RESUMO
Inflammation is a hallmark of some of today's most life-threatening diseases such as arteriosclerosis, cancer, diabetes and Alzheimer's disease. Herbal medicines (HMs) are re-emerging resources in the fight against these conditions and for many of them, anti-inflammatory activity has been demonstrated. However, several aspects of HMs such as their multi-component character, natural variability and pharmacodynamic interactions (e.g. synergism) hamper identification of their bioactive constituents and thus the development of appropriate quality control (QC) workflows. In this study, we investigated the potential use of Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy as a tool to rapidly and non-destructively assess different anti-inflammatory properties of ethanolic extracts from various species of the Genus Lonicera (Caprifoliaceae). Reference measurements for multivariate calibration comprised in vitro bioactivity of crude extracts towards four key players of inflammation: Nitric oxide (NO), interleukin 8 (IL-8), peroxisome proliferator-activated receptor ß/δ (PPAR ß/δ), and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB). Multivariate analysis of variance (MANOVA) revealed a statistically significant, quantitative pattern-activity relationship between the extracts' ATR-FTIR spectra and their ability to modulate these targets in the corresponding cell models. Ensemble orthogonal partial least squares (OPLS) discriminant models were established for the identification of extracts exhibiting high and low activity with respect to their potential to suppress NO and IL-8 production. Predictions made on an independent test set revealed good generalizability of the models with overall sensitivity and specificity of 80% and 100%, respectively. Partial least squares (PLS) regression models were successfully established to predict the extracts' ability to suppress NO production and NF-κB activity with root mean squared errors of cross-validation (RMSECV) of 8.7% and 0.05-fold activity, respectively.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Lonicera/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Animais , Anti-Inflamatórios/isolamento & purificação , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Interleucina-8/antagonistas & inibidores , Interleucina-8/imunologia , Camundongos , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/imunologia , Extratos Vegetais/isolamento & purificação , Células RAW 264.7 , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
PURPOSE: Neurolaena lobata is a Caribbean medicinal plant used for the treatment of several conditions including inflammation. Recent data regarding potent anti-inflammatory activity of the plant and isolated sesquiterpene lactones raised our interest in further pharmacological studies. The present work aimed at providing a mechanistic insight into the anti-inflammatory activity of N. lobata and eight isolated sesquiterpene lactones, as well as a structure-activity relationship and in vivo anti-inflammatory data. METHODS: The effect of the extract and its compounds on the generation of pro-inflammatory proteins was assessed in vitro in endothelial and monocytic cells by enzyme-linked immunosorbent assay. Their potential to modulate the expression of inflammatory genes was further studied at the mRNA level. In vivo anti-inflammatory activity of the chemically characterized extract was evaluated using carrageenan-induced paw edema model in rats. RESULTS: The compounds and extract inhibited LPS- and TNF-α-induced upregulation of the pro-inflammatory molecules E-selectin and interleukin-8 in HUVECtert and THP-1 cells. LPS-induced elevation of mRNA encoding for E-selectin and interleukin-8 was also suppressed. Furthermore, the extract inhibited the development of acute inflammation in rats. CONCLUSIONS: Sesquiterpene lactones from N. lobata interfered with the induction of inflammatory cell adhesion molecules and chemokines in cells stimulated with bacterial products and cytokines. Structure-activity analysis revealed the importance of the double bond at C-4-C-5 and C-2-C-3 and the acetyl group at C-9 for the anti-inflammatory activity. The effect was confirmed in vivo, which raises further interest in the therapeutic potential of the compounds for the treatment of inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Asteraceae/química , Lactonas/farmacologia , Sesquiterpenos/farmacologia , Animais , Selectina E/metabolismo , Edema/tratamento farmacológico , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Interleucina-8/metabolismo , Masculino , Estrutura Molecular , Monócitos/efeitos dos fármacos , Componentes Aéreos da Planta/química , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
BACKGROUND AND PURPOSE: The transcription factor NF-κB orchestrates many pro-inflammatory signals and its inhibition is considered a promising strategy to combat inflammation. Here we report the characterization of the natural product plumericin as a highly potent inhibitor of the NF-κB pathway with a novel chemical scaffold, which was isolated via a bioactivity-guided approach, from extracts of Himatanthus sucuuba, an Amazonian plant traditionally used to treat inflammation-related disorders. EXPERIMENTAL APPROACH: A NF-κB luciferase reporter gene assay was used to identify NF-κB pathway inhibitors from H. sucuuba extracts. Monitoring of TNF-α-induced expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin by flow cytometry was used to confirm NF-κB inhibition in endothelial cells, and thioglycollate-induced peritonitis in mice to confirm effects in vivo. Western blotting and transfection experiments were used to investigate the mechanism of action of plumericin. KEY RESULTS: Plumericin inhibited NF-κB-mediated transactivation of a luciferase reporter gene (IC50 1 µM), abolished TNF-α-induced expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin in endothelial cells and suppressed thioglycollate-induced peritonitis in mice. Plumericin exerted its NF-κB pathway inhibitory effect by blocking IκB phosphorylation and degradation. Plumericin also inhibited NF-κB activation induced by transfection with the constitutively active catalytic subunit of the IκB kinase (IKK-ß), suggesting IKK involvement in the inhibitory action of this natural product. CONCLUSION AND IMPLICATIONS: Plumericin is a potent inhibitor of NF-κB pathways with a new chemical scaffold. It could be further explored as a novel anti-inflammatory lead compound.
Assuntos
Anti-Inflamatórios/farmacologia , Indenos/farmacologia , Mediadores da Inflamação/antagonistas & inibidores , Inflamação/prevenção & controle , Iridoides/farmacologia , NF-kappa B/antagonistas & inibidores , Extratos Vegetais/farmacologia , Animais , Apocynaceae , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Tioglicolatos , TransfecçãoRESUMO
Melampyrum pratense L. (Koch) is used in traditional Austrian medicine for the treatment of different inflammation-related conditions. In this work, we show that the extracts of M. pratense stimulated peroxisome proliferator-activated receptors- (PPARs-) α and - γ that are well recognized for their anti-inflammatory activities. Furthermore, the extract inhibited the activation of the proinflammatory transcription factor NF- κ B and induction of its target genes interleukin-8 (IL-8) and E-selectin in vitro. Bioassay-guided fractionation identified several active flavonoids and iridoids including melampyroside and mussaenoside and the phenolic compound lunularin that were identified in this species for the first time. The flavonoids apigenin and luteolin were distinguished as the main components accountable for the anti-inflammatory properties. Apigenin and luteolin effectively inhibited tumor necrosis factor α (TNF- α )-induced NF- κ B-mediated transactivation of a luciferase reporter gene. Furthermore, the two compounds dose-dependently reduced IL-8 and E-selectin protein expression after stimulation with lipopolysaccharide (LPS) or TNF- α in endothelial cells (ECs). The iridoids melampyroside and mussaenoside prevented the elevation of E-selectin in LPS-stimulated ECs. Lunularin was found to reduce the protein levels of the proinflammatory mediators E-selectin and IL-8 in ECs in response to LPS. These data validate the ethnomedical use of M. pratense for the treatment of inflammatory conditions and point to the constituents accountable for its anti-inflammatory activity.
RESUMO
Stromal derived factor 1 (SDF-1) is a CXC chemokine important in the homing process of stem cells to injured tissue. It has been implicated in healing and tissue repair. Growing evidence suggests that the glycoprotein-130 (gp130) ligand family is involved in repair processes in the heart. The aim of our study was to determine whether gp130 ligands could affect SDF-1 expression in cardiac cells. Human adult cardiac myocytes (HACMs) and fibroblasts (HACFs) were treated with gp130 ligands. Protein and mRNA levels of SDF-1 were determined using ELISA and RT-PCR, respectively. mRNA levels of SDF-1 were determined in human and mouse heart samples by RT-PCR. HACMs and HACFs constitutively express SDF-1, which was significantly up-regulated by the gp130 ligand oncostatin M (OSM). This effect was counteracted by a p38 inhibitor and to a lesser extent by a PI3K inhibitor. mRNA expression of SDF-1 in hearts of mice injected with OSM increased significantly. Levels of OSM and SDF-1 mRNA correlated significantly in human failing hearts. Our data, showing that OSM induces SDF-1 protein secretion in human cardiac cells in vitro and murine hearts in vivo, suggest that OSM via the induction of SDF-1 might play a key role in repair and tissue regeneration.
Assuntos
Quimiocina CXCL12/metabolismo , Inflamação/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Oncostatina M/metabolismo , Adulto , Animais , Células Cultivadas , Quimiocina CCL1/genética , Quimiocina CCL1/metabolismo , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Flavonoides/farmacologia , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oncostatina M/administração & dosagem , Oncostatina M/genética , Fatores de Tempo , Regulação para CimaRESUMO
Previous studies demonstrated that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine inhibits inflammatory effects of the bacterial lipopolisacharide (LPS, endotoxin). In this work we have characterized the anti-endotoxin activity of other classes of oxidized phospholipids with different polar head groups and fatty acid residues. LPS-induced expression of E-selectin on human endothelial cells was inhibited by oxidized phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidic acids. The anti-endotoxin effect insignificantly depended on the type of polyunsaturated fatty acids. Unoxidized phospholipids did not suppress effects of LPS. Thus, the anti-endotoxin activity of oxidized phospholipids crucially depends on the presence of oxidatively modified fatty acid residue.
Assuntos
Inflamação/prevenção & controle , Lipopolissacarídeos/farmacologia , Fosfolipídeos/uso terapêutico , Células Cultivadas , Selectina E/biossíntese , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Escherichia coli , Humanos , Inflamação/metabolismo , Oxirredução , Relação Estrutura-Atividade , Veias Umbilicais/citologiaRESUMO
The polymorphism of cytokine genes (11-1beta, IL-10, IL-6, and TGF-beta1) in patients with chronic C hepatitis and in healthy individuals was studied. The study found that the polymorphism of IL-1beta, IL-6, and TGF-beta1 may at least partially explain the genetic predisposition to an aggressive course of the disease with the development of terminal stage.
Assuntos
DNA/genética , Hepatite C Crônica/genética , Interleucina-10/genética , Interleucina-1beta/genética , Interleucina-6/genética , Polimorfismo Genético , Fator de Crescimento Transformador beta1/genética , Adolescente , Adulto , Progressão da Doença , Feminino , Predisposição Genética para Doença , Hepatite C Crônica/sangue , Hepatite C Crônica/complicações , Humanos , Interleucina-10/sangue , Interleucina-1beta/sangue , Interleucina-6/sangue , Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta1/sangueRESUMO
UNLABELLED: Ischemic heart disease (IHD) develops in patients with familial hypercholesterolemia (FHC) 15-20 years earlier than in general population. However age of onset of the disease, its clinical manifestations are variable and not completely determined by cholesterol level and class of low density lipoprotein receptor mutations. AIM: To elucidate associations of some auxiliary genetic factors -- such as C151565T, C677T, R353Q polymorphisms of glycoprotein IIIa (GPIIIa), methylenetetrahydrofolate reductase (MTHFR) and coagulation factor VII genes, respectively, -- with the presence of IHD in patients with FHC. MATERIAL: Patients with clinical diagnosis of heterozygous FHC (n=198) with (n=106) and without (n=92) IHD. RESULTS: Patients with compared with those without IHD had similar frequency of T-allele of MTHFR gene (p=0.519), more often had T-allele of GPIIIa gene (23 and 12.5%, respectively, p=0.009), and less often -- Q-allele of factor VII gene (13 and 21%, respectively, p=0.048). Multifactorial analysis showed that risk of IHD was higher in patients with TT compared with CC genotype of the GPIIIa gene (OR 1.53, 95%CI 1.12-2.3), and lower in patients with RQ and QQ compared with RR genotype of factor VII gene (OR 0.41, 95%CI 0.19-0.75). CONCLUSION: In patients with FHC polymorphisms in factor VII and GPIIIa genes but not C677T polymorphism of MTHFR gene were associated with the presence of IHD.
Assuntos
Hiperlipoproteinemia Tipo II/genética , Isquemia Miocárdica/etiologia , Adulto , Colesterol/sangue , Fator VII/genética , Feminino , Genótipo , Heterozigoto , Humanos , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/complicações , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Pessoa de Meia-Idade , Isquemia Miocárdica/sangue , Isquemia Miocárdica/genética , Polimorfismo Genético , Fatores de Risco , Fumar/efeitos adversos , Fatores de TempoRESUMO
In recent decades, studies have shown that lipoproteid physiological role goes beyond just their part in lipid transport. Database is accumulating on connection of lipoproteid pathological effects upon cardiovascular system with their ability to interfere in the signal transmission processes necessary for normal control of the vascular homeostasis. There is some evidence that lipoproteids are capable of affecting vascular tonus, coagulation balance, and inflammatory processes in the vascular wall. These effects will be realized through activation of the blood cell and vascular wall signal systems and can accelerate development of atherosclerosis and prompt hypertension and arterial thrombophilia.
Assuntos
Plaquetas/fisiologia , Vasos Sanguíneos/fisiopatologia , Lipoproteínas/fisiologia , Transdução de Sinais , Animais , Arteriosclerose/etiologia , Vasos Sanguíneos/patologia , Endotélio Vascular/fisiopatologia , Humanos , Hiperlipidemias/complicações , Hiperlipidemias/patologia , Hiperlipidemias/fisiopatologia , Hipertensão/etiologia , Hipertensão/fisiopatologia , Lipoproteínas/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Trombose/sangue , Trombose/etiologia , Trombose/fisiopatologiaRESUMO
UNLABELLED: Low density lipoprotein receptor (LDLR) gene mutations cause familial hypercholesterolemia which is associated with elevated risk of ischemic heart disease. AIM: To define LDLR gene mutations in unrelated patients with heterozygous familial hypercholesterolemia in Russia. METHODS: PCR- single-strand conformation polymorphism analysis, automated DNA sequencing, and test for the presence of the apolipoprotein (apo) B-3500 mutation known to induce hereditary defect in apo-B-100. RESULTS: We found 6 novel mutations of LDLR gene designated E8X, 230insG, 671_679dupGACAAATCT, W422R, D461Y, and V698L. We also identified three missense mutations - C139G, E207K and R395W, which were previously described in FH patients from western populations. None of the studied persons had apo-B-3500 mutation. CONCLUSION: These findings broaden knowledge on mutations responsible for development of familial hypercholesterolemia and confirm molecular heterogeneity of this disease in Russia.
Assuntos
Hiperlipoproteinemia Tipo II , Receptores de LDL , Apolipoproteína B-100 , Apolipoproteínas B , Humanos , Hiperlipoproteinemia Tipo II/genética , Mutação , Receptores de LDL/genéticaRESUMO
T-cadherin is an unusual glycosilphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. In contrast to classical cadherins, tissue distribution of T-cadherin so far remained unknown. We examined tissue distribution of T-cadherin in rats using Western blotting and immunohistochemical method. Our results show that T-cadherin is expressed in all types of muscles (cardiac, striated, and smooth muscles), in brain neurons, and spinal cord, in the vessel endothelium, at the apical pole of intestinal villar epithelium, in the basal layer of skin, and eosophagal epithelium. Blood-derived and lymphoid cells as well as connective tissue were T-cadherin-negative. The highest level of T-cadherin expression was revealed in the cardiovascular system. Although T-cadherin was detected in smooth muscle cells, its role in the intimal thickening and restenosis is not known. We examined T-cadherin expression within 1-28 days after balloon injury of rat left carotid arteries. T-cadherin expression was valued immunohistochemically with semiquantitative method. In uninjured arteries, T-cadherin was expressed in endothelial (vWF-positive) cells, and smooth muscle (alpha-actin-positive) cells (SMCs). After denudation of arterial wall, T-cadherin was present both in the media and neointima. We revealed dynamics of T-cadherin expression in the media of injured artery: an essential increase being registered at the stage of cell migration and proliferation in the media and neointima (1-7 days), followed by its decrease to the baseline level (10-28 days). The high upregulation of T-cadherin expression in the media and neointima during migration and proliferation of vascular cells after vessel injury enables us to suggest the involvement of T-cadherin in vessel remodeling after balloon catheter injury.
Assuntos
Caderinas/metabolismo , Lesões das Artérias Carótidas/metabolismo , Angioplastia Coronária com Balão , Animais , Western Blotting , Encéfalo/metabolismo , Caderinas/análise , Artérias Carótidas/metabolismo , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Epitélio/metabolismo , Esôfago/metabolismo , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Masculino , Músculo Esquelético/metabolismo , Músculo Liso/metabolismo , Miocárdio/metabolismo , Ratos , Ratos Wistar , Medula Espinal/metabolismo , Fatores de TempoAssuntos
Adesão Celular/fisiologia , Dinoprosta/farmacologia , Endotélio Vascular/metabolismo , Monócitos/metabolismo , Vasoconstritores/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprosta/análogos & derivados , Selectina E/metabolismo , Endotélio Vascular/citologia , F2-Isoprostanos , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Modelos Biológicos , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Receptores de Tromboxanos/antagonistas & inibidores , Tromboxanos/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Alterations in expression of surface adhesion molecules on resident vascular and blood-derived cells play a fundamental role in the pathogenesis of cardiovascular disease. Smooth muscle cells (SMCs) have been shown to express T-cadherin (T-cad), an unusual GPI-anchored member of the cadherin family of adhesion molecules. Particular relevance for T-cad in cardiovascular tissues is indicated by our present screen (immunoblotting) of human tissues and organs whereby highest expression of T-cad was found in aorta, carotid, iliac and renal arteries and heart. To explore the (patho)physiological role for T-cad in the vasculature we performed an immunohistochemical analysis of T-cad expression in normal human aorta and atherosclerotic lesions of varying severity. T-cad was present both in the intima and media and was expressed in endothelial cells (ECs), SMCs and pericytes, but not in monocytes/macrophages, foam cells and lymphocytes. In the adventitia T-cad was present in the wall of vasa vasorum and was expressed in ECs, SMCs and pericytes. T-cad was differentially expressed in SMCs from distinct vascular layers of normal aorta (for example, high in the subendothelial (proteoglycan) layer of the intima, low in the musculoelastic intimal layer and in the media), as well as at different stages of lesion progression. In SMCs there was an apparent inverse relationship between the intensities of T-cad and smooth muscle alpha-actin expression, this being most prominent in lesions. The findings suggest a phenotype-associated expression of T-cad which may be relevant to control of the normal vascular architecture and its remodelling during atherogenesis.
Assuntos
Arteriosclerose/metabolismo , Caderinas/metabolismo , Músculo Liso Vascular/metabolismo , Aorta/metabolismo , Células Cultivadas , Humanos , Immunoblotting , Imuno-Histoquímica , Túnica Íntima/metabolismo , Túnica Média/metabolismoRESUMO
AIM: To determine occurrence of apolipoprotein B-100 mutation in codon 3500 (apoB3500) in patients with primary hypercholesterolemia in Russia. MATERIALS AND METHODS: The study included 71 patients with clinical diagnosis of familial hypercholesterolemia (FH) and 24 relatives. All the subjects were tested for the presence of apoB3500 mutation using polymerase chain reaction and cleavage with restriction enzyme HhaII. Samples demonstrating anomalous pattern were further analysed by automatic DNA sequencing. RESULTS: Apob3500 mutation was detected in two (2.8%) female patients. In both cases cholesterol levels were severely increased although clinical features were different. CONCLUSION: Some cases of primary hypercholesterolemia in Russia may be due to familial defective apoB-100. Further screening of FH patients is required for a precise estimation of the incidence rate of familial defective apoB-100 in this country.
Assuntos
Apolipoproteínas B/genética , Análise Mutacional de DNA , DNA/análise , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/genética , Adulto , Apolipoproteína B-100 , Apolipoproteínas B/sangue , Colesterol/sangue , Códon/genética , Primers do DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/sangue , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Diagnóstico Diferencial , Feminino , Marcadores Genéticos , Humanos , Hiperlipoproteinemia Tipo II/sangue , Masculino , Mutação Puntual , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
T-cadherin (T-cad) is an unusual glycosylphosphatidylinositol-anchored member of the cadherin family of cell adhesion molecules. Binding of low density lipoproteins (LDLs) to T-cad can be demonstrated on Western blots of smooth muscle cell lysates, membranes and purified proteins. Using HEK293 cells transfected with human T-cad cDNA (T-cad+), we have investigated the adhesion properties of expressed mature and precursor proteins and examined the postulate that LDL represents a physiologically relevant ligand for T-cad. T-cad+ exhibits an increased Ca(2+)-dependent aggregation (vs. control) that was reduced by selective proteolytic cleavage of precursor T-cad and abolished after either proteolytic or phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of both mature and precursor proteins, indicating that both proteins function in intercellular adhesion. T-cad+ exhibited a significantly increased specific cell surface-binding of [(125)I]-LDL that was sensitive to PI-PLC pre-treatment of cells. Ca(2+)-dependent intercellular adhesion of T-cad+ was significantly inhibited by LDL. Our results support the suggestion that LDL is a physiologically relevant ligand for T-cad.
Assuntos
Caderinas/metabolismo , Lipoproteínas LDL/metabolismo , Caderinas/genética , Cálcio/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Humanos , Immunoblotting , Cinética , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Ligação Proteica , Transdução de Sinais , Fatores de Tempo , Transfecção , Fosfolipases Tipo C/metabolismoRESUMO
Potential antigenic determinants of the atypical lipoprotein-binding proteins T-cadherin (p105) and its precursor (p130) from cells of human smooth muscles were synthesized by the solid phase method according to the Fmoc-scheme. These corresponded to the 51-61, 140-160, 161-179, 260-271, 340-352, 350-362, and 370-385 sequences of p130 and were chosen on the basis of computer analysis of its antigenic structure. The conjugates of the peptides with horseradish peroxidase were used for the immunization of mice and rabbits. Antisera against the peptides corresponding to the 140-160, 161-179, and 260-271 sequences of p105 were shown by immunoblotting to react with p105, which we isolated from the vascular cells of smooth muscles and earlier identified as T-cadherin. These antisera inhibited the binding of low density lipoproteins with p105 in a dose-dependent manner. These results confirmed the identification of the p105 protein as T-cadherin and demonstrated the fundamental possibility of studying the interaction of this protein with low density lipoproteins by using antipeptide antibodies that inhibit binding.
Assuntos
Anticorpos/metabolismo , Caderinas/imunologia , Epitopos/imunologia , Lipoproteínas LDL/metabolismo , Fragmentos de Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Western Blotting , Caderinas/isolamento & purificação , Humanos , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/isolamento & purificação , Precursores de Proteínas/imunologia , Precursores de Proteínas/isolamento & purificação , CoelhosRESUMO
Atypical cell surface lipoprotein-binding proteins of 105 kDa and 130 kDa are present in membranes of vascular smooth muscle cells. We recently identified the 105 kDa protein from human aortic media as T-cadherin, an unusual glycosylphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. The goal of the present study was to determine the identity of 130 kDa lipoprotein-binding protein of smooth muscle cells. We applied different approaches that included protein sequencing of purified protein from human aortic media, the use of human T-cadherin peptide-specific antisera, and enzymatic treatment of cultured cells with trypsin and GPI-specific phospholipase C. Our results indicate that the 130 kDa protein is a partially processed form of T-cadherin which is attached to the membrane surface of smooth muscle cells via a GPI anchor and contains uncleaved N-terminal propeptide sequence. Our data disclose that, in contrast to classical cadherins, T-cadherin is expressed on the cell surface in both its precursor (130 kDa) and mature (105 kDa) forms.
Assuntos
Caderinas/análise , Membrana Celular/metabolismo , Músculo Liso Vascular/metabolismo , Precursores de Proteínas/análise , Receptores de LDL/análise , Aorta , Caderinas/imunologia , Células Cultivadas , Epitopos/imunologia , Humanos , Soros Imunes/imunologia , Immunoblotting , Peso Molecular , Receptores de LDL/químicaRESUMO
The expression level of two new lipoprotein-binding proteins p105 and p130 was maximal in inactive VSMC and could be suppressed by activators of proliferation. Both proteins were detected by antisera against three synthetic fragments of T-cadherin and were rendered soluble by GPI-specific phospholipase C. The findings suggest that the 105 kDa lipoprotein-binding protein is T-cadherin whereas p130 is a partially processed GPI-anchored precursor of T-cadherin.
Assuntos
Músculo Liso Vascular/metabolismo , Receptores de Lipoproteínas/genética , Adolescente , Adulto , Sequência de Aminoácidos , Aorta/metabolismo , Caderinas/genética , Caderinas/metabolismo , Membrana Celular/metabolismo , Humanos , Immunoblotting , Pessoa de Meia-Idade , Dados de Sequência Molecular , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Receptores de Lipoproteínas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The atypical low density lipoprotein (LDL) binding proteins (Mr 105 and 130 kDa; p105 and p130) in human aortic medial membranes and cultured human and rat aortic smooth muscle cells (SMC) have recently been identified as the cell adhesion glycoprotein T-cadherin. Although cadherins are generally recognized to be important regulators of morphogenesis, the function of T-cadherin in the vasculature is poorly understood. This study has examined the relationship between expression of T-cadherin and the density and proliferation status of SMC. T-cadherin (p105 and p130) levels in SMC lysates were measured on Western blots using ligand-binding techniques. T-cadherin expression was dependent upon cell density, and maximal levels were achieved at confluency. T-cadherin levels were reversibly modulated by switching cultures between serum-free (upmodulation) and serum-containing (downmodulation) conditions. Platelet-derived growth factor (PDGF)-BB, epidermal growth factor (EGF) or insulin-like growth factor (IGF) elicited a dose- and time-dependent downmodulation that was reversible after transfer of SMC to growth factor-free medium. Our results support the hypothesis that T-cadherin may function as a negative determinant of cell growth.
Assuntos
Caderinas/biossíntese , Glicoproteínas/biossíntese , Músculo Liso Vascular/metabolismo , Animais , Contagem de Células , Divisão Celular , Células Cultivadas , Meios de Cultura Livres de Soro , Humanos , Lipoproteínas/metabolismo , Músculo Liso Vascular/citologia , RatosRESUMO
Cadherins are a family of cellular adhesion proteins mediating homotypic cell-cell binding. In contrast to classical cadherins, T-cadherin does not possess the transmembrane and cytosolic domains known to be essential for tight mechanical coupling of cells, and is instead attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. This study explores the hypothesis that T-cadherin might function as a signal-transducing protein. Membranes from human and rat vascular smooth muscle cells were fractionated using Triton X-100 solubilization and density gradient centrifugation techniques. We demonstrate that T-cadherin is enriched in a minor detergent-insoluble low-density membrane domain and co-distributes with caveolin, a marker of caveolae. This domain was enriched in other GPI-anchored proteins (CD-59, uPA receptor) and signal-transducing molecules (G alpha s protein and Src-family kinases), but completely excluded cell-cell and cell-matrix adhesion molecules (N-cadherin and beta1-integrin). Coupling of T-cadherin with signalling molecules within caveolae might enable cellular signal transduction.