5 CD 40 Ligand and Its Receptors in Atherothrombosis

Atherothrombosis is the main underlying determinant of cardiovascular diseases, which remain the leading cause of death in developed countries. Multiple lines of evidence now support the concept of atherothrombosis as a chronic inflammatory disease of the arterial wall.1, 2 This process involves a complex interplay between modified lipids and cells of the immune and vascular system, which usually evolves into the formation of atherosclerotic lesions yielding a stable necrotic plaque. If left untreated, plaque rupture and thrombosis may ensue, leading to important clinical manifestations, such as acute coronary syndromes and sudden death.3


Introduction
Atherothrombosis is the main underlying determinant of cardiovascular diseases, which remain the leading cause of death in developed countries.Multiple lines of evidence now support the concept of atherothrombosis as a chronic inflammatory disease of the arterial wall. 1,2 his process involves a complex interplay between modified lipids and cells of the immune and vascular system, which usually evolves into the formation of atherosclerotic lesions yielding a stable necrotic plaque.If left untreated, plaque rupture and thrombosis may ensue, leading to important clinical manifestations, such as acute coronary syndromes and sudden death. 3 the incorporation of modified low-density lipoproteins in the arterial wall represents a important step in the onset of atherothrombosis, the subsequent recruitment and activation of inflammatory cells, including monocytes, B-and T-lymphocytes, neutrophils and platelets play a critical role in the pathogenesis of this disease. 4These cells exhibit proatherogenic functions through multiple co-stimulatory and immune molecules present on their cell surface.][10][11] While CD40L is known to mainly interact with its classical receptor CD40, additional binding partners have been described, namely the integrins IIb 3 , M 2 and 5 1 .This chapter discusses the role of CD40L and its receptors in the pathophysiology of atherothrombosis, while highlighting its therapeutic potentials in the treatment of this chronic inflammatory disease.

CD40-TRAF dependent signaling
T h e e n g a g e m e n t o f C D 4 0 b y C D 4 0 L p r o m o t e s t h e c l u s t e r i n g o f C D 4 0 a n d i n d u c e s t h e association of TRAFs to the cytoplasmic domain of CD40. 30These adapter proteins are essential for the activation of different signaling pathways including the canonical and noncanonical nuclear factor κB (NF-κB)-signaling pathways and the activation of mitogenactivated protein kinases (MAPKs). 30The TRAF family comprises six known members, among which TRAF-1, -2, -3, -5 and -6 have been shown to drive CD40-dependent cellular responses.
TRAF-1 can only bind weakly to the cytoplasmic tail of CD40 and therefore regulates the signaling of others TRAF members, in particular TRAF-2. 30,31 ndeed, TRAF-1 deficiency in antigen presenting cells and B-lymphocytes leads to a significant reduction in the recruitment of TRAF-2 to CD40, indicating that TRAF-1 facilitates the association of TRAF-2 to the cytoplasmic domain of CD40. 32,33 n agreement with these results, it has been shown that the recruitment of both TRAF-1 and TRAF-2 are required for complete activation of NF-κB in B-cells, since the knockout of both genes results in a greater inhibition of the NF-κB signaling pathway, in comparison to single knockouts. 33AF-2 is an important contributor to CD40 signaling and its major role resides in the activation of the NF-κB signaling pathway, as well as the activation of the p38, Akt and JNK MAPKs.CD40 bears a direct binding site for TRAF-2 and blockage of this interaction leads to immune deficiencies such as B-cell proliferation and isotype switching. 24,34 espite its significant implications in CD40 signaling, TRAF-2 deficiency may be overcome by TRAF-6 activation.[37] TRAF-3 functions as a negative regulator of CD40 signaling through its constitutive association with TRAF-2. 38,39 n absence of stimulation, TRAF-3 interacts with TRAF-2, which allows the degradation of the NF-κB inducing kinase (NIK) protein, a critical stimulator of NF-κB. 40TRAF-3 deficiency in B cells exacerbates NF-κB and JNK activation, primarily through cytosolic accumulation of NIK, thus confirming the negative regulatory functions of TRAF-3 in B cells. 41ry little information is available regarding the role of TRAF-5 in CD40 signaling.Nevertheless, it appears that TRAF-5 can interact with TRAF-3 to modulate NF-κB activation in B cells.This was shown by experiments in which TRAF-5 deficiency diminishes NF-κB activation, causing a reduction in cell activation, expression of co-stimulatory molecules and antibody production. 42,43 F-6 plays a significant role in the activation of key CD40-dependent signaling pathways, such as NF-κB, p38, JNK and Akt. 44As discussed above, TRAF-6 synergizes with TRAF-2 in order to regulate the activation of NF-κB.Although TRAF-6 contains a direct binding site for CD40, specific inhibition of this domain shows lesser inhibitory effects than ablation of the complete protein, indicating that TRAF-6 may still have a functional role in CD40 signaling without binding directly to CD40. 30,45 ndeed, one of the main functions of TRAF-6 resides in its ability to interact with TRAF-2, which is already bound to CD40, and facilitate the activation of downstream targets.
Recently, a study aiming at evaluating the implication of TRAF members in neointima formation, a critical step of atherothrombosis, was conducted.Using a CD40 transgenic mouse model, in which mutations at the TRAF2/3/5, TRAF6 or TRAF2/3/5/6 binding sites were carried out, the authors conclude that the CD40-TRAF6 axis is a key regulator of inflammatory cell infiltration and neointima formation at sites of vascular injury. 46though most vascular complications associated to CD40L, including atherothrombosis, have been largely attributed to its interaction with CD40, recently identified additional receptors merit attention.These include the integrins IIb 3 , 5 1 and M 2 and (Figure 1).Fig. 1.CD40L and its receptors.The binding of CD40L to its classical CD40 counterreceptor regulates numerous critical biological responses.These mainly include B-cell dependent isotype switching, cell-mediated immunity (production of cytokines, chemokines, adhesion molecules, growth factors, MMPs and procoagulants) and apoptosis.The CD40L/CD40 interaction is at the forefront of the pathogenesis of multiple inflammatory disorders, including atherothrombosis.The interaction of CD40L with the IIb 3 platelet integrin is involved in thrombus stabilization and may provide a novel outside-in signaling pathway by which platelets can be activated.CD40L can also bind to the inactive conformation 5 1 and this interaction was shown to induce activation of the human monocytic U937 cell line.Finally, M 2 can mediate CD40L-dependent inflammatory responses, in particular leukocyte adhesion and neointimal formation.The pathophysiological relevance of these novel CD40L-mediated interactions in inflammation remains elusive and additional studies will be required to address this issue.

IIb 3
The IIb 3 integrin is the most abundant receptor of the surface of platelets and mediates platelet adhesion and aggregation.Like all molecules of the integrin family, it will change conformation upon inside-out cellular activation, thereby allowing binding to its natural ligands (fibrinogen, fibronectin, vWF...). 47These ligands contain KGD sequences and binding is mediated through the KGD recognition domain present on the IIb 3 molecule.Interestingly, CD40L also contains a KGD sequence making its interaction with IIb 3 possible.Binding of CD40L to IIb 3 was shown to induce phosphorylation of tyrosine residues within the cytoplasmic domain of the 3 subunit and appears essential for thrombus stabilization in vivo. 48,49 ndeed, CD40L -/-mice exhibit unstable thrombi, which can be overcome by infusion of wild type recombinant human CD40L and not CD40L specifically mutated at the site of interaction with IIb 3. 48

1
The 5 1 integrin was also shown to act as a CD40L receptor. 50Indeed, sCD40L can bind and activate cells of the undifferentiated human monocytic U937 cell line in a CD40-and IIb 3-independent manner.Binding to this cell line was reversed by an anti-5 1 antibody, as well as in the presence of soluble 5 1, thus confirming 5 1 specificity.Moreover, this interaction is unaffected by pre-treatment of CD40L with soluble CD40, indicating that CD40L can bind both CD40 and 5 1 concomitantly. 50Interestingly, CD40L binds to inactive 5 1, contrary to most ligands of the integrin family.However, the physiological relevance of this interaction remains unexplored and additional studies are needed to fully characterize the interplay that might take place between CD40L and 5 1 in inflammatory disorders.

M 2
The M 2 (Mac-1) integrin mediates firm adhesion of leukocytes to inflamed vessels by interacting with its endothelial cell counterreceptor intercellular adhesion molecule 1 (ICAM-1). 51CD40L was also recently shown to bind to active M 2 and this interaction may represent an alternative pathway for CD40L-mediated inflammation. 52Indeed, inhibition of this novel CD40L binding partner significantly attenuates leukocyte accumulation at sites of inflammation and reduces atherogenesis, indicating that CD40L may promote, at least in part, atherosclerotic lesions in a M 2-dependent manner.Again, the relative contribution of this CD40L receptor (in comparison to CD40) in the development of inflammatory disorders such as atherothrombosis remains unknown.Perhaps, each of these CD40L receptors may interfere at different stages of the disease, thus contributing to proinflammatory reactions and atherogenesis in their own way.

CD40L in atherothrombosis
The involvement of CD40L in the pathogenesis of atherothrombosis is supported by numerous studies.Targeting of CD40L by both pharmacological and genetic approaches has highlighted the importance of this molecule in all stages of the disease.In 1998, Mach et al. showed that treatment of hyperlipidemic LDLR -/-mice with an anti-CD40L antibody significantly ameliorates the size and lipid contents of atherosclerotic lesions. 53These results were further confirmed by a genetic approach, which showed that CD40L -/-/ApoE -/-mice exhibit considerably smaller plaque area than control ApoE -/-mice. 8Moreover, these animals display enhanced collagen fibrils within the fibrous cap of lesions, a key component of plaque stability.In an additional study, the administration of an anti-CD40L antibody in ApoE -/-mice, at the onset of lesions or once atherosclerotic lesions are fully established, reduces lipid contents and increases plaque stability. 54Taken together, these studies support the contribution of CD40L in plaque initiation, progression and stability (Figure 2).Fig. 2. Role of CD40L in atherothrombosis.The incorporation of oxidized LDLs, among other factors, may upregulate the expression of the CD40L system on the endothelium, thereby promoting the recruitment of platelets and leukocytes at the sites of injury.The CD40L-dependent adhesion of T-lymphocytes and platelets to the endothelium induces an important inflammatory response characterized by the secretion of various cytokines and the upregulation of additional endothelial adhesion molecules.This favors in turn the incorporation and transmigration of additional leukocytes, in particular monocytes.Once in the sub-endothelial space, CD40L/CD40 interactions between foam cells (macrophages which have undergone phagocytosis of oxidized LDL particules), T-lymphocytes and smooth muscle cells take place.These cross talks ultimately lead in part to the proliferation of smooth muscle cells into the intima and promote vascular angiogenesis, primarily through the secretion of key inflammatory and angiogenic cytokines and chemokines.This process eventually yields a stable lipid-enriched atherosclerotic plaque surrounded by a fibrous cap.Plaque stability is threatened by the production of MMPs, which are directly responsible for collagen degradation and rupture of the fibrous cap.The binding of CD40 on endothelial cells, macrophages and smooth muscle cells can provoke the secretion of a long list of MMPs.Following rupture, platelets rapidly adhere to the surface of the highly prothrombotic contents of the atherosclerotic plaque, thereby leading to thrombus formation and arterial occlusion.CD40L may also be involved in thrombus formation and procoagulant activity.CD40L binding to the endothelium promotes tissue factor expression, while the binding of CD40L (soluble and membrane-bound forms) to CD40 and IIb 3 on platelets enhances platelets aggregation and thrombus stabilization, respectively.

Initiation of lesions and leukocyte recruitment
Plaque initiation is normally characterized by the accumulation of low-density lipoproteins (LDL) in the arterial wall and the subsequent recruitment and transmigration of leukocytes within the sub-endothelial space. 1 The initial trigger of CD40L (and CD40) expression within cells of the developing atherosclerotic plaque (endothelial cells, lymphocytes, platelets, monocytes/macrophages, and smooth muscle cells) remains elusive.Possible candidates include oxidized LDL, infectious pathogens and alterations in vascular hemodynamic forces. 55- 57For instance it has been demonstrated that lipid lowering reduces CD40L expression in atheroma. 55In addition, oxidized LDL were reported to induce the expression of CD40 on endothelial cells, which can then bind CD40L from activated T-lymphocytes adherent to the site of injury. 58CD40 activation on endothelial cells provides a critical proinflammatory signal for the initiation of lesions.Indeed, the CD40L/CD40 interaction favors the up-regulation of adhesion molecules (E-selectin, P-selectin, vascular cell adhesion molecule-1 [VCAM-1] and ICAM-1) and leads to the secretion of proinflammatory cytokines (IL-6, IL-8, IL-15, monocytes chemotactic protein-1 [MCP-1], macrophage inflammatory protein-1 [MIP-1 / ] and regulated on activation normal T cell expressed and secreted [61][62] These reactions induce in turn the incorporation and accumulation of additional leukocytes, in particular monocytes, at the sites of developing lesions.
As discussed above, M 2, an integrin expressed on neutrophils and monocytes/ macrophages, has been identified as a receptor for CD40L.This interaction may also mediate adhesion and migration of inflammatory cells at sites of plaque initiation.In agreement with this hypothesis, M 2 deficiency attenuates lesion development and reduces lesional macrophage accumulation in LDLR -/-mice, supporting the implication of this integrin in atherothrombosis. 52However, additional studies will be required to specifically establish the importance of the CD40L/ M 2 in plaque initiation.
In addition, platelets have been shown to play a crucial role in the initiation of atherothrombosis.Platelets are among the first inflammatory cells at the site of injury and their adhesion to the endothelium provides a fundamental mechanism by which leukocytes are recruited. 63,64 ecause the surface of activated platelets contains a higher density of Pselectin than activated endothelial cells, significantly more leukocytes will incorporate at the sites of injury in their presence. 65Interestingly, CD40L from activated platelets can also induce a proinflammatory response on endothelial cells, in a similar fashion to that of Tlymphocytes.Henn et al. demonstrated that CD40 ligation on endothelial cells by CD40L from activated platelets induces the expression of numerous adhesion molecules, cytokines, and matrix metalloproteinases involved in the initiation of inflammatory reactions. 16

Plaque development and progression
The progression of the atherosclerotic plaque is typically highlighted by the proliferation and migration of smooth muscle cells into the intima, as well as the formation neovessels (angiogenesis), which supports the growth of lesions.This process will eventually yield a stable lipid-enriched necrotic plaque surrounded by a fibrous cap.Once in the subendothelial space, macrophages (originally monocytes) undergo phagocytosis of oxidized LDL particles, leading to the formation of foam cells. 66Thereafter, CD40L from infiltrated T-lymphocytes will bind to CD40 on the surface of differentiated foam cells, favoring the release of further proinflammatory cytokines (IL-1, IL-6 and IL-12), growth factors (vascular endothelial growth factor [VEGF]) and MMPs (MMP-1 and MMP-3). 7,67 hese responses are intimately involved in the proliferation and migration of smooth muscle cells into the intima layer.In parallel, cross talks between smooth muscle cells and T-lymphocytes may also take place, in which CD40 activation on the former initiates a positive feedback loop enhancing the inflammatory reactions already in place. 6Indeed, CD40 signaling in smooth muscle cells has been shown to induce the secretion of the cytokines IL-8 and MCP-1. 67,68 oreover, the accumulation of migrating fibroblasts within the intima layer exacerbates the atherosclerotic lesions in development and the CD40L/CD40 axis might also takes part in this process. 69timulation of fibroblasts with CD40L was reported to up-regulate the expression of cell surface adhesion molecules, thus facilitating their interaction with immune cells at the site of lesions. 702][73] Hence, the CD40L/CD40 interaction is at the forefront of a plethora of key inflammatory reactions involved in neointima formation and plaque accumulation.Lesions with eventually develop into the formation of a stable necrotic core consisting of infiltrated leukocytes, foam cells, proliferating smooth muscle cells, extracellular matrix proteins and lipids.
The formation of neovessels or angiogenesis plays an integral part in plaque progression and several reports have highlighted the importance of CD40L in this process.][76] These responses are tightly linked to tubule formation and angiogenesis, essential elements of plaque support and growth.Interestingly, the 5 1 integrin is upregulated on angiogenesis-prone endothelial cells and could also provide a novel mechanism by which CD40L modulates pathological angiogenesis. 77It would be worthwhile investigating this issue in further details.

Plaque instability and thrombosis
Plaque stability is regulated by a tight balance between extracellular matrix proteins such as collagen fibers and MMP production.A thin fibrous cap protects the highly thrombotic components of the atherosclerotic plaque.However, upon secretion of MMPs by macrophages and other inflammatory cells present, plaque rupture may ensue following digestion of the collagen fibers within the fibrous cap. 78,79 his process leads to thrombus formation and may cause complete obstruction of the artery.CD40L mediates several of the processes that set the stage for plaque rupture and its clinical sequelae.CD40L stimulation on endothelial cells, macrophages and smooth muscle cells can provoke the secretion of a long list of MMPs (MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13), the main digestive enzymes of the collagen-rich fibrous cap. 18,80,81 Patelets, in addition to their pivotal role in thrombosis, also participate in this process.Indeed, membrane-bound CD154 expressed on the surface of activated platelets can induce MMP upregulation in endothelial cells. 82MMP secretion and proteolytic activity can be abrogated by physical hindrance of platelet-endothelial contacts, IIb 3 interfering agents or anti-CD40L antibodies, thus highlighting in part the importance of platelets and CD40L in this phenomenon.
Following rupture, platelets rapidly adhere to the surface of the highly pro-thrombotic contents of the atherosclerotic plaque, thereby leading to thrombus formation and arterial occlusion. 83,84 ccumulating evidence also support a role for CD40L in platelet function and thrombus formation, albeit some of the data remain conflicting.For instance, Andre et al. have shown that CD40L plays a role in thrombus stabilization by interacting with IIb 3, while we and others have demonstrated that CD40L enhances platelet aggregation and thrombus formation through a CD40-mediated TRAF-2/Rac1/p38 signaling pathway. 27,28,48 Ieed, enhanced levels of circulating sCD40L exacerbate thrombus formation in vivo, also in a CD40-dependent fashion. 27Nevertheless, these studies all support the concept of CD40L as a pro-thrombotic agent, predisposing platelets to enhanced cell function.CD40L may also enhance the coagulation system through the induction of tissue factor release from various vascular cells.][87] Besides its important role in the induction of the extrinsic coagulation cascade, tissue factor also represents a powerful platelet agonist.

Soluble CD40L and coronary syndromes
Given the pivotal contribution of the CD40L system in atherothrombosis, multiple clinical studies have evaluated the association between levels of circulating sCD40L and cardiovascular risk, in particular acute coronary syndrome (ACS) such as acute myocardial infarction (AMI) and unstable angina (UA).These studies can be divided into two main categories.The first type of clinical studies has investigated the link between levels of sCD40L and ACS, while the second has determined the link between levels of sCD40L and prognosis and risk prediction.
9][90][91][92] Indeed, it appears that a gradual increase in sCD40L levels occurs with ACS progression, with peaks as early as 9 hours following the onset of AMI or UA. 89,93,94 Fr instance, patients suffering from AMI or UA present with levels ranging from 5-25 ng/mL, depending on the study. 88Moreover, sCD40L levels are independent from other important inflammatory markers, such as IL-6, sICAM-1, sVCAM-1, C-reactive protein and troponin, indicating that sCD40L may represent a more reliable risk factor, as compared to others. 92Because sCD40L in circulation almost exclusively originates from the shedding of membrane-bound CD40L at the surface of activated platelets, its measuring levels may reflect a state of platelet activation rather than an inflammatory condition.More importantly, some clinical studies have evaluated the relationship between sCD40L levels and disease prognosis.In the CAPTURE (c7E3 Fab Anti-Platelet Therapy in unstable Refractory Angina) trial, patients with high levels of sCD40L were 3-fold more at risk of developing cardiovascular death or AMI. 21Moreover, in the MIRACL (Myocardial Ischemia Reduction with Aggressive Cholesterol Lowering) study, sCD40L levels were an independent risk factor for recurrent cardiovascular events, such as death, nonfatal myocardial infarction, cardiac arrest and worsening angina requiring rehospitalization. 95nterestingly, individuals carrying the -3459A>G polymorphism on the CD40L gene, are more at risk of developing AMI. 96hether enhanced levels of sCD40L seen in patients with ACS are a consequence of increased platelet activation or a predetermining cause of these complications (or perhaps both) is still unknown.Recently, we have shown that enhancing levels of circulating sCD40L in mice to approximately 45 ng/mL exacerbates thrombus formation in a CD40-dependent manner. 27This observation supports the idea that increased levels of sCD40L in patients may drive, at least in part, the development of certain cardiovascular complications.It would be tempting to speculate the existence of a positive feedback loop taking place in these patients, where disease initiation correlates with platelet activation and release of sCD40L in the circulation.This in turn could further exacerbate pre-existing complications through enhancement of platelet function and thrombus formation.

Disruption of the CD40L system as a therapeutic target in atherothrombosis
In light of all the aforementioned data supporting the contribution of CD40L in inflammation, disruption of this system as a therapeutic strategy for the treatment of atherothrombosis and its clinical manifestations has been investigated.Unfortunately, clinical trials using an anti-CD40L antibody were put on hold due to thromboembolic complications. 97,98 nteractions between CD40 and CD40L-immune complexes at the surface of platelets have been suggested as a possible mechanism by which CD40L therapy induces these complications. 97nce circulating levels of sCD40L result from platelet activation, indirect targeting of the CD40L system through anti-platelet therapy may represent an alternative approach to suppress this important component.Clopidogrel, a potent inhibitor of the platelet adenosine diphosphate (ADP) receptor, has been reported to block sCD40L release from ADPstimulated platelets. 99Interestingly, clopidogrel regiment significantly reduces platelet CD40L expression and sCD40L levels in patients with stable CAD. 100 Moreover, IIb 3 inhibitors, such as abciximab, inhibit platelet aggregation and sCD40L release from activated platelets. 101In the CAPTURE trial, abciximab significantly reduces sCD40L levels and cardiovascular risk in high-risk ACS patients, confirming a link between IIb 3 signaling and platelet sCD40L release. 21atins exert multiple pleiotropic anti-inflammatory effects, in addition to their lipid lowering properties.Several reports have investigated the effects of these drugs on inflammatory markers, including CD40L.Particularly, they have been shown to reduce cytokine-induced CD40L expression on endothelial cells, smooth muscle cells and macrophages. 102Notably, atorvastatin treatment in the MIRACL trial reduces the risk of recurrent cardiovascular events, which are associated with sCD40L levels. 95st of these agents indirectly target the CD40L system, perhaps through inhibition of platelet activation and the subsequent release of sCD40L.Specific disruption of CD40L or its receptors remains a promising approach for the treatment of atherothrombosis.Although clinical studies using anti-CD40L antibodies have been unsuccessful, alternative targets of this system may render better clinical outcomes.For example, novel anti-CD40L agents that specifically target the interaction of CD40L with its different receptors or inhibition of critical intracellular signaling elements, such as TRAFs, represent valuable approaches.