Effect of PAI-1 Specific RNA Aptamers On Cell Adhesion and Motility.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2135-2135
Author(s):  
Yolanda Fortenberry ◽  
Charlene M Blake ◽  
Bruce A Sullenger
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Cell Adhesion ◽  
Binding Site ◽  
Plasminogen Activator ◽  
Umbilical Vein ◽  
Rna Aptamers ◽  
Type Plasminogen Activator ◽  
Pai 1

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.

Proteolysis, Cell Adhesion, Chemotaxis, and Invasiveness Are Regulated by the u-PA-u-PAR-PAI-1 System

1999 ◽  
Vol 82 (08) ◽  
pp. 298-304 ◽  
Author(s):  
Francesco Blasi
Keyword(s):  
Cell Adhesion ◽  
Plasminogen Activator ◽  
Tyrosine Kinases ◽  
Plasminogen Activator Inhibitor ◽  
Prognostic Indicators ◽  
Type Plasminogen Activator ◽  
Cell Adhesion And Migration ◽  
And Migration ◽  
Pai 1

IntroductionHigh levels of urokinase-type plasminogen activator (u-PA), of its inhibitor (plasminogen activator inhibitor (PAI)-1), or of its receptor (u-PAR, CD87) are strong prognostic indicators of relapse in human cancers. In addition, many in vitro data show that u-PA, PAI-1, and u-PAR have a profound influence on cell migration. This set of molecules regulates surface proteolysis, cell adhesion, and chemotaxis through different mechanisms. Binding to u-PAR strongly stimulates the activation of pro-u-PA and, hence, of plasminogen, resulting in localized production of the broad-spectrum serine protease, plasmin, which can digest extracellular matrix proteins or activate latent motogenic factors. Chemotaxis is induced through an u-PA-dependent conformational change in u-PAR, which uncovers a very potent chemotactic epitope(s) that acts through a pertussis toxin-sensitive step and activates intracellular tyrosine kinases. In addition, cell adhesion is affected by an u-PA-dependent exposure of u-PAR epitope(s), which interact with vitronectin (VN), integrins, and caveolin, thus modifying the substrate specificity. Thus, u-PA binding can transform u-PAR from a simple receptor for u-PA into a pleiotropic ligand for other surface molecules.All of these processes are regulated by the u-PA inhibitor, PAI-1. Inhibition of cell adhesion and migration by PAI-1 on VN occurs because the same region of VN is required for interaction with PAI-1, u-PAR, and integrins. PAI-1, however, also affects u-PAR occupancy by triggering the internalization of the u-PA-u-PAR complex, the degradation of u-PA, and the recycling of free u-PAR. Available data suggest that cells respond to a “stop” signal, due to the PAI-1-dependent internalization and degradation of u-PA. Cells also respond to a “go” signal through the stimulation of surface-proteolysis, exposure of chemotactic epitopes, and recycling of u-PAR to novel surface positions. Finally, cells respond to a “pause” signal through transient u-PAR-dependent adhesion stages, thus shifting the cells between an “adhesion-mode” and a “migration-mode.”

scholarly journals Type 1 plasminogen activator inhibitor synthesis of endothelial cells is downregulated by smooth muscle cells

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1277-1283 ◽  
Author(s):  
G Christ ◽  
D Seiffert ◽  
P Hufnagl ◽  
A Gessl ◽  
J Wojta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Type Plasminogen Activator ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Plasminogen activator inhibitor type 1 (PAI-1), the physiologic inhibitor of both tissue-type plasminogen activator (tPA) and urokinase- type plasminogen activator (uPA), is a major biosynthetic product of endothelial cells in vitro; endothelial cells in vivo, in contrast, do not appear to produce significant amounts of PAI-1 as made evident by in situ-hybridization studies in normal mice. This suggests that the high rate of PAI-1 synthesis of endothelial cells in vitro might be a result of the culture conditions. When human umbilical vein endothelial cells (HUVEC) were grown on human amniotic membranes, resembling the natural growth support instead of coated plastic, their morphology was changed from the cobblestone-like appearance on plastic to an in vivo like flagstone pattern. However, this morphological change had no significant effect on the synthesis and secretion of PAI-1. When smooth muscle cell (SMC) conditioned media (CM) were added to HUVEC cultures, PAI-1 antigen secretion of HUVEC was reduced by 40% to 60% as measured by enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation experiments using 36S-methionine metabolically labeled HUVEC and Northern blot analysis of HUVEC PAI-1 mRNA indicate that this reduction was attributable to decreased PAI-1 synthesis and reduced steady-state levels of both the 3.2 kb and 2.2 kb form of PAI-1 mRNA. This effect was dose-dependent and observed under serum-containing as well as serum- free conditions, in the absence or presence of endothelial cell growth supplement (ECGS, 0 to 100 micrograms/mL) and attributable to a nondialyzable factor. Our data suggest that the high level of PAI-1 biosynthesis of endothelial cells in vitro may be attributable to the lack of a soluble factor produced by SMC, which controls and suppresses PAI-1 biosynthesis of endothelial cells in vivo.

T cell lymphokines modulate bFGF-induced smooth muscle cell fibrinolysis and migration

1997 ◽  
Vol 272 (2) ◽  
pp. C392-C398 ◽  
Author(s):  
W. Wang ◽  
H. J. Chen ◽  
A. Schwartz ◽  
P. J. Cannon ◽  
L. E. Rabbani
Keyword(s):  
Smooth Muscle ◽  
T Cell ◽  
Smooth Muscle Cell ◽  
Plasminogen Activator ◽  
Muscle Cell ◽  
Ifn Gamma ◽  
Type Plasminogen Activator ◽  
And Migration ◽  
Pai 1

Smooth muscle cell (SMC) fibrinolysis is necessary for SMC migration. To determine whether the T cell lymphokines interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) modulate SMC fibrinolysis and migration induced by basic fibroblast growth factor (bFGF), we examined the effects of IL-4 and IFN-gamma on human SMC tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (UPA), and plasminogen activator inhibitor 1 (PAI-1) antigen production, determined by enzyme-linked immunosorbent assays. Although IL-4 had no effects on SMC tPA, UPA, and PAI-1 production, it potentiated bFGF-induced tPA, UPA, and PAI-1 antigens. IL-4 plus bFGF resulted in a net increase in SMC fibrinolytic activity. IFN-gamma did not significantly affect bFGF induction of SMC tPA and PAI-1 antigens. However, IFN-gamma significantly decreased bFGF-mediated induction of SMC UPA antigen. IFN-gamma decreased the IL-4 plus bFGF induction of both tPA and UPA antigens. IL-4 increased and IFN-gamma abrogated bFGF induction of in vitro SMC migration through a modified micro-Boyden chamber. Therefore, IL-4 and IFN-gamma modulate bFGF-mediated induction of in vitro vascular SMC fibrinolysis and migration.

scholarly journals Type 1 plasminogen activator inhibitor synthesis of endothelial cells is downregulated by smooth muscle cells

Blood ◽  
1993 ◽  
Vol 81 (5) ◽  
pp. 1277-1283
Author(s):  
G Christ ◽  
D Seiffert ◽  
P Hufnagl ◽  
A Gessl ◽  
J Wojta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Type Plasminogen Activator ◽  
Activator Inhibitor ◽  
Pai 1

Plasminogen activator inhibitor type 1 (PAI-1), the physiologic inhibitor of both tissue-type plasminogen activator (tPA) and urokinase- type plasminogen activator (uPA), is a major biosynthetic product of endothelial cells in vitro; endothelial cells in vivo, in contrast, do not appear to produce significant amounts of PAI-1 as made evident by in situ-hybridization studies in normal mice. This suggests that the high rate of PAI-1 synthesis of endothelial cells in vitro might be a result of the culture conditions. When human umbilical vein endothelial cells (HUVEC) were grown on human amniotic membranes, resembling the natural growth support instead of coated plastic, their morphology was changed from the cobblestone-like appearance on plastic to an in vivo like flagstone pattern. However, this morphological change had no significant effect on the synthesis and secretion of PAI-1. When smooth muscle cell (SMC) conditioned media (CM) were added to HUVEC cultures, PAI-1 antigen secretion of HUVEC was reduced by 40% to 60% as measured by enzyme-linked immunosorbent assay (ELISA). Immunoprecipitation experiments using 36S-methionine metabolically labeled HUVEC and Northern blot analysis of HUVEC PAI-1 mRNA indicate that this reduction was attributable to decreased PAI-1 synthesis and reduced steady-state levels of both the 3.2 kb and 2.2 kb form of PAI-1 mRNA. This effect was dose-dependent and observed under serum-containing as well as serum- free conditions, in the absence or presence of endothelial cell growth supplement (ECGS, 0 to 100 micrograms/mL) and attributable to a nondialyzable factor. Our data suggest that the high level of PAI-1 biosynthesis of endothelial cells in vitro may be attributable to the lack of a soluble factor produced by SMC, which controls and suppresses PAI-1 biosynthesis of endothelial cells in vivo.

Localization of a Vitronectin Binding Region of Plasminogen Activator Inhibitor-1

1995 ◽  
Vol 73 (05) ◽  
pp. 829-834 ◽  
Author(s):  
Jaya Padmanabhan ◽  
David C Sane
Keyword(s):  
Binding Site ◽  
Plasminogen Activator ◽  
Inhibitory Activity ◽  
Plasminogen Activator Inhibitor ◽  
Structural Homology ◽  
Binding Region ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

SummaryThe PAI-1 binding site for VN was studied using two independent methods. PAI-1 was cleaved by Staph V8 protease, producing 8 fragments, only 2 of which bound to [125I]-VN. These fragments were predicted to overlap between residues 91-130. Since PAI-2 has structural homology to PAI-1, but does not bind to vitronectin, chimeras of PAI-1 and PAI-2 were constructed. Four chimeras, containing PAI-1 residues 1-70,1-105,1-114, and 1-167 were constructed and expressed in vitro. PAI-1, PAI-2, and all of the chimeras retained inhibitory activity for t-PA, but only the chimera containing PAI-1 residues 1-167 formed a complex with VN. Together, these results predict that the VN binding site of PAI-1 is between residues 115-130.

Abstract T P75: Temporal Profiles of Plasminogen Activator Inhibitor-1 Concentration and Activity Changes in Circulation after Focal Stroke and Tissue Type Plasminogen Activator Administration in Rats

Stroke ◽  
2014 ◽  
Vol 45 (suppl_1) ◽  
Author(s):  
Qi Liu ◽  
Xiang Fan ◽  
Helen Brogren ◽  
Ming-Ming Ning ◽  
Eng H Lo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Temporal Profiles ◽  
Activator Inhibitor ◽  
Pai 1

Aims: Plasminogen activator inhibitor-1 (PAI-1) is the main and potent endogenous tissue-type plasminogen activator (tPA) inhibitor, but an important question on whether PAI-1 in blood stream responds and interferes with the exogenously administered tPA remains unexplored. We for the first time investigated temporal profiles of PAI-1 concentration and activity in circulation after stroke and tPA administration in rats. Methods: Permanent MCAO focal stroke of rats were treated with saline or 10mg/kg tPA at 3 hours after stroke (n=10 per group). Plasma (platelet free) PAI-1 antigen and activity levels were measured by ELISA at before stroke, 3, 4.5 (1.5 hours after saline or tPA treatments) and 24 hours after stroke. Since vascular endothelial cells and platelets are two major cellular sources for PAI-1 in circulation, we measured releases of PAI-1 from cultured endothelial cells and isolated platelets after direct tPA (4 μg/ml) exposures for 60 min in vitro by ELISA (n=4 per group). Results: At 3 hours after stroke, both plasma PAI-1 antigen and activity were significantly increased (3.09±0.67, and 3.42±0.57 fold of before stroke baseline, respectively, all data are expressed as mean±SE). At 4.5 hours after stroke, intravenous tPA administration significantly further elevated PAI-1 antigen levels (5.26±1.24), while as expected that tPA neutralized most elevated PAI-1 activity (0.33±0.05). At 24 hours after stroke, PAI-1 antigen levels returned to the before baseline level, however, there was a significantly higher PAI-1 activity (2.51±0.53) in tPA treated rats. In vitro tPA exposures significantly increased PAI-1 releases into culture medium in cultured endothelial cells (1.65±0.08) and platelets (2.02±0.17). Conclution: Our experimental results suggest that tPA administration may further elevate stroke-increased blood PAI-1 concentration, but also increase PAI-1 activity at late 24 hours after stroke. The increased PAI-1 releases after tPA exposures in vitro suggest tPA may directly stimulate PAI-1 secretions from vascular walls and circulation platelets, which partially contributes to the PAI-1 elevation observed in focal stroke rats. The underlying regulation mechanisms and pathological consequence need further investigation.

Abstract 188: Selective Inhibition of Vascular Smooth Muscle Cell Migration by Targeting Plasminogen Activator Inhibitor-1

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Neha Goyal ◽  
Zhen Weng ◽  
Philip Fish ◽  
Tammy Strawn ◽  
Samantha Myears ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Plasminogen Activator ◽  
Intimal Hyperplasia ◽  
Differential Effect ◽  
Plasminogen Activator Inhibitor ◽  
Dependent Manner ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

Introduction: Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of mammalian plasminogen activators and an important regulator of cell migration. We have shown that tiplaxtinin, a small molecule, specific inhibitor of PAI-1, inhibits intimal hyperplasia in a murine vein graft model. However, little is known about the effects of pharmacological inhibition of PAI-1 on vascular cell migration under physiologically relevant conditions. Methods: We studied the effects of tiplaxtinin on migration of smooth muscle cells (SMCs) and endothelial cells (ECs). Results: Tiplaxtinin significantly inhibited migration of murine SMCs through 3-dimensional (3-D) collagen matrix in a concentration-dependent manner. Tiplaxtinin did not inhibit SMC proliferation, and it did not inhibit migration of PAI-1-deficient SMCs, suggesting that tiplaxtinin’s effect on SMCs was non-toxic and PAI-1-dependent. The anti-migratory effect of tiplaxtinin on SMCs was preserved in collagen 3-D matrix containing vitronectin and other extracellular matrix molecules, further supporting the physiological significance of the effect. In contrast to SMCs, tiplaxtinin did not inhibit migration of human aortic ECs in vitro or murine ECs in vivo, the latter assessed in a murine carotid injury model. To study the basis for the differential effect of tiplaxtinin on SMCs vs. ECs, we compared expression of LDL receptor-related protein 1 (LRP1), a motogenic receptor for PAI-1, between cell types by RT-PCR and found that LRP1 gene expression was significantly lower in ECs than in SMCs. Furthermore, recombinant PAI-1 stimulated the migration of wild-type mouse embryonic fibroblasts (MEFs), but not LRP1-deficient MEFs. Conclusions: Tiplaxtinin, a pharmacological inhibitor of PAI-1, inhibits SMC migration under physiological conditions, while having no inhibitory effect on EC migration. The differential effect of PAI-1 inhibition on SMCs vs. ECs appears to be mediated by LRP1 and may be of clinical significance, as it is advantageous to prevent intimal hyperplasia by inhibiting SMC migration without inhibiting EC migration, which is key to preserving an intact, anti-thrombotic vascular endothelium.

Inactivation of Plasminogen Activator Inhibitor-1 by RNA Aptamer Molecules

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1107-1107
Author(s):  
Yolanda Fortenberry ◽  
Jared Damare
Keyword(s):  
Plasminogen Activator ◽  
Active Site ◽  
Plasminogen Activator Inhibitor ◽  
Rna Aptamers ◽  
Plasminogen Activator Inhibitor 1 ◽  
Rna Molecules ◽  
Type Plasminogen Activator ◽  
Antiproteolytic Activity ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Abstract 1107 Introduction: The serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1), binds and inhibits the following plasminogen activators: tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). This decreases plasmin production and triggers the dissolution of fibrin clots. Elevated levels of PAI-1 have been correlated with an increased risk for cardiovascular disease, as well as obesity and metabolic syndrome. Consequently, pharmacologically suppressing PAI-1 might prevent, or successfully treat vascular disease. Several PAI-1 small molecule inhibitors have recently been studied (PAI-039 is the best characterized). Since PAI-1 is a multifunctional protein, completely inhibiting PAI-1 may hinder its other functions. Therefore, it is important to independently develop inhibitors to the various regions of PAI-1. This can be accomplished by using small RNA molecules (aptamers) that bind with high affinity and specificity to individual protein domains. We recently published a paper showing how PAI-1 specific RNA aptamers bind to the heparin/vitronectin binding site of PAI-1 (Blake et al., 2009). We demonstrated that PAI-1 specific aptamers prevent cancer cells from detaching from vitronectin (in the presence of PAI-1), resulting in increased cell adhesion. These aptamers had no effect on PAI-1's other functions, particularly its antiproteolytic activity. Objective: This study's goal was to develop RNA aptamers to the active site of PAI-1; thereby, preventing the ability of PAI-1 to interact with plasminogen activators (tPA and uPA). Methods: The aptamers were generated by the systematic evolution of ligands by exponential enrichment (SELEX). Adopting the SELEX in vitro selection technique ensures the creation of nuclease-resistant RNA molecules that will bind to target proteins. We used in vitroassays to determine the effect of the aptamers on the interaction of PAI-1 with both tPA and uPA. Results: We isolated a family of aptamers that bind to wild-type PAI-1 with affinities in the nanomolar range. From this family, two aptamer clones (10–2 and 10–4) exhibited reduced binding to elastase cleaved PAI-1 and the PAI-1/tPA complex. This suggests that they bind to, or in the vicinity of, the active site. Using a chromogenic assay, we showed that the aptamer clone 10–4, and (to a lesser extent) the aptamer clone 10–2, inhibited PAI-1's antiproteolytic activity against tPA, further suggesting that these clones bind to PAI-1 within its active site region. Interestingly, neither clone was able to prevent PAI-1 from inhibiting uPA activity. Both aptamer clones disrupted PAI-1's ability to form a stable covalent complex with tPA. Increasing aptamer concentrations positively correlated with an increase in cleaved PAI-1, suggesting that these aptamer clones convert PAI-1 from an inhibitor to a substrate. Furthermore, we showed that both aptamer clones are able to inhibit PAI-1's activity in the presence of vitronectin. Conclusions: We have shown that we are able to inhibit one of PAI-1's functions without hindering its other functions. To our knowledge, this is the first report of an RNA molecule that is able to inhibit the antiproteolytic activity of PAI-1. We have generated two specific RNA aptamer molecules that hinder the ability of PAI-1 to interact with tPA, which has the potential to be used as an antithrombotic agent. Disclosures: No relevant conflicts of interest to declare.

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