Plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism and endometriosis. Influence of PAI-1 polymorphism on PAI-1 antigen and mRNA expression.

INTRODUCTION: Endometriosis is a benign gynecologic disease with a high prevalence. It is a multifactorial and polygenic entity in which the fibrinolytic system may be implicated. The objective of this study was to evaluate the plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism in a group of women with and without endometriosis and to analyze the influence of this polymorphism in PAI-1 expression in endometrial tissue and peritoneal fluid. MATERIAL AND METHODS: In 389 women (170 patients with endometriosis and 219 controls) PAI-1 4G/5G polymorphism was determined by PCR amplification using allele-specific primers. Quantitative real-time RT-PCR assay was used to quantify PAI-1 mRNA and PAI-1 antigen (ag) levels were quantified by ELISA. RESULTS: The genotype and allele frequencies of PAI-1 4G/5G polymorphism did not differ significantly between patients and controls. Control women with the 4G/4G genotype had higher endometrial PAI-1ag (P=0.026) and mRNA (P=0.014) levels than those with the 5G/5G genotype. Control carrying the 4G/4G genotype tended to have higher peritoneal fluid PAI-1ag levels than those carrying the 5G/5G genotype. Moreover, PAI-1ag levels in peritoneal fluid were higher in patients than in controls (P=0.003). CONCLUSIONS: The PAI-1 genotype distribution was similar in patients and controls. PAI-1 levels in endometrial tissue and peritoneal fluid seem to be associated with PAI-1 4G/5G polymorphism in controls. The increased PAI-1ag levels observed in peritoneal fluid from patients could contribute to increase the peritoneal adhesions observed in endometriosis.

Fibrinolytic inhibitor levels and polymorphisms in Behçet disease and their association with thrombosis.

This study aimed to assess the fibrinolytic inhibitors and their association with thrombosis in Behçet disease. Thrombin activatable fibrinolysis inhibitor (TAFI) (P < 0.001) and plasminogen activator inhibitor-1 (PAI-1) levels (P = 0.022) were significantly higher in 79 patients than in 84 controls. No significant differences were observed in CPB2 (TAFI) Thr325Ile and SERPINE1 (PAI1) 4G/5G polymorphism distribution between patients and controls. TAFI activity levels were significantly higher in patients with thrombosis than in those without thrombosis (P = 0.024). In conclusion, the increased TAFI levels in Behçet disease could contribute to the increased risk of thrombosis observed in these patients.

Plasminogen activator inhibitor-1 levels in severe and morbid obesity. Effect of weight loss and influence of 4G/5G polymorphism.

INTRODUCTION: An association between an increase in plasminogen activator inhibitor type 1 and obesity has been described. It has also been shown that a decrease in adiposity has beneficial effects. However, less information is available regarding morbid obesity and hypofibrinolysis. The aim of the present study was to evaluate the effect of weight loss and the influence of the plasminogen activator inhibitor type 1 promoter 4G/5G genotype on plasminogen activator inhibitor type 1 levels in severe and morbid obesity. MATERIALS AND METHODS: Sixty-seven obese patients were studied before and three months after a weight reduction program, and compared with 67 controls. We determined plasminogen activator inhibitor type 1 antigen and activity levels, tissue type plasminogen activator antigen levels, 4G/5G genotype and biochemical parameters in both groups. RESULTS: A significant increase in plasminogen activator inhibitor type 1 antigen and activity was observed in obese patients in comparison with the control group (P<0.001). No significant differences in plasminogen activator inhibitor type 1 levels among 4G/5G genotypes were obtained. After weight loss, a significant decrease in plasminogen activator inhibitor type 1 antigen and activity was observed (P<0.001). A significant and positive correlation was observed in percentage changes in plasminogen activator inhibitor type 1 and body mass index (P=0.02). CONCLUSIONS: A decrease in body mass index in severe and morbid obesity shows a favourable effect on the fibrinolytic system due to a decrease in plasminogen activator inhibitor type 1 levels. However, no influence of 4G/5G polymorphism has been observed in this setting.

Expression of plasminogen activator inhibitors type 1 and type 3 and urokinase plasminogen activator protein and mRNA in breast cancer.

BACKGROUND: The urokinase plasminogen activator (uPA) system has been involved in cancer cell invasion and in metastasis. uPA activity is controlled by its principal inhibitor, the PA inhibitor type-1 (PAI-1), but it can also be inhibited by PAI-3. Increased levels of uPA and PAI-1 are known to be associated with a poor prognosis in breast cancer. To our knowledge this is the first study of the expression and role of PAI-3 in human breast cancer tissue. MATERIALS AND METHODS: Protein and mRNA levels were evaluated for uPA, PAI-1 and PAI-3 in breast cancer tissues from 70 different patients. The localization of antigen and mRNA of these proteins was studied by immunohistochemistry and in situ hybridization, respectively. RESULTS: No significant differences were observed for PAI-3 mRNA or protein levels between the nodal status groups or the different post-surgical tumor-node-metastasis (pTNM) stages. However, uPA and PAI-1 mRNA and antigen levels significantly increased at the pTNM stage and in node-positive patients. PAI-3 antigen levels were significantly higher in early relapse-free patients, whereas PAI-1 antigen levels were significantly higher in patients who suffered a relapse. PAI-3 protein and mRNA were localized in stromal cells. PAI-1 and uPA protein were detected in cancer, endothelial and stromal cells and their mRNA mainly in stromal cells. CONCLUSIONS: Our results indicate that PAI-3 is expressed in human breast cancer tissues, and that elevated levels of PAI-3 could be a positive prognostic factor in this disease. A potential mechanism for the contribution of PAI-3 to a positive long-term outcome may involve suppression of tumor invasion through protease inhibition in stroma.

Endorepellin in vivo: targeting the tumor vasculature and retarding cancer growth and metabolism.

BACKGROUND: The antiangiogenic approach to controlling cancer requires a better understanding of angiogenesis and the discovery of new compounds that modulate this key biological process. Here we investigated the role of endorepellin, an angiostatic protein fragment that is derived from the C-terminus of perlecan, a heparan sulfate proteoglycan, in controlling tumor angiogenesis in vivo. METHODS: We administered human recombinant endorepellin systemically to mice bearing orthotopic squamous carcinoma xenografts or syngeneic Lewis lung carcinoma tumors. We monitored tumor growth, angiogenesis, metabolism, hypoxia, and mitotic index by using quantitative immunohistochemistry and positron emission tomography scan imaging. In addition, we determined the localization of injected endorepellin using near-infrared labeling and immunohistochemistry of frozen tumor sections. Finally, we isolated tumor-derived endothelial cells and tested whether endorepellin could interact with these cells and disrupt in vitro capillary morphogenesis. All statistical tests were two-sided. RESULTS: Endorepellin specifically targeted the tumor vasculature as determined by immunohistochemical analysis and accumulated in the tumor perivascular zones where it persisted for several days as discrete deposits. This led to inhibition of tumor angiogenesis (as measured by decreased CD31-positive cells, mean control = 1902 CD31-positive pixels, mean endorepellin treated = 343.9, difference between means = 1558, 95% confidence interval [CI] = 1296 to 1820, P<.001), enhanced tumor hypoxia, and a statistically significant decrease in tumor metabolism and mitotic index (as measured by decreased Ki67-positive cells, mean control Ki67 pixels = 5970, mean endorepellin-treated Ki67 pixels = 3644, difference between means = 2326, 95% CI = 1904 to 2749, P<.001) compared to untreated controls. Endorepellin was actively internalized by tumor-derived endothelial cells causing a redistribution of alpha2beta1 integrin such that both proteins colocalized to punctate deposits in the perivascular region. Endorepellin treatment inhibited in vitro capillary morphogenesis of both normal and tumor-derived endothelia. CONCLUSIONS: Our results provide support for the hypothesis that endorepellin is an effective antitumor vasculature agent that could be used as a therapeutic modality to combat cancer.

Lipoprotein (a) in young individuals as a marker of the presence of ischemic heart disease and the severity of coronary lesions.

The purpose of this study was to evaluate whether high levels and small isoforms of lipoprotein (a) [Lp(a)] are markers of risk of early myocardial infarction and markers of the severity of coronary atherosclerosis. Lp(a) levels and small apo(a) isoforms were higher in 222 patients than in 199 controls (p<0.001). In patients, Lp(a)> or =30 mg/dL was associated with the presence of coronary lesions (p=0.007) and the severity of coronary atherosclerosis (p=0.002). The present study suggests that Lp(a) levels and small isoforms are markers of early myocardial infarction and that Lp(a) levels > or =30 mg/dL are associated with severe patterns of coronary atherosclerosis.

Plasminogen activator inhibitor-1 4G/5G polymorphism in breast cancer patients and its association with tissue PAI-1 levels and tumor severity.

BACKGROUND: The plasminogen activator inhibitor type 1 (PAI-1) 4G/5G polymorphism may have significance for PAI-1 expression. High levels of PAI-1 in breast cancer patients are associated with a poor prognosis. In this study, we analyzed the influence of the PAI-1 4G/5G polymorphism on tissue PAI-1 levels and its association with tumor severity in women with breast cancer. MATERIAL AND METHODS: We studied 104 women with breast carcinoma (patient group) and 104 healthy age-matched women (control group). In patients and controls, the PAI-1 4G/5G polymorphism was determined by PCR amplification using allele-specific primers. In patients, PAI-1 levels were quantified in breast cancer tissue by using an ELISA. RESULTS: The frequency of the PAI-1 4G allele tended to be higher in patients than in controls (p=0.062). The presence of the 4G allele (4G/5G plus 4G/4G genotypes) was significantly higher among patients with histological grade 3 tumors than among those with grade 1 tumors (p=0.026). Furthermore, patients with the 4G/4G genotype had significantly higher tissue PAI-1 levels than those with the 5G/5G genotype. Moreover, tissue PAI-1 antigen levels were significantly and positively correlated with tumor severity (p=0.003) and tumor size (p=0.009). However, no significant differences in PAI-1 level were observed in relation to menopause, hormone receptor or nodal status. CONCLUSION: Tissue PAI-1 antigen levels and tumor severity seem to be associated with the PAI-1 4G/5G polymorphism. Further studies with a larger number of patients are needed to clarify the influence of this polymorphism in breast cancer.

Plasminogen activators and plasminogen activator inhibitors in endometriosis.

Endometriosis is one of the most frequent benign gynecological diseases that affect women. Little is known about the pathogenesis and etiology of endometriosis, despite the numerous studies performed in this field. Although endometriosis is a benign disease, the endometrial tissue, after attachment to the peritoneum, has the ability to grow and invade the surrounding tissues. Similar to neoplastic growth, local extracellular proteolysis might take place, and therefore, the fibrinolytic system may be involved. An altered expression of several components of the fibrinolytic system in the endometrium and peritoneal fluid of women with the disease has been suggested as a key factor in the establishment of the endometriotic lesions. There is evidence of increased fibrinolytic activity in the eutopic endometrium of these women, resulting in endometrial fragments with a high potential to degrade the extracellular matrix and facilitate implantation. The peritoneum possesses an inherent fibrinolytic activity that is responsible for the degradation of the fibrin deposits originated after an injury. This physiological function allows a correct repair of the mesothelium, and therefore, prevents the formation of adhesions. Peritoneal fluid of women with endometriosis and pelvic adhesions has shown to have an increased fibrinolytic activity that may be implicated in reducing the formation of new adhesions. Endometriotic tissue has abnormal proteolytic capacity, which is determined by modifications of the fibrinolytic parameters in this tissue. Proteolytic status is determined by the imbalance between plasminogen activators and plasminogen activator inhibitors, which are expressed differently depending on the type of lesion considered and the stage of the disease. The aim of the present study is to review the role of the plasminogen activator system in endometriosis, consider the clinical implications and focus on possible further research efforts and therapeutic applications in this disease.

Thrombin-activatable fibrinolysis inhibitor in young patients with myocardial infarction and its relationship with the fibrinolytic function and the protein C system.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a fibrinolytic inhibitor. Studies in coronary artery disease have reported increased TAFI activity (TAFI Act) and low TAFI antigen (TAFI Ag) levels. This controversy might be explained by the polymorphisms of its gene. Only the Thr325Ile polymorphism modulates both TAFI Ag and Act levels in vitro. This study assessed TAFI Ag and Act levels, TAFI Thr325Ile polymorphism, the fibrinolytic and protein C systems and some prothrombotic mutations in a young patient group (n = 127, aged < 51 years, with myocardial infarction) and a control group (n = 99) with similar characteristics. Patients exhibited hypofibrinolysis and higher plasminogen activator inhibitor-1 (PAI-1) levels. Although TAFI Ag was lower, TAFI Act level was significantly higher in patients and positively correlated with PAI-1, protein C inhibitor and the euglobulin lysis time. No differences between groups were found according to the Thr325Ile polymorphism. Irrespective of the genotype, patients had higher TAFI Act levels. The Ile-325 variant exhibited lower TAFI Ag levels. We suggest that the hypofibrinolysis observed in these patients results from an increase in both PAI-1 and TAFI Act, which is not related to the Thr325Ile polymorphism. Patients have high TAFI Act with low TAFI Ag levels, probably because of an increased stability of TAFI related to a fibrinolytic hypofunction.

Quantitative real-time reverse transcription-PCR assay for urokinase plasminogen activator, plasminogen activator inhibitor type 1, and tissue metalloproteinase inhibitor type 1 gene expressions in primary breast cancer.

BACKGROUND: The plasminogen activation system and matrix metalloproteinases (MMPs) play a key role in the degradation of basement membrane and extracellular matrix in tissue remodeling, cancer cell invasion, and metastasis. METHODS: Quantitative real-time reverse-transcription-PCR (RT-PCR) assays were developed to quantify urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor type 1 (PAI-1), and tissue metalloproteinase inhibitor type 1 (TIMP-1) mRNA in 54 breast cancer tissues. Gene fragments were amplified in a LightCycler real-time PCR system using gene-specific primers and SYBR Green I. The results were normalized to beta-actin mRNA. We also quantified antigen and functional concentrations of these components. RESULTS: The intra- and interassay variabilities for mRNA quantification showed mean SDs for the crossing point of 0.12 and 0.15 cycles, respectively. PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and PAI-1 and uPA functional concentrations increased with tumor severity; the increase was statistically significant for PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and for uPA functional concentrations. Node-positive patients showed significantly higher PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations than those who were node negative. CONCLUSIONS: Quantitative real-time RT-PCR is a highly sensitive, reproducible, and fast method for measuring gene expression of PAI-1, uPA, and TIMP-1 in breast cancer. These components may be involved in breast cancer development, and increased mRNA expression may be associated with a worse prognosis.