Antichemokine Treatments in Acute Ischaemic Cardiovascular Diseases

Abstract

Inflammatory processes have been shown to be major pathophysiological determinants of patient vulnerability for acute ischaemic cardiovascular diseases. Among soluble inflammatory mediators, chemokines have been investigated as potential proatherosclerotic factors in both humans and animal models. In particular, several chemokines were shown to be related to plaque vulnerability and to predict independently the risk of ischaemic events. Moreover, chemokines are under investigation in secondary prevention. Considering the pathophysiological relevance of chemokines in atherogenesis, the development of therapeutic compounds selectively targeting their bioactivities might represent a promising approach to the prevention of both plaque rupture and adverse evolution of ischaemic injury. Although several compounds have been investigated in animal models with some promising results, at present there is no experimental evidence for the use of antichemokine mediators in clinics. Some potential safety concerns (immunosuppression and allergic reactions) have been indicated as potential limitations. The aim of this narrative review is to provide an update of the role of chemokines as biomarkers and promising therapeutic tools in acute ischaemic cardiovascular diseases.

1. Introduction

Atherosclerosis is a progressive disease affecting nearly all individuals all over the world. Atherogenesis slowly progresses from childhood until forming advanced lesions in adulthood [1]. Advanced lesions may remain stable or evolve into rupture-prone plaques (also termed vulnerable plaques). This acute event is the leading cause of lifethreatening ischaemic events, such as myocardial infarction and ischaemic stroke.

Since traditional risk factors, which emerged from Framingham heart study, failed to predict precisely the risk of plaque rupture, a new approach has been explored in the last decade [2]. Naghavi and co-workers proposed a novel paradigm of “vulnerable” patients focusing on three parameters (systemic, intraplaque and peripheral tissue vulnerabilities) and emphasising the role of inflammation as the driving force leading to “global” patient vulnerability [3]. Combined with the general concept of atherosclerosis as a systemic disease, this approach highlighted the importance of systemic biomarkers (including mediators of inflammation, prothrombotic factors and markers of matrix degradation) potentially to identify vulnerable plaques [4]. Many biomarkers have been investigated, but several studies are supporting a potential predictive role of chemokines for acute ischaemic events (

Table 1. Summary of studies investigating chemokine circulating levels as predictors of acute ischemic events in humans.

) [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22].

Although circulating levels of several chemokines have been shown to predict future ischaemic cardiovascular (CV) events, their use as potential clinical biomarkers is still unvalidated.

2. Chemokines in Acute Myocardial Infarction and Stroke

Chemokines (chemotactic cytokines) are small heparin-binding proteins that regulate leucocyte trafficking to sites of inflammation. The systematic nomenclature and classification of currently known chemokines (almost 50) relies on the different spacing of two conserved cysteine residues at the N-terminus. The different spacing establishes chemokine quaternary structure, function and also their classification into four families: CC-, CXC-, CX3C- and XC-chemokines [23]. CC-chemokines attract mainly mononuclear cells to inflammatory sites. CXC-chemokines recruit primarily polymorphonuclear leukocytes to sites of acute inflammation. CX3CL1/fractalkine is the only member of CX3C family; XCL1/lymphotactin and XCL2/SCL-1β are members of the XC family [23].

Chemokine intracellular signalling is transduced by binding to specific G-protein-coupled seven-transmembrane receptors (a superfamily of 20 members) categorised on the basis of their specificity for chemokine family (CCR and CXCR) [23]. Since several chemokines bind to multiple receptors and vice versa, different combinations of chemokine and chemokine receptor expression are available on the cell surface, thus enabling “tailor-made” cell recruitment. In addition, although certain chemokines are constitutively expressed, others are inducible and up-regulated by environmental stimuli, further enhancing leucocyte recruitment [24].

In the ischaemic myocardium, several overlapping pathways might up-regulate chemokine expression, including oxidative stress, cytokines, the complement cascade, toll-like receptor and NF-κB [25]. However, chemokine bioactivities are not limited to neutrophil recruitment during the first inflammatory phase. For instance, the chemokines CXCL12 and CCL2 have been shown to protect cardiomyocytes directly [26,27]. In addition, the chemokines CXCL12, CXCL1, CXCL2 and CCL2 were shown to induce angiogenesis and cell differentiation [28].

Also in the central nervous system, chemokines were shown to regulate both physiological and pathological processes. The CXCL12-CXCR4 axis might promote not only the inflammatory response [29], but also neural progenitor/stem-cell migration, proliferation and differentiation, both in neurogenesis [30] and after ischaemic stroke. We will discuss in the next paragraphs the specific role of CXC and CC chemokines in the pathophysiology of acute cardiovascular events.

3. CXC Chemokines

CXC chemokines have been associated with both atherosclerotic plaque instability and ischaemia/reperfusion injury within heart and brain, owing to their potent attraction of neutrophils and monocytes [31]. In humans, CXCL8 is the prototype of the glutamate-leucine-arginine (ELR+) subfamily and the most investigated CXC chemokine. Its homolog chemokine in mice is CXCL2. Oxidised low density lipoproteins (oxLDL) strongly induced CXCL8 expression by monocytes [32]. In addition, CXCL8 has been shown to be released by other cells colonising atherosclerotic plaques, such as foam [33] and endothelial cells [34]. In experimental ischaemia/reperfusion injury, CXCL8 was detected in the border zone of the infarct [35], closely linked to neutrophil infiltration. Accordingly, treatment with recombinant CXCL8 [35] or anti-CXCL8 [36] antibodies enhances or prevents neutrophil infiltration, respectively. We recently suggested a direct role for CXCL8 in human carotid plaque vulnerability. In fact, patients with symptoms of ischaemic stroke had higher intraplaque levels of CXCL8 messenger ribonucleic acid (mRNA) as compared with asymptomatic subjects [37]. In addition, CXCL8 levels were also increased in serum [38] and cerebrospinal fluid [39] after an ischaemic stroke. CXCL1 was shown to enhance not only vascular inflammation [40], but also angiogenesis and endothelial progenitor cell (EPC) recruitment [41] together with CXCL8 [42], even if there is no agreement about their activities [43]. In the cerebrospinal fluid, CXCL1 levels positively correlate with the volume of cerebral hypodense areas (assessed with computed tomography [CT]), suggesting an involvement of this chemokine during early inflammatory phases after ischaemic stroke [44].

On the other hand, CXC chemokines lacking the ELR motif (such as CXCL9, CXCL10) have been shown to block the early healing phases after ischaemic injury. In addition, these chemokines have been described as are active angiostatic factors [45,46] and inhibitors of fibroblast migration [47]. In contast to other CXC chemokines, CXCL12 and its receptors CXCX4 and CXCR7 were clearly shown to induce beneficial effects. CXCL12 is expressed in atherosclerotic plaques [48] as well as in myocardium [49] and brain [50] after ischaemia, and it was shown to promote tissue recovery through EPC recruitment [51,52]. It should be noted that modified CXCL12 may also have detrimental effects. In fact, the cleavage of CXCL12 by matrix metalloproteinase (MMP) 2 was shown to create a neurotoxic molecule that did not bind CXCR4, but had an increased affinity for CXCR3 [53].

4. CC Chemokines

CCL2 was shown to increase plaque vulnerability, recruiting proinflammatory monocytes in both mouse [54] and human [55] atherosclerotic plaques. In an experimental model of myocardial ischaemia/reperfusion injury, CCL2 inhibition [56] or deletion [57] were shown to reduce infarct size. Similar findings were observed in mice deficient of CCR2 (CCL2 receptor) [58]. In addition to monocyte recruitment, CCL2 was shown to play a pivotal role in infarct healing, modulating macrophage differentiation and cytokine expression [59] and promoting fibroblast progenitor recruitment and differentiation [60]. In mouse models of stroke, CCL2 [61] or CCR2 [62] knockout mice resulted in a smaller infarct size. On the other hand, CCL2 was shown potentially to contribute to cerebral recovery, promoting recruitment of bone marrow-derived stromal cells [63] and newly formed neuroblasts from the neurogenic region [64]. Interestingly, these latter findings suggest a double role of CCL2 in ischaemic stroke, harmful in the earlier stages and protective later.

Another CC chemokine (CCL5) was recently shown to have detrimental effects in ischaemia/reperfusion injuries [65]. CCL5 orchestrates the recruitment of several inflammatory cell subsets, such as monocytes, neutrophils, dendritic cells, and lymphocytes, to the inflammatory site, through the binding to various transmembrane receptors (CCR1, 3, 4, 5). As showed by our research group, treatment with antibodies neutralising CCL5 bioactivity reduced both infarct size and postinfarction heart failure in a mouse model of chronic cardiac ischaemia [66]. On the other hand, in experimental models of focal cerebral ischaemia, deletion of CCL5 was associated with reduction in leucocyte infiltration, infarct size and blood-brain barrier permeability [67]. In addition, Tokami and coworkers, starting from the observation that CCL5 serum levels were increased in ischaemic stroke patients as compared with healthy controls (p<0.001), reported that CCL5 expression was up-regulated in mouse neurones after middle cerebral artery occlusion [68]. In the same study, the authors observed that treatment with CCL5 triggered salvation intracellular pathways in experimental neuronal cells [68].

5. Update on Treatments Targeting Chemokines

Selective chemokine inhibitors currently under investigation were synthesized in accordance with different strategy approaches:

• modified chemokines;
• synthetic small molecules acting as antagonist or inverse agonist at chemokine receptors;
• neutralising antibodies targeting chemokines or their receptors;
• chemokine-binding proteins.

In addition, other drugs were shown to interfere indirectly with chemokine bioactivities.

6. Selective Chemokines Inhibitors

As reported in

Table 2. Summary of selective anti-chemokine treatments for acute ischemic events in animal models.

selective inhibitors of both CC and CXC chemokines have been recently tested in animal models of acute ischaemic cardiovascular diseases [65,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86].

Liehn and coworkers investigated a nonagonist CCL2/MCP-1 mutant (PA508) with increased affinity for glycosaminoglycans, thus competing with CCL2 in binding CCR2. This molecule reduced myocardial ischaemia/reperfusion injury and limited neointima formation in experimental carotid artery injury [74,86]. On the other hand, CCL5 inhibition was recently shown to be a very promising treatment against plaque vulnerability and acute myocardial infarction in mice. Braunersreuther and coworkers showed that treatment with [44AANA47]-RANTES (a mutated variant of CCL5/RANTES that inhibits chemokine oligomerisation on endothelial cell surface) reduced histological features of plaque vulnerability and infarct size in mice by impairing inflammatory cell recruitment [82,87].

Related to the CXC chemokines, plerixafor (formerly AMD3100; Mozobil™) is a small bicyclam molecule originally developed for treatment of human immunodeficiency virus (HIV) infection and currently approved by US Food and Drug Administration and European Medicines Evaluation Agency (EMEA) for bone marrow-derived stem cell (BMSC) mobilisation in autologous stem cell transplantation. Plerixafor reversibly disrupts the interaction between chemokine receptor CXCR4 and its ligand CXCL12 [88]. The enhanced BMSC mobilisation improves haematological outcome but several insights suggest beneficial effects of plerixafor also in healing of ischaemia and ischaemia/reperfusion injury.

First in 2007, Proulx and coworkers reported that pulse therapy with AMD3100 in a rodent model of myocardial infarction reduced infarct size, improving systolic function [69]. Other research groups confirmed these findings in experimental models of both myocardial infarction [71,75] and myocardial ischaemia/reperfusion injury [73], also reporting the key role played by BMSC recruitment in the recovery after ischaemic injury [71,74]. AMD3100 has also proved to be effective in a mouse model of ischaemic stroke, where it reduced neutrophil recruitment, cytokine production and brain swelling, thus improving neurological outcome [76]. In contrast, when continuously infused, AMD3100 was shown to increase infarct size and impair cardiac remodelling, so worsening ventricular function [70,71,72,73]. These conflicting results might depend on the pharmacological properties of AMD3100 (especially reversible binding and the short plasma half-life [0.9 h in rodents]). Thus, compared with pulse therapy, continuous infusion of AMD3100 would compromise BMSCs homing in to the injured myocardium, which is largely dependent on local expression of CXCL12.

Reparixin (an inhibitor of CXCR1 and CXCR2) attenuates neutrophil recruitment (assessed as myeloperoxidase activity) in a rodent model of stroke. However, the promising preliminary results have been weakened by more recent conflicting results on the pathophysiological relevance of neutrophils in cerebral infarction [77,78,79].

Evasins (chemokine-binding proteins secreted in the saliva of bloodsucking parasites, such as ticks) have been recently isolated and tested in acute cardiovascular diseases [89]. We showed that treatment with evasin-3 (an inhibitor of CXC chemokines) was able to reduce the recruitment of leucocytes in the injured tissues in mouse models of myocardial infarction [83] and ischaemic stroke [84]. However, the potent anti-inflammatory properties of evasin-3 were associated with improvements in infarct size only in acute myocardial ischaemia. Conversely, the selective inhibition of CXCL1 failed to reduce neutrophil recruitment or infarct size in a mouse model of chronic myocardial ischaemia [85].

7. Nonselective Chemokine Inhibitors

Different drugs were shown to modulate indirectly chemokines and their cognate receptors in ischaemic tissues, thus interfering with post-infarction inflammation and ischaemia/reperfusion injury [8,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105] (

Table 3. Nonselective treatments for acute ischemic events impacting the expression of chemokines and their receptors.

).

For instance, Di Filippo and coworkers showed an improvement in a model of ischaemia/reperfusion myocardial injury under treatment with the cannabinoid receptor type 2 (CBR2) agonist WIN-55,212-2, associated with a decreased CXCL2 expression [90]. Accordingly, we and other researchers [91,92] showed that treatment with the CBR2 agonist JWH-133 was able to reduce myocardial infarct size and the associated increase of the chemokines CXCL1, CXCL2 and CCL3. Dipeptidyl peptidase-4 ([DPP-4], a serine protease that cleaves off N-terminal dipeptides from peptide substrates) was shown to improve cardiac function after myocardial ischaemia, increasing CXCL12-mediated BMSC recruitment 80,94].

Finally, the inhibition of chemokine up-regulation after acute myocardial infarction was also induced by the reactive oxygen species (ROS) scavenger edaravone [104] and FK866 (a nicotinamide phosphoribosyltransferase [Nampt] inhibitor) [105].

8. Limitations of Antichemokine Treatments in Humans

To date, only two chemokine receptor antagonists have been approved by the US Food and Drug Administration and the EMEA: the CCR5 antagonist maraviroc for treatment of HIV and the CXCR4 antagonist plerixafor for stem cell mobilisation. There were many disappointments in clinical testing of potential inhibitors of chemokines and their receptors. The compounds might have failed for several reasons, especially during clinical evaluation, that point out the differences between animal models and humans. Although the redundancy in the chemokine system can explain lack of efficiency or adverse drug reactions [106], the greatest concerns arise from immunological side effects that impair host defenses. The pivotal role of chemokines in immune the response against pathogens has been well established [107]. Similarly, impairment in immune responses was observed after inhibition of the CCL2–CCR2 axis [107] or after Met-CCL5 administration [108]. In addition, animals used for experiments are usually maintained in a pathogen-free environment, an uncommon situation in human life. It is conceivable that side effects are comparable to those caused by prolonged treatment with tumor necrosis factor blockers [109] or corticosteroids [110].

Moreover, both modified chemokines and synthetic peptides have poor bioavailability orally and require subcutaneous or intravenous administration. This might increase the risk of developing allergic reactions or antibodies that would hamper long-term treatment.

9. Conclusions

It is well established that the chemokine system plays a pivotal pathophysiological role in cardiac and cerebral ischaemic injuries, modulating a wide range of biological processes (especially leucocyte recruitment, but also angiogenesis and BMSC infiltration). However, the biological consequences of their pharmacological inhibition require further basic research before the clinical use. As biomarkers, chemokines might also play a critical role in the better assessment of cardiovascular risk. In that case, additional clinical studies are also needed to validate their potential to predict acute ischAemic cardiovascular events in both primary and secondary prevention.