Endothelial cell functions, such as arachidonic acid metabolism, may be modulated by membrane stresses induced by blood flow. The production of prostacyclin by primary human endothelial cell cultures subjected to pulsatile and steady flow shear stress was measured. The onset of flow led to a sudden increase in prostacyclin production, which decreased to a steady rate within several minutes. The steady-state production rate of cells subjected to pulsatile shear stress was more than twice that of cells exposed to steady shear stress and 16 times greater than that of cells in stationary culture.

Using DNA microarray screening (GeneFilter 211, Research Genetics, Huntsville, AL) of mRNA from primary human umbilical vein endothelial cells (HUVEC), we identified 52 genes with significantly altered expression under shear stress [25 dynes͞cm 2 for 6 or 24 h (1 dyne ‫؍‬ 10 N), compared with matched stationary controls]; including several genes not heretofore recognized to be shear stress responsive. We examined mRNA expression of nine genes by Northern blot analysis, which confirmed the results obtained on DNA microarrays. Thirty-two genes were up-regulated (by more than 2-fold), the most enhanced being cytochromes P450 1A1 and 1B1, zinc finger protein EZF͞GKLF, glucocorticoid-induced leucine zipper protein, argininosuccinate synthase, and human prostaglandin transporter. Most dramatically decreased (by more than 2-fold) were connective tissue growth factor, endothelin-1, monocyte chemotactic protein-1, and spermidine͞spermine N1-acetyltransferase. The changes observed suggest several potential mechanisms for increased NO production under shear stress in endothelial cells. During the past 15 years, over 40 genes have been identified as being regulated by shear stress in endothelial cells (1-4). Shear stress responsive genes are involved in cell proliferation, differentiation, maintenance of vascular tone, thrombosis, cellmatrix and cell-cell adhesion, and modulation of the inflammatory͞immune system. The identification of such genes is important not only for developing a fundamental understanding of how endothelial cells work, but also for understanding and treating pathological conditions that are influenced by shear stress, such as thrombosis, restenosis, and atherosclerosis (5, 6).Most of the genes that have been shown to be regulated by shear stress were identified by using traditional techniques such as Northern blot analysis or reverse transcriptase PCR (7-9). The main limitation of these techniques is that only one gene or at best a handful of genes can be studied in one experiment. When multiple genes are studied by using traditional methods, the experiments usually require a reiteration of the detection procedure for each gene. Investigators must therefore be very selective in the genes they choose to study, necessitating a priori information linking the chosen genes to shear stress. Thus, these experiments generally tend to validate or disprove specific hypotheses and do not lead to the discovery of unexpected differentially expressed genes. However, DNA microarray technology allows researchers to study several thousands of genes at one time. In addition to identifying unexpected genes, this technology also has the power to lead to the development of new hypotheses concerning how cells respond to shear stress and identification of coregulated pathways responsive to the mechanical environment of the cell.We used DNA GeneFilter GF211 from Research Genetics (Huntsville, AL; ref. 10), which contains over 4,000 named human genes, to identify genes altered by shear stress in primary human umbilical vein en...

A flow apparatus has been developed f o r t h e study of t h e metabolic response o f anchorage-dependent c e l l s t o a wide range o f s t e a d y and p u l s a t i l e s h e a r stresses under well-controlled conditions. Human umb i l i c a l vein e n d o t h e l i a l c e l l monolayers were s u b j e c t e d t o s t e a d y shear stresses of up t o 24 dynes/cm2, and t h e production o f p r o s t a c y c l i n was determined. The onset of flow l e d t o a b u r s t i n p r o s t a c y c l i n production which decayed t o a long-term s t e a d y s t a t e rate (SSR). The SSR of c e l l s exposed t o flow was greater than the basal release l e v e l , and increased l i n e a r l y w i t h i n c r e a s i n g shear stress.T h i s s t u d y demonstrates t h a t shear stress i n c e r t a i n ranges may not be d e t r i m e n t a l t o mammalian c e l l metabolism.I n f a c t , throughout t h e range o f shear stresses s t u d i e d , metabolite production is maximized by maximizing shear stress.

As a key element in the cytoskeleton, actin filaments are highly dynamic structures that constantly sustain forces. However, the fundamental question of how force regulates actin dynamics is unclear. Using atomic force microscopy force-clamp experiments, we show that tensile force regulates G-actin/G-actin and G-actin/F-actin dissociation kinetics by prolonging bond lifetimes (catch bonds) at a low force range and by shortening bond lifetimes (slip bonds) beyond a threshold. Steered molecular dynamics simulations reveal force-induced formation of new interactions that include a lysine 113(K113):glutamic acid 195 (E195) salt bridge between actin subunits, thus suggesting a molecular basis for actin catch-slip bonds. This structural mechanism is supported by the suppression of the catch bonds by the single-residue replacements K113 to serine (K113S) and E195 to serine (E195S) on yeast actin. These results demonstrate and provide a structural explanation for actin catchslip bonds, which may provide a mechanoregulatory mechanism to control cell functions by regulating the depolymerization kinetics of force-bearing actin filaments throughout the cytoskeleton.single-molecule force spectroscopy | mechanotransduction | mechanosensing | nemaline myopathy T he actin cytoskeleton, primarily a force-bearing structure, controls the morphology, motility, and adhesion of the cell (1-4). Its core filamentous component, assembled from actin monomers via noncovalent interactions (5), undergoes rapid and controlled polymerization and depolymerization, allowing the dynamic reorganization of the actin cytoskeleton (1, 2).In cells, this dynamic process can be modulated by forces, and this is crucial to mechanosensitivity, mechanotransduction, and cellular adaptations to mechanical stresses (3, 6-8). For example, the assembly, stabilization, and reorganization of the actin stress fiber and the focal adhesion, where actin filaments constantly sustain tension, are induced by externally applied forces (9-12) dependent on myosin-generated contractility (4,8,13,14) and sensitive to substrate rigidity (3, 15, 16). These observations led us to investigate the molecular mechanism by which actin dynamics are regulated by force.The force-regulated kinetics of several molecular interactions important to adhesion and force-bearing functions of cells are governed by catch-slip bonds, in which the interaction is stabilized by tensile force in a low range and destabilized when force exceeds a threshold (17)(18)(19)(20)(21)(22). Various mechanisms, such as the allosteric model based on intramolecular conformational change under forces (23,24) and the sliding-rebinding model based on force-induced formation of new interactions due to intermolecular interface sliding (18,25), have been proposed to provide structural explanations for catch-slip bonds in different molecular interactions.Here we use atomic force microscopy (AFM) force-clamp experiments to determine how force regulates the off-rate of actin depolymerization and to elucidate the structural ...