VEGF (vascular endothelial growth factor) overproduction has been identified as a major factor underlying pathological angiogenesis in vivo, including such conditions as psoriasis, macular degeneration, and tumor proliferation. Endothelial cell tyrosine kinase receptors, KDR and Flt-1, have been implicated in VEGF responses including cellular migration, proliferation, and modulation of vascular permeability. Therefore, agents that limit VEGF-cellular interaction are likely therapeutic candidates for VEGF-mediated disease states (particularly agents blocking activity of VEGF165, the most frequently occurring VEGF isoform). To that end, a nuclease-resistant, VEGF165-specific aptamer NX1838 (2'-fluoropyrimidine, RNA-based oligonucleotide/40-kDa-PEG) was developed. We have assessed NX1838 inhibition of a variety of cellular events associated with VEGF, including cellular binding, signal transduction, calcium mobilization, and induction of cellular proliferation. Our data indicate that NX1838 inhibits binding of VEGF to HUVECs (human umbilical vein endothelial cells) and dose-dependently prevents VEGF-mediated phosphorylation of KDR and PLCgamma, calcium flux, and ultimately VEGF-induced cell proliferation. NX1838-inhibition of VEGF-mediated cellular events was comparable to that observed with anti-VEGF monoclonal antibody, but was ineffective as an inhibitor of VEGF121-induced HUVEC proliferation. These findings, coupled with nuclease stability of the molecule, suggest that NX1838 may provide therapeutic utility in vivo.

No abstract

Thromboxane (Tx) A2 generation and subsequent selective pulmonary sequestration of neutrophils (PMNs) is characteristic of several forms of the adult respiratory distress syndrome (ARDS). Therefore, we examined PMN-dependent adhesion to cultured pulmonary microvessel and aortic endothelium (EC) in response to U46,619 (Tx mimic). Nonstimulated PMNs were two fold more adherent to pulmonary microvessel EC than to aortic EC (P less than 0.01). PMN pretreatment with Tx mimic (10(-6) M) increased adhesion to both types of EC (P less than 0.01). The Tx mimic-induced adhesion was blocked by receptor antagonists to Tx (SQ29,548) and to leukotrienes (FPL55,712), and by the anti-CD18 mAb TS1/18 (P less than 0.01, all cases). Baseline PMN adhesion also was modulated by Tx, leukotrienes, and CD18, for both EC types. These results indicate pulmonary microvessel EC is intrinsically more adhesive for both nonstimulated and stimulated PMNs than aortic EC and that Tx mediates PMN-dependent adhesion by coupled interaction of Tx and LT receptors via CD18 activation.