Epigenetic clock analysis and increased plasminogen activator inhibitor-1 in high-functioning autism spectrum disorder.

BACKGROUND: Autism spectrum disorder (ASD) is characterized by impaired social communication and behavioral problems. An increased risk of premature mortality has been observed in individuals with ASD. Therefore, we hypothesized that biological aging is accelerated in individuals with ASD. Recently, several studies have established genome-wide DNA methylation (DNAm) profiles as 'epigenetic clocks' that can estimate biological aging. In addition, ASD has been associated with differential DNAm patterns. METHODS: We used two independent datasets from blood samples consisting of adult patients with high-functioning ASD and controls: the 1st cohort (38 ASD cases and 31 controls) and the 2nd cohort (6 ASD cases and 10 controls). We explored well-studied epigenetic clocks such as HorvathAge, HannumAge, SkinBloodAge, PhenoAge, GrimAge, and DNAm-based telomere length (DNAmTL). In addition, we investigated seven DNAm-based age-related plasma proteins, including plasminogen activator inhibitor-1 (PAI-1), and smoking status, which are the components of GrimAge. RESULTS: Compared to controls, individuals with ASD in the 1st cohort, but not in the 2nd cohort, exhibited a trend for increased GrimAge acceleration and a significant increase of PAI-1 levels. A meta-analysis showed significantly increased PAI-1 levels in individuals with ASD compared to controls. CONCLUSION: Our findings suggest there is no epigenetic age acceleration in the blood of individuals with ASD. However, this study provides novel evidence regarding increased plasma PAI-1 levels in individuals with high-functioning ASD. These findings suggest PAI-1 may be a biomarker for high-functioning ASD, however, larger studies based on epigenetic clocks and PAI-1 will be necessary to confirm these findings.

Silencing of ELK3 Induces S-M Phase Arrest and Apoptosis and Upregulates SERPINE1 Expression Reducing Migration in Prostate Cancer Cells.

ELK3, an ETS domain-containing transcription factor, participates in various physiological and pathological processes including cell proliferation, migration, angiogenesis, and malignant progression. However, the role of ELK3 in prostate cancer cells and its mechanism are not fully understood. The contribution of ELK3 to prostate cancer progression was investigated in the present study. We showed that silencing of ELK3 by siRNA in prostate cancer cell DU145 induced S-M phase arrest, promoted apoptosis, inhibited cell proliferation and migration in vitro, and suppressed xenograft growth in mice in vivo. In accordance with its ability to arrest cells in S-M phase, the expression of cyclin A and cyclin B was downregulated. In addition, the expression of p53 was upregulated following ELK3 knockdown, while that of antiapoptotic Bcl-2 was decreased. The migration inhibition may partly due to upregulation of SERPINE1 (a serine protease inhibitor) followed ELK3 knockdown. Consistently, downregulation of SERPINE1 resulted in a modest elimination of migration inhibition resulted from ELK3 knockdown. Furthermore, we found that the AKT signaling was activated in ELK3 knockdown cells, and treatment these cells with AKT inhibitor attenuated SERPINE1 expression induced by ELK3 silencing, suggesting that activation of AKT pathway may be one of the reasons for upregulation of SERPINE1 after ELK3 knockdown. In conclusion, modulation of ELK3 expression may control the progression of prostate cancer partly by regulating cell growth, apoptosis, and migration.

Expression, clinicopathologic and prognostic significance of plasminogen activator inhibitor 1 in hepatocellular carcinoma.

OBJECTIVES: Thus far, biological roles of plasminogen activator inhibitor 1 (PAI1) in hepatocellular carcinoma (HCC) remain controversial. Moreover, its expression, clinicopathologic and prognostic significance in HCC have not been comprehensively investigated, therefore needing further evidence. METHODS: PAI1 expression was measured, using tissue microarray-based immunohistochemical staining, in matched HCC and adjacent liver samples from 178 patients with HCC after curative resection. The correlations of PAI1 H-scores with clinicopathologic variables and survival were further evaluated. Its prognostic value was finally confirmed in some public databases. RESULTS: It was found that PAI1 expression was significantly higher in HCC than in adjacent liver tissues. Moreover, high PAI1 expression was more frequent in those with multiple lesions. Univariate analyses showed that PAI1 expression was negatively associated with both overall and relapse-free survival. Although PAI1 expression was not statistically significant in multivariate Cox regression test, combination of it with TNM stage effectively distinguished survival and relapse, and served as an independent prognostic factor. In the online available datasets of HCC and liver cancer used, SERPINE1, the gene encoding PAI1, was also revealed to be prognostic. CONCLUSIONS: Our data suggested that high PAI1 expression was predictive for unfavorable biological behavior and long-term prognosis in HCC.

Expression of urokinase-type plasminogen activator system in non-metastatic prostate cancer.

PURPOSE: To investigate the prognostic role of expression of urokinase-type plasminogen activator system members, such as urokinase-type activator (uPA), uPA-receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1), in patients treated with radical prostatectomy (RP) for prostate cancer (PCa). METHODS: Immunohistochemical staining for uPA system was performed on a tissue microarray of specimens from 3121 patients who underwent RP. Cox regression analyses were performed to investigate the association of overexpression of these markers alone or in combination with biochemical recurrence (BCR). Decision curve analysis was used to assess the clinical impact of these markers. RESULTS: uPA, uPAR, and PAI-1 were overexpressed in 1012 (32.4%), 1271 (40.7%), and 1311 (42%) patients, respectively. uPA overexpression was associated with all clinicopathologic characteristics of biologically aggressive PCa. On multivariable analysis, uPA, uPAR, and PAI-1 overexpression were all three associated with BCR (HR: 1.75, p < 0.01, HR: 1.22, p = 0.01 and HR: 1.20, p = 0.03, respectively). Moreover, the probability of BCR increased incrementally with increasing cumulative number of overexpressed markers. Decision curve analysis showed that addition of uPA, uPAR, and PAI-1 resulted in a net benefit compared to a base model comparing standard clinicopathologic features across the entire threshold probability range. In subgroup analyses, overexpression of all three markers remained associated with BCR in patients with favorable pathologic characteristics. CONCLUSION: Overexpression of uPA, uPAR, and PAI-1 in PCa tissue were each associated with worse BCR. Additionally, overexpression of all three markers is informative even in patients with favorable pathologic characteristics potentially helping clinical decision-making regarding adjuvant therapy and/or intensified follow-up.

Catheter tips are a possible resource for biological study on catheter failure.

Few studies have investigated the molecular mechanisms of catheter failure (CF). Herein, we performed histological and molecular biological analyses of the catheter tip to demonstrate its potential as a resource for biological investigation. Additionally, we searched for risk factors for the development of inflammation and coagulation, which are pathological conditions clarified by biological analysis. The CF group included 30 failed catheters involving thrombus and subcutaneous edema identified by ultrasonography. The No-CF group included 26 catheters with no complications. The removed catheter tips were fixed for hematoxylin-eosin (HE) staining with the application of a real-time reverse transcriptase polymerase chain reaction for eukaryotic 18S ribosomal RNA (rRNA), interleukin 1ß, tumor necrosis factor α, tissue plasminogen activator, and plasminogen activator inhibitor 1 (SERPINE1). HE staining identified attached nuclear cells on the inner surfaces of both CF and No-CF catheters. The 18S rRNA was amplified in all samples. The expression level of SERPINE1 was significantly higher in the CF group than in the No-CF group (p = 0.01), whereas the expression levels of other genes did not differ between the groups. Symptoms of CF associated with the expression of SERPINE1 were analyzed. The catheter being in contact with blood vessels during placement was a suggested factor related to the high expression of SERPINE1 (p = 0.04). Catheter tips are a potential resource for biological investigation, and expression analysis of the attached cells can reflect the pathological condition of the catheterized tissue. Further studies using catheter tips are required to elucidate the molecular mechanisms of CF.

Overexpression of CEP72 Promotes Bladder Urothelial Carcinoma Cell Aggressiveness via Epigenetic CREB-Mediated Induction of SERPINE1.

A vital constituent of the centrosome involved in regulating the activity of the organelle during the cell cycle is centrosomal protein (CEP)-72, whose function in the case of human cancer yet lacks clarity. The expression dynamics of CEP72 and its clinical impact were examined in a large cohort of bladder tissues. Several experiments at both the in vitro and in vivo levels on urothelial carcinoma of the bladder (UCB) cells were conducted to understand the role of this molecule along with the mechanisms. Overexpression of CEP72 in UCB was linked with the acquisition of an aggressive phenotype, which was associated with poor prognosis. In UCB cell lines, knockdown of CEP72 using shRNA was sufficient to inhibit cell invasiveness/metastasis, whereas ectopic overexpression of CEP72 promoted cell invasiveness and/or metastasis both in vitro and in vivo. CEP72 was demonstrated to induce UCB cell aggressiveness via up-regulation of an important target downstream, the serpin family member 1 gene (SERPINE1) (alias plasminogen activator inhibitor, PAI1), ultimately leading to increased cancer cell invasiveness. Particularly, overexpression of CEP72 was associated with a sizable increase in cAMP response element-binding protein binding at the SERPINE1 promoter, leading to increased SERPINE1 transcription. Such observations are suggestive of the potential use of CEP72 as a therapeutic tool for UCB.

The association between IUGR and maternal inherited thrombophilias: A case-control study.

One of the risk factors for vascular obstetric complications, such as intrauterine growth restriction (IUGR), is inherited thrombophilias. Nevertheless, routine screening for thrombophilias is not endorsed in pregnant women due to their low prevalence and conflicting results of published studies regarding the usefulness of screening in these patients. The cause of IUGR remains unknown in almost 1 quarter of cases. There are no published studies evaluating the association of inherited thrombophilias and IUGR in patients with IUGR of unknown origin. Understanding and preventing IUGR is an important public health concern, as IUGR has been associated with fetal mortality and neonatal morbidity, as well as adverse long-standing consequences. This study aimed to evaluate the prevalence of inherited thrombophilias in IUGR of unknown cause and to test the association between the inherited thrombophilias and IUGR of unknown cause.This study included 33 cases of IUGR of unknown cause tested for inherited thrombophilias and 66 controls individually matched for age, ethnicity, and smoking status.Patients with plasminogen activator inhibitor 1 (PAI-1) and methylenetetrahydrofolate reductase (MTHFR) had significantly higher odds for IUGR of unknown cause (P < .001 and P = .002, respectively) with OR 13.546 (CI 95% 3.79-48.37) and 8.139 (CI 95% 2.20-30.10), respectively. A positive association between other inherited thrombophilias (homozygous 20210 prothrombin gene mutation and homozygous factor V Leiden) and IUGR of unknown cause was also found, P = .096, OR 6.106 (CI 95% 0.72-51.30), although it was not statistically significant (P = .096, OR = 6.106, CI 95% 0.72-51.30).Our results indicate that PAI-1 and MTHFR thrombophilias represent risk factors for IUGR of otherwise unidentified cause.

Changes in the expression level of IL-17A and p53-fibrinolytic system in smokers with or without COPD.

COPD is a chronic airway inflammatory disease characterized mainly by neutrophil airway infiltrations. The neutrophil airway inflammation is mainly mediated through a key player like the pro-inflammatory cytokine IL-17A which is involved in the modulation of p53-fibrinolytic system. This study was undertaken to examine the molecular changes for the expressions of IL-17A and p53-fibrinolytic system in smokers with or without COPD. Blood and serum samples were collected from ten patients of smokers having COPD and ten samples from smokers without COPD and ten healthy control subjects. Western blot analyses were performed to evaluate the expressions of IL-17A, p53 and PAI-1. Apoptosis was assessed by immunoblot for cleaved caspase-3. In addition, FEV% was also determined of these patients. qRT-PCR was done to detect the gene expression study from the blood samples on p53-fibrinolytic components. A significant difference was found in the expression levels of IL-17A in smokers with COPD patient when compared to smokers without COPD and the control subjects. Similarly the smokers with COPD showed significant increase in the fibrinolytic component PAI-1 as well as in expression levels of p53 when compared to smokers without COPD and normal subjects. Increased cleaved caspase-3 may also promote apoptosis.The expression pattern of the IL-17A in chronic obstructive pulmonary distress syndrome samples was increased as compared of those of normal samples, and their main role in the regulation of and p53-fibrinolytic system makes these components as a predictive prominent component in smokers with COPD.

PAI-1, CAIX, and VEGFA expressions as prognosis markers in oral squamous cell carcinoma.

BACKGROUND: In oral squamous cell carcinoma (OSCC), the HIF-1 complex promotes the expression of genes involved in specific mechanisms of cell survival under hypoxic conditions, such as plasminogen activator inhibitor-1 (PAI-1), carbonic anhydrase 9 (CAIX), and vascular endothelial growth factor A (VEGFA). The study aimed to investigate the presence and prognostic value of PAI-1, CAIX, and VEGFA in OSCC. MATERIALS AND METHODS: Immunohistochemistry was used to analyze the expressions of these proteins in 52 tumoral tissue samples of patients with OSCC, surgically treated and followed by a minimum of 24 months after surgery. The correlations between protein expressions and clinicopathological parameters and prognosis were analyzed. RESULTS: Positive PAI-1 membrane expression was significantly associated with local disease relapse (P = .027). Multivariate analysis revealed that the positive PAI-1 membrane expression is an independent marker for local disease relapse, with approximately 14-fold increased risk when compared to negative expression (OR = 14.49; CI = 1.40-150.01, P = .025). Strong PAI-1 cytoplasmic expression was significantly associated with the less differentiation grade (P = .027). Strong CAIX membrane expression was significantly associated with local disease-free survival (P = .038). Positive CAIX cytoplasmic expression was significantly associated with lymph node affected (P = .025) and with disease-specific survival (P = .022). Multivariate analysis revealed that the positive CAIX cytoplasmic expression is an independent risk factor for disease-related death, increasing their risk approximately 3-fold when compared to negative expression (HR = 2.84; CI = 1.02-7.87, P = .045). Positive VEGFA cytoplasmic expression was significantly associated with less differentiation grade (P = .035). CONCLUSION: Our results suggest a potential role for these expressions profiles as tumor prognostic markers in OSCC patients.

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

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

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

Intra-abdominal adhesion is a common complication after laparotomy. Conventional therapeutic strategies still cannot safely and effectively prevent this disorder. In this study, a combination of chitosan, cellulose, and seaweed polysaccharide (thereafter referred as CCS) was developed to significantly alleviate the formation of postoperative adhesion in rats with abdominal trauma. Transforming growth factor ß1 (TGF-ß1, an important promoter of fibrosis) and its downstream factors-namely, alpha-smooth muscle actin and plasminogen activator inhibitor-1 (PAI-1)-were effectively suppressed by CCS in vivo, and as a result, the activation of tissue plasminogen activator (tPA, may generate plasmin that is a fibrinolytic factor capable of breaking down fibrin) was significantly promoted, presenting antifibrosis effects of CCS. In addition, the activity of kinases [e.g., transforming growth factor-activated kinase 1 (TAK1), c-Jun N-terminal kinase (JNK)/Stress-activated Protein Kinase (SAPK), and p38] in the mitogen-activated protein kinase (MAPK) inflammation signaling pathway was also significantly suppressed by CCS in vivo, demonstrating anti-inflammatory functions of CCS. The histologic studies further confirmed the role of CCS in the inhibition of fibrosis, collagen deposition, inflammation, and vascular proliferation. These results indicate the clinical potential of CCS in the treatment of postoperative intra-abdominal adhesion. CCS may induce both antifibrosis and anti-inflammatory effects, potentially inhibiting the postoperative intra-abdominal adhesion. For antifibrosis effects, the expression of PAI-1 (a key factor for the adhesion formation) can be regulated by different TGF-ß1-associated signaling pathways, such as the Smads/p53 pathway, metalloproteinase/tissue inhibitor of matrix metalloproteinases pathway, Mitogen-activated Extracellular signal-regulated Kinase (MEK)/extracellular regulated protein kinase (ERK) pathway, and Yes-associated protein/transcriptional coactivator with PDZ-binding motif pathway. Following the downregulation of PAI-1 achieved by CCS, the activation of tPA (which may generate plasmin that is a fibrinolytic factor capable of breaking down fibrin) is significantly promoted. For anti-inflammation effects, CCS may suppress the phosphorylation of classic kinases (e.g., TAK1, JNK, and p38) in the MAPK signaling pathway. In addition to the MAPK pathway, inflammatory pathways, such as Nuclear Factor-κ-gene Binding(NF-κB), MEK/ERK, and Ras homologue protein/Rho associated coiled coil forming protein, are associated with the formation of intra-abdominal adhesion. Therefore, the prevention mechanisms of CCS will be further investigated in the future, with a hope of fully understanding of antiadhesion effects.

Alpha mangostin Inhibits Hepatic Stellate Cells Activation Through TGF-ß/Smad and Akt Signaling Pathways: An in vitro Study in LX2.

BACKGROUND: Alpha mangostin has been reported to have activity for the treatment of liver fibrosis in the rats. However, the mechanisms of action are poorly understood. This study was aimed to investigate the effect of alpha mangostin on hepatic stellate cells (HSC) activation and proliferation through TGF-ß/Smad and Akt signaling pathways. METHODS: Immortalized HSC, LX2 cells, were incubated with TGF-ß with or without alpha mangostin (5 or 10 µM). Sorafenib 10 µM was used as positive control. LX2 viability was counted using trypan blue exclusion method. The effect of alpha mangostin on TGF-ß concentrations, and the expressions of proliferation and fibrogenic markers were evaluated. RESULTS: Alpha mangostin treatment resulted in a reduced proliferation of HSC, decreased Ki-67 and p-Akt expressions. These findings were followed with decreased concentrations of TGF-ß in the medium of cells treated with alpha mangostin, decreased expressions of COL1A1, TIMP1, PAI1, α-SMA, and p-Smad3 as fibrogenic markers. These effects were shown to be dose-dependent. CONCLUSIONS: Alpha mangostin inhibits hepatic stellate cells proliferation and activation through TGF-ß/Smad and Akt signaling pathways in dose dependent manner.

Blocking fibrotic signaling in fibroblasts from patients with carpal tunnel syndrome.

Fibrosis of the subsynovial connective tissue (SSCT) in carpal tunnel syndrome (CTS) patients is increasingly recognized as an important aspect of CTS pathophysiology. In this study, we evaluated the effect of blocking profibrotic pathways in fibroblasts from the SSCT in CTS patients. Fibroblasts were stimulated with transforming growth factor ß1 (TGF-ß1), and then treated either with a specific fibrosis pathway inhibitor targeting TGF-ß receptor type 1 (TßRI), platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), or vascular endothelial growth factor receptor (VEGFR). Fibrosis array and quantitative real-time polymerase chain reaction of fibrotic genes were evaluated. Array gene expression analysis revealed significant down-regulation of multiple fibrotic genes after treatment with TßRI, PDGFR, and VEGFR inhibitors. No array fibrotic genes were significantly down-regulated with EGFR inhibition. Further gene expression analysis of known CTS fibrosis markers collagen type I A2 (Col1), collagen type III A1 (Col3), connective tissue growth factor (CTGF), and SERPINE1 showed significantly down-regulation after TßRI inhibition. In contrast, VEGFR inhibition significantly down-regulated CTGF and SERPINE1, whereas, PDGFR and EGFR inhibition significantly down-regulated Col3. Taken together the inhibition of TßRI appears to be the primary mediator of fibrotic gene expression in fibroblasts from CTS patients. TGF-ß/Smad activity was further evaluated, and as expected inhibition of Smad activity was significantly down-regulated after inhibition of TßRI, but not with PDGFR, VEGFR, or EGFR inhibition. These results indicate that local therapies specifically targeting TGF-ß signaling alone or in combination offer the potential of a novel local antifibrosis therapy for patients with CTS.

Differential effect of the ultra-low dose and standard estrogen plus dydrogesterone therapy on thrombin generation and fibrinolysis in postmenopausal women.

INTRODUCTION: The objective of this study was to estimate the effects of different doses of oral hormone therapy (HT) on thrombin generation and fibrinolytic activity in postmenopausal women after 12 months of treatment. MATERIAL AND METHODS: Thrombin generation, fibrinolysis activators and inhibitors were determined before and after 12 months of treatment. Participants (180) were assigned (1:1:1) as follows: (1) standard HT group, 17ß-estradiol (1 mg/day) with dydrogesterone (5 mg/day); (2) ultra-low dose HT group, 17ß-estradiol (0.5 mg/day) with dydrogesterone (2.5 mg/day); (3) control group, no treatment. RESULTS: The standard HT led to a higher concentration of prothrombin 1 + 2 fragment (by 5.8%) with lower antithrombin activity (by 6.1%). Compared with baseline, we observed a reduction in mean antithrombin activity in the standard HT group and increases in mean levels of prothrombin 1 + 2 fragment in two HT groups. We found decreases after treatment in both standard and ultra-low dose HT groups in plasminogen activator inhibitor-1 (PAI-1) activity (-32.4% and -19.6%, respectively) and PAI-1 antigen (-9.9% and -7.8%, respectively). Intergroup analysis revealed reduction in both mean PAI-1 activities and PAI-1 antigen levels in the two treatment groups when compared with the control. CONCLUSION: Contrary to the standard estrogen plus dydrogesterone treatment, ultra-low dose HT revealed positive effects on hemostasis by intensifying fibrinolysis through a decrease in both PAI-1 activity and antigen levels, and with no impact on thrombin generation.

Pentoxifylline Regulates Plasminogen Activator Inhibitor-1 Expression and Protein Kinase A Phosphorylation in Radiation-Induced Lung Fibrosis.

Purpose. Radiation-induced lung fibrosis (RILF) is a serious late complication of radiotherapy. In vitro studies have demonstrated that pentoxifylline (PTX) has suppressing effects in extracellular matrix production in fibroblasts, while the antifibrotic action of PTX alone using clinical dose is yet unexplored. Materials and Methods. We used micro-computed tomography (micro-CT) and histopathological analysis to evaluate the antifibrotic effects of PTX in a rat model of RILF. Results. Micro-CT findings showed that lung density, volume loss, and mediastinal shift are significantly increased at 16 weeks after irradiation. Simultaneously, histological analysis demonstrated thickening of alveolar walls, destruction of alveolar structures, and excessive collagen deposition in the irradiated lung. PTX treatment effectively attenuated the fibrotic changes based on both micro-CT and histopathological analyses. Western analysis also revealed increased levels of plasminogen activator inhibitor- (PAI-) 1 and fibronectin (FN) and PTX treatment reduced expression of PAI-1 and FN by restoring protein kinase A (PKA) phosphorylation but not TGF-ß/Smad in both irradiated lung tissues and epithelial cells. Conclusions. Our results demonstrate the antifibrotic effect of PTX on radiation-induced lung fibrosis and its effect on modulation of PKA and PAI-1 expression as possible antifibrotic mechanisms.

The Association of Plasminogen Activator Inhibitor Type 1 (PAI-1) Level and PAI-1 4G/5G Gene Polymorphism with the Formation and the Grade of Endometrial Cancer.

Plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (Serpine 1), and it inhibits both tissue plasminogen activator and urokinase plasminogen activator which are important in fibrinolysis. We aimed to find whether there is a possible association between PAI-1 level, PAI-1 4G/5G polymorphism, and endometrial cancer. PAI-1 levels in peripheral blood were determined in 82 patients with endometrial carcinoma and 76 female healthy controls using an enzyme-linked immunoassay (ELISA). Then, the genomic DNA was extracted and screened by reverse hybridization procedure (Strip assay) to detect PAI 1 4G/5G polymorphism. The levels of PAI-1 in the patients were higher statistically in comparison to controls (P < 0.001). The distribution of PAI-1 4G/5G polymorphism was quite different between patients and controls (P = 0.008), and 4G allelic frequency was significantly higher in the patients of endometrial cancer than in controls (P = 0.026). We found significant difference between Grade 1 and Grade 2+3 patients in terms of the PAI-1 levels (P = 0.047). There was no association between PAI-1 4G/5G polymorphism and the grades of endometrial cancer (P = 0.993). Our data suggest that the level of PAI-1 and PAI-1 4G/5G gene polymorphism are effective in the formation of endometrial cancer. PAI-1 levels are also associated with the grades of endometrial cancer.

Vasohibin-2 is required for epithelial-mesenchymal transition of ovarian cancer cells by modulating transforming growth factor-ß signaling.

Vasohibin-2 (VASH2) is a homolog of VASH1, an endothelium-derived angiogenesis inhibitor. Vasohibin-2 is mainly expressed in cancer cells, and has been implicated in the progression of cancer by inducing angiogenesis and tumor growth. Although VASH2 has been recently reported to be involved in epithelial-mesenchymal transition (EMT), its precise roles are obscure. The aim of the present study was to clarify the role of VASH2 in the EMT of cancer cells in relation to transforming growth factor-ß (TGF-ß) signaling, which is a major stimulator of EMT. Decreased expression of VASH2 in ovarian cancer cells significantly repressed the expression of TGF-ß type I receptor, namely activin receptor-like kinase 5. Transforming growth factor-ß1-induced phosphorylation of Smad2 and Smad3 was markedly decreased in VASH2 knockdown cells while the expression of Smad2 and Smad3 was unchanged. Accordingly, the responses to TGF-ß1 shown by promoter assay and plasminogen activator inhibitor type 1 expression were significantly attenuated in VASH2 knockdown cells. Furthermore, knockdown of VASH2 in cancer cells abrogated the TGF-ß1-induced reduced expression of epithelial markers including E-cadherin, and the elevated expression of mesenchymal markers including fibronectin, ZEB2, and Snail2, suggesting that endogenous VASH2 is required for TGF-ß1-induced EMT. In accordance with these results, the effects of TGF-ß1 on cell morphology, migration, invasion, and MMP2 expression were also abrogated when VASH2 was knocked down. These results indicate that VASH2 played a significant role in the EMT by modulating the TGF-ß signaling. We propose that VASH2 would be a novel molecular target for the prevention of EMT in cancers.

Cholest-4-en-3-one attenuates TGF-ß responsiveness by inducing TGF-ß receptors degradation in Mv1Lu cells and colorectal adenocarcinoma cells.

PURPOSE: The transforming growth factor-beta (TGF-ß) pathway is an important in the initiation and progression of cancer. Due to a strong association between an elevated colorectal cancer risk and increase fecal excretion of cholest-4-en-3-one, we aim to determine the effects of cholest-4-en-3-one on TGF-ß signaling in the mink lung epithelial cells (Mv1Lu) and colorectal cancer cells (HT29) in vitro. METHODS: The inhibitory effects of cholest-4-en-3-one on TGF-ß-induced Smad signaling, cell growth inhibition, and the subcellular localization of TGF-ß receptors were investigated in epithelial cells using a Western blot analysis, luciferase reporter assays, DNA synthesis assay, confocal microscopy, and subcellular fractionation. RESULTS: Cholest-4-en-3-one attenuated TGF-ß signaling in Mv1Lu cells and HT29 cells, as judged by a TGF-ß-specific reporter gene assay of plasminogen activator inhibitor-1 (PAI-1), Smad2/3 phosphorylation and nuclear translocation. We also discovered that cholest-4-en-3-one suppresses TGF-ß responsiveness by increasing lipid raft and/or caveolae accumulation of TGF-ß receptors and facilitating rapid degradation of TGF-ß and thus suppressing TGF-ß-induced signaling. CONCLUSIONS: Our results suggest that cholest-4-en-3-one inhibits TGF-ß signaling may be due, in part to the translocation of TGF-ß receptor from non-lipid raft to lipid raft microdomain in plasma membranes. Our findings also implicate that cholest-4-en-3-one may be further explored for its potential role in colorectal cancer correlate to TGF-ß deficiency.

Thrombospondin-1 Affects Bovine Luteal Function via Transforming Growth Factor-Beta1-Dependent and Independent Actions.

Thrombospondin-1 (THBS1) and transforming growth factor-beta1 (TGFB1) are specifically up-regulated by prostaglandin F2alpha in mature corpus luteum (CL). This study examined the relationship between the expression of THBS1 and TGFB1 and the underlying mechanisms of their actions in luteal endothelial cells (ECs). TGFB1 stimulated SMAD2 phosphorylation and SERPINE1 levels in dose- and time-dependent manners in luteal EC. THBS1 also elevated SERPINE1; this effect was abolished by TGFB1 receptor-1 kinase inhibitor (SB431542). The findings here further imply that THBS1 activates TGFB1 in luteal ECs: THBS1 increased the effects of latent TGFB1 on phosphorylated SMAD (phospho-SMAD) 2 and SERPINE1. THBS1 silencing significantly decreased SERPINE1 and levels of phospho-SMAD2. Lastly, THBS1 actions on SERPINE1 were inhibited by LSKL peptide (TGFB1 activation inhibitor); LSKL also counteracted latent TGFB1-induced phospho-SMAD2. We found that TGFB1 up-regulated its own mRNA levels and those of THBS1. Both compounds generated apoptosis, but THBS1 was significantly more effective (2.5-fold). Notably, this effect of THBS1 was not mediated by TGFB1. THBS1 and TGFB1 also differed in their activation of p38 mitogen-activated protein kinase. Whereas TGFB1 rapidly induced phospho-p38, THBS1 had a delayed effect. Inhibition of p38 pathway by SB203580 did not modulate TGFB1 effect on cell viability, but it amplified THBS1 actions. THBS1-stimulated caspase-3 activation coincided with p38 phosphorylation, suggesting that caspase-induced DNA damage initiated p38 phosphorylation. The in vitro data suggest that a feed-forward loop exists between THBS1, TGFB1, and SERPINE1. Indeed all these three genes were similarly induced in the regressing CL. Their gene products can promote vascular instability, apoptosis, and matrix remodeling during luteolysis.

Interferon gamma effect on immune mediator production in human nerve cells infected by two strains of Toxoplasma gondii.

Interferon gamma (IFN-γ) is the major immune mediator that prevents toxoplasmic encephalitis in murine models. The lack of IFN-γ secretion causes reactivation of latent T. gondii infection that may confer a risk for severe toxoplasmic encephalitis. We analyse the effect of IFN-γ on immune mediator production and parasite multiplication in human nerve cells infected by tachyzoites of two T. gondii strains (RH and PRU). IFN-γ decreased the synthesis of MCP-1, G-CSF, GM-CSF and Serpin E1 in all cell types. It decreased IL-6, migration inhibitory factor (MIF) and GROα synthesis only in endothelial cells, while it increased sICAM and Serpin E1 synthesis only in neurons. The PRU strain burden increased in all nerve cells and in contrast, RH strain replication was controlled in IFN-γ-stimulated microglial and endothelial cells but not in IFN-γ-stimulated neurons. The proliferation of the PRU strain in all stimulated cells could be a specific effect of this strain on the host cell.