Shirin Feghhi

Flow mechanotransduction regulates traction forces, intercellular forces, and adherens junctions

Endothelial cells respond to fluid shear stress through mechanotransduction responses that affect their cytoskeleton and cell-cell contacts. Here, endothelial cells were grown as monolayers on arrays of microposts and exposed to laminar or disturbed flow to examine the relationship among traction forces, intercellular forces, and cell-cell junctions. Cells under laminar flow had traction forces that were higher than those under static conditions, whereas cells under disturbed flow had lower traction forces. The response in adhesion junction assembly matched closely with changes in traction forces since adherens junctions were larger in size for laminar flow and smaller for disturbed flow. Treating the cells with calyculin-A to increase myosin phosphorylation and traction forces caused an increase in adherens junction size, whereas Y-27362 cause a decrease in their size. Since tugging forces across cell-cell junctions can promote junctional assembly, we developed a novel approach to measure intercellular forces and found that these forces were higher for laminar flow than for static or disturbed flow. The size of adherens junctions and tight junctions matched closely with intercellular forces for these flow conditions. These results indicate that laminar flow can increase cytoskeletal tension while disturbed flow decreases cytoskeletal tension. Consequently, we found that changes in cytoskeletal tension in response to shear flow conditions can affect intercellular tension, which in turn regulates the assembly of cell-cell junctions.

Thermal fracture of oxidized polydimethylsiloxane during soft lithography of nanopost arrays

During the fabrication of nanopost arrays for measuring cellular forces, we have observed surface cracks in the negative molds used to replicate the arrays from a silicon master. These cracks become more numerous and severe with each replication such that repeated castings lead to arrays with missing or broken posts. This loss in pattern fidelity from the silicon master undermines the spatial resolution of the nanopost arrays in measuring cellular forces. We hypothesized that these cracks are formed because of a mismatch in the coefficient of thermal expansion (CTE) of polydimethylsiloxane (PDMS) and its oxidized surface layer. To study the fracture of PDMS due to thermal effects, we treated circular test samples of PDMS with oxidizing plasma and then heated them to cause surface cracks. These cracks were found to be more abundant at 180 °C than at lower temperatures. Finite element analysis of a bilayer material with a CTE mismatch was used to validate that thermal stresses are sufficient to overcome the fracture toughness of oxidized PDMS. As a consequence, we have ascertained that elevated temperatures are a significant detriment to the reproducibility of nanoscale features in PDMS during replica molding.

Mechanobiology of platelets: techniques to study the role of fluid flow and platelet retraction forces at the micro- and nano-scale.

Coagulation involves a complex set of events that are important in maintaining hemostasis. Biochemical interactions are classically known to regulate the hemostatic process, but recent evidence has revealed that mechanical interactions between platelets and their surroundings can also play a substantial role. Investigations into platelet mechanobiology have been challenging however, due to the small dimensions of platelets and their glycoprotein receptors. Platelet researchers have recently turned to microfabricated devices to control these physical, nanometer-scale interactions with a higher degree of precision. These approaches have enabled exciting, new insights into the molecular and biomechanical factors that affect platelets in clot formation. In this review, we highlight the new tools used to understand platelet mechanobiology and the roles of adhesion, shear flow, and retraction forces in clot formation.

Glycoprotein Ib-IX-V Complex Transmits Cytoskeletal Forces That Enhance Platelet Adhesion

Platelets bind to exposed vascular matrix at a wound site through a highly specialized surface receptor, glycoprotein (GP) Ib-IX-V complex, which recognizes von Willebrand factor (VWF) in the matrix. GPIb-IX-V is a catch bond for it becomes more stable as force is applied to it. After attaching to the wound site, platelets generate cytoskeletal forces to compact and reinforce the hemostatic plug. Here, we evaluated the role of the GPIb-IX-V complex in the transmission of cytoskeletal forces. We used arrays of flexible, silicone nanoposts to measure the contractility of individual platelets on VWF. We found that a significant proportion of cytoskeletal forces were transmitted to VWF through GPIb-IX-V, an unexpected finding given the widely held notion that platelet forces are transmitted exclusively through its integrins. In particular, we found that the interaction between GPIbα and the A1 domain of VWF mediates this force transmission. We also demonstrate that the binding interaction between GPIbα and filamin A is involved in force transmission. Furthermore, our studies suggest that cytoskeletal forces acting through GPIbα are involved in maintaining platelet adhesion when external forces are absent. Thus, the GPIb-IX-V/VWF bond is able to transmit force, and uses this force to strengthen the bond through a catch-bond mechanism. This finding expands our understanding of how platelets attach to sites of vascular injury, describing a new, to the best of our knowledge, mechanism in which the catch bonds of GPIb-IX-V/VWF can be supported by internal forces produced by cytoskeletal tension.

Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase

Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosins ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis.

Micropatterning on Micropost Arrays

Micropatterning of cells can be used in combination with microposts to control cell shape or cell-to-cell interaction while measuring cellular forces. The protocols in this chapter describe how to make SU8 masters for stamps and microposts, how to use soft lithography to replicate these structures in polydimethylsiloxane, and how to functionalize the surface of the microposts for cell attachment.

Effect of Silanization Film Thickness in Soft Lithography of Nanoscale Features

Soft lithography was used to replicate nanoscale features made using electron beam lithography on a polymethylmethacrylate (PMMA) master. The PMMA masters were exposed to fluorinated silane vapors to passivate its surfaces so that polydimethylsiloxane (PDMS) did not permanently bond to the master. From scanning electron microscopy, the silanization process was found to deposit a coating on the master that was a few hundreds of nanometers thick. These silane films partially concealed the nanoscale holes on the PMMA master, causing the soft lithography process to produce PDMS features with dimensions that were significantly reduced. The thickness of the silane films was directly measured on silicon or PMMA masters and was found to increase with exposure time to silane vapors. These findings indicate that the thickness of the silane coatings is a critical parameter when using soft lithography to replicate nanoscale features, and caution should be taken on how long a master is exposed to silane vapors. [DOI: 10.1115/1.4005665]

E-Beam Nanopost Arrays Reveal That Glycoprotein Ib-IV-X Complex and Von Willebrand Factor Interactions Transmit Platelet Cytoskeletal Forces

We have developed a new tool to measure the contractility of single platelets. This tool consists of an array of nano-scale polydimethylsiloxane (PDMS) posts. Von Willebrand factor (VWF) and a recombinant protein encompassing the GPIb-IX-V binding region of VWF (A1 domain) were used to study the role of GPIb-VWF interactions in platelet contractility. Platelets were treated with AK2 and 7E3 antibodies to block platelet adhesion through receptors GPIb and αIIbβ3, respectively. Platelets treated with these antibodies showed reduced spread area and forces in comparison to untreated platelets on VWF. Furthermore, platelets were able to generate contractile forces on substrates coated with A1 domain of VWF. These results suggest that platelet contractile forces can be transmitted through a non-integrin receptor, such as GPIb.Copyright

Platelet Retraction Forces Induced Under High Shear Gradient Activation

Hemodynamics play an important role in the activity of platelets. High shear rate gradients can occur at locations where blood vessels bend, branch, or narrow and can arise at a vascular stent or artificial valve. These shear gradients have been observed to cause platelets to adhere to the vessel wall, leading to their activation and aggregation [1, 2]. High shear gradients can cause a self-sustaining process where platelet aggregation increases the local shear gradient, further causing platelets to adhere and aggregate. This process can become dangerous if the aggregate grows large enough to obstruct the vessel or if it detaches and clogs a vessel downstream.Copyright

The Role of Nonmuscle Myosin IIA Regulation in Platelet Forces Using Microposts and Multiphysics Modeling

A multi-physics model has been developed that closely matches with the biochemical regulation of platelet forces. The model is based on measurements of platelet forces using arrays of microposts. Different concentrations of thrombin or myosin inhibitors were added to the platelets to reduce their forces on the posts. The platelet forces obtained from the model have good agreement with those measured in the inhibition studies.Copyright