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Abstract
The neovascular form of age-related macular degeneration (AMD), called wet-AMD or choroidal neovascularisation, begins with damage to the outer retinal cells and retinal pigment epithelium (RPE), which elicits a cascade of inflammatory and angiogenic responses leading to neovascularisation under the macula. Studies showed that oxidative damage, chronic inflammation of the RPE and complement misregulation work at different steps of this disease. After established neovascularisation, several pro- and antiangiogenic agents start to play an important role. Vascular endothelial growth factors (VEGFs) are the most specific and potent regulators of angiogenesis, which are inhibited by intravitreal injections of ranibizumab, bevacizumab, VEGF Trap, pegaptanib sodium and other agents under investigation. Pigment epithelium-derived factor, on the other hand, shows neuroprotective and antiangiogenic activities. Hepatocyte growth factor (HGF) has a mitogenic effect on a wide range of epithelial and endothelial cells, and it is inhibited by an anti-HGF monoclonal antibody. Platelet-derived growth factor is a potent chemoattractant and mitogen for both fibroblasts and retinal RPE cells, which has been inhibited experimentally by VEGF Trap and human anti-platelet-derived growth factor-D monoclonal antibody. Fibroblast growth factor-2 has pleiotropic effects in different cell and organ systems, and it is blocked by anti-FGF antibodies, with a greater benefit regarding antiangiogenesis when combined treatment with anti-VEGF is performed. Tumour necrosis factor alpha is expressed in the retina and the choroid, and its blockade in choroidal neovascularisation includes the use of monoclonals such as infliximab. This paper reviews the most important cytokines involved in the pathogenesis of wet-AMD, with emphasis on potential combined therapies for disease control.
- Age-related macular degeneration (AMD)
- choroidal neovascularisation (CNV)
- cytokines
- vascular endothelial growth factor (VEGF)
- angiogenesis
- choroid
- retina
- neovascularisation
- pharmacology
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- Age-related macular degeneration (AMD)
- choroidal neovascularisation (CNV)
- cytokines
- vascular endothelial growth factor (VEGF)
- angiogenesis
- choroid
- retina
- neovascularisation
- pharmacology
Introduction
Age-related macular degeneration (AMD) causes visual loss in around 10% of the older population, and these numbers are expected to increase over the next decade. In wet-AMD or choroidal neovascularisation (CNV), damage to the outer retinal cells and retinal pigment epithelium (RPE) elicits a cascade of inflammatory and angiogenic responses leading to neovascularisation under the macula.1 Significant progress was made in identifying the cytokines or growth factors that promote and inhibit angiogenesis in CNV (tables 1, 2).2 Several other types of agents and receptors participate in CNV formation, including the ephrins, angiopoietins, complement proteins, interleukins and chemokines, among others.1 This review will focus on the current state-of-the-art literature knowledge of the most relevant pro- and antiangiogenic cytokines involved in CNV formation. The secondary objective of this study is to point out the therapeutic drugs that selectively target the various cytokines in wet-AMD.
Proangiogenic and angiogenic cytokines involved in choroidal neovascularisation resembling age-related macular degeneration
Subtypes, molecular weight, year of discovery, ocular neovascular diseases, action, cellular origin in eye, systemic diseases and inhibitors regarding the cytokines involved in choroidal neovascularisation (CNV)
Vascular endothelial growth factors
The vascular endothelial growth factors (VEGFs) are the most specific and crucial regulators of angiogenesis.3 VEGF-A is a 45 kDa homodimeric glycoprotein belonging to the family that also includes VEGF-B through VEGF-E, platelet-derived growth factor (PDGF) and placental growth factor (PlGF).4–6 These regulators exert their biological effect through interaction with transmembrane tyrosine kinase receptors that activate a cascade of downstream proteins. The VEGF receptors identified so far are designated VEGFR1, VEGFR2, VEGFR3 and the neuropilins.7 VEGF-A is the most potent pro-angiogenic protein described to date; it induces proliferation, sprouting and tube formation of endothelial cells, playing a major role in CNV.8
A variety of agents have been evaluated to block VEGF-A action, thereby inhibiting CNV. An anti-VEGF aptamer, pegaptanib sodium (Macugen, Eyetech Pharmaceuticals/Pfizer, New York) was the first anti-VEGF-A165 isoform aptamer FDA-licensed for treatment of neovascular-AMD, but the drug has been removed from the market by the commercial provider.9 The humanised monoclonal antibody (mAb) bevacizumab (Avastin, Genentech/Roche, San Francisco) binds to and neutralises all human VEGF-A isoforms, with an affinity superior to that of the original antibody, but does not neutralise other members of the VEGF family. Ranibizumab (Lucentis, Genentech/Novartis, San Francisco), a high-affinity Fab variant of the rhumab VEGF-A, has been approved by the FDA for the treatment of wet AMD.10 Monthly injections of the intravitreal ranibizumab resulted in substantial improvement (15 or more letters) in 33 and 41% of those treated in the MARINA trial and ANCHOR trial, respectively, and a minority of patients (∼10%) experienced substantial vision loss.11–13 The past, current and future clinical trials with either bevacizumab or ranibizumab have been thoroughly reviewed.14–17
VEGF Trap (Aflibercept, Bayer HealthCare AG, Leverkusen, Germany; and Regeneron Pharmaceuticals, Tarrytown, New York), currently in phase III clinical trials for wet-AMD, is a recombinant soluble VEGF-receptor protein in which the binding domains of VEGFR1 and VEGFR2 are combined with the Fc portion of immunoglobulin G. The receptor portion of the molecule has a high affinity to all VEGF-A isoforms, PlGF 1/2, VEGF-B, VEGF-C and VEGF-D.18 The findings of phase II trials with VEGF Trap revealed that the most intense dosing regimen studied, 2 mg monthly intravitreal injection, resulted in a gain of more than 10 letters after 12 weeks.19 Additional promising broader VEGF blockers against CNV include drugs which act on the tyrosine kinase cascade, as PTK787 (Vatalanib, Bayer Schering Pharma AG/Novartis, Berlin, Germany), TG100801 (TargeGen, San Diego, California), GW786034B (Pazopanib, GlaxoSmithKline, Brentford, UK), AG-013958 (Pfizer, New York), SU11248 (Sunitinib, Sutent, Pfizer, New York), AL39324 (Alcon, Fort Worth, Texas) or SU5416 (Semaxanib, Pfizer, New York).20 21 Small interfering RNA (siRNA) targeting VEGF-A or its receptor VEGFR1 represents another therapeutic option.22 Despite the large number of drugs available, therapy alone may not be sufficient to cause permanent vessel regression in advanced stages of aberrant angiogenesis and may have a limited ability to impact established disease.23 In addition, strong and constant inhibition of VEGF-A may interfere with the paracrine communication between the choroid and the RPE cells. Parallel studies in mice showed that excessive VEGF-inhibition led to an increase in the number of dying cells of the inner and outer nuclear layers.24 Therefore, additional specific antiangiogenic agents are necessary for greater angiogenesis control, probably to be used in conjunction with anti-VEGFs.
The roles of VEGF-B, -C and -D in pathological angiogenesis remain a matter of debate. VEGF-B has been shown to affect cell adhesion by regulating plasminogen activator activity through VEGFR1, to have a mitogenic effect on ECs, and to modulate VEGF-A activity by the formation of heterodimers.25 26 Otani et al demonstrated strong immunoreactivity for VEGF-B in extracted CNV secondary to various causes.27 However, a recent study suggested that the angiogenic activity of VEGF-B is restricted to the ischaemic heart and has no effects on retinal neovascularisation.28 Two novel drugs, BiCentis and BiVastin (PhiloGene, New Jersey), are under evaluation in wet-AMD, as they have been claimed to be dual-functioning, both antiangiogenic and cytoprotective (http://www.philogene-inc.com/newsinfo3.html).
VEGF-C belongs to the PDGF-related growth factors and is a ligand for VEGFR2 and VEGFR3, which was reported to induce in vitro vascular EC migration, in vivo angiogenesis by means of VEGFR2 mitogenesis and EC survival.25 29 30 Otani et al described strong VEGF-C-staining in most pigment-containing cells in all CNV specimens.27 It may indicate that VEGF-B to -D contribute greatly to CNV and also modify permeability as a paracrine factor in CNV formation. VEGF Trap may bind all VEGF classes, including VEGF-C; however, no specific VEGF-C inhibitor is available yet.31
Recently, VEGF-F, was identified from viper snake venom. VEGF-F binds selectively to VEGFR2 and exhibits potent biological activity both in vitro and in vivo when compared with VEGF-A165.32 However, no consistent data are available regarding its ability to inhibit CNV.
Although PlGF-deficient mice develop normally, loss of PlGF blocks pathological angiogenesis and vascular leakage in cancer, ischaemia and wound healing.33 PlGF was highly detected in CNV specimens obtained during surgery, but VEGF-D was detected inconsistently27 In one study, deficiency or neutralisation of PlGF receptor induced a significant reduction in the incidence and in the severity of laser-induced CNV, suggesting that PlGF may have an important role in CNV.34 Tyrosine kinase inhibitors may inhibit all VEGF members including PlGF; however, no specific PlGF inhibitor is available yet.21
Pigment epithelium-derived factor
Pigment epithelium-derived factor (PEDF) has been found in a variety of ocular tissues including retinal and choroidal cells.35 36 The PEDF activities can be divided into two broad categories, neurotrophic/neuroprotective and antiangiogenic.37 There is an indirect correlation between PEDF and the extent of ocular neovascularisation, and an equilibrium shift between PEDF and VEGF in the uncontrolled growth of blood vessels in the eye.38 39 Under hypoxic conditions and in wet-AMD, secretion of PEDF is decreased, thereby allowing the endothelial mitogenic activity of VEGF to go unchecked.40–42
Retinal neovascularisation in preclinical models has been inhibited by intravitreal PEDF or by the expression of a transgene.43–45 In a phase I study, patients with neovascular AMD were given a single intravitreous injection of an adenoviral vector expressing human PEDF; there was no increase in lesion size in the high-dose group.46 A further study showed that subconjunctival administration of recombinant human PEDF in murine models decreased laser-induced CNV lesions.47 However, until the inconsistencies found in animal models with regard to dose-related induction of CNV are resolved, particular caution is required in the clinical use of PEDF.37
Hepatocyte growth factor
Hepatocyte growth factor (HGF) is a plasminogen-derived multifunctional polypeptide produced by mesenchymal cells that has a mitogenic effect and has been shown to be more effective at stimulating vascular EC proliferation than fibroblast growth factor (FGF) or VEGF.48–52 Although HGF and VEGF are considered to operate in parallel and may possibly evoke synergistic actions during angiogenesis, HGF may function as a VEGF inhibitor under certain circumstances.53 54
A study showed that a single blocking anti-HGF mAb, called L2G7, can inhibit biological activities induced by HGF in vitro and has profound neuronal antitumour activities.55 To inhibit tumour growth, a combination of three anti-HGF mAbs may be necessary, which may imply that multiple HGF epitopes should be targeted for cytokine inhibition, a difficult approach for drug development.56 Hu et al showed in the laser-induced CNV model that there was a 25% increase in VEGF expression which remained at a low-level plateau and that the neovascularisation corresponded to HGF and FGF expansion into the CNV.57 These molecular and immunocytochemical results suggest that FGF and HGF may be important as initial regulators of neovascularisation in the CNV model.
Platelet-derived growth factor
PDGF is a potent chemoattractant, dedifferentiator and mitogen for both fibroblasts and RPE cells.58 59 PDGF has five isoforms, PDGF-AA, -BB, -AB, -CC and –DD, that interact differentially with structurally related receptors designated α and β receptors.60 61 A study showed that retinal angiogenesis was inhibited by blocking PDGF-B alone without blocking VEGF164; however, combined blockade provided greater inhibition. Conversely, another study demonstrated that VEGF blockage alone inhibited the development of laser-induced CNV, whereas blocking PDGF-B signalling was ineffective on its own; again, greater inhibition occurred if both pathways were blocked.23 62
VEGF Trap and tyrosine kinase inhibitors act on numerous growth factors that mediate both neovascularisation and inflammation, including PDGF.63 Potential therapies directed against PDGF members include CR002 (CuraGen Corporation, Branford, Connecticut), a human anti-PDGF-D mAb whose safety was investigated in a phase I study in healthy subjects, and anti-PDGFRα antibody (mAb 3G3, ImClone Systems, Branchburg, New Jersey / Eli Lilly, Indianapolis, Indiana), which decreased cell proliferation and survival in several hepatoma cell lines.64 65 However, these targeted therapies have not undergone investigation for CNV therapy.
Fibroblast growth factor-2
Fibroblast growth factor-2 (FGF-2), also known as basic FGF or bFGF, is a heparin-binding growth factor, member of the FGF family that comprises nine members.66 67 FGF is a potent angiogenic molecule in vivo and is produced by a variety of cell types including vascular ECs of the choriocapillaris, fibroblasts, astrocytes and RPE cells.68 69 Basic FGF induces the secretion of VEGF and HGF by Müller glial cells and stimulates cell proliferation.69–71
As with VEGF and HGF, neovascularisation involves integrated VEGF and FGF signalling.69–72 Maximum co-labelling of VEGF and bFGF in rat RPE and choriocapillaris cells occurs within CNV sites between 2 and 7 days after laser photocoagulation.73 Rat bFGF typically occurs in three isoforms that are well expressed in retinal tissues, and a less typical 14 kDa low-molecular-weight isoform was detected in retinal tissue samples between 6 and 24 h after laser photocoagulation.57 One study compared the antiangiogenic effect of bevacizumab alone and combined with anti-bFGF antibodies. The main observation was that EC sprouting could not be fully inhibited by single inhibition of either anti-VEGF or anti-bFGF antibodies alone. However, combination treatment reduced RPE-induced EC sprouting.74 FGF is known to exert its angiogenic effect via VEGF-dependent as well as VEGF-independent pathways.75–78 Thus, targeting FGF in addition to VEGF could show synergistic effects in CNV treatment. It is important to note, however, that even combined inhibition did not fully reduce CNV-RPE-induced EC sprouting to baseline levels and that genetic ablation of the bFGF gene did not inhibit the formation of laser-induced CNV.72 This finding suggests that additional angiogenic pathways beyond VEGF and bFGF are involved in CNV RPE-induced angiogenesis.74
Tumour necrosis factor alpha
Tumour necrosis factor alpha (TNF-α) is the prototypical member of a family of cytokines that also include FasL, CD40L and TRAIL, involved in apoptosis, differentiation and cell activation.79 TNF-α receptors are expressed on many cell types in the retina and choroid, including RPE, Müller and choroidal vascular cells.80 81
TNF inhibitors in ophthalmology are currently being investigated in clinical trials for uveitis, especially infliximab (Remicade, Centocor, Horsham, Pennsylvania), a chimeric human immunoglobulin IgG1 with a mouse Fv variable fragment with high TNF-α affinity and neutralising capacity.82 Recent studies reported improvement of ocular inflammatory signs and visual acuity with infliximab in patients with uveitis.83–86 Interestingly, intravenous administration of infliximab for treatment of rheumatoid arthritis caused regression of CNV in patients with AMD.87 Etanercept (Enbrel, Amgen, Thousand Oaks, California) is an engineered recombinant dimeric protein generated by the fusion of the ligand-binding portion of human TNFR-2 linked to the Fc portion of human IgG1, used subcutaneously in children with juvenile idiopathic arthritis and associated uveitis.88–90 In a murine model study, mice treated intraperitoneally with etanercept or infliximab after laser induced-CNV showed a reduction in CNV size compared with the control group.91 A recent murine study showed that intravitreous infliximab injection reduced angiogenesis and glycosaminoglycan expression at low doses, whereas opposite effects were observed at high doses.92 A pilot study using intravitreal infliximab in patients with CNV secondary to AMD showed that a low dose of the drug was not well tolerated and was both immunogenic and retinotoxic.93 It should be acknowledged that CNV inhibition with anti-TNF-α agents still cannot be safely used in clinical practice.
Interleukin-8 and interferon-gamma
Two other factors involved in CNV formation are the interleukins (IL-6, -8 and-10) and the interferon γ (IFN-γ), involved in inflammation, oxidative stress, altered cholesterol metabolism and impaired function of the RPE.94–96
IL-8, a potent chemo-attractant and activator of neutrophils, is primarily involved in the initiation and amplification of acute inflammatory reactions and in chronic inflammatory processes.97 Concerning AMD, the response to reactive oxygen species in photoreceptor outer segments or inflammatory injury can lead to increased expression and secretion of IL-8.98 99 Individuals carrying the rs4073 variant may exacerbate RPE damage and favour progression towards advanced AMD.99
The interferon γ (IFN-γ), an antiangiogenic cytokine, has been shown to induce expression of Complement Factor H (CFH) in the human ARPE-19 cell line, and upregulate expression of major histocompatibility complex-II in primary human RPE cells.100 101 In contrast, Chen et al showed that CFH production in a cultured murine RPE cell line was not affected by IFN-γ.102 No specific therapy to date is available targeting those cytokines.
Vitreous and acqueous cytokines in CNV
Vitreous concentration in CNV due to AMD has been studied. Holekamp et al found that the PEDF level was higher in a control group than in a CNV/AMD group (16.4±7.1 vs 2.8±1.3 ng/μl, respectively), but there was no significant difference in VEGF levels (27.4±41.0 vs 39.3±28.9 pg/ml, respectively).42 In another study, on the other hand, the difference between PEDF in the CNV/AMD group and the control group was not statistically significant (1219 ng/ml vs 1469 ng/ml, respectively), and VEGF was undetectable in both groups.103
The aqueous cytokines have been assessed. Roh et al found that the mean aqueous VEGF concentration in AMD eyes was similar to that found in control cataract patients.104 Funk et al, by contrast, noticed an increased aqueous VEGF expression and a decreased PDGF expression in neovascular AMD eyes, but a decrease in VEGF was observed after intravitreal injection of ranibizumab.105 Lai et al showed a reduction in the mean aqueous VEGF level after intravitreal bevacizumab injections. Additionally, three monthly intravitreal injections of 1.25 mg bevacizumab seemed to be more effective than 2.5 mg in the treatment of neovascular AMD.106 Other studies also showed a significantly decreased level of VEGF with consecutive bevacizumab intravitreal injections.104 107 Those results suggest that measuring the concentration of cytokines from the aqueous humour might be a new pathway to detect treatment response.
Cytokine gene polymorphisms in neovascular AMD
Genetic polymorphisms in several cytokine genes have been described and demonstrated to influence gene transcription.108 Consistent with the role of inflammation in AMD, studies identified a strong association between a common variant (Y402H) of the gene for CFH and the risk of AMD.109–111 Chen et al found the risk-allele (C allele) in 5.8% of the subjects with wet-AMD and in 3.9% of control subjects in a Chinese population.112
The relation between interleukin gene polymorphisms (IL-6, -8 and -10) and AMD has also been studied. A study found that only the presence of the IL-8+781T allele was significantly associated with wet-AMD, disagreeing with another study that only found association for the –251A allele.108 113 Hull et al showed that the functional allele of IL-8 may lie on chromosomes of the −251A/+781T haplotype, and −251A may not be functional.114 However, the relation has been controversial, and it is unclear which one is the functional variant that directly affects IL-8 production.
Further biological and/or functional evidence is needed to confirm the role of cytokine gene polymorphisms in AMD.
Final remarks
The application of anti-VEGF drugs for CNV-therapy has markedly enhanced standards in terms of improvements in the control of disease. Bevacizumab and ranibizumab may undoubtedly induce clinical regression. Challenges in the research of VEGF therapy will lie in the clarification of which patients are most likely to respond to anti-VEGF drugs. Further studies should evaluate the efficacy of other types of VEGF inhibitors such as VEGF Trap or tyrosine kinase inhibitors.
Greater benefits may be achieved if angiogenesis, scarring and inflammation are simultaneously targeted. Inflammatory and angiogenic pathways become more numerous and redundant as disease progresses. Considering this, it is unlikely that inhibiting one factor or pathway will produce a sustained clinical effect in patients with previously treated, highly refractory disease. The fact that inflammation appears early in AMD pathology may explain why anti-inflammatory agents are beneficial as preventive or adjunctive therapies for patients who do not respond to conventional anti-VEGF therapy. However, more studies are required to test the efficacy and safety of systemic, periocular and intravitreal applications of each anti-inflammatory agent.115 Combination approaches may not only increase overall efficacy but also reduce the potential for side effects by allowing relatively low doses to yield a greater level of efficacy than higher doses of a single agent. Vessels with pericyte coverage do not regress with VEGF withdrawal, apparently because of Ang-1/Tie2 signalling between the pericytes, mediating survival mechanisms.116 117 Blocking the extravascular component and its products may help reduce disease morbidity in CNV as well. Molecular targeting of the inflammatory cytokines TNF, PDGF or FGF is possible via targeted biological therapies, and these cytokines are the most likely targets for effective combination treatments of wet-AMD.118 119
References
Footnotes
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.