PAI-1 Inhibitor Research Articles

A Plasminogen Activator Inhibitor-1 Inhibitor Reduces Airway Remodeling in a Murine Model of Chronic Asthma

We previously reported that plasminogen activator inhibitor (PAI)-1 deficiency prevents collagen deposition in the airways of ovalbumin (OVA)-challenged mice. In this study, we explored the therapeutic utility of blocking PAI-1 in preventing airway remodeling, using a specific PAI-1 inhibitor, tiplaxtinin. C57BL/6J mice were immunized with intraperitoneal injections of OVA on Days 0, 3, and 6. Starting on Day 11, mice were challenged with phosphate-buffered saline or OVA by nebulization three times per week for 4 weeks. Tiplaxtinin was mixed with chow and administered orally from 1 day before the phosphate-buffered saline or OVA challenge. Lung tissues were harvested after challenge and characterized histologically for infiltrating inflammatory cells, mucus-secreting goblet cells, and collagen deposition. Airway hyperresponsiveness was measured using whole-body plethysmography. Tiplaxtinin treatment significantly decreased levels of PAI-1 activity in bronchoalveolar lavage fluids, which indicates successful blockage of PAI-1 activity in the airways. The number of infiltrated inflammatory cells was reduced by tiplaxtinin treatment in the lungs of the OVA-challenged mice. Furthermore, oral administration of tiplaxtinin significantly attenuated the degree of goblet cell hyperplasia and collagen deposition in the airways of the OVA-challenged mice, and methacholine-induced airway hyperresponsiveness was effectively reduced by tiplaxtinin in these animals. This study supports our previous findings that PAI-1 promotes airway remodeling in a murine model of chronic asthma, and suggests that PAI-1 may be a novel target of treatment of airway remodeling in asthma.

Abstract 2579: tPA Contributes To Aggravation of Endothelin and Thromboxane Induced Cerebrovasoconstriction After Hypoxia/Ischemia Through Upregulation of ERK MAPK

Introduction: The sole FDA approved treatment for acute stroke is tissue type plasminogen activator (tPA). However, endogenous tPA is upregulated and potentiates impairment of pial artery dilation in response to hypotension after hypoxia/ischemia (H/I) in pigs. Mitogen activated protein kinase (MAPK), a family of at least 3 kinases, ERK, p38 and JNK, is also upregulated after H/I, with ERK contributing to vasodilator impairment. Impairment of stimulus induced vasodilation may result from tonic withdrawal and/or impairment of a vasodilator influence or upregulation of a vasoconstrictor. This study examined the effect of H/I on the vascular response to two important spasmogens released during CNS ischemic disorders, endothelin-1 (ET-1) and thromboxane, and the influence of tPA and ERK MAPK in such responses. Methods: Cerebral ischemia (20 min) was induced via global increase in intracranial pressure via saline infusion into a hollow bolt placed in the cranium (dura intact) while hypoxia (10 min, pO2 35 mm Hg) was produced by decreasing the inspired O2 via inhalation of N2. Vascular responses to topical ET-1 (10-10, 10-8 M) and U 46619 (1, 10 ng/ml) were obtained at 1h post insult. Phosphorylated and total ERK MAPK were measured by ELISA in the CSF of piglets equipped with a closed cranial window. Data (n=5) were analyzed by ANOVA, with significance at p less than 0.05. Results: H/I aggravated pial artery vasconstriction induced by ET-1 and the thromboxane mimic U 46619, which was blocked by EEIIMD, an inhibitor of PAI-1’s vascular activity and signaling of tPA, but not its fibrinolytic action, but unchanged by its inactive analogue EEIIMR. CSF ERK MAPK was increased by H/I and potentiated by tPA. The ERK MAPK antagonist U 0126 blocked H/I induced aggravation of ET-1 and U 46619 vasoconstriction. Discussion: These data indicate that H/I aggravates ET-1 and thromboxane mediated cerebral vasoconstriction through upregulation of tPA and ERK MAPK. These data suggest that thrombolytic therapy for treatment of CNS ischemic disorders can dysregulate cerebrohemodynamics by augmenting vasoconstriction induced by spasmogens co-released during CNS pathology.

A67 Deficiency in Plasminogen-Activator-Inhibitor-1 (PAI-1) decreases Brain Damage after Transient Focal Cerebral Ischemia in MCAO Model in Mice

Background: Focal cerebral ischemia is characterized by prompt cessation of perfusion by vascular occlusion and delayed microclot formation as a result of hypoxic induction of procoagulatory pathways. Therefore, research efforts focus primarily on lysis of clots and rescuing the ischemic penumbra. Recent data indicate that fibrinolytic pathways are suppressed following cerebral ischemia. Plasminogen activator inhibitors (PAI) are the primary regulators of the fibrinolytic system and modulate clot stability by inhibiting urokinase and tissue type plasminogen activators (uPA / tPA). uPA and tPA cleave plasminogen to plasmin which finally degrades fibrin of formed clots. Suppression of endogenous lysis of micro embolism or spontaneous clot formation might contribute to brain damage following cerebral ischemia. This study therefore investigated the role of PAI in an experimental stroke model by use of PAI-1 and PAI-2 deficient mice. Methods: Brain ischemia was induced for 60 min by temporary filament occlusion of the middle cerebral artery (tMCAO). PAI-1-/-, PAI-2-/-, and wild-type (WT; PAI-1+/+; PAI-2+/+) mice were randomized to tMCAO (n = 10 each group) or sham surgery (n = 4 each group). Brain lesion was quantified 24 h after reperfusion in 2,3,5-triphenyltetrazolium chloride (TTC)-stained sections. Results: In PAI-1-/- mice lesion volume was significantly lower, while infarct size of PAI-2-/- mice was not different compared to WT animals (PAI-1-/-: *34 ± 10 %; PAI-2-/-: 48 ± 13 %; WT: 51 ± 11 %; *P < 0.05 vs. WT). Conclusion: Although both PAI-1 and PAI-2 are up-regulated following stroke, only PAI-1 deficiency demonstrated protection. The data therefore indicate a PAI-1 dependent impairment of the thrombolytic tPA / uPA function following tMCAO. The present study suggests that PAI-1 inhibition might be a promising therapeutic strategy to limit secondary brain damage after ischemic stroke.

Low Molecular Weight Antagonists of Plasminogen Activator Inhibitor-1: Therapeutic Potential in Cardiovascular Disease

Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) is the major physiologic regulator of the plasmin-based pericellular proteolytic cascade, a modulator of vascular smooth muscle cell (VSMC) migration and a causative factor in cardiovascular disease and restenosis, particularly in the context of increased vessel transforming growth factor- β1 (TGF-β1) levels. PAI-1 limits conversion of plasminogen to plasmin (and, thereby, fibrin degradation) by inhibiting its protease targets urokinase and tissue-type plasminogen activators (uPA, tPA). PAI-1 also has signaling functions and binds to the low density lipoprotein receptor-related protein 1 (LRP1) to regulate LRP1-dependent cell motility that, in turn, contributes to neointima formation. PAI-1/uPA/uPA receptor/LRPI/integrin complexes are endocytosed with subsequent uPAR/LRP1/integrin redistribution to the leading edge, initiating an "adhesion-detachment-readhesion" cycle to promote cell migration. PAI-1 also interacts with LRP1 in a uPA/uPAR-independent manner triggering Jak/Stat1 pathway activation to stimulate cell motility. PAI-1 itself is a substrate for extracellular proteases and exists in a "cleaved" form which, while unable to interact with uPA and tPA, retains LRP1-binding and migratory activity. These findings suggest that there are multiple mechanisms through which inhibition of PAI-1 may promote cardiovascular health. Several studies have focused on the design, synthesis and preclinical assessment of PAI-1 antagonists including monoclonal antibodies, peptides and low molecular weight (LMW) antagonists. This review discusses the translational impact of LMW PAI-1 antagonists on cardiovascular disease addressing PAI-1-initiated signaling, PAI-1 structure, the design and characteristics of PAI-1-targeting drugs, results of in vitro and in vivo studies, and their clinical implications.

Glibenclamide Induces Collagen IV Catabolism in High Glucose-Stimulated Mesangial Cells

We have shown the full prevention of mesangial expansion in insulin-deficient diabetic rats by treatment with clinically-relevant dosages of glibenclamide (Glib). Studies in mesangial cells (MCs) also demonstrated reduction in the high glucose (HG)-induced accumulation of collagens, proposing that this was due to increased catabolism. In the present study, we investigated the signaling pathways that may be implicated in Glib action. Rat primary MCs were exposed to HG for 8 weeks with or without Glib in therapeutic (0.01 μM) or supratherapeutic (1.0 μM) concentrations. We found that HG increased collagen IV protein accumulation and PAI-1 mRNA and protein expression, in association with decreased cAMP generating capacity and decreased PKA activity. Low Glib increased collagen IV mRNA but fully prevented collagen IV protein accumulation and PAI-1 overexpression while enhancing cAMP formation and PKA activity. MMP2 mRNA, protein expression and gelatinolytic activity were also enhanced. High Glib was, overall, ineffective. In conclusion, low dosage/concentration Glib prevents HG-induced collagen accumulation in MC by enhancing collagen catabolism in a cAMP-PKA-mediated PAI-1 inhibition.

Application of frontal affinity chromatography combined on-line with mass spectrometry to screening PAI-1 inhibitors from traditional Chinese medicine

Frontal affinity chromatography combined on-line with mass spectrometry (FAC-MS) is an efficient method for screening traditional Chinese medicine (TCM). This paper reports the application of FAC-MS to screening PAI-1 inhibitor from TCM. Novel small molecule inhibitors derived from natural products are particularly desirable as inhibitors of PAI-1. Several promising inhibitors of PAI-1 from TCM were identified by FAC-MS. These inhibitors included chrysophanol, rhein, corilagin, and geraniin, with 50% inhibitory concentrations (IC50) of 1.06, 37.32, 30.38, and 10.48 μM, respectively. The results indicate that FAC-MS is a direct and fast method for screening active compounds from complex molecular systems. The sample in this method is a mixture, so it is suitable for the screening Chinese herb extracts.

Discovery of inhibitors of plasminogen activator inhibitor-1: Structure–activity study of 5-nitro-2-phenoxybenzoic acid derivatives

Two novel series of 5-nitro-2-phenoxybenzoic acid derivatives are designed as potent PAI-1 inhibitors using hybridization and conformational restriction strategy in the tiplaxtinin and piperazine chemo types. The lead compounds 5a, 6c, and 6e exhibited potent PAI-1 inhibitory activity and favorable oral bioavailability in the rodents.

The antifibrinolytic function of factor XIII is exclusively expressed through α2-antiplasmin cross-linking

AbstractFactor XIII (FXIII) generates fibrin-fibrin and fibrin-inhibitor cross-links. Our flow model, which is sensitive to cross-linking, was used to assess the effects of FXIII and the fibrinolytic inhibitor, α2-antiplasmin (α2AP) on fibrinolysis. Plasma model thrombi formed from FXIII or α2AP depleted plasma lysed at strikingly similar rates, 9-fold faster than pooled normal plasma (PNP). In contrast, no change was observed on depletion of PAI-1 or thrombin activatable fibrinolysis inhibitor (TAFI). Inhibition of FXIII did not further enhance lysis of α2AP depleted thrombi. Addition of PNP to FXIII or α2AP depleted plasmas normalized lysis. Lysis rate was strongly inversely correlated with total cross-linked α2AP in plasma thrombi. Reconstitution of FXIII into depleted plasma stabilized plasma thrombi and normalized γ-dimers and α-polymers formation. However, the presence of a neutralizing antibody to α2AP abolished this stabilization. Our data show that the antifibrinolytic function of FXIII is independent of fibrin-fibrin cross-linking and is expressed exclusively through α2AP.

Characterization of a panel of monoclonal antibodies toward mouse PAI-1 that exert a significant profibrinolytic effect in vivo

IntroductionPAI-1 is the main physiological inhibitor of t-PA and u-PA. Elevated PAI-1 levels have been implicated in the pathogenesis of several thrombotic and non-thrombotic diseases. The effect of PAI-1 inhibition can be studied in mouse models, when appropriate immunological tools are available. The majority of the available monoclonal antibodies against PAI-1 have been raised against human PAI-1. Even though some of these antibodies cross-react with non-glycosylated PAI-1 from different species, these antibodies often do not cross-react sufficiently with glycosylated mouse PAI-1. Moreover, the antibodies that cross-react with glycosylated mouse PAI-1 often have decreased inhibitory properties in the presence of vitronectin. Our objective was the generation of a panel of monoclonal antibodies reacting with vitronectin-bound glycosylated mouse PAI-1. ResultsFive monoclonal antibodies revealed binding to glycosylated mouse PAI-1 and exerted a strong (i.e. 58-80% inhibition of PAI-1 activity) inhibitory effect toward mouse PAI-1. Similar inhibitory effects were seen in the presence of a 33-fold molar excess of vitronectin. The PAI-1 inhibitory potential of the antibodies in vivo was demonstrated in a thromboembolism model, in which the evaluated antibodies significantly increased the percentage of mice with normal physical activity in comparison to mice treated with negative control antibody. ConclusionsTo the best of our knowledge this is the first panel of monoclonal antibodies that can inhibit mouse PAI-1 in the presence of vitronectin and that show a profibrinolytic effect in vivo. Therefore these antibodies provide excellent immunological tools to further investigate the role of PAI-1 in mouse models.

Investigation into the mechanism(s) of antithrombotic effects of carbon monoxide releasing molecule-3 (CORM-3)

Carbon monoxide (CO) like nitric oxide (NO) has been recognized as activator of soluble guanylate cyclase (sGC) in many physiological functions. Studies, which demonstrate the mechanisms by which CO inhibits platelet aggregation in in vivo models, are few. Here we investigated the possible involvement of sGC, NO, plasminogen activator inhibitor (PAI-1) and p38 MAP Kinase in antithrombotic effects of CO released by a novel, water-soluble, CO releasing molecule-3 (CORM-3) using rat. The effects of CORM-3 on in vitro and ex vivo platelet aggregation induced by thrombin as well as in in vivo thrombosis models were studied. When added to rat washed platelets in in vitro study, CORM-3 (100 and 200 μM) inhibited thrombin-induced platelet aggregation. Similarly, antiplatelet effect was also observed when 3 mg/kg i.v. infusion of CORM-3 administered for 10 minutes in ex vivo study using rat. Interestingly, in presence of inhibitor of sGC (ODQ, 10 mg/kg, i.p.) and inhibitor of nitric oxide synthase (L-NAME, 30 mg/kg, i.p.), inhibition of thrombin-induced aggregation by CORM-3 was significantly blocked. Notably, in presence of inhibitor of K ATP channel (glibenclamide, 10 mg/kg, i.p.) and p38 MAP Kinase (SCIO-469, 1 mg/kg, i.p.), inhibition of aggregation by CORM-3 was not blocked. In in vivo studies using animal models of thrombosis, we found that CORM-3-mediated antithrombotic effect was dependent on activation of sGC, NO and suppression of PAI-1 in arterial thrombosis and Arterio-Venous (A-V) shunt models. Therefore, we concluded that antithrombotic activity of CORM-3 may be mediated by activation of sGC, NO and inhibition of PAI-1.

Diverse inhibition of plasminogen activator inhibitor type 1 by theaflavins of black tea

Fruits, vegetables, spices and a variety of teas are suggested for the prevention of many diseases. They encompass active, non-nutritional ingredients called nutraceuticals which are defined as food products that provide health benefits. Many nutraceuticals have been tested to identify inhibitors of plasminogen activator inhibitor (PAI-1). PAI-1 is the major and fast acting physiological inhibitor of fibrinolysis. However, preclinical studies of PAI-1 inhibitors have revealed an additional role of PAI-1 in the pathogenesis of vascular remodeling, renal injury, diabetes, obesity, Alzheimer's disease and cancer. Thus PAI-1 is a potential therapeutic target in some of these diseases. Our previous study revealed that a black tea extract (containing mostly theaflavins) inhibits PAI-1. In this study we report results for four pure (>98%) theaflavins. Inactivation of PAI-1 was tested by clot formation and by its lysis using thromboelastometry and measurements of human plasma turbidity. Among four tested theaflavins, theaflavin-3'-gallate was the most potent in PAI-1 inhibition trailed by theaflavin-3,3'-digallate, while the other two i.e., theaflavin and theaflavin-3-gallate did not show inhibitory activity.

A Specific PAI-1 Inhibitor, PAI-039, Reduced Airway Inflammation And Collagen Deposition In An Ova-challenged Murine Model Of Chronic Asthma

A Specific PAI-1 Inhibitor, PAI-039, Reduced Airway Inflammation And Collagen Deposition In An Ova-challenged Murine Model Of Chronic Asthma

The Effects of Pharmacologic Plasminogen Activator Inhibitor-1 Inhibition in Acute and Chronic Rejection in Murine Cardiac Allografts

Acute rejection and graft arterial disease (GAD) in cardiac transplantation limit the long-term survival of recipients; these processes are enhanced by inflammation and thrombus formation. Plasminogen activator inhibitor (PAI)-1 is critical in the inflammation and thrombus formation. However, little is known about the effect of PAI-1 in heart transplantation. Thus, the objective was to clarify the role of PAI-1 in the progression of cardiac rejection. Murine hearts were heterotopically transplanted using major mismatch combinations for evaluation of acute rejection and class II mismatch combinations for the GAD. We administered the specific PAI-1 inhibitor (IMD-1622) into murine recipients after cardiac allografts. Nontreated allografts of the major mismatch group were acutely rejected, whereas the PAI-1 inhibitor prolonged their survival. Although severe cell infiltration and intimal thickening with enhancement of inflammatory factors were observed in untreated allografts of class II mismatch group on day 60, the PAI-1 inhibitor attenuated these changes. The PAI-1 inhibitor is potent for the suppression of both acute rejection and GAD.

Inhibition of Plasminogen Activator Inhibitor (PAI)-1 Corrects Diabetic CD34+ Dysfunction.

AbstractAbstract 1601 Objective:The dysfunction of human diabetic CD34+ endothelial progenitor cells limits their utility in autologous cell therapy for vascular complications. Previously, we showed that transient inhibition of transforming growth factor-beta 1 (TGF-β1) enhances vascular reparative function of human CD34+ cells isolated from diabetics (Bhatwadekar et al, 2010). Expression of PAI-1, the major gene product of TGF-β1 activation, is increased by high glucose and insulin exposure in endothelial cells and PAI-1 has been shown to be increased in the serum of diabetics. We asked whether the beneficial effects of TGF-β1 blockade on CD34+ cells function were mediated by inhibition of PAI-1 and whether blocking of PAI-1 could correct diabetes associated dysfunction of these cells. Research Design and Methods:Plasma determinations of PAI-1 and TGF-β1 (both measured by ELISA) were compared in type 2 (n=17) and type 1 (n=7) diabetic patients. CD34+ cells from these individuals were isolated and analyzed for cell survival (in the presence and absence of growth factors), cell proliferation, cell cycle analysis and migration. The effect of TGF-β1 phosphorodiamidate morpholino oligomers (PMO) treatment on PAI-1 level was determined in CD34+ cells. In CD34+ cells, PAI-1 was blocked using either lentivirus expressing PAI-1 shRNA or PAI-1 siRNA. In vivo homing ability of PAI-1 inhibited CD34+ cells was assessed using an ocular model of ischemia/reperfusion (I/R) Injury. Results:Plasma PAI-1 level was increased in type 2 diabetic patients compared to type 1 (p<0.05) and directly correlated with TGF-β1 plasma levels (r= 0.44). TGF-β1 PMO treatment resulted in a reduction of PAI-1 mRNA expression (p=0.0018 in diabetic, p=0.05 in non-diabetic). PAI-1 blockade promoted EPC proliferation in vitro and bypassed the inhibitory effect of TGF-β1 on cell survival (p<0.001) even in the absence of growth factors. PAI-1 blockade enhanced the migration of these cells in response to SDF-1α in (p<0.01) compared to cells treated with scrambled siRNA and improved the in vivo re-endothelialization by CD34+ cells in the I/R model. Conclusions:Our results suggest that the cytostatic activity of TGF-β1 in CD34+ cells is mediated largely through PAI-1. Blocking PAI-1 corrects multiple defects in CD34+ cells from type 2 diabetic patients. This approach may offer a promising therapeutic strategy for restoring vascular reparative function in diabetic cells and facilitate their use in autologous cell therapy. Disclosures:No relevant conflicts of interest to declare.

Urokinase and Tissue Plasminogen Activators and Their PAI-1 Inhibitor in Tumors of Patients with Oral Mucosal Cancer: Relationship with the Main Clinical Morphological Factors

Enhanced activation of plasminogen by the urokinase pathway (uPA elevation) in patients with cancer of the oral mucosa paralleled by an increase of PAI-1 level in the tumor compared to the adjacent mucosa was shown by enzyme immunoassay. No statistically significant associations of the level of the studied proteins in the tumor with such prognostic factors as location, growth form, histological structure, differentiation degree, size, and dissemination of the primary tumor, involvement of the regional lymph nodes, and stage of the disease were detected.

Ovarian cancer G protein-coupled receptor 1-dependent and -independent vascular actions to acidic pH in human aortic smooth muscle cells

Atherosclerosis is a chronic inflammation disease characterized by acidic micromilieu and the accumulation of numerous bioactive lipid mediators, such as lysophosphatidic acid (LPA) and prostaglandins, in the atherosclerotic lesion. Chronic acidification induced various effects on vascular smooth muscle cells, but the molecular mechanisms underlying these effects remain unknown. In this study, we examine the role of proton-sensing ovarian cancer G protein-coupled receptor 1 (OGR1) in extracellular acidification-induced regulation of cyclooxygenase (COX)-2 induction, PGI(2) production, MAPK phosphatase (MKP)-1 expression, and plasminogen activator inhibitor (PAI)-1 expression and proliferation in human aortic smooth muscle cells (AoSMCs). Experiments with knockdown with small interfering RNA specific to OGR1 and specific inhibitors for G proteins showed that acidification-induced COX-2 expression, PGI(2) production, and MKP-1 expression, but not PAI-1 expression and inhibition of proliferation, were dependent on OGR1 and mainly mediated by G(q/11) protein. LPA remarkably enhanced, through the LPA(1) receptor/G(i) protein, the OGR1-mediated vascular actions to acidic pH. In conclusion, acidic pH-induced vascular actions of AoSMCs can be dissected to OGR1-dependent and -independent pathways: COX-2 expression, PGI(2) production, and MKP-1 expression are mediated by OGR1, but PAI-1 expression and inhibition of proliferation are not. LPA, which is usually thought to be a proatherogenic lipid mediator, may exert antiatherogenic actions under acidic micromilieu through cross-talk between LPA(1)/G(i) protein and OGR1/G(q/11) protein.

Theaflavin digallate inactivates plasminogen activator inhibitor: Could tea help in Alzheimer's disease and obesity?

Proteolysis in general and particularly the serine proteases are causally involved in many physiological processes and different diseases. Recently it was reported that plasminogen activator inhibitor type one (PAI-1) inactivation can alleviate the symptoms of Alzheimer's disease and reduce the body weight of obese individuals. In our broad search for natural compounds and their derivatives that can inhibit PAI-1, we include the polyphenols of teas since teas (green and black) or their components have been reported to alleviate the symptoms of both obesity and Alzheimer's disease. Inactivation of PAI-1 was measured in human plasma using thromboelastography. We used known PAI-1 inhibitor PAI039 [{1-benzyl-5-[4-(trifluoromethoxy) phenyl]-1H-indol-3-yl}(oxo)acetic acid] as a positive control and (-)-epigallo-catechin-3-gallate (EGCG), its prodrug octaacetate EGCG (OcAc EGCG) and theaflavin digallate [TH(2)] as potential PAI-1 inhibitors. We found that inactivation of PAI-1 in plasma by EGCG and OcAc EGCG was low or very low. However, TH(2) inactivated PAI-1 in a concentration-dependent manner with an IC50 of 18 microM which is equal to or better than the IC50 reported for known PAI-1 inhibitor PAI039. Clearly TH(2) inhibits PAI-1 and might play a role in slowing down the progression of Alzheimer's disease or obesity by a PAI-1-dependent pathway. While the clinical value of TH(2) has not been proven, long-term prospective studies assessing its efficacy are warranted due to the benign nature of the substance.

Characterization of a Novel Class of Polyphenolic Inhibitors of Plasminogen Activator Inhibitor-1

Plasminogen activator inhibitor type 1, (PAI-1) the primary inhibitor of the tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, has been implicated in a wide range of pathological processes, making it an attractive target for pharmacologic inhibition. Currently available small-molecule inhibitors of PAI-1 bind with relatively low affinity and do not inactivate PAI-1 in the presence of its cofactor, vitronectin. To search for novel PAI-1 inhibitors with improved potencies and new mechanisms of action, we screened a library selected to provide a range of biological activities and structural diversity. Five potential PAI-1 inhibitors were identified, and all were polyphenolic compounds including two related, naturally occurring plant polyphenols that were structurally similar to compounds previously shown to provide cardiovascular benefit in vivo. Unique second generation compounds were synthesized and characterized, and several showed IC(50) values for PAI-1 between 10 and 200 nm. This represents an enhanced potency of 10-1000-fold over previously reported PAI-1 inactivators. Inhibition of PAI-1 by these compounds was reversible, and their primary mechanism of action was to block the initial association of PAI-1 with a protease. Consistent with this mechanism and in contrast to previously described PAI-1 inactivators, these compounds inactivate PAI-1 in the presence of vitronectin. Two of the compounds showed efficacy in ex vivo plasma and one blocked PAI-1 activity in vivo in mice. These data describe a novel family of high affinity PAI-1-inactivating compounds with improved characteristics and in vivo efficacy, and suggest that the known cardiovascular benefits of dietary polyphenols may derive in part from their inactivation of PAI-1.

A Novel Inhibitor of Plasminogen Activator Inhibitor-1 Provides Antithrombotic Benefits Devoid of Bleeding Effect in Nonhuman Primates

Inhibition of plasminogen activator inhibitor (PAI)-1 is useful to treat several disorders including thrombosis. An inhibitor of PAI-1 (TM5275) was newly identified by an extensive study of structure-activity relationship based on a lead compound (TM5007) which was obtained through virtual screening by docking simulations. Its antithrombotic efficacy and adverse effects were tested in vivo in rats and nonhuman primates (cynomolgus monkey). TM5275, administered orally in rats (1 to 10 mg/kg), has an antithrombotic effect equivalent to that of ticlopidine (500 mg/kg) in an arterial venous shunt thrombosis model and to that of clopidogrel (3 mg/kg) in a ferric chloride-treated carotid artery thrombosis model. TM5275 does not modify activated partial thromboplastin time and prothrombin time or platelet activity and does not prolong bleeding time. Combined with tissue plasminogen activator, TM5275 improves the latter's therapeutic efficacy and reduces its adverse effect. Administered to a monkey model of photochemical induced arterial thrombosis, TM5275 (10 mg/kg) has the same antithrombotic effect as clopidogrel (10 mg/kg), without enhanced bleeding. This study documents the antithrombotic benefits of a novel, more powerful, PAI-1 inhibitor in rats and, for the first time, in nonhuman primates. These effects are obtained without adverse effect on bleeding time.

Modulation of Inflammatory Markers by miR-146a during Replicative Senescence in Trabecular Meshwork Cells

To investigate the alterations in microRNA (miRNA) expression during replicative senescence (RS) in human trabecular meshwork (HTM) cells. Two HTM cell lines were serially passaged until they reached RS. Changes in expression of 30 miRNAs were assessed by real-time quantitative (q)-PCR. The effects of miR-146a on gene expression were analyzed with gene arrays and the results confirmed by real-time q-PCR. Protein levels of IRAK1 and PAI-1 were analyzed by Western blot and those of IL6 and IL8 by ELISA. Senescence-associated markers were monitored by flow cytometry and cell proliferation by BrdU incorporation. RS of HTM cells was associated with significant changes in expression of 18 miRNAs, including the upregulation of miR-146a. miR-146a downregulated multiple genes associated with inflammation, including IRAK1, IL6, IL8, and PAI-1, inhibited senescence-associated beta-galactosidase (SA-beta-gal) activity and production of intracellular reactive species (iROS), and increased cell proliferation. Overexpression of either IRAK1 or PAI-1 inhibited the effects of miR-146a on cell proliferation and iROS production in senescent cells. RS in HTM cells was associated with changes in miRNA expression that could influence the senescent phenotype. Upregulation of the anti-inflammatory miR-146a may serve to restrain excessive production of inflammatory mediators in senescent cells and limit their deleterious effects on the surrounding tissue. Among the different proteins repressed by miR-146a, the inhibition of PAI-1 may act to minimize the effects of senescence on the generation of iROS and growth arrest and prevent alterations of the extracellular proteolytic activity of the TM.