Release Of Plasminogen Activator Inhibitor-1 Research Articles

Tumour necrosis factor-mediated release of plasminogen activator inhibitor 1 by human peritoneal mesothelial cells.

Human mesothelial cells synthesize plasminogen activator inhibitor (PAI) 1 in inflamed peritoneal tissue. The role of tumour necrosis factor (TNF) in the mediation of this response was studied. Postoperative peritoneal drain fluid contained both TNF and PAI-1. Peak levels of TNF at 4 h (median 271 pg/ml) preceded a rise in PAI-1 concentration, which peaked at 18 h (median 943.1 ng/ml). Thus TNF may mediate increased PAI-1 release in inflamed peritoneum. TNF significantly increased the mean(s.e.m.) release of PAI-1 by human peritoneal mesothelial cells in vitro at 4 h (control 1.84(0.17) ng/micrograms versus TNF 2.37(0.17) ng/micrograms, P < 0.05), 6 h (2.53(0.09) versus 3.88(0.46) ng/micrograms, P < 0.05), 18 h (0.50(0.02) versus 1.04(0.11) ng/micrograms, P < 0.05) and 24 h (0.87(0.05) versus 1.35(0.11) ng/micrograms, P < 0.05). TNF may be an important mediator of PAI-1 production by human mesothelial cells during peritoneal inflammation.

Cardiac fibrinolytic capacity is markedly increased after brief periods of local myocardial ischemia, but declines following successive periods in anesthetized pigs

Fibrinolysis in blood is mainly reflected by the activities of tissue plasminogen activator (tPA) and of plasminogen activator inhibitor-1 (PAI-1). The effect of myocardial ischemia on their activities in the coronary circulation is, however, not established. With an improved experimental model, we therefore examined the effect of a brief period of myocardial ischemia on their activities. Furthermore, the consequences of repeated periods of ischemia, mimicking the situations in patients with unstable angina, were investigated. In six anesthetized pigs, we occluded the distal left anterior descending coronary artery (LAD) four times for 10 min with 40 min intervals and determined the activities of tPA and PAI-1 in arterial and coronary venous blood. By simultaneously recording LAD flow, we could estimate cardiac release of these factors at baseline conditions and during reperfusion. Neither net cardiac release of PAI-1 nor alterations in plasma PAI-1 levels were demonstrated during the experiment. However, a significant net release of tPA activity of 10.4 +/- 3.2 IU mL(-1) (P < 0.005) was recorded during baseline conditions. During reperfusion following the first period of ischemia, the cardiac release of tPA activity increased to a peak of 103 +/- 30-fold baseline release, but declined progressively after repeated periods of ischemia. After the fourth period, tPA release did not exceed an estimated baseline accumulation during ischemia and early reperfusion. In this porcine model, a substantial local increase in fibrinolytic capacity was observed after brief periods of ischemia, but declined subsequently by repeated periods of ischemia.

Glycation Does Not Alter LDL‐Induced Secretion of Tissue Plasminogen Activator and Plasminogen Activator Inhibitor‐1 from Human Aortic Endothelial Cells

Diabetes may induce both quantitative and qualitative changes in lipoproteins, especially low-density lipoprotein (LDL). Effects of LDL glycation on endothelial cell secretion of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) have not been fully elucidated. Human aortic endothelial cell (HAEC) tPA and PAI-1 production were determined after incubation with LDL (50 to 500 microg/mL protein, 24 h) from three sources: (1) nondiabetic LDL (N-LDL) modified in vitro to form six preparations: native, nonmodified (N); glycated (G); minimally oxidized (MO); minimally oxidized and glycated (MOG); heavily oxidized (HO); and heavily oxidized and glycated (HOG); (2) in vivo glycated and relatively nonglycated LDL subfractions from type 1 diabetic patients; (3) LDL from type 1 diabetic patients and matched controls, which was subfractionated using density gradient ultracentrifugation. In experiments using LDL modified in vitro, the rate of tPA release by HAECs incubated with N-LDL (83 +/- 4 ng/mg cell protein/24 h) did not differ significantly from those incubated with G-LDL (73 +/- 7), MO-LDL (74 +/- 13), or MOG-LDL (66 +/- 15) and was not influenced by LDL concentration. The rate of PAI-1 release was similar in HAECs incubated with N-LDL (5.7 +/- 0.6 mug/mg cell protein/24 h), G-LDL (5.7 +/- 0.7), MO-LDL (5.5 +/- 0.8), or MOG-LDL (5.7 +/- 0.9) and was not influenced by LDL concentration. In contrast, tPA release was significantly decreased in cells incubated with LDL (10 microg/mL) modified extensively by oxidation, and averaged 45.2 +/- 5.0 and 43.7 +/- 9.9 ng/mg/24 h for HO-LDL and HOG-LDL, respectively, and was further decreased with increasing concentrations of the heavily oxidized LDL preparations. PAI-1 release was not significantly decreased relative to N-LDL in cells incubated with low concentrations (5 to 50 microg/mL) of HO-LDL and HOG-LDL, but was decreased to 3.2 +/- 0.5 and 3.1 +/- 0.7 microg/mg/24 h for HO-LDL and HOG-LDL at 200 microg/mL, respectively. Results using in vivo glycated versus nonglycated LDL showed that tPA and PAI-1 release did not differ between subfractions. Release of tPA averaged 5.11 +/- 0.6 and 5.12 +/- 0.7 ng/mg/24 h, whereas release of PAI-1 averaged 666 +/- 27 ng/mg/24 h and 705 +/- 30 ng/mg/24 h for nonglycated and glycated LDL subfractions, respectively. Using LDL of different density subclasses, tPA and PAI-1 release in response to LDL from diabetic patients compared with control subjects did not differ when HAECs were incubated with LDLs of increasing density isolated from each subject pair. We conclude that oxidation of LDL, but not glycation, may contribute to the altered fibrinolysis observed in diabetes.

Basal release of urokinase plasminogen activator, plasminogen activator inhibitor-1, and soluble plasminogen activator receptor from separated and cultured endometriotic and endometrial stromal and epithelial cells

To investigate whether separated and cultured endometriotic and endometrial stromal and epithelial cells release urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and soluble plasminogen activator receptor (suPAR) antigens in vitro. In vitro study. University hospital clinic. Regularly menstruating women with and without endometriosis. Tissue samples were collected at surgery performed for clinical reasons. The antigen concentrations of uPA, PAI-1, and suPAR in culture medium were assayed by enzyme-linked immunosorbent assay. Both stromal and epithelial cells from endometriotic and endometrial tissue released the three types of antigens, but the release of PAI-1 was significantly higher from stromal cells in the three types of tissue than from epithelial cells. Furthermore, the release of PAI-1 was significantly higher from endometriotic cells than from endometrial stromal cells. This study has demonstrated the basic capacity of separated epithelial and stromal cells from all three types of tissue to release uPA, PAI-1, and suPAR without any paracrine influence, as in vivo. The higher release of PAI-1 from endometriotic stromal cells might have importance for the invasive growth.

Transforming growth factor beta-1 released from platelets contributes to hypercoagulability in veno-occlusive disease following hematopoetic stem cell transplantation

Background Hepatic veno-occlusive disease (VOD) is one of the most disastrous complications after allogeneic hematopoetic stem cell transplantation (HSCT). Thrombocytopenia with refractoriness to platelet transfusions suggests an increased platelet consumption in these patients. Interactions between platelets and endothelial cells might contribute to the hypercoagulable state at the sinusoidal endothelium as a central mechanism in the pathogenesis of VOD. Study design The influence of activated platelets on cultured human endothelial cells was investigated in vitro. We focused on the release of plasminogen activator inhibitor-1 (PAI-1) from endothelial cells which has earlier been found to be significantly elevated in plasma of VOD patients. Endothelial cells isolated from human umbilical cords (HUVEC) were incubated with activated platelets. The release of PAI-1 in the presence or absence of specific antibodies was determined by ELISA technique. Tissue factor (TF) expression on endothelial cells was observed by flowcytometric analysis. Results HUVEC incubated with activated platelets were found to release significantly more PAI-1 compared to untreated cultures. The endothelial PAI-1-secretion after incubation of HUVEC with activated platelets was completely inhibited by an IgG monoclonal antibody against human transforming growth factor beta-1 (TGF beta-1). In contrast, PAI-1 production was not suppressed after inhibition of HUVEC–platelet-interaction by an IgG monoclonal antibody against CD154 (CD40L) expressed on the surface of activated platelets. An increased release of PAI-1 and an increased expression of tissue factor (TF) on the endothelial cell surface were observed after stimulation with TGF beta-1. Conclusion TGF beta-1 released from activated platelets contributes to the hemostatic imbalance at the sinusoidal endothelium in patients with hepatic VOD by increase of endothelial cell PAI-1 production and TF expression. As a potent profibrotic cytokine, TGF beta-1 might further be involved in phlebosclerosis and sinusoidal fibrosis occurring in VOD.

Insulin resistance causes impaired vasodilation and hypofibrinolysis in young women with polycystic ovary syndrome

Introduction Insulin resistance, a novel cardiovascular risk factor, is often associated with increased plasminogen activator inhibitor-1 levels and impaired vasodilation. Insulin infusion in the forearm induces plasminogen activator inhibitor-1 and tissue plasminogen activator expression and endothelium-dependent vasodilation in normal subjects. The present study explores the relationship between insulin-induced vasodilatory and fibrinolytic properties of the endothelium in women with polycystic ovary syndrome, frequently affected by insulin resistance and early atherosclerosis. Materials and methods Metabolic, hormonal and fibrinolytic parameters were evaluated in 64 patients with polycystic ovary syndrome (19 insulin-resistant and 45 insulin-sensitive) and in 25 controls. In 16 women with polycystic ovary syndrome, 8 insulin-resistant and 8 insulin-sensitive, blood flow, plasminogen activator inhibitor-1 and tissue plasminogen activator expression were evaluated during insulin infusion into the forearm. Results Elevated basal plasminogen activator inhibitor-1 levels were found in women with polycystic ovary syndrome, correlating directly with insulin levels. Plasminogen activator inhibitor-1 expression increased during insulin infusion in all women with polycystic ovary syndrome, but was delayed and sustained in insulin-resistant patients ( p<0.01). Vasodilatory response to insulin was blunted ( p<0.01) and tissue plasminogen activator expression abolished in insulin-resistant patients ( p<0.01). Conclusion Our study demonstrates that women with polycystic ovary syndrome and insulin resistance show a blunted endothelial-dependent vasodilation. The impaired endothelial release of tissue–plasminogen activator and the sustained plasminogen activator inhibitor-1 release during insulin infusion suggest a hypofibrinolytic state in PCOS patients with insulin resistance. This hemodynamic and fibrinolytic derangement may contribute to the pathogenesis of early atherosclerosis in insulin resistance.

Angiotensin II and IV stimulate expression and release of plasminogen activator inhibitor-1 in cultured human coronary artery endothelial cells.

There is increasing evidence that angiotensin II influences thrombogenesis by regulating the expression of plasminogen activator inhibitor-1 (PAI-1). In this study, the effects of angiotensin II and its receptors on the expression and release of PAI-1 and tissue-type plasminogen activator (t-PA) were examined in human coronary artery endothelial cells (HCAECs). As control, cells were treated with angiotensin IV. HCAECs incubated with angiotensin II increased the expression of PAI-1 mRNA in a concentration (10-9-10-5 M)- and time (6-24 h)-dependent manner. PAI-1 protein release was also increased in the culture medium of HCAECs treated with angiotensin II. The effects of angiotensin II (10-6 M) were blocked completely by the AT1 receptor blocker losartan (10-6 M) but not by the AT2 receptor blocker PD123319 (10-6 M). Angiotensin II pretreatment also slightly, but significantly, increased t-PA mRNA expression. This effect of angiotensin II on t-PA mRNA was blocked by losartan but not by PD123319. HCAECS treated with angiotensin II revealed large amounts of the lipid peroxidation product, malonaldehyde (MDA). The effects of angiotensin II on PAI-1 expression and MDA release were blocked by pretreatment of cells with alpha-tocopherol (10-5 M). In control experiments, treatment of HCAECs with angiotensin IV markedly increased PAI-1 mRNA expression and protein release. This effect of angiotensin IV was blocked by the AT4 receptor blocker divalinal (10-6 M). These observations indicate that AT1 receptor activation plays an important role in the stimulation of PAI-1 expression and release in response to angiotensin II. Upregulation of t-PA gene may reflect autoregulation in response to PAI-1 release. Angiotensin II-mediated activation of oxidation pathways may relate to uupregulation of PAI-1. This study also confirms that angiotensin IV upregulates PAI-1 expression in HCAECs.

L-158,809 and ( d-Ala 7)-angiotensin I/II (1–7) decrease PAI-1 release from human umbilical vein endothelial cells

The endothelium is a major source of plasminogen activator inhibitor-1 (PAI-1), which plays a critical role in the regulation of fibrinolysis. There are many reports on the increase in the expression of PAI-1 by angiotensin II (Ang II). In the present study, we investigated the effects of angiotensin-related substances on the release of PAI-1 from human umbilical vein endothelial cells (HUVECs). Ang II increased PAI-1 and tissue plasminogen activator (t-PA) release, while its metabolite angiotensin-(1–7) (Ang-(1–7)) amino acid fragment decreased them. Angiotensin Type 1 (AT1) receptor antagonist, l-158,809 ( l-1), and Ang-(1–7) receptor antagonist, ( d-Ala 7)-angiotensin I/II (1–7) ( d-Ala), decreased PAI-1 and t-PA release; angiotensin Type 2 (AT2) antagonist, PD123,319 (PD), however, did not have any effects on the release of PAI-1 and t-PA. The addition of the equal concentration or 10-times-higher concentration of l-1 to Ang II did not change PAI-1 release compared to that by Ang II. Although Ang-(1–7) and l-1 decreased PAI-1 release, there were no additional effects on the decrease of the amounts of PAI-1 by the mixture of Ang-(1–7) and the equal concentration or 10-times-higher concentration of l-1 compared to those by Ang-(1–7). The equal concentration of d-Ala to Ang II did not change the amounts of PAI-1, but the addition of the 10-times-higher concentration of d-Ala to Ang II resulted in significant decrease of the amounts of PAI-1 compared to those by Ang II. The addition of equal concentration or 10-times-higher concentration of d-Ala to Ang-(1–7) showed the significant decrease of the amounts of PAI-1 compared to those by Ang-(1–7). In conclusion, l-158,809 and ( d-Ala 7)-angiotensin I/III (1–7) may be used as profibrinolytic drugs.

Impact of Diabetes-Associated Lipoproteins on Generation of Fibrinolytic Regulators from Vascular Endothelial Cells

Increased thrombotic tendency and decreased fibrinolytic activity have been frequently found in patients with diabetes mellitus (DM). Previous studies by our group indicated that nonenzymatically glycated low density lipoprotein (LDL) increased plasminogen activator inhibitor-1 (PAI-1) production and decreased the generation of tissue plasminogen activator (tPA) from cultured human umbilical vein endothelial cells (HUVEC). The present study demonstrates that plasma levels of PAI-1 or PAI-1/tPA were significantly increased in patients with type 1 (n = 10) and type 2 DM (n = 14) compared with those in healthy controls (n = 10; P < 0.05 or 0.01). LDL from patients with type 1 or type 2 DM, and very low density lipoprotein (VLDL) from patients with type 2 DM induced significantly greater increases in the release of PAI-1 and more profound reduction in tPA generation from HUVEC compared with corresponding lipoproteins from healthy controls (P < 0.05 or 0.01). HDL from diabetic patients did not significantly alter the generation of PAI-1 or tPA from endothelial cells (EC) compared with HDL from controls. Comparable effects of lipoproteins from DM patients on the generation of PAI-1 and tPA were found in human coronary artery EC. LDL and VLDL from patients with type 2 DM enhanced the activation of PAI-1 promoter (-1528/+55)/luciferase reporter gene transiently transfected in HUVEC (P < 0.01). The results of the present study suggest that LDL and VLDL from patients with DM reduce the generation of tPA and increase PAI-1 production through the activation of the PAI-1 promoter in vascular EC.

Erythropoietin and granulocyte colony-stimulating factor increase plasminogen activator inhibitor-1 release in HUVEC culture

Recombinant human erythropoietin (rHuEPO) is considered to be a mitogenic and chemotactic agent in cultured endothelial cells (ECs). The effect of recombinant granulocyte–macrophage (rGM-CSF) and granulocyte (rG-CSF) colony-stimulating factors on the proliferation of human umbilical vein endothelial cells (HUVECs) has been controversial. Methods: HUVEC proliferation and the release of endothelial markers in HUVEC culture stimulated by rHuEPO, rGM-CSF and G-CSF were measured. Results: We found the dose-dependent stimulating effect of rHuEPO and rGM-CSF on HUVEC proliferation, but we did not achieve this with rG-CSF. rHuEPO like rG-CSF was an effective agent in stimulating the plasminogen activator inhibitor (PAI)-1 release from HUVECs to a cultured medium, while rGM-CSF failed. Conclusion: We suggest that rHuEPO showed prothrombotic changes in HUVEC culture. Our results support the idea of suspected rHuEPO direct prothrombotic role in haemodialysed patients treated with rHuEPO.

Deficient release of plasminogen activator inhibitor-1 from astrocytes triggers apoptosis in neuronal cells

Plasminogen activator inhibitor-1 (PAI-1) plays an important role in the processes of peripheral tissue remodeling and fibrinolysis through the regulation of plasminogen activation. We found that cultured human astrocytes efficiently released PAI-1, and that both mRNA expression and protein release of PAI-1 were suppressed by pretreatment of the cells with daunorubicin. To examine the role of PAI-1 in the nervous system, neuronally differentiated PC-12 cells (PC-12 neurons) were maintained in a PAI-1-deficient culture medium derived from daunorubicin-pretreated astrocytes. The deficiency of PAI-1 in the medium caused a significant reduction in Bcl-2 and Bcl-X L mRNAs and an increase in Bcl-X S and Bax mRNAs in PC-12 neurons at 3 h. The changes in balance between mRNA expressions of the anti- and pro-apoptotic Bcl-2 family proteins caused caspase-3 activation following the release of cytochrome c from mitochondria. Apoptotic morphological change and DNA fragmentation were also observed in the neuronal cells at 24 h. Addition of exogenous PAI-1 protein to the inhibitor-deficient medium blocked the apoptotic changes in PC-12 neurons. However, addition of PAI-1 antibodies to control medium caused similar apoptotic changes in PC-12 neurons. During the apoptotic processes, plasminogen activator (PA) activity in the PAI-1-deficient medium was as low as the control level. The present data suggest that PAI-1 has physiological functions other than its role as PA inhibitor for the survival of neurons.

Effect of angiotensin peptides on PAI-1 expression and production in human adipocytes.

Angiotensin (Ang) II is the active component of the renin-angiotensin-system (RAS), but its degradation products have also been shown to exhibit biological activity. This system, which mainly controls blood pressure and electrolyte homeostasis, was recently found to be completely expressed in human adipose tissue. The major determinant in the fibrinolytic system is the plasminogen activator inhibitor-1 (PAI-1). Both PAI-1 and components of the RAS are over-expressed in the obese state. We have recently shown that Ang II is able to induce PAI-1 expression and release via the AT1-receptor in human fat cells in primary culture, and have provided the first evidence that two metabolites, Ang III and Ang IV, may have a similar stimulatory effect on PAI-1 release. We have now performed additional experiments to further characterize the role of the angiotensin peptides in the production of PAI-1. Ang III and Ang IV showed a time- and dose-dependent stimulation of PAI-1 protein release. Concomitantly, mRNA-levels were markedly elevated. Using specific receptor blockers, all angiotensin peptides seem to induce PAI-1 expression via the angiotensin receptor subtype 1. However, components of the renin-angiotensin-system seem to play an important role in the control of fibrinolysis in adipose tissue. We conclude that PAI-1 production by adipose tissue may contribute to the elevated thromboembolic risk in obesity.

Daunorubicin attenuates tumor necrosis factor-α-induced biosynthesis of plasminogen activator inhibitor-1 in human umbilical vein endothelial cells

The anthracycline antibiotic daunorubicin is reported to induce apoptosis in cells by triggering ceramide generation through de novo synthesis or sphingomyelin hydrolysis. Treatment of human umbilical vein endothelial cells (HUVEC) with daunorubicin markedly decreased the mRNA expression and protein release of plasminogen activator inhibitor-1 (PAI-1). This cellular event was accompanied by a significant increase in the total ceramide content in HUVEC. On the other hand, tumor necrosis factor (TNF)-α treatment of HUVEC led to an increase in both PAI-1 mRNA expression and protein release, and an enhancement of total ceramide content was also observed. The stimulating effect of TNF-α on PAI-1 synthesis was attenuated by the pretreatment of HUVEC with daunorubicin. Interestingly, the daunorubicin-induced increase in ceramide content was blocked by addition of the potent ceramide synthase inhibitor fumonisin B 1, while the TNF-α-induced ceramide increase was not affected by this drug. Fumonisin B 1 treatment restored the daunorubicin-induced decrease in PAI-1 release to approximately 70% of the control, but did not affect the TNF-α-induced increase in PAI-1 release. Thus, these data imply the possibility that the subcellular topology of ceramide production determines its lipid mediator function in the regulation of PAI-1 synthesis in HUVEC, because both TNF-α and daunorubicin could increase the ceramide levels.

Release of PAI-1 by Human Preadipocytes and Adipocytes Independent of Insulin and IGF-1

The effect of insulin and insulin-like growth factor-1 (IGF-1) on plasminogen activator inhibitor-1 (PAI-1) release from primary cultures of human preadipocytes and adipocytes has been investigated. Initial experiments measuring basal PAI-1 release (ng/ml) indicated variability between individual cultures. Using a novel technique for adipocyte quantitation, additional experiments were performed to determine PAI-1 release per cell, indicating a significant reduction with differentiation. Insulin and IGF-1 over a range of concentrations had no effect on PAI-1 release, and RT-PCR of PAI-1 mRNA following treatment with insulin and IGF-1 also indicated similar expression between treatments. The cultures did exhibit insulin-stimulated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and leptin synthesis following differentiation to the adipocyte phenotype. This is the first report of PAI-1 secretion by primary cultures of human preadipocytes and adipocytes, indicating PAI-1 release independent of insulin and IGF-1 and implicating other factors in the elevated plasma PAI-1 observed with insulin resistance.

Vascular release of plasminogen activator inhibitor-1 impairs fibrinolysis during acute arterial thrombosis in mice

The role of plasminogen activator inhibitor-1 (PAI-1) in the plasma, blood platelets, and vessel wall during acute arterial thrombus formation was investigated in gene-deficient mice. Photochemically induced thrombosis in the carotid artery was analyzed via transillumination. In comparison to thrombosis in C57BL/6J wild-type (wt) mice (113 +/- 19 x 10(6) arbitrary light units [AU] n = 15, mean +/- SEM), thrombosis in PAI-1(-/-) mice (40 +/- 10 x 10(6) AU, n = 13) was inhibited (P <.01), indicating that PAI-1 controls fibrinolysis during thrombus formation. Systemic administration of murine PAI-1 into PAI-1(-/-) mice led to a full recovery of thrombotic response. Occurrence of fibrinolytic activity was confirmed in alpha(2)-antiplasmin (alpha(2)-AP)-deficient mice. The sizes of thrombi developing in wt mice, in alpha(2)-AP(+/-) and alpha(2)-AP(-/-) mice were 102 +/- 35, 65 +/- 8.1, and 13 +/- 6.1 x 10(6) AU, respectively (n = 6 each) (P <.05), compatible with functional plasmin inhibition by alpha(2)-AP. In contrast, thrombi in wt mice, t-PA(-/-) and u-PA(-/-) mice were comparable, substantiating efficient inhibition of fibrinolysis by the combined PAI-1/alpha(2)-AP action. Platelet depletion and reconstitution confirmed a normal thrombotic response in wt mice, reconstituted with PAI-1(-/-) platelets, but weak thrombosis in PAI-1(-/-) mice reconstituted with wt platelets. Accordingly, murine (wt) PAI-1 levels in platelet lysates and releasates were 0.43 +/- 0.09 ng/10(9) platelets and plasma concentrations equaled 0.73 +/- 0.13 ng/mL. After photochemical injury, plasma PAI-1 rose to 2.9 +/- 0.7 ng/mL (n = 9, P <.01). The plasma rise was prevented by ligating the carotid artery. Hence, during acute thrombosis, fibrinolysis is efficiently prevented by plasma alpha(2)-AP, but also by vascular PAI-1, locally released into the circulation after endothelial injury.

Vascular release of plasminogen activator inhibitor-1 impairs fibrinolysis during acute arterial thrombosis in mice

AbstractThe role of plasminogen activator inhibitor-1 (PAI-1) in the plasma, blood platelets, and vessel wall during acute arterial thrombus formation was investigated in gene-deficient mice. Photochemically induced thrombosis in the carotid artery was analyzed via transillumination. In comparison to thrombosis in C57BL/6J wild-type (wt) mice (113 ± 19 × 106 arbitrary light units [AU] n = 15, mean ± SEM), thrombosis in PAI-1−/− mice (40 ± 10 × 106 AU, n = 13) was inhibited (P < .01), indicating that PAI-1 controls fibrinolysis during thrombus formation. Systemic administration of murine PAI-1 into PAI-1−/− mice led to a full recovery of thrombotic response. Occurrence of fibrinolytic activity was confirmed in α2-antiplasmin (α2-AP)–deficient mice. The sizes of thrombi developing in wt mice, in α2-AP+/− and α2-AP−/− mice were 102 ± 35, 65 ± 8.1, and 13 ± 6.1 × 106 AU, respectively (n = 6 each) (P < .05), compatible with functional plasmin inhibition by α2-AP. In contrast, thrombi in wt mice, t-PA−/− and u-PA−/−mice were comparable, substantiating efficient inhibition of fibrinolysis by the combined PAI-1/α2-AP action. Platelet depletion and reconstitution confirmed a normal thrombotic response in wt mice, reconstituted with PAI-1−/− platelets, but weak thrombosis in PAI-1−/− mice reconstituted with wt platelets. Accordingly, murine (wt) PAI-1 levels in platelet lysates and releasates were 0.43 ± 0.09 ng/109 platelets and plasma concentrations equaled 0.73 ± 0.13 ng/mL. After photochemical injury, plasma PAI-1 rose to 2.9 ± 0.7 ng/mL (n = 9, P < .01). The plasma rise was prevented by ligating the carotid artery. Hence, during acute thrombosis, fibrinolysis is efficiently prevented by plasma α2-AP, but also by vascular PAI-1, locally released into the circulation after endothelial injury.

Role of cytokines in the regulation of plasminogen activator inhibitor-1 expression and secretion in newly differentiated subcutaneous human adipocytes.

Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are characteristic for obesity and are associated with increased risk of thromboembolic complications. PAI-1 recently was reported to be expressed and secreted by human adipocytes, but little is known about regulation of PAI-1 in human adipose tissue. Therefore, we examined the effects of selected cytokines present in adipose tissue on expression and secretion of PAI-1 in in vitro, differentiated subcutaneous human adipocytes in primary culture. Transforming growth factor-beta1 (TGF-beta1) increased PAI-1 secretion in a dose- and time-dependent manner. PAI-1 protein increased by 3.2-fold and PAI-1 mRNA by 1.9-fold after a 6-hour exposure to 400 pmol/L TGF-beta1. This effect is probably mediated by TGF-beta1 type 2 and 3 receptors, which were found to be expressed in cultured human adipocytes. Moreover, TNF-alpha and interkeukin-1beta (IL-1beta) also exerted a stimulatory effect on PAI-1 release and increased PAI-1 mRNA levels. As assessed by a semiquantitative reverse transcription-polymerase chain reaction technique, TGF-beta1 mRNA is expressed by differentiation of human preadipocytes and is moderately upregulated by TNF-alpha and IL-1beta. In conclusion, our results clearly indicate that TGF-beta1 is a potent inducer of PAI-1 production in subcutaneous human adipocytes. In addition, data suggest that TNF-alpha and IL-1beta also have stimulatory effects on PAI-1 protein secretion and may contribute to the elevated PAI-1 levels observed in obesity.

Impact of antioxidants and HDL on glycated LDL-induced generation of fibrinolytic regulators from vascular endothelial cells.

Hyperglycemia and dyslipoproteinemia are biochemical markers of diabetes mellitus (DM). Elevated levels of plasminogen activator inhibitor-1 (PAI-1) with and without reduction of tissue plasminogen activator (tPA) in plasma have been frequently found in patients with DM. Our previous studies indicated that glycation enhances low density lipoprotein (LDL)-induced production of PAI-1 and further decreases tPA generation in vascular endothelial cells (ECs). The present study demonstrated that treatment with antioxidants, butylated hydroxytoluene or vitamin E, blocked native LDL- and glycated LDL-induced changes in PAI-1 and tPA generation in ECs. Native or glycated high density lipoprotein (HDL) did not significantly alter tPA generation in ECs. Glycated but not native HDL (>/=100 microg/mL) moderately increased PAI-1 release from ECs. Cotreatment with native or glycated HDL inhibited LDL-induced or glycated LDL-induced changes in PAI-1 and tPA generation in ECs. The abundance of conjugated dienes was increased in glycated or EC-modified LDL. Treatment with butylated hydroxytoluene, vitamin E, or HDL reduced the abundance of conjugated dienes in glycated or EC-modified LDL. The effects of antioxidants and HDL on LDL-induced or its glycated LDL-induced changes in the generation of PAI-1 and tPA were also found in cultured human coronary artery ECs. The findings of the present study suggest that antioxidants and HDL may attenuate native LDL- or glycated LDL-induced changes in the generation of fibrinolytic regulators from vascular ECs, which possibly results from their inhibition on the lipid peroxidation of LDL particles. Treatment with antioxidants or hypolipidemic agents potentially improves fibrinolytic activity and reduces thrombotic tendencies in patients with DM.

Oversulfated fucoidan inhibits the basic fibroblast growth factor-induced tube formation by human umbilical vein endothelial cells: its possible mechanism of action

We have previously demonstrated that chemically oversulfated fucoidan (OSF) but not native fucoidan (NF) effectively suppresses the tube structure formation by human umbilical vein endothelial cells (HUVEC) on the basement membrane preparation, Matrigel. In this study, using more defined systems where basic fibroblast growth factor (bFGF) induces the tube formation by HUVEC on collagen gel, we investigated the mechanism responsible for the inhibition of angiogenesis by OSF in vitro. Unlike NF and desulfated fucoidan (desF), OSF potently inhibited the bFGF-induced HUVEC migration and tube formation. ELISA for tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in the culture media indicated that OSF increased the bFGF-induced release of PAI-1 antigen, but not of t-PA antigen. Analyses of the binding of bFGF to HUVEC surfaces and the following protein tyrosine phosphorylation revealed that OSF could promote the cell binding and autophosphorylation of 140 and 160 kDa receptors. In heparitinase-treated HUVEC, contrarily, the bFGF binding and PAI-1 release were decreased by OSF. These results suggest that OSF is a highly sulfated unique polysaccharide that can promote the binding of bFGF to the heparan sulfate molecules required for binding to the high affinity receptors with tyrosine kinase activity. The resultant increase in PAI-1 release may play a key role for the prevention of cell migration accompanied by matrix proteolysis.

Effect of tumor necrosis factor alpha and transforming growth factor beta 1 on plasminogen activator inhibitor-1 secretion from subcutaneous and omental human fat cells in suspension culture

Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are characteristic of the obese state and may contribute to the association between obesity and cardiovascular disease. In this study, we measured the rate of secretion of PAI-1 antigen in isolated subcutaneous and omental abdominal adipocytes from severely obese and non-obese individuals and studied the effect of selected cytokines on PAI-1 release using a suspension culture technique. PAI-1 secretion was approximately 2-fold greater in isolated fat cells from the obese versus non-obese subjects. In addition, PAI-1 mRNA levels were higher in adipose tissue samples from obese versus non-obese individuals (P < .05). PAI-1 release was also approximately 2-fold greater in omental versus subcutaneous adipocytes from both obese and non-obese subjects (each P < .05). A 24-hour exposure to 1 nmol/L tumor necrosis factor alpha (TNF-alpha) slightly increased PAI-1 release from both subcutaneous and omental adipocytes (30% +/- 21% and 17% +/- 18%, respectively, nonsignificant [NS]). Transforming growth factor beta 1 (TGF-beta1) induced a significant dose-dependent increase of PAI-1 release into the medium. Exposure to 400 pmol/L TGF-beta1 of subcutaneous and omental fat cells from both obese and non-obese individuals elevated PAI-1 secretion by 2-fold. These data provide evidence that human fat cells release a substantial amount of PAI-1 in a depot-specific manner and that TGF-beta1 particularly contributes to the regulation of PAI-1 secretion.