Smooth Muscle Cells Promote Adhesion of Platelets to Cocultured Endothelial Cells
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循环研究杂志 2006年第1期
The Centre for Cardiovascular Sciences, The Medical School, The University of Birmingham, Edgbaston, Birmingham, United Kingdom.
Abstract
Although platelets do not ordinarily bind to endothelial cells (EC), pathological interactions between platelets and arterial EC may contribute to the propagation of atheroma. Previously, in an in vitro model of atherogenesis, where leukocyte adhesion to EC cocultured with smooth muscle cells was greatly enhanced, we also observed attachment of platelets to the EC layer. Developing this system to specifically model platelet adhesion, we show that EC cocultured with smooth muscle cells can bind platelets in a process that is dependent on EC activation by tumor necrosis factor (TNF)- and transforming growth factor (TGF)-1. Recapitulating the model using EC alone, we found that a combination of TGF-1 and TNF- promoted high levels of platelet adhesion compared with either agent used in isolation. Platelet adhesion was inhibited by antibodies against GPIb-IX-V or IIb3 integrin, indicating that both receptors are required for stable adhesion. Platelet activation during interaction with the EC was also essential, as treatment with prostacyclin or theophylline abolished stable adhesion. Confocal microscopy of the surface of EC activated with TNF- and TGF-1 revealed an extensive matrix of von Willebrand factor that was able to support the adhesion of flowing platelets at wall shear rates below 400 seC1. Thus, we have demonstrated a novel route of EC activation which is relevant to the atherosclerotic microenvironment. EC activated in this manner would therefore be capable of recruiting platelets in the low-shear environments that commonly exist at points of atheroma formation.
Key Words: smooth muscle cells endothelial cells coculture transforming growth factor-1 platelet adhesion
Introduction
Adhesion of platelets to the artery wall and formation of mural thrombi occur in the late stages of atherosclerosis and underlie cardiovascular pathology.1 This process requires exposure of thrombogenic subendothelial materials which contact arterial blood on rupture of mechanically compromised, "mature" plaques.1eC3 The idea that platelets might additionally adhere to endothelial cells (EC) during earlier stages of plaque development and contribute to disease progression has also been proposed.1,4,5 Further, animal models show that endothelium supports adhesion of platelets at sites prevalent to formation of atherosclerosis through a mechanism that is not understood.6,7 The platelet receptors GPIb-IX-V and integrin IIb3 are implicated in this process,6 but the events that initiate platelet adhesion are unclear.
Ordinarily, EC present an antithrombotic surface to flowing blood.4,5 This is achieved by constitutive production of NO and the lipid prostanoid prostacyclin.8eC10 However, a substantial number of studies report that platelets bind EC with compromised antithrombotic properties, although the pathophysiological significance of many of these reports is unclear, as powerful stimulatory agents were used to activate EC or platelets.4,11eC22 Recently, experiments in mice report platelet adhesion to mesenteric venules following treatment with calcium ionophore.23 The adhesion of platelets to arteries at sites of atheroma formation has also been visualized in apolipoprotein E (apoE) knockout mice.6,24 The major route of platelet adhesion in these models is via bridging of platelet IIb3 integrin16eC18,20,21 to endothelial cell v3 integrin18,19 by von Willebrand factor (VWF), with a possible contribution from P-selectin.25 However, the molecular basis of the change in EC reactivity that supports platelet adhesion has not been identified and cannot be readily mapped using animal models.
We and others have previously shown that cells known to be present within the atherosclerotic environment interact with EC, so that their inflammatory phenotype is markedly altered.26eC29 For example, crosstalk between monocytes and EC may establish a self-perpetuating and escalating cycle of EC activation and leukocyte recruitment.26,27 Additionally, crosstalk between secretory smooth muscle cells (SMC) and EC "primes" EC, so that they are hypersensitive to inflammatory stimulation by tumor necrosis factor (TNF) and can support significantly increased levels of leukocyte adhesion.28,29 Under the latter conditions, platelets, which were present in low numbers in the preparations of monocytes, also adhered to TNF-stimulated, cocultured EC. Thus, here we set out to examine the hypothesis that transcellular cross talk between EC and SMC altered the ability of EC to bind platelets, a result that has important implications for the events that initiate atheroma formation.
Materials and Methods
Platelet Isolation and Preparation
Human platelet-poor plasma and washed red blood cells were prepared from blood anticoagulated with 5 U/mL heparin. Washed human platelets were prepared from platelet-rich plasma produced from blood anticoagulated with citrate phosphate dectrose adenine in the presence of theophylline. Platelets were fluorescently labeled with calcein-acetylmethylester (5 e/mL), washed, and resuspended in medium 199 (Invitrogen, Paisley, UK) containing 20% autologous platelet-poor plasma and 5 U/mL heparin. In some experiments, autologous washed red blood cells were added to obtain a hematocrit of 20%. Where stated, platelets were activated with 5 eol/L ADP immediately before addition to adhesion assay. For details, see Section 1.1 in the online data supplement available at http://circres.ahajournals.org.
Culture and Coculture of EC and SMC
HUVEC were isolated and cultured as described.30 Human SMC were explanted from the arteries of umbilical cords as previously described.28,29 Each experiment used first passage EC from a different donor. EC were cocultured with SMC on the opposite sides of porous polyethylene terephthalate culture plastic inserts.28,29 Alternatively, EC were cultured in gelatinized 24-well tissue culture plates or in gelatinized glass capillaries (microslides) until confluent.30 For details, see Sections 1.2 and 1.3 in the online data supplement.
Platelet Adhesion Assays
Adhesion of platelets to EC cultures on filters or in plastic dishes or to EC cocultured with SMC was quantified under static conditions. Calcein-acetylmethylester-labeled platelets were added to the EC surface and allowed to adhere for 1 hour at 37°C. Nonadherent cells were removed by washing with PBS/BSA and the EC monolayers fixed. Platelets were observed in situ by fluorescent microscopy and video recordings made for analysis of platelet adhesion.
The adhesion of flowing platelets was assayed in microslides at a wall shear rate of 100 or 400 seC1. In some experiments, video recordings of platelets binding to EC were made in real time during platelet perfusion. In other experiments, the system was not illuminated until nonadherent cells had been removed with wash buffer. Platelet adhesion was quantified using Image Pro Plus software (Media Cybernetics).
In some experiments platelets were treated with antibodies against GPIb, aIIb3, or control antibody against vascular cell adhesion molecule (VCAM)-1. Alternatively, platelet activation was inhibited with prostacyclin or theophylline. In coculture experiments, function-neutralizing antibody against transforming growth factor (TGF)-1 was included in the culture medium on the addition of EC to the insert. For details, see Section 1.4 in the online data supplement.
Visualization and Quantification of VWF on EC
To visualize VWF, confluent monolayers of EC were grown on glass coverslips in 24-well plates. Labeled VWF was detected on live cells using confocal microscopy and fluorescence quantified by integrated pixel intensity determination over an entire field. For details, see Section 1.5 in the online data supplement.
Results
Endothelial Cells Cocultured With Secretory Smooth Muscle Cells Support the Adhesion of Platelets in the Presence of TNF
Unstimulated EC cultured in isolation or cocultured with SMC on porous transwells for 48 hours did not support adhesion of isolated washed platelets (Figures 1a, 1b, and 2a). Moreover, the addition of TNF to EC cultured alone did not promote platelet adhesion (Figure 2a). However, when EC cocultured with SMC for 24 hours were stimulated for a further 24 hours with TNF, they supported significant levels of platelet adhesion (Figures 1c and 2 a). We have previously shown that biologically active TGF-1, generated by the proteolytic action of plasmin in cocultures, regulates the inflammatory phenotype of EC.28 Taken with the current data, this raised the possibility that exogenous TNF combined with released TGF-1, promoted platelet adhesion. Consistent with this hypothesis, we found that neutralizing the activity of TGF-1 using an antibody abolished platelet adhesion (Figures 1d and 2b), whereas a control antibody had no effect. Moreover, platelet adhesion was greatly reduced if aprotinin, a plasmin inhibitor, was added to coculture supernatants (Figure 2b). Thus, coculture promoted interactions between platelets and EC that depended on a novel route of EC activation, which required a combination of TGF-1 and TNF-.
Antibodies against the platelet receptors GPIb or IIb3 inhibited adhesion of platelets to cocultured EC indicating that both adhesion molecules were required for binding (Figure 2c). Platelet adhesion to the cocultured EC also depended on activation of the platelets, as prostacyclin inhibited the response (Figure 2c).
TGF-1 and TNF Promote Platelet Adhesion to EC Cultured Alone
The role of TGF-1 and TNF was confirmed using purified recombinant reagents to reconstitute the coculture environment. Untreated EC grown in 24 well plates (Figures 1e and 3) or cells exposed to TNF (Figures 1f and 3), interleukin (IL)-1, or a combination of these proinflammatory cytokines did not support adhesion of unactivated platelets or platelets that had been activated with ADP (Figure 3). However, after treatment with TGF-1, platelets bound to EC and the level of this was greatly increased if platelets were activated with ADP (Figures 1g and 3). When the combination of TGF-1 and TNF was used to stimulate EC, we found that adhesion of platelets increased dramatically to levels comparable with those seen on cocultured EC (Figures 1h and 3). Interestingly, there was no requirement for exogenous activation of the platelets when EC were activated with the combination of TGF-1 and TNF, as activating platelets with ADP did not cause increased adhesion (Figure 3). We verified that TNF, TGF-1, or a combination of these agents did not directly activate platelets by conducting aggregation assays in their presence (supplemental Figures I and II).
To confirm that the same receptors were used for platelet adhesion, we blocked GPIb and IIb3. Platelet adhesion to EC stimulated with TGF-1 and TNF was abolished in the presence of antibody against either receptor, but a control antibody had no consistent affect (Figure 4). Platelet activation by the EC was essential for binding in this system, because treatment with either prostacyclin or theophylline inhibited platelet adhesion (Figure 4). To determine the nature of the activating stimulus we conducted experiments using indomethacin and antagonists of ADP receptors. Both strategies significantly reduced platelet adhesion, strongly implying that thromboxane and ADP were necessary for stable platelet adhesion (supplemental Figure III).
These observations imply that exposure of EC to TGF-1 induces expression of receptor(s) that supports platelet adhesion in the presence of an exogenous platelet agonist such as ADP. However, in the presence of TGF-1 and TNF, EC also provide an endogenous activator of platelets, which leads to stabilization of adhesion.
Combined Stimulation With TGF-1 and TNF Induces the Expression of a Matrix of VWF on the Surface of Endothelial Cells
As VWF is a ligand for both GPIb and IIb3 and is abundant within EC, we determined whether it was expressed on the surface of EC exposed to TGF-1 and TNF. Using immunofluorescent staining and confocal microscopy, we could not detect VWF on unstimulated (Figure 5a) or on TNF stimulated EC (Figure 5b). Exposure of EC to TGF-1 induced a small amount of surface VWF (Figure 5c). However, coexposure of EC to TGF-1 and TNF induced the expression of a matrix of VWF across the EC monolayer (Figure 5d), which was statistically significant (Figure 6).
High molecular weight VWF is an effective ligand for platelet GPIb at both venous and arterial rates of shear. Surprisingly, we found that stimulated EC were only able to support platelet adhesion at modest wall shear rates (100 seC1, Figures 7 and 8 a), and we verified that this was supported by both GPIb and IIb3 integrin (supplemental Figure IV). At 400 seC1, platelet adhesion was not observed (Figure 8a). It is possible that GPIb-VWF interactions, which usually support platelet tethering and rolling, were occurring in our system but that stable adhesion via IIb3 was not being achieved under flow. To investigate this, we measured the number of platelet-EC interactions that lasted for greater than 40 ms in our experiments. This analysis demonstrated that even tethering interactions between platelets and VWF were evident at a much reduced incidence on EC exposed to flowing platelets at 400 seC1 (Figure 8b). Thus, although we could induce EC coverage with VWF, this matrix of protein did not bind platelets at higher rates of shear. Interestingly, platelets did bind to EC at a shear rate of 400 seC1 when experiments were conducted in the absence of the plasma borne protease, ADAMTS-13, which has been described to process ultra-large VWF into less adhesive units. Thus, in the absence of autologous plasma or in the presence of heat inactivated plasma (which neutralizes ADAMTS-13) or an antibody against ADAMTS-13, we saw the formation of platelet "strings" which have been recently described31 (supplemental Figures V and VI).
Discussion
Over recent years, the concept that platelets bind to the EC of the intact artery wall and promote atherogenesis has received considerable attention, although the cellular changes that support platelet adhesion have not been described. Here, we identify a novel route of EC activation that supports platelet adhesion using a multicellular model of the artery wall, which may be relevant to the process of atherogenesis. In cocultures of EC and SMC, TGF-1 and TNF- promoted platelet adhesion, an observation that could be recapitulated using EC alone and recombinant proteins. Once localized at the site of atheroma, platelets might contribute to disease progression by inducing SMC mitogenesis and migration1,32eC34 by activating EC to support leukocyte recruitment35eC38 or by supporting leukocyte recruitment and activation themselves.39eC47
Previous studies have identified other conditions where EC support platelet adhesion. For example, platelets bind EC activated with TNF-48 or a combination of TNF- and IL-1.14 However, we were unable reproduce these observations. The reason for this discrepancy is unclear, although our results indicate the need for an additional stimulus to TNF or IL-1 to promote platelet adhesion. The formation of ultralarge VWF strings on EC in response to TNF, IL-8, IL-6, or histamine has been reported.49 Strings bind platelets at arterial rates of shear via GPIb.31 We could neither detect exposure of VWF nor adhesion of flowing platelets to TNF stimulated EC in the absence of TGF-1 or SMC coculture. Furthermore, we did not observe the phenomenon of strings of VWF in the presence of ADAMTS-13, which degraded the ultrahigh molecular weight multimers, which form VWF strings. In the absence of ADAMTS-13 we did observe string formation at high shear. It has also been reported that platelets activated with thrombin can bind to confluent unstimulated monolayers of EC.18,50,51 In our hands, activation of platelets with a relatively weak agonists, ADP, did not result in adhesion to unstimulated or cytokine stimulated EC, although it did potentiate adhesion of platelets in response to EC activation with TGF-1. It should also be noted that the interpretation of results using a strong agonist such as thrombin may be hampered by the formation of thrombi in suspension.
Here, TNF and TGF-1 promoted the expression of VWF that covered the EC monolayer. It is likely that this VWF was the ligand for platelet adhesion to EC, as platelet GPIb and IIb3 integrin were both required in our system and are known to be receptors for VWF.52 Additionally, VWF supports platelet adhesion to EC in a number of in vitro studies that report VWF bridged platelet IIb3 integrin and endothelial cell v3 integrin or P-selectin.16eC18,20,21 Several in vivo studies implicate VWF in adhesion of platelets to EC. For example, crossing mice lacking VWF with mice lacking low-density lipoprotein receptors significantly reduced the size of atherosclerotic lesions.7 Platelet adhesion may also play a key role in the process of very early atherogenesis in the apoE knockout mouse.6 Using intravital techniques, these authors showed that fluorescent platelets bound preferentially to the EC of atherosclerosis prone areas of the carotid artery. Furthermore, chronic treatment with antibodies against GPIb or IIb3 inhibited platelet adhesion and significantly reduced lesion formation. We also found that GPIb and IIb3 were essential for platelet binding to EC in static and flow based systems. However, GPIb-VWF interactions do not directly support platelet immobilization. Rather, platelets tether to VWF through GPIb, which results in the mobilization of intracellular calcium stores that activate IIb3 and thereby promote "stable" integrin mediated adhesion.54,55 Our observations that platelet adhesion can be blocked with antibodies against either receptor imply that integrin mediated adhesion is essential for stable adhesion and that this is disrupted by directly hindering IIb3eCVWF interactions or by removing the integrin activating signal by blocking interactions between GPIb and VWF.
When we conducted flow experiments, we were surprised that the VWF on the surface of EC was not an efficient ligand for platelets at high shear rate. In fact, EC activated with TGF-1 and TNF- did not support adhesion of flowing platelets above a wall shear rate of 100 seC1. This strongly implies that VWF on EC was not the large molecular weight multimeric form reported to be an efficient ligand for rapidly flowing platelets (wall shear rates >1000 seceC1).56 Similar observations have been reported in an intravital model (the microvasculature of the mouse cremaster muscle) where topical application of calcium ionophore induced expression of VWF on venous EC.23 This supported GPIb-mediated platelet adhesion at shear rates up to 100 seC1. As stated above, EC also express strings of high molecular weight VWF in response to histamine, TNF-, IL-6, or IL-8,49 which support the adhesion of flowing platelets at high shear rates. However, these experiments were conducted in the absence of plasma. If plasma was added, VWF was rapidly processed by the proteolytic activity of ADAMTS-13 to lower molecular weight units, which did not support platelet adhesion at high shear.31 It is probable, therefore, that were plasma is present, VWF is rapidly degraded so that it less efficiently supports platelet adhesion and can only do so at low wall shear rates. This would not preclude the adhesion of platelets to developing or established atheroma, as blood flow is often disturbed so that flow separation, eddies, flow reversal, and even stasis of flow can occur where shear rates are markedly reduced.57,58
The observation that TGF-1 regulates platelet adhesion to EC indicates a complex role for this agent in the evolution of atheroma. For example, TGF-1 can inhibit the responses of cultured EC to cytokines,59eC62 although we and others have reported that in multicellular culture systems or whole tissues, TGF-1 primes EC for increased sensitivity to TNF- or lipopolysaccharide.28,63,64 Interestingly, in rodent models of atherosclerosis and in human atheroma, the overexpression of TGF-1 appears to stabilize complex plaques.65,66 Additionally the loss of TGF- signaling via TGF--RII in murine T cells greatly exacerbates atheroma formation in apoE knockout mice.67,68 Thus, it is possible that TGF-1 has antiinflammatory, proinflammatory, and prothrombotic roles in atherogenesis. The balance of these signals may vary at different stages of plaque formation and may be critical in orchestrating the evolution of the plaque.
In conclusion, we have demonstrated that SMC in a phenotype relevant to the atherosclerotic microenvironment can activate EC to support platelet adhesion. This novel route of EC activation promotes the expression of a matrix of VWF on the EC surface, which is the adhesive substrate. We have previously demonstrated that the process of EC/SMC coculture hypersensitizes EC to inflammatory stimulation by TNF and greatly increases leukocyte adhesion. Thus, the process of transcellular crosstalk between secretory SMC and EC may confer a prothrombotic as well as a proinflammatory phenotype on EC. A mechanism of EC activation that promotes the adhesion of platelets and leukocytes may be critical for the development of atheromatous disease.
Acknowledgments
This work was supported by a Project grant (PG/03/015/15068) and a Nonclinical Senior Lectureship (BS/97001) (to G.E.R.) from British Heart Foundation and a National Health and Medical Research Council (Aust) CJ Martin Fellowship (to S.C.H.).
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Abstract
Although platelets do not ordinarily bind to endothelial cells (EC), pathological interactions between platelets and arterial EC may contribute to the propagation of atheroma. Previously, in an in vitro model of atherogenesis, where leukocyte adhesion to EC cocultured with smooth muscle cells was greatly enhanced, we also observed attachment of platelets to the EC layer. Developing this system to specifically model platelet adhesion, we show that EC cocultured with smooth muscle cells can bind platelets in a process that is dependent on EC activation by tumor necrosis factor (TNF)- and transforming growth factor (TGF)-1. Recapitulating the model using EC alone, we found that a combination of TGF-1 and TNF- promoted high levels of platelet adhesion compared with either agent used in isolation. Platelet adhesion was inhibited by antibodies against GPIb-IX-V or IIb3 integrin, indicating that both receptors are required for stable adhesion. Platelet activation during interaction with the EC was also essential, as treatment with prostacyclin or theophylline abolished stable adhesion. Confocal microscopy of the surface of EC activated with TNF- and TGF-1 revealed an extensive matrix of von Willebrand factor that was able to support the adhesion of flowing platelets at wall shear rates below 400 seC1. Thus, we have demonstrated a novel route of EC activation which is relevant to the atherosclerotic microenvironment. EC activated in this manner would therefore be capable of recruiting platelets in the low-shear environments that commonly exist at points of atheroma formation.
Key Words: smooth muscle cells endothelial cells coculture transforming growth factor-1 platelet adhesion
Introduction
Adhesion of platelets to the artery wall and formation of mural thrombi occur in the late stages of atherosclerosis and underlie cardiovascular pathology.1 This process requires exposure of thrombogenic subendothelial materials which contact arterial blood on rupture of mechanically compromised, "mature" plaques.1eC3 The idea that platelets might additionally adhere to endothelial cells (EC) during earlier stages of plaque development and contribute to disease progression has also been proposed.1,4,5 Further, animal models show that endothelium supports adhesion of platelets at sites prevalent to formation of atherosclerosis through a mechanism that is not understood.6,7 The platelet receptors GPIb-IX-V and integrin IIb3 are implicated in this process,6 but the events that initiate platelet adhesion are unclear.
Ordinarily, EC present an antithrombotic surface to flowing blood.4,5 This is achieved by constitutive production of NO and the lipid prostanoid prostacyclin.8eC10 However, a substantial number of studies report that platelets bind EC with compromised antithrombotic properties, although the pathophysiological significance of many of these reports is unclear, as powerful stimulatory agents were used to activate EC or platelets.4,11eC22 Recently, experiments in mice report platelet adhesion to mesenteric venules following treatment with calcium ionophore.23 The adhesion of platelets to arteries at sites of atheroma formation has also been visualized in apolipoprotein E (apoE) knockout mice.6,24 The major route of platelet adhesion in these models is via bridging of platelet IIb3 integrin16eC18,20,21 to endothelial cell v3 integrin18,19 by von Willebrand factor (VWF), with a possible contribution from P-selectin.25 However, the molecular basis of the change in EC reactivity that supports platelet adhesion has not been identified and cannot be readily mapped using animal models.
We and others have previously shown that cells known to be present within the atherosclerotic environment interact with EC, so that their inflammatory phenotype is markedly altered.26eC29 For example, crosstalk between monocytes and EC may establish a self-perpetuating and escalating cycle of EC activation and leukocyte recruitment.26,27 Additionally, crosstalk between secretory smooth muscle cells (SMC) and EC "primes" EC, so that they are hypersensitive to inflammatory stimulation by tumor necrosis factor (TNF) and can support significantly increased levels of leukocyte adhesion.28,29 Under the latter conditions, platelets, which were present in low numbers in the preparations of monocytes, also adhered to TNF-stimulated, cocultured EC. Thus, here we set out to examine the hypothesis that transcellular cross talk between EC and SMC altered the ability of EC to bind platelets, a result that has important implications for the events that initiate atheroma formation.
Materials and Methods
Platelet Isolation and Preparation
Human platelet-poor plasma and washed red blood cells were prepared from blood anticoagulated with 5 U/mL heparin. Washed human platelets were prepared from platelet-rich plasma produced from blood anticoagulated with citrate phosphate dectrose adenine in the presence of theophylline. Platelets were fluorescently labeled with calcein-acetylmethylester (5 e/mL), washed, and resuspended in medium 199 (Invitrogen, Paisley, UK) containing 20% autologous platelet-poor plasma and 5 U/mL heparin. In some experiments, autologous washed red blood cells were added to obtain a hematocrit of 20%. Where stated, platelets were activated with 5 eol/L ADP immediately before addition to adhesion assay. For details, see Section 1.1 in the online data supplement available at http://circres.ahajournals.org.
Culture and Coculture of EC and SMC
HUVEC were isolated and cultured as described.30 Human SMC were explanted from the arteries of umbilical cords as previously described.28,29 Each experiment used first passage EC from a different donor. EC were cocultured with SMC on the opposite sides of porous polyethylene terephthalate culture plastic inserts.28,29 Alternatively, EC were cultured in gelatinized 24-well tissue culture plates or in gelatinized glass capillaries (microslides) until confluent.30 For details, see Sections 1.2 and 1.3 in the online data supplement.
Platelet Adhesion Assays
Adhesion of platelets to EC cultures on filters or in plastic dishes or to EC cocultured with SMC was quantified under static conditions. Calcein-acetylmethylester-labeled platelets were added to the EC surface and allowed to adhere for 1 hour at 37°C. Nonadherent cells were removed by washing with PBS/BSA and the EC monolayers fixed. Platelets were observed in situ by fluorescent microscopy and video recordings made for analysis of platelet adhesion.
The adhesion of flowing platelets was assayed in microslides at a wall shear rate of 100 or 400 seC1. In some experiments, video recordings of platelets binding to EC were made in real time during platelet perfusion. In other experiments, the system was not illuminated until nonadherent cells had been removed with wash buffer. Platelet adhesion was quantified using Image Pro Plus software (Media Cybernetics).
In some experiments platelets were treated with antibodies against GPIb, aIIb3, or control antibody against vascular cell adhesion molecule (VCAM)-1. Alternatively, platelet activation was inhibited with prostacyclin or theophylline. In coculture experiments, function-neutralizing antibody against transforming growth factor (TGF)-1 was included in the culture medium on the addition of EC to the insert. For details, see Section 1.4 in the online data supplement.
Visualization and Quantification of VWF on EC
To visualize VWF, confluent monolayers of EC were grown on glass coverslips in 24-well plates. Labeled VWF was detected on live cells using confocal microscopy and fluorescence quantified by integrated pixel intensity determination over an entire field. For details, see Section 1.5 in the online data supplement.
Results
Endothelial Cells Cocultured With Secretory Smooth Muscle Cells Support the Adhesion of Platelets in the Presence of TNF
Unstimulated EC cultured in isolation or cocultured with SMC on porous transwells for 48 hours did not support adhesion of isolated washed platelets (Figures 1a, 1b, and 2a). Moreover, the addition of TNF to EC cultured alone did not promote platelet adhesion (Figure 2a). However, when EC cocultured with SMC for 24 hours were stimulated for a further 24 hours with TNF, they supported significant levels of platelet adhesion (Figures 1c and 2 a). We have previously shown that biologically active TGF-1, generated by the proteolytic action of plasmin in cocultures, regulates the inflammatory phenotype of EC.28 Taken with the current data, this raised the possibility that exogenous TNF combined with released TGF-1, promoted platelet adhesion. Consistent with this hypothesis, we found that neutralizing the activity of TGF-1 using an antibody abolished platelet adhesion (Figures 1d and 2b), whereas a control antibody had no effect. Moreover, platelet adhesion was greatly reduced if aprotinin, a plasmin inhibitor, was added to coculture supernatants (Figure 2b). Thus, coculture promoted interactions between platelets and EC that depended on a novel route of EC activation, which required a combination of TGF-1 and TNF-.
Antibodies against the platelet receptors GPIb or IIb3 inhibited adhesion of platelets to cocultured EC indicating that both adhesion molecules were required for binding (Figure 2c). Platelet adhesion to the cocultured EC also depended on activation of the platelets, as prostacyclin inhibited the response (Figure 2c).
TGF-1 and TNF Promote Platelet Adhesion to EC Cultured Alone
The role of TGF-1 and TNF was confirmed using purified recombinant reagents to reconstitute the coculture environment. Untreated EC grown in 24 well plates (Figures 1e and 3) or cells exposed to TNF (Figures 1f and 3), interleukin (IL)-1, or a combination of these proinflammatory cytokines did not support adhesion of unactivated platelets or platelets that had been activated with ADP (Figure 3). However, after treatment with TGF-1, platelets bound to EC and the level of this was greatly increased if platelets were activated with ADP (Figures 1g and 3). When the combination of TGF-1 and TNF was used to stimulate EC, we found that adhesion of platelets increased dramatically to levels comparable with those seen on cocultured EC (Figures 1h and 3). Interestingly, there was no requirement for exogenous activation of the platelets when EC were activated with the combination of TGF-1 and TNF, as activating platelets with ADP did not cause increased adhesion (Figure 3). We verified that TNF, TGF-1, or a combination of these agents did not directly activate platelets by conducting aggregation assays in their presence (supplemental Figures I and II).
To confirm that the same receptors were used for platelet adhesion, we blocked GPIb and IIb3. Platelet adhesion to EC stimulated with TGF-1 and TNF was abolished in the presence of antibody against either receptor, but a control antibody had no consistent affect (Figure 4). Platelet activation by the EC was essential for binding in this system, because treatment with either prostacyclin or theophylline inhibited platelet adhesion (Figure 4). To determine the nature of the activating stimulus we conducted experiments using indomethacin and antagonists of ADP receptors. Both strategies significantly reduced platelet adhesion, strongly implying that thromboxane and ADP were necessary for stable platelet adhesion (supplemental Figure III).
These observations imply that exposure of EC to TGF-1 induces expression of receptor(s) that supports platelet adhesion in the presence of an exogenous platelet agonist such as ADP. However, in the presence of TGF-1 and TNF, EC also provide an endogenous activator of platelets, which leads to stabilization of adhesion.
Combined Stimulation With TGF-1 and TNF Induces the Expression of a Matrix of VWF on the Surface of Endothelial Cells
As VWF is a ligand for both GPIb and IIb3 and is abundant within EC, we determined whether it was expressed on the surface of EC exposed to TGF-1 and TNF. Using immunofluorescent staining and confocal microscopy, we could not detect VWF on unstimulated (Figure 5a) or on TNF stimulated EC (Figure 5b). Exposure of EC to TGF-1 induced a small amount of surface VWF (Figure 5c). However, coexposure of EC to TGF-1 and TNF induced the expression of a matrix of VWF across the EC monolayer (Figure 5d), which was statistically significant (Figure 6).
High molecular weight VWF is an effective ligand for platelet GPIb at both venous and arterial rates of shear. Surprisingly, we found that stimulated EC were only able to support platelet adhesion at modest wall shear rates (100 seC1, Figures 7 and 8 a), and we verified that this was supported by both GPIb and IIb3 integrin (supplemental Figure IV). At 400 seC1, platelet adhesion was not observed (Figure 8a). It is possible that GPIb-VWF interactions, which usually support platelet tethering and rolling, were occurring in our system but that stable adhesion via IIb3 was not being achieved under flow. To investigate this, we measured the number of platelet-EC interactions that lasted for greater than 40 ms in our experiments. This analysis demonstrated that even tethering interactions between platelets and VWF were evident at a much reduced incidence on EC exposed to flowing platelets at 400 seC1 (Figure 8b). Thus, although we could induce EC coverage with VWF, this matrix of protein did not bind platelets at higher rates of shear. Interestingly, platelets did bind to EC at a shear rate of 400 seC1 when experiments were conducted in the absence of the plasma borne protease, ADAMTS-13, which has been described to process ultra-large VWF into less adhesive units. Thus, in the absence of autologous plasma or in the presence of heat inactivated plasma (which neutralizes ADAMTS-13) or an antibody against ADAMTS-13, we saw the formation of platelet "strings" which have been recently described31 (supplemental Figures V and VI).
Discussion
Over recent years, the concept that platelets bind to the EC of the intact artery wall and promote atherogenesis has received considerable attention, although the cellular changes that support platelet adhesion have not been described. Here, we identify a novel route of EC activation that supports platelet adhesion using a multicellular model of the artery wall, which may be relevant to the process of atherogenesis. In cocultures of EC and SMC, TGF-1 and TNF- promoted platelet adhesion, an observation that could be recapitulated using EC alone and recombinant proteins. Once localized at the site of atheroma, platelets might contribute to disease progression by inducing SMC mitogenesis and migration1,32eC34 by activating EC to support leukocyte recruitment35eC38 or by supporting leukocyte recruitment and activation themselves.39eC47
Previous studies have identified other conditions where EC support platelet adhesion. For example, platelets bind EC activated with TNF-48 or a combination of TNF- and IL-1.14 However, we were unable reproduce these observations. The reason for this discrepancy is unclear, although our results indicate the need for an additional stimulus to TNF or IL-1 to promote platelet adhesion. The formation of ultralarge VWF strings on EC in response to TNF, IL-8, IL-6, or histamine has been reported.49 Strings bind platelets at arterial rates of shear via GPIb.31 We could neither detect exposure of VWF nor adhesion of flowing platelets to TNF stimulated EC in the absence of TGF-1 or SMC coculture. Furthermore, we did not observe the phenomenon of strings of VWF in the presence of ADAMTS-13, which degraded the ultrahigh molecular weight multimers, which form VWF strings. In the absence of ADAMTS-13 we did observe string formation at high shear. It has also been reported that platelets activated with thrombin can bind to confluent unstimulated monolayers of EC.18,50,51 In our hands, activation of platelets with a relatively weak agonists, ADP, did not result in adhesion to unstimulated or cytokine stimulated EC, although it did potentiate adhesion of platelets in response to EC activation with TGF-1. It should also be noted that the interpretation of results using a strong agonist such as thrombin may be hampered by the formation of thrombi in suspension.
Here, TNF and TGF-1 promoted the expression of VWF that covered the EC monolayer. It is likely that this VWF was the ligand for platelet adhesion to EC, as platelet GPIb and IIb3 integrin were both required in our system and are known to be receptors for VWF.52 Additionally, VWF supports platelet adhesion to EC in a number of in vitro studies that report VWF bridged platelet IIb3 integrin and endothelial cell v3 integrin or P-selectin.16eC18,20,21 Several in vivo studies implicate VWF in adhesion of platelets to EC. For example, crossing mice lacking VWF with mice lacking low-density lipoprotein receptors significantly reduced the size of atherosclerotic lesions.7 Platelet adhesion may also play a key role in the process of very early atherogenesis in the apoE knockout mouse.6 Using intravital techniques, these authors showed that fluorescent platelets bound preferentially to the EC of atherosclerosis prone areas of the carotid artery. Furthermore, chronic treatment with antibodies against GPIb or IIb3 inhibited platelet adhesion and significantly reduced lesion formation. We also found that GPIb and IIb3 were essential for platelet binding to EC in static and flow based systems. However, GPIb-VWF interactions do not directly support platelet immobilization. Rather, platelets tether to VWF through GPIb, which results in the mobilization of intracellular calcium stores that activate IIb3 and thereby promote "stable" integrin mediated adhesion.54,55 Our observations that platelet adhesion can be blocked with antibodies against either receptor imply that integrin mediated adhesion is essential for stable adhesion and that this is disrupted by directly hindering IIb3eCVWF interactions or by removing the integrin activating signal by blocking interactions between GPIb and VWF.
When we conducted flow experiments, we were surprised that the VWF on the surface of EC was not an efficient ligand for platelets at high shear rate. In fact, EC activated with TGF-1 and TNF- did not support adhesion of flowing platelets above a wall shear rate of 100 seC1. This strongly implies that VWF on EC was not the large molecular weight multimeric form reported to be an efficient ligand for rapidly flowing platelets (wall shear rates >1000 seceC1).56 Similar observations have been reported in an intravital model (the microvasculature of the mouse cremaster muscle) where topical application of calcium ionophore induced expression of VWF on venous EC.23 This supported GPIb-mediated platelet adhesion at shear rates up to 100 seC1. As stated above, EC also express strings of high molecular weight VWF in response to histamine, TNF-, IL-6, or IL-8,49 which support the adhesion of flowing platelets at high shear rates. However, these experiments were conducted in the absence of plasma. If plasma was added, VWF was rapidly processed by the proteolytic activity of ADAMTS-13 to lower molecular weight units, which did not support platelet adhesion at high shear.31 It is probable, therefore, that were plasma is present, VWF is rapidly degraded so that it less efficiently supports platelet adhesion and can only do so at low wall shear rates. This would not preclude the adhesion of platelets to developing or established atheroma, as blood flow is often disturbed so that flow separation, eddies, flow reversal, and even stasis of flow can occur where shear rates are markedly reduced.57,58
The observation that TGF-1 regulates platelet adhesion to EC indicates a complex role for this agent in the evolution of atheroma. For example, TGF-1 can inhibit the responses of cultured EC to cytokines,59eC62 although we and others have reported that in multicellular culture systems or whole tissues, TGF-1 primes EC for increased sensitivity to TNF- or lipopolysaccharide.28,63,64 Interestingly, in rodent models of atherosclerosis and in human atheroma, the overexpression of TGF-1 appears to stabilize complex plaques.65,66 Additionally the loss of TGF- signaling via TGF--RII in murine T cells greatly exacerbates atheroma formation in apoE knockout mice.67,68 Thus, it is possible that TGF-1 has antiinflammatory, proinflammatory, and prothrombotic roles in atherogenesis. The balance of these signals may vary at different stages of plaque formation and may be critical in orchestrating the evolution of the plaque.
In conclusion, we have demonstrated that SMC in a phenotype relevant to the atherosclerotic microenvironment can activate EC to support platelet adhesion. This novel route of EC activation promotes the expression of a matrix of VWF on the EC surface, which is the adhesive substrate. We have previously demonstrated that the process of EC/SMC coculture hypersensitizes EC to inflammatory stimulation by TNF and greatly increases leukocyte adhesion. Thus, the process of transcellular crosstalk between secretory SMC and EC may confer a prothrombotic as well as a proinflammatory phenotype on EC. A mechanism of EC activation that promotes the adhesion of platelets and leukocytes may be critical for the development of atheromatous disease.
Acknowledgments
This work was supported by a Project grant (PG/03/015/15068) and a Nonclinical Senior Lectureship (BS/97001) (to G.E.R.) from British Heart Foundation and a National Health and Medical Research Council (Aust) CJ Martin Fellowship (to S.C.H.).
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