June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Short-term exposure to thrombin induces long lasting disruption of barrier properties and proangiogenic signals in RPE
Author Affiliations & Notes
  • Tami Livnat
    Laboratory of Eye Research, Felsenstein Medical Research Center, PetachTikva, Israel
  • Omer Bialer
    Department of Ophthalmology, Rabin Medical Center, Petah Tiqwa, Israel
  • Yael Nisgav
    Laboratory of Eye Research, Felsenstein Medical Research Center, PetachTikva, Israel
  • Mor Dachbash
    Laboratory of Eye Research, Felsenstein Medical Research Center, PetachTikva, Israel
  • Rima Dardik
    Laboratory of Eye Research, Felsenstein Medical Research Center, PetachTikva, Israel
    The Israeli National Hemophilia Center and Institute of Thrombosis and Hemostasis, Sheba Medical Center, Tel Hashomer, Israel
  • Dov Weinberger
    Department of Ophthalmology, Rabin Medical Center, Petah Tiqwa, Israel
    Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
  • Footnotes
    Commercial Relationships Tami Livnat, None; Omer Bialer, None; Yael Nisgav, None; Mor Dachbash, None; Rima Dardik, None; Dov Weinberger, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3655. doi:
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      Tami Livnat, Omer Bialer, Yael Nisgav, Mor Dachbash, Rima Dardik, Dov Weinberger; Short-term exposure to thrombin induces long lasting disruption of barrier properties and proangiogenic signals in RPE. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3655.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Thrombin is a multifunctional protease playing a central role in coagulation. Apart from its major function in vein and artery occlusion, thrombin was found to play a significant role in inflammatory diseases. Thrombin exerts cellular effects through its membrane receptor (PAR). In vivo testing of thrombin is difficult since its existence in plasma is short-term and transient. The retinal pigment epithelium (RPE) forms the outer blood retina barrier (BRB) and its integrity is essential for normal function of the retina. Retinal pathologies involving BRB disruption, such as diabetic retinopathy, inflammation, vascular occlusions and tumors may be accompanied by elevation in thrombin levels. In the present work, we aimed to explore the impact of thrombin on the integrity and function of the RPE blood barrier. We induced in-vitro a short-term exposure of RPE to pathological levels of thrombin and studied the immediate and long lasting changes in the RPE permeability and angiogenic balance.

Methods: ARPE-19 cells were grown for a month to achieve definite polarity properties. Following a short (10 minutes) exposure to thrombin, the cells were washed and covered with new medium until measurements were performed. Permeability was evaluated based on spectrophotometric monitoring of the leakage of labeled dextran molecules. The expression of pro- and anti- angiogenic genes was evaluated using real time PCR. Protein levels were measured by ELISA or by FlowCytomix™. MMPs activity was examined by zymography.

Results: Short-term exposure to thrombin induced a long lasting (for hours) increase in RPE permeability to 10, 40 and 70 KD dextran. Decrease in PEDF mRNA and increase in VEGF and HGF mRNA expression and protein levels were detected even 24 hours after the 10 minute exposure to thrombin. MMPs 2 and 9 activities were also increased hours after the short-term exposure to thrombin.

Conclusions: Short-term exposure to thrombin induces proangiogenic signals in RPE and disruption of barrier properties. The data indicate that changes in permeability, gene expression, proteins levels and activity persisted for hours after short-term exposure to thrombin. Based on our findings, we suggest that accumulation of short-term exposures to thrombin over years contributes to the pathological processes involved in CNV development in the elderly.

Keywords: 701 retinal pigment epithelium • 700 retinal neovascularization • 694 retinal culture  
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