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 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.     

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.     

Plasminogen activator inhibitor-1 (PAI-1) 4G/5G promoter polymorphism and levels in subjects with cerebrovascular disease

The exact role of the fibrinolytic system in the pathogenesis of stroke remains to be established. Elevated circulating levels of plasminogen activator inhibitor-1, the principle inactivator of the fibrinolytic system, have been related to the development of myocardial infarction. There is evidence that a polymorphism in the promoter region of the PAI-1 gene is associated with circulating PAI-1 levels. We studied a common single nucleotide insertion/deletion (4G/5G) polymorphism by PCR in 558 patients with stroke, the pathological type of which was established by cranial computed tomography, and in 172 controls. 4G/5G genotype and PAI-1 activity were investigated in relation to 1) stroke type and 2) mortality occurring within four weeks, three months and six months of the stroke. No difference in genotype frequency was observed when all cases of stroke were compared with controls nor between the clinically determined subtypes of cerebral infarction. PAI-1 activity was significantly higher in patients with stroke (n = 245) both at presentation (11.6 U/ml) and after three months (11.8 U/ml), in paired samples, than in control subjects (8.8 U/ml, p < 0.0001). Thirty-seven (6.2%), 86 (14.5%) and 122 (20.5%) patients had died within one, three and six months of admission, respectively. PAI-1 activity was independently associated with all-cause mortality at one and three months after stroke (p = 0.02 and p = 0.03 respectively), but not after six months. In this population the 4G/5G promoter polymorphism is not associated with an increased risk of stroke. PAI-1 levels were elevated at the time of acute stroke which persisted after three months. PAI-1 level but not genotype was associated with early mortality following stroke.     

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.     

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.     

Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition

PURPOSE: In a rabbit model, transposition of a muscle pedicle flap to an ischemic hind limb has been shown to result in the development of new blood vessels that connect the arterial circulation of the flap to the circulation of the limb. The hypothesis that exogenous recombinant basic fibroblast growth factor (bFGF) would enhance the development of this new blood supply was examined and the regulation of bFGF in this process was investigated. METHODS: The right common iliac artery was ligated in 12 male New Zealand white rabbits. An abdominal wall muscle flap based on the left inferior epigastric artery was transposed to the right thigh. bFGF in phosphate-buffered saline (PBS) at 3 ng/h (n = 6), or PBS alone (n = 6), was infused for 7 days via mini-osmotic pumps with an infusion catheter positioned at the flap-muscle interface. The flap-muscle interface was immunostained with anti-alpha-actin antibody to determine blood vessel density (number of vessels/mm) and with anti-bFGF antibody to evaluate bFGF distribution. RNA was isolated from these sections, and polymerase chain reaction (PCR) was used to examine endogenous bFGF messenger RNA (mRNA) expression. RESULTS: Blood vessel density was significantly increased in animals receiving exogenous bFGF (22. 0 +/- 10.6 vessels/mm vs. 10.7 +/- 8.8 vessels/mm, P =.009). In the controls, neovessels were arranged in clusters with endogenous bFGF concentrated around these clusters. In bFGF-treated animals, vessels were diffusely scattered throughout the flap-limb interface, corresponding to the distribution pattern of infused bFGF. There was no difference in bFGF mRNA expression between the control and the bFGF-treated groups. CONCLUSION: Exogenous bFGF infusion significantly augmented new blood vessel development at the flap-limb interface. Endogenous bFGF was up-regulated around the newly developed microvessels in control animals, and vessel growth correlated with the diffuse distribution of exogenous bFGF, implicating bFGF as an important factor in angiogenesis. Exogenous bFGF did not affect bFGF mRNA expression, suggesting that the regulation of bFGF is not under autocrine control.     

Regulation of tissue-type plasminogen activator (tPA) and type-1 plasminogen activator inhibitor (PAI-1) gene expression in rat hepatocytes in primary culture

We have performed a comparative study on tPA and PAI-1 mRNA expression in primary cultures of rat hepatocytes and elucidated the possible regulation of these factors by certain hormonal stimulation. The tPA mRNA increased 2- to 4-fold in the presence of cholera toxin (CT), dibutyryl cyclic AMP (dbcAMP), or 3-isobutyl-1-methyl xanthine (IBMX), but slightly decreased in the presence of dexamethasone. The tPA activity was also changed by these agents in a similar fashion. On the contrary, PAI-1 mRNA decreased with CT, dbcAMP, or IBMX, but increased transiently with dexamethasone. From results obtained with cycloheximide, ongoing protein synthesis was judged to be required for both PAI-1 induction with dexamethasone and PAI-1 suppression with IBMX, but not for the tPA induction with IBMX. Dexamethasone exerted opposite regulatory effects on the tPA mRNA expression depending on its concentration: at 10(-8) to 10(-6) M, it suppressed the expression; whereas at 10(-10) M, it elevated the expression.     

Lipoprotein(a) enhances the expression of intercellular adhesion molecule-1 in cultured human umbilical vein endothelial cells

Quite a substantial number of human disorders have been associated with a primary or a secondary impairment of one or several of the dopaminergic pathways. Among disorders associated with a primary impairment of dopaminergic transmission are Parkinson's disease, striatonigral degeneration, progressive supranuclear palsy, and possibly schizophrenia. Diseases of secondary dopamine dysfunction are chiefly represented by Huntington's disease in which dopaminergic transmission is being interrupted by progressive loss of the striatal neurons bearing the postsynaptic D1- and D2-dopamine receptors. Central dopaminergic systems have anatomical as well as organizational properties that render them unique by comparison to other neurotransmission systems, making them able to play a pivotal role in the modulation of various important brain functions such as locomotor activity, attention, and some cognitive abilities. These properties of dopamine neurons have obviously several implications in the clinical expression of human disorders involving dopamine neuron dysfunction. In addition, they can greatly influence the clinical/behavioral consequences of experimental lesions in animal models of dopamine dysfunctions.     

Human plasminogen activator inhibitor-1 (PAI-1) deficiency: characterization of a large kindred with a null mutation in the PAI-1 gene

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue- and urokinase-type plasminogen activators, is considered a critical regulator of the fibrinolytic system. We previously reported a child with abnormal bleeding and complete PAI-1 deficiency caused by a frame-shift mutation in exon 4 of the PAI-1 gene. The purpose of this study was to provide genetic and clinical data on the extended pedigree of the original proband to better define the phenotype associated with PAI-1 deficiency. Allele-specific oligonucleotide hybridization was used to genotype individuals, and serum PAI-1 antigen was measured by enzyme-linked immunosorbent assay. By this approach we have identified 19 individuals who are heterozygous for the PAI-1 null allele and 7 homozygous individuals with complete PAI-1 deficiency. Clinical manifestations of PAI-1 deficiency were restricted to abnormal bleeding, which was observed only after trauma or surgery in homozygous affected individuals. A spectrum of bleeding patterns was observed, including intracranial and joint bleeding after mild trauma, delayed surgical bleeding, severe menstrual bleeding, and frequent bruising. Fibrinolysis inhibitors, including epsilon-aminocaproic acid and tranexamic acid, were effective in treating and preventing bleeding episodes. Other than abnormal bleeding, no significant developmental or other abnormalities were observed in homozygous PAI-1-deficient individuals. Heterozygous PAI-1 deficiency was not associated with abnormal bleeding, even after trauma or surgery. These observations define the clinical spectrum of PAI-1 deficiency and provide additional evidence to support the hypothesis that the primary function of plasminogen activator inhibitor-1 in vivo is to regulate vascular fibrinolysis.     

Involvement of calcium and G proteins in the acute release of tissue-type plasminogen activator and von Willebrand factor from cultured human endothelial cells

In this study, we investigated the role of Ca2+ and G proteins in thrombin-induced acute release (regulated secretion) of tissue-type plasminogen activator (TPA) and von Willebrand factor (vWF), using a previously described system of primary human umbilical vein endothelial cells (HUVECs). The acute release of TPA and vWF, as induced by alpha-thrombin, was almost zero after chelation of Ca2+i, showing that an increase in [Ca2+]i was required. It did not matter whether the increase in [Ca2+]i came from an intracellular or extracellular Ca2+ source. Thrombin-induced release of TPA and vWF already started at low [Ca2+]i, around 100 nmol/L. Half-maximal release was found at a [Ca2+]i, of 261 nmol/L for TPA and at 222 nmol/L for vWF. The Ca2+ signal was transduced to calmodulin, as calmodulin inhibitors inhibited TPA and vWF release. The Ca2+ ionophore ionomycin dose dependently released vWF; half-maximal vWF release occurred at a [Ca2+]i of 311 nmol/L. In contrast, no TPA release was found at all below a [Ca2+]i of 500 nmol/L. Thus, below 500 nmol/L [Ca2+]i, an increase in [Ca2+]i alone was sufficient to induce vWF release but not sufficient to induce TPA release. Protein kinase C did not appear to be involved in TPA or vWF release, as neither an activator nor an inhibitor of protein kinase C significantly influenced release. Inhibition of phospholipase A2 also did not reduce thrombin-induced TPA and vWF release. The involvement of G proteins was studied by using both saponin-permeabilized and intact cells. GDP-beta-S, which inhibits heterotrimeric and small G proteins, significantly inhibited thrombin-induced vWF and TPA release from permeabilized cells. AlF-4, which activates heterotrimeric G proteins, induced TPA and vWF release in both intact and permeabilized HUVECs. Preincubation of HUVECs with pertussis toxin significantly inhibited thrombin-induced vWF release, due to inhibition of thrombin-induced Ca2+ influx. Pertussis toxin did not affect ionomycin-induced release. The inhibitory effect of pertussis toxin was less obvious in thrombin-induced TPA release, because it was counterbalanced by a positive effect of the toxin on TPA release. Thus, both inhibitory and stimulatory (pertussis toxin-sensitive) G proteins were involved in TPA release. Therefore, thrombin-induced acute release of TPA and vWF differed in two respects. First, below a [Ca2+]i of 500 nmol/L, an increase in Ca2+ was sufficient for vWF release but not for TPA release. Second, pertussis toxin-sensitive G proteins were differentially involved in acute TPA and vWF release.     

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.     

The 4G/5G genetic polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene is associated with differences in plasma PAI-1 activity but not with risk of myocardial infarction in the ECTIM study. Etude CasTemoins de I'nfarctus du Mycocarde

We have investigated the interrelationships of plasma PAI-1 activity, the PAI-1 4G/5G polymorphism and risk of myocardial infarction (MI) in the ECTIM study, a case-control study of MI based in Belfast, Lille, Strasbourg and Toulouse. Mean PAI-1 levels in cases were similar across all centres but in controls, levels in the French centres were significantly higher. Only in Belfast were PAIl1 levels higher in cases (11.7 AU/ml) than controls (10.5 AU/ml). The PAI-1 4G allele frequency was similar in cases and controls (0.55 and 0.54). In all groups, 4G homozygotes had the highest mean plasma PAI-1 level (4G4G vs 5G5G; cases overall: 14.2 vs 12.1AU/ml; controls overall: 15.0 vs 12.6AU/ml), with the heterozygotes generally intermediate. The data from Belfast are consistent with the literature implicating PAI-1 level as an MI risk factor. In ECTIM, the PAI-1 4G/5G polymorphism is not a genetic risk factor for MI but is associated with PAI-1 activity. Thus homozygosity for the 4G allele may predispose to elevated PAI-1 and impaired fibrinolysis, perhaps requiring interaction with other genetic or environmental factors to influence MI risk.     

Pretreatment plasminogen activator inhibitor-1 (PAI-1) levels and the outcome of thrombolysis with streptokinase in patients with acute myocardial infarction

We have evaluated the potential usefulness of radiolabelled [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide as radiopharmaceuticals for somatostatin receptor-targeted scintigraphy and radiotherapy. In vitro somatostatin receptor binding and in vivo metabolism in rats of the compounds were investigated in comparison with [111In-DTPA0] octreotide. Comparing different peptide-chelator constructs, [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide were found to have a higher affinity than [DTPA0]octreotide for subtype 2 somatostatin receptors (sst2) in mouse AtT20 pituitary tumour cell membranes (all IC50 values obtained were in the low nanomolar range). In vivo studies in CA20948 tumor-bearing Lewis rats revealed a significantly higher uptake of both 111In-labelled [DOTA0,Tyr3]octreotide and [DTPA0,Tyr3]octreotide in sst2-expressing tissues than after injection of [111In-DTPA0]octreotide, showing that substitution of Tyr for Phe at position 3 in octreotide results in an increased affinity for its receptor and in a higher target tissue uptake. Uptake of 111In-labelled [DTPA0]octreotide, [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide in pituitary, pancreas, adrenals and tumour was decreased to less than 7% of control by pre-treatment with 0.5 mg unlabelled octreotide/rat, indicating specific binding to sst2. Comparing different radionuclides, [90Y-DOTA0,Tyr3]octreotide had the highest uptake in sst2-positive organs, followed by the [111In-DOTA0,Tyr3]octreotide, whereas [DOTA0,125I-Try3]octreotide uptake was low compared to that of the other radiopharmaceuticals, when measured 24 hr after injection. Renal uptake of 111In-labelled [DTPA0]octreotide, [DTPA0,Tyr3]octreotide and [DOTA0,Tyr3]octreotide was reduced over 50% by an i.v. injection of 400 mg/kg D-lysine, whereas radioactivity in blood, pancreas and adrenals was not affected.     

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.     

4G/5G polymorphism of PAI-1 gene promoter and fibrinolytic capacity in patients with deep vein thrombosis

A deletion/insertion polymorphism (4G or 5G) in the promoter of the plasminogen activator inhibitor type 1 gene has been suggested to be involved in regulation of the synthesis of the inhibitor, the 4G allele being associated with enhanced gene expression. A relationship between 4G/5G polymorphism and PAI-1 levels was found in patients with cardiovascular and metabolic diseases, but not in healthy subjects. In the present work we studied the distribution of PAI-1 4G/5G genotype and its relation to fibrinolytic capacity in 70 unrelated patients with deep vein thrombosis. Each patient was assayed before and after 20 min. Venous occlusion for euglobulin lysis time, t-PA antigen and activity, and PAI-1 antigen and activity. The prevalence of 5G homozygous carriers was significantly lower in patients than in controls (10% vs. 26%, p=0.009). The 5G allele frequency was reduced, even though not significantly, in DVT patients compared to healthy subjects (0.40 vs. 0.51, respectively). In the patient group, the mean PAI-1 antigen and activity levels were significantly higher than among controls and related to the 4G/5G polymorphism. In patients with 4G/5G and 4G/4G genotype a significant correlation was found between PAI-1 levels and the global fibrinolytic activity as evaluated by euglobulin lysis time. The prevalence of a reduced fibrinolytic potential due to PAI-1 excess was 45.7% among DVT patients. Moreover, the prevalence of PAI-1 induced hypofibrinolysis was strongly related to PAI-1 polymorphism, since it was significantly lower in 5G homozygous patients (28.6%) than in both 4G/5G carriers (55.3%, p <0.001) and 4G homozygous patients (57.9%, p <0.001). In conclusion, in patients with deep vein thrombosis the 4G polymorphism of PAI-1 gene promoter may influence the expression of PAI-1 and it should be taken into consideration as a facilitating condition for pathological fibrinolysis together with other environmental and genetic factors. Whether this has any significance in regard to the pathogenesis of venous thrombosis remains to be proven.     

Immunoelectron microscopic localization of plasminogen activator inhibitor type 1 (PAI-1) in smooth muscle cells from morphologically normal and atherosclerotic human arteries

Vascular wall fibrinolytic system proteins are believed to play a pivotal role in atherogenesis. Tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) influence persistence of luminal thrombi and proteolysis of extracellular matrix, respectively. The major physiologic inhibitor of t-PA and u-PA is plasminogen activator inhibitor type 1 (PAI-1). All three of these fibrinolytic system proteins have been detected in vascular endothelial cells, smooth muscle cells, and macrophages by light microscopic immunohistochemistry. This study was undertaken to delineate, by immunoelectron microscopy, the loci of PAI-1 in smooth muscle cells from intact morphologically normal and atherosclerotic human arteries as well as in isolated and cultured smooth muscle cells from arteries. In intact vessels, PAI-1 immunoreactivity was associated with contractile filaments in cells in both normal and atherosclerotic tissues. Lipid-laden smooth muscle cells in atherosclerotic vessels were mainly of the synthetic phenotype and displayed lesser amounts of PAI-1 associated with rough endoplasmic reticulum and contractile filaments. Isolated smooth muscle cells exhibited either a contractile or synthetic phenotype. In the cells with a contractile phenotype, PAI-1 was associated with the contractile elements, whereas in the cells with a synthetic phenotype, the PAI-1 was associated predominantly with elements of the endoplasmic reticulum. Because PAI-1 is associated predominantly with contractile filaments in smooth muscle cells, the net amount of immunodetectable PAI-1 appears to be greater in contractile compared with synthetic phenotype cells.