Effect of valsartan on angiotensin II-induced plasminogen activator inhibitor-1 biosynthesis in arterial smooth muscle cells

Previous studies have shown that angiotensin II stimulates the synthesis of plasminogen activator inhibitor-1 in cultured vascular cells, which suggests that activation of the renin-angiotensin system may impair fibrinolysis. We have investigated the effects of angiotensin II and of valsartan, a recently developed angiotensin II antagonist that is highly specific and selective for the angiotensin II subtype 1 receptor, on plasminogen activator inhibitor-1 secretion by smooth muscle cells isolated from rat and human vessels. Angiotensin II induced a time- and concentration-dependent increase of plasminogen activator inhibitor activity in supernatants of rat aortic cells, which reached a plateau after 6 hours of incubation with 100 nmol/L angiotensin II (2.4+/-0.6-fold over control value; P:<0.001). The angiotensin II-induced plasminogen activator inhibitor activity was inhibited, in a concentration-dependent manner, by valsartan with an IC(50) value of 21 nmol/L. Valsartan fully prevented the angiotensin II-induced increase in plasminogen activator inhibitor-1 protein and mRNA. Furthermore, angiotensin II doubled the secretion of plasminogen activator inhibitor-1 by smooth muscle cells obtained from human umbilical and internal mammary arteries, and valsartan fully prevented it. Angiotensin II did not affect the secretion of tissue plasminogen activator antigen by any of the cell systems tested. Thus, valsartan effectively inhibits angiotensin II-induced plasminogen activator inhibitor-1 secretion without affecting that of tissue plasminogen activator in arterial rat and human smooth muscle cells.     

糖基化终产物对平滑肌细胞增殖及纤溶酶原激活物抑制剂1基因表达的影响

目的探讨糖基化终产物对大鼠平滑肌细胞增殖以及纤溶酶原激活物抑制剂1基因表达的影响。方法体外分离培养大鼠平滑肌细胞,以不同浓度(50、100、200和400mg/L)糖基化终产物作用于平滑肌细胞不同时间(0、4、8、16、24、48、72和96h),MTT法观察糖基化终产物对平滑肌细胞增殖的影响,逆转录聚合酶链反应测定纤溶酶原激活物抑制剂1基因的表达。结果不同浓度的糖基化终产物作用8h均对平滑肌细胞增殖无影响,以后各时间点不同浓度的糖基化终产物均导致平滑肌细胞增殖,与对照组比较差异有显著性(P<0.05)。糖基化终产物作用不同时间均可使平滑肌细胞表达纤溶酶原激活物抑制剂1基因水平升高,且随着浓度升高而增加(分别为0.85±0.05、0.97±0.05、1.08±0.12和1.41±0.05),与对照组(0.80±0.03)比较差异有显著性(P<0.05)。结论糖基化终产物诱导血管平滑肌细胞增殖并增加纤溶酶原激活物抑制剂1基因表达是糖尿病患者易患动脉粥样硬化的可能原因。     

Effect of Probucol on Smooth Muscle Cell Proliferation and Dedifferentiation After Vascular Injury in Rabbits: Possible Role of PDGF

We previously reported a clinical study in which probucol reduced the restenosis rate. The mechanism of this effect is unclear. Restenosis is characterized by neointimal hyperplasia caused by proliferation of smooth muscle cells (SMCs), which increases the expression of Platelet-derived growth factor (PDGF)-A and SMemb. SMemb, a non–muscle-type myosin heavy chain most predominantly expressed in embryonic smooth muscle, can be used as a good molecular marker for dedifferentiated SMC. The aim of this study was to analyze the effect of probucol on neointimal proliferation and the level of expression of PDGF-A and SMemb after balloon injury in rabbits. Probucol was given orally 1.3 g/d from 2 weeks prior to carotid balloon injury to the time of killing (2 or 4 weeks after balloon injury). Intimal area was determined histologically using a computerized morphometry program. For quantification of SMC proliferation, alpha-actin–positive cells and proliferating cell nuclear antigen (PCNA)-labeled cells were counted. The expression of PDGF-A and SMemb mRNA was analyzed by the RNase protection assay. SMemb expression was also examined by immunohistochemistry. Probucol remarkably decreased intimal area by 70% and the number of SMC and PCNA-labeled cells in the intima. The expression of PDGF-A mRNA was significantly increased after balloon injury in untreated rabbits, whereas it was markedly suppressed with probucol treatment. The expression of SMemb was significantly increased in injured arteries at mRNA and protein levels. However, probucol did not suppress SMemb expression. Probucol is effective in preventing SMC proliferation, which is possibly due to a decrease in the expression of PDGF.     

Endothelin-mediated remodeling in aortas of diabetic rats

BACKGROUND: Smooth muscle cells proliferation and extracellular matrix (ECM) protein deposition are key features of diabetic macroangiopathy. In the present study, we have studied the role of endothelin(A) (ET(A)) receptor, the predominant receptor on smooth muscle cells, in diabetes-induced vascular hypertrophy and remodeling. METHODS: Streptozotocin-induced diabetic rats were administrated a selective ET(A) receptor antagonist, TBC3214, for 26 weeks. Following treatment, aortas were harvested and subjected to gene expression and morphometric analyses. We quantified fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) expression as indicators of increased ECM protein synthesis. ET-1, ET-3, transforming growth factor-beta1 (TGF-beta1) and angiotensinogen mRNA levels were measured to elucidate genes involved in FN expression. We have investigated an embryonic splice variant of FN, oncofetal FN, and nonmuscle myosin heavy chain (SMemb) as vascular remodeling indicators. RESULTS: Our results show that diabetes leads to upregulation of FN, PAI-1, ET-1, ET-3, TGF-beta1 and angiotensinogen mRNA levels in association with increased medial thickness. Immunohistochemical analyses revealed concurrent protein level changes. Diabetes also upregulated oncofetal FN and SMemb mRNA levels. Treatment with TBC3214 attenuated the mRNA levels of several genes and prevented increased medial thickness. CONCLUSIONS: These results indicate that diabetes-induced vascular hypertrophy and remodeling is associated with reexpression of embryonic forms of FN and myosin heavy chain. Such changes are ET-dependent and may be mediated via TGF-beta1 and angiotensin.     

Interleukin-4 stimulates expression of urokinase-type-plasminogen activator in cultured human foreskin microvascular endothelial cells

The effect of interleukin-4 (IL-4) on the fibrinolytic system of human microvascular and macrovascular endothelial cells in culture was studied. Only foreskin microvascular endothelial cells (EC) responded to IL-4 treatment with a dose- and time-dependent increase in urokinase- type plasminogen activator (u-PA) (control: 3.0 +/- 0.8 ng/10(5) cells/24 h; 200 U/mL IL-4: 6.7 +/- 0.8 ng/10(5) cells/24 h), whereas human macrovascular EC remained unaffected. A maximum effect was achieved with 200 U/mL IL-4. Little u-PA activity was detected in the conditioned media of human foreskin microvascular EC (HFMEC) treated without and with IL-4 before plasmin treatment (control: 0.03 +/- 0.003 IU/10(5) cells/20 h; 200 U/mL IL-4: 0.09 +/- 0.007 IU/10(5) cells/20 h). These values increased to 0.18 +/- 0.02 IU/10(5) cells/20 h and 0.53 +/- 0.04 IU/10(5) cells/20 h, respectively, after plasmin treatment, indicating that u-PA is released by HFMEC predominantly in its inactive precursor form single-chain u-PA (scu-PA). u-PA activity increased also in the cell lysates of HFMEC up to 2.5-fold after IL-4 treatment. Plasminogen activator inhibitor type-1 (PAI-1) levels produced by HFMEC remained unaffected by IL-4, whereas tissue-type plasminogen activator (t-PA) levels were slightly decreased when HFMEC were treated with IL-4. These findings were also reflected in the specific mRNA levels as determined by Northern blotting. u-PA-specific mRNA increased significantly in HFMEC in the presence of IL-4, whereas t-PA mRNA and PAI-1-specific mRNA in HFMEC and u-PA specific mRNA in human saphenous vein EC (HSVEC) remained unaffected by IL-4 treatment. Our findings suggest a role for IL-4 in the process of angiogenesis, in addition to its known proliferative effect on human microvascular EC, by increasing the fibrinolytic potential of such EC, thereby facilitating extracellular proteolysis and cell migration.     

Endogenous NO regulates plasminogen activator inhibitor-1 during angiotensin-converting enzyme inhibition

To test the hypothesis that NO contributes to effects of angiotensin-converting enzyme inhibitors on fibrinolysis, fibrinolytic balance was assessed in 17 normal subjects during placebo and after randomized, double-blind 4-week treatment with the NO precursor L-arginine (3 g TID), ramipril (10 mg QD), or L-arginine+ramipril. Neither L-arginine nor ramipril alone affected basal plasminogen activator inhibitor-1 or tissue-type plasminogen activator (t-PA) antigen in these salt-replete subjects in whom plasma renin activity was suppressed (mean+/-SD 0.7+/-0.5 ng angiotensin I/mL per hour). In contrast, L-arginine+ramipril reduced morning plasminogen activator inhibitor-1 antigen (10.8+/-9.5 ng/mL) and the molar ratio of plasminogen activator inhibitor-1:t-PA (2.3+/-1.6) compared with placebo (13.5+/-10.8 ng/mL, P=0.006; ratio 2.9+/-2.1, P=0.015) or ramipril alone (15.2+/-13.2 ng/mL, P=0.009; ratio 3.7+/-3.3, P=0.005). L-arginine and ramipril synergistically increased d-dimers (23.1+/-31.5, 29.7+/-50.0, 35.1+/-50.0, and 57.1+/-144.8 ng/mL during placebo, L-arginine, ramipril, and L-arginine+ramipril, respectively; P<0.05 for L-arginine+ramipril versus any other group). During ramipril, the NO synthase inhibitor L-NG-nitro-arginine-methyl-ester (2 mg/kg) significantly increased plasminogen activator inhibitor-antigen after 2 hours (from 9.4+/-8.6 ng/mL during vehicle to 13.5+/-11.0 ng/mL during L-NG-nitro-arginine-methyl-ester; P=0.020), consistent with an effect on expression but rapidly increased t-PA activity (from 0.4+/-0.3 to 0.5+/-0.4 IU/mL; P=0.031), consistent with an effect on release. Both effects of L-NG-nitro-arginine-methyl-ester were reversed by L-arginine. During angiotensin-converting enzyme inhibition, endogenous NO decreases plasminogen activator inhibitor-1 antigen and improves fibrinolytic balance in normotensive salt-replete subjects.     

Fibrinopeptide A levels indicative of pulmonary vascular thrombosis in patients with primary pulmonary hypertension

Although the mechanisms involved in the pathophysiology of primary pulmonary hypertension have not yet been delineated, thrombosis has been implicated. This study was designed to determine whether thrombin activity as reflected by plasma concentrations of fibrinopeptide A (FPA), a marker of the action of thrombin on fibrinogen, is increased in patients with primary pulmonary hypertension. To evaluate fibrinolytic activity, we measured plasma concentrations of tissue-type plasminogen activator, plasminogen activator inhibitor-1, and cross-linked fibrin degradation products. We studied 31 patients with primary pulmonary hypertension. Plasma FPA concentrations measured by radioimmunoassay, were elevated to 87.4 +/- 36.9 ng/ml (mean +/- SEM). Fifteen minutes after administration of heparin (5,000 U), FPA concentrations decreased to 6.8 +/- 1.4 ng/ml (p less than 0.001 compared with preheparin levels). In 21 of 30 patients (70%), FPA concentrations after heparin administration were less than half the preheparin levels, a response consistent with inhibition of thrombin by heparin and the short half-life of FPA. Despite evidence for marked thrombin activity, plasma concentrations of cross-linked fibrin degradation products were normal in all but four patients. Plasminogen activator inhibitor-1 activity was elevated in 19 of the 27 patients in whom it was measured, potentially limiting the fibrinolytic response. The elevations of FPA indicate that thrombin activity is increased in vivo in patients with primary pulmonary hypertension. Thus, sequential assays of plasma markers of thrombosis and fibrinolysis in vivo may help identify those patients who may benefit from treatment with anticoagulants.     

The effect of plasminogen activator inhibitor-1 -675 4G/5G polymorphism on PAI-1 gene expression and adipocyte differentiation

Obesity is a complex, multifactorial chronic disease frequently associated with cardiovascular risks, hypertriglyceridemia, low high-density lipoprotein-cholesterol, high blood pressure, and the insulin resistance that appears to be central to the pathogenesis of Type II diabetes. Plasminogen activator inhibitor-1 expression induced in differentiating adipose tissue, but its role in adipogenesis and obesity is poorly understood. Circulating plasminogen activator inhibitor-1 levels are elevated at an early stage of impaired glucose tolerance, resulting in diabetes and metabolic syndrome. Plasminogen activator inhibitor-1 levels are also significantly elevated in the plasma of obese individuals and in adipose tissues of obese mice and humans. Some investigators proposed that the -675 4G/5G polymorphism in plasminogen activator inhibitor-1 promoter caused overexpression of this gene and predisposed carriers to obesity. In this study, we investigated the role of -675 4G/5G polymorphism in plasminogen activator inhibitor-1 promoter in the expression of this gene and the contribution of plasminogen activator inhibitor-1 to adipogenesis. Using a dual-luciferase promoter assay, we determined that the -675 4G/5G polymorphism contributes significantly to overexpression of plasminogen activator inhibitor-1 in the course of adipogenesis. The antidiabetic agents troglitazone and ciglitazone inhibited reporter gene expression driven by wild-type and -675 4G/5G mutant promoter, as well as the expression of endogenous plasminogen activator inhibitor-1, indicating that suppression of plasminogen activator inhibitor-1 expression may contribute to antidiabetic effects of these agents. The results indicate that absence of plasminogen activator inhibitor-1 in adipocytes may protect the cells against insulin resistance by promoting glucose uptake and adipocyte differentiation via a decrease in the peroxisome proliferator activated receptor-gamma expression that modulates the adipocyte differentiation.     

纤维蛋白原、纤维蛋白及其降解产物对共培养血管内皮细胞纤溶活性的影响

目的研究纤维蛋白原（Fg）、纤维蛋白（Fb）及其降解产物（FDP）对共培养体系中人脐静脉内皮细胞组织型纤溶酶原激活物和纤溶酶原激活物抑制剂表达的影响。方法应用Transwell膜建立人脐静脉内皮细胞-兔主动脉平滑肌细胞共培养体系，在不同浓度（0、0．5、1．5、3．0、4．5和6．0g／L）Fg、Fb和FDP干预24h后，分别检测该共培养体系中人脐静脉内皮细胞组织型纤溶酶原激活物和纤溶酶原激活物抑制剂mRNA水平（RT-PCR法）以及培养上清中组织型纤溶酶原激活物和纤溶酶原激活物抑制剂抗原含量（ELISA法）与活性（发色底物法）的变化情况。结果Fg对组织型纤溶酶原激活物的表达没有显著影响，较高浓度的Fg（3．0～4．5g／L）可明显促进纤溶酶原激活物抑制剂mRNA表达、抗原含量及活性升高，但过高浓度的Fg（6．0g／L）却抑制纤溶酶原激活物抑制剂的表达。3．0-4．5g／L的Fb对组织型纤溶酶原激活物mRNA和抗原含量都起上调作用，同时显著下调组织型纤溶酶原激活物活性。较高浓度的Fb（1．5-4．5g／L）则可明显上调纤溶酶原激活物抑制剂的表达，且在mRNA、蛋白和活性水平趋势基本一致。3．0-6．0g／L的FDP均可明显下调组织型纤溶酶原激活物mRNA、蛋白和活性水平，1．5-6．0mg／ml的FDP均可促进纤溶酶原激活物抑制剂的高表达。结论Fg、Fb和FDP可以通过影响组织型纤溶酶原激活物和纤溶酶原激活物抑制剂的表达，引起纤溶活性降低，参与动脉粥样硬化的发展进程。     

Guggulsterone, an anti-inflammatory phytosterol, inhibits tissue factor and arterial thrombosis

Background  The phytosterol guggulsterone is a potent anti-inflammatory mediator with less side effects than classic steroids. This study assesses the impact of guggulsterone on tissue factor (TF) expression and thrombus formation. Methods and results  Guggulsterone inhibited TNF-α-induced endothelial TF protein expression and surface activity in a concentration-dependent manner; in contrast, dexamethasone did not affect TNF-α-induced TF expression. Guggulsterone enhanced endothelial tissue factor pathway inhibitor and impaired plasminogen activator inhibitor-1 as well as vascular cell adhesion molecule-1 protein. Real-time polymerase chain reaction revealed that guggulsterone inhibited TNF-α-induced TF mRNA expression; moreover, it impaired activation of the MAP kinases JNK and p38, while that of ERK remained unaffected. In vivo, guggulsterone inhibited TF activity and photochemical injury induced thrombotic occlusion of mouse carotid artery. Guggulsterone also inhibited TF expression, proliferation, and migration of vascular smooth muscle cells in a concentration-dependent manner. Conclusions  Guggulsterone inhibits TF expression in vascular cells as well as thrombus formation in vivo; moreover, it impairs vascular smooth muscle cell activation. Hence, this phytosterol offers novel therapeutic options, in particular in inflammatory diseases associated with an increased risk of thrombosis.     

Serotonin induces the expression of tissue factor and plasminogen activator inhibitor-1 in cultured rat aortic endothelial cells

Serotonin (5-hydroxytryptamine, or 5-HT), released from activated platelets, not only accelerates aggregation of platelets but also is known to promote mitosis, migration, and contraction of vascular smooth muscle cells (VSMCs). These effects are considered to contribute to thrombus formation and atherosclerosis. The aim of this study was to investigate the effects of 5-HT on the expressions of coagulative and fibrinolytic factors in rat aortic endothelial cells. Endothelial cells were stimulated with various concentrations of 5-HT (0.1 approximately 10 microM), and the expressions of tissue factor (TF), tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), and tissue-type plasminogen activator (TPA) messenger RNAs (mRNAs) were evaluated by Northern blot analysis. The activities of TF and PAI-1 were also measured. TF and PAI-1 mRNA were increased significantly in a concentration- and time-dependent manner. However, TFPI and TPA mRNA expression did not change. The inductions of TF and PAI-1 mRNAs were inhibited by a 5-HT1/5-HT2 receptor antagonist (methiothepin) and a selective 5-HT2A receptor antagonist (MCI-9042). These results indicate that 5-HT increases procoagulant activity and reduces fibrinolytic activities of endothelial cells through the 5-HT2A receptor. It was concluded that the modulation of procoagulant and hypofibrinolytic activities of endothelial cells by 5-HT synergistically promotes thrombus formation at the site of vessel injury with the platelet aggregation, VSMC contraction, and VSMC proliferation.     

Plasma plasminogen activator inhibitor-1 activity in normoglycemic hypertriglyceridemic north Asian Indian subjects: a preliminary case-control study.

BACKGROUND: Recent evidence suggests that increased activity of plasma plasminogen activator inhibitor-1, an important component of the insulin resistance syndrome, plays a crucial role in the pathogenesis of atherosclerosis. METHODS AND RESULTS: In this case-control study, relationships between plasma plasminogen activator inhibitor-1 activity, serum triglyceride levels and hyperinsulinemia were explored in 40 non-diabetic patients with primary hypertriglyceridemia (Group 1) and 40 non-diabetic normotriglyceridemic controls (Group 2) matched for potential confounders like smoking and physical activity. Mean values of fasting serum insulin levels were increased in Group 1 (p>0.05). Hyperinsulinemia was observed in 14 (17.5%) individuals in Group 1 and 11 (13.8%) individuals in Group 2. Mean plasma plasminogen activator inhibitor-I activity in Group 1 (9.8+/-8.4 IU) was higher than in Group 2 (7.0+/-7.7 IU), though the difference was not significant (p>0.05). However, when only subjects with elevated levels of plasma plasminogen activator inhibitor-1 activity were taken into account, mean values were significantly higher in Group 1 (p<0.05). The plasma plasminogen activator inhibitor-1 activity was higher in subjects with body mass index >25 in both the groups, significantly so in males (p=0.05). Hyperinsulinemic subjects with a body mass index >25 and raised serum triglyceride levels had higher mean values of plasma plasminogen activator inhibitor-1 activity (18.42+/-11.15 IU) than subjects with similar characteristics and normal triglyceride levels (14.22+/-8.20 IU, p<0.05). CONCLUSIONS: Though in the current study a trend for hyperinsulinemia and high plasma plasminogen activator inhibitor-1 activity was observed in hypertriglyceridemic subjects, a larger study is needed to achieve significant differences and correlations. Obese male subjects, irrespective of their lipid profile, are at risk for thrombotic events in view of their significantly higher plasma plasminogen activator inhibitor-1 values. Procoagulant tendency is further enhanced if hypertriglyceridemia and hyperinsulinemia are added on to obesity.     

Plasmin-mediated fibroblast growth factor-2 mobilisation supports smooth muscle cell proliferation in human saphenous vein

The focus of this study was identification of the contribution of the plasminogen activator-plasmin system to smooth muscle cell proliferation and migration in human saphenous vein. Segments of human saphenous vein were grown in organ culture for up to 14 days. Smooth muscle cell proliferation and migration were measured by incubating vein segments in bromodeoxyuridine, and smooth muscle cell death was detected by in situ end-labelling. Tissue-type (tPA) and urokinase-type (uPA) plasminogen activator enzymic activities were detectable in cultured saphenous vein segments, and were concentrated in focal zones. Inhibition of plasmin activity with alpha-N-acetyl-L-lysine methyl ester (NALME) or of uPA activity with a neutralising antibody caused significant decreases in smooth muscle cell proliferation in the media and the intima, but no significant changes in smooth muscle cell migration. Intimal thickness was also significantly decreased. Incubation with plasminogen or plasmin caused fibroblast growth factor-2 (FGF2) to be released into the medium. Addition of FGF2 to segments cultured with NALME reversed the inhibition of smooth muscle cell proliferation, and blocking the activity of FGF2 with a neutralising antibody caused a significant decrease in medial smooth muscle cell proliferation. These data suggest that plasmin mobilises FGF2 bound to the extracellular matrix of human saphenous vein, so that it can support smooth muscle cell proliferation and intimal thickening.     

Angiostatin binds to smooth muscle cells in the coronary artery and inhibits smooth muscle cell proliferation and migration In vitro.

Angiostatin is an inhibitor of angiogenesis that is known to reduce endothelial cell proliferation and consequently prevent the progression of tumor metastases. However, the modest effect of angiostatin on endothelial cell proliferation raises the possibility that angiostatin might exert its effects on other cells. To determine the cellular distribution of angiostatin binding in tissues with neovasculature (atherosclerotic coronary arteries), we developed a fusion protein consisting of placental alkaline phosphatase and the first 3 kringles of plasminogen. Angiostatin binding colocalized with smooth muscle cells and could be inhibited by a 50-fold molar excess of plasminogen and 10 mmol/L epsilon-amino-n-caproic acid. The fusion protein also bound to smooth muscle cells in culture. Angiostatin inhibited hepatocyte growth factor-induced proliferation and migration of smooth muscle cells, suggesting that they are a target for the antiangiogenic effect of angiostatin.     

Growth factor-induced expression of macrophage migration inhibitory factor in osteoblasts: relevance to the plasminogen activator system

Macrophage migration inhibitory factor (MIF) is a cytokine that mediates inflammatory processes in a variety of tissues. In this study, we examined the expression of MIF mRNA in the mouse osteoblastic cell line MC3T3-E1, whose proliferation is promoted by various growth factors. In the subconfluent state, transforming growth factor-beta, basic fibroblast growth factor, insulin-like growth factor-II, and fetal calf serum markedly upregulated MIF mRNA expression. The upregulation of MIF mRNA was less extensive when the cells were stimulated by the same growth factors in the overconfluent state. We also investigated the expression of MIF mRNA through a whole cell cycle from G0 phase when the osteoblastic cells were synchronized by serum starvation. The MIF mRNA expression, which gradually increased from the G0 and reached its maximum at the S phase, was nonperiodic. Moreover, human recombinant MIF upregulated the expression of urokinase plasminogen activator inhibitor-1 (PAI-1) precursor mRNA in human osteoblastic Saos-2 cells. Plasminogen activator (PA) is known to play an important role in bone metabolism, for example, in activation of procollagenase or growth factors indirectly via the formation of plasmin, and in mitogenic activity for osteoblastic cells. Our results suggest that MIF modulates the proliferation of osteoblasts and, moreover, bone tissue remodeling through the PA and plasmin system.     

Lack of effect of granulocyte-macrophage and granulocyte colony-stimulating factors on cultured human endothelial cells.

The hematopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), enhance the effector functions of mature myeloid cells, including the interaction with vascular endothelium. We examined the direct effect of recombinant human GM-CSF (rhGM-CSF) and recombinant human G-CSF (rhG-CSF) on the growth and function of cultured human umbilical vein endothelial cells (HUVEC). Endothelial cell growth supplement (ECGS) increased the proliferation of passaged and primary cells by 305% +/- 45% (mean +/- SEM, n = 5, P less than .01) over control cells at 4 days; GM-CSF and G-CSF had no effect. Endothelial cell procoagulant activity was increased after 4-hour incubation with recombinant interleukin-1 beta (IL-1 beta) 10 U/mL and recombinant tumor necrosis factor (TNF) 10 U/mL to 1,721% +/- 376% (n = 7, P less than .005) and 247% +/- 71% (n = 4) of control levels, respectively. gamma-Interferon (gamma-IFN) 50 U/mL had no direct effect of its own but was able to prime the response to IL-1 beta. There was no direct or priming effect of GM-CSF (1 ng to 1 microgram/mL) on the expression of procoagulant activity in endothelial cells. GM-CSF and G-CSF (1 ng/mL to 1 microgram/mL) had no effect on the expression of either tissue plasminogen activator (tPA) or plasminogen activator inhibitor-1 (PAI-1) by endothelial cells. The secretion of tPA by endothelial cells was increased, however, after 24-hour incubation with thrombin 4 U/mL (314% +/- 72% of control levels, n = 5, P less than .025). The production of PAI-1 was increased by TNF 200 U/mL (241% +/- 44% of control, n = 3, P less than .005), thrombin 4 U/mL (180% +/- 12% of control, n = 5, P less than .0005) and IL-1 beta 10 U/mL (275% +/- 44% of controls, n = 5, P less than .0005). In four experiments, endothelial cells showed no specific binding of 125I-GM-CSF, whereas peripheral blood (PB) neutrophils demonstrated the presence of 802 +/- 78 high-affinity receptors for GM-CSF. Thus, we found no effect of rhGM-CSF or rhG-CSF on the proliferation activities by these cells. These findings are in accordance with the lack of demonstrable receptors for GM-CSF on cultured HUVEC.     

Helicobacter pylori neutrophil-activating protein stimulates tissue factor and plasminogen activator inhibitor-2 production by human blood mononuclear cells

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a virulence factor that activates phagocytic NADPH-oxidase. The effect of HP-NAP on the production of tissue factor (TF), plasminogen activator inhibitor-2 (PAI-2), and urokinase-type plasminogen activator (u-PA) by human blood mononuclear cells (MNC) was evaluated by using functional and immunological assays and mRNA analysis. HP-NAP induced time- and dose-dependent increases in TF and PAI-2, with a maximal effect at 300 nmol/L (>15-fold increase in antigens). No changes in u-PA were observed. When whole bacteria were used, an H. pylori mutant lacking HP-NAP was significantly less active than the wild-type strain. MNC from a patient with chronic granulomatous disease behaved as do normal cells, which indicates that HP-NAP effects can occur independently of NADPH-oxidase. HP-NAP, by inducing the coordinate expression of cell procoagulant and antifibrinolytic activities, might favor fibrin deposition and contribute to the inflammatory reaction of gastric mucosa elicited by H. pylori.