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Endothelial cells (ECs) release ATP in response to shear stress, a mechanical force generated by blood flow, and the ATP released modulates EC functions through activation of purinoceptors. The molecular mechanism of the shear stress-induced ATP release, however, has not been fully elucidated. In this study, we have demonstrated that cell surface ATP synthase is involved in shear stress-induced ATP release. Immunofluorescence staining of human pulmonary arterial ECs (HPAECs) showed that cell surface ATP synthase is distributed in lipid rafts and co-localized with caveolin-1, a marker protein of caveolae. Immunoprecipitation indicated that cell surface ATP synthase and caveolin-1 are physically associated. Measurement of the extracellular metabolism of [[sup.3]H]ADP confirmed that cell surface ATP synthase is active in ATP generation. When exposed to shear stress, HPAECs released ATP in a dose-dependent manner, and the ATP release was markedly suppressed by the membrane-impermeable ATP synthase inhibitors angiostatin and piceatannol and by an anti-ATP synthase antibody. Depletion of plasma membrane cholesterol with methyl-[beta]-cyclodextrin (M[beta]CD) disrupted lipid rafts and abolished co-localization of ATP synthase with caveolin-1, which resulted in a marked reduction in shear stress-induced ATP release. Pretreatment of the cells with cholesterol prevented these effects of M[beta]CD. Downregulation of caveolin-1 expression by transfection of caveolin-1 siRNA also markedly suppressed ATP-releasing responses to shear stress. Neither M[beta]CD, M[beta]CD plus cholesterol, nor caveolin-1 siRNA had any effect on the amount of cell surface ATP synthase. These results suggest that the localization and targeting of ATP synthase to caveolae/lipid rafts is critical for shear stress-induced ATP release by HPAECs.

Partial hepatectomy causes hemodynamic changes that increase portal blood flow in the remaining lobe, where the expression of immediate-early genes, including plasminogen activator inhibitor-1 (PAL-1), is induced. We hypothesized that a hyperdynamic circulatory state occurring in the remaining lobe induces immediate-early gene expression. In this study, we investigated whether the mechanical force generated by flowing blood, shear stress, induces PAI-1 expression in hepatocytes. When cultured rat hepatocytes were exposed to flow, PAI-1 mRNA levels began to increase within 3 h, peaked at levels significantly higher than the static control levels, and then gradually decreased. The flow-induced PAI-1 expression was shear stress dependent rather than shear rate dependent and accompanied by increased hepatocyte production of PAI-1 protein. Shear stress increased PAI-1 transcription but did not affect PAI-1 mRNA stability. Functional analysis of the 2.1-kb PAI-1 5'-promoter indicated that a 278-bp segment containing transcription factor Sp1 and Ets-1 consensus sequences was critical to the shear stress-dependent increase of PAI-1 transcription. Mutations of both the Sp1 and Ets-1 consensus sequences, but not of either one alone, markedly prevented basal PAI-1 transcription and abolished the response of the PAI-1 promoter to shear stress. EMSA and chromatin immunoprecipitation assays showed binding of Sp1 and Ets-1 to each consensus sequence under static conditions, which increased in response to shear stress. In conclusion, hepatocyte PAI-1 expression is flow sensitive and transcriptionally regulated by shear stress via cooperative interactions between Sp1 and Ets-1. portal flow; hemodynamic force; immediate-early gene; partial hepatectomy; liver regeneration

Author(s): Kimiko Yamamoto [1, 6]; Takaaki Sokabe [1, 6]; Takahiro Matsumoto [2]; Kimihiro Yoshimura [2]; Masahiro Shibata [1]; Norihiko Ohura [1]; Toru Fukuda [2]; Takashi Sato [2]; Keisuke Sekine [2]; [...]

To study the role of nitric oxide (NO) in regulating oxygen consumption by vessel walls, the oxygen consumption rate of arteriolar walls in rat cremaster muscle was measured in vivo during flow-induced vasodilation and after inhibiting NO synthesis. The oxygen consumption rate of arteriolar walls was calculated based on the intra- and perivascular P[o.sub.2] values measured by phosphorescence quenching laser microscopy. The perivascular P.sub.[o.sub.2] value of the arterioles during vasodilation was significantly higher than under control conditions, although the intravascular P[o.sub.2] values under both conditions were approximately the same. Inhibition of NO synthesis, on the other hand, caused a significant increase in arterial blood pressure and a significant decrease in arteriolar diameter. Inhibition of NO synthesis also caused a significant decrease in both the intra- and perivascular P[o.sub.2] values of the arterioles. Inhibition of NO synthesis increased the oxygen consumption rate of the vessel walls by 42%, whereas enhancement of flow-induced NO release decreased it by 34%. These results suggest that NO plays an important role not only as a regulator of peripheral vascular tone but also as a modulator of tissue oxygenation by reducing oxygen consumption by vessel walls. In addition, enhancement of NO release during exercise may facilitate efficient oxygen supply to the surrounding high metabolic tissue. flow-induced vasodilation; [[N.sup.[omega]]-nitro-L-arginine methyl ester; oxygen transport; vascular tone; vascular endothelial cells

To examine the effects of vascular tone reduction on [O.sub.2] consumption of the vascular wall, we determined the [O.sub.2] consumption rates of arteriolar walls under normal conditions and during vasodilation induced by topical application of papaverine. A phosphorescence quenching technique was used to quantify intra- and perivascular P [O.sub.2] in rat cremaster arterioles with different branching orders. Then, the measured radial P[O.sub.2] gradients and a theoretical model were used to estimate the [O.sub.2] consumption rates of the arteriolar walls. The vascular [O.sub.2] consumption rates of functional arterioles were > 100 times greater than those observed in in vitro experiments. The vascular [O.sub.2] consumption rate was highest in first-order (1A) arterioles, which are located upstream, and sequentially decreased downstream in 2A and 3A arterioles under normal conditions. During papaverine-induced vasodilation, on the other hand, the [O.sub.2] consumption rates of the vascular walls decreased to similar levels, suggesting that the high [O.sub.2] consumption rates of 1A arterioles under normal conditions depend in part on the workload of the vascular smooth muscle. These results strongly support the hypothesis that arteriolar walls consume a significant amount of [O.sub.2] compared with the surrounding tissue. Furthermore, the reduction of vascular tone of arteriolar walls may facilitate an efficient supply of [O.sub.2] to the surrounding tissue. oxygen diffusion; vascular wall; vascular tone; vasodilation; papaverine

Pluripotent embryonic stem (ES) cells are capable of differentiating into all cell lineages, but the molecular mechanisms that regulate ES cell differentiation have not been sufficiently explored. In this study, we report that shear stress, a mechanical force generated by fluid flow, can induce ES cell differentiation. When Flk-1-positive (Flk-[1.sup.+]) mouse ES cells were subjected to shear stress, their cell density increased markedly, and a larger percentage of the cells were in the S and [G.sub.2]-M phases of the cell cycle than Flk-[1.sup.+] ES cells cultured under static conditions. Shear stress significantly increased the expression of the vascular endothelial cell-specific markers Flk-1, Flt-1, vascular endothelial cadherin, and PECAM-1 at both the protein level and the mRNA level, but it had no effect on expression of the mural cell marker smooth muscle [alpha]-actin, blood cell marker CD3, or the epithelial cell marker keratin. These findings indicate that shear stress selectively promotes the differentiation of Flk-[1.sup.+] ES cells into the endothelial cell lineage. The shear stressed Flk-[1.sup.+] ES cells formed tubelike structures in collagen gel and developed an extensive tubular network significantly faster than the static controls. Shear stress induced tyrosine phosphorylation of Flk-1 in Flk-[1.sup.+] ES cells that was blocked by a Flk-1 kinase inhibitor, SU1498, but not by a neutralizing antibody against VEGF. SU1498 also abolished the shear stress-induced proliferation and differentiation of Flk-[1.sup.+] ES cells, indicating that a ligand-independent activation of Flk-1 plays an important role in the shear stress-mediated proliferation and differentiation by Flk-[1.sup.+] ES cells. hemodynamic force; blood vessel; vascular endothelial growth factor; mechanical stress; neovascularization

Differential regulation of urokinase-type plasminogen activator expression by fluid shear stress in human coronary artery endothelial cells. Am J Physiol Heart Circ Physiol 287: H2027-H2034, 2004. First published July l, 2004; doi:10.1152/ajpheart.00260.2004.--Atherosclerotic plaques preferentially localize at arterial regions exposed to turbulent low-shear flow. Urokinase-type plasminogen activator (uPA) plays a role in vascular remodeling by facilitating smooth muscle cell migration and proliferation in addition to the proteolysis of extracellular matrix, and the expression of uPA is elevated in atherosclerotic lesions. In this study, we analyzed the effects of laminar and turbulent shear stress on uPA expression in cultured human coronary artery endothelial cells. The application of laminar shear stress (1.5 or 15 dyn/[cm.sup.2]) significantly decreased the amount of uPA mRNA as well as the secretion of uPA protein. In contrast, turbulent shear stress (average intensity, 1.5 dyn/[cm.sup.2]) markedly increased uPA gene expression and protein secretion. Laminar shear stress downregulated uPA gene expression transcriptionally and post-transcriptionally; laminar shear stress activated transcription factor GATA6, which binds to a GATA consensus element located between -692 and -687 bp in the uPA promoter, thereby inhibiting uPA gene transcription. Laminar shear stress also accelerated the degradation of uPA mRNA; the half-life of uPA mRNA decreased to about half of the static control's half-life. Although turbulent shear stress had no effect on the transcription of uPA, it significantly increased uPA mRNA stability; the half-life of uPA mRNA increased by about two times the static control's half-life. Our results suggest that endothelial uPA expression is flow sensitive and differentially regulated by laminar and turbulent shear stress in vitro. We speculate that this effect may contribute to the local nature of atherosclerosis. hemodynamic force: atherosclerosis; GATA; mRNA stability

The mechanisms by which flow-imposed shear stress elevates intracellular [Ca.sup.2+] in cultured endothelial cells (ECs) are not fully understood. Here we report finding that endogenously released ATP contributes to shear stress-induced [Ca.sup.2+] responses. Application of flow of Hanks' balanced solution to human pulmonary artery ECs (HPAECs) elicited shear stress-dependent increases in [Ca.sup.2+] concentrations. Chelation of extracellular [Ca.sup.2+] with EGTA completely abolished the [Ca.sup.2+] responses, whereas the phospholipase C inhibitor U-73122 or the [Ca.sup.2+]-ATPase inhibitor thapsigargin had no effect, which thereby indicates that the response was due to the influx of extracellular [Ca.sup.2+]. The [Ca.sup.2+] influx was significantly suppressed by apyrase, which degrades ATP, or antisense oligonucleotide targeted to [P2X.sub.4] purinoceptors. A luciferase luminometric assay showed that shear stress induced dose-dependent release of ATP. When the ATP release was inhibited by the ATP synthase inhibitors angiostatin or oligomycin, the [Ca.sup.2+] influx was markedly suppressed but was restored by removal of these inhibitors or addition of extracellular ATP. These results suggest that shear stress stimulates HPAECs to release ATP, which activates [Ca.sup.2+] influx via [P2X.sub.4] receptors. hemodynamic force; mechanotransduction; ATP synthase; purinoceptors; and [P2X.sub.4] receptor

Stimulation of endothelial cells (ECs) with ATP evoked an increase in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]). In a single bovine aortic EC, the [[[Ca.sup.2+]].sub.i] rise started at a specific peripheral locus and propagated throughout the entire cell as a [Ca.sup.2+] wave. The initiation locus was constant upon repeated stimulation with ATP or other agonists (bradykinin and thrombin). The [Ca.sup.2+] wave was unaffected by the removal of extracellular [Ca.sup.2+], demonstrating its dependence on intracellular [Ca.sup.2+] release. Microinjection of heparin into the cell inhibited the ATP-induced [Ca.sup.2+] responses, indicating that the [Ca.sup.2+] wave is at least partly mediated by the inositol 1,4,5-trisphosphate receptor. Immunofluorescence staining revealed that caveolin, a marker protein for caveolae, is distributed heterogeneously in the cell and that [Ca.sup.2+] waves preferentially originate at caveolin-rich cell edges. In contrast to caveolin, internalized transferrin and subunits of the clathrin-associated adaptor complexes such as adaptor protein-1 and -2 were diffusely distributed. Disruption of microtubules by Colcemid led to redistribution of caveolin away from the edges into the perinuclear center of the cell, and the ATP-induced [[[Ca.sup.2+]].sub.i] increase was initiated on the rim of the centralized caveolin. Thus, caveolae may be involved in the initiation of ATP-induced [Ca.sup.2+] waves in ECs.

The molecular mechanism of the downregulation of vascular cell adhesion molecule 1 (VCAM-1) gene expression by shear stress was examined by applying nuclear run-on assay and luciferase assay. A reporter gene consisting of the VCAM-1 promoter was coupled with luciferase. A cis-acting element in the promoter was also localized to study its function in the negative regulation by shear stress. Results suggest that VCAM-1 mRNA response is shear stress dependent rather than shear rate dependent.

Application of medium flow in a parallel plate flow chamber to monolayers of endothelial cells (EC) from mice to study the effects of shear stress on the adhesion of EC to lymphocytes and identify the adhesive molecules affected by shear stress reveals that adhesiveness is inhibited by shear stress in a time- and magnitude of stress-dependent manner. Adhesion of stressed EC to lymphocytes decreases to 68.8 plus or minus 12.8% of that observed in control EC. Flow cytometric analysis reveals that flow loading reduces the surface expression of vascular cell adhesion molecule-1 to 48.5 plus or minus 15.8% of control.