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 |
|---|---|
| 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 |
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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)
Cite this
Immunotargeting of glucose oxidase : Intracellular production of H2O2 and endothelial oxidative stress. / Gow, Andrew J.; Branco, Frank; Christofidou-Solomidou, Melpo; Black-Schultz, Linda; Albelda, Steven M.; Muzykantov, Vladimir R.
In: American Journal of Physiology - Lung Cellular and Molecular Physiology, Vol. 277, No. 2 21-2, 08.1999.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Immunotargeting of glucose oxidase
T2 - Intracellular production of H2O2 and endothelial oxidative stress
AU - Gow, Andrew J.
AU - Branco, Frank
AU - Christofidou-Solomidou, Melpo
AU - Black-Schultz, Linda
AU - Albelda, Steven M.
AU - Muzykantov, Vladimir R.
PY - 1999/8
Y1 - 1999/8
N2 - 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.
AB - 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.
KW - Bioconjugation
KW - CD31
KW - Drug delivery
KW - Hydrogen peroxide
KW - Platelet-endothelial cell adhesion molecule-1
UR - http://www.scopus.com/inward/record.url?scp=0032848760&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032848760&partnerID=8YFLogxK
M3 - Article
VL - 277
JO - American Journal of Physiology - Lung Cellular and Molecular Physiology
JF - American Journal of Physiology - Lung Cellular and Molecular Physiology
SN - 1040-0605
IS - 2 21-2
ER -