Abstract
Abstract 2135
Poster Board II-112
Introduction:
The serine protease inhibitor (serpin), plasminogen activator inhibitor-1 (PAI-1) binds to and inhibits the plasminogen activators tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). This results in both a decrease in plasmin production, as well as a decrease in the dissolution of fibrin clots. PAI-1 is also associated with the pathophysiology of several diseases, including cancer and cardiovascular disease. Both experimental and clinical studies have shown that increasing the plasma and vessel wall PAI-1 levels positively correlates with an increased risk of cardiovascular-related events. Consequently, the pharmacological suppression of PAI-1 might prevent or treat vascular disease. Unfortunately, since PAI-1 is a multifunctional protein, complete inhibition of PAI-1 might hinder its ability to regulate fibrinolysis, which can provoke bleeding. However, eliminating the pathological functions of PAI-1 without hindering its physiological functions might be beneficial in treating a variety of diseases. Extracellular matrix vitronectin (VN) increases at sites of vessel injury and is also present in fibrin clots. In response to injury, vitronectin facilitates cell adhesion, thereby increasing vascular cell migration by binding to integrins and to surface-bound uPA. PAI-1 competes with integrins and the urokinase-type plasminogen activator receptor (uPAR) for VN binding, resulting in the detachment of cells from the extracellular matrix. The binding of PAI-1 to VN prevents integrins from binding to VN, and inhibits cell adhesion and migration.
Objective:
The goal of this study was to develop RNA aptamers to interfere with a single PAI-1 function, without obstructing its other functions. The present study concentrated on developing PAI-1 aptamers to the vitronectin binding site of PAI-1. Aptamers are single-stranded nucleic acids, either DNA or RNA, that bind to their target protein with high affinity and specificity.
Methods:
Our aptamers were generated by the systematic evolution of ligands by exponential enrichment (SELEX). Adopting the SELEX in vitro selection technique ensures creation of nuclease-resistant RNA molecules that will bind to target proteins. We used in vitro assays to determine the effect of the aptamers on the adhesion and migration of smooth muscle (SM) and human umbilical vein endothelial cells (EC).
Results:
Recently, we published a paper that showed the generation of PAI-1 specific RNA aptamers that bind to the heparin/vitronectin binding site of PAI-1 (Blake et al., 2009). We showed that PAI-1 specific aptamers prevented the detachment of cancer cells from vitronectin in the presence of PAI-1, resulting in an increase in cell adhesion. We have expanded these studies to include smooth muscle (SMC) and human umbilical vein endothelial cells (EC). We demonstrated that the PAI-1 specific aptamers (SM-20 and WT-15) dose dependently increase SMC and EC attachment in the presence of vitronectin (compared to the control aptamer). Interestingly, SM-20 (the aptamer to stable PAI-1) was more effective than WT-15 (aptamer to wild-type PAI-1). Whereas PAI-1 significantly inhibited cell migration (in the presence of vitronectin), the PAI-1 specific aptamers were able to restore migration of both SMC and EC cells. Additionally, the PAI-1 aptamers were unable to bind to the PAI-1 vitronectin binding mutant, further suggesting that these aptamers bind to the PAI-1's vitronectin binding site. Importantly, these aptamers did not affect the antiprotease activity of PAI-1.
Conclusions:
We have shown that we are able to inhibit one of PAI-1's functions without hindering its other functions. By promoting smooth muscle and endothelial cell migration, these aptamers can potentially eliminate the adverse effects of elevated PAI-1 levels in the pathogenesis of vascular disease.
Disclosures:
Sullenger: Regado Biosciences Inc.: Equity Ownership, Scientific Founder.
Inhibition of PAI-1 Antiproteolytic Activity Against tPA by RNA Aptamers
