Surgical removal of inflamed epididymal white adipose tissue attenuates the development of non-alcoholic steatohepatitis in obesity.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is strongly associated with abdominal obesity. Growing evidence suggests that inflammation in specific depots of white adipose tissue (WAT) has a key role in NAFLD progression, but experimental evidence for a causal role of WAT is lacking. METHODS: A time-course study in C57BL/6J mice was performed to establish which WAT depot is most susceptible to develop inflammation during high-fat diet (HFD)-induced obesity. Crown-like structures (CLS) were quantified in epididymal (eWAT), mesenteric (mWAT) and inguinal/subcutaneous (iWAT) WAT. The contribution of inflamed WAT to NAFLD progression was investigated by surgical removal of a selected WAT depot and compared with sham surgery. Plasma markers were analyzed by enzyme-linked immunosorbent assay (cytokines/adipokines) and lipidomics (lipids). RESULTS: In eWAT, CLS were formed already after 12 weeks of HFD, which coincided with maximal adipocyte size and fat depot mass, and preceded establishment of non-alcoholic steatohepatitis (NASH). By contrast, the number of CLS were low in mWAT and iWAT. Removal of inflamed eWAT after 12 weeks (eWATx group), followed by another 12 weeks of HFD feeding, resulted in significantly reduced NASH in eWATx. Inflammatory cell aggregates (-40%; P<0.05) and inflammatory genes (e.g., TNFα, -37%; P<0.05) were attenuated in livers of eWATx mice, whereas steatosis was not affected. Concomitantly, plasma concentrations of circulating proinflammatory mediators, viz. leptin and specific saturated and monounsaturated fatty acids, were also reduced in the eWATx group. CONCLUSIONS: Intervention in NAFLD progression by removal of inflamed eWAT attenuates the development of NASH and reduces plasma levels of specific inflammatory mediators (cytokines and lipids). These data support the hypothesis that eWAT is causally involved in the pathogenesis of NASH.

Pathogenesis of postoperative adhesion formation.

BACKGROUND: Current views on the pathogenesis of adhesion formation are based on the 'classical concept of adhesion formation', namely that a reduction in peritoneal fibrinolytic activity following peritoneal trauma is of key importance in adhesion development. METHODS: A non-systematic literature search (1960-2010) was performed in PubMed to identify all original articles on the pathogenesis of adhesion formation. Information was sought on the role of the fibrinolytic, coagulatory and inflammatory systems in the disease process. RESULTS: One unifying concept emerged when assessing 50 years of studies in animals and humans on the pathogenesis of adhesion formation. Peritoneal damage inflicted by surgical trauma or other insults evokes an inflammatory response, thereby promoting procoagulatory and antifibrinolytic reactions, and a subsequent significant increase in fibrin formation. Importantly, peritoneal inflammatory status seems a crucial factor in determining the duration and extent of the imbalance between fibrin formation and fibrin dissolution, and therefore in the persistence of fibrin deposits, determining whether or not adhesions develop. CONCLUSION: Suppression of inflammation, manipulation of coagulation as well as direct augmentation of fibrinolytic activity may be promising antiadhesion treatment strategies.

Human visceral adipose tissue and the plasminogen activator inhibitor type 1.

OBJECTIVE: The objective of this study was to systematically evaluate the molecular basis of the association between visceral fat mass and plasma plasminogen activator inhibitor-1 (PAI-1) levels in man. DESIGN: A comprehensive approach comprising observational, in vitro, and human intervention studies. MEASUREMENTS AND RESULTS: We confirmed an exclusive relationship between visceral fat and plasma PAI-1 levels (r=0.79, P<0.001) and corroborated preferential PAI-1 release from adipose tissue explants. Yet, messenger RNA analysis and in vivo measurement of PAI-1 release from visceral fat (AV-differences over the omentum) not only excluded visceral adipose tissue as a relevant source of circulating PAI-1, but also excluded visceral fat as a significant source of proinflammatory mediators such as tumor necrosis factor-alpha, IL-1 or transforming growth factor-beta that could induce PAI-1 expression in tissues other than visceral fat. Short-term interventions with acipimox and growth hormone (GH) as well as statistical evaluation excluded free fatty acids and GH as metabolic links. Further analysis of the metabolic data in a stepwise regression model indicated that plasma PAI-1 levels and visceral fat rather are co-correlates that both relate to impaired lipid handling. CONCLUSION: Our PAI-1 studies show that visceral fat mass and plasma PAI-1 levels are co-correlated rather than causatively related, with lipid load as common denominator.

Low-grade inflammation may play a role in the etiology of the metabolic syndrome in patients with coronary heart disease: the HIFMECH study.

Risk of coronary heart disease has been related to insulin resistance, but the mechanism for this is incompletely understood. Variables attributed to insulin resistance are associated with low-grade inflammation. A case-control study was performed of 469 male myocardial infarction (MI) survivors aged < 60 years and 575 control subjects recruited from centers in northern and southern Europe. Principal factor analysis was used to explore correlations between insulin resistance and inflammatory variables. Three factors resulted: (a) "Metabolic Syndrome" (insulin/proinsulin/ triglyceride/body mass index [BMI]); (b) "Inflammation" (fibrinogen/C-reactive protein [CRP]/interleukin-6 [IL-6]); and (c) "Blood Pressure" (systolic and diastolic blood pressure). The "Metabolic Syndrome" factor was related to the "Inflammation" factor (largely independently of obesity), the "Blood Pressure" factor, smoking, and south location (all P < or = .0002). There were significant relationships between all 3 factors and case status (P < or = .0002). Markers of low-grade inflammation are strongly related to metabolic syndrome variables independently of obesity. This raises the possibility that links between insulin resistance and cardiovascular disease could, in part, represent common consequences of low-grade inflammation.

Time course of peritoneal tissue plasminogen activator after experimental colonic surgery: effect of hyaluronan-based antiadhesive agents and bacterial peritonitis.

BACKGROUND: This study assessed the peritoneal fibrinolytic response during the first week after colonic surgery in rats with and without bacterial peritonitis, and possible modulation of the response by two hyaluronan-based antiadhesive agents. METHODS: A colonic anastomosis was constructed in 90 male Wistar rats. Peritonitis was induced in another 108 rats and a colonic anastomosis was constructed after 24 h. Rats in both groups were randomized into an untreated group or one of two groups treated with hyaluronan-based agents. One-third of each group was killed at each of days 1, 3 and 7 after operation, and tissue plasminogen activator (tPA) antigen and activity were measured in peritoneal biopsies. RESULTS: One day after colonic surgery in normal rats, tPA antigen concentration was significantly (P < 0.005) increased, whereas tPA activity levels were normal. By day 3 after operation tPA antigen had returned to baseline values while tPA activity was significantly increased (P < 0.05). One day after inducing peritonitis tPA antigen was significantly increased (P < 0.001), while tPA activity was significantly reduced (P < 0.05). Three and seven days after colonic surgery in rats with peritonitis tPA activity was increased (P < 0.001) while tPA antigen had returned to baseline values. Neither of the hyaluronan-based agents affected peritoneal tPA antigen levels or activity after colonic surgery. CONCLUSION: Both abdominal surgery and infection caused an early increase in peritoneal tPA antigen levels, followed by an increase in tPA activity. Peritonitis severely depressed early tPA activity. Application of hyaluronan-based agents did not affect the peritoneal fibrinolytic response to surgery and/or infection.

Negative regulation of human fibrinogen gene expression by peroxisome proliferator-activated receptor alpha agonists via inhibition of CCAAT box/enhancer-binding protein beta.

Fibrinogen is a coagulation factor and an acute phase reactant up-regulated by inflammatory cytokines, such as interleukin 6 (IL-6). Elevated plasma fibrinogen levels are associated with coronary heart diseases. Fibrates are clinically used hypolipidemic drugs that act via the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha). In addition, most fibrates also reduce plasma fibrinogen levels, but the molecular mechanism is unknown. In this study, we demonstrate that fibrates decrease basal and IL-6-stimulated expression of the human fibrinogen-beta gene in human primary hepatocytes and hepatoma HepG2 cells. Fibrates diminish basal and IL-6-induced fibrinogen-beta promoter activity, and this effect is enhanced in the presence of co-transfected PPAR alpha. Site-directed mutagenesis experiments demonstrate that PPAR alpha activators decrease human fibrinogen-beta promoter activity via the CCAAT box/enhancer-binding protein (C/EBP) response element. Co-transfection of the transcriptional intermediary factor glucocorticoid receptor-interacting protein 1/transcriptional intermediary factor 2 (GRIP1/TIF2) enhances fibrinogen-beta gene transcription and alleviates the repressive effect of PPAR alpha. Co-immunoprecipitation experiments demonstrate that PPAR alpha and GRIP1/TIF2 physically interact in vivo in human liver. These data demonstrate that PPAR alpha agonists repress human fibrinogen gene expression by interference with the C/EBP beta pathway through titration of the coactivator GRIP1/TIF2. We observed that the anti-inflammatory action of PPAR alpha is not restricted to fibrinogen but also applies to other acute phase genes containing a C/EBP response element; it also occurs under conditions in which the stimulating action of IL-6 is potentiated by dexamethasone. These findings identify a novel molecular mechanism of negative gene regulation by PPAR alpha and reveal the direct implication of PPAR alpha in the modulation of the inflammatory gene response in the liver.

Adenovirus-mediated transfer of the 39 kD receptor-associated protein increases fibrinolytic capacity.

BACKGROUND: The mesothelium has an important role in maintaining an adequate fibrinolytic capacity in the peritoneal cavity and thus in preventing the formation of fibrinous peritoneal adhesions by secreting the fibrinolytic enzyme tissue-type plasminogen activator (t-PA). The fibrinolytic activity of human mesothelial cells (HMCs) is counteracted by rapid uptake of t-PA via the low-density lipoprotein receptor-related protein (LRP). The 39 kD receptor-associated protein (RAP) is an inhibitor of binding of t-PA to LRP, but RAP itself is also rapidly degraded via LRP. METHODS: Adenovirus-mediated RAP gene transfer technology was used to evaluate the effect of prolonged overexpression of RAP on t-PA accumulation in conditioned medium of HMCs under basal and inflammatory conditions. RESULTS: Infection of HMCs with a recombinant adenovirus carrying the RAP cDNA resulted within one day in t-PA levels that were maximally twofold to threefold increased as compared with noninfected or adenovirus-beta-galactosidase-infected cells. Whereas upon prolonged incubation, t-PA levels in the conditioned medium of uninfected cells leveled off because of rapid uptake and degradation via LRP, t-PA concentrations in the medium of adenovirus-RAP-infected cells continued to increase, reaching fivefold control levels after 72 hours. The increased t-PA accumulation persisted for seven days and then slowly returned to control values over the next few weeks. In contrast, the production of a specific inhibitor of t-PA, plasminogen activator inhibitor-1 (PAI-1), was not affected by adenoviral RAP gene transfer. Northern blotting analysis showed that t-PA, PAI-1, and LRP mRNA concentrations were not changed after adenoviral infection, underlining that the elevated t-PA levels are the result of RAP-blocked uptake and degradation of t-PA rather than increased t-PA synthesis. RAP gene transfer also restored diminished fibrinolytic activity of cytokine-treated mesothelial cells. CONCLUSIONS: Adenovirus-mediated transfer of the RAP gene provides an efficient way of transiently increasing the fibrinolytic capacity of mesothelial cells.

Aberrant fibrin formation and cross-linking of fibrinogen Nieuwegein, a variant with a shortened Aalpha-chain, alters endothelial capillary tube formation.

A congenital dysfibrinogenemia, fibrinogen(Nieuwegein), was discovered in a young man without any thromboembolic complications or bleeding. A homozygous insertion of a single nucleotide (C) in codon Aalpha 453 (Pro) introduced a stop codon at position 454, which resulted in the deletion of the carboxyl-terminal segment Aalpha 454-610. The ensuing unpaired cysteine at Aalpha 442 generated fibrinogen-albumin complexes of different molecular weights. The molecular abnormalities of fibrinogen(Nieuwegein) led to a delayed clotting and a fibrin network with a low turbidity. Electron microscopy confirmed that thin fibrin bundles were organized in a fine network. The use of fibrinogen(Nieuwegein)-derived fibrin (fibrin(Nieuwegein)) in an in vitro angiogenesis model resulted in a strong reduction of tube formation. The ingrowth of human microvascular endothelial cells (hMVEC) was independent of alpha(v)beta(3), indicating that the reduced ingrowth is not due to the absence of the RGD-adhesion site at position Aalpha 572-574. Rather, the altered structure of fibrin(Nieuwegein) is the cause, since partial normalization of the fibrin network by lowering the pH during polymerization resulted in an increased tube formation. Whereas factor XIIIa further decreased the ingrowth of hMVEC in fibrin(Nieuwegein), tissue transglutaminase (TG), which is released in areas of vessel injury, did not. This is in line with the absence of the cross-linking site for TG in the alpha-chains of fibrinogen(Nieuwegein). In conclusion, this newly discovered congenital dysfibrinogenemia has a delayed clotting time and leads to the formation of an altered fibrin structure, which could not be cross-linked by TG and which is less supportive for ingrowth of endothelial cells.

Steroids and cytokines in endometrial angiogenesis.

Angiogenesis, the formation of new blood vessels from pre-existing ones, occurs physiologically in the endometrium and pathologically e.g. during tumour growth. Sex-steroid hormones affect angiogenesis in the endometrium during the menstrual cycle and might be implicated in cancer angiogenesis. Little information is presently available regarding the exact mechanisms by which these steroids exert their function on the process of both physiological and pathological angiogenesis. In this overview a survey is given of factors important for angiogenesis and of the effects of steroids on the expression of these factors. We have focused on endometrial angiogenesis, because the endometrium is unique in its cyclic growth pattern for which angiogenesis is indispensable. Stimulators and inhibitors of endometrial angiogenesis, that have been found (or suggested) to respond to ovarian steroids, are discussed These factors include vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs), tumour necrosis factor a (TNF-alpha), erythropoietin (Epo) and trombospondin-1 (TSP-1). Also, the influence of steroids on the expression of matrixdegrading proteases, in particular the plasminogen activator/plasmin system and matrixdegrading metalloproteinases (MMPs) are reviewed, because these proteases play an important role in the migration and invasion of endothelial cells during the process of angiogenesis. An insight into the effects of steroids on endometrial angiogenesis may be helpful to understand and anticipate the potential stimulatory and inhibitory effect of various steroids on angiogenesis in other tissues, in particular tumours.

Use of fibrinolytic agents in the prevention of postoperative adhesion formation.

OBJECTIVE: To review the events leading to the formation of adhesions, to describe the development of fibrinolytic agents, to review more than a century of research on the use of fibrinolytic agents in adhesion prevention, and to look at future aspects of adhesion prevention. RESULTS: A better understanding of the pathogenesis of adhesion formation has resulted in the use of fibrinolytic agents in their prevention. Fibrinolytic agents promote fibrinolytic activity during the early period after peritoneal trauma during which an increased formation of fibrin is seen in combination with a deficiency of endogenous fibrinolytic activity. Initially, chemical attacks on fibrin (fibrolysin and hypertonic glucose), foreign digestive ferments (pepsin, trypsin, and papain), and stimulation of intraperitoneal leukocytosis (amniotic fluid) were used. Development of new thrombolytic agents was soon followed by experiments in animal adhesion models and clinical studies to examine their antiadhesion properties. Plasmin preparations (plasmin, actase, and fibrinolysin) and plasmin activators (streptokinase, urokinase, and tissue-type plasminogen activator) were found to be efficacious in preventing adhesion formation in the greater part of reviewed animal and clinical studies. CONCLUSION(S): From the current literature, it can be concluded that postoperative intraperitoneal administration of thrombolytic agents can significantly decrease adhesion formation. Given the large number of experimental studies in animals, future studies should focus on the clinical use of fibrinolytic agents in the prevention of postsurgical adhesion formation.

Oral, but not transdermal, administration of estrogens lowers tissue-type plasminogen activator levels in humans without affecting endothelial synthesis.

Oral estrogen administration decreases plasma levels of tissue-type plasminogen activator (tPA), which may be explained by a decrease in endothelial tPA synthesis, an increase in its hepatic clearance, or both. In the present study, we determined (1) differences between oral (ie, via the liver) ethinyl estradiol and transdermal (ie, systemic) 17beta-estradiol administration on plasma antigen levels of tPA and plasminogen activator inhibitor type-1 before and after 4 months of hormone administration and (2) effects on endothelial tPA synthesis, by measuring the local increase in plasma tPA during venous occlusion of the upper extremity. Thirty transsexual males (median age 32 years, range 20 to 44 years ) were randomly assigned to either oral ethinyl estradiol (n=15) or transdermal 17beta-estradiol (n=15); both treatments included the antiandrogen cyproterone acetate (CA). Ten males were treated with CA alone. Seventeen transsexual females (median age 27 years, range 18 to 37 years) were treated with intramuscular testosterone esters. Only oral ethinyl estradiol plus CA but neither transdermal 17beta-estradiol plus CA, nor oral CA, nor parenteral testosterone lowered plasma tPA and plasminogen activator inhibitor-1 (P<0.001 for both). tPA release during venous occlusion was not affected by oral ethinyl estradiol plus CA in males (P=0.52) or by parenteral testosterone in females (P=0.89). These data are consistent with a previous observation, in rodents, that the decrease in tPA after oral estrogen administration can be explained by an increase in hepatic tPA clearance, leaving endothelial tPA synthesis unchanged, and suggest that these mechanisms also explain the decrease in tPA in humans.

Increased clearance explains lower plasma levels of tissue-type plasminogen activator by estradiol: evidence for potently enhanced mannose receptor expression in mice.

Several clinical studies have demonstrated an inverse relationship between circulating levels of estrogen and tissue-type plasminogen activator (t-PA). The present study was designed to test the hypothesis that estrogens lower plasma levels of t-PA by increasing its clearance from the bloodstream. 17alpha-Ethinyl estradiol (EE) treatment resulted in a significant increase in the clearance rate of recombinant human t-PA in mice (0.46 mL/min in treated mice v 0. 32 mL/min in controls; P <.01). The clearance of endogenous, bradykinin-released t-PA in rats was also significantly increased after EE treatment (area under the curve [AUC], 24.9 ng/mL. min in treated animals v 31.9 ng/mL. min in controls; P <.05). Two distinct t-PA clearance systems exist in vivo: the low-density lipoprotein receptor-related protein (LRP) on liver parenchymal cells and the mannose receptor on mainly liver endothelial cells. Inhibition of LRP by intravenous injection of receptor-associated protein (RAP) as a recombinant fusion protein with Salmonella japonicum glutathione S-transferase (GST) significantly retarded t-PA clearance in control mice (from 0.41 to 0.25 mL/min; n = 5, P <.001) and EE-treated mice (from 0.66 to 0.35 mL/min; n = 5, P <.005), but did not eliminate the difference in clearance capacity between the 2 experimental groups. Similar results were obtained in mice in which LRP was inhibited via overexpression of the RAP gene in liver by adenoviral gene transduction. In contrast, administration of mannan, a mannose receptor antagonist, resulted in identical clearances (0.22 mL/min in controls and 0.24 mL/min in EE-treated mice). Northern blot analysis showed a 6-fold increase in mannose receptor mRNA expression in the nonparenchymal liver cells of EE-treated mice, whereas the parenchymal LRP mRNA levels remained unchanged. These findings were confirmed at the protein level by ligand blotting and Western blotting analysis. Our results demonstrate that EE treatment results in increased plasma clearance rate of t-PA via induction of the mannose receptor and could explain for the inverse relationship between estrogen status and plasma t-PA concentrations as observed in humans.

Hormone replacement therapy.

Hormone replacement therapy is increasingly being used for purposes unrelated to the alleviation of menopausal symptoms, such as the prevention of osteoporosis and cardiovascular disease. Clinical trials, however, suggest that the one drug/many purposes concept may be too optimistic. The availability of new estrogen-like compounds and the discovery of a second estrogen receptor have opened new possibilities for more specific drug development.

Fibrinolytic activity of human mesothelial cells is counteracted by rapid uptake of tissue-type plasminogen activator.

BACKGROUND: Human mesothelial cells (HMCs) have an important role in maintaining an adequately functioning fibrinolytic system in the peritoneal cavity by secreting the fibrinolytic enzymes tissue-type and urokinase-type plasminogen activator (t-PA and u-PA), as well as a specific PA inhibitor, PA inhibitor type 1 (PAI-1). In this study, we investigated whether the fibrinolytic capacity of HMCs is further counterbalanced by rapid uptake of t-PA and u-PA from the medium. METHODS: Cultured HMCs were used to study the uptake and degradation of radiolabeled t-PA and u-PA in the absence or presence of an inhibitor of cellular protein degradation, chloroquine, and of specific receptor antagonists. Northern blotting and ligand-blotting techniques were applied to demonstrate the presence of specific receptors for binding of t-PA and u-PA. RESULTS: At 37 degreesC, HMCs rapidly internalized and degraded 125I-t-PA and 125I-u-PA, which could be inhibited by an excess of unlabeled t-PA and u-PA, respectively, and by the lysosomotropic agent chloroquine. Northern blot analysis showed the expression of low-density lipoprotein (LDL) receptor-related protein (LRP), very low density lipoprotein (VLDL) receptor, and u-PA receptor. The addition of recombinant 39 kDa receptor-associated protein (RAP; an inhibitor of LRP and VLDL receptor) almost completely blocked the degradation of t-PA and partly that of u-PA. RAP ligand blotting demonstrated predominantly the presence of LRP, suggesting a major role for the LRP in mediating uptake and degradation of t-PA in HMCs. Endocytosis of u-PA occurs via two different pathways. After binding to u-PA receptor, a RAP-inhibitable and a non-RAP-inhibitable route for u-PA degradation was demonstrated. Tumor necrosis factor alpha (TNFalpha) diminished the fibrinolytic activity of HMCs by decreasing t-PA and increasing PAI-1 synthesis. The fall in t-PA levels could be counteracted by inhibiting t-PA degradation by either RAP or chloroquine. Interestingly, chloroquine also quenched the TNFalpha-induced changes in t-PA and PAI-1 mRNA levels. Using TNFalpha mutants and agonistic or blocking monoclonal antibodies specific for the TNF receptors p55 and p75, we found evidence that chloroquine interfered with the activation of the TNF receptor p55 and/or its intracellular signaling route. CONCLUSIONS: Receptor-mediated endocytosis plays a crucial role in regulating the fibrinolytic capacity of HMCs by its participation in the degradation of t-PA and u-PA, and in the TNFalpha-induced decrease in t-PA and the increase in PAI-1 expression.

On the role of c-Jun in the induction of PAI-1 gene expression by phorbol ester, serum, and IL-1alpha in HepG2 cells.

We have characterized the regulation of plasminogen activator inhibitor-1 (PAI-1) gene expression by phorbol 12-myristate 13-acetate (PMA), serum, and interleukin-1alpha (IL-1alpha) in the human hepatoma cell line HepG2. PMA, serum, and IL-1alpha induced a rapid and transient 28-fold (PMA), 9-fold (serum), and 23-fold (IL-1alpha) increase in PAI-1 mRNA, peaking after approximately 4 hours. These inductions of PAI-1 mRNA accumulation were reduced by pretreatment of the HepG2 cells with the protein tyrosine kinase inhibitor genistein. Conversely, stimulation of tyrosine phosphorylation by sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, caused an increase in PAI-1 mRNA levels. The effects of PMA, serum, and IL-1alpha on PAI-1 mRNA expression have been compared with their ability to modulate the expression of a chloramphenicol acetyltransferase (CAT) reporter plasmid, which was under control of the -489 to +75 region of the PAI-1 promoter, and stably transfected into HepG2 cells. This region of the PAI-1 promoter was previously found to contain a tetradecanoyl phorbol acetate-response element (TRE; between -58 and -50) necessary for PMA responsiveness and with a high affinity for c-Jun homodimers. Whereas incubation of these transfected HepG2 cells with PMA and serum showed an induction profile of CAT mRNA similar to that of PAI-1 mRNA, hardly any induction of CAT mRNA was found with IL-1alpha. In line with these findings, IL-1alpha poorly induced c-Jun homodimer binding to the PAI-1 TRE in gel mobility-shift assays. Pretreatment of HepG2 cells with the protein kinase C inhibitor Ro 31-8220 or the mitogen-activated protein kinase kinase (MAPKK)1,2 activity blocker PD98059 selectively suppressed the induction of PAI-1 (and CAT) expression by PMA, but not that by IL-1alpha. In contrast, the protein tyrosine kinase inhibitor herbimycin A blocked PAI-1 mRNA induction by IL-1 alpha only. We propose 2 separate PAI-1 inductory pathways for PMA and IL-1alpha in HepG2, both involving protein tyrosine kinase activation; the serum-induced signaling pathway may (partially) overlap with the PMA-activated protein kinase C/mitogen-activated protein kinase kinase pathway, leading to c-Jun homodimer binding to the PAI-1 TRE.

Retinoids increase human apo C-III expression at the transcriptional level via the retinoid X receptor. Contribution to the hypertriglyceridemic action of retinoids.

Hypertriglyceridemia is a metabolic complication of retinoid therapy. In this study, we analyzed whether retinoids increase the expression of apo C-III, an antagonist of plasma triglyceride catabolism. In men, isotretinoin treatment (80 mg/d; 5 d) resulted in elevated plasma apo C-III, but not apo E concentrations. In human hepatoma HepG2 cells, retinoids increased apo C-III mRNA and protein production. Transient transfection experiments indicated that retinoids increase apo C-III expression at the transcriptional level. This increased apo C-III transcription is mediated by the retinoid X receptor (RXR), since LG1069 (4-[1-(5,6,7,8-tetrahydro-3,5,5,8, 8-pentamethyl-2-naphtalenyl)ethenyl]benzoic acid), a RXR-specific agonist, but not TTNPB ((E)- 4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethyl-2-naphtalenyl)propenyl]benzoic acid), a retinoic acid receptor (RAR)-specific agonist, induced apo C-III mRNA in HepG2 cells and primary human hepatocytes. Mutagenesis experiments localized the retinoid responsiveness to a cis-element consisting of two imperfect AGGTCA sequences spaced by one oligonucleotide (DR-1), within the previously identified C3P footprint site. Cotransfection assays showed that RXR, but not RAR, activates apo C-III transcription through this element either as a homo- or as a heterodimer with the peroxisome proliferator-activated receptor. Thus, apo C-III is a target gene for retinoids acting via RXR. Increased apo C-III expression may contribute to the hypertriglyceridemia and atherogenic lipoprotein profile observed after retinoid therapy.

Effect of steroid hormones and retinoids on the formation of capillary-like tubular structures of human microvascular endothelial cells in fibrin matrices is related to urokinase expression.

Angiogenesis, the formation of new capillary blood vessels, is a feature of a variety of pathological processes. To study the effects of a specific group of hormones (all ligands of the steroid/retinoid/thyroid hormone receptor superfamily) on the angiogenic process in humans, we have used a model system in which human microvascular endothelial cells from foreskin (hMVEC) are cultured on top of a human fibrin matrix in the presence of basic fibroblast growth factor and tumor necrosis factor-alpha. This model mimics the in vivo situation where fibrin appears to be a common component of the matrix present at sites of chronic inflammation and tumor stroma. Our results show that testosterone and dexamethasone are strong inhibitors and all-trans retinoic acid (at-RA) and 9-cis retinoic acid (9-cis RA) are potent stimulators of the formation of capillary-like tubular structures. These effects are mediated by their respective nuclear hormone receptors as demonstrated by the use of specific synthetic receptor agonists and antagonists. 17beta-estradiol, progesterone, and 1,25-dihydroxyvitamin D3 did not affect or only weakly affected in vitro angiogenesis, which may be related to the lack of significant nuclear receptor expression. Although hMVEC express both thyroid hormone receptors alpha and beta, no effect of thyroid hormone on tube formation was found. The effects of testosterone, dexamethasone, at-RA, and 9-cis RA on tube formation were accompanied by parallel changes in urokinase-type plasminogen activator (u-PA) expression, at both mRNA and antigen levels. Exogenous suppletion of the medium with single chain u-PA enhances tube formation in our in vitro model, whereas quenching of u-PA activity (but not of tissue-type plasminogen activator activity) or of u-PA binding to u-PA receptor by specific antibodies suppressed basal and retinoid-stimulated tube formation. Moreover, addition of scu-PA to testosterone- or dexamethasone-treated hMVEC restored the suppressed angiogenic activity for a substantial part. Aprotinin, an inhibitor of plasmin activity, completely inhibited tube formation, indicating that the proteolytic properties of the u-PA/u-PA receptor complex are crucial in this process. Our results show that steroid hormones (testosterone and dexamethasone) and retinoids have strong, but opposite effects on tube formation in a human in vitro model reflecting pathological angiogenesis in the presence of fibrin and inflammatory mediators. These effects can be explained by hormone-receptor-mediated changes in u-PA expression, resulting in enhanced local proteolytic capacity of the u-PA/u-PA receptor complex.

Effects of gemfibrozil and ciprofibrate on plasma levels of tissue-type plasminogen activator, plasminogen activator inhibitor-1 and fibrinogen in hyperlipidaemic patients.

Evaluation of fibrate treatment in humans has focused primarily on its anti-lipidaemic effects. A potentially favourable haemostasis-modulating activity of fibrates has also been recognized but the data are not consistent. We sought to learn more about this variability by examining the effects of gemfibrozil and ciprofibrate on plasma levels of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and fibrinogen in primary hyperlipidaemic patients after six and twelve weeks of treatment using different assay systems for PAI-1 and fibrinogen. Although both fibrates effectively lowered triglyceride and cholesterol levels, no effect on the elevated baseline antigen levels of t-PA and PAI-1 was observed after fibrate treatment. However, both fibrates influenced plasma fibrinogen levels, albeit in a different way. Fibrinogen antigen levels were elevated by 17.6% (p <0.05) and 24.3% (p <0.001) with gemfibrozil after six and twelve weeks, respectively, whereas with ciprofibrate there was no effect. Using a Clauss functional assay with either a mechanical end point or a turbidity-based end point, no significant change in fibrinogen levels was seen after six weeks of gemfibrozil treatment. However, after twelve weeks, gemfibrozil enhanced functional fibrinogen levels by 7.2% (p <0.05) as assessed by the Clauss mechanical assay, but decreased functional fibrinogen levels by 12.5% (p <0.0001) when a Clauss assay based on turbidity was used. After six or twelve weeks of ciprofibrate treatment, functional fibrinogen levels were decreased by 10.1% (p <0.001) and 10.5% (p <0.0001), respectively on the basis of Clauss mechanical and by 14.2% (p <0.001) and 28.2% (p <0.0001), respectively with the Clauss turbidimetric assay. A remarkable and consistent finding with both fibrates was the decrease in functionality of fibrinogen as assessed by the ratio of functional fibrinogen (determined by either of the two Clauss assays) to fibrinogen antigen. Taken together, our results indicate that at least part of the variability in the effects of fibrates on haemostatic parameters can be explained by intrinsic differences between various fibrates, by differences in treatment period and/or by the different outcomes of various assay systems. Interestingly, the two fibrates tested both reduced the functionality of fibrinogen.

Differences in metabolism and isomerization of all-trans-retinoic acid and 9-cis-retinoic acid between human endothelial cells and hepatocytes.

Retinoic acid stimulates the expression of tissue-type plasminogen activator (t-PA) in vascular endothelial cells in vitro and enhances t-PA levels in plasma and tissues in vivo. Compared with the in vivo situation, high retinoic acid concentrations are required to induce optimally t-PA expression in vitro. These findings led us to study retinoic acid metabolism in cultured human endothelial cells. For comparison, these studies were also performed in the human hepatoma cell line, HepG2, and key experiments were repeated with human primary hepatocytes. Both hepatocyte cultures gave very similar results. Human endothelial cells were shown to possess an active retinoic acid metabolizing capacity, which is quantitatively comparable to that of hepatocytes, but different from that of hepatocytes in several qualitative aspects. Our results demonstrate that all-trans-retinoic acid is quickly metabolized by both endothelial cells and hepatocytes. All-trans-retinoic acid induces its own metabolism in endothelial cells but not in hepatocytes. 9-cis-Retinoic acid is degraded slowly by endothelial cells, whereas hepatocytes metabolize 9-cis-retinoic acid very quickly. Furthermore, our data show that hepatocytes, but not endothelial cells, detectably isomerise all-trans-retinoic acid to 9-cis-retinoic acid and vice versa. In both endothelial cells and hepatocytes all-trans-retinoic acid metabolism was inhibitable by the cytochrome P-450 inhibitors liarozole (10 microM) and ketoconazole (10 microM), albeit to different extents and with different specificities. In the presence of the most potent retinoic acid metabolism inhibitor in endothelial cells, liarozole, at least 10-fold lower all-trans-retinoic acid concentrations were required than in the absence of the inhibitor to obtain the same induction of t-PA. In conclusion, our results clearly demonstrate that all-trans-retinoic acid and 9-cis retinoic acid are actively but differently metabolized and isomerised by human endothelial cells and hepatocytes. The rapid metabolism of retinoic acid explains the relatively high concentrations of retinoic acid required to induce t-PA in cultured endothelial cells.