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Plasminogen activator inhibitor type-2 (PAI-2), a member of the serpin family, is dramatically upregulated during pregnancy and in response to inflammation. Although PAI-2 exists in glycosylated and non-glycosylated forms in vivo, the majority of in vitro studies of PAI-2 have exclusively involved the intracellular non-glycosylated form. This study shows that exposure to inflammation-associated hypochlorite induces the oligomerisation of PAI-2 via a mechanism involving dityrosine formation. Compared to plasminogen activator inhibitor type-1 (PAI-1), both forms of PAI-2 are more resistant to hypochlorite-induced inactivation of its protease inhibitory activity. Holdase-type extracellular chaperone activity plays a putative non-canonical role for PAI-2. Our data demonstrate that glycosylated PAI-2 more efficiently inhibits the aggregation of Alzheimer’s disease and preeclampsia-associated amyloid beta peptide (Aβ), compared to non-glycosylated PAI-2 in vitro. However, hypochlorite-induced modification of non-glycosylated PAI-2 dramatically enhances its holdase activity by promoting the formation of very high-molecular-mass chaperone-active PAI-2 oligomers. Both PAI-2 forms protect against Aβ-induced cytotoxicity in the SH-SY5Y neuroblastoma cell line in vitro. In the villous placenta, PAI-2 is localised primarily to syncytiotrophoblast with wide interpersonal variation in women with preeclampsia and in gestational-age-matched controls. Although intracellular PAI-2 and Aβ staining localised to different placental cell types, some PAI-2 co-localised with Aβ in the extracellular plaque-like aggregated deposits abundant in preeclamptic placenta. Thus, PAI-2 potentially contributes to controlling aberrant fibrinolysis and the accumulation of misfolded proteins in states characterised by oxidative and proteostasis stress, such as in Alzheimer’s disease and preeclampsia.

Endometrial stem cells are located in the basal layer of the endometrium, and they are responsible for the cyclic regeneration of the uterus during the menstrual cycle. Recent studies have revealed that recurrent pregnancy loss is associated with an age-related stem cell deficiency in the endometrium. Therefore, intensive study of endometrial stem cell aging may provide new insights for preventing recurrent pregnancy loss. Sonic hedgehog (SHH) signaling has been identified as a morphogen during the embryonic development processes. In addition to this canonical function, we found that the age-associated decline in regenerative potential in the endometrium may be due to decreased SHH-signaling integrity in local stem cells with aging. Importantly, the current study also showed that SHH activity clearly declines with aging both in vitro and in vivo, and exogenous SHH treatment significantly alleviates various aging-associated declines in multiple endometrial stem cell functions, suggesting that SHH may act as an endogenous anti-aging factor in human endometrial stem cells. Moreover, we found that stem cell senescence may enhance SERPINB2 expression, which in turn mediates the effect of SHH on alleviating senescence-induced endometrial stem cell dysfunctions, suggesting that SERPINB2 is a master regulator of SHH signaling during the aging process.

Plasminogen activator inhibitor 2 (PAI2) has been shown to be associated with invasive phenotypes and prognosis in hepatocellular carcinoma (HCC). However, its biological roles and underlying mechanisms in invasion of HCC have not been explored. The present study aimed to address the issues. First, sub-lines in that PAI2 was stably overexpressed and silenced were established based on MHCC97H and BEL7402 cell lines, respectively. Wound-healing and transwell assays were applied to evaluate cell migration and invasion. Urokinase-type plasminogen activator (uPA) activity was measured using an ELISA kit. Real-time RT-PCR and western blotting were used to show gene expression at mRNA and protein levels. E2F1 expression in human specimens was determined by tissue microarray-based immunohistochemical staining. The sub-lines, MHCC97H-PAI2 and BEL7402-siPAI2, were successfully established. The two sub-lines carried much lower and higher migration and invasion powers, respectively, in contrast to the controls. In MHCC97H-PAI2 sub-line, intra-medium uPA activity was significantly decreased, while RB expression was obviously elevated, compared with the controls. The BEL7402-siPAI2 sub-line presented the opposite trend. To identify the role of RB/E2F1 pathway, we transiently overexpressed E2F1 in MHCC97H-PAI2 sub-line, and largely reversed the inhibitory effects of PAI2 on cell migration and invasion, through regulating multiple matrix metalloproteinases and epithelial–mesenchymal transition. In HCC specimens, E2F1 expression was much higher in tumor than in non-tumor tissues, and was significantly related to Edmondson–Steiner grade, overall as well as tumor-free survival. Our data suggest that PAI2 inhibits invasive potential of HCC cells via uPA- and RB/E2F1-related mechanisms.

Objectives Preeclampsia (PE) is a serious complication of pregnancy. The fibrinolytic system play crucial roles regarding placentation and evolution of PE. Aim To study comprehensively components of the fibrinolytic system and fibrin lysability in women with PE. Design and Methods 117 women with PE and matched controls were included. Tissue type plasminogen activator (t-PA), plasminogen, PAI-1, plasmin inhibitor (PI), D-dimer, the fibrinolytic potential of dextran sulphate euglobulin fraction (DEF), PAI-2, polymere PAI-2, fibrin clot lysability, thrombin activatable fibrinolysis inhibitor (TAFI) and fibrinogen were assessed. Results Women with PE had significantly increased concentrations of t-PA and PAI-1, whereas the plasma concentration of PAI-2 was significantly lower compared to controls, p Conclusion This study demonstrates that PE is associated with an affected t-PA/PAI-1 system, decreased PAI-2 and increased fibrin lysability. Furthermore, PAI-2 has the potential to polymerize during pregnancy.

There is abundant evidence that the urokinase-type plasminogen activator (uPA), its inhibitors PAI-1 and PAI-2 (plasminogen activator inhibitor type-1 and type-2) and its cells surface receptor (uPA-R, CD87) play a fundamental role in tumor invasion and metastasis and are of significant prognostic significance for many tumor types. We performed a systematic Med-line search on uPA, PAI, uPA-R and (epithelial) ovarian cancer (EOC). The majority of malignant EOC specimens show moderate to strong immunostating of tumor and stromal cells. Overexpression of u-PA and PAI-1 can be found in more the 75% of primary ovarian carcinomas, in most metastatic EOC samples and all examined epithelial ovarian cancer cell lines. uPA overexpression in primary specimens was significantly associated with tumor stage, grade, residual disease status after cytoreductive surgery, and poor clinical outcome. This may be explained by increased chemoresistance, a lower resectability and more aggressive tumor biology and tumor dissemination in patients with high uPA and PAI-1. Several therapeutical approaches aimed at inhibiting the uPA/uPAR functions have shown to possess anti-tumor effects in vitro and in animal models. When treating a patient with advanced ovarian cancer it may to be assumed that inhibiting the progression of established (micro) metastases may be more therapeutically relevant than trying to destroy all tumor cells which is not possible in most cases with current systemic treatment modalities. Taking into account the role of uPA and PAI in cell detachment, formation of new stroma, tumor cell reimplantation and metastasis uPA inhibition should be further investigated as maintenance treatment in patients with advanced EOC.

Background/Aim: Placental syndromes (PS) are characterized by endothelial dysfunction complicating placental dysfunction. Possible markers for endothelial dysfunction and amount of trophoblast are fibronectin and plasminogen activator inhibitor-2 (PAI-2), respectively. We aimed (1) to determine whether in women with recurrent PS (rPS), this complication is preceded by deviating fibronectin- and PAI-2-levels, and (2) whether this is dependent on pre-pregnant plasma volume (PV). Methods: In 36 former patients, we determined fibronectin- and PAI-2-levels in blood-samples collected preconceptionally and at 12-16 weeks in their next pregnancy. Differences were analyzed between pregnancies with rPS (n = 12) and without rPS (non-rPS, n = 24) using linear mixed models, with subanalyses based on pre-pregnant normal or subnormal PV. Results: We observed higher fibronectin-levels at 12-16 weeks (p < 0.05 and p < 0.01, respectively) and lower PAI-2-levels at 16 weeks (p < 0.01) in the rPS subgroup, the intergroup differences being larger in women with subnormal PV. Conclusion: We showed that former PS patients who developed rPS have raised fibronectin- and reduced PAI-2-levels already in early/mid pregnancy. These deviations are even more prominent in women with subnormal pre-pregnant PV, supporting development of a 2-step screening program for former patients to identify the high-risk subgroup of women who may benefit from closer surveillance.

The urokinase-type plasminogen activator (uPA) system is closely related to the occurrence and progression of cancer in many aspects. Previous studies demonstrated that the conclusions about the prognosis value of uPA, plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2) in lung cancer are controversial, so this study was performed for the exploration of the predictive effect of uPA, PAI-1 and PAI-2 on the overall survival (OS) of resectable pulmonary adenocarcinoma patients.UPA, PAI-1 and PAI-2 expression levels were assayed by immunohistochemical staining based on tissue microarray (TMA) that is composed of formalin-fixed paraffin-embedded specimens from 84 resectable lung adenocarcinoma patients from July 2004 to June 2009. The relationship of IHC, mRNA expression levels of three molecules were investigated respectively. The three molecules' relationship with clinicopathologic parameters and OS was explored by Chi-square, Kaplan-Meier, and Cox regression analyses. The Cancer Genome Atlas (TCGA) database was used to analyze differential gene expressions of RNA-sequencing data of pulmonary adenocarcinoma and normal tissues, and Kaplan-Meier methods were adopted to confirm the prognostic value of uPA, PAI-1 and PAI-2 in resectable lung adenocarcinoma in TCGA database and the R package MethylMix was used to conduct an analysis integrating methylation data and gene expression of RNA-sequencing data based on TCGA.UPA, PAI-1 and PAI-2 had much higher IHC expression levels in tumor than those in the normal tissues (uPA, Z = -10.511; PAI-1, Z = -4.836; PAI-2, Z = -6.794; all P0.0001). High DNA methylation level of gene uPA resulted in the decrease of its expression. In addition, expression level of PAI-2 was positively associated with tumor size (χOur data show that, the expression levels of uPA, PAI-1 and PAI-2 are significantly up-regulated in resectable lung adenocarcinoma. Besides, this study highlights PAI-1 as a latent adverse prognostic factor in resectable adenocarcinoma of lung.

Purpose To investigate the effect of miR-200c/PAI-2 on macrophage polarization into M2-type TAMs in TNBC. Methods and materials PAI-2 expression in MDA-MB-231con, MDA-MB-231miR-200ab and MDA-MB-231miR-200c breast cancer cells was evaluated by RT-PCR and immunofluorescence (IF), while the expression of the TAM marker F4/80 and the M2-type TAM marker CD206 in MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 mouse lung metastatic tumor tissues was examined with immunohistochemistry (IHC). The effects of RAW264.7 cells on MDA-MB-231con, MDA-MB-231miR-200c and MDA-MB-231miR-200c/siPAI-2 were examined by transwell co-culture. CD206 expression in RAW264.7 cells were confirmed by immunostaining. The level of PAI-2 and IL-10 in the co-culture supernatants were assessed using ELISA. Results 1. RT-PCR and IF analysis showed that PAI-2 was upregulated in MDA-MB-231miR-200c cells. 2. IHC assays analysis showed that the numbers of F4/80 and CD206 positive cells were increased in MDA-MB-231miR-200c tumor tissues, while in MDA-MB-231miR-200c/siPAI-2 tumor tissues were decreased. 3. Transwell co-culture assays analysis showed that MDA-MB-231miR-200c cells significantly promoted the cell migration ability compared with the control group, while knockdown PAI-2 significantly inhibited the cell migration ability (P Conclusions miR-200c promotes the malignant progressions of TNBC by PAI-2 upregulation and M2 phenotype macrophages polarization.

In cancer, cellular senescence is a complex process that leads to inhibition of proliferation of cells that may develop a neoplastic phenotype. A plethora of signaling pathways, when dysregulated, have been shown to elicit a senescence response. Two well-known tumor suppressor pathways, controlled by the p53 and retinoblastoma proteins, have been implicated in maintaining the cellular senescence phenotype. Kindlin-2, a member of an actin cytoskeleton organizing and integrin activator proteins, has been shown to play a key role in the regulation of several hallmarks of several cancers, including breast cancer (BC). The molecular mechanisms whereby Kindlin-2 regulates cellular senescence in BC tumors remains largely unknown. Here we show that Kindlin-2 regulates cellular senescence in part through its interaction with p53, whereby it regulates the expression of the p53-responsive genes; i.e., SerpinB2 and p21, during the induction of senescence. Our data show that knockout of Kindlin-2 via CRISPR/Cas9 in several BC cell lines significantly increases expression levels of both SerpinB2 and p21 resulting in the activation of hallmarks of cellular senescence. Mechanistically, interaction between Kindlin-2 and p53 at the promotor level is critical for the regulated expression of SerpinB2 and p21. These findings identify a previously unknown Kindlin-2/p53/SerpinB2 signaling axis that regulates cellular senescence and intervention in this axis may serve as a new therapeutic window for BCs treatment.

Background The proliferation of pulmonary arterial smooth muscle cells (PASMCs) and subsequent pulmonary vascular remodeling leads to pulmonary arterial hypertension (PAH). Understanding the underlying mechanisms and identifying molecules that can suppress PASMCs proliferation is critical for developing effective pharmacological treatment. We previously showed that plasminogen activator inhibitor-2 (PAI-2) inhibited human PASMC (hPASMCs) proliferation in vitro. However, its inhibitory effect on PAH remains to be determined, and the mechanism remains to be illustrated. Methods We compared serum PAI-2 levels between PAH patients and healthy controls, and examined the correlation between PAI-2 level and disease severity. In monocrotaline-induced PAH rats, we examined the effects of exogenous PAI-2 administration on pulmonary vascular remodeling and PAH development. The effect of PAI-2 and potential mechanisms was further examined in cultured hPASMCs. Results The serum PAI-2 was decreased in PAH patients compared with controls. PAI-2 level was negatively correlated with mean pulmonary arterial pressure and estimated systolic pulmonary arterial pressure in ultrasonic cardiogram, while positively correlated with 6-min walking distance. In rats, administration of exogenous PAI-2 significantly reversed monocrotaline-induced PAH, as indicated by the decrease in right ventricle systolic pressure, right ventricular hypertrophy index and percent media thickness of pulmonary arterioles. Further mechanistic investigation in hPASMCs showed that PAI-2 inhibited cell proliferation by preventing the activation of PI3K/Akt and ERK pathways. Conclusion PAI-2 is downregulated in PAH patients. PAI-2 attenuates PAH development by suppressing hPASMCs proliferation via the inhibition of PI3K/Akt and ERK pathways. PAI-2 may serve as a potential biomarker and therapeutic target for PAH.

SerpinB2 (plasminogen activator inhibitor type 2) is constitutively expressed at high levels by differentiating keratinocytes in mice and humans; however, the physiological function of keratinocyte SerpinB2 remains unclear. Herein, we show that SerpinB2 −/− mice are more susceptible to contact dermatitis after topical application of dinitrofluorobenzene, and show enhanced inflammatory lesions after topical applications of phorbol ester. Untreated SerpinB2 −/− mice showed no overt changes in epithelial structure, and we were unable to find evidence for a role for keratinocyte SerpinB2 in regulating immunity, apoptosis, IL-1β production, proteasomal activity, or wound healing. Instead, the phenotype was associated with impaired skin barrier function and a defective stratum corneum, with SerpinB2 −/− mice showing increased transepidermal water loss, increased overt loss of stratum corneum in inflammatory lesions, and impaired stratum corneum thickening after phorbol ester treatment. Immunoblotting suggested that SerpinB2 (cross-linked into the cornified envelope) is present in the stratum corneum and retains the ability to form covalent inhibitory complexes with urokinase. Data suggest that the function of keratinocyte SerpinB2 is protection of the stratum corneum from proteolysis via inhibition of urokinase, thereby maintaining the integrity and barrier function of the stratum corneum, particularly during times of skin inflammation. Implications for studies involving genetically modified mice treated with topical agents and human dermatological conditions, such as contact dermatitis, are discussed.

SerpinB2, also known as plasminogen activation inhibitor type 2 (PAI-2), is classically viewed as an inhibitor of fibrinolysis. However, we show herein a distinct, hitherto unrecognized role for SerpinB2 in hemostasis. Mice deficient in SerpinB2 expression and mice with an active site mutation in SerpinB2, both showed significant reductions in tail bleeding times. This hemostatic phenotype was associated with platelets, with SerpinB2 and SerpinB2-urokinase complexes clearly present in platelet fractions, and immunohistochemistry of blood clots suggesting SerpinB2 is associated with platelet aggregates. Thromboelastography illustrated faster onset of clot formation in blood from SerpinB2 deficient mice, whereas clotting of platelet-free plasma was unaffected. The results appear consistent with the low circulating SerpinB2 levels and hypercoagulation seen during pre-eclampsia; however, SerpinB2 was not detected in human platelets.

Introduction: Accurate early risk prediction for gestational diabetes mellitus (GDM) would target intervention and prevention in women at the highest risk. We evaluated novel biomarker predictors to develop a first-trimester risk prediction model in a large multiethnic cohort. Methods: Maternal clinical, aneuploidy and pre-eclampsia screening markers (PAPP-A, free hCGβ, mean arterial pressure, uterine artery pulsatility index) were measured prospectively at 11–13+6 weeks’ gestation in 980 women (248 with GDM; 732 controls). Nonfasting glucose, lipids, adiponectin, leptin, lipocalin-2, and plasminogen activator inhibitor-2 were measured on banked serum. The relationship between marker multiples-of-the-median and GDM was examined with multivariate regression. Model predictive performance for early (< 24 weeks’ gestation) and overall GDM diagnosis was evaluated by receiver operating characteristic curves. Results: Glucose, triglycerides, leptin, and lipocalin-2 were higher, while adiponectin was lower, in GDM (p < 0.05). Lipocalin-2 performed best in Caucasians, and triglycerides in South Asians with GDM. Family history of diabetes, previous GDM, South/East Asian ethnicity, parity, BMI, PAPP-A, triglycerides, and lipocalin-2 were significant independent GDM predictors (all p < 0.01), achieving an area under the curve of 0.91 (95% confidence interval [CI] 0.89–0.94) overall, and 0.93 (95% CI 0.89–0.96) for early GDM, in a combined multivariate prediction model. Conclusions: A first-trimester risk prediction model, which incorporates novel maternal lipid markers, accurately identifies women at high risk of GDM, including early GDM.

Background Thalidomide is an effective therapy in children with inflammatory bowel disease refractory to standard treatments, but thalidomide-induced peripheral neuropathy (TiPN) limits its long-term use. We aimed to investigate the risk factors and the outcome of TiPN in children with inflammatory bowel disease. Methods Within a retrospective multicenter cohort study, we evaluated prevalence and evolution of TiPN. Clinical data and candidate genetic profiles of patients with and without TiPN were compared with detect predisposing factors. Results One hundred forty-two patients were identified. TiPN was found in 72.5% of patients (38.7% clinical and instrumental alterations, 26.8% exclusive electrophysiological anomalies, and 7.0% exclusive neurological symptoms). Median TiPN-free period of treatment was 16.5 months; percentage of TiPN-free patients was 70.0% and 35.6% at 12 and 24 months of treatment, respectively. The risk of TiPN increased depending on the mean daily dose (50-99 mg/d adjusted hazard ratio 2.62; 95% confidence interval [CI], 1.31-5.21; 100-149 mg/d adjusted hazard ratio 6.16; 95% CI, 20.9-13.06; >150 mg/d adjusted hazard ratio 9.57; 95% CI, 2.6-35.2). Single nucleotide polymorphisms in ICAM1 (rs1799969) and SERPINB2 (rs6103) genes were found to be protective against TiPN (odds ratio 0.15; 95% CI, 0.03-0.82 and 0.36; 95% CI, 0.14-0.88, respectively). TiPN was the cause of drug suspension in 41.8% of patients. Clinical symptoms resolved in 89.2% of cases, whereas instrumental alteration persisted in more than half of the patients during a short follow-up. Conclusions In children with inflammatory bowel disease, TiPN is common but mild and generally reversible. Cumulative dose seems to be the most relevant risk factor, whereas polymorphisms in genes involved in neuronal inflammation may be protective.

Impaired fibrinolytic activity is implicated in the pathogenesis of recurrent spontaneous abortion (RSA). This case-control study assessed the prevalence of polymorphisms in fibrinolytic system genes in RSA. Cases comprised 202 Sinhalese women who had experienced at least two first-trimester spontaneous abortions and had no living children; controls were 202 women with no history of spontaneous abortion and two or more living children. The groups were matched for age and ethnicity. DNA was genotyped using the Sequenom MassARRAY system. The PLAUR rs4251923 A (OR 95% CI 2.3 [1.3 to 4.0]), SERBP2 rs6098 A (OR 95% CI 1.4 [1.1 to 1.9]) and SERBP2 rs6103 C alleles (OR 95% CI 1.4 [1.1 to 1.9]) were increased in the RSA group compared with controls. The prevalence of PLAUR rs4251923/ SERBP2 rs6098/ SERBP2 rs6103 GG/AA/CC (OR 95% CI 2.4 [1.2 to 4.9], GA/GA/GC(OR 95% CI 3.9 [1.3 to 11.2]), GA/AA/CC (OR 95% CI 2.9 [1.0 to 8.6] and GA/GG/GG (OR 95% CI 21.3 [1.1 to 410.3]) genotypes were also increased in cases. Polymorphisms in the fibrinolytic system genes are associated with RSA in Sinhalese women. These likely impair implantation.

Introduction Our objective was to assess fetal sex specific differences in first trimester placental biomarkers of both physiological and pathological pregnancies and their interaction with environmental influences. This study is embedded in the Generation R Study, a prospective cohort study. Methods Only live singleton births were included. Linear regression was performed to assess the effect of sex on first trimester placental biomarkers. Interaction analyses were performed to assess interaction of fetal sex with environmental influences. First trimester soluble fms-like tyrosine kinase (s-Flt1), placental growth factor (PLGF), plasminogen activator inhibitor (PAI-2) and homocysteine levels were assessed. Results Significant fetal sex specific differences in placental biomarkers were observed. S-Flt1, PAI-2 and PLGF log transformated concentrations were 0.08 ng/mL (95% CI 0.05; 0.11), 0.07 ng/mL (95% CI 0.06; 0.09) and 0.04 pg/mL (95% CI 0.01; 0.06) higher in case of female as compared to male placentas. In pregnancies complicated by pre-eclampsia (PE), preterm birth (PTB) or a newborn being small for gestational age (SGA) no fetal sex specific differences were observed. Interaction analyses suggest that concentrations of s-Flt1, PLGF and PAI-2 decrease in male placentas in the case of hyperhomocysteinemia but remain equal in female placentas. Discussion Fetal sex affects early placentation processes with discrepancies regarding pregnancies complicated by PE, PTB or a newborn being SGA. This suggests that other mechanisms causing these complications may dominate the fetal sex effect. The differences concerning homocysteine suggest that fetal sex dependent placental gene–environment interactions exist. Conclusion Fetal sex specific differences in placental biomarkers exist.

SummaryBrain metastasis is an ominous complication of cancer, yet most cancer cells that infiltrate the brain die of unknown causes. Here, we identify plasmin from the reactive brain stroma as a defense against metastatic invasion, and plasminogen activator (PA) inhibitory serpins in cancer cells as a shield against this defense. Plasmin suppresses brain metastasis in two ways: by converting membrane-bound astrocytic FasL into a paracrine death signal for cancer cells, and by inactivating the axon pathfinding molecule L1CAM, which metastatic cells express for spreading along brain capillaries and for metastatic outgrowth. Brain metastatic cells from lung cancer and breast cancer express high levels of anti-PA serpins, including neuroserpin and serpin B2, to prevent plasmin generation and its metastasis-suppressive effects. By protecting cancer cells from death signals and fostering vascular co-option, anti-PA serpins provide a unifying mechanism for the initiation of brain metastasis in lung and breast cancers.

Although preeclampsia remains a major cause of maternal and fetal morbidity and mortality, its pathogenesis is not fully understood. Coagulation and fibrinolysis changes were suggested to have a role in the pathogenesis of preeclampsia.A case-control study was conducted in Medani Hospital, Sudan, to investigate thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen-activated inhibitor (PAI) in women with severe preeclampsia. Obstetrics and medical history was gathered using questionnaire. TAFI, PAI-1, and PAI-2 levels were measured using ELISA.In comparison with the controls, women with severe preeclampsia had significantly higher levels [mean (SD)] of TAFI [3.4 (1.1) vs. 3.0(0.7) ng/ml, P = 0.019], PAI-1 [3.2 (1.3) vs. 2.5(1.0), IU/ml, P = 0.001], and significantly lower PAI-2 level [4.2(1.3) vs. 5.8(2.6) ng/ml, P0.001]. In linear regression, severe preeclampsia was significantly associated with TAFI (0.408 ng/ml, P = 0.038), PAI-1 (0.722, IU/ml P = 0.003), and PAI-2 (-1.745, ng/ml, P0.001).The current study revealed a significant increase level of TAFI and PAI-1, coupled with a decrease in PAI-2 in women with severe preeclampsia in comparison with the control group.

The urokinase-plasminogen activator (u-PA), its receptor (u-PAR) and the inhibitors of u-PA (PAI-1 and PAI-2) provide a multi-molecular system in leucocytes that exerts pleiotropic functions influencing the development of inflammatory and immune responses. The objective of the present study was to examine the ability of water soluble extracts (WSE) obtained from traditional Greek yoghurt made from bovine or ovine milk to modulate the expression of u-PA, u-PAR, PAI-1 and PAI-2 in ovine monocytes and neutrophils. WSE were obtained from 8 commercial traditional type Greek yoghurts made from ovine or bovine milk. WSE upregulated the expression of all 4 u-PA related genes in monocytes but the upregulation was much higher in the PAI-1 (10-fold) than in u-PA and u-PAR (3–4 fold) thus, shifting the system towards inhibition. In line with this observation, WSE reduced total and membrane-bound u-PA activity in monocytes. In neutrophils, WSE caused small (50–60%) but significant (P < 0·05) reductions in expression of u-PAR and PAI-2 but had no effect on expression of u-PA, PAI-1 and on total cell-associated and membrane-bound u-PA activity. WSE from yoghurts made from bovine or ovine milk were essentially equally effective in affecting the u-PA system except for the u-PAR gene in ovine neutrophils that was affected (reduced) by the ovine and not the bovine WSE. In conclusion, peptides present in WSE modulated the expression of u-PA related genes but the effect was much more prominent in monocytes than in neutrophils.

WOS: 000399955500030 PubMed ID: 27781272 Background and ObjectiveThe aim of this study was to evaluate the effect of nonsurgical periodontal therapy on clinical parameters and gingival crevicular fluid levels of tissue/blood vessel-type plasminogen activator (t-PA) and plasminogen activator inhibitor-2 (PAI-2) in patients with periodontitis, with or without rheumatoid arthritis (RA). Material and MethodsFifteen patients with RA and chronic periodontitis (RA-P), 15 systemically healthy patients with chronic periodontitis (H-P) and 15 periodontally and systemically healthy volunteers (C) were included in the study. Plaque index, gingival index, probing pocket depth, clinical attachment level, bleeding on probing, gingival crevicular fluid t-PA and PAI-2 levels, erythrocyte sedimentation rate, serum C-reactive protein and disease activity score were evaluated at baseline and 3 mo after mechanical nonsurgical periodontal therapy. ResultsAll periodontal clinical parameters were significantly higher in the RA-P and H-P groups compared with the C group (p < 0.001) and decreased significantly after treatment (p < 0.001). Pretreatment t-PA levels were highest in the RA-P group and significantly decreased post-treatment (p = 0.047). Pre- and post-treatment PAI-2 levels were significantly lower in controls compared with both periodontitis groups (p < 0.05). Gingival crevicular fluid volume and the levels of t-PA and PAI-2 were significantly correlated. ConclusionIn patients with periodontitis and RA, nonsurgical periodontal therapy reduced the pretreatment gingival crevicular fluid t-PA levels, which were significantly correlated with gingival crevicular fluid PAI-2 levels. The significantly higher t-PA and PAI-2 gingival crevicular fluid levels in periodontal patients, regardless of systemic status, suggest that the plasminogen activating system plays a role in the disease process of periodontitis. Ankara University Scientific Research Projects Office, Ankara, Turkey [08B3334005] This study was supported by the Ankara University Scientific Research Projects Office, Ankara, Turkey (08B3334005). The authors declare that they have no conflicts of interest.

// Tiefeng Jin 1, 2, * , Hoe Suk Kim 1, * , Sul Ki Choi 1, 3 , Eun Hye Hwang 1 , Jisu Woo 1 , Han Suk Ryu 4 , Kwangsoo Kim 5 , Aree Moon 6 and Woo Kyung Moon 1, 3 1 Department of Radiology, Seoul National University Hospital, Jongno-gu, Seoul 03080, Korea 2 Department of Pathology & Cancer Research Center, Yanbian University Medical College, Yanji 133002, China 3 Department of Biomedical Science, Seoul National University College of Medicine, Seoul National University, Jongno-gu, Seoul 03080, Korea 4 Department of Pathology, Seoul National University Hospital, Jongno-gu, Seoul 03080, Korea 5 Division of Clinical Bioinformatics, Biomedical Research Institute, Seoul National University Hospital, Jongno-gu, Seoul 03080, Korea 6 Duksung Innovative Drug Center College of Pharmacy, Duksung Women's University, Dobong-gu, Seoul 01369, Korea * These authors have contributed equally to this work Correspondence to: Woo Kyung Moon, email: moonwk@snu.ac.kr Keywords: SerpinB2, SerpinE1, metastasis, microRNA-200c/141, triple negative breast cancer Received: June 06, 2016 Accepted: February 12, 2017 Published: February 24, 2017 ABSTRACT The microRNA-200 (miR-200) family is associated with tumor metastasis and poor patient prognosis. We found that miR-200c/141 cluster overexpression upregulated SerpinB2 in the MDA-MB-231 triple-negative (TN) breast cancer cell line. We observed transcription factor (c-Jun, c-Fos, and FosB) upregulation, nuclear localization of c-Jun, and increased SerpinB2 promoter-directed chloramphenicol acetyltransferase activity in miR-200c/141 cluster-overexpressing cells relative to controls. Additionally, miR-124a and miR-26b, which directly target SepinB2, were downregulated compared to controls. In mouse xenograft models, miR-200c/141 cluster overexpression promoted lymph node and lung metastasis, and siRNA-mediated SerpinB2 knockdown decreased lung metastasis, suggesting that SerpinB2 mediates miR-200c/141-induced lung metastasis. We also explored the clinical significance of SerpinB2 protein status through analysis of primary breast tumor samples and The Cancer Genome Atlas (TCGA) data. High SerpinB2 levels were associated with reduced survival and increased lymph node metastasis in breast cancer patients. SerpinB2 was overexpressed in the TN breast cancer subtype as compared to the luminal subtype. The present study demonstrates that SerpinB2 promotes miR-200c/141 cluster overexpression-induced breast cancer cell metastasis, and SerpinB2 overexpression correlates with increased metastatic potential and unfavorable outcomes in breast cancer patients. SerpinB2 may be a useful biomarker for assessing metastasis risk in breast cancer patients.

Angiotropism/pericytic mimicry and vascular co-option involve tumor cell interactions with the abluminal vascular surface. These two phenomena may be closely related. However, investigations of the two processes have developed in an independent fashion and different explanations offered as to their biological nature. Angiotropism describes the propensity of tumor cells to spread distantly via continuous migration along abluminal vascular surfaces, or extravascular migratory metastasis (EVMM). Vascular co-option has been proposed as an alternative mechanism by which tumors cells may gain access to a blood supply. We have used a murine brain melanoma model to analyze the interactions of GFP human melanoma cells injected into the mouse brain with red fluorescent lectin-labeled microvascular channels. Results have shown a striking spread of melanoma cells along preexisting microvascular channels and features of both vascular co-option and angiotropism/pericytic mimicry. This study has also documented the perivascular expression of Serpin B2 by angiotropic melanoma cells in the murine brain and in human melanoma brain metastases. Our findings suggest that vascular co-option and angiotropism/pericytic mimicry are closely related if not identical processes. Further studies are needed in order to establish whether EVMM is an alternative form of cancer metastasis in addition to intravascular cancer dissemination.

Item does not contain fulltext OBJECTIVE: To estimate whether the imbalance of angiogenic factors (soluble fms-like tyrosine kinase-1, placental growth factor) and fibrinolytic factors (plasminogen activator inhibitor-2 [PAI-2]) might affect placentation in early pregnancy. METHODS: We studied the associations of maternal soluble fms-like tyrosine kinase-1, placental growth factor, and PAI-2 concentrations in the first trimester (before 18 weeks of gestation) and soluble fms-like tyrosine kinase-1 and placental growth factor concentrations in the second trimester (18-25 weeks of gestation) with placental function and adverse pregnancy outcomes. This study was embedded in a population-based prospective cohort study. Data were used from 7,519 women. Biomarker concentrations were divided into deciles and evaluated in multivariable linear and logistic regression models. RESULTS: First-trimester high soluble fms-like tyrosine kinase-1 was associated with a 5.2% lower uterine artery index in the second-trimester and a 1.6% higher birth weight (55 g, confidence interval [CI] 15-95). Neither in the first nor in the second trimester were soluble fms-like tyrosine kinase-1 concentrations significantly associated with preeclampsia. First-trimester low placental growth factor was associated with a 6.1% higher uterine artery index and a 3.4% lower birth weight (-115 g, CI -157 to -74). First-trimester low placental growth factor was associated with fetal growth restriction (odds ratio [OR] 2.62, CI 1.68-4.08) and preeclampsia (OR 2.46, CI 1.49-4.08). First-trimester low PAI-2 was associated with a 1.9% higher uterine artery index and a 2.7% lower birth weight (-94 g, CI -136 to -51). First-trimester low PAI-2 was associated with a higher risk of fetal growth restriction (OR 2.22, CI 1.39-3.55). CONCLUSION: First-half-of-pregnancy concentrations of soluble fms-like tyrosine kinase-1, placental growth factor, and PAI-2 are associated with uteroplacental vascular resistance, placental weight, and birth weight. Moreover, first-trimester placental growth factor and PAI-2 are associated with an increased risk of adverse pregnancy outcomes. LEVEL OF EVIDENCE: II. 01 juni 2012

Plasminogen activator inhibitor (PAI)-2 expression is acutely upregulated in pregnancy, inflammation, infection, and other pathophysiological conditions. Circumstances that prevent PAI-2 upregulation are associated with chronic pathology. Altogether this strongly suggests that PAI-2 is one of the many proteins that maintain homeostasis during damage or stress. However, several functions ranging from a classical serpin to various intracellular roles have been ascribed to PAI-2 and, because none of these have been definitively proven in vivo, to this day its precise role or roles remains an enigma. This review readdresses the evidence supporting a role for PAI-2 in fibrinolysis and proteolysis within extracellular environments and includes a review of the many potential intracellular functions attributed to PAI-2.

Urokinase (uPA) plays an essential role in the activation of plasminogen to plasmin, and together with its receptor (uPAR), tissue activator (tPA) and urokinase inhibitors (PAI 1, PAI 2, PAI 3 and protease nexin) forms the plasminogen activator system (PAS), a component of metastatic cascade importantly contributing to the invasive growth and angiogenesis of malignant tumours. In our project we examined the expression of uPA, uPAR, PAI 1 and PAI 2 in tumor tissue and we also studied the plasma levels of PAI 1 before and after the initiation of therapy in patients with colorectal carcinoma in relationship to grade of tumor and the treatment response. In our prospective evaluation we included 80 patients treated for adenocarcinoma of the colon and rectum. Analysis of collected data revealed statistically significant evidence of a relationship between the level of PAI 1 in plasma before treatment and grade of the tumor, which increases with tumor grade (p=0.025). We demonstrated that there exists a statistically significant relationship between the expression of PAI 2 (p0.001) and uPAR (p=0.031) and grade of tumor. We also confirmed a statistically significant relationship between soluble levels of PAI 1 before treatment and therapeutic response (p=0.021). In our group of patients the expression of uPA, uPAR, PAI 1 and 2 in tumor tissue in relation to response to treatment was also assessed. Our results suggest that the greater expression of these parameters in tumor tissue is linked to a worse response to therapy. In conclusion, PAS factors help as a prognostic indicators and could also act as a predictive factor in colorectal carcinoma.

The main objective of the present study was to examine whether genes implicated in the plasminogen activating cascade: urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR) and plasminogen activator inhibitors type 1 (PAI-1) and type 2 (PAI-2) are expressed in a differential manner in ovine blood monocytes and neutrophils obtained from healthy and mastitic dairy ewes. A total of 48 blood samples were collected from 8 healthy and 8 mastitic dairy ewes over a period of 3 weeks. Streptococcus agalactiae was detected in milk samples isolated from mastitic animals. Results indicated that expression of all four genes was very low in monocytes and neutrophils isolated from healthy animals. In contrast, there was a 2- to 5-fold increase (P

To evaluate initial periodontal treatment effects on gingival crevicular fluid (GCF) interleukin-6 (IL-6), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-2 (PAI-2), albumin levels in type 2 diabetic patients.GCF samples were collected from 20 type 2 diabetic, 22 non-diabetic non-smokers all with chronic periodontitis at baseline, 1-, 3-months following initial periodontal treatment. Biochemical analysis was performed by ELISA. Data were tested by Mann-Whitney U, Wilcoxon tests.The total amounts of albumin, IL-6, tPA, PAI-2 decreased significantly in diabetics after treatment (1- and 3-months) whereas, only PAI-2 decreased in non-diabetic group at 3-months (p0.05). There were statistically significant differences between the diabetics and non-diabetics at all time points for albumin, PAI-2 and at 1-, 3-months for GCF volume (p0.050) but only at baseline for IL-6 (p0.050).Present data suggest clinical improvements are less apparent in diabetic chronic periodontitis patients as reflected by disease markers in GCF and by an increase in concentrations of inflammatory proteins IL-6, tPA, and PAI-2 in GCF of this patient group following initial periodontal treatment.

In characterizing mice with targeted disruption of the SerpinB2 gene, we observed animals that were small at birth with delayed growth and decreased life expectancy. Although this phenotype cosegregated with homozygosity for the inactive SerpinB2 allele, analysis of homozygous SerpinB2 -deficient mice derived from two additional independent embryonic stem (ES) cell clones exhibited no growth abnormalities. Examination of additional progeny from the original SerpinB2 -deficient line revealed recombination between the small phenotype ( smla ) and the SerpinB2 locus. The locus responsible for smla was mapped to a 2.78-Mb interval approximately 30 Mb proximal to SerpinB2 , bounded by markers D1Mit382 and D1Mit216. Sequencing of Irs1 identified a nonsense mutation at serine 57 (S57X), resulting in complete loss of IRS1 protein expression. Analysis of ES cell DNA suggests that the S57X Irs1 mutation arose spontaneously in an ES cell subclone during cell culture. Although the smla phenotype is similar to previously reported Irs1 alleles, mice exhibited decreased survival, in contrast to the enhanced longevity reported for IRS1 deficiency generated by gene targeting. This discrepancy could result from differences in strain background, unintended indirect effects of the gene targeting, or the minimal genetic interference of the S57X mutation compared with the conventionally targeted Irs1 -KO allele. Spontaneous mutations arising during ES cell culture may be a frequent but underappreciated occurrence. When linked to a targeted allele, such mutations could lead to incorrect assignment of phenotype and may account for a subset of markedly discordant results from experiments independently targeting the same gene.

In the colon, the urokinase-type plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitors, PAI-1 and PAI-2, are implicated in the transition from mucosa to adenoma and tumour progression. However, expression in the mucosa adjacent, or distant, to an adenoma has not yet been investigated. Three biopsies from mucosae adjacent (20 cm, ipsilateral) and distant (contralateral) to an isolated tubular adenoma were analysed in 14 patients and 8 controls. Laser microdissection isolated stromal and epithelial crypt components, and quantitative RT-PCR analyses of uPA, uPAR, PAI-1 and PAI-2 mRNA levels were performed. Among controls, no significant differences in the markers were noted. With left colon isolated tubular adenoma, uPA, uPAR, and PAI-2 mRNA levels were significantly increased in the adjacent mucosal stroma compared to epithelial crypt levels (p < 0.05). In right colon adenoma, the mRNA levels of these 3 molecular markers were significantly increased only in the adjacent mucosal stromal samples (p < 0.05). Isolated tubular adenoma in the colon increases significantly the mRNA levels of 3 proteolysis-associated molecular markers in the stromal, but not in the epithelial, components of adjacent mucosa. These results suggest the presence of regional and dynamic interactions in apparently non-involved mucosae.

Fluctuations in sex steroid hormones, which are also noticeable through the menstrual cycle of women, may impact periodontal health. The aim of this study is to evaluate the effect of hormonal changes occurring in the menstrual cycle on gingival inflammation and the gingival crevicular fluid (GCF) levels of interleukin 6 (IL-6), prostaglandin E(2) (PGE(2)), tissue plasminogen activator (t-PA), and plasminogen activator inhibitor-2 (PAI-2).Twenty-five gingivitis patients and 25 periodontally healthy subjects having regular menstrual cycles were seen at menstruation (ME) (1 to 2 days of menstruation), ovulation (OV) (12 to 14 days), and premenstrual phases (PM) (22 to 24 days). GCF and saliva samples were collected and clinical parameters including plaque index and bleeding on probing were recorded at each menstrual phase. Salivary estrogen and progesterone levels were analyzed to determine exact menstrual cycle days. GCF levels of IL-6, PGE(2), t-PA, and PAI-2 were measured by enzyme-linked immunosorbent assay.The percentages of sites with bleeding on probing were significantly higher in ME (60.85 +/- 18.36) and OV (58.92 +/- 25.04) than in the PM (40.12 +/- 20.10) phase in the gingivitis group (P0.001; repeated measures analysis of variance), whereas it was similar for all phases in the healthy group (P0.05; repeated measures analysis of variance). GCF levels of IL-6 were significantly elevated in gingivitis patients compared to healthy subjects in all phases (P = 0.004, P = 0.041, and P = 0.046 for ME, OV, and PM, respectively; Mann-Whitney U test). GCF levels of IL-6, PGE(2), t-PA, and PAI-2 were unchanged in different menstrual phases in both groups (P0.05; Friedman test).The present study suggests that changes in the sex steroid hormones during menstrual cycles might have a limited effect on the inflammatory status of gingiva, but GCF cytokine levels were not affected.

Two serine proteases, urokinase and tissue type, control the activation of plasminogen to plasmin. These proteases are in turn specifically inhibited by plasminogen activator inhibitors type 1 and 2 (PAI-1 and PAI-2), both of which belong to the serine protease inhibitor (serpin) superfamily. Very little information is available on the role of PAI-1 and PAI-2 in ruminants, in mammary gland involution and in the adipose tissue. In this paper we describe the isolation of the full-length cDNAs of ovine PAI-1 and PAI-2 using a polymerase chain reaction based strategy. The ovine PAI-1 cDNA comprised of 1460bp and it is characterized by a coding region of 1209bp, and 5'- and 3'-UTR regions of 147 and 104bp, respectively. The deduced amino acid sequence consists of 402 amino acids. The ovine PAI-2 cDNA is comprised of 2128bp and it is characterized by a coding region of 957bp and 5'- and 3'-UTR regions of 58 and 819bp respectively. The deduced amino acid sequence consists of 416 amino acids. Three-dimensional models of the putative protein products of both cDNAs showed that the proteins bear a high similarity with their human counterparts. Real-time PCR revealed that the two inhibitors were predominantly expressed in the ovine mammary gland and adipose tissue. Furthermore, PAI-1 and PAI-2 mRNA levels were higher in the involuting mammary tissue and the adipose tissue obtained from non-lactating ewes compared to the corresponding values in tissues obtained from lactating ewes. These data are consistent with the notion that the plasminogen activation cascade plays a key role in involution of the mammary gland. The upregulation of expression of both inhibitors in the adipose tissue during the non-lactating period is a rather enigmatic observation. However, the expression of both inhibitors (PAI-1 and PAI-2) together with that of urokinase type plasminogen activator and its receptor previously reported by our group, strengthen the suggestion that the adipose tissue functions as an endocrine besides an energy storage organ.

Objective. In patients with preeclampsia maternal plasma concentration of plasminogen activator inhibitor-2 (PAI-2) is reduced. The objective of the study was to determine if the altered levels of PAI-2 precede the onset of the disease. Methods. Plasma PAI-2 was measured at 11–13 weeks of gestation in 119 pregnancies that developed preeclampsia, 85 that developed gestational hypertension and 204 controls. Results. There were no significant differences in PAI-2 between the preeclampsia, gestational hypertension and controls (1.07 MoM, 1.08 MoM and 0.96 MoM). Conclusion. The decrease in plasma PAI-2 observed in preeclampsia does not precede the clinical onset of the disease.

Background Cholangiocarcinoma (CCA) has an abundance of tumor stroma which plays an important role in cancer progression via tumor-promoting signals. This study aims to explore the microRNA (miRNA) profile of CCA-associated fibroblasts (CCFs) and the roles of any identified miRNAs in CCA progression. Methods miRNA expression profiles of CCFs and normal skin fibroblasts were compared by microarray. Identified downregulated miRNAs and their target genes were confirmed by real-time PCR. Their binding was confirmed by a luciferase reporter assay. The effects of conditioned-media (CM) of miRNA mimic- and antagonist-transfected CCFs were tested in CCA migration in wound healing assays. Finally, the levels of miRNA and their target genes were examined by real-time PCR and immunohistochemistry in clinical CCA samples. Results miR-15a was identified as a downregulated miRNA in CCFs. Moreover, PAI-2 was identified as a novel target gene of miR-15a. Recombinant PAI-2 promoted migration of CCA cells. Moreover, CM from miR-15a mimic-transfected CCFs suppressed migration of CCA cells. Lower expression of miR-15a and higher expression of PAI-2 were observed in human CCA samples compared with normal liver tissues. Importantly, PAI-2 expression correlated with poor prognosis in CCA patients. Conclusions These findings highlight the miR-15a/PAI-2 axis as a potential therapeutic target in CCA patients. Electronic supplementary material The online version of this article (10.1186/s12943-018-0760-x) contains supplementary material, which is available to authorized users.

Protease inhibitor serpin-B2 (plasminogen activator inhibitor-2 [PAI-2]) protects pRb from degradation in human papillomavirus (HPV)-18+ HeLa cells. Our objective was to assess whether the pRb-mediated HPV-suppressive effect of PAI-2 in cancer cell lines has implications in the outcome of HPV infections. Cervical biopsy specimens from 225 women were analyzed for PAI-2 expression to assess its value as a predictor of cervical intraepithelial neoplasia (CIN) grade, high-risk (HR) HPV at baseline, outcomes of HR-HPV infections, and the development of incident CIN. PAI-2 expression increased in parallel with lesion grade. Nuclear PAI-2 expression was significantly related to HR-HPV detection and had a linear relationship with HR-HPV load. PAI-2 expression was of no value in predicting the outcomes of HR-HPV infections. The same was true for PAI-2 as a predictor of surrogate end points (incident CIN 1+, CIN 2+) of progressive disease. PAI-2 expression is up-regulated on transition from CIN 2 to CIN 3. The HR-HPV suppressive effects of PAI-2 were not related to more favorable outcomes of HR-HPV infections or lower risk of disease progression to CIN., Supported by the European Commission, INCO-DEV Programme (contract ICA4-CT-2001-10013).

Summary. Background: The mechanisms leading to pregnancy-related hypertensive disorders, and pregnancy-induced hypertension (PIH) and pre-eclampsia (PE) in particular, are still not clear. Diagnostic criteria are clinical because specific markers of the condition are lacking. A role of the fibrinolytic system has been suggested. Objectives: We aimed to evaluate the behavior of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1), PAI-2, and the placental hormone inhibin-A in women with a normal pregnancy vs. women with pregnancies complicated by PIH or PE. Methods: Blood samples were drawn between the 25th and 30th gestational week (GW) and between the 31st and 36th GW in order to assay t-PA, PAI-1, PAI-2 and inhibin-A; routine biochemical exams, ultrasonography umbilical artery pulsatility index (PI), placental weight and newborn weight were measured. Results: In pregnancies complicated by hypertensive disorders, PAI-1 levels were higher than in controls and increased significantly after the 25th GW, especially in PE, as did inhibin-A. PAI-2 levels were significantly lower after the 30th GW in patients with PIH and PE. The PAI-1/PAI-2 ratio was significantly higher in PE patients than in controls as of the 25th GW, but only after the 30th GW in patients with PIH. Inhibin-A was significantly correlated with fibrinolytic parameters, and inversely with newborn weight. Receiver–operator characteristic curves for PAI-1 and inhibin-A showed a high sensitivity and specificity for PE. PAI-2 correlated with newborn and placental weight, and inversely with PI of the umbilical artery. Conclusions: Fibrinolytic tests (especially PAI-1) and inhibin-A monitoring during pregnancy may help in the early diagnosis of pregnancy-related hypertensive disorders.

Few studies have been carried out to investigate whether distinct areas of full term placenta express different amounts of markers involved in the placental haemostasis and angiogenesis. A possible relationship between the expression of genes involved in the haemostasis and angiogenesis of human placenta has not been investigated.Twenty-eight fresh human placentas (35-41 weeks of gestation) from uneventful pregnancies were dissected with two different methods. Quantitative mRNA expression of the tissue factor (TF), TF pathway inhibitor (TFPI), TFPI-2, plasminogen activator inhibitor (PAI-2), annexin V (Anx V), vascular endothelial growth factor (VEGF), and thrombomodulin (TM) genes was evaluated by quantitative real time PCR system. Histology of each sample was graded.Gene expression of all the considered markers was not significantly different in each area, using both the different methods of dissection. A significant correlation (p0.05) was found between the expression of TF and TFPI-2. TF and TFPI-2 were significantly (p0.05) associated with VEGF, whereas a stronger association (p0.01) was found between TFPI and TFPI-2. TFPI and TFPI-2 were strongly associated with PAI-2 expression (p0.01).In placentas with central cord insertion, gene expression is not dependent on the method of sampling site. A significant relationship between haemostasis and angiogenesis in at term placentas was shown.

The corpus luteum (CL) offers the opportunity to study high proliferative processes during its development and degradation processes during its regression. We examined the mRNA expression of matrix metalloproteases (MMP)-1, MMP-2, MMP-9, MMP-14, MMP-19, tissue inhibitor of MMP (TIMP)-1, TIMP-2, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), uPA-receptor (uPAR), PA-inhibitors (PAI)-1, PAI-2 in follicles 20 h after GnRH application, CLs during days 1–2, 3–4, 5–7 and 8–12 of the oestrous cycle as well as after induced luteolysis. Cows in the mid-luteal phase were injected with Cloprostenol and the CLs were collected at 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2α injection. Real-time RT-PCR determined mRNA expressions. Expression from 20 h after GnRH to day 12: MMP-1, MMP-2, MMP-14 and tPA showed a clear expression, but no regulation. TIMP-1 and uPAR mRNA increased when compared with the follicular phase. TIMP-2, MMP-9, MMP-19 and uPA increased from the follicular phase to days 8–12. PAI-1 and PAI-2 expression increased from days 1–7 and decreased to days 8–12. Induced luteolysis: MMP-1, MMP-2, MMP-9, MMP-14, MMP-19 and TIMP-1 all increased at different time points and intensities, whereas TIMP-2 was constantly decreased from 24 to 64 h. The plasminogen activator system and their inhibitors were up-regulated from 2 to 64 h, tPA was already increased after 0.5 h. Immunohistochemistry for MMP-1, MMP-2, MMP-14: an increased staining for MMP-1 and MMP-14 was seen in large luteal cells beginning 24 h after PGF2α application. MMP-2 showed a strong increase in staining in endothelial cells at 48 h.

The plasminogen activator inhibitor type 2 (PAI2) when labelled with (213)Bi forms the (213)Bi-PAI2 alpha conjugate (AC). This AC has been shown to be efficacious in preclinical studies with breast, ovarian, prostate and pancreatic cancers. The objectives of this study were to investigate the pharmacokinetics and in vivo stability of (213)Bi-PAI2 in mice, its toxicity in mice and rabbits; and to determine whether a prior injection of a metal chelator (Ca-DTPA) or lysine can reduce toxicity by decreasing renal uptake.Two chelators (CHX-A"-DTPA and cDTPA) were used for preparation of the (213)Bi-PAI2 conjugate, for intraperitoneal administration in mice and ear vein injection in rabbits. The mice were sacrificed at different time points for pharmacokinetic studies. Blood and organs were collected for toxicity studies for all groups.Both chelators were found to have similar %ID/g in the kidneys over four hours. Mice and rabbits did not show any short term toxicity over 13 weeks at 1420 MBq/kg and 120 MBq/kg (213)Bi-PAI2 respectively. Kidney uptake was decreased three fold by lysine. Radiation nephropathy was observed at 20-30 weeks in mice, leading to severe weight loss, whereas severe and widespread renal tubular necrosis was observed at 13 weeks in rabbits.Radiation nephropathy is the dose limiting toxicity observed in mice and rabbits. Lysine can reduce kidney uptake by three fold. Based on long-term monitoring, the maximum tolerance doses (MTD) are 350 and 120 MBq/kg for mice and rabbits respectively.

Plasminogen activator inhibitors (PAIs) play critical roles in regulating cellular invasion and fibrinolysis. An increase in the ratio of PAI-1/PAI-2 in placenta and maternal serum is suggested to result in excessive intervillous fibrin deposition and placental infarction in pregnancies complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR). In the current study we used dual (maternal and fetal) perfusion of human term placentas to examine the release of PAIs to the intervillous space. ELISA revealed a significant time-dependent increase in total PAI-1 levels in maternal perfusate (MP) between 1 and 7h of perfusion. Conversely, PAI-2 levels decreased resulting in a 3-fold increase in the PAI-1/PAI-2 ratio in MP. Levels of PAI-1, but not PAI-2, in placental tissue extracts increased during perfusion. In perfusions carried out with xanthine and xanthine oxidase (X + XO), compounds used to generate reactive oxygen species (ROS), no time-dependent increase in total PAI-1 levels was observed. In addition, X + XO treatment promoted a 3-fold reduction in active PAI-1 levels in MP, indicating that ROS decrease PAI-1 release to MP. The finding of a time-dependent change in patterns of PAI expression and response to ROS indicates the utility of dual perfusion as a model to dissect mechanism(s) promoting aberrant fibrinolysis in pregnancies complicated by PE and IUGR.

Objectives: To assess possible relationships between peri-implant crevicular fluid (PICF) volumes, biochemical markers of the peri-implant immune response, and periodontitis-associated genotype. Material and methods: PICF samples from 29 implant maintenance patients, 24 wearing overdentures, five having single crowns and bridgework (11 patients with peri-implantitis and 18 individuals with healthy peri-implant conditions), were analyzed for per site and per crevicular-fluid-volume concentrations of interleukin-1β, plasminogen activator inhibitor type 2, and prostaglandin E2 by ELISA. Associations between the three substance concentrations and to crevicular fluid flow rate were analyzed by linear regression analysis. The possible differentiating influence of the composite interleukin-1A and -1B genotype on the patients' peri-implant health and biochemical inflammatory status was checked formally with t-test statistics and the Wilcoxon' test. One implant per patient was chosen for analysis. Results: In patients with healthy peri-implant conditions, genotype-positive individuals showed elevated crevicular fluid flow rates and at the same time reduced mediator concentrations. In patients with an implant affected from peri-implantitis, no statistically significant influence of the periodontitis-associated genotype around the fixture can be stated. There was no statistical difference between per site and per crevicular-fluid-volume concentration analyses. All three mediator concentrations were positively related to each other, while there was a strong negative correlation between crevicular fluid volume and plasminogen activator inhibitor 2 or prostaglandin E2. Conclusions: The Interleukin-1 polymorphism investigated exerted only little influence on the peri-implant crevicular immune response, and this influence appeared to be of limited impact in sites with established peri-implantitis lesions.

Recent findings suggest that the urokinase-type plasminogen activator (uPA), its receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and -2 (PAI-2) play key roles in cancer invasion. The prognostic value of components of this system is well established in breast cancer. However, little is known of its involvement in pancreatic cancer (PC).Quantitative real-time polymerase chain reaction (Q-RT-PCR) was used on tissue-banked specimens and immunohistochemistry (IHC) on paraffin specimens was used to measure expression of uPA, uPAR, PAI-1, and PAI-2 proteins in 46 PC and 12 cystadenoma specimens. Results were related to survival using Cox's proportional hazards testing.Increased expression of uPA, uPAR, and PAI-1 in PC tissue were independently associated with a higher Union Internationale Contre le Cancer [International Union Against Cancer (UICC)] tumor stage (P0.001) and were intercorrelated (P0.001). Overexpression of uPAR indicated reduced survival (P = 0.03). Conversely, PAI-2 messenger ribonucleic acid (mRNA) overexpression, which occurred in 21 of 46 tumors, negatively correlated with tumor size (P = 0.008) and survival (P0.007) but not with uPA, uPAR, or tumor stage. There was good agreement between PAI-2 mRNA value and IHC score (P0.001). Using Cox's stepwise analysis, PAI-2 mRNA value (HR = 0.24; P = 0.001) and UICC tumor stage (HR = 2.014; P = 0.001) independently predicted survival. An IHC score for PAI-2 of 3+ or 4+ also independently predicted improved survival (HR = 2.72; P = 0.025).The uPA/uPAR/PAI-1 system is activated in advanced pancreatic cancer and may account for the tumor's aggressive behavior, whereas PAI-2 expression appears to be independent of uPA/uPAR/PAI-1 and is associated with improved prognosis. Because of its intercorrelation with mRNA expression, PAI-2 IHC may be used as an indicator of survival.

Contains fulltext : 53585.pdf (Publisher’s version ) (Closed access) OBJECTIVE: The plasminogen activator system and beta-hCG may affect neural crest cells and angiogenesis, and thereby embryogenesis. Therefore, we investigated these parameters in amniotic fluids of pregnancies with a complex congenital malformation. STUDY DESIGN: In a case-control study amniotic fluid samples were collected from 62 pregnancies with a complex congenital malformation and from 110 healthy control pregnancies at an obstetric department of a large university hospital in the Netherlands. We determined concentrations of tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), plasminogen activator inhibitors (PAI-1, PAI-2), tPA approximately PAI-1 and uPA approximately PAI-1 complexes, and beta-hCG with enzyme-linked immunosorbent assays. Mann-Whitney U-tests and analysis of covariance, adjusting for gestational and maternal age, were performed for data comparisons. RESULTS: Compared with controls, cases demonstrated significantly lower adjusted geometric mean levels of uPA (24%), tPA (> or =19%) and tPA approximately PAI-1 (35%). Cases showed significantly higher adjusted mean levels of beta-hCG (> or =48%) and PAI-2 (10 ng/mL) than controls. Mean PAI-1 and uPA approximately PAI-1 levels were comparable between both groups. CONCLUSIONS: Disturbances in the plasminogen activator system and beta-hCG levels are suggested to be involved in the pathogenesis of complex congenital malformations by affecting neural crest cell migration and angiogenesis.

Background Plasminogen activator inhibitor −2 (PAI-2) is an important molecular that plays a crucial role in vascular homeostasis and constitutes a critical response mechanism to cardiovascular injury, such as atherosclerosis, coronary artery disease (CAD). Methods The aim of the current study was to explore the association between the variants in PAI-2 gene and CAD and its prognoses. The three variants (rs8093048, rs9946657, rs9320032) of the PAI-2 gene were detected in 407 patients with CAD and 518 control subjects. All patients with CAD underwent one-year follow-up for major adverse cardiac events (MACE). Results The frequencies of the TT genotype and T allele of rs8093048 was significantly higher in CAD patients than that in control subjects (7.6 % vs.3.5 %, P = 0.003, 28.1 % vs.21.7 %, P

Background and aims Evidence continues to accumulate that athero-protective effects of high-density lipoprotein (HDL) depend to some degree on effective HDL functionality and that such functionality can become degraded in the setting of chronic inflammation. To investigate this issue, we have studied a group of post-myocardial infarction patients with high levels of C-reactive protein as an indicator of chronic inflammation and with concurrently high levels of HDL cholesterol. For these patients we have demonstrated high-risk for recurrent cardiac events as well as a strong association of risk with a polymorphism of the gene ( SERPINB2 ) for plasminogen activator inhibitor-2 (PAI-2) presumptively reflective of an important role for fibrinolysis in risk. However, additional processes might be involved. The current work sought to characterize processes underlying how PAI-2 might be involved in the generation of risk. Methods Multivariate population data were leveraged using Bayesian network modeling, a graphical probabilistic approach for knowledge discovery, to generate networks reflective of influences on PAI-2 polymorphism-associated risk. Results Modeling results revealed three individual networks centering on the PAI-2 polymorphism with specific features providing information relating to how the polymorphism might associate with risk. These included racial dependency, platelet clot initiation and propagation, oxidative stress, inflammation effects on HDL metabolism and coagulation, and induction and termination of fibrinolysis. Conclusions Beyond direct association of a PAI-2 polymorphism with recurrent risk in post-myocardial infarction patients, results suggest that PAI-2 likely plays a key role leading to risk through multiple pathophysiologic processes. Such knowledge could potentially be valuable with individualization of patient care.

To investigate expression of gene markers for the plasminogen system, inflammation, and bone resorption/remodelling in peri-implant crevicular fluid samples from healthy subjects, subjects with mucositis and subjects with peri-implantitis. A possible inhibitory effect of suppuration on the analysis of gene expression in samples from subjects with peri-implantitis was also analysed.Peri-implant crevicular fluid (PICF) was sampled from 25 healthy subjects (H), 25 subjects with mucositis (M) and 25 subjects with peri-implantitis (P) using paper points and suction tips. The samples were analysed by quantitative polymerase chain reaction (qPCR). The following biomarkers associated with the plasminogen system, inflammation and bone resorption/ remodelling were investigated: interleukin-1 beta (IL-1β), interleukin 8 (IL-8), tissue plasminogen activator (tPA), plasminogen activator inhibitor 2 (PAI-2), tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CatK).IL-1β and IL-8 were significantly upregulated in the P group, and tPA and PAI-2 were significantly upregulated in the M group. These four genetic markers were oppositely regulated in samples from the subjects in the mucositis compared with the peri-implantitis group. TRAP and CatK showed no differences between the groups. The presence of suppuration did not have a detectable effect on gene analysis in samples from subjects with peri-implantitis.Markers for the plasminogen system and inflammation could be used to distinguish between mucositis and peri-implantitis. The results suggested that the plasminogen system was sufficiently upregulated allowing for resolution of inflammation and healing at the inflamed implant site in subjects with mucositis, whereas such upregulation was insufficient resulting in impaired healing and prolonged inflammation in subjects with peri-implantitis. The combination of tissue inflammation and low levels of tPA was a strong predictor of marginal bone loss in this study. It may be an interesting candidate for the unambiguous diagnosis of mucositis and peri-implantitis independent of radiographs and could possibly constitute a powerful future tool for rapid assessment of the periimplant tissue condition and the effect of subject treatment.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers in the world. Epidemiological survey studies have verified that the development of ESCC relates to a complex interactive process between multiple genetic susceptibilities and environmental exposure. Serpins are a broadly distributed family of protease inhibitors and have been recognized as tumor suppressors in multiple cancer types. While previous studies have reported that Serpin polymorphisms are associated with tumorigenesis, the genetic and functional single nucleotide polymorphisms (SNP) in these genes appear to be complex and remain to be elucidated. In this study, a total of 500 ESCC cases and 500 matched controls in a Southwest China population were evaluated for six SNPs in the exons of three Serpin genes (SerpinB5, SerpinB2, and SerpinE1). Among the six SNPs, the C allele of rs2289519 and rs2289520 in SerpinB5 showed decreased risk of ESCC and the variants might interact with smoking status. Haplotype analysis showed that the T-G haplotype (corresponding to rs2289519-rs2289520) increased the risk of ESCC, while the C-C haplotype decreased the risk. We also found that SerpinB5 gene mRNA expression was significantly downregulated in ESCC cell lines and patient specimen while there is no change in protein structure with different haplotypes. Our results demonstrated that the expression of SerpinB5 was downregulated in ESCC, and the positive SNPs might be associated with a risk of ESCC development.

Background In lung cancer, uPA, its receptor (uPAR), and the inhibitors PAI-1 and PAI-2 of the plasminogen activator family interact with MMP-2 and MMP-9 of the MMP family to promote cancer progression. However, it remains undetermined which of these markers plays the most important role and may be the most useful indicator to stratify the patients by risk. Methods We determined the individual prognostic value of these 6 markers by analyzing a derivation cohort with 98 non-small cell lung cancer patients by immunohistochemical staining. The correlation between the IHC expression levels of these markers and disease prognosis was investigated, and an immunohistochemical panel for prognostic prediction was subsequently generated through prognostic model analysis. The value of the immunohistochemical panel was then verified by a validation cohort with 91 lung cancer patients. Results In derivation cohort, PAI-2 is the most powerful prognostic factor (HR = 2.30; P = 0.001), followed by MMP-9 (HR = 2.09; P = 0.019) according to multivariate analysis. When combining PAI-2 and MMP-9, the most unfavorable prognostic group (low PAI-2 and high MMP-9 IHC expression levels) showed a 6.40-fold increased risk of a poor prognosis compared to the most favorable prognostic group (high PAI-2 and low MMP-9 IHC expression levels). PAI-2 and MMP-9 IHC panel could more precisely identify high risk patients in both derivation and validation cohort. Conclusions We revealed PAI-2 as the most powerful prognostic marker among PA and MMP protease family even after considering their close relationships with each other. By utilizing a combination of PAI-2 and MMP-9, more precise prognostic information than merely using pathological stage alone can be obtained for lung cancer patients.

This study was undertaken to compare periodontal conditions, gingival crevicular fluid (GCF) levels of tissue-type plasminogen activator (t-PA), its inhibitor plasminogen activator inhibitor-2 (PAI-2), interleukin-1beta (IL-1beta), prostaglandin E(2) (PGE(2)) in rheumatoid arthritis (RA) patients and control groups.Twenty-three RA patients, 17 systemically healthy patients with periodontal disease (PD), and 17 systemically and periodontally healthy subjects were recruited. GCF samples were obtained from two single-rooted teeth. Full-mouth clinical periodontal measurements were recorded at six sites/tooth. GCF samples were analysed using relevant ELISA kits. Data were tested statistically by appropriate tests.Total amounts of t-PA, PAI-2 and PGE(2) in GCF samples of the healthy control group were significantly lower than the other groups (p0.05). The RA group exhibited a higher total amount of t-PA in GCF samples than the PD group (p0.05). PAI-2, IL-1beta and PGE(2) total amounts were similar in RA and PD groups (p0.05).The coexistence of RA and periodontitis does not seem to affect clinical periodontal findings or systemic markers of RA. Similar inflammatory mediator levels in RA and PD groups, despite the long-term usage of corticosteroids, non-steroidal anti-inflammatory drugs, suggest that RA patients may have a propensity to overproduce these inflammatory mediators.