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Guido Hartmann; Barrier function studies of VEGF and ANG-2 in human endothelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3217.
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© ARVO (1962-2015); The Authors (2016-present)
Breakdown of the endothelial blood retinal barrier is key to pathophysiological changes in age-related macular degeneration and diabetic macular edema. VEGF-A (Vascular Endothelial Growth Factor-A) is involved in mediating breakdown of the endothelial barrier resulting in enhanced vascular leakage and subsequent edema in retinal tissue. Angiopoietins (ANG-1 and ANG-2) also modulate endothelial barrier function. We investigated a cellular model of endothelial barrier breakdown in vitro. VEGF-A, ANG-1 and ANG-2 and their respective neutralizing reagents were tested for the modulation of vascular barrier function.
Microvascular endothelial cells were cultured on transwell filters and stimulated with VEGF-A and/or ANG-1/ANG-2 in the presence and absence of neutralizing antibodies to VEGF-A and ANG-2. Measurement of transendothelial resistance (TEER) was followed by immunostainings for key molecules of adherens junctions and tight junctions. Immunocytochemistry was used to determine the expression of endothelial cell junctional proteins (VE-cadherin, a key component of endothelial cell-to-cell adherens junctions, ZO-1 adapter molecule, a component of tight junctions and the adhesion molecule PECAM-1).
VEGF-A induced a dose dependent break down of barrier function in human microvascular cells. This effect was inhibited with an anti-VEGF antibody given preventative or interventative. ANG-1 increased barrier function as demonstrated by increased TEER values. Simultaneous incubation of VEGF-A and ANG-1 resulted in reduced barrier breakdown by VEGF-A. Endothelial cells in culture constitutively secrete high levels of ANG-2. Stimulation with VEGF-A enhanced this secretion further. Addition of neutralizing anti-ANG2 antibody reduced VEGF-A induced barrier breakdown while combined addition of ANG-1 and anti-ANG-2 was most efficacious. Distortion of the typical cell-cell boundary of VE-cadherin, ZO-1 and PECAM-1 was demonstrated when cells were treated with VEGF-A and prevented by coincubation with ANG1 and ANG1/anti-VEGF-A.
VEGF-A is a well-known inducer of barrier breakdown of endothelial cells. We describe in vitro experiments of endothelial barrier breakdown induced by VEGF-A and modulation by ANG-1 and anti-ANG-2. Combined inhibition of VEGF-A and ANG-2 might be an promising approach to enhance the efficacy of anti-VEGF-A treatments.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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