Thrombin induction of plasminogen activator inhibitor mRNA in human umbilical vein endothelial cells in culture.

We have previously reported that incubation of human umbilical vein endothelial cells (HUVECs) with human alpha-thrombin causes a time- and concentration-dependent increase in secreted plasminogen activator inhibitor type 1 (PAI-1) activity (Gelehrter TD, Sznycer-Laszuk R. J Clin Invest 1986;77:165-9). Here we report that the regulation of PAI-1 activity by thrombin is secondary to the thrombin-induced increase in PAI-1 mRNA accumulation. Incubation of HUVECs for 6 to 24 hours with 0.3 to 1.0 U/ml thrombin causes a 1.8-fold to 10-fold increase in PAI-1 activity and a 1.5-fold to threefold increase in accumulation of both the 3.2 and the 2.2 kilobase PAI-1 mRNAs. These effects are prevented by the prior addition of hirudin, a specific thrombin inhibitor. Inhibition of RNA synthesis with actinomycin D blocks the thrombin induction of PAI-1 mRNA accumulation. The protein synthesis inhibitor, cycloheximide, which itself markedly stimulates the accumulation of PAI-1, appears to prevent the induction by thrombin, suggesting that thrombin may act by inducing another effector such as interleukin-1. Consistent with this hypothesis is our observation that simultaneous addition of antibodies to interleukin-1-alpha prevents the thrombin induction of PAI-1 activity and mRNA.     

Thrombin suppresses matrix metalloproteinase 2 activity and increases tissue inhibitor of metalloproteinase 1 synthesis in cultured human peritoneal mesothelial cells.

OBJECTIVE: Recently, high levels of intraperitoneally generated thrombin were found in the effluent of patients treated with continuous ambulatory peritoneal dialysis (CAPD). The aim of the present study was to investigate in human peritoneal mesothelial cells (HMCs) the effect of thrombin on the activity and synthesis of matrix metalloproteinases (MMPs), which regulate the degradation of basement membrane collagen. METHODS: Cultured HMCs were isolated from omental tissue and used at confluence for the experiments. Conditioned media were obtained by incubating cells with serum-free M199 containing the relevant doses of thrombin. Activity of MMP-2 and MMP-9 were determined by an activity assay system. The antigen levels of MMPs and of the specific tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by ELISA. Northern blot analysis was applied to analyze mRNA expression of MMP-2 and TIMP-1. RESULTS: Incubation of HMCs with increasing doses of thrombin resulted in a concentration- and time-dependent suppression of MMP-2 activity. No changes in MMP-9 activity were seen. After a 48-hour stimulation period with thrombin (5 U/mL), MMP-2 activity decreased to 53% of that seen in control conditions. Antigen measurements revealed that this decrease was paralleled by a slight reduction in MMP-2 levels, which became significant at a thrombin dose of 5 U/mL [50.65 +/- 7.5 ng/10(5) cells (48 hours, 5 U/mL) vs 64.6 +/- 10.1 ng/10(5) cells (control)]. Under the same conditions, TIMP-1 levels were considerably increased [3.9 +/- 0.46 microg/10(5) cells (48 hours, 5 U/mL) vs 1.2 +/- 0.14 microg/10(5) cells (control)]. Hirudin (10 U/mL) completely inhibited the thrombin-induced effects on MMP-2 and TIMP-1 synthesis. These results were also reflected by Northern blot hybridization, where a slight decrease in MMP-2 and an increase in TIMP-1 mRNA expression were observed in response to thrombin. CONCLUSIONS: Our results suggest that high thrombin levels suppress MMP-2 activity through decreased MMP-2 and increased TIMP-1 synthesis. Thus, thrombin may promote the accumulation of basement membrane collagen. In addition to fibrin formation, this mechanism may represent a further contribution by thrombin to peritoneal thickening during CAPD.     

Thrombin stimulates tissue plasminogen activator release from cultured human endothelial cells

The effect of thrombin on the release of tissue plasminogen activator from endothelial cells was studied in primary cultures of human umbilical vein endothelial cells. Tissue plasminogen activator concentration in conditioned medium was measured by a two-site radioimmunometric assay. The addition of increasing concentrations (0.01 to 10 U/ml) of thrombin to confluent cultures produced a saturable, dose-dependent increase in the rate of release of tissue plasminogen activator. A sixfold increase in tissue plasminogen activator concentration (from 2 to 12 ng/ml) occurred after the addition of 1 U/ml thrombin (8 X 10(-9) M) to cultures containing 5 X 10(4) cells/cm2. Enhanced release was not observed until 6 h after thrombin addition, reached a maximum rate of 1.3 ng/ml per h between 8 and 16 h, and then declined to 0.52 ng/ml per h after 16 h. The 6-h lag period before increased tPA release was reproducible and independent of thrombin concentration. Thrombin inactivated with diisopropylfluorophosphate or hirudin did not induce an increase in tissue plasminogen activator levels. A 50-fold excess of diisopropylfluorophosphate-treated thrombin, which inhibits binding of active thrombin to endothelial cell high affinity binding sites, did not inhibit the thrombin-induced increase. It is concluded that proteolitically active thrombin causes an increase in the rate of release of tissue plasminogen activator from cultured human endothelial cells. The 6-h interval between thrombin treatment and enhanced tissue plasminogen activator release may reflect a delaying mechanism that transiently protects hemostatic plugs from the sudden increase in the local concentration of this fibrinolytic enzyme.     

Pregnancy complicated by plasminogen activator inhibitor type 1 deficiency

We present the case of a patient with a history of hemorrhage following prior surgery whose pregnancy was complicated by plasminogen activator inhibitor type 1 deficiency. To our knowledge, this is the first reported case of a pregnancy complicated by plasminogen activator inhibitor type 1 (PAI-1) deficiency.     

Interleukin-1beta stimulates the production of extracellular matrix in cultured human peritoneal mesothelial cells.

OBJECTIVE: To investigate the effect of interleukin-1beta (IL-1beta) on the production of extracellular matrix in cultured human peritoneal mesothelial cells (HPMCs). DESIGN: Cultured HPMCs were treated with or without IL-1beta. Cell morphology was observed. The expression of fibronectin, alpha1(I) procollagen, and transforming growth factor-beta1 (TGFbeta1) mRNAs was measured by Northern blot analysis. The cell surface expression of fibronectin and type I collagen was evaluated by immunofluorescent staining. Fibronectin and type I collagen in culture supernatant were measured by inhibition ELISA. RESULTS: Interleukin-1beta induced morphologic change in HPMCs from a cuboidal epithelioid shape into an elongated fibroblastoid shape.The elongated cells were positive for cytokeratin although they had a fibroblastoid appearance. Treatment of HPMCs with IL-1beta resulted in increased expression of both fibronectin and alpha1(I) pro-collagen mRNA in dose- and time-dependent manners. Immunofluorescent staining showed strong and diffuse cytoplasmic expression of fibronectin and type I collagen in the cells treated with IL-1beta, whereas only weak perinuclear cytoplasmic staining was noted in the cells on media alone. The concentrations of secreted fibronectin and type I collagen in culture supernatant were significantly higher in the cells treated with IL-1beta than in the control cells. IL-1beta also stimulated the expression of TGFbeta1 mRNA. However, IL-1beta-induced fibronectin mRNA expression was only partially blocked by neutralizing anti-TGFbeta antibody. CONCLUSION: Interleukin-1beta stimulated the production of extracellular matrix in cultured HPMCs along with the induction of morphologic changes.This may play a role in the development of peritoneal fibrosis caused by peritonitis or bioincompatible dialysate in continuous ambulatory peritoneal dialysis patients.     

Role of plasminogen activator inhibitor type 1 in tumor angiogenesis]

The plasminogen/plasmin system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumour cell migration. High levels of components of the plasminogen activation system, and paradoxically also its inhibitor, plasminogen activator inhibitor 1 (PAI-1), have been correlated with a poor prognosis for patients with cancers of different types. Recent findings clearly suggest that PAI-1 is essential for capillary sprouting during tumour angiogenesis. Moreover, there is accumulating evidence that both the urokinase receptor and PAI-1 are multifunctional proteins involved not only in extracellular matrix proteolysis but also in cellular adhesion and migration through their binding site for vitronectin. The understanding of whether PAI-1 plays a regulatory role in angiogenesis by tightly controlling proteolytic activity or by influencing cell migration could allow a new anti-angiogenic approach for tumour therapy.     

Protein composition of plasminogen activator inhibitor type 1-derived endothelial microparticles

Endothelial microparticles (EMPs) are small vesicles released from the plasma membrane of endothelial cells in response to cell injury, apoptosis, or activation. Low levels of MPs are shed into the blood from the endothelium, but in some pathologic states, the number of EMPs is elevated. The mechanism of MP formation and the wide-ranging effects of elevated EMPs are poorly understood. Here, we report the protein composition of EMPs derived from human umbilical cord endothelial cells stimulated with plasminogen activator inhibitor type 1 (PAI-1). Two-dimensional gel electrophoresis followed by mass spectrometry identified 58 proteins, of which some were verified by Western blot analysis. Gene Ontology database searches revealed that proteins identified on PAI-1-derived EMPs are highly diverse. Endothelial microparticles are composed of proteins from different cellular components that exhibit multiple molecular functions and are involved in a variety of biological processes. Important insight is provided into the generation and protein composition of PAI-1-derived EMPs.     

纤溶酶原激活物抑制剂-1的研究进展

纤溶酶原激活物抑制剂-1(plasminogen activator inhibitor 1,PAI-1)作为体内组织型纤溶酶原激活物(tissue-type plasminogen activator,t-PA)和尿激酶型纤溶酶原激活物(urokinase-type plasminogen activator,u-PA)的主要抑制剂,与动静脉血栓、出血和凝血异常、细胞迁移密切相关,进而引起缺血性脑卒中、冠心病、静脉血栓、肿瘤转移、出血、股骨头坏死、流产等一系列疾病的发生发展。同时,体内血浆PAI-1活性水平又受血脂、血糖等调节,进一步参与肥胖、糖尿病、高脂血症等疾病的进程,又影响着上述相关疾病的预后。     

Bleeding diathesis due to decreased functional activity of type 1 plasminogen activator inhibitor.

We evaluated an elderly patient with a lifelong history of severe bleeding after surgery or trauma and with evidence of persistent hyperfibrinolysis. Routine coagulation studies were normal. Serum plasminogen (40%, normal 72-128%) and alpha 2-antiplasmin (55%, normal 70-145%) activities were decreased. Euglobulin clot lysis was abnormally shortened (50 min) and normalized in vitro with epsilon-aminocaproic acid (EACA). The patient was treated with EACA with prompt cessation of bleeding. Patient tissue-plasminogen activator (t-PA) levels in serum were normal (4.7 ng/ml, control 3.5-7.2) as detected by a two-site immunoradiometric assay (IRMA). Patient fibrinolytic inhibitor activities were assessed by incubating 125I-labeled t-PA with either whole blood or serum followed by SDS-PAGE and autoradiography to identify the resultant protease/protease inhibitor complexes. In comparison to blood samples obtained from normal donors, patient plasma and serum demonstrated reduced binding of a fast-acting plasminogen activator inhibitor to 125I-labeled t-PA. Immunoprecipitation experiments indicated diminished complex formation between type 1 plasminogen activator inhibitor (PAI-1) in patient serum and 125I-labeled t-PA. Low patient PAI-1 activity was confirmed in serum (0.36 U/ml, control 0.87-1.81; n = 3) and in platelet lysates using a functional IRMA to quantitate PAI-1 binding to immobilized t-PA. However, patient serum PAI-1 antigen was within the normal range when analyzed by IRMA (31.8 ng/ml, control 19.6-42.2); this result was confirmed in both serum and platelets by Western blot (n = 3). Mixing experiments using purified PAI-1 as well as patient and control sera did not show evidence for an inhibitor against PAI-1. We conclude that this patient's bleeding diathesis was due to hyperfibrinolysis and defective PAI-1. This patient provides the first demonstration of a link between decreased in vivo PAI-1 activity and disordered hemostasis, and supports a role for PAI-1 in control of vivo fibrinolysis.     

Thrombin induction of plasminogen activator-inhibitor in cultured human endothelial cells.

We have examined the effect of thrombin on the activity of plasminogen activator (PA) and plasminogen activator-inhibitor (PA-I) in medium conditioned by primary cultures of human umbilical vein endothelial cells. PA activity was measured by fibrinolytic and esterolytic assays, and total tissue-type PA (tPA) antigen by radioimmunoassay. Net PA-I activity was assayed by titration of human urokinase esterolytic activity. Incubation of confluent endothelial cell cultures with thrombin for 24 h caused a sixfold increase in PA-I activity. The effect of thrombin was half-maximal at approximately 0.4 U/ml (less than 4 nM), and required concomitant RNA and protein synthesis. The stimulation of PA-I activity required active alpha-thrombin and was not obtained with gamma-thrombin nor with thrombin catalytically inactivated with hirudin. Because of the excess of PA-I, PA activity was not measurable in either control or thrombin-treated cells. Thrombin did, however, increase medium concentration of tPA antigen by approximately fourfold. The thrombin-induced PA-I inhibited both tPA and urokinase, did not lose activity upon acidification, and was stable to sodium dodecyl sulfate and thiol reduction. We conclude that physiologic concentrations of thrombin increase both PA-I activity and tPA antigen in medium conditioned by human umbilical vein endothelial cells. Because there was always a several-fold increase in the net activity of free PA-I, these observations suggest that the net effect of thrombin is to decrease fibrinolytic activity in human endothelial cells. Thus, thrombin, in addition to its role in coagulation, may protect clots from premature lysis by increasing the amount of a specific fibrinolytic inhibitor.     

Tumor necrosis factor-alpha and troglitazone regulate plasminogen activator inhibitor type 1 production through extracellular signal-regulated kinase- and nuclear factor-kappaB-dependent pathways in cultured human umbilical vein endothelial cells

Plasminogen activator inhibitor type 1 (PAI-1) plays a role in the development of atherosclerosis in diabetic patients. PAI-1 is produced by endothelial cells stimulated with various inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha, which induces insulin resistance. In diabetic patients, troglitazone, a thiazolidinedione, can lower the concentration of PAI-1. We investigated the TNF-alpha-induced signaling pathway that leads to PAI-1 synthesis and the target step of troglitazone in this pathway. TNF-alpha induced PAI-1 mRNA expression and protein production in human umbilical vein endothelial cells (HUVECs). A specific inhibitor for p38 mitogen-activated protein kinase, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB 203580), and a protein kinase C inhibitor, calphostin C, had no inhibitory effects on TNF-alpha-induced PAI-1 secretion. A protein tyrosine kinase inhibitor, genistein, completely inhibited TNF-alpha-induced PAI-1 secretion, whereas an inhibitor of extracellular signal-regulated kinase (ERK) kinase, 2'-amino-3'-methoxyflavone (PD98059), and a nuclear factor-kappaB (NF-kappaB) inhibitor, emodin, partly inhibited TNF-alpha-induced PAI-1 secretion. Together, PD98059 and emodin completely inhibited TNF-alpha-induced PAI-1 secretion, suggesting that both NF-kappaB-dependent and NF-kappaB-independent pathways are involved in TNF-alpha-induced signal pathway to PAI-1 production and that the latter pathway is mediated by activation of ERK. Furthermore, we have shown that troglitazone inhibited both TNF-alpha-induced PAI-1 protein secretion and mRNA in HUVECs. Genistein, but neither PD98059 nor emodin, was additive to the inhibitory effect of troglitazone on TNF-alpha-induced PAI-1 secretion. These results indicate That ERK and NF-kappaB are possible targets of TNF-alpha and troglitazone in the regulation of PAI-1 production.     

ACE inhibitor and AT1-receptor blocker attenuate the production of VEGF in mesothelial cells.

OBJECTIVE: Human mesothelial cells (HMC) are a major source of intraperitoneal vascular endothelial growth factor (VEGF) and by that are presumably involved in complications of long-term peritoneal dialysis (PD), such as ultrafiltration failure. This prompted us to look for agents that reduce basic mesothelial VEGF production and abrogate VEGF-overproduction induced by proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1alpha (IL-1alpha). Angiotensin-converting enzyme (ACE) inhibition was found to preserve peritoneal function and ameliorate morphologic changes in a rat PD model. The present in vitro study was designed to investigate the effect of the ACE inhibitors captopril and enalapril, and the angiotensin II type 1-receptor (AT1) antagonist losartan on mesothelial VEGF synthesis. METHODS: HMC were isolated from omental tissue and taken into culture. VEGF antigen concentrations were measured in the cell supernatant by ELISA. VEGF mRNA levels were determined by real-time polymerase chain reaction. RESULTS: Incubation of HMC with captopril (100-1000 micromol/L) resulted in a concentration-dependent attenuation of VEGF synthesis. Incubation with captopril (500-1000 micromol/L), enalapril (100-1000 micromol/L), and losartan (1-100 micromol/L) significantly decreased inflammatory mediator (TNF-alpha, IL-1alpha)-induced mesothelial VEGF overproduction. CONCLUSION: The results indicate that ACE inhibitors and AT1-receptor blockers are capable of effectively attenuating the overproduction of VEGF due to proinflammatory cytokine stimuli. These data suggest that locally produced angiotensin II in the peritoneal cavity may be a potential therapeutic target in ultrafiltration failure during longterm PD.     

Expression of urokinase-type plasminogen activator and plasminogen activator inhibitor in colon disease

Surveillance colonoscopy and biopsy are inaccurate methods of predicting the likelihood of ulcerative colitis patients to develop colon carcinoma. We examined uPA and PAI-1 as potential markers for assessing these patients and those with familial polyposis who are at risk of developing colon cancer. For comparison, biopsies of normal colon and Crohn's disease were evaluated. We examined 77 colonic mucosa specimens taken from patients undergoing elective resection for benign and malignant colonic disease. uPA and PAI-1 were measured using a monoclonal antibody-based ELISA kit (American Diagnostica, Greenwich, CT) and expressed as ng/mg extract protein. Intra- and interassay controls of uPA gave CV = 3–4% and CV = 8–9%, respectively, while those for PAI-1 were 6–7% and 10–11%, respectively. The Mann-Whitney test showed that both uPA and PAI-1 expression were significantly higher in colon cancer, chronic ulcerative colitis, and Crohn's disease than in normal colon. uPA in familial polyposis samples was similar to that of normal colon, while PAI-1 was much lower than in normal colon. Neither patient age nor sex appeared to influence the expression of these potential markers in any tissue. The pattern of uPA and PAI-1 expression in normal, benign and malignant colon suggests these proteins deserve further consideration as markers for assessing colon carcinoma risk.     

Effect of iron sucrose on human peritoneal mesothelial cells

BACKGROUND: Iron supplementation is often required in uraemic patients with anaemia. Peritoneal cavity was proposed as an alternative intravenous route for iron infusion in patients treated with peritoneal dialysis. We studied the effect of iron sucrose (Venofer) on the function of human peritoneal mesothelial cells maintained in in vitro culture. MATERIALS AND METHODS: In in vitro experiments on human peritoneal, the mesothelial effect of elemental iron (in conc. 0.0001-1 mg mL-1) present in Venofer on their viability, growth and synthesis of IL-6 was studied. Additionally we evaluated with a fluorescent probe (2',7'-dichlorodihydro-fluorescein diacatate) generation of reactive oxygen species in cells exposed to iron sucrose. We also measured accumulation of iron in the cytoplasm of mesothelial cells after their in vitro exposure to Venofer. RESULTS: In in vitro conditions iron induces a dose-dependent inhibition of viability of the mesothelial cells as reflected by inhibition of the cells growth by 34% at Fe 0.1 mg mL-1 vs. control (P < 0.05) increased release of lactate dehydrogenase (LDH) from the cytosol: 67.1 +/- 30.3 mU mL-1 at Fe 1 mg mL-1 vs. 7.9 +/- 6.4 in control group (P < 0.001), and reduced synthesis of IL-6: 209 +/- 378 pg mg-1 cell protein at Fe 1 mg mL-1 vs. 38674 +/- 4146 pg mg-1 cell protein in controls (P < 0.001). Cytotoxicity of iron towards mesothelial cells was enhanced in vitro when it was tested in presence of the dialysis fluid. Iron used in vitro at concentration 0.0001 mg mL-1 and greater induces generation of oxygen-derived free radicals in mesothelial cells. Furthermore, iron is taken by these cells and stored in their cytosol, resulting in stimulation of the intracellular generation of free radicals. CONCLUSIONS: We conclude that iron used in the form of iron sucrose is cytotoxic to human peritoneal mesothelial cells. Accumulation of iron sucrose within cytoplasm of these cells may lead to induction of its chronic cytotoxic effect.     

A mutant,noninhibitory plasminogen activator inhibitor type 1 decreases matrix accumulation in experimental glomerulonephritis

In fibrotic renal disease, elevated TGF-beta and angiotensin II lead to increased plasminogen activator inhibitor type 1 (PAI-1). PAI-1 appears to reduce glomerular mesangial matrix turnover by inhibiting plasminogen activators, thereby decreasing plasmin generation and plasmin-mediated matrix degradation. We hypothesized that therapy with a mutant human PAI-1 (PAI-1R) that binds to matrix vitronectin but does not inhibit plasminogen activators, would enhance plasmin generation, increase matrix turnover, and decrease matrix accumulation in experimental glomerulonephritis. Three experimental groups included normal, untreated disease control, and PAI-1R-treated nephritic rats. Plasmin generation by isolated day 3 glomeruli was dramatically decreased by 69%, a decrease that was reversed 43% (P < 0.02) by in vivo PAI-1R treatment. At day 6, animals treated with PAI-1R showed significant reductions in proteinuria (48%, P < 0.02), glomerular staining for periodic acid-Schiff positive material (33%, P < 0.02), collagen I (28%, P < 0.01), collagen III (34%, P < 0.01), fibronectin (48%, P < 0.01), and laminin (41%, P < 0.01), and in collagen I (P < 0.01) and fibronectin mRNA levels (P < 0.02). Treatment did not alter overexpression of TGF-beta1 and PAI-1 mRNAs, although TGF-beta1 protein was significantly reduced. These observations strongly support our hypothesis that PAI-1R reduces glomerulosclerosis by competing with endogenous PAI-1, restoring plasmin generation, inhibiting inflammatory cell infiltration, decreasing local TGF-beta1 concentration, and reducing matrix accumulation.     

Electrophysiology and glucose transport of human peritoneal mesothelial cells: implications for peritoneal dialysis.

OBJECTIVE: To elucidate ionic and glucose transport across human peritoneal mesothelium, we utilized an Ussing chamber setup and studied the electrophysiological characteristics and tissue permeabilities of human peritoneal mesothelial cells (HPMC) to L- and D-glucose. METHODS: Human mesothelial cells were grown on polyester filters (snapwell; Costar, Cambridge, MA, U.S.A.) that, upon confluence, were fitted into Ussing chambers. Transmesothelial resistance and resting potential were determined using electrophysiological techniques. Radiolabeled glucose was added to one side of the chamber and the permeabilities determined by serial sampling in the receptive compartment. RESULTS: The transmesothelial potential and resistance were 0.54 +/- 0.07 mV (apical positive) and 20.4 +/- 3.2 ohms x cm2 respectively (mean +/- SEM, n = 36). The course of overall transfer of D- and L-glucose was examined using L-glucose as a positive diffusion-plus-leak marker. The permeabilities of HPMC to D-glucose were 3.00 +/- 0.26 cm/sec (apical-to-basolateral) and 3.25 +/- 0.27 cm/sec (basolateral-to-apical) [n = 6 experiments, p = not significant (NS)], which were not different from those of L-glucose: 3.00 +/- 0.30 cm/sec (apical-to-basolateral) and 2.71 +/- 0.24 (basolateral-to-apical) (n = 6 experiments, p = NS). CONCLUSIONS: The transepithelial resistance of HPMC is low and the ionic gradient, although it exists, is small and inconsequential. Passive paracellular flow accounts for the majority of transmesothelial glucose transport. The existence of a large paracellular shunt precludes the mesothelial membrane as a clinically relevant osmotic barrier.