Luis A. Ramón

Fibrinolysis: the key to new pathogenetic mechanisms.

The fibrinolytic system includes a broad spectrum of proteolytic enzymes with physiological and pathophysiological functions in several processes, such as haemostatic balance, tissue remodeling, tumor invasion, angiogenesis and reproduction. The main enzyme of the plasminogen activator system is plasmin, which is responsible for the degradation of fibrin into soluble degradation products. The activation of plasminogen into plasmin is mediated by two types of activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). The activity of both is regulated by specific plasminogen activator inhibitors (PAIs). There are 3 types of PAIs described so far but the most important fibrinolytic inhibitor in vivo is PAI type 1 (PAI-1). Among others, the presence of metabolic syndrome and the -675 4G/5G promoter polymorphism are known to be modulators of PAI-1 levels. Besides their fibrinolytic profile, plasmin and plasminogen activators are implicated in tissue proliferation and cellular adhesion, as they can proteolytically degrade the extracellular matrix and regulate the activation of both growth factors and matrix metalloproteinases. By all these means, the fibrinolytic system is also involved in physiological processes, and in pathological situations such as thrombosis, arteriosclerosis, endometriosis and cancer. PAI 1 has been studied in different settings with thrombotic pathophysiology, such as coronary artery disease and ischaemic stroke. Controversial results have been published and concerns about study designs or presence of confounders have been claimed to be responsible of them. Recently, its involvement in adverse thrombotic events related to the modern drug-eluting coronary stents has renewed the interest of its study. PAI-1 also plays an important role in signal transduction, cell adherence, and migration. Indeed, studies of several types of cancers, including breast cancer, have shown that increased uPA and PAI-1 levels are associated with aggressive tumor behavior and poor prognosis. Endometriosis is defined by the presence of endometrial glands and stroma outside the uterus with marked ability to attach and invade the peritoneum. It is one of the most frequent benign gynecological diseases that affect women with pelvic pain or infertility during their reproductive age. Immune system disorders, genetic predisposition, altered peritoneal environment and endometrial alterations are believed to increase the susceptibility to endometriosis. The plasminogen activator system may be involved in this process, where local extracellular proteolysis plays a crucial role. Altered expression of several components of the fibrinolytic system in both eutopic and ectopic endometrium and peritoneal fluid of women with the disease has been implicated not only in the onset, but also in the progression of the endometriotic lesions.

microRNAs expression in endometriosis and their relation to angiogenic factors

BACKGROUND Endometriosis is a common, multifactorial disease in which angiogenesis may be involved in the growth of endometrium outside the uterus. microRNAs (miRNAs) are 21-22 nucleotide non-coding RNAs that regulate gene expression and play fundamental roles in biological processes. The objective of this study was to analyze several miRNAs related to angiogenesis and the angiogenic factors, vascular endothelial growth factor-A (VEGF-A) and thrombospondin-1 (TSP-1), in endometriotic lesions (ovarian endometrioma, peritoneal lesion and rectovaginal nodule) and eutopic endometrium from women with endometriosis. METHODS TaqMan real-time PCR was used to assess the expression of the miRNAs (miR-15b, -16, -17-5p, -20a, -21, -125a, -221 and -222), while VEGF-A and TSP-1 mRNA were assessed by real-time PCR, with SYBR Green I and VEGF-A and TSP-1 protein levels were quantified by ELISA. Included in the study were 58 women with endometriosis and 38 control women. RESULTS In paired samples, ovarian endometrioma showed significantly lower VEGF-A mRNA (P = 0.02) and protein (P = 0.002) expression than eutopic endometrium and higher expression of miR-125a (P = 0.003) and miR-222 (P <0.001). However, ovarian endometrioma had significantly higher expression of the angiogenic inhibitor TSP-1 and lower expression of miR-17-5p than eutopic endometrium (P < 0.001). Moreover, a significant inverse correlations between miR-222 and VEGF-A protein levels (-0.267, P = 0.018) and between miR-17-5p and TSP-1 protein levels (-0.260, P=0.022) were observed. Peritoneal lesions showed a significant increase in VEGF-A in comparison with ovarian endometrioma (P < 0.01). CONCLUSIONS Expression levels of miRNAs related to angiogenesis were different in eutopic endometrium from that observed in ovarian endometrioma. This could influence the expression of angiogenic factors and play a role in the pathogenesis of endometriosis.

Vascular endothelial growth factor polymorphisms (-460C/T, +405G/C, and 936C/T) and endometriosis: their influence on vascular endothelial growth factor expression.

OBJECTIVE To analyze three functional vascular endothelial growth factor (VEGF) polymorphisms (-460C/T, +405G/C, and 936C/T) in women with and without endometriosis and their correlation with VEGF expression in endometrial tissue and peritoneal fluid (PF). DESIGN Case-control study. SETTING University-based hospital. PATIENT(S) One hundred eighty-six women with endometriosis and 180 controls without the disease. INTERVENTION(S) Endometrial biopsies were performed by aspiration and PF samples were obtained at laparoscopy. MAIN OUTCOME MEASURE(S) VEGF polymorphisms (-460C/T, +405G/C, and 936C/T) were determined using a polymerase chain reaction (PCR)-restriction fragment length polymorphism assay. Quantitative real-time reverse transcriptase (RT)-PCR assay was used to quantify VEGF-A messenger RNA (mRNA) and VEGF-A antigen levels were quantified by ELISA. RESULT(S) Patients with endometriosis showed a higher VEGF 936T allele frequency than controls. However, the distribution of genotypes and allele frequencies in the other two VEGF (-460C/T, +405G/C) polymorphisms was similar in the endometriosis and control groups. Endometrium and PF from women with endometriosis showed an increase in VEGF levels, but no association was found between the VEGF polymorphisms studied and VEGF expression in endometrial tissue and PF. CONCLUSION(S) These findings suggest that the VEGF 936C/T polymorphism may be associated with the risk of endometriosis in a Caucasian population, but the increased VEGF levels observed in endometriosis do not appear to be associated with this polymorphism.

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.

Peritoneal fluid reduces angiogenesis-related microRNA expression in cell cultures of endometrial and endometriotic tissues from women with endometriosis.

Endometriosis, defined as the presence of endometrium outside the uterus, is one of the most frequent gynecological diseases. It has been suggested that modifications of both endometrial and peritoneal factors could be implicated in this disease. Endometriosis is a multifactorial disease in which angiogenesis and proteolysis are dysregulated. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the protein expression and may be the main regulators of angiogenesis. Our hypothesis is that peritoneal fluid from women with endometriosis could modify the expression of several miRNAs that regulate angiogenesis and proteolysis in the endometriosis development. The objective of this study has been to evaluate the influence of endometriotic peritoneal fluid on the expression of six miRNAs related to angiogenesis, as well as several angiogenic and proteolytic factors in endometrial and endometriotic cell cultures from women with endometriosis compared with women without endometriosis. Methods Endometrial and endometriotic cells were cultured and treated with endometriotic and control peritoneal fluid pools. We have studied the expression of six miRNAs (miR-16, -17-5p, -20a, -125a, -221, and -222) by RT-PCR and protein and mRNA levels of vascular endothelial growth factor-A, thrombospondin-1, urokinase plasminogen activator and plasminogen activator inhibitor-1 by ELISA and qRT-PCR respectively. Results Control and endometriotic peritoneal fluid pools induced a significant reduction of all miRNAs levels in endometrial and endometriotic cell cultures. Moreover, both peritoneal fluids induced a significant increase in VEGF-A, uPA and PAI-1 protein levels in all cell cultures without significant increase in mRNA levels. Endometrial cell cultures from patients treated with endometriotic peritoneal fluid showed lower expression of miRNAs and higher expression of VEGF-A protein levels than cultures from controls. In conclusion, this “in vitro” study indicates that peritoneal fluid from women with endometriosis modulates the expression of miRNAs that could contribute to the angiogenic and proteolytic disequilibrium observed in this disease.

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.

A simplified assay for the quantification of circulating activated protein C

Available assays for circulating levels of activated protein C (APC) are either time-consuming or difficult to use in a routine laboratory, or have a detection limit above normal levels. We have developed a simplified assay that measures both the in vivo free APC and the in vivo APC complexed to PC inhibitor (PCI). We measured APC levels, with both assays, in 339 plasma samples, 165 from patients with venous thromboembolism (VTE) and 174 from healthy individuals. The mean APC level in the 339 samples was 0.038±0.010 nM, using a previous assay that measures only the in vivo APC level, and 0.041±0.010 nM with the present new assay. The coefficient of correlation between assays was r=0.954 (P<0.001). The mean APC level in VTE patients was 0.034±0.009 nM (previous assay) and 0.037±0.009 nM (new assay), significantly lower than those in controls (P<0.001). In both groups there was a significant correlation between the levels obtained by the two assays (P<0.001). These results show that both assays are equivalent, and confirm that the APC level is lower in VTE patients than in healthy individuals. Therefore, the new simplified assay, which measures the sum of circulating free APC and APC complexed to PCI, may be used to estimate the level of circulating APC, and will allow its use in routine laboratories.

C0081 Tissue factor expression and micrornas −17-5p and –20a in endometrial cell cultures from women with endometriosis: Its modulation by peritoneal fluid

Background: Endothelial colonies forming cells (ECFC) are good candidates for cell-based therapy in cardiovascular diseases. Concerns have been raised about the potential risks of ECFC-based cell therapy, in terms of thrombogenicity. We recently published that cord blood ECFC (cb-ECFC) express Tissue Factor (TF). The objective of the present study is to detect TF in two different cell therapy products (CTP). As we previously reported a co-regulation of TF with proangiogenic MMPs, we also focused on the expression of MMP-2 and MMP-9 in CTP. Methods: ECFC were cultured from cord blood after isolation of CD34+ cells. CTP were obtained from an ongoing clinical trial evaluating cell therapy to improve vascularization in chronic critical limb ischemia (NIH NCT00533104). Two different CTP were analyzed: 1Peripheral blood mononuclear cells (PB-MNC) obtained by cytapheresis; 2Bone marrow mononuclear cells (BM-MNC). Results: Full length TF mRNA was detected in both CTP. The alternatively spliced form (as-TF) was also detected but represented a very minor part of total TF mRNA. TF mRNA was estimated to be a 2 fold higher in BM-MNC than in cb-ECFC. On the contrary, TF mRNA was a 5 fold lower in PB-MNC than in cb-ECFC. TF mRNA levels was also compared with CD34+ cells and found respectively 3 fold lower in BM-MNC and 27 fold lower in PB-MNC. The source of TF in CTP could be monocytes or CD34+ cells. Indeed, the percentage of CD14+ cells was respectively 17% and 26% in BM-MNC and in PBMNC whereas the percentage of CD34+ cells was 2.6% in BM-MNC and 0.1% in PB-MNC. TF protein was found in both CTP at a very low range. Still, in the clinical trial, one patient who had received PB-MNC developed a deep vein thrombosis at the site of injection. In BM-MNC, MMP-2 and MMP-9 were expressed at a very high level. In PB-MNC, MMP-2 and MMP-9 could be detected but at a very low level. In comparison, cb-ECFC strongly expressed MMP-2 whereas the expression of MMP-9 was weak. Comment: The profile of TF and MMPs is different in BM-MNC and in PB-MNC, suggesting a higher proangiogenic potential for BM-MNC. The different cell composition of CTP may explain these differences. However, the prothrombotic potential of CTP has to be considered.