May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Apoptotic Bodies From Retinal Endothelial Cells Promote Endothelial Progenitor Cell Recruitment
Author Affiliations & Notes
  • A. D. Bhatwadekar
    Dept of Ophthalmology, Centre for Vision Science, Queen's University Belfast, Belfast, United Kingdom
  • D. P. Dash
    Dept of Ophthalmology, Centre for Vision Science, Queen's University Belfast, Belfast, United Kingdom
  • T. A. Gardiner
    Dept of Ophthalmology, Centre for Vision Science, Queen's University Belfast, Belfast, United Kingdom
  • T. M. Curtis
    Dept of Ophthalmology, Centre for Vision Science, Queen's University Belfast, Belfast, United Kingdom
  • A. W. Stitt
    Dept of Ophthalmology, Centre for Vision Science, Queen's University Belfast, Belfast, United Kingdom
  • Footnotes
    Commercial Relationships A.D. Bhatwadekar, None; D.P. Dash, None; T.A. Gardiner, None; T.M. Curtis, None; A.W. Stitt, None.
  • Footnotes
    Support Insight-Trust for the Visually Impaired
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1363. doi:
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      A. D. Bhatwadekar, D. P. Dash, T. A. Gardiner, T. M. Curtis, A. W. Stitt; Apoptotic Bodies From Retinal Endothelial Cells Promote Endothelial Progenitor Cell Recruitment. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1363.

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

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Abstract

Purpose:: Progressive degeneration of the retinal vasculature is a feature of diabetic retinopathy (DR). Bone marrow derived endothelial progenitor cells (EPCs) can contribute to microvascular repair although the precise stimulus attracting these cells to sites of endothelial injury is largely unknown. Using a unique model whereby apoptosis was induced in defined regions of retinal microvascular endothelial cell (RMEC) monolayers we have investigated the factors that recruit EPCs to "wound" sites.

Methods:: Following uptake of the photoreactive drug verteporfin (VPF; 0.5-1.0µg/ml), RMEC monolayers were exposed to collimated red light to create highly delineated regions of apoptosis; wounding by this method maintains the integrity of the subendothelial matrix. VPF-treated monolayers were also used to induce blanket apoptosis for the generation of apoptotic cells. Human EPCs were isolated from peripheral blood and characterised for VEGFR2, CD34, CD31, UEA-1 lectin and uptake of acLDL-DiI. EPCs following labelling by DiI were added to wounded monolayers, with or without residual apoptotic cells at the wound site. In separate experiments apoptotic bodies were fed to non-wounded RMEC monolayers for 6 & 24 hours and mRNA levels for ICAM, VCAM and E-Selectin quantified by real time RT-PCR. Finally conditioned medium obtained from apoptotic RMECs was assayed for cytokines and tested for EPC chemotaxis with a Dunn chamber assay

Results:: Apoptotic bodies left attached to the VPF-induced wounds enhanced EPC recruitment by 5-fold (p<0.05) as compared to controls in which apoptotic cells had been washed off. Immunostaining showed enhanced expression of ICAM and VCAM at wound sites and application of apoptotic bodies to unwounded cultures produced a 5-15 fold upregulation in expression of ICAM (p<0.001), VCAM (p<0.05) and E-selectin (P<0.001). EPCs showed a strong migratory response (p<0.05) to conditioned medium obtained from apoptotic RMEC while ELISA of the medium showed increased levels of IL-6, IL-8, and TNF-α. SDF-1 remained unchanged when compared to non-apoptotic control cultures.

Conclusions:: This investigation suggests that apoptotic endothelial cells are an important recruitment stimulus for EPCs, possibly by up-regulating adhesion molecule expression on nearby endothelial cells and by increasing chemoattractant gradients of pro-inflammatory cytokines and chemokines

Keywords: diabetic retinopathy • vascular cells 
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