Normal tissue cells are generally not viable when suspended in a fluid and are therefore said to be anchorage dependent. Such cells must adhere to a solid, but a solid can be as rigid as glass or softer than a baby's skin. The behavior of some cells on soft materials is characteristic of important phenotypes; for example, cell growth on soft agar gels is used to identify cancer cells. However, an understanding of how tissue cells-including fibroblasts, myocytes, neurons, and other cell types-sense matrix stiffness is just emerging with quantitative studies of cells adhering to gels (or to other cells) with which elasticity can be tuned to approximate that of tissues. Key roles in molecular pathways are played by adhesion complexes and the actinmyosin cytoskeleton, whose contractile forces are transmitted through transcellular structures. The feedback of local matrix stiffness on cell state likely has important implications for development, differentiation, disease, and regeneration.

/pdf/tissue-cells-feel-and-respond-to-the-stiffness-of-their-6dx0rumap6.pdf

Tissue Cells Feel and Respond to the Stiffness of Their Substrate

Atherosclerosis, the leading cause of death in the developed world and
nearly the leading cause in the developing world, is associated with systemic
risk factors including hypertension, smoking, hyperlipidemia, and diabetes
mellitus, among others. Nonetheless, atherosclerosis remains a geometrically
focal disease, preferentially affecting the outer edges of vessel bifurcations.
In these predisposed areas, hemodynamic shear stress, the frictional force
acting on the endothelial cell surface as a result of blood flow, is weaker
than in protected regions. Studies have identified hemodynamic shear stress
as an important determinant of endothelial function and phenotype. Arterial-level
shear stress (>15 dyne/cm2) induces endothelial quiescence and
an atheroprotective gene expression profile, while low shear stress (<4
dyne/cm2), which is prevalent at atherosclerosis-prone sites, stimulates
an atherogenic phenotype. The functional regulation of the endothelium by
local hemodynamic shear stress provides a model for understanding the focal
propensity of atherosclerosis in the setting of systemic factors and may help
guide future therapeutic strategies.

/pdf/hemodynamic-shear-stress-and-its-role-in-atherosclerosis-1u0na41ojd.pdf

Hemodynamic shear stress and its role in atherosclerosis.

A central question in cell biology is how membrane-spanning receptors transmit extracellular signals inside cells to modulate cell adhesion and motility. Focal adhesion kinase (FAK) is a crucial signalling component that is activated by numerous stimuli and functions as a biosensor or integrator to control cell motility. Through multifaceted and diverse molecular connections, FAK can influence the cytoskeleton, structures of cell adhesion sites and membrane protrusions to regulate cell movement.

Focal adhesion kinase: in command and control of cell motility

During development and in the context of different morphogenetic events, epithelial cells undergo a process called epithelial to mesenchymal transition or transdifferentiation (EMT). In this process, the cells lose their epithelial characteristics, including their polarity and specialized cell-cell contacts, and acquire a migratory behavior, allowing them to move away from their epithelial cell community and to integrate into surrounding tissue, even at remote locations. EMT illustrates the differentiation plasticity during development and is complemented by another process, called mesenchymal to epithelial transition (MET). While being an integral process during development, EMT is also recapitulated under pathological conditions, prominently in fibrosis and in invasion and metastasis of carcinomas. Accordingly, EMT is considered as an important step in tumor progression. TGF-β signaling has been shown to play an important role in EMT. In fact, adding TGF-β to epithelial cells in culture is a convenient way to induce EMT in various epithelial cells. Although much less characterized, epithelial plasticity can also be regulated by TGF-β-related bone morphogenetic proteins (BMPs), and BMPs have been shown to induce EMT or MET depending on the developmental context. In this review, we will discuss the induction of EMT in response to TGF-β, and focus on the underlying signaling and transcription mechanisms.

/pdf/tgf-beta-induced-epithelial-to-mesenchymal-transition-xww3aw4gv3.pdf

TGF-beta-induced epithelial to mesenchymal transition.

The shapes of eukaryotic cells and ultimately the organisms that they form are defined by cycles of mechanosensing, mechanotransduction and mechanoresponse Local sensing of force or geometry is transduced into biochemical signals that result in cell responses even for complex mechanical parameters such as substrate rigidity and cell-level form These responses regulate cell growth, differentiation, shape changes and cell death Recent tissue scaffolds that have been engineered at the micro- and nanoscale level now enable better dissection of the mechanosensing, transduction and response mechanisms

https://faculty.washington.edu/nsniadec/ME599/S09/private/25Vogel.pdf

Local force and geometry sensing regulate cell functions.

The mechanical environment crucially influences many cell functions. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin-cytoskeleton interaction, which is essential for the transduction of mechanical stimuli. Using fluorescent resonance energy transfer (FRET), here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells (HUVECs) by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed (mean +/- s.e.m.) of 18.1 +/- 1.7 nm s(-1). This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations.

/pdf/visualizing-the-mechanical-activation-of-src-188ghmqaxs.pdf

Visualizing the mechanical activation of Src

Effects of hematocrit and plasma proteins on human blood rheology at low shear rates.

The small GTPase Rho regulates the formation of actin stress fibers in adherent cells through activation of its effector proteins Rho-kinase and mDia. We found in bovine aortic endothelial cells that inhibitions of Rho, Rho-kinase, and mDia (with C3, Y27632, and F1F2Δ1, respectively) suppressed stress fiber formation, but fibers appeared after 10% cyclic uniaxial stretch (1-Hz frequency). In contrast to the predominately perpendicular alignment of stress fibers to the stretch direction in normal cells, the stress fibers in cells with Rho pathway inhibition became oriented parallel to the stretch direction. In cells with normal Rho activity, the extent of perpendicular orientation of stress fibers depended on the magnitude of stretch. Expressing active RhoV14 plasmid in these cells enhanced the stretch-induced stress fiber orientation by an extent equivalent to an additional ≈3% stretch. This augmentation of the stretch-induced perpendicular orientation by RhoV14 was blocked by Y27632 and by F1F2Δ1. Thus, the activity of the Rho pathway plays a critical role in determining both the direction and extent of stretch-induced stress fiber orientation in bovine aortic endothelial cells. Our results demonstrate that the stretch-induced stress fiber orientation is a function of the interplay between Rho pathway activity and the magnitude of stretching.

https://www.pnas.org/content/pnas/102/44/15895.full.pdf

Cooperative effects of Rho and mechanical stretch on stress fiber organization

The focal distribution of atherosclerotic lesions in the arterial tree is related to the local shear stress generated by blood flow, but the molecular basis of the atherogenic response of endothelial cells in these lesion-prone areas is still unclear. We report that shear stress mediates a biphasic response of monocyte chemotactic protein 1 (MCP-1) gene expression in vascular endothelial cells (EC). Northern blot analysis indicated that the level of MCP-1 mRNA in human umbilical vein EC (HUVEC) subjected to a shear stress of 16 dynes/cm2 (1 dyne = 10 microN) for 1.5 hr increased by 2- to 3-fold when compared with static cells. The MCP-1 gene expression decreased to the basal level at 4 hr and then declined further to become completely quiescent at 5 hr after the onset of shear. Once the gene expression was fully suppressed, it remained quiescent even after static incubation for 1.5 hr and would not respond to reshearing after this static incubation. However, if the postshearing incubation extended from 1.5 to 24 hr, the MCP-1 mRNA returned to the basal level and was then able to increase after the reapplication of shear stress. Nuclear run-on experiments showed that the shear-induced increased MCP-1 mRNA in HUVEC was regulated at the transcriptional level. By using cycloheximide, it was shown that de novo protein synthesis was not necessary for the induction of MCP-1 by shear stress. The biphasic response of MCP-1 gene expression was found in experiments in which the applied shear stress was 6, 16, or 32 dynes/cm2, and it was observed not only in HUVEC but also in HeLa cells, glioma cell lines, and skin fibroblasts. This in vitro study demonstrates that the response of MCP-1 gene to shear stress represents an immediate early gene activation and suggests that this gene is probably suppressed in EC that have been exposed to a constant shear stress.

https://www.pnas.org/content/pnas/91/11/4678.full.pdf

Shunichi Usami

Papers

Visualizing the mechanical activation of Src

Effects of hematocrit and plasma proteins on human blood rheology at low shear rates.

Cooperative effects of Rho and mechanical stretch on stress fiber organization

Fluid shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium

In vivo measurements of “apparent viscosity” and microvessel hematocrit in the mesentery of the cat