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T Kooistra

Publications and source records attributed to T Kooistra.

At least 37 records · Page 2Linked to original sources
PubMed1999

D-glucose increases the synthesis of tissue-type plasminogen activator (t-PA) in human peritoneal mesothelial cells.

Physical and chemical irritation of the peritoneum through glucose-based hyperosmolar dialysis solutions results in a nonbacterial serositis with fibrinous exudation. Thereby, human peritoneal mesothelial cells (HMC) play an important role in maintaining the balance between the peritoneal generation and degradation of fibrin by expressing the fibrinolytic enzyme tissue-type plasminogen activator (t-PA) as well as the specific plasminogen activator inhibitor-1 (PAI-1). In this study, we analyzed the effect of D-glucose and metabolically inert monosaccharides on the synthesis of t-PA and PAI-1 in cultured HMC. Incubation of HMC with D-glucose or the metabolically inert monosaccharides mannitol and L-glucose (5-90 mM) resulted in a time- and concentration-dependent increase in t-PA mRNA expression and antigen secretion without affecting PAI-1 synthesis. A similar effect was evident when HMC were first exposed sequentially to pooled spent peritoneal dialysis effluent for up to 4 hours, and subsequently incubated for 20 hours in control medium. The stimulating effect of high D-glucose on t-PA expression in HMC was prevented by treating the cells with different protein kinase C (PKC) inhibitors (Ro 31-8220, Gö 6976), but could not be mimicked by the PKC-activating phorbol ester PMA, indicating that this effect of high glucose is dependent on PKC activity, but not mediated through PKC activation. Also, using specific inhibitors (PD 98059, SB 203580) and activators (PMA, anisomycin, IL-1alpha) of the major routes of the mitogen-activated protein kinases (MAPKs) cascade, we found no evidence for a role of this cascade in regulating t-PA expression in HMC. We conclude that hyperosmolarity induces t-PA (but not PAI-1) in HMC via a regulatory mechanism that requires active PKC, but that does not involve a major pathway in the MAPK cascade.

Cells, Cultured↗
PubMed1999

Relationship between visceral fat and PAI-1 in overweight men and women before and after weight loss.

This study was aimed at evaluating the relationship between visceral fat accumulation and plasma plasminogen activator inhibitor-1 (PAI-1) levels in healthy, obese men and women undergoing weight loss therapy. The subjects, 25 men and 25 premenopausal women, aged between 26 and 49 years, with an initial body mass index between 28 and 38 kg/m2, received a controlled diet for 13 weeks providing a 4.2 MJ/day energy deficit. Magnetic resonance imaging was used to measure visceral and subcutaneous abdominal fat. Our results show that before weight loss visceral fat was significantly correlated with PAI-1 in men (r = 0.45; p<0.05), but not in women (r = -0.15; ns). The association between visceral fat and PAI-1 in men remained significant after adjustment for age and total fat mass, and multiple linear regression analysis showed a significant independent contribution of visceral fat to plasma PAI-1 levels. Both visceral fat areas and PAI-1 levels decreased significantly with weight loss in both men and women. Changes in visceral fat area were related to changes in PAI-1 in women (r = -0.43; p = 0.05) but not in men (r = -0.01; ns); however, this association in women disappeared after adjustment for total fat mass. We conclude that there is a relationship between visceral fat and PAI-1 in obese men but not in obese women, and that PAI-1 levels decrease substantially (52%) by weight loss, but this change is not related to changes in visceral fat mass per se.

Abdomen↗
PubMed1998

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.

Adult↗
PubMed1998

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.

Aprotinin↗
PubMed1998

Visceral fat accumulation is an important determinant of PAI-1 levels in young, nonobese men and women: modulation by cross-sex hormone administration.

Increased plasminogen activator inhibitor type-1 (PAI-1) levels, leading to impaired fibrinolysis, are associated with increased visceral fat in middle-aged and obese subjects. It is unknown, however, whether this association is independent of other disturbances clustered in the insulin resistance syndrome. We analyzed this association in young, nonobese transsexual men and women before and after administration of cross-sex steroids, which potentially influence many elements of the insulin resistance syndrome, including PAI-1 levels and visceral fat accumulation. We assessed the visceral fat area (by MRI); total body fat; insulin sensitivity (with a glucose clamp technique); and plasma levels of PAI-1, insulin, and triglycerides in young (<37 years old), nonobese (body mass index <28 kg/m2), healthy men (n=18) and women (n=15) before and after 12 months of cross-sex hormone administration. Men were treated with ethinyl estradiol 100 microgram/d plus cyproterone acetate 100 mg/d, and women were treated with testosterone esters 250 mg IM every 2 weeks. At baseline, only visceral fat area was significantly correlated with plasma PAI-1 levels in both men (r=0.57, P=0.03) and women (r=0.59, P=0.03). In multivariate linear regression analysis, this association was independent of total body fat, insulin sensitivity, and plasma levels of triglycerides and insulin. After 12 months of cross-sex hormone administration, the plasma PAI-1 levels were no longer correlated with visceral fat (which had increased). We conclude that in young, nonobese men and women, visceral fat area is an important determinant of plasma PAI-1 levels. After cross-sex hormone administration, this association was no longer demonstrable.

Adipose Tissue↗
PubMed1998

The effects of sex steroids on plasma levels of marker proteins of endothelial cell functioning.

We studied thirteen male-to-female (M-->F) and ten female-to-male (F-->M) transsexuals who, for four months, received cross-sex treatment with, respectively, ethinylestradiol and cyproterone acetate, and with testosterone esters. We assessed the effects of treatment on plasma levels of tissue-type plasminogen activator (tPA), von Willebrand factor (vWF), vWF-propeptide (vWF:AgII) and big-endothelin-1 (big-ET-1), four proteins that are markers of endothelial cell functioning. We also measured urokinase-type PA (uPA) and plasminogen activator inhibitor-type 1 (PAI-1), which may not be endothelium-derived but share major clearance pathways with tPA. In M-->F transsexuals, mean plasma levels of tPA (minus 4.4 ng/ml), big-ET-1 (minus 0.8 pg/ml), uPA (minus 0.5 ng/ml) and PAI-1 (minus 26 ng/ml) decreased (all Ps < or =0.02). The level of vWF increased (plus 24%; P = 0.005), while vWF: AgII did not change (P = 0.49). In F-->M transsexuals, levels of big-ET-1 increased (plus 0.4 pg/ml; P = 0.02), while tPA, uPA and PAI-1 did not change (all Ps >0.25). In this group vWF decreased (minus 14%; P = 0.06), but vWF:AgII did not change (P = 0.38). Estrogens and androgens have clear effects on plasma levels of endothelial marker proteins. The mechanisms behind these effects are complex and appear to involve both altered secretion (big-ET-1) and processing and/or clearance (vWF and possibly tPA). Therefore, effects of hormones on the levels of endothelial marker proteins do not necessarily reflect changes in endothelial cell functioning, at least with regard to changes in vWF level associated with the oral administration of high doses of ethinylestradiol and cyproterone acetate to healthy men and the parenteral administration of testosterone to healthy women.

Biomarkers↗
PubMed1998

Fibrate-modulated expression of fibrinogen, plasminogen activator inhibitor-1 and apolipoprotein A-I in cultured cynomolgus monkey hepatocytes -- role of the peroxisome proliferator-activated receptor-alpha.

Fibrates are used to lower plasma triglycerides and cholesterol levels in hyperlipidemic patients. In addition, fibrates have been found to alter the plasma concentrations of fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and apolipoprotein A-I (apo A-I). We have investigated the in vitro effects of fibrates on fibrinogen, PAI-1 and apo A-I synthesis and the underlying regulatory mechanisms in primary monkey hepatocytes. We show that fibrates time- and dose-dependently increase fibrinogen and apo A-I expression and decrease PAI-1 expression in cultured cynomolgus monkey hepatocytes, the effects demonstrating different potency for different fibrates. After three consecutive periods of 24 h the most effective fibrate. ciprofibrate (at 1 mmol/l), increased fibrinogen and apo A-I synthesis to 356% and 322% of control levels, respectively. Maximum inhibition of PAI-1 synthesis was about 50% of control levels and was reached by 1 mmol/l gemfibrozil or ciprofibrate after 48 h. A ligand for the retinoid-X-receptor (RXR), 9-cis retinoic acid, and specific activators of the peroxisome proliferator-activated receptor-alpha (PPARalpha), Wy14,643 and ETYA, influenced fibrinogen, PAI-1 and apo A-I expression in a similar fashion, suggesting a role for the PPARalpha/RXRalpha heterodimer in the regulation of these genes. When comparing the effects of the various compounds on PPARalpha transactivation activity as determined in a PPARalpha-sensitive reporter gene system and the ability of the compounds to affect fibrinogen, PAI-1 and apo A-I antigen production, a good correlation (r=0.80; p <0.01) between PPARalpha transactivation and fibrinogen expression was found. Apo A-I expression correlated only weakly with PPARalpha transactivation activity (r=0.47; p=0.24), whereas such a correlation was absent for PAI-1 (r=0.03; p=0.95). These results strongly suggest an involvement of PPARalpha in the regulation of fibrinogen gene expression.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1997

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.

Alitretinoin↗
PubMed1997

Cell-type specific DNA-protein interactions at the tissue-type plasminogen activator promoter in human endothelial and HeLa cells in vivo and in vitro.

Tissue-type plasminogen activator (t-PA) gene expression in human endothelial cells and HeLa cells is stimulated by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) at the level of transcription. To study the mechanism of transcriptional regulation, we have characterized a segment of the t-PA gene extending from -135 to +100 by in vivo footprinting analysis [dimethyl sulphate (DMS) method] and gel mobility shift assay. In vivo footprinting analysis revealed changes in cleavage pattern in five distinct promoter elements in both endothelial cells and HeLa cells, including a PMA-responsive element (TRE), a CTF/NF-1 binding site and three GC-boxes, and an altered cleavage pattern of the TRE and CTF/NF-1 element after PMA treatment of HeLa cells. Although endothelial cells and HeLa cells differed in the exact G residues protected by nuclear proteins,in vitro bandshift analysis showed that nuclear protein binding to the t-PA promoter was qualitatively and quantitatively very similar in both cell types, except for the TRE. Protein binding to the TRE under non- stimulated conditions was much higher in human endothelial cells than in HeLa cells, and this TRE-bound protein showed a lower dissociation rate in the endothelial cells than in HeLa cells. In endothelial cells, the proteins bound to the TRE consisted mainly of the AP-1 family members JunD and Fra-2, while in HeLa cells predominantly JunD, FosB and Fra-2 were bound. The proteins bound to the other protected promoter elements were identified as SP-1 (GC-box II and III) and CTF/NF-1 (CTF/NF-1 binding site). After PMA treatment of the cells, AP-1 and SP-1 binding was increased two-fold in endothelial cell nuclear extracts and >20-fold in HeLa nuclear extracts. In the endothelial cells, all Jun and Fos forms (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 and Fra-2) were part of the AP-1 complex after PMA induction. In HeLa cells, the complex consisted predominantly of c-Jun and the Fos family members FosB and Fra-2. In the light of previous studies involving mutational analysis of the human and murine t-PA promoter our results underline an important role of the five identified promoter regions in basal and PMA-stimulated t-PA gene expression in intact human endothelial cells and HeLa cells. The small differences in DMS protection pattern and differences in the individual AP-1 components bound in endothelial cells and HeLa cells point to subtle cell-type specific differences in t-PA gene regulation.

CCAAT-Enhancer-Binding Proteins↗
PubMed1997

Studies on the mechanism of fibrate-inhibited expression of plasminogen activator inhibitor-1 in cultured hepatocytes from cynomolgus monkey.

Fibrates are widely used drugs in hyperlipidemic disorders. In addition to lowering serum triglyceride levels, fibrates have also been shown to reduce elevated plasma plasminogen activator inhibitor-1 (PAI-1) levels in vivo. We demonstrate that fibrates suppress PAI-1 synthesis in cultured cynomolgus monkey hepatocytes in a concentration-dependent way (0.1 to 1.0 mmol/L) and independent of their lipid-lowering effect. Different fibrates showed different potency in suppressing PAI-1 production: gemfibrozil and clofibric acid, at a concentration of 1 mmol/L, reduced PAI-1 synthesis over 24 hours to 52 +/- 20% and 60 +/- 5%, while clofibrate and bezafibrate lowered PAI-1 synthesis to only 86 +/- 17% and 84 +/- 15% of control values, respectively. These changes in PAI-1 production by fibrates correlated with changes in PAI-1 mRNA levels and were also visible at the level of gene transcription. Fibrates did not lower basal PAI-1 synthesis but attenuated an acceleration of PAI-1 production during culture. The suppressing effect of fibrates on PAI-1 synthesis could not be mimicked with activators or inhibitors of protein kinase C (PKC). Furthermore, fibrates did not inhibit the increase in PAI-1 synthesis induced by epidermal growth factor or transforming growth factor-beta. These results make mechanisms involving PKC modulation or growth factor receptor inactivation as a mode of action of fibrates unlikely. The suppressing effect of fibrates on PAI-1 synthesis could involve the nuclear receptor peroxisome proliferator-activated receptor (PPAR) and its heterodimeric partner, the retinoid X receptor (RXR). The alpha forms of PPAR and RXR were both found to be expressed in cynomolgus monkey hepatocytes. the ligand for RXR alpha, 9-cis retinoic acid, suppressed PAI-1 synthesis to the same extent as gemfibrozil, while a combination of gemfibrozil and 9-cis retinoic acid had no more effect on PAI-1 synthesis than any of these compounds alone at optimal concentrations. In conclusion, fibrates downregulate an induced PAI-1 production in cynomolgus monkey hepatocytes independent of a decrease in triglyceride levels. A possible involvement of PPAR alpha/RXR alpha in this down-regulation is discussed.

Animals↗
PubMed1997

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.

Calcium↗
PubMed1997

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.

Adult↗
PubMed1997

Mechanism of the slow induction of apolipoprotein A-I synthesis by retinoids in cynomolgus hepatocytes: involvement of retinoic acid and retinoid X receptors.

We showed previously that retinoids stimulate apolipoprotein A-I (apoA-I) synthesis in cultured cynomolgus hepatocytes only after a 24-h lag phase. Here we report on the biochemical background of the slow response, the requirement for high retinoic acid concentrations, and the involvement of different retinoid receptors. The time course of the effect of 10 microM all-trans retinoic acid (at-RA) on apoA-I mRNA levels and protein secretion were comparable, i.e., minor increases were observed after a 24-h incubation and mRNA levels were increased 2.2- and 3.5-fold after 48 h and 72 h, respectively. In contrast, apoA-I gene transcription was already increased (2.6-fold) after a 4-h incubation with 10 microM at-RA. At-RA disappeared rapidly from the cultures: after 2 h of incubation 40% of the added amount was left and after 24 h only 2%. RAR beta mRNA and gene expression were increased after incubation with 10 microM at-RA, whereas RAR alpha and RXR alpha mRNA levels and expression remained unchanged. No transcriptional activity and mRNA for other retinoid receptors were detectable. Both RAR-selective (TTNPB) and RXR-selective (3-methyl-TTNEB) agonists induced apoA-I synthesis at 1 and 10 microM. These results show that i) the slow increase in apoA-I secretion is caused by a slow increase of its mRNA level; ii) the apoA-I gene transcription in cynomolgus hepatocytes is induced rapidly by retinoids; iii) the added at-RA disappeared rapidly from the cultures, explaining the necessity for high initial concentrations; iv) RLR alpha and/or RAR beta and RXR alpha are involved in the activation of apoA-I expression by retinoids.

Alitretinoin↗
PubMed1996

Role of c-Jun and proximal phorbol 12-myristate-13-acetate-(PMA)-responsive elements in the regulation of basal and PMA-stimulated plasminogen-activator inhibitor-1 gene expression in HepG2.

Experiments were designed to clarify the role of c-Jun/c-Fos and of putative phorbol 12-myristate-13-acetate-(PMA)-responsive elements (TREs) in the induction of plasminogen-activator inhibitor 1 (PAI-1) gene transcription in the human hepatoma cell line HepG2 by activators of protein kinase C (PKC). Treatment of HepG2 cells with the phorbol ester PMA or serum rapidly and transiently increased c-Jun and c-Fos mRNA and protein levels prior to PAI-1 induction. This induction of PAI-1 gene transcription was found to be dependent on ongoing protein synthesis. An essential role of c-Jun and c-Fos in basal and PMA-stimulated transcription of the PAI-1 gene is demonstrated by our finding that antisense c-jun and c-fos oligodeoxynucleotides both strongly reduced basal and PMA-stimulated PAI-1 synthesis. Since it has already been shown that two TREs between positions -58 and -50 and between -79 and -72 of the PAI-1 promoter are essential for basal and PMA-induced PAI-1 promoter activity ([16]), we examined binding of nuclear proteins to these elements. The protein-binding activity to the TRE between positions -79 and -72 shows very strong PMA induction of an unknown factor, which is not related to c-Jun or c-Fos. The TRE binding between positions -58 and -50 forms two complexes, both containing c-Jun protein. The faster migrating complex primarily contains c-Jun homodimers. The amount of the faster migrating complex is enhanced more than 30-fold in PMA-treated cells, due to a strongly increased binding of c-Jun homodimers and, to a minor extent, to binding of c-Jun/c-Fos heterodimers. Dissociation experiments suggest that the c-Jun/c-Fos heterodimers bind with much lower affinity compared to binding of c-Jun homodimers. Together with the finding that both antisense c-jun and antisense c-fos oligodeoxynucleotides reduced the amount of c-Jun homodimer, we conclude that binding of c-Jun homodimer to the TRE at positions -58 to -50 is important in the basal activity and PMA activation of the PAI-1 promoter in HepG2 cells.

Base Sequence↗
PubMed1996

Stimulation of tissue-type plasminogen activator expression by retinoic acid in human endothelial cells requires retinoic acid receptor beta 2 induction.

We previously showed the involvement of retinoic acid receptor alpha (RAR alpha) in the induction of tissue-type plasminogen activator (t-PA) synthesis by RA in human umbilical vein endothelial cells (HUVECs). However, the rather slow onset of this induction of t-PA synthesis suggested an indirect role of RAR alpha. Here, we show that the protein synthesis inhibitor, cycloheximide completely blocks the induction of t-PA by RA, which points to the need of an intermediary protein in t-PA stimulation. This intermediary protein is likely to be RAR beta 2 on the basis of the following findings: (1) the induction of RAR beta by RA exactly precedes that of t-PA; (2) HUVECs with elevated RAR beta mRNA levels show an undelayed t-PA induction on stimulation with RA, and this response can be almost completely inhibited with an RAR antagonist; and (3) an antisense oligodeoxynucleotide against the translation initiation site of RAR beta 2 mRNA greatly reduces the t-PA induction by RA. Thus, induction of t-PA by RA in HUVECs involves a 2-step mechanism requiring induction of RAR beta 2 via RAR alpha, followed by induction of t-PA synthesis via RAR beta 2. Each of these steps is shown to have a different activation profile with RA and 9 cis RA.

Base Sequence↗
PubMed1996

Modulation of procoagulant and fibrinolytic system components of mesothelial cells by inflammatory mediators.

Human peritoneal mesothelial cells (HMC) play a critical role in maintaining the intraperitoneal balance between fibrinolysis and coagulation by expressing the fibrinolytic enzyme tissue-type plasminogen activator (t-PA) as well as a specific plasminogen activator inhibitor, PAI-1, and the procoagulant protein tissue factor (TF). Of three compounds known to stimulate t-PA synthesis in cultured human endothelial cells, i.e., retinoic acid, the protein kinase C activator 4 beta-phorbol 12-myristate 13-acetate (PMA), and sodium butyrate, only butyrate (1 mM) caused about a threefold increase in t-PA synthesis and mRNA expression in HMC after 24 h of incubation, without markedly affecting PAI-1 synthesis. PMA (10 nM) induced a threefold increase in urokinase-type plasminogen activator (u-PA) mRNA, but u-PA antigen levels in the HMC conditioned media remained below the detection level (0.5 ng/ml), possibly as a result of rapid uptake and degradation by the u-PA receptor. The u-PA receptor mRNA levels were about fivefold enhanced above control levels after PMA treatment of the cells. An increase in intracellular adenosine 3',5'-cyclic monophosphate levels by forskolin (10 microM) diminished t-PA and PAI-1 levels 43 and 17%, respectively. Among the inflammatory mediators tested [tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha, and bacterial lipopolysaccharide], TNF-alpha (10-1,000 U/ml) showed the strongest procoagulant effects. We found that the isoflavone compound genistein (25 micrograms/ml) prevented the TNF-alpha-induced expression of PAI-1 and TF while also slightly counteracting the decrease in t-PA synthesis. The protein kinase C inhibitor R0-318220 (3 microM) only moderately opposed the TNF-alpha-induced changes in t-PA and PAI-1 synthesis but completely prevented the induction of TF mRNA. In summary, our results demonstrate that t-PA synthesis in HMC is relatively insensitive to pharmacological stimulation. To restore the balance between fibrinolysis and coagulation under inflammatory conditions, attempts to interfere with the TNF-alpha-signaling pathway were more successful.

Blood Coagulation↗
PubMed1995

Involvement of retinoic acid receptor alpha in the stimulation of tissue-type plasminogen-activator gene expression in human endothelial cells.

Retinoids stimulate tissue-type plasminogen-activator (t-PA) gene expression in human endothelial cells, and are likely to do so by binding to one or more nuclear retinoid receptors. The present study was initiated to identify the retinoid receptor(s) involved in this process. Expression and regulation of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) were analyzed by Northern-blot analysis of total or poly(A)-rich RNA prepared from cultured human umbilical vein endothelial cells (HUVEC). Prior to any exposure to retinoids, HUVEC express two transcripts for RAR-alpha (3.6 kb and 2.8 kb), and low levels of transcripts for RAR-beta (3.4 kb and 3.2 kb) and RAR-gamma (3.3 kb and 3.1 kb). Two RXR subtypes were identified, RXR-alpha (4.8 kb) and, at a much lower concentration, RXR-beta (2.4 kb); no evidence for the presence of RXR-gamma was found. Furthermore, HUVEC express cellular retinol-binding protein I (CRBP-I) and cellular retinoic-acid-binding protein I (CRABP-I) mRNA. Exposure of HUVEC to 1 microM retinoic acid or the retinobenzoic acid, Ch55, led to the induction of the two RAR-beta mRNAs, RXR-alpha mRNA and CRBP-I mRNA, whereas the expression of the other receptor and CRABP-I transcripts did not change appreciably. Using retinoid analogues that bind preferentially to one of the RAR or RXR subtypes, we found evidence that RAR-alpha is involved in the retinoid-induced t-PA expression in HUVEC. This conclusion was strengthened by experiments in which blocking of RAR-alpha with a specific RAR-alpha antagonist, Ro 41-5253, was demonstrated to suppress the induction of t-PA by retinoids.

Benzoates↗