CC Chemokine receptor-3 is involved in angiogenic processes. Recently, CCR3 was reported to play an important role in choroidal neovascularization development and CCR3 targeting was documented to be superior to standard vascular endothelial growth factor-A (VEGF-A) treatment when tested in an artificially induced CNV in animals. As CCR3 studies are lacking in the human age-related macular degeneration (AMD) patients we sought to determine if CCR3 has any association with inflammatory processes that occur in CNV. Total 176 subjects were included on the basis of inclusion criteria. Real time PCR was used to analyze the single nucleotide polymorphism in CCR3 of AMD (115) and normal controls (n= 61). Genotype frequency was adjusted for possible confounders like cigarette smoking, alcohol, meat consumption and other risk factors. Chi-square test was used for analysis of polymorphism. The genotype distribution of CCR3 (rs3091250)
polymorphism was significantly different in AMD patients as compared to controls in the Indian population. GT (Heterozygous) and TT (homozygous) at the rs3091250 SNP increased risk of AMD as compared to the GG genotypes (OR =4.8, CI95%= 2.2-10.8 and OR = 4.1, CI 95% = 1.6-10.1 respectively). Subgroup analysis of AMD patients in wet and dry revealed no significant differences. There was no significant difference for rs3091312 in AMD and control group. A significant association between AMD and CCR3 (rs3091250) polymorphism localized on chromosome 3p21.3 was detected. The results suggest the possible contribution of rs3091250, a new predisposing allele in AMD.
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Key Words: CCR3, inflammation, chemokines, single nucleotide polymorphism, population study, AMD
Age-related macular degeneration (AMD) is a leading cause of central vision loss in the elderly in industrialized countries (Cook et al., 2008). Symptoms of AMD may appear in one or in both eyes. Early symptoms include metamorphopsia or blurring of central vision. AMD is characterized by the formation of drusen in Bruch's membrane, the degeneration of photoreceptors and the underlying retinal pigment epithelium in the macula, geographic atrophy, and choroidal neovascularization.
According to clinical age-related maculopathy staging system, age-related maculopathy grades are: no drusen, several small drusen and no retinal pigment epithelial changes, retinal pigment epithelial alteration but no drusen, both small drusen and retinal pigment epithelial changes, several intermediate-size and large drusen, drusenoid retinal pigment epithelial detachment, geographic atrophy and choroidal neovascular membrane with disciform scar (Seddon et al., 2006). The geographic atrophy, also known as the ''dry'' form of AMD, is characterized by atrophy of the central retina. The exudative or ''wet'' AMD, a major cause of severe vision loss, is identified by the presence of choroidal neovascularization (CNV), new blood vessels originated from the choroid that invade the macula area.
It is widely accepted that there is a complex involvement of both genetic and environmental factors in the pathogenesis of AMD. Studies have identiï¬ed numerous AMD-associated genes in mechanistic pathways related to complement system activation, inï¬‚ammation, microglial recruitment, DNA repair, extracellular matrix function, and neovascularization (Ding et al., 2009).
Much of the previous work on genetic factors inï¬‚uencing AMD has focused on single nucleotide polymorphisms (SNPs). Although highly signiï¬cant statistical associations have been discovered between various SNPs and AMD, they do not account for the entire genetic component of the disease. It is believed that complement activation resulting from dysfunction of CC chemokines may contribute to inflammation. The infiltration of monocytes is accompanied by inflammatory chemokines as key mediators.
Studies recently indicated that inflammation plays a fundamental role in the development of CNV (Rohrer et al., 2009). Additionally, genetic evidence has identified variations in multiple genes (Sharma et al., 2009). Studies had also investigated the role of Asthma with AMD and found that asthma could be a risk factor for the development of AMD (Sun et al., 2012).
We previously described the spontaneous development of CNV in senescent mice deficient in monocyte chemoattractant protein-1 (CCL2, also known as MCP-1) or its CCR2 receptor postulating its key role in AMD pathogensis (Ambati et al., 2003). We hypothesized that CCR3 is also involved in similar processes and any polymorphism in this gene may result in chronic inflammation by sustained activation of the complement system contributing to the pathogenesis of AMD.
Antivascular endothelial growth factor treatment is currently used for wet AMD patients (The CATT Research Group et al., 2011). Even though Eghoj et al (2012) showed that out of the 1076 eyes, a total of 20 (2%) eyes met the criteria for tachyphylaxis i.e. drug did not respond at the time of reactivation of CNV in AMD patients who had responded to the treatment initially anti-VEGF-A therapy induces dysfunction in and damage to the inner and outer murine retina (Nishijima et al., 2007; Saint-Geniez et al., 2008), raising a question of potential retinal toxicity or recurrent choroidal neovascularization (CNV). In this context it is pertinent to review the report of Takeda et al who reported that the G-protein-coupled receptor (GPCR), CCR3 is important in neovascular AMD showing that CCR3 neutralizing antibodies are more effective than VEGF-A neutralizing antibodies in inhibiting the CNV in mice model (Takeda et al., 2010). Furthermore, genetically engineered mice that were deficient in CCR3 or its ligands were also protected to some extent from the effect of laser injury on the choroidal vasculature (Takeda et al., 2010). Additional evidence also supports its role in CNV(Ahmad et al., 2011). However, a recent study reported that CCR3 was not important in CNV development when using a Matrigel CNV model (Li et al., 2011). Therefore, further study regarding the potential role of CCR3 in AMD is needed.
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CCR3 is a receptor for eotaxin found on the surface of a variety of cells, including white blood cells. It is most frequently associated with eosinophils and mast cells that plays a major role in allergic reactions (Pope et al., 2005) as well as angiogenesis (Li et al., 2012). CCR3 gene is located on the short arm of chromosome 3. AMD is a complex disease, inï¬‚uenced by environmental and genetic factors. The absence of any such genetic association studies of CCR3 and AMD prompted us to explore the role of this chemokin in these patients. We therefore wanted to determine the polymorphism of CCR3 in the human AMD patients.
Materials and Methods
The study population included 176 subjects, which include 115 AMD patients and 61 normal healthy controls from advanced eye centre, Post-Graduate Institute of Medical Education and Research, Chandigarh, India. 50 years or older AMD patients with the diagnosis of advanced AMD as defined by geographic atrophy and/or choroidal neovascularization with drusen more than five in at least one eye were included in the study. The controls in the study included those above 50 years with no drusen and absence of other diagnostic criteria for AMD.
The exclusion criteria included the retinal diseases involving the photoreceptors and/or outer retinal layers other than AMD loss such as high myopia, retinal dystrophies, central serous retinopathy, vein occlusion, diabetic retinopathy, uveitis or similar outer retinal diseases that have been present prior to the age of 50 and opacities of the ocular media, limitations of pupillary dilation or other problems sufficient to preclude adequate stereo fundus photography. These conditions include occluded pupils due to synechiae, cataracts and opacities due to ocular diseases. Ethical clearance was obtained for this study by the Institute Ethics Committee, Post-Graduate Institute of Medical Education and Research, Chandigarh, India vide letter No Micro/10/1411. Informed consent was obtained in the prescribed format endorsed by the Institute Ethical Committee.
Patients underwent complete clinical ophthalmic examination by a retina specialist for best corrected visual acuity, slit lamp biomicroscopy of anterior segment and dilated fundus examination. All AMD patients were subjected to optical coherence tomography (OCT) and fluorescein fundus angiography (FFA). The diagnosis of AMD was based on ophthalmoscopic and FFA findings.
All the subjects were fully informed of the purpose and procedures involved in the study and interviewed. A written informed consent was taken from each participant. The risk factor questionnaire included information about demographic characteristics such as cigarette smoking, alcohol intake, food habit, comorbidity etc. Smokers were defined as those having smoked at least three cigarettes per day or 54 boxes for at least 6 months and were segregated further into smokers and never smokers. Non vegetarian patients were defined as those having chicken, meat or fish for at least 6 months and alcoholic patients were defined as those having whiskey, rum, wine or home made alcohol for at least 6 months. Co-morbidity was determined based on the participants responses to whether a physician had ever told them for diagnosis of stroke, migraine or any heart diseases. Subjects were also asked to report any prior diagnosis of any neurological, cardiovascular or metabolic disorders etc.
Selection of single-nucleotide polymorphisms
The selected single-nucleotide polymorphisms (SNPs) used in our study have been previously examined for other allergic and inflammatory diseases like asthma, because like CNV, asthma is a multifactorial disorder with both environmental and genetic factors contributing to its development (Mizutani et al., 2009). Because some of the mechanisms of development for both CNV and asthma are similar (Sun et al., 2012) , we hypothesized that there is a relationship between these two diseases. During the past decade, several population-based studies have reported that asthma is associated with a high risk of developing CNV.
DNA was extracted from commercially available DNA extraction kit (Qiagen) as per the instructions provided by manufacturer. Extracted DNA was stored at 4oC to further investigate the polymorphism in CCR3 gene.
Real Time PCR
SNP (Single nucleotide polymorphism) was analyzed by using real time PCR, and was performed in the 48 wells model Step OneTM (Applied Biosystems Inc., Foster city, CA) using published TaqMan® SNP Genotyping Assays. Real time PCR was carried out for 20.0µl containing 10ul master mix, 5ul Assay (Applied Biosystems), 20ng DNA and molecular biology grade water was added to make the volume 20.0µl. All reactions were carried out using TaqMan® SNP Genotyping Assays (Applied Biosystems) according to manufacturer's recommendations. Two reporter dyes VIC and FAM were used to label the Allele 1 and 2 probes and 5Í Nuclease Assay was carried out. Negative controls included the PCR mix without DNA. Software StepOneTM v 2.0 (Applied Biosystems Inc., Foster city, CA) was used to perform amplification and to estimate SNP. After PCR amplification the Sequence Detection System (SDS) Software was used to import the fluorescence measurements made during the plate read to plot fluorescence (Rn) values.
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The real time PCR estimated genotypes for each mutation were stratified for heterozygosity, and homozygosity for the respective allelic variant. Pearson's Chi-square test was applied to study the association between various groups. Genotype distributions were analyzed by logistic regression, integrating adjustments. Genotypic associations and odds ratios (ORs) with 95% confidence intervals (CI) were estimated by binary logistic regression. The p ≤0.05 was considered to be significant. Statistical analysis was performed with the help of SPSS 20.0 software.
Summary statistics of all important variables were obtained and reported in Table 1. In a case-control study of 176 subjects, common genetic variants in CCR3 were analysed. Table 2 and 3 shows the SNPs of CCR3 in AMD and normal controls. While examining rs3091250 loci a significant difference was observed for the genotype and allele frequency. Both the heterozygous (GT) and homozygous (TT) genotypes at rs3091250 SNP were found to be significantly more frequent in AMD patients as compared to controls (Table 2, Figure 1A, OR =4.8, CI95%= 2.2-10.8, p=0.001 and OR = 4.1, CI 95% = 1.6-10.1, p=0.002 respectively). While examining rs3091312 loci no significant difference was found for AMD and controls (Table 2, Figure 1A). However, the T allele frequency was significantly higher in AMD patients for both SNPs (Table 3, Figure 1C). We did not find any significant difference in genotype and allele frequency for wet and dry AMD patients (Table 2, 3, Figure 1B and 1D). Logistic regression analysis in both SNPs for food habit, smoking, alcohol and comorbidity did not show any difference. (Table 4, Figure 2). The difference was also not significant when compared between familial patients, number of eyes affected, gender and wet AMD patients ie minimally classic, predominantly classic and occult (data not shown).
Logistic regression analysis was used to analyze the association of genetic polymorphism and other risk factors. We analyzed age, gender and smoking as risk factors which have been shown to be associated with AMD previously. To account for gender, age and smoking effects, we did a logistic regression analysis with gender, age, smoking and genotypes as independent variables. The results confirmed the significant association between the GT and TT genotype of rs3091250 and AMD (Table 2).
AMD is a serious progressive irreversible disease influenced by genetic and other environmental factors. Our earlier studies showed that Ccl2/Ccr2 deficient mice exhibit features of AMD. Combined with recent studies establishing CCR3 as an emerging candidate for AMD pathogenesis the focus has shifted on its role in AMD. CCR3 is the major chemokine receptor on basophils and eosinophils (Heath et al., 1997) and its ligands are important elements for the basophils, chemotaxis and activation of eosinophils at the place of allergic inflammation (Grimaldi et al., 1999).
In the present study we examined the two CCR3 SNPs and discovered that the GT and TT genotypes from rs3091250 could be responsible for AMD. This is the first study showing the relationship of rs3091250 with AMD, importantly from Indian subcontinent. Mutation can affect the effectiveness of gene transcription, which in turn can change levels of mRNA and, thus, protein levels in general. Mutations in CCR3 may alter the levels of CCR3 in blood. It has been earlier reported that activated CCR3 promotes choroidal endothelial cell (CEC) migration, thus causing Rac 1 and VEGFR2 activation resulting to neovascular AMD (Haibo et al., 2011). The function of CCR3 is to activate and recruit eosinophils to the site of inflammation and stimulate macrophage activation. Activated eosinophils can release reactive oxygen species which contribute to host tissue damage during chronic inflammatory responses.
We believe these observations to have strong implications for evolving new strategies for targeting CCR3 in AMD. These results indirectly support the results of Takeda et al who showed that CCR3 is a potential therapeutic agent as compared to others for treating AMD, (Takeda et al., 2009) indicating that CCR3 may impact the pathogenesis of the disease. CCR3 blockade had been shown to be more effective at inhibiting CNV development than VEGF-A neutralization. However, Li et al., (Li et al., 2011) showed that CCR3 was not involved in CNV induced by gelatinous protein presumably because the model used did not employ aged mice, a known factor associated with AMD.
The mechanism of development of both AMD and asthma appear to share a common mechanism. During the past decade, several population-based studies reported that a history of asthma is associated with a high risk of developing CNV (Sun et al., 2012). However, other studies have shown no association between asthma and CNV (Moorthy et al., 2011). Therefore, the association between these two diseases remains unknown. After analyzing the asthma related SNPs in AMD patients, we speculate that the mechanism of development of both diseases is far from being dissimilar. Previously, whole-genome scans have also suggested that the chromosome region 3p21-24, which contains a gene cluster of CC chemokine receptors, including CCR3, is linked to asthma (Ober et al., 2000).
Interestingly, rs3091250 mutation is also reported in aspirin-intolerant asthma patients (Kim et al., 2008). Mutation in CCR3 gene was associated with asthma in a British population (Fukunaga et al., 2001) and not in Japanese,( Kim et al., 2008) Taiwanese,( Wang et al., 2007) and Korean populations. Three intronic CCR3 SNPs (-22557G/A, -520T/G, -174C/ T) were identified in a Korean population (Lee et al., 2007).
Our study showed the non-redundant role of CCR3 in pathogenesis of AMD and may encourage multi-ethnic studies to verify the evidence presented here.
It is important to note that many of the important risk factors were investigated in the present study, for instance, many epidemiology studies have found an association between smoking, alcohol, comorbidity, other risk factors and increased risk of AMD (Pons et al., 2011; Barbara et al., 2001), but our results did not show any association with smoking, alcohol, food habit and comorbidity.
For early detection of CNV, CCR3 could represent a novel biomarker with potential to be targeted as a new therapeutic entity through future studies. Abundant levels of CCR3-specific binding molecule spotted in the retina could be indicative of AMD. Detection of AMD during initial stages may allow treatment to be initiated early. Additional immunohistochemical and biochemical investigations in autopsy specimens are needed to verify the claims held by this study. Besides, future studies can focus on the association of CCR3 polymorphism in various populations.