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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.

We studied the response of porcine vascular smooth muscle cells (PVSMCs) to cyclic sinusoidal stretch at a frequency of 1 Hz. Cyclic stretch with an area change of 25% caused an increase in PVSMC apoptosis, which was accompanied by sustained activation of c-Jun N[H.sub.2]-terminal kinases (JNK) and the mitogen-activated protein kinase p38. Cyclic stretch with an area change of 7% had no such effect. Infection of PVSMCs with recombinant adenoviruses expressing constitutively active forms of upstream molecules that activate JNK and p38 also led to apoptosis. The simultaneous blockade of both JNK and p38 pathways with adenovirus-mediated expression of dominant-negative mutants of c-Jun and p38 caused a significant decrease (to 1/2) of the apoptosis induced by 25% cyclic stretch. The 25% stretch also caused sustained clustering of tumor necrosis factor-[alpha] (TNF-[alpha]) receptor-1 and its association with TNF-[alpha] receptor-associated factor-2 (TRAF-2). Overexpressing the wild-type TRAF-2 in PVSMCs caused an increase in apoptosis. In contrast, the expression of a dominant-negative mutant of TRAF-2 attenuated stretch-induced apoptois. These results support the hypothesis that circumferential overload under hypertensive conditions induces a clustering of death receptors that cause vascular smooth muscle cell apoptosis. c-Jun N[H.sub.2]-terminal kinases; p38; mechanotransduction; mechanical overload; vascular wall

To study the roles of anisotropic cell morphology and directionality of mechanical force in apoptosis, the spreading of human umbilical vein endothelial cells (HUVECs) was constrained by growing on micropatterned (MP) strips of fibronectin (FN, 20 µg/cm²) with widths of 15, 30, and 60 µm on silicone membrane. Cells on 30- and 60-µm strips, like cells on a nonpatterned (NP) surface coated with FN, showed clear actin stress fibers with anchoring spots of phosphorylated focal adhesion kinase (p-FAK) and no significant apoptosis. On 15-em strips, cells had few stress fibers, no p-FAK, and significant apoptosis. After seeding for 12 h, the cells were subjected to pulsatile shear stress (12 ± 4 dyn/cm²) parallel or perpendicular to MP strips, or kept under static condition. Parallel flow caused cell elongation with enhanced stress fibers and p-FAK, and a reduction in apoptosis, but perpendicular flow did not. The Rho inhibitory C3 exoenzyme abolished the effects of parallel flow. RhoV14, the constitutively active Rho, enhanced stress fibers and p-FAK, and prevented apoptosis of HUVECs on 15-µm strips under static condition. RhoV14 also reduced cell apoptosis under both parallel and perpendicular flows. Our results indicate that cell apoptosis can be modulated by changes in ECM micropatterning, anisotropic cell morphology, and mechanical forces. These extracellular microenvironment factors affect cell survival through alterations in Rho GTPase activity, stress fiber organization, and FAK phosphorylation. [ABSTRACT FROM AUTHOR]

Vascular endothelial cells (ECs) are subjected to shear stress and cytokine stimulation. We studied the interplay between shear stress and cytokine in modulating the expression of adhesion molecule genes and the adhesive function of ECs. Shear stress (20 dynes/cm2) was applied to ECs prior to or following the addition of tumor necrosis factor (TNF)-α. Shear stress increased the TNF-α-induced expression of intercellular adhesion molecule-1 (ICAM-1) at both mRNA and surface protein levels, but decreased the TNF-α-induced expression of vascular adhesion molecule-1 (VCAM-1). The TNF-α-induced increase in EC adhesiveness for monocytic THP-1 cells was reduced by shear stress. After 24-h pre-shearing followed by 1 h of static incubation, the effect of pre-shearing on TNF-α-induced ICAM-1 mRNA expression vanished. The recovery of the TNF-α-induced VCAM-1 mRNA expression following pre-shearing, however, required a static incubation time of >6 h (completely recovery at 24 h). Pre- and post-shearing caused a reduction in the nuclear factor (NF)-κB-DNA binding activity induced by TNF-α in the EC nucleus. Our findings suggest that shear stress plays differential roles in modulating the TNF-α-induced EC expressions of ICAM-1 and VCAM-1 genes, which serve similar functions in vascular biology. [ABSTRACT FROM AUTHOR]

Describes the role of the dynamics of local adhesion kinase in the mechanotaxis of endothelial cells. Modulation of endothelial cells through shear stress; Migration of vascular endothelial cells; Induction of lamellipodial protrusions.

The migration of vascular endothelial cells (ECs) plays an important role in vascular remodeling. Here we studied the effects of cell morphology on the migration of bovine aortic ECs by culturing cells on micropatterned strips of collagen matrix (60-, 30-, and 15-μm wide). The spreading areas of the cells on 15- and 30-μm wide strips were 30% lower than those on 60- μm wide strips and unpatterned collagen. The cells on 15-μm wide strips completely aligned in the direction of the strip, and had significantly lower shape index than those in all other groups. On strips of all widths, ECs tended to migrate in the direction of strips. ECs on 15-μm wide strips had highest speed, particularly in the direction of the strip. Vinculin staining showed that the leading edge of ECs on 15-μm wide strips had focal adhesions that were oriented with their lamellipodial protrusion and the direction of cell migration; this arrangement of the focal adhesions may promote EC migration. The present study provides direct evidence on the role of cell morphology in EC migration, and will help us to understand the mechanisms of EC migration during angiogenesis and wound healing. [ABSTRACT FROM AUTHOR]

New viscometers for blood viscometry, improved intravital microscope, and related instruments, which are capable of measuring important rheological factors for microcirculatory research were developed and applied for hemorheological studies. As the results, four major determinants of the suspension viscosity were determined and the role of suspension viscosity of the blood as a function of microcirculatory flow was clarified. Meanwhile, the use of fluorescence microscopy and digitized video microscopic techniques has allowed the investigation of the structure and function of cells at the level of the single intact living cell. In addition, the developments and applications of laser confocal microscopy provided for not only three-dimensional image construction, but also four-dimensional image construction. In recent years, the dramatic advances in molecular biology and instrumental technology have made it possible to probe the structural and functional bases of cellular events at the molecular level. The ultimate understanding of biorheological behavior is dependent upon a molecular basis. In the future, the use of molecular biological techniques and nanotechnology (e.g., laser tweezers, atomic force microscopy and near-field microscopy, etc.) will allow the understanding of normal and abnormal rheological properties of cells, tissues, organs, and systems at the molecular level, and will contribute to progress in biomedical science. [ABSTRACT FROM AUTHOR]