Abstract-Cyclic mechanical strain produced by pulsatile blood flow regulates the orientation of endothelial cells lining blood vessels and influences critical processes such as angiogenesis. Mechanical stimulation of stretch-activated calcium channels is known to mediate this reorientation response; however, the molecular basis remains unknown. Here, we show that cyclically stretching capillary endothelial cells adherent to flexible extracellular matrix substrates activates mechanosensitive TRPV4 (transient receptor potential vanilloid 4) ion channels that, in turn, stimulate phosphatidylinositol 3-kinase-dependent activation and binding of additional ␤1 integrin receptors, which promotes cytoskeletal remodeling and cell reorientation. Inhibition of integrin activation using blocking antibodies and knock down of TRPV4 channels using specific small interfering RNA suppress strain-induced capillary cell reorientation. Thus, mechanical forces that physically deform extracellular matrix may guide capillary cell reorientation through a strain-dependent "integrin-to-integrin" signaling mechanism mediated by force-induced activation of mechanically gated TRPV4 ion channels on the cell surface. Key Words: mechanical strain Ⅲ integrin Ⅲ TRPV4 Ⅲ endothelial cell Ⅲ reorientation Ⅲ cytoskeleton M echanical forces regulate vascular growth and development by influencing endothelial cell growth, survival, differentiation and migration. 1,2 Local mechanical cues conveyed by extracellular matrix (ECM) attributable to cyclic deformation of blood vessels, hemodynamic forces, or cellgenerated traction forces are also potent inducers of directional capillary blood vessel growth and vascular remodeling in vitro and in vivo. [3][4][5][6][7][8][9][10] For example, the initial step in neovascularization involves reorientation of a subset of capillary endothelial (CE) cells that spread and migrate perpendicular to the main axis of the preexisting vessel toward the angiogenic stimulus 11 ; however, the molecular mechanism responsible for this CE cell reorientation response is unknown. Many cell types, including large vessel endothelial cells, realign perpendicular to the direction of the applied force when they experience cyclic stretching (mechanical strain). [12][13][14][15] In the case of macrovascular endothelium, this reorientation response can be prevented by treatment with chemical inhibitors of stretch-activated (SA) ion channels. 15 But neither the identity of these channels nor the mechanism by which they elicit cell reorientation is known.Endothelial cells express most members of the transient receptor potential (TRP) family of ion channels 16 -18 and TRP vanilloid (TRPV)4 has been reported to mediate flowinduced vasodilation in large vessel endothelium. 19 -22 Here, we show that calcium influx through TRPV4 channels stimulated by mechanically stretching CE cells through their integrin-extracellular matrix (ECM) adhesions promotes cell reorientation by activating phosphatidylinositol 3-kinase (PI3K), thereby stimulating activ...

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Integrins are ubiquitous transmembrane mechanoreceptors that elicit changes in intracellular biochemistry in response to mechanical force application, but these alterations generally proceed over seconds to minutes. Stress-sensitive ion channels represent another class of mechanoreceptors that are activated much more rapidly (within msec), and recent findings suggest that calcium influx through Transient Receptor Potential Vanilloid-4 (TRPV4) channels expressed in the plasma membrane of bovine capillary endothelial cells is required for mechanical strain-induced changes in focal adhesion assembly, cell orientation and directional migration. However, whether mechanically stretching a cell’s extracellular matrix (ECM) adhesions might directly activate cell surface ion channels remains unknown. Here we show that forces applied to β1 integrins result in ultra-rapid (within 4 msec) activation of calcium influx through TRPV4 channels. The TRPV4 channels were specifically activated by mechanical strain in the cytoskeletal backbone of the focal adhesion, and not by deformation of the lipid bilayer or submembranous cortical cytoskeleton alone. This early-immediate calcium signaling response required the distal region of the β1 integrin cytoplasmic tail that contains a binding site for the integrin-associated transmembrane CD98 protein, and external force application to CD98 within focal adhesions activated the same ultra-rapid calcium signaling response. Local direct strain-dependent activation of TRPV4 channels mediated by force transfer from integrins and CD98 may therefore enable compartmentalization of calcium signaling within focal adhesions that is critical for mechanical control of many cell behaviors that underlie cell and tissue development.