Transduction of mechanical forces and chemical signals affect every cell in the human body. Fluid flow in systems such as the lymphatic or circulatory systems modulates not only cell morphology, but also gene expression patterns, extracellular matrix protein secretion and cell-cell and cell-matrix adhesions. Similar to the role of mechanical forces in adaptation of tissues, shear fluid flow orchestrates collective behaviours of adherent cells found at the interface between tissues and their fluidic environments. These behaviours range from alignment of endothelial cells in the direction of flow to stem cell lineage commitment. Therefore, it is important to characterize quantitatively fluid interface-dependent cell activity. Common macro-scale techniques, such as the parallel plate flow chamber and vertical-step flow methods that apply fluid-induced stress on adherent cells, offer standardization, repeatability and ease of operation. However, in order to achieve improved control over a cell's microenvironment, additional microscale-based techniques are needed. The use of microfluidics for this has been recognized, but its true potential has emerged only recently with the advent of hybrid systems, offering increased throughput, multicellular interactions, substrate functionalization on 3D geometries, and simultaneous control over chemical and mechanical stimulation. In this review, we discuss recent advances in microfluidic flow systems for adherent cells and elaborate on their suitability to mimic physiologic micromechanical environments subjected to fluid flow. We describe device design considerations in light of ongoing discoveries in mechanobiology and point to future trends of this promising technology.

P2X7 is a ligand-gated ion channel which is activated by ATP and displays secondary permeability characteristics. The mechanism of development of the secondary permeability pathway is currently unclear, although a role for the hemichannel protein pannexin-1 has been suggested. In this study we investigated the role of pannexin-1 in P2X7-induced dye uptake and ATP-induced IL-1β secretion from human monocytes. We found no pharmacological evidence for involvement of pannexin-1 in P2X7-mediated dye uptake in transfected HEK-293 cells with no inhibition seen for carbenoxolone and the pannexin-1 mimetic inhibitory peptide, 10Panx1. However, we found that probenecid inhibited P2X7-induced cationic and anionic dye uptake in stably transfected human P2X7 HEK-293 cells. An IC50 value of 203 μM was calculated for blockade of ATP-induced responses at human P2X7. Probenecid also reduced dye uptake and IL-1β secretion from human CD14+ monocytes whereas carbenoxolone and 10Panx1 showed no inhibitory effect. Patch clamp and calcium indicator experiments revealed that probenecid directly blocks the human P2X7 receptor.

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While the mechanisms by which chemical signals control cell fate have been well studied, how mechanical inputs impact cell fate decisions are not well understood. Here, using the well-defined system of keratinocyte differentiation in the skin, we examine whether and how direct force transmission to the nucleus regulates epidermal cell fate. Using a molecular biosensor, we find that tension on the nucleus through Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes requires integrin engagement in undifferentiated epidermal stem cells, and is released during differentiation concomitant with decreased tension on A-type lamins. LINC complex ablation in mice reveals that LINC complexes are required to repress epidermal differentiation in vivo and in vitro and influence accessibility of epidermal differentiation genes, suggesting that force transduction from engaged integrins to the nucleus plays a role in maintaining keratinocyte progenitors. This work reveals a direct mechanotransduction pathway capable of relaying adhesion-specific signals to regulate cell fate.

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

Extracellular ATP induces cation fluxes in and impairs the growth of murine erythroleukemia (MEL) cells in a manner characteristic of the purinergic P2X7 receptor, however the presence of P2X7 in these cells is unknown. This study investigated whether MEL cells express functional P2X7. RT-PCR, immunoblotting and immunofluorescence staining demonstrated the presence of P2X7 in MEL cells. Cytofluorometric measurements demonstrated that ATP induced ethidium+ uptake into MEL cells in a concentration-dependent fashion and with an EC(50) of approximately 154 microM. The most potent P2X7 agonist 2'- and 3'-0(4-benzoylbenzoyl) ATP, but not ADP or UTP, induced ethidium+ uptake. ATP-induced ethidium+ and YO-PRO-1(2+) uptake were impaired by the P2X7 antagonist, A-438079. A colourmetric assay demonstrated that ATP impaired MEL cell growth. A cytofluorometric assay showed that ATP induced MEL cell death and that this process was impaired by A-438079. Finally, cytofluorometric measurements of Annexin-V binding and bio-maleimide staining demonstrated that ATP could induce rapid phosphatidylserine exposure and microparticle release in MEL cells respectively, both of which were impaired by A-438079. These results demonstrate that MEL cells express functional P2X7, and indicate that activation of this receptor may be important in the death and release of microparticles from red blood cells in vivo.

Corneal wound healing is a complex process that consists of cellular integration of multiple soluble biochemical cues and cellular responses to biophysical attributes associated with the matrix of the wound space. Upon corneal stromal wounding, the transformation of corneal fibroblasts to myofibroblasts is promoted by transforming growth factor-β (TGFβ). This process is critical for wound healing; however, excessive persistence of myofibroblasts in the wound space has been associated with corneal fibrosis resulting in severe vision loss. The objective of this study was to determine the effect of hepatocyte growth factor (HGF), which can modulate TGFβ signaling, on corneal myofibroblast transformation by analyzing the expression of α-smooth muscle actin (αSMA) as a marker of myofibroblast phenotype particularly as it relates to biomechanical cues. Human corneal fibroblasts were cultured on tissue culture plastic (>1 GPa) or hydrogel substrates mimicking human normal or wounded corneal stiffness (25 and 75 kPa) in media containing TGFβ1 ± HGF. The expression of αSMA was analyzed by quantitative PCR, Western blot and immunocytochemistry. Cellular stiffness, which is correlated with cellular phenotype, was measured by atomic force microscopy (AFM). In primary human corneal fibroblasts, the mRNA expression of αSMA showed a clear dose response to TGFβ1. The expression was significantly suppressed when cells were incubated with 20 ng/ml HGF in the presence of 2 ng/ml of TGFβ1. The protein expression of αSMA induced by 5 ng/ml TGFβ1 was also decreased by 20 ng/ml of HGF. Cells cultured on hydrogels mimicking human normal (25 kPa) and fibrotic (75 kPa) cornea also showed an inhibitory effect of HGF on αSMA expression in the presence or absence of TGFβ1. Cellular stiffness was decreased by HGF in the presence of TGFβ1 as measured by AFM. In this study, we have demonstrated that HGF can suppress the myofibroblast phenotype promoted by TGFβ1 in human corneal stromal cells. These data suggest that HGF holds the potential as a therapeutic agent to improve wound healing outcomes by minimizing corneal fibrosis.

Purpose: Transforming growth factor β1 (TGFβ1) is elevated in wounds after injury and promotes the transdifferentiation of quiescent cells in the stroma (keratocytes, to activated fibroblasts and subsequently myofibroblasts-KFM transformation). Coactivators of transcription, YAP (Yes-associated protein) and TAZ (Transcriptional coactivator with PDZ-binding motif), are mechanotransducers that intersect with the TGFβ pathway via interactions with Smad proteins. Here, we examined the distinct role of YAP and TAZ on TGFβ1 induced myofibroblast transformation of primary human corneal fibroblasts (HCFs). Methods:A knockdown approach was used to silence YAP and TAZ individually in HCFs. Forty-eight hours post siRNA transfection, cells were cultured in the presence or absence of 2 ng/ml TGFβ1 for 24h. The cells were subjected to nuclear and cytoplasmic fractionation. The expression of α-smooth muscle actin (αSMA), Smad 2, 3 and 4, CTGF and phospho-Smad2, 3, and 4 were assessed by qPCR and Western blotting.