RESUMEN
BACKGROUND: Inappropriate inflammation is a key mechanism in the development of atherosclerosis. Antibodies against components of the atherosclerotic lesion, in particular, oxidised low density lipoprotein, have been described. OBJECTIVE: To determine whether a systemic autoimmune response, characterised by the presence of high titres of antinuclear antibodies, is associated with the presence of coronary atherosclerosis. METHODS: Serum was prepared from 40 subjects (aged 53-76) with at least 50% stenoses of three main coronary arteries (TVD subjects), and 30 subjects (aged 48-74) with no evidence of coronary atherosclerosis (NCA subjects) determined by coronary angiography. RESULTS: Antinuclear antibodies (ANA), characterised by immunofluorescent detection of human antibodies bound to HEp-2000 cells, were detected at a titre of at least 1/40 in 28 (70%) of the TVD subjects, but only five (17%) of the NCA patients (odds ratio 11.67 (95% confidence interval (CI) 3.91 to 17.82; p<0.001)). Most ANA positive TVD subjects had a pattern typical of antibodies directed against nucleolar antigens. The antigen has not yet been identified, but several common extractable antigens were excluded. The presence of ANA was not associated with incidence of prior myocardial infarction among the TVD group. CONCLUSION: The presence of ANA, commonly associated with autoimmune diseases, is substantially more prevalent among subjects with severe coronary atherosclerosis than those with normal coronary arteries. This association merits further assessment as a potentially useful indicator of increased risk of coronary heart disease.
Asunto(s)
Anticuerpos Antinucleares/sangre , Enfermedad de la Arteria Coronaria/inmunología , Anciano , Estudios de Casos y Controles , Nucléolo Celular/inmunología , Estenosis Coronaria/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Persona de Mediana Edad , Estadística como AsuntoRESUMEN
Smooth muscle cells from neonatal rats and from injured blood vessels grow with a characteristic cobblestone morphology that distinguishes them from adult smooth muscle cells. This has led to the proposition that there are two distinct types of smooth muscle cells with different proliferative capacity. Here we systematically compare the properties of subcultured adult smooth muscle cells in culture and clonal lines of cobblestone smooth muscle cells from both neonatal rats and injured vessels. The cobblestone smooth muscle cells have a significantly smaller average cell volume, estimated using two different flow cytometry measurements. However, the two types of smooth muscle cells have indistinguishable protein expression patterns when the levels of more than 20 different proteins (including cytoskeletal proteins, matrix proteins, cytokines, cytokine receptors, adhesion molecules and enzymes) are measured by quantitative immunofluorescence. Furthermore, in contrast to previous observations, we demonstrate that both types of smooth muscle cells secrete a powerful mitogenic activity. The higher cell density achieved by the cobblestone smooth muscle cells in culture was responsible for the earlier reports that this mitogenic activity was secreted only by cobblestone smooth muscle cells. We conclude that many of the differences seen between cobblestone smooth muscle cells and adult smooth muscle cells in vitro (proliferation rate, morphology, protein expression pattern, secretion of mitogenic activity) could be attributable to a stable difference in the median cell volume of the cultures.
Asunto(s)
Envejecimiento/fisiología , División Celular/fisiología , Músculo Liso Vascular/citología , Proteínas/metabolismo , Células 3T3 , Animales , Animales Recién Nacidos , Aorta , Recuento de Células , Tamaño de la Célula , Células Cultivadas , Técnicas de Cocultivo , Cinética , Masculino , Ratones , Músculo Liso Vascular/fisiología , Ratas , Ratas Wistar , Timidina/metabolismo , Túnica Íntima/citología , Túnica Íntima/fisiologíaRESUMEN
Cerebral ischemia-reperfusion injury is associated with a developing inflammatory response with pathologic contributions from vascular leukocytes and endogenous microglia. Signaling chemokines orchestrate the communication between the different inflammatory cell types and the damaged tissue leading to cellular chemotaxis and lesion occupation. Several therapies aimed at preventing this inflammatory response have demonstrated neuroprotective efficacy in experimental models of stroke, but to date, few investigators have used the chemokines as potential therapeutic targets. In the current study, the authors investigate the neuroprotective action of NR58-3.14.3, a novel broad-spectrum inhibitor of chemokine function (both CXC and CC types), in a rat model of cerebral ischemia-reperfusion injury. Rats were subjected to 90 minutes of focal ischemia by the filament method followed by 72 hours of reperfusion. Both the lesion volume, measured by serial magnetic resonance imaging, and the neurologic function were assessed daily. Intravenous NR58-3.14.3 was administered, 2 mg/kg bolus followed by 0.5 mg/kg hour constant infusion for the entire 72-hour period. At 72 hours, the cerebral leukocytic infiltrate, tumor necrosis factor-alpha (TNF-alpha), and interleukin-8 (IL-8)-like cytokines were analyzed by quantitative immunofluorescence. NR58-3.14.3 significantly reduced the lesion volume by up to 50% at 24, 48, and 72 hours post-middle cerebral artery occlusion, which was associated with a marked functional improvement to 48 hours. In NR58-3.14.3-treated rats, the number of infiltrating granulocytes and macrophages within perilesional regions were reduced, but there were no detectable differences in inflammatory cell numbers within core ischemic areas. The authors reported increased expression of the cytokines, TNF-alpha, and IL-8-like cytokines within the ischemic lesion, but no differences between the NR58-3.14.3-treated rats and controls were reported. Although chemokines can have pro- or antiinflammatory action, these data suggest the overall effect of chemokine up-regulation and expression in ischemia-reperfusion injury is detrimental to outcome.
Asunto(s)
Quimiocinas/antagonistas & inhibidores , Ataque Isquémico Transitorio , Fármacos Neuroprotectores/uso terapéutico , Péptidos Cíclicos/uso terapéutico , Daño por Reperfusión/prevención & control , Animales , Encéfalo/patología , Arterias Cerebrales , Constricción , Técnica del Anticuerpo Fluorescente , Granulocitos/patología , Interleucina-8/análisis , Leucocitos/patología , Receptores de Lipopolisacáridos/análisis , Macrófagos/inmunología , Macrófagos/patología , Imagen por Resonancia Magnética , Masculino , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/patología , Factor de Necrosis Tumoral alfa/análisisRESUMEN
The introduction of a range of different genetic modifications in mice results in altered lipoprotein metabolism and the development of vascular lipid lesions. At present, however, it is unclear to what extent the molecular events underlying lipid lesion formation are similar in these different mouse models of atherosclerosis. The aim of this study was to compare the protein expression pattern of lipid lesions from seven different mouse lines with varying susceptibility to vascular lipid lesion development, to determine to what extent lesions induced by different genetic interventions have a similar composition. The proteins we have measured, using quantitative immunofluorescence, are proteins whose expression is known to be modulated during atherogenesis in humans, including plasminogen activator inhibitor (PAI)-1, transforming growth factor (TGF)-beta 1, osteopontin and the macrophage marker CD11b. In all the mice lines we have investigated, PAI-1 was elevated wherever lesions developed. Active TGF-beta was depressed in the vessel wall of mice which developed lipid lesions, particularly in the intima. In contrast, TGF-beta 1 antigen (active plus latent TGF-beta 1) was increased at lesion sites. Accumulation of osteopontin and, with the marked exception of apolipoprotein(a) transgenic mice, tissue macrophages occurred at sites of lipid deposition in the vessel wall. Each lesion, irrespective of its size and the mouse strain in which it developed, had similar amounts of PAI-1, active TGF-beta and osteopontin per unit area of lesion. These data are consistent with a common phenotype accompanying atherogenesis, irrespective of the genetic basis of susceptibility.
Asunto(s)
Apolipoproteínas/genética , Arteriosclerosis/etiología , Animales , Apolipoproteínas A/genética , Apolipoproteínas B/genética , Apolipoproteínas E/genética , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Femenino , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Monocitos , Osteopontina , Fenotipo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Sialoglicoproteínas/metabolismo , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1RESUMEN
Chemokines participate in the regulation of leucocyte recruitment in a wide variety of inflammatory processes, including host defence and diseases such as asthma, atherosclerosis and autoimmune disorders. We have previously described the properties of Peptide 3, the first broad-specificity chemokine inhibitor in vitro. Here, we report the properties of NR58-3.14.3, a retroinverso analogue of Peptide 3. NR58-3.14.3 inhibited leucocyte migration induced by a range of chemokines, including monocyte chemoattractant protein-1 (MCP-1) (2.5 nM), macrophage inflammatory protein-1alpha (MIP-1alpha) (5 nM), regulated on activation, normal T-cell expressed and presumably secreted (RANTES) (20 nM), stromal cell-derived factor-1alpha (SDF-1alpha) (25 nM) and interleukin-8 (IL-8) (30 nM), but did not affect migration induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or complement C5a (> 100 microM). NR58-3.14.3 is therefore approximately 1000-fold more potent than Peptide 3 but retains the broad-spectrum chemokine inhibitory activity of the parent peptide. In vivo, pretreatment with a systemic dose of 10 mg of NR58-3.14.3, but not the inactive derivative NR58-3.14.4, abolished leucocyte recruitment in response to intradermal injection of 500 ng of MCP-1 into rat skin. This suggests that NR58-3.14.3 is a functional chemokine inhibitor in vivo as well as in vitro. We utilized NR58-3.14.3 as a tool to investigate the role of chemokine activity during leucocyte recruitment in response to lipopolysaccharide (LPS) in vivo. NR58-3.14.3, but not NR58-3.14.4, abolished leucocyte recruitment in response to intradermal injection of 50 ng of LPS into rat skin. Furthermore, NR58-3.14.3 completely inhibited LPS-induced accumulation of tumour necrosis factor-alpha (TNF-alpha). This data is consistent with a model in which multiple chemokines act in parallel upstream of TNF-alpha. NR58-3.14.3 is therefore a powerful anti-inflammatory agent in vivo, suppressing proinflammatory cytokine production and leucocyte recruitment in response to endotoxin stimulus in rat skin.
Asunto(s)
Dermatitis/prevención & control , Leucocitos/inmunología , Lipopolisacáridos/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/inmunología , Quimiocinas/antagonistas & inhibidores , Dermatitis/inmunología , Diseño de Fármacos , Femenino , Humanos , Lipopolisacáridos/inmunología , Péptidos Cíclicos/química , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Células Tumorales CultivadasRESUMEN
Thrombin has been proposed to play a key role in the development of atherosclerosis, both by promoting fibrin deposition into the atherosclerotic vessel wall and also by signalling through thrombin receptors. Unfortunately, mice homozygous for a deletion of the prothrombin gene (FII) die in utero, making a direct assessment of the role of thrombin during atherogenesis difficult. We have assessed the contribution of thrombin-dependent processes to vascular lipid lesion formation in the atherosclerosis-prone apolipoprotein E (ApoE)-deficient mice by inhibiting thrombin generation with warfarin. ApoE-/- mice were treated with warfarin at a dose that increased the prothrombin time (PT) more than 10-fold (250-375 microg/kg body weight/day) for 12 weeks from the age of 12 weeks onwards. The extent and composition of the vascular lipid lesions that developed were assessed using oil red O to measure neutral lipid in the vessel wall and quantitative immunofluoresence to measure fibrin(ogen) levels as well as macrophage and smooth muscle cell numbers. Mice treated with warfarin developed lesions both in the aortic sinus and the descending aorta to the same degree as mice receiving no treatment (28,351+/-350 microm2/mouse treated with warfarin versus 27,952+/-750 micro2/control mouse; P = .86). However, the amount of fibrin(ogen) deposited in the vessel wall was decreased by more than 60% (34+/-11 arbitrary units in warfarin treated mice versus 92+/-11 arbitrary units in control mice; P < .01). Staining of macrophage and for smooth muscle cell markers was unaltered by treatment with warfarin. We conclude that suppressing thrombin generation does not alter the development of vascular lipid lesions in mice with a severe disorder of lipid metabolism, despite a marked reduction in fibrin(ogen) deposition.
Asunto(s)
Arteriosclerosis/etiología , Trombina/biosíntesis , Animales , Anticoagulantes/farmacología , Aorta/efectos de los fármacos , Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Arteriosclerosis/patología , Depresión Química , Modelos Animales de Enfermedad , Fibrina/efectos de los fármacos , Fibrina/metabolismo , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/patología , Tiempo de Protrombina , Trombina/antagonistas & inhibidores , Trombina/farmacología , Warfarina/farmacologíaRESUMEN
In early development much of the cellular diversity and pattern formation of the embryo is believed to be set up by morphogens. However, for many morphogens, including members of the TGF-beta superfamily, the mechanism(s) by which they reach distant cells is unknown. We have used immunofluorescence to detect, at single cell resolution, a morphogen gradient formed across vertebrate tissue. The TGF-beta ligand is distributed in a gradient visible up to 7 cell diameters (about 150-200 microm) from its source, and is detectable only in the extracellular space. This morphogen gradient is functional, since we demonstrate activation of a high response gene (Xeomes) and a low-response gene (Xbra) at different distances from the TGF-beta source. Expression of the high affinity type II TGF-beta receptor is necessary for detection of the gradient, but the shape of the gradient formed only depends in part on the spatial variation in the amount of receptor. Finally, we demonstrate that the molecular processes that participate in forming this functional morphogen gradient are temperature independent, since the gradient forms to a similar extent whether the cells are maintained at 4 degrees C or 23 degrees C. In contrast, TGF-beta1 internalisation by cells of the Xenopus embryo is a temperature-dependent process. Our results thus suggest that neither vesicular transcytosis nor other active processes contribute to a significant extent to the formation of the morphogen gradient we observe. We conclude that, in the model system used here, a functional morphogen gradient can be formed within a few hours by a mechanism of passive diffusion.
Asunto(s)
Proteínas de Xenopus , Xenopus/embriología , Animales , Tipificación del Cuerpo , Difusión , Regulación del Desarrollo de la Expresión Génica , Ligandos , Morfogénesis , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas de Dominio T Box/genética , Temperatura , Factor de Crecimiento Transformador beta/metabolismo , Xenopus/genética , Xenopus/metabolismoRESUMEN
The cytokine transforming growth factor beta1 (TGF-beta1) is secreted in a latent form that has no known biological activity. The conversion of latent TGF-beta1 into its biologically active 25 kDa form is thought to be an important step in the regulation of TGF-beta activity both in cell culture and in vivo. Thrombospondin (TSP)-1, a 360 kDa platelet alpha-granule and extracellular matrix protein, has been shown to participate in TGF-beta1 activation. We have used a chemically defined system to examine the mechanism of TSP-1-mediated TGF-beta1 activation. However, the addition of two different preparations of TSP-1 to recombinant small latent TGF-beta1 in the test tube resulted in only a very small increase in the proportion of the TGF-beta1 able to bind to the TGF-beta type II receptor: from 0.1% to a maximum of 0.4%. This small effect was not specific for TSP-1: matrix metalloproteinase 2, tissue inhibitor of matrix metalloproteinase 2 and active plasminogen activator inhibitor 1, but not transglutaminase, human serum albumin or immunoglobulin, had quantitatively similar effects on latent TGF-beta1. Furthermore, no change in the activity associated with small latent TGF-beta1 was noted in either mink lung epithelial cell or rat aortic smooth-muscle cell culture systems in the presence of TSP-1 (or TSP-1-derived peptides). We conclude that TSP-1, either alone or in the presence of cultured smooth-muscle cells (a cell type known to activate latent TGF-beta in vitro and in vivo) is unable to activate latent TGF-beta1. Any TSP-mediated activation of TGF-beta1 must depend on additional factor(s) not present in our systems.
Asunto(s)
Músculo Liso/metabolismo , Trombospondina 1/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Células Cultivadas , Músculo Liso/citología , RatasRESUMEN
Transforming growth factor-(beta) (TGF(beta)) has a wide range of activities on vascular cells and inflammatory cells, suggesting it may have different functions during various stages of atherogenesis. We report that mice heterozygous for the deletion of the tgfb1 gene (tgfb1(+/-) mice) have reduced levels of TGF(beta)1 in the artery wall until at least 8 weeks of age. On a normal mouse chow diet, the vascular endothelium of tgfb1(+/-) mice is indistinguishable from wild-type littermates, assessed by morphology and intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression. In contrast, levels of the smooth muscle isoforms of actin and myosin in medial smooth muscle cells of tgfb1(+/-) mice are significantly reduced. Following feeding a cholesterol-enriched diet for 12 weeks, high levels of ICAM-1 and VCAM-1 were detected in the vascular endothelial cells of tgfb1(+/-) mice, but not wild-type mice. Furthermore, marked deposition of lipid into the artery wall was only observed in the tgfb1(+/-) mice on the cholesterol-enriched diet. These vascular lipid lesions were accompanied by local invasion of macrophages. We conclude that deletion of a single allele of the tgfb1 gene results in a reduced level of TGFbeta1 antigen in the aorta together with reduced smooth muscle cell differentiation, whereas the addition of a high fat dietary challenge is required to activate the vascular endothelium and to promote the formation of fatty streaks resembling early atherosclerosis in humans.
Asunto(s)
Arteriosclerosis/etiología , Arteriosclerosis/fisiopatología , Grasas de la Dieta/efectos adversos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Metabolismo de los Lípidos , Músculo Liso Vascular/metabolismo , Factor de Crecimiento Transformador beta/deficiencia , Animales , Aorta/metabolismo , Aorta/patología , Aorta/fisiopatología , Arteriosclerosis/patología , Endotelio Vascular/patología , Inflamación/patología , Inflamación/fisiopatología , Lipoproteínas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/patología , Músculo Liso Vascular/fisiopatologíaRESUMEN
The cytokine transforming growth factor-beta (TGF-beta) was initially purified from human platelets, a rich source of this protein. In addition to platelets, TGF-beta1 is also found in other blood fractions, including plasma and the circulating leukocytes. However, more than 15 years after the initial isolation of TGF-beta1, there remains no consensus on how much TGF-beta1 is present in normal human plasma. Here we review the difficulties associated with measuring TGF-beta concentrations in complex biological fluids, and discuss the current state of knowledge on the distribution of TGF-beta isoforms in various blood fractions as well as the nature of the TGF-beta-containing protein complexes.
Asunto(s)
Factor de Crecimiento Transformador beta/sangre , Animales , Plaquetas/metabolismo , Cromatografía Liquida/métodos , Ensayo de Inmunoadsorción Enzimática , Humanos , Valores de Referencia , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/orinaRESUMEN
A method for quantifying an intramolecularly linked all-d-amino acid peptide, NR58-3.14.3, in rat serum by LC-MS using selected ion monitoring with inclusion of a diastereomer as internal standard was developed. The reproducible quantitation of multiply charged compounds by LC-MS using single ion or selective reaction monitoring is often a challenge as the intensity ratio of the ions in a series of different charge states can vary. Good precision was obtained in the selected ion monitoring mode by integrating the summed ion currents of the singly, doubly, and triply charged molecular ions. Since stable isotope analogs are costly and integration of residual unlabeled material can be of concern, a diastereomer of NR58-3.14.3, NR58-3.14.5, was used as internal standard. The diastereomers were indistinguishable by electrospray MS, but fully separated by reversed-phase LC. Consequently, interference due to isotopic impurities or coelution was not encountered. The calibration plot was linear throughout a concentration range of 0.2 to 200.0 microg/ml (r(2) = 0.9996). Intraday precision of the standards analyzed was less than 12% RSD over the calibration range and the accuracy within +/-11% RE. Serum pharmacokinetics were in good agreement with the pharmacokinetic profiles of small, ionic, and polar molecules.
Asunto(s)
Quimiocina CCL2/análogos & derivados , Péptidos Cíclicos/sangre , Animales , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/normas , Femenino , Espectrometría de Masas/métodos , Espectrometría de Masas/normas , Péptidos Cíclicos/farmacocinética , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , EstereoisomerismoRESUMEN
Osteoporosis is a major public health problem characterized by low bone mineral density (BMD) that presently has no biochemical test useful for its diagnosis. The cytokine TGF-beta has been postulated to play a role in controlling bone density by regulating the fine balance between bone matrix deposition by osteoblasts and its resorption by osteoclasts. We explored whether measurement of serum levels of different TGF-beta isoforms could be useful as a clinical tool in osteoporosis. We measured the concentration of TGF-beta1 antigen using the BDA19 capture sandwich enzyme-linked immunosorbent assay (ELISA), TGF-beta2 antigen concentration using a Quantikine sandwich ELISA kit and TGF-beta3 antigen concentration using a modified version of the TGF-beta1 Quantikine sandwich ELISA kit. Subjects were 41 women with osteoporosis (with nontraumatic vertebral fracture or lumbar spine BMD Z-score <-1.5 SD) and a total of 199 control women from different sources. Serum concentrations of TGF-beta1 and TGF-beta2 were similar in all groups. However, detectable levels of TGF-beta3 (>0.2 ng/ml) were found in 35 of 41 patients with osteoporosis (median 7.2 (5.2-8.9) ng/ml) compared with 11 of 36 controls or 24 of 89 healthy women of unknown bone density. Differences among the groups could not be accounted for by age, weight, medications, use of hormone replacement therapy or the presence of osteoarthritis. Using the optimal cut-off of >/=2 ng/ml, the test was able to detect an individual with low spine BMD (Z-score <-1.5) with a sensitivity of 84% and a specificity of 53%, with similar results for the femoral neck. The odds ratio for osteoporosis associated with a positive test at this level was 5.93 (95% CI 2.41-11.59), and 4.1 (95% CI 1.66-10.11) using the WHO cut-off of T-score <-2.5. Serum TGF-beta3 concentration is raised in osteoporotic women and the test appears to have potential as a marker for osteoporosis. The underlying mechanisms and the relationships between TGF-beta3 and bone turnover and fractures remain to be explored.
Asunto(s)
Osteoporosis Posmenopáusica/diagnóstico , Factor de Crecimiento Transformador beta/sangre , Análisis de Varianza , Biomarcadores/sangre , Densidad Ósea , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Persona de Mediana Edad , Oportunidad Relativa , Osteoporosis Posmenopáusica/sangre , Isoformas de Proteínas/sangre , Curva ROC , Sensibilidad y EspecificidadRESUMEN
The presence of active transforming growth factor-beta (TGF-beta) in serum has not been widely accepted. In particular, although at least five studies have concluded that active TGF-beta is present in normal human plasma and serum, assays that use the extracellular domain of the TGF-beta type II receptor as a capture agent have given contradictory results. We show that there is an antagonist present in normal human serum which inhibits the binding of active TGF-beta to the extracellular domain of the TGF-beta type II receptor when it is coated on the well of an ELISA plate. This antagonist activity is due to a pool of immunoglobulins of the G2, D and M classes. Moreover, we show that this same pool of immunoglobulins also recognizes the extracellular domain of the platelet-derived growth factor alpha-receptor, insulin-like growth factor-1 receptor and interleukin-3 receptor, by serial transfer of serum over the different receptors. In addition, the same immunoglobulin pool inhibits the binding of platelet-derived growth factor-AA to its receptor, in an analogous way to the inhibition of binding of TGF-beta to its type II receptor. Circumstantial evidence suggests that the pool of immunoglobulins is recognizing a carbohydrate residue that is attached to the protein when it is synthesized by the mouse myeloma cell line, NSO, in which it is made. If the cytokine receptors are similarly glycosylated in vivo, then the presence of these antibodies in normal human serum may modulate physiological cytokine signalling.
Asunto(s)
Anticuerpos/inmunología , Inmunoglobulinas/inmunología , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismo , Animales , Anticuerpos/sangre , Anticuerpos/aislamiento & purificación , Humanos , Inmunoglobulinas/sangre , Inmunoglobulinas/aislamiento & purificación , Ratones , Unión Proteica , Proteínas Serina-Treonina Quinasas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
We have identified an amino acid sequence, termed peptide 3, corresponding to amino acids 51-62 of the mature human monocyte chemoattractant protein-1 (MCP-1), which inhibits human mononuclear-cell and THP-1-cell migration induced by a wide range of chemokines. For example, peptide 3 inhibited MCP-1-induced THP-1 migration in a transwell assay with an ED50 of approx. 8 microM. Peptide 3 binds directly to THP-1 cells with an association constant of approx. 10 microM, and is therefore likely to be a direct receptor antagonist for CC and CXC chemokine receptors. By performing a structure-function analysis of this peptide, we have identified a sequence variant that shows an approx. 3-4-fold greater potency as an inhibitor of chemokine-induced migration [Leu4Ile11 peptide 3 (1-12)]. Furthermore, unlike peptide 3, which binds to the Duffy antigen receptor for chemokines on human erythrocytes with a similar affinity to the specific chemokine receptors on THP-1 cells, the Leu4Ile11 peptide 3 (1-12) sequence variant shows at least 20-fold greater selectivity for the specific receptors. Derivatives of Leu4Ile11 peptide 3 (1-12) are therefore the best candidates among the molecules we have investigated for use as a chemokine inhibitor in vivo.
Asunto(s)
Antígenos de Protozoos , Quimiocinas/antagonistas & inhibidores , Quimiotaxis de Leucocito/efectos de los fármacos , Oligopéptidos/farmacología , Proteínas Protozoarias , Adulto , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Células Cultivadas , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/química , Quimiocina CCL2/metabolismo , Quimiocina CCL2/farmacología , Quimiocinas/química , Quimiocinas/metabolismo , Quimiocinas/farmacología , Secuencia Conservada/genética , Relación Dosis-Respuesta a Droga , Eritrocitos/citología , Eritrocitos/metabolismo , Femenino , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/genética , Oligopéptidos/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Receptores de Superficie Celular/metabolismo , Receptores de Quimiocina/antagonistas & inhibidores , Receptores de Quimiocina/metabolismo , Receptores de Quimiocina/fisiología , Alineación de Secuencia , Relación Estructura-Actividad , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
BACKGROUND: Apolipoprotein (apo)(a) transgenic mice and C57BL/6 mice fed a high fat diet develop similar-sized lipid lesions, but lesions in apo(a) mice are devoid of macrophages. We used this observation to identify which proinflammatory proteins might be involved in mediating monocyte recruitment during atherogenesis. METHODS AND RESULTS: Macrophage-deficient apo(a) transgenic mouse lesions contained similar levels of several different proinflammatory proteins, both adhesion molecules (intercellular adhesion molecule-1 [ICAM-1] and vascular cell adhesion molecule-1 [VCAM-1]) and cytokines (tumor necrosis factor-alpha [TNF-alpha] and macrophage inflammatory protein-1alpha [MIP-1alpha]), similar to the macrophage-rich lesions of C57BL/6 mice. CONCLUSIONS: From this we conclude that ICAM-1, VCAM-1, TNF-alpha, and MIP-1alpha may all be necessary for vascular monocyte recruitment in vivo, but they cannot be sufficient. Monocyte chemoattractant protein-1 (MCP-1) protein was undetectable in the vessel wall taken from apo(a) transgenic mice fed a high fat diet compared with high expression in mice with lipid lesions (C57BL/6 and apoE knockout mice). Therefore elevated expression of MCP-1 but not TNF-alpha, MIP-1alpha, ICAM-1, or VCAM-1 is correlated with vascular macrophage accumulation. To test the hypothesis that monocyte infiltration during atherogenesis is MCP-1 dependent, it will be necessary to develop specific pharmacological inhibitors of MCP-1 activity.
Asunto(s)
Arteriosclerosis/patología , Quimiocina CCL2/análisis , Grasas de la Dieta/toxicidad , Lipoproteína(a) , Macrófagos/patología , Monocitos/fisiología , Factor de Necrosis Tumoral alfa/análisis , Animales , Apolipoproteínas/fisiología , Apoproteína(a) , Femenino , Molécula 1 de Adhesión Intercelular/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Molécula 1 de Adhesión Celular Vascular/análisisRESUMEN
The concentration of transforming growth factor beta (TGF-beta) in plasma has been correlated with the development of several diseases, including atherosclerosis and certain forms of cancer. However, the mechanisms that control the concentration of TGF-beta in plasma are poorly understood. In a study of 170 pairs of female twins (average age 57.7 years) we show that the concentration of active plus acid-activatable latent TGF-beta1 [(a+l) TGF-beta therefore is predominantly under genetic control (heritability estimate 0.54). Single strand conformation polymorphism (SSCP) mapping of the TGF-beta1 gene promoter has identified two single base substitution polymorphisms. The two polymorphisms (G-->A at position -800 bp and C-->T at position -509 bp) are in linkage disequilibrium (correlation coefficient Delta = 0.215, P < 0.01). The C-509T polymorphism is significantly associated with the plasma concentration of (a+l) TGF-beta1, explaining 8.2% of the additive genetic variance of (a+l) TGF-beta1 concentration. It is therefore possible that predisposition to atherosclerosis, bone diseases or various forms of cancer may be correlated with the presence of particular alleles at the TGFB1 locus.
Asunto(s)
Factor de Crecimiento Transformador beta/sangre , Factor de Crecimiento Transformador beta/genética , Adulto , Anciano , Alelos , Arteriosclerosis/sangre , Arteriosclerosis/genética , Secuencia de Bases , Enfermedades Óseas/sangre , Enfermedades Óseas/genética , ADN/genética , Cartilla de ADN/genética , Femenino , Variación Genética , Humanos , Desequilibrio de Ligamiento , Persona de Mediana Edad , Modelos Genéticos , Neoplasias/sangre , Neoplasias/genética , Polimorfismo Conformacional Retorcido-Simple , Regiones Promotoras Genéticas , Gemelos Dicigóticos , Gemelos MonocigóticosRESUMEN
1. Transforming growth factor-beta1 is a cytokine with a very wide spectrum of biological activities. Previous studies have shown that it is involved in a number of physiological and pathological processes including heart disease. In our study we aimed to scan the transforming growth factor-beta1 locus for polymorphisms and to identify haplotypes significantly associated with a predisposition to coronary atherosclerosis.2. Two patient groups comprising 244 angiographically normal individuals and 655 patients with coronary artery disease were recruited from London and Sheffield. DNA samples from these subjects were screened for mutations in the transforming growth factor-beta1 locus and all subjects were genotyped by a coupled polymerase chain reaction-restriction enzyme digestion method.3. Five polymorphisms have been identified in the transforming growth factor-beta1 gene at positions G-800A, C-509T in the promoter region, Leu10-->Pro, Arg25-->Pro in exon 1 and Thr263-->Ile in exon 5. No significant difference in frequencies for any of the five polymorphisms was found between controls and patients with coronary artery disease. Similarly, there was no correlation between these polymorphisms and hypertension.4. The genotypes of all the individuals participating in the study were assigned to seven main haplotypes of the transforming growth factor-beta1 locus. Based on species comparison data we propose that GCCGC is the ancestral haplotype in humans.5. Our data suggest that these transforming growth factor-beta1 polymorphisms are not associated with coronary artery disease and therefore their presence alone would not be a genetic risk factor for predisposition to coronary artery disease.
Asunto(s)
Enfermedad Coronaria/genética , Predisposición Genética a la Enfermedad , Polimorfismo Genético , Factor de Crecimiento Transformador beta/genética , Estudios de Casos y Controles , Distribución de Chi-Cuadrado , Femenino , Marcadores Genéticos , Genotipo , Haplotipos , Humanos , Desequilibrio de Ligamiento , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Factores de RiesgoRESUMEN
Whether the post-prandial lipemic response is linked to potentially pro-atherogenic and/or prothrombotic changes in plasminogen activator inhibitor (PAI) and transforming growth factor-beta (TGF-beta) is uncertain. The aim of our study was to determine whether PAI-1 antigen and PAI activity were elevated during post-prandial lipemia following a standard fat tolerance test. We also investigated changes in TGF-beta1 antigen and TGF-beta activity, to determine whether changes in TGF-beta activity were associated with changes in PAI measurements. Lastly, the influence of genotype at a common insertion/deletion polymorphism in the PAI-1 promoter on changes in PAI activity and PAI-1 antigen was examined. Fat tolerance tests were undertaken in 57 healthy middle-aged men to investigate associations between plasma concentrations of lipoproteins, PAI (antigen and activity) and TGF-beta. PAI-1 concentration increased by 76% after 8 h (P < 0.0001). PAI activity also increased by 64% (P = 0.0054) and TGF-beta activity decreased by 10% (P < 0.0001). Increases in PAI-I antigen and PAI activity varied markedly between individuals. To investigate these heterogeneous responses we examined whether genotype at the common insertion/deletion polymorphism of the PAI-1 promoter accounted for these differences. Individuals with at least one 4G (deletion) allele showed potentially pro-atherogenic changes in both PAI-1 and TGF-beta, compared to individuals who were homozygous for the 5G (insertion) allele. In conclusion, increased PAI and decreased TGF-beta activity occur during a fat tolerance test and this effect may be modulated by a common insertion/deletion polymorphism in the PAI-1 promoter.
Asunto(s)
Grasas de la Dieta/administración & dosificación , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inactivadores Plasminogénicos/metabolismo , Factor de Crecimiento Transformador beta/sangre , Anciano , Alelos , Índice de Masa Corporal , Grasas de la Dieta/farmacología , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Inhibidor 1 de Activador Plasminogénico/genética , Periodo Posprandial , Regiones Promotoras Genéticas , Triglicéridos/sangreRESUMEN
Variations in the levels of smooth muscle-specific isoforms of contractile proteins have been reported to occur in many different vascular diseases. However, although much work has been done in vitro to investigate the regulation of smooth muscle cell differentiation, the molecular mechanisms which regulate the differentiation of vascular smooth muscle tissue in vivo are unknown. Using quantitative immunofluorescence, we show that in rat arteries levels of smooth muscle differentiation markers correlate with the levels of the cytokine TGF-beta. In young mice with one allele of the TGF-beta1 gene deleted, the levels of both TGF-beta1 and smooth muscle differentiation markers are reduced compared to wild-type controls. This regulation of smooth muscle differentiation by TGF-beta during post-natal development also occurs dynamically in the adult animal. Following various pharmacological or surgical interventions, including treatment of mice with tamoxifen and balloon injury of rat carotid arteries, there is a strong correlation between the changes in the levels of TGF-beta and changes in the levels of smooth muscle differentiation markers (r=0. 9, P<0.0001 for n=26 experiments). We conclude that TGF-beta dynamically regulates smooth muscle differentiation in rodent arteries in vivo.