PAI-1 mRNA Levels Research Articles

Interferon γ and plasminogen activator inhibitor 1 regulate adhesion formation after partial hepatectomy

The pathophysiology of intra-abdominal adhesions has not been studied extensively. The aim of this study was to elucidate the molecular mechanisms underlying adhesion formation in a murine model and in patients undergoing hepatectomy. Partial hepatectomy was performed using bipolar forceps in mice. Wild-type mice, antibodies to CD4 and interferon (IFN) γ, IFN-γ, natural killer T (NKT) cells and plasminogen activator inhibitor (PAI) 1 knockout (KO) mice were used. Recombinant hepatocyte growth factor (HGF) was tested for its ability to prevent adhesions. Liver specimens were obtained during surgery from patients undergoing hepatectomy. Adhesion formation was evaluated using a scoring system that ranged from 0 (no adhesions) to 5 (severe adhesions). Levels of IFN-γ and PAI-1 mRNA, and protein concentration of PAI-I were measured, and fluorescence immunostaining was performed. Adhesion formation depended on IFN-γ produced by NKT cells, and NKT KO mice developed few adhesions (mean(s.d.) 1·7(0·3) versus 4·6(0·4) in wild-type mice; P = 0·037). In wild-type mice, the level of PAI-1 mRNA increased after hepatectomy, followed by a decrease in the tissue plasminogen activator (tPA) mRNA level. Adhesion formation was inhibited completely in PAI-1 KO mice (0(0) versus 4·1(0·8) in wild-type mice; P = 0·002). HGF inhibited formation of abdominal adhesions after hepatectomy by reducing IFN-γ and PAI-1 levels, and increasing tPA levels compared with those in mice treated with phosphate-buffered saline (P < 0·001, P = 0·002 and P = 0·035 respectively). In human liver specimens, NKT cells accumulated in the liver after hepatectomy, and PAI-1 expression was increased 5·25-fold (P = 0·030). IFN-γ is a key molecule for abdominal adhesion formation after hepatectomy, acting via the reciprocal balance of PAI-1 and tPA. This molecular mechanism may also regulate adhesion formation in patients following hepatectomy. HGF inhibited formation of adhesions by regulating IFN-γ and PAI-1, suggesting that it may be an important target for prevention of adhesions after hepatectomy. SURGICAL RELEVANCE: Postoperative intra-abdominal adhesions can be asymptomatic or cause significant morbidity and mortality. Adhesion formation after hepatectomy has not been studied extensively. In the present study, the molecular mechanisms underlying intra-abdominal adhesions after hepatectomy were investigated in a murine model and in patients. Interferon (IFN) γ produced by natural killer T cells is a key molecule for adhesion formation after hepatectomy in mice, acting via the reciprocal balance between plasminogen activator inhibitor (PAI) 1 and tissue plasminogen activator, the pivotal factors in fibrinolytic activity. This mechanism was also involved in the regulation of adhesions in human tissue samples. Hepatocyte growth factor (HGF) strongly inhibited adhesion formation by regulating IFN-γ and PAI-1. These results indicate that IFN-γ and PAI-1 are possible therapeutic targets, and HGF could prevent postoperative adhesion formation after hepatectomy.

Abstract 192: Activation of Kidney Renin-angiotensin System (RAS) and Renoprotective Effects of Direct Renin Inhibition (DRI) in Glomerulonephritis

The development of glomerulonephritis causes glomerular injury and reduced renal function and is associated with increased renin release. The aims of this study were to demonstrate activation of intrarenal RAS in glomerulonephritis and determine the effects of DRI with aliskiren on progression of glomerulonephritis associated renal injury induced by administration of an anti-Thy1.1 antibody. We divided rats into 3 groups; control group, anti-Thy1.1 glomerulonephritis group and anti-Thy1.1 glomerulonephritis treated with aliskiren (30 mg/kg/day) group. In the anti-Thy1.1 rats, protein excretion and micro albumin excretion levels increased markedly after anti-Thy1.1 antibody injection. The increases in protein (94.7 ± 13.0 mg/day) and micro albumin (7.5 ± 2.6 mg/day) excretions were attenuated by aliskiren treatment (43.6 ± 4.5 mg/day of protein and 2.6 ± 0.7 mg/day of micro albumin). The glomerular expansion score was significantly higher in the anti-Thy1.1 group compared to the control group. The anti-Thy1.1 group also showed elevation of intrarenal TGF-β and PAI-1 mRNA levels compared with control rats. The progression of glomerular expansion and elevation of these mRNA levels was prevented by aliskiren. Importantly, the anti-Thy1.1 group had markedly increased urinary angiotensinogen (AGT) excretion at days 3 (3102.9 ± 683.8 ng/day), but these changes were markedly suppressed by aliskiren treatment (441.3 ± 104.8 ng/day). AGT protein expression in the kidney was significantly lower in the aliskiren treatment group compared with anti-Thy1.1 group (western blot, 0.8 ± 0.04 and immunohistochemistry, 0.7 ± 0.11 relative to anti-Thy1.1 group). Furthermore, plasma renin activity (PRA) and plasma angiotensin (Ang) II levels were significantly increased in the anti-Thy1.1 group and aliskiren treatment prevented the elevation of PRA and plasma Ang II. Anti-Thy1.1 antibody also elicited left ventricular hypertrophy and fibrosis, which were not ameliorated by aliskiren. These results demonstrate that DRI prevents or reduces intrarenal RAS activation and glomerular injury in progressive glomerulonephritis. Accordingly, DRI may be an effective approach to treat glomerulonephritis as well as other intrarenal RAS associated kidney diseases.

Plasminogen Activator Inhibitor-1 Is Involved in Streptozotocin-Induced Bone Loss in Female Mice

In diabetic patients, the risk of fracture is high because of impaired bone formation. However, the details of the mechanisms in the development of diabetic osteoporosis remain unclear. In the current study, we investigated the role of plasminogen activator inhibitor (PAI)-1 in the pathogenesis of type 1 diabetic osteoporosis by using PAI-1–deficient mice. Quantitative computed tomography analysis showed that PAI-1 deficiency protected against streptozotocin-induced bone loss in female mice but not in male mice. PAI-1 deficiency blunted the changes in the levels of Runx2, osterix, and alkaline phosphatase in tibia as well as serum osteocalcin levels suppressed by the diabetic state in female mice only. Furthermore, the osteoclast levels in tibia, suppressed in diabetes, were also blunted by PAI-1 deficiency in female mice. Streptozotocin markedly elevated the levels of PAI-1 mRNA in liver in female mice only. In vitro study demonstrated that treatment with active PAI-1 suppressed the levels of osteogenic genes and mineralization in primary osteoblasts from female mouse calvaria. In conclusion, the current study indicates that PAI-1 is involved in the pathogenesis of type 1 diabetic osteoporosis in females. The expression of PAI-1 in the liver and the sensitivity of bone cells to PAI-1 may be an underlying mechanism.

Noninvasive Prognostication of Polyomavirus BK Virus–Associated Nephropathy

BK virus-associated nephropathy (BKVN) is associated with an increased risk of graft failure. Levels of mRNAs encoding proteins implicated in inflammation and fibrosis were measured in urine collected at the time of biopsy diagnosis of BKVN in 29 kidney graft recipients and analyzed for prognosticating graft failure using logistic regression. Ten of 29 BKVN patients had graft failure within 36 months of BKVN diagnosis and the remaining 19 patients did not. Serum creatinine level, BKVN biopsy stage, and urinary cell levels of mRNA for plasminogen activator inhibitor (PAI)-1, vimentin, tissue inhibitor of metalloproteinase-1, fibronectin, granzyme B, or perforin were associated with graft failure. A combination of PAI-1 mRNA level, BKVN biopsy stage, and creatinine level (P = 0.0015, by logistic regression) and a combination of PAI-1 mRNA and creatinine levels (P = 0.001) were the best-fitting models for prognosticating graft failure, and PAI-1 mRNA level was the only independent predictor (odds ratio, 2.8; 95% confidence interval [CI], 1.1-6.8; P = 0.03) by multivariable analysis. The area under the curve for the combination of PAI-1 mRNA, biopsy, and creatinine was 0.92 (95% CI, 0.80-1.0; P < 0.001) by receiver operating characteristic curve analysis, and the area under the curve was 0.92 (95% CI, 0.80-1.0; P < 0.001) for the combination of PAI-1 mRNA and creatinine. Graft outcome was correctly predicted in 27 of 29 BKVN patients by either model. Urinary cell level of PAI-1 mRNA, measured at the time of BKVN diagnosis, is an independent prognosticator of graft failure and a prediction model of serum creatinine and PAI-1 mRNA is as accurate as the model that includes the biopsy result.

AB0119 BMP-9 induces both SMAD1/5/8 and SMAD2/3 phosphorylation, and specific response genes, in chondrocytes

BackgroundOsteoarthritis is characterized by loss of articular cartilage. The main cartilage degrading enzyme is Matrix metalloproteinase 13 (MMP13). Expression of MMP13 is positively correlated with Activin receptor-like kinase 1 (ALK1)...

Hypoxia and cytokines differentially regulate the expression of the tissue plasminogen activator (tPA) and plasminogen activator inhibitor‐1 (PAI‐1) genes in cultured human mesothelial cells (HMCs): Implications for post‐surgical peritoneal fibrinolytic dysregulation

Abdominal surgery induces mesothelial hypoxia and inflammation, both implicated in the dysregulation of the peritoneal fibrinolytic system and intraabdominal adhesion formation. Our objective examined the contribution of each using cultured HMCs. Cells were treated with either a cytokine (C) cocktail (TGFβ+IL‐1β+IFN‐γ), the hypoxia mimetic dimethyloxaloylglycine (D) or both, and the expression of tPA and PAI‐1 mRNA was measured at 1, 3, 6, 12, and 18 hours (n=4/group) by RT‐PCR. C alone and C+D increased PAI‐1 mRNA levels at 6 hr (2.4±0.1 fold; p&lt;0.001 and 2.1±0.17 fold; p&lt;0.001 vs controls) and 12 hr (4.1±0.4 fold; p&lt;0.001 and 4.1±0.7 fold; p&lt;0.001 vs controls) while D alone elevated PAI‐1 mRNA levels at 6 and 12 hr (p&lt;0.05). D increased tPA mRNA levels at 12 hr (2.2±0.16 fold; p&lt;0.001) and 18 hr (3.2±0.7 fold; p&lt;0.001) vs control while C+D paralleled closely at both time points (p&lt;0.001). The tPA/PAI‐1 mRNA ratio, a predictor of fibrinolytic capacity, was reduced 40% on average (p&lt;0.001) at 6 hr in all groups vs control. However, it rose thereafter in D and C+D (2.2±0.4 fold; p&lt;0.001 and 3.8±0.7 fold; p&lt;0.001 vs 6 hr) while C continued to drop at 12 hr. Our results show that cytokines appear to regulate PAI‐1 while hypoxia regulates tPA, suggesting that both contribute to peritoneal fibrinolytic dysregulation and adhesiogenesis. Funded by Cooper‐Tyler Endowment, Department of Surgery, Boston University Medical Center.

PAI-1 and IFN-γ in the regulation of innate immune homeostasis during sublethal yersiniosis

Plasminogen activator inhibitor type 1 (PAI-l), a key part of the fibrinolytic system, plays a critical host protective role during the acute phase of infection by regulating interferon(IFN)-γ release. IFN-γ regulates PAI-1 expression, which suggests an intricate interplay between PAI-1 and IFN-γ. Here, using the notion of a feedback loop, we report the complicated regulatory relationship between PAI-1 and IFN-γ. Mice were inoculated intravenously with 1×10(3) colony forming units of Yersinia enterocolitica; PAI-1 deficiency enhanced lethality (p<0.0001) and increased bacterial growth and dissemination (p=0.08 on day 3, p=0.004 on day 5, respectively). PAI-1 significantly increased the levels IFN-γ mRNA (p<0.005), which may increase survival and decrease bacterial burden. Simultaneously, we showed that IFN-γ increased PAI-1 mRNA levels in vivo (p<0.05). Next, we investigated the transduction signal pathway. After mice were inoculated intraperitoneally with 50μg lipopolysaccharide (LPS), both levels of IFN-γ mRNA (p=0.05) and levels of PAI-1 mRNA (p<0.0001) decreased in MyD88-deficient mice. The same trend was also found in mice treated with 1000μg LPS. As a result of correlations of IFN-γ and PAI-1 in wild-type mice, we delineated the transduction signal pathway, namely MyD88-IFN-γ-PAI-1. The in vivo LPS-injected animal model further confirmed that PAI-1 feedback controlled IFN-γ in a direct or indirect manner. New perspectives of the relationship between PAI-1 and IFN-γ should help in understanding the complex and often conflicting results that have been reported in different infection models. Thus, the feedback loop between PAI-1 and IFN-γ is part of the dynamic equilibrium of coagulation and inflammation that helps maintain innate immune homeostasis.

TGFβ Signaling Regulates Hepcidin Gene Expression

AbstractAbstract 992 Introduction:Hepcidin regulates iron metabolism by reducing duodenal iron absorption and iron release from macrophages and hepatocytes. In inflammatory states, including infection, neoplasia, and heart failure, cytokines induce hepcidin synthesis leading to the development of anemia of inflammation. The regulation of hepcidin gene expression by bone morphogenetic proteins (BMPs), members of the TGFβ family of growth factors, has been extensively investigated. In contrast, less is known about the regulation of hepcidin gene expression by other stimuli, including TGFβ itself. Although TGFβ expression is increased in inflammatory states, the role of TGFβ in the induction of hepcidin gene expression is controversial. To further elucidate the role TGFβ in iron metabolism, we investigated the regulation of hepcidin gene expression in the hepatoma cell line, HepG2. Methods:HepG2 cells were incubated with TGFβ (0.1, 0.5, 1, 2.5, and 5 ng/ml) for varying durations. RNA was extracted for measurement of levels of mRNAs encoding hepcidin, PAI-1 (a TGFβ-target gene), and Id-1 (a BMP-target gene). Cellular proteins were extracted to measure levels of phosphorylated TGFβ-responsive SMADs (using antibodies directed against phosphorylated SMAD2 or SMAD3) and levels of phosphorylated BMP-responsive SMADs (using antibodies directed to phosphorylated SMADs 1 and 5, SMAD1/5). The mechanisms by which TGFβ regulates hepcidin were investigated by pretreating cells with cycloheximide, an inhibitor of protein synthesis (50 μg/mL); Noggin (250 ng/mL) or LDN-193189 (100 nM), inhibitors of BMP signaling; or SB-431542 (5 μM), an inhibitor of the TGFβ type 1 receptor, Alk5. In additional experiments, HepG2 cells were transfected with an siRNA directed against Alk5, 72 hours before exposure to TGFβ. Results:In HepG2 cells, TGFβ induced hepcidin gene expression in a time- and dose-dependent manner: hepcidin mRNA levels were maximal at 2 hours after stimulation with TGFβ (1 ng/ml) and declined thereafter. Incubation of HepG2 cells increased PAI-1 and Id-1 mRNA levels, although increased PAI-1 mRNA levels persisted for at least 8 hours whereas Id-1 mRNA levels peaked at 2 hours. Cycloheximide did not block the ability of TGFβ to induce expression of genes encoding hepcidin, PAI-1, or Id-1. TGFβ induced phosphorylation of SMADs 2 and 3, as well as SMAD1/5. Pretreatment of HepG2 cells with LDN-193189 (at concentrations that inhibit all four BMP type I receptors, as well as Alk1 which is a target of both BMPs and TGFβ) did not block the ability of TGFβ to induce hepcidin or Id-1 gene expression or phosphorylation of SMADs 2, 3, or 1/5. Pretreatment with Noggin gave similar results. Inhibition of Alk5 with SB-421542 blocked the ability of TGFβ to induce expression of genes encoding hepcidin, PAI-1, and Id-1, as well as phosphorylation of SMADs 2, 3, or 1/5. TGFβ-stimulated hepcidin gene expression was inhibited by siRNA-mediated knockdown of Alk5. Conclusion:In HepG2 cells, TGFβ induces hepcidin gene expression via a mechanism which requires Alk5. Although, in addition to phosphorylation of SMADs 2 and 3, TGFβ induces phosphorylation of BMP-responsive SMADs, the failure of cycloheximide to inhibit the induction of hepcidin gene expression by TGFβ suggests that synthesis of BMPs is not required. Moreover, the inability of LDN-193189 to inhibit TGFβ-stimulated hepcidin gene expression suggests against a role for activation of Alk1 by TGFβ. Taken together our findings suggest that TGFβ stimulates hepcidin gene expression via a mechanism that requires Alk5 and may be mediated by signaling either via SMADs 2 and 3 or SMAD1/5. Targeting the regulation of hepcidin gene expression by TGFβ may offer a novel therapeutic approach to the anemia of inflammation. Disclosures:No relevant conflicts of interest to declare.

Genetic variation in the circadian rhythm genes, clock and clif, and possible association with coronary artery disease in individuals of Chinese Han descent

Clock is a core, well-established, circadian gene, and clif is a newly discovered circadian gene, both of which not only maintain the circadian rhythm of cells but also regulate some organic functions. The purpose of this study was to determine whether genetic variations (single nucleotide polymorphisms – SNPs) in clock and/or clif genes associate with coronary artery disease (CAD). We silenced the clock and clif genes in cells and mice. The mRNA levels of clock, clif, pai-1, and tm of mice were examined using Reverse transcription polymerase chain reaction (RT-PCR). Plasminogen activator inhibitor 1 (PAI-1) and thrombomodulin (TM) levels will be examined using enzyme-linked immunosorbent assay. The clock SNPs (rs3840267, rs3749474, and rs1402963), and clif SNPs (rs62758860 and rs2289709) were analyzed in 116 patients with CAD and 95 healthy controls by sequence-specific primer-polymerase chain reaction. Data were analyzed by χ2 analysis. The mRNA levels of pai-1 and tm was decreased in the clock or clif gene silenced groups, and their protein levels were accordingly decreased. The blood serum PAI-1 and TM levels were significantly lower in CAD patients than that in healthy control. There showed significant association between clock SNPs (rs3840267) and CAD. And there was no significant association between clock SNPs (rs3749474 and rs1402963), clif SNPs (rs62758860 and rs2289709) and CAD. The data support an association between clock/clif and CAD. The clock SNPs (rs3840267) is associated with CAD. The clock gene could influence the expressions of PAI-1and TM in both cell and mice, and the blood levels had differences of PAI-1 and TM between CAD human recruitments and health controls. The process of PAI-1/TM influencing CAD may be under control by clock and clif gene.

Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

BackgroundPlasminogen activator inhibitor type 1 (PAI-1) is the primary inhibitor of urokinase type plasminogen activators (uPA) and tissue type plasminogen activators (tPA), which mediate fibrinolysis. PAI-1 is also involved in the innate immunity by regulating cell migration and phagocytosis. However, little is known about the role of PAI-1 in the central nervous system.MethodsIn this study, we identified PAI-1 in the culture medium of mouse mixed glial cells by liquid chromatography and tandem mass spectrometry. Secretion of PAI-1 from glial cultures was detected by ELISA and western blotting analysis. Cell migration was evaluated by in vitro scratch-wound healing assay or Boyden chamber assay and an in vivo stab wound injury model. Phagocytic activity was measured by uptake of zymosan particles.ResultsThe levels of PAI-1 mRNA and protein expression were increased by lipopolysaccharide and interferon-γ stimulation in both microglia and astrocytes. PAI-1 promoted the migration of microglial cells in culture via the low-density lipoprotein receptor-related protein (LRP) 1/Janus kinase (JAK)/signal transducer and activator of transcription (STAT)1 axis. PAI-1 also increased microglial migration in vivo when injected into mouse brain. PAI-1-mediated microglial migration was independent of protease inhibition, because an R346A mutant of PAI-1 with impaired PA inhibitory activity also promoted microglial migration. Moreover, PAI-1 was able to modulate microglial phagocytic activity. PAI-1 inhibited microglial engulfment of zymosan particles in a vitronectin- and Toll-like receptor 2/6-dependent manner.ConclusionOur results indicate that glia-derived PAI-1 may regulate microglial migration and phagocytosis in an autocrine or paracrine manner. This may have important implications in the regulation of brain microglial activities in health and disease.

Synergistic and Multidimensional Regulation of Plasminogen Activator Inhibitor Type 1 Expression by Transforming Growth Factor Type β and Epidermal Growth Factor

The major physiological inhibitor of plasminogen activator, type I plasminogen activator inhibitor (PAI-1), controls blood clotting and tissue remodeling events that involve cell migration. Transforming growth factor type β (TGFβ) and epidermal growth factor (EGF) interact synergistically to increase PAI-1 mRNA and protein levels in human HepG2 and mink Mv1Lu cells. Other growth factors that activate tyrosine kinase receptors can substitute for EGF. EGF and TGFβ regulate PAI-1 by synergistically activating transcription, which is further amplified by a decrease in the rate of mRNA degradation, the latter being regulated only by EGF. The combined effect of transcriptional activation and mRNA stabilization results in a rapid 2-order of magnitude increase in the level of PAI-1. TGFβ also increases the sensitivity of the cells to EGF, thereby recruiting the cooperation of EGF at lower than normally effective concentrations. The contribution of EGF to the regulation of PAI-1 involves the MAPK pathway, and the synergistic interface with the TGFβ pathway is downstream of MEK1/2 and involves phosphorylation of neither ERK1/2 nor Smad2/3. Synergism requires the presence of both Smad and AP-1 recognition sites in the promoter. This work demonstrates the existence of a multidimensional cellular mechanism by which EGF and TGFβ are able to promote large and rapid changes in PAI-1 expression.

BMP-9 induces both smad1/5/8 and Smad2/3 phosphorylation, and specific response genes, in chondrocytes

Background: Osteoarthritis is characterized by loss of articular cartilage. The main cartilage degrading enzyme is Matrix metalloproteinase 13 (MMP13). Expression of MMP13 is positively correlated with Activin receptor-like kinase 1 (ALK1) expression. Moreover, terminal differentiation of chondrocytes is induced by ALK1 via phosphorylation of Smad1/5/8 (Smad1/5/8p). BMP-9, also known as GDF-2, has recently been characterized as a potent, high-affinity ALK1 ligand, that is present in serum in high amounts. We wanted to further investigate intracellular BMP-9 signaling in chondrocytes to clarify its role in cartilage maintenance and degradation. Purpose: To study Smad1/5/8 and Smad2/3 phosphorylation kinetics in response to addition of exogenous BMP-9, to investigate BMP-9’s potency on chondrocytes, and to investigate downstream signaling responses on mRNA level. Methods: Both primary bovine chondrocytes, isolated from the metacarpophalangeal joint of 2 year old animals, as well as the murine H4 chondrocyte cell line were used in this study. Chondrocytes were cultured to near confluency, and subsequently incubated with BMP-9. After short term stimulation (1 h), Smad phosphorylation was analyzed by specific Smad2/3p and Smad1/5/8p staining of Western blots. After long term stimulation (24-48 h), expression of PAI-1, a downstream marker of Smad2/3 signaling, and ID-1, a downstream marker of Smad1/5/8 signaling, were analyzed by quantitative real time PCR (qPCR). Results: In both the murine cell line, as well as the primary bovine chondrocytes, addition of BMP-9 resulted in phosphorylation of Smad 1/5/8, that was maximal at very low concentrations (100 pg / ml). Unexpectedly, Smad2/3p was also observed at this concentration. Further investigations showed that BMP-9-induced Smad2/3p was maximal after 1 h, at the physiological relevant concentration of 5 ng/ml. Exposure of chondrocytes to BMP9 not only resulted in both Smad1/5/8p and Smad2/3p but also in transcription of pathway specific response genes, as observed by a significant increase in PAI-1 and ID-1 mRNA levels after both 24 and 48h. In both cell types, collagen type 2A1 and aggrecan mRNA levels were also up regulated by BMP9. In the described experiments, no significant changes in MMP13 expression were observed after 48 h of BMP-9 stimulation. Conclusion: In chondrocytes, BMP9 is capable of inducing both Smad1/5/8p and Smad2/3p. By doing so it can up regulate both PAI-1 and ID-1 simultaneously. BMP-9 is very potent in inducing Smad phosphorylation, as very low concentrations (pg/ml) are sufficient for signal induction. The induction of Smad2/3p and subsequent PAI-1 activity sets BMP-9 apart from other BMPs, like BMP-2 and BMP-6, that only induce Smad1/5/8 phosphorylation. The lack of MMP13 expression after BMP-9 stimulation can be explained by the induced Smad2/3p expression, as this potently blocks MMP13 transcription. Based on these results we propose that exposure of young cartilage to BMP-9 does not result in MMP13 expression due to its ability to induce Smad2/3p. However, we previously reported that in ageing cartilage the ability to phosphorylate Smad2/3 is reduced, and therefore BMP-9 exposure will most likely result in MMP13 production in old cartilage.

Ginsenoside Rb1 Reverses H2O2-induced Senescence in Human Umbilical Endothelial Cells

Senescence of endothelial cells has been implicated in endothelial dysfunction and atherogenesis. This study investigated the effects of Rb1, a major ginsenoside in ginseng, on H2O2-induced senescence in primary human umbilical vein endothelial cells (HUVECs). Real-time PCR and Western blot were used to detect the mRNA and protein expression, respectively. H2O2 (40∼100 μmol/L) effectively increased SA-β-gal activity and PAI-1 mRNA levels, two important senescence related biomarkers, in HUVECs, which were dramatically inhibited by Rb1 pre-incubation. Furthermore, Rb1 administration reversed the H2O2-decreased protein and mRNA levels of eNOS and its phosphorylation at Ser-1177, and the increased eNOS phosphorylation at Thr-495. As a result, Rb1 pretreatment restored the NO generation, as assayed by nitrate reductase method. However, pretreatment with L-NAME, a NOS inhibitor, abolished all the inhibitory effects of Rb1 on senescence. Importantly, Rb1 modulated the H2O2-altered caveolin-1 and pAkt, two most important regulators of eNOS expression and activity, in HUVECs. We showed that Rb1 effectively protects HUVECs from senescence through eNOS modulation.

Hormonal regulation of tissue-type plasminogen activator and plasminogen activator inhibitor type-1 gene expression in cultured mouse Sertoli cells.

We have demonstrated for the first time that (i) mouse Sertoli cells predominantly secrete tPA under the action of FSH and cAMP-generating agents, whereas Leydig cells mainly produce uPA; (ii) Sertoli cells are also capable of secreting PAI-1, as well as FSH, growth factors and GnRH increase PAI-1 gene expression; (iii) the increases in tPA and PAI-1 activities by different hormones in the conditioned media of Sertoli cells correspond to the increases in the levels of tPA and PAI-1 mRNA in the cultured cells, suggesting that the synthesis of the activator-inhibitor in mouse Sertoli cells is regulated at a transcriptional level and the tPA secreted by Sertoli cells may be involved in the spermatogenesis.

The effects of tea polyphenols on the injury of fibrinolytic functions induced by high-methionine dietary in rats

To study the protective impact of tea polyphenols (TP) on the injury of fibrinolytic functions induced by high-methionine dietary in rats. 50 male Wistar rats were divided by stratified based on body weight into 5 groups with 10 in each group: namely control group, model group, low-dose TP group, medium-dose TP group and high-dose TP group. The rats in model group and TP groups were fed with 3% methionine dietary, control group rats with routine diet. In addition, rats in low-dose, medium-dose and high-dose TP groups were treated with TP at 50, 100 and 200 mg/kg dosage respectively by gavages every day, control group and model group rats were given with same amount distilled water. The animals were sacrificed after 8 weeks. The levels of tissue-type plasminogen activator (t-PA) and type-1 plasminogen activator inhibitor (PAI-1) in plasma were determined by ELISA assays, mRNA levels of t-PA and PAI-1 in aortic arch were detected by RT-PCR, t-PA and PAI-1 expression in aortic arch were detected by immunohistochemistry strept-avidin-biotin complex (SABC). After experiment, the t-PA expression of aortic arch in control group, model group, low-dose TP group, medium-dose TP group and high-dose TP group were 133.03 ± 10.14, 95.46 ± 11.08, 111.97 ± 11.91, 130.23 ± 10.80, 139.39 ± 9.41 (F = 14.15, P < 0.01), respectively, and the PAI-1 expression were 90.91 ± 8.67, 166.76 ± 12.18, 139.63 ± 12.71, 134.66 ± 13.19, 109.49 ± 10.82 (F = 31.44, P < 0.01). The t-PA concentration of plasma were (10.69 ± 1.26), (6.13 ± 0.92), (8.56 ± 1.19), (9.69 ± 0.92), (11.97 ± 1.08) ng/ml, respectively (F = 41.98, P < 0.01), and the PAI-1 concentration of plasma were (6.31 ± 0.81), (16.98 ± 1.27), (11.39 ± 0.82), (8.46 ± 0.67), (8.08 ± 0.91) ng/ml, respectively (F = 207.74, P < 0.01). The mRNA levels of t-PA in aortic arch were 1.12 ± 0.02, 0.75 ± 0.14, 1.01 ± 0.09, 0.95 ± 0.08, 1.05 ± 0.13 (F = 5.77, P < 0.05), and the mRNA levels of PAI-1 in aortic arch were 1.25 ± 0.11, 1.74 ± 0.06, 1.23 ± 0.05, 1.09 ± 0.14, 1.23 ± 0.04 (F = 23.56, P < 0.01). The results indicate that TP seems to have regulatory function on transcription and protein levels of t-PA and PAI-1, in addition to maintaining the balance between PAI-1 and t-PA and healing the injury of fibrinolytic functions in rats induced by high-methionine dietary.

Particulate Matter-Induced Lung Inflammation Increases Systemic Levels of PAI-1 and Activates Coagulation Through Distinct Mechanisms

BackgroundExposure of human populations to ambient particulate matter (PM) air pollution significantly contributes to the mortality attributable to ischemic cardiovascular events. We reported that mice treated with intratracheally instilled PM develop a prothrombotic state that requires the release of IL-6 by alveolar macrophages. We sought to determine whether exposure of mice to PM increases the levels of PAI-1, a major regulator of thrombolysis, via a similar or distinct mechanism.Methods and Principal FindingsAdult, male C57BL/6 and IL-6 knock out (IL-6−/−) mice were exposed to either concentrated ambient PM less than 2.5 µm (CAPs) or filtered air 8 hours daily for 3 days or were exposed to either urban particulate matter or PBS via intratracheal instillation and examined 24 hours later. Exposure to CAPs or urban PM resulted in the IL-6 dependent activation of coagulation in the lung and systemically. PAI-1 mRNA and protein levels were higher in the lung and adipose tissue of mice treated with CAPs or PM compared with filtered air or PBS controls. The increase in PAI-1 was similar in wild-type and IL-6−/− mice but was absent in mice treated with etanercept, a TNF-α inhibitor. Treatment with etanercept did not prevent the PM-induced tendency toward thrombus formation.ConclusionsMice exposed to inhaled PM exhibited a TNF-α-dependent increase in PAI-1 and an IL-6-dependent activation of coagulation. These results suggest that multiple mechanisms link PM-induced lung inflammation with the development of a prothrombotic state.

Study of the mechanisms of aldosterone prothrombotic effect in rats

We investigated the role of primary haemostasis, fibrinolysis, nitric oxide (NO) and oxidative stress as well as mineralocorticoid receptors (MR) in acute aldosterone prothrombotic action. Venous thrombosis was induced by stasis in Wistar rats. Aldosterone (ALDO; 10, 30, 100 µg/kg/h) was infused for 1 h. Eplerenone (EPL; 100 mg/kg, p.o.), a selective MR antagonist, was administered before ALDO infusion. Bleeding time (BT) and platelet adhesion to collagen were evaluated. The expression of nitric oxide synthase (NOS), NADPH oxidase, superoxide dismutase (SOD) and plasminogen activator inhibitor (PAI-1) was measured. NO, malonyl dialdehyde (MDA) and hydrogen peroxide (H(2)O(2)) plasma levels were assayed. Significant enhancement of venous thrombosis was observed after ALDO infusion. ALDO shortened BT and increased platelet adhesion. Marked increases were observed in PAI-1, NADPH oxidase and SOD mRNA levels. MDA and H(2)O(2) levels were augmented in ALDO-treated groups, and NOS expression and NO level were decreased. EPL reduced ALDO effects on thrombus formation, primary haemostasis, PAI-1 expression and MDA level. Short-term ALDO infusion enhances experimental venous thrombosis in the mechanism involving primary haemostasis, fibrinolysis, NO and oxidative stress-dependent pathways. The MR antagonist only partially diminished the ALDO effects, suggesting the involvement of additional mechanisms.

E0147 Mechanism of different activators of PPARαregulating plasminogen activator inhibitor-1 expression

ObjectiveTo observe the effects of fenofibrate and linoleic acid which was the different activators of PPARα on the expression of PAI-1 and PPARα in HepG2 cells. To investigate the relationship...

The Plasminogen System in Microdissected Colonic Mucosa Distant from an Isolated Adenoma

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.

Increased expression of urokinase plasminogen activator and its cognate receptor in human seminomas

BackgroundThe urokinase plasminogen activating system (uPAS) is implicated in neoplastic progression and high tissue levels of uPAS components correlate with a poor prognosis in different human cancers. Despite that, relative few studies are available on the expression and function of the uPAS components in human seminomas. In the present study we characterized the expression of the urokinase plasminogen activator (uPA), its cognate receptor (uPAR) and the uPA inhibitors PAI-1 and PAI-2 in normal human testis and seminomas.MethodsThe expression of the above genes was evaluated by means of quantitative RT-PCR, western blot, zymographic analysis and immunohistochemistry.ResultsQuantitative RT-PCR analysis of 14 seminomas demonstrated that uPA and uPAR mRNAs were, with respect to control tissues, increased in tumor tissues by 3.80 ± 0.74 (p < 0.01) and 6.25 ± 1.18 (p < 0.01) fold, respectively. On the other hand, PAI-1 mRNA level was unchanged (1.02 ± 0.24 fold), while that of PAI-2 was significantly reduced to 0.34 ± 0.18 (p < 0.01) fold. Western blot experiments performed with protein extracts of three seminomas and normal tissues from the same patients showed that uPA protein levels were low or undetectable in normal tissues and induced in tumor tissues. On the same samples, zymographic analysis demonstrated increased uPA activity in tumor tissue extracts. Western blot experiments showed that also the uPAR protein was increased in tumor tissues by 1.83 ± 0.15 fold (p < 0.01). The increased expression of uPA and uPAR was further confirmed by immunohistochemical staining performed in 10 seminomas and autologous uninvolved peritumoral tissues. Finally, variation in the mRNA level of PAI-1 significantly correlated with tumor size.ConclusionsWe demonstrated the increased expression of uPA and uPAR in human seminomas with respect to normal testis tissues, which may be relevant in testicular cancer progression.