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Dr.Santosh Gopi Krishna Gadde, G16678, Dr.Rohit Shetty, Dr.Naresh Kumar Yadav

Introduction

Age related macular degeneration (AMD) is a progressive disease leading to vision loss in more than 35 million people. An estimated 8.69% of the global population are affected by AMD and It is considered as third major cause of age-related blindness [1]. Pathogenesis of AMD is led by dysfunction of retinal pigment epithelial (RPE) cells which are important to maintain normal photoreceptor homeostasis and visual cycle on [2]. Impaired RPE results in the deposition of clinically detectable yellow aggregates between the basement membrane of RPE and bruch’s membrane and known as drusen. In the advanced stage of the disease confluent drusenthickens the bruch’s membrane and impairs its permeability which results in localized hypoxia and inflammation causing new leaky blood vessels to appear and causes choroidal neovascularisation.

The only targeted treatments for this advanced disease stage currently available are anti-VEGF therapies. However, in many patients, the disease progresses from dry to wet stand and despite repeated anti-VEGF injections some patients fail to respond favourably indicating that we need to understand the underlying disease mechanisms better and find new therapeutic targets. Hence, in the study we investigate the regulatory role of an enzyme (Telomerase) which intricately involved in aging process and a well-known pro-angiogenic factor (Angiogenin).

Angiogenin (ANG), the first angiogenic factor isolated is also known as ribonuclease 5 (RNASE5) is the fifth member of ribonuclease super-family encoded by ANG gene located at chromosome 14q11 [3, 4]. It is 14kDa small vertebrate specific secreted protein first isolated from the conditioned medium of cultured HT-29 human colon carcinoma cell lines and characterised by Vallee and co-workers in 1985 at Harvard [4, 5]. Angiogenin was the first isolated from cancer and found to induce angiogenesis as well as growth of cancer cells [6, 7].

Telomerase is an enzyme present in all cells that helps determine the lifespan of the cells by regulating the length and integrity of the ‘telomeres’-ends of our chromosomes. We have previously shown that Telomerase can in fact also regulate essential functions in a cell such as response to stress and inflammation. In this study we show how Telomerase and Angiogeninregulate each other and alteration to their dynamic balance might be contributing to AMD pathology. Thus, modulating Telomerase-Angiogeninbalance present new targets for treatment of the disease.

Methods:

Patient recruitment and soluble factor profiling:

AMD patient with choroidal neovascularisation (n=78) and age-sex matched cataract controls (n=87) undergoing cataract surgery without any retinal disorder were recruited in this study with informed consent. Study was approved by the Institutional

Review Board (IRB) Narayana Nethralaya as per the guidelines of Indian council of medical research (ICMR).

Aqueous humorwas 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 aqueous humour. 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 bymultiplex ELISA usingCytometric 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.

Telomere repeat amplification protocol (TRAP)

To evaluate the difference in the telomerase activity in the leukocyte of wAMD, dAMD and control samples. Leukocytes were isolated form the whole blood by RBC lysis. Protein isolated using NP40 lysis buffer and incubated with TS primer followed by PCR amplification using Phusion hot flex polymerase. Amplified product was run on the acrylamide gel.

In vitro studies

In vitro studies were carried out in human adult retinal pigmented epithelium (ARPE-19) cells.  Briefly ARPE-19 weregrown in Dulbecco modified essential medium (DMEM)media supplemented with 10 % FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. Prior to experiment cells were grown in DMEM with reduced serum (2% FBS). Cobalt chloride (100 uM) was used for to induce hypoxia and TNF-α (10ng/mL) was used to induced inflammation. Recombinant angiogenin (1 ug/mL) treatment was given prior to hypoxia of inflammation. Angiogeninand TERT overexpression were achieved by transient transfection using lipofectamine LTX PLUS reagent. Briefly ARPE-19 cells were transfected at a confluency of 70% using 3 ug of plasmid DNA using manufacture’s protocol. Cells were harvested after 48h post transfection.

In vitro tube formation: Human vein endothelial cells (HUVEC) cells were used to study tube formation, an in vitro model to study angiogenesis. Briefly 30000 cells were seeded in 96 well plate coated with growth factor reduced Matrigel in EBM2 media supplemented with 2% FBS. Cells were treated either with 100 ng/mL rhVEGF, rhANG (1 ug/mL), neamine (200 uM), avastin (1mg/mL), MST312 (4uM)

and combination of them. Cells were incubated at 37 C in 5% CO2 for 12 to 16 h. Images were capture and analysed using ImageJ software

Isolation of RNA, cDNA Synthesis and Real-Time PCR

Cells/isolated PBMCs were washed twice with PBS. RNA was extracted using TRIZOL method as per manufacturer’s protocol. Complementary DNA was synthesized using iScript cDNA conversion kit (Biorad).Quantitative PCR cycle included preincubation at 958C for 5 minutes,45 amplification cycles at 958C for 10 seconds, 608C for 15seconds, and 728C for 30 seconds using a CFX Connect realtimePCR detection system (Bio-Rad, Philadelphia, PA, USA).

Western Blotting: for western blotting, protein was isolated using NP40 lysis buffer form the harvested cells. Isolated protein lysate was run on a 10% SDS PAGE followed by transfer on a PVDF membrane. The membrane was probed for TERT, AKT, phosphor-AKT, ERK1/2, phosphor ERK1/2, HIF-1α, GAPDH.

Results:

Aqueous sample profiling of wAMD (n=78) samples showed significantly elevated high level of VEGF, ANG, L-selectin, VCAM, MCP1, MIP1beta, MIG, IP-10, ITAC and bFGFas compared to controls (n=87).To validate our findings regarding ANG and VEGF, through functional assay, we performed tube formation assay with rhANG, rhVEGF, neamine (angiogenin inhibitor), avastin (anti-VEGF) with various combinations. rhANG and rhVEGF induces tube formation in HUVEC (vascular endothelial cells) which were inhibited by their respective inhibitors. However, tube formation was increases when ANG was added in presence of avastin. This indicate the critical role of ANG in retinal angiogenic conditions, including wet AMD

Molecular investigation revealed an increased gene expression of ANG and VEGF in ARPE19 cells under hypoxic stress and inflammatory stress. The cells also showed increased level of ANG and VEGF in the secreted fraction. To study the molecular pathways regulating ANG in our in-vitro model, we compared protein expression through western blotting. Increased phospho-AKT expression (AKT activation) was observed under hypoxic stress and inflammatory stimuli. We have also observed Akt activation and VEGF production was induced by recombinant ANG treatment and by hypoxia. TERT overexpression inhibited ANG gene expression as well as secreted protein levels and ANG overexpression inhibited TERT expression, indicating the negative regulatory role of these factors. The above results clearly indicate an inverse interplay between TERT and ANG. Ourresults also demonstrate that ANG acts through AKT pathway to regulate genes which might be involved in AMD pathogenesis.

Discussion:

AMD is reported to exhibit localised hypoxia and higher expression of proangiogenic, proinflammatory and cell adhesion molecules [8-13]which was also similar in our patient cohort with high level of VEGF, bFGF, L-selectin, VCAM, MCP1, MIP1beta, MIG, IP-10, ITAC. We have also observed higher level of ANG in the aqueous humor of wet AMD patients. In vitro studiesfrom the literature as well as this projectsupport the hypoxia mediated expression of proangiogenic factors such as ANG and

VEGF[14, 15]. ANG is a potent pro-angiogenic factor reported in various cancer studies[16]. High levels of which in the aqueous humor from wet-AMD patients indicates its possible role in the choroidal neovascularization and macular edema in addition to VEGF. ANG is also reported to induce VEGF and other pro-angiogenic factors[6].

Telomeraseis an enzyme whose conventional role is to maintain telomere length and its activity is believed to decrease with the Age. Its non-classical role (extra-telomeric role) is also well documented, which can be critical in maintaining cellular function and homeostasis.  We have seen an inverse regulation between ANG and TERT in our in vitro studies but how this regulation takes place is yet to be known. The possible mechanism could be through the transcription regulation which needs further investigation. Functional relevance of ANG is validated by an in vitro tube formation assay in endothelial cells which clearly revealed that ANG can induce tube formation independent of VEGF which could further contribute to progression of disease and/or poor response or resistance to the anti-VEGF therapy.

Conclusion:Our data suggest negative regulatory association betweenangiogeninand telomerase and its implication in AMD.

 References

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