Plasminogen Activator Inhibitor-1 Expression Research Articles

Abstract 362: Plasminogen Activator Inhibitor 1 Forms a Complex With FXIa on the Surface of Endothelial Cells, Blocking Its Activity and Inducing the Clearance, Internalization and Degradation of FXIa

Background: The activation of coagulation factor (F) XI by activated coagulation FXII (FXIIa) is a prothrombotic process. The endothelium is known to play an antithrombotic role by preventing thrombin generation and platelet activation. It is unknown whether the antithrombotic role of the endothelium includes regulation of activated FXI (FXIa) activity. Hypothesis: Endothelial cells (ECs) express anticoagulant properties that block the procoagulant activity of FXIa. Methods: We used a chromogenic assay to measure FXIa, kallikrein or FXIIa activity on cultured human umbilical vein ECs (HUVECs). To detect FXIa-inhibitor complexes in either the supernatant (SN) or cell lysate (CL), HUVECs were exposed to FXIa for 2 hrs at 4°C, then washed and lysed, followed by immunoprecipitation and western blot (WB) using an anti-FXIa mAb to the catalytic domain. To assess FXIa binding and internalization, HUVECs were exposed to FXIa for 2 hrs a 4C, washed and incubated at 37C for 5 min to 2 hrs, followed by fixation, and immunostaining with an anti-FXI antibody. Results: HUVECs block the activity of FXIa but not kallikrein or FXIIa activity. The catalytic domain of FXIa (30 kDa) formed a covalent bond with one inhibitor generating one band at 80 kDa. The formation of FXIa-inhibitor complexes appeared in the SN when ECs were transferred to 37C. Mass spectrometry analysis of the 80kDa band revealed that FXIa forms a complex with plasminogen activator inhibitor 1 (PAI-1). WB for PAI-1 from the SN and CL detected a band at 80 kDa, while the expression of PAI-1 on the HUVEC surface decreased after incubation with FXIa. FXIa bound to the EC surface at 4C. After 5 mins at 37C, FXIa was internalized and localized beneath the plasma membrane, and after 30 mins to 2 hr at 37C, FXIa accumulated in the early and late endosomes and lysosomes. The serine protease inhibitor PPACK blocked FXIa-PAI-1 complex formation and internalization of FXIa. A blocking anti-PAI-1 antibody increased the cleavage of the chromogenic substrate by FXIa and the capacity of FXIa to promote fibrin formation on ECs in recalcified plasma. Summary: FXIa forms a complex with PAI-1 on the surface of ECs blocking its activity. Inhibition of FXIa on the endothelium may support internalization and clearance of FXIa from the circulation.

Sonic hedgehog-c-Jun N-terminal kinase-zinc finger protein Gli1 signaling protects against high glucose concentration-induced reactive oxygen species generation in human fibroblasts.

Diabetes mellitus (DM) complications affect patients and cause varying damage. Skin ulcers exhibit difficulties in wound healing, and the regulatory basis for this remains unclear. High glucose concentration (HG) was utilized to mimic DM in cultured cells. Reverse transcription-quantitative polymerase chain reaction, western blotting and fluorescence dye analyses were performed to analyze the effects of hedgehog signaling in regulation of HG or diabetes in fibroblasts. HG-stress suppressed hedgehog-signaling gene expression, whereas the apoptosis and inflammatory response markers, Caspase-3 and plasminogen activator inhibitor-1 (PAI1), respectively, were induced. In addition, HG-stress inhibited the fibroblast proliferation rate. In parallel, treatment with Sonic hedgehog (Shh), an activator of hedgehog signaling, together with HG eliminated effects of HG on expression of hedgehog-signaling genes, Caspase-3 and PAI1, and rescued the cell proliferation rate in fibroblasts. In addition, Shh application activated c-Jun N-terminal kinase (JNK), which was inhibited by HG stress. sp600125, a JNK specific inhibitor, treatment inhibited the effect of Shh on fibroblast proliferation and hedgehog-signaling marker gene expression. Furthermore, zinc finger protein Gli1 (Gli1) overexpression partially eliminated the effect of HG and sp600125 on fibroblast proliferation, and reduced HG-induced ROS generation in fibroblasts. Together, these results indicate that HG stress inhibits hedgehog signaling, and Shh-JNK-Gli1 pathway positively regulates HG-induced damage on fibroblasts.

PAI-1 is a novel component of the miR-17~92 signaling that regulates pulmonary artery smooth muscle cell phenotypes.

We have previously reported that miR-17~92 is critically involved in the pathogenesis of pulmonary hypertension (PH). We also identified two novel mR-17/20a direct targets, PDZ and LIM domain protein 5 (PDLIM5) and prolyl hydroxylase 2 (PHD2), and elucidated the signaling pathways by which PDLIM5 and PHD2 regulate functions of pulmonary artery smooth muscle cells (PASMCs). In addition, we have shown that plasminogen activator inhibitor-1 (PAI-1) is also downregulated in PASMCs that overexpress miR-17~92. However, it is unclear whether PAI-1 is a direct target of miR-17~92 and whether it plays a role in regulating the PASMC phenotype. In this study, we have identified PAI-1 as a novel target of miR-19a/b, two members of the miR-17~92 cluster. We found that the 3'-untranslated region (UTR) of PAI-1 contains a miR-19a/b binding site and that miR-19a/b can target this site to suppress PAI-1 protein expression. MiR-17/20a, two other members of miR-17~92, may also indirectly suppress PAI-1 expression through PDLIM5. PAI-1 is a negative regulator of miR-17~92-mediated PASMC proliferation. Silencing of PAI-1 induces Smad2/calponin signaling in PASMCs, suggesting that PAI-1 is a negative regulator of the PASMC contractile phenotype. We also found that PAI-1 is essential for the metabolic gene expression in PASMCs. Furthermore, although there is no significant change in PAI-1 levels in PASMCs isolated from idiopathic pulmonary arterial hypertension and associated pulmonary arterial hypertension patients, PAI-1 is downregulated in hypoxia/Sugen-induced hypertensive rat lungs. These results suggest that miR-17~92 regulates the PASMC contractile phenotype and proliferation coordinately and synergistically by direct and indirect targeting of PAI-1.

MicroRNA-30b protects myocardial cell function in patients with acute myocardial ischemia by targeting plasminogen activator inhibitor-1.

The aim of the present study was to determine the expression of plasminogen activator inhibitor-1 (PAI-1) and microRNA (miR)-30b in the blood of patients with acute myocardial ischemia (AMI) and in the blood and myocardial tissue of mice with AMI. In addition, the present study aimed to identify the mechanism of action of miR-30b in AMI. A total of 36 patients with AMI were included in the present study and 28 healthy subjects were included as a control. Peripheral blood was collected from all subjects. For animal experiments, mice in the AMI group received an intraperitoneal injection of pituitrin (20 U/kg), whereas mice in the negative control group received an intraperitoneal injection of the same volume of saline. Blood and myocardial tissue was collected from all mice for analysis. Reverse transcription-quantitative polymerase chain reaction was performed to determine the expression of PAI-1 mRNA and miR-30b in the serum and myocardial tissue. An enzyme-linked immunosorbent assay was performed to measure the expression of PAI-1 protein in the serum of humans and mice, whereas western blotting was performed to determine the expression of PAI-1 protein in mouse myocardial tissue. Catalase, glutathione peroxidase and superoxide dismutase activity was measured using an automatic biochemical analyzer. A dual luciferase assay was performed to identify the interactions between PAI-1 mRNA and miR-30b. The results indicated that patients with AMI have higher PAI-1 levels and lower miR-30b expression in the peripheral blood compared with healthy subjects. AMI damaged the myocardium tissue of mice and reduced catalase, glutathione peroxidase and superoxide dismutase activity. Mice that have undergone AMI exhibit increased PAI-1 levels but decreased miR-30b expression in the peripheral blood and myocardial tissues. It was also demonstrated that miR-30b is able to bind to the 3'-untranslated region of PAI-1 mRNA to regulate its expression. The present study demonstrates that patients with AMI exhibit decreased miR-30b expression and elevated PAI-1 expression in the peripheral blood. miR-30b may therefore inhibit the damage to myocardial cells that occurs following AMI and protect myocardial cell function by targeting PAI-1 expression.

Anti-high mobility group box-1 monoclonal antibody treatment of brain edema induced by influenza infection and lipopolysaccharide.

Encephalopathy is a major cause of influenza-associated child death and severe neurological sequelae in Japan, highlighting the urgent need for new therapeutic strategies. In this study, we evaluated the effects of anti-high mobility group box-1 monoclonal antibody (α-HMGB1) treatment on brain edema induced by influenza A virus (IAV) and lipopolysaccharide in 4-week-old BALB/c female mice. The results showed that administration of 7.5 mg/kg α-HMGB1 1 h after IAV (A/Puerto Rico/8/34) inoculation significantly alleviated brain edema at 48 h after IAV inoculation, as confirmed by the suppression of Evans Blue dye leakage and matrix metallopeptidase-9 mRNA expression in the brain. Moreover, we also observed suppression of oxidative stress and different cytokines in IAV-inoculated mice. The expression of plasminogen activator inhibitor-1 was also attenuated following treatment with α-HMGB1. Notably, α-HMGB1 treatment had no effect on virus propagation in the lung. In summary, anti-HMGB1 treatment may improve the prognosis in cases with influenza-associated encephalopathy by attenuating brain edema and reducing the inflammatory responses induced by HMGB1.

Leukotriene D4 induces cellular senescence in osteoblasts

Aging is associated with the development of osteoporosis, in which cellular senescence in osteoblasts plays a key role. Leukotriene D4 (LTD4), an important cysteinyl leukotriene (cysLT), is a powerful pro-inflammatory mediator formed from arachidonic acid. However, little information regarding the effects of LTD4 on the pathogenesis of osteoporosis has been reported before. In the present study, we defined the physiological roles of LTD4 in cellular senescence in osteoblasts. Our results indicate that LTD4 treatment decreased the expression of SIRT1 in a dose-dependent manner in MC3T3-E1 osteoblastic cells. Additionally, LTD4 significantly increased the expression of p53, p21 and plasminogen activator inhibitor-1 (PAI-1). LTD4 was also found to elevate the activity of β-galactosidase (SA-β-Gal) but to prevent BrdU incorporation. Our results indicate that cysteinyl leukotriene receptor 1 (cysLT1R) could be detected in MC3T3-E1 osteoblastic cells at both the mRNA and protein levels. However, cysLT2R was not expressed in these cells. Interestingly, we found that knockdown of cysLT1R or use of the selective cysLT1R antagonist montelukast abolished the LTD4-induced reduction in SIRT1 and increase in p53, p21, and PAI-1. Notably, knockdown of cysLT1R by transfection with cysLT1R siRNA or treatment with montelukast attenuated the LTD4-induced increase in SA-β-Gal activity. Our study shows for the first time that LTD4 has a significant impact on cellular senescence in osteoblasts.

Treadmill exercise alleviates diabetic cardiomyopathy by suppressing plasminogen activator inhibitor expression and enhancing eNOS in streptozotocin-induced male diabetic rats.

ObjectiveTo investigate the biological mechanism of the effect of different intensity exercises on diabetic cardiomyopathy.Methods87 raise specific pathogen SPF healthy 6-week-old male Sprague–Dawley rats, fed 6 weeks with high-fat diet for rats were used, and a diabetic model was established by intraperitoneal injection of streptozotocin – randomly selected 43 rats were divided into Diabetic control group (DCG, n = 10), Diabetic exercise group 1 (DEG1, n = 11), Diabetic exercise group 2 (DEG2, n = 11) and Diabetic exercise group 3 (DEG3, n = 11). The rats in DEG1 were forced to run on a motorized treadmill, the exercise load consisted of running at a speed of 10 m/min, the exercise load of the rats in DEG2 were running at a speed of 15 m/min, the exercise load of the rats in DEG3 were running at a speed of 20 m/min, for one hour once a day for 6 weeks. After 6 weeks of exercise intervention, glucose metabolism-related indexes in rats such as blood glucose (FBG), glycosylated serum protein (GSP) and insulin (FINS); cardiac fibrinolytic system parameters such as PAI-1 (plasminogen activator inhibitor 1), Von Willebrand factor (vWF), protein kinase C (PKC) and diacylglycerol (DAG); and serum level of NO, eNOS and T-NOS were measured.ResultCompared with DCG, fasting blood glucose and GSP were decreased, while insulin sensitivity index and insulin level were increased in all rats of the three exercise groups. FBG decrease was statistically significant (P < 0.01), only GSP decrease was statistically significant (P < 0.05) in DEG1 and DEG2, PAI-1 in three exercise groups were significantly reduced (P < 0.05), plasma vWF levels in the three exercise groups were significantly lower than those in the DCG group (P < 0.01); PKC levels decreased dramatically in the three exercise groups and DAG levels decrease slightly (P < 0.05), but with no significant difference. Compared with DCG, the serum level of NO was significantly higher (P < 0.05), and eNOS level was significantly elevated (P < 0.05). T-NOS elevation was statistically significant in DEG1 (P < 0.05).ConclusionsLow- and moderate-intensity exercise can better control blood glucose level in diabetic rats; myocardial PAI-1 in DEG1, DEG2 and DEG3 rats decreased significantly (P < 0.05), serum NO increased (P < 0.05) and eNOS increased (P < 0.05) significantly. Therefore, it is inferred that exercise improves the biological mechanism of diabetic cardiomyopathy by affecting the levels of PAI-1 and eNOS, and there is a dependence on intensity.

Combined treatment with benzo[a]pyrene and 1α,25-dihydroxyvitamin D3 induces expression of plasminogen activator inhibitor 1 in monocyte/macrophage-derived cells

Benzo[a]pyrene (BaP) is an environmental pollutant found in cigarette smoke and is implicated as a causative agent of tobacco-related diseases, such as arteriosclerosis. In contrast, vitamin D signaling, which is principally mediated by conversion of vitamin D to the active form, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], decreases cardiovascular disease risk. However, combined treatment with BaP and 1,25(OH)2D3 enhances BaP toxicity, including BaP-DNA adduct formation. We further investigated the cross-talk between BaP and 1,25(OH)2D3 signaling pathways, and found that combined treatment with these compounds induces mRNA and protein expression of plasminogen activator inhibitor 1 (PAI-1) in monocyte/macrophage-derived THP-1 and U937 cells. Protein synthesis inhibitor treatment did not inhibit induction of the PAI-1 gene (SERPINE1) in these cells. BaP plus 1,25(OH)2D3 induced differentiation markers, inhibited cellular proliferation, and induced apoptosis and oxidative stress in these cells. Reactive oxygen species scavenger treatment suppressed apoptosis but not SERPINE1 induction in cells treated with BaP plus 1,25(OH)2D3. Thus, combined treatment with BaP and 1,25(OH)2D3 induced SERPINE1 mRNA expression in these cells through a mechanism that does not require de novo protein synthesis or reactive oxygen species production. These findings suggest that induction of the proinflammatory factor PAI-1 plays a role in BaP toxicity. Interestingly, PAI-1 knockdown decreased expression of the cell surface antigen CD14, a monocytic differentiation marker, in THP-1 cells treated with BaP plus 1,25(OH)2D3. PAI-1 induction may also be related to a function of monocytes/macrophages in response to xenobiotic and vitamin D signaling.

SK-216, a Novel Inhibitor of Plasminogen Activator Inhibitor-1, Suppresses Lung Metastasis of Human Osteosarcoma.

Lung metastasis constitutes the leading cause of the death in patients with osteosarcoma. We have previously reported that plasminogen activator inhibitor-1 (PAI-1) regulates the invasion and lung metastasis of osteosarcoma cells in a mouse model and as well as in clinical samples. In the present study, we examined the anti-metastatic effect of SK-216, a small compound PAI-1 inhibitor, in human 143B osteosarcoma cells. An in vitro study showed that SK-216 treatment suppressed invasion activity by inhibiting PAI-1 expression in 143B cells, but had no influence on their proliferation or migration. 143B cells treated with SK-216 exhibited reduced matrix metalloproteinase-13 (MMP-13) secretion in a dose-dependent manner. Moreover, intraperitoneal injection of SK-216 into mouse models resulted in downregulation of PAI-1 expression levels in the primary tumors and showed suppression of lung metastases without influencing the proliferative activity of the tumor cells in the primary lesions. These results indicate that SK-216, a PAI-1 inhibitor, may serve as a novel drug to prevent lung metastasis in human osteosarcoma.

Antioxidant and anti-inflammatory activities of alpha lipoic acid protect against indomethacin-induced gastric ulcer in rats

Little is known about the role of tumor necrosis factor-alpha (TNF-α), plasminogen activator inhibitor-1 (PAI-1), and inducible nitric oxide synthase (iNOS) in the gastric ulcer and the effect of alpha lipoic acid (ALA) in their modulation. Hence, this experimental study was designed to assess the possible protective effect of ALA against indomethacin (IND)-induced gastric ulcer in rats, as well as to determine the possible underlying mechanisms with a special focus on TNF-α, PAI-1, and iNOS. Adult male rats (n = 28) were divided into four equal groups: the control group received distilled water, the vehicle group received 0.5% carboxymethylcellulose, the ulcer group received a single oral dose of IND (50 mg/kg) and the ALA-treated group received ALA (100 mg/kg) orally for 3 days before ulcer induction. Four hours after IND administration, all rats were sacrificed. The ulcer index, and gastric tissue homogenate contents of total antioxidant capacity (TAC), malondialdehyde (MDA), TNF-α, and PAI-1 were evaluated. Immunohistochemical evaluation of iNOS protein expression and histopathological examination of gastric tissue were investigated. The results revealed that ALA pretreatment significantly decreased the ulcer index, the gastric levels of MDA, TNF-α, PAI-1, and iNOS protein expression while increased the gastric levels of TAC as well as improved the histopathological appearance of gastric tissues. In conclusion, ALA ameliorated the IND-induced gastric ulceration. This could be attributed to its antioxidant and anti-inflammatory activities via suppression of TNF-α-induced elevation of both PAI-1 level and iNOS expression in the gastric tissue.

Hyperglycemia-induced mouse trophoblast spreading is mediated by reactive oxygen species.

During embryo implantation, the outer layer of the blastocyst interacts with the endometrium giving rise to the development of the trophoblast cell lineage. The cells in this lineage participate in the penetration of endometrium due to their motility and invasive properties. The mechanisms that regulate the differentiation and invasive ability of these cells are essential for the establishment and maintenance of an efficient exchange between maternal and fetal tissues during pregnancy. In this context, hyperglycemia can induce oxidative stress causing alterations in the placenta. This study evaluated the role of reactive oxygen species (ROS) in the actions of high glucose concentration (HG) on trophoblast spreading and the expression of extracellular proteases in cultured mouse conceptuses. Blastocysts from gestational day 4 (GD4) were cultured until GD7 in HAM-F10 medium and further treated for 48 hr with HG (25 mM glucose) from GD7 to GD9. This treatment induced larger trophoblast outgrowths and increased ROS concentration, which was associated with increased expression levels of urokinase-type plasminogen activator (PLAU), plasminogen activator inhibitor 1 (PAI-1), and matrix metalloproteinase 9 (MMP-9). These effects were prevented by treatment with the non-specific antioxidant N-acetylcysteine (NAC) or apocynin, an inhibitor of NADPH oxidase. Our data suggest that the HG-induced trophoblast spreading and the expression of PLAU, PAI-1, and MMP-9 were mediated by the production of ROS via NADPH oxidase activity. Our results shed light on placental alterations in gestational diabetes mellitus.

Plasminogen Activator Inhibitor-1 Reduces Tissue-Type Plasminogen Activator–Dependent Fibrinolysis and Intrahepatic Hemorrhage in Experimental Acetaminophen Overdose

Acetaminophen (APAP)-induced liver injury in mice is associated with activation of the coagulation cascade and deposition of fibrin in liver. Plasminogen activator inhibitor-1 (PAI-1) is an important physiological inhibitor of tissue-type plasminogen activator (tPA) and plays a critical role in fibrinolysis. PAI-1 expression is increased in both experimental APAP-induced liver injury and patients with acute liver failure. Prior studies have shown that PAI-1 prevents intrahepatic hemorrhage and mortality after APAP challenge, but the downstream mechanisms are not clear. We tested the hypothesis that PAI-1 limits liver-related morbidity after APAP challenge by reducing tPA-dependent fibrinolysis. Compared with APAP-challenged (300 mg/kg) wild-type mice, hepatic deposition of cross-linked fibrin was reduced, with intrahepatic congestion and hemorrhage increased in PAI-1-deficient mice 24 hours after APAP overdose. Administration of recombinant wild-type human PAI-1 reduced intrahepatic hemorrhage 24 hours after APAP challenge in PAI-1-/- mice, whereas a mutant PAI-1 lacking antiprotease function had no effect. Of interest, tPA deficiency alone did not affect APAP-induced liver damage. In contrast, fibrinolysis, intrahepatic congestion and hemorrhage, and mortality driven by PAI-1 deficiency were reduced in APAP-treated tPA-/-/PAI-1-/- double-knockout mice. The results identify PAI-1 as a critical regulator of intrahepatic fibrinolysis in experimental liver injury. Moreover, the results suggest that the balance between PAI-1 and tPA activity is an important determinant of liver pathology after APAP overdose.

Effects of C1 inhibitor on endothelial cell activation in a rat hind limb ischemia-reperfusion injury model

ObjectiveIschemia-reperfusion (I/R) injury is a major clinical problem linked to vascular surgery. Currently, no drugs to prevent or to treat I/R injury are approved for clinical use. C1 inhibitor (C1 INH) is known to reduce activation of the plasma cascade systems that are involved in the pathophysiologic process of I/R injury. The aim of this study was therefore to investigate the effect of C1 INH on complement deposition and endothelial cell activation in a rat model of hind limb I/R injury. MethodsMale Wistar rats (wild type, bred at the central animal facility, University of Bern), weighing 250 to 320 g, were used. The rats underwent 2-hour ischemia and 24-hour reperfusion by unilateral clamping of the femoral artery and additional use of a tourniquet. Five groups were divided according to intravenous treatment 5 minutes before ischemia: 50 IU/kg C1 INH (n = 5); 100 IU/kg C1 INH (n = 7); vehicle control (n = 5); nontreated control (n = 7); and normal, healthy control without intervention (n = 4). At the end, muscle edema, tissue viability, and histologic features were assessed. Deposition of immunoglobulin M, C1r, C4d, and fibrin and expression of plasminogen activator inhibitor 1, heparan sulfate (HS), E-selectin, and vascular cell adhesion molecule 1 were evaluated by fluorescence staining. In addition, high-mobility group box 1 protein was measured in plasma. ResultsEdema formation was reduced by C1 INH at two dosages, mirrored by improved histologic injury scores and preserved muscle viability. Deposition of immunoglobulin M, C4d, and fibrin was significantly decreased by 100 IU/kg C1 INH compared with nontreated controls. Pretreatment with 100 IU/kg C1 INH also significantly reduced HS shedding and expression of plasminogen activator inhibitor 1 as well as plasma levels of high-mobility group box 1 protein. ConclusionsPretreatment with both 50 and 100 IU/kg C1 INH attenuated reperfusion injury of rat hind limbs. Pretreatment with 100 IU/kg also preserved the endothelial HS layer as well as the natural, profibrinolytic phenotype of the endothelium. Prevention of endothelial cell activation by C1 INH may therefore be a promising strategy to prevent I/R injury in the clinical setting of peripheral vascular diseases and elective surgery on extremities.

Plasminogen activator inhibitor-1: a risk factor for deep vein thrombosis after total hip arthroplasty

BackgroundThe onset of deep vein thrombosis (DVT) in patients after total hip arthroplasty (THA) may expand or enlarge and subsequently lead to significant mortality. The objective of this study was to investigate potential risk factors for DVT in patients after THA.MethodsEligible patients with hip joint diseases who were scheduled for unilateral primary THA at our hospital were prospectively included into this study. The demographic and clinical features, preoperative plasma biomarkers were detailed, recorded, and compared. The multiple logistic regression analysis was used to evaluate potential risk factors for DVT.ResultsA total of 214 subjects were enrolled into our study cohort for the final analysis, and 23 of them have suffered DVT with an incidence of 9.5%. The performance of logistic regression analysis showed that preoperative expression of plasminogen activator inhibitor-1 (PAI-1) was an independent risk factor for the onset of DVT in patients after THA (OR 1.18, 95% CI 1.04–1.29; p = 0.011).ConclusionsOur study indicated preoperative plasma PAI-1 expression as an independent risk factor for DVT in patients who underwent THA.

Role of plasminogen activator inhibitor-1 gene polymorphisms in osteoporosis: A study in Chinese post-menopausal women

Osteoporosis is a chronic multifactorial disease characterized by deterioration of bone mass and is vulnerable to bone fracture. Plasminogen activator inhibitor-1 (PAI-1) is an important molecule for maintenance of optimum bone mass. Several single-nucleotide polymorphisms (SNPs) in PAI-1 have been reported to alter PAI-1 expression and/or the translational level. In this report, we explored the possible role of common PAI-1 gene polymorphisms on predisposition to osteoporosis in a Chinese cohort. A total of 364 post-menopausal Chinese women diagnosed of having osteoporosis and 350 healthy females hailing from similar areas were enrolled in this study. Five common SNPs (−844G &gt; A, −6754G/5G, +43G &gt; A, +9785G &gt; A and +11053T &gt; G) were genotyped by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). Relative expression of PAI-1 mRNA and plasma PAI-1 levels were quantified by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Prevalence of homozygous mutant (5G/5G) and minor allele (5G) of PAI-1 (−675 4G/5G) polymorphism was significantly more frequent in patients than in healthy controls (5G/5G: P &lt; 0.0001, odds ratio (OR) = 3.18; 5G: P &lt; 0.0001, OR = 1.65). Both plasma PAI-1 and relative mRNA expression levels were significantly lower in patients compared to healthy controls. Interestingly, the quantity of plasma PAI-1 and mRNA expression was correlated with PAI-1 (−675 4G/5G) polymorphism: subjects with 4G/4G genotype had elevated PAI-1 in comparison to homozygous mutant, and displayed lower quantity of PAI-1 protein and mRNA values. PAI-1 (−675 4G/5G) mutant is associated with susceptibility to development of osteoporosis in post-menopausal Chinese women. Furthermore, this variant in the promoter region alters plasma protein levels and relative expression of PAI-1.

Pulmonary Arterial Hypertension and Endothelial Dysfunction Is Linked to NADPH Oxidase-Derived Superoxide Formation in Venous Thrombosis and Pulmonary Embolism in Mice.

Pulmonary embolism (PE) results from deep vein thrombosis (DVT) and can lead to chronic thromboembolic pulmonary hypertension (CTEPH) involving vascular dysfunction. Mechanisms are incompletely understood, in part due to lack of mouse models. We induced PE in C57BL/6 mice by intravenous injection of thrombin (166 U/kg BW), confirmed by a sudden bradycardia, bradypnea, and an increase in pulmonary artery (PA) pressure observed by high-frequency ultrasound. While symptoms resolved rapidly after single thrombin application, repeated PEs resulted in sustained PA-pressure increase, increased PA superoxide formation assessed by oxidative fluorescent microtopography, increased PA gp91phox expression, and endothelial dysfunction assessed by isometric tension studies of isolated PA segments after 24 hours. DVT was modeled in C57BL/6 mice by ligation of the inferior vena cava (IVC). Importantly, small pulmonary emboli could be detected along with a mild phenotype of PA endothelial dysfunction and oxidative stress in the absence of PA-pressure elevation. mRNA expression of plasminogen activator inhibitor-1 was increased in PAs of mice with recurrent PE after repetitive thrombin injections and to a lesser extent in DVT mice. In summary, our data suggest that PA endothelial dysfunction, induced by gp91phox-derived ROS, is an early event upon repetitive PE. This phenomenon might help to elucidate the mechanisms of PA dysfunction in the pathogenesis of CTEPH.

A Combination of Chitosan, Cellulose, and Seaweed Polysaccharide Inhibits Postoperative Intra-abdominal Adhesion in Rats.

Intra-abdominal adhesion is a common complication after laparotomy. Conventional therapeutic strategies still cannot safely and effectively prevent this disorder. In this study, a combination of chitosan, cellulose, and seaweed polysaccharide (thereafter referred as CCS) was developed to significantly alleviate the formation of postoperative adhesion in rats with abdominal trauma. Transforming growth factor β1 (TGF-β1, an important promoter of fibrosis) and its downstream factors-namely, alpha-smooth muscle actin and plasminogen activator inhibitor-1 (PAI-1)-were effectively suppressed by CCS in vivo, and as a result, the activation of tissue plasminogen activator (tPA, may generate plasmin that is a fibrinolytic factor capable of breaking down fibrin) was significantly promoted, presenting antifibrosis effects of CCS. In addition, the activity of kinases [e.g., transforming growth factor-activated kinase 1 (TAK1), c-Jun N-terminal kinase (JNK)/Stress-activated Protein Kinase (SAPK), and p38] in the mitogen-activated protein kinase (MAPK) inflammation signaling pathway was also significantly suppressed by CCS in vivo, demonstrating anti-inflammatory functions of CCS. The histologic studies further confirmed the role of CCS in the inhibition of fibrosis, collagen deposition, inflammation, and vascular proliferation. These results indicate the clinical potential of CCS in the treatment of postoperative intra-abdominal adhesion. CCS may induce both antifibrosis and anti-inflammatory effects, potentially inhibiting the postoperative intra-abdominal adhesion. For antifibrosis effects, the expression of PAI-1 (a key factor for the adhesion formation) can be regulated by different TGF-β1-associated signaling pathways, such as the Smads/p53 pathway, metalloproteinase/tissue inhibitor of matrix metalloproteinases pathway, Mitogen-activated Extracellular signal-regulated Kinase (MEK)/extracellular regulated protein kinase (ERK) pathway, and Yes-associated protein/transcriptional coactivator with PDZ-binding motif pathway. Following the downregulation of PAI-1 achieved by CCS, the activation of tPA (which may generate plasmin that is a fibrinolytic factor capable of breaking down fibrin) is significantly promoted. For anti-inflammation effects, CCS may suppress the phosphorylation of classic kinases (e.g., TAK1, JNK, and p38) in the MAPK signaling pathway. In addition to the MAPK pathway, inflammatory pathways, such as Nuclear Factor-κ-gene Binding(NF-κB), MEK/ERK, and Ras homologue protein/Rho associated coiled coil forming protein, are associated with the formation of intra-abdominal adhesion. Therefore, the prevention mechanisms of CCS will be further investigated in the future, with a hope of fully understanding of antiadhesion effects.

Reactive oxygen species modulate HIF-1 mediated PAI-1 expression: involvement of the GTPase Rac1.

The hypoxia-inducible transcription factor HIF-1 mediates upregulation of plasminogen activator inhibitor-1 (PAI-1) expression under hypoxia. Reactive oxygen species (ROS) have also been implicated in PAI-1 gene expression. However, the role of ROS in HIF-1-mediated regulation of PAI-1 is not clear. We therefore investigated the role of the GTPase Rac1 which modulates ROS production in the pathway leading to HIF-1 and PAI-1 induction. Overexpression of constitutively activated (RacG12V) or dominant-negative (RacT17N) Rac1 increased or decreased, respectively, ROS production. In RacG12V-expressing cells, PAI-1 mRNA levels as well as HIF-alpha nuclear presence were reduced under normoxia and hypoxia whereas expression of RacT17N resulted in opposite effects. Treatment with the antioxidant pyr-rolidinedithiocarbamate or coexpression of the redox factor-1 restored HIF-1 and PAI-1 promoter activity in RacG12V-cells. In contrast, NFkappaB activation was enhanced in RacG12V-cells, but abolished by RacT17N. Thus, these findings suggest a mechanism explaining modified fibrinolysis and tissue remodeling in an oxidized environment.

Sickle Mice Are Sensitive to Hypoxia/Ischemia-Induced Stroke but Respond to Tissue-Type Plasminogen Activator Treatment.

The effects of lytic stroke therapy in patients with sickle cell anemia are unknown, although a recent study suggested that coexistent sickle cell anemia does not increase the risk of cerebral hemorrhage. This finding calls for systemic analysis of the effects of thrombolytic stroke therapy, first in humanized sickle mice, and then in patients. There is also a need for additional predictive markers of sickle cell anemia-associated vasculopathy. We used Doppler ultrasound to examine the carotid artery of Townes sickle mice tested their responses to repetitive mild hypoxia-ischemia- and transient hypoxia-ischemia-induced stroke at 3 or 6 months of age, respectively. We also examined the effects of tPA (tissue-type plasminogen activator) treatment in transient hypoxia-ischemia-injured sickle mice. Three-month-old sickle cell (SS) mice showed elevated resistive index in the carotid artery and higher sensitivity to repetitive mild hypoxia-ischemia-induced cerebral infarct. Six-month-old SS mice showed greater resistive index and increased flow velocity without obstructive vasculopathy in the carotid artery. Instead, the cerebral vascular wall in SS mice showed ectopic expression of PAI-1 (plasminogen activator inhibitor-1) and P-selectin, suggesting a proadhesive and prothrombotic propensity. Indeed, SS mice showed enhanced leukocyte and platelet adherence to the cerebral vascular wall, broader fibrin deposition, and higher mortality after transient hypoxia-ischemia. Yet, post-transient hypoxia-ischemia treatment with tPA reduced thrombosis and mortality in SS mice. Sickle mice are sensitive to hypoxia/ischemia-induced cerebral infarct but benefit from thrombolytic treatment. An increased resistive index in carotid arteries may be an early marker of sickle cell vasculopathy.

Inhibition of CD147 (Cluster of Differentiation 147) Ameliorates Acute Ischemic Stroke in Mice by Reducing Thromboinflammation.

Inflammation and thrombosis currently are recognized as critical contributors to the pathogenesis of ischemic stroke. CD147 (cluster of differentiation 147), also known as extracellular matrix metalloproteinase inducer, can function as a key mediator of inflammatory and immune responses. CD147 expression is increased in the brain after cerebral ischemia, but its role in the pathogenesis of ischemic stroke remains unknown. In this study, we show that CD147 acts as a key player in ischemic stroke by driving thrombotic and inflammatory responses. Focal cerebral ischemia was induced in C57BL/6 mice by a 60-minute transient middle cerebral artery occlusion. Animals were treated with anti-CD147 function-blocking antibody (αCD147) or isotype control antibody. Blood-brain barrier permeability, thrombus formation, and microvascular patency were assessed 24 hours after ischemia. Infarct size, neurological deficits, and inflammatory cells invaded in the brain were assessed 72 hours after ischemia. CD147 expression was rapidly increased in ischemic brain endothelium after transient middle cerebral artery occlusion. Inhibition of CD147 reduced infarct size and improved functional outcome on day 3 after transient middle cerebral artery occlusion. The neuroprotective effects were associated with (1) prevented blood-brain barrier damage, (2) decreased intravascular fibrin and platelet deposition, which in turn reduced thrombosis and increased cerebral perfusion, and (3) reduced brain inflammatory cell infiltration. The underlying mechanism may include reduced NF-κB (nuclear factor κB) activation, MMP-9 (matrix metalloproteinase-9) activity, and PAI-1 (plasminogen activator inhibitor-1) expression in brain microvascular endothelial cells. Inhibition of CD147 ameliorates acute ischemic stroke by reducing thromboinflammation. CD147 might represent a novel and promising therapeutic target for ischemic stroke and possibly other thromboinflammatory disorders.