Roles of cell confluency and fluid shear in 3-dimensional intracellular forces in endothelial cells

Abstract

We use a novel 3D inter-/intracellular force microscopy technique based on 3D traction force microscopy to measure the cell-cell junctional and intracellular tensions in subconfluent and confluent vascular endothelial cell (EC) monolayers under static and shear flow conditions. We found that z-direction cell-cell junctional tensions are higher in confluent EC monolayers than those in subconfluent ECs, which cannot be revealed in the previous 2D methods. Under static conditions, subconfluent cells are under spatially nonuniform tensions, whereas cells in confluent monolayers are under uniform tensions. The shear modulations of EC cytoskeletal remodeling, extracellular matrix (ECM) adhesions, and cell-cell junctions lead to significant changes in intracellular tensions. When a confluent monolayer is subjected to flow shear stresses with a high forward component comparable to that seen in the straight part of the arterial system, the intracellular and junction tensions preferentially increase along the flow direction over time, which may be related to the relocation of adherens junction proteins. The increases in intracellular tensions are shown to be a result of chemomechanical responses of the ECs under flow shear rather than a direct result of mechanical loading. In contrast, the intracellular tensions do not show a preferential orientation under oscillatory flow with a very low mean shear. These differences in the directionality and magnitude of intracellular tensions may modulate translation and transcription of ECs under different flow patterns, thus affecting their susceptibility for atherogenesis.cell alignment | endothelial monolayer | finite element method | fluid shear stress | junctional force B lood vessels are constantly exposed to hemodynamic forces imposed by the blood flow and pressure. Vascular endothelial cells (ECs), which line the inner blood vessel wall, bear the shear stress resulting from the blood flow. Responses of ECs to hemodynamic forces play significant roles in vascular homeostasis in health and disease. Atherosclerotic lesions are preferentially localized in regions, such as arterial branch points, where the ECs are subjected to disturbed flow consisting of flow separation, reversal, and reattachment (1-3). The reattachment area, which is exposed to a low shear stress magnitude and significant oscillatory reversal (i.e., the flow oscillates back and forth with little net direction), has random EC morphology and cytoskeletal organization, incomplete intercellular junctions, and pro-inflammatory and pro-atherogenic phenotypes (1). In contrast, ECs in the straight part of the arterial tree, which is generally spared from atherosclerosis, are exposed to high shear flow with a large net mean direction and have parallel cell orientation, aligned cytoskeletal fibers, and intact junctions. Studies on cultured ECs have advanced the knowledge of how different flow conditions regulate EC functions (1, 4, 5) and provided evidence for the biomedical importance of EC responses to flow shear. The b...

Flow-dependent cellular mechanotransduction in atherosclerosis

“…Assuming the adhesions occupy 5% of the surface, the forces from adhesions spread over the whole cell would be 250 Pa. Traction forces that originate in myosin are therefore ,100 times larger than the forces from moderate physiological shear stress on the cell.] A more recent report determined that the forces within the cytoskeleton that are induced by exposing ECs to long-term shear stress were nearly one order of magnitude larger than the value required to passively balance the force from shear stress (Hur et al, 2012). Taken together, these observations suggest that the response of ECs to shear stress cannot be explained as a passive response to applied force (e.g.…”

P2Y2 and Gq/G11 control blood pressure by mediating endothelial mechanotransduction

“…VEGFR-2 in turn becomes tyrosine phosphorylated and activates downstream signaling events including the activation of AKT kinases (13,16). Biophysical evidence indicated that PECAM-1 is not a primary mechanosensor, but that it is controlled by upstream mechanisms that have remained elusive (17,18). Other mechanotransducers suggested to date include mechanosensitive ion channels (19)(20)(21), the endothelial glycocalyx layer (22,23), and the primary cilium (24).…”

Shear-induced force transmission in a multicomponent, multicell model of the endothelium

“…It is well known that hypertension, diabetes and hypercholesterolemia promote atherosclerosis by disrupting the ability of the endothelium to respond to shear stress [1][2][3][4][5][6][7][8][9]. Therefore, elucidation of the mechanisms of shear-mediated signal transduction will greatly advance our understanding of atherosclerosis.…”

“…Responses of endothelial cells (ECs) to haemodynamic forces play a significant role in vascular health and disease [4][5][6][7][8][9]. It is well known that ECs transduce the fluid shear stress (FSS) resulting from blood flow into intracellular signals that affect gene expression and cellular functions such as proliferation, apoptosis, migration, permeability, cell alignment and mechanical properties [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The activation of signalling pathways by shear forces arises at discrete locations in ECs by force amplification and forceinduced directional biasing of signal propagation [1][2][3][4][10][11][12][16][17][18][20][21][22][23].…”