Platelet-Endothelial Cell Adhesion Molecule-1-Directed Endothelial Targeting of Superoxide Dismutase Alleviates Oxidative Stress Caused by Either Extracellular or Intracellular Superoxide

Abstract

ABSTRACT. However, another reactive oxygen species, superoxide anion, is also involved in many forms of vascular oxidative stress, including ischemia/reperfusion, hypertension, and inflammation. To protect endothelium against superoxide attack, we designed and tested antibody-directed targeting of superoxide dismutase (SOD) to the endothelial surface determinant, platelet-endothelial cell adhesion molecule (PECAM)-1. We synthesized anti-PECAM/SOD conjugates that retained 70% of enzymatic activity (superoxide anion dismutation) and specifically bound to endothelial cells, but not PECAM-negative cells. The effect of anti-PECAM/SOD delivery to cells was tested in two distinct models of oxidative stress induced by either extracellular or intracellular generation of superoxide anion. In the first model, anti-PECAM/SOD, but not unconjugated SOD, protected endothelial cells against injury caused by superoxide produced in the medium by hypoxanthine-xanthine oxidase. At the optimal dose, anti-PECAM/SOD provided up to 40 to 50% protection against cell death in this model. In the second model, anti-PECAM/SOD at the optimal dose provided complete protection against necrosis caused by paraquat-induced intracellular superoxide generation. Endothelial targeting of SOD represents a new molecular antioxidant approach that could be used for the management of vascular oxidative stress.

Targeted Detoxification of Selected Reactive Oxygen Species in the Vascular Endothelium

“…The size of prepared conjugates was measured by dynamic light scattering (DLS) apparatus 90Plus Particle Sizer (Brookhaven Instruments Corp., Holtsville, NY). Our previous studies in vitro and in vivo documented that anti-PECAM conjugates of this size optimally bind to endothelial cells, enter cells via cell adhesion molecule-mediated endocytosis, circulate in vivo, and deliver cargoes to endothelium in the vasculature (Muzykantov et al, 1999;Christofidou-Solomidou et al, 2003;Kozower et al, 2003;Shuvaev et al, 2007b).…”

“…Conjugates were prepared by use of amino-based cross-linking chemistry as described previously (Shuvaev et al, 2007b). Number of corresponding reactive groups introduced per molecule of IgG, antibody, and AOE (catalase or SOD) has been adjusted individually for each type of conjugate to maintain functional activity of both antibody and AOE moieties and produce conjugates with diameter of ϳ300 nm.…”

“…125 I-radiolabeled anti-PECAM/SOD and IgG/SOD conjugates were prepared as described previously (Shuvaev et al, 2007b) with final conjugate labeling at a density of ϳ10,000 cpm/g total SOD. Ten micrograms of radiolabeled immunoconjugated SOD were injected intravenously in normal C57BL/65 mice (The Jackson Laboratory, Bar Harbor, ME) via tail vein.…”

“…Intravenous injection of glucose oxidase conjugated with thrombomodulin antibody (anti-TM/ GOX) leads to anti-TM/GOX uptake in the pulmonary vasculature, generation of H 2 O 2 in endothelium, and acute edematous lung injury in mice . Exposure of mice injected with anti-TM/GOX to elevated levels of oxygen (98% O 2 , hyperoxia) further aggravates lung injury (Shuvaev et al, 2007b). This "double-hit" model imitates pulmonary pathological conditions caused by vascular ROS toxicity aggravated by iatrogenic effects of oxygen ventilation that may happen, for example, in treatment of acute lung injury (Matthay et al, 2003).…”

“…Therefore, selective scavenging of superoxide will inhibit peroxynitrite formation and may help preserve endothelial NO production. Accordingly, we have designed anti-PECAM/SOD conjugates and found that endothelial targeting of anti-PECAM/SOD protects against cell damage caused by either extracellular or intracellular superoxide anion production in cell culture (Shuvaev et al, 2007b).…”