Joji Ando

Impaired flow-dependent control of vascular tone and remodeling in P2X4-deficient mice

The structure and function of blood vessels adapt to environmental changes such as physical development and exercise. This phenomenon is based on the ability of the endothelial cells to sense and respond to blood flow; however, the underlying mechanisms remain unclear. Here we show that the ATP-gated P2X4 ion channel, expressed on endothelial cells and encoded by P2rx4 in mice, has a key role in the response of endothelial cells to changes in blood flow. P2rx4−/− mice do not have normal endothelial cell responses to flow, such as influx of Ca2+ and subsequent production of the potent vasodilator nitric oxide (NO). Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2rx4−/− mice. Furthermore, P2rx4−/− mice have higher blood pressure and excrete smaller amounts of NO products in their urine than do wild-type mice. Moreover, no adaptive vascular remodeling, that is, a decrease in vessel size in response to a chronic decrease in blood flow, was observed in P2rx4−/− mice. Thus, endothelial P2X4 channels are crucial to flow-sensitive mechanisms that regulate blood pressure and vascular remodeling.

Cytoplasmic calcium response to fluid shear stress in cultured vascular endothelial cells.

SummaryVascular endothelial cells modulate their structure and functions in response to changes in hemodynamic forces such as fluid shear stress. We have studied how endothelial cells perceive the shearing force generated by blood flow and the substance(s) that may mediate such a response. We identify cytoplasmic-free calcium ion (Ca++), a major component of an internal signaling system, as a mediator of the cellular response to fluid shear stress. Cultured monolayers of bovine aortic endothelial cells loaded with the highly fluorescent Ca++-sensitive dye Fura 2 were exposed to different levels of fluid shear stress in a specially designed flow chamber, and simultaneous changes in fluorescence intensity, reflecting the intracellular-free calcium concentration ([Ca++]i), were monitored by photometric fluorescence microscopy. Application of shear stress to cells by fluid perfusion led to an immediate severalfold increase in fluorescence within 1 min, followed by a rapid decline for about 5 min, and finally a plateau somewhat higher than control levels during the entire period of the stress application. Repeated application of the stress induced similar peak and plateau levels of [Ca++]i but at reduced magnitudes of response. These responses were observed even in Ca++-free medium. Thus, a shear stress transducer might exist in endothelial cells, which perceives the shearing force on the membrane as a stimulus and mediates the signal to increase cytosolic free Ca++.

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.

The effect of fluid shear stress on the migration and proliferation of cultured endothelial cells

We have examined the effect of shear stress on the regenerative response of cultured vascular endothelial cells by using a fluid shear apparatus designed in our laboratory. The shear stress was created on the endothelial cell layer of a fetal calf and grown confluently in a culture dish by whirling the medium, with a rotating disk placed on the fluid surface. The effect of the shear load (0.3-1.7 dyn/cm2) over 24 hr was evaluated by counting the number of regenerated cells in a denuded area that had been created by mechanically removing some cells before rotating the medium. The cell number observed in the denuded area after the exposure to shear stress was about twice as great as that of the static control. The difference was statistically significant (P less than 0.01 to P less than 0.05). Cell migration and proliferation occurred more prominently in the downstream portion of the flow than in the upstream part. The cell number in the downstream portions correlated significantly with the intensity of the applied shear stress (P less than 0.05). These results indicate that shear stress can stimulate the migration and proliferation of endothelial cells.

Effects of shear stress and stretch on endothelial function.

Vascular endothelial cells (ECs) play a central role in the control of blood vessel function and circulatory system homeostasis. It is well known that that EC functions are regulated by chemical mediators, including hormones, cytokines, and neurotransmitters, but it has recently become apparent that EC functions are also controlled by hemodynamic forces such as shear stress and stretch (cyclic strain). ECs recognize shear stress and cyclic strain as mechanical stimuli, and transmit the signal into the interior of the cells, thereby triggering a variety of cellular responses that involve alterations in cell morphology, cell function, and gene expression. Impaired EC responses to shear stress and cyclic strain lead to vascular diseases, including hypertension, thrombosis, and atherosclerosis. A great deal of research has already been conducted on the mechanotransduction of shear stress and cyclic strain, and its molecular mechanisms are gradually coming to be understood. However, much remains unclear, and further studies of mechanotransduction should increase our understanding of the molecular basis of the hemodynamic-force-mediated control of vascular functions.

Shear Stress Increases Expression of the Arterial Endothelial Marker EphrinB2 in Murine ES Cells via the VEGF-Notch Signaling Pathways

Objective—Arterial-venous specification in the embryo has been assumed to depend on the influence of fluid mechanical forces, but its cellular and molecular mechanisms are still poorly understood. Our previous in vitro study revealed that fluid shear stress induces endothelial cell (EC) differentiation by murine embryonic stem (ES) cells. In the present study we investigated whether shear stress regulates the arterial-venous specification of ES-cell-derived ECs. Methods and Results—When murine ES cell–derived VEGFR2+ cells were exposed to shear stress, expression of the arterial EC marker protein ephrinB2 increased dose-dependently. The ephrinB2 mRNA levels also increased in response to shear stress, whereas the mRNA levels of the venous EC marker EphB4 decreased. Notch cleavage and translocation of the Notch intracellular domain (NICD) into the nucleus occurred as early as 30 minutes after the start of shear stress and increased with time. Gamma-Secretase inhibitors (DAPT and L685 458) and the recombinant extracellular domain of the Notch ligand DLL4 abolished the shear stress–induced NICD translocation, and that, in turn, blocked the shear stress–induced upregulation of ephrinB2 expression. In addition, the VEGF receptor kinase inhibitor SU1498 was found to suppress both the shear-stress-induced Notch cleavage and up-regulation of ephrinB2 expression. Conclusion—Exposure to shear stress induces an increase in expression of ephrinB2 in murine ES cells via VEGF-Notch signaling pathways.

Cyclic strain induces mouse embryonic stem cell differentiation into vascular smooth muscle cells by activating PDGF receptor β

Embryonic stem (ES) cells are exposed to fluid-mechanical forces, such as cyclic strain and shear stress, during the process of embryonic development but much remains to be elucidated concerning the role of fluid-mechanical forces in ES cell differentiation. Here, we show that cyclic strain induces vascular smooth muscle cell (VSMC) differentiation in murine ES cells. Flk-1-positive (Flk-1+) ES cells seeded on flexible silicone membranes were subjected to controlled levels of cyclic strain and examined for changes in cell proliferation and expression of various cell lineage markers. When exposed to cyclic strain (4-12% strain, 1 Hz, 24 h), the Flk-1+ ES cells significantly increased in cell number and became oriented perpendicular to the direction of strain. There were dose-dependent increases in the VSMC markers smooth muscle alpha-actin and smooth muscle-myosin heavy chain at both the protein and gene expression level in response to cyclic strain, whereas expression of the vascular endothelial cell marker Flk-1 decreased, and there were no changes in the other endothelial cell markers (Flt-1, VE-cadherin, and platelet endothelial cell adhesion molecule 1), the blood cell marker CD3, or the epithelial marker keratin. The PDGF receptor beta (PDGFR beta) kinase inhibitor AG-1296 completely blocked the cyclic strain-induced increase in cell number and VSMC marker expression. Cyclic strain immediately caused phosphorylation of PDGFR beta in a dose-dependent manner, but neutralizing antibody against PDGF-BB did not block the PDGFR beta phosphorylation. These results suggest that cyclic strain activates PDGFR beta in a ligand-independent manner and that the activation plays a critical role in VSMC differentiation from Flk-1+ ES cells.

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).