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Detection and evaluation of VEGF/HIF-1A angiogenesis signaling pathway in patients with primary ophthalmic pterygium

Poster Details

First Author: C.Dimitriou UK

Co Author(s):    E. Tsiambas   G. Vilaras   A. Kandarakis   A. Sharma   D. Brouzas   D. Papaconstantinou

Abstract Details



Purpose:

The purpose of this study is to detect and evaluate the potential involvement of VEGF/HIF-1α angiogenesis signalling pathway in the pathogenesis of primary ophthalmic pterygium. Protein HIF-1α is a precursor of VEGF molecule and a vital mediator of cell oxygen homeostasis. It is a hypoxia-dependent regulatory transcriptional factor that promotes angiogenesis through a feedback interaction with protein VEGF. Under normal oxygen supply conditions (normoxia) HIF-1α undergoes rapid catabolism. Under hypoxia or anoxia conditions HIF-1α is activated through parallel systems of signal transduction triggering expression of gene promoters responsible for angiogenesis, e.g. VEGF. Our study is designed to reveal the actual VEGF and HIF-1A expression levels in patients with ophthalmic pterygium as compared to control groups. This research may lead to development of novel anti-VEGF and HIF-1a inhibition molecules as new treatment strategies for halting progression of primary pterygium or preventing recurrence following primary surgical excision.

Setting:

Samples were collected on a semi-sterile preparation/resuscitation ward of the operating theatres of both Ophthalmiatreion Athens Eye Infirmary and 'Georgios Gennimatas' General University Hospital of Athens, which were then immediately stored as suspension in a specialised preservative fluid for liquid-based cytology (FDA cleared Liqui-PREPTM, LGM International Inc., Melbourne, FL, USA). The collection of the cytological smears was performed by a senior specialist registrar of Ophthalmiatreion Athens Eye Infirmary (C.D.) at both Ophthalmiatreion Athens Eye Infirmary and 'Georgios Gennimatas' General University Hospital of Athens with masked coding of the obtained vials of Liqui-PREPTM and anonymised samples' delivery to a specialised and certified clinical laboratory technician of the National and Kapodistrian University of Athens, Medical School, Department of Pathology and Laboratory Studies, Greece (G.V.). Both bright-field optical microscopy and digital image analysis were performed by a specialised and certified academic research scientist of the National and Kapodistrian University of Athens, Medical School, Department of Pathology and Laboratory Studies, Greece (Ε.Τ.). Research ethical approval has been obtained for the use of pterygial tissue samples through the scientific and research ethics committees of both Ophthalmiatreion Athens Eye Infirmary and 'Georgios Gennimatas' General University Hospital of Athens, Greece.

Methods:

In this controlled prospective cohort study eight patients with ophthalmic pterygium and sixteen healthy individuals with no manifest ocular surface disease have been recruited up to date. Smear from the nasal and temporal conjunctiva was obtained from the above patients, with a minimally traumatic technique, utilizing a special micro-brush (Rovers® Cervex-Brush® Combi) and stored in a specialised fixative solution for cytology specimen (Liqui-PREPTM). Subsequently liquid-based cytology was applied following centrifugation (1000g x 10min) of each specimen, coating of glass microscope slides and immuno-histo-chemical (IHC) analysis for protein molecules VEGF and HIF-1α. Visualisation of the primary antibodies that were used in our study was accomplished with the aid of polymer detection systems, which utilise a new technology of controlled polymerisation for the production of conjugated horseradish peroxidase polymers and antibody-binder. Brightfield microscopy image analysis through an advanced research technology of digital processing (Nikon® NIS-Elements Ar v.3.1®) were applied to determine the expression of VEGF and HIF-1α. The range of staining intensity levels based on grey scale analysis according to the red-green-blue protocol per image pixel lie within the spectrum of 0 to 255: 0 = absolute dark, meaning overexpression of biomarkers, whereas 255 = absolute white, meaning total loss of expression.

Results:

In patients with nasal ophthalmic pterygium, the expression of VEGF factor was greater at the nasal site compared to the temporal site from which cytological smear was obtained, with borderline statistical significance (0.1>p>0.05). The mean value of the mean intensity (M.I.) of IHC stain was Μ.Ι.VEGF/PN=134.99375 nasally and Μ.Ι.VEGF/PT=147.67625 temporally. In the same group the expression of factor HIF-1α was even higher in the nasal compared with temporal site with statistically high significance (p<0.01), Μ.Ι.HIF-1α/PN=128.47375 and Μ.Ι.HIF-1α/PT=142.94, respectively. In the control group: Μ.Ι.VEGF/CN=143.64812 and Μ.Ι.VEGF/CT=149.04937 with limited statistical significant of overexpression of VEGF at the nasal as compared to the nasal site (p<0.1), whereas HIF-1α was found to be remarkably elevated nasally compared to the temporal site with high statistical significance (p<0.01), Μ.Ι.ΗΙF-1α/CN=142.67875 and Μ.Ι.ΗΙF-1α/CT=151.31312 respectively. At the nasal site VEGF was higher with limited statistical significance (p<0.1) in eyes with nasal pterygium compared to the control group, whereas HIF-1α was increased in a statistically significant degree (p<0.01) in eyes with ophthalmic pterygium compared to controls. At the temporal site VEGF did not show any statistically significant difference (p>0.2) between eyes with nasal pterygium and controls, whereas HIF-1α was statistically significantly elevated (p<0.05) in eyes with pterygium compared to controls.

Conclusions:

In patients with ophthalmic pterygium we recorded an overexpression of HIF-1α particularly nasally (p < 0.01) but also temporally (p < 0.05) in relation to the corresponding sites of eyes in the control group. Higher expression was also noted with regards to VEGF particularly at the nasal site, but this finding was of limited statistical significance. In the control group there was also an increased expression of HIF-1α (p < 0.01) nasally in comparison with the temporal site, but corresponding VEGF overexpression did not reach statistical significance. We postulated that our methodology demonstrates greater sensitivity in the identification of angiogenic factor HIF-1α as opposed to VEGF. The present study describes a novel method of obtaining and processing cytological sample from conjunctival and pterygium layers that may inspire further exploration of ocular surface diseases. With the previously described method we achieved visualization and quantification of the levels of expression of protein factors through IHC staining, brightfield microscopy and digital image analysis. As a VEGF precursor, HIF-1α may play a primary prognostic role in pterygium growth and development and may also lead to the discovery of potential new methods for inhibition of angiogenic mechanisms that are involved in both primary and recurrent pterygia. Our study is likely to provide a platform which could aid the development of novel strategies that combine surgical excision and administration of anti-VEGF and/or HIF-1α inhibition molecules in eliminating pterygium recurrence. FINANCIAL INTEREST: NONE

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