Immune responses regulating the response to vascular injury

Paul C. Dimayuga, Kuang-Yuh Chyu and Bojan Cercek

Oppenheimer Atherosclerotic Research Center, Division of Cardiology, Cedars-Sinai Heart Institute Los Angeles, California, USA Correspondence to Paul C. Dimayuga, PhD, Davis 1064, Cedars-Sinai Heart Institute, Los Angeles, CA 90048, USA Tel: +1 310 423 7605; fax +1 310 423 0299; email: dimayugap@cshs.org Current Opinion in Lipidology 2010, 21:416-421

Purpose of review Immune modulation of neointimal formation after vascular injury had been investigated for several decades but the complexities involved continue to obscure a clearer understanding of the process. The rapidly changing field of immunology makes this knowledge imperative. Recent findings The review discusses immune factors involved in the response to vascular injury. Although innate immune responses play a predominantly detrimental role, the adaptive immune response is more complex. Mechanisms of T-cell activation, recruitment, as well as possible regulation are highlighted. Summary Progress in understanding the role of the immune system in the response to arterial injury has been impressive. However, recent findings underscore the need to unravel the intricacies involved such as the kinetics and specific pathways of activation, specificity of immune cell involvement, and the identification of targets for therapy. This is relevant in light of the increasing reports of immune factors involved in vascular disease and intervention in the clinical setting. Keywords immune response, neointima, vascular injury Curr Opin Lipidol 21:416-421

C 2010 Wolters Kluwer Health I Lippincott Williams & Wilkins 0957-9672

Introduction Percutaneous transluminal coronary angioplasty (PTCA) was first performed over 30 years ago [1], yet the underlying mechanisms of the process of restenosis remain unresolved. Advances in the technology used have improved outcomes, and it is intriguing that immune-suppressive agents are among the drugs under investigation to reduce in-stent restenosis. The intricacy of the process and the rapidly changing field of immunology underscore the complexity of the immune response to vascular injury. The current review will attempt to summarize certain key aspects using a short historical narrative and discussion of more recent developments. The immune response to vascular injury The link between injury of endothelial cells lining the arterial wall and atherosclerotic plaques was already under investigation [2,3] when performed. Within this framework of studies came the report immunoglobulin (Ig)G presence in the injured endothelial cells of the implicating the humoral immune response in arterial injury.

the presence of PTCA was first that described rabbit aorta [4]

T lymphocytes were not implicated until much later, when they were found in injured vascular tissue [5]. Subsequently, it was reported that neointimal thickening after injury was increased in T-cell-depleted or athymic nude rats [6]. These early report confirmed the role of the immune system in the response to arterial injury. The availability of genetically manipulated mice and different forms of vascular injury show that not only responses but also on the various methods of arterial injury as well. Tissue injury and immune response Increase neointimal formation in immune-deficient animals [6-10] provides a clear link between injury and the immune response to it. However, the specific antigens involved remain unknown. Various reports on the roles of specific cell types and immune factors provide some insight. In addition, cell injury alone can result in the release of intracellular material, including uric acid [11] and heat-shock proteins that can act as adjuvants resulting in immune activation [12]. Sometimes referred to as damageassociated molecular patterns (Fig.1), these endogenous molecules, produced by cell apoptosis or necrosis, signal through pathogen-associated molecular pattern receptors[13]. Therefore, it is not unreasonable to speculate that injury will result in both antigen-specific and nonspecific immune response. The specific arm of the immune system responding to vascular injury may be as important determining factors. Figure 1 Schematic diagram of immune response to arterial injury

Arterial injury Innate immunity Adaptive immunity Macrophage (DAMPs, TLRs) B cells (natural antibodies) Complement T cells (subset specificity?) CRP ( thrombosis) NKT Tregs? Neointimal formation ? Co-stimulation: ICOS;4-1BB;CD40;sCD40L CD40L (T cells) Pathways include adaptive immunity regulating innate immune responses, which predominantly promotes neointimal formation. Unknown factors include T-cell subtype, dendritic cell function, the role of regulatory T cells, and whether neointimal cells amplify or suppress further immune signaling. CRP, C-reactive protein; DAMPs, damageassociated molecular patterns; DCs, dendritic cells; NKT, natural killer T cells; TLRs, Tolllike receptor; Tregs, regulatory T cells. Innate immunity Mice lacking a functional adaptive immune system have compensatory changes in innate immune fuctions [14]. Recombination-activating gene-deficient (Rag-/-) mice have a severe combined immune-deficiency phenotype [15]. The exaggerated neointimal formation in Rag-/- mice [8-10,16] suggest that innate immunity without the inherent control by adaptive immune responses [17] is detrimental after vascular injury (Fig. 1). Macrophage infiltration promotes neointimal formation after injury. Mice deficient in granulocyte-macrophage colony-stimulating factor had reduced macrophage infiltration and neointimal formation [18]. Various growth factors and proteases have been implicated in the process but of particular interest in immune function of macrophages is the Toll-like receptor (TLR) family of proteins (Fig. 1). TLR-2 and TLR-4 have both been shown to contribute to neointimal formation and vessel remodeling after arterial injury. However, it would appear that endogenous TLR ligands after vascular injury are insufficient triggers as the studies [19,20] used exogenous ligands to stimulate the signaling cascade. Common to the signaling cascade of various TLRs is the myeloid differentiation protein-88, which regulates vascular remodeling [21], but its role specifically in neointimal formation remains unknown. Although it is not yet clear which pathway(s) of complement activation is involved in vascular injury, it is known that complement activation is detrimental to the outcome (Fig.1). Neointimal formation is reduced by C1esterase inhibition in hypercholesterolemic mice [22]. In addition, exuberant neointimal thickening in immune-deficient Rag-1-/- mice is inhibited by complement depletion using cobra venom factor [16]. Interestingly, serum C3 is higher in immune-deficient mice [16,23], supporting the notion of a compensatory increase in innate immune response in mice deficient in adaptive immune responses or of a regulatory role of the adaptive immune response to innate immunity [14,17].

Not as straightforward in its apparent role in vascular injury is C-reactive protein (CRP). A phenotype of mice transgenic for human CRP is increased thrombosis after wire injury of the artery. Treatment of wire-injured male CRP-transgenic mice with antithrombotic drugs, to uncouple its prothrombotic role in injury, reduced neointimal formation [24]. However, neointimal formation is dependent on Fc RI. This seemingly opposite results are perhaps indicative of the prothrombotic role of CRP in vascular injury and neointimal formation (Fig.1). The flow-cessation model causes thrombus formation in the immediate vicinity of the ligature [26], whereas the wire injury model normally does not. As mentioned above, transgenic CRP expression increased thrombosis in the wireinjured mice. Accordingly, when thrombosis was inhibited by drug treatment, neointima was less. Further studies are needed to clarify this issue. Interestingly, deficiency in the tissue factor pathway inhibitor, a regulator of the coagulation cascade, increased neointimal formation in the flow-cessation model [27]. Adaptive immunity Although a generalization can be made that innate immune responses are predominantly detrimental in vascular injury, the adaptive immune response appears to be more complex. B cells and natural antibodies B lymphocytes inhibit neointimal formation [8], likely through the production of immunoglobulin as direct administration of serum-derived, nonimmune IgG or IgMreduced neointimal formation in Rag-1-/- mice [16]. The effect is polyreactive from natural antibodies (Fig.1) as the immunoglobulin isotypes were derived from nonimmunized mice. Natural antibodies are produced in the absence of overt specific antigenic stimulation and found in serum of normal mice [28]. Interestingly, the effects were mediated at least in part by regulating complement activation, deposition in injured arteries, or both [16]. Antibodies reactive to specific epitopes may have specific effects on the injured vascular wall. A prominent antigen reported to elicit specific IgM response to apoptotic cells is phosphorycholine. Arterial injury did not affect phosphorycholine-IgG or phosphorycholine-IgM levels, and treatment of injured Rag-1-/- mice with a phosphorycholine-specific monoclonal IgM did not affect neointimal formation [16], suggesting that the phosphorycholine response may not be an important factor in vascular injury. In contrast, phosphorycholine-IgM treatment reduced neointimal formation in a model of syngeneic vein graft arteriosclerosis [29], suggesting that the immune response may have some degree of specificity to the type of vascular injury or insult. T cells

Evidence points to a likely role of T lymphocytes in the response to vascular injury [6,7,9]. Deficiency of the C-C chemokine receptor 5 (CCR5) in apolipoprotein E-/- mice was associated with reduced presence of CD3+ T lymphocytes and reduced neointimal formation after arterial injury [30]. Reduced T-cell infiltration in mice deficient in chemokine (C-C motif) ligand 5 (CCL5) (also known as RANTES Regulated upon Activation, Normal T-cell Expressed, and Secreted; a known ligand of CCR5) was also observed early after arterial injury [31*]. Transfer of wild-type bone marrow cells into irradiated CCL5-/- mice significantly reduced T-cell infiltration and neointimal formation, confirming the pivotal role of CCR5-CCL5 interaction that recruits T cells in the injured vessel wall. But the results also underscore the complexity of T-cell involvement in the response to injury. Prior studies [6,7,9] using T-cell depletion, T-cell deficiency, or T-cell transfer experiments all suggest an inhibitory role in neointimal formation after arterial injury. Yet, the results from CCR5-CCL5 studies cited above suggest otherwise. Memory T cells are thought to have high CCR5 expression [32] and CD4+ T cells with impaired CCR5 signaling have reduced CD25 expression [33]. One can speculate that global depletion on T cells seems to be detrimental but selective inhibition of specific T-cell subsets may reduce neointimal formation. It is likely that the specific T-cell type involved dictates downstream interaction and signaling with other cells that results in specific outcomes (Fig.1). For example, self-lipids and glycolipids are important immune signaling molecules in the response to arterial injury [34]. CD1d signals a unique T-cell population called natural killer T cells that respond to lipid antigens [35]. Cell injury and death releases various molecules including lipid neoantigens that may elicit immune responses. Deficiency of the major histocompatibility complex-related molecule CD1d in mice results in reduced neointimal thickening [34]. It is currently unknown what others T-cell subtypes are selectively involved in the response to vascular injury. A recent report has directly linked a specific signaling pathway induced by arterial injury with T-cell response. Selective deletion of hypoxia-inducible factor-1-alpha (Hif-1 ) in T cells resulted in significantly increased neointimal formation. The main findings of the study indicates that Hif-1 is an important regulator of T-cell function and response [36**], as had been previously reported [37,38], the significance of which will be discussed later. What will be emphasized at this points is that the report by Kurobe et al. [36**] has identified a signaling pathway that links the immune response to arterial injury. HIF-1 is one of the mechanisms cells use to adjust to low oxygen tension [39], which occurs in the injured artery [36**]. However, HIF-1 signaling can be induced by both hypoxic and nonhypoxic pathways [40,41], and arterial injury induces both. What is intriguing is that T-cell activation by T-cell receptor (TCR) signaling increases HIF-1 expression [37]. The report by Kurobe et al. [36**] provides evidence that arterial injury induces specific T-cell activation pathways that play a significant role in the response to injury (Fig.1). Dendritic cells and co-stimulation

T-cell activation and response after injury likely involves stimulatory and co-stimulatory signals. Dendritic cells, the classic antigen-presenting cells involve in immune activation, have been observed in the vascular wall [42], yet their specific role in the response to injury remains to be clucidated (Fig.1). Dendritic cells presence in the neointimal layer increased within the first week of injury in rats [43], although no specific immunerelated signaling was defined and instead was linked with antiapoptotic functions. On the contrary, dendritic cells may have the capacity to induce autoimmune response in T cells [44] that could exacerbate the inflammatory response after vascular injury. However, other cells such as macrophage, endothelial cells, and smooth muscle cells may act as sources of co-stimulatory signals that could participate in priming or sustaining the response to injury. Inducible co-stimulator (ICOS) is a member of the CD28/cytotoxic T lymphocyte antigen family of co-stimulatory molecules and blocking of ICOS signaling reduced neointimal formation after arterial injury [45]. CD137 (4-1BB) is a member of the tumor necrosis factor (TNF)-receptor superfamily expressed on activated T cells, which, when bound by its ligand, initiates signaling through adaptor molecules called TNF-receptor associated factors (TRAFs). Blocking the 4-1BB/4-1BBL pathway with a soluble 4-1BB immunoglobulin -fusion protein significantly reduced neointimal formation in a wireinjury model [46]. The study further showed that expression of both 4-1BB and its ligand increased in the vascular wall after injury, suggesting that the observed results are not mediated exclusively through immune functions. This is especially important given the recent findings that 4-1BB/4-1BBL signaling may actually have dual immunologic functions [47]. CD40-CD40L interaction, also a member of the TNF superfamily, is an important component of adaptive immune signaling. Neointimal formation after ligation injury was significantly attenuated in CD40-/- mice, predominantly through TRAF6 signaling [48]. There was significantly less lymphocyte infiltration of the injured arterial segment, again suggesting that T cells contribute to neointimal formation. Interestingly, thare was a neosignificant trend for a similar neointimal reduction in CD40L-/- mice. In contrast, Remskar et al. [7] had previously reported that CD40L-/- mice had significantly increased neointimal formation compared with wild-type mice. As was suggested by Donners et al. [48], the difference in outcome was likely a result of the different injury models used, again suggesting the importance of thrombosis in the response to vascular injury. CD40 deficiency in mice does not affect thrombus formation [48]. On the contrary, CD40L-/mice have reduced thrombosis in 3 integrin-dependent manner [49]. Indeed, neointimal formation was reduced in 3 integrin-deficient mice using a carotid ligation injury model, nut not a modified wire-injury model [50]. Thrombus formation in large arteries is 3 integrin dependent [51]. Thus, the results again suggest that the propensity to form a thrombus will significantly affect the outcome of neointimal formation after arterial injury.

Interestingly, Remskar et al. [7] showed that rescuing CD40L deficiency on T lymphocytes by adoptive T-cell transfer reduced neointimal formation, suggesting that the CD40-CD40L signaling in that model is predominantly through T lymphocytes. On the contrary, soluble CD40L increased leukocyte recruitment [52] and impaired endothelial cell regrowth [53] after injury, leading to increased neointimal formation. Thus, soluble CD40L may signal differently, presumably beyond immune cell interaction and result in a different outcome. These findings support the notion of selective signaling by specific Tcell subsets in response to vascular injury (Fig.1). Regulation of the immune response to injury Renewed interest in how the immune system regulates itself ahs provided evidence that regulatory T cells (Treg) play an important role in modulating the immune response to atherosclerosis disease [54-57]. However, clear evidence for a role in the response to injury is practically nonexistent. But the study by Kurobe et al. [36**] cited above suggests a role for T-cell regulation in the response to injury. HIF-1 regulates T lymphocyte function [38] and HIF-1 deficiency in T cells results in increased neointimal formation [36**], indicating that unregulated T-cell response to injury results in a detrimental outcome. It also suggests that immune intervention targeted to enhancing inherent Treg fuctions may be a therapeutic avenue that warrants further investigation. Immune-suppressive drug-coated drug-eluting stents The wide use of PTCA with the inherent risk of arterial recoil and shrinkage, and the migration and proliferation of vascular cells associated with restenosis led to the development of drug-eluting stents (DES). One of the principal agents used is an immune-suppressive drug, sirolimus and its analogues [58,59]. Its effects in reducing, restenosis and attributed to inhibiting the transition of cycling cells from G1 to S phase. However, reports [58,59] of complications increasingly raised concerns that the treatment led to late stent thrombosis attributed to incomplete repair of the injured artery. Paradoxically, very few studies have been conducted to investigate T lymphocytes in DES in spite of the use of immune-suppressive drugs. One study [60] showed two out of six atherectomy samples from restenotic stents over 8 months after DES implantation with significant T lymphocyte infiltration. Another study [61] showed that effector-memory T cells were increased in blood sampled from the coronary sinus by sirolimus but not paclitaxel DES. Furthermore, PTCA increased T helper (Th)1-cell frequency in nonstatin-treated stable angina patients [62] and decreased Th2 cytokines in unstable angina patients [63]. Thus, a clear understanding of the role of lymphocytes in restenosis remains elusive. The importance of this issue is underscored by the reported persistence of immune activation after PTCA [64] (Fig.1). Conclusion Although substantial experimental evidence supports a role for the immune system in response to arterial injury, little is known about the kinetics of the response. This is of course changing at a measured pace, and clinical correlates are intriguing. Yet, it remains

unclear what the dominant response is cellular or humoral? (Fig.1). How is the response activated and how is it resolved? What are the implications of the use of different models of experimental arterial injury? The answers will no doubt be forthcoming, and an approach that includes the notion that at least a part of the immune response to arterial injury is a response to the self, with self-antigens and self intriguing results and lead to a better understanding of the interaction between biological process in the vascular wall and the immune system. Acknowledgements The authors are supported by funding from the Spielberg Family Cardiovascular Research Fund and the Heart Foundation at Cedars-Sinai Medical Center.

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