Abstract
Reactive oxygen species (ROS) superoxide anion (O2⨪) and hydrogen peroxide (H2O2) produced by activated leukocytes and endothelial cells in sites of inflammation or ischemia cause endothelial barrier dysfunction that may lead to tissue edema. Antioxidant enzymes (AOEs) catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet-endothelial cell adhesion molecule-1 (PECAM-1) specifically bind to endothelium, quench the corresponding ROS, and alleviate vascular oxidative stress and inflammation. In the present work, we studied the effects of anti-PECAM/catalase and anti-PECAM/SOD conjugates on the abnormal permeability manifested by transendothelial electrical resistance decline, increased fluorescein isothiocyanate-dextran influx, and redistribution of vascular endothelial-cadherin in human umbilical vein endothelial cell (HUVEC) monolayers. Anti-PECAM/catalase protected HUVEC monolayers against H2O2-induced endothelial barrier dysfunction. Polyethylene glycol-conjugated catalase exerted orders of magnitude lower endothelial uptake and no protective effect, similarly to IgG/catalase. Anti-PECAM/catalase, but not anti-PECAM/SOD, alleviated endothelial hyperpermeability caused by exposure to hypoxanthine/xanthine oxidase, implicating primarily H2O2 in the disruption of the endothelial barrier in this model. Thrombin-induced endothelial permeability was not affected by treatment with anti-PECAM/AOEs or the NADPH oxidase inhibitor apocynin or overexpression of AOEs, indicating that the endogenous ROS play no key role in thrombin-mediated endothelial barrier dysfunction. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase, inhibited a vascular endothelial growth factor (VEGF)-induced increase in endothelial permeability, identifying a key role of endogenous O2⨪ in the VEGF-mediated regulation of endothelial barrier function. Therefore, AOEs targeted to endothelial cells provide versatile molecular tools for testing the roles of specific ROS in vascular pathology and may be translated into remedies for these ROS-induced abnormalities.
Footnotes
This work was supported in part by the National Institutes of Health National Heart, Lung, and Blood Institute [Grant R01-HL073940] (to V.R.M.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.111.180620.
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The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- VEGF
- vascular endothelial growth factor
- ROS
- reactive oxygen species
- AOE
- antioxidant enzyme
- SOD
- superoxide dismutase
- PECAM-1
- platelet-endothelial cell adhesion molecule-1
- PEG
- polyethylene glycol
- NOX
- NADPH oxidase
- HUVEC
- human umbilical vein endothelial cell
- VE
- vascular endothelial
- HX
- hypoxanthine
- XO
- xanthine oxidase
- NO
- nitric oxide
- Ab
- antibody
- ELISA
- enzyme-linked immunosorbent assay
- DPI
- diphenylene iodonium
- FITC
- fluorescein isothiocyanate
- TEER
- transendothelial electrical resistance
- HBSS
- Hanks' balanced salt solution.
- Received February 16, 2011.
- Accepted April 6, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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