Size and targeting to PECAM vs ICAM control endothelial delivery, internalization and protective effect of multimolecular SOD conjugates.

Controlled endothelial delivery of SOD may alleviate abnormal local surplus of superoxide involved in ischemia-reperfusion, inflammation and other disease conditions. Targeting SOD to endothelial surface vs. intracellular compartments is desirable to prevent pathological effects of external vs.

Anti-inflammatory effect of targeted delivery of SOD to endothelium: mechanism, synergism with NO donors and protective effects in vitro and in vivo.

Pro-inflammatory activation of vascular endothelium is implicated in pathogenesis of severe conditions including stroke, infarction and sepsis. We have recently reported that superoxide dismutase (SOD) conjugated with antibodies (Ab/SOD) that provide targeted delivery into endothelial endosomes mitigates inflammatory endothelial activation by cytokines and agonists of Toll-like receptors (TLR). The goal of this study was to appraise potential utility and define the mechanism of this effect.

Targeting recombinant thrombomodulin fusion protein to red blood cells provides multifaceted thromboprophylaxis.

Thrombin generates fibrin and activates platelets and endothelium, causing thrombosis and inflammation. Endothelial thrombomodulin (TM) changes thrombin's substrate specificity toward cleavage of plasma protein C into activated protein C (APC), which opposes its thrombotic and inflammatory activities. Endogenous TM activity is suppressed in pathologic conditions, and antithrombotic interventions involving soluble TM are limited by rapid blood clearance.

Modulation of endothelial targeting by size of antibody-antioxidant enzyme conjugates.

Endothelial targeting of antioxidant enzymes attenuates acute vascular oxidative stress in animal studies. Superoxide dismutase (SOD) and catalase conjugated with antibodies to Platelet-Endothelial Cell Adhesion Molecule-1 (anti-PECAM/SOD and anti-PECAM/catalase) bind to endothelium, accumulate in the pulmonary vasculature, and detoxify reactive oxygen species. In order to define the role of conjugate size in the efficacy and specificity of endothelial targeting, we synthesized anti-PECAM/enzyme conjugates of controlled size (40nm-10,000nm).

Sustained thromboprophylaxis mediated by an RBC-targeted pro-urokinase zymogen activated at the site of clot formation.

Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T).

Targeting of a mutant plasminogen activator to circulating red blood cells for prophylactic fibrinolysis.

Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity.

Platelet-endothelial cell adhesion molecule-1-directed endothelial targeting of superoxide dismutase alleviates oxidative stress caused by either extracellular or intracellular superoxide.

Targeting of the antioxidant enzyme catalase to endothelial cells protects against vascular oxidative stress induced by hydrogen peroxide (H(2)O(2))(Am J Physiol 285:L283-L292, 2003; Nat Biotechnol 21:392-398, 2003; Am J Physiol 293:L162-L169, 2007). 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.

Factors modulating the delivery and effect of enzymatic cargo conjugated with antibodies targeted to the pulmonary endothelium.

Vascular drug targeting may improve therapies, yet a thorough understanding of the factors that regulate effects of drugs directed to the endothelium is needed to translate this approach into the clinical domain. To define factors modulating the efficacy and effects of endothelial targeting, we used a model enzyme (glucose oxidase, GOX) coupled with monoclonal antibodies (anti-TM(34) or anti-TM(201)) to distinct epitopes of thrombomodulin, a surface determinant enriched in the pulmonary endothelium. GOX delivery results in conversion of glucose and oxygen into H(2)O(2) leading to lung damage, a clear physiologic endpoint.

Tumor necrosis factor-alpha differentially regulates the expression of proinflammatory genes in human airway smooth muscle cells by activation of interferon-beta-dependent CD38 pathway.

Recent evidence suggests that CD38, an ectoenzyme that converts NAD(+) to cyclic ADP-ribose (cADPr), may play a role in cytokine-induced airway smooth muscle (ASM) cell hyper-responsiveness, a key feature associated with chronic asthma. In the present study, we investigated the major signaling pathways by which tumor necrosis factor-alpha (TNFalpha) induces CD38 expression and its role in regulating gene expression in human ASM cells. Using flow cytometry analyses, TNFalpha enhanced CD38 expression in a manner that was time-(0-24 h), concentration-(0.

Tumor necrosis factor alpha modulates airway smooth muscle function via the autocrine action of interferon beta.

Current evidence suggests that tumor necrosis factor alpha (TNFalpha) and the family of interferons (IFNs) synergistically regulate many cellular responses that are believed to be critical in chronic inflammatory diseases, although the underlying mechanisms of such interaction are complex, cell-specific, and not completely understood. In this study, TNFalpha in a time-dependent manner activated both janus tyrosine kinase 1 and Tyk2 tyrosine kinase and increased the nuclear translocation of interferon-regulatory factor-1, STAT1, and STAT2 in human airway smooth muscle cells. In cells transfected with a luciferase reporter, TNFalpha stimulated gamma-activated site-dependent gene transcription in a time- and concentration-dependent manner.