Plasminogen activator inhibitor-1 regulation in cultured rat peritubular cells by basic fibroblast growth factor and transforming growth factor-alpha

In the present study we examined the in vitro regulation of plasminogen activator inhibitor I (PAI-1) expression in peritubular cells recovered from 20-day-old rat testes. We tested two growth factors, basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF alpha). They are synthesized by Sertoli cells, and peritubular cells exhibit the corresponding high affinity receptors. After exposure to bFGF or TGF alpha (0.1-30 ng/ml), PAI-1 messenger RNA levels, as determined by Northern hybridization analysis, increased in a dose-dependent manner. The first significant effects were noted after 2-h exposure to bFGF or TGF alpha (10 ng/ml), and PAI-1 messenger RNA levels were maximally stimulated approximately 12-fold (bFGF) and 8-fold (TGF alpha) after 4 h. The two growth factors increased the amount of immunoreactive (Western blots) and biologically active (Stachrom) PAI-1 measured in the culture medium. Actinomycin D inhibited the effects of these factors, whereas cycloheximide augmented them. Phorbol myristate acetate, an activator of protein kinase C, mimicked the effects of bFGF and TGF alpha. Interestingly, long term (24-h) pretreatment with phorbol myristate acetate resulted in a severe loss of responsiveness to bFGF or TGF alpha. Staurosporine, an inhibitor of protein kinase C, also significantly reduced the effects of bFGF and TGF alpha. Given that PAI-1 inhibits Sertoli cell plasminogen activator activity and that bFGF and TGF alpha are synthesized by Sertoli cells, these factors are likely to interact to regulate protease activity in localized regions of the seminiferous tubule.     

Up-regulation of a serine protease inhibitor in astrocytes mediates the neuroprotective activity of transforming growth factor beta1

Serine proteases play a key role in the fundamental biology of the central nervous system (CNS), and recent data suggest their involvement in the pathophysiology of neurodegenerative diseases. Little is known about the physiological regulation of these proteases in the CNS. Among the multiple growth factors present in the brain, transforming growth factor beta1 (TGF-beta1) has been described as an injury-related growth factor. However, its beneficial or deleterious role remains unclear. In the present study, we investigated the influence of TGF-beta1 in apoptosis and necrosis, two mechanisms involved in ischemic neuronal death. We show that TGF-beta1 exerts a neuroprotective role restricted to necrosis induced by N-methyl-D-aspartate. This effect is observable only in the obligatory presence of TGF-beta1-responsive astrocytes. We demonstrate that this neuroprotective activity is mediated through an up-regulation of a serine protease inhibitor (PAI-1) in astrocytes. These results underline the involvement of serine proteases and extracellular matrix components such as the PAI-1/t-PA axis in the excitotoxic cascade. Moreover, regardless of the underlying mechanisms of t-PA involvement in excitotoxic injury, our observations might warn against the use of tissular plasminogen activator as an alternative therapy for the treatment of hypoxic-ischemic injury in the brain.     

Enhanced urokinase plasminogen activation in chronic pancreatitis suggests a role in its pathogenesis

BACKGROUND & AIMS: Urokinase plasminogen activator (uPA) regulates plasmin generation from plasminogen. The aim of this study was to analyze the role of the plasminogen activator/plasmin system in chronic pancreatitis (CP). METHODS: Using Northern blot analysis, in situ hybridization, and immunohistochemistry, the expression of uPA, its receptor (uPAR), plasminogen activator inhibitor 1 (PAI-1), and transforming growth factor beta 1 (TGF-beta 1) was studied in 14 patients undergoing pancreatic resection for CP. Normal control pancreatic tissue was obtained through an organ donor program. RESULTS: Eight of 14 CP samples showed concomitant increased expression (P < 0.001) of uPA (5.2-fold), uPAR (5.9-fold), and TGF-beta 1 (8.8-fold) messenger RNA (mRNA) compared with normal controls. PAI-1 mRNA expression was increased (6.5-fold; P < 0.001) in all CP samples. By in situ hybridization, moderate to strong mRNA staining of all four factors was present in acinar cells, some ductal cells, and areas with ductal metaplasia in CP samples. A similar staining pattern was found by immunohistochemistry. Intense mRNA and immunostaining for all of these factors in CP samples was associated with a higher degree of pancreatic damage. CONCLUSIONS: uPA and its receptor may contribute to the lytic damage observed in CP by plasmin generation. Similarly, increased amounts of plasmin may activate latent TGF-beta, thereby leading to the accumulation of fibrotic tissue.     

The significance of colocalization of plasminogen activator inhibitor-1 and vitronectin in hepatic fibrosis

BACKGROUND: We examined the relationships among vitronectin (VN), plasminogen activator inhibitor-1 (PAI-1), and transforming growth factor beta 1 (TGF-beta 1) in liver diseases to evaluate the presence of plasmin cascade in human hepatic fibrosis. METHODS: Blood and liver tissues were obtained from 57 patients with liver disease. Plasma VN, PAI-1 antigen, and PAI-1 activity levels were evaluated. Biopsied liver specimens were observed by light and electron microscopy after immunohistochemical staining. Morphometric analysis was performed on these specimens. RESULTS: Plasma VN and PAI-1 activity levels decreased significantly with the progression of hepatic fibrosis and were particularly marked in the liver cirrhosis group. Plasma PAI-1 antigen level increased significantly. The immunolocalization of the active form of TGF-beta became more intense with the progression of hepatic fibrosis, whereas that of the dual-stained positive areas of PAI-1 and VN (PAI-1.VN) decreased. There was a positive correlation between TGF-beta and PAI-1, whereas there was a negative correlation between TGF-beta and PAI-1.VN. Immunoelectron microscopy showed the localization of PAI-1-VN in the extracellular space around the sinusoidal cells or surface of aggregating platelets, TGF-beta mainly in Ito cells, and VN in hepatocytes near the focal necrotic area or fibrous septa. CONCLUSIONS: These findings suggest that VN and PAI-1 are related to the active form of TGF-beta and that it is possible that the plasmin cascade is present in the human liver.     

Enhanced expression of PAI-1 in visceral fat: possible contributor to vascular disease in obesity

The presence of obesity increases the risk of thrombotic vascular diseases. The role of fat accumulation and its effect on plasminogen activator inhibitor-1 (PAI-1) levels was investigated in humans and animals. Plasma PAI-1 levels were closely correlated with visceral fat area but not with subcutaneous fat area in human subjects. PAI-1 mRNA was detected in both types of fat tissue in obese rats but increased only in visceral fat during the development of obesity. These data suggest that an enhanced expression of the PAI-1 gene in visceral fat may increase plasma levels and may have a role in the development of vascular disease in visceral obesity.     

Induction of plasminogen activator inhibitor type 1 and type 1 collagen expression in rat cardiac microvascular endothelial cells by interleukin-1 and its dependence on oxygen-centered free radicals

BACKGROUND: Ischemia with or without reperfusion induces the release of diverse products from monocytes, including cytokines such as interleukin-1 (IL-1). To determine whether these phenomena modulate fibrinolysis and potentially exacerbate impairment of the macrocirculation, microcirculation, or both, we characterized the effects of IL-1 on the expression of fibrinolytic system and matrix proteins in rat cardiac microvascular endothelial cells (CMECs). METHODS AND RESULTS: Confluent CMECs were exposed to IL-1 in serum-free medium for 24 hours, and cell-conditioned medium was assayed for plasminogen activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of plasminogen activators, and for type 1 collagen with Western blotting. IL-1 (2 ng/mL) specifically increased the accumulation of PAI-1 (4.4 +/- 0.6-fold; mean +/- SD; n = 9) without affecting tissue plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA) levels, which remained unchanged. IL-1 increased the accumulation of collagen in conditioned media by 3.5 +/- 0.7-fold (n = 6). Conversely, the accumulation of both PAI-1 and collagen induced by IL-1 was inhibited with an IL-1 receptor antagonist (200 ng/mL; n = 6) and with cycloheximide (10 micrograms/mL; n = 6), implying that protein synthesis was a requirement for the effect. To determine whether the IL-1 effect was mediated by induction of oxygen-centered free radical production, known to be induced by IL-1, we exposed the cells to the hydroxyl radical scavenger tetramethylthiourea (10 mmol/L) and observed abolition of the IL-1-induced increase in the expression of PAI-1 and collagen (n = 6). Conversely, superoxides (generated with 10 mU/mL xanthine oxidase plus 0.6 mmol/L hypoxanthine, and 100 mumol/L hydrogen peroxide) induced the accumulation of PAI-1 and collagen (n = 6). IL-1 (1 microgram/kg body wt) and lipopolysaccharide (50 micrograms/kg body wt) administered in vivo increased PAI-1 protein in rat hearts as detected with Western blotting and PAI-1 immunostaining of rat heart microvessels, indicating the effects delineated in vitro were paralleled by effects in vivo. CONCLUSIONS: These results indicate that IL-1-induced oxygen-centered free radicals stimulate elaboration of PAI-1 and collagen by CMECs. Accordingly, microvascularly mediated inhibition of fibrinolysis may predispose to the persistence of microvascular thrombi, thereby contributing to impaired microcirculatory function, the no-reflow phenomenon, and cardiac dysfunction after ischemia and reperfusion.     

PAF-antagonists with lipid structure. 6. Alkylpropandiol lipids with acyl- and ether- structures at the C-3 position and heterocyclic head groups; synthesis, characterization and structure-activity relationship

OBJECTIVE: To investigate whether nonenzymatic glycated end products (AGEs) have effects on the expression and bioactivity of plasminogen activator inhibitor-1 (PAI-1), one of the seripin proteinases, which lead to extracellular matrix (ECM) degradation in cultured human mesangial cells. METHODS: Human mesangial cells (HMC) were cultured. Cell proliferation, fibronectin production, mRNA expression and bioactivity of PAI-1 were determined after exposure to AGE-BSA for 24 hours and 48 hours in vitro. RESULTS: HMC stimulated by AGE-BSA exhibited inhibition in HMC proliferation, increase in fibronectin production, and PAI-1 bioactivity. These changes were pronounced with prolongation of experimental time. PAI-1 mRNA expression increased significantly at 24 hr (0.45% +/- 0.06% vs 0.65% +/- 0.08%, P < 0.05), however more marked increase of PAI-1 mRNA expression at 48 hr (0.51 +/- 0.08% vs 0.92 +/- 0.10%, P < 0.01). CONCLUSIONS: Increase of mRNA expression and bioactivity of PAI-1 induced by AGEs decreased ECM degradation and play an important role in the pathogenesis of ECM accumulation and glomerulosclerosis.     

Long term changes in brain cholinergic markers and nerve growth factor levels after partial immunolesion

The pathophysiology of central nervous system (CNS) inflammatory disease is dependent, in part, on leukocyte recruitment across the blood-brain barrier. The expression of cytokines and chemokines by astrocytes may contribute to this process. Astrocytes express monocyte chemoattractant protein-1 (MCP-1), an activator of monocytes and a chemoattractant for monocytes and activated T cells. We examined the regulation of MCP-1 expression in human fetal astrocytes following cytokine treatment in the presence and absence of transforming growth factor beta (TGF-beta). TGF-beta, TNFalpha and IL-1beta, but not IFNgamma, induced MCP-1 mRNA and protein. TGF-beta, in cotreatment with TNFalpha caused an additive increase in MCP-1 mRNA, but not protein. In combination with IFNgamma, TGF-beta significantly increased MCP-1 mRNA and protein, as compared to either untreated, TGF-beta- or IFNgamma-treated astrocytes. However, TGF-gamma in cotreatment with IL-1beta decreased MCP-1 mRNA and protein, as compared to IL-1beta alone. Treatment of astrocytes with TGF-beta prior to TNFalpha, IFNgamma or IL-1beta treatment significantly increased MCP-1 expression. The kinetics of cytokine expression in the CNS may differentially regulate astrocyte-derived MCP-1 expression and subsequent recruitment and activation of leukocytes.     

Novel low-molecular-weight inhibitor of PAI-1 (XR5118) promotes endogenous fibrinolysis and reduces postthrombolysis thrombus growth in rabbits

BACKGROUND: Elevated levels of plasminogen activator inhibitor 1 (PAI-1) have been associated with the occurrence of thrombotic disease, and inhibition of PAI-1 activity in vivo resulted in enhanced thrombolysis and a reduction in reocclusion. Besides monoclonal antibodies and peptides, no suitable agents that are able to block PAI-1 activity are available to date. The present study was designed to test the interaction between a nonantibody, nonpeptide, diketopiperazine-based inhibitor of PAI-1, XR5118, and PAI-1 and to assess the effect of XR5118 on PAI-1 activity in vitro and on in vivo thrombolysis and thrombus growth in an experimental thrombosis model in rabbits. METHODS AND RESULTS: The binding site of XR5118 on the PAI-1 molecule was studied by competitive binding experiments with mapped anti-PAI-1 monoclonal antibodies by use of surface plasmon resonance experiments. XR5118 selectively and competitively inhibited binding of the PAl-1-inhibiting monoclonal antibody CLB-2C8, indicating that binding of XR5118 to PAI-1 takes place at the area between amino acids 110 and 145 of the PAI-1 molecule, which is known to be involved with the binding of PAI-1 to tissue plasminogen activator (TPA). Incubation of plasma or platelet releasate with XR5118 resulted in a dose-dependent inhibition of PAI-1 activity. Systemic infusion of XR5118 induced a significant reduction in plasma PAI-1 activity levels from 23.7+/-4.9 to 10.9+/-3.4 IU/mL. Administration of XR5118 resulted in a significant, twofold increase in endogenous thrombolysis compared with the control. Thrombus growth in rabbits receiving both XR5118 and rTPA was significantly attenuated compared with rabbits receiving rTPA alone (13.5+/-2.7% versus 19.9+/-3.8%, respectively). CONCLUSIONS: XR5118 binds to PAI-1 and reduces plasma PAI-1 activity levels. Furthermore, XR5118 promotes endogenous thrombolysis and inhibits thrombus accretion and is the first nonpeptide compound with significant anti-PAI-1 activity in vivo in these models.     

Thrombin stimulates expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 in cultured human vascular smooth muscle cells

The effect of thrombin on the fibrinolytic potential of human vascular smooth muscle cells (SMC) in culture was studied. SMC of different origin responded to thrombin treatment with a dose and time dependent increase in tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) levels in both cell lysates and conditioned media with maximum effects achieved at 10-20 IU/ml thrombin. PAI-1 antigen levels also increased in the extracellular matrix of thrombin treated SMC. PAI-2 levels in cell lysates of such SMC were not affected by thrombin. The effect was restricted to active thrombin, since DFP-thrombin and thrombin treated with hirudin showed no increasing effect on t-PA and PAI-1 levels in SMC. Enzymatically active thrombin also caused a four-fold increase in specific PAI-1 mRNA and a three-fold increase in t-PA mRNA. Furthermore we demonstrated the presence of high and low affinity binding sites for thrombin on the surface of SMC with a KD = 4.3 x 10(-10)M and 9.0 x 10(4) sites per cell and a KD = 0.6 x 10(-8) M and 5.8 x 10(5) sites per cell respectively. Thrombin could come in contact with SMC in case of vascular injury or following gap formation between endothelial cells. Our data support the idea that besides its known proliferative effect for SMC, thrombin could also modulate their fibrinolytic system.     

Characterization of mutated transforming growth factor-beta s which possess unique biological properties

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell growth and differentiation. On the basis of the crystal structure of TGF-beta 2, we have designed and synthesized two mutant TGF-beta s, TGF-beta 1 (71 Trp) and TGF-beta 1 (delta 69-73). Although both of these molecules inhibited the growth of Mv1Lu mink lung epithelial cells and LS1034 colorectal cancer cells, which are affected equally by TGF-beta 1 and TGF-beta 2, TGF-beta 1 (delta 69-73) was much less potent than TGF-beta 1 or TGF-beta 1 (71 Trp) at inhibiting the growth of LS513 colorectal cancer cells which are growth-inhibited by TGF-beta 1 but not TGF-beta 2. Both TGF-beta 1 (71 Trp) and TGF-beta 1 (delta 69-73) increased levels of mRNAs for fibronectin and plasminogen activator inhibitor with Mv1Lu cells, whereas only TGF-beta 1 (71 Trp) and not TGF-beta 1 (delta 69-73) up-regulated the mRNA level of carcinoembryonic antigen in LS513 cells. The expression level of carcinoembryonic antigen mRNA in LS1034 cells was not altered by either wild-type or mutant TGF-beta s. Receptor labeling experiments demonstrated that TGF-beta 1 (71 Trp) bound with high affinity to the cell-surface receptors of Mv1Lu, LS1034, and LS513 cells while TGF-beta 1 (delta 69-73) bound effectively to the receptors of Mv1Lu and LS1034 cells but much less to the receptors on LS513 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dexamethasone regulates basic fibroblast growth factor, nerve growth factor and S100beta expression in cultured hippocampal astrocytes

Glucocorticoids regulate hippocampal neuron survival during fetal development, in the adult, and during aging; however, the mechanisms underlying the effects are unclear. Since astrocytes contain adrenocortical receptors and synthesize and release a wide variety of growth factors, we hypothesized that glucocorticoids may alter neuron-astrocyte interactions by regulating the expression of growth factors in hippocampal astrocytes. In this study, three growth factors, which are important for hippocampal neuron development and survival, were investigated: basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and S100beta. Enriched type I astrocyte cultures were treated with 1 microM dexamethasone (DEX), a synthetic glucocorticoid, for up to 120 h. Cells and culture medium were collected and total RNA and protein were measured at 6, 12, 24, 48, 72, 96 and 120 h after the initiation of hormone treatment. Growth factor mRNA levels were measured and quantified using solution hybridization-RNase protection assays and protein levels were quantified using ELISA methods. We report that DEX stimulates the bFGF mRNA levels over the 120-h treatment. In contrast, DEX suppresses NGF mRNA continuously over the same period of treatment. DEX induces a biphasic response in S100beta mRNA levels. In addition, some of the changes in gene expression are translated into parallel changes in protein levels of these growth factors. Our results demonstrate that dexamethasone can differentially regulate the expression of growth factors in hippocampal astrocytes in vitro. This suggests that one of the mechanisms through which glucocorticoids affect hippocampal functions may be by regulating the expression of astrocyte-derived growth factors.     

Sphingomyelinase and cell-permeable ceramide analogs increase the release of plasminogen activator inhibitor-1 from cultured endothelial cells

We investigated the effect of exogenous staphylococcal sphingomyelinase (SMase) on the release of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) from cultured human umbilical vein endothelial cells (HUVEC). Addition of SMase (2 units/ml) to the culture medium induced an approx. 15-fold increase in the extracellular level of PAI-1 antigen at 3 h. No significant increase in the level of t-PA antigen was detected. Treatment of HUVEC with SMASE (2 units/ml) for 3 h resulted in a significant decrease in the cellular sphingomyelin (SM) level, accompanied by a corresponding increase in the ceramide level. Cell-permeable ceramide analogs also enhanced the release of PAI-1 from cultured HUVEC in concentration- and time-dependent manners. A 6-fold increase in PAI-1 antigen level was observed after incubation for 3 h with 10 microM N-acetylsphingosine. Similar effect was noted as early as 2 h with 10 microM N-hexyanoylsphingosine. Addition of sphingosine failed to affect the release of PAI-1 from cultured HUVEC, indicating that the effects of ceramide analogs were independent of sphingosine generation. Pretreatment with cycloheximide or actinomycin D abated the response of HUVEC to N-acetylsphingosine in the increased levels of both extracellular and intracellular PAI-1. These results suggest that ceramide, generated via "SM cycle", acts as a lipid mediator of PAI-1 release from vascular endothelial cells, and may contribute to a better understanding of the pathogenesis of the PAI-1-associated thrombotic disorders.     

Angiotensin II regulates the expression of plasminogen activator inhibitor-1 in cultured endothelial cells. A potential link between the renin-angiotensin system and thrombosis

Plasminogen activator-inhibitor C-1 (PAI-1) plays a critical role in the regulation of fibrinolysis, serving as the primary inhibitor of tissue-type plasminogen activator. Elevated levels of PAI-1 are a risk factor for recurrent myocardial infarction, and locally increased PAI-1 expression has been described in atherosclerotic human arteries. Recent studies have shown that the administration of angiotensin converting enzyme inhibitors reduces the risk of recurrent myocardial infarction in selected patients. Since angiotensin II (Ang II) has been reported to induce PAI-1 production in cultured astrocytes, we have hypothesized that one mechanism that may contribute to the beneficial effect of angiotensin converting enzyme inhibitors is an effect on fibrinolytic balance. In the present study, we examined the interaction of Ang II with cultured bovine aortic endothelial cells (BAECs) and the effects of this peptide on the production of PAI-1. 125I-Ang II was found to bind to BAECs in a saturable and specific manner, with an apparent Kd of 1.4 nM and Bmax of 74 fmol per mg of protein. Exposure of BAECs to Ang II induced dose-dependent increases in PAI-1 antigen in the media and in PAI-1 mRNA levels. Induction of PAI-1 mRNA expression by Ang II was not inhibited by pretreating BAECs with either Dup 753 or [Sar1, Ile8]-Ang II, agents that are known to compete effectively for binding to the two major angiotensin receptor subtypes. These data indicate that Ang II regulates the expression of PAI-1 in cultured endothelial cells and that this response is mediated via a pharmacologically distinct form of the angiotensin receptor.     

The fat mouse: a powerful genetic model to study elevated plasminogen activator inhibitor 1 in obesity/NIDDM

Plasminogen activator inhibitor-1 is elevated in obesity and may be a risk factor for obesity/NIDDM related cardiovascular disease. In spite of this, little is known about the tissue and cellular origin of elevated PAI-1 in obesity or of the mediators and molecular mechanisms that regulate it. We have begun to systematically address these issues using genetically obese (ob/ob, db/db) mice. Plasma PAI-1 levels were 5-fold higher in obese mice compared to their lean counterparts. Subsequent RT-PCR and in situ hybridization studies suggest that the increased plasma PAI-1 originates primarily from the adipocyte in response to chronically elevated levels of tumor necrosis factor-alpha (TNF-alpha), insulin, and transforming growth factor-beta (TGF-beta). Thus, the signals and mechanisms that lead to elevated plasma PAI-1 observed in obesity are complex, and appear to involve interactions between multiple mediators and the adipose tissue itself.