Risa Korenaga

Fluid Shear Stress Transcriptionally Induces Lectin-like Oxidized LDL Receptor-1 in Vascular Endothelial Cells

Fluid shear stress has been shown to modulate various endothelial functions, including gene expression. In this study, we examined the effect of fluid shear stress on the expression of lectin-like oxidized LDL receptor-1 (LOX-1), a novel receptor for atherogenic oxidized LDL in cultured bovine aortic endothelial cells (BAECs). Exposure of BAECs to the physiological range of shear stress (1 to 15 dyne/cm2) upregulated LOX-1 protein and mRNA in a time-dependent fashion. LOX-1 mRNA levels peaked at 4 hours, and LOX-1 protein levels peaked at 8 hours. Inhibition of de novo RNA synthesis by actinomycin D totally abolished shear stress-induced LOX-1 mRNA expression. Furthermore, nuclear runoff assay showed that shear stress directly stimulates transcription of the LOX-1 gene. Chelation of intracellular Ca2+ with quin 2-AM completely reduced shear stress-induced LOX-1 mRNA expression; furthermore, the treatment of BAECs with ionomycin upregulated LOX-1 mRNA levels in a dose-dependent manner. Taken together, physiological levels of fluid shear stress can regulate LOX-1 expression by a mechanism dependent on intracellular Ca2+ mobilization. Inducible expression of LOX-1 by fluid mechanics may play a role in localized expression of LOX-1 and atherosclerotic lesion formation in vivo.

Shear Stress Augments Expression of C-Type Natriuretic Peptide and Adrenomedullin

Shear stress is known to dilate blood vessels and exert antiproliferative effects on vascular walls: these effects have been ascribed to shear stress-induced upregulation of endothelium-derived vasoactive substances, mainly nitric oxide and prostacyclin. We have demonstrated the significance of C-type natriuretic peptide (CNP) as a novel endothelium-derived relaxing peptide (EDRP) that shares a cGMP pathway with nitric oxide. Adrenomedullin is a recently isolated EDRP that elevates intracellular cAMP as prostacyclin does. To elucidate the possible role of these EDRPs under shear stress, we examined the effect of physiological shear stress on CNP mRNA expression in endothelial cells derived from the human umbilical vein (HUVECs), bovine aorta (BAECs), and murine lymph nodes (MLECs) as well as adrenomedullin mRNA expression in HUVECs. CNP mRNA was stimulated prominently in HUVECs under shear stress of 15 dyne/cm2 in a time-dependent manner (4 hours, sixfold increase compared with that in the static condition; 24 hours, 30-fold increase). Similar results were obtained in BAECs (4 hours, twofold increase; 24 hours, threefold increase) and MLECs (4 hours, threefold increase; 24 hours, 10-fold increase). Augmentation of CNP mRNA expression that was dependent on shear stress intensity was also observed (5 dyne/cm2, 2.5-fold increase of static; 15 dyne/cm2, 4.5-fold increase). Increased CNP secretion was also confirmed by the specific radioimmunoassay for CNP. Adrenomedullin mRNA expression in HUVECs increased under shear stress of 15 dyne/cm2 in a time-dependent manner (4 hours, 1.2-fold increase of static: 24 hours, threefold increase) and shear stress intensity-dependent manner (15 dyne/cm2, threefold increase compared with that at 5 dyne/cm2). These results suggest that the coordinated augmentation of mRNA expression of these novel EDRPs may constitute shear stress-dependent vasodilator and antiproliferative effects.

Fluid Shear Stress Activates Ca2+ Influx Into Human Endothelial Cells via P2X4 Purinoceptors

Ca2+ signaling plays an important role in endothelial cell (EC) responses to shear stress generated by blood flow. Our previous studies demonstrated that bovine fetal aortic ECs showed a shear stress–dependent Ca2+ influx when exposed to flow in the presence of extracellular ATP. However, the molecular mechanisms of this process, including the ion channels responsible for the Ca2+ response, have not been clarified. Here, we demonstrate that P2X4 purinoceptors, a subtype of ATP-operated cation channels, are involved in the shear stress–mediated Ca2+ influx. Human umbilical vein ECs loaded with the Ca2+ indicator Indo-1/AM were exposed to laminar flow of Hanks’ balanced salt solution at various concentrations of ATP, and changes in [Ca2+]i were monitored with confocal laser scanning microscopy. A stepwise increase in shear stress elicited a corresponding stepwise increase in [Ca2+]i at 250 nmol/L ATP. The shear stress–dependent increase in [Ca2+]i was not affected by phospholipase C inhibitor (U-73122) but disappeared after the chelation of extracellular Ca2+ with EGTA, indicating that the Ca2+ increase was due to Ca2+ influx. Antisense oligonucleotides designed to knockout P2X4 expression abolished the shear stress–dependent Ca2+ influx seen at 250 nmol/L ATP in human umbilical vein ECs. Human embryonic kidney 293 cells showed no Ca2+ response to flow at 2 &mgr;mol/L ATP, but when transfected with P2X4 cDNA, they began to express P2X4 purinoceptors and to show shear stress–dependent Ca2+ influx. P2X4 purinoceptors may have a “shear-transducer” property through which shear stress is perceived directly or indirectly and transmitted into the cell interior via Ca2+ signaling.

Contribution of Sustained Ca Elevation for Nitric Oxide Production in Endothelial Cells and Subsequent Modulation of Ca Transient in Vascular Smooth Muscle Cells in Coculture

To elucidate the intracellular Ca (Ca) transient responsible for nitric oxide (NO) production in endothelial cells (ECs) and the subsequent Ca reduction in vascular smooth muscle cells (VSMCs), we administrated four agonists with different Ca-mobilizing mechanisms for both cells in iso- or coculture. We monitored the Ca of both cells by two-dimensional fura-2 imaging, simultaneously measuring NO production as NO. The order of potency of the agonists in terms of the peak Ca in ECs was bradykinin (100 nM) > ATP (10 μM) > ionomycin (50 nM) > thapsigargin (1 μM). In contrast, the order in reference to both the extent of Ca reduction in cocultured VSMCs and the elevation in NO production over the level of basal release in ECs completely matched and was ranked as thapsigargin > ionomycin > ATP > bradykinin. Treatment by N-monomethyl-L-arginine monoacetate but not indomethacin or glybenclamide restored the Ca response in cocultured VSMCs to the isoculture level. In ECs, when the Ca influx was blocked by Ni or by chelating extracellular Ca, all four agonists markedly decreased NO production, the half decay time of the Ca degenerating phase, and the area under the Ca curve. The amount of produced NO hyperbolically correlated to the half decay time and the area under the Ca curve but not to the Ca peak level. Thus, the sustained elevation of Ca in ECs, mainly a result of Ca influx, determines the active NO production and subsequent Ca reduction in adjacent VSMCs. Furthermore, L-arginine but not D-arginine or L-lysine at high dose (5 mM) without agonist enhanced the NO production, weakly reduced the Ca in ECs, and markedly decreased the Ca in VSMCs, demonstrating the autocrine and paracrine effects of NO (Shin, W. S., Sasaki, T., Kato, M., Hara, K., Seko, A., Yang, W. D., Shimamoto, N., Sugimoto, T., and Toyo-oka, T.(1992) J. Biol. Chem. 267, 20377-20382).

Fluid Shear Stress Increases the Production of Granulocyte-Macrophage Colony-Stimulating Factor by Endothelial Cells via mRNA Stabilization

To investigate whether the production of colony-stimulating factors (CSFs) by vascular endothelial cells is regulated by hemodynamic force, we exposed cultured human umbilical vein endothelial cells (HUVECs) to controlled levels of shear stress in a flow-loading apparatus and examined changes in the production of CSFs at both the protein and mRNA level. Exposure of HUVECs to a shear stress of 15 and 25 dyne/cm2 markedly increased the release of granulocyte-macrophage CSF (GM-CSF) detected by ELISA to 5.0 and 9.5 times, respectively, the amount released by the static controls at 24 hours, but it had no significant influence on the release of granulocyte CSF or macrophage CSF. The results of reverse transcriptase-polymerase chain reaction demonstrated that GM-CSF mRNA began to increase as early as 2 hours after initiation of 15 dyne/cm2 shear stress and continued to increase with time, reaching a peak of about four times the control levels at 24 hours. This increase in GM-CSF mRNA levels in response to shear stress depended on protein synthesis, because it was blocked by cycloheximide. Neither nuclear run-on assay or luciferase assay using a reporter gene containing GM-CSF gene promoter showed any significant change in transcription of the GM-CSF gene even after 24-hour exposure to a shear stress of 15 dyne/cm2. Actinomycin D chase experiments using a competitive polymerase chain reaction showed that shear stress extended the half-life of GM-CSF mRNA from approximately 23 to 42 minutes in HUVECs. These findings suggest that fluid shear stress increases the production of GM-CSF in HUVECs via mRNA stabilization.

Effect of extracellular ATP level on flow-induced Ca++ response in cultured vascular endothelial cells

Cultured vascular endothelial cells loaded with the highly fluorescent Ca(++)-sensitive dye Fura-2 were exposed to the flow of a fluid containing various concentrations of ATP (0, 0.5, 1, 5 microM) in an apparatus designed on the basis of fluid dynamics, and simultaneous changes in intracellular free Ca++ concentration were monitored by photometric fluorescence microscopy. The flow rate of the perfusate was altered from 0 to 6.3 to 22.8 to 39.0 cm/sec, inducing shear stress on the cell surface of 0, 2.9, 10.4, and 17.9 dynes/cm2, respectively. Although no significant change in intracellular Ca++ level was observed at ATP levels below 100 nM, at an ATP level of 500 nM, the intracellular Ca++ level increased together with an increase in the flow rate of the perfusate. At this level of ATP, the intracellular Ca++ levels at flow rates of 0, 6.3, 22.8, and 39.0 cm/sec were 44.8 +/- 7.3, 60.3 +/- 10.7, 74.0 +/- 5.8 and 89.4 +/- 6.4 nM (mean +/- SD; n = 8), respectively. At ATP levels over 1 microM, the flow-rate dependency of Ca++ response became less clear than that observed at the ATP level of 500 nM. These Ca++ responses to changes in flow rate disappeared when extracellular Ca++ was chelated by adding 2 mM of EGTA to the perfusate. These results suggest that the vascular endothelial cell has a mechanism that elevates the intracellular Ca++ level in accord with the flow rate at appropriate ATP concentrations, and that changes in intracellular Ca++ level under this mechanism seem to be chiefly caused by the influx of extracellular Ca++ into cells.

Down-Regulation of Vascular Adhesion Molecule-1 by Fluid Shear Stress in Cultured Mouse Endothelial Cellsa

This study was undertaken to determine whether blood flow modulates the adhesive property of vascular endothelial cells to lymphocytes and, if it does, what adhesion molecules are involved. Cultured mouse endothelial cells were exposed to medium flow in a parallel plate chamber, and binding assay using fluorescence-labeled lymphocytes was carried out. The adhesion rate of endothelial cells to lymphocytes, which was high in the static control state, decreased when exposed to shear stress (1.5 dynes/cm2) for 6 h. The treatment of static endothelial cells with a monoclonal antibody of vascular cell adhesion molecule-1 (VCAM-1) depressed the adhesion rate to the same extent as that caused by flow, while monoclonal antibodies of CD44 and intercellular adhesion molecule-1 had no effect on it. Flow cytometric analysis revealed that the application of flow decreased markedly the amount of VCAM-1 expressed on the cell surface. A reverse transcriptase-polymerase chain reaction of mRNA showed that flow depressed VCAM-1 mRNA levels. These results suggest that blood flow can modulate the adhesive property of endothelial cells to lymphocytes via affecting the surface expression of adhesion molecules, e.g., down-regulation of VCAM-1.

Physiologic shear stress suppresses endothelin-converting enzyme-1 expression in vascular endothelial cells

Shear stress dilates blood vessels and exerts an antiproliferative effect on vascular walls. These effects are ascribed to shear stress-induced, endothelium-derived vasoactive substances. Endothelin-converting enzymes (ECEs), the enzymes that convert big endothelin-1 (ET-1) to ET-1, have recently been isolated and the corresponding proteins have been termed ECE-1 and ECE-2. Furthermore, two isoforms of human ECE-1 have been demonstrated and termed ECE-1 alpha and ECE-1 beta. In this study, to elucidate the role of ECE-1 under shear stress we examined the effect of physiologic shear stress on the mRNA expression of ECE-1 and ET-1 in cultured bovine carotid artery endothelial cells (BAECs) and human umbilical veins (HUVECs), and also ECE-1 alpha mRNA expression in HUVECs. ECE-1 mRNA expression was significantly downregulated by shear stress in 24 h, both in BAECs and HUVECs, in a shear stress intensity-dependent manner. The expression of ECE-1 alpha mRNA was also attenuated by shear stress in HUVECs. ET-1 mRNA expression showed a concordant decrease with ECE-1 mRNA expression. These results suggest that shear stress-induced gene regulation of ET-1 and ECE-1 mRNA expression can contribute to the decrease of ET-1 peptide level by shear stress.

Intracellular calcium response to directly applied mechanical shearing force in cultured vascular endothelial cells.

We studied the responses of cultured endothelial cells to mechanical shearing force directly applied to those cells in vitro to determine changes in the concentration of intracellular calcium ion (Ca++), one of the factors that transfers information within the cell. Cultured bovine fetal aortic endothelial cells containing the Ca++ fluorescence indicator, Fura-2, were rubbed with a latex balloon in a specially designed system, and changes in the fluorescence of Fura-2 caused by this shear stimulation were determined by photometric fluorescence microscopy. Immediately after shear stimulation, the concentration of Ca++ in the cells was increased and reached a peak (511 +/- 165 nM, n = 12) within 15 seconds after stimulation. After the peak, the concentration was gradually restored to the resting level (55 +/- 17 nM, n = 12). The magnitude of the Ca++ response was dependent on the intensity of the shear force applied. Analysis of fluorescence images of Fura-2 revealed that the cells showed this Ca++ reaction without being injured or desquamated, although there were slight differences in the degree and duration of reaction among cells. This reaction appeared even when the cells were placed in the air with no contact with the fluid. This result suggests that neither the fluid flow associated with the balloon movement nor chemical substances in the fluid are involved in the reaction, but that pure physical force alone is responsible for the Ca++ reaction. Further, it suggests that endothelial cells have the ability to perceive such physical stimulation as shear force and to transfer this information to the interior of the cell via changes in the intracellular Ca++ concentration.

The effect of flow on the neutrophil-mediated Ca2+ responses in human vascular endothelial cells stimulated by endotoxin.

Leukocyte-vascular endothelial cell (EC) interactions which promote inflammatory and immune reactions involved bidirectional signaling between two cell types. We investigated the effects of flow on neutrophil-mediated changes in endothelial intracellular Ca2+ levels ([Ca2+]i). Cultured human umbilical vein ECs stimulated by endotoxin were labeled with Fura-2 and exposed to fluid flow with neutrophils. The individual changes in [Ca2+]i were monitored. The application of flow with neutrophils to stimulated ECs led to an increase [Ca2+]i although either flow without neutrophils or neutrophils without flow rarely induced a rise in [Ca2+]i. Furthermore, flow application with neutrophils to unstimulated ECs also rarely promoted a rise in [Ca2_]i. These findings suggest that the flow might thus induce or enhance the inflammatory process by the induction of Ca2+ signaling in endotoxin-stimulated endothelium facing neutrophils in the blood flow.