FP1540 : Correlation of Macular Edema Resolution in CNVM with Aqueous Humour Levels of Soluble Factors.

Dr. Bharathi Bavaharan, B12665, Dr. Rohit Shetty, Dr. Naresh
Kumar Yadav, Dr. Santosh Gopi Krishna Gadde

INTRODUCTION:

Age related macular degeneration (AMD) is one of the leading causes of blindness among the older age group^1,2. The disease has two stages, dry and wet stage. Formation of drusen, Retinal Pigment Epithelium (RPE) dysfunction, geographical atrophy of RPE and photoreceptors, accumulation of fluids in the intra-retinal and/or sub-retinal space, choroidal neovascularization and sub-retinal fibrosis are the pathological developments in the disease.² In retina, the outer blood retinal barrier function is maintained by the RPEwhich controls the transport of water as well as nutrients between the neurosensory retina and the fenestrated choroid ^3,4. Dysfunction and atrophy of the RPE layer causes the breakdown of this barrier function resulting into the accumulation of fluids into the sub-retinal and intra-retinal space. In the severe stage (wet-AMD), there is development of choroidal neovascular membranes (CNVM) which further contribute to the accumulation of fluids into the retina. VEGF is one of the most well studied and potent factor driving the pathogenesis in AMD^5,6 and hence served as the first molecular therapeutic target.

The more severe stages of the disease are managed by using single or multiple therapeutic approaches which include the use of laser mediated photocoagulation, intravitreal corticosteroids and intravitreal anti-VEGF injections^7-9. Despite multiple and/or targeted molecular therapeutic options available, there is a subset of patients (19.6% to 67.4% as per different studies testing the efficiency of the different anti-VEGF drugs and dosages like CATT, HORIZON, VISION, IVAN, ANCHOR, MARINA,FOCUS)  which does not respond to the therapy or develops resistance to it^10-13.

Studies suggest the involvement of alternate molecules (FGF, PDGF) and pathways involved in the development of resistance to anti-VEGF or persistence of clinical pathology despite treatment^14-16. Hence, identifying additional factors which impacts disease prognosis is essential. Various body fluids such as plasma, tears, aqueous humor (AH) and vitreous humor (VH) are being studied at various disease stages to determine novel factors and their possible role in mediating the choroidal neovascularization and edema in AMD patients^17-19. The identification of systemically dysregulated soluble factors such as from serum/plasma may not be representatives of the retinal conditions in the disease. Vitreous humor being in closest proximity to the affected area would serve as more appropriate choice of sample, however it may not be available for all the stages of the disease and/or for multiple sampling and the collection method is more invasive and risky²⁰. However, these studies have not been conclusive and there is a need to determine dysregulated molecules in the eye at closer proximity to the retina and further study their association with the treatment response. Further, there is still a dearth of knowledge as to the list of molecular factors that could possibly impact treatment outcomes in CNVM. Hence, this study address to fill this gap and pave way for potential additional therapeutics in the management of CNVM.

OBJECTIVE:

The objective of this study is to determine the association between the soluble factors in aqueous humor samples and the treatment response in CNVM patients in terms of edema resolution

METHODS:

The cross-sectional study following approval by the Institutional Review Board was performed as per guidelines stipulated by the Indian Council for Medical Research (ICMR). Subjects were recruited for the study post informed written consent as per institutional and ethics board guidelines and as referred to our institute. CNVM patients receiving therapy were classified as persistent and resolving based on the measurement of the macular edema volume. Resolving:were the patients with reduction in macular edema volume in the 2^nd visit than the 1^st visit/baseline after intravitreal injection. Persistent: were the patients with increased or no reduction in macular edema volume in the 2^nd visit than the 1^st visit after intravitreal injections.

Aqueous humour (AH):

was collected by anterior chamber paracentesis where a syringe with 30 mm gauge needle was used to access the anterior chamber through the peripheral cornea to aspirate at least 50μL of AH. This procedure was performed in subjects underdoing surgical intervention (as part of standard of care) that would require access into anterior chamber of the eye. All the samples were stored at -80°C until further processed.

Soluble factors:

The selection of the soluble factors was based on the available literature on the involvement of soluble factors in mediating the pathological features such as neovascularization and edema.The concentrations of the mentioned chemokines in aqueous humor samples were measured with Cytometric Bead Array (CBA). The acquisition of processed samples was done on FACS Canto-II (BD Biosciences) and the quantification was performed with FCAP Array software (Version 3.0).

Statistical analysis:

Relevant statistical analysis (distribution analysis, t-test, Mann Whitney test, Pearson correlation coefficient) was performed following experiments using GraphPad Prism software.

Clinical parameters: The clinical parameters such as measurement of edema volume, Central Subfield Thickness (CST) and retina volume were measured from Spectral Domain-Optical Coherence Tomography (SD-OCT). Heidelberg OCT software (Spectralis HRA+OCT) was used to measure CST and retina volume. The edema volume was calculated using MATLAB 8.5, The Mathworks, Inc., Massachusetts, USA software.

RESULTS:

Statistical significance was calculated for the aqueous humor soluble factor concentrations between the resolving and the persistent group of patients. There was a marked reduction in the concentration of VEGF in the persistent group than the resolving edema group, which can be the result of the anti-VEGF injections administered. sICAM-1, sVCAM, sL-selectin and CXCL10 are soluble cell adhesion molecules which play a role in the inflammatory response through aiding the transmigration of immune cells, were found to be significantly higher in the persistent group than the resolving group. CXCL9 and IL-8 was observed to have an inverse relationship with difference in the central foveal thickness between the visits. sICAM, sVCAM, sL-selectin and IL-2 exhibited positive correlation with difference in the edema volume between two visits. The higher levels of these factors indicate the possible role of these soluble factors in mediating the pathogenesis and/or poor response to the therapy.The reduction in CFT for the resolving group correlated with lower levels of soluble factors suggesting that a subgroup of soluble immune factors may be responsible for poor therapeutic outcomes or preventing the resolution of edema

DISCUSSION:

Metabolic dysregulations, hypoxiaand oxidative stress drive the pathogenesis of AMD^21,22. As a consequence of the stressed retina, the inflammatory pathways are activated, which further contribute to the development of AMD^17,21-23. In order to determine the molecular factors that are instrumental in the disease pathogenesis and patients response to the administered drug, several studies have been directed towards identification of dysregulated soluble factors in the retina having a potential to contribute to the neovascularization and vascular permeability or edema^17,18,24-27.The studies have been directed towards identification of the dysregulated factors in the disease than control subjects^25,26 and/or assessing their associations with the disease severity in AMD²⁴. Previous studies have also profiled AH soluble factors to assess their potential in mediating macular edema in diabetic macular edema patients, however it has not been studied in AMD.^28,29Cell adhesion molecules such as ICAM, VCAM are known to play a vital role in the inflammatory response by mediating the arrest and transmigration of the activated circulating immune cells to the site of injury³⁰.Higher levels of these factors are seen in retinal diseases such as diabetic retinopathy, ³¹AMD, diabetic macular edema.^25,32 Previous studies have shown the higher levels of sICAM-1 are associated with the sub-retinal fluid height as well as disease severity in diabetic macular edema^32-34.Concentrations of these inflammatory markers in aqueous humor and their association with the treatment response is studied in diabetic macular edema^28,29. However, the association was not studied in AMD. Our current findings with high levels of certain molecular factors in patient group with lack of edema resolution will be explored for its causal role in rending this resistance to therapy. Following which further evaluation of these soluble factors influencing the treatment response needs to be studied for its potential use as therapeutic targets.

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