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1.
Biotechnol Lett ; 45(10): 1265-1277, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37606752

RESUMO

OBJECTIVES: Gene therapy using viral vectors and antibody-based therapies continue to expand within the pharmaceutical market. We evaluated whether Cellhesion® VP, a chitin-based material, can be used as 3D culture platform for cell lines used for the production of antibodies and viral vectors. RESULTS: The results of Cell Counting Kit-8 assay and LDH assay revealed that Cellhesion® VP had no adverse effect to Human Embryonic Kidney (HEK) 293, A549 and Chinese hamster ovary (CHO) DG44-Interferon-ß (IFN) cells. Cell growth analyses showed that Cellhesion® VP supported the 3D culture of HEK293, A549 and CHO DG44- IFN-ß cells with a spherical morphology. Importantly, subculture of these cell lines on Cellhesion® VP was easily performed without trypsinization because cells readily transferred to newly added scaffold. Our data also suggest that CHO DG44-IFNß, cultured on Cellhesion® VP secreted IFNß stably and continuously during the culture period. CONCLUSIONS: Cellhesion® VP provides a simple and streamlined expansion culture system for the production of biopharmaceuticals.


Assuntos
Produtos Biológicos , Animais , Cricetinae , Humanos , Células HEK293 , Quitina , Células CHO , Cricetulus , Técnicas de Cultura de Células
2.
In Vitro Cell Dev Biol Anim ; 59(7): 505-513, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37507645

RESUMO

Human vascular smooth muscle cells (SMCs) are adherent cells, and they cannot survive without scaffolds in suspension culture. Here, we aimed to establish a suspension culture of SMCs using the functional biopolymer FP003 and to investigate the proliferation status of the cells. When SMCs were suspension cultured with FP003, their proliferation was inhibited with a viability of 75% until day 15. When SMCs were re-plated on plastic plates after suspension culture with FP003 for 48 h, the SMCs proliferated as in a normal plate culture. The SMCs cultured in suspension with FP003 showed a relatively low phosphorylation of retinoblastoma protein, low expression of cyclin D1, high proportion of G0/G1 phase cells, low proportion of S phase cells, and no obvious signs of apoptosis, indicating that this culture system inhibited progression from the G1 to S phase. This growth arrest was a reversible property that showed no significant changes in the expressions of the marker proteins α-smooth muscle actin and smooth muscle myosin heavy chain. These results suggest that human SMCs can be stably cultured in suspension with FP003 without losing their characteristics when they are cultured on plastic plates again.


Assuntos
Músculo Liso Vascular , Proteínas , Humanos , Animais , Células Cultivadas , Proteínas/metabolismo , Biopolímeros/metabolismo , Miócitos de Músculo Liso/metabolismo
3.
Biomaterials ; 288: 121696, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36038421

RESUMO

Currently, there are many challenges in the culture of human induced pluripotent stem (iPS) cell-derived intestinal organoids (HIOs) for use in drug discovery, disease research, and regenerative medicine. For example, the main culture method, embedding culture, makes industrial large-scale culture difficult, and Matrigel, which is used for almost all HIO cultures, is not respected for its application in regenerative medicine. To overcome these challenges, we herein propose a new culture method using low concentrations of natural polysaccharides in a suspension culture. In the present study, five natural polysaccharides free from heterologous animal-derived components were used, and HIOs were successfully cultured in suspension with FP001 and FP003, which are microbial exopolysaccharide analogs of gellan gum. The fabricated HIOs were similar to living intestinal tracts with respect to their gene expression, microstructure, and protein expression. The observed activities of the drug metabolizing enzymes and drug transporters in the generated HIOs suggested that they have pharmacokinetic functions. We believe that suspension culture of HIOs using FP001 or FP003 can be widely applied to not only drug discovery research but also disease research and regenerative medicine.


Assuntos
Células-Tronco Pluripotentes Induzidas , Organoides , Animais , Diferenciação Celular/genética , Humanos , Mucosa Intestinal/metabolismo , Intestinos , Polissacarídeos/metabolismo , Compostos de Enxofre
4.
Cells ; 11(6)2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35326446

RESUMO

Mesenchymal stem cell (MSC) transplantation, in particular allogeneic transplantation, is a promising therapy for a variety of diseases. However, before performing allograft treatment it is necessary to find suitable donors, establish culture methods that maintain cell quality, and reduce cell production costs. Here, we present a new method of producing allogeneic MSCs combining human umbilical cord-derived mesenchymal stem cells (UCMSCs) and chitin-based polysaccharide fibers (Cellhesion® MS). UCMSC numbers significantly increased, and cells grew as dispersed spheres on Cellhesion® MS. Subsequent biological analyses showed that the expression levels of stemness-related and migration-related genes were significantly upregulated, including octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), and C-X-C chemokine receptor type 4 (CXCR4). The secretion levels of paracrine factors such as prostaglandin E2 (PGE2), TNFα-stimulating gene (TSG)-6, fibroblast growth factor 2 (bFGF), and Angiogenin (Ang) from UCMSCs using Cellhesion® MS were significantly higher than with microcarrier and U-bottom plate culture. In addition, culture supernatant from UCMSCs with Cellhesion® MS had better angiogenic potential than that from monolayer cultured UCMSCs. Furthermore, we succeeded in a scaled-up culture of UCMSCs with Cellhesion® MS using a closed culture bag. Therefore, Cellhesion® MS is a key material for producing high-quality UCMSCs in a three-dimensional (3D) culture system.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Quitina/metabolismo , Quitina/farmacologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Cordão Umbilical
5.
Biomaterials ; 271: 120742, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33706111

RESUMO

Mesenchymal stem cell (MSC) transplantation is a promising therapy for regenerative medicine. However, MSCs grown under two-dimensional (2D) culture conditions differ significantly in cell shape from those in the body, with downregulated stemness genes and secretion of paracrine factors. Here, we evaluated the effect of 3D culture using Cellhesion VP, a water-insoluble material composed of chitin-based polysaccharide fibers, on the characteristics of human Wharton's jelly-derived MSCs (hMSCs). Cellhesion VP significantly increased cell proliferation after retrieval. Transcriptome analyses suggested that genes involved in cell stemness, migration ability, and extracellular vesicle (EV) production were enhanced by 3D culture. Subsequent biochemical analyses showed that the expression levels of stemness genes including OCT4, NANOG, and SSEA4 were upregulated and migration capacity was elevated in 3D-cultured hMSCs. In addition, EV production was significantly elevated in 3D cells, which contained a distinct protein profile from 2D cells. Gene and drug connectivity analyses revealed that the 2D and 3D EVs had similar functions as immunomodulators; however, 3D EVs had completely distinct therapeutic profiles for various infectious and metabolic diseases based on activation of disease-associated signaling pathways. Therefore, EVs from Cellhesion VP-primed hMSCs offer a new treatment for immune and metabolic diseases.


Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Geleia de Wharton , Humanos , Fatores Imunológicos , Medicina Regenerativa
6.
Pharmaceutics ; 12(10)2020 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-33050367

RESUMO

The evaluation of drug pharmacokinetics in the small intestine is critical for developing orally administered drugs. Caucasian colon adenocarcinoma (Caco-2) cells are employed to evaluate drug absorption in preclinical trials of drug development. However, the pharmacokinetic characteristics of Caco-2 cells are different from those of the normal human small intestine. Besides this, it is almost impossible to obtain primary human intestinal epithelial cells of the same batch. Therefore, human iPS cell-derived enterocytes (hiPSEs) with pharmacokinetic functions similar to human intestinal epithelial cells are expected to be useful for the evaluation of drug absorption. Previous studies have been limited to the use of cytokines and small molecules to generate hiPSEs. Dietary fibers play a critical role in maintaining intestinal physiology. We used gellan gum (GG), a soluble dietary fiber, to optimize hiPSE differentiation. hiPSEs cocultured with GG had significantly higher expression of small intestine- and pharmacokinetics-related genes and proteins. The activities of drug-metabolizing enzymes, such as cytochrome P450 2C19, and peptide transporter 1 were significantly increased in the GG treatment group compared to the control group. At the end point of differentiation, the percentage of senescent cells increased. Therefore, GG could improve the differentiation efficiency of human iPS cells to enterocytes and increase intestinal maturation by extending the life span of hiPSEs.

7.
Org Biomol Chem ; 17(19): 4835-4842, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31033986

RESUMO

For the improvement of nuclease resistance, four kinds of new modifications through a carbamoylethyl linker were designed. Among them, the 2'-O-[2-N-{2-(benzimidazol-1-yl)ethyl}carbamoylethyl] modification showed 20-fold longer half-life when treated with a 3' to 5' exonuclease compared to the 2'-O-methoxyethyl (MOE) modification, which is used in approved drugs. In addition, this large modification did not disturb the binding affinity or RNase H-dependent antisense activity. From these findings, it could be concluded that an adequate linker, such as carbamoylethyl in this study, could extend the utility of 2'-O-modification without loss of the properties of nucleic acids. This strategy would be useful for the development of nucleic acid therapeutics.


Assuntos
Oligonucleotídeos/química , Ribonucleases/química , Animais , Camundongos , Conformação de Ácido Nucleico , Oligonucleotídeos/síntese química , Ribonucleases/metabolismo
8.
PLoS One ; 13(9): e0200515, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30240390

RESUMO

Scalable production of avian cell lines exhibits a valuable potential on therapeutic application by producing recombinant proteins and as the substrate for virus growth due to the special glycosylation occurs in avian species. Chicken primordial germ cells (cPGCs), a germinal pluripotent avian cell type, present the ability of self-renewal, an anchorage-independent cell growth and the ability to be genetically modified. This cell type could be an interesting bioreactor system for industrial purposes. This study sought to establish an expandable culture system with defined components for three-dimensional (3D) culture of cPGCs. cPGCs were cultured in medium supplemented with the functional polymer FP003. Viscoelasticity was low in this medium but cPGCs did not sediment in culture and efficiencies of space and nutrient utilization were thus enhanced and consequently their expansion was improved. The total number of cPGCs increased by 17-fold after 1 week of culture in 3D-FAot medium, an aseric defined medium containing FP003 polymer, FGF2 and Activin A as growth factors and Ovotransferrin as protein. Moreover, cPGC cell lines stably expressed the germline-specific reporter VASA:tdTOMATO, as well as other markers of cPGCs, for more than 1 month upon culture in 3D-FAot medium, indicating that the characteristics of these cells are maintained. In summary, this novel 3D culture system can be used to efficiently expand cPGCs in suspension without mechanical stirring, which is available for long-term culture and no loss of cellular properties was found. This system provides a platform for large-scale culture of cPGCs.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Meios de Cultura/farmacologia , Células Germinativas/citologia , Células Germinativas/metabolismo , Animais , Embrião de Galinha , Galinhas
9.
Nucleic Acid Ther ; 28(5): 307-311, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30020852

RESUMO

An RNase H-dependent antisense oligonucleotide (ASO), having the 2'-O-(2-N-methylcarbamoylethyl) (MCE) modification, was evaluated in vitro and in vivo. The antisense activities of an ASO having the MCE modification were comparable with those of an ASO having the 2'-O-methoxyethyl (MOE) modification in both in vitro and in vivo experiments. In contrast, the hepatotoxic potential of the ASO having the MCE modification was lower than that of the ASO having the MOE modification. Thus, these findings suggested that the MCE modification could be used as an alternative to the MOE modification.


Assuntos
Oligodesoxirribonucleotídeos/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , RNA Mensageiro/administração & dosagem , Ribonuclease H/genética , Animais , Humanos , Fígado/efeitos dos fármacos , Camundongos , Nucleotídeos/efeitos adversos , Nucleotídeos/química , Nucleotídeos/genética , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Oligonucleotídeos Antissenso/efeitos adversos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Ribonuclease H/administração & dosagem , Ribonuclease H/química
10.
In Vitro Cell Dev Biol Anim ; 53(3): 191-198, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27924445

RESUMO

The proliferation of vascular smooth muscle cells (SMCs) causes restenosis in biomaterial vascular grafts. The purposes of this study were to establish a suspension culture system for SMCs by using a novel substrate, low-acyl gellan gum (GG) and to maintain SMCs in a state of growth inhibition. When SMCs were cultured in suspension with GG, their proliferation was inhibited. Their viability was 70% at day 2, which was maintained at more than 50% until day 5. In contrast, the viability of cells cultured in suspension without GG was 5.6% at day 2. By cell cycle analysis, the ratio of SMCs in the S phase when cultured in suspension with GG was lower than when cultured on plastic plates. In SMCs cultured in suspension with GG, the ratio of phosphorylated retinoblastoma (Rb) protein to Rb protein was decreased and p27Kip1 expression was unchanged in comparison with SMCs cultured on plastic plates. In addition, SMCs could be induced to proliferate again by changing the culture condition from suspension with GG to plastic plates. These results suggest that our established culturing method for SMCs is useful to maintain SMCs in a state of growth inhibition with high viability.


Assuntos
Proliferação de Células/efeitos dos fármacos , Meios de Cultura/farmacologia , Músculo Liso Vascular/citologia , Polissacarídeos Bacterianos/farmacologia , Animais , Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Polissacarídeos Bacterianos/química , Coelhos , Proteína do Retinoblastoma/biossíntese , Proteína do Retinoblastoma/genética , Suspensões/química , Suspensões/farmacologia
11.
Cancer Sci ; 107(12): 1858-1866, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27699918

RESUMO

Anticancer drug discovery efforts have used 2-D cell-based assay models, which fail to forecast in vivo efficacy and result in a lower success rate of clinical approval. Recent 3-D cell culture models are expected to bridge the gap between 2-D and in vivo models. However, 3-D cell culture methods that are available for practical anticancer drug screening have not yet been fully attained. In this study, we screened several polymers for their ability to suspend cells or cell spheroids homogeneously in a liquid medium without changing the viscosity behavior, and identified gellan gum (FP001), as the most potent polymer. FP001 promoted cell dispersion in the medium and improved the proliferation of a wide range of cancer cell lines under low attachment conditions by inhibiting the formation of large-sized spheroids. In addition, cancer cells cultured with FP001-containing medium were more susceptible to inhibitors of epidermal growth factor (EGF) signaling than those cultured under attachment conditions. We also showed that ligands of the EGF receptor family clearly enhance proliferation of SKOV3 ovarian carcinoma cells under anchorage-independent conditions with FP001. Consistent with this result, the cells grown with FP001 showed higher EGF receptor content compared with cells cultured under attachment conditions. In conclusion, we developed a novel 3-D cell culture system that is available for high throughput screening of anticancer agents, and is suitable for evaluation of molecular-targeted anticancer drugs. Three-dimensional cell culture using FP001 will be of value in the development of useful technologies for anticancer drug discovery.


Assuntos
Antineoplásicos/farmacologia , Técnicas de Cultura de Células , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Humanos , Esferoides Celulares , Células Tumorais Cultivadas
12.
Hepatol Res ; 46(10): 1045-57, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26724677

RESUMO

AIM: The hepatoma-derived cell line HepaRG is regarded as an in vitro model of drug metabolism because fully differentiated HepaRG cells demonstrate functional metabolic responses comparable to those of primary human hepatocytes. Recently, it was demonstrated that the 3D culture of HepaRG cells enhanced their metabolic functions and toxicological responses. We approached the mechanisms underlying these enhancement effects. METHODS: We compared 2D-cultured HepaRG cells with 3D-cultured HepaRG spheroids in the gene expression patterns and the metabolic functions. In the present study, we performed 3D culture of HepaRG cells using functional polymers (FP). To reveal the in vivo differentiation ability, we transplanted the 3D-cultured HepaRG spheroids into TK-NOG mice. RESULTS: A comparison between 2D and 3D cultures revealed that 3D-cultured HepaRG spheroids demonstrated reductions in bile duct marker expression, accelerated expression of cytochrome P450 3A4, and increases in the ratio of albumin-expressing hepatocytes. Furthermore, catalytic activities of cytochrome P450 3A4 were modified by omeprazole and rifampicin in the 3D-cultured HepaRG spheroids. Transplantation analysis revealed that 3D-cultured HepaRG spheroids formed hepatocyte-like colonies rather than cholangiocytes in vivo. CONCLUSION: Our results indicated that the enhancement of hepatic functions in 3D-cultured HepaRG cells was induced by selective hepatocyte differentiation and accelerated hepatocyte maturation. HepaRG spheroids reproduced the metabolic responses of human hepatocytes. Therefore, FP-dependent 3D-cultured HepaRG cells may serve as an excellent in vitro model for evaluating the hepatic metabolism and toxicity.

13.
Br J Pharmacol ; 159(7): 1418-28, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20233214

RESUMO

BACKGROUND AND PURPOSE: It is not clear if the new 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor pitavastatin prevents atherogenesis by a direct effect. Statins have a cholesterol-lowering effect, so an accessible animal model of atherosclerosis showing only moderate hypercholesterolaemia as in humans, is needed. The effects of pitavastatin were evaluated on atherosclerotic lesions accumulating foam cells derived from macrophages, produced in rabbits with moderate hypercholesterolaemia by chronic inhibition of nitric oxide synthase (NOS). EXPERIMENTAL APPROACH: White New Zealand rabbits were fed a 0.2% cholesterol diet with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) in the same diet. Pitavastatin (0.1 and 0.3 mg x kg(-1)) was given orally once a day for 8 weeks. The aortic arch and thoracic aorta were analysed by histochemistry and atherosclerotic lesions were quantified. The effect of pitavastatin on adhesion of THP-1 cells to endothelial cells, and cholesterol content in RAW264.7 cells incubated with oxidized or acetylated LDL were also investigated. KEY RESULTS: Atherosclerotic lesions containing foam cells were induced in a model of atherosclerosis in rabbits with moderate hypercholesterolaemia by chronic inhibition of NOS. The area of atherosclerotic lesions was diminished by pitavastatin administration. The adhesion of THP-1 cells and cholesteryl ester content in RAW macrophages were decreased by pitavastatin treatment. CONCLUSION: Atherosclerosis induced by chronic inhibition of NOS in moderately hypercholesterolaemic rabbits was suppressed by pitavastatin via inhibition of macrophage accumulation and macrophage foam cell formation.


Assuntos
Aterosclerose/etiologia , Inibidores Enzimáticos/farmacologia , Hipercolesterolemia/metabolismo , Óxido Nítrico/antagonistas & inibidores , Quinolinas/farmacologia , Animais , Aterosclerose/prevenção & controle , Adesão Celular/efeitos dos fármacos , Endotélio Vascular/citologia , Humanos , Hipercolesterolemia/complicações , Imuno-Histoquímica , Masculino , Monócitos/citologia , Óxido Nítrico/biossíntese , Coelhos
14.
J Clin Invest ; 118(8): 2733-46, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18618022

RESUMO

Medial-to-intimal migration of SMCs is critical to atherosclerotic plaque formation and remodeling of injured arteries. Considerable amounts of the shed soluble form of the LDL receptor relative LR11 (sLR11) produced by intimal SMCs enhance SMC migration in vitro via upregulation of urokinase-type plasminogen activator receptor (uPAR) expression. Here, we show that circulating sLR11 is a novel marker of carotid intima-media thickness (IMT) and that targeted disruption of the LR11 gene greatly reduces intimal thickening of arteries through attenuation of Ang II-induced migration of SMCs. Serum concentrations of sLR11 were positively correlated with IMT in dyslipidemic subjects, and multivariable regression analysis suggested sLR11 levels as an index of IMT, independent of classical atherosclerosis risk factors. In Lr11-/- mice, femoral artery intimal thickness after cuff placement was decreased, and Ang II-stimulated migration and attachment of SMCs from these mice were largely abolished. In isolated murine SMCs, sLR11 caused membrane ruffle formation via activation of focal adhesion kinase/ERK/Rac1 accompanied by complex formation between uPAR and integrin alphavbeta3, a process accelerated by Ang II. Overproduction of sLR11 decreased the sensitivity of Ang II-induced activation pathways to inhibition by an Ang II type 1 receptor blocker in mice. Thus, we demonstrate a requirement for sLR11 in Ang II-induced SMC migration and propose what we believe is a novel role for sLR11 as a biomarker of carotid IMT.


Assuntos
Angiotensina II/farmacologia , Biomarcadores/sangue , Movimento Celular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Receptores de LDL/sangue , Receptores de LDL/fisiologia , Animais , Movimento Celular/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados , Relação Dose-Resposta a Droga , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Knockout , Modelos Biológicos , Receptores de LDL/genética , Solubilidade , Fatores de Tempo
15.
Biochem Biophys Res Commun ; 348(4): 1367-77, 2006 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-16919601

RESUMO

Statins, inhibitors of HMG-CoA reductase, elicit various actions on vascular cells including the modulation of proliferation and migration of smooth muscle cells (SMCs). Here, we have elucidated the mechanism by which statins, in particular pitavastatin, attenuate the migration activity of SMCs. The expression of LR11, a member of the LDL receptor family and an enhancer of cell surface localization of urokinase-type plasminogen activator receptor (uPAR), is increased in cultured SMCs by treatment with PDGF-BB. Pitavastatin attenuates the PDGF-BB -induced surface expression of LR11 and uPAR. The increased migration of SMCs observed both upon overexpression of LR11 and via stimulation of secretion of soluble LR11 is not reversed by pitavastatin. In vivo studies showed that the SMCs expressing LR11 in plaques are almost congruent with intimal cells expressing nonmuscle myosin heavy chain (SMemb). Pitavastatin reduced the expression of LR11 and SMemb, and the levels of LR11, uPAR, and SMemb in cultured intimal SMCs were reduced to those seen in medial SMCs. We propose that this statin reduces PDGF-induced migration through the attenuation of the LR11/uPAR system in SMCs. Modulation of the LR11/uPAR system with statins suggests a novel treatment strategy for atherogenesis based on suppression of intimal SMC migration.


Assuntos
Movimento Celular/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Quinolinas/farmacologia , Receptores de LDL/antagonistas & inibidores , Animais , Becaplermina , Células Cultivadas , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Coelhos , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Receptores de LDL/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase
16.
Metabolism ; 54(5): 573-8, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15877285

RESUMO

4-(5 H -2,3-(2,5-Dimethyl-2,5-hexano)-5-methyl-8-nitrodibenzo[b,e][1,4]diazepin-11-yl)benzoic acid (HX531) is a novel retinoid X receptor antagonist. This study provides evidence that HX531 improves leptin resistance without increasing plasma leptin levels in KK-A y mice, an animal model with high plasma leptin levels and leptin resistance. Under normal dietary conditions, 3 weeks of treatment with HX531 (0.03% and 0.06% food admixture) in KK-A y mice decreased plasma leptin levels in a dose- and time-dependent manner, in addition to decreasing body weight and mesenteric fatty tissue weight. To evaluate the effect of HX531 on leptin resistance, leptin was injected intraperitoneally in the KK-A y mice for 4 days after 1 week of treatment with HX531 (0.06% food admixture). This pretreatment with HX531 resulted in exogenously administered leptin causing a significant decrease in food intake. These results suggested that HX531 decreased plasma leptin levels accompanied by a decrease in fatty tissue content in the KK-A y mice and a simultaneous improvement in leptin resistance. This is the first report that HX531 improves leptin resistance without increasing plasma leptin level in KK-A y mice, under normal dietary conditions.


Assuntos
Benzoatos/farmacologia , Compostos de Bifenilo/farmacologia , Diabetes Mellitus Tipo 2/fisiopatologia , Dieta , Leptina/metabolismo , Receptores X de Retinoides/antagonistas & inibidores , Ácido 3-Hidroxibutírico/sangue , Tecido Adiposo/patologia , Animais , Temperatura Corporal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Resistência a Medicamentos/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Privação de Alimentos , Leptina/sangue , Leptina/farmacologia , Masculino , Mesentério , Camundongos , Camundongos Endogâmicos , Reto/fisiopatologia
17.
Arterioscler Thromb Vasc Biol ; 24(8): 1422-8, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15166012

RESUMO

OBJECTIVE: Studies on the involvement of low-density lipoprotein receptor relatives (LRs) in atherosclerosis have recently gained new focus because of the specific expression of certain of these receptors in the thickened intima. Here, we show that LRP1B, a member of LRs, modulates the migration of smooth muscle cells (SMCs) by increasing the degradation of membrane receptors, urokinase-type plasminogen activator receptor (uPAR), and platelet-derived growth factor receptor (PDGFR) beta. METHODS AND RESULTS: Immunohistochemistry showed that LRP1B expression in human coronary arteries is localized to the intimal SMCs near the plaque surface as well as to medial SMCs. LRP1B expression levels in cultured SMCs increase at the late phase of proliferation. Cell surface and internalization assays, in combination with coimmunoprecipitation experiments, showed that LRP1B binds and internalizes uPAR. Metabolic labeling analysis demonstrated that anti-LRP1B IgY decreased the catabolism of uPAR. In addition, the anti-LRP1B antibody raised PDGFRbeta protein and PDGFR-mediated phosphorylation levels of ERK1/2. Finally, the anti-LRP1B IgY enhanced the migration and invasion of SMCs in the presence of PDGF-BB. CONCLUSIONS: LRP1B modulates the catabolism of uPAR and PDGFR, affecting the migration of SMCs. This functional characterization of LRP1B opens novel avenues for elucidating the (patho)physiological significance of SMC migration in atheromatous plaques.


Assuntos
Membrana Celular/metabolismo , Vasos Coronários/patologia , Músculo Liso Vascular/citologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de LDL/fisiologia , Idoso , Sequência de Aminoácidos , Angina Instável/patologia , Animais , Becaplermina , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Galinhas , Vasos Coronários/química , Vasos Coronários/ultraestrutura , Humanos , Imunoglobulinas/imunologia , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Músculo Liso Vascular/metabolismo , Infarto do Miocárdio/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/ultraestrutura , Fosforilação , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-sis , Receptor beta de Fator de Crescimento Derivado de Plaquetas/efeitos dos fármacos , Receptores de LDL/genética , Receptores de LDL/imunologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Túnica Íntima/patologia
18.
Circ Res ; 94(6): 752-8, 2004 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-14764453

RESUMO

LR11, a member of the LDL receptor family, is highly expressed in vascular smooth muscle cells (SMCs) of the hyperplastic intima, and induces enhanced migration of SMCs in vitro via its upregulation of urokinase-type plasminogen activator receptor (uPAR) expression. In this study, we have delineated the mechanism by which LR11 elevates the expression levels of uPAR in SMCs. Secretion of soluble LR11 is induced in SMCs during the rapidly proliferating phase, and the secreted LR11 induces the migration activities of SMCs. Both the cell-anchored and secreted forms of LR11 have the capacity to bind to and form complexes with uPAR. LR11-overexpressing cells show significantly enhanced uPAR binding, but decreased uPAR internalization. LR11 colocalizes with uPAR on the cell surface and inhibits the LDL receptor-related protein (LRP)-mediated binding and internalization of uPAR. Thus, LR11 mediates the uPAR localization to the plasma membrane. LR11 is highly expressed in the atheromatous plaque areas of apoE knockout mice, particularly in the intimal SMCs at the border between intima and media. The neutralization of LR11 function with anti-LR11 antibody reduced cuff-induced intimal thickness in mice. The novel mechanism of regulation of uPAR localization in SMCs accompanied with enhanced migration activity possibly constitutes an important factor in the process of atherosclerosis and arterial remodeling.


Assuntos
Proteínas de Membrana Transportadoras/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Receptores de LDL/fisiologia , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Células COS , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas/citologia , Chlorocebus aethiops , Colágeno , Meios de Cultivo Condicionados/farmacologia , DNA Complementar/genética , Endocitose , Ligantes , Proteínas de Membrana/fisiologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Coelhos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de LDL/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Transfecção
19.
Metabolism ; 52(12): 1633-7, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14669169

RESUMO

NIP-222 is a novel peroxisome proliferator-activated receptor (PPAR)gamma agonist. This study provides evidence that NIP-222 decreases urinary albumin excretion (UAE) in diabetic mice independent of its PPARgamma activation. We compared the effect of NIP-222 and another PPARgamma agonist, troglitazone, on UAE, plasma glucose level, blood pressure, and creatinine clearance (C(cr)) in streptozotocin (STZ)-induced diabetic mice. Treatment for 3 weeks with NIP-222 (30 mg/kg) was associated with a significant decrease in UAE without any change in blood pressure, creatinine clearance, or plasma glucose level. In contrast, UAE did not decrease in mice treated with troglitazone (300 mg/kg). These results indicate that NIP-222 has PPARgamma independent effects on UAE in diabetic mice and suggest that this agent may have potential to minimize the development and progression of diabetic nephropathy.


Assuntos
Albuminúria/tratamento farmacológico , Diabetes Mellitus Experimental/complicações , Hipoglicemiantes/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/agonistas , Fatores de Transcrição/metabolismo , Albuminúria/complicações , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Glicemia/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Captopril/farmacologia , Cromanos/farmacologia , Diabetes Mellitus Experimental/urina , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Genes Reporter/genética , Rim/efeitos dos fármacos , Rim/patologia , Luciferases/genética , Masculino , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Plasmídeos/genética , Tiazolidinedionas/farmacologia , Troglitazona , Urodinâmica/efeitos dos fármacos
20.
Biochem Biophys Res Commun ; 294(2): 231-7, 2002 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-12051699

RESUMO

Platelet-derived growth factor (PDGF) stimulates expression of matrix metalloproteinases (MMPs), including stromelysin-1 (MMP-3). Induction of these expressions is known to occur during the course of atherosclerosis, tumor invasion, and metastasis. We investigated PDGF-alpha receptor (alphaR)- and beta receptor (betaR)-mediated signaling pathways for the expression of MMP-3 and invasion activity using porcine aortic endothelial (PAE) cells with stable expression of normal or mutated PDGF receptors. RT-PCR and Western blot analyses revealed that PDGF-BB induces MMP-3 expression in PAE cells that exclusively express either the PDGF-alphaR or the -betaR, but not in non-transfected control cells. To identify the signals necessary for PDGF receptor-mediated induction of MMP-3 expression, several lines of PAE cells expressing mutant PDGF receptors were further analyzed. Cells expressing mutant PDGF receptors unable to associate with Src or PLCgamma, retained the ability to induce MMP-3 expression as a result of PDGF-BB stimulation. However, incubation with PDGF-BB did not induce MMP-3 expression in cells expressing a mutant PDGF-betaR unable to associate with phosphatidylinositol 3(')-kinase (PI3K). LY294002, a PI3K inhibitor, reduced PDGF-BB-stimulated MMP-3 expression in PAE cells expressing wild-type PDGF receptors. In contrast, PDGF-BB induced MMP-3 expression in the presence of U-73122, a PLCgamma inhibitor. Moreover, PDGF-BB enhanced the invasiveness of cells expressing wild type PDGF-beta receptors, but not of cells expressing mutant PDGF-betaRs impaired in their ability to associate with PI3K. In light of these results, it appears that PDGF-BB is capable of inducing MMP-3 expression through both the PDGF-alphaR and the -betaR, and the effects are contributed by the PI3K-mediated transduction pathways.


Assuntos
Endotélio Vascular/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Mutação , Receptores do Fator de Crescimento Derivado de Plaquetas/biossíntese , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Animais , Aorta , Becaplermina , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Metaloproteinase 3 da Matriz/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosfolipase C gama , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , RNA Mensageiro/biossíntese , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/biossíntese , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossíntese , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Suínos , Fosfolipases Tipo C/antagonistas & inibidores
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