Elevated Angiogenin levels associated with both forms of age-related macular degeneration is regulated by hypoxia and telomerase

Abstract

Purpose : Angiogenin (ANG) is a potent pro-angiogenic factor with ribonuclease activity shown to regulate a variety of cellular functions including the formation of stress granules, neovascularization, survival and proliferation. Advanced forms of AMD present with geographic atrophy and neovascularization that leads to irreversible visual loss, necessitating deeper understanding of molecular mechanisms and identification of novel therapeutic targets.

Methods : All human samples for study were obtained with written patient consent and approved by the Institutional Ethics Committee. Plasma samples were obtained from 20 age-matched control, 20 wet AMD and 30 dry AMD subjects. Levels of VEGF, ANG and a panel of 12 pro-angiogenic and inflammatory factors were determined in plasma (20 age-matched control, 20 wet AMD and 30 dry AMD) and aqueous humor (6 control and 7 wet AMD) by cytometric bead array (CBA). Blood leukocytes were obtained from 11 healthy adults. Gene expression in cultured cells and blood leukocytes was determined by quantitative PCR. TERT (Telomerase) was ectopically expressed in human RPE and HeLa cells. Hypoxia was induced by culturing cells in a hypoxic chamber (2% O₂, 72 hrs).

Results : Amongst the 12 factors tested by CBA array, plasma levels of ANG were higher in both dry AMD(16592+4442, p=0.003) and wet AMD(23789+4775, p<0.0001) compared to controls(3321+1550). However, VEGF was significantly higher in wet AMD(109+19, p=0.04), but not in dry AMD(63.8+7.8) compared to controls (55.5+4.6). In patient aqueous humor, both VEGF levels (625+257; p=0.001) and ANG (9007+1386; p=0.02) were significantly higher than controls (50.5+5.5 and 4161+620 respectively). In cells with 11-fold TERT overexpression, ANG levels were reduced by more than 50%. TERT and ANG levels in leukocytes correlated inversely (r= -0.618; p=0.04). RPE cells under hypoxic stress induced ANG by 4.5-fold and VEGF by 10-fold.

Conclusions : ANG is elevated in the plasma of both dry and wet AMD in comparison to VEGF which is high only in wet, suggesting an early role for ANG in disease progression. The in vitro data support a hypoxia mediated mechanism of ANG induction which may be a possible pathway driving the effects of ANG on the human retina. In addition, the inverse relationship of TERT with ANG in human samples may be a novel regulatory axis in this age-related disease.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.