Dickkopf-1 Enhances Inflammatory Interaction Between Platelets and Endothelial Cells and Shows Increased Expression in Atherosclerosis
Objective— Based on the emerging importance of the wingless (Wnt) pathways in inflammation and vascular biology, we hypothesized a role for Dickkopf-1 (DKK-1), a major modulator of Wnt signaling, in atherogenesis and plaque destabilization.
Methods and Results— We report increased levels of DKK-1 in experimental (ApoE−/− mice) and clinical (patients with coronary artery disease [n=80] and patients with carotid plaque [n=47]) atherosclerosis, both systemically (serum) and within the lesion, with particularly high levels in advanced and unstable disease. We identified platelets as an important cellular source of DKK-1 as shown by in vitro experiments and by immunostaining of thrombus material obtained at the site of plaque rupture in patients with acute ST-elevation myocardial infarction, with strong immunoreactivity in platelet aggregates. Our in vitro experiments identified a role for platelet- and endothelial-derived DKK-1 in platelet-dependent endothelial activation, promoting enhanced release of inflammatory cytokines. These inflammatory effects of DKK-1 involved inhibition of the Wnt/β-catenin pathway and activation of nuclear factor κB.
Conclusion— Our findings identify DKK-1 as a novel mediator in platelet-mediated endothelial cell activation. The demonstration of enhanced DKK-1 expression within advanced carotid plaques may suggest that this DKK-1-driven inflammatory loop could be operating within the atherosclerotic lesion.
Proteins from the wingless (Wnt) signaling pathways are involved in diverse developmental and physiological processes, including cardiac and vascular development. Wnt signals are transduced to the canonical and the noncanonical pathways for control of cell fate, cell movement, and tissue polarity.1 The Wnt pathways are regulated by multiple families of secreted antagonists including soluble frizzled related receptors and dickkopfs (DKK). The best studied of these is DKK-1, which dampens Wnt signaling by binding to the low-density lipoprotein receptor-related protein (LRP)5/6 and a cell surface coreceptor, Kremen-1, promoting internalization of the receptor complex.2 In adults, DKK-1 has been implicated in bone disease, cancer, and brain ischemia, and most recently, the destructive effect of tumor necrosis factor α (TNFα) on joints in rheumatoid arthritis was found to involve DKK-1.2,3 Also, serum levels of DKK-1 give prognostic information in patients with multiple myeloma and other malignancies as well as in patients with osteoarthritis.4,5
Recent evidence points to an important role of the Wnt signaling pathways in the regulation of inflammation. Thus, activation of the canonical Wnt/β-catenin pathway induces proliferation and survival of endothelial cells, enhances monocyte adhesion, and regulates transendothelial migration of monocytes.6–9 Moreover, activation of the noncanonical pathway has been shown to regulate inflammatory responses of human monocytes and macrophages in vitro.10,11 However, the interaction between the different proteins in the Wnt family is rather complex, and the role of Wnt signaling and their inhibitors in inflammation is far from clear.
Atherosclerosis is a chronic disease characterized by lipid accumulation and inflammation.12 Although the concept that inflammation plays a major role in atherogenesis is no longer controversial, the identification and characterization of the different actors are not fulfilled. Recently, Christman et al reported Wnt5a expression in human and murine atherosclerotic lesions,13 but at present, the role of the Wnt-related families of secreted proteins in atherogenesis is rather unknown. Based on the emerging importance of the Wnt signaling pathways in inflammation and vascular biology, we hypothesized a role for DKK-1, as a major modulator of Wnt signaling, in atherogenesis and plaque destabilization. Here, this hypothesis was investigated by various experimental approaches including in vivo studies in clinical and experimental atherosclerosis as well as in vitro studies in endothelial cells, particularly focusing on the role of DKK-1 in the inflammatory interaction between platelets and endothelial cells.
Patients and Controls
Patients with angina pectoris undergoing clinically indicated coronary angiography were consecutively recruited into the study (supplemental Table I, available online at http://atvb.ahajournals.org).
Tissue Sampling From Carotid Plaque
Human plaque tissue was obtained from the Biobank of Karolinska Endarterectomies study.14
Tissue Sampling of Thrombus Materials
In 6 patients with acute ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI), thrombus material at the site of the occlusion were aspirated immediately after crossing the lesion with the guide wire as previously described.15
Apolipoprotein E-deficient (ApoE−/−) mice on the C57BL/6 background (strain C57BL/6H-ApoETM1UNC129) were used for experiments as previously described (M&B, Ry, Denmark).16
Platelet Preparation and Stimulation
Preparation and stimulation of citrated platelet-rich plasma (PRP) were performed as previously described.17
Endothelial Cell Culture
Human umbilical vein endothelial cells (HUVECs) were obtained from umbilical cord veins by digestion with 0.1% collagenase A (Roche Diagnostics GmbH, Mannheim, Germany) and cultured as previously described.18
Western blotting was performed as previously described.19
Concentrations of DKK-1, monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6, and IL-8 were measured by enzyme immunoassays (EIA; R&D Systems).
Further detailed experimental procedures are provided in supplemental materials, available online at http://atvb.ahajournals.org.
Circulating Levels of DKK-1 in Angina Patients and Healthy Controls
Both patients with stable (n=40) and particularly those with unstable (n=40) angina had significantly increased serum levels of DKK-1 compared to healthy controls (n=20; Figure 1A). Some of the patients had additional risk factors that could interfere with inflammatory responses (eg, diabetes and hypertension), but the same pattern of DKK-1 levels was observed even if these patients were excluded from the study. There was a higher proportion of women in the unstable compared with the stable angina group and healthy controls (supplemental Table I), but we found the same pattern of DKK-1 levels in both genders.
In contrast to serum levels, we found no significant differences between angina patients and healthy controls when analyzing DKK-1 concentrations in platelet-poor plasma (Figure 1B), suggesting that the coagulation procedure could influence DKK-1 levels. Certainly, although there was no difference between serum and plasma levels of DKK-1 in healthy controls, DKK-1 levels were markedly higher in serum than in plasma of angina patients, particularly in those with unstable disease, suggesting increased release of DKK-1 during platelet aggregation in CAD patients (Figure 1C).
Platelets, but Not PBMCs and Neutrophils, Release DKK-1 on Activation
Our findings may suggest that platelets could release DKK-1 during activation, and on thrombin receptor stimulation (SFLLRN, 100 μmol/L), platelets rapidly released large amount of DKK-1 reaching maximum within a few minutes (Figure 2A). When added to PRP 20 minutes before SFLLRN stimulation, PGE1 (20 μmol/L), a potent inhibitor of platelet activation through its ability to increase intracellular cAMP levels, markedly attenuated the release of DKK-1 in SFLLRN-activated PRP (Figure 2B and 2C). In contrast, inhibitors of calcium-dependent metalloproteinases (ie, GM6001, a hydroxamate inhibitor of matrix metalloproteinases; 30 μmol/L) and inhibitors of actin polymerization (cytochalasin D; 60 μmol/L) did not affect DKK-1 release (Figure 2B). The release of DKK-1 from platelets seems not to be restricted to thrombin receptor activation as also collagen (8.4 μg/mL) and ADP (9.4 μmol/L) significantly enhanced the release of DKK-1, although the responses were less prominent than on SFLLRN activation (Figure 2D). It is well known that platelets retain a small but functionally significant amount of megakaryocyte-derived RNA as well as the proteins and molecular machinery necessary for translation and, indeed, by using real-time RT-PCR, we showed expression of mRNA for DKK-1 in highly purified platelets (Figure 2E). Aspirin is the prototypical platelet inhibitor that is widely used in clinical medicine, and aspirin (1 mmol/L) significantly reduced the SFLLRN-induced release of DKK-1 in PRP (Figure 2F). Also, aspirin (160 mg qd) significantly downregulated serum levels of DKK-1 when given for 7 days in 12 healthy controls (Figure 2G). However, even if the proportion of patients using aspirin, clopidogrel, and GP IIb/IIIa inhibitors was higher in those with unstable as compared with those with stable angina (supplemental Table I), they still had raised DKK-1 levels compared with stable angina patients, suggesting that additional platelet inhibition is needed to downregulate DKK-1 levels. Finally, in contrast to the release from activated platelets, neutrophils and PBMCs that had been activated by LPS (10 ng/mL), SFLLRN (100 μmol/L), or PMA (100 ng/mL) did not release any significant amounts of DKK-1 after culturing for 2, 5, and 24 hours (data not shown). In line with this, whereas serum levels of DKK-1 were significantly correlated with platelet counts within the total CAD population (r=0.43, P<0.001), there was no correlation between serum levels of DKK-1 and total leukocyte counts (r=0.11, P=0.37).
Neutralizing Antibodies Against DKK-1 Abrogate Platelet-Induced Cytokine Production in Endothelial Cells
The enhanced release of DKK-1 in angina patients during coagulation could be an ex vivo phenomenon, but it could also be relevant to the in vivo situation within an atherosclerotic lesion, where the interaction between platelets and an activated endothelium could result in markedly enhanced platelet degranulation and thrombus formation. Such interactions could also promote inflammation.20 Chemokines like MCP-1 and IL-8 are known to promote atherogenesis,21 and as depicted in Figure 3A and 3B, platelet releasates markedly enhanced the mRNA and protein expression of IL-8 and MCP-1 in HUVECs. DKK-1 is known to inhibit Wnt signaling by binding to LRP5/6 and Kremen 1, and transcripts of these proteins were expressed in HUVECs (supplemental Figure I). More importantly, whereas an unspecific antibody had no significant effect on the expression of these chemokines, a neutralizing antibody against DKK-1 totally abolished production (mRNA levels in cell pellets, Figure 3C) and release (protein levels in supernatants, Figure 3D) of IL-8 and MCP-1 in HUVECs that were activated for 24 hours by platelet releasates.
Silencing Endothelial-Derived DKK-1 Attenuates the Inflammatory Interaction Between Platelets and Endothelial Cells
To examine whether also endothelial-derived DKK-1 could modulate the inflammatory interactions between platelets and endothelial cells, we transfected HUVECs with siRNA probes to silence DKK-1. HUVECs spontaneously expressed large amount of DKK-1 at both the mRNA and protein levels, and we found successful silencing of DKK-1 as assessed by real-time RT-PCR (≈90%) and by EIA (≈95%) 48 hours posttransfection (Figure 4A). Moreover, silencing DKK-1 markedly attenuated the expression of MCP-1 and IL-8 in HUVECs that were activated in 24 hours by platelet releasates as assessed by mRNA levels in cell pellets and protein levels in cell supernatants (Figure 4B and 4C). The effect of DKK-1 was not restricted to IL-8 and MCP-1 as silencing DKK-1 also markedly attenuated the production and release of platelet-induced IL-6 (Figure 4D). Of note, IL-6 is released through a different mechanism than IL-8 and MCP-1 which depend on release from storage granules,22 suggesting that the effect of DKK-1 on cytokines in HUVECs is not related to a particular release mechanism of these mediators. Although silencing DKK-1 markedly downregulated the platelet-induced release of IL-8 and MCP-1 (Figure 4B and 4C), the downregulatory effect of anti-DKK-1 was even more pronounced (Figure 3B and 3D; IL-8: 107±33% versus 49±0.07% inhibition [P=0.12]; MCP-1: 100±14% versus 56±0.03% inhibition [P<0.03], anti-DKK-1 and siRNA DKK-1, respectively), underscoring the ability of anti-DKK-1 to block both platelet- and endothelial-derived DKK-1.
The Enhancing Effect of DKK-1 on Platelet-Mediated Endothelial Cell Activation Involves the Wnt/β-Catenin and NFκB Pathways
DKK-1 is thought to inhibit Wnt signaling through the canonical Wnt pathway resulting in decreased intracellular accumulation of β-catenin.2 As shown in supplemental Figure IIA and IIB, platelet releasates enhanced phosphorylation of β-catenin, known to result in degradation and impaired nuclear translocation of this factor,9 and silencing DKK-1 in HUVECs nearly abolished this inhibition of the Wnt/β-catenin pathway. Some Wnt ligands can also trigger signaling through noncanonical pathways involving activation of JNK, Akt, and NFκB.23 We therefore analyzed the effect of platelet releasates on HUVECs by multiplex suspension array technology in DKK-1 siRNA and scramble siRNA-treated cells. As depicted in supplemental Figure IIC, platelets promoted enhanced phosphorylation of IκB, a marker of NFκB activation, and DKK-1 silencing attenuated activation of this inflammatory pathway in HUVECs. As for Akt, ATF-2, ERK1/2, JNK, p38 MAPK, and STAT3, we found no significant activation in HUVECs after being exposed to releasates from activated platelets for 15 minutes (data not shown).
DKK-1 Expression in Clinical and Experimental Atherosclerosis
Our findings suggest that platelet- and endothelial-derived DKK-1 could contribute to the inflammatory interaction between platelets and endothelial cells, involving the Wnt/β-catenin and NFκB pathways. To elucidate the in vivo relevance of these findings, we examined the expression of DKK-1 within atherosclerotic lesions in clinical and experimental atherosclerosis.
In human carotid plaques (n=6), DKK-1 immunoreactivity was seen in core regions with predominantly calprotectin-positive macrophages and in vWF- and CD31-positive endothelial cells (Figure 5A). In contrast, only weak DKK-1 immunostaining was seen in the vessel wall from nonatherosclerotic human iliac arteries (data not shown). DKK-1 has recently been shown to mobilize vasculogenic progenitors,24 and interestingly, we found DKK-1 immunostaining in neovessel-rich areas within the carotid plaques (Figure 5B).
Our findings have related DKK-1 to activated platelets, and indeed, immunohistochemical staining of the material removed from the site of plaque rupture in 6 patients with acute STEMI undergoing primary PCI showed DKK-1 immunostaining corresponding to CD41-positive aggregated platelets and calprotectin-positive monocytes/macrophages (Figure 5C). Although we could not measure any detectable amounts of DKK-1 in neutrophils as assessed by EIA and real-time RT-PCR measurements in cell pellets (data not shown), we found DKK-1 immunoreactivity in relation to CD177-positive neutrophils in the thrombus material, potentially suggesting that DKK-1 could bind to these cells (Figure 5D).
Similar to the finding in clinical atherosclerosis, atherosclerotic lesions of aorta of ApoE−/− mice (n=6) showed strong DKK-1 immunostaining in vWF-positive endothelial cells and in regions with predominantly CD68-positive macrophages (Figure 5D). However, only parts of the area with predominantly CD68 staining, indicating macrophages, displayed DKK-1 immunoreactivity, and these regions were mainly outside the acellular and necrotic regions.
Enhanced DKK-1 Expression in Human Carotid Plaques
To achieve a quantitative assessment of DKK-1 expression in human atherosclerotic lesion, we analyzed the mRNA levels of DKK-1 in atherosclerotic tissue, collected from patients undergoing carotid endarterectomy (n=47), and in control tissue, obtained from iliac arteries of organ donors (n=10), by means of Affymetrix Gene Array analysis.14 The carotid plaques showed significantly elevated mRNA levels of DKK-1 as compared with control samples from nonatherosclerotic arteries (supplemental Figure III). The mRNA expression of DKK-1 within the atherosclerotic plaques was significantly correlated with the degree of stenosis as measured by carotid duplex ultrasound and CT angiography (r=0.36, P=0.02; supplemental Figure III).
Abnormal Wnt signaling has been associated with many human diseases, ranging from cancer to degenerative disorders. The Wnt pathway has also been suggested to play a distinct role in inflammation and immunity,10,11 and more recently, Christman et al reported high expression of Wnt5a in the macrophage-rich regions of murine and human atherosclerotic plaques.13 In the present study we report increased levels of DKK-1, a regulatory molecule of the Wnt pathway, in experimental and clinical atherosclerosis, both systemically and within the atherosclerotic lesion, with particularly high levels in advanced and unstable disease. Within the atherosclerotic plaques, high DKK-1 expression was found in macrophages and endothelial cells, and immunostaining of thrombus material obtained at the site of plaque rupture showed strong immunoreactivity in platelet aggregates. Our in vitro findings identified a role for DKK-1 in platelet-dependent endothelial activation, and taken together, the current data suggest the involvement of Wnt signaling pathways in platelet-mediated inflammation as well as in atherogenesis and plaque destabilization.
Several lines of evidence support a role for platelets as inflammatory cells. On activation they release and express inflammatory mediators and induce an inflammatory response in adjacent leukocytes and endothelial cells. Such platelet-mediated inflammation seems to play a pathogenic role in atherogenesis and plaque destabilization.25 Herein we show that DKK-1 is rapidly released from platelets on activation. Although we have no data on the intracellular localization of DKK-1, this pattern may suggest α-granule release where the granule components are fully released within minutes. Moreover, we demonstrated markedly increased serum levels of DKK-1 in angina patients with particularly high levels in those with unstable disease. In contrast, there was no difference in DKK-1 levels between angina patients and healthy controls when measuring DKK-1 in platelet-poor plasma. In general, caution is needed when interpreting serum levels of platelet-derived mediators in various clinical samples, and it may be argued that the high serum levels of DKK-1 in CAD is an ex vivo phenomenon reflecting enhanced release of DKK-1 during coagulation. However, serum levels may reflect the capacity of platelets to release various molecules on massive activation, and this may be relevant to the in vivo situation within the atherosclerotic lesion, where platelets are exposed to several potent activators, particular within an unstable plaque.26 The demonstration of positive DKK-1 immunostaining in platelet aggregates at the site of plaque rupture in STEMI patients may further support platelet-mediated DKK-1 release during plaque destabilization. Our findings underscore that platelets from CAD patients are different from platelets in healthy controls, contributing to the inflammatory arm of atherogenesis.
The interaction between stimulated platelets and an activated endothelium are of major importance in atherogenesis.25,27 On activation, platelets release the contents of their secretory granules, and this platelet releasate comprises of a multitude of inflammatory and vasoactive substances, which can attract atherogenic leukocytes from the circulation and activate endothelial cells.28 Our current data suggest that Wnt signaling could play an important role in this inflammatory cross-talk between platelet releasate and endothelial cells. Thus, by blocking DKK-1, platelet-mediated endothelial release of chemokines and IL-6 was markedly downregulated, with platelet- as well as endothelial-derived DKK-1 involved in the process. Based on our demonstration of strong DKK-1 immunoreactivity in platelets at the site of plaque rupture in STEMI patients as well as in the endothelium of symptomatic carotid plaques, such inflammatory effects of DKK-1 could potentially also be operating in vivo within an atherosclerotic lesion. Our finding of significantly enhanced DKK-1 expression in symptomatic carotid plaques, further support such a notion.
As DKK-1 is only 1 of several mediators that are released from platelets on activation, the ability of anti-DKK-1 to totally abolish the platelet-mediated up-regulation of IL-8 and MCP-1 in endothelial cells may apparently seem in conflict with a role for other platelet-derived mediators in this process. However, we have previously shown a marked attenuating effect of anti-LIGHT (≈75% reduction) on the release of IL-8 and MCP-1 in platelet-activated HUVECs.18 It is tempting to hypothesize that blocking of one of the platelet-derived mediators may not only influence the effect of this particular molecule, but also attenuate the effects of other platelet-derived mediators through lack of coactivation and synergistic effects. However, the attenuating effect of anti-DKK-1 seems to be even more pronounced than the downregulatory effect of anti-LIGHT. This may reflect that in addition to neutralizing platelet-derived DKK-1, anti-DKK-1 also inhibits endothelial-derived DKK-1 that is released from HUVECs on platelet activation. Indeed, the inhibitory effect of anti-DKK-1 was more pronounced than the attenuating effect of silencing endothelial-derived DKK-1, underscoring the ability of anti-DKK-1 to block both platelet- and endothelial-derived DKK-1.
Wnt signaling has been linked to inflammation, but there are also some reports indicating that this pathway could be involved in antiinflammatory responses. Thus, β-catenin stabilization, as a result of activation of the canonical Wnt pathway, has been shown to promote enhanced survival of existing regulatory T cells with potentially powerful antiinflammatory net effects.29 Moreover, activation of the canonical Wnt/β-catenin pathway has been reported to downregulate receptor activator of NFκB ligand (RANKL),30 a member of the TNF superfamily that is involved in atherogenesis and plaque destabilization.15 Phosphorylation results in destabilization of β-catenin,9 and our demonstration of increased phosphorylation of endothelial-related β-catenin on platelet activation in scramble siRNA-treated, but not in DKK-1 siRNA-treated cells, suggest that the enhancing effect of DKK-1 on platelet-mediated endothelial cell activation involves inhibition of the canonical Wnt/β-catenin pathway. Furthermore, we showed that platelets induced NFκB activation in endothelial cells, known to be a potent inducer of IL-8 and MCP-1, and the platelet-mediated activation of this pathway was markedly attenuated when silencing endothelial-derived DKK-1. Our findings, showing a DKK-1-mediated inhibition of the canonical Wnt/β-catenin pathway, potentially resulting in downregulation of antiinflammatory responses, as well as a DKK-1-mediated enhancement of the inflammatory NFκB pathway in platelet-activated endothelial cells, could be of importance for the inflammatory interaction between platelets and endothelium. Whether our finding of inhibitory effects of DKK-1 on the Wnt/β-catenin pathway, combined with enhancing effects on NFκB activation, represent distinct effects on the canonical and noncanonical pathway, respectively, or if these effects reflect the recently described cross-talk between Wnt/β-catenin and NFκB pathways,31 is at present unclear. Nonetheless, the present study suggests that DKK-1 and Wnt signaling pathways could play a role in platelet-mediated endothelial cell activation.
Recent studies have revealed that Wnt signaling occurs downstream of additional signaling cascades, including responses to inflammatory cytokines such as TNFα and IL-6.32 Herein we show that the inflammatory interaction between platelets and endothelial cells may be operating through this pathway. Inflammatory cytokines such as TNFα can induce the expression of DKK-1,3 a mechanism likely to be operating within an atherosclerotic plaque. Furthermore, we show that both endothelial- and platelet-derived DKK-1 could enhance the inflammatory interaction between platelets and endothelial cells, conceivably representing a self-perpetuating pathogenic mechanism within an inflammatory microenvironment. Our demonstration of enhanced expression of DKK-1 within symptomatic carotid plaques suggests that this DKK-1-driven inflammatory loop could be operating in the atherosclerotic lesion, potentially contributing to atherogenesis and plaque destabilization.
Sources of Funding
This work was supported by grants from the Norwegian Council of Cardiovascular Research, Research Council of Norway, Helse Sør, Rikshospitalet, Swedish Medical Research Council, and Swedish Heart-Lung Foundation.
Received January 2, 2009; revision accepted May 10, 2009.
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