Abstract
Extracellular and intracellular reactive oxygen species attack different targets and may, therefore, result in different forms of oxidative stress. To specifically study an oxidative stress induced by a regulated intracellular flux of a defined reactive oxygen species in endothelium, we used immunotargeting of the H2O2-generating enzyme glucose oxidase (GOX) conjugated with an antibody to platelet-endothelial cell adhesion molecule (PECAM)-1, an endothelial surface antigen. Anti-PECAM-125I-GOX conjugates specifically bind to both endothelial and PECAM-transfected cells. Approximately 70% of cell-bound anti-PECAM-125I-GOX was internalized. The cell-bound conjugate was enzymatically active and generated H2O2 from glucose. Use of the fluorescent dye dihydrorhodamine 123 revealed that 70% of H2O2 was generated intracellularly, whereas 30% of H2O2 was detected in the cell medium. Catalase added to the cells eliminated H2O2 in the medium but had little effect on the intracellular generation of H2O2 by anti- PECAM-GOX. Both H2O2 added exogenously to the cell medium (extracellular H2O2) and that generated by anti-PECAM-GOX caused oxidative stress manifested by time-and dose-dependent irreversible plasma membrane damage. Inactivation of cellular catalase by aminotriazole treatment augmented damage caused by either extracellular H2O2 or anti-PECAM-GOX. Catalase added to the medium protected either normal or aminotriazole-treated cells against extracellular H2O2, yet failed to protect cells against injury induced by anti-PECAM-GOX. Therefore, treatment of PECAM-positive cells with anti-PECAM- GOX leads to conjugate internalization, predominantly intracellular H2O2 generation and intracellular oxidative stress. These results indicate that anti-PECAM-GOX 1) provides cell-specific intracellular delivery of an active enzyme and 2) causes intracellular oxidative stress in PECAM-positive cells.
Original language | English |
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Journal | American Journal of Physiology - Lung Cellular and Molecular Physiology |
Volume | 277 |
Issue number | 2 21-2 |
Publication status | Published - Aug 1999 |
Externally published | Yes |
Keywords
- Bioconjugation
- CD31
- Drug delivery
- Hydrogen peroxide
- Platelet-endothelial cell adhesion molecule-1
ASJC Scopus subject areas
- Pulmonary and Respiratory Medicine
- Cell Biology
- Physiology
- Physiology (medical)