Effect of the small molecule plasminogen activator inhibitor‐1 (PAI‐1) inhibitor,PAI‐749, in clinical models of fibrinolysis

Summary. Background: The principal inhibitor of fibrinolysis in vivo is plasminogen activator inhibitor‐1 (PAI‐1). PAI‐749 is a small molecule inhibitor of PAI‐1 with proven antithrombotic efficacy in several preclinical models. Objective: To assess the effect of PAI‐749, by using an established ex vivo clinical model of thrombosis and a range of complementary in vitro human plasma‐based and whole blood‐based models of fibrinolysis. Methods: In a double‐blind, randomized, crossover study, ex vivo thrombus formation was assessed using the Badimon chamber in 12 healthy volunteers during extracorporeal administration of tissue‐type plasminogen activator (t‐PA) in the presence of PAI‐749 or control. t‐PA‐mediated lysis of plasma clots and of whole blood model thrombi were assessed in vitro. The role of vitronectin was examined by assessing lysis of fibrin clots generated from purified plasma proteins. Results: There was a dose‐dependent reduction in ex vivo thrombus formation by t‐PA (P < 0.0001). PAI‐749 had no effect on in vitro or ex vivo thrombus formation or fibrinolysis in the presence or absence of t‐PA. Inhibition of PAI‐1 with a blocking antibody enhanced fibrinolysis in vitro (P < 0.05). Conclusions: Despite its efficacy in a purified human system and in preclinical models of thrombosis, the current study suggests that PAI‐749 does not affect thrombus formation or fibrinolysis in a range of established human plasma and whole blood‐based systems.     

The PAI‐1 4G/5G polymorphism is not associated with an increased risk of adverse pregnancy outcome in asymptomatic nulliparous women

Summary. Background: Plasminogen activator inhibitor type 1 (PAI‐1) is an important regulator of fibrinolysis. A common deletion polymorphism that results in a sequence of 4G instead of 5G in the promoter region of the gene is associated with a small increase in the risk of venous thromboembolism. Its potential association with adverse pregnancy events remains controversial. Objective: We aimed to assess the impact of the 4G PAI‐1 polymorphism on pregnancy outcomes in women who had no prior history of adverse pregnancy outcomes or personal or family history of venous thromboembolism. Patients/methods: This study represents a secondary investigation of a prior prospective cohort study investigating the association between inherited thrombophilias and adverse pregnancy events in Australian women. Healthy nulliparous women were recruited to this study prior to 22 weeks gestation. Genotyping for the 4G/5G PAI‐1 gene was performed using Taqman assays in an ABI prism 7700 Sequencer several years after the pregnancy was completed. Pregnancy outcome data were extracted from the medical record. The primary outcome was a composite comprising development of severe pre‐eclampsia, fetal growth restriction, major placental abruption, stillbirth or neonatal death. Results: Pregnancy outcome data were available in 1733 women who were successfully genotyped for this polymorphism. The primary composite outcome was experienced by 139 women (8% of the cohort). Four hundred and fifty‐nine women (26.5%) were homozygous for the 4G deletion polymorphism, while 890 (51.4%) were heterozygous. Neither homozygosity nor heterozygosity for the PAI‐1 4G polymorphism was associated with the primary composite outcome (homozygous OR = 1.30, 95% CI = 0.81–2.09, P = 0.28, heterozygous OR = 0.84, 95% CI = 0.53–1.31, P = 0.44) or with the individual pregnancy complications. Conclusion: The PAI‐1 4G polymorphism is not associated with an increase in the risk of serious adverse pregnancy events in asymptomatic nulliparous women.     

Treatment with either COX‐2 inhibitor or 5‐LOX inhibitor causes no compensation between COX‐2 pathway and 5‐LOX pathway in chronic aluminum overload‐induced liver injury in rats

This study was designed to observe the compensation between cyclooxygenase‐2 pathway and 5‐lipoxygenase pathway in chronic aluminum overload‐induced liver injury rats. A rat hepatic injury model of chronic aluminum injury was established by the intragastric administration of aluminum gluconate (Al3 + 200 mg/kg per day, 5 days a week for 20 weeks). The COX‐2 inhibitor [meloxicam (1 mg/kg)] and 5‐LOX inhibitor [caffeic acid (30 mg/kg)] were intragastrically administered 1 h after aluminum administration. The histopathology was detected by hematoxylin‐eosin staining. A series of biochemical indicators were measured with biochemistry assay or ELISAs. The expressions of COX‐2 and 5‐LOX were measured by immunohistochemistry. Our experimental results showed that aluminum overload caused a significant damage to the liver and also significantly increased the expressions of COX‐2, 5‐LOX and the levels of inflammation and oxidative stress. The administration of meloxicam and caffeic acid significantly protected livers against histopathological injury, significantly decreased plasma ALT, AST, and ALP levels, significantly decreased TNF‐α, IL‐6, IL‐1β levels, and oxidative stress. However, the administration of caffeic acid did not significantly increase the expression of COX‐2 compared with the model group. On the other hand, the administration of meloxicam also did not significantly increase the expression of 5‐LOX compared with the model group. Our results indicate that there is no compensation between COX‐2 pathway and 5‐LOX pathway by inhibiting either COX‐2 or 5‐LOX in chronic aluminum overload‐induced liver injury rat.     

The hyperfibrinolytic state of mice with combined thrombin‐activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor‐1 gene deficiency is critically dependent on TAFI deficiency

Summary. Background: Mice with single gene deficiency of thrombin‐activatable fibrinolysis inhibitor (TAFI) or plasminogen activator inhibitor‐1 (PAI‐1) have an enhanced fibrinolytic capacity. Objectives: To unravel the function and relevance of both antifibrinolytic proteins through the generation and characterization of mice with combined TAFI and PAI‐1 gene deficiency. Results: Mating of TAFI knockout (KO) mice with PAI‐1 KO mice resulted in the production of TAFI/PAI‐1 double‐KO mice that were viable, were fertile, and developed normally. In a tail vein bleeding model, the bleeding time and hemoglobin content of the TAFI/PAI‐1 double‐KO mice did not deviate significantly from those of the single‐KO mice or of the wild‐type (WT) counterparts. Interestingly, in ex vivo rotational thromboelastometry measurements with whole blood samples, TAFI KO mice and TAFI/PAI‐1 double‐KO mice were more sensitive to fibrinolytic activation with tissue‐type plasminogen activator than WT or PAI‐1 KO mice. This enhanced fibrinolytic capacity was confirmed in vivo in a mouse thromboembolism model, as shown by decreased fibrin deposition in the lungs of TAFI KO mice and TAFI/PAI‐1 double‐KO mice as compared with WT or PAI‐1 KO mice.Conclusions: TAFI gene inactivation predominantly contributes to the increased fibrinolytic capacity of TAFI and PAI‐1 double‐gene‐deficient mice, as observed in some basic thrombosis models.     

Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor α2‐antiplasmin

Summary. Background and objective: Resistance of thrombi to plasmin digestion depends primarily on the amount of α₂‐antiplasmin (α₂AP) incorporated within fibrin. Circulating prolyl‐specific serine proteinase, antiplasmin‐cleaving enzyme (APCE), a homologue of fibroblast activation protein (FAP), cleaves precursor Met‐α₂AP between ‐Pro12‐Asn13‐ to yield Asn‐α₂AP, which is crosslinked to fibrin approximately 13× more rapidly than Met‐α₂AP and confers resistance to plasmin. We reasoned that an APCE inhibitor might decrease conversion of Met‐α₂AP to Asn‐α₂AP and thereby enhance endogenous fibrinolysis. Methods and results: We designed and synthesized several APCE inhibitors and assessed each vs. plasma dipeptidyl peptidase IV (DPPIV) and prolyl oligopeptidase (POP), which have amino acid sequence similarity with APCE. Acetyl‐Arg‐(8‐amino‐3,6‐dioxaoctanoic acid)‐d ‐Ala‐l ‐boroPro selectively inhibited APCE vs. DPPIV, with an apparent K_i of 5.7 nm vs. 6.1 μm , indicating that an approximately 1000‐fold greater inhibitor concentration is required for DPPIV than for APCE. An apparent K_i of 7.4 nm was found for POP inhibition, which is similar to 5.7 nm for APCE; however, the potential problem of overlapping FAP/APCE and POP inhibition was negated by our finding that normal human plasma lacks POP activity. The inhibitor construct caused a dose‐dependent decrease of APCE‐mediated Met‐α₂AP cleavage, which ultimately shortened plasminogen activator‐induced plasma clot lysis times. Incubation of the inhibitor with human plasma for 22 h did not lessen its APCE inhibitory activity, with its IC₅₀ value in plasma remaining comparable to that in phosphate buffer. Conclusion: These data establish that inhibition of APCE might represent a therapeutic approach for enhancing thrombolytic activity.     

Aberrant methylation of mutL homolog 1 is associated with increased risk of non‐small cell lung cancer

Background

Non‐small cell lung cancer (NSCLC) is a common malignant tumor. DNA hypermethylation in the promoter region has been served as a potential molecular marker for several tumors. The goal of the current study was to assess the diagnostic ability of mutL homolog 1 (MLH1) promoter methylation in NSCLC.

Methods

A total of 111 NSCLC patients’ paired tissue samples were obtained to explore the association between MLH1 promoter methylation and NSCLC by methylation‐specific polymerase chain reaction (MSP) method. Public databases including The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were used to verify our findings.

Results

Our results showed a significantly higher MLH1 methylation frequency in tumor tissue samples than their paired adjacent tissues (P = .008). ROC curve indicated that MLH1MSP assay was a sensitive but not a specific method in the diagnosis for NSCLC (sensitivity = 0.964, specificity = 0.135, AUC = 0.550). And the association between the methylation level and clinical characteristics has no statistical significance. TCGA cohort evinced a higher methylation probability in tumor group compared with nontumor group (the mean β value: −0.449 [−0.467, −0.437] vs −0.466 [−0.472, −0.437], P = .011), which was consistent with our results. Meanwhile, an inverse correlation between MLH1 methylation and MLH1 expression was detected in TCGA and GEO databases.

Conclusions

The MSP method for MLH1 methylation was a sensitive but not a specific diagnostic method for NSCLC.

     

Renal tubulointerstitial expansion is associated with endothelial dysfunction and inflammation in type 1 diabetes

Objective. Diabetic nephropathy has been considered to be primarily of glomerular origin, but there is now compelling evidence that disruption of the tubulointerstitial architecture determines the outcome of diabetic nephropathy in interplay with the glomerular damage. We investigated whether reactive oxidative species, pro‐inflammatory cytokines and endothelial dysfunction were implicated in the progression of tubulointerstitial damage in young subjects with type 1 diabetes. Material and methods. In a prospective study, we investigated 18 young subjects (mean age 21 years) with type 1 diabetes and microalbuminuria. Quantitative morphometry concerning glomerular and tubulointerstitial changes was performed at baseline (i.e. mean duration of diabetes 10 years) and 2.5 and 8 years later. Markers of endothelial activation and inflammation, intercellular adhesion molecule‐1, vascular cell adhesion molecule‐1, tumour necrosis factor‐α, interleukin‐6, interleukin‐8 and highly sensitive C‐reactive protein were measured at baseline and after 8 years. Tissue plasminogen activator antigen and plasminogen activator inhibitor (PAI‐1 activity) and asymmetric dimethylargine (ADMA) were measured at baseline and after 2.5 years. Results. PAI‐1 activity at baseline was a significant independent variable of the 8‐year increment in interstitial volume fraction (Vv(Int/cortex)). ADMA/L‐arginine ratio at baseline was associated with the increment in Vv(Int/cortex) during 2.5 years (p<0.01), still significant after adjustment for covariates (p = 0.02). No associations between Vv(Int/cortex) and glomerular parameters, HaemoglobinA1c and urinary albumin excretion were observed. Conclusions. Biomarkers involved in interstitial volume expansion seem to be different from those of mesangial expansion in early diabetic nephropathy. PAI‐1 activity may have a predictive role in the development of the tubulointerstitial expansion.     

The PKB inhibitor Akti‐1/2 potentiates PAR‐1‐mediated platelet function independently of its ability to block PKB

Summary. Background: The role of PKB in platelet function is poorly defined due to the lack of genuinely selective small‐molecule inhibitors and limiting genetic models. Recently, a selective, non‐ATP‐competitive PKB inhibitor, Akti‐1/2 has been reported, but the efficacy and specificity against PKB activation in platelet function is unknown. Objective and methods: To determine the effect of the PKB inhibitor Akti‐1/2 on PKB activation and platelet function by Western blotting and aggregometry/flow cytometry, respectively. Results: Akti‐1/2 potently inhibited thrombin‐mediated PKB phosphorylation on Thr³⁰⁸ and Ser⁴⁷³ and phosphorylation of its downstream substrate GSK3β, with a negligible effect on the phosphorylation of pleckstrin, p38, ERK and JNK. Surprisingly, Akti‐1/2 strongly potentiated PAR‐1‐mediated platelet aggregation. This effect persisted in the presence of PI3 kinase inhibitors, indicating a mechanism of action that is independent of PKB. Potentiation of aggregation by Akti‐1/2 was associated with increased [Ca⁺⁺]_i, PKC activation and degranulation and was ablated by agents that antagonized this pathway. Conclusions: The PKB inhibitor Akti‐1/2 increases PAR‐1‐mediated platelet responses in a PKB‐independent, Ca⁺⁺/PKC‐dependent manner. This effect is strong and rapid and may impact on the therapeutic application of Akti‐1/2 and structurally related compounds.     

Plasminogen activator inhibitor type‐1 is an independent marker of metabolic disorders in young adults born small for gestational age

Summary. Background: Metabolic syndrome (MS) has been associated with being born small for gestational age (SGA). In epidemiological studies plasminogen activator inhibitor type‐1 (PAI‐1) levels have been associated with MS. Few studies have examined this association in subjects born SGA. Patients and methods: Five hundred and fifty‐seven SGA adults (birth weight < 10th percentile) were compared with 671 subjects with a birth weight between the 25th and 75th percentiles (control group). MS was defined using the World Health Organization (WHO) definition. Active PAI‐1 was measured on citrated plasma with bio‐immunoassay. Results: MS was more prevalent in the SGA group (8.7%) than in the control group (5.5%; P = 0.03). In both groups, PAI‐1 concentrations were significantly correlated with waist circumference, plasma triglycerides, homeostatic model assessment‐insulin resistance (HOMA‐IR) and associated with male sex and MS. PAI‐1 concentrations were significantly increased in the SGA group (12.2 ± 21.2 vs. 10.0 ± 13.5 IU mL^?1, P = 0.03) and this remained after adjustment of metabolic variables (P = 0.009). PAI‐1 concentrations above 4.9 IU mL^?1 (= median of PAI‐1 concentration in the control group) were present in 94% of the subjects with MS. Moreover, the adjusted odds ratio (OR) for having elevated PAI‐1 was 1.48 (1.08; 1.95) in the SGA group in comparison with the control group (P = 0.005). Conclusions: PAI‐1 plasma concentrations were significantly increased in SGA subjects independently of MS. These data suggest that elevation of PAI‐1 concentrations might be an indication of an abnormal secretion at the level of the adipose tissue, endothelial cells or liver and implicated in metabolic disorders reported in SGA subjects.     

Platelet endothelial cell adhesion molecule‐1 regulates collagen‐stimulated platelet function by modulating the association of phosphatidylinositol 3‐kinase with Grb‐2‐associated binding protein‐1 and linker for activation of T cells

Summary. Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)γ‐chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3‐kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb‐2‐associated binding protein‐1 (Gab1), which is regulated by binding of the Src homology 2 domain‐containing protein tyrosine phosphatase‐2 (SHP‐2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule‐1 (PECAM‐1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcRγ‐chain signaling via recruitment of SHP‐2 to phosphorylated immunoreceptor tyrosine‐based inhibitory motifs in PECAM‐1. Objective: To investigate the possibility that PECAM‐1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI‐mediated platelet activation in platelets. Methods: The ability of PECAM‐1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM‐1‐associated SHP‐2 in collagen‐stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen‐stimulated PI3K signaling. We therefore propose that PECAM‐1‐mediated inhibition of GPVI‐dependent platelet responses result, at least in part, from recruitment of SHP‐2–p85 complexes to tyrosine‐phosphorylated PECAM‐1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.     

High‐fat diet selectively decreases bone marrow lin−/CD117+ cell population in aging mice through increased ROS production

Bone marrow (BM) stem cells (BMSCs) are an important source for cell therapy. The outcome of cell therapy could be ultimately associated with the number and function of donor BMSCs. The present study was to evaluate the effect of long‐term high‐fat diet (HFD) on the population of BMSCs and the role of reactive oxygen species (ROS) in aging mice. Forty‐week‐old male C57BL/6 mice were fed with HFD for 3 months with regular diet as control. Experiments were repeated when ROS production was reduced in mice treated with N‐acetylcysteine (NAC) or using mice overexpressing antioxidant enzyme network (AON) of superoxide dismutase (SOD)1, SOD3, and glutathione peroxidase. BM and blood cells were analyzed with flowcytometry for lineage negative (lin^?) and Sca‐1⁺, or lin^?/CD117⁺, or lin^?/CD133⁺ cells. Lin^?/CD117⁺ cell population was significantly decreased with increased intracellular ROS and apoptosis and decreased proliferation in BM, not in blood, in HFD‐treated mice without change for Sca‐1⁺ or CD133⁺ cell populations in BM or blood. NAC treatment or AON overexpression effectively prevented HFD‐induced intracellular ROS production and reduction of BM lin^?/CD117⁺ population. These data suggested that long‐term HFD selectively decreased BM lin^?/CD117⁺ cell population in aging mice through increased ROS production.