RESUMO
OBJECTIVE: To demonstrate, quantify, and mechanistically dissect antiatherosclerotic effects of fenofibrate besides lowering plasma cholesterol per se. METHODS AND RESULTS: ApoE*3Leiden transgenic mice received either a high-cholesterol diet (HC) or HC containing fenofibrate (HC+FF) resulting in 52% plasma cholesterol-lowering. In a separate low-cholesterol diet (LC) control group, plasma cholesterol was adjusted to the level achieved in the HC+FF group. Low plasma cholesterol alone (assessed in LC) resulted in reduced atherosclerosis (lesion area, number and severity) and moderately decreased plasma serum amyloid-A (SAA) concentrations. Compared with LC, fenofibrate additively reduced lesion area, number and severity, and the total aortic plaque load. This additional effect in HC+FF was paralleled by an extra reduction of aortic inflammation (macrophage content; monocyte adhesion; intercellular adhesion molecule-1 [ICAM-1], soluble vascular cell adhesion molecule-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), MCP-1, and NF-kappaB expression), systemic inflammation (plasma SAA and fibrinogen levels), and by an upregulation of plasma apoE levels. Also, enhanced expression of ABC-A1 and SR-B1 in aortic macrophages may contribute to the antiatherosclerotic effect of fenofibrate by promoting cholesterol efflux. CONCLUSIONS: Fenofibrate reduces atherosclerosis more than can be explained by lowering total plasma cholesterol per se. Impaired recruitment of monocytes/macrophages, reduced vascular and systemic inflammation, and stimulation of cholesterol efflux may all contribute to these beneficial effect of fenofibrate.
Assuntos
Apolipoproteínas E/metabolismo , Aterosclerose/sangue , Aterosclerose/patologia , Colesterol/sangue , Fenofibrato/farmacologia , Hipolipemiantes/farmacologia , Animais , Aorta/patologia , Valva Aórtica/patologia , Apolipoproteína E3 , Apolipoproteínas E/genética , Feminino , Inflamação/patologia , Lipídeos/sangue , Lipoproteínas/sangue , Camundongos , Camundongos TransgênicosRESUMO
Tissue-type plasminogen activator (t-PA) and its inhibitor, plasminogen activator inhibitor 1 (PAI-1), play an important role in regulating the fibrinolytic capacity of plasma. Both t-PA and PAI-1 are synthesized by the endothelium. We report that retinoic acid (vitamin A acid) and other retinoids rather specifically stimulate the production of t-PA by cultured human umbilical vein endothelial cells. Effective retinoids induced a dose-dependent (range: 0.01-50 microM) increase in the production of t-PA of maximally about six-fold, while simultaneously causing no or only a small increase (less than two-fold) of PAI-1. The effects on t-PA synthesis were apparent by 4-8 h, and reached maximal values after about 24-48 h of incubation with retinoid. The retinoid effect on t-PA production was accompanied by increased t-PA mRNA levels, without any parallel change in PAI-1 or GAPDH mRNA concentrations. The study also shows that modifications at the carboxyl group of retinoic acid are associated with a decrease in stimulatory potency. The stimulatory pathway appears to be identical for all retinoids but distinct from a pathway by which another strong inducer, sodium butyrate, induces t-PA synthesis in endothelial cells. The induction of t-PA by retinoids might involve protein kinase C (PKC) as judged by an experiment using a specific PKC inhibitor. The effect of retinoids on the fibrinolytic system in vivo was assessed by feeding rats with a vitamin A deficient diet or a diet with excess of vitamin A or other retinoids.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Endotélio Vascular/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Retinoides/farmacologia , Ativador de Plasminogênio Tecidual/biossíntese , Animais , Butiratos/farmacologia , Ácido Butírico , Células Cultivadas , Sondas de DNA , Endotélio Vascular/metabolismo , Humanos , Estrutura Molecular , Inativadores de Plasminogênio/metabolismo , Proteína Quinase C/fisiologia , Ratos , Ratos Endogâmicos BN , Trombose/prevenção & controle , Extratos de Tecidos , Deficiência de Vitamina A/metabolismoRESUMO
In our search for compounds that can stimulate endogenous fibrinolysis, we have found that certain triazolobenzodiazepines enhance the production of tissue-type plasminogen activator (t-PA) by vascular endothelial cells maintained in vitro, with no or even a lowering effect on plasminogen activator inhibitor type-1 (PAI-1) production. The most active compounds tested, U-34599, U-46195 and U-51477, were studied in more detail and showed a time- and dose-dependent increase in the production of t-PA by human umbilical vein endothelial cells. At optimal stimulatory concentrations (about 10 microM), the three compounds stimulated t-PA expression about 2-fold after 24 hr and maximally about 4-fold after 48 hr of incubation; this maximal increase in t-PA synthesis was sustained at prolonged incubations of 72 or 96 hr. The triazolobenzodiazepine effects on t-PA production were accompanied by parallel increases in t-PA mRNA levels, without marked changes in PAI-1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA concentrations. Numerous analogues of the three lead compounds were then tested to determine the relationship between benzodiazepine structure and the ability to stimulate t-PA production. No positive correlation was found between the ability of the various triazolobenzodiazepines to stimulate t-PA production and their affinity for the benzodiazepine receptor. In agreement with this, no specific binding of [3H]flunitrazepam, a specific ligand for benzodiazepine receptors, to endothelial cell membrane preparations was observed. Thus, it is unlikely that the triazolobenzodiazepines act through central-type benzodiazepine receptors to stimulate t-PA production. Similarly, no evidence was found for the presence of peripheral-type benzodiazepine receptors on endothelial cell membranes. The ability of the benzodiazepines to stimulate t-PA production, however, appeared to be related to their platelet-activating factor (PAF) antagonist activity. Despite this finding, several non-benzodiazepine PAF antagonists did not stimulate t-PA production. While the precise mechanism of action is not yet clear, selected benzodiazepine analogues possessing PAF antagonist activity stimulate the production of t-PA by endothelial cells in vitro.
Assuntos
Benzodiazepinas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/biossíntese , Triazóis/farmacologia , Células Cultivadas/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Fator de Ativação de Plaquetas/antagonistas & inibidores , RNA Mensageiro/biossíntese , Relação Estrutura-AtividadeRESUMO
Matrix metalloproteinases (MMPs) play an important role in many pathologic processes, but their activities are difficult to determine since no simple specific and/or chromogenic substrate exists. We have developed a novel MMP activity assay using a modified urokinase as a substrate. Protein engineering enabled the plasmin activation site in this urokinase to be substituted by a specific activation site recognized by MMPs. In this way the MMP activity can be monitored via urokinase activity as measured by a simple chromogenic assay. The assay was made specific for MMP-9 by a capture step using MMP-9-specific antibodies that do not interfere with MMP-activity. This assay monitors the amount of active enzyme as well as the latent, but potentially active proform. Using this assay the levels of MMP-9 were investigated in urine from patients with various kinds of carcinoma. High levels of both active and latent MMP-9 were detected in urine from patients with carcinoma of the bladder, whereas hardly any activity was observed in urine from healthy controls. MMP-9 in urine was present in its intact form. Surprisingly, MMP-9 was also increased in the urine of patients with nonurogenital carcinoma. Therefore, measurement of urinary MMP-9 activity levels may be a convenient diagnostic tool for various types of carcinoma.
Assuntos
Biomarcadores Tumorais/urina , Colagenases/urina , Neoplasias/urina , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/urina , Neoplasias Esofágicas/urina , Feminino , Humanos , Neoplasias Pulmonares/urina , Masculino , Metaloproteinase 9 da Matriz , Pessoa de Meia-Idade , Neoplasias/enzimologia , Neoplasias/terapia , Neoplasias Ovarianas/urina , Valores de Referência , Especificidade por SubstratoAssuntos
Materiais Biocompatíveis , Transplante de Células , Fator 1 de Crescimento de Fibroblastos/farmacologia , Sobrevivência de Enxerto/efeitos dos fármacos , Fígado/irrigação sanguínea , Fígado/citologia , Politetrafluoretileno , Animais , Sobrevivência Celular/efeitos dos fármacos , Transplante de Células/métodos , Fígado/efeitos dos fármacos , Macaca mulatta , Transplante Homólogo/imunologiaRESUMO
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.
Assuntos
Gordura Intra-Abdominal/metabolismo , Inibidor 1 de Ativador de Plasminogênio/sangue , Adiposidade/fisiologia , Adulto , Antropometria , Citocinas/biossíntese , Feminino , Hormônio do Crescimento Humano/deficiência , Humanos , Mediadores da Inflamação/metabolismo , Gordura Intra-Abdominal/anatomia & histologia , Gordura Intra-Abdominal/patologia , Metabolismo dos Lipídeos , Pessoa de Meia-Idade , Obesidade/metabolismo , Obesidade/patologia , Inibidor 1 de Ativador de Plasminogênio/genética , RNA Mensageiro/genética , Técnicas de Cultura de Tecidos , Fator de Necrose Tumoral alfa/biossínteseRESUMO
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.
Assuntos
Expressão Gênica/efeitos dos fármacos , Interleucina-1/farmacologia , Fígado/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Proteínas Proto-Oncogênicas c-jun/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Sítios de Ligação , Sangue , Northern Blotting , Carcinoma Hepatocelular , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Humanos , Técnicas de Imunoadsorção , Neoplasias Hepáticas , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas , Vanadatos/farmacologiaRESUMO
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.
Assuntos
Células Epiteliais/metabolismo , Fibrinólise/fisiologia , Lectinas Tipo C , Lectinas de Ligação a Manose , Ativador de Plasminogênio Tecidual/metabolismo , Anticorpos/farmacologia , Transporte Biológico Ativo , Proteínas de Transporte/farmacologia , Células Cultivadas , Cloroquina/farmacologia , Meios de Cultivo Condicionados , Células Epiteliais/efeitos dos fármacos , Expressão Gênica , Glutationa Transferase/farmacologia , Humanos , Radioisótopos do Iodo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Receptor de Manose , Inibidor 1 de Ativador de Plasminogênio/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes de Fusão/farmacologia , Ativador de Plasminogênio Tecidual/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
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.
Assuntos
Receptores do Ácido Retinoico/genética , Ativador de Plasminogênio Tecidual/genética , Benzoatos/farmacologia , Células Cultivadas , Cromanos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Expressão Gênica , Humanos , Inibidor 1 de Ativador de Plasminogênio/genética , RNA Mensageiro/genética , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores do Ácido Retinoico/classificação , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Proteínas de Ligação ao Retinol/genética , Proteínas Celulares de Ligação ao Retinol , Estereoisomerismo , Fatores de Transcrição/genética , Tretinoína/química , Tretinoína/farmacologiaRESUMO
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.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Mediadores da Inflamação/farmacologia , Peritônio/metabolismo , Células Epiteliais , Epitélio/metabolismo , Genisteína , Humanos , Isoflavonas/farmacologia , Peritônio/citologia , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/farmacologia , Estimulação Química , Tromboplastina/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
We have previously shown that the pleiotropic agent sodium butyrate strongly stimulates tissue-type plasminogen activator (t-PA) expression in human umbilical vein endothelial cells (HUVEC). Here we provide the following evidence that the butyrate-induced t-PA expression in HUVEC involves histone H4 acetylation. (1) t-PA induction by butyrate occurs at the transcriptional level and does not require new protein synthesis, indicating a direct effect. (2) t-PA induction by butyrate can be fully mimicked by a specific, structurally unrelated, histone deacetylase inhibitor, trichostatin A. (3) At optimally stimulatory conditions, a combination of butyrate and trichostatin A does not enhance t-PA production more than each of the compounds alone, indicating that both compounds act through a common regulatory mechanism. (4) Induction of t-PA transcription by butyrate and trichostatin A was found to be preceded by histone H4 acetylation; at suboptimal inducing concentrations of butyrate and trichostatin A, the degree of acetylation of histone H4 caused by each agent was similarly reduced. These results are consistent with a role for histone H4 acetylation in t-PA induction by butyrate in HUVEC.
Assuntos
Butiratos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Ativador de Plasminogênio Tecidual/genética , Acetilação , Ácido Butírico , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases , Humanos , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/genética , RNA Mensageiro/genética , Ativador de Plasminogênio Tecidual/biossíntese , Transcrição Gênica , Tretinoína/farmacologiaRESUMO
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.
Assuntos
Proteínas de Transporte/farmacologia , Fibrinólise/efeitos dos fármacos , Glicoproteínas/farmacologia , Peritônio/metabolismo , Adenoviridae/genética , Proteínas de Transporte/genética , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Regulação para Baixo , Estabilidade de Medicamentos , Células Epiteliais/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Glicoproteínas/genética , Humanos , Immunoblotting , Proteína Associada a Proteínas Relacionadas a Receptor de LDL , Peritônio/citologia , Peritonite/metabolismo , Peritonite/patologia , Fatores de Tempo , Extratos de Tecidos/metabolismo , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
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.
Assuntos
Congêneres do Estradiol/farmacologia , Etinilestradiol/farmacologia , Fibrinólise , Lectinas Tipo C , Lectinas de Ligação a Manose , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tecidual/sangue , Animais , Endotélio Vascular/metabolismo , Humanos , Fígado/metabolismo , Receptor de Manose , Camundongos , Ratos , Proteínas Recombinantes/sangue , Proteínas Recombinantes/farmacocinéticaRESUMO
Activation of protein kinase C leads to a strong induction of tissue-type plasminogen activator (t-PA) expression in endothelial cells. Using endothelial cells from human umbilical vein (HUVECs) and human aorta (HAECs), we have studied this regulation of t-PA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), at the mRNA level and have compared their induction with the expression of platelet-derived growth factors A and B (PDGF-A and PDGF-B) and the proto-oncogenes c-jun and c-fos. Treatment of HUVECs with exogenous bacterial phospholipase C or the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol led to a threefold and a twofold increase, respectively, in t-PA concentrations in 24-hour-conditioned medium. Similarly, the more stable protein kinase C activator 4 beta-phorbol-12-myristate-13-acetate (PMA) caused about a 10-fold increase in t-PA antigen levels. This effect of PMA is maximal between 8 and 16 hours at a concentration of 10 nM and is fully accounted for by parallel increases in t-PA mRNA levels. An increase in intracellular cyclic adenosine monophosphate levels by forskolin (10 microM) slightly diminished t-PA expression but further enhanced the PMA-induced increases in t-PA synthesis and mRNA levels by at least twofold. PMA also enhanced the mRNA levels of two other important endothelium-expressed genes, PDGF-A and PDGF-B, with a time profile similar to that of t-PA, with peak values about fivefold higher than control values. Forskolin did not further stimulate this PMA-induced PDGF expression in HUVECs, which suggests a regulatory mechanism different from that of t-PA. Qualitatively very similar induction patterns of t-PA, PDGF-A, and PDGF-B were seen with HAECs. In contrast to t-PA and PDGF, PAI-1 mRNA and antigen levels increased only slightly after PMA treatment of HUVECs or HAECs; forskolin alone or in combination with PMA diminished the expression of PAI-1. The induction of t-PA mRNA by PMA was dependent on protein synthesis and was preceded by a strong transient increase in c-jun and c-fos mRNA levels; the induction of c-fos but not of c-jun was potentiated by forskolin. Because the products of these two proto-oncogenes form dimeric complexes for which specific binding sites are present in the t-PA promoter region, they may mediate the protein kinase C-dependent increase in t-PA gene expression, including the stimulating action of cyclic adenosine monophosphate.
Assuntos
AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/metabolismo , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Aorta/efeitos dos fármacos , Células Cultivadas , Colforsina/farmacologia , Sondas de DNA , Diglicerídeos/farmacologia , Endotélio Vascular/citologia , Humanos , Inativadores de Plasminogênio/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-jun , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Ativador de Plasminogênio Tecidual/metabolismo , Fosfolipases Tipo C/farmacologia , Veias Umbilicais/efeitos dos fármacosRESUMO
Matrix metalloproteinases (MMP) types 2 and 9 (also known as gelatinase A and B) are thought to be causally involved in cancer invasion and metastasis. In normal as well as in malignant tissue, both these MMPs occur in multiple forms such as inactive precursors, active enzymes and enzyme-inhibitor complexes. Using newly developed quantitative activity assays, the levels of active MMP-2, total (active and activatable) MMP-2 and total MMP-9 were found to be significantly higher in breast carcinomas than in fibroadenomas. In addition, active MMP-2 and MMP-9 were detected more frequently in malignant than in benign breast carcinoma. These new quantitative activity assays are likely to be of use in studying the mechanism of action of both MMP-2 and -9, assessing their potential prognostic value in different cancers and in the design of MMP inhibitors for preventing cancer metastasis.