, FSS stimulation enhances the RVD of osteoblast cell by increasing the expression of the ClC-3 and enhancing the chloride channel activity.

Prostaglandin E&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt; modulates F-actin stress fiber in FSS-stimulated MC3T3-E1 cells in a PKA-dependent manner

The effect of prostaglandin E2 (PGE2) on bone mass has been well-established in vivo. Previous studies have showed that PGE2 increases differentiation, proliferation, and regulates cell morphology through F-actin stress fiber in statically cultured osteoblasts. However, the effect of PGE2 on osteoblasts in the presence of fluid shear stress (FSS), which could better uncover the anabolic effect of PGE2 in vivo, has yet to be examined. Here, we hypothesized that PGE2 modulates F-actin stress fiber in FSS-stimulated MC3T3-E1 osteoblastic cells through protein kinase A (PKA) pathway. Furthermore, this PGE2-induced F-actin remodeling was associated with the recovery of cellular mechanosensitivity. Our data showed that treatment with 10 nM dmPGE2 for 15 min significantly suppressed the F-actin stress fiber intensity in FSS-stimulated cells in a PKA-dependent manner. In addition, dmPGE2 treatment enhanced the cells' calcium peak magnitude and the percentage of responding cells in the second FSS stimulation, though these effects were abolished and attenuated by co-treatment with phalloidin. Our results demonstrated that 10 nM dmPGE2 was able to accelerate the 'reset' process of F-actin stress fiber to its pre-stimulated level partially through PKA pathway, and thus promoted the recovery of cellular mechanosensitivity. Our finding provided a novel cellular mechanism by which PGE2 increased bone formation as shown in vivo, suggesting that PGE2 could be a potential target for treatments of bone formation-related diseases.

The interaction betweenβ1 integrins and ERK1/2 in osteogenic differentiation of human mesenchymal stem cells under fluid shear stress modelled by a perfusion system

Fluid shear stress (FSS) is an important biomechanical factor regulating the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and is therefore widely used in bone tissue engineering. However, the mechanotransduction of FSS in hMSCs remains largely unknown. As β1 integrins are considered to be important mechanoreceptors in other cells, we suspect that β1 integrins should also be important for hMSCs to sense the stimulation of FSS. We used a perfusion culture system to produce FSS loading on hMSCs seeded in PLGA three-dimensional (3D) scaffolds and investigated the roles of β1 integrins, FAK and ERK1/2 in FSS-induced osteogenic differentiation of hMSCs. Our results showed that FSS not only markedly increased ALP activity and the expression of ALP, OCN, Runx2 and COLIα genes but also significantly enhanced the phosphorylation of ERK1/2, Runx2 and FAK. FSS-induced activation of ERK1/2 and FAK was inhibited by blockade of the connection between β1 integrins and ECM with RGDS peptide and integrins β1 monoclonal antibody. Our study also found that FSS could upregulate the expression level of β1 integrins and that this upregulation could be abolished by PD98059. Further investigation indicated that FSS-activated ERK1/2 led to the phosphorylation of IκBα and NFκB p65. The activation of NFκB p65 resulted in the upregulation of β1 integrin expression. Therefore, it could be inferred that β1 integrins should sense the stimulation of FSS and thus activate ERK1/2 through activating of FAK, and FSS-activated ERK1/2 feedback to upregulate the expression of β1 integrins through activating NFκB.

Mechanical Stress Stimulates Conformational Changes in 5-Hydroxytryptamine Receptor 1B in Bone Cells

In this study we describe the conformational transitions of hydroxytryptamine receptor 1B (5HT1b) in response to ligand and fluid shear stress (FSS) stimulation. Fluorescence resonance energy transfer (FRET) and picosecond time-resolved fluorescence microscopy were employed to detect conformational changes in a genetically engineered 5HT1b receptor sensor containing an intramolecular GPCR conformation-sensitive FRET pair. Stimulation of 5HT1b sensor by a cognate ligand, 5-hydroxytryptamine (5HT), or the FSS led to distinct conformational changes characterized by an increase or decrease in FRET efficiency, respectively. The response to the FSS stimulation was inhibited in the presence of 5HT in the flow medium. Fluidization of the plasma membrane using benzyl alcohol stimulated 5HT1b conformational transitions characterized by decreased FRET levels. Our study provides the first evidence of (1) ligand-independent conformational response of the 5HT1b to mechanical perturbation and (2) modulation of such mechanosensitive response by the cognate ligand.

Fluid flow stress induced contraction and re-spread of mesenchymal stem cells: a microfluidic study

Mesenchymal stem cells (MSCs), the multipotent progenitor cells, are sensitive to fluid shear stress (FSS). MSCs can migrate through the blood stream by intravasation into the circulatory system to transfer to distant positions through the blood stream. During the transferring process, MSCs may differentiate into cells of corresponding tissues for repair, or remain undifferentiated and initiate ectopic tissue formation, lipid accumulation, or calcification, which are closely related to the pathology of atherosclerosis. However, how the MSCs sense and respond to vascular FSS stimulation and lead to subsequent biological effects remains elusive. In this study, by using an in situ time-lapse microfluidic cell culture and observation system, we found that rat mesenchymal stem cells (rMSCs) presented a contraction and re-spread (CRS) process when they were initially subjected to a physiological FSS (1.3 Pa). Our subsequent studies demonstrated that integrin and cilia played key roles in sensing FSS. Calcium, F-actin, and Rho-kinase were key molecules in the mechanotransduction of the CRS of the rMSCs. Our study revealed the immediate response of the rMSCs to FSS. It will be helpful for the understanding of MSC-related tissue repair and the role of MSCs in the initiation of atherosclerosis.

Cerebral Arteriogenesis is Enhanced by Pharmacological as Well as Fluid-Shear-Stress Activation of the Trpv4 Calcium Channel

Objectives This study aimed to determine the importance of the shear-stress-sensitive calcium channels Trpc1, Trpm7, Trpp2, Trpv2 (transient receptor potential cation channel, subfamily V, member 2) and Trpv4 for cerebral arteriogenesis. The expression profiles were analysed, comparing the stimulation of collateral growth by target-specific drugs to that achieved by maximum increased fluid shear stress (FSS). Design A prospective, controlled study wherein rats were subjected to bilateral carotid artery ligature (BCL), or BCL + arteriovenous fistula, or BCL + drug application. Methods Messenger RNA (mRNA) abundance and protein expression were determined in FSS-stimulated cerebral collaterals by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. Drugs were applied via osmotic mini pumps and arteriogenesis was evaluated by post-mortem angiograms and Ki67 immunostaining. Results Trpv4 was the only mechanosensitive Trp channel showing significantly increased mRNA abundance and protein expression after FSS stimulation. Activation of Trpv4 by 4α-phorbol-12,13-didecanoate caused significantly enhanced collateral growth (length: 4.43 ± 0.20 mm and diameter: 282.6 ± 8.1 μm) compared with control (length: 3.80 ± 0.06 mm and diameter: 237.3 ± 5.3 μm). Drug application stimulated arteriogenesis to almost the same extent as did maximum FSS stimulation (length: 4.61 ± 0.07 mm and diameter: 327.4 ± 12.6 μm). Conclusions Trpv4 showed significantly increased expression in FSS-stimulated cerebral collaterals. Pharmacological Trpv4 activation enhanced cerebral arteriogenesis, pinpointing Trpv4 as a possible candidate for the development of new therapeutic concepts.