Constitutively expressed determinants are good candidates for targeting either normal or pathologically altered endothelium. For example, platelet-endothelial adhesion molecule-1 (PECAM, CD31) is a panendothelial transmembrane Ig superfamily glycoprotein, predominantly localized in the sites of cellular contacts in the endothelial monolayer. Platelets and white blood cells also express PECAM, but at levels that are orders of magnitude lower than endothelial cells. PECAM is involved in the cellular recognition, adhesion, signaling, and transendothelial migration of leukocytes, key in pathogenesis of many disease conditions. Blocking PECAM by anti-PECAM suppresses inflammation and protects organs against leukocyte-mediated oxida-tive stress. Therefore, anti-PECAM targeting may provide secondary benefits for management of inflammation, per haps by attenuation of leukocyte transmigration. PECAM is abundant in endothelial cells, which express millions of anti-PECAM binding sites. In addition, PECAM is a stable endothelial cell antigen: cytokines and ROS do not down-regulate its expression and surface density on the endothe-lium. This permits a robust PECAM-targeted drug delivery to either normal or pathologically altered vasculature, for either prophylaxis or therapies. Endothelial cells bind anti-PECAM without internalization, but readily internalize multimeric anti-PECAM complexes within 100- to 350-nm diameter via a novel internalization pathway different from clathrin- and caveoli-mediated endocytosis, namely CAMmediated endocytosis (common for PECAM-1 and ICAM-1). Thus, by rational design of anti-PECAM conjugates of a proper size, one can achieve either intracellular or surface targeting to endothelium . An active reporter enzyme, b-galactosidase, conjugated to anti-PECAM has been shown to accumulate intracellularly in the pulmonary endothelium as soon as 10 minutes after IV injection in mice and pigs. Targeting of anti-PECAM/catalase conjugate protects pulmonary vasculature against acute oxidant stress in mice and rats .
Intercellular adhesion molecule-1 (ICAM-1, CD54) is another Ig superfamily surface glycoprotein with a short cytoplasmic domain, a transmembrane domain, and a large extracellular domain. It is normally expressed by endothe-lial cell at relatively high surface density (2 x 104 to 2 x 105 surface copies per cell). Some other cell types also express ICAM, yet the major fraction of blood-accessible ICAM is exposed on the luminal surface of endothelium. Robust and specific binding of ICAM antibodies and anti-ICAM conjugates to vascular endothelium after intravenous administration has been documented in animals. Pathological stimuli, such oxidants, cytokines, abnormal shear stress, and hypoxia, stimulate surface expression of ICAM by endothelial cells. Thus, inflammation and other pathological conditions do not suppress, but rather markedly facilitate ICAM targeting. ICAM, a counter-receptor for integrins on leukocytes, supports their adhesion to endothelial cells and thus contributes to inflammation. In addition, ICAM serves as a natural ligand for certain viruses. ICAM may also serve as a signaling molecule, yet the significance of this signaling remains to be more fully elucidated. ICAM antibodies suppress leukocytes adhesion, thus producing anti-inflammatory effects in animal models and clinical pathological settings associated with vascular injury, such as acute inflammation, ischemia-reperfusion, and oxidative stress. The anti-inflammatory effect of multivalent anti-ICAM conjugates may be even more potent because of the higher affinity/valency of the conjugate binding and down-regulation of surface ICAM due to internalization via CAMmediated endocytosis (which was described earlier for PECAM). Thus, subcellular localization of anti-ICAM conjugates can be controlled by their size: Anti-ICAM carriers permit targeted delivery of antithrombotic agents to the endothelial surface and antioxidant enzymes to the endothe-lial interior .
Angiotensin-converting enzyme (ACE) is a transmembrane glycoprotein expressed on the endothelial luminal surface, which converts Ang I into Ang II to induce vasoconstricting, pro-oxidant, and proinflammatory activities. Pulmonary vasculature is enriched in ACE: Nearly 100 percent of endothelial cells in the alveolar capillaries are ACE positive, whereas 10 to 15 percent in the extrapulmonary capillaries are ACE-positive. Radiolabeled anti-ACE and reporter compounds conjugated with anti-ACE accumulate in the lungs after injections via diverse routes in animals and humans. Anti-ACE does not cause acute harmful reactions in animals and humans. ACE antibodies suppress its activity; this might enhance therapeutic utility of the targeting and provide beneficial effects in conditions associated with vascular oxidative stress, ischemia, and inflammation. Endothelial cells internalize anti-ACE that may deliver drugs intracellularly. Anti-ACE-conjugated antioxidant enzymes, such as catalase, accumulate in rat lungs in vivo and protect perfused rat lungs against H2O2. Proinflammatory agents suppress ACE expression and may inhibit therapeutic targeting directed to ACE, yet anti-ACE is a good candidate for targeting to the pulmonary endothe-lium for a prophylactic use and for gene delivery [8, 9].
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