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Biomedical subjects

F Lupu

Publications and source records attributed to F Lupu.

At least 55 records · Page 3Linked to original sources
PubMed1997

Urokinase but not tissue plasminogen activator mediates arterial neointima formation in mice.

To define the role of the plasminogen activators (PAs) tissue PA (t-PA) and urokinase PA (u-PA) in vascular wound healing, neointima formation and reendothelialization were evaluated after electric or mechanical arterial injury in mice with a single or combined deficiency of t-PA (t-PA-/-) and/or u-PA (u-PA-/-). In both models, neointima formation and neointimal cell accumulation were reduced in u-PA-/- and in t-PA-/-/u-PA-/- arteries but not in t-PA-/- arteries. The electric injury model was used to characterize the underlying cellular mechanisms. Topographic analysis of vascular wound healing in electrically injured wild-type and t-PA-/- arteries revealed a similar degree of migration of smooth muscle cells from the noninjured borders into the necrotic center. In contrast, in u-PA-/- and t-PA-/-/u-PA-/- arteries, smooth muscle cells accumulated at the uninjured borders but failed to migrate into the necrotic center. Cultured u-PA-/- but not t-PA-/- smooth muscle cells also failed to migrate in vitro after scrape wounding. Proliferation of smooth muscle cells was not affected by PA deficiency. Reendothelialization after electric injury was similar in all genotypes. In situ analysis revealed markedly elevated u-PA zymographic activity, mRNA, and immunoreactivity in smooth muscle cells, endothelial cells, and leukocytes within 1 week after injury, eg, when cells migrated into the wound. Thus, u-PA plays a significant role in vascular wound healing and arterial neointima formation after injury, most likely by affecting cellular migration.

Animals↗
PubMed1996

Effects of an Igf1 gene null mutation on mouse reproduction.

Both sexes of adult mice homozygous for a targeted mutation of the Igf1 gene, encoding insulin-like growth factor 1, are infertile dwarfs (approximately 30% of normal size). The testes are reduced in size less than expected from the degree of dwarfism but sustain spermatogenesis only at 18% of the normal level. The epididymides are overall nearly allometric to the reduced body weight, but the distal regions of the duct, vas deferens, seminal vesicles, and prostate are vestigial. Despite the mutational impact on the epididymis, capacitated sperm are able to fertilize wild type eggs in vitro. It is hypothesized that the infertility of male mutants is caused by failure of androgenization resulting in absence of mating behavior, due to drastically reduced levels of serum testosterone (18% of normal). This hormonal deficiency was correlated with an ultrastructural analysis of mutant Leydig cells revealing a significant developmental delay, while assays in organ culture showed that the basal and LH-stimulated production of testosterone by testicular parenchyma is reduced in comparison with wild type controls. The female mutants fail to ovulate even after administration of gonadotropins, which is apparently the primary cause of their infertility, and possess an infantile uterus that exhibits a dramatic hypoplasia especially in the myometrium. The phenotypic manifestations of the mutation were correlated with the localization of transcripts for insulin-like growth factor I and its cognate receptor in wild type reproductive tissues by in situ hybridization.

Animals↗
PubMed1995

Prothrombin cleavage by human vascular smooth muscle cells: a potential alternative pathway to the coagulation cascade.

Thrombin is a potent mitogen for human vascular smooth muscle cells (HVSMC) and its enzymatic activity is required for this function. The present study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs upon incubation of prothrombin with the cells. Analysis by SDS-PAGE, immunoblotting, and amino acid sequencing revealed that prothrombin incubated with HVSMC undergoes limited proteolysis. Prethrombin 1 was formed through cleavage at R155-S156. Cleavage at R271-T272 generated fragment 1.2 and prethrombin 2 whilst cleavage at R284-T285 yielded truncated prothrombin 2 (prethrombin 2'). However, cleavage at R320-I321 which, during prothrombin activation produces two-chain alpha-thrombin, was not detectable. Studies on HVSMC-conditioned medium revealed that a similar pattern of prothrombin cleavage occurred by a cell-secreted factor(s). Amidolytic activity analysis indicated that 1-3% catalytically active thrombin-like activity was generated upon incubation of prothrombin with HVSMC-conditioned medium. By treating conditioned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R271-S272 and by alpha-thrombin at R155-S156 and R284-T285. Antibodies neutralising the activity of either urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This activity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak with apparent molecular mass of 30-40 kDa.

Adolescent↗
PubMed1995

A novel L23-related gene 40 kb downstream of the imprinted H19 gene is biallelically expressed in mid-fetal and adult human tissues.

The closely linked IGF2 and H19 genes on human chromosome 11p15.5 are monoallelically expressed as a result of genomic imprinting and show altered expression in Wilms' tumors (WTs). To map regional imprinting we have sought to isolate additional human genes close to IGF2/H19 and to characterize their allelic expression patterns. Here we report a novel gene, provisionally named L23MRP [L23 (mitochondrial)-related protein], which is oriented 'tail-to-tail' with H19 and is transcribed to within 40 kb of the last H19 exon. L23MRP is expressed biallelically in many mid-fetal and adult human tissues. This gene is also expressed at normal levels in WTs which have lost expression of H19 either via loss of the maternal chromosome 11p15.5 or via an epigenetic pathway involving site-specific DNA hypermethylation. These data indicate that, at least in post-embryonic stages, L23MRP is functionally insulated from the IGF2/H19 imprinted domain.

Adult↗
PubMed1995

Thrombin induces the redistribution and acute release of tissue factor pathway inhibitor from specific granules within human endothelial cells in culture.

Tissue factor pathway inhibitor (TFPI) is a vascular anticoagulant that regulates the tissue (TF)-dependent pathway of coagulation. The majority of intravascular TFPI is thought to be noncovalently bound to the vessel wall. Our immunolocalization studies in cultures of human umbilical vein endothelial cells (HUVEC) and immortalized EA.hy926 cells that TFPI is located in well-defined granules evenly spread over the cell surface and with apical polarization within the cytoplasm. These granules are smaller than and distinct from Weibel-Palade bodies. Upon treatment of cultured cells with low concentrations of thrombin (0.01 to 1 NIH U/mL), a marked redistribution of TFPI, occurred with patching in focal points and increased exposure of both TFPI antigen and anticoagulant activity on the surface of the stimulated endothelial cells. This redistribution was paralleled by an acute release of TFPI in the cell medium. EA.hy926 cells responded more readily to thrombin stimulation than HUVECs. The process was inhibited by both hirudin and anti-thrombin receptor antibody. Our findings demonstrate a novel mechanism by which thrombin may exert a negative feedback control on blood coagulation. Therefore, this pathway can be physiological importance in controlling TF-mediated thrombin generation.

Biological Transport↗
PubMed1995

Plasminogen activator expression in human atherosclerotic lesions.

The plasminogen activator (PA) system may participate in the pathogenesis of atherosclerosis by modulating the turnover of intimal fibrin and extracellular matrix deposits and by contributing to intimal cell migration. We present an analysis of tissue-type PA (tPA) and urokinase-type PA (uPA) expression at three levels: mRNA by in situ hybridization, antigen by immunohistochemistry, and enzymatic activity by histoenzymology and zymography. For PA colocalization with cellular or matrix components, we used double immunofluorescence labeling in conjunction with confocal microscopy. In normal arteries, tPA antigen and mRNA were detected in endothelial cells and smooth muscle cells (SMCs). In atherosclerotic arteries, tPA antigen and mRNA were increased in intimal SMCs and in macrophage-derived foam cells of fibro-fatty lesions. Part of the tPA was detected in the extracellular space and colocalized with fibrin deposits. uPA antigen and mRNA were detected in association with the intimal macrophages and SMCs. A particularly high uPA expression was noted on macrophages localized on the rims of the necrotic core. Moreover, using a novel histoenzymological assay as well as classic zymography, we revealed uPA-dependent lytic activity in the advanced lesions, whereas in normal arteries, only tPA-dependent activity was detected, mainly over the vasa vasorum. Also, strong tPA and uPA staining was detected in neomicrovessels of the plaques, suggesting that PAs may play a role in plaque angiogenesis. Our results suggest a local dynamic process of PA-dependent proteolysis in lesion areas that is associated with macrophages and SMCs. A better comprehension of these proteolytic mechanisms in advanced atherosclerotic plaques may provide the basis for therapeutic approaches for plaque stabilization.

Aorta↗
PubMed1995

Thrombin receptor activating peptide (TRAP) stimulates mitogenesis, c-fos and PDGF-A gene expression in human vascular smooth muscle cells.

The synthetic peptide SFLLRNPNDKYEPF, identical in sequence to the new amino-terminus of the thrombin receptor generated following cleavage of thrombin, acts a thrombin receptor agonist/activating peptide (TRAP). In this study, Northern blot analysis showed that cultured human vascular smooth muscle cells (HVSMC) express a thrombin receptor transcript. TRAP, in contrast to thrombin was shown to be a weak mitogen for HVSMC. A combination of TRAP and enzymatically-inactivated thrombin (PPACK-thrombin) which provides receptor occupancy, did not potentiate TRAP-induced mitogenesis, indicating that TRAP and PPACK-thrombin do not reproduce the mitogenic effect of enzymatically-active thrombin. Both thrombin and TRAP, induced the expression of c-fos and the PDGF-A gene in a pertussis toxin (PTX)-insensitive manner. Examination of thrombin and TRAP-treated cells by immunofluorescence staining followed by computer assisted image analysis revealed that thrombin and to a lesser extent TRAP induced PDGF-AA protein expression. Antibodies to PDGF-AA partially inhibited thrombin but not TRAP-induced mitogenesis in HVSMC. This study indicates that in addition to the common signalling pathways utilised by thrombin and TRAP, enzymatically-active thrombin activates other signalling pathways and hence TRAP does not mimic fully the biological effect of thrombin on HVSMC.

Cells, Cultured↗
PubMed1994

4-Hydroxynonenal induces membrane perturbations and inhibition of basal prostacyclin production in endothelial cells, and migration of monocytes.

Cultured bovine aortic endothelial cells (BAEC) were incubated for 5 days with 10(-5) 4-hydroxynonenal (HN). HN treated BAEC and controls were either (i) further incubated with 125I-polymyxin B (IPxB) or with radioiodinated, inactivated coagulation factor Xa (IFXai) as markers of membrane phospholipid perturbation, or (ii) assayed for the synthesis of prostacyclin (PGI2) and thromboxane A2 (TXA2). Rabbit blood mononuclear cells enriched in monocytes (MC) were isolated and assayed for chemotactic response to HN. The results showed six - fold increases of IPxB and IFXai binding to BAEC treated with HN, as compared to untreated controls. We also found in HN treated cells a marked inhibition of PGI2 synthesis, but an unmodified TXA2 production. In addition, HN in the 10(-5)-10(-10) M range induced oriented migration of MC.

Aldehydes↗
PubMed1994

Expression of LDL receptor-related protein/alpha 2-macroglobulin receptor in human normal and atherosclerotic arteries.

Low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP/alpha 2-MR) is a multifunctional cell-surface receptor that is responsible for the clearance of lipoprotein remnants, proteases, or cytokines/growth factors in complex with alpha 2-macroglobulin as well as of plasminogen activators complexed with inhibitors. We investigated the expression of LRP/alpha 2-MR in healthy and atherosclerotic human arteries by in situ hybridization using an LRP/alpha 2-MR mRNA-specific riboprobe and immunocytochemistry using specific monoclonal antibodies. The cell types expressing LRP/alpha 2-MR were identified by immunolabeling of antigens specific for endothelial cells, smooth muscle cells, and macrophages. In normal arteries, LRP/alpha 2-MR mRNA and protein were found in smooth muscle cells of the media and vasa vasorum and in adventitial fibroblasts. Endothelial cells were negative for LRP/alpha 2-MR protein but positive for its mRNA. Atherosclerotic arteries exhibited a strong labeling for LRP/alpha 2-MR mRNA and protein that was observed in intimal smooth muscle cells exhibiting normal or foam cell characteristics and in lipid-laden cells positive for macrophage markers. A particularly high expression was detected in macrophages located in the cap of the lipid-rich necrotic core. These results suggest that cellular components of the atherosclerotic plaque express LRP/alpha 2-MR. This receptor may play an important local scavenger role for lipoprotein remnants, for growth factors/cytokines, and for extracellular protease-inhibitor complexes.

Arteries↗
PubMed1993

Enhanced prothrombin and intrinsic factor X activation on blood platelets from diabetic patients.

The present work was conducted to bring information about the enhanced procoagulant activity of blood platelets in diabetes mellitus, as compared to normal control platelets. The inquiry was performed on platelets from either human patients or hamsters with streptozotocin-induced diabetes. The experiments comprised functional assays of factor Xa and thrombin generation in the presence of normal or diabetic platelets, using saturating amounts of coagulation factors. The prothrombinasic and tenasic complexes activation was measured by using discontinuous amidolytic tests with chromogenic substrates (S-2238 for FXa and S-2222 for thrombin). The results showed that, in contrast to the low prothrombin and factor X converting activity of normal platelets, the thrombin and FXa generation on diabetic platelets was increased by 3-7 times for either humans or hamsters. These findings may be relevant for the pathogenesis of thrombotic complications known to occur in diabetes mellitus.

Aged↗
PubMed1993

Differentiation-stage specific self-peptides bound by major histocompatibility complex class I molecules.

We have tested the hypothesis that phenotypic changes of development are accompanied by expression of differentiation-stage specific peptides bound to major histocompatibility complex (MHC) class I molecules. The U937 cell line, when cultured in the presence of phorbol myristate acetate (PMA), undergoes differentiation from monoblasts to macrophage-like cells. The high-performance liquid chromatography profile of peptides eluted from purified human histocompatibility leukocyte antigen class I molecules expressed by U937 treated with PMA differs from that obtained from control, untreated U937 cells. Chemical sequencing of eluted peptides identified a peptide derived from cytomegalovirus in both treated and untreated cells. PMA-treated, but not untreated cells, displayed an additional peptide derived from interleukin 1 beta. Hence, differentiation-induction of U937 is accompanied by the presentation of at least one differentiation-stage specific peptide. Our results indicate that, similar to viral infection, cellular development and transformation is accompanied by the de novo synthesis of proteins which are processed and presented on MHC class I molecules.

Amino Acid Sequence↗
PubMed1993

Localization and production of plasminogen activator inhibitor-1 in human healthy and atherosclerotic arteries.

High plasma levels of plasminogen activator inhibitor type-1 (PAI-1), the principal inhibitor of the fibrinolytic system, have been associated with thrombotic and arterial disease. To study PAI-1 expression in healthy and atherosclerotic human arteries, a detailed analysis was made by light and electron microscopy immunocytochemistry and by in situ hybridization. In healthy arteries PAI-1 was found both at the level of endothelial cells and of smooth muscle cells (SMCs) of the arterial media. In early atherosclerotic lesions PAI-1 was also detected in intimal SMCs and in extracellular areas in association with vitronectin. Immunogold analysis by electron microscopy revealed PAI-1 in vesicular structures in endothelial cells and in SMCs with normal or foam cell characteristics. In advanced atheromatous plaques, PAI-1 mRNA expression in SMCs within the fibrous cap was increased compared with SMCs located in the adjacent media or in normal arterial tissue. PAI-1 mRNA was also detected in macrophages located at the periphery of the necrotic core. The increased synthesis of PAI-1 by cellular components of the atherosclerotic plaque and the extracellular accumulation of PAI-1 may contribute to the thrombotic complications associated with plaque rupture and possibly play a role in the accumulation of extracellular matrix deposits.

Arteries↗
PubMed1993

Intrinsic procoagulant surface induced by hypercholesterolaemia on rabbit aortic endothelium.

The effect of hyperlipidaemia on endothelial cell haemostatic properties was examined using ex vivo studies on aortic segments obtained from fat-fed Chinchilla rabbits, mounted in a template device which exposed the luminal surface. Exposure of arterial endothelium to lipids resulted in marked enhancement of externally exposed anionic phospholipids, detected using either fluorescence microscopy with the probe merocyanine 540 or by binding of 125I-polymyxin B and 125I-Annexin V. Consistent with the known procoagulant properties of anionic phospholipid, following the lipid and cholesterol-rich diet intake, intact endothelial cells demonstrated enhanced binding of radioiodinated factors IX/IXa and Xa, and enhanced factor IXa/VIII-dependent factor X activation and factor Xa-factor Va-mediated prothrombin activation. Both factor Xa and thrombin formation were blocked, in large part, by polymyxin B, suggesting dependence of the reaction on anionic phospholipids. Consistent with these results, evidence of increased activation of the coagulation mechanism in vivo was observed in hyperlipidaemic animals, as assessed by a three-fold increase in levels of circulating antithrombin-protease complexes, compared with normolipidaemic controls.

Animals↗
PubMed1992

Population and family studies of HLA-DR4 by use of oligonucleotide typing.

Using PCR and DR4 group-specific primers and SSO's we have examined DRB1*04 nucleotide polymorphisms in a population of 123 DR4-positive individuals (86 NAC, 27 Hispanics and 10 African Americans) from New York carrying a total of 134 DR4 haplotypes. We found that the distribution of DRB1*04 alleles on DR4 haplotypes differs in these three ethnic groups. In this relatively small population, certain alleles such as DRB1*0406 and 0411 were encountered only in Hispanics, while others such as DRB1*0403, 0408 and 0409 were found only in NAC (North American Caucasians). Such differences may be important in studies of HLA-DR4 and disease associations. Evidence from MLC and PLT studies of an HLA-B/DR crossover family, which was informative for the segregation of HLA-DRB1*0406 and DRB1*0407, supports the concept that subtypes of HLA-DR4 and/or associated HLA-DP alleles elicit T-cell alloreactivity, and may thus play a role in transplantation.

Amino Acid Sequence↗
PubMed1992

Polymorphism of HLA in the Romanian population.

We have investigated the HLA-class I and class II polymorphism in a population of 83 Romanians using conventional serology together with PCR amplification and oligonucleotide typing of HLA-class II genes. Romanians show a higher frequency of HLA-A11, B13, B18, B37, B39, B51 and DR2 than other European populations. HLA-DRB1*1501 and 1601 account for the high frequency of the serologic specificity DR2. In Romanians, HLA-DR2 is in linkage disequilibrium with HLA-B18 and HLA-Bw52 rather than with HLA-B7 as in the case in other Europeans. Unexpected HLA-DR2 haplotypes include HLA-DRB1*1502, DQA1*0102, DQB1*0601; HLA-DRB1*1602, DQA1*0102, DQB1*0502. Other unusual haplotypes include HLA-DRB1*0405, DQA1*03, DQB1*0302; HLA-DRB1*1305, DQA1*0103, DQB1*0603; and HLA-DRB1*1405, DQA1*0101, DQB1*05032. Analysis of the genetic distance between Romanians and other Europeans who have been studied serologically are consistent with the hypothesis that Romanians descend from Roman ancestors who colonized Dacia between the 1st century B.C. and 1st century A.D.

Base Sequence↗
PubMed1991

A new class of potential carcinogenesis inhibitors: hindered p-benzoquinones.

The investigation of the mechanisms of action by which phenolic antioxidants (i.e. BHA, BHT, etc.) protect DNA against interaction with activated BaP, led us to the finding that oxidated aminophenols are much more potent in this respect, being also powerful inhibitors of cytochrome P-450 dependent monooxygenases. However, the quinoneimine structures probably involved in this effect are chemically unstable reactive species and therefore difficult to handle. Based on these observations, we extended this study on several types of benzoquinones. We demonstrated that 2,6-di-t-butylbenzoquinone exerted a very good protective effect at DNA level (in standard conditions) as compared to the "classical" BHA (i. e. 91.3% and 34.4%, respectively). This hindered quinone is nontoxic (DL50 = 3085 mg/kg b. w.) and also did not exhibit inhibitory activity against GST. In contrast, the nonsubstituted p-benzoquinone is a powerful inhibitor of this last enzyme. Recently, 2,6-di-t-butylbenzoquinone was isolated from mutagenic depressing food and was considered one of the factors responsible for this effect.

Animals↗