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Biomedical subjects

F Niculescu

Publications and source records attributed to F Niculescu.

At least 19 recordsLinked to original sources
PubMed1999

Inhibition of oligodendrocyte apoptosis by sublytic C5b-9 is associated with enhanced synthesis of bcl-2 and mediated by inhibition of caspase-3 activation.

We have previously shown that generation of sublytic C5b-9, the membrane attack complex of complement, induces oligodendrocytes to enter cell cycle and reduces apoptotic cell death in vitro. In the present study, the cellular factors involved in apoptosis of oligodendrocyte progenitor cells and oligodendrocytes, and the inhibitory effect of C5b-9 on apoptotic process were investigated. Oligodendrocyte progenitor cells identified by mAb A2B5 that were isolated from neonatal rat brains were differentiated into oligodendrocytes in serum-free defined medium. The differentiation, which occurs simultaneously with apoptotic cell death, was associated with a rapid loss of bcl-2 mRNA and increased expression of caspase-3 mRNA. Activation of caspase-3 in differentiating cells was demonstrated by the generation of 17- and 12-kDa fragments of caspase-3 proenzyme and by cleavage of poly(ADP-ribose) polymerase, a specific caspase-3 substrate. Cell death associated with differentiation was inhibited by the caspase-3 inhibitor DEVD-CHO in a dose-dependent manner. Assembly of sublytic C5b-9 resulted in inhibition of caspase-3 activation. In addition, synthesis of BCL-2 protein in oligodendrocytes was significantly increased by C5b-9. The TNF-alpha-induced apoptosis of oligodendrocytes was also inhibited by C5b-9. These results indicate that up-regulation of BCL-2 protein and inhibition of caspase-3 activation are potential mechanisms by which C5b-9 increases survival of oligodendrocyte in vitro and possibly in vivo during inflammation and immune-mediated demyelination affecting the CNS.

Animals↗
PubMed1999

Sublytic C5b-9 induces proliferation of human aortic smooth muscle cells: role of mitogen activated protein kinase and phosphatidylinositol 3-kinase.

Proliferation of vascular smooth muscle cells contributes to initimal hyperplasia during atherogenesis, but the factors regulating their proliferation are not well known. In the present study we report that sublytic C5b-9 assembly induced proliferation of differentiated human aortic smooth muscle cells (ASMC) in culture. Cell cycle re-entry occurred through activation of cdk4, cdk2 kinase and the reduction of p21 cell cycle inhibitor. We also investigated if C5b-9 cell cycle induction is mediated through activation of mitogen activated protein kinase (MAPK) pathways. Extracellular signal regulated kinase (ERK) 1 activity was significantly increased, while c-jun NH2-terminal kinase (JNK) 1 and p38 MAPK activity were only transiently increased. Pretreatment with wortmannin inhibits ERK1 activation by C5b-9, suggesting the involvement of phosphatidylinositol 3-kinase (PI 3-kinase). Both PI 3-kinase and p70 S6 kinase were activated by C5b-9 but not by C5b6. C5b-9 induced DNA synthesis was abolished by pretreatment with inhibitors of ERK1 and PI 3-kinase, but not by p38 MAPK. These data indicated that ERK1 and PI 3-kinase play a major role in C5b-9 induced ASMC proliferation.

Androstadienes↗
PubMed1999

Tyrosine phosphorylation and activation of Janus kinase 1 and STAT3 by sublytic C5b-9 complement complex in aortic endothelial cells.

The pathway involving Janus kinase (JAK) and signal transducers and activators of transcription (STATs) plays an important role in differentiation and proliferation of cells initiated by receptor activation. In the present study we identified the JAK and STAT proteins activated by C5b-9 in human aortic endothelial cells (AEC). JAK1 but not JAK2 was tyrosine phosphorylated in response to sublytic C5b-9. STAT3 was rapidly tyrosine phosphorylated also by C5b-9. Pertussis toxin inhibited the C5b-9 induced JAK1 activation. However, phosphorylation of STAT3 was not inhibited by Pertussis toxin, although C5b-9 induced a time-dependent nuclear translocation of STAT3. These observations indicated that JAK1 is phosphorylated by C5b-9 through activation of trimeric G proteins of the Gi/Go family. Raf-1 and ERK1 were also activated by C5b-9 in human AEC in a G protein dependent manner. Therefore, JAK1 activity may be involved in activation of Raf-1 and ERK1 via G proteins activated by C5b-9. This study demonstrates the ability of membrane-inserted C5b-9 to activate JAK1 and STAT3 proteins, thus defining new signalling pathway by which C5b-9 may regulate gene activation.

Adult↗
PubMed1997

Activation of Ras and mitogen-activated protein kinase pathway by terminal complement complexes is G protein dependent.

Assembly of terminal complement complexes (TCC) C5b-7, C5b-8, and C5b-9 on target cells during acute and chronic inflammation induces hydrolysis of plasma membrane phospholipids and heterotrimeric G protein activation. TCC also stimulate a variety of cellular activities, which include cytokine synthesis, proto-oncogene activation, and mitotic signaling. Now we report that sublytic TCC induced Ras, Raf-1, and extracellular signal-regulated kinase (ERK) 1 activation in JY25 B cell line. When cells were exposed to C5b-9, GTP-bound Ras in anti-C5b-9 immunoprecipitates was increased 3.2-fold at 2 min, while GTP-bound Ras in anti-Ras immunoprecipitates was increased 2-fold at 10 min. Both C5b-9 and C5b-7, but not C5b6, increased Raf-1 kinase activity maximum 3.3-fold at 2 min and 2.8-fold at 5 min, respectively. ERK1 activity was 2-fold increased by C5b-9 at 2 min and by C5b-7 at 10 min, over the C5b6 level. The role of mitogen-activated protein kinase (MAPK) pathway on TCC-inducible mitotic signaling was evaluated by assessing DNA synthesis and activator protein 1 (AP-1) DNA-binding activity. The MAPK/ERK-specific inhibitor PD 098,059 abolished the C5b-9-induced DNA synthesis. Involvement of G protein in the activation of MAPK pathway by TCC was indicated by inhibition of Raf-1 and ERK1 kinase activity, as well as the DNA synthesis by pretreatment of cells with pertussis toxin. Overexpression of beta-adrenergic receptor kinase 1 carboxyl-terminal peptide in JY25 cells also inhibited Raf-1 and ERK1 activity, indicating a direct involvement of G betagamma subunits in the signal transduction generated through activation of MAPK pathway by TCC assembly in the plasma membrane.

B-Lymphocytes↗
PubMed1997

Terminal complement complexes induce cell cycle entry in oligodendrocytes through mitogen activated protein kinase pathway.

Sublytic complement attack through C5b-9 assembly induces oligodendrocytes (OLG) to express proto-oncogenes and to enter the cell cycle from resting G0/G1 phase to S phase. We have investigated whether cell cycle induction by C5b-9 is mediated by mitogen activated protein kinase (MAPK) pathway in OLG. C5b-9 but not C5b6 induced activation of both ERK1 and c-jun NH2 terminal kinases 1 (JNK1) in OLG. The increased ERK1 and JNK1 activities are transient, reaching a maximum around 20 min following exposure to C5b-9. Activation of Raf-1 and MEK1, upstream kinases of ERK1, was shown by increased Raf-1 kinase activity in anti-Raf-1 immunoprecipitates of OLG treated with C5b-9 and ERK1 activity that can be inhibited by PD098,059, a specific MEK1 inhibitor. Requirement for the ERK1 pathway in DNA synthesis was then evaluated using PD098,059. Enhanced DNA synthesis induced by serum complement was completely abolished when OLG were pretreated with PD098,059. On the other hand, c-fos mRNA expression induced by complement was inhibited only 50% by PD098,059, while the c-jun mRNA level was not affected by this MEK1 inhibitor. Interestingly, p70 S6 kinase, an important ribosomal kinase in mitogenesis, was also activated by C5b-9. These findings indicated that the MAPK pathways appears to play a major role in inducing OLG to enter the S phase of the cell cycle from the resting G1/G0 phase.

Animals↗
PubMed1996

Interleukin-6 and interleukin-8 protein and gene expression in human arterial atherosclerotic wall.

Interleukin 6 (IL-6) and interleukin 8 (IL-8) are present in the human arterial atherosclerotic wall as cellular and extracellular deposits in the connective tissue matrix. Quantitative determinations of IL-6 by ELISA showed mean values of 27.6 +/- 3.3 ng/100 mg protein in normal intima, 37.3 +/- 2.1 ng/100 mg protein in fibrous plaque and 25.7 +/- 4.3 ng/100 mg total extracted protein in media. IL-8 levels were 3.5 +/- 0.6 ng/100 mg protein in normal intima, 11.3 +/- 2.1 ng/100 mg protein in fibrous plaque and 8.5 +/- 1.4 ng/100 mg total extracted protein in media. Fibrous plaques presented statistically significant higher levels of both IL-6 and IL-8. IL-6 and IL-8 gene transcripts were present in human iliac fibrous plaque and media prelevated at surgery indicating that a local production by the cells of the arterial wall participate to their accumulation. We also tested the role of complement activation in induction of IL-6 and IL-8 protein synthesis as well as the subsequent activation of endothelial cells. Only IL-8 was induced by complement activation and this may contribute to increased IL-8 levels found in the atherosclerotic wall. When exposed to terminal complement complexes, endothelial cells in culture also showed an increase of both DNA-synthesis and p70 S6 kinase activity indicating that complement is able to induce not only IL-8 synthesis but also cell activation. The presence of IL-6 and IL-8 in the arterial wall where complement activation also occurred, clearly show the involvement of inflammatory events in initiation and progression of atherosclerosis.

Aorta↗
PubMed1996

Sublytic complement attack induces cell cycle in oligodendrocytes.

Sublytic complement attack on oligodendrocytes (OLG) by activation of terminal complement complexes (TCC) selectively enhances the decay of myelin protein mRNAs. We have investigated whether TCC also stimulate differentiated OLG to enter the cell cycle and whether the cell cycle induction is related to the oncogene expression. Complement activation and TCC assembly induced expression of c-jun, JunD, and c-fos mRNAs, increased AP-1 DNA-binding activity within 1 h, and increased [3H]thymidine uptake. The c-jun NH2-terminal kinase activity was increased to the maximum level 20 min after TCC assembly. The increase in thymidine uptake was inhibited by pretreatment of OLG with antisense c-jun oligonucleotides. Studies on cyclin-dependent kinase (cdk) activation revealed that complement increased cyclin-dependent cell cycle associated kinase-2 activity in G1, while cdk2 and cdk4 showed low activity during G1 progression. However, the activity of cdk4 complexed with cyclin D2 showed a marked increase in G1/S transition. Our data provide evidence that sublytic TCC stimulate OLG to enter the cell cycle by induction of c-jun through activation of the c-jun NH2-terminal kinase pathway. In addition, sublytic TCC assembly significantly reduced the number of OLG undergoing apoptotic cell death, which occurs spontaneously in defined medium. These changes together with enhanced degradation of myelin protein mRNA may represent a mechanism for differentiated primary OLG to respond to limited complement activation in inflammation.

Animals↗
PubMed1994

Receptor-independent activation of guanine nucleotide-binding regulatory proteins by terminal complement complexes.

Activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) by terminal complement complexes (TCC) was investigated on human lymphoblastoid B-cell line JY25 and its mutant JY5 deficient in glycosylphosphatidylinositol-anchored proteins. TCC assembly achieved by antibody-dependent activation of C7-deficient serum reconstituted with C7 increased specific guanosine-5'-(gamma-thio)triphosphate (GTP gamma S) binding, 4- and 8-fold, in JY25 and JY5 membranes, respectively, between 2 and 10 min, over the level without C7. TCC also increased GTPase activity 5- and 4-fold in JY25 and JY5, respectively, between 5 and 10 min. Increased GTPase activity was noted first with C5b-7 assembly, which increased further with C5b-8 and C5b-9. The presence of G proteins in anti-TCC immunoprecipitates of cell lysates was investigated by demonstration of G alpha subunit that can be ADP-ribosylated by pertussis toxin (PTX). Immunoprecipitated TCC complexes contained a PTX-sensitive 41-kDa Gi alpha/Go alpha subunit, as shown by SDS-PAGE and Western blotting. These complexes were functionally active as determined by GTP gamma S binding. We have further shown that enhanced TCC elimination from the plasma membrane induced by TCC-generated signals was inhibited by PTX. In conclusion the biological activities induced by TCC in nucleated cells may be mediated in part by activation of PTX-sensitive G proteins.

B-Lymphocytes↗
PubMed1993

Generation of diacylglycerol and ceramide during homologous complement activation.

Formation of sublytic terminal complement complexes (TCC) on nucleated cells produces transient increase in [Ca2+]i and activates protein kinase C. The present study is to evaluate whether TCC can generate endogenous signal messengers other than Ca2+ that regulate cell activities by measuring mass-levels of sn-1,2-diacylglycerol (DAG) and ceramide. As targets, lymphoblastoid human B cell lines JY25 and its mutant JY5 were used. JY5, cells deficient in glycosylphosphatidylinositol-anchored proteins with higher lytic susceptibility to human complement, are four times more efficient in forming C5b-9. When cells sensitized with limited anti-class II IgG were exposed to human serum to generate sublytic TCC, a sustained increase in DAG and ceramide was observed with a maximum 3.6-fold DAG increase over basal level in JY25 and 2.8-fold in JY5, and 6.3-fold ceramide increase in JY25 and 2.8-fold in JY5. The effect of TCC was evaluated with C7-deficient human serum (C7D) +/- C7 and also with C5b6, C7, C8, and C9 proteins. The DAG and ceramide increase by C7D + C7 over C7D control were 1.6- and 1.8-fold, respectively, in JY25, and 2.3-, and two-fold in JY5. TCC activation also induced an increased hydrolysis of sphyingomyelin and phosphatidylcholine. In addition, DAG increase by TCC was primarily achieved by C5b-7 and preincubation of cells with pertussis toxininhibited DAG increase, suggesting an involvement of a pertussis toxin-sensitive GTP-binding protein. As important signal transduction molecules, DAG and ceramide generated in response to TCC assembly, could participate in cell activation during inflammation and repair.

Animals↗
PubMed1992

Persistent complement activation on tumor cells in breast cancer.

The neoantigens of the C5b-9 complement complex, IgG, C3, C4, S-protein/vitronectin, fibronectin, and macrophages were localized on 17 samples of breast cancer and on 6 samples of benign breast tumors using polyclonal or monoclonal antibodies and the streptavidin-biotin-peroxidase technique. All the tissue samples with carcinoma in each the TNM stages presented C5b-9 deposits on the membranes of tumor cells, thin granules on cell remnants, and diffuse deposits in the necrotic areas. When chemotherapy and radiation therapy preceded surgery, C5b-9 deposits were more intense and extended. The C5b-9 deposits were absent in all the samples with benign lesions. S-protein/vitronectin was present as fibrillar deposits in the connective tissue matrix and as diffuse deposits around the tumor cells, less intense and extended than fibronectin. IgG, C3, and C4 deposits were present only in carcinoma samples. The presence of C5b-9 deposits is indicative of complement activation and its subsequent pathogenetic effects in breast cancer.

Blood Proteins↗
PubMed1991

Tumor necrosis factor-alpha in human arterial wall with atherosclerosis.

Tumor necrosis factor-alpha (TNF-alpha) was demonstrated in human normal and atherosclerotic aorta, iliac and femoral arteries by immunohistochemistry using monoclonal and polyclonal antibodies. TNF was present in the cells of the arterial wall and as granular and diffuse extracellular deposits in the connective tissue matrix. Quantitative determinations of TNF by ELISA showed mean values of 21.7 +/- 0.7 ng/100 mg total extracted protein in normal intima, 38.2 +/- 0.5 in intimal thickenings, 25.5 +/- 1.1 in fibrous plaques and 16.8 +/- 0.2 ng/100 mg total extracted protein in media. Intimal thickenings presented the highest amounts of TNF with a statistically significant difference when compared to normal intima (P less than 0.05) and media (P less than 0.01). TNF-alpha concentrations in arterial eluates were about 200 times higher than in the corresponding serum samples. Western blotting analysis confirmed TNF-alpha eluted from the arterial wall to be about 17 kDa similar to human recombinant TNF-alpha. TNF-alpha in human atherosclerotic wall could be actively involved in the inflammatory events associated with atherosclerosis.

Aorta↗
PubMed1990

Decay-accelerating factor regulates complement-mediated damage in the human atherosclerotic wall.

Decay-accelerating factor (DAF) is an intrinsic membrane inhibitor that regulates the activity of C3 and C5 convertases of the classical and alternative complement pathways. Using two monoclonal antibodies, IC6 and IA10, DAF was localized by immunohistochemistry using streptavidin-biotin-peroxidase complex or silver-intensified immunogold techniques in aortic, iliac and femoral samples obtained at surgery and autopsy from 32 patients. DAF was localized on the cells and in the connective tissue matrix of the arterial wall. Fibrous plaques and intimal thickenings presented larger amounts than fatty streaks, intimae and normal areas. By Western blotting analysis, DAF extracted from the arterial wall had a molecular weight of about 67 kDa. Using a double-labeling technique, DAF and C5b-9 complexes were co-localized on nucleated cells and on cell debris. The cells isolated after enzyme digestion of the arterial wall were tested for the protective role of DAF to complement-mediated damage. When DAF of the sensitized cells was blocked by monoclonal antibodies, complement-mediated cell lysis was enhanced from 10-15% to 60-70%. The effect of anti-DAF antibodies was dose-dependent. DAF blocking in the absence of antibodies used for sensitization led to a lysis under 10%. These data suggest a protective role of DAF against autologous complement activation, however insufficient to prevent complement activation in the human atherosclerotic wall.

Adult↗
PubMed1989

Tissue-type plasminogen activator (t-PA) and dilute blood clot lysis time in nephrotic patients.

When compared to normal weight normolipidemic control subjects, dilute blood clot lysis time was found to be obviously (p less than 0.001) prolonged in hypertriglyceridemic patients without proteinuria and slightly (p less than 0.05) accelerated in hyperlipidemic nephrotic patients in spite of their very high levels of plasma fibrinogen. As a result the ratio plasma fibrinogen (mg/dl) per clot lysis time (minutes) was 1.241 +/- 0.08 (X +/- SEM) in control subjects, 0.574 +/- 0.07 in hypertriglyceridemic patients and 2.69 +/- 0.172 in nephrotic patients. This finding suggesting that a larger amount of fibrin is rather readily dispersed from dilute blood clots of nephrotic patients was associated with higher levels of plasma t-PA:Ag (9.45 ng/ml +/- 1.18 in nephrotic patients versus 5.8 ng/ml +/- 1.23 in controls before venous occlusion and respectively 33.1 ng/ml +/- 3.83 versus 20.3 +/- 3.40 in controls after venous occlusion). Plasminogen activator activity of the euglobulins as assessed by the bovine fibrin-agarose plate was significantly higher in nephrotic patients only after venous occlusion. Plasma samples of nephrotic patients exerted a more potent inhibition of fibrinolysis in a urokinase activated system. This effect was, however, mainly due to the high levels of alpha 2 macroglobulin in nephrotic plasma which apparently have little influence on dilute blood clot lysis time.

Adolescent↗
PubMed1989

Immunoelectron-microscopic localization of S-protein/vitronectin in human atherosclerotic wall.

S-protein/vitronectin is a multifunctional glycoprotein interacting with both complement activation and coagulation pathways. Its presence was investigated in 5 femoral and 5 iliac atherosclerotic human arteries, obtained at surgery, by immunoelectron microscopy using an affinity purified rabbit IgG specific for human S-protein/vitronectin. The immunoelectron dense specific deposits were found in both intimal thickenings and fibrous plaques in association with elastic fibers, collagen bundles and cell debris in the vicinity of elastin. Cell debris embedded in the collagen matrix were S-protein/vitronectin negative. S-protein/vitronectin was also absent on intact cells, lipid droplets and cholesterol clefts. All cell debris, however, was positive for C5b-9 deposits suggesting that complement activation had occurred at these sites with or without S-protein/vitronectin interaction. S-protein/vitronectin may play a role in the arterial wall defence by restricting the extent of complement activation.

Arteriosclerosis↗
PubMed1989

Cells carrying C5b-9 complement complexes in human atherosclerotic wall.

Fibrous plaques and intimal thickenings of 5 femoral and 5 iliac human arteries obtained at surgery were processed for indirect and double-labeling immunoelectron microscopy using an affinity purified rabbit IgG anti-C5b-9 neoantigen and the EBM 11 monoclonal antibody anti-human macrophages. The C5b-9 complexes were localized in intact cells, disintegrated cells and cell debris enmeshed in the connective tissue matrix. Some of the cell debris bearing C5b-9 deposits was found to be of macrophage origin. Endocyted or exocyted pieces of membrane with pore-forming C5b-9 complexes were also identified. Damage of cells by complement in atherosclerotic lesions may contribute to atherogenesis.

Antibodies, Monoclonal↗
PubMed1988

The relationship between macrophages and C5b-9 complement complexes in human atherosclerosis.

The relationship between macrophages and the terminal C5b-9 complement complexes was investigated in human arteries affected with atherosclerosis by using monoclonal antibodies and indirect immunoperoxidase, immunogold silver staining, and double-labeling immunohistochemical techniques. Macrophages were found in all the atherosclerotic arteries as immunoreactive deposits with a nucleus, considered as intact cells, or without a nucleus, considered as cell remnants. The double-labeling technique shows C5b-9 deposits partially colocalized on the intact macrophages or on the cell debris of macrophage origin. These data suggest that C5b-9 complement complex may be formed on activated or dying macrophages with subsequent promotion of inflammatory events and progression of the atherosclerotic lesions.

Adult↗