Integral Role of RhoA Activation in Monocyte Adhesion–Triggered Tissue Factor Expression in Endothelial Cells
Objective— The role of Rho activation in the regulation of tissue factor (TF) is not clear. This study was undertaken to investigate this in endothelial cells induced by monocyte adhesion.
Methods and Results— Isolated human peripheral blood monocytes were added to cultured human coronary endothelial cells. Monocyte adhesion to endothelial cells increased the levels of TF antigen in the endothelial cells. The results of transient transfection of the human TF promoter/luciferase gene into endothelial cells indicated that the increase in endothelial expression of the TF gene caused by monocyte adhesion occurred at the transcriptional level. The upregulation of TF was inhibited by statins, and the suppressive effect of statins was reversed by geranylgeranylpyrophosphate. Monocyte adhesion rapidly upregulated the membrane translocation and GTP/GDP exchange of RhoA, but not of Cdc42 or Rac, in endothelial cells. Rho inhibition by C3 exoenzyme or adenovirus-mediated expression of N19RhoA prevented the endothelial upregulation of TF caused by monocyte adhesion, and this was mimicked by Rho-kinase inhibitors. Moreover, monocyte adhesion increased the phosphorylation of nuclear factor-κB p65 in endothelial cells, and this was prevented by statins and Rho inhibition.
Conclusions— Our study shows that RhoA activation plays an integral role in TF expression in endothelial cells.
Tissue factor (TF), the physiological initiator of the blood coagulation cascade, plays a pivotal role in determining plaque thrombogenicity in coronary artery disease.1,2 ⇓ Vulnerable plaques, characterized by endothelial dysfunction and macrophage accumulation, have increased expression of TF, and the exposed atheroma content to blood by way of plaque rupture appears to be a primary cause of thrombus formation in the onset of acute coronary syndromes.1,3 ⇓ However, the significance of endothelial TF expression in thrombogenicity remains to be elucidated, although the expression of TF in endothelial cells has been shown in human coronary atherosclerotic lesions.1,4 ⇓ The recent results by Day et al5 with a TF-deficient mouse model suggest that thrombosis after arterial injury is driven primarily by vascular wall TF expression. This strongly suggests that TF expression in endothelial cells also plays an important role in thrombosis.
Circulating monocytes adhere to dysfunctional endothelial cells, not only in the early stage of atherosclerosis, but also in the vulnerable plaques.3,6 ⇓ Monocyte adhesion to endothelial cells seems to accelerate endothelial dysfunction, including procoagulant activity. However, whether monocyte adhesion to endothelial cells plays a role in the expression of TF in endothelial cells is poorly understood.
Recently, we showed that lysophosphatidylcholine (LPC), an atherogenic compound of oxidized LDL, rapidly activates RhoA in endothelial cells and that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) suppress the activation of RhoA induced by LPC by inhibiting geranylgeranylation, suggesting that RhoA activation might play a role in endothelial dysfunction.7 Using cultured human monocytes, we also found evidence that RhoA activation might be involved in TF expression.8 However, it is not clear whether RhoA activation is induced in endothelial cells by monocyte adhesion or is involved in signaling in endothelial TF expression.
In the present study, we have shown that monocyte adhesion induced the rapid activation of RhoA, accompanied by increased expression of TF in endothelial cells and that Rho inhibition by C3 exoenzyme or adenovirus-mediated expression of a dominant-negative form of RhoA, N19RhoA, prevented the upregulation of TF caused by monocyte adhesion. These findings suggest that RhoA activation might play an integral role in endothelial TF expression when monocytes adhere to endothelial cells and that increased TF expression in endothelial cells has pathophysiological significance in arterial thrombus formation.
The sources of most of the conventional reagents were described previously.7,8 ⇓ Pravastatin and cerivastatin were kindly provided by Sankyo Pharmaceutical Co (Tokyo, Japan) and Bayer Ltd (Leverkusen, Germany), respectively. C3 exoenzyme, a specific Rho inhibitor, was purchased from Upstate Biotechnology. Y-27632 and fasudil, Rho-kinase inhibitors, were generously provided by Welfide Co (Osaka, Japan) and Asahi Chemical Industry Co (Osaka, Japan), respectively.
Isolation of Human Peripheral Blood Monocytes
Human monocytes were isolated as described previously.8,9 ⇓ Informed consent for experimental examination of peripheral blood was obtained from all of the volunteers, and ethical approval for the study was obtained from Fukushima Medical University.
Preparation of Endothelial Cells
Human coronary artery endothelial cells were cultured according to the supplier’s instructions (Clonetics, Inc, Walkersville, Md, and Sanko Junyaku Co Ltd, Tokyo, Japan) as described previously.7 They were used for experiments after 5 to 10 passages.
Experimental Design of TF Assay
We added monocytes (5×105 cells) to near-confluent endothelial cells in 60-mm culture dishes, and the dishes were washed with phosphate-buffered saline (PBS) 3 times at various time intervals before Western blotting, pull-down assays, and membrane translocation assays. Most of the added monocytes appeared to have been washed out when the dishes were washed before 4 hours of coculture, whereas some residual monocytes were present after washing when the cocultures were continued for 18 hours. Thus, we essentially determined TF antigen levels and intercellular adhesion molecule-1 (ICAM-1) in the endothelial cells, although there were residual monocytes present, which seemed to depend on the time of culture. In addition, 5 μmol/L geranylgeranylpyrophosphate (GGPP) or 5 μmol/L farnesylpyrophosphate (FPP) was added to endothelial cells just before the additions of monocytes. For Rho inhibition studies, endothelial cells were treated with 0.25 μg/mL C3 exoenzyme overnight or infected with adenoviruses encoding N19RhoA, and then monocytes were added. A Rho-kinase inhibitor, Y-27632 or fasudil, was added to endothelial cells simultaneously with monocytes. The effect of monocyte culture medium on endothelial TF expression was also examined.
Western Blotting of TF and ICAM-1
Western blotting of cell extracts for TF and ICAM-1 was performed as described previously.8,9 ⇓ We used a 1:500 dilution of a mouse monoclonal antibody to human TF (Enzyme Research Laboratories) and a 1:500 dilution of a rabbit polyclonal antibody to human ICAM-1 (sc-7891, Santa Cruz Biotechnology Inc). The signals from immunoreactive bands were visualized by fluorography with an Amersham enhanced chemiluminescence system (ECL system, Amersham Pharmacia Biotech UK Ltd).
DNA Transfection and Luciferase Assay
The luciferase reporter construct (TF/Luc) contains the human TF promoter region between −383 and +121, as described previously.10 DNA transfection of TF into cultured endothelial cells was performed by using Tfx (Promega) according to the manufacture’s procedure. Cells in 35-mm culture dishes were transfected with 2 μg of TF promoter/luciferase construct with Tfx-50 (Promega) under serum starvation. After transfection, the medium was replaced with 2% fetal bovine serum– (GIBCO) containing medium, and the cells were cultured for 24 hours. Monocytes (2.5×105 cells) were added to the transfected endothelial cells, and the mixture was incubated for 6 hours. Cells were washed with ice-cold PBS 3 times and lysed in 120 μL of cell culture lysis reagent (Promega). The luciferase activity of each lysate was measured with a luminometer (Monolight 401, Analytical Lumininescence Laboratory Inc) and was normalized to the concentration of cellular protein, which was measured with a Bio-Rad DC protein assay system (Bio-Rad Laboratories).11
Membrane Translocation of RhoA, Cdc42, and Rac
The levels of RhoA, Cdc42, and Rac in membrane and cytosol fractions were determined by Western blotting, as described previously.7,12 ⇓ Monocytes (106 cells) were added to endothelial cells in 100-mm culture dishes. The supernatants were discarded after 15, 30, and 60 minutes, and the cells were washed 3 times with ice-cold PBS and lysed with a hypotonic buffer (10 mmol/L Tris, 2 mmol/L EDTA, 20 μg/mL antipain, 20 μg/mL leupeptin, 1 μmol/L dithiothreitol, and 1 μmol/L phenylmethylsulfonyl fluoride). We used a mouse monoclonal antibody to RhoA (Santa Cruz) diluted 1:500, a polyclonal antibody to Cdc42 (Santa Cruz) diluted 1:250, and a mouse monoclonal antibody to Rac (Upstate Biotechnology) diluted 1:1000.
GTP/GDP Exchange of RhoA, Cdc42, and Rac
Monocytes (5×105 cells) were added to endothelial cells in 60-mm culture dishes. The supernatants were discarded after 15, 30, and 60 minutes, and the cells were washed with ice-cold PBS 3 times and lysed in a lysis buffer (50 mmol/L Tris, pH 7.2, 500 mmol/L NaCl, 10 mmol/L MgCl2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 20 μg/mL antipain, 20 μg/mL leupeptin, and 1 μmol/L phenylmethylsulfonyl fluoride). After centrifugation at 18 000g at 4°C for 10 minutes, the extracts were incubated at 4°C for 45 minutes with glutathione-Sepharose 4B beads coupled with glutathione-S-transferase (GST)–rhotekin fusion protein for determination of Rho activity or GST–p21-activated kinase (PAK) for determination of Cdc42 and Rac activities, as described previously.7,13 ⇓ Bound RhoA, Cdc42, and Rac proteins were quantified by Western blotting, as described above.
Determination of NF-κB Activation
Nuclear factor (NF)-κB activation was determined by Western blotting by using rabbit polyclonal antibodies against phosphorylated NF-κB p65 (Ser536) and unphosphorylated NF-κB p65 (Cell Signaling Technology Inc), 1 of the heterodimers of NF-κB, according to the manufacture’s instructions. Immunoblots were analyzed as described above.
Adenovirus Gene Transfer
Endothelial cells were infected with adenoviruses encoding N19RhoA or LacZ at a multiplicity of infection of approximately 50, as described previously,14 and allowed to recover for 4 hours in medium containing 2% fetal bovine serum. After transfection, cells were washed with PBS 3 times and incubated for 24 hours in medium containing 2% fetal bovine serum, and then monocytes were added. The mixture was incubated for another 24 hours. This procedure resulted in the expression of LacZ as a marker gene in nearly 100% of transfected cells.
After scanning blots onto a computer (Epson GT5500ART, Tokyo), the optical densities of individual bands of Western blots were analyzed by using the National Institutes of Health (Bethesda, Md) image program. The area of each band analyzed was kept constant for each blot analyzed. Background density was subtracted from the densitometric data of each band.
Statistical analyses were performed by using ANOVA with Scheffé’s post hoc test when appropriate. A value of P<0.05 was considered significant. Data are expressed as mean±SD.
ICAM-1 and TF Expression
To confirm that added monocytes adhered to endothelial cells in the cocultures, we examined the expression of ICAM-1 in endothelial cells in response to monocyte addition. Figure 1A shows the time course of the expression of ICAM-1 after monocyte addition, as determined by Western blotting (lane 1, control; lane 2, 1 hour; lane 3, 4 hours; lane 4, 8 hours; and lane 5, 18 hours). A significant increase in ICAM-1 expression was observed at 4 hours after monocyte addition, and it increased further after 8 hours. Both pravastatin and cerivastatin did not alter the increased expression of ICAM-1 observed 8 hours after monocyte addition, at least with this culture condition (Figure 1B). Western blots showed the time course of the appearance of TF in response to monocyte addition (Figure 1C, lane 1, control; lane 2, 1 hour; lane 3, 4 hours; and lane 4, 18 hours). The added 5×105 monocytes showed a negligible immunoblot of TF under the same culture conditions (data not shown). After 4 hours, the coculture showed increased expression of TF, and this increase continued (Figure 1C). To examine the effect of monocyte-derived humoral factors, we added the monocyte culture medium to endothelial cells and found that medium from 18-hour monocyte cultures did not change the levels of TF antigen in endothelial cells, as determined by Western blotting (data not shown).
To determine the role of endothelial cells in the increased expression of TF in the cocultures of monocytes and endothelial cells, we performed promoter analyses of the human TF gene in endothelial cells with or without coculturing them with monocytes. Figure 1D shows that the promoter activities of TF in endothelial cells were increased by monocyte adhesion 6 hours after their addition (n=4, P<0.005), indicating that upregulation of the TF gene in endothelial cells caused by coculturing them with monocytes occurs at the transcriptional level.
Suppressive Effect of Statins on TF Antigen via Inhibition of the GGPP Pathway
Figure 2 shows the levels of TF antigen in cells measured by densitometric analysis of Western blots after 18 hours of coculturing. As described above, 5×105 monocytes alone showed negligible expression of TF (data not shown). Coculturing (lane 2) induced an increase in the levels of TF antigen compared with the endothelial cells alone (Figure 2, lane 2; n=4, P<0.05). Cerivastatin (0.01 μmol/L) prevented the upregulation of TF expression (Figure 2, lane 3; n=4, P<0.05). To determine which isoprenylation was inhibited by cerivastatin, we added 5 μmol/L GGPP or 5 μmol/L FPP to the cultures in the presence of 0.01 μmol/L cerivastatin. Western blotting demonstrated that GGPP (lane 4) but not FPP (lane 5) reversed the suppressive effect of cerivastatin on the expression of TF. Similar results were obtained in cells treated with 20 μmol/L pravastatin (data not shown). Preliminary experiments revealed no significant differences in the levels of TF antigen when 20 μmol/L pravastatin and 0.01 μmol/L cerivastatin were used (data not shown). These observations suggest that the suppressive effect of statins on TF synthesis is mediated by inhibition of geranylgeranylation of a signaling molecule(s), which appears to play a role in TF regulation.
Membrane Translocation of RhoA
To determine the involvement of geranylgeranylated small G proteins in the increased expression of TF in endothelial cells in response to monocyte addition, we measured the membrane translocation of RhoA, Cdc42, and Rac in endothelial cells after monocyte addition. Figure 3A demonstrates a time-dependent dramatic increase in membrane translocation of RhoA from 15 to 60 minutes after monocyte addition (top panels), but not that of Cdc42 or Rac (middle and bottom panels). As a result of the membrane translocation of RhoA in endothelial cells, a decrease in the levels of cytoplasmic RhoA was clearly induced by monocyte adhesion (Figure 3A, top panels, lanes 2, 3, and 4). A similar degree of membrane translocation during 60 minutes was observed after 18 hours of coculturing (Figure 3B, bottom panel, lane 2). We also determined the effect of statins on the activity of RhoA induced by monocyte addition because statins suppressed the synthesis of TF in the coculture. Cerivastatin (0.05 μmol/L) was added to endothelial cells just before the addition of monocytes. Figure 3B shows that the increased membrane translocation of RhoA was suppressed by cerivastatin 1 hour after the addition of monocytes (top panel) and that after 18 hours of coculture, cerivastatin prevented the membrane translocation of RhoA (bottom panel). Similar results were obtained with cells treated with 20 μmol/L pravastatin (data not shown). The medium from 18-hour monocyte cultures did not alter the levels of membrane translocation in endothelial cells (data not shown).
In addition, monocyte adhesion increased the levels of NF-κB p65 phosphorylation 1 hour after their addition (Figure 3B, middle panel, lane 2), although the levels of nonphosphorylated NF-κB p65 were not changed by monocyte addition (data not shown). Cerivastatin prevented the activation of NF-κB p65 caused by monocyte addition in endothelial cells (Figure 3B, middle panel, lane 3).
Pull-down assays revealed that monocyte adhesion induced an increase in the levels of the GTP-bound form of RhoA in endothelial cells 15 to 60 minutes after the addition of monocytes (Figure 3C, top panel), whereas the levels of RhoA in whole-cell (total) lysates were not changed by monocyte adhesion. However, the levels of the GTP-bound forms of Cdc42 and Rac were not altered by monocyte adhesion (Figure 3C, middle and bottom panels). There were still high levels of the GTP-bound form of RhoA in endothelial cells induced by monocyte adhesion after 18 hours of coculture (Figure 3D, lane 2). The effect of statins on RhoA GTP/GDP exchange was also examined. Pravastatin (20 μmol/L) markedly reduced the levels of the GTP-bound form of RhoA caused by monocyte addition after 18 hours of culture (Figure 3D, top panel, lane 3), although the levels of RhoA in whole-cell lysates (total RhoA) were not significantly changed by pravastatin. Similar results were obtained with cerivastatin (data not shown).
Effect of Rho Inhibition on TF Synthesis
To clarify the relation between RhoA activation and TF synthesis during monocyte adhesion to endothelial cells, we treated endothelial cells with 0.25 μg/mL C3 exoenzyme overnight to inhibit Rho and then added monocytes. Promoter assays revealed that the increase in promoter activity of the TF gene in endothelial cells caused by monocyte adhesion was prevented by pretreatment of endothelial cells with C3 exoenzyme (Figure 4A, n=3, lane 3). Western blotting demonstrated that treatment with the C3 exoenzyme prevented upregulation of the TF antigen (Figure 4B). In addition, monocyte addition to endothelial cells induced the activation of NF-κB p65 (Figure 4C, lane 2), and Rho inhibition by C3 exoenzyme prevented the phosphorylation of NF-κB p65, suggesting that Rho is upstream of NF-κB in the signaling pathway involved in monocyte adhesion to endothelial cells and that RhoA activation might play an integral role in TF expression in endothelial cells. To further confirm the role of Rho activation in the expression of TF induced by monocyte adhesion to endothelial cells, we transfected the endothelial cells with N19RhoA or LacZ. Densitometric analyses of Western blots revealed that Rho inhibition by N19RhoA transfection prevented upregulation of TF expression in response to monocyte adhesion (n=3, lane 1, LacZ: lane 2, LacZ + Mo; lane 3, N19RhoA + Mo). Representative immunoblots are shown in the top panel of Figure 5.
Effect of a Rho-Kinase Inhibitor on the Synthesis of TF
To study the role of Rho-kinase, 1 of the effectors of Rho, in the monocyte-endothelial cell interaction, we examined the effects of Y-27632 and fasudil (0.1, 1, and 10 μmol/L) on the synthesis of TF in coculture. Western blotting demonstrated that Y-27632 and fasudil suppressed the upregulation of TF induced by monocyte adhesion in dose-dependent manners (Figures 6, lanes 3, 4, and 5).
In this study, we found that monocyte adhesion rapidly induced the activation of RhoA in human coronary endothelial cells in vitro. Our data also showed that endothelial RhoA activation induced by monocyte adhesion was associated with the upregulation of TF expression, which was prevented by inhibiting Rho activation by C3 exoenzyme treatment or dominant-negative Rho gene transfection. Our findings suggest that RhoA activation plays a crucial role in the regulation of TF expression in endothelial cells induced by monocyte adhesion and that the increased endothelial TF expression caused by monocyte adhesion might contribute to thrombus formation.
The activation of Rho consists of geranylgeranylation, a GTP/GDP exchange reaction, and translocation into membranes.7,15 ⇓ In this study, microscopic observation revealed that most of the added monocytes adhered to endothelial cells approximately 15 minutes after their addition to the cultures. Western blots of ICAM-1 indicated the adhesion of monocytes to endothelial cells. Before performing the pull-down and membrane translocation assays after 15, 30, and 60 minutes of incubation, the dishes were washed 3 times with PBS, and most of the monocytes appeared to have been washed out. Our results showed that within 15 minutes, monocyte adhesion markedly induced an increase in the GTP/GDP exchange and the membrane translocation of RhoA, but not those of Cdc42 or Rac, in endothelial cells. A limitation of our study is that we could not determine whether RhoA activation also occurred in contaminating monocytes. Nevertheless, we think this is the first study to demonstrate the induction of RhoA activation in endothelial cells as early as 15 minutes after the addition of monocytes. Our previous study showed that LPC activates RhoA in endothelial cells within 1 minute.7 Taken together, these studies strongly suggest that the activation of RhoA, as judged by GTP/GDP exchange and membrane translocation, occurs promptly in endothelial cells in response to atherogenic stimuli, such as monocyte adhesion and LPC.
Because GTP/GDP exchange is believed to be controlled mainly by the activation of guanine nucleotide exchange factors (Rho GEFs),15 our findings suggest that endothelial activation of RhoA takes place rapidly via the activation of GEF in response to monocyte adhesion. However, the details of GEF activation in the process of GTP loading in this monocyte-endothelium model were not clarified by this study, and this aspect should be investigated further. Furthermore, statins prevented the endothelial RhoA activation induced by monocyte adhesion, probably by inhibiting the geranylgeranylation of RhoA. This suggests that the activation of geranylgeranylation plays an important role in RhoA activation induced by monocyte adhesion to endothelial cells. The mechanism of this should also be elucidated.
Previous studies, including ours, in which a Rho inhibitor and statins were used, provide indirect evidence that Rho activation is involved in TF synthesis.8,16 ⇓ The present study demonstrated that the increase in promoter activity of the TF gene was induced by monocyte adhesion, indicating that monocyte adhesion increased TF expression in endothelial cells at the transcriptional level. Furthermore, inactivation of endothelial RhoA by C3 exoenzyme or dominant-negative Rho gene transfection prevented the upregulation of TF expression caused by monocyte adhesion. These findings suggest that activation of endothelial cell RhoA plays an integral role in the upregulation of TF expression induced by monocyte adhesion to endothelial cells.
In addition, inhibition of Rho-kinase also attenuated the upregulation of TF synthesis in endothelial cells induced by monocyte adhesion, indicating that the Rho/Rho-kinase pathway is involved in endothelial TF expression induced by monocyte adhesion. Taken together, our results suggest that the Rho/Rho-kinase pathway has an integral role in the synthesis of TF in the vasculature.
TF expression does not always accompany thrombus formation in atherosclerotic lesions.1,4 ⇓ Thus, it is very important to elucidate the pathological significance of endothelial TF expression in thrombogenicity. Kaikita et al4 reported that TF was expressed by endothelial cells in all types of coronary atherosclerotic lesions without plaque disruption, suggesting that this might reflect procoagulant activity in the circulation and in plaques as basal conditions of coronary artery disease. A recent in vivo study with a TF-deficient mouse model showed that TF expression in the vascular wall is critical for thrombus formation.5 Our study clearly shows that monocyte adhesion dramatically causes an increase in endothelial TF expression via RhoA activation. This suggests that monocyte adhesion upregulates the procoagulant activity of endothelial cells in the vascular wall. In addition, further study with an impaired endothelial model, such as cells treated with L-NAME, will be of interest to evaluate TF expression in monocyte adhesion to endothelial cells.
To address the issue underlying transcriptional activation of TF, we attempted to clarify the cross-talk between RhoA and NF-κB p65. Monocyte adhesion to endothelial cells increased the phosphorylation of NF-κB p65 after 1 hour of coincubation, and inactivation of endothelial cell RhoA by C3 exoenzyme prevented the activation of NF-κB p65 induced by monocyte adhesion (Figure 4). These results suggest that RhoA is upstream of NF-κB p65 in the signaling from monocyte adhesion to endothelial cells. However, details of the correlation between TF expression and NF-κB remain to be elucidated.
In clinical trials, pravastatin has been shown to prevent primary and secondary cardiovascular events.17,18 ⇓ The present study demonstrated that statins, including hydrophilic pravastatin, suppressed the upregulation of TF synthesis by RhoA inactivation as well as NF-κB activation in the endothelium induced by monocyte adhesion. Our recent study showed that pravastatin suppresses TF synthesis in cultured human monocytes with the inactivation of RhoA.8 Thus, the present study supports the beneficial effect of pravastatin on cardiovascular events in terms of TF expression in vascular cell components.
In conclusion, the present study shows that increased TF expression in endothelial cells is induced by monocyte adhesion via RhoA activation, suggesting that RhoA activation might be an attractive target for treating thrombogenicity in coronary artery disease.
This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science (13670732 and 14370232) and a Research Grant for Cardiovascular Diseases from the Ministry of Health, Labor and Welfare of Japan.
- Received January 17, 2003.
- Accepted February 17, 2003.
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