RESUMEN
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.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Neovascularización Coroidal/tratamiento farmacológico , Desarrollo de Medicamentos/métodos , Fragmentos Fab de Inmunoglobulinas/farmacología , Ingeniería de Proteínas , Secuencia de Aminoácidos/genética , Animales , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Biespecíficos/ultraestructura , Becaplermina/antagonistas & inhibidores , Sitios de Unión de Anticuerpos/genética , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Fragmentos Fab de Inmunoglobulinas/ultraestructura , Concentración 50 Inhibidora , Inyecciones Intravítreas , Masculino , Modelos Moleculares , Prueba de Estudio Conceptual , Conformación Proteica , Ratas , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Autofagia/efectos de los fármacos , Becaplermina/farmacología , Indoles/farmacología , Músculo Liso Vascular/efectos de los fármacos , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Becaplermina/antagonistas & inhibidores , Western Blotting , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Músculo Liso Vascular/citología , FenotipoRESUMEN
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.
Asunto(s)
Arterias/metabolismo , Aterosclerosis/metabolismo , Becaplermina/antagonistas & inhibidores , Receptor 2 Celular del Virus de la Hepatitis A/biosíntesis , Receptor 2 Celular del Virus de la Hepatitis A/sangre , Músculo Liso Vascular/metabolismo , Anciano , Arterias/citología , Arterias/crecimiento & desarrollo , Arteriosclerosis Obliterante/sangre , Aterosclerosis/inducido químicamente , Becaplermina/efectos adversos , Línea Celular , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Interleucina-6/metabolismo , Extremidad Inferior/irrigación sanguínea , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/citología , Músculo Liso Vascular/crecimiento & desarrollo , FN-kappa B/metabolismo , Análisis por Matrices de Proteínas , Transcriptoma , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Pulmonary hypertension (PH) is characterized by a thickening of the distal pulmonary arteries caused by medial hypertrophy, intimal proliferation and vascular fibrosis. Low density lipoprotein receptor-related protein 1 (LRP1) maintains vascular homeostasis by mediating endocytosis of numerous ligands and by initiating and regulating signaling pathways. Here, we demonstrate the increased levels of LRP1 protein in the lungs of idiopathic pulmonary arterial hypertension (IPAH) patients, hypoxia-exposed mice, and monocrotaline-treated rats. Platelet-derived growth factor (PDGF)-BB upregulated LRP1 expression in pulmonary artery smooth muscle cells (PASMC). This effect was reversed by the PDGF-BB neutralizing antibody or the PDGF receptor antagonist. Depletion of LRP1 decreased proliferation of donor and IPAH PASMC in a ß1-integrin-dependent manner. Furthermore, LRP1 silencing attenuated the expression of fibronectin and collagen I and increased the levels of α-smooth muscle actin and myocardin in donor, but not in IPAH, PASMC. In addition, smooth muscle cell (SMC)-specific LRP1 knockout augmented α-SMA expression in pulmonary vessels and reduced SMC proliferation in 3D ex vivo murine lung tissue cultures. In conclusion, our results indicate that LRP1 promotes the dedifferentiation of PASMC from a contractile to a synthetic phenotype thus suggesting its contribution to vascular remodeling in PH.
Asunto(s)
Becaplermina/genética , Desdiferenciación Celular/genética , Hipertensión Pulmonar Primaria Familiar/genética , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Miocitos del Músculo Liso/metabolismo , Actinas/genética , Actinas/metabolismo , Adulto , Animales , Anticuerpos Neutralizantes/farmacología , Becaplermina/antagonistas & inhibidores , Becaplermina/metabolismo , Estudios de Casos y Controles , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animales de Enfermedad , Hipertensión Pulmonar Primaria Familiar/inducido químicamente , Hipertensión Pulmonar Primaria Familiar/metabolismo , Hipertensión Pulmonar Primaria Familiar/patología , Femenino , Fibronectinas/genética , Fibronectinas/metabolismo , Regulación de la Expresión Génica , Homeostasis/genética , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/antagonistas & inhibidores , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Monocrotalina/administración & dosificación , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal , Técnicas de Cultivo de Tejidos , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
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.
Asunto(s)
Antiinflamatorios/farmacología , Apolipoproteínas E/deficiencia , Aterosclerosis/prevención & control , Melatonina/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Animales , Aorta , Becaplermina/antagonistas & inhibidores , Becaplermina/farmacología , Proliferación Celular/efectos de los fármacos , Masculino , Ratones , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/patología , FN-kappa B/análisis , Fosforilación/efectos de los fármacos , Células RAW 264.7 , Ratas , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/análisisRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Becaplermina/antagonistas & inhibidores , Endostatinas/farmacología , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Quinasas Asociadas a rho/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Animales , Becaplermina/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Ratas , Transducción de Señal/genética , Factor de Crecimiento Transformador beta1/metabolismo , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
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⯵M) and PDGF-BB (20â¯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.