Human fibroblasts downregulate plasminogen activator inhibitor type-1 in cultured human macrovascular and microvascular endothelial cells

We have previously reported that plasminogen activator inhibitor type-1 (PAI-1) expression in endothelial cells (ECs) can be modulated differently by smooth muscle cells depending on their origin. Human pulmonary artery smooth muscle cells (HPASMCs) strongly downregulated PAI-1 expression in ECs. Fibroblasts (FBs) are another cell type that could come in close contact with ECs. Therefore, it was the aim of this study to investigate whether FBs could also influence the fibrinolytic potential of ECs. As in the case of HPASMCs, PAI-1 antigen produced by human umbilical vein ECs (HUVECs) cocultured with human skin FBs (HSFBs) was significantly lower as compared with the sum of PAI-1 secreted by the respective cell types cultured separately. Not only HUVECs but also human skin microvascular ECs (HSMECs) responded in a dose-dependent way to serum-free conditioned media (CM) from HSFBs from one individual donor. Similar results were obtained when CM from HSFBs from four other individual donors were used. PAI-1 mRNA decreased in HUVECs incubated for 6 hours with HSFB-CM to 24% to 55% of control, depending on the preparation of HSFBs used. A significant PAI-1 downregulatory effect was only observed when CM from low-passage HSFBs (up to passage no. 5) was used, whereas no reduction in EC PAI-1 production was observed with CM obtained from HSFBs in passage no. 8. This PAI-1 downregulatory activity present in HSFB-CM was heat-labile and had a molecular mass of approximately 5 kD. When CM from HPASMCs was analyzed in the same way, an almost identical elution profile was found. In conclusion, our data showed that FBs can decrease the expression of PAI-1 in ECs. Such an effect could be operative during wound-healing and at other capillary sites where FBs could render ECs profibrinolytic, thereby facilitating processes requiring an increase in proteolytic activity such as EC migration and proliferation.     

Modulation by endothelin-1 of tissue plasminogen activator and plasminogen activator inhibitor-1 release from cultured human vascular endothelial cells: interaction of endothelin-1 with cytokines

The interaction of endothelin-1 (ET-1) with either interleukin-1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF alpha) on the release of tissue plasminogen activator antigen (t-PA:Ag) and plasminogen activator inhibitor-1 antigen (PAI-1:Ag) was investigated in a culture system of vascular endothelial cells derived from human umbilical vein. The t-PA:Ag release was significantly decreased by either IL-1 beta or TNF alpha; ET-1 intensified the suppressive effect of the cytokines. In contrast, PAI-1:Ag release was significantly increased by either IL-1 beta or TNF alpha; ET-1 significantly reduced the stimulatory effect of the cytokines. The data suggest that endothelial cell-mediated fibrinolysis may be modulated by ET-1.     

Production of plasminogen activator inhibitor-1 by human saphenous vein endothelial cells seeded onto endarterectomy surfaces in vitro

PURPOSE: Endothelial cells produce many biologically important factors that may be used as functional markers, including plasminogen activators and their inhibitors (PAI). PAI-1 is a common peptide with a central role in the balance of thrombosis and fibrinolysis in vivo, and its production by vascular endothelial cells has been demonstrated for many in vitro cell lines. METHODS: The basal rate of PAI-1 release from cultured human adult endothelial cells was studied in both a well-plate-seeding model and after seeding onto human endarterectomy specimens. The effect of nonspecific stimulation with thrombin on PAI-1 release was examined in well-plate cultures. PAI-1 was measured by enzyme-linked immunosorbent assay. RESULTS: Cultured human saphenous endothelial cells release PAI-1 constitutively at a steady rate, which can be increased in the short term by the addition of thrombin. CONCLUSION: After seeding onto endarterectomy specimens, seeded endothelial cells release significant amounts of PAI-1, which suggests that they retain functional integrity and may potentially influence thrombosis in vivo after seeding.     

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.     

In-vitro effect of oncostatin M on the release by endothelial cells of von Willebrand factor, tissue-type plasminogen activator and plasminogen activator inhibitor-1

Epidemiological studies have demonstrated that levels of plasma fibrinogen, von Willebrand factor (vWf), plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (tPA) are associated with the incidence of vascular disease. Since oncostatin M dramatically induces fibrinogen biosynthesis by hepatocytes and could be implicated in vascular injury leading to atherosclerosis, we have analyzed the effect of oncostatin M on PAI-1, vWf and tPA secretion by endothelial cells. A 2-h incubation of human umbilical vein endothelial cells with oncostatin M increases thrombin-induced secretion of vWf to the same extent as tumour necrosis factor-alpha or interleukin-1 (137+/-26% of control for 5 ng/ml oncostatin M, P < 0.001, n=5). The effects on tPA and PAI-1 secretion were different depending on the type of endothelial cells tested. On human umbilical vein endothelial cells, oncostatin M induced an increase in PAI-1 and a decrease in tPA secretion, which could explain the thrombogenicity of oncostatin M on large vessels. On a human microvasculature endothelial cell line, oncostatin M did not modify PAI-1 but induced an increase in tPA secretion. This observation of the effects of oncostatin M on both macro- and microvasculature could explain the increased levels of vWf, PAI-1 and tPA in the plasma of atherosclerotic subjects identified in epidemiological studies, suggesting that oncostatin M could play a key role in the development of atherosclerotic lesions.     

Production of urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor-1 (PAI-1) in human brain tumours

We investigated the role of plasminogen activators (PAs) and their inhibitor (plasminogen activator inhibitor-1, PAI-1) in human brain tumours. The amounts of urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor-1 (PAI-1), and the activity of u-PA and t-PA were determined by enzyme-linked immunosorbent assay (ELISA), and u-PA and PAI-1 were immunolocalized using monoclonal antibodies in human brain tumours and normal brain tissues. The tissues were surgically removed from 64 patients; normal brain tissue (5 cases), low-grade glioma (4 cases), high-grade glioma (17 cases), metastatic tumour (9 cases), meningioma (benign 12 cases, malignant 6 cases), acoustic schwannoma (11 cases). u-PA activity and u-PA and PAI-1 antigen levels were significantly elevated in malignant brain tumours (malignant meningiomas, high-grade gliomas, and metastatic tumours) and acoustic schwannomas but very low in benign meningiomas, low-grade gliomas and normal brain. There was no difference in t-PA antigen levels among normal and malignant tissues, however levels of t-PA activity were markedly decreased in metastastic tumours. All malignant brain tumour tissues showed positive immunostaining for u-PA and PAI-1, however, some tumour cells showed negative intensity while others showed strong intensity for these antibodies. This contrasts to the homogeneous staining pattern found in acoustic schwannoma. These findings indicate that malignancy in human brain tumours is associated with elevated levels of u-PA and PAI-1 and that an imbalance between these proteins in a micro-environment contributes (ascribes) to tumour cell invasion.     

Prognostic significance of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in primary breast cancer

The uPA-mediated pathway of plasminogen activation is central to cancer metastasis. Whether uPA and PAI-1 are related to local recurrence, metastatic spread or both is not clear. We present a retrospective study of 429 primary breast cancer patients with a median follow-up of 5.1 years, in which the levels of uPA and PAI-1 in tumour extracts were analysed by means of an enzyme-linked immunosorbent assay. The median values of uPA and PAI-1, which were used as cut-off points, were 4.5 and 11.1 ng mg(-1) protein respectively. The levels of uPA and PAI-1 were correlated with tumour size, degree of anaplasia, steroid receptor status and number of positive nodes. Patients with high content of either uPA or PAI-1 had increased risk of relapse and death. We demonstrated an independent ability of PAI-1 to predict distant metastasis (relative risk 1.7, confidence limits 1.22 and 2.46) and that neither uPA nor PAI-1 provided any information regarding local recurrence.     

Localization of vitronectin binding domain in plasminogen activator inhibitor-1

Plasminogen activator inhibitor type 1 (PAI-1) is the rapid physiologic inhibitor of tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). In plasma and the extracellular matrix, PAI-1 is associated with the adhesive glycoprotein vitronectin. In order to characterize the PAI-1 structural domain responsible for binding to vitronectin, the segment of the PAI-1 cDNA encoding amino acids 13-147 (nucleotides 248-650) was randomly mutagenized and subcloned into a bacterial expression vector containing the mature PAI-1 coding sequence. Recombinant PAI-1 mutants were expressed in Escherichia coli and bacterial lysates assayed in duplicate for uPA inhibitory activity and vitronectin binding. Of 190 clones screened, six consistently demonstrated decreased vitronectin binding relative to uPA inhibitory activity. DNA sequence analysis of four of these clones identified 10 unique missense mutations, all located between base pairs 298 and 641, with each clone containing between one and four substitutions. Each substitution was expressed independently by site-directed mutagenesis and again analyzed for uPA inhibitory activity and vitronectin binding. Five point mutations that selectively disrupt vitronectin binding were identified. All 5 residues are located on the exterior of the PAI-1 structure. These findings appear to define a complex binding surface that bridges alpha-helices C and E to beta-strand 1A and includes amino acids 55, 109, 110, 116, and 123. These results suggest that vitronectin binding may stabilize the active conformation of PAI-1 by restricting the movement of beta-sheet A and thereby preventing insertion of the reactive center loop.     

In vitro production of plasminogen activator by human granulosa cells

Plasminogen activator (PA) production by granulosa cells has been demonstrated in several species. In the human ovary, tissue-type PA and urokinase-type PA antigens have been found in the follicular fluids, but neither PA activity nor mRNA for both enzymes was found in granulosa cells of preovulatory follicles. All of these studies were performed on granulosa cells collected from follicles immediately before ovulation, when the cells were already in the luteal phase. In the attempt to better characterize the PA/plasminogen system in the human ovary, we examined PA and PA inhibitor (PAI) production in cultures of granulosa cells obtained from normally cycling untreated women at different stages of the cycle. In addition, we analyzed granulosa-luteal cells obtained from hormonally stimulated women undergoing gamete intrafallopian tube transfer, as a model of late phase follicular development. Zymographic analysis as well as immunoprecipitation with specific antisera revealed that granulosa cells from follicles at early phases of antral stages secreted high levels of PA of the urokinase type in the medium. No free tissue-type PA activity was found in any of the examined samples. On the contrary, free PAI was undetectable in medium obtained from granulosa cell cultures, and it was abundant in granulosa-luteal cell cultures, where it was found in two forms. These data show that in the human ovary as in that of the rat, PAs and PAIs are tightly time regulated. The timing of PA production in human granulosa cells suggests a role for PA activity at early stages of follicular maturation.     

Regulation of arterial thrombolysis by plasminogen activator inhibitor-1 in mice

BACKGROUND: Platelet-rich arterial thrombi are resistant to lysis by plasminogen activators. However, the mechanisms underlying thrombolysis resistance are poorly defined. Plasminogen activator inhibitor-1 (PAI-1), which is present in plasma, platelets, and vascular endothelium, may be an important determinant of the resistance of arterial thrombi to lysis. However, in vitro studies examining the regulation of platelet-rich clot lysis by PAI-1 have yielded inconsistent results. METHODS AND RESULTS: We developed a murine arterial injury model and applied it to wild-type (PAI-1 [+/+]) and PAI-1-deficient (PAI-1 [-/-]) animals. FeCl3 was used to induce carotid artery thrombosis. Thrombi consisted predominantly of dense platelet aggregates, consistent with the histology of thrombi in large-animal arterial injury models and human acute coronary syndromes. To examine the role of PAI-1 in regulating endogenous clearance of platelet-rich arterial thrombi, thrombi were induced in 22 PAI-1 (+/+) mice 14 PAI-1 (-/-) mice. Twenty-four hours later, the amount of residual thrombus was determined by histological analysis of multiple transverse sections of each artery. Residual thrombus was detected in 55 of 85 sections (64.7%) obtained from PAI-1 (+/+) mice compared with 19 of 56 sections (33.9%) from PAI-1 (-/-) mice (P=.009). Computer-assisted planimetry analysis revealed that mean thrombus cross-sectional area was 0.033+/-0.0271 mm2 in PAI-1 (+/+) mice versus 0.016+/-0.015 mm2 in PAI-1 (-/-) mice (P=.048). CONCLUSIONS: PAI-1 is an important determinant of thrombolysis at sites of arterial injury. Application of this model to other genetically altered mice should prove useful for studying the molecular determinants of arterial thrombosis and thrombolysis.     

Gene polymorphisms for plasminogen activator inhibitor-1/tissue plasminogen activator and development of allograft coronary artery disease

BACKGROUND: Impaired fibrinolytic activity has been linked to the presence and severity of allograft vasculopathy (Tx CAD). This impairment may be associated with the presence of certain fibrinolytic protein gene polymorphisms. METHODS AND RESULTS: To investigate the relation between donor-specific fibrinolytic protein genotypes and Tx CAD, we identified donor plasminogen activator inhibitor-1 (PAI-1) HindIII and tissue plasminogen activator (TPA) EcoRI restriction fragment length polymorphisms-based genotypes by Southern blot analysis in 48 recipients of cardiac allografts and correlated these genotypes with the development of CAD. No association was found between donor TPA genotypes and the presence of Tx CAD. Among the 48 patients, 17% were homozygous for the 1/1 PAI-1 genotype, 51% for the 2/2 PAI-1 genotype, and 32% for the 1/2 PAI-1 genotype. The actuarial freedom from any CAD for the recipients with each respective donor PAI-1 genotype at 12 and 24 months was 100% and 100% for the 1/1 PAI-1 genotype, 92% and 92% for the 1/2 PAI-1 genotype, and 75% and 45% for the 2/2 PAI-1 genotype (P=0.03). Recipients with a diseased 2/2 PAI-1 genotyped allograft had longer ischemic times (P=0.02) than those recipients with a Tx CAD-free allograft. CONCLUSIONS: These data suggest that recipients with a 2/2 PAI-1 genotype are at a significant risk of developing Tx CAD. This genotype may serve as a useful screening tool for predicting the future development of Tx CAD.     

Advanced glycation endproducts stimulate the MAP-kinase pathway in tubulus cell line LLC-PK1

Advanced glycation endproducts (AGEs) are suggested to play an important role in diabetic nephropathy. They induce specific cellular responses such as the release of cytokines in different cell lines. The effect of AGEs on signal transduction pathways was investigated in the renal tubulus cell line LLC-PK1. Using a serine-phosphate-specific antibody AGE-induced cellular responses associated with phosphorylation/dephosphorylation events were demonstrated. In particular, the p42MAP kinase and its downstream target, the AP-1 complex, are shown to be activated by AGE-BSA but not by BSA. In contrast, only partial phosphorylation is observed for the p70S6-kinase. Thus, AGEs appear to induce specific signal transduction pathways.     

Regulation of tissue plasminogen activator production in cultured human fetal mesangial cells

This study was designed to determine the persistence of culturable bacteria versus DNA in the presence of a middle ear effusion in a chinchilla model of otitis media. Cohorts of animals were either infected with an ampicillin-resistant Haemophilus influenzae strain or injected with a tripartite inoculum consisting of freeze-thawed Streptococcus pneumoniae; pasteurized Moraxella catarrhalis; and DNA from H influenzae. The H influenzae-infected animals displayed culture positivity and polymerase chain reaction positivity through day 35. In the chinchillas infected with the low-copy number inocula of S pneumoniae, DNA was not detectable after day 1 from the co-inoculated pasteurized M catarrhalis bacteria or the purified H influenzae DNA; however, amplifiable DNA from the live low-copy number bacteria persisted through day 21 even though they were not culture-positive past day 3. These results demonstrate that DNA, and DNA from intact but nonviable bacteria, does not persist in an amplifiable form for more than a day in the presence of an effusion; however, live bacteria, while not culturable, persist in a viable state for weeks.     

Initiation of a fibrinolytic system in hepatic resection: the roles of tissue-type plasminogen activator and plasminogen activator inhibitor-1

The factors related to the initiation of fibrinolysis, especially with regard to the tissue-type plasminogen activator (tPA) and the plasminogen activator inhibitor-1 (PAI-1), were investigated in 15 patients who underwent hepatic resection, and the findings were compared between those with normal livers and those with diseased livers. It was found that tPA increased before hepatic division, whereas PAI-1 increased after hepatic division and reached a peak immediately following the operation. Plasminogen decreased during hepatectomy, reaching its lowest point on postoperative day 1, and increasing later. Decreased levels of both plasminogen and the alpha 2-plasmin inhibitor were considered to be partly due to plasmin formation in the blood. Patients with a diseased liver tended to have higher intraoperative values of euglobulin lysis activity and higher postoperative values of plasminogen activator, but significantly lower postoperative values of alpha 2-plasmin inhibitor than those with a normal liver. The results of this study suggest that activation of the fibrinolytic system occurs both during hepatectomy and in the early postoperative period, and that patients with a diseased liver are prone to develop hyperfibrinolysis during hepatectomy. Moreover, the increased levels of both tPA and PAI-1 can serve as one of the most sensitive markers for the vital reaction against surgical stress.     

Advanced glycation endproducts stimulate mitogen-activated protein kinase and proliferation in rabbit vascular smooth muscle cells

Two patients with plasma cell leukemia (PCL) with a t(11;14)(q13;q32) translocation are reported. Case 1 is a 64-year-old woman diagnosed as having primary PCL (IgA/lambda, Stage III) with high serum LDH and beta 2-microglobulin (beta 2MG) levels. She was treated with combination chemotherapy but died of gastrointestinal bleeding on the 45th hospital day. Case 2 is a 52-year-old man, initially diagnosed with multiple myeloma (IgG/kappa, Stage III) in August 1993. Relapse several months after primary chemotherapy was characterized by a rapid increase in plasma cells in peripheral blood, high serum LDH and beta 2MG levels, and resistance to further chemotherapy. Both cases showed complex karyotypic abnormalities including t(11;14), and Northern analysis revealed overexpression of the PRAD1/ cyclin D1 gene. The PRAD1 gene is found on chromosome band 11q13 and encodes cyclin D1. Cyclin D1 plays an important role in control of the cell cycle, and overexpression of PRAD1/cyclin D1 may be involved in disease progression in these cases.     

A pathological role of increased expression of plasminogen activator inhibitor-1 in human or animal disorders

Plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of plasminogen activators and may be the principal regulator of plasminogen activation in vivo. Abnormal expression of PAI-1 has been reported in various types of human disorders and in animal models for the diseases in relation to thrombosis. For example, plasma PAI-1 activity was elevated in patients with endotoxemia, and a dramatic induction of PAI-1 mRNA was observed in tissues of endotoxin-treated animals, resulting in tissue microthrombosis. It has also been demonstrated that PAI-1 expression levels are increased in the kidneys of mice with glomerulonephritis, in the adipose tissue of obese subjects or mice, and in human atherosclerotic arteries. This PAI-1 induction may be relevant to pathological processes in these diseases because PAI-1 not only regulated fibrin dissolution in vivo but also inhibited degradation of extracellular matrix by reducing plasmin generation. The responsible cells for abnormal expression of PAI-1 have been identified in each tissue under pathological conditions and PAI-1 synthesis appears to be regulated in a tissue-specific manner. These observations suggest that PAI-1 could play an important role in the progression of tissue pathologies in a variety of human diseases by controlling the rate of plasmin formation.     

Dynamic structural and functional relationships in recombinant plasminogen activator inhibitor-1 (rPAI-1)

The conformational characteristics of active, latent, and denatured recombinant plasminogen activator inhibitor-1 (rPAI-1) were compared using UV spectroscopy, spectrofluorimetry and circular dichroism (CD) techniques. The UV absorbance wavelength maxima in all preparations approximated 280 nm, while the extinction coefficients of active and latent rPAI-1 differed by up to 60%. When incubated at 37 degrees C, the A280 of latent rPAI-1 was quite stable while the A280 of active rPAI-1 spontaneously increased, eventually approximating that of latent rPAI-1. Alkali difference spectroscopy yielded markedly divergent titration patterns for active and latent rPAI-1, suggesting that the tyrosine residues present in active and latent rPAI-1 differ in terms of solvent exposure. At an excitation wavelength of 280 nm, active rPAI-1 exhibited the greatest relative fluorescence quantum yield. The relative fluorescence of latent and denatured rPAI-1 were less than that of active PAI-1, and the emission maxima of both species were slightly red-shifted in comparison to that of active rPAI-1, suggesting that at least one of the four tryptophan residues present in rPAI-1 is less exposed to the aqueous environment in the active form of the molecule. In contrast, the derived secondary structures based on CD of active and latent rPAI-1 were nearly identical, with both moieties exhibiting approx. 40% alpha-helix and 15% beta-sheet. Taken together, these spectroscopic data provide evidence supporting the hypothesis that active and latent PAI-1 differ in terms of their tertiary conformation and aromatic residue exposure, while their secondary structures appear generally comparable. Furthermore, denaturant-induced reactivation of latent rPAI-1 produces a partially active rPAI-1 with spectroscopic properties similar to that of latent rPAI-1, suggesting that denatured rPAI-1 more closely resembles the latent rPAI-1 conformation after refolding. The spontaneous spectroscopic changes observed in rPAI-1 may reflect conformational transitions that are critical to the regulation of endogenous PAI-1 activity.     

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.