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

Background Accumulating evidence suggests that plasminogen activator inhibitor-1 (PAI-1) plays an important role in bladder tumorigenesis by regulating cell cycle. However, it remains unclear whether and how inhibition of PAI-1 suppresses bladder tumorigenesis. Methods To elucidate the therapeutic effect of PAI-1 inhibition, we tested its tumorigenicity in PAI-1 knockout (KO) mice exposed to a known bladder carcinogen. Results PAI-1 deficiency did not inhibit carcinogen-induced bladder cancer in mice although carcinogen-exposed wild type mice significantly increased PAI-1 levels in bladder tissue, plasma and urine. We found that PAI-1 KO mice exposed to carcinogen tended to upregulate protein C inhibitor (PAI-3), urokinase-type plasminogen activator (uPA) and tissue-type PA (tPA), and significantly increased PAI-2, suggesting a potential compensatory function of these molecules when PAI-1 is abrogated. Subsequent studies employing gene expression microarray using mouse bladder tissues followed by post hoc bioinformatics analysis and validation experiments by qPCR and IHC demonstrated that SERPING1 is further downregulated in PAI-1 KO mice exposed to BBN, suggesting that SERPING1 as a potential missing factor that regulate PAI-2 overexpression (compensation pathway). Conclusions These results indicate that serpin compensation pathway, specifically PAI-2 overexpression in this model, supports bladder cancer development when oncoprotein PAI-1 is deleted. Further investigations into PAI-1 are necessary in order to identify true potential targets for bladder cancer therapy.

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

In 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.

Tumour expression of the urokinase plasminogen activator correlates with invasive capacity. Consequently, inhibition of this serine protease by physiological inhibitors should decrease invasion and metastasis. However, of the two main urokinase inhibitors, high tumour levels of the type 1 inhibitor actually promote tumour progression, whereas high levels of the type 2 inhibitor decrease tumour growth and metastasis. We propose that the basis of this apparently paradoxical action of two similar serine protease inhibitors lies in key structural differences controlling interactions with components of the extracellular matrix and endocytosis-signalling co-receptors.

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

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

Extracellular plasminogen activator inhibitor type-2 (PAI-2) is a potent inhibitor of urokinase-type plasminogen activator (u-PA) and also acts as a multifunctional protein. However, the biological activity of intracellular PAI-2, as well as its intracellular targets, until now remain an enigma. Here, we show that pRb2/p130 and Rb1/p105, but not p107, interact with PAI-2 in both the cytoplasm and nucleus of normal primary human corneal and conjunctival epithelial cells. We provided the first in vivo evidence that a specific fragment of the PAI-2 promoter is bound simultaneously by pRb2/ p130, PAI-2, E2F5, histone deacetylase 1 (HDAC1), DNA methyltransferase 1 (DNMT1), and histone methyltransferase (SUV39H1), in normal primary human corneal epithelial cells, and by pRb2/p130, PAI-2, E2F5, HDAC1, and DNMT1, in normal primary human conjunctiva epithelial cells. Our results strongly indicate a physiological interaction between pRb family members and PAI-2, suggesting the hypothesis that pRb2/p130 and PAI-2 may cooperate in modulating PAI-2 gene expression by chromatin remodeling, in normal corneal and conjunctival cells.

The urokinase plasminogen activator(uPA) system plays important roles in tumor cell invasion and metastasis. In the present study, we evaluated the effects of ATF-PAI2CD, a hybrid protein of the amino-terminal fragment of urokinase and mutant plasminogen activator inhibitor-2, on 95D cells in vitro and in vivo. Furthermore, our results support a current hypothesis that fusion protein blocks tumor invasion and motility by inhibiting localized pericellular proteolysis. Treatment of 95D cells with ATF-PAI2CD resulted in a dose-dependent decrease in tumor-cell invasion through matrigel, and ATF-PAI2CD was much more effective than PAI-2CD. In addition, extracellular regular protein kinase (ERK1/2) expression was downregulated and the adhesion ability to fibronectin was increased in 95D cells treated with the fusion protein, which was confirmed by cell adhesion assay. A high-concentration of ATF-PAI2CD caused a significant reduction in tumor volume and weight in BALB/c (nu/nu) mice female inoculated with human 95D cells (5 x 10(6)); the antitumor effects were significant, which demonstrated a 67.9+/-4.2% reduction in tumor growth compared with control mice. The number of lymphatic metastasis was significantly reduced in mice treated with high- and middle- concentrations of ATF-PAI2CD, whereas a low-concentration of ATF-PAI2CD failed to exhibit any antimetastatic effects. In conclusion, the results suggested that the hybrid protein has therapeutic potential for lung carcinoma and other tumors to inhibit tumor invasion and metastasis.

The penetration and permeation of the recombinant protein plasminogen activator inhibitor type 2 (PAI-2) in two formulations, one containing a penetration enhancer, into the psoriatic and uninvolved skin of eight patients with plaque-type psoriasis were investigated. Penetration and permeation of PAI-2 were measured by gamma counting and imaging following radiolabelling of a fraction of the applied PAI-2 with (123)I. The feasibility of topical delivery of drug to psoriatic plaques was confirmed by the finding that the permeability of psoriatic plaques to radiolabelled PAI-2 (P=0.007) and free (123)I (P=0.001) was approximately tenfold higher than the permeability of uninvolved skin. The addition of a penetration enhancer improved the permeation of PAI-2 into psoriatic plaques from an average of 35% to 46% (P=0.005). Occlusion decreased the permeation amount of PAI-2 from 46% to 15% due to losses on the occlusive dressing (P=0.001).

Suppression subtractive hybridization, a PCR-based method for cDNA subtraction, was used to identify differentially expressed genes in keratinocytes. Differentiation was induced by elevating the calcium level in the cell culture medium. Using HaCaT immortalized keratinocytes cultured in the presence of a high calcium concentration, we isolated 60 clones representing 48 different genes. By reverse Northern analysis, 13 genes were scored as overexpressed in these HaCaT cells. Northern blot analysis was used to confirm differential gene expression. Six genes, keratin 1, plasminogen activator inhibitor type 2 (PAI-2), ferritin H, peroxiredoxin 5 (PRDX5), insulin-like growth factor binding protein-3 (IGFBP-3), and one EST gene, were differentially expressed in HaCaT cells cultured in the presence of a high calcium concentration. Two of these genes, keratin 1 and PAI-2, are differentially expressed during keratinocyte terminal differentiation. IGFBP-3, which has reduced expression during epidermal differentiation, was increased after culture in a high-calcium medium for 2 or 5 days. Overexpression of the ferritin H and PRDX5 genes due to elevated calcium has not been reported in keratinocytes. We demonstrated the expression of IGFBP-3, ferritin H, PRDX5, and one gene of a matching sequence from the EST database during differentiation in primary cultured normal human keratinocytes. The EST gene expressed two transcripts of 1.8 kb and 2.5 kb in HaCaT cells, and the transcripts were confirmed to increase in keratinocytes cultured in a high-calcium medium.

The expression of plasminogen- and colony-stimulating factor-1-associated markers was first investigated in seven bladder carcinoma cell lines and in 15 primary bladder tumors using RT-PCR (mRNAs), zymography (protein activity), ELISA and immunocytochemistry analysis (ICC) (protein levels). The mRNAs expression, the activity and the levels of the secreted proteins were not informative. Only urokinase plasminogen activator receptor (uPA-R/CD87) and possibly plasminogen activator inhibitor type-2 (PAI2) antigen expression at the cellular levels seem to be useful markers. uPA-R antigen expression correlated with the secretion of hepatocyte growth factor (HGF) ( P=0.016) and the motility of the bladder tumor cells ( P=0.014), two markers associated with a poor prognosis in bladder carcinoma. To validate our technique and confirm these preliminary results, uPA-R and PAI2 antigen expression was determined in the imprints from 129 resected bladder carcinoma fragments. uPA-R correlated with the grade ( P=0.002), tumor invasion ( P=0.003) and the ploidy ( P=0.05) of the bladder carcinomas and with the low overall survival ( P=0.045) of the patients. PAI2 correlated only with the stage ( P=0.02) and low overall survival ( P=0.038). We conclude that in bladder carcinomas, studying the transcripts of PAs, PAIs, CSF-1 and its receptor, as well as measuring their concentration or activity in culture supernatants was of no clinical interest in terms of diagnostic or prognostic value. Only the ICC of uPA-R, which correlated with the major histopathological parameters of tumors and the low overall survival, proved to be a diagnostic and prognostic marker.

Metastasis is the principal cause of death in breast cancer patients. New and improved treatments for eradicating micrometastases are needed. To this end, a novel alpha-emitting protein construct, 213Bi-labelled plasminogen activator inhibitor type-2 (PAI-2) (alpha-PAI-2), was evaluated in vitro. This construct exploits: (a) the overexpression of the cell-surface receptor bound urokinase plasminogen activator (uPA) in the metastatic spread of breast cancer cells; (b) the binding and inhibition of receptor-bound uPA by PAI-2; and (c) the high cytotoxicity of alpha radiation. High labeling efficiencies and stability of 213Bi bound to human recombinant PAI-2 conjugated with cyclic diethylenetriaminepentaacetic acid anhydride were achieved (greater than 90%). The uPA inhibitory activity of the chelated PAI-2 was maintained as determined by complex formation with uPA and by inhibition of uPA activity. Furthermore, the reactivity of alpha-PAI-2 was confirmed in a cell assay as this construct was highly cytotoxic to breast cancer cell lines that express active, receptor bound uPA. The specificity of alpha-PAI-2 targeting was shown using several controls. Firstly, an active uPA blocking agent that limits PAI-2 binding significantly improved cell survival by a factor greater than three. Secondly, a non-specific alpha-BSA construct had minimal cytotoxic effect. Moreover, alpha-PAI-2 was not cytotoxic to freshly isolated normal human leukocytes, confirming that cells which do not contain active, receptor bound uPA cannot be targeted by alpha-PAI-2. In conclusion, we have validated, in vitro, the potential of alpha-PAI-2 as a novel therapeutic agent for breast cancer.

The plasminogen activating system is important in extracellular proteolysis. Plasmin degrades tissues and activates proteases. Plasminogen activators (tissue type; t-PA and urokinase type; u-PA) and plasminogen activator inhibitors (PAI-1, PAI-2) are found in high concentrations in gingival crevicular fluid (GCF). Previous findings indicate the significance of PAI-2 in gingival inflammation. When PAI-2 inhibits a plasminogen activator its conformation relaxes and neoepitopes can be detected with a monoclonal antibody (#2H5). Our aim was to study if and where in the gingival region PAI-2 has acted as an inhibitor. Methodological studies were performed on GCF with western blotting. Frozen sections of human gingiva were studied immunohistochemically. The methodological studies showed that our antibody #2H5 selectively detects relaxed low molecular weight non-glycosylated PAI-2. Total PAI-2 and relaxed PAI-2 were found in all gingival epithelia with a honeycomb-like staining. Relaxed PAI-2 showed the most pronounced staining in the cell layers near the surface of the epithelium and no staining in the suprabasal layers, while total PAI-2 was found throughout the epithelium, often more pronounced suprabasally. The results showed that PAI-2 indeed has acted as an inhibitor of a protease in gingival tissues, primarily in the epithelia. The results also suggest primarily an intracellular localization and thus the interaction of PAI-2 with a protein other than t-PA.

The growth factor receptor-dependent protein kinase Raf-1 is activated by GTP-bound Ras, thereby activating the mitogen-activated protein kinase pathway. To study the role of Raf in transformation we transduced Rat-1 cells with a tetracycline-regulatable retroviral vector encoding the constitutively active oncogenic C-terminal fragment of the human Raf-1 protein. Using subtractive hybridization of mRNAs from induced and noninduced cells and robot-assisted screening by complex hybridization, Raf-induced genes with various different characteristics of induction were investigated. Among the strongly induced genes were those involved in carcinogenesis such as metalloproteinases 3, 10 and 13, cathepsin L, ornithine decarboxylase, and putative tumor-suppressing genes such as monocyte chemoattracting protein 1, interferon-induced protein 10, a recently identified 2'-5' oligoadenylate synthetase-like protein, and plasminogen activator inhibitor type 2. Other components of the plasminogen activator system were not induced. Plasminogen activator inhibitor type 2 is a down-regulator of the proteolytic cascade consisting of various metalloproteinases, some of which are induced by a carboxy-terminal Raf mutant (RafCT). In conclusion, RafCT induces factors which act in a conflicting manner in respect of carcinogenesis, especially within the proteolytic system of the extracellular matrix.

The regulation of plasminogen activation involves genes for two plasminogen activators (tissue type and urokinase type), two specific inhibitors (type 1 and type 2), and a membrane-anchored urokinase-type plasminogen-activator-specific receptor. This system plays an important role in various biological processes involving extracellular proteolysis. Recent studies have revealed that the system, through interplay with integrins and the extracellular matrix protein vitronectin, is also involved in the regulation of cell migration and proliferation in a manner independent of proteolytic activity. The genes are expressed in many different cell types and their expression is under the control of diverse extracellular signals. Gene expression reflects the levels of the corresponding mRNA, which should be the net result of synthesis and degradation. Thus, modulation of mRNA stability is an important factor in overall regulation. This review summarizes current understanding of the biology and regulation of genes involved in plasminogen activation at different levels.

In stratified squamous epithelia a critical balance among cell proliferation, differentiation, and death must be maintained in order for these tissues to fulfill their barrier function. Previous studies have demonstrated that plasminogen activator inhibitor 2 (PAI-2) is a product of differentiating epidermal keratinocytes, suggesting a role for this inhibitor during squamous differentiation. Furthermore, in certain tumor cell lines, overexpression of PAI-2 confers resistance to the induction of programmed cell death, suggesting cytoprotective function(s). In the present study we demonstrate that PAI-2 mRNA and protein are constitutively and uniquely expressed in differentiating cells of murine stratified squamous epithelia, including epidermis, esophagus, vagina, oral mucosa, and tongue. PAI-2 immunohistochemical localization patterns suggest a predominantly cytosolic distribution, consistent with biochemical identification of the major PAI-2 species as a 43-kDa, presumably non-glycosylated protein. Functional analysis shows that the majority of epithelial PAI-2 is active. In contrast to the high levels of PAI-2 expression in stratified squamous epithelia, little or no PAI-2 is detectable in simple epithelia. These findings suggest that epithelial PAI-2 may mediate inhibition of intracellular proteinases associated with events during terminal differentiation and death that are unique to stratified squamous epithelia.

The levels of uPA, its inhibitors PAI-1 and PAI-2, and the uPA receptor (uPAR) have prognostic value in breast cancer. However, different extraction methods and assays kits are used in different laboratories and may directly influence the levels observed. To define a buffer suitable for both PAI-2 and uPAR extraction from breast cancer tissue compatible with hormone receptors and other cytosolic prognosticator assays, we compared PAI-2 and uPAR values obtained by immunoenzymatic assays (American Diagnostica, Green-which, USA) in several extraction conditions: 1) cytosol obtained with the standard hormone receptor buffer; 2) solubilized pellets obtained by Triton X100 extraction of the pelleted membranes obtained with standard hormone receptor buffer; 3) cytosol obtained by direct extraction in the buffer (containing Triton X100) recommended by the manufacturer, after 2 hours or 12 hours of incubation. Cytosol extracts prepared using the standard procedure recommended for hormone receptors gave the highest PAI-2 values. The highest uPAR values were obtained in the subsequent detergent extraction of the pelleted membranes. PAI-2 levels obtained with the kit manufacturer's method after 12 hours of incubation were lower than those obtained after 2 hours of incubation, whereas uPAR levels were similar. We conclude that the most suitable extraction protocol employs standard hormone receptor extraction buffer to obtain a supernatant cytosol fraction for PAI-2 assay, and subsequent detergent extraction of the pelleted membranes to obtain an extract suitable for uPAR.

Mechanical injury of tissues is followed by the formation of a provisional fibrin matrix, which is later replaced by granulation tissue. The fibrinolytic proteinase, plasmin, is thought to contribute to the displacement of the primary matrix. Plasmin is generated from the ubiquitous proenzyme plasminogen by plasminogen activators. The system of plasminogen activation is controlled at several levels: plasminogen activator inhibitors (PAI-1 and PAI-2) counteract the activity of plasminogen activators and alpha 2-antiplasmin inhibits the activity of plasmin. In order to elucidate the mechanisms that regulate the plasminogen activator system in healing human skin wounds, we performed the immunohistological study reported here. The plasmin inhibitor alpha 2-antiplasmin and PAI-2 were found in the primary fibrin-rich matrix and in the granulation tissue. alpha 2-Antiplasmin was diffusely distributed in the tissue and its distribution correlated with the presence and localization of plasmin(ogen) except that, in contrast to plasmin(ogen), the alpha 2-antiplasmin was apparently not cell-associated. The stainings for PAI-2 increased with time and paralleled the development of the cellular infiltrate. PAI-2 was found in association with cells, which were identified by double immunofluorescence stainings as monocytes/macrophages and fibroblasts. In line with the immunohistological data, polymerase chain reaction after reverse transcription revealed mRNA for PAI-2 in healing human skin wounds. Taken together, our findings indicate that in healing human skin wounds, PAI-2 is the primary regulator of plasminogen activators, whereas alpha 2-antiplasmin may serve to control plasmin activity.

In patients with colorectal cancer, profound alterations of the plasminogen activator system have been described at the tumor level, but conflicting results have been obtained for fibrinolytic parameters in plasma. Components of the fibrinolytic system, including tissue-type and urokinase-type plasminogen activators and their inhibitors type 1 and 2, were measured in tissue and/or plasma from 41 patients with colorectal cancer and in 40 controls. Procoagulant activity of freshly isolated mononuclear cells (basal activity) and the procoagulant activity and fibrinolytic proteins produced by the cells after incubation for 18 h without exogenous stimulation were also evaluated. Malignant tissue extracts had significantly higher levels of urokinase-type plasminogen activator and plasminogen activator inhibitor-1, but lower levels of tissue-type plasminogen activator than normal mucosa. Plasminogen activator inhibitor-1 alone was higher in advanced (Dukes' stages C + D) than limited (B) tumors. Plasminogen activator inhibitor-2 was not different in malignant tissue and normal mucosa. Plasma levels of plasminogen activator inhibitor-1 antigen were significantly increased in cancer patients compared with controls, but there were no differences in tissue-type and urokinase-type plasminogen activator, in plasminogen activator inhibitor-2, and D-dimer levels. Intra-patient analysis revealed no significant correlation between tumor and plasma levels of plasminogen activators or type 1 inhibitor. Tissue-type plasminogen activator, but not the urokinase type or inhibitor type 1, was higher in venous than in arterial blood collected at the tumor site during surgery. Basal procoagulant activity of mononuclear cells and the procoagulant activity and inhibitor type-2 produced by the cells after short-term culture were comparable in patients and controls. These findings indicate that, at least in our patients with colorectal cancer, the profound changes occurring at tumor level are barely detectable in the blood. Thus, the clinical relevance of plasma fibrinolytic parameters, especially urokinase-type plasminogen activator antigen, as tumor markers in colorectal cancer remains to be established.

The plasminogen activator (PA) proteolytic cascade has been implicated in the regulation of cell activities, including proliferation and differentiation, both of which occur continuously in normal human epidermis and are aberrant in psoriatic epidermis. To elucidate further the mechanisms by which PA is regulated in epidermis, we evaluated the levels of PA inhibitors type 1 (PAI-1) and type 2 (PAI-2) in normal and psoriatic epidermis. PAI-2, but not PAI-1, was detectable by mRNA, antigen, and activity assays, indicating that PAI-2 is the predominant epidermal PA inhibitor. In situ hybridization revealed that PAI-2 mRNA occurred throughout normal epidermis, although the signal was most intense in the granular layers. Similarly, PAI-2 antigen was most prominent in the granular layers; its distribution in these differential layers was along the cell periphery. Diffuse, fainter staining for PAI-2 was also detected in the basal cells and in some spinous layers of normal epidermis. Extracts of normal epidermis contained PA inhibitory activity identified as PAI-2 by immunoprecipitation with specific antibody. In psoriatic epidermis, PAI-2 mRNA and antigen were most prominent in the more superficial layers beneath the cornified cells. As with normal epidermis, PAI-2 assumed a pericellular distribution in the psoriatic cells. These data demonstrate that PAI-2 is constitutively expressed in vivo by keratinocytes in human epidermis and indicate that this protein is the predominant inhibitor of PA activity in normal and psoriatic human epidermis.

It is now clearly established that proteolytic enzymes, including plasminogen activator (uPA), play an important role in breaking down the extracellular matrix, which is considered to be a step in metastasis formation. Plasminogen activators are controlled at various levels. Two inhibitors, PAI-1 and PAI-2, have been identified, the latter being more specific for uPA. In attempts to determine their prognostic value, it is essential to investigate the relative importance of these parameters and their interactions. We used an immunoenzymatic method to assay uPA, PAI-1 and PAI-2 antigens in cytosols prepared from 314 primary breast tumours. The patients were followed up for a minimum of 6 years and all relevant clinical and laboratory findings were recorded. Univariate analysis confirmed the poor outcome of patients whose tumours contained large amounts of uPA and PAI-1. In addition, low levels of PAI-2 correlated with shorter disease-free survival in the overall population (P = 0.02), post-menopausal women (P = 0.02) and women without lymph node involvement (P = 0.02). Multivariate analysis in the 'main effects' Cox model identified node involvement, macroscopic tumour size and PAI-2 as significant variables. The 'interactive' model, taking into account interactions between uPA and its two inhibitors, identified a first subgroup with a very poor prognosis associating either high levels of PAI-1 with low levels of PAI-2 in the overall population and the women with no node involvement or high levels of uPA with low levels of PAI-2 in the group of menopausal women. We conclude that PAI-1 provides the same prognostic information as uPA, and does not appear to play a role as an inhibitor. In contrast, PAI-2 increases the prognostic value of uPA, particularly in post-menopausal women, and PAI-1 in patients with no node involvement.

Short-term co-cultivation of blastemal cells from 12-day-old mouse limb buds and human rheumatoid synovial fluid cells in high density cultures (Trowell culture system) resulted, depending on when co-cultivation started, either in (1) an inhibition of chondrogenesis (co-cultivation right from the start) or in (2) an extensive breakdown of cartilaginous matrix (co-cultivation after formation of embryonic cartilage). These synovial effects were markedly impeded if Avarol (a dioxygenase inhibitor) was applied singly or in combination with PAI-2 (a u-PA-inhibitor). PAI-2 alone, however, had no effect on the synovial-induced inhibition of chondrogenesis, but produced a pronounced inhibitory effect on matrix breakdown. The effects of both inhibitors were studied electron microscopically and biochemically (determination of sulfated-glycosaminoglycans in the high density cultures by Alcian Blue binding assay). The results of this study are consistent with the presumption that rheumatoid synovial cells are capable of inhibiting chondrogenesis and enhancing the breakdown of the cartilaginous matrix. Amongst others, the possible mediators involved are prostaglandins and plasminogen activators. The response to the inhibitors Avarol and PAI-2 is compatible with their mode of action. The chondroprotective action of these substances may be useful in developing potential antirheumatic drugs.

Aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, is known to mediate a wide variety of pharmacological and toxicological effects caused by polycyclic aromatic hydrocarbons. Recent studies have revealed that AhR is involved in the normal development and homeostasis of many organs. Here, we demonstrate that AhR knockout (AhR KO) mice are hypersensitive to lipopolysaccharide (LPS)-induced septic shock, mainly due to the dysfunction of their macrophages. In response to LPS, bone marrow-derived macrophages (BMDM) of AhR KO mice secreted an enhanced amount of interleukin-1beta (IL-1beta). Since the enhanced IL-1beta secretion was suppressed by supplementing Plasminogen activator inhibitor-2 (Pai-2) expression through transduction with Pai-2-expressing adenoviruses, reduced Pai-2 expression could be a cause of the increased IL-1beta secretion by AhR KO mouse BMDM. Analysis of gene expression revealed that AhR directly regulates the expression of Pai-2 through a mechanism involving NF-kappaB but not AhR nuclear translocator (Arnt), in an LPS-dependent manner. Together with the result that administration of the AhR ligand 3-methylcholanthrene partially protected mice with wild-type AhR from endotoxin-induced death, these results raise the possibility that an appropriate AhR ligand may be useful for treating patients with inflammatory disorders.