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.

Validation of an immunoassay to measure plasminogen-activator inhibitor-1 concentrations in human saliva

Introduction We have previously shown that the concentrations of D-dimer are significantly elevated in saliva compared with plasma. Saliva offers several advantages compared with blood analysis. We hypothesised that human saliva contains plasminogen activator inhibitor-1 (PAI-1) and that the concentrations are not affected by the time of saliva collection. The aim was to adopt and validate an immunoassay to quantify PAI-1 concentrations in saliva and to determine whether saliva collection time has an influence in the measurement. Materials and methods Two saliva samples (morning and afternoon) from the same day were collected from healthy subjects (N = 40) who have had no underlying heart conditions. A customized AlphaLISA® immunoassay (PerkinElmer®, MA, USA) was adopted and used to quantify PAI-1 concentrations. We validated the analytical performance of the customized immunoassay by calculating recovery of known amount of analyte spiked in saliva. Results: The recovery (95.03%), intra- (8.59%) and inter-assay (7.52%) variations were within the acceptable ranges. The median salivary PAI-1 concentrations were 394 pg/mL (interquartile ranges (IQR) 243.4-833.1 pg/mL) in the morning and 376 (129.1-615.4) pg/mL in the afternoon and the plasma concentration was 59,000 (24,000-110,000) pg/mL. Salivary PAI-1 did not correlate with plasma (P = 0.812). Conclusions The adopted immunoassay produced acceptable assay sensitivity and specificity. The data demonstrated that saliva contains PAI-1 and that its concentration is not affected by the time of saliva collection. There is no correlation between salivary and plasma PAI-1 concentrations. Further studies are required to demonstrate the utility of salivary PAI-1 in CVD risk factor studies.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines : targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

Serine protease inhibition and mitochondrial dysfunction associated with cisplatin resistance in human tumor cell lines: targets for therapy

Indicators of mitochondrial function were studied in two different cell culture models of cis-diamminedichloroplatinum-II (CDDP) resistance: the intrinsically resistant human ovarian cancer cell line CI-80-13S, and resistant clones (HeLa-S1a and HeLa-S1b) generated by stable expression of the serine protease inhibitor—plasminogen activator inhibitor type-2 (PAI-2), in the human cervical cancer cell line HeLa. In both models, CDDP resistance was associated with sensitivity to killing by adriamycin, etoposide, auranofin, bis[1,2-bis(diphenylphosphino)ethane]gold(I) chloride {[Au(DPPE)2]Cl}, CdCl2 and the mitochondrial inhibitors rhodamine-123 (Rhl23), dequalinium chloride (DeCH), tetraphenylphosphonium (TPP), and ethidium bromide (EtBr) and with lower constitutive levels of ATP. Unlike the HeLa clones, CI-80-13S cells were additionally sensitive to chloramphenicol, 1-methyl-4-phenylpyridinium ion (MPP+), rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, and showed poor reduction of 1-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), suggesting a deficiency in NADH dehydrogenase and/or succinate dehydrogenase activities. Total platinum uptake and DNA-bound platinum were slightly lower in CI-80-13S than in sensitive cells. The HeLa-S1a and HeLa-S1b clones, on the other hand, showed poor reduction of triphenyltetrazolium chloride (TTC), indicative of low cytochrome c oxidase activity. Total platinum uptake by HeLa-S1a was similar to HeLa, but DNA-bound platinum was much lower than for the parent cell line. The mitochondria of CI-80-13S and HeLa-S1a showed altered morphology and were fewer in number than those of JAM and HeLa. In both models, CDDP resistance was associated with less platinum accumulation and with mitochondrial and membrane defects, brought about one case with expression of a protease inhibitor which is implicated in tumor progression. Such markers may identify tumors suitable for treatment with gold phosphine complexes or other mitochondrial inhibitors.

The expression of plasminogen activator system in a rat model of periodontal wound healing

BACKGROUND: The plasminogen activator system has been proposed to play a role in proteolytic degradation of extracellular matrices in tissue remodeling, including wound healing. The aim of this study was to elucidate the presence of components of the plasminogen activator system during different stages of periodontal wound healing. METHODS: Periodontal wounds were created around the molars of adult rats and healing was followed for 28 days. Immunohistochemical analyses of the healing tissues and an analysis of the periodontal wound healing fluid by ELISA were carried out for the detection of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and 2 plasminogen activator inhibitors (PAI-1 and PAI-2). RESULTS: During the early stages (days 1 to 3) of periodontal wound healing, PAI-1 and PAI-2 were found to be closely associated with the deposition of a fibrin clot in the gingival sulcus. These components were strongly associated with the infiltrating inflammatory cells around the fibrin clot. During days 3 to 7, u-PA, PAI-1, and PAI-2 were associated with cells (particularly monocytes/macrophages, fibroblasts, and endothelial cells) in the newly formed granulation tissue. During days 7 to 14, a new attachment apparatus was formed during which PAI-1, PAI-2, and u-PA were localized in both periodontal ligament fibroblasts (PDL) and epithelial cells at sites where these cells were attaching to the root surface. In the periodontal wound healing fluid, the concentration for t-PA increased and peaked during the first week. PAI-2 had a similar expression to t-PA, but at a lower level over the entire wound-healing period. CONCLUSIONS: These findings indicate that the plasminogen activator system is involved in the entire process of periodontal wound healing, in particular with the formation of fibrin matrix on the root surface and its replacement by granulation tissue, as well as the subsequent formation of the attachment of soft tissue to the root surface during the later stages of wound repair.

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.

Effects of inhibitors of plasminogen activator, serine proteinases, and collagenase IV on the invasion of basement membranes by metastatic cells

Using both human and murine cell lines, we show that malignant cells are able to invade through basement membrane and also secrete elevated amounts of collagenase IV, an enzyme implicated in the degradation of basement membranes. Using serine proteinase inhibitors and antibodies to plasminogen activators as well as a newly described collagenase inhibitor we demonstrate that a protease cascade leads to the activation of an enzyme(s) that cleaves collagen IV. Inhibition at each step reduces the invasion of the tumor cells through reconstituted basement membrane in vitro. Treatment with a collagenase inhibitor reduced the incidence of lung lesions in mice given i.v. injections of malignant melanoma cells.

Canagliflozin : something new for type 2 diabetes, but is it safe and efficacious?

Evaluation of Inagaki N, Kondo K, Yoshinari T, et al. Efficacy and safety of canagliflozin in Japanese patients with type 2 diabetes: a randomized, double-blind, placebo-controlled, 12-week study. Diabetes Obes Metab 2013. [Epub ahead of print] and Cefalu WT, Leiter LA, Yoon KH, et al. Efficacy and safety of canagliflozin versus glimepiride in patients with type 2 diabetes inadequately controlled with metformin (CANTATA-SU): 52 week results from a randomized, double-blind, phase 3 non-inferiority trial. Lancet 2013;382:941-50 INTRODUCTION Inhibition of the sodium-glucose cotransporter 2 (SGLT2), to promote the excretion of glucose, is a new paradigm in the treatment of type 2 diabetes. AREAS COVERED Canagliflozin is an SGLT2 inhibitor, which has been the subject of two recent clinical trials, which are evaluated. EXPERT OPINION Studies with canagliflozin, in subjects with type 2 diabetes, have shown that its use is associated with reductions in HbA1c and body weight and small reductions in blood pressure and triglycerides, while increasing high-density lipoprotein cholesterol and low-density lipoprotein cholesterol. As monotherapy in Japanese subjects, or in comparison with glimepiride in CANTATA-SU (CANagliflozin Treatment and Trial Analysis versus SUlphonylurea), canagliflozin causes a low incidence of hypoglycemia, and this is an advantage over glimepiride. However, one of the disadvantages with canagliflozin, which was also highlighted in CANTATA-SU, is that canagliflozin can cause urogenital infections, which are not observed with other antidiabetic drugs. The Federal Drug Administration has recently approved canagliflozin for use in type 2 diabetes, while directing that a clinical outcome safety trial be undertaken. We are concerned that canagliflozin has been approved for use in type 2 diabetes prior to a clinical outcome study of efficacy being undertaken and without the outcome of further safety testing.

Dapagliflozin – do we need it registered for type 2 diabetes?

INTRODUCTION: Inhibitors of the sodium-glucose co-transporter 2 (SGLT2) promote the excretion of glucose to reduce glycated hemoglobin (HbA1c) levels. Canagliflozin was the first SGLT2 inhibitor to be approved by the US FDA for use in the treatment of type 2 diabetes, and recently dapagliflozin has also been approved. AREAS COVERED: We evaluated a recent Phase III clinical trial with dapagliflozin. EXPERT OPINION: Dapagliflozin was studied as add-on therapy to sitagliptin with or without metformin, and was shown to lower HbA1c levels and body weight. The incidence of hypoglycaemia was low with dapagliflozin, but it did increase the incidence of urogenital infections. As no clear benefits have been identified for dapagliflozin over canagliflozin, which was the first gliflozin registered by the FDA, we do not fully understand why it was necessary to register dapagliflozin. Given that there are no completed cardiovascular/clinical outcome studies with dapagliflozin, and therefore no evidence of beneficial effect, it also seems premature to be using it extensively or considering it as an alternative to the clinically proven metformin.

Is liraglutide or exenatide better in type 2 diabetes?

Background: Subjects with type 2 diabetes have high circulating levels of glucose. Glucagon-like peptide-1 (GLP-1) is an intestinal hormone that has a major role in glucose homeostasis. Exenatide and liraglutide are both agonists at the GLP-1 receptor, and are effective at reducing circulating glucose levels (measured as HbA1c levels), but they have not been compared. Objectives/methods: This evaluation is of a clinical trial comparing liraglutide once a day with exenatide twice a day in subjects with type 2 diabetes. Results: In the Liraglutide Effect and Action in Diabetes (LEAD)-6 trial, subcutaneous liraglutide 1.8 mg once a day was compared with exenatide 10 μg twice a day. The primary efficacy outcome was change in HbA1c levels, and this was significantly greater with liraglutide (1.12%) than with exenatide (0.79%). Liraglutide and exenatide had similar small abilities to reduce body weight, blood pressure and LDL-cholesterol. Conclusions: Liraglutide was more effective than exenatide for overall glycaemic control in subjects with type 2 diabetes. However, this is only true for the preparations and doses tested, that is liraglutide 1.8 mg once weekly and exenatide 10 μg b.i.d., and may not apply when the comparison is undertaken with the new longer-lasting preparation of exenatide once weekly.

Resistance training for obese, type 2 diabetic adults: A review of the evidence

In both developed and developing countries, increased prevalence of obesity has been strongly associated with increased incidence of type 2 diabetes mellitus (T2DM) in the adult population. Previous research has emphasized the importance of physical activity in the prevention and management of obesity and T2DM, and generic exercise guidelines originally developed for the wider population have been adapted for these specific populations. However, the guidelines traditionally focus on aerobic training without due consideration to other exercise modalities. Recent reviews on resistance training in the T2DM population have not compared this modality with others including aerobic training, or considered the implications of resistance training for individuals suffering from both obesity and T2DM. In short, the optimal mix of exercise modalities in the prescription of exercise has not been identified for it benefits to the metabolic, body composition and muscular health markers common in obesity and T2DM. Similarly, the underlying physical, social and psychological barriers to adopting and maintaining exercise, with the potential to undermine the efficacy of exercise interventions, have not been addressed in earlier reviews. Because it is well established that aerobic exercise has profound effects on obesity and T2DM risk, the purpose of this review was to address the importance of resistance training to obese adults with T2DM.

Self-efficacy, outcome expectations and self-care behaviour in people with type 2 diabetes in Taiwan

Aims. To explore differences in self-care behaviour according to demographic and illness characteristics; and relationships among self-care behaviour and demographic and illness characteristics, efficacy expectations and outcome expectations of people with type 2 diabetes in Taiwan. Background. Most people with diabetes do not control their disease appropriately in Taiwan. Enhanced self-efficacy towards managing diseases can be an effective way of improving disease control as proposed by the self-efficacy model which provides a useful framework for understanding adherence to self-care behaviours. Design and methods. The sample comprised 145 patients with type 2 diabetes aged 30 years or more from diabetes outpatient clinics in Taipei. Data were collected using a self-administered questionnaire for this study. One-way anova, t-tests, Pearson product moment correlation and hierarchical regression were analysed for the study. Results. Significant differences were found: between self-care behaviour and complications (t = −2·52, p < 0·01) and patient education (t = −1·96, p < 0·05). Self-care behaviour was significantly and positively correlated with duration of diabetes (r = 0·36, p < 0·01), efficacy expectations (r = 0·54, p < 0·01) and outcome expectations (r = 0·44, p < 0·01). A total of 39·1% of variance in self-care behaviour can be explained by duration of diabetes, efficacy expectations and outcome expectations. Conclusions. Findings support the use of the self-efficacy model as a framework for understanding adherence to self-care behaviour. Relevance to clinical practice. Using self-efficacy theory when designing patient education interventions for people with type 2 diabetes will enhance self-management routines and assist in reducing major complications in the future.

Factors that affect health outcomes in adults with type 2 diabetes : a cross-sectional study

Background: Factors that individually influence blood sugar control, health-related quality of life, and diabetes self-care behaviors have been widely investigated; however, most previous diabetes studies have not tested an integrated association between a series of factors and multiple health outcomes. ---------- Objectives: The purposes of this study are to identify risk factors and protective factors and to examine the impact of risk factors and protective factors on adaptive outcomes in people with type 2 diabetes.---------- Design: A descriptive correlational design was used to examine a theoretical model of risk factors, protective factors, and adaptive outcomes.---------- Settings: This study was conducted at the endocrine outpatient departments of three hospitals in Taiwan. Participants A convenience sample of 334 adults with type 2 diabetes aged 40 and over.---------- Methods: Data were collected by a self-reported questionnaire and physiological examination. Using the structural equation modeling technique, measurement and structural regression models were tested.---------- Results: Age and life events reflected the construct of risk factors. The construct of protective factors was explained by diabetes symptoms, coping strategy, and social support. The construct of adaptive outcomes comprised HbA1c, health-related quality of life, and self-care behaviors. Protective factors had a significant direct effect on adaptive outcomes (β = 0.68, p < 0.001); however, risk factors did not predict adaptive outcomes (β = − 0.48, p = 0.118).---------- Conclusions: Identifying and managing risk factors and protective factors are an integral part of diabetes care. This theoretical model provides a better understanding of how risk factors and protective factors work together to influence multiple adaptive outcomes in people living with type 2 diabetes.