RESUMO
Atherosclerosis is an inflammatory disease process associated with elevated levels of plasma cholesterol, especially low-density lipoproteins. The latter become trapped within the arterial wall and are oxidized and taken up by macrophages to form foam cells. This process is an initiating event for atherosclerosis. Fatty acid binding proteins (FABP) are involved in fatty acid metabolism and cellular lipid transport, and adipocyte FABP (aP2) is also expressed in macrophages. We recently generated mice lacking both apolipoprotein (Apo)E and aP2 (ApoE-/-aP2-/-) and found that these mice, compared with ApoE-/- mice, developed markedly smaller atherosclerotic lesions that contained fewer macrophages. Here we investigated the mechanism(s) responsible for this prevention of atherosclerotic lesion formation. Bone marrow transplantations were performed in ApoE-/- mice, receiving cells from either ApoE-/- or ApoE-/-aP2-/- mice. The lack of aP2 in donor marrow cells led to the development of smaller (5.5-fold) atherosclerotic lesions in the recipient mice. No differences were found in plasma cholesterol, glucose, or insulin levels between recipients of bone marrow cells from ApoE-/- or ApoE-/-aP2-/- mice. However, the expression of chemoattractant and inflammatory cytokines was decreased in macrophages from ApoE-/-aP2-/- mice compared with ApoE-/- mice, which may contribute to the decrease in atherosclerotic lesion formation. Taken together, we demonstrate the importance of macrophage aP2 in the development of atherosclerotic lesions.
Assuntos
Arteriosclerose/patologia , Proteínas de Transporte/fisiologia , Hipercolesterolemia/complicações , Macrófagos/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Arteriosclerose/sangue , Arteriosclerose/complicações , Glicemia/metabolismo , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Proteínas de Transporte/genética , Colesterol/sangue , Citocinas/sangue , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Insulina/sangue , Camundongos , Camundongos Knockout , Óxido Nítrico/sangueAssuntos
Arteriosclerose/prevenção & controle , Proteínas de Transporte/fisiologia , Hipercolesterolemia/complicações , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Animais , Aorta/metabolismo , Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Proteínas de Transporte/genética , Colesterol/sangue , Colágeno/metabolismo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Metabolismo dos Lipídeos , Macrófagos/patologia , Camundongos , Camundongos Knockout , Triglicerídeos/sangueRESUMO
This study examined the premise that the atherogenic lipoprotein, beta-migrating very low density lipoprotein (betaVLDL), might activate the mitogen-activated protein (MAP) kinases ERK1/ERK2, thereby contributing to the induction of smooth muscle cell proliferation in atherosclerosis. The data show that betaVLDL activates rabbit smooth muscle cell ERK1/ERK2. Interestingly, ERK1/ERK2 activation is mediated by G protein-coupled receptors that transactivate the epidermal growth factor (EGF) receptor. betaVLDL-induced MAP kinase activation depends on Ras and Src activity as well as protein kinase C. The inhibition of lysosomal degradation of betaVLDL has no effect on ERK1/ERK2 activation. The contribution of betaVLDL-induced activation of ERK1/ERK2 to smooth muscle cell proliferation was also explored. betaVLDL induces expression of egr-1 and c-fos mRNA. Despite its ability to stimulate early gene expression, betaVLDL alone is unable to inspire quiescent cells into S phase. When added in conjunction with EGF, however, stimulation of [(3)H]thymidine incorporation into DNA and an increase in histone gene expression are observed. Moreover, betaVLDL plus EGF synergistically induce cyclin D1 expression and down-regulate p27(KIP1) expression. The addition of either betaVLDL or EGF stimulates a robust activation of ERK1/ERK2, but the addition of both agents simultaneously sustains the activation for a longer time period. Inhibition of MAP kinase kinase, pertussis toxin-sensitive G proteins, the EGF receptor, or protein kinase C blocks betaVLDL plus EGF-induced proliferation, demonstrating that activation of the betaVLDL-induced signaling pathway results in smooth muscle cell proliferation.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Lipoproteínas VLDL/farmacologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Músculo Liso/enzimologia , Receptores de Superfície Celular/fisiologia , Ativação Transcricional , Animais , Divisão Celular/efeitos dos fármacos , Ciclina D1/biossíntese , Proteínas de Ligação a DNA/biossíntese , Ativação Enzimática , Lipoproteínas LDL/farmacologia , Sistema de Sinalização das MAP Quinases , Músculo Liso/citologia , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas c-fos/biossíntese , Coelhos , Fatores de Transcrição/biossínteseRESUMO
Immunohistochemical localization of the injury specific apolipoprotein, acute phase serum amyloid A (A-apoSAA), was compared in brains of patients with neuropathologically confirmed Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease (PD); Pick's disease (Pick's), dementia with Lewy bodies (DLB), coronary artery disease (CAD), and schizophrenia. Affected regions of both AD and MS brains showed intense staining for A-apoSAA in comparison to an unaffected region and non-AD/MS brains. The major site of A-apoSAA staining in both diseases was the myelin sheaths of axons in layers V and VI of affected cortex. A-apoSAA contains a cholesterol binding site near its amino terminus and is likely to have a high affinity for cholesterol-rich myelin. These findings, along with our recent evidence that A-apoSAA can inhibit lipid synthesis in vascular smooth muscle cells suggest that A-apoSAA plays a role in the neuronal loss and white matter damage occurring in AD and MS.
Assuntos
Doença de Alzheimer/metabolismo , Apolipoproteínas/metabolismo , Encéfalo/metabolismo , Bainha de Mielina/metabolismo , Terminações Pré-Sinápticas/metabolismo , Proteína Amiloide A Sérica/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Encéfalo/patologia , Membrana Celular/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Precursores de Proteínas/metabolismoRESUMO
Serum amyloid A apolipoproteins (apoSAA) appear to compromise the ability of high density lipoprotein to protect against atherosclerosis and it is of interest to determine whether aortic smooth muscle cells can contribute to local pools of apoSAA in the presence of cytokines that are known to stimulate acute phase apoSAA (A-apoSAA) synthesis in the liver. In this study, the regulation of A-apoSAA synthesis was monitored in cultured neonatal rabbit aortic smooth muscle cells. Constitutive apoSAA3 gene expression was minimal, and only detectable by amplification of the mRNA by reverse transcriptase-polymerase chain reaction. ApoSAA3 gene expression and protein synthesis were stimulated by IL-1 alpha; as little as 0.01 ng/ml of IL-1 alpha stimulated an increase in steady state levels of apoSAA3 mRNA. Interestingly, IL-6 (which is required in addition to IL-1 alpha for the optimal synthesis of A-apoSAA by human hepatoma cells) had little if any effect on apoSAA3 synthesis by the smooth muscle cells. In a time course, it was shown that the stimulation of apoSAA3 mRNA levels was apparent by 1-2 h after the addition of cytokine, and that levels remained elevated in the presence of the cytokine for at least 48 h. Immunoprecipitation using an antiserum directed against apoSAA3 revealed that IL-1 alpha stimulated the synthesis and secretion of apoSAA3 protein in a manner that was consistent with apoSAA3 mRNA expression. The implications of these findings in atherogenesis are discussed.
Assuntos
Apolipoproteínas/biossíntese , Regulação da Expressão Gênica , Interleucina-1/farmacologia , Músculo Liso Vascular/metabolismo , Proteína Amiloide A Sérica/biossíntese , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Aorta , Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Células Cultivadas , Primers do DNA/química , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-6/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Dados de Sequência Molecular , Músculo Liso Vascular/efeitos dos fármacos , Reação em Cadeia da Polimerase , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , RNA Mensageiro/biossíntese , Coelhos , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismoRESUMO
The human apoSAA proteins comprise both acute phase (apoSAA1, apoSAA2) and constitutive (apoSAA4) isoforms; all are expressed in human atherosclerotic lesions as well as in liver. Recombinant acute phase apoSAA binds cholesterol with an affinity of approximately 170 nM and enhances cholesterol uptake by HepG2 cells (J. Lipid Res. 1995. 36:37-46). In the present study, we sought to define the region of acute phase apoSAA involved in cholesterol binding and to investigate the ability of constitutive apoSAA4 to bind cholesterol. Binding of [3H]cholesterol to apoSAAp was inhibited by unlabeled cholesterol (1-100 nM), but not significantly by vitamin D and estradiol. Direct binding of acute phase, but not constitutive, apoSAA to the surfaces of polystyrene microtiter wells was strongly diminished in the presence of cholesterol. The ability of apoSAAp to bind cholesterol was inhibited by antibodies to human apoSAA1 and to peptide 1-18 of apoSAA1. There was only slight inhibition of cholesterol binding by antibodies to peptide 40-63, and no inhibition by antibodies to peptides spanning regions containing amino acid residues 14-44 and 59-104. [3H]cholesterol uptake by neonatal rabbit aortic smooth muscle and HepG2 cells was enhanced by a synthetic peptide corresponding to amino acids 1-18 of hSAA1, but not by peptides corresponding to amino acids 1-18 of hSAA4. [3H]cholesterol uptake by HepG2 cells was slightly increased by a peptide corresponding to amino acids 40-63 of hSAA1. These findings suggest that apoSAA modulates the local flux of cholesterol between cells and lipoproteins during inflammation and atherosclerosis.
Assuntos
Aorta/metabolismo , Colesterol/metabolismo , Fígado/metabolismo , Músculo Liso Vascular/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteína Amiloide A Sérica/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sítios de Ligação , Transporte Biológico , Linhagem Celular , Estradiol/metabolismo , Humanos , Fragmentos de Peptídeos/química , Ligação Proteica , Estrutura Secundária de Proteína , Coelhos , Proteínas Recombinantes/metabolismo , Proteína Amiloide A Sérica/química , Vitamina D/metabolismoRESUMO
We have investigated the synthesis and transport of apoE, the major apolipoprotein of the central nervous system, in the retina of the living rabbit. Four hours after the injection of [35S]methionine/cysteine into the vitreous, 44% of [35S]Met/Cys-labeled apoE is in soluble and membrane-enclosed retinal fractions, while 50% is in the vitreous. A significant amount of intact [35S]Met/Cys-labeled apoE is rapidly transported into the optic nerve and its terminals in the lateral geniculate and superior colliculus within 3-6 h in two distinguishable vesicular compartments. Müller glia in cell culture also synthesize and secrete apoE. Taken together, these results suggest that apoE is synthesized by Müller glia and secreted into the vitreous. ApoE is also internalized by retinal ganglion cells and/or synthesized by these cells and rapidly transported into the optic nerve and brain as an intact molecule. We discuss the possible roles of retinal apoE in neuronal dynamics.
Assuntos
Apolipoproteínas E/biossíntese , Neuroglia/metabolismo , Nervo Óptico/metabolismo , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Apolipoproteínas E/isolamento & purificação , Apolipoproteínas E/metabolismo , Autorradiografia , Transporte Axonal , Células Cultivadas , Cisteína/metabolismo , Eletroforese em Gel de Poliacrilamida , Corpos Geniculados/metabolismo , Imuno-Histoquímica , Cinética , Metionina/metabolismo , Neuroglia/citologia , Coelhos , Retina/citologia , Radioisótopos de Enxofre , Colículos Superiores/metabolismo , Fatores de Tempo , Corpo Vítreo/metabolismoRESUMO
The anticancer activity of synthetic ether lipids may depend in part upon their ability to activate cells of the monocyte/macrophage lineage. In the present study, we have sought to determine whether 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and related ether lipids enhance superoxide production by mouse peritoneal macrophages. Ether lipids were administered intraperitoneally to C57BL/6 mice 4 d after injection with thioglycollate broth. Elicited peritoneal macrophages were harvested and purified one day later, and superoxide production was detected by measuring the superoxide dismutase inhibitable reduction of cytochrome c. Low levels of superoxide were secreted by macrophages in the absence of phorbol 12-myristate 13-acetate (PMA). When PMA was added in vitro to macrophages from ET-18-OMe-treated mice, these cells secreted 194.2 nmol superoxide/mg protein in comparison to 53.5 nmol superoxide/mg protein for PMA-treated control cells. The in vitro treatment of the macrophages with ET-18-OMe was not effective in stimulating superoxide secretion. Macrophages harvested from mice treated with a series of ether lipids (with and without phosphorus) were examined, and superoxide secretion was found to vary with structure. AM-18-OEt and CP-7 were the most effective compounds, secreting 8.6 and 11.9 times more superoxide, respectively, than PMA-stimulated control cells. Moreover, direct cytotoxicity of the compounds for HL60 human promyelocytic leukemic cells did not necessarily correlate with the ability of each drug to increase macrophage superoxide production.
Assuntos
Macrófagos Peritoneais/metabolismo , Éteres Fosfolipídicos/farmacologia , Superóxidos/metabolismo , Animais , Antineoplásicos/farmacologia , Grupo dos Citocromos c/metabolismo , Humanos , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Superóxido Dismutase/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
This report describes the enhancement of growth potentiating activity produced by mononuclear phagocytes that were incubated with beta-migrating very low density lipoproteins (beta-VLDL). Conditioned media harvested from cultured human peripheral blood monocytes incubated in the presence or absence of the lipoprotein were evaluated for their ability to stimulate DNA synthesis ([3H]thymidine incorporation) of sparsely seeded quiescent BHK-21 (BHK) cells as well as neonatal rat aortic smooth muscle cells (NRSMC). Conditioned media from monocytes incubated in the presence of beta-VLDL enhanced [3H]thymidine incorporation into DNA of both BHK and NRSMC, when compared to conditioned media harvested from monocytes incubated in the absence of beta-VLDL. Studying NRSMC, this effect was evident using media collected from monocytes incubated with lipoprotein for 2 days; however, a longer incubation of monocytes plus lipoprotein was necessary to induce changes in growth potentiating activity for BHK cells. Likewise, the effect of beta-VLDL treatment of thioglycollate broth elicited BALB/c mouse peritoneal macrophages was evaluated. Conditioned media from lipoprotein-treated macrophages exhibited greater growth-stimulating activity for both BHK cells and NRSMC when compared to conditioned media from macrophages incubated in the absence of the lipoprotein. beta-VLDL did not affect viability of the mononuclear cells. These findings further implicate the involvement of the monocyte-derived foam cell in the development of atherosclerosis.