Vortex-mediated mechanical stress induces integrin-dependent cell adhesion mediated by inositol 1,4,5-trisphosphate-sensitive Ca2+ release in THP-1 cells.

In the downstream regions of stenotic vessels, cells are subjected to a vortex motion under low shear forces, and atherosclerotic plaques tend to be localized. It has been reported that such a change of shear force on endothelial cells has an atherogenic effect by inducing the expression of adhesion molecules. However, the effect of vortex-induced mechanical stress on leukocytes has not been investigated. In this study, to elucidate whether vortex flow can affect the cell adhesive property, we have examined the effect of vortex-mediated mechanical stress on integrin activation in THP-1 cells, a monocytic cell line, and its signaling mechanisms. When cells are subjected to vortex flow at 400-2,000 rpm, integrin-dependent cell adhesion to vascular cell adhesion molecule-1 or fibronectin increased in a speed- and time-dependent manner. Next, to examine the role of Ca(2+) in this integrin activation, various pharmacological inhibitors involved in Ca(2+) signaling were tested to inhibit the cell adhesion. Pretreatment of cells with BAPTA-AM, thapsigargin +NiCl(2), or U-73122 (a phospholipase C inhibitor) inhibited cell adhesion induced by vortex-mediated mechanical stress. We also found that W7 (a calmodulin inhibitor) blocked the cell adhesion. However, pretreatment of cells with GdCl(3), NiCl(2), or ryanodine did not affect the cell adhesion. These data indicate that vortex-mediated mechanical stress induces integrin activation through calmodulin and inositol 1,4,5-trisphosphate-mediated Ca(2+) releases from intracellular Ca(2+) stores in THP-1 cells.

In the downstream regions of stenotic vessels, cells are subjected to a vortex motion under low shear forces, and atherosclerotic plaques tend to be localized. It has been reported that such a change of shear force on endothelial cells has an atherogenic effect by inducing the expression of adhesion molecules. However, the effect of vortex-induced mechanical stress on leukocytes has not been investigated. In this study, to elucidate whether vortex flow can affect the cell adhesive property, we have examined the effect of vortex-mediated mechanical stress on integrin activation in THP-1 cells, a monocytic cell line, and its signaling mechanisms. When cells are subjected to vortex flow at 400 -2,000 rpm, integrin-dependent cell adhesion to vascular cell adhesion molecule-1 or fibronectin increased in a speed-and time-dependent manner. Next, to examine the role of Ca 2؉ in this integrin activation, various pharmacological inhibitors involved in Ca 2؉ signaling were tested to inhibit the cell adhesion. Pretreatment of cells with BAPTA-AM, thapsigargin ؉NiCl 2 , or U-73122 (a phospholipase C inhibitor) inhibited cell adhesion induced by vortex-mediated mechanical stress. We also found that W7 (a calmodulin inhibitor) blocked the cell adhesion. However, pretreatment of cells with GdCl 3 , NiCl 2 , or ryanodine did not affect the cell adhesion. These data indicate that vortex-mediated mechanical stress induces integrin activation through calmodulin and inositol 1,4,5-trisphosphate-mediated Ca 2؉ releases from intracellular Ca 2؉ stores in THP-1 cells.
The nature of blood flow patterns and shear forces within blood vessels may be very variable depending upon vessel size, shape, branching, and partial obstructions (1). Biomechanical forces induced within the cardiovascular system affect gene expression in cells of blood vessel walls (2,3) and functions of the cells in the vessel wall and in the fluid phase (4 -8). Changes of shear forces occur in bifurcated or stenotic regions where atherosclerotic regions are prone to develop.
According to the multistep theory in cell transmigration, monocytes roll on the endothelial cells, interact with selectins, adhere to the endothelial cells by firm adhesion to ICAM-1 1 and vascular cell adhesion molecule-1 (VCAM-1), and then migrate into the subendothelium (9). Rolling of monocytes on endothelial cells is dependent on the binding of E-selectin and sialyl Lewis X, and adhesion to the endothelium is dependent on the interaction between integrins on monocytes and adhesion molecules on the endothelial cells, such as VCAM-1 and ICAM-1.
Integrins consist of several subtypes, and each subtype is specific for each ligand. For example, ␣4␤1 integrin, VLA-4, binds to VCAM-1, and ␤2 integrins bind to ICAM-1. Fibronectin, one of the extracellular matrix proteins, is also known to bind to ␤1 integrins, mainly to ␣5␤1 integrin. Thus activation of adhesion molecules in endothelial cells and leukocytes is important for the cell migration process.
In this study, we hypothesized that leukocyte adhesion might be increased at bifurcations and in the downstream of the restricted vessels. In normal laminar flow, it has been reported that human leukocytes respond to fluid shear stress by retracting pseudopods and down-regulation of integrins (10,11), which is a requirement for normal passage of circulating leukocytes through the microcirculation. In the downstream of the region where the vessel lumen is partially occluded, however, a backward vortex can be observed where cells in the fluid phase are subjected to a vortex motion under low shear forces (1,(12)(13)(14). Although such a change of shear force on endothelial cells can regulate the expression of adhesion molecules resulting in the progression of atherosclerosis (15,16), the effect of vortex-mediated mechanical stress on leukocytes has not yet been determined. If vortex-induced mechanical stress can induce cell adhesion in leukocytes, leukocytes would be more prone to attach to the endothelial lining in the turbulent flow because the residence time of leukocytes in the regions with nonlaminar flow is longer than in those with laminar flow (12,13).
A variety of signaling systems are induced by a mechanosensor in endothelial cells. As a mechanosensor, stretch-activated channels have been reported to regulate Ca 2ϩ influx induced by flow stress in cells such as endothelial cells or smooth muscle cells (17). There is much evidence that stretch increases intracellular Ca 2ϩ levels (4,17). Thus the importance of Ca 2ϩ signaling in endothelial mechanotransduction has been established. However, the role of Ca 2ϩ in cell response to the mechanical stress in leukocytes has not been examined so far. Therefore, the aim of this study was to examine the effect of * This study was supported by Grants-in-aid 13307034 and 14570657 and Center of Excellence Grant 12CE2006 from the Japanese Ministry of Education, Science, Sports, and Culture (12CE2006) and a research grant for health sciences from the Japanese Ministry of Health and Welfare. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mechanical stress on integrin-dependent cell adhesion in human monocytic THP-1 cells and to elucidate the role of Ca 2ϩ signaling involved in this process.
Cell Lines-The monocytic cell line THP-1 was a generous gift from Dr. K. Nishida (Daiichi Pharmaceuticals Co. Ltd., Tokyo) and was cultured in RPMI supplemented with L-glutamine and penicillin/streptomycin plus 10% fetal calf serum in an atmosphere of 95% air and 5% CO 2 at 37°C.
Cell Adhesion Assay-Cell adhesion assays were carried out essentially as described (18). Briefly, polystyrene 96-well flat-bottomed microtiter plates (Costar 3595, Corning Inc., Corning, NY) were coated with 50 l of soluble VCAM-1 (2.5 g/ml), soluble ICAM-1 (2.5 g/ml), or fibronectin (10 g/ml) for 1 h at room temperature. After incubation, wells were blocked by incubation with 200 l of 10 mg/ml heat-denatured bovine serum albumin for 30 min at room temperature. Control wells were filled with 10 mg/ml heat-denatured bovine serum albumin. One hundred l of THP-1 cells suspended at a concentration of 10 6 /ml in 10% fetal calf serum-RPMI were incubated for the indicated times in a CO 2 incubator at 37°C after exposure to vortex flow by vortex machine (MS1 minishaker from IKA Works, Wilmington, NC). After incubation, nonadherent cells were removed by centrifugation (top side down) at 48 ϫ g for 5 min. The plates were then centrifuged inversely at 80 ϫ g for 5 min. Attached cells were fixed with 5% glutaraldehyde for 30 min at room temperature. Cells were washed three times with water, and 100 l of 0.1% crystal violet in 200 mM MES (pH 6.0) was added to each well and incubated at room temperature for 20 min. Excess dye was removed by washing with water three times, and the bound dye was solubilized with 100 l of 10% acetic acid. The absorbance of each well at 595 nm was then measured using a multiscan enzyme-linked immunosorbent assay reader (SPECTRA classic, Tecan, Maennedorf, Austria). Each sample was assayed in triplicate. The absorbance was linear to the cell number up to OD of 1.9 (data not shown). For example, 0.05 of OD. represents adhesion of about 2,000 cells, and 0.5 of OD represents adhesion of about 25,000 cells.

Vortex-mediated Mechanical Stress Increased Adhesion of THP-1 Cells to VCAM-1 and Fibronectin-
To determine the regulation of integrin avidity or affinity by mechanical stress mediated by vortex flow, we studied adhesion of THP-1 cells to purified adhesion molecules. Cell adhesion to soluble VCAM-1, soluble ICAM-1, and fibronectin was determined after cells were exposed to vortex flow for 5 s at 1,500 rpm to mimic vortices that may occur in the cardiovascular system (12,13,19). Vortex-mediated mechanical stress increased adhesion of THP-1 cells to VCAM-1 and fibronectin by approximately fivefold but not to ICAM-1 (Fig. 1).
Vortex-mediated cell adhesion to VCAM-1 and fibronectin increased in a speed-dependent manner (Fig. 2). To show that this cell adhesion is dependent on ␣4␤1 and ␣5␤1 integrins, we preincubated the cells with anti-␣4 antibody and RGDS peptides. Preincubation of the cells with anti-␣4 antibody inhibited vortex-mediated cell adhesion to VCAM-1 by about 80%, but not with control IgG (Fig. 3A). Preincubation with RGDS, but not with REDS peptides, inhibited vortex-mediated cell adhesion to fibronectin (Fig. 3B). We also studied the change of ␤1 integrin expression on THP-1 cells induced by vortex-mediated mechanical stress, but we could not find any change of the expression by flow cytometry (data not shown). These data indicate that cell adhesion in our assay depends on the inter-action between integrins and their ligands and that vortexmediated mechanical stress increased the avidity or affinity of both ␣4␤1 and ␣5␤1 integrins in THP-1 cells.
Transient Integrin Activation after Vortex-mediated Mechanical Stress-Next, we studied the time-dependent effect of vortex flow on cell adhesion to VCAM-1 and fibronectin. We found that vortex-mediated mechanical stress increased cell adhesion to both VCAM-1 and fibronectin quite rapidly, reaching a peak at 2-5 s of stimulation, indicating that such a brief vortex stimulation is enough to activate ␤1 integrin (Fig. 4). To examine reversibility of this integrin activation, cells were vortexed at 1,500 rpm for 5 s and left static for the indicated minutes. Cell adhesion to VCAM-1 or fibronectin was then determined. After the cells were left static for only 4 min, the cell adhesion induced by vortex flow was rapidly reduced to ϳ50% (Fig. 5), showing that this integrin activation induced by vortex-mediated mechanical stress is quite transient and reversible. Integrin Activation Induced by Vortex-mediated Mechanical Stress Depends on IP 3 -sensitive Ca 2ϩ Release from Intracellular Stores-Calcium signals are reported to be important for various cell responses such as integrin activation leading to cell adhesion (20). To determine whether Ca 2ϩ is involved in integrin activation induced by vortex-mediated mechanical stress, we next pretreated the cells with BAPTA-AM, an intracellular Ca 2ϩ chelator. Pretreatment of the cells with BAPTA-AM inhibited vortex-mediated cell adhesion to fibronectin (Fig. 6A) and VCAM-1 (data not shown), indicating that intracellular Ca 2ϩ is necessary for this integrin activation. To determine whether a stretch-activated Ca 2ϩ channel, a well known sensing system for mechanical stress (17), or Ca 2ϩ influx from the extracellular space is involved in integrin activation induced by vortex-mediated mechanical stress, we next pretreated the cells with GdCl 3 ⅐6H 2 O, a specific stretch-activated channel inhibitor, or NiCl 2 ⅐6H 2 O, a nonspecific Ca 2ϩ influx inhibitor. Pretreatment of cells with these inhibitors did not affect vortex-mediated cell adhesion to fibronectin (Fig. 6B) or VCAM-1 (data not shown), indicating that this integrin activation does not depend on stretch-activated channels or Ca 2ϩ influx from outside of the cells. These data indicate that Ca 2ϩ release from intracellular Ca 2ϩ stores such as endoplasmic reticulum may play a key role for this phenomenon.
Ca 2ϩ is released from the intracellular Ca 2ϩ stores via two known channels, one sensitive to inositol 1,4,5-trisphosphate (IP 3 ) and the other sensitive to ryanodine. Therefore, to determine the mechanism of Ca 2ϩ release from intracellular Ca 2ϩ stores, we pretreated the cells with thapsigargin, an inhibitor of Ca 2ϩ -ATPase that inhibits IP 3 -dependent Ca 2ϩ release from intracellular stores (21,22). Because thapsigargin itself induces sustained elevation of intracellular calcium mediated by capacitative Ca 2ϩ influx (23, 24), we added NiCl 2 to block this Ca 2ϩ influx. Pretreatment of THP-1 cells with thapsigargin and NiCl 2 inhibited vortex-mediated mechanical stress-induced cell adhesion to fibronectin (Fig. 6B)  not shown). We also pretreated the cells with U-73122, a specific PLC inhibitor, because mechanical stimulation of a single cell can activate PLC to elevate IP 3 (25). Pretreatment of the cells with U-73122 inhibited vortex-mediated cell adhesion to fibronectin and VCAM-1 (data not shown) in a dose-dependent manner (Fig. 6C).
To examine the role of ryanodine-sensitive Ca 2ϩ release from intracellular Ca 2ϩ stores, we next pretreated the cells with ryanodine, which can inhibit ryanodine-sensitive Ca 2ϩ release (26). Pretreatment of THP-1 cells with ryanodine up to 10 M did not affect vortex-mediated cell adhesion to fibronectin (Fig.  6D). These data indicate that IP 3 -dependent Ca 2ϩ release from intracellular Ca 2ϩ stores plays a key role in this phenomenon.
Calmodulin Is Also Necessary for Integrin Activation Induced by Vortex-mediated Mechanical Stress-We also examined the potential role of Ca 2ϩ -calmodulin in integrin activation induced by vortex-mediated mechanical stress. To determine the involvement of calmodulin in integrin activation induced by vortex-mediated mechanical stress, we pretreated the cells with W-7, a calmodulin inhibitor, before vortexing the cells. Pretreatment of cells with W-7 inhibited vortex-mediated cell adhesion to VCAM-1 and fibronectin in a dose-dependent manner (Fig. 7), indicating that calmodulin is also involved in integrin activation induced by vortex-mediated mechanical stress.

DISCUSSION
In this study we have examined the effect of vortex-mediated mechanical stress on integrin-dependent cell adhesion in human monocytic THP-1 cells and have clearly shown that a brief period of vortex-mediated mechanical stress activated ␤1 integrin, resulting in cell adhesion to VCAM-1 and fibronectin in a transient and reversible manner. We have also shown that IP 3 -dependent Ca 2ϩ release from intracellular Ca 2ϩ stores and calmodulin are involved in this integrin activation. This mechanism might explain why atherosclerosis is prone to progress in bifurcated or stenotic regions, and this may be a novel aspect of atherosclerosis and inflammation.
Most of the studies on mechanotransduction in the cardiovascular field have been done in endothelial cells and smooth muscle cells. The endothelial cells are normally subjected to mechanical stimuli from shear stress and from strain associated with stretch of the vessel wall. These stimuli can be detected by a mechanosensor that initiates a variety of signal transduction cascades (17,27). For example, in response to the change in shear stress the endothelium can change the gene expression of various cytokines and adhesion molecules (15,16,28) that would be related to the promotion of atherosclerosis, thrombosis, and inflammation. Few studies, however, have been conducted to elucidate the changes in the adhesive property of leukocytes in the vortex flow, which might be also related to the induction of atherosclerosis. Fukuda et al. (11) have reported that human leukocytes respond to fluid shear stress by retracting pseudopods and down-regulate the integrin expression under the laminar flow condition, which would help leukocytes to run in the vessel wall. However, in the tortuous cardiovascular system, such as branching of the vessels and downstream of partially occluded vessels, leukocytes and plate- lets can be subjected to differing shear forces under nonlaminar flow patterns (1,12,13). In this study, therefore, we exposed cells to vortex flow in order to mimic vortices that may occur in the cardiovascular system. In the study of platelet aggregation, a stirring bar has been used to expose platelets to vortex flow (19). Because it is important to expose whole cells to vortex flow instantaneously to mimic the in vivo situation, vortexing the cells in a vortex machine would be more reasonable to stimulate the cells in vitro. Establishing an in vivo model would be more important to show the relevance of this data to in vivo situations.
In previous studies, the endothelial intracellular Ca 2ϩ concentration in response to mechanical stress is biphasic, consisting of an initial transient rise that depends on Ca 2ϩ release from IP 3 -sensitive stores, followed by a sustained elevation mediated by Ca 2ϩ influx (22,29,30). However, in this report we have shown that Ca 2ϩ influx from the extracellular space is not necessary for integrin activation induced by vortex-mediated mechanical stress on THP-1 cells. Our data also clearly indicate that IP 3 -dependent Ca 2ϩ release from intracellular Ca 2ϩ stores plays a key role in this mechanism. Although the reason why only Ca 2ϩ release from intracellular stores is required for vortex-mediated integrin activation remains unclear, it might be because of the shortness of vortex stimulation and integrin activation.
Calmodulin is a Ca 2ϩ binding protein and is reported to be important for various cell responses, such as integrin activation leading to T cell adhesion (20) and aggregation (31). Our study clearly demonstrates that calmodulin also plays an essential role in regulating integrin activation induced by vortexmediated mechanical stress as shown in various cell responses (32,33). However, at present it is not clear how Ca 2ϩ release from intracellular stores can be linked to the activation of calmodulin and integrin activation in THP-1 cells. Further studies, therefore, are required to clarify this mechanism.
In this study we have not been able to identify the sensing mechanism for vortex-induced mechanical stress in THP-1 cells. There is a possibility that a mechanosensor itself is not involved in this process. The forces applied at the cell surface might be transmitted to other locations via cytoskeleton. This kind of mechanotransduction is shown in the area of mechanical stretch (34). Therefore, an explanation of the sensing mechanism would be required to understand this process. Further understanding of how leukocyte adhesion functions in the tortuous cardiovascular system would enhance our knowledge of the nuances of the atherosclerotic and inflammatory process and should facilitate the development of drugs to regulate the process.
In summary, we have provided clear evidence that vortexmediated mechanical stress on THP-1 cells quickly induces Ca 2ϩ -and calmodulin-dependent integrin activation, and IP3-dependent Ca 2ϩ release from intracellular Ca 2ϩ stores is involved in its mechanism. These findings might enlighten another aspect of increased atherosclerosis at stenotic or bifurcated regions.