Apoptosis of erythroid precursors under stimulation with thrombopoietin: contribution to megakaryocytic lineage choice.
(17/1441)
Although the effect of thrombopoietin (TPO) on megakaryocyte production is well established, its role in the commitment of multipotential hematopoietic progenitors to the megakaryocytic lineage remains to be determined. In the present study, we attempted to clarify the determination process of megakaryocytic lineage as a terminal differentiation pathway under stimulation with TPO. Day 7 cultured cells grown by TPO derived from cord blood CD34+ cells were divided into four subpopulations on the basis of CD34 and CD41 expression. The CD34-/CD41- cells showed the labeling pattern of anti-CD42b and anti-CD9 antibodies closer to that of the CD34+/CD41- cells than the CD34+/CD41+ cells. Replating experiments revealed that approximately 40% of the CD34-/CD41- cells proliferated in response to a combination of growth factors, and more than 80% of them were pure erythroid precursors. However, this subpopulation failed to grow/survive and fell into apoptosis in the presence of TPO alone. In contrast, the CD34+/CD41+ cells, which predominantly contained megakaryocytic precursors, exerted a low but significant proliferative potential in the presence of TPO. The insufficient response to TPO of the CD34-/CD41- cells may result from the apparently low expression of c-MpI, as determined by flow cytometric analysis and reverse transcription-polymerase chain reaction analysis. Therefore, these results suggest that the apoptosis of hematopoietic precursors other than megakaryocytic precursors is related to the determination of the terminal differentiation under the influence of TPO. (+info)
Comparison of expression and regulation of the high-density lipoprotein receptor SR-BI and the low-density lipoprotein receptor in human adrenocortical carcinoma NCI-H295 cells.
(18/1441)
In rodents, cholesterol for adrenal steroidogenesis is derived mainly from high-density lipoproteins (HDL) via the HDL receptor, scavenger receptor-BI (SR-BI). In humans cholesterol for steroidogenesis is considered to be derived from the low-density lipoprotein (LDL) receptor pathway, and the contribution of SR-BI to that is unknown. In the present study SR-BI expression and regulation by steroidogenic stimuli was analysed in human adrenocortical cells and compared with LDL receptor expression. In addition, the functional contribution of both receptors for cholesteryl ester delivery to human adrenocortical cells was compared. Northern blot and reverse transcription-PCR amplification and sequence analysis demonstrated the presence of SR-BI mRNA in foetal and adult human adrenal cortex. Furthermore, SR-BI mRNA was expressed to similar levels in human primary adrenocortical and adrenocortical carcinoma NCI-H295 cells, indicating its presence in the steroid-producing cells. Treatment of NCI-H295 cells with 8Br-cAMP, a stimulator of glucocorticoid synthesis via the protein kinase A second messenger signal transduction pathway, resulted in an increase of both SR-BI and LDL receptor mRNA levels in a time- and dose-dependent manner. The induction of SR-BI and LDL receptor by cAMP was independent of ongoing protein synthesis and occurred at the transcriptional level. Ligand blot experiments indicated that a protein of similar size to SR-BI is the major HDL-binding protein in NCI-H295 cells. Western blot analysis demonstrated that cAMP treatment increased the levels of LDL receptor and, to a lesser extent, SR-BI protein in NCI-H295 cells. Binding and uptake of cholesterol was quantitatively smaller from HDL than from LDL, both in basal as well as in cAMP-stimulated cells. Scatchard analysis under basal conditions indicated that NCI-H295 cells express twice as many specific binding sites for LDL than for HDL. Dissociation constant values (Kd; in nm) were approximately five times higher for HDL than for LDL, indicating a lower affinity of HDL compared with LDL. The combined effects of these two parameters and the low cholesteryl ester content of HDL subfraction 3 (HDL3) contributes to a lower cholesteryl ester uptake from HDL than from LDL by the NCI-H295 cells. In conclusion, both the SR-BI and LDL receptor genes are expressed in the human adrenal cortex and coordinately regulated by activators of glucocorticoid synthesis. In contrast to rodents, in human adrenocortical cells the HDL pathway of cholesterol delivery appears to be of lesser importance than the LDL pathway. Nevertheless, the SR-BI pathway may become of major importance in conditions of functional defects in the LDL receptor pathway. (+info)
Cell cholesterol efflux: integration of old and new observations provides new insights.
(19/1441)
Numerous studies using a variety of cell/acceptor combinations have demonstrated differences in cholesterol efflux among cells. These studies also show that different acceptors, ranging from simple molecules like cyclodextrins to serum, stimulate efflux through a variety of mechanisms. By combining early observations with data derived from recent studies, it is now possible to formulate a model for cell cholesterol efflux which proposes that an array of different mechanisms, including aqueous diffusion, lipid-free apolipoprotein membrane microsolubilization, and SR-BI-mediated cholesterol exchange contribute to cholesterol flux. In this model the relative importance of each mechanism would be determined both by the cell type and the nature of the extracellular cholesterol acceptor. (+info)
Cellular cholesterol regulates expression of the macrophage type B scavenger receptor, CD36.
(20/1441)
CD36, the macrophage type B scavenger receptor, binds and internalizes oxidized low density lipoprotein (OxLDL), and may potentially play a role in the development of atherosclerosis. We reported that the native and modified low density lipoproteins increased CD36 mRNA and protein ( J. Biol. Chem. 272: 21654-21659). In this study, we investigated the effect of alterations of cellular cholesterol content on macrophage expression of CD36. Depletion of cholesterol by treatment with beta-cyclodextrins (beta-cyclodextrin [beta-CD] and methylated beta-cyclodextrin [MebetaCD]) significantly decreased CD36 mRNA and 125I-labeled OxLDL binding. Conversely, loading macrophages with cholesterol or cholesteryl ester (acetate) with MebetaCD:cholesterol complexes increased CD36 mRNA, 125I-labeled OxLDL binding, and CD36 surface expression as determined by fluorescence activated cell sorting. Thus, CD36 expression paralleled cellular cholesterol levels after removal of cholesterol with beta-cyclodextrins or addition of cholesterol with MebetaCD:cholesterol complexes. Neither cholesterol depletion nor loading altered expression of type A scavenger receptor mRNA. Kinetics studies showed that changes in CD36 mRNA occurred after changes of cellular cholesterol. Neither beta-cyclodextrins nor MebetaCD:cholesterol altered CD36 mRNA half-life in the presence of actinomycin D, suggesting that alterations in CD36 expression by cholesterol occur at the transcriptional level. These experiments demonstrate that CD36 expression is enhanced by cholesterol and down-regulated by cholesterol efflux, and imply that macrophage expression of CD36 and foam cell formation in atherosclerotic lesions may be perpetuated by a cycle in which lipids drive expression of CD36 in a self-regulatory manner. (+info)
Lovastatin decreases the receptor-mediated degradation of acetylated and oxidized LDLs in human blood monocytes during the early stage of differentiation into macrophages.
(21/1441)
3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors are used therapeutically to upregulate the LDL receptor-mediated removal of plasma cholesterol by the liver. Several lines of evidence indicate that these drugs also exert direct effects on the metabolism of native and modified LDL in extrahepatic cells. We studied the effects of lovastatin (LOV) on the degradation of native, acetylated, and oxidized LDL, and on levels of mRNA encoding for the LDL, types I and II class A macrophage scavenger, and CD36 receptors in human blood monocytes at different stages of their maturation into adherent macrophages. LOV (10 micromol/L) reduced the degradation of acetylated LDL when added to freshly isolated cells cultured for 2 (81+/-4% of control, P<0.05) and 5 (76+/-6%, of control, P<0.05) days. The degradation of oxidized LDL was also reduced in cells treated with LOV for 2 days after seeding (51+/-3% of control, P<0. 001) but not in 5-day-old cells. LOV had no significant effect on the degradation of either acetylated or oxidized LDL when added to fully matured macrophages allowed to differentiate under control conditions for 7 days before incubations with 10 micromol/L LOV for an additional 2 days. In contrast, LOV increased the degradation of native LDL in these cells at all 3 stages of cell differentiation. LOV also reduced class A types I and II macrophage scavenger receptor and CD36 mRNA levels in 2- and 5-day-old cells but not in the more mature macrophages. These data suggest that 3-hydroxy-3-methylglutaryl-coenzyme A inhibitors may reduce the expression and function of the class A types I and II macrophage scavenger receptor and CD36 in monocytes, during the early stages of their differentiation into adherent macrophages. These effects, if operative in vivo, may slow down the development of the atherosclerotic plaque and thus contribute to the beneficial effects of these drugs. (+info)
Probucol enhances selective uptake of HDL-associated cholesteryl esters in vitro by a scavenger receptor B-I-dependent mechanism.
(22/1441)
Recently, the class B, type I scavenger receptor (SR-BI) has been shown to mediate the selective uptake of high density lipoprotein (HDL) cholesteryl esters (CEs), ie, lipid uptake independent of HDL holoparticle uptake. In vivo, this selective uptake delivers CEs to the liver for excretion and to steroidogenic tissues for hormone synthesis. Probucol, a hydrophobic antioxidant drug, lowers plasma cholesterol in humans and rodents and may inhibit progression of atherosclerosis and postangioplasty restenosis. In this study, the effect of probucol on HDL selective CE uptake was investigated in mice and in cells expressing SR-BI. Probucol feeding lowered plasma HDL cholesterol and markedly increased selective CE uptake from HDL in the liver and adrenal glands. However, probucol did not alter SR-BI protein levels in membranes from these organs. When incubated with control Chinese hamster ovary (CHO) cells, HDL isolated from probucol-treated mice (P-HDL) and HDL from control mice (C-HDL) showed similar low selective uptake of CEs. However, when incubated with SR-BI-transfected CHO cells, P-HDL showed a 2-fold increase in selective uptake compared with C-HDL. In an adrenal cell line (Y1-BS1), which expresses SR-BI in an adrenocorticotropic hormone-inducible manner, P-HDL showed significantly greater selective CE uptake than did C-HDL, and the differential response was amplified by adrenocorticotropic hormone treatment. In contrast to P-HDL, incorporation of this compound into HDL in vitro did not result in stimulation of selective CE uptake by SR-BI-transfected CHO cells, even though a significant mass of probucol could be detected in the HDL preparation. The specific interaction of P-HDL with SR-BI in cell culture could be observed after only 24 hours of probucol feeding, when there were minimal changes in HDL size and composition. Thus, probucol or one of its metabolites increases selective CE uptake in vivo by modifying HDL in a way that causes enhanced interaction with SR-BI. The increased interaction of P-HDL with SR-BI in the liver and arterial wall may be partly responsible for the effects of probucol on atherosclerosis and restenosis. (+info)
CD36, a novel receptor for oxidized low-density lipoproteins, is highly expressed on lipid-laden macrophages in human atherosclerotic aorta.
(23/1441)
CD36 has been reported to be a receptor for oxidized LDL (Ox-LDL). In our previous study, the uptake of Ox-LDL in CD36-deficient macrophages was reduced by approximately 50% compared with that in control macrophages, suggesting an important role of CD36 as a receptor for Ox-LDL in humans. In the current study, we examined the immunohistochemical localization of CD36 in human aorta in comparison with that of scavenger receptor class A type I and type II (SRA). Cryostat sections were made from aortic tissues. For immunohistochemical staining, the following antibodies were used: (1) FA6-152, anti-CD36 antibody, and (2) SRI-2, which recognizes both type I and type II SRAs. Immunohistochemical staining for CD36 and SRA was performed using labeled streptavidin method. In macrophages scattered in aortic walls without atherosclerotic lesions, the expression of CD36 was hardly observed, whereas that of SRA was detected weakly but consistently. In contrast, in atherosclerotic lesions, macrophages around the core region showed a weak immunoreactivity to CD36 and a strong immunoreactivity to SRA. Furthermore, lipid-laden macrophages, which mainly existed in the core region, had a strongly positive immunoreactivity to CD36, but a low or moderate level of immunoreactivity to SRA. The distributions of CD36 and SRA were different from each other, and especially foamed, large-sized macrophages in atherosclerotic plaques tended to more abundantly express CD36 protein. These data demonstrate, for the first time, that the expression of both CD36 and SRA might be differentially regulated in aortic walls, and might play different roles in the formation of foam cells in atherosclerotic lesions. (+info)
Expression of thrombospondin receptor (CD36) in B-cell chronic lymphocytic leukemia as an indicator of tumor cell dissemination.
(24/1441)
BACKGROUND AND OBJECTIVE: The expression of CD36 antigen has not been conclusively associated with human B-lymphocytes although CD36 was recently detected in a human B-cell angiotropic lymphoma where it might be involved in lymphoblast-endothelial cell adhesion. We investigated the expression of CD36 in B-cell chronic lymphocytic leukemia (CLL) by multiparameter flow cytometry; results were correlated with clinical features. DESIGN AND METHODS: CD36 expression was evaluated on peripheral blood and bone marrow samples from 24 patients affected by CD5+ B-CLL. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation, were labeled with fluorochrome-conjugated monoclonal antibodies under standard experimental conditions and were analyzed by flow cytometry. CD36 expression was quantified both in terms of frequency of CD19+CD36+ cells and of mean fluorescence intensity (MFI-R) of CD36+ cell populations. The intensity of CD36 expression was arbitrarily classified as weak (MFI-R ranging from 3 to 6; score 0), moderate (MFI-R ranging from 6 to 9; score 1), intermediate (MFI-R ranging from 9 to 11; score 2) or strong (MFI-R ranging from 11 to 17; score 3). RESULTS: CD36 could be detected on 3% (range 2-5) of normal CD19+ B-lymphocytes and on 45% (range 30-75) of neoplastic CD19+ B-cells. When CLL patients were stratified according to CD36 staining intensity, higher hemoglobin levels (Hb) were recorded in patients assigned to score 0 (Hb = 14.3 g/dL; range 13.9-15.1) compared to patients scoring 1-2 (Hb = 11.2; range 10.3-12.2) or 3 (Hb = 9.8; range 9.6-11.6; p=0.0053). Similarly, higher platelet counts (Plt) were found in patients scoring 0 (Plt = 282x10(3)/microL; range 244-319), compared to patients with intermediate (Plt = 175x10(3)/microL; range 144-238) and high scores (Plt = 149x10(3)/microL; range 103-230; p=0.044); lymphocyte count (Ly) was significantly higher in patients assigned to score 3-4 (Ly = 23.3x10(3)/microL, range 13-30) compared to score 0-2 (Ly = 9.8x10(3)/microL, range 8.5-10.8; p=0.045). CLL patients expressing CD36 at intermediate-to-strong intensity (MFI-R = 14, range 9-16) were more frequently assigned to Rai stages III-IV than stages I-II (CD36 MFI-R = 9, range 6.5-11; p=0.005) and stage 0 (CD36 MFI-R = 6, range 4-7.3; p<0.001). Interestingly, bone marrow diffuse histology was strongly associated with higher CD36 expression (MFI-R = 8.7; range 4.7-13.9) compared to non-diffuse patterns of bone marrow infiltration (MFI-R = 6.7; range 5.2-9.3; p=0.0019). In multivariate regression analysis, CD36 staining intensity significantly and independently correlated with diffuse BM histology (p=0.033). INTERPRETATION AND CONCLUSIONS: The present report provides the first evidence of CD36 expression on CD19+ B-cells from CLL; the correlations with clinical parameters strongly support the view that CD36 might favor tumor cell spreading. Whether high CD36 expression levels on CLL CD19+ B-cells identify an aggressive disease subset remains to be further confirmed in larger series of patients. (+info)