Effect of six-month use of oral contraceptive pills on plasminogen activator inhibitor-1 & factor VIII among women with polycystic ovary syndrome: An observational pilot study.

Background & objectives: Polycystic ovary syndrome (PCOS) is an endocrinopathy warranting lifelong individualized management by lifestyle and pharmacological agents mainly oral contraceptive pills (OCPs). This study was aimed to report the impact of six-month OCP use on plasminogen activator inhibitor-1 (PAI-1) and factor VIII (FVIII) in women with PCOS. Methods: PCOS women diagnosed on the basis of Rotterdam 2003 criteria, either treated with OCPs (ethinyl estradiol-0.03 mg, levonorgestrel-0.15 mg) for a period of six months (n=40) or drug-naïve (n=42), were enrolled in this study. Blood was drawn to estimate glucose, insulin levels and lipid profile. Chemiluminescence immunoassays were used to measure hormones (LH, FSH, PRL, T4). Plasma levels of PAI-I and FVIII were measured by commercially available kits. Results: Menstrual regularity, Ferriman-Gallwey score and serum total testosterone significantly improved in the OCP group compared to drug-naïve group (P<0.01). No significant difference was observed in PAI-1 levels of the two groups; however, significant decrease in FVIII levels was observed in OCP group as compared to drug-naïve group. PAI-1 levels of OCP group correlated positively with blood glucose two hours, triglycerides and insulin two hours, while FVIII levels of OCP group correlated negatively with fasting insulin and homoeostatic model assessment-insulin resistance. Interpretation & conclusions: OCPs use has differential effect on pro-coagulant markers among women with PCOS. Well-designed, long-term, prospective, large-scale studies are prerequisite to elucidate the efficacy and safety of OCP in the treatment of PCOS.

Tissue plasminogen activator involvement in experimental autoimmune myasthenia gravis: aggravation and therapeutic potential.

Tissue plasminogen activator (tPA), a component of the PA/plasmin system, is elevated in inflammatory areas and plays a role in inflammatory neurological disorders. In the present study we explored the involvement of tPA and the potential immunomodulatory activity of tPA in experimental autoimmune myasthenia gravis (EAMG). Mice deficient in tPA (tPA(-/-)) present with a markedly more severe disease than wild type EAMG mice. In an attempt to treat EAMG with an 18aa peptide derived from the PA system inhibitor (PAI-1), designed to tether out the endogenous inhibitor, a significant suppression of disease severity was demonstrated. The more severe disease in tPA(-/-) mice was accompanied by a higher level of anti-AChR antibodies and increased expression of B-cell markers. In view of the essential role of B-cell activating factor (BAFF) in B-cell maturation, the expression of BAFF family components was tested. An increase in BAFF and BAFF receptor was observed in EAMG tPA(-/-) mice, whereas BCMA expression was reduced, consistent with the increased level of pathogenic antibodies and the more severe disease. Given the importance of T regulatory cells (Tregs) in EAMG, they were evaluated and their number was reduced in tPA(-/-) mice, in which EAMG was aggravated, whereas following PAI-1dp treatment, Tregs were replenished and the disease was ameliorated. The results show the involvement of tPA in EAMG, implying a protective role for tPA in EAMG pathogenesis. The amelioration of EAMG by PAI-1dp treatment suggests that the PA system may be considered a potential site for therapeutic intervention in neuroimmune diseases.

THR-18, a 18-mer peptide derived from PAI-1, is neuroprotective and improves thrombolysis by tPA in rat stroke models.

OBJECTIVE: The thrombolytic treatment of stroke is limited by a narrow therapeutic time window and is associated with significant adverse side effects. To improve this situation, the modulation of tissue-type plasminogen activator (tPA) activity by a synthetic plasminogen activator inhibitor-1-derived 18-mer peptide (THR-18) was examined in two models of stroke in rats. METHODS: In the first model (thromboembolic), stroke was induced by intra-carotid injection of micro-clots to rats, and tPA (6 mg/kg) was intravenously infused for 30 minutes with or without THR-18 (1 mg/kg) at 4 hours post-induction. In the second model [transient middle cerebral artery occlusion (tMCAO)], stroke was induced for 2 hours by a transient mechanical occlusion. tPA and/or THR-18 (0.02, 0.1, and 1 mg/kg) were intravenously infused for 60 minutes at the time of reperfusion. RESULTS: In the thromboembolic model, cerebral blood flow, measured before and up to 5.5 hours post-induction, revealed that tPA administration caused reperfusion of flow at 30 minutes post-infusion. Later on, an additional increase in reperfusion was seen in the tPA+THR-18 group, and not with tPA alone. In both models, the frequency of intracranial hemorrhage in the tPA-treated group was found to be significantly higher than the control, and this tPA effect was attenuated by THR-18. In the thromboembolic study, infarct size and brain edema were similar in the control and tPA-treated rats. However, the combination of tPA and THR-18 caused a statistically significant reduction in both parameters (infarct size 17.8 versus 25.0%, brain edema 5 versus 8%, tPA+THR-18 versus control, respectively). In the tMCAO mechanical model, infarct size and brain edema were both increased by tPA treatment as compared to the control group, and this increase was markedly diminished by THR-18 co-administration. Neurobehavioral assessment of the tMCAO animals performed at 72 hours post-stroke induction revealed significant improvements (P<0.05-0.01) in neuroscores in all groups of animals treated with peptide-tPA, as compared to the tPA monotherapy group. A significant (P<0.05) improvement in the neurological outcome was also seen in the THR-18 monoterapy group, as compared to the control animals, thus demonstrating a clear neuroprotective effect by the peptide on its own. DISCUSSION: The results support the use of THR-18 together with tPA in the thrombolytic therapy of stroke, in order to achieve better patency, less tPA-induced damage, and possibly a widening of tPA therapeutic time window.

Intravesical administration of plasminogen activator inhibitor type-1 inhibits in vivo bladder tumor invasion and progression.

PURPOSE: The potent effects of PAI-1 on tumorigenesis and angiogenesis in various experimental models are complex, complicated and at times contradictory. We determined the therapeutic potential of PAI-1 for inhibiting bladder tumor invasion under conditions that closely mimic the clinical setting. MATERIALS AND METHODS: An orthotopic rat bladder tumor model was established by implanting AY-27 rat transitional carcinoma cells into the bladder lumen of syngeneic Fischer F344 rats. Three weeks after implantation 1 microM PAI-1 was administrated directly into the bladder lumen twice weekly for 2 weeks. Two days after the final treatment tumor size, total bladder weight, tumor stage and angiogenesis were assessed. To assess the uPA axis the levels of active and total uPA, and active and total PAI-1 in tumor extracts were determined 0, 2, 24 and 48 hours after intravesical PAI-1 administration. RESULTS: Intravesical PAI-1 bound and inactivated its molecular target, tumor uPA. There was significant inhibition of bladder tumor progression, as manifested by 53%, 37% and 57% reductions in tumor size, total bladder weight and angiogenesis, respectively. Only 22% of PAI-1 treated tumors invaded muscle vs 79% in controls. No PAI-1 toxicity was detected. CONCLUSIONS: To our knowledge this study is the first to demonstrate that intravesical treatment with PAI-1 significantly inhibits tumor progression in an in vivo model of bladder cancer. Further clinical development is warranted for using PAI-1 directly or in combination with current standards, such as bacillus Calmette-Guerin or interferon.

Systemic inhibition of the angiotensin-converting enzyme limits lipopolysaccharide-induced lung neutrophil recruitment through both bradykinin and angiotensin II-regulated pathways.

Recruitment of neutrophils to the lung is a sentinel event in acute lung inflammation. Identifying mechanisms that regulate neutrophil recruitment to the lung may result in strategies to limit lung damage and improve clinical outcomes. Recently, the renin angiotensin system (RAS) has been shown to regulate neutrophil influx in acute inflammatory models of cardiac, neurologic, and gastrointestinal disease. As a role for the RAS in LPS-induced acute lung inflammation has not been described, we undertook this study to examine the possibility that the RAS regulates neutrophil recruitment to the lung after LPS exposure. Pretreatment of mice with the angiotensin-converting enzyme (ACE) inhibitor enalapril, but not the anti-hypertensive hydralazine, decreased pulmonary neutrophil recruitment after exposure to LPS. We hypothesize that inhibition of LPS-induced neutrophil accumulation to the lung with enalapril occurred through both an increase in bradykinin, and a decrease in angiotensin II (ATII), mediated signaling. Bradykinin receptor blockade reversed the inhibitory effect of enalapril on neutrophil recruitment. Similarly, pretreatment with bradykinin receptor agonists inhibited IL-8-induced neutrophil chemotaxis and LPS-induced neutrophil recruitment to the lung. Inhibition of ATII-mediated signaling, with the ATII receptor 1a inhibitor losartan, decreased LPS-induced pulmonary neutrophil recruitment, and this was suggested to occur through decreased PAI-1 levels. LPS-induced PAI-1 levels were diminished in animals pretreated with losartan and in those deficient for the ATII receptor 1a. Taken together, these results suggest that ACE regulates LPS-induced pulmonary neutrophil recruitment via modulation of both bradykinin- and ATII-mediated pathways, each regulating neutrophil recruitment by separate, but distinct, mechanisms.

Dose-dependent modulation of choroidal neovascularization by plasminogen activator inhibitor type I: implications for clinical trials.

PURPOSE: To explain the conflicting reports about the influence of plasminogen activator inhibitor type (PAI-1) on pathologic angiogenesis, such as occurs during the exudative form of age-related macular degeneration. METHODS: The expression of PAI-1 mRNA was analyzed in human and murine choroidal neovascularization (CNV) by RT-PCR. The influences of increasing doses of recombinant PAI-1 were evaluated by daily intraperitoneal injections in PAI-1(-/-) and wild-type animals with a model of laser-induced CNV. The double mechanism of action of PAI-1 (proteolytic activity inhibition versus vitronectin binding) was explored by immunohistochemical localization of fibrinogen/fibrin and by injection of recombinant PAI-1 protein defective for vitronectin binding or with adenoviral vectors bearing a mutated binding-deficient PAI-1 gene. RESULTS: PAI-1 expression was present in human CNV and strongly induced in the course of experimental subretinal neovascularization. Daily injections of recombinant PAI-1 proteins in control and PAI-1(-/-) animals demonstrated that PAI-1 could exhibit both pro- and antiangiogenic effects, dependent on the dose. PAI-1 mutants defective for vitronectin binding were used to show that PAI-1 promotes choroidal pathologic angiogenesis merely through its antiproteolytic activity. CONCLUSIONS: These observations may help to reconcile reports with opposite results regarding the effects of PAI-1 on angiogenesis and certainly warn against uncontrolled use of PAI-1-modulating drugs in clinical trials.

Plasminogen activator inhibitor type 1 increases neointima formation in balloon-injured rat carotid arteries.

BACKGROUND: Elevated plasma levels of plasminogen activator inhibitor type 1 (PAI-1) are associated with myocardial infarction, atherosclerosis, and restenosis. PAI-1 is increased in atherosclerotic arteries and failed vein grafts. No experimental data, however, support a causal relationship between elevated PAI-1 expression and vascular lesions. Paradoxically, data generated in PAI-1 knockout mice suggest that PAI-1 might decrease lesion formation after arterial injury and that PAI-1 gene transfer might prevent restenosis. METHODS AND RESULTS: Using the rat carotid balloon injury model and a PAI-1-expressing adenoviral vector, we tested whether elevated arterial PAI-1 expression would alter neointima formation. Compared with control-transduced arteries, neointima formation in PAI-1-transduced arteries was initially retarded. By 14 days, however, the intimas of PAI-1-transduced arteries were significantly larger than intimas of control-transduced arteries (1.6+/-0.1x10(5) versus 1.2+/-0.1x10(5) micrometer(2), n=18 to 19, P<0.03). PAI-1 expression in individual arteries correlated with increased cell proliferation at 4 and 8 days after injury (R=0.6, P<0.02 and P<0.006). PAI-1 expression also correlated with fibrin(ogen) accumulation (R=0.77, P<0.001), and fibrin(ogen) accumulation correlated strongly with proliferation (R=0.86, P<0.00001). CONCLUSIONS: Increased expression of PAI-1 in the artery wall promotes neointima growth after balloon injury. Therefore, despite encouraging data generated in other animal models, PAI-1 is not a promising agent for gene therapy to prevent restenosis. Moreover, our data associate elevated PAI-1 expression with fibrin(ogen) accumulation and increased cell proliferation. These data suggest a mechanism to explain the association between elevated PAI-1 expression and the progression of arterial disease.

Reduction of experimental adhesion formation by inhibition of plasminogen activator inhibitor type 1.

BACKGROUND: Adhesion formation is a common cause of complications following surgery. A reduction in peritoneal fibrinolytic capacity during operation is a key mechanism in the early formation of adhesions. An increase in the main inhibitor of fibrinolysis, plasminogen activator inhibitor type 1 (PAI-1), is a major factor in the loss of fibrinolytic activity. The aim of this study was to investigate if inhibition of PAI-1 could reduce the formation of adhesions after surgery. METHODS: Mice (n = 53) were subjected to a standard surgical procedure in order to induce adhesion formation to the abdominal side wall. At the conclusion of the operation, fragments for antigen binding of polyclonal rabbit antibody against PAI-1 (PRAP-1) were injected intraperitoneally, at two different concentrations. Control animals received an equal volume of the vehicle (saline). One week after operation adhesion formation was quantified. RESULTS: Both doses of PRAP-1 significantly reduced adhesion formation compared with the saline control (P = 0.003 and P = 0.002). There were no signs of bleeding in the postoperative period or at reoperation. CONCLUSION: The present observations lend further support to the hypothesis of a pivotal role of fibrinolysis in the early formation of adhesions, and open up new possibilities for adhesion reduction by inhibiting PAI-1.

Inhibition of metastasis of intraocular melanomas by adenovirus-mediated gene transfer of plasminogen activator inhibitor type 1 (PAI-1) in an athymic mouse model.

Uveal melanoma is the most common intraocular malignancy in adults and results in the death of 50% of the patients. Plasminogen activators (PA) are believed to facilitate tumor metastasis by promoting invasion of tissue barriers. The present study explored the possibility of preventing the metastasis of intraocular melanomas by disrupting plasminogen activator function through gene transfer. A replication-deficient adenovirus vector was used for the in vivo transfer of plasminogen activator inhibitor type 1 (PAI-1) cDNA. Intraocular injection of an adenovirus vector (AdCMV-PAI-1) expressing plasminogen activator inhibitor-1 resulted in: (1) the transduction of more than 95% of human and murine uveal melanoma cells in the eyes of nude mice; (2) a 50% reduction in the number of animals developing liver metastases; and (3) a 78% reduction in the metastatic tumor burden in animals that eventually developed metastases. In other experiments intravenous injections of AdCMV-PAI-1 resulted in transduction of normal liver cells and culminated in a sharp reduction in the incidence of metastases and a significant prolongation of host survival. The results support the feasibility of disruption of PA function through gene transfer as a therapeutic strategy for preventing metastases and prolonging host survival.