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1.
Nat Commun ; 12(1): 708, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514724

RESUMO

We report the development of a platform of dual targeting Fab (DutaFab) molecules, which comprise two spatially separated and independent binding sites within the human antibody CDR loops: the so-called H-side paratope encompassing HCDR1, HCDR3 and LCDR2, and the L-side paratope encompassing LCDR1, LCDR3 and HCDR2. Both paratopes can be independently selected and combined into the desired bispecific DutaFabs in a modular manner. X-ray crystal structures illustrate that DutaFabs are able to bind two target molecules simultaneously at the same Fv region comprising a VH-VL heterodimer. In the present study, this platform is applied to generate DutaFabs specific for VEGFA and PDGF-BB, which show high affinities, physico-chemical stability and solubility, as well as superior efficacy over anti-VEGF monotherapy in vivo. These molecules exemplify the usefulness of DutaFabs as a distinct class of antibody therapeutics, which is currently being evaluated in patients.


Assuntos
Anticorpos Biespecíficos/farmacologia , Neovascularização de Coroide/tratamento farmacológico , Desenvolvimento de Medicamentos/métodos , Fragmentos Fab das Imunoglobulinas/farmacologia , Engenharia de Proteínas , Sequência de Aminoácidos/genética , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Biespecíficos/ultraestrutura , Becaplermina/antagonistas & inibidores , Sítios de Ligação de Anticorpos/genética , Cristalografia por Raios X , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/uso terapêutico , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Concentração Inibidora 50 , Injeções Intravítreas , Masculino , Modelos Moleculares , Estudo de Prova de Conceito , Conformação Proteica , Ratos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores
2.
Life Sci ; 259: 118397, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32896557

RESUMO

There is increasing evidence that Bazedoxifene, as an FDA-approved selective estrogen inhibitor, approved by FDA, not only inhibits estrogen receptors, but also has other pharmacological effects. The purpose of this study was to investigate the effects of Bazedoxifene on the functional changes of vascular smooth muscle cells (VSMCs) after PDGF-BB stimulation. VSMCs were divided into control group, PDGF-BB treatment group, and PDGF-BB treatment group with different concentrations of Bazedoxifene. CCK-8 and EdU staining were used to determine the VSMCs viability and proliferation. Western blot was used to detect the expressions of vimentin, SMA, ERK, p-ERK, STAT3, p-STAT3, AKT, p-AKT, and LC3 I/II. Wound healing method was used to detect the migration of VSMCs. PDGF-BB treatment significantly enhanced the viability and proliferation of VSMCs as indicated by CCK-8 and EdU assays (P < 0.01), while Bazedoxifene pretreatment could reduce the increased viability and proliferation of VSMCs caused by PDGF-BB (P < 0.05). Wound healing test also showed Bazedoxifene significantly attenuated the migration in the PDGF-BB stimulated VSMCs (P < 0.01). PDGF-BB also induced the phenotypic switch and decreased the autophagy level in VSMCs, manifested as a reduction in vimentin, SMA, and LC3 II (P < 0.01). These effects of PDGF-BB were partially reversed by Bazedoxifene (P < 0.05). Bazedoxifene may inhibit the proliferation and migration of VSMCs through up-regulate the autophagy level after PDGF-BB stimulation.


Assuntos
Autofagia/efeitos dos fármacos , Becaplermina/farmacologia , Indóis/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Becaplermina/antagonistas & inibidores , Western Blotting , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Músculo Liso Vascular/citologia , Fenótipo
3.
Mol Med Rep ; 22(2): 886-894, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32467985

RESUMO

Increasing evidence suggests that T­cell immunoglobulin and mucin domain 3 (TIM­3) displays anti­atherosclerotic effects, but its role in vascular smooth muscle cells (VSMCs) has not been reported. The present study aimed to investigate the function of TIM­3 and its roles in human artery VSMCs (HASMCs). A protein array was used to investigate the TIM­3 protein expression profile, which indicated that TIM­3 expression was increased in the serum of patients with lower extremity arteriosclerosis obliterans disease (LEAOD) compared with healthy individuals. Immunohistochemistry and western blotting of arterial tissue further revealed that TIM­3 expression was increased in LEAOD artery tissue compared with normal artery tissue. Additionally, platelet­derived growth factor­BB (PDGF­BB) displayed a positive correlation with TIM­3 expression in HASMCs. TIM­3 decreased the migration and proliferation of PDGF­BB­induced HASMCs, and anti­TIM­3 blocked the effects of TIM­3. The effect of TIM­3 on the proliferation and migration of HASMCs was further investigated using LV­TIM­3­transduced cells. The results revealed that TIM­3 also inhibited PDGF­BB­induced expression of the inflammatory factors interleukin­6 and tumor necrosis factor­α by suppressing NF­κB activation. In summary, the present study revealed that TIM­3 displayed a regulatory role during the PDGF­BB­induced inflammatory reaction in HASMCs, which indicated that TIM­3 may display anti­atherosclerotic effects.


Assuntos
Artérias/metabolismo , Aterosclerose/metabolismo , Becaplermina/antagonistas & inibidores , Receptor Celular 2 do Vírus da Hepatite A/biossíntese , Receptor Celular 2 do Vírus da Hepatite A/sangue , Músculo Liso Vascular/metabolismo , Idoso , Artérias/citologia , Artérias/crescimento & desenvolvimento , Arteriosclerose Obliterante/sangue , Aterosclerose/induzido quimicamente , Becaplermina/efeitos adversos , Linhagem Celular , Movimento Celular , Proliferação de Células , Feminino , Humanos , Interleucina-6/metabolismo , Extremidade Inferior/irrigação sanguínea , Masculino , Pessoa de Meia-Idade , Músculo Liso Vascular/citologia , Músculo Liso Vascular/crescimento & desenvolvimento , NF-kappa B/metabolismo , Análise Serial de Proteínas , Transcriptoma , Fator de Necrose Tumoral alfa/metabolismo
4.
J Agric Food Chem ; 67(7): 1889-1901, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30661353

RESUMO

Chronic inflammation and proliferation play important roles in atherosclerosis progression. This study aimed to identify the mechanisms responsible for the anti-inflammatory and antiproliferative effects of melatonin on tumor necrosis factor-α (TNF-α)- and platelet-derived growth factor-BB (PDGF-BB)-treated rat aortic smooth muscle cells (RASMCs). Melatonin reduced TNF-α-induced RASMC inflammation by decreasing vascular cell adhesion molecule-1 (VCAM-1) expression and nuclear factor-kappa B (NF-κB) P65 activity by inhibiting P38 mitogen-activated protein kinase phosphorylation ( P < 0.05). Additionally, melatonin inhibited PDGF-BB-induced RASMC proliferation by reducing mammalian target of rapamycin (mTOR) phosphorylation ( P < 0.05) but not migration in vitro. Melatonin also reduced TNF-α- and PDGF-BB-induced reactive oxygen species (ROS) production ( P < 0.05). Furthermore, melatonin treatment (prevention and treatment groups) significantly repressed high cholesterol diet-stimulated atherosclerotic lesions in vivo (19.59 ± 4.11%, 20.28 ± 5.63%, 32.26 ± 12.06%, respectively, P < 0.05). Taken together, the present study demonstrated that melatonin attenuated TNF-α-induced RASMC inflammation and PDGF-BB-induced RASMC proliferation in cells and reduced atherosclerotic lesions in mice. These results showed that melatonin has anti-inflammatory and antiproliferative properties and may be a novel therapeutic target in atherosclerosis.


Assuntos
Anti-Inflamatórios/farmacologia , Apolipoproteínas E/deficiência , Aterosclerose/prevenção & controle , Melatonina/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Animais , Aorta , Becaplermina/antagonistas & inibidores , Becaplermina/farmacologia , Proliferação de Células/efeitos dos fármacos , Masculino , Camundongos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/química , Miócitos de Músculo Liso/patologia , NF-kappa B/análise , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , Ratos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/análise
5.
Drug Des Devel Ther ; 13: 285-290, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30666090

RESUMO

AIM: To testify the hypothesis that endostatin exerts antifibrotic effects in hepatic stellate cells (HSCs) by modulating RhoA (ras homolog gene family, member A)/ROCK 1 (Rho-associated protein kinase 1) signal pathways. MATERIALS AND METHODS: HSCs-T6 of passages 3-5 were cultured in DMEM and serum starved for 48 hours. HSCs were grouped as follows: control group, TGF-ß1 (transforming growth factor ß1) group, endostatin+TGF-ß1 group, PDGF-BB (platelet-derived growth factor-BB) group, and endostatin+PDGF-BB group. In the PDGF-BB group, HSCs were treated with PDGF-BB (200 ng/mL) for 72 hours; in the TGF-ß1 group, they were treated with TGF-ß1 (10 ng/mL) for 72 hours. In the Endostatin+TGF-ß1 group or Endostatin+PDGF-BB group, HSCs were treated with TGF-ß1 (10 ng/mL) or PDGF-BB (200 ng/mL) for 72 hours after pretreatment with endostatin (5 µg/mL) for 1 hour. In the control group, HSCs were only treated with serum-free DMEM for 72 hours. Collagen I was analyzed with ELISA. F-actin was detected with immunofluorescent staining. The mRNAs and proteins of α-smooth muscle actin, RhoA, and ROCK1 were analyzed by using real-time PCR and Western blot, respectively. RESULTS: TGF-ß1 and PDGF-BB promote the proliferation of HSCs significantly at 48 and 72 hours. Endostatin inhibits the proliferation effect induced by TGF-ß1 or PDGF-BB significantly (P<0.01). The expression of collagen I and F-actin was significantly upregulated in both TGF-ß1 and PDGF-BB groups than in the control group (P<0.01). Both the collagen I and F-actin expression were downregulated significantly in the endostatin-treated groups (P<0.05). Endostatin significantly inhibited the upregulated expression of α-smooth muscle actin, RhoA, and ROCK1 induced by TGF-ß1 or PDGF-BB (P<0.01). CONCLUSION: These results suggested that endostatin inhibited TGF-ß1- or PDGF-BB-induced fibrosis in HSCs by modulating RhoA/ROCK signal pathways.


Assuntos
Antineoplásicos/farmacologia , Becaplermina/antagonistas & inibidores , Endostatinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Animais , Becaplermina/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Ratos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
6.
Eur J Pharmacol ; 842: 329-337, 2019 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-30395849

RESUMO

In proliferative vitreoretinopathy (PVR), the proliferation and migration of retinal pigment epithelial (RPE) cells are important to pathogenesis. Platelet-derived growth factor (PDGF) is an important factor in the underlying mechanism. Several studies have shown that PDGF induced the proliferation and migration effects on RPE cells in PVR. Crocetin-anantioxidant carotenoid that is abundant in saffron-has been shown to suppress the migration and proliferation of many cell types, but studies of the effects on RPE cell migration and proliferation are incomplete. Therefore, we investigated the inhibitory effect of crocetin on the proliferation and migration of ARPE-19 cells induced by PDGF-BB, an isoform of PDGF. The proliferation of cells was assessed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays. The apoptosis of cells was assessed by flow cytometric analysis. The migration of RPE cells was detected by a Transwell migration assay and an in vitro scratch assay. The levels of main regulatory proteins for apoptosis and the PDGF-BB-induced signaling pathway were determined by western blot analysis. The proliferation and migration of ARPE-19 cells treated with crocetin (100-400 µM) and PDGF-BB (20 ng/ml) were significantly inhibited in a concentration- and time-dependent manner. Crocetin exhibited potent inducing effects on the apoptosis of PDGF-BB-induced ARPE-19 cells via the modulation of Bcl-2 family regulators in a concentration-dependent manner. The inhibitory effects of crocetin on PDGF-BB-induced platelet-derived growth factor receptor ß (PDGFRß) and the underlying pathways of PI3K/Akt and ERK, p38, JNK activation were identified. The results showed that crocetin is an effective inhibitor of PDGF-BB-induced proliferation and migration of ARPE-19 cell through the downregulation of regulatory signaling pathways.


Assuntos
Becaplermina/antagonistas & inibidores , Becaplermina/farmacologia , Carotenoides/farmacologia , Movimento Celular/efeitos dos fármacos , Epitélio Pigmentado da Retina/citologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Vitamina A/análogos & derivados
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