Plasminogen Activator Inhibitor Type-1 Expression Research Articles

Carbamylated LDL induces a pro-thrombotic state via the LOX-1 receptor and arterial thrombus formation: a novel mechanism of cardiovascular events in end-stage renal disease

Background: Carbamylation of low-density lipoproteins (cLDL) alters protein structure and is thought to promote vascular inflammation and dysfunction during end-stage renal disease (ESRD). However, it remains unknown whether cLDL exerts prothrombotic effects in vascular cells and platelets and whether cLDL affects arterial thrombus formation in vivo. Methods: LDL was isolated from healthy subjects or patients with ESRD by sequential ultra-centrifugation. Ex vivo carbamylation of LDL (cLDL) from healthy subjects was induced with potassium cyanate. Arterial thrombus formation was analysed in a murine carotid artery photochemical injury model. Analysis of tissue factor (TF) and plasminogen activator inhibitor type-1 (PAI-1) expression was performed by Western blot and real-time PCR analysis. Platelet aggregation was analysed by impedance aggregometry. Results: Intravenous administration of cLDL (2mg/kg body weight) accelerated arterial thrombus formation in a murine photochemical carotid artery injury model as compared to treatment with native LDL (2mg/kg body weight) or vehicle (PBS) (n=8; p<0.05 versus nLDL and control). Analyses of tissue lysates demonstrated that cLDL induced the expression of TF and PAI-1 mRNA levels in carotid arteries (n=5, p<0.01). In human aortic smooth muscle cells (AoSMC) cLDL (10-300 μg/ml) induced TF and PAI-1 expression by 3.5- and 3-fold, as compared with nLDL (n=4; p<0.01). cLDL also enhanced basal and TNF-α induced expression of TF and PAI-1 in human aortic endothelial cells (n=4; p<0.02). In contrast, native LDL (nLDL) had no effect on endothelial expression or activity of TF and PAI-1. In both cell types, cLDL activated the mitogen-activated protein kinases p38 and ERK (n=3; p<0.05) as well as the transcription factor NFkB (n=4, p<0.05). Silencing of lectin-like oxidized LDL receptor-1 (LOX-1) using specific siRNA prevented the induction of TF and PAI-1 expression in vascular cells. In platelets cLDL activated the p38 MAP kinase and induced platelet aggregation. Finally, LDL isolated from patients with ESRD mimicked the prothrombotic effects of cLDL on vascular cells and platelets. Conclusions: These data demonstrate that cLDL at physiologically relevant concentrations,via activation of the LOX-1 receptor induces potent pro-thrombotic effects in vascular cells and platelets leading to enhanced thrombus formation in vivo. This observation may explain the markedly increased incidence of fatal acute thrombotic events in patients with end-stage renal disease and lead to the development of novel LDL targeting strategies in this population.

PI3K/p110alpha inhibition selectively interferes with arterial thrombosis and neointima formation, but not re-endothelialization: a new concept in drug-eluting stent design

Background: Impaired re-endothelialization and stent thrombosis are a safety concern associated with drug-eluting stents (DES) as mTOR inhibitors reduce restenosis rates, but impair endothelialization and facilitate thrombus formation. PI3K/p110α controls cellular wound healing pathways, thereby representing an emerging drug target to modulate vascular homeostasis after injury. However, its effect on arterial thrombosis and neointima formation remains unknown. Methods: PI3K/p110α was inhibited by treatment with the small molecule inhibitor PIK75 or a specific siRNA. Arterial thrombosis, neointima formation and re-endothelialization studies were performed in a murine carotid artery injury model. Proliferation and migration of VSMC and EC were assessed by cell number and Boyden chamber, respectively. Endothelial senescence was evaluated by β-galactosidase assay, endothelial dysfunction by organ chambers for isometric tension. Tissue factor (TF), plasminogen activator inhibitor type 1 (PAI-1), and eNOS expression was determined by Western blot and real-time PCR analysis. Results: Male C57Bl6 mice were either treated with PIK75 (10 mg/kg/d for 7 days) or vehicle. Arterial thrombus formation was delayed in mice treated with PIK75 as compared to controls (n=8; p<0.005). PIK 75 impaired arterial expression and activity of TF and PAI-1 as well as NFκB activity (n=8; p<0.05); in contrast, plasma clotting and tail bleeding times did not differ (n=8; p=NS). In human vascular smooth muscle and endothelial cells, PIK75 inhibited expression and activity of TF and PAI-1 (n=4; p<0.01). These effects occurred at the transcriptional level via the RhoA signaling cascade and the transcription factor NFkB. Furthermore, inhibition of PI3K/p110α with PIK75 or a specific siRNA selectively impaired proliferation and migration of VSMC while sparing EC completely. In line with this observation, treatment with PIK75 selectively inhibited neointima formation without affecting re-endothelialization following vascular injury. In contrast to rapamycin and paclitaxel, treatment with PIK75 did not induce endothelial senescence nor inhibit eNOS expression or endothelium-dependent relaxations. Conclusions: Following vascular injury, PI3K/p110α inhibition selectively interferes with arterial thrombosis and neointima formation, but not reendothelialization. Hence, PI3K/p110α represents an attractive new target in DES design.

Beware the low HDAC11: males at risk for ischemic kidney injury

plasminogen activator inhibitor type 1 (PAI-1) is a member of the serine protease inhibitor (serpin) family. As such, it is well known as a physiological inhibitor of fibrinolysis, but PAI-1 also plays a key role in inflammation and production of extracellular matrix proteins (reviewed in Ref. 1). PAI-1 is implicated in the pathology of fibrosis in the heart, lung, kidney, liver, and skin. In pathologic states, excessive PAI-1 contributes to accumulation of collagen and other extracellular matrix proteins, while lack of PAI-1 protects from fibrosis in response to injury-related profibrotic signals. Involvement of PAI-1 has been implicated in disease states that include insulin resistance, diabetes, cardiovascular disease, kidney fibrosis in unilateral ureteral obstruction, and aldosterone-induced glomerular injury. Regulation of PAI-1 levels is complicated and occurs through transcriptional, posttranscriptional, and posttranslational mechanisms (reviewed in Ref. 1). Multiple cytokines and growth factors, including transforming growth factor (TGF)-β, epidermal growth factor, and insulin, and endotoxin and oxidative stress impact local PAI-1 production through these mechanisms. Thus PAI-1 may have a role in outcomes in chronic kidney disease and especially acute kidney injury. In an issue of the American Journal of Physiology-Renal Physiol, Kim et al. (4) examined the effect of both PAI-1 expression and gender in outcome of kidney ischemia/reperfusion (I/R)-induced injury in mice. Male gender and male hormone testosterone increase susceptibility to renal I/R injury (5); mechanisms that underlie these effects were elucidated by Kim et al. (4). They noticed an unimpressive increase in renal PAI-1-encoding gene (Serpine1) expression in females with or without ovariectomy 24 h after a 30-min I/R injury. In contrast, PAI-1-encoding gene expression in orchiectomized males was also low, but administration of dihydrotestosterone (DHT) triggered an increase to levels comparable to those seen in males without orchiectomy. Because I/R injury represents a well-established model of gender dimorphism in acute kidney injury (AKI) outcomes and since pharmacological PAI-1 inhibition attenuated adverse renal I/R effects, the authors then examined the gender-specific mechanisms regulating PAI-1 expression in this model. They observed that renal expression of several histone deacetylase (HDAC)-encoding genes (i.e., Hdac1, 5, 8, 9, and 11) decreased following 30 min of renal I/R injury. However, decreased expression of Hdac9 and Hdac11 did not occur in orchiectomized males exposed to kidney I/R injury. In addition, among the five studied HDAC isotypes, only HDAC11 inhibited PAI-1 expression (using an activated monocyte/macrophage cell line and treatment with HDAC11 small interfering RNA). Based on chromatin immunoprecipitation assay, this inhibition was mediated by direct HDAC11 interaction with the PAI-1 gene (Serpine1) promoter. The I/R-induced release of HDAC11 from PAI-1 promoter then led to increased histone H3 acetylation. This effect was observed only in males without orchiectomy but not in orchiectomized males or intact or ovariectomized females. This observation, coupled with the fact that DHT reversed the effect of orchiectomy on I/R-induced decreased HDAC11 expression, pointed to a testosterone-HDAC11-PAI-1 axis as a likely pathway regulating gender-induced differences in I/R-induced renal injury (Fig. 1). Fig. 1. Schema of the effects of testosterone on plasminogen activator inhibitor type 1 (PAI-1)-enhanced renal injury. Testosterone increases severity of kidney ischemia/reperfusion (I/R)-induced renal injury in mice by inhibiting histone deacetylase 11 (HDAC11). ... Gender is an important regulator of susceptibility to AKI in a wide range of renal insults (reviewed in Ref. 3). These gender-induced differences have been attributed to biological effects of male and female sex hormones. While some of these differences may be explained by testosterone-enhanced susceptibility to endoplasmic reticulum stress (2), additional specific mechanisms underlying the sex hormone effects in AKI have remained elusive. The current study by Kim et al. (4) points to testosterone-induced HDAC11-regulated histone H3 acetylation as a key mechanism responsible for gender-related susceptibility to AKI. This new concept offers a novel avenue for development of gender-based therapeutic strategies, an important component of emerging personalized medical care (7). While such strategies hold potential for reducing adverse outcomes in clinical care (e.g., in males with high risk of AKI), first it is necessary to confirm that the above concept is not murine specific but can be also applied to humans and other mammals (e.g., unlike the outcome of the current study on mice, testosterone protected kidneys from I/R-induced AKI in rats; Ref. 6). In addition, the findings in the paper also raise questions of potentially unpredictable adverse effect of non-selective HDAC inhibitors in AKI. Unfortunately, specific HDAC11 activity-enhancing therapeutics have not been described; their development may hasten potential translation of this discovery to clinical care.

Effects of black cohosh on the plasminogen activator system in vascular smooth muscle cells

ObjectiveThe rhizome of the Cimicifuga racemosa plant (commonly known as black cohosh) has been used for menopausal complaints. Studies regarding the cardiovascular effects of black cohosh are lacking. We investigated the effect of black cohosh on the plasminogen activator system in cultured vascular smooth muscle cells (VSMCs). MethodsVSMCs were isolated from rat aortae. Expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) proteins were evaluated by Western blot analysis and enzyme-linked immunosorbent assay, respectively. The activities of PAI-1 and t-PA in the conditioned media were assessed by fibrin overlay zymography. A 40% 2-propanol extract of black cohosh was used. ResultsBlack cohosh extract (BcEx) stimulated the protein expression of PAI-1, but it did not affect that of t-PA. Vitamin E, a potent antioxidant, inhibited the BcEx-induced increase in PAI-1 expression, while ICI 182,780, an estrogen receptor antagonist, had no effect. Fibrin overlay zymography revealed that BcEx increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA by inducing a binding to PAI-1. ConclusionsBcEx induces PAI-1 protein expression in the VSMCs likely via an oxidant mechanism. It also stimulates the enzyme activity of PAI-1 and reduces that of free t-PA. These findings suggest that black cohosh might exert a negative influence on fibrinolysis.

Gender-specific role of HDAC11 in kidney ischemia- and reperfusion-induced PAI-1 expression and injury

Male gender and the male hormone testosterone increase susceptibility to kidney ischemia and reperfusion (I/R) injury, which is associated with inflammatory responses. Possible involvement of histone deacetylase (HDAC) in inflammatory responses has been suggested. We investigated the gender-specific role of HDACs in plasminogen activator inhibitor type-1 (PAI-1) expression and I/R injury. PAI-1 inhibition protected the kidney from I/R-induced inflammation and functional loss. Among HDACs, only HDAC11 negatively regulated PAI-1 expression in I/R-subjected kidney gender specifically and lipopolysaccharide (LPS)-stimulated mouse monocytes/macrophages. HDAC11 gene silencing increased PAI-1 expression. Chromatin immunoprecipitation assay confirmed binding of HDAC11 to the promoter region of PAI-1 and then release by I/R insult or LPS treatment. I/R-induced HDAC11 release was inhibited by orchiectomy and reversed by dihydrotestosterone treatment. Release of HDAC11 increased acetylation of histone H3. In conclusion, male gender and male hormones accelerate I/R-induced decreases in expression and binding of HDAC11, resulting in an increase in PAI-1 expression. These data provide important insight into gender dimorphism offering HDAC11 as a novel target for I/R injury.

Abstract 4683: Correlation of transcript levels of members of the urokinase-type plasminogen activator system with clinico-pathological parameters in prostate cancer .

Abstract Background: Members of the urokinase-type plasminogen activator (uPA) system play an important role in tumor progression and angiogenesis in different human malignancies including breast, kidney, colon, stomach and prostate cancer (PCa). Aim of the study was to characterize mRNA expression levels of members of the uPA system in tissue specimens from primary PCa and their potential association with clinico-pathological parameters. Methods: Transcript levels of members of the uPA system, i.e., uPA, uPA receptor (uPAR) and plasminogen activator inhibitor type 1 (PAI-1) were analyzed in a cohort of 132 corresponding pairs of malignant and adjacent non-malignant prostate tissue samples. Relative gene expression levels were determined by quantitative PCR (qPCR) and normalized to the reference gene TATA-box-binding protein (TBP). Potential correlations between the three markers themselves and the clinico-pathological parameters were assessed by Spearman's Rho test and by Fisher's exact test. Results: There were no significant differences in expression levels of uPA system genes between the corresponding malignant and non-malignant tissue specimens. Transcript levels of all members of the uPA system displayed a significant correlation with each other in the tumor tissues. At stratifying uPA, uPAR and PAI-1 expression levels by the median a significant correlation between an elevated expression of uPA and PAI-1 was correlated with higher Gleason score (P = 0.001 and P = 0.007). In addition, a significant positive correlation between uPAR transcript levels and tumor stage was detected (P = 0.025). Conclusion: Our results demonstrate that mRNA expression levels of members of the uPA system correlated with each other and with clinico-pathological parameters. We suggest that transcript levels of uPA system members can be helpful markers for the study of tumor behavior of prostate cancer. Citation Format: Omar Al-Janabi, Helge Taubert, Sven Wach, Robert Stöhr, Maximilian Burger, Wolf Ferdinand Wieland, Andrea Lohse-Fischer, Michael Fröhner, Manfred P. Wirth, Bernd Wullich, Viktor Magdolen, Matthias Kotzsch, Susanne Fuessel. Correlation of transcript levels of members of the urokinase-type plasminogen activator system with clinico-pathological parameters in prostate cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4683. doi:10.1158/1538-7445.AM2013-4683

Abstract 1922: Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression.

Abstract Introduction: Bladder cancer (BCa) is among the five most common malignancies worldwide and has the highest recurrence rate of any tumor type. BCa biology is poorly understood and early detection remains one of the most urgent issues in BCa research. Previously we have identified a preliminary molecular signature of BCa in voided urine samples from BCa patients. Our results revealed aberrant overexpression of the serine protease inhibitor (serpin) plasminogen activator inhibitor type-1 (PAI-1), both at the genomic and protein level. Increasing evidence indicate that elevated levels of PAI-1 correlates to a poor prognosis in many tumor types including breast, colorectal, gastric, ovarian, and more recently bladder cancer. PAI-1 has emerged as a potential target for BCa treatment and the aim of this study is to provide a greater understanding of its role in bladder cancer progression. Methods: PAI-1 expression levels in UROtsa (benign urothelial cell), KU7 (low-grade papillary urothelial cancer cell), and UMUC14 and T24 (high-grade invasive urothelial cancer cell lines) was tested by real-time PCR (RTPCR) and Western blot analysis. Secreted PAI-1 levels were measured by ELISA. KU7 stable clones overexpressing PAI-1 was established by transfection of plasmid DNA encoding human PAI-1. T24 and UMUC14 stable clones with shRNA knockdown of PAI-1 were established by infection with retroviral particles. Cell proliferation, migration, invasion, and apoptosis assays were performed in stable clones. Results: RTPCR analysis revealed an increase of PAI-1 in T24 and UMUC14 cells (27.9 and 5.2 times, respectively) while KU7 cells exhibited significantly reduced levels as compared to the UROtsa control. Similar results were observed for PAI-1 protein concentrations in cell culture supernatants and western blot analysis. While overexpression of PAI-1 in KU7 cells promoted an increase in proliferation, migration and invasion, these were significantly reduced upon inactivation of PAI-1 in T24 and UMUC14 cells stably expressing shRNA PAI-1. Loss of PAI-1 function also led to a substantial increase in apoptotic cell death. Conclusions: These results suggest that PAI-1 overexpression in BCa promotes tumor progression and therapeutic strategies targeting PAI-1 may serve clinically beneficial outcomes. Funding provided by research grants from Florida Department of Health James and Esther King Team Science Award 10KT-01 (CJR) and National Cancer Institute RO1 CA116161 (SG). Citation Format: Evan Gomes, Steven Goodison, Makito Miyake, Shanti Ross, Ge Zhang, Charles Rosser. Plasminogen activator inhibitor type-1 (PAI-1) enhances bladder cancer tumor progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1922. doi:10.1158/1538-7445.AM2013-1922

Plasminogen activator inhibitor-1 in kidney pathology

Plasminogen activator inhibitor type-1 (PAI-1) inhibits tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which convert plasminogen to plasmin, a strong proteolytic enzyme. Thus, PAI-1 is a primary and negative regulator of plasmin-driven proteolysis. In addition to its main role as an inhibitor of fibrinolysis, PAI‑1 has been implicated as a mediator in other processes, including fibrosis, rheumatoid arthritis, atherosclerosis, tumor angiogenesis and bacterial infections. It also significantly modulates cellular adhesion or migration, wound healing, angiogenesis and tumor cell metastasis. However, in the present study, we have reviewed the literature in relation to different kidney diseases where PAI-1 regulates fibrinolysis and acts independently of proteolysis. PAI-1 is normally produced in trace amounts in healthy kidneys but is synthesized in a wide variety of both acute and chronic diseased kidneys. We reviewed the role of PAI-1 in diabetic kidney nephropathy, chronic kidney disease, hemodialysis, peritoneal dialysis and in kidney transplantation. Increased PAI-1 expression results in accumulation of extracellular matrix (ECM) leading to numerous kidney diseases. Predisposition to some diseases is due to the genetic role of PAI-1 in their development. A number of studies demonstrated that the inhibition of PAI-1 activity or therapy with a mutant PAI-1 increases matrix turnover and reduces glomerulosclerosis by competing with endogenous PAI-1. This strongly suggests that PAI-1 is a valid target in the treatment of fibrotic renal disease. However, net proteolytic activity depends on the delicate balance between its negative regulation by PAI-1 and activation by uPA and tPA. Also, plasmin activated by its inhibitors upregulates activity of other enzymes. Thus, assessment of prognosis for the diseased kidney should include a variety of proteolysis regulators and enzymes.

Cell Density-dependent Nuclear Accumulation of ELK3 is Involved in Suppression of PAI-1 Expression

Cell-cell contact regulates the proliferation and differentiation of non-transformed cells, e.g., NIH/3T3 cells show growth arrest at high cell density. However, only a few reports described the dynamic behavior of transcription factors involved in this process. In this study, we showed that the mRNA levels of plasminogen activator inhibitor type 1 (PAI-1) decreased drastically at high cell density, and that ELK3, a member of the Ets transcription factor family, repressed PAI-1 expression. We also demonstrated that while ELK3 was distributed evenly throughout the cell at low cell density, it accumulated in the nucleus at high cell density, and that binding of DNA by ELK3 at the A domain facilitated its nuclear accumulation. Furthermore, we found that ETS1, a PAI-1 activator, occupied the ELK3-binding site within the PAI-1 promoter at low cell density, while it was released at high cell density. These results suggest that at high cell density, the switching of binding of transcription factors from ETS1 to ELK3 occurs at a specific binding site of the PAI-1 promoter, leading to the cell-density dependent suppression of PAI-1 expression.

Expression of urokinase plasminogen activator and plasminogen activator inhibitor type-1 in ovarian cancer and its clinical significance

The urokinase plasminogen activator system, which consists of urokinase plasminogen activator (uPA), plasminogen activator inhibitor type-1 (PAI-1) and urokinase plasminogen activator receptor (uPAR), plays an important role in tumor invasion and metastasis, and it may be a potential diagnostic biomarker and therapeutic target in cancer. It has been found that the expression of uPA and PAI-1 in ovarian cancer is related to clinical pathologies, while their effects on the biological behavior of tumor cells and their clinical significance are still unknown. In this study, 100 tissue samples (60 samples from malignant tumors, 20 from benign tumors and 20 from controls) and 147 blood samples (49 samples each from patients with malignant tumors, benign tumors and control group, respectively) were analyzed. The positive expression levels of uPA and PAI-1 in the malignant tumor samples and their serum concentrations in the malignant group were all significantly higher than these levels in the benign tumors and controls. In addition, the levels in patients with poorly differentiated and stageIII-IV cancers, cancers with metastases as well as residual tumors >2cm after surgery, were all obviously increased, consistent with their concentrations in serum. The Cox model analysis showed that expression of uPA at the transcription level had significant associations with prognosis. In addition, uPA greatly enhanced the abilities of cell invasion, migration and adhesion through its overexpression in SKOV3 cells. Collectively, our results showed that uPA and PAI-1 play important roles in ovarian cancer development; therefore, their expression in tissues and their concentrations in serum would greatly assist the diagnosis and prediction of the prognosis in ovarian cancer.

Antifibrotic Effects of Pirfenidone in Rat Proximal Tubular Epithelial Cells

Objective: Renal fibrosis is a common cause of renal dysfunction with chronic kidney disease. We previously investigated the renoprotective effects of the antifibrotic agent pirfenidone in a rat model of subtotal nephrectomy. Here, we further evaluated the antifibrotic effects of pirfenidone in rat proximal tubular epithelial cells. Methods: NRK52E cells were incubated in a medium containing either transforming growth factor (TGF)-β1 (3 ng/mL) or platelet-derived growth factor (PDGF)-BB (5 Ang/mL) or both, with or without pirfenidone (0.1–1 mmol/L), for 24 h to assess mRNA expression, for 48 h to assess protein production, and for 1 h or various time (5–120 min) to assess phosphorylation of signal kinase. Results: TGF-β1, a key mediator in renal fibrosis, induced increases in the mRNA expression of various profibrotic factors and extracellular matrix, including plasminogen activator inhibitor type 1 (PAI-1), fibronectin, type 1 collagen, and connective tissue growth factor (CTGF)—increases which pirfenidone significantly inhibited. Specifically, pirfenidone potently inhibited TGF-β1-induced increases in the mRNA expression and protein secretion of PAI-1, an effect mediated, at least in part, via the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling. Further, PDGF-BB, which has been implicated in renal interstitial fibrosis, potently activated PAI-1 expression under TGF-β1 stimulation, and pirfenidone significantly inhibited TGF-β1- and PDGF-BB-induced increases in PAI-1 expression. Conclusions: Taken together, these results suggest that TGF-β1 closely correlates with renal fibrosis in cooperation with several fibrosis-promoting molecules, such as PAI-1 and PDGF, in rat proximal tubular epithelial cells, and pirfenidone inhibits TGF-β1-induced fibrosis cascade and will therefore likely exert antifibrotic effects under pathological conditions.

Transforming growth factor-1 promotes the transcriptional activation of plasminogen activator inhibitor type 1 in carcinoma-associated fibroblasts

Carcinoma-associated fibroblasts (CAFs) play a pivotal role in promoting the growth, invasion and metastasis of tumor cells. However, to date little is known about the oncogenic mechanisms of CAFs. This study aimed to identify the microenvironmental factors involved in tumor development and progression directed by CAFs in liver metastases. Tissue samples collected from 20 patients with colorectal liver metastases were used in this study. Histological and morphological characterization of the samples was performed using hybridization and immunohistological assays. The mRNA expression of α-smooth muscle actin (α-SMA) was measured by northern blotting. The expression of plasminogen activator inhibitor type1 (PAI-1) was measured by enzyme-linked immunosorbent assay (ELISA). As a result, co-expression of Thy-1 (CD90) and α-SMA was identified in CAFs, while normal liver samples were negative for α-SMA and Thy-1. Compared with epidermal growth factor (EGF) and tumor necrosis factor (TNF) incubation, the expression of α-SMA increased significantly following transforming growth factor-1 (TGF-1) incubation (P<0.05), while platelet-derived growth factor (PDGF) caused a significant suppression of α-SMA expression (P<0.05). PAI-1 expression was significantly lower in unstimulated fibroblasts compared to TGF-1-treated fibroblasts (P<0.01). The levels of PAI-1 transcription were significantly higher in CAFs from the patient samples compared with the healthy controls. Taken together, our findings suggest that CAFs may be important in migration, matrix degradation, invasion and angiogenesis of tumors, and TGF-1 may promote the activation of PAI-1 transcription in CAFs.

Posttranscriptional regulation of expression of plasminogen activator inhibitor type-1 by sphingosine 1-phosphate in HepG2 liver cells

Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a major physiologic inhibitor of fibrinolysis, is implicated in the progression of atherosclerosis. Sphingosine 1-phosphate (S1P) regulates expression of diverse genes and alters expression of PAI-1 in several types of cells. However, the nature of posttranscriptional regulation of expression of PAI-1 by S1P has not yet been thoroughly elucidated. The present study was undertaken to determine whether S1P has important effects on the posttranscriptional regulation of PAI-1 expression. To evaluate this possibility, we determined promoter activity, mRNA levels, 3′-untranslated region (UTR) activity, and protein levels of PAI-1 in HepG2 cells. S1P increased PAI-1 promoter activity and the expression of PAI-1 mRNA within 4h of exposure. It decreased the expression of PAI-1 mRNA and the accumulation of PAI-1 protein into the media in 24h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2kb). S1P decreased the baseline luciferase activity of the 1kb fragment of the 3′ terminus (+2177 to 3176nt) of the 3′-UTR of the 3.2kb PAI-1 mRNA [3′-UTR (+2177–3176)]. S1P decreased expression of PAI-1 protein, presumably by regulating PAI-1 expression at the posttranscriptional level thereby affecting mRNA stability. SERPINE1 mRNA binding protein (SERBP1) and ARE3 in the 3′-UTR were involved in the posttranscriptional regulation by S1P. Our data suggest that S1P can destabilize 3.2kb PAI-1 mRNA through specific effects on the 3′-UTR. These effects appear to involve SERBP1 leading to decreased expression of PAI-1 protein.

Monitoring PAI-1 and VEGF Levels in 6 Human Squamous Cell Carcinoma Xenografts During Fractionated Irradiation

Previous studies have shown that the plasminogen activator inhibitor type-1 (PAI-1) and vascular endothelial growth factor (VEGF) are regulated by hypoxia and irradiation and are involved in neoangiogenesis. The aim of this study was to determine invivo whether changes in PAI-1 and VEGF during fractionated irradiation could predict for radiation resistance. Six xenografted tumor lines from human squamous cell carcinomas (HSCC) of the head and neck were irradiated with 0, 3, 5, 10, and 15 daily fractions of 2 Gy. The PAI-1 and VEGF antigen levels in tumor lysates were determined by enzyme-linked immunosorbent assay kits. The amounts of PAI-1 and VEGF were compared with the dose to cure 50% of tumors (TCD(50)). Colocalization of PAI-1, pimonidazole (hypoxia), CD31 (endothelium), and Hoechst 33342 (perfusion) was examined by immunofluorescence. Human PAI-1 and VEGF (hVEGF) expression levels were induced by fractionated irradiation in UT-SCC-15, UT-SCC-14, and UT-SCC-5 tumors, and mouse VEGF (msVEGF) was induced only in UT-SCC-5 tumors. High hVEGF levels were significantly associated with radiation sensitivity after 5 fractions (P=.021), and high msVEGF levels were significantly associated with radiation resistance after 10 fractions (P=.007). PAI-1 staining was observed in the extracellular matrix, the cytoplasm of fibroblast-like stroma cells, and individual tumor cells at all doses of irradiation. Colocalization studies showed PAI-1 staining close to microvessels. These results indicate that the concentration of tumor-specific and host-specific VEGF during fractionated irradiation could provide considerably divergent information for the outcome of radiation therapy.

Impact of Lysophosphatidylcholine on the Plasminogen Activator System in Cultured Vascular Smooth Muscle Cells

The balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) regulates fibrinolysis. PAI-1 expression increases in atherosclerotic arteries and vascular smooth muscle cells (VSMCs) are one of major constituents of atheroma. We investigated the impact of lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, on the plasminogen activator system of the rat VSMCs. The lysoPC stimulated the protein and gene expressions of PAI-1 but did not affect the protein expression of t-PA. Fibrin overlay zymography revealed that lysoPC increased the activity of PAI-1 in the conditioned media, while concurrently decreasing that of free t-PA. Vitamin E inhibited the lysoPC-induced PAI-1 expression. Further, lysoPC increased the intracellular reactive oxygen species (ROS) formation. Caffeic acid phenethyl ester, an inhibitor of NF-κB, blocked this lysoPC effect. Indeed, lysoPC induced the NF-κB-mediated transcriptional activity as measured by luciferase reporter assay. In addition, genistein, an inhibitor of protein-tyrosine kinase (PTK), diminished the lysoPC effect, while 7,12-dimethylbenz[a]anthracene, a stimulator of PTK, stimulated PAI-1 production. In conclusion, lysoPC does not affect t-PA expression but induces PAI-1 expression in the VSMC by mediating NF-κB and the genistein-sensitive PTK signaling pathways via oxidative stress. Importantly, lysoPC stimulates the enzyme activity of PAI-1 and suppresses that of t-PA.

High level of urokinase plasminogen activator contributes to cholangiocarcinoma invasion and metastasis.

To investigate the role of urokinase plasminogen activator (uPA) in cholangiocarcinoma (CCA) invasion and its correlation with clinicopathological parameters. uPA expression in CCA tissue was determined by immunohistochemistry. The level of uPA from two CCA cell lines (HuCCA-1 and KKU-M213) and a non-cancer immortalized cholangiocyte cell line (H69) was monitored by plasminogen-gelatin zymography and western blotting, whereas that of plasminogen activator inhibitor type 1 (PAI-1) protein and uPA receptor (uPAR) mRNA was monitored by western blotting and quantitative real-time reverse transcriptase polymerase chain reaction, respectively. Two independent methods were employed to suppress uPA function: a synthetic uPA inhibitor (B428) and silencing of uPA gene expression using siRNA. In vitro invasion of the uPA-disrupted cells was assessed by Matrigel-coated Transwell assay. The immunohistochemical study showed that 75.3% (131/174) of CCA tissues expressed uPA. High uPA expression was correlated with lymphatic invasion and metastasis of CCA patients. Plasminogen-gelatin zymography of the conditioned media and cell-surface eluates showed that both CCA cell lines, but not H69, expressed both secreted and membrane-bound forms of uPA. Although the two CCA cell lines, HuCCA-1 and KKU-M213, expressed a relatively high level of uPA and uPAR, the latter exhibited a much lower degree of in vitro invasiveness, correlating with a high expression of PAI-1 in the latter, but not in the former. Suppressing uPA function with a specific uPA inhibitor, B428, or with siRNA against uPA reduced in vitro invasiveness of KKU-M213 cells, demonstrating the requirement for uPA in the invasiveness of CCA cells. Therefore, our in vivo and in vitro studies suggest that uPA is an important requirement for the invasion process of CCA. uPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro, suggesting its potential as a therapeutic target.

Modulators of induction of plasminogen activator inhibitor type-1 in HepG2 cells by transforming growth factor-β

An increased expression of plasminogen activator inhibitor type-1 (PAI-1) has been implicated in accelerating atherogenesis and coronary artery disease in patients with type 2 diabetes. Transforming growth factor (TGF)-β increases its expression. An increased PAI-1 appears to predispose also to augmented fibrosis potentially contributing to negative left ventricular remodeling and heart failure after myocardial infarction. Diabetes is well known to induce oxidative stress. To elucidate molecular mechanisms underlying an increased PAI-1 production, the effects of TGF-β and oxidative stress implicated as agonists of PAI-1 synthesis were characterized with the use of human liver-derived HepG2 cells. PAI-1 mRNA was assayed by real-time PCR, and PAI-1 protein was assayed by western blotting. PAI-1 promoter (-825 -+42 bp) activity was assessed with the luciferase assay. The role of the 3'-untranslated region was delineated with the use of luciferase constructs containing the 3'-untranslated region. Oxidative stress was measured after loading carboxy-2,7-dichlorodihydrofluorescein into cells. TGF-β increased oxidative stress, which was accompanied by increases in NADPH oxidase 3 mRNA and membrane translocation of Rac proteins. TGF-β-inducible increases in the PAI-1 promoter activity involved Smad-binding elements and a nuclear factor-κB-binding site. TGF-β did not increase the activity of the PAI-1 mRNA 3'-untranslated region. TGF-β-inducible PAI-1 expression was attenuated by simvastatin and curcumin, a natural polyphenol. TGF-β can increase the expression of PAI-1 through multiple mechanisms involving Smad and nuclear factor-κB pathways and oxidative stress. As both oxidative stress and PAI-1 production were reduced by simvastatin and curcumin, modulation of oxidative stress and PAI-1 production are attractive targets for pharmacotherapy of cardiovascular disorders associated with an increased PAI-1 including type 2 diabetes and its associated consequences including accelerated coronary artery disease and an increased fibrosis that may exacerbate adverse left ventricular remodeling after myocardial infarction.

Ascochlorin suppresses TGF-β1-induced PAI-1 expression through the inhibition of phospho-EGFR in rat kidney fibroblast cells

Fibrosis is induced by the excessive and abnormal deposition of extracellular matrix (ECM) with various growth factors in tissues. Transforming growth factor-β1 (TGF-β1), the growth factor involved in fibrosis, modulates ECM synthesis and accumulation. TGF-β1 enhances the production of stimulators of ECM synthesis such as plasminogen activator inhibitor type 1 (PAI-1). As such, PAI-1 expression directly influences the proteolysis, invasion, and accumulation of ECM. It was shown in this study that ascochlorin, a prenylpenl antiobiotic, prevents the expression of profibrotic factors, such as PAI-1 and collagen type I, and that the TGF-β1-induced PAI-1 promoter activity is inhibited by ascochlorin. Ascochlorin abolishes the phosphorylation of the EGFR-MEK-ERK signaling pathway to regulate the TGF-β1-induced expression of PAI-1 without the inhibition of TβRII phosphorylation. Furthermore, the MEK inhibitor and EGFR siRNA block PAI-1 expression, and the Raf-1, MEK, and ERK signaling pathways for the regulation of PAI-1 expression. Ascochlorin suppresses the matrix metalloproteinases (MMPs) activity to activate the heparin-binding EGF-like growth factor (HB-EGF), to induce the phosphorylation of EGFR, and the MMPs inhibitor suppresses EGFR phosphorylation and the PAI-1 mRNA levels. These results suggest that ascochlorin prevents the expression of PAI-1 via the inhibition of an EGFR-dependent signal transduction pathway activated by MMPs.

Posttranscriptional regulation of expression of plasminogen activator inhibitor type-1 by cAMP in HepG2 liver cells

Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a physiologic fibrinolysis inhibitor, is implicated in atherosclerosis. Cyclic adenosine monophosphate (cAMP) alters PAI-1 expression in several cells. Nevertheless, posttranscriptional regulation of PAI-1 has not been elucidated. To determine whether cAMP affects PAI-1 expression at posttranscriptional level, we determined promoter activity, mRNA levels, 3'-untranslated region (UTR) activity and protein levels of PAI-1 using HepG2 cells. cAMP decreased PAI-1 promoter activity at 24 h and mRNA expression at 4 h while it increased mRNA expression and accumulation of PAI-1 protein into media at 24 h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2 kb), and cAMP increased baseline luciferase activity of 3'-UTR of the 3.2 kb PAI-1 mRNA [3'-UTR (+1358-3176)] and 1 kb fragment of 3'-terminus of 3'-UTR of 3.2 kb mRNA [3'-UTR (+2177-3176)]. cAMP increased PAI-1 protein expression despite decrease in promoter activity, presumably by regulating PAI-1 expression at the posttranscriptional level and thereby affecting mRNA stability. The 53-nt fragment in 3'-UTR (+2591 to +2643 nt) was involved in posttranscriptional regulation by cAMP. Thus, cAMP can stabilize 3.2 kb PAI-1 mRNA mediated by specific effects on 3'-UTR, and these effects are associated with increased expression of PAI-1 protein.

Dual diverse dynamic reversible actions of Ankaferd on EPCR and PAI-1 inside vascular endothelial cells with and without LPS

Objective: Ankaferd blood stopper (ABS) is comprised of a mixture of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. ABS is used as a topical hemostatic agent due to its antihemorrhagic effect, yet its hemostatic mechanism of action remains to be investigated. ABS does not affect the levels of coagulation factors II, V, VII, VIII, IX, X, XI and XII. The aim of this study was to investigate the effects of ABS on endothelium and immune response. As such, we evaluated changes in endothelial cell protein C receptor (EPCR) and plasminogen activator inhibitor type-1 (PAI-1) expression inside human umbilical vein endothelial cells (HUVECs) in the presence and absence of lipopolysaccharides (LPSs). Material and Methods: We exposed HUVECs to 10 μL and 100 μL of ABS for 5 min, 25 min, 50 min, 6 h, and 24 h. Additionally, 10 μg mL–1 of LPS was administered for 1 h to observe the effects of LPS challenge on HUVECs, and then the cells were treated with ABS for 5 min, 25 min, 50 min, and 6 h to observe the effects of ABS on HUVECs. Total RNA was isolated from HUVECs and then the level of expression of EPCR and PAI-1 mRNA was measured. Results: Cells were microscopically observed to arise from the surface and adhere to each other following the administration of ABS to HUVECs. Additionally, after 24 h the cells had normal growth and physiology, which suggests that the adhesive cellular effects of ABS might be reversible. ABS had a negative effect on EPCR and PAI-1 expression; the effect in response to 100 µL was greater than that to 10 µL. EPCR and PAI-1 expression increased over time in response to LPS and 10 µL of ABS. EPCR and PAI-1 expression was very low during the first hour of exposure to LPS and 100 µL of ABS, but after 6 h increased to levels similar to those observed in response to LPS and 10 µL of ABS. Conclusion: It was observed that ABS had dual diverse dynamic reversible effects on EPCR and PAI-1 expression in HUVECs, which were dependent on dose and concentration. ABS might play a role in numerous cellular mechanisms, in addition to having hemostatic effects.Conflict of interest:None declared.