title: PECAM-directed immunotargeting of catalase: specific, rapid and transient protection against hydrogen peroxide.
abstract: Vascular immunotargeting to Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM) facilitates drug delivery to endothelium. We used human PECAM-transfected REN cells (REN/PECAM) as a model to compare targeting of antioxidant enzyme catalase conjugated with PECAM antibody (anti-PECAM/catalase) with adenoviral catalase delivery. Anti-PECAM/(125)I-catalase bound to REN/PECAM, but not to REN cells (70 vs. 1 ng/well vs. < 2 ng/well of unmodified catalase). At a virus-to-cell ratio of 1, elevated levels of catalase protein were detected by immunoblotting after adenoviral transfection of REN/PECAM and REN cells alike; H(2)O(2)-degrading activity of cell lysates was elevated at ratios of 10 and higher. REN/PECAM cells internalize 66% of cell-bound anti-PECAM/(125)I-catalase. Confocal microscopy localized anti-PECAM/catalase to intracellular vesicles, while catalase expressed by adenovirus was distributed in vesicles and throughout the cytosol. Within 15 min of delivery, anti-PECAM/catalase augmented H(2)O(2)-degrading activity and survival of H(2)O(2)-exposed REN/PECAM cells. The effects of conjugate delivery reached a plateau within 1 h and declined to the basal level within 12 h. In contrast, adenoviral delivery required several hours for transduction and development of the effects, but permitted much longer duration of protection (at least 48 h). Simultaneous exposure of REN/PECAM cells to anti-PECAM/catalase and catalase-encoding adenovirus afforded protection against H(2)O(2) with a rapid onset and a prolonged duration. Therefore, PECAM-directed immunotargeting provides a specific, antigen-directed intracellular delivery of catalase that affords a rapid but transient protection against H(2)O(2) and may complement gene delivery strategies for antioxidant protection.
authors.affiliation: Institute of Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, PA 19104-6068, USA.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Sweitzer
authors.forename: Anu P
authors.initials: AP
authors.lastname: Thomas
authors.forename: Rainer
authors.initials: R
authors.lastname: Wiewrodt
authors.forename: Marian T
authors.initials: MT
authors.lastname: Nakada
authors.forename: Francisco
authors.initials: F
authors.lastname: Branco
authors.forename: Vladimir R
authors.initials: VR
authors.lastname: Muzykantov
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000975:Antioxidants / Q000378:metabolism; D019921:Biotinylation; D002374:Catalase / Q000378:metabolism*; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems; D004730:Endothelium, Vascular / Q000378:metabolism; D005609:Free Radicals; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D015151:Immunoblotting; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018384:Oxidative Stress; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
chemical_list: D000975:Antioxidants; D005609:Free Radicals; D008081:Liposomes; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D006861:Hydrogen Peroxide; D002374:Catalase