Detection of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2(PAI-2) in gingival crevicular fluid from healthy, gingivitis and periodontitis patients

Background: The regulation of plasminogen activation is a key element in controlling proteolytic events in the extracellular matrix. Our previous studies had demonstrated that in inflamed gingival tissues, tissue-type plasminogen activator (t-PA) is significantly increased in the extracellular matrix of the connective tissue and that interleukin 1β (IL-1β) can up regulate the level of t-PA and plasminogen activator inhibitor-2 (PAI-2) synthesis by human gingival fibroblasts. Method: In the present study, the levels of t-PA and PAI-2 in gingival crevicular fluid (GCF) were measured from healthy, gingivitis and periodontitis sites and compared before and after periodontal treatment. Crevicular fluid from106 periodontal sites in 33 patients were collected. 24 sites from 11 periodontitis patients received periodontal treatment after the first sample collection and post-treatment samples were collected 14 days after treatment. All samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for t-PA and PAI-2. Results: The results showed that significantly high levels of t-PA and PAI-2 in GCF were found in the gingivitis and periodontitis sites. Periodontal treatment led to significant decreases of PAI-2, but not t-PA, after 14 days. A significant positive linear correlation was found between t-PA and PAI-2 in GCF (r=0.80, p<0.01). In the healthy group, different sites from within the same subject showed little variation of t-PA and PAI-2 in GCF. However, the gingivitis and periodontitis sites showed large variation. These results suggest a good correlation between t-PA and PAI-2 with the severity of periodontal conditions. Conclusion: This study indicates that t-PA and PAI-2 may play a significant rôle in the periodontal tissue destruction and tissue remodeling and that t-PA and PAI-2 in GCF may be used as clinical markers to evaluate the periodontal diseases and assess treatment.

Effect of lipopolysaccharide from periodontal pathogens on the production of tissue plasminogen activator and plasminogen activator inhibitor 2 by human gingival fibroblasts.

Both tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2 (PAI-2) are important proteolysis factors present in inflamed human periodontal tissues. The aim of the present study was to investigate the effect of lipopolysaccharide (LPS) on the synthesis of t-PA and PAI-2 by human gingival fibroblasts (HGF). LPS from different periodontal pathogens including Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum were extracted by the hot phenol water method. The levels of t-PA and PAI-2 secreted into the cell culture media were measured by enzyme-linked immunosorbent assays (ELISA). The mRNA for t-PA and PAI-2 were measured by RT-PCR. The results showed t-PA synthesis was increased in response to all types of LPS studied and PAI-2 level was increased by LPS from A. actinomycetemcomitans and F. nucleatum, but not P. gingivalis. When comparing the effects of LPS from non-periodontal bacteria (Escherichia coli and Salmonella enteritidis) with the LPS from periodontal pathogens, we found that the ratio of t-PA to PAI-2 was greater following exposure of the cells to LPS from periodontal pathogens. The highest ratio of t-PA to PAI-2 was found in those cells exposed to LPS from P. gingivalis. These results indicate that LPS derived from periodontal pathogens may cause unbalanced regulation of plasminogen activator and plasminogen activator inhibitor by HGF and such an effect may, in part, contribute to the destruction of periodontal connective tissue through dysregulated pericellular proteolysis.

Detection of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 2(PAI-2) in gingival crevicular fluid from healthy, gingivitis and periodontitis patients

Background: The regulation of plasminogen activation is a key element in controlling proteolytic events in the extracellular matrix. Our previous studies had demonstrated that in inflamed gingival tissues, tissue-type plasminogen activator (t-PA) is significantly increased in the extracellular matrix of the connective tissue and that interleukin 1 beta (IL-1 beta) can up regulate the level of t-PA and plasminogen activator inhibitor-2 (PAI-2) synthesis by human gingival fibroblasts. Method: In the present study, the levels of t-PA and PAI-2 in gingival crevicular fluid (GCF) were measured from healthy, gingivitis and periodontitis sites and compared before and after periodontal treatment. Crevicular fluid from 106 periodontal sites in 33 patients were collected. 24 sites from 11 periodontitis patients received periodontal treatment after the first sample collection and post-treatment samples were collected 14 days after treatment. All samples were analyzed by enzyme-linked immunosorbent assay (ELISA) for t-PA and PAI-2. Results: The results showed that significantly high levels of t-PA and PAI-2 in GCF were found in the gingivitis and periodontitis sites. Periodontal treatment led to significant decreases of PAI-2, but not t-PA, after 14 days. A significant positive linear correlation was found between t-PA and PAI-2 in GCF (r=0.80, p

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.

Evaluation of Alpha 1-Antitrypsin and the Levels of mRNA Expression of Matrix Metalloproteinase 7, Urokinase Type Plasminogen Activator Receptor and COX-2 for the Diagnosis of Colorectal Cancer

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A NOVEL PLASMINOGEN-ACTIVATOR FROM SNAKE-VENOM - PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native GIu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg(561)-Val-(562). Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-gIycosylation site at Asn(1G1). The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Aghistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Monocyte urokinase-type plasminogen activator up-regulation reduces thrombus size in a model of venous thrombosis

Background - Our previous studies showed that the direct injection of an adenovirus construct expressing urokinase-type plasminogen activator (uPA) into experimental venous thrombi significantly reduces thrombus weight. The systemic use of adenovirus vectors is limited by inherent hepatic tropism and inflammatory response. As macrophages are recruited into venous thrombi, it is reasonable to speculate that these cells could be used to target the adenovirus uPA (ad-uPA) gene construct to the thrombus. The aims of this study were to determine whether macrophages transduced with ad-uPA have increased fibrinolytic activity and whether systemic injection of transduced cells could be used to target uPA expression to the thrombus and reduce its size. Methods - The effect of up-regulating uPA was examined in an immortalized macrophage cell line (MM6) and macrophages differentiated from human blood monocyte-derived macrophages (HBMMs). Cells were infected with ad-uPA or blank control virus (ad-blank). Fibrinolytic mediator expression, cell viability, and cytokine expression were measured by activity assays and enzyme-linked immunosorbent assays. Monocyte migration was measured using a modified Boyden chamber assay. A model of venous thrombosis was developed and characterized in mice with severe combined immunodeficiency (SCID). This model was used to study whether systemically administered macrophages over-expressing uPA reduced thrombus size. Uptake of HBMMs into the thrombus induced in these mice was confirmed by a combination of PKH2-labeled cell tracking and colocalization with human leukocyte antigen (HLA) by immunohistology. Results - Compared with ad-blank, treated HBMMs transduction with ad-uPA increased uPA production by >1000-fold (P = .003), uPA activity by 150-fold (P = .0001), and soluble uPA receptor (uPAR) by almost twofold (P = .043). Expression of plasminogen activator inhibitor (PAI-1) and PAI-2 was decreased by about twofold (P = .011) and threefold (P = .005), respectively. Up-regulation of uPA had no effect on cell viability or inflammatory cytokine production compared with ad-blank or untreated cells. Ad-uPA transduction increased the migration rate of HBMMs (about 20%, P = .03) and MM6 cells (>twofold, P = .005) compared with ad-blank treated controls. Human macrophage recruitment into the mouse thrombus was confirmed by the colocalization of HLA with the PKH2-marked cells. Systemic injection of uPA-up-regulated HBMMs reduced thrombus weight by approximately 20% compared with ad-blank (P = .038) or sham-treated controls (P = .0028). Conclusion - Transduction of HBBM with ad-uPA increases their fibrinolytic activity. Systemic administration of uPA up-regulated HBBMs reduced thrombus size in an experimental model of venous thrombosis. Alternative methods of delivering fibrinolytic agents are worth exploring.

L-mimosine and dimethyloxaloylglycine decrease plasminogen activation in periodontal fibroblasts

BACKGROUND The use of prolyl hydroxylase inhibitors such as l-mimosine (L-MIM) and dimethyloxaloylglycine (DMOG) to improve angiogenesis is a new approach for periodontal regeneration. In addition to exhibiting pro-angiogenic effects, prolyl hydroxylase inhibitors can modulate the plasminogen activator system in cells from non-oral tissues. This study assesses the effect of prolyl hydroxylase inhibitors on plasminogen activation by fibroblasts from the periodontium. METHODS Gingival and periodontal ligament fibroblasts were incubated with L-MIM and DMOG. To investigate whether prolyl hydroxylase inhibitors modulate the net plasminogen activation, kinetic assays were performed with and without interleukin (IL)-1. Moreover, plasminogen activators and the respective inhibitors were analyzed by casein zymography, immune assays, and quantitative polymerase chain reaction. RESULTS The kinetic assay showed that L-MIM and DMOG reduced plasminogen activation under basal and IL-1-stimulated conditions. Casein zymography revealed that the effect of L-MIM involves a decrease in urokinase-type plasminogen activator activity. In agreement with these findings, reduced levels of urokinase-type plasminogen activator and elevated levels of plasminogen activator inhibitor 1 were observed. CONCLUSION L-MIM and DMOG can reduce plasminogen activation by fibroblasts from the gingiva and the periodontal ligament under basal conditions and in the presence of an inflammatory cytokine.

Thrombospondin 1 expression in transformed endothelial cells restores a normal phenotype and suppresses their tumorigenesis.

Murine endothelial cells are readily transformed in a single step by the polyomavirus oncogene encoding middle-sized tumor antigen. These cells (bEND.3) form tumors (hemangiomas) in mice which are lethal in newborn animals. The bEND.3 cells rapidly proliferate in culture and express little or no thrombospondin 1 (TS1). To determine the role of TS1 in regulation of endothelial cell phenotype, we stably transfected bEND.3 cells with a human TS1 expression vector. The cells expressing human TS1 were readily identified by their altered morphology and exhibited a slower growth rate and lower saturation density than the parental bEND.3 cells. The TS1-expressing cells also formed aligned cords of cells instead of clumps or cysts in Matrigel. Moreover, while the bEND.3 cells formed large tumors in nude mice within 48 hr, the TS1-expressing cells failed to form tumors even after 1 month. The TS1-transfected cells expressed transforming growth factor beta mRNA and bioactivity at levels similar to those of the parental or vector-transfected bEND.3 cells, indicating that the effects of TS1 expression are not due to the activation of transforming growth factor beta by TS1. TS1 expression resulted in a > 100-fold decrease in net fibrinolytic (urokinase-type plasminogen activator, uPA) activity due to more plasminogen-activator inhibitor 1 and less uPA secretion. TS1 thus appears to be an important regulator of endothelial cell phenotype required for maintaining the quiescent, differentiated state.

The urokinase plasminogen activating system in thyroid cancer: clinical implications

The urokinase plasminogen activator (uPA) system (uPAS) comprises the uPA, its cell membrane receptor (uPAR) and two specific inhibitors, the plasminogen activator inhibitor 1 (PAI-1) and 2 (PAI-2). The uPA converts the plasminogen in the serine protease plasmin, involved in a number of physiopathological processes requiring basement membrane (BM) or extracellular matrix (ECM) remodelling, including tumor progression and metastasis. The tumor-promoting role of PAS is not limited to the degradation of ECM and BM required for local diffusion and spread to distant sites of malignant cells, but widens to tumor cell proliferation, adhesion and migration, intravasation, growth at the metastatic site and neoangiogenesis. The relevance of uPAS in cancer progression has been confirmed by several studies which documented an increased expression of uPA, uPAR and PAI-1 in different human malignancies, and a positive correlation between the levels of one or more of them and a poor prognosis. For these reasons, the uPAS components have aroused considerable interest as suitable targets for anticancer therapy, and several pharmacological approaches aimed at inhibiting the uPA and/or uPAR expression or function in preclinical and clinical settings have been described. In the present manuscript, we will first glance at uPAS biological functions in human cancer progression and its clinical significance in terms of prognosis and therapy. We will then review the main findings regarding expression and function of uPAS components in thyroid cancer tissues along with the experimental and clinical evidence suggesting its potential value as molecular prognostic marker and therapeutic target in thyroid cancer patients.

Molecular markers of obesity and diabetes

Recently it has been shown that the consumption of a diet high in saturated fat is associated with impaired insulin sensitivity and increased incidence of type 2 diabetes. In contrast, diets that are high in monounsaturated fatty acids (MUFAs) or polyunsaturated fatty acids (PUFAs), especially very long chain n-3 fatty acids (FAs), are protective against disease. However, the molecular mechanisms by which saturated FAs induce the insulin resistance and hyperglycaemia associated with metabolic syndrome and type 2 diabetes are not clearly defined. It is possible that saturated FAs may act through alternative mechanisms compared to MUFA and PUFA to regulate of hepatic gene expression and metabolism. It is proposed that, like MUFA and PUFA, saturated FAs regulate the transcription of target genes. To test this hypothesis, hepatic gene expression analysis was undertaken in a human hepatoma cell line, Huh-7, after exposure to the saturated FA, palmitate. These experiments showed that palmitate is an effective regulator of gene expression for a wide variety of genes. A total of 162 genes were differentially expressed in response to palmitate. These changes not only affected the expression of genes related to nutrient transport and metabolism, they also extend to other cellular functions including, cytoskeletal architecture, cell growth, protein synthesis and oxidative stress response. In addition, this thesis has shown that palmitate exposure altered the expression patterns of several genes that have previously been identified in the literature as markers of risk of disease development, including CVD, hypertension, obesity and type 2 diabetes. The altered gene expression patterns associated with an increased risk of disease include apolipoprotein-B100 (apo-B100), apo-CIII, plasminogen activator inhibitor 1, insulin-like growth factor-I and insulin-like growth factor binding protein 3. This thesis reports the first observation that palmitate directly signals in cultured human hepatocytes to regulate expression of genes involved in energy metabolism as well as other important genes. Prolonged exposure to long-chain saturated FAs reduces glucose phosphorylation and glycogen synthesis in the liver. Decreased glucose metabolism leads to elevated rates of lipolysis, resulting in increased release of free FAs. Free FAs have a negative effect on insulin action on the liver, which in turn results in increased gluconeogenesis and systemic dyslipidaemia. It has been postulated that disruption of glucose transport and insulin secretion by prolonged excessive FA availability might be a non-genetic factor that has contributed to the staggering rise in prevalence of type 2 diabetes. As glucokinase (GK) is a key regulatory enzyme of hepatic glucose metabolism, changes in its activity may alter flux through the glycolytic and de novo lipogenic pathways and result in hyperglycaemia and ultimately insulin resistance. This thesis investigated the effects of saturated FA on the promoter activity of the glycolytic enzyme, GK, and various transcription factors that may influence the regulation of GK gene expression. These experiments have shown that the saturated FA, palmitate, is capable of decreasing GK promoter activity. In addition, quantitative real-time PCR has shown that palmitate incubation may also regulate GK gene expression through a known FA sensitive transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), which upregulates GK transcription. To parallel the investigations into the mechanisms of FA molecular signalling, further studies of the effect of FAs on metabolic pathway flux were performed. Although certain FAs reduce SREBP-1c transcription in vitro, it is unclear whether this will result in decreased GK activity in vivo where positive effectors of SREBP-1c such as insulin are also present. Under these conditions, it is uncertain if the inhibitory effects of FAs would be overcome by insulin. The effects of a combination of FAs, insulin and glucose on glucose phosphorylation and metabolism in cultured primary rat hepatocytes at concentrations that mimic those in the portal circulation after a meal was examined. It was found that total GK activity was unaffected by an increased concentration of insulin, but palmitate and eicosapentaenoic acid significantly lowered total GK activity in the presence of insulin. Despite the fact that total GK enzyme activity was reduced in response to FA incubation, GK enzyme translocation from the inactive, nuclear bound, to active, cytoplasmic state was unaffected. Interestingly, none of the FAs tested inhibited glucose phosphorylation or the rate of glycolysis when insulin is present. These results suggest that in the presence of insulin the levels of the active, unbound cytoplasmic GK are sufficient to buffer a slight decrease in GK enzyme activity and decreased promoter activity caused by FA exposure. Although a high fat diet has been associated with impaired hepatic glucose metabolism, there is no evidence from this thesis that FAs themselves directly modulate flux through the glycolytic pathway in isolated primary hepatocytes when insulin is also present. Therefore, although FA affected expression of a wide range of genes, including GK, this did not affect glycolytic flux in the presence of insulin. However, it may be possible that a saturated FA-induced decrease in GK enzyme activity when combined with the onset of insulin resistance may promote the dys-regulation of glucose homeostasis and the subsequent development of hyperglycaemia, metabolic syndrome and type 2 diabetes.

Ethnic differences in lipid metabolism in two groups of obese South African women

There is a higher prevalence of ischemic heart disease (IHD) in South African white than black women. The objective of this study was to determine biochemical explanations for this prevalence. The study group contained 15 obese black women (OBW) and 14 obese white women (OWW), ah premenopausal, who were examined after an overnight fast. Anthropometric measurements and blood concentrations of glucose, non-esterified fatty acids (NEFAs), catecholamines, plasminogen activator inhibitor-1, C-peptide, proinsulin, lipograms, cortisol, growth hormone, and post-heparin Lipoprotein Lipase activity were measured during an oral glucose tolerance test (OGTT), Body composition was measured using bioelectrical impedance analysis, and subcutaneous and visceral fat mass were assessed with CT-scans. Visceral fat area was higher in OWW (139.7 +/- 10.7 cm(2)) than in OBW (72.3 +/- 3.9 cm(2)) (P < 0.01), as were fasting and 3 h triglyceride concentrations (P < 0.05 for all). OWW also had higher NEFA levels than OBW at 3 and 4 h compared, with OBW (P < 0.05 for both). Fasting cortisol (266 +/- 24 vs. 197 +/- 19 nmol/l; P < 0.05) was higher in OWW than in OBW. These data demonstrate that OWW have higher visceral fat mass than OBW, which may lead to a more atherogenic fasting and postprandial Lipid profile. The higher cortisol levels of the OWW may promote visceral fat deposition. - Punyadeera, C., M-T. van der Merwe, N.J. Crowther, M. Toman, C. P. Schlaphoff, and I. P. Gray. Ethnic differences in lipid metabolism in two groups of obese South African women.

Substrate Selectivity and Molecular Adaptation in the Outer Membrane Proteases of Yersinia pestis and Salmonella enterica

Proteolysis is important in bacterial pathogenesis and colonization of animal and plant hosts. In this work I have investigated the functions of the bacterial outer membrane proteases, omptins, of Yersinia pestis and Salmonella enterica. Y. pestis is a zoonotic pathogen that causes plague and has evolved from gastroenteritis-causing Yersinia pseudotuberculosis about 13 000 years ago. S. enterica causes gastroenteritis and typhoid fever in humans. Omptins are transmembrane β-barrels with ten antiparallel β-strands and five surface-exposed loops. The loops are important in substrate recognition, and variation in the loop sequences leads to different substrate selectivities between omptins, which makes omptins an ideal platform to investigate functional adaptation and to alter their polypeptide substrate preferences. The omptins Pla of Y. pestis and PgtE of S. enterica are 75% identical in their amino acid sequences. Pla is a multifunctional protein with proteolytic and non-proteolytic functions, and it increases bacterial penetration and proliferation in the host. Functions of PgtE increase migration of S. enterica in vivo and bacterial survival in mouse macrophages, thus enhancing bacterial spread within the host. Mammalian plasminogen/fibrinolytic system maintains the balance between coagulation and fibrinolysis and participates in several cellular processes, e.g., cell migration and degradation of extracellular matrix proteins. This system consists of activation cascades, which are strictly controlled by several regulators, such as plasminogen activator inhibitor 1 (PAI-1), α2-antiplasmin (α2AP), and thrombin-activatable fibrinolysis inhibitor (TAFI). This work reveals novel interactions of the omptins of Y. pestis and S. enterica with the regulators of the plasminogen/fibrinolytic system: Pla and PgtE inactivate PAI-1 by cleavage at the reactive site peptide bond, and degrade TAFI, preventing its activation to TAFIa. Structure-function relationship studies with Pla showed that threonine 259 of Pla is crucial in plasminogen activation, as it prevents degradation of the plasmin catalytic domain by the omptin and thus maintains plasmin stability. In this work I constructed chimeric proteins between Pla and Epo of Erwinia pyrifoliae that share 78% sequence identity to find out which amino acids and regions in Pla are important for its functions. Epo is neither a plasminogen activator nor an invasin, but it degrades α2AP and PAI-1. Cumulative substitutions towards Pla sequence turned Epo into a Pla-like protein. In addition to threonine 259, loops 3 and 5 are critical in plasminogen activation by Pla. Turning Epo into an invasin required substitution of 31 residues located at the extracellular side of the Epo protein above the lipid bilayer, and also of the β1-strand in the N-terminal transmembrane region of the protein. These studies give an example of how omptins adapt to novel functions that advantage their host bacteria in different ecological niches.