Plasminogen Activator Inhibitor-1 Deficient Mice Research Articles

Abstract 2023: Plasminogen Activator Inhibitor-1 Deficiency Augments Angiotensin II-induced Cardiac Fibrosis, But Not Aortic Aneurysm Formation In Mice

Background: Plasminogen activator inhibitor-1 (PAI-1) is highly abundant in human thoracic aortic aneurysms (TAA). This high abundance is mimicked in TAAs created by chronic infusion of angiotensin II (AngII) into mice. The purpose of this study was to determine whether deletion of PAI-1 influenced the development of thoracic aortic aneurysms during AngII infusion. Methods and Results: To determine the role of PAI-1 in acute and chronic phases of AngII-induced pathology, whole-body PAI-1 deficient mice (PAI-1 -/-) and wild type littermates (PAI-1 +/+) were infused with AngII (1,000 ng/kg/min) for 7 or 28 days, respectively. Aortic diameters were measured by ultrasonography and in situ measurement. PAI-1 deficiency did not alter maximal luminal or external aortic diameters at 7 or 28 days of AngII. At 7 days of AngII, ventricular hemorrhage was visibly evident in PAI-1 -/- mice. Mid-ventricular heart sections were next examined by hematoxylin and eosin staining, and red blood cell accumulation was found primarily within the epicardium. Masson’s trichrome staining revealed interstitial fibrosis in the epicardium, myocardium, and posterior septum of PAI-1 -/- mice. At 28 days of AngII, PAI-1 -/- mice displayed primarily epicardial fibrosis evident by trichrome staining and gross appearance. The posterior septum presented less fibrosis after 28 days of AngII relative to 7 days of AngII. Conclusions: PAI-1 deficiency did not alter aortic dilatation, indicating that PAI-1 is not a key contributor to AngII-induced TAAs. Paradoxically, PAI-1 deficiency augmented AngII-induced cardiac fibrosis with distinct distributions between acute and chronic phases.

Plasminogen activator inhibitor-1 stimulates macrophage activation through Toll-like Receptor-4

While inflammation is often associated with increased Plasminogen Activator Inhibitor-1 (PAI-1), the functional consequences of PAI-1 in inflammation have yet to be fully determined. The aim of this study was to establish the in vivo relevance of PAI-1 in inflammation. A mouse model of systemic inflammation was employed in wild-type (WT) and PAI-1 deficient (PAI-1−/−) mice. Mice survival, macrophage infiltration into the lungs, and plasma levels of pro-inflammatory cytokines were assessed after lipopolysaccharide (LPS) infusion. In vitro experiments were conducted to examine changes in LPS-induced inflammatory responses after PAI-1 exposure. PAI-1 was shown to regulate inflammation, in vivo, and affect macrophage infiltration into lungs. Further, PAI-1 activated macrophages, and increased pro-inflammatory cytokines at both the mRNA and protein levels in these cells. The effect of PAI-1 on macrophage activation was dose-dependent and LPS-independent. Proteolytic inhibitory activity and Lipoprotein Receptor-related Protein (LRP) and vitronectin (VN) binding functions, were not involved in PAI-1-mediated activation of macrophages. However, the effect of PAI-1 on macrophage activation was partially blocked by a TLR4 neutralizing antibody. Furthermore, PAI-1-induced Tumor Necrosis Factor-alpha (TNF-α) and Macrophage Inflammatory Protein-2 (MIP-2) expression was reduced in TLR4−/− macrophages compared to WT macrophages. These results demonstrate that PAI-1 is involved in the regulation of host inflammatory responses through Toll-like Receptor-4 (TLR4)-mediated macrophage activation.

Abstract 566: Functional Analysis of Inducible Pluripotent Stem Cell-derived Vascular Endothelial Cells From Plasminogen Activator Inhibitor-1 Deficient Patients

Background: Plasminogen Activator inhibitor-1 (PAI-1) is the principle regulator of PA system and its enhanced expression levels are related to many pathological conditions including thrombosis and malignant tumors. We have identified two distinct PAI-1 deficient patients having apparent phenotypes of severe bleeding and impaired wound healing, both of which are not seen in the PAI-1 deficient mice. With employing inducible Pluripotent Stem (iPS) cells established from the patients, we investigated the intrinsic function of PAI-1 in endothelial cells. Methods: iPS cells, generated from PAI-1 deficient patients and control subject, were differentiated to endothelial cells. Endothelial cells were isolated by magnetic sorting with anti-VEGFR-2 antibody, and their functions and the related gene expressions were analyzed. Results: (1) Endothelial cells derived from PAI-1 deficient iPS cells (PAI-1 iPS-ECs) detached more easily from gelatin-coated dishes. (2) PAI-1 iPS-ECs migrated faster in migration assay. (3) Tube formation activities of PAI-1 iPS-ECs, especially vessel branching, were impaired compared with control iPS-ECs. (4) The expression of Dll4 gene, known to be mainly observed at the region of angiogenic-edge, was reduced in PAI-1 iPS-ECs. Conclusion: PAI-1 appeared to play important roles in the function of endothelial cells and to contribute to angiogenesis.

Abstract 890: Effects of Lewis lung carcinoma on trabecular microstructural changes in wild-type and plasminogen activator inhibitor-1 deficient mice fed a high-fat diet

Abstract Bone is a major target organ of metastasis. The present study investigated the effects of Lewis lung carcinoma (LLC) on trabecular microstructural changes, using tomographic analysis, in distal femur and lumbar 4 vertebra from LLC-bearing wild-type and plasminogen activator inhibitor-1 (PAI-1) deficient mice (C57BL/6J background) fed the AIN93G diet or a high-fat diet; a group of AIN93G-fed non-tumor-bearing wild-type mice were used as controls. Compared to AIN93G-fed non-tumor-bearing wild-type mice, LLC significantly reduced bone volume fraction (BV/TV), connective density (Conn.D), trabecular number (Tb.N), trabecular thickness (Tb.Th) and bone mineral density (BMD) and significantly increased trabecular separation (Tb.Sp) in distal femurs of AIN93G-fed wild-type mice. In high-fat diet-fed mice, compared to the AIN93G-fed ones, LLC resulted in significantly greater reductions BV/TV, Conn.D, Tb.N and BMD and significantly greater increases in structure model index (SMI) and Tb.Sp. Deficiency in PAI-1, compared to wild-type mice, significantly increased BV/TV, Conn.D, Tb.N and significantly reduced SMI. In vertebra, LLC resulted in similar trabecular changes compared to the non-tumor-bearing wild-type controls, the high-fat diet reduced BV/TV and BMD and increased SMI, whereas PAI-1 deficiency did not affect trabecular microstructure in vertebrae. These results demonstrated (1) that LLC is detrimental to trabecular bone which may make it more vulnerable to malignant invasion, (2) that high-fat diet enhances LLC-induced trabecular deterioration and (3) that the improvement in trabecular microstructure by PAI-1 deficiency indicates that PAI-1 may play a role in trabecular microarchitectural deterioration during malignant progression. Citation Format: Lin Yan. Effects of Lewis lung carcinoma on trabecular microstructural changes in wild-type and plasminogen activator inhibitor-1 deficient mice fed a high-fat diet. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 890. doi:10.1158/1538-7445.AM2015-890

Plasminogen Activator Inhibitor-1 Antagonist TM5484 Attenuates Demyelination and Axonal Degeneration in a Mice Model of Multiple Sclerosis.

Multiple sclerosis (MS) is characterized by inflammatory demyelination and deposition of fibrinogen in the central nervous system (CNS). Elevated levels of a critical inhibitor of the mammalian fibrinolitic system, plasminogen activator inhibitor 1 (PAI-1) have been demonstrated in human and animal models of MS. In experimental studies that resemble neuroinflammatory disease, PAI-1 deficient mice display preserved neurological structure and function compared to wild type mice, suggesting a link between the fibrinolytic pathway and MS. We previously identified a series of PAI-1 inhibitors on the basis of the 3-dimensional structure of PAI-1 and on virtual screening. These compounds have been reported to provide a number of in vitro and in vivo benefits but none was tested in CNS disease models because of their limited capacity to penetrate the blood-brain barrier (BBB). The existing candidates were therefore optimized to obtain CNS-penetrant compounds. We performed an in vitro screening using a model of BBB and were able to identify a novel, low molecular PAI-1 inhibitor, TM5484, with the highest penetration ratio among all other candidates. Next, we tested the effects on inflammation and demyelination in an experimental allergic encephalomyelitis mice model. Results were compared to either fingolimod or 6α-methylprednisolone. Oral administration of TM5484 from the onset of signs, ameliorates paralysis, attenuated demyelination, and axonal degeneration in the spinal cord of mice. Furthermore, it modulated the expression of brain-derived neurotrophic factor, which plays a protective role in neurons against various pathological insults, and choline acetyltransferase, a marker of neuronal density. Taken together, these results demonstrate the potential benefits of a novel PAI-1 inhibitor, TM5484, in the treatment of MS.

Inhibition of PAI-1 Via PAI-039 Improves Dermal Wound Closure in Diabetes.

Diabetes impairs the ability to heal cutaneous wounds, leading to hospitalization, amputations, and death. Patients with diabetes experience elevated levels of plasminogen activator inhibitor 1 (PAI-1), regardless of their glycemic control. It has been demonstrated that PAI-1-deficient mice exhibit improved cutaneous wound healing, and that PAI-1 inhibition improves skeletal muscle repair in mice with type 1 diabetes mellitus, leading us to hypothesize that pharmacologically mediated reductions in PAI-1 using PAI-039 would normalize cutaneous wound healing in streptozotocin (STZ)-induced diabetic (STZ-diabetic) mice. To simulate the human condition of variations in wound care, wounds were aggravated or minimally handled postinjury. Following cutaneous injury, PAI-039 was orally administered twice daily for 10 days. Compared with nondiabetic mice, wounds in STZ-diabetic mice healed more slowly. Wound site aggravation exacerbated this deficit. PAI-1 inhibition had no effect on dermal collagen levels or wound bed size. PAI-039 treatment failed to improve angiogenesis in the wounds of STZ-diabetic mice and blunted angiogenesis in the wounds of nondiabetic mice. Importantly, PAI-039 treatment significantly improved epidermal cellular migration and wound re-epithelialization compared with vehicle-treated STZ-diabetic mice. These findings support the use of PAI-039 as a novel therapeutic agent to improve diabetic wound closure and demonstrate the primary mechanism of its action to be related to epidermal closure.

Plasminogen Activator Inhibitor-1 Deficiency Ameliorates Insulin Resistance and Hyperlipidemia But Not Bone Loss in Obese Female Mice

We previously demonstrated that plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, is involved in type 1 diabetic bone loss in female mice. PAI-1 is well known as an adipogenic factor induced by obesity. We therefore examined the effects of PAI-1 deficiency on bone and glucose and lipid metabolism in high-fat and high-sucrose diet (HF/HSD)-induced obese female mice. Female wild-type (WT) and PAI-1-deficient mice were fed with HF/HSD or normal diet for 20 weeks from 10 weeks of age. HF/HSD increased the levels of plasma PAI-1 in WT mice. PAI-1 deficiency suppressed the levels of blood glucose, plasma insulin, and total cholesterol elevated by obesity. Moreover, PAI-1 deficiency improved glucose intolerance and insulin resistance induced by obesity. Bone mineral density (BMD) at trabecular bone as well as the levels of osterix, alkaline phosphatase, and receptor activator of nuclear factor κB ligand mRNA in tibia were decreased by HF/HSD in WT mice, and those changes by HF/HSD were not affected by PAI-1 deficiency. HF/HSD increased the levels of plasma TNF-α in both WT and PAI-1-deficient mice, and the levels of plasma TNF-α were negatively correlated with trabecular BMD in tibia of female mice. In conclusion, we revealed that PAI-1 deficiency does not affect the trabecular bone loss induced by obesity despite the amelioration of insulin resistance and hyperlipidemia in female mice. Our data suggest that the changes of BMD and bone metabolism by obesity might be independent of PAI-1 as well as glucose and lipid metabolism.

Plasminogen activator inhibitor-1 deficient mice are protected from angiotensin II-induced fibrosis

PAI-1 has been shown to be both profibrotic and antifibrotic in animal models of hepatic fibrosis. Although these models have similarities to human fibrotic liver disease, no rodent model completely recapitulates the clinical situation; indeed, transaminase values in most models of hepatic fibrosis are much higher than in chronic liver diseases in humans. Here, wild-type and PAI-1 −/− mice were administered AngII (500 ng/kg/min) for 4 weeks. ECM accumulation was evaluated by Sirius red staining, hydroxyproline content, and fibrin and collagen immunostaining. Induction of pro-fibrotic genes was assessed by real-time RT-PCR. Despite the absence of any significant liver damage, AngII infusion increased the deposition of hepatic collagen and fibrin ECM, with a perisinusoidal pattern. PAI-1 −/− mice were protected from these ECM changes, indicating a causal role of PAI-1 in this fibrosis model. Protection in the knockout strain correlated with a blunted increase in αSMA, and elevated activities of matrix metalloproteinases (MMP2, MMP9). These data suggest that PAI-1 plays a critical role in mediating fibrosis caused by AngII and lends weight-of-evidence to a pro-fibrotic role of this protein in liver. Furthermore, the current study proposes a new model of ‘pure’ hepatic fibrosis in mice with little inflammation or hepatocyte death.

Genetic Deficiency of Plasminogen Activator Inhibitor-1 Promotes Cardiac Fibrosis in Aged Mice

Background— Elevated levels of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of urokinase plasminogen activator and tissue plasminogen activator, are implicated in the pathogenesis of tissue fibrosis. Paradoxically, lack of PAI-1 in the heart is associated with the development of cardiac fibrosis in aged mice. However, the molecular basis of cardiac fibrosis in aged PAI-1–deficient mice is unknown. Here, we investigated the molecular and cellular bases of myocardial fibrosis. Methods and Results— Histological evaluation of myocardial tissues derived from aged PAI-1–deficient mice revealed myocardial fibrosis resulting from excessive accumulation of collagen. Immunohistochemical characterization revealed that the levels of matrix metalloproteinase-2, matrix metalloproteinase-9, and transforming growth factor-β1/2 and the number of Mac3-positive and fibroblast specific protein-1–positive cells were significantly elevated in aged PAI-1–deficient myocardial tissues compared with controls. Zymographic analysis revealed that matrix metalloproteinase-2 enzymatic activity was elevated in PAI-1–deficient mouse cardiac endothelial cells. Real-time quantitative polymerase chain reaction analyses of RNA from myocardial tissues revealed the upregulation of profibrotic markers in aged PAI-1–deficient mice. The numbers of phosphorylated Smad2–, phosphorylated Smad3–, and phosphorylated ERK1/2 MAPK–, but not pAkt/PKB–, positive cells were significantly increased in PAI-1–deficient myocardial tissues. Western blot and immunocytochemical analysis revealed that PAI-1–deficient mouse cardiac endothelial cells were more susceptible to endothelial-to-mesenchymal transition in response to transforming growth factor-β2. Conclusions— These results indicate that spontaneous activation of both Smad and non-Smad transforming growth factor-β signaling may contribute to profibrotic responses in aged PAI-1–deficient mice hearts and establish a possible link between endothelial-to-mesenchymal transition and cardiac fibrosis in PAI-1–deficient mice.

Abstract 1331: Myocardial Function Alterations Correlate to Cardiac Fibrosis and Upregulation of Profibrotic Signaling in Plasminogen Activator Inhibitor-1 Deficient Mice

Cardiac fibrosis is a common sequelae of cardiac injury and has deleterious functional consequences impacting cardiac filling, function and rhythm. Plasminogen activator inhibitor-1 (PAI-1) has been implicated in the pathogenesis of tissue fibrosis in mice. To investigate the longitudinal effect of PAI-1 on cardiac structure and function, M-mode echocardiography was employed to examine cardiac function in PAI-1 deficient (PAI-1 −/− ) and wild-type (WT) control mice in four age groups (6,12,18, 24 months). Eighteen month old PAI-1 −/− mice exhibited reduced left ventricular (LV) diastolic internal dimension ( p =0.0118) and a trend towards increased LV posterior wall (LVPW) thickness, compared to WT. Two year old PAI-1 −/− mice showed increased diastolic and systolic LVPW thickness ( p =0.0127 and p =0.0212, respectively), reduced diastolic and systolic LV internal dimension ( p =0.0486 and p =0.0124), but with preserved LV fractional shortening compared to WT. Histological examination of cardiac sections revealed fibrosis on the anterior epicardial surface of the hearts in 18 month old PKO, which in 26 month old mice had become confluent with extensive (10 –17% by area) epicardial, perivascular, and interstitial distribution (compared to none in WT). Real time polymerase chain reaction (RT-PCR) revealed upregulation of transforming growth factor beta (TGF-β) and fibroblast growth factor 2 in PAI-1 −/− compared to WT ( p =0.0234 and p =0.037, respectively). Immunofluoresence confirmed this finding with bright TGF-β staining localized in the media of intra-myocardial arterioles, and phosphorylated SMAD2/3, the downstream TGF-β signaling mediator, in areas of fibrosis. Thoracic aortic cells from aged (18 –24 month) PKO and WT mice were grown in culture, with RT-PCR revealing 4 fold increased TGF-β and 17 fold increased SMAD3 ( p <0.05 for both) RNA levels in PAI-1 −/− , supplying additional evidence for upregulation of a profibrotic TGF-β/SMAD tissue signaling pathway. The present study is one of the first to elucidate some of the functional consequences and relevant molecular signaling pathways related to aging and PAI-1 deficiency mediated cardiac fibrosis.

No functional role of plasminogen activator inhibitor-1 in murine adipogenesis or adipocyte differentiation.

To substantiate a potential role of plasminogen activator inhibitor-1 (PAI-1) in adipogenesis, we have studied its effects on in vitro adipocyte differentiation and on in vivo adipose tissue formation. Our in vitro data do not support a functional role of PAI-1, as substantiated by our findings that: (i) inhibition of PAI-1 with a neutralizing antibody did not affect differentiation of 3T3-F442A preadipocytes; (ii) overexpression of murine PAI-1 in 3T3-F442A cells had no effect on differentiation; and (iii) differentiation of PAI-1-deficient murine embryonic fibroblasts into mature adipocytes was comparable to wild-type (WT) cells. Furthermore, our in vivo studies did not reveal an important role for PAI-1, as suggested by our findings that: (i) de novo fat pad formation in NUDE mice following injection of 3T3-F442A cells was not affected by a PAI-1 neutralizing antibody; and (ii) adipose tissue formation following combined injection of Matrigel and basic fibroblast growth factor was comparable in WT and in PAI-1 deficient mice. Taken together, these in vitro and in vivo studies in murine model systems do not support an important functional role of PAI-1 in adipogenesis.

Bone Marrow Plasminogen Activator Inhibitor-1 Influences the Development of Obesity

Plasma levels of plasminogen activator inhibitor-1 (PAI-1) are elevated in obesity and correlate with body mass index. The increase in PAI-1 associated with obesity likely contributes to increased cardiovascular risk and may predict the development of type 2 diabetes mellitus. Although adipocytes are capable of synthesizing PAI-1, the bulk of evidence indicates that cells residing in the stromal fraction of visceral fat are the primary source of PAI-1. We hypothesized that bone marrow-derived PAI-1, e.g. derived from macrophages located in visceral fat, contributes to the development of diet-induced obesity. To test this hypothesis, male C57BL/6 wild-type mice and C57BL/6 PAI-1 deficient mice were transplanted with either PAI-1(-/-), PAI-1(+/-), or PAI-1(+/+) bone marrow. The transplanted animals were subsequently fed a high fat diet for 24 weeks. Our findings show that only the complete absence of PAI-1 protects from the development of diet-induced obesity, whereas the absence of bone marrow-derived PAI-1 protects against expansion of the visceral fat mass. Remarkably, there is a link between the PAI-1 levels, the degree of inflammation in adipose tissue, and the development of obesity. Based on these findings we suggest that bone marrow-derived PAI-1 has an effect on the development of obesity through its effect on inflammation.

Impairment of adipose tissue development by hypoxia is not mediated by plasminogen activator inhibitor-1

Hypoxia in rodents and humans is associated with a reduction of body fat on the one hand, and with enhanced expression of plasminogen activator inhibitor-1 (PAI-1), the main inhibitor of the fibrinolytic system, on the other hand. It was the objective of this study to investigate whether impairment of adipose tissue development by hypoxia may be mediated by PAI-1. Five week old male wild-type (WT) C57Bl/6 mice were fed a standard (SFD) or high fat (HFD) diet and kept under normoxic or hypoxic (10% O(2)) conditions. In addition, PAI-1 deficient mice and WT littermates were kept on HFD under normoxia or hypoxia. In vitro, the effect of hypoxia (2% O(2)) was investigated on differentiation of 3T3-L1 cells into adipocytes. Hypoxia induced a significant reduction of weight gain in WT mice on either SFD or HFD, accompanied by lower weights of subcutaneous (SC) and gonadal (GON) fat. Under hypoxic conditions, adipocytes in the adipose tissues were significantly smaller, whereas blood vessel size and density were larger. Serum PAI-1 levels were enhanced in hypoxic mice on SFD but not on HFD, and overall did not correlate with the observed changes in adipose tissue composition. Furthermore, the effects of hypoxia on adipose tissue in mice on HFD were not affected by deficiency of PAI-1. The inhibiting effect of hypoxia on in vitro preadipocyte differentiation was not mediated by PAI-1 activity. In conclusion, impairment of in vivo adipose tissue development and in vitro differentiation of preadipocytes by hypoxia is not mediated by PAI-1.

Pivotal role of PAI-1 in a murine model of hepatic vein thrombosis

Hepatic veno-occlusive disease (VOD) is a common complication of high-dose chemotherapy associated with bone marrow transplantation. While the pathogenesis of VOD is uncertain, plasminogen activator inhibitor-1 (PAI-1) has emerged as a diagnostic marker and predictor of VOD in humans. In this study, we investigated the role of PAI-1 in a murine model of VOD produced by long-term nitric oxide synthase inhibition using L-NAME. After 6 weeks, wild-type (WT) mice developed extensive fibrinoid hepatic venous thrombi and biochemical evidence of hepatic injury and dysfunction. In contrast, PAI-1-deficient mice were largely protected from the development of hepatic vein thrombosis. Furthermore, WT mice that received tiplaxtinin, an antagonist of PAI-1, were effectively protected from L-NAME-induced thrombosis. Taken together, these data indicate that NO and PAI-1 play pivotal and antagonistic roles in hepatic vein thrombosis and that PAI-1 is a potential target in the prevention and treatment of VOD in humans.

Prevention of obesity and insulin resistance in mice lacking plasminogen activator inhibitor 1.

Increased plasminogen activator inhibitor 1 (PAI-1) has been linked to not only thrombosis and fibrosis but also to obesity and insulin resistance. Increased PAI-1 levels have been presumed to be consequent to obesity. We investigated the interrelationships of PAI-1, obesity, and insulin resistance in a high-fat/high-carbohydrate (HF) diet-induced obesity model in wild-type (WT) and PAI-1-deficient mice (PAI-1(-/-)). Obesity and insulin resistance developing in WT mice on an HF diet were completely prevented in mice lacking PAI-1. PAI-1(-/-) mice on an HF diet had increased resting metabolic rates and total energy expenditure compared with WT mice, along with a marked increase in uncoupling protein 3 mRNA expression in skeletal muscle, likely mechanisms contributing to the prevention of obesity. In addition, insulin sensitivity was enhanced significantly in PAI-1(-/-) mice on an HF diet, as shown by euglycemic-hyperinsulinemic clamp studies. Peroxisome proliferator-activated receptor (PPAR)-gamma and adiponectin mRNA, key control molecules in lipid metabolism and insulin sensitivity, were maintained in response to an HF diet in white adipose tissue in PAI-1(-/-) mice, contrasting with downregulation in WT mice. This maintenance of PPAR-gamma and adiponectin may also contribute to the observed maintenance of body weight and insulin sensitivity in PAI-1(-/-) mice. Treatment in WT mice on an HF diet with the angiotensin type 1 receptor antagonist to downregulate PAI-1 indeed inhibited PAI-1 increases and ameliorated diet-induced obesity, hyperglycemia, and hyperinsulinemia. PAI-1 deficiency also enhanced basal and insulin-stimulated glucose uptake in adipose cells in vitro. Our data suggest that PAI-1 may not merely increase in response to obesity and insulin resistance, but may have a direct causal role in obesity and insulin resistance. Inhibition of PAI-1 might provide a novel anti-obesity and anti-insulin resistance treatment.

Aging Accelerates Endotoxin-Induced Thrombosis: Increased Responses of Plasminogen Activator Inhibitor-1 and Lipopolysaccharide Signaling with Aging

Although older subjects are susceptible to thrombosis under septic conditions, the underlying molecular mechanisms have not been fully elucidated. Since elevated plasminogen activator inhibitor-1 (PAI-1) primarily contributes to endotoxin-induced thrombosis, we first compared the induction of PAI-1 by lipopolysaccharide (LPS) between young and aged mice. The higher induction of PAI-1 antigen and mRNA with increased renal glomerular fibrin deposition was observed in LPS-treated aged mice compared to young mice. In situ hybridization analysis showed that the aging-associated induction of PAI-1 mRNA by LPS was pronounced in hepatocytes and in renal glomerular cells. The increased magnitude of the response of aged mice to lower doses of LPS was observed in terms of renal glomerular fibrin deposition and PAI-1 mRNA induction in the tissues. Furthermore, older PAI-1 deficient mice treated with LPS developed much less fibrin deposition in kidneys. Importantly, a larger induction of receptor molecules for LPS (eg, CD14 and Toll-like receptor 4) was demonstrated in LPS-treated aged mice as compared with young mice. The enhanced LPS signaling in aged mice was also demonstrated by the marked induction of nuclear factor-kappaB in the tissues after endotoxin treatment. As a consequence, increases in an inflammatory cytokine, tumor necrosis factor-alpha, were pronounced in plasma and tissues of LPS-treated aged mice. These results emphasize the key role played by PAI-1 in aging-associated deterioration in this thrombosis model, and suggest that the hyperresponse of PAI-1 gene to LPS results from the enhanced LPS signaling and the subsequent inflammatory response in aged mice.

Possible role of the 4G/5G polymorphism of the plasminogen activator inhibitor 1 gene in the development of asthma

findings show that PAI-1 is closely associated with fibrosis and the accumulation of ECM after lung injury or inflammation. We previously reported that the PAI1 gene is induced in lung mast cells in asthmatic subjects.3 These findings suggest that PAI-1 could contribute to tissue remodeling and subsequent fibrosis in asthma. The PAI1 gene has variation in the promoter region on the basis of a single guanosine insertion-deletion (5G or 4G),4,5 and the *4G allele is correlated with increased plasma PAI-1 levels.5,6 In vitro experiments have initially shown that the *5G allele contains an additional binding site for a protein likely related to the nuclear factor κB group of transcription factors, and this binding site is absent in the *4G allele.4 After stimulation with IL-1, HepG2 cells transfected with the *4G allele produce 6 times more PAI-1 mRNA than those with the *5G allele. These data suggest a functional role of the 4G/5G polymorphism in the response to cytokines, with the *4G allele being associated with enhanced gene expression. In the present study we demonstrate that the *4G allele is preferentially transmitted to asthmatic children. These data suggest a possible role of the PAI1 gene and the 4G polymorphism in the pathophysiology of asthma.

PAI-1 deficiency attenuates the fibrogenic response to ureteral obstruction

Progressive renal disease is characterized by the induction of plasminogen activator inhibitor-1 (PAI-1), suggesting that impaired activity of the renal plasmin cascade may play a role in renal fibrosis. To test this hypothesis, the severity of renal fibrosis caused by unilateral ureteral obstruction (UUO) was compared in PAI-1 wild-type (+/+) and PAI-1 deficient (-/-) mice. The extent of interstitial inflammation and fibrosis, renal plasminogen activator and plasmin activity, and renal expression of profibrotic genes was evaluated after 3, 7, and 14 days of UUO. Renal PAI-1 mRNA levels increased 8- to 16-fold in the +/+ mice after UUO surgery, and PAI-1 protein was detected in kidney homogenates. Interstitial fibrosis was significantly attenuated in -/- mice compared with +/+ mice at day 7 and day 14, based on the interstitial area stained with picrosirius red and total kidney collagen content. However, neither the mean renal plasminogen activator nor plasmin activities were increased in -/- mice compared with +/+ mice. The number of interstitial macrophages were significantly lower in the -/- mice three and seven days after UUO; interstitial myofibroblasts were significantly fewer at three days. At the same time points, this altered interstitial cellularity was associated with a significant reduction in renal mRNA levels for transforming growth factor-beta and procollagens alpha 1(I) and alpha 1(III). These studies establish an important fibrogenic role for PAI-1 in the renal fibrogenic response. The results demonstrate that one important fibrosis-promoting function of PAI-1 is its role in the recruitment of fibrosis-inducing cells, including myofibroblasts and macrophages.

A Fas pathway to pulmonary fibrosis.

A Fas pathway to pulmonary fibrosis.

Plasminogen Activator Inhibitor-1 Is a Major Determinant of Arterial Thrombolysis Resistance

Platelet-rich thrombi are resistant to lysis by tissue plasminogen activator (tPA). Plasminogen activator inhibitor-1 (PAI-1), a rapid inhibitor of tPA, may contribute to arterial thrombolysis resistance. However, few data are available regarding the effect of PAI-1 on arterial thrombolysis in animals. We used a murine carotid injury model to test the hypothesis that PAI-1 inhibits thrombolysis mediated by pharmacological concentrations of tPA. Platelet-rich thrombi were induced in wild-type mice (PAI-1 +/+; n=11) and PAI-1-deficient mice (PAI-1 -/-; n=11) with ferric chloride. Baseline carotid blood flows and mean occlusion times did not differ between PAI-1 +/+ and PAI-1 -/- mice. Clot lysis was induced by infusion of heparin (200 U/kg bolus, 70 U. kg-1. h-1 drip), human plasminogen (50 mg/kg), and tPA at 20 (n=10) or 100 (n=12) microg. kg-1. min-1. Mean plasma tPA antigens were 2.7 microg/mL (tPA infusion, 20 microg. kg-1. min-1) and 5.5 microg/mL (tPA infusion, 100 microg. kg-1. min-1), with no significant differences between PAI-1 +/+ mice and PAI-1 -/- mice. Reperfusion after tPA 20 microg. kg-1. min-1 occurred in 1 of 5 PAI-1 +/+ mice versus 5 of 5 PAI-1 -/- mice (P=0.0006). Reperfusion occurred in all mice that received tPA 100 microg. kg-1. min-1, but reperfusion times were significantly shorter in PAI-1 -/- mice (17. 8+/-2.6 minutes, n=6) than in PAI-1 +/+ mice (35.7+/-5.1 minute, n=6; P=0.01). Histological analyses confirmed that carotid thrombi were platelet rich and that PAI-1 was distributed uniformly throughout thrombi from PAI-1 +/+ mice. Lysates of PAI-1 +/+ platelets inhibited human tPA, whereas PAI-1 -/- platelet lysates did not. PAI-1 is a major determinant of the resistance of platelet-rich arterial thrombi to lysis by pharmacological concentrations of tPA. Strategies to inhibit or resist PAI-1 may enhance thrombolysis.