Expression of VEGFA‐regulating miRNAs and mortality in wet AMD

Abstract MicroRNAs (miRNAs) regulate gene expression; many of them act in the retinal pigment epithelium (RPE), and RPE degeneration is known to be a critical factor in age‐related macular degeneration (AMD). Repeated injections with anti‐VEGFA (vascular endothelial growth factor A) are the only effective therapy in wet AMD. We investigated the correlation between the expression of 18 miRNAs involved in the regulation of the VEGFA gene in serum of 76 wet AMD patients and 70 controls. Efficacy of anti‐VEGFA treatment was evaluated by counting the number of injections delivered up to 12 years. In addition, we compared the relative numbers of deaths in patient with AMD and control groups. We observed a decreased expression of miR‐34‐5p, miR‐126‐3p, miR‐145‐5p and miR‐205‐5p in wet AMD patients as compared with controls. These miRNAs are involved in the regulation of angiogenesis, cytoprotection and protein clearance. No miRNA was significantly correlated with the treatment outcome. Wet AMD patients had greater mortality than controls, and their survival was inversely associated with the number of anti‐VEGFA injections per year. No association was observed between miRNA expression and mortality. Our study emphasizes the need to clarify the role of miRNA regulation in AMD pathogenesis.


| INTRODUC TI ON
Age-related macular degeneration (AMD) is a complex eye disease; it is the leading cause of legal blindness in the elderly in the developed countries. AMD occurs in either dry or wet (neovascular) forms. Wet AMD is characterized by the sprouting of new vessels from choriocapillaris through the Bruch's membrane into the sub-retinal space or the retina layers. Vascular endothelial growth factor A (VEGFA) and its receptor are crucial regulators of choroidal neovascularization (CNV) in wet AMD. 1 The vessels produced in CNV are fragile, and their contents tend to leak into the retina layers, promoting fibrogliosis which results in the formation of a disciform scar and severe loss of vision if not properly treated.
Targeting VEGFA by anti-VEGFA agents significantly improved the treatment of wet AMD outcome and, in fact, led to its removal from the list of incurable diseases. 2 Bevacizumab (Avastin ® ), ranibizumab (Lucentis ® ) and aflibercept (Eylea ® ) are used to prevent CNV activity. However, not all patients display a positive response to these drugs, and some of them are unresponsive to therapy. 3 Moreover, the relatively short half-life of anti-VEGFA drugs means that they need to be administered on a monthly basis, with the treatment often lasting for the patient's life-time. Therefore, new modes of treatments of wet AMD with the currently used and new drugs are being investigated to find compounds with greater efficacy and better safety than the present anti-VEGFA-based therapies.
The goal of anti-VEGFA therapy in wet AMD is to decelerate the worsening of visual acuity and to prevent the loss of vision. Although the use of VEGFA inhibitors revolutionized wet AMD therapy, several issues still need to be resolved in the treatment of this disease.
First, VEGFA is not the only protein involved in neovascularization.
Second, neovascularization is not the only unwanted process ongoing in affected eyes. Third, individual susceptibility to therapy is influenced by genotype, epigenotype and environmental factors, all of which determine the expression of genes whose products are important for the therapeutic response.
Wet AMD is frequently associated with other systemic conditions, primarily vascular complications that can be linked with increased mortality. [4][5][6][7] Therefore, although the question of whether wet AMD may be an independent risk factor for death is complex, it should be addressed as advanced age is the main factor in the pathogenesis of AMD. Moreover, some reports suggest that therapy with intravitreous anti-VEGFA injections can influence the mortality of wet AMD patients undergoing this kind of therapy. [8][9][10] Several genetic factors, mainly related to the complement system, have been identified as playing either a documented or a putative role in the pathogenesis of AMD, but the role of epigenetic control in AMD is much less clear. [11][12][13] As epigenetic microRNAs (miRNAs) are an important element in the regulation of gene expression, a panel of miRNA species involved in the progression of wet AMD or the conversion of dry AMD into wet AMD should be clarified in order to better understand AMD pathogenesis and to personalize wet AMD therapy. miRNAs now have an emerging role in the regulation of the expression of eukaryotic genes, and they are known to be involved in the pathogenesis of many human diseases. 14 Their main function is to trigger the RNA interference (RNA i ) pathway, either to degrade mRNA produced by the target gene or to repress its translation. However, several other functions have been attributed to miRNAs, including transcriptional gene activation. 15 Moreover, a single miRNA can be involved in the control of multiple genes belonging to a single molecular pathway or multiple pathways. A recent study has identified as many as 416 miRNAs which are expressed in RPE and choroid. 16 Targeting these miRNAs in order to modulate their expression seems to be a promising strategy in AMD treatment as supported by experiments conducted in animal models of AMD. 17 In the present work, we investigated the expression of VEGFA gene-regulating miRNAs in the serum of wet AMD patients. In addition, we analysed mortality in the wet AMD patients and control groups without AMD.

| Patients
A total of 76 patients with wet AMD and 70 controls were enrolled in this study. The mean age of the patients was 79.5 years (range 74.5-84.5), whereas the mean age of controls was 73.8 years (68.6-78.7). The patient group contained 18 males and 58 females; the numbers in the control group were 37 males and 33 females.
Because of the differences in age and sex, all calculations were adjusted for these parameters. The controls were individuals without AMD or any other retinal disease who were undergoing cataract operation and were selected as described earlier. 18 The criteria for patient selection were based on CNV in optical coherent tomography (OCT) and/or fluorescein angiography (FAG). No patient reported any genetic disease, and diabetes mellitus was an exclusion criterion. All patients with AMD were subjected to an examination in the Department of Ophthalmology of Kuopio University Hospital, involving best-corrected visual acuity (VA), intraocular pressure, slit lamp, fundus and biomicroscopy examination, fundus photographs (Canon CX-1 Hybrid Retinal Camera, Canon), FAG (Canon CX-1) and/ or OCT (SPECTRALIS OCT2, Heidelberg Engineering). Real-world data (RWD) were monitored for up to 12 years. Finnish national guidelines for modified PRN (pro re nata) were applied in the followup and treatments of wet AMD patients. [19][20][21][22] Ethics Committee of the Kuopio University Hospital has approved the study, and the tenets of the Declaration of Helsinki are followed. All participants have been asked to sign an informed consent form. org). Only those miRNAs were selected which had been experimentally validated with evidence emerging from reliable methods. They are listed in Table 1.  (Tables S1 and   S2). HRs were adjusted for confounders including patient age and sex. P-values .05 or less were considered as statistically significant.

| RE SULTS
Four out of 18 miRNAs regulating the expression of the VEGFA gene displayed a lower level of expression in patients with AMD than in controls: miR-34a-5p, miR-126-3p, miR-145-5p and miR-205-5p ( Figure 1). We observed a significantly elevated mortality ratio in wet AMD patients in comparison with controls (P = .021, Table 2).
The wet AMD patients who died during the treatment had received a higher number (>3 injections/year (P = .0013) of anti-VEGFA injections than those who survived (Table 3). In Cox regression analysis adjusted for age and sex, cataract surgery patients with wet AMD and anti-VEGFA injections (mean number of 9.8 ± 8.9 anti-VEGFA injections during the follow-up) had HR 2.05 for death, 95% CI 1.59-2.64, when compared to the cataract surgery patients without wet AMD (P < .001, Figure 2). No association was observed between mortality and the expression of any miRNA (data not shown).
TA B L E 1 miRNAs targeting the VEGFA (vascular endothelial growth factor A) gene miRNA miRBase/miRTarBase entry

| D ISCUSS I ON
MiRNAs are an emerging topic, for example as biomarkers and therapeutic targets of diseases, including AMD. We observed decreased serum levels of miR-34-5p, miR-126-3p, miR-145-5p and miR-205-5p in wet AMD patients as compared with non-AMD controls.
The expression of miR-34a-5p may occur in response to oxidative stress, a major factor of AMD pathogenesis. 26   cellular proliferation. [35][36][37][38] Therefore, miR-126-3p may be an important epigenetic regulator in the development of wet AMD. miR-145-5p is known to be a negative regulator of angiogenesis. 39 Decreased levels miR-145-5p levels enhance the secretion of IL-1β, TNF-α and IL-6 during hypoxia. 40 Therefore, miR-145-5p can be considered as a therapeutic target to suppress the inflammatory response and to prevent the apoptosis occurring in hypoxic conditions, a characteristic of wet AMD. 41,42 miR-205-5p was shown to regulate EMT through the PI3K/ AKT pathway. 43,44 Autophagy, a key lysosomal clearance mechanism, has been linked to the PI3K/AKT signalling. 45 52 The complexity of AMD pathogenesis increases when non-genetic data are analysed with genetic and epigenetic data. 53 However, a bioinformatics analysis of epigenetics, pharmacogenetics, comorbidities and genetic counselling in AMD is a promising way to open new perspectives for personalized medicine and help to identify phenotype differences between dry and wet AMD. [53][54][55][56] Anti-VEGFA therapy in wet AMD is considered to be safe for patients, even although some side-effects have been reported. 12,57 Moreover, Papudesu et al showed that AREDS2 participants with wet AMD in one eye at baseline had a statistically significant increased risk for mortality compared with patients having no or only a few drusen. 58 Moreover, a visual acuity less than 20/40 was associated with a reduced survival. In our material, 93% of patients had a visual acuity equal or less than 20/40 in the treated eye after follow-up of one year. Those patients who had received anti-VEGFA therapy had a greater risk of mortality than non-treated individuals. In another population-based study, Dalvin et al concluded that anti-VEGFA therapy in wet AMD patients was not associated with mortality as compared with non-treated individuals, including patients with AMD. 59 Gopinah et al detected a positive correlation between advanced AMD and mortality. 60 Hanhart et al noted an increased mortality in wet AMD patients after myocardial infarction (MI) treated with bevacizumab as compared to non-treated individuals. 9,10 Previously, these authors also observed increased F I G U R E 2 Registry of cataract surgery patients operated after 3 September 2007. Patients with wet AMD (ICD code: H35.31) and intravitreal injections (ICD code: CKD05) (blue) were compared to the cataract cohort (red). Kaplan-Meier curves were generated, and Cox regression was used to estimate hazard ratios (HR) for death between wet AMD patients with anti-VEGFA injections and cataract cohort. All patients were 65 years or older. In wet AMD patients, after adjusting for age and sex, HR for death was 2.05; 95% CI 1.59-2.64; P < .001 when compared to cataract surgery patients without wet AMD. N = 330 for wet AMD patients and N = 15 364 for the cataract cohort mortality in bevacizumab-treated wet AMD patients as compared to a control cohort without AMD. 8 Recent meta-analyses on mortality of wet AMD patients treated with bevacizumab intravitreal injections have pointed to a less than 2% mortality ratio in the first year of therapy. 61 A study of over 20 000 participants from the Melbourne Collaborative Cohort Study led to the conclusion that late AMD was linked with an increased mortality. 62 We detected a relatively high mortality ratio in the wet AMD group, and this did Furthermore, we have demonstrated the not unexpected significant association between mortality and the patient's age. However, when age was included as a confounding variable and used to standardize (adjust) the association between mortality and number of anti-VEGFA injections, this association became greatly reduced (al- though it yet remained statistically significant). In our opinion, this is the most elegant statistical proof of what we have stated in our paper. Specifically, as our analysis was adjusted for the age of the patients with AMD, we may hypothesize that it is unlikely that the observed effect was not purely coincidental but instead related to an advanced age of the patients. It is noteworthy however that although advanced age is the main factor in the pathogenesis of AMD, it is not the only significant factor and not all AMD-related phenomena can be attributed simply to ageing.
Although our samples are unique as they represent an observation period of up to 12 years, their number is not impressive from the statistical point of view. That is why we applied a bootstrapped estimate with 10 000 iterations to minimize the chance of introducing bias into our analyses. We emphasize that this is a common way to validate conclusions drawn from clinical studies evaluating not very large groups. With such an approach, we attempted to minimize the risk that we would be too eager to reject the null hypothesis when identifying a significant outcome in our inference or association tests. Our study has several limitations, which should be addressed Ponczek for an initial statistical analysis.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N
JB wrote the paper; CW performed statistical analysis; RT performed mortality analysis and wrote the paper; NK gathered clinical data; AK, HU-J and AT designed experiments and wrote the paper; MW, JM and SZ analysed the data; AF designed experiments and wrote the paper; KK conceived the concept of this work, collected the samples, did clinical examinations, interpreted data, organized financial support and wrote the paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available because of privacy and ethical restrictions and are stored at www.kuh.fi.